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Antiviral agents targeting the influenza virus: a review and publication analysis
L. Eyer, K. Hruska
Veterinary Research Institute, Brno, Czech Republic
ABSTRACT: Influenza is a serious infectious disease, which is life-threatening especially in children, seniors and immunocompromised patients. In addition to vaccination, the development of new anti-influenza agents represents a crucial defence strategy to combat seasonal and pandemic influenza strains. At present most attention is paid to the development of inhibitors of influenza neuraminidase, which has been established as a key drug target for the prophylaxis and treatment of influenza infections. However, the emergence of drug-resistant influenza vari-ants highlights the need of continuously innovative strategies for the development of new drugs with improved antiviral effects, higher safety and increased tolerability. In this review article, an analysis of publications describ-ing anti-influenza agents indexed in the Web of Science® database has been carried out. The most important publications are presented in tables and are characterised by several key words, abstracts and references. The presented publications have been sorted according to five basic criteria: (i) review articles, (ii) design, synthesis and evaluation of new anti-influenza drugs, (iii) major classes of anti-influenza drugs, (iv) combination therapy of influenza infections and (v) influenza drug resistance. The design of this review article allows us to offer a complex overview of known antiviral agents targeting influenza viruses, facilitates easy and rapid orientation in numerous publications written on this subject, and aids the gathering of required data.
Keywords: influenza; virus; antiviral; agents; drug; resistance; therapy; structure-based drug design
Contents
1. Introduction1.1. Database used as the source of information
Table 1.1. Analysis of publications: search profiles and numbers of results retrieved1.2. Basic analysis of publications on antiviral agents targeting the influenza virus
Figure 1. The number of publications on anti-influenza agents during the period from 1990 to 2012Table 1.2. Analysis of publications: TopTen authors, institutions and countries according to the number
of published papers (Web of Science®, publications in total 14 458)2. Review articles
Table 2. Review articles3. Design, synthesis and evaluation of new anti-influenza drugs
Table 3. Design, synthesis and evaluation of new anti-influenza drugs4. Major classes of anti-influenza drugs
Figure 2. Life-cycle of the influenza virus (according to Beigel and Bray 2008)Figure 3. Structure of the influenza virus (according to Ludwig et al. 2003) Figure 4. Chemical structures of the most important anti-influenza drugs
4.1. Inhibitors of haemagglutininTable 4.1. Inhibitors of haemagglutinin
Supported by the Ministry of Education, Youth and Sports, Czech Republic (AdmireVet; Grant No. CZ 1.05/2.1.00/01.0006-ED 0006/01/01) and the Ministry of Agriculture of the Czech Republic (Grant No. MZe 0002716202).
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1. InTRoduCTIon
Influenza is considered to be one of the life-threatening infectious diseases. In some countries seasonal influenza affects annually up to 40% of the population and 500 million people die from it worldwide every year. New highly-virulent in-fluenza strains can arise unexpectedly to cause world-wide pandemics with markedly increased morbidity and mortality, such as the “avian flu” in 1997 and “swine flu” in 2009. At present, the development of antiviral drugs represents a crucial strategy in the control and prevention of seasonal and pandemic influenza infections. Antiviral drugs can overcome the limitations of vaccination strate-gies, such as the time-consuming vaccine design, insufficient protection for immunocompromised patients and the unpredictable antigenic changes in influenza strains which render vaccination inef-fective. In general, the anti-influenza agents can be divided into two basic groups, i.e., synthetic analogues of biomolecules required during virus infection and substances derived from natural plant extracts. With regard to the considerable genetic and antigenic variability of the influenza virus, research has been predominantly focused on broad-spectrum antiviral drugs, which are effective against a large variety of influenza strains. The de-velopment of antivirals targeting host-cell proteins, which play an important role in viral replication, has also gathered pace recently. Moreover, combi-nation therapy based on the application of two or more different antivirals represents a promising approach to combat influenza infections.
The aim of this review is to offer an overview of known antiviral agents targeting influenza viruses and to discuss their characteristic properties, modes of action and advantages or limitations of their ther-apeutic use. Publications which contain key informa-tion concerning the issues of anti-influenza agents are in this review presented in tables and character-ised with descriptive words, full or shortened ab-stracts and relevant references. The text in the tables contains several format imperfections, which exist in the Web of Science® database and are caused by transmission and copying of data between various information sources. The loss of cursive typeface in Latin names in titles of references and in abstracts is such an example. The presented publications have been classified according to five basic criteria: (i) re-view articles, (ii) design, synthesis and evaluation of new anti-influenza drugs, (iii) major classes of anti-influenza drugs, (iv) combination therapy of in-fluenza infections and (v) influenza drug resistance. This review article should facilitate orientation in numerous publications dealing with anti-influenza drugs and enable rapid and easy search for specific data, information and protocols. The previous re-views in this format have been well received (Vass et al. 2008; Eyer and Hruska 2012; Hruska and Kaevska 2012; Hruska and Franek 2012).
1.1. database used as the source of information
The publications were retrieved from the Web of Science® database using the general search profile:
4.2. M2 ion channel blockersTable 4.2. M2 ion channel blockers
4.3. Inhibitors of viral RNA polymeraseTable 4.3. Inhibitors of viral RNA polymerase
4.4. Inhibitors of neuraminidaseTable 4.4. Inhibitors of neuraminidase
4.5. Host cell factor targetingTable 4.5. Host cell factor targeting
4.6. Other anti-influenza agentsTable 4.6. Other anti-influenza agents
5. Combination therapy of influenza infectionsTable 5. Combination therapy of influenza infections
6. Influenza drug resistanceTable 6. Influenza drug resistance
7. Acknowledgement8. References
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Table 1.1. Analysis of publications: search profiles and numbers of results retrieved
Timespan All years 2010–2012General search profileTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*)Results 14 458 4 673Review articles cited in Table 2 17design, synthesis and evaluation of new anti-influenza drugsTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND (design OR synthesis OR evaluation)Results 3083 1048Cited in Table 3 19Haemagglutinin inhibitorsTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND (hemagglutinin OR haemagglutinin)Results 2616 742Cited in Table 4.1 18M2 ion channel blockersTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND (adamantane* OR amantadine OR rimantadine)Results 897 249Cited in Table 4.2 15Inhibitors of viral RnA polymeraseTopic= (influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND polymerase*Results 824 344Cited in Table 4.3 22Inhibitors of neuraminidaseTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND neuraminidase*Results 2658 930Cited in Table 4.4 21Host cell factor targetingTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND (“host factor*” OR “host protein*”)Results 43 22Cited in Table 4.5 14other anti-influenza agentsTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND (nucleoprotein* OR “non-structural protein” OR siRNA* OR plant* OR herbal* OR antibod*)Results 3899 1382Cited in Table 4.6 16Combination therapy of influenza infectionsTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND “combination therapy”Results 102 38Cited in Table 5 11Influenza drug resistanceTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND resistanceResults 1757 700Cited in Table 6 19
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Topic = (influenza OR flu) AND (antivir* OR vi-rostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*), Timespan = all years. A total of 14 458 publications were obtained, demonstrating the huge number of papers on the subject pub-lished during the period from 1945 to 2012. To ease orientation through the numerous publications, the results were subsequently refined using more specific search profiles as described in Table 1.1. A selection of the most important publications has been based on reading abstracts and hundreds of available full papers.
1.2. Basic analysis of publications on antiviral agents targeting the influenza virus
The Web of Science® utilities were employed for search result analysis. Regarding the high num-ber of retrieved publications, attention has been predominantly paid to papers published in the pe-riod 2010 to 2012; however, some older key papers are also included in the tables. As is evident from Figure 1, publication activity on the topic has con-tinuously increased since the early 1990s. In the last three years, 4673 papers were published, with 1529 papers in 2012. The oldest papers were published in the late 1940s and describe the research on the first influenza haemagglutinin inhibitors in pigs. In 1960s, the antiviral effects of the first ion channel blockers were reported. The latest publications are focused on computer-aided structure-based design of new neuraminidase and viral polymerase inhibi-tors to combat pandemic influenza strains. Of all the published papers, original research articles pre-vail (82.7%). Our analysis shows that 2938 institu-tions from 112 countries are concerned with the
Table 1.2. Analysis of publications: TopTen authors, institutions and countries according to the number of published papers (Web of Science®, publications in total 14 458)
Item Number of publications
Authors (12 596 in total)
Garcia-Sastre A 156
Webster RG 112
Suzuki Y 103
Hayden FG 95
Kawaoka Y 85
Gubareva LV 74
Suzuki T 66
Palese P 62
Kochs G 61
Li Y 61
Institutions (2938 in total)
Centers for Disease Control and Prevention 307
Harvard University 197
Chinese Academy of Sciences 175
Icahn School of Medicine at Mount Sinai, New York 165
National Institute of Allergy and Infectious Diseases 164
University of Virginia 155
University of Wisconsin 150
University of Washington 149
University of Oxford 133
Emory University 125
Countries (112 in total)
USA 5695
Japan 1344
England 1169
People’s Republic of China 1097
Germany 963
Canada 710
France 653
Australia 637
Italy 561
Netherlands 484
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Figure 1. The number of publications on anti-influenza agents during the period from 1990 to 2012
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The designed structures that are predicted to show high affinity to the viral target are then syn-thesised using various chemical procedures (Shie et al. 2011; Rawat et al. 2012) and their anti-influ-enza effects are evaluated using standardised in vitro screening methods. These methods include biochemical assays (Hung et al. 2012) and cell-based antiviral screens (Schmidtke et al. 2001), such as the plaque reduction assay to monitor viral replication efficiency, dye-uptake assays for measuring virus cytopathic effect, and yield-re-duction assays for quantification of specific virus antigens. Through the combination of bioinfor-matic approaches, applicative robotics and min-iaturisation strategies, most of these techniques can be adapted to high-throughput screen for-mats, which are applicable for testing large drug libraries containing millions of unique chemical structures (Dai et al. 2012). As an alternative to experimental screening methods, virtual (com-putational) screening can be used to select the desired chemical structure from large molecular databases (Yamada et al. 2012).
Besides in vitro tests, potential influenza virus inhibitors are further studied using animal models, such as the ferret, laboratory mouse, and chicken (Yoshimoto et al. 2000; Sauerbrei et al. 2006). Such in vivo studies play a crucial role in the preclini-cal evaluation of new drugs. The final step in the development of new medications is represented by clinical trials, which focus particularly on clinical pharmacokinetic studies and evaluation of antiviral efficacy, safety and tolerability in human volunteers (He et al. 1999; Cao et al. 2012). For key publica-tions describing the design, synthesis and evalua-tion of anti-influenza drugs see Table 3.
4. Major classes of anti-influenza drugs
The anti-influenza drugs are usually classified ac-cording to their target in the viral life-cycle, which is schematically depicted in Figure 2. Such antiviral molecules are particularly used as inhibitors of the following processes: attachment of the virus to host cell receptors, endocytosis and fusion of viral and cell membranes, replication and transcription of the viral genome, synthesis of viral proteins, as-sembly of the viral progeny and release of the new virions into the outside environment. The following paragraphs are focused on the description of basic classes of influenza virus inhibitors.
subject of antiviral agents targeting the influenza virus. The authors, institutions and countries which show the highest publication activities (TopTen) are listed in Table 1.2.
2. Review articles
Review articles represent 9.2% of all publications retrieved from the Web of Science® database us-ing the above mentioned search profile. In Table 2, seventeen key review articles are presented and characterised with a few descriptive words (left column), abstract (in the middle), and source ref-erence (right column). These papers offer overall reviews on all important topics, especially regard-ing structure-based drug design techniques (Du et al. 2012), chemical synthesis of antiviral drugs (Gong and Xu 2008; Marra et al. 2008), biochemi-cal and cell-based antiviral screen assays (Tisdale 2000; Atkins et al. 2012), animal models for study of influenza virus inhibitors (Sidwell and Smee 2000), structure, chemical properties and modes of ac-tion of the major groups of anti-influenza drugs (Gong et al. 2009; Chintakrindi et al. 2012; Grienke et al. 2012), influenza virus-neutralising antibod-ies (Martinez et al. 2009), natural products with antiviral activity (Grienke et al. 2012), combina-tion therapy of influenza infections (Kaihatsu and Barnard 2012) and mechanisms of influenza drug resistance (Pizzorno et al. 2011). All the topics are also discussed in detail in the original papers pre-sented in Tables 3 to 6.
3. design, synthesis and evaluation of new anti-influenza drugs
The first anti-influenza drugs were usually iden-tified using large-scale screening methods or by chance and their chemical structure and modes of action were not completely understood (Davies et al. 1964). In contrast, the current development of new antivirals is based on detailed knowledge of the X-ray crystallography-derived structure of influenza proteins as drug targets. Such an inven-tive process of finding new drugs, which is termed structure-based drug design, involves the devel-opment of organic molecules or macromolecular scaffolds that are complementary in shape and charge to potential ligand-binding pockets of the viral target protein (Kim et al. 1997; Lv et al. 2011).
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(Figure 4), have been licensed for influenza control and are commercially available under the trade-marks Symmetrel® and Flumadine®, respectively (Galvao et al. 2012). The adamantanes are relatively cheap, highly stable in storage and show strong anti-influenza activity at micromolar concentra-tions. At present, the application of adamantanes for prevention and treatment of influenza infec-tions is, however, not recommended because of the rapid emergence of drug-resistant virus vari-ants that retain full virulence and transmissibility (Bright et al. 2005; Barr et al. 2008). Moreover, seri-ous gastrointestinal and neurological side effects were observed in patients undergoing adamantane therapy (Galvao et al. 2012). Another disadvantage of adamantanes is their strong specificity against influenza A strains only (Rosenberg and Casarotto 2010). Selected publications describing the struc-ture, chemical properties and application of ada-mantanes, adamantane-derivatives and other M2 ion channel blockers are presented in Table 4.2.
4.3. Inhibitors of viral RnA polymerase
Transcription and replication of the influenza virus genome is carried out by the influenza RNA polymerase holoenzyme, which is characterised by two catalytic activities. Polymerase activity is
needed for the elongation of nascent RNA chains, whereas endonuclease activity is essential for cleavage of the 5’-capped primer sequence of the host mRNA. The cap is the terminal 7-methyl-guanosin bound through a triphosphate group to the host mRNA. This “cap snatching” process is needed for the initiation of viral RNA transcrip-tion (Lv et al. 2011). Influenza RNA polymerase is an extremely suitable target for the develop-ment of new broad-specific antivirals because of its highly conserved structure among influenza strains. It is thought that the influenza polymerase plays a crucial role in virus adaptation to human-to-human transmission and, consequently, in the formation of pandemic influenza variants (Miotto et al. 2008; Boivin et al. 2010; Aggarwal et al. 2011; Ping et al. 2011).
Two basic classes of RNA polymerase inhibitors have been described based on different mecha-nisms of action. The first group is represented by nucleoside analogues for the blocking of viral RNA chain elongation (Tisdale et al. 1995). A typ-ical member of this group is favipiravir (T-705, Figure 4), which is an inhibitor of influenza A, B and C strains, including variants resistant to amantadine or oseltamivir. This compound is currently in the stage of clinical testing (Furuta et al. 2005). Other nucleoside analogs with anti-influenza activity include ribavirin (Virazole®)
Figure 4. Chemical structures of the most important anti-influenza drugs; (a) amantadine, (b) riman-tadine, (c) favipiravir (T-705), (d) ribavirin, (e) viramidine, (f ) zan-amivir, (g) oseltamivir
(a) (b)
(c) (d) (e)
(f ) (g)
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and its derivative viramidine, originaly licensed for treatment of hepatitis C infections (Figure 4). Their application is, however, sometimes con-nected with the development of haemolytic anae-mia (Sidwell et al. 2005). The second class of antiviral molecules targeting the influenza poly-merase is represented by compounds which block the endonuclease and cap-binding domains of the polymerase holoenzyme. These antivirals include cap analogues (Lv et al. 2011), short capped oligo-nucleotides (Tado et al. 2001), and small organic compounds, such as 4-substitued 2,4-phenylbu-tanoic acid (Hastings et al. 1996) and flutimide isolated from the fungus Delitschia confertaspora (Tomassini et al. 1996). For the representative publications concerning viral RNA polymerase inhibitors, see Table 4.3.
4.4. Inhibitors of neuraminidase
Neuraminidase, also referred to as sialidase, is an antigenic glycoprotein anchored in the surface en-velope of the influenza virions, which hydrolytically cleaves the terminal sialic acid from the host cell receptors (Figure 3). Thus, it plays a crucial role in the release of viral progeny from the membranes of infected cells, prevents self-aggregation of virions and facilitates the movement of the infectious viral particles in the mucus of the respiratory epithelia (Matrosovich et al. 2004; Suzuki et al. 2005). Influenza neuraminidase has been established as a key drug target for the prophylaxis and treatment of influenza infections, predominantly for the following reasons: Firstly, the structure of the influenza neuraminidase active site is highly conserved between influenza A and B strains, making neuraminidase an attractive target for the development of broad-spectrum in-hibitors (Yen et al. 2006). Secondly, resistance to neuraminidase inhibitors develops less commonly than to other anti-influenza drugs. Nevertheless, the intensive application of neuraminidase inhibitors for influenza treatment results in a permanently in-creasing number of drug-resistant strains (Garcia et al. 2009). Thirdly, in contrast to adamantanes, neuraminidase inhibitors are mostly well tolerated in patients under therapy (Cao et al. 2012). Finally, neuraminidase protein is a freely accessible target for antiviral molecules with an extracellular mode of action.
The development of neuraminidase inhibi-tors started in the middle 1970s, when the first
structural analogues of sialic acid were described and denoted as DANA (2-deoxy-2,3-didehydro-N-acetyl neuraminic acid) and its trifluoroacetyl derivative FANA (Schulman and Palese 1975). At present, several licensed anti-influenza medica-tions are available on the market, most notably the inhalant zanamivir with the trademark Releza®, and the orally administered oseltamivir (Tamiflu®) having excellent bioavailability and relatively long half-life in vivo (He et al. 1999; Greengard et al. 2000) (Figure 4). In response to the emergence of some oseltamivir-resistant influenza strains, peramivir and laninamivir have been recently developed (Bantia et al. 2006; Kubo et al. 2010). New-generation neuraminidase inhibitors are cur-rently under investigation, e.g., multimeric forms of zanamivir (Watson et al. 2004), dual-targeted bifunctional antivirals (Liu et al. 2012), and several herbal remedies, such as flavonols, alkaloids and saponins (Jeong et al. 2009). The key publications on the structure, synthesis and therapeutic appli-cation of neuraminidase inhibitors are presented in Table 4.4.
4.5. Host cell factor targeting
Many human host cell molecules play a crucial role in influenza virus propagation and, there-fore, represent promising targets for the design of new generation inhibitors of the virus-cell interaction. Muller et al. (2012) describes in his review 35 cellular factors essential for influenza virus infection for which 57 inhibitors with ap-parent anti-influenza activity are available. The most intensively studied are the compounds which effectively inhibit intracellular signalling cascades with a resulting negative influence on the establishment of viral infection (Nacken et al. 2012). Studies have also focused on inhibi-tors of vacuolar proton-ATPase which render the viral M2 ion channels inactive (Guinea and Carrasco 1994), inhibitors of cellular proteases which block the proteolytic activation of haemag-glutinin (Zhirnov et al. 2011), and blockers of the cellular ubiquitin-proteasome system (Dudek et al. 2010). Although the development of host fac-tor inhibitors is a promising research strategy to limit the emergence of drug-resistant mutants, their possible toxic side-effects in vivo need to be carefully studied. Selected papers on the topic are presented in Table 4.5.
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4.6. other anti-influenza agents
During the last decades, a large variety of chemical compounds with anti-influenza activity have been investigated. Several examples of such novel drugs are the inhibitors of viral nucleoprotein (Hung et al. 2012), blockers of influenza non-structural pro-teins (Basu et al. 2009) and short interfering oligo-nucleotides (siRNAs) used for viral RNA silencing (Stewart et al. 2011). Using large-scale screening techniques, new antiviral molecules which show significant anti-influenza effects have been identi-fied; however, their chemical structure and mecha-nism of action remains unknown. These include, for instance, natural substances isolated from plants in chemical and pharmaceutical studies (He et al. 2012; Jiao et al. 2012). Another important group of prospective therapeutics are monoclonal antibod-ies and recombinant antibody fragments with high virus-neutralising activities (Hanson et al. 2006; Wei et al. 2011). Sixteen publications describing alterna-tive anti-influenza agents are presented in Table 4.6.
5. Combination therapy of influenza infections
Recent in vitro and in vivo studies have demon-strated that the simultaneous application of two or more anti-influenza drugs with different modes of action, e.g. oseltamivir and amantadine, results in increased virus inhibition and enhanced thera-peutic efficiency (Masihi et al. 2007). Similar find-ings were made with the combination of influenza virus inhibitors and immunomodulatory agents, especially corticosteroids (Zheng et al. 2008; Quispe-Laime et al. 2010). These observations are in accordance with the hypothesis that the applied drugs exert additive or synergistic anti-influenza effects in the infected cells. Such a combination regimen enables a reduction in the concentration of the individual inhibitory drugs, resulting in de-creased drug toxicity and a reduced risk of antivi-ral resistance emergence in seasonal and pandemic influenza viruses (Govorkova et al. 2004; Smee et al. 2010; Nguyen et al. 2012). The principals of the combination therapy can in the future become a crucial strategy not only in the treatment of influ-enza infections, but also in the therapy of other se-rious viral, bacterial and parasitic diseases. Selected publications, which discuss combination antiviral therapy, are presented in Table 5.
6. Influenza drug resistance
As with all antimicrobials, propagation of vi-ruses in the presence of antiviral drugs increases the selection pressure for mutations in the viral target proteins, which results in the induction of virus drug resistance. As an example, adamantane-resistant strains are typically characterised by a single substitution in the transmembrane region of the M2 ion channel (Saito et al. 2003; Shiraishi et al. 2003). On the other hand, resistance to neu-raminidase inhibitors can result from mutations in the neuraminidase active cavity, but also from amino acid substitutions on the molecular surface of the neuraminidase protein (Yen et al. 2006; Du et al. 2010). It is noteworthy that resistance to adamantanes is acquired rapidly and by a high number of virus strains (Bright et al. 2005). In contrast to adamantane resistance, neuraminidase inhibitor resistance has developed over a longer time period and occurs with a relatively lower fre-quency (Garcia et al. 2009). This may be due to the fact that some mutations significantly affect viral infectivity and ability to replicate in the host cell. While amantadine-resistant strains do not show growth or virulence impairment (Sweet et al. 1991), influenza variants resistant to oseltamivir exhibit reduced neuraminidase activity and viral fitness in vitro (Yen et al. 2006), and decreased transmissibility in ferret models (Herlocher et al. 2004). The increasing emergence of drug-resistant influenza strains highlights the need to search continuously for innovative strategies for the de-velopment of new drugs with improved antiviral effects, higher safety and better tolerability. For key publications describing the mechanisms of viral drug resistance, as well as prevalence and clinical impact of drug resistant influenza strains see Table 6.
7. Acknowledgement
We wish to thank A. Durisova (Veterinary Research Institute, Brno) for her excellent graphical service.
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Received: 2013–03–25Accepted after corrections: 2013–04–05
Corresponding Author:
Mgr. Ludek Eyer, Ph.D., Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech RepublicTel. +420 533 331 911, E-mail: [email protected]
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
130
Tabl
e 2.
Rev
iew
art
icle
s
Stru
ctur
e-ba
sed
drug
de
sign
Neu
ram
inid
ase
inhi
bito
rs
Dru
g re
sist
ance
Rece
nt e
mer
genc
e of
influ
enza
A H
5N1
and
H1N
1 st
rain
s ha
s he
ight
ened
con
cern
, esp
ecia
lly a
s a
resu
lt of
thei
r dr
ug re
sist
ance
. The
life
cyc
le o
f inf
luen
za v
irus
es h
as b
een
wel
l stu
died
and
nea
rly
all t
he v
iral
pro
tein
s ar
e be
com
-in
g po
tent
ial t
hera
peut
ic ta
rget
s. In
this
revi
ew, w
e pr
esen
t an
over
view
of r
ecen
t pro
gres
s in
str
uctu
re-b
ased
ant
i-in
fluen
za d
rug
desi
gn, p
ayin
g cl
ose
atte
ntio
n to
the
incr
easi
ng ro
le o
f com
puta
tion
and
stra
tegi
es fo
r ov
erco
min
g dr
ug
resi
stan
ce.
Du
et a
l. 20
12
Stru
ctur
e-ba
sed
drug
de
sign
Hae
mag
glut
inin
inhi
bito
rN
eura
min
idas
e in
hibi
tor
M2
inhi
bito
rPo
lym
eras
e in
hibi
tor
Kin
ase
inhi
bito
r
In th
is re
view
, we
will
dis
cuss
dru
g de
sign
bas
ed o
n pr
oven
and
pot
entia
l ant
i-in
fluen
za d
rug
targ
ets
incl
udin
g vi
ral
hem
aggl
utin
in (H
A),
neur
amin
idas
e (N
A),
M2
ion
chan
nel,
3P p
olym
eras
e co
mpl
ex, a
nd h
ost f
acto
rs s
uch
as k
inas
es.
We
have
sum
mar
ized
influ
enza
inhi
bito
rs b
ased
on
thei
r m
ode
of a
ctio
ns. F
or in
stan
ce, i
nclu
ded
are
desc
ript
ions
of
(1) i
nhib
itors
of H
A c
leav
age,
suc
h is
naf
amos
tat,
cam
osta
t, ga
bexa
te, e
psilo
n-am
inoc
apro
nic
acid
and
apr
otin
in,
(2) i
nhib
itors
of f
usio
n an
d en
try,
suc
h as
ben
zoqu
inon
es a
nd h
ydro
quir
ione
s, C
L 38
5319
, BM
Y-27
709,
sta
chyf
lin,
and
thei
r an
alog
ues,
(3) i
nhib
itors
of v
iral
RN
Ps/p
olym
eras
e/en
donu
clea
se, s
uch
as T
-705
, L-7
35,8
22, f
lutim
ide
and
thei
r an
alog
ues,
(4) i
nhib
itors
of M
EK, s
uch
as P
D 0
3259
01, C
I-10
40 a
nd A
RRY-
1428
86, a
nd (5
) inh
ibito
rs o
f NA
su
ch a
s D
AN
A, F
AN
A, z
anam
ivir,
and
ose
ltam
ivir,
etc
. Alth
ough
am
anta
dine
and
rim
anta
dine
are
not
reco
mm
ende
d fo
r tr
eatin
g in
fluen
za v
irus
infe
ctio
ns b
ecau
se o
f dru
g re
sist
ance
pro
blem
, the
se v
iral
M2
ion
chan
nel b
lock
ers
esta
b-lis
hed
a pr
oof-
of-c
once
pt th
at th
e en
docy
tosi
s of
vir
ion
into
hos
t cel
ls c
an b
e a
valid
dru
g ta
rget
bec
ause
M2
prot
ein
is in
volv
ed in
the
endo
cyto
sis
proc
ess.
The
influ
enza
pol
ymer
ase
com
plex
not
onl
y ca
taly
zes
RNA
pol
ymer
izat
ion
but
also
enc
odes
the
“cap
sna
tchi
ng” a
ctiv
ity. A
fter
bei
ng e
xpor
ted
from
the
nucl
eus
to th
e cy
topl
asm
, the
new
ly s
ynth
e-si
zed
vRN
Ps a
re a
ssem
bled
into
vir
ions
at t
he p
lasm
a m
embr
ane.
The
pro
geny
vir
ions
will
then
leav
e th
e ho
st c
ells
th
roug
h th
e ac
tion
of N
A. T
he s
trat
egie
s fo
r di
scov
ery
of s
mal
l mol
ecul
e in
hibi
tors
of i
nflu
enza
vir
us re
plic
atio
n ba
sed
on e
ach
part
icul
ar m
echa
nism
will
be
disc
usse
d. F
inal
ly, t
he le
sson
s le
arne
d fr
om th
e de
sign
of N
A in
hibi
tors
(NA
I) a
re
also
incl
uded
. Man
y ex
citin
g op
port
uniti
es a
wai
t the
cad
re o
f vir
olog
ists
, med
icin
al c
hem
ists
, and
pha
rmac
olog
ists
to
desi
gn n
ovel
influ
enza
dru
gs w
ith fa
vora
ble
phar
mac
olog
ical
and
pha
rmac
okin
etic
pro
pert
ies
to c
omba
t thi
s th
reat
en-
ing
infe
ctio
us d
isea
se.
Hsi
eh a
nd H
su
2007
Hig
h-th
roug
hput
scr
een
tech
niqu
esIn
trod
uctio
n: In
fluen
za a
ntiv
iral
hig
h-th
roug
hput
scr
eens
hav
e be
en e
xten
sive
, and
yet
no
appr
oved
influ
enza
ant
ivi-
rals
hav
e be
en id
entif
ied
thro
ugh
high
-thr
ough
put s
cree
ning
. Thi
s un
ders
core
s th
e id
ea th
at d
evel
opm
ent o
f suc
cess
-fu
l scr
eens
sho
uld
focu
s on
the
expl
oita
tion
of th
e un
derr
epre
sent
ed v
iral
targ
ets
and
nove
l, th
erap
eutic
hos
t tar
gets
. A
reas
cov
ered
: The
aut
hors
revi
ew c
onve
ntio
nal s
cree
ning
app
licat
ions
and
em
ergi
ng te
chno
logi
es w
ith th
e po
tent
ial
to e
nhan
ce in
fluen
za a
ntiv
iral
dis
cove
ry. R
eal-
wor
ld e
xam
ples
from
the
auth
ors’
wor
k in
bio
cont
aine
d en
viro
nmen
ts
are
also
pro
vide
d. F
utur
e in
nova
tions
are
dis
cuss
ed, i
nclu
ding
the
use
of ta
rget
ed li
brar
ies,
mul
tiple
xed
assa
ys, p
rox-
imity
-bas
ed e
ndpo
int m
etho
ds, n
on-l
abor
ator
y-ad
apte
d vi
rus
stra
ins,
and
pri
mar
y ce
lls, f
or im
med
iate
phy
siol
ogic
al
rele
vanc
e an
d tr
ansl
atio
nal a
pplic
atio
ns. E
xper
t opi
nion
: The
lack
of s
ucce
ssfu
l ant
i-in
fluen
za d
rug
disc
over
y us
ing
high
-thr
ough
put s
cree
ning
sho
uld
not d
eter
futu
re e
ffort
s. In
crea
sed
unde
rsta
ndin
g of
the
func
tions
of v
iral
targ
ets
and
host
-pat
hoge
n in
tera
ctio
ns h
as b
road
ened
the
targ
et re
serv
oir.
Futu
re s
cree
ning
effo
rts
shou
ld fo
cus
on id
entif
y-in
g ne
w d
rugs
aga
inst
une
xplo
ited
vira
l and
hos
t tar
gets
usi
ng c
urre
ntly
dev
elop
ed a
ssay
s, a
nd o
n th
e de
velo
pmen
t of
nove
l, in
nova
tive
assa
ys to
dis
cove
r ne
w d
rugs
with
nov
el m
echa
nism
s. In
nova
tive
scre
ens
mus
t be
desi
gned
to id
entif
y co
mpo
unds
that
spe
cific
ally
inhi
bit p
rote
in-p
rote
in o
r pr
otei
n-RN
A in
tera
ctio
ns o
r ot
her
viru
s/ho
st fa
ctor
inte
ract
ions
th
at a
re c
ruci
al fo
r vi
ral r
eplic
atio
n. F
inal
ly, t
he u
se o
f rec
ent v
iral
isol
ates
, inc
reas
ed b
ioco
ntai
nmen
t (fo
r hi
ghly
-pa
thog
enic
str
ains
), pr
imar
y ce
ll lin
es, a
nd ta
rget
ed c
ompo
und
libra
ries
mus
t con
verg
e in
eff
icie
nt h
igh-
thro
ughp
ut
prim
ary
scre
ens
to g
ener
ate
high
-con
tent
, phy
siol
ogic
ally
-rel
evan
t dat
a on
com
poun
ds w
ith ro
bust
ant
ivir
al a
ctiv
ity.
Atk
ins e
t al.
2012
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
131
Susc
epti
bilit
y m
onit
orin
gPl
aque
-red
uctio
n as
say
Yiel
d-re
duct
ion
assa
yD
ye-u
ptak
e as
say
Neu
ram
inid
ase
Hae
mag
glut
inin
With
the
clin
ical
dev
elop
men
t of a
nti-
vira
l age
nts,
mon
itori
ng fo
r the
con
tinue
d su
scep
tibili
ty o
f wild
-typ
e st
rain
s has
be
com
e im
port
ant i
n di
seas
e m
anag
emen
t. Va
riou
s met
hods
hav
e be
en u
sed
to m
onito
r vir
al su
scep
tibili
ty; t
he a
dvan
-ta
ges a
nd d
isad
vant
ages
of w
hich
dep
end
on th
e vi
rus,
the
targ
et a
nd th
e sc
ale
of th
e re
sear
ch b
eing
und
erta
ken.
The
plaq
ue-r
educ
tion
assa
y is
val
uabl
e fo
r mea
suri
ng su
scep
tibili
ty o
f mos
t vir
uses
but
is n
ot id
eal f
or la
rge-
scal
e m
onito
ring
. Yi
eld-
redu
ctio
n, m
easu
ring
spec
ific
viru
s ant
igen
s, a
nd d
ye-u
ptak
e as
says
, mea
suri
ng v
irus
cyt
opat
hic
effec
ts, a
re m
ore
suita
ble
for h
igh-
thro
ughp
ut re
quir
emen
ts, b
ut th
e IC
50 v
alue
(the
con
cent
ratio
n th
at in
hibi
ts 5
0% o
f vir
us) v
arie
s with
th
e vi
ral i
nocu
lum
. Sur
veill
ance
of i
nflue
nza
susc
eptib
ility
to ri
man
tadi
ne/a
man
tadi
ne in
the
clin
ic h
as p
redo
min
antly
us
ed E
IA-b
ased
ass
ays,
sinc
e pl
aqui
ng o
f infl
uenz
a cl
inic
al is
olat
es is
var
iabl
e. W
ith d
evel
opm
ent o
f the
influ
enza
NA
in
hibi
tors
it b
ecam
e ap
pare
nt th
at c
urre
nt c
ell-b
ased
ass
ays w
ere
unsu
itabl
e fo
r mon
itori
ng su
scep
tibili
ty to
this
new
cl
ass o
f dru
gs. V
aria
bilit
y m
ay re
sult
from
vir
us sp
read
dir
ectly
from
cel
l to
cell
in c
ultu
re b
y-pa
ssin
g th
e N
A fu
nctio
n.
Furt
herm
ore,
mut
atio
ns se
lect
ed in
the
HA
, whi
le n
ot a
ppar
ently
con
trib
utin
g to
phe
noty
pic
resi
stan
ce in
viv
o, m
ay
resu
lt in
cel
l-cul
ture
bas
ed re
sist
ance
, and
may
mas
k N
A re
sist
ance
in c
ell c
ultu
re b
y m
odify
ing
rece
ptor
-bin
ding
spec
i-fic
ity. O
ne im
port
ant d
istin
ctio
n be
twee
n N
A in
hibi
tors
and
oth
er a
ntiv
iral
enz
yme
inhi
bito
rs is
that
bot
h ta
rget
enz
yme
and
inhi
bito
r wor
k ex
trac
ellu
larl
y. N
A a
ssay
s are
ther
efor
e m
ost r
epre
sent
ativ
e of
the
in v
ivo
situ
atio
n fo
r mon
itori
ng su
s-ce
ptib
ility
, sup
port
ed b
y H
A se
quen
cing
. As t
he c
linic
al u
se o
f NA
inhi
bito
rs e
scal
ates
, a m
ajor
cha
nge
will
be
requ
ired
in
appr
oach
es u
sed
to m
onito
r sus
cept
ibili
ty o
f infl
uenz
a is
olat
es in
vir
olog
y la
bora
tori
es w
orld
-wid
e.
Tisd
ale
2000
Synt
heti
c st
rate
gies
Ose
ltam
ivir
pho
spha
teO
selta
miv
ir p
hosp
hate
(Tam
iflu)
is th
e on
ly o
rally
act
ive
anti-
influ
enza
dru
g th
at p
oten
tly in
hibi
t neu
ram
inid
ase.
The
rece
nt e
mer
genc
e of
avi
an fl
u, e
spec
ially
the
H5N
1 ty
pe, m
akes
the
situ
atio
n of
Tam
iflu
supp
ly a
nd d
eman
d in
crea
sing
ly
seri
ous.
Fur
ther
opt
imiz
atio
n of
the
curr
ent c
omm
erci
al a
ppro
ach
and
expl
orat
ion
of n
ew sy
nthe
tic ro
utes
are
urg
ent.
Her
e, d
iffer
ent s
ynth
etic
stra
tegi
es o
f ose
ltam
ivir
pho
spha
te a
re re
view
ed, i
nclu
ding
dis
cove
ry a
nd im
prov
ed sy
nthe
tic
rout
e fr
om (–
)-qu
inic
aci
d or
(–)-
shik
imic
aci
d, n
ew a
sym
met
ric
synt
hesi
s via
cat
alyt
ic d
esym
met
riza
tion
of a
mes
oazi
ri-
dine
(CD
MA
), D
iels
-Ald
er R
eact
ion
and
from
oth
er a
vaila
ble
mat
eria
ls.
Gon
g an
d X
u 20
08
Synt
heti
c st
rate
gies
Tetr
a- a
nd o
ctav
alen
t si
alos
ide
clus
ters
Tetr
a- a
nd o
ctav
alen
t sia
losi
de c
lust
ers w
ere
prep
ared
in g
ood
yiel
ds e
xplo
iting
for t
he fi
rst t
ime
the
mul
tiple
cop
per-
cata
lyze
d cy
cloa
dditi
on o
f a p
ropa
rgyl
thio
sial
osid
e w
ith c
alix
[4]a
rene
pol
yazi
des.
The
cycl
oadd
ucts
feat
ured
the
hydr
o-ly
tical
ly st
able
car
bon-
sulfu
r bon
d at
the
anom
eric
pos
ition
and
the
1,4-
disu
bstit
uted
tria
zole
ring
as t
he sp
acer
bet
wee
n th
e si
alic
aci
d m
oiet
ies a
nd th
e pl
atfo
rm. I
t was
dem
onst
rate
d th
at th
ese
unna
tura
l mot
ifs d
id n
ot h
ampe
r the
des
ired
bi
olog
ical
act
ivity
of t
he si
aloc
lust
ers.
In fa
ct, t
hey
wer
e ab
le to
inhi
bit,
at su
bmill
imol
ar c
once
ntra
tions
, the
hem
aggl
uti-
natio
n an
d th
e vi
ral i
nfec
tivity
med
iate
d bo
th b
y BK
and
influ
enza
A v
irus
es.
Mar
ra e
t al.
2008
Ant
ivir
al a
ctiv
ity
eval
uati
onIn
vitr
o an
tivir
al a
ssay
sA
nim
al m
odel
s
Eval
uatio
n of
pot
entia
l infl
uenz
a vi
rus i
nhib
itors
may
util
ize
mul
tiple
step
s. F
irst
wou
ld b
e to
det
erm
ine
if th
e vi
ral t
arge
t (e
.g. i
nflue
nza
viru
s neu
ram
inid
ase)
bei
ng fo
cuse
d up
on w
ill b
e in
hibi
ted
in th
e ap
prop
riat
e as
say.
Stan
dard
in v
itro
antiv
iral
ass
ays,
use
d ne
xt in
ant
ivir
al e
valu
atio
ns, m
ay u
tiliz
e in
hibi
tion
of v
iral
pla
ques
, vir
al c
ytop
athi
c eff
ect (
CPE
), an
d vi
ral h
emag
glut
inin
or o
ther
pro
tein
, with
inhi
bitio
n of
vir
al y
ield
use
d in
follo
w-u
p ev
alua
tions
. The
CPE
can
be
dete
rmin
ed v
isua
lly a
nd b
y dy
e up
take
, Ani
mal
mod
els u
sed
for s
tudy
of p
oten
tial i
nflue
nza
viru
s inh
ibito
rs in
clud
e th
e fe
rret
, the
labo
rato
ry m
ouse
, and
the
chic
ken,
with
a v
arie
ty o
f par
amet
ers u
sed
to in
dica
te th
e se
veri
ty o
f the
infe
ctio
n an
d its
inhi
bitio
n by
ther
apy.
Mul
tiple
par
amet
ers a
re re
com
men
ded
in a
ny in
viv
o an
tivir
al e
valu
atio
n. Th
e fe
rret
and
th
e m
ouse
infe
ctio
n m
odel
s hav
e be
en u
sefu
l in
stud
ying
the
deve
lopm
ent o
f dru
g re
sist
ance
and
the
rela
tive
viru
lenc
e of
dru
g-re
sist
ant v
irus
es. Th
e in
fluen
za m
ouse
mod
el h
as a
lso
been
of v
alue
for t
he e
valu
atio
n of
imm
unom
odul
atin
g eff
ects
of t
est c
ompo
unds
and
for t
he st
udy
of th
e ut
ility
of a
ntiv
iral
dru
gs fo
r use
aga
inst
influ
enza
vir
us in
fect
ions
in th
e im
mun
ocom
prom
ised
hos
t. In
con
side
ring
the
use
of a
ny a
nim
al m
odel
, spe
cies
diff
eren
ces i
n dr
ug p
harm
acol
ogy
and
met
abol
ism
mus
t be
take
n in
to a
ccou
nt. Th
is re
view
has
des
crib
ed th
e sy
stem
s whi
ch h
ave
been
use
d m
ost f
requ
ently
by
ant
ivir
al in
vest
igat
ors,
usi
ng, a
s exa
mpl
es, r
ecen
t stu
dies
with
the
clin
ical
ly a
ppro
ved
influ
enza
vir
us n
eura
min
idas
e in
hibi
tors
ose
ltam
ivir
and
zan
amiv
ir.
Sidw
ell a
nd S
mee
20
00
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
132
Ant
i-in
fluen
za a
gent
sIn
fluen
za is
a d
isea
se fo
r de
eply
affe
ctin
g m
illio
ns o
f peo
ple
ever
y ye
ar. R
ecen
tly, t
here
has
bee
n co
nsid
erab
le c
once
rn
rega
rdin
g th
e hi
ghly
pat
hoge
nic
H5N
1 av
ian
influ
enza
vir
us, a
nd it
s hu
man
pan
dem
ic p
oten
tial.
With
dev
elop
men
ts
in v
iral
bio
logy
, the
re a
re m
ore
nove
l ant
ivir
al s
trat
egie
s ta
rget
ing
thes
e vi
ruse
s. In
this
revi
ew, w
e w
ill d
iscu
ss s
ever
al
prov
en a
nd p
oten
tial a
nti-
influ
enza
targ
ets,
incl
udin
g vi
ral f
acto
rs (s
uch
as h
emag
glut
inin
(HA
), M
2 io
n ch
anne
l pro
-te
in, R
NA
-dep
ende
nt R
NA
pol
ymer
ase
(RdR
p), n
ucle
opro
tein
(NP)
, non
-str
uctu
ral p
rote
in (N
S) a
nd n
eura
min
idas
e (N
A))
and
hos
t fac
tors
(suc
h as
v-A
TPa
se, p
rote
ase,
inos
ine
mon
opho
spha
te d
ehyd
roge
nase
(IM
PDH
) and
intr
acel
lula
r si
gnal
ling
casc
ades
), an
d th
eir
rele
vant
inhi
bito
rs.
Gon
g et
al.
2009
neu
ram
inid
ase
Nat
ural
pro
duct
sSe
cond
ary
plan
t m
etab
olite
s
Influ
enza
neu
ram
inid
ase
(NA
), a
key
enzy
me
in v
iral
repl
icat
ion,
spr
ead,
and
pat
hoge
nesi
s, is
con
side
red
to b
e on
e of
th
e m
ost p
rom
isin
g ta
rget
s fo
r co
mba
ting
influ
enza
. Des
pite
the
subs
tant
ial m
edic
al p
oten
tial o
f NA
inhi
bito
rs (N
AIs
), on
ly th
ree
of th
ese
drug
s ar
e cu
rren
tly o
n th
e m
arke
t (za
nam
ivir,
ose
ltam
ivir,
and
per
amiv
ir).
Mor
eove
r, su
dden
ch
ange
s in
NA
I sus
cept
ibili
ty re
veal
ed th
e ur
gent
nee
d in
the
disc
over
y/id
entif
icat
ion
of n
ovel
inhi
bito
rs. N
atur
e of
fers
an
abu
ndan
ce o
f bio
synt
hesi
zed
com
poun
ds c
ompr
isin
g ch
emic
al s
caffo
lds
of h
igh
dive
rsity
, whi
ch p
rese
nt a
n in
finite
po
ol o
f che
mic
al e
ntiti
es fo
r ta
rget
-ori
ente
d dr
ug d
isco
very
in th
e ba
ttle
aga
inst
this
hig
hly
cont
agio
us p
atho
gen.
Thi
s re
view
illu
min
ates
the
incr
easi
ng re
sear
ch e
ffort
s of
the
past
dec
ade
(200
0–20
11),
focu
sing
on
the
stru
ctur
e, fu
nctio
n an
d dr
ugga
bilit
y of
influ
enza
NA
, as
wel
l as
its in
hibi
tion
by n
atur
al p
rodu
cts.
Fol
low
ing
a cr
itica
l dis
cuss
ion
of p
ubli-
catio
ns d
escr
ibin
g so
me
150
seco
ndar
y pl
ant m
etab
olite
s te
sted
for
thei
r in
hibi
tory
pot
entia
l aga
inst
influ
enza
NA
, the
im
pact
of t
hree
diff
eren
t str
ateg
ies
to id
entif
y an
d de
velo
p no
vel N
AIs
is p
rese
nted
: (i)
bioa
ctiv
ity s
cree
ning
of h
erba
l ex
trac
ts, (
ii) e
xplo
itatio
n of
em
piri
cal k
now
ledg
e, a
nd (i
ii) c
ompu
tatio
nal a
ppro
ache
s. T
his
wor
k ad
dres
ses
the
late
st
deve
lopm
ents
in th
eore
tical
and
exp
erim
enta
l res
earc
h on
pro
pert
ies
of N
A th
at a
re a
nd w
ill b
e dr
ivin
g an
ti-in
fluen
za
drug
dev
elop
men
t now
and
in th
e ne
ar fu
ture
.
Gri
enke
et a
l. 20
12
neu
ram
inid
ase
inhi
bito
rsN
eura
min
idas
e cr
ysta
l st
ruct
ure
150-
and
430
-loop
regi
on
Neu
ram
inid
ase
inhi
bito
rs s
uch
as o
selta
miv
ir a
nd z
anam
ivir
hav
e be
en w
idel
y us
ed in
the
trea
tmen
t and
hav
e ga
ined
re
mar
kabl
e su
cces
s. A
lthou
gh, t
hey
are
effe
ctiv
e in
pre
vent
ion
of in
fluen
za; t
he c
once
rn fo
r dr
ug re
sist
ance
stil
l re
mai
ns a
que
stio
n. R
ecen
tly, t
he a
vaila
bilit
y of
cry
stal
str
uctu
res
of th
e en
zym
e ga
ve a
new
tren
d to
the
stru
ctur
e ba
sed
drug
des
igni
ng o
f neu
ram
inid
ase
inhi
bito
rs. T
he a
rtic
le re
view
s a
deta
iled
unde
rsta
ndin
g of
the
stru
ctur
al fe
a-tu
res
with
in n
eura
min
idas
e en
zym
e w
hich
turn
outs
to b
e cr
ucia
l for
futu
re d
rug
deve
lopm
ent.
In d
epth
ana
lysi
s fo
r th
e ne
wly
pro
pose
d sp
ots
with
in th
e 15
0 an
d 43
0-lo
op re
gion
s in
N1
mak
es it
dis
tingu
isha
ble
amon
g th
e su
btyp
es. F
urth
er
we
have
dis
cuss
ed th
e va
riou
s co
mpu
tatio
nal s
tudi
es c
arri
ed o
ut in
opt
imiz
ing
the
desi
gnin
g of
neu
ram
inid
ase
inhi
bi-
tors
ther
eby
prov
idin
g ne
w c
lues
to m
odify
the
curr
ently
ava
ilabl
e dr
ugs.
Chi
ntak
rind
i et
al. 2
012
Cel
lula
r dr
ug ta
rget
sT
his
revi
ew s
umm
ariz
es c
urre
nt k
now
ledg
e on
influ
enza
A v
irus
repl
icat
ion
with
a fo
cus
on e
mer
ging
cel
lula
r dr
ug
targ
ets.
Inte
rest
ingl
y, fo
r m
any
of th
ese
targ
ets,
com
poun
ds fo
r w
hich
saf
ety
test
ing
has
been
car
ried
out
in h
uman
s ar
e av
aila
ble.
It is
pos
sibl
e th
at s
ome
of th
ese
com
poun
ds, s
uch
as in
hibi
tors
of h
eat s
hock
pro
tein
90,
pro
teas
ome,
impo
r-tin
0/5
or
prot
ein
kina
se C
, will
be
used
for
trea
tmen
t of I
AV in
fect
ions
aft
er c
aref
ul e
valu
atio
n in
hum
an p
rim
ary
cells
an
d se
vere
ly il
l flu
pat
ient
s.
Mul
ler e
t al.
2012
Influ
enza
neu
tral
izin
g an
tibo
dies
The
hum
an a
ntib
ody
resp
onse
to in
fluen
za v
irus
infe
ctio
n pl
ays
a pr
otec
tive
role
aga
inst
re-i
nfec
tion,
yet
litt
le m
olec
u-la
r de
tail
is a
vaila
ble
rega
rdin
g ho
w h
uman
ant
ibod
ies,
whe
n ch
arac
teri
zed
at th
e m
onoc
lona
l lev
el, n
eutr
aliz
e th
is
impo
rtan
t hum
an p
atho
gen.
Rec
ent s
tudi
es, u
sing
a d
iver
se a
rray
of s
trat
egie
s, h
ave
isol
ated
and
cha
ract
eriz
ed h
uman
an
ti-vi
rus
neut
raliz
ing
antib
odie
s an
d sh
ed li
ght n
ot o
nly
on th
e sp
ecifi
city
and
ori
gin
of th
ese
antib
odie
s bu
t on
thei
r po
tent
ial f
or th
erap
eutic
use
aga
inst
influ
enza
vir
us in
fect
ion.
Mar
tinez
et a
l. 20
09
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
133
Trad
itio
nal C
hine
se
med
icin
e dr
ugs
Swin
e flu
Aft
er n
ew h
uman
tran
smis
sibl
e H
1N1
(sw
ine
flu) v
irus
es w
ere
repo
rted
in M
exic
o an
d th
e U
nite
d St
ates
in A
pril
2009
, th
e W
orld
Hea
lth O
rgan
izat
ion
(WH
O) a
nnou
nced
the
emer
genc
e of
a n
ovel
influ
enza
A v
irus
. Mos
t gov
ernm
ents
in
the
wor
ld h
ave
been
ale
rted
and
are
mon
itori
ng th
e si
tuat
ion
clos
ely.
As
one
of th
e of
ficia
l res
pons
es to
the
H1N
1 pa
ndem
ic, t
he C
hine
se g
over
nmen
t has
rele
ased
thre
e ed
ition
s of
a d
ocum
ent e
ntitl
ed “R
ecom
men
ded
Sche
mes
for
Pand
emic
Influ
enza
A D
iagn
oses
and
Tre
atm
ents
”. T
he th
ird
editi
on re
com
men
ded
the
use
of n
ot o
nly
two
targ
eted
an
ti-flu
dru
gs, o
selta
miv
ir a
nd z
anam
ivir,
but
als
o fo
ur a
nti-
flu T
CM
(Tra
ditio
nal C
hine
se M
edic
ine)
pre
scri
ptio
ns.
Sinc
e th
en, T
CM
has
pla
yed
a si
gnifi
cant
role
in fi
ghtin
g th
e pa
ndem
ic. T
CM
dru
gs c
ompr
ise
mul
tiple
com
poun
ds
regu
latin
g m
ultip
le ta
rget
s fo
r a
give
n cl
ass
of m
edic
al in
dica
tions
, and
are
tuna
ble
to th
e sy
mpt
oms
of th
e in
divi
dual
. T
his
revi
ew s
umm
ariz
es a
nti-
influ
enza
age
nts
from
TC
M, i
nclu
ding
com
poun
ds, h
erbs
, and
TC
M p
resc
ript
ions
, and
su
gges
ts th
at, b
y fu
rthe
r in
vest
igat
ing
TC
M th
eory
and
min
ing
TC
M d
atab
ases
, a b
ette
r dr
ug d
isco
very
par
adig
m m
ay
aris
e –
one
that
can
be
bene
ficia
l to
both
TC
M a
nd m
oder
n m
edic
ine.
Ge
et a
l. 20
10
Com
bina
tion
ther
apy
The
pand
emic
pot
entia
l of i
nflue
nza
viru
ses h
as e
ngag
ed a
larg
e po
rtio
n of
the
antiv
iral
dru
g di
scov
ery
rese
arch
com
mu-
nity
in th
e de
velo
pmen
t of n
umer
ous a
ntiv
iral
age
nts,
with
the
ultim
ate
goal
to su
pple
men
t effe
ctiv
e im
mun
izat
ion
whe
n ne
w st
rain
s ari
se, e
spec
ially
aft
er a
n an
tigen
ic sh
ift. A
ntiv
iral
age
nts a
gain
st in
fluen
za A
targ
ets d
iffer
ent r
eplic
atio
n st
eps
of th
e vi
rus l
ife c
ycle
s. S
ome
of th
e ag
ents
are
ana
logu
es o
f bio
mol
ecul
es re
quir
ed d
urin
g vi
rus i
nfec
tion
and
othe
rs a
re
insp
ired
from
nat
ural
pla
nt e
xtra
cts.
In th
is re
view
, we
sum
mar
ize
thei
r mec
hani
sms o
f act
ion
duri
ng th
e in
fluen
za li
fe
cycl
e in
vitr
o an
d th
e effi
caci
es o
f com
bina
tiona
l the
rapi
es w
ith th
ese
agen
ts a
gain
st th
e in
fluen
za v
irus
infe
ctio
ns in
viv
o.
Kai
hats
u an
d Ba
rnar
d 20
12
Com
bina
tion
ther
apy
Cur
rent
ly a
vaila
ble
agen
tsTh
e em
erge
nce
of p
ande
mic
H1N
1 in
fluen
za v
irus
es in
Apr
il 20
09 a
nd th
e co
ntin
uous
evo
lutio
n of
hig
hly
path
ogen
ic
H5N
1 in
fluen
za v
irus
es u
nder
scor
e th
e ur
genc
y of
nov
el a
ppro
ache
s to
chem
othe
rapy
for h
uman
influ
enza
infe
ctio
n.
Ant
i-infl
uenz
a dr
ugs a
re c
urre
ntly
lim
ited
to th
e ne
uram
inid
ase
inhi
bito
rs (o
selta
miv
ir a
nd z
anam
ivir
) and
to M
2 io
n ch
anne
l blo
cker
s (am
anta
dine
and
rim
anta
dine
), al
thou
gh re
sist
ance
to th
e la
tter
cla
ss d
evel
ops r
apid
ly. P
oten
tial t
arge
ts
for t
he d
evel
opm
ent o
f new
ant
i-infl
uenz
a ag
ents
incl
ude
the
vira
l pol
ymer
ase
(and
end
onuc
leas
e), t
he h
emag
glut
inin
, an
d th
e no
n-st
ruct
ural
pro
tein
NS1
. The
limita
tions
of m
onot
hera
py a
nd th
e em
erge
nce
of d
rug-
resi
stan
t var
iant
s mak
e co
mbi
natio
n ch
emot
hera
py th
e lo
gica
l the
rape
utic
opt
ion.
Her
e w
e re
view
the
expe
rim
enta
l dat
a on
com
bina
tion
chem
o-th
erap
y w
ith c
urre
ntly
ava
ilabl
e ag
ents
and
the
deve
lopm
ent o
f new
age
nts a
nd th
erap
y ta
rget
s.
Gov
orko
va a
nd
Web
ster
201
0
Prot
ease
inhi
bito
rsA
mbr
oxol
Cla
rith
rom
ycin
Neu
ram
inid
ase
inhi
bito
rsC
ombi
natio
n th
erap
y
Influ
enza
A v
irus
(IAV
) is o
ne o
f the
mos
t com
mon
infe
ctio
us p
atho
gens
in h
uman
s. S
ince
IVA
gen
ome
does
not
hav
e th
e pr
oces
sing
pro
teas
e fo
r the
vir
al m
embr
ane
fusi
on g
lyco
prot
ein
prec
urso
rs, e
ntry
of’
this
vir
us in
to c
ells
is d
eter
min
ed
prim
arily
by
host
cel
lula
r, tr
ypsi
n-ty
pe, p
roce
ssin
g pr
otea
ses t
hat p
rote
olyt
ical
ly a
ctiv
ate
the
fusi
on g
lyco
prot
ein
prec
ur-
sors
of I
AV. A
t lea
st fi
ve d
iffer
ent p
roce
ssin
g pr
otea
ses h
ave
been
iden
tified
in th
e ai
rway
s of a
nim
als a
nd h
uman
s. Th
ese
prot
ease
s det
erm
ine
the
infe
ctio
us o
rgan
trop
ism
of I
AV in
fect
ion
as w
ell a
s the
effi
cien
cy o
f vir
al m
ultip
licat
ion
in th
e ai
rway
, and
som
etim
es in
the
brai
n. P
rote
ases
in th
e up
per r
espi
rato
ry tr
act a
re su
ppre
ssed
by
secr
etor
y le
ukop
rote
ase
inhi
bito
r, an
d th
ose
in th
e lo
wer
resp
irat
ory
trac
t are
supp
ress
ed b
y pu
lmon
ary
surf
acta
nt w
hich
, by
adso
rptio
n, in
hibi
ts
the
inte
ract
ion
betw
een
the
prot
ease
s and
vir
al m
embr
ane
prot
eins
. Sin
ce p
rote
ase
activ
ities
pre
dom
inat
e ov
er th
ose
of
endo
geno
us in
hibi
tory
com
poun
ds u
nder
nor
mal
air
way
con
ditio
ns, a
dmin
istr
atio
n of
pro
teas
e in
hibi
tors
in th
e ea
rly-
stag
e of
infe
ctio
n si
gnifi
cant
ly su
ppre
sses
vir
al e
ntry
and
vir
al m
ultip
licat
ion.
Sev
eral
vir
al n
eura
min
idas
e in
hibi
tors
are
us
ed c
linic
ally
as a
nti-i
nflue
nza
viru
s age
nts,
bas
ed o
n th
eir i
nhib
itory
act
ion
oil v
iral
rele
ase
from
infe
cted
cel
ls. F
ur-
ther
mor
e, p
rote
ase
inhi
bito
rs o
f vir
al e
ntry
cou
ld b
e po
tent
ially
use
ful a
gain
st in
fluen
za v
irus
as w
ell a
s neu
ram
inic
lase
in
hibi
tor-
resi
stan
t vir
uses
. We
also
foun
d th
at a
mbr
oxol
, a m
ucol
ytic
and
ant
i-oxi
dant
age
nt, u
p-re
gula
tes t
he le
vels
of
endo
geno
us p
rote
ase
inhi
bito
ry c
ompo
unds
in th
e ai
rway
flui
ds in
ear
ly-p
hase
infe
ctio
n, a
nd th
at c
lari
thro
myc
in, a
mac
-ro
tide
antib
iotic
, inc
reas
es Ig
A le
vels
and
muc
osal
imm
unity
thro
ugh
augm
enta
tion
of in
terl
euki
n-12
leve
ls in
the
airw
ay.
The
com
bina
tion
of n
eura
min
idas
e in
hibi
tors
and
pro
teas
e in
hibi
tors
, cla
rith
rom
ycin
or a
mbr
oxol
, cou
ld b
e po
tent
ially
us
ed a
s a p
oten
t ant
i-infl
uenz
a th
erap
y to
min
imiz
e th
e em
erge
nce
of d
rug-
resi
stan
t mut
ant v
irus
es.
Kid
o et
al.
2007
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
134
Tabl
e 3.
des
ign,
syn
thes
is a
nd e
valu
atio
n of
new
ant
i-in
flue
nza
drug
s
dru
g de
sign
and
an
tivi
ral a
ctiv
ity
eval
uati
onD
rug
data
base
of
400
0 co
mpo
unds
Doc
king
sim
ulat
ion
Viab
ility
ass
ayPl
aque
ass
ayBe
nzbr
omar
one
Dic
lazu
ril
RNA
pol
ymer
ase
We
perf
orm
ed a
doc
king
sim
ulat
ion
targ
etin
g th
e in
terf
ace
of P
A in
tera
ctin
g w
ith P
B1 u
sing
a d
rug
data
base
incl
ud-
ing
sim
ilar
to 4
000
com
poun
ds. W
e th
en c
ondu
cted
cel
l via
bilit
y as
say
and
plaq
ue a
ssay
usi
ng IA
V/W
SN/3
3. F
inal
ly
we
exam
ined
thei
r an
ti-IA
V m
echa
nism
by
surf
ace
plas
mon
reso
nanc
e an
d IA
V re
plic
on a
ssay
. Res
ults
: We
foun
d th
at
benz
brom
aron
e, d
icla
zuri
l, an
d tr
enbo
lone
ace
tate
had
str
ong
anti-
IAV
act
iviti
es. W
e co
nfir
med
that
ben
zbro
mar
one
and
dicl
azur
il bo
und
with
PA
sub
unit
, and
dec
reas
ed th
e tr
ansc
ript
iona
l act
ivity
of t
he v
iral
RN
A p
olym
eras
e. C
on-
clus
ions
: Ben
zbro
mar
one
and
dicl
azur
il ha
d st
rong
ant
i-IA
V a
ctiv
ities
with
nov
el a
ctio
n m
echa
nism
, i.e
. inh
ibiti
on o
f vi
ral R
NA
pol
ymer
ase.
Gen
eral
sig
nific
ance
: Sin
ce b
enzb
rom
aron
e an
d di
claz
uril
are
alre
ady
used
in p
ublic
as
med
i-ci
nes,
thes
e co
uld
be th
e ca
ndid
ates
for
alte
rnat
ives
in c
ase
of a
n ou
tbre
ak o
f IAV
whi
ch is
resi
stan
t to
pre-
exis
ting
anti-
IAV
dru
gs.
Fuku
oka
et a
l. 20
12
Stru
ctur
e-ba
sed
drug
de
sign
Com
puta
tiona
l m
etho
dolo
gies
Vir
tual
scre
enin
gD
ocki
ngPh
arm
acop
hore
mod
elin
g20
0 ne
w d
esig
ned
ligan
ds
The
M2
chan
nel p
rote
in o
n th
e in
fluen
za A
vir
us m
embr
ane
has
beco
me
the
mai
n ta
rget
of t
he a
nti-
flu d
rugs
am
an-
tadi
ne a
nd r
iman
tadi
ne. T
he s
truc
ture
of t
he M
2 ch
anne
l pro
tein
s of
the
H3N
2 (P
DB
code
2RL
F) a
nd 2
009-
H1N
1 (G
enba
nk a
cces
sion
num
ber
GQ
3853
83) v
irus
es m
ay h
elp
rese
arch
ers
to s
olve
the
drug
-res
ista
nt p
robl
em o
f the
se tw
o ad
aman
tane
-bas
ed d
rugs
and
dev
elop
mor
e po
wer
ful n
ew d
rugs
aga
inst
influ
enza
A v
irus
. In
the
pres
ent s
tudy
, we
sear
ched
for
new
M2
chan
nel i
nhib
itors
thro
ugh
a co
mbi
natio
n of
diff
eren
t com
puta
tiona
l met
hodo
logi
es, i
nclu
ding
vi
rtua
l scr
eeni
ng w
ith d
ocki
ng a
nd p
harm
acop
hore
mod
elin
g. V
irtu
al s
cree
ning
was
per
form
ed to
cal
cula
te th
e fr
ee
ener
gies
of b
indi
ng b
etw
een
rece
ptor
M2
chan
nel p
rote
ins
and
200
new
des
igne
d lig
ands
. Aft
er th
at, p
harm
acop
hore
an
alys
is w
as u
sed
to id
entif
y th
e im
port
ant M
2 pr
otei
n-in
hibi
tor
inte
ract
ions
and
com
mon
feat
ures
of t
op b
indi
ng
com
poun
ds w
ith M
2 ch
anne
l pro
tein
s. F
inal
ly, t
he tw
o m
ost p
oten
tial c
ompo
unds
wer
e de
term
ined
as
nove
l lea
ds to
in
hibi
t M2
chan
nel p
rote
ins
in b
oth
H3N
2 an
d 20
09-H
1N1
influ
enza
A v
irus
.
Tran
et a
l. 20
11
dru
g re
sist
ance
Ada
man
tane
s and
ne
uram
inid
ase
inhi
bito
rs
Influ
enza
vir
uses
are
maj
or h
uman
pat
hoge
ns w
ith a
glo
bal d
istr
ibut
ion,
acc
ount
ing
for
mor
e th
an 5
00 0
00 a
nnua
l de
aths
wor
ldw
ide
and
with
con
side
rabl
e im
pact
on
the
qual
ity o
f life
and
pro
duct
ivity
of t
he s
ocie
ty. D
ue to
the
limite
d ef
ficac
y of
vac
cina
tion,
ant
ivir
al d
rugs
con
stitu
te a
com
plem
enta
ry a
ppro
ach
in th
e co
ntro
l and
pre
vent
ion
of in
fluen
za
infe
ctio
ns a
nd th
us p
lay
an im
port
ant r
ole
in th
e m
anag
emen
t of i
nflu
enza
out
brea
ks a
nd p
ande
mic
s. C
urre
ntly
, ada
-m
anta
nes
and
neur
amin
idas
e in
hibi
tors
(NA
Is) a
re th
e on
ly tw
o cl
asse
s of
ant
i-in
fluen
za a
gent
s ap
prov
ed fo
r cl
inic
al
use.
How
ever
, the
wor
ldw
ide
emer
genc
e an
d hi
gh p
reva
lenc
e of
ada
man
tane
-res
ista
nt v
irus
var
iant
s ha
s di
scou
rage
d th
e us
e of
the
form
er d
rugs
. NA
Is h
ave
prov
ed to
be
very
effe
ctiv
e ag
ains
t inf
luen
za A
and
B v
irus
es. N
ever
thel
ess,
os
elta
miv
ir-r
esis
tant
str
ains
hav
e al
so b
een
repo
rted
qui
te fr
eque
ntly
, as
in th
e ca
se o
f sea
sona
l H1N
1 vi
ruse
s th
at
circ
ulat
ed b
etw
een
2007
and
200
9. In
deed
, the
em
erge
nce
of d
rug-
resi
stan
t vir
us v
aria
nts
is a
lway
s a
mat
ter
of c
once
rn
beca
use
it co
uld
sign
ifica
ntly
com
prom
ise
the
usef
ulne
ss o
f suc
h in
terv
entio
n. T
his
high
light
s th
e ne
ed fo
r co
ntin
uous
m
onito
ring
of r
esis
tanc
e m
arke
rs, a
s w
ell a
s th
e de
velo
pmen
t of n
ew a
nti-
influ
enza
dru
gs a
nd c
ombi
natio
n th
erap
ies.
Pizz
orno
et a
l. 20
11
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
135
Inte
ract
ion
of
adam
anta
ne w
ith
M2
prot
ein
Pore
doc
king
mod
el
Inte
ract
ion
of a
min
oada
man
tane
s w
ith th
e in
fluen
za A
vir
us M
2 pr
oton
cha
nnel
was
ana
lyze
d by
doc
king
sim
ulat
ions
of
a s
erie
s of
syn
thet
ic a
min
oada
man
tane
der
ivat
ives
, of d
iffer
ing
bind
ing
affin
ity, i
nto
the
crys
tal s
truc
ture
of t
he
tran
smem
bran
e (M
2TM
) por
e. T
he p
ore
bloc
king
mod
el te
sted
in th
e ‘g
as p
hase
’ des
crib
es q
ualit
ativ
ely
the
chan
ges
on th
e re
lativ
e bi
ndin
g af
finiti
es o
f the
com
poun
ds (a
lthou
gh a
ser
ies
of h
ighl
y hy
drop
hobi
c lig
ands
whi
ch s
eem
to
have
litt
le c
apac
ity fo
r di
ffere
nt s
peci
fic in
tera
ctio
ns w
ith th
eir
rece
ptor
). T
he d
ocki
ng c
alcu
latio
ns p
redi
cted
pos
es
in w
hich
the
adam
anta
ne r
ing
is s
urro
unde
d m
ainl
y by
the
alky
l sid
e ch
ains
of V
al27
or
Ala
30 a
nd th
e lig
and’
s am
ino
grou
p is
gen
eral
ly h
ydro
gen
bond
ed w
ith h
ydro
xyls
of S
er31
or
carb
onyl
s of
Val
27 o
r ca
rbon
yls
of A
la30
, the
form
er
(Ser
31) b
eing
the
mos
t sta
ble
and
mos
t fre
quen
tly o
bser
ved.
The
bin
ding
of t
he li
gand
is a
com
prom
ise
betw
een
hydr
ogen
bon
ding
abi
lity,
whi
ch is
ele
vate
d by
a p
rim
ary
amin
o gr
oup,
and
apo
lar
inte
ract
ions
, whi
ch a
re in
crea
sed
by
the
abili
ty o
f the
lipo
phili
c m
oiet
y to
ade
quat
ely
fill a
hyd
roph
obic
poc
ket w
ithin
the
M2T
M p
ore.
A d
elic
ate
bala
nce
of th
ese
hydr
opho
bic
cont
ribu
tions
is re
quir
ed fo
r op
timal
inte
ract
ion.
Elef
ther
atos
et a
l. 20
10
Stru
ctur
e-ba
sed
drug
de
sign
Poly
mer
ase
basi
c
prot
ein
2Po
lym
eras
e in
hibi
tors
Influ
enza
vir
uses
cau
se a
sig
nific
ant l
evel
of m
orbi
dity
and
mor
talit
y in
the
popu
latio
n ev
ery
year
. The
ir re
sist
ance
to
curr
ent a
nti-
influ
enza
dru
gs in
crea
ses
the
diff
icul
ty o
f flu
trea
tmen
t. T
hus,
dev
elop
men
t of n
ew a
nti-
influ
enza
dru
gs
is n
eces
sary
in re
gard
s of
pre
vent
the
trag
edy
of in
fluen
za p
ande
mic
. The
Pol
ymer
ase
basi
c pr
otei
n 2
(PB2
) sub
unit
of in
fluen
za v
irus
RN
A p
olym
eras
e is
one
of p
oten
tial t
arge
ts fo
r ne
w d
rugs
bec
ause
the
bind
ing
of P
B2 w
ith th
e 5’
ca
p of
the
host
pre
-mRN
As
is th
e in
itial
ste
p of
the
viru
s’ pr
otei
n sy
nthe
sis.
In th
is s
tudy
, we
com
pare
d th
e bi
ndin
g po
tenc
y of
PB2
cap
bin
ding
dom
ain
with
two
smal
l mol
ecul
es, i
.e.,
RO a
nd P
PT28
, with
that
of P
B2 w
ith c
ap a
nalo
g m
7GT
P. T
he c
alcu
late
d bi
ndin
g en
ergi
es s
how
ed th
at R
O a
nd P
PT28
had
hig
her
bind
ing
affin
ity w
ith P
B2. F
urth
er
inte
ract
ion
anal
ysis
sho
wed
that
the
impo
rtan
t par
ts fo
r bi
ndin
g w
ere
the
five-
and
six-
mem
ber
hete
rocy
clic
rin
gs (t
he
6/5-
mem
ber
ring
s) o
f sm
all m
olec
ules
, as
wel
l as
the
hydr
opho
bic
part
s of
RO
and
PPT
28 w
hich
had
goo
d in
tera
c-tio
ns w
ith th
e hy
drop
hobi
c re
sidu
es in
the
bind
ing
cavi
ty. T
hus,
RO
and
PPT
28 a
re tw
o po
tent
ial a
nti-
influ
enza
dru
gs
targ
eted
PB2
, whi
ch m
ay in
hibi
t the
gro
wth
of i
nflu
enza
vir
us b
y co
mpe
titiv
ely
bind
ing
with
the
cap
stru
ctur
e bi
ndin
g do
mai
n of
PB2
.
Lv e
t al.
2011
des
ign,
syn
thes
is, i
n vi
tro
eval
uati
onTr
ansi
tion-
stat
e-ba
sed
neur
amin
idas
e in
hibi
tors
X-r
ay c
ryst
allo
grap
hic
stru
ctur
eH
ydro
phob
ic p
ocke
t
The
des
ign,
syn
thes
is, a
nd in
vitr
o ev
alua
tion
of th
e no
vel c
arbo
cycl
es a
s tr
ansi
tion-
stat
e-ba
sed
inhi
bito
rs o
f inf
luen
za
neur
amin
idas
e (N
A) a
re d
escr
ibed
. The
dou
ble
bond
pos
ition
in th
e ca
rboc
yclic
ana
logu
es p
lays
an
impo
rtan
t rol
e in
N
A in
hibi
tion
as d
emon
stra
ted
by th
e an
tivir
al a
ctiv
ity o
f 8 (I
C50
= 6
.3 µ
M) v
s 9
(IC
50 >
200
µM
). St
ruct
ure-
activ
ity
stud
ies
of a
ser
ies
of c
arbo
cycl
ic a
nalo
gues
6a-
i ide
ntifi
ed th
e 3-
pent
ylox
y m
oiet
y as
an
appa
rent
opt
imal
gro
up a
t the
C
-3 p
ositi
on w
ith a
n IC
50 v
alue
of 1
nM
for
NA
inhi
bitio
n. T
he X
-ray
cry
stal
logr
aphi
c st
ruct
ure
of 6
h bo
und
to N
A
reve
aled
the
pres
ence
of a
larg
e hy
drop
hobi
c po
cket
in th
e re
gion
cor
resp
ondi
ng to
the
glyc
erol
sub
site
of s
ialic
aci
d.
The
hig
h an
tivir
al p
oten
cy o
bser
ved
for
6h a
ppea
rs to
be
attr
ibut
ed to
a h
ighl
y fa
vora
ble
hydr
opho
bic
inte
ract
ion
in
this
poc
ket.
The
pra
ctic
al s
ynth
esis
of 6
sta
rtin
g fr
om (–
)-qu
inic
aci
d is
als
o de
scri
bed.
Kim
et a
l. 19
97
Enan
tios
elec
tive
sy
nthe
sis
(–)-
osel
tam
ivir
free
bas
e(–
)-m
ethy
l 3-e
pi-
shik
imat
e
A n
ew e
nant
iose
lect
ive
synt
hesi
s of
the
anti-
influ
enza
age
nt (–
)-os
elta
miv
ir fr
ee b
ase
(7.1
% o
vera
ll yi
eld;
98%
ee)
and
(–
)-m
ethy
l 3-e
pi-s
hiki
mat
e (1
6% o
vera
ll yi
eld;
98%
ee)
has
bee
n de
scri
bed
from
read
ily a
vaila
ble
raw
mat
eria
ls. S
harp
-le
ss a
sym
met
ric
epox
idat
ion
and
dias
tere
osel
ectiv
e Ba
rbie
r al
lyla
tion
of a
n al
dehy
de a
re th
e ke
y re
actio
ns e
mpl
oyed
in
the
inco
rpor
atio
n of
chi
ralit
y, w
hile
the
cycl
ohex
ene
carb
oxyl
ic e
ster
cor
e w
as c
onst
ruct
ed th
roug
h a
ring
clo
sing
m
etat
hesi
s re
actio
n.
Raw
at e
t al.
2012
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
136
Synt
heti
c st
rate
gies
4-am
ino-
1-ph
osph
ono-
DA
NA
Phos
phon
o-za
nam
ivir
Two
phos
phon
ate
com
poun
ds la
(4-a
min
o-1-
phos
phon
o-D
AN
A) a
nd 1
b (p
hosp
hono
-zan
amiv
ir) a
re s
ynth
esiz
ed a
nd
show
n m
ore
pote
nt th
an z
anam
ivir
aga
inst
the
neur
amin
idas
es o
f avi
an a
nd h
uman
influ
enza
vir
uses
, inc
ludi
ng th
e os
elta
miv
ir-r
esis
tant
str
ains
. For
the
first
tim
e, th
e pr
actic
al s
ynth
esis
of t
hese
pho
spho
nate
com
poun
ds is
real
ized
by
conv
ersi
on o
f sia
lic a
cid
to p
erac
etyl
ated
pho
spho
no-D
AN
A d
ieth
yl e
ster
(5) a
s a
key
inte
rmed
iate
in th
ree
step
s by
a
nove
l app
roac
h. In
com
pari
son
with
zan
amiv
ir, th
e hi
gh a
ffin
ity o
f la
and
1b c
an b
e pa
rtly
att
ribu
tabl
e to
the
stro
ng
elec
tros
tatic
inte
ract
ions
of t
heir
pho
spho
nate
, gro
up’s
with
the
thre
e ar
gini
ne re
sidu
es (A
rg11
8, A
rg29
2, a
nd A
rg37
1)
in th
e ac
tive
site
of n
eura
min
idas
es. T
hese
pho
spho
nate
s ar
e no
ntox
ic to
the
hum
an 2
93T
cel
ls; t
hey
prot
ect c
ells
from
in
fluen
za: v
irus
infe
ctio
n w
ith E
C50
val
ues.
in lo
w-n
anom
olar
ran
ge; i
nclu
ding
the
wild
-typ
e W
SN (H
1N1)
, the
200
9 pa
ndem
ic (H
1N1)
, the
ose
ltam
ivir
-res
ista
nt H
274Y
(H1N
1), R
G14
(H5N
1); a
nd U
dorn
(H3N
2) in
fluen
za s
trai
ns.
Shie
et a
l. 20
11
Synt
heti
c st
rate
gies
(+)-
stac
hyfli
nA
nov
el a
nd p
oten
t hem
aggl
utin
in in
hibi
tor,
(+)-
stac
hyfli
n, w
as e
ffic
ient
ly s
ynth
esiz
ed in
an
enan
tiose
lect
ive
man
ner
star
ting
from
the
(+)-
5-m
ethy
l-Wie
land
-Mie
sche
r ke
tone
. The
syn
thet
ic m
etho
d fe
atur
es a
BF(
3) c
ente
r do
t Et(
2)O
-ind
uced
cas
cade
epo
xide
-ope
ning
/rea
rran
gem
ent/
cycl
izat
ion
reac
tion
to s
tere
osel
ectiv
ely
cons
truc
t the
requ
isite
pe
ntac
yclic
rin
g sy
stem
in o
ne s
tep.
In o
rder
to r
atio
naliz
e th
e m
echa
nism
of t
he c
asca
de re
actio
n, q
uant
um c
hem
ical
ca
lcul
atio
ns o
f the
pos
sibl
e in
term
edia
ry c
arbo
catio
ns a
nd tr
ansi
tion
stat
es in
the
mod
el s
ynth
esis
wer
e ca
rrie
d ou
t. A
n al
tern
ativ
e ap
proa
ch to
syn
thes
ize
(+)-
stac
hyfli
n by
em
ploy
ing
a si
mila
r ca
scad
e re
actio
n w
as a
lso
desc
ribe
d.
Saku
rai e
t al.
2011
nuc
leop
rote
in s
cree
ning
as
say
Tryp
toph
an fl
uore
scen
ce
quen
chin
g
Rece
nt s
tudi
es h
ave
show
n th
at N
P (n
ucle
opro
tein
), w
hich
pos
sess
es m
ultip
le fu
nctio
ns in
the
vira
l life
cyc
le, i
s a
new
po
tent
ial a
nti-
influ
enza
dru
g ta
rget
. NP
inhi
bito
rs re
liabl
y in
duce
con
form
atio
nal c
hang
es in
NPs
, and
thes
e ch
ange
s m
ay c
onfe
r in
hibi
tion
of th
e in
fluen
za v
irus
. The
six
con
serv
ed tr
ypto
phan
resi
dues
in N
P ca
n be
use
d as
an
intr
insi
c pr
obe
to m
onito
r th
e ch
ange
in fl
uore
scen
ce o
f the
tryp
toph
an re
sidu
es in
the
prot
ein
upon
bin
ding
to a
n N
P in
hibi
-to
r. In
the
pres
ent s
tudy
, we
foun
d th
at th
e flu
ores
cenc
e of
reco
mbi
nant
NP
prot
eins
was
que
nche
d fo
llow
ing
the
bind
ing
of a
vaila
ble
NP
inhi
bito
rs (s
uch
as n
ucle
ozin
) in
a co
ncen
trat
ion-
and
tim
e-de
pend
ent m
anne
r, w
hich
sug
gest
s th
at th
e in
hibi
tor
indu
ced
conf
orm
atio
nal c
hang
es in
the
NPs
. The
min
imal
fluo
resc
ence
-que
nchi
ng e
ffect
and
wea
k bi
ndin
g co
nsta
nt o
f nuc
leoz
in to
the
swin
e-or
igin
influ
enza
vir
us H
1N1p
dm09
(SO
IV) N
P re
veal
ed th
at th
e SO
IV is
re
sist
ant t
o nu
cleo
zin.
We
have
use
d th
e flu
ores
cenc
e-qu
ench
ing
prop
erty
of t
rypt
opha
ns in
NPs
that
wer
e bo
und
to
ligan
ds in
a 9
6-w
ell-
plat
e-ba
sed
drug
scr
een
to a
sses
s th
e ab
ility
of p
rom
isin
g sm
all m
olec
ules
to in
tera
ct w
ith N
Ps a
nd
have
iden
tifie
d on
e ne
w a
nti-
influ
enza
dru
g, C
SV0C
0010
18, w
ith a
hig
h SI
val
ue. T
his
conv
enie
nt m
etho
d fo
r dr
ug
scre
enin
g m
ay fa
cilit
ate
the
deve
lopm
ent o
f ant
ivir
al d
rugs
that
targ
et v
irus
es o
ther
than
the
influ
enza
vir
us, s
uch
as
HIV
and
HBV
.
Hun
g et
al.
2012
neu
ram
inid
ase
enzy
mat
ic a
ssay
Reco
mbi
nant
ne
uram
inid
ase
expr
essi
onRe
com
bina
nt b
acul
ovir
us
Con
text
: Dev
elop
men
t of i
nexp
ensi
ve a
nd s
afe
enzy
mat
ic a
ssay
s to
scr
een
for
puta
tive
neur
amin
idas
e in
hibi
tors
. O
bjec
tive:
Val
idat
e th
e us
e of
reco
mbi
nant
neu
ram
inid
ase
expr
esse
d in
bac
ulov
irus
loca
ted
on th
e vi
ral s
urfa
ce
caps
ule
to d
evel
op a
neu
ram
inid
ase
inhi
bito
r sc
reen
ing
assa
y. M
ater
ials
and
met
hods
: Rec
ombi
nant
bac
ulov
irus
pa
rtic
les
disp
layi
ng n
eura
min
idas
e N
1 an
d N
3 w
ere
used
as
enzy
me
sour
ces.
The
ass
ay s
et-u
p re
quir
ed th
e us
e of
2’
-(4-
met
hylu
mbe
llife
ryl)-
alph
a-D
-ace
tyl n
eura
min
ic a
cid
as s
ubst
rate
and
ose
ltam
ivir
car
boxy
late
as
benc
hmar
k in
hibi
tor.
Resu
lts: T
he a
ssay
was
set
up
in a
sta
ndar
d 96
-wel
l pla
te. T
he w
ithin
- and
bet
wee
n-as
say
coef
ficie
nts
of v
ari-
atio
n w
ere,
on
aver
age,
less
than
10%
. The
50%
inhi
bito
ry c
once
ntra
tion
valu
es o
f the
inhi
bito
r w
ere
in g
ood
agre
e-m
ent w
ith th
ose
dete
rmin
ed b
y in
depe
nden
t kin
etic
exp
erim
ents
. Dis
cuss
ion
and
conc
lusi
ons:
The
ass
ay s
how
ed s
atis
-fa
ctor
y w
ithin
- and
bet
wee
n-as
say
repe
atab
ility
. The
obt
aine
d re
sults
sug
gest
that
reco
mbi
nant
bac
ulov
irus
exp
ress
ing
neur
amin
idas
e lo
cate
d on
the
viru
s m
embr
ane
caps
ule
can
be u
sed
to s
et u
p af
ford
able
and
relia
ble
neur
amin
idas
e in
hibi
tors
scr
eeni
ng a
ssay
s.
Kon
gkam
nerd
et
al. 2
012
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
137
Fluo
resc
ence
po
lari
sati
on a
ssay
Endo
nucl
ease
inhi
bito
rs
The
end
onuc
leas
e ac
tivity
of t
he in
fluen
za v
irus
PA
(N) p
rote
in is
ess
entia
l for
vir
us re
plic
atio
n an
d is
a p
rom
isin
g ta
rget
for
nove
l ant
i-in
fluen
za d
rugs
. To
faci
litat
e th
e di
scov
ery
of e
ndon
ucle
ase
inhi
bito
rs, w
e ha
ve d
evel
oped
a h
igh-
tluou
ghpu
t flu
ores
cenc
e po
lari
zatio
n (F
P) a
ssay
, util
izin
g a
nove
l flu
ores
cein
-lab
eled
com
poun
d (K
–d =
0.3
78 m
u M
) an
d a
PA(N
) con
stru
ct, t
o id
entif
y sm
all m
olec
ules
that
bin
d to
the
PA(N
) end
onuc
leas
e ac
tive
site
. Sev
eral
kno
wn
4-su
bstit
uted
2,4
-dio
xobu
tano
ic a
cid
inhi
bito
rs w
ith h
igh
and
low
aff
initi
es h
ave
been
eva
luat
ed in
this
FP-
base
d,
com
petit
ive
bind
ing
assa
y, a
nd th
ere
was
a g
ener
al c
orre
latio
n be
twee
n bi
ndin
g an
d th
e re
port
ed in
hibi
tion
of e
ndo-
nucl
ease
act
ivity
. Add
ition
ally
, we
have
dem
onst
rate
d th
e ut
ility
of t
his
assa
y fo
r id
entif
ying
end
onuc
leas
e in
hibi
tors
in
a s
mal
l div
erse
targ
eted
frag
men
t lib
rary
. The
se fr
agm
ent h
its w
ere
used
to b
uild
a fo
llow
-up
libra
ry th
at th
at le
d to
new
act
ive
com
poun
ds th
at d
emon
stra
te F
P bi
ndin
g an
d an
ti-in
fluen
za a
ctiv
ities
in p
laqu
e in
hibi
tion
assa
ys. T
he
assa
y of
fers
sig
nific
ant a
dvan
tage
s. o
ver
prev
ious
ly re
port
ed a
ssay
s an
d is
sui
tabl
e fo
r hi
gh-t
hrou
ghpu
t and
frag
men
t-ba
sed
scre
enin
g st
udie
s. A
dditi
onal
ly th
e de
mon
stra
tion
of th
e ap
plic
abili
ty o
f a m
echa
nism
-bas
ed “t
arge
ted
frag
men
t”
libra
ry s
uppo
rts
the
gene
ral p
oten
tial o
f thi
s no
vel a
ppro
ach
for
othe
r en
zym
e ta
rget
s. T
hese
resu
lts s
erve
as
a so
und
foun
datio
n fo
r th
e de
velo
pmen
t of n
ew th
erap
eutic
lead
s ta
rget
ing
influ
enza
end
onuc
leas
e.
Baug
hman
et a
l. 20
12
Hig
h-th
roug
hput
sc
reen
ing
Cyt
opat
hic
effec
t in
hibi
tory
ass
ayC
ryst
al v
iole
t upt
ake
assa
yIn
fluen
za v
irus
AC
oxsa
ckie
vir
us B
3H
erpe
s sim
plex
vir
us-1
In o
rder
to id
entif
y ne
w p
oten
tial a
ntiv
iral
dru
gs, s
mal
l am
ount
s of
ext
ract
s or
com
poun
ds h
ave
to b
e ex
amin
ed fo
r cy
toto
xici
ty a
nd a
ntiv
iral
act
ivity
in p
rim
ary
scre
enin
g us
ing
a ra
pid,
eas
y, in
expe
nsiv
e, a
nd h
ighl
y st
anda
rdis
ed te
st
syst
em. I
n th
is s
tudy
, hig
h-th
roug
hput
cyt
opat
hic
effe
ct (C
PE) i
nhib
itory
ass
ays
wer
e es
tabl
ishe
d fo
r co
xsac
kie
viru
s B3
on
HeL
a O
hio
cells
, inf
luen
za v
irus
A o
n M
adin
-Dar
by c
anin
e ki
dney
cel
ls, a
nd h
erpe
s si
mpl
ex v
irus
type
(HSV
-1)
on
gree
n m
onke
y ki
dney
cel
ls th
at m
eet t
hese
requ
irem
ents
. The
cyt
otox
ic a
nd th
e an
tivir
al e
ffect
s w
ere
quan
ti-fie
d us
ing
a cr
ysta
l vio
let u
ptak
e as
say
allo
win
g au
tom
ated
han
dlin
g of
larg
e nu
mbe
rs o
f can
dida
te a
gent
s. T
o en
sure
co
mpa
rabl
e re
sults
with
pla
que
redu
ctio
n as
says
. the
50
and
90%
pla
que
inhi
bito
ry c
once
ntra
tions
of g
uani
dine
, am
anta
dine
, and
pho
spho
nofo
rmic
aci
d w
ere
used
to s
tand
ardi
se th
e an
ti-co
xsac
kie
viru
s B3
, ant
i-in
fluen
za v
irus
A,
and
anti-
HSV
-l te
sts,
resp
ectiv
ely.
The
str
ong
corr
elat
ion
betw
een
the
antiv
iral
act
ivity
det
erm
ined
by
CPE
-inh
ibito
ry
assa
ys a
nd p
laqu
e re
duct
ion
assa
y w
as fu
rthe
r pr
oved
for
othe
r an
tivir
als.
In s
umm
ary,
low
am
ount
s of
larg
e nu
mbe
rs
of c
ompo
unds
may
be
test
ed in
expe
nsiv
ely
and
stan
dard
ised
with
in 2
4 h
(cox
sack
ie v
irus
B3
and
influ
enza
vir
us A
) or
48 h
(her
pes
sim
plex
vir
us ty
pe 1
) pos
t-in
fect
ion
usin
g C
PE in
hibi
tory
ass
ays.
Schm
idtk
e et
al.
2001
Hig
h-th
roug
hput
sc
reen
ing
Plaq
ue in
hibi
tion
assa
y Ti
me-
of-a
dditi
on
expe
rim
ent
Trad
ition
al C
hine
se
med
icin
ePr
ocya
nidi
n
In th
is re
sear
ch, w
e ha
ve e
stab
lishe
d a
high
-thr
ough
put s
cree
ning
(HT
S) p
latf
orm
bas
ed o
n th
e in
fluen
za A
vir
us
(IAV
) vRN
A p
rom
oter
. Usi
ng th
is H
TS
plat
form
, we
sele
cted
35
med
icin
al p
lant
s ou
t of 8
3 ex
ampl
es o
f tra
ditio
nal
Chi
nese
med
icin
e an
d fo
und
that
7 e
xam
ples
had
not
bee
n re
port
ed. A
fter
exa
min
ing
man
y pr
evio
us re
port
s, w
e fo
und
that
Vac
cini
um a
ngus
tifol
ium
Ait
., V
itis
vini
fera
L, a
nd C
inna
mom
um c
assi
a Pr
esl h
ad a
com
mon
act
ive
com
poun
d,
proc
yani
din,
and
then
det
erm
ined
the
anti-
IAV
effe
ct o
f pro
cyan
idin
and
exp
lore
d its
mec
hani
sm o
f act
ion.
With
a
plaq
ue in
hibi
tion
assa
y an
d a
time-
of-a
dditi
on e
xper
imen
t, w
e fo
und
that
pro
cyan
idin
cou
ld in
hibi
t the
IAV
repl
ica-
tion
at s
ever
al s
tage
s of
the
life
cycl
e. In
the
Wes
tern
blo
t and
EG
FP-L
C3
loca
lizat
ion
assa
ys, w
e fo
und
that
pro
cyan
i-di
n co
uld
inhi
bit t
he a
ccum
ulat
ion
of L
C3I
I and
the
dot-
like
aggr
egat
ion
of E
GFP
-LC
3. In
the
RT-P
CR
and
Wes
tern
bl
ot a
ssay
s, w
e fo
und
proc
yani
din
coul
d in
hibi
t the
exp
ress
ion
of A
tg7,
Atg
5, a
nd A
tg12
. Fin
ally
, by
the
bim
olec
ular
flu
ores
cenc
e co
mpl
emen
tatio
n-flu
ores
cenc
e re
sona
nce
ener
gy tr
ansf
er a
nd c
o-im
mun
opre
cipi
tatio
n as
says
, we
foun
d th
at p
rocy
anid
in c
ould
inhi
bit t
he fo
rmat
ion
of th
e A
tg5-
Atg
12/A
tg16
het
erot
rim
er a
nd th
e di
ssoc
iatio
n of
the
becl
in1/
bcl2
het
erod
imer
. In
conc
lusi
on, w
e ha
ve e
stab
lishe
d an
HT
S pl
atfo
rm a
nd id
entif
ied
proc
yani
din
as a
nov
el
and
prom
isin
g an
ti-IA
V a
gent
.
Dai
et a
l. 20
12
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
138
Com
puta
tion
al/
expe
rim
enta
l sc
reen
ing
Flow
cyt
omet
ry a
naly
sis
Cas
pase
-3 a
ssay
Min
i-gen
ome
assa
yTi
me-
of-a
dditi
on
expe
rim
ent
Ribo
nucl
eopr
otei
n co
mpl
ex
In th
is s
tudy
, com
puta
tiona
l and
exp
erim
enta
l scr
eeni
ng o
f an
exte
nsiv
e co
mpo
und
libra
ry id
entif
ied
TH
C19
, whi
ch
was
abl
e to
sup
pres
s in
fluen
za v
irus
repl
icat
ion.
Thi
s co
mpo
und
had
no c
ytot
oxic
effe
cts
and
did
not d
isru
pt c
ell
cycl
e pr
ogre
ssio
n or
indu
ce a
popt
osis
in M
DC
K c
ells
as
conf
irm
ed b
y W
ST-1
ass
ays,
flow
cyt
omet
ry a
naly
sis,
and
ca
spas
e-3
assa
ys. T
ime-
of-a
dditi
on e
xper
imen
ts s
how
ed th
at T
HC
19 a
cts
at a
rela
tivel
y ea
rly
stag
e of
the
vira
l life
-cy
cle.
Sub
sequ
ent m
ini-
geno
me
assa
ys re
veal
ed th
at T
HC
19 in
hibi
ted
vira
l gen
ome
repl
icat
ion
and/
or tr
ansc
ript
ion,
su
gges
ting
that
it in
terf
eres
with
one
or
mor
e of
the
vira
l com
pone
nts
that
form
the
ribo
nucl
eopr
otei
n co
mpl
exes
, na
mel
y po
lym
eras
e ba
sic
2 (P
B2),
poly
mer
ase
basi
c 1
(PB1
), po
lym
eras
e ac
idic
(PA
), nu
cleo
prot
ein
(NP)
and
vir
al
RNA
. Fin
ally
, min
i-ge
nom
e as
says
whe
re P
B2, P
B1, P
A o
r N
P fr
om A
/WSN
/33
(H1N
1) v
irus
wer
e re
plac
ed w
ith th
ose
from
A/U
dorn
/307
/197
2 (H
3N2)
vir
us e
ffect
ivel
y de
mon
stra
ted
that
TH
C19
inhi
bite
d vi
ral m
ultip
licat
ion
in a
man
ner
depe
nden
t upo
n th
e PA
sub
unit
. Tak
en to
geth
er, t
hese
resu
lts s
ugge
st th
at in
fluen
za v
irus
PA
pro
tein
is a
pot
entia
l ta
rget
for,
and
may
aid
the
deve
lopm
ent o
f, no
vel c
ompo
unds
that
inhi
bit i
nflu
enza
A v
irus
repl
icat
ion.
Yam
ada
et a
l. 20
12
Ferr
et/m
ouse
mod
elIn
tran
asal
adm
inis
trat
ion
Stac
hyfli
n de
riva
tives
The
in-v
ivo
anti-
influ
enza
-vir
us a
ctiv
ity o
f Sta
chyf
lin d
eriv
ativ
es (I
II a
nd it
s ph
osph
ate
este
r, II
I-Ph
os),
a ne
w c
lass
of
haem
aggl
utin
in fu
sion
inhi
bito
r, an
d th
e im
prov
emen
t of t
heir
abs
orpt
ion
afte
r or
al o
r in
tran
asal
adm
inis
trat
ion
wer
e st
udie
d in
mic
e, r
ats,
and
ferr
ets.
The
abs
orpt
ion
of II
I in
PEG
400
0 an
d II
I-Ph
os a
queo
us s
olut
ion
incr
ease
d ab
out
thre
e an
d fo
ur fo
ld in
AU
C a
fter
ora
l adm
inis
trat
ion
to u
ninf
ecte
d m
ice
com
pare
d w
ith th
at o
f 0.5
% H
PMC
(hyd
roxy
-pr
opyl
-met
hylc
ellu
lose
) sus
pens
ion.
Usi
ng a
mou
se in
fluen
za v
irus
infe
ctio
n m
odel
, sig
nific
ant a
nti-
influ
enza
-vir
us
activ
ity w
as o
bser
ved
in in
fect
ed m
ice
trea
ted
oral
ly w
ith th
ese
com
poun
ds d
isso
lved
in P
EG 4
000
or d
istil
led
wat
er,
resp
ectiv
ely,
but
not
in m
ice
trea
ted
with
0.5
% H
PMC
. The
in-v
ivo
anti-
influ
enza
-vir
us a
ctiv
ity in
ferr
ets,
a g
ood
mod
el fo
r in
fluen
za v
irus
infe
ctio
n in
man
, was
als
o st
udie
d. A
lthou
gh th
e co
ncen
trat
ion
of II
I in
plas
ma
was
abo
ve
the
IC50
aga
inst
the
influ
enza
vir
us s
trai
n us
ed fo
r 6h
aft
er th
e or
al a
dmin
istr
atio
n of
III i
n PE
G 4
00 to
uni
nfec
ted
ferr
ets,
no
in-v
ivo
antii
nflu
enza
-vir
us a
ctiv
ity w
as o
bser
ved
at th
e sa
me
dosa
ge g
iven
4 ti
mes
dai
ly fo
r 3
days
. The
in
tran
asal
adm
inis
trat
ion
of II
I-Ph
os, w
hich
was
exp
ecte
d to
hav
e a
mor
e no
tabl
e in
-viv
o an
ti-in
fluen
za-v
irus
act
iv-
ity, w
as e
xam
ined
. III
-Pho
s, w
hose
intr
anas
al a
bsor
ptio
n ha
d be
en im
prov
ed b
y th
e m
odifi
catio
n of
III w
ith p
hosp
hate
es
ter
in r
ats,
inhi
bite
d vi
ral r
eplic
atio
n in
the
nasa
l cav
ity a
nd s
uppr
esse
d in
fluen
za-v
irus
-ind
uced
feve
r w
hen
adm
inis
-te
red
intr
anas
ally
to in
fect
ed fe
rret
s. T
his
stud
y de
mon
stra
tes
that
intr
anas
ally
adm
inis
tere
d co
mpo
unds
with
ant
i-in
fluen
za-v
irus
act
ivity
mus
t per
mea
te th
e na
sal m
embr
anes
to p
rodu
ce th
eir
anti-
influ
enza
-vir
us e
ffect
.
Yosh
imot
o et
al.
2000
Chi
cken
egg
mod
elC
ompa
rativ
e st
udy
Am
anta
dine
Rim
anta
dine
Ose
ltam
ivir
Z
anam
ivir
Prev
ious
stud
ies h
ave
show
n th
at e
mbr
yona
ted
egg
prov
ides
a c
onve
nien
t and
eas
y to
use
syst
em fo
r in
vivo
scre
enin
g of
an
ti-in
fluen
za v
irus
inhi
bito
rs. H
owev
er, i
t is n
ot k
now
n w
heth
er th
is m
odel
is su
itabl
e fo
r tes
ting
neur
amin
idas
e (N
A)
inhi
bito
rs, t
oo. Th
eref
ore,
the
pres
ent s
tudy
des
crib
es th
e ev
alua
tion
of th
e io
n-ch
anne
l blo
cker
s am
anta
dine
and
rim
an-
tadi
ne in
com
pari
son
with
the
NA
inhi
bito
rs o
selta
miv
ir a
nd z
anam
ivir
by
usin
g th
e in
fluen
za A
vir
us h
en’s
egg
mod
el.
The
trea
tmen
t was
star
ted
imm
edia
tely
bef
ore
or a
fter
the
chal
leng
e do
se w
as p
lace
d on
the
chor
ioal
lant
oic
mem
bran
e (C
AM
). D
iffer
ence
s bet
wee
n th
e su
rviv
al ra
te o
f tre
ated
and
unt
reat
ed c
hick
em
bryo
s inf
ecte
d w
ith in
fluen
za A
vir
us
wer
e an
alyz
ed st
atis
tical
ly. A
s res
ult,
the
surv
ival
rate
of c
hick
em
bryo
s cou
ld b
e si
gnifi
cant
ly in
crea
sed
whe
n th
e tr
eat-
men
t with
am
anta
dine
, rim
anta
dine
, ose
ltam
ivir,
or z
anam
ivir
was
star
ted
befo
re th
e C
AM
was
inoc
ulat
ed w
ith o
ne e
gg
infe
ctiv
e do
se 5
0% (E
ID50
) infl
uenz
a A
vir
us. W
hen
the
drug
s wer
e ad
min
iste
red
shor
tly a
fter
vir
al in
ocul
atio
n, si
gnifi
-ca
nt a
ntiv
iral
effi
cacy
was
show
n fo
r rim
anta
dine
, ose
ltam
ivir,
and
zan
amiv
ir. A
ntiv
iral
effi
cacy
cou
ld b
e de
mon
stra
ted
excl
usiv
ely
for b
oth
osel
tam
ivir
and
zan
amiv
ir a
fter
the
embr
yos w
ere
infe
cted
with
hig
her c
halle
nge
dose
s of 1
02 EID
50
influ
enza
A v
irus
. In
conc
lusi
on, t
he N
A in
hibi
tors
ose
ltam
ivir
and
zan
amiv
ir h
ave
a si
gnifi
cant
ly b
ette
r ant
ivir
al a
ctiv
ity
agai
nst i
nflue
nza
A v
irus
than
am
anta
dine
and
rim
anta
dine
test
ed in
em
bryo
nate
d he
n’s e
ggs.
Ther
efor
e, th
is m
odel
can
be
a v
alua
ble
alte
rnat
ive
appr
oach
for i
n vi
vo p
re-t
estin
g an
ti-in
fluen
za v
irus
act
ivity
of N
A in
hibi
tors
.
Saue
rbre
i et a
l. 20
06
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
139
Phar
mac
okin
etic
stu
dies
Ose
ltam
ivir
Bioa
vaila
bilit
yRe
nal c
lear
ance
Stea
dy-s
tate
pla
sma
conc
entr
atio
n
Ose
ltam
ivir
is a
n et
hyl e
ster
pro
drug
of P
o 64
-080
2, a
sel
ectiv
e in
hibi
tor
of in
fluen
za v
irus
neu
ram
inid
ase.
Ora
l ad
min
istr
atio
n of
ose
ltam
ivir
del
iver
s th
e ac
tive
antiv
iral
Po
64-0
802
to th
e bl
oods
trea
m, a
nd th
us a
ll si
tes
of in
fluen
za
infe
ctio
n (lu
ng, n
asal
muc
osa,
mid
dle
ear)
are
acc
essi
ble.
The
pha
rmac
okin
etic
pro
file
of o
selta
miv
ir is
sim
ple
and
pre-
dict
able
, and
twic
e da
ily tr
eatm
ent r
esul
ts in
effe
ctiv
e an
tivir
al p
lasm
a co
ncen
trat
ions
ove
r th
e en
tire
adm
inis
trat
ion
inte
rval
. Aft
er o
ral a
dmin
istr
atio
n, o
selta
miv
ir is
read
ily a
bsor
bed
from
the
gast
roin
test
inal
trac
t and
ext
ensi
vely
con
-ve
rted
to th
e ac
tive
met
abol
ite. T
he a
bsol
ute
bioa
vaila
bilit
y of
the
activ
e m
etab
olite
from
ora
lly a
dmin
iste
red
osel
tam
i-vi
r is
80%
. The
act
ive
met
abol
ite is
det
ecta
ble
in p
lasm
a w
ithin
30
min
utes
and
reac
hes
max
imal
con
cent
ratio
ns a
fter
3
to 4
hou
rs. A
fter
pea
k pl
asm
a co
ncen
trat
ions
are
att
aine
d, th
e co
ncen
trat
ion
of th
e ac
tive
met
abol
ite d
eclin
es w
ith
an a
ppar
ent h
alf-
life
of 6
to 1
0 ho
urs.
Ose
ltam
ivir
is e
limin
ated
pri
mar
ily b
y co
nver
sion
to a
nd re
nal e
xcre
tion
of th
e ac
tive
met
abol
ite. R
enal
cle
aran
ce o
f bot
h co
mpo
unds
exc
eeds
glo
mer
ular
filtr
atio
n ra
te, i
ndic
atin
g th
at re
nal t
ubul
ar
secr
etio
n co
ntri
bute
s to
thei
r el
imin
atio
n vi
a th
e an
ioni
c pa
thw
ay. N
eith
er c
ompo
und
inte
ract
s w
ith c
ytoc
hrom
e P4
50
mix
ed-f
unct
ion
oxid
ases
or
gluc
uron
osyl
tran
sfer
ases
. The
pha
rmac
okin
etic
pro
file
of th
e ac
tive
met
abol
ite is
line
ar
and
dose
-pro
port
iona
l, w
ith le
ss th
an 2
-fol
d ac
cum
ulat
ion
over
a d
osag
e ra
nge
of o
selta
miv
ir 5
0 to
500
mg
twic
e da
ily.
Stea
dy-s
tate
pla
sma
conc
entr
atio
ns a
re a
chie
ved
with
in 3
day
s of
twic
e da
ily a
dmin
istr
atio
n, a
nd a
t a d
osag
e of
75m
g tw
ice
daily
the
stea
dy-s
tate
pla
sma
trou
gh c
once
ntra
tions
of a
ctiv
e m
etab
olite
rem
ain
abov
e th
e m
inim
um in
hibi
tory
co
ncen
trat
ion
for
all i
nflu
enza
. str
ains
test
ed. E
xpos
ure
to th
e ac
tive
met
abol
ite a
t ste
ady
stat
e is
app
roxi
mat
ely
25%
hi
gher
in e
lder
ly c
ompa
red
with
you
ng in
divi
dual
s; h
owev
er, n
o do
sage
adj
ustm
ent i
s ne
cess
ary.
In p
atie
nts
with
rena
l im
pair
men
t, m
etab
olite
cle
aran
ce d
ecre
ases
line
arly
with
cre
atin
ine
clea
ranc
e. A
dos
age
redu
ctio
n to
75m
g on
ce d
aily
is
reco
mm
ende
d fb
r pa
tient
s w
ith c
reat
inin
e cl
eara
nce
<30
ml/
min
(1.8
l/h)
. The
pha
rmac
okin
etic
s in
pat
ient
s w
ith
influ
enza
are
qua
litat
ivel
y si
mila
r to
thos
e in
hea
lthy
youn
g ad
ults
. In
vitr
o an
d in
viv
o st
udie
s in
dica
te n
o cl
inic
ally
si
gnifi
cant
dru
g in
tera
ctio
ns. N
eith
er p
arac
etam
ol (a
ceta
min
ophe
n) n
or c
imet
idin
e al
tere
d th
e ph
arm
acok
inet
ics
of
Ro 6
4-08
02. C
oadm
inis
trat
ion
of p
robe
neci
d re
sulte
d in
a 2
.5-f
old
incr
ease
in e
xpos
ure
to R
o 64
-080
2, h
owev
er, t
his
com
petit
ion
is u
nlik
ely
to re
sult
in c
linic
ally
rele
vant
effe
cts.
The
se p
rope
rtie
s m
ake
osel
tam
ivir
a. s
uita
ble
cand
idat
e fo
r us
e in
the
prev
entio
n an
d tr
eatm
ent o
f inf
luen
za.
He
et a
l. 19
99
Mul
tice
nter
clin
ical
st
udy
Zan
amiv
irA
ntiv
iral
act
ivity
Safe
tyTo
lera
bilit
yC
hina
201
0–20
11A
dole
scen
ts/a
dults
Back
grou
nd It
is th
e fir
st m
ultic
ente
r cl
inic
al s
tudy
in C
hina
to in
vest
igat
e za
nam
ivir
use
am
ong
Chi
nese
ado
lesc
ents
an
d ad
ults
with
influ
enza
-lik
e ill
ness
(ILI
) sin
ce 2
009,
whe
n in
hale
d za
nam
ivir
(REL
ENZ
A (R
)) w
as m
arke
ted
in
Chi
na. M
etho
ds A
n un
cont
rolle
d op
en-l
abel
, mul
ticen
tre
stud
y to
eva
luat
e th
e an
tivir
al a
ctiv
ity, a
nd s
afet
y of
inha
led
zana
miv
ir (a
s Ro
tadi
sk v
ia D
iskh
aler
dev
ice)
; 10
mg
adm
inis
tere
d tw
ice
daily
for
5 da
ys in
sub
ject
s ≥
12 y
ears
old
w
ith IL
I. Pa
tient
s w
ere
enro
lled
with
in 4
8 ho
urs
of o
nset
and
follo
wed
for
eigh
t day
s. P
atie
nts
wer
e de
fined
as
bein
g in
fluen
za-p
ositi
ve if
the
real
-tim
e re
vers
e tr
ansc
ript
ase-
poly
mer
ase
chai
n re
actio
n (r
RT-P
CR)
test
had
pos
itive
resu
lts.
Resu
lts A
tota
l of 4
00 p
atie
nts
≥ 12
yea
rs o
ld w
ere
scre
ened
from
11
cent
ers
in s
even
pro
vinc
es fr
om M
arch
201
0 to
Ja
nuar
y 20
11. T
hree
hun
dred
and
nin
ety-
two
patie
nts
who
took
at l
east
one
dos
e of
zan
amiv
ir w
ere
ente
red
into
the
safe
ty a
naly
sis.
The
mea
n ag
e w
as 3
3.8
year
s an
d 50
% w
ere
mal
e. C
ardi
ovas
cula
r di
seas
es a
nd d
iabe
tes
wer
e th
e m
ost
com
mon
com
orbi
ditie
s. A
ll th
e re
port
ed a
dver
se e
vent
s, s
uch
as r
ash,
nas
al a
che,
mus
cle
ache
, nau
sea,
dia
rrhe
a, h
ead-
ache
, occ
urre
d in
less
than
1%
of s
ubje
cts.
Mild
sin
us b
rady
cadi
a or
arr
hyth
mia
occ
urre
d in
four
sub
ject
s (1
%).
Mos
t of
the
adve
rse
even
ts w
ere
mild
and
did
not
requ
ire
any
chan
ge o
f tre
atm
ent.
No
seve
re a
dver
se e
vent
s (S
AE)
or
fata
l ca
ses
wer
e re
port
ed. B
ronc
hosp
asm
was
foun
d in
a 3
8 ye
ars
old
wom
an w
hose
sym
ptom
s di
sapp
eare
d af
ter
stop
ping
za
nam
ivir
and
with
out a
dditi
onal
trea
tmen
t. A
ll th
e 61
influ
enza
vir
us is
olat
es (4
3 be
fore
enr
ollm
ent,
18 d
urin
g tr
eat-
men
t) p
rove
d to
be
sens
itive
to z
anam
ivir.
Con
clus
ions
Zan
amiv
ir is
wel
l tol
erat
ed b
y C
hine
se a
dole
scen
ts a
nd a
dults
w
ith IL
Is. T
here
is n
o ev
iden
ce fo
r th
e em
erge
nce
of d
rug-
resi
stan
t iso
late
s du
ring
trea
tmen
t with
zan
amiv
ir.
Cao
et a
l. 20
12
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
140
Tol
erab
ility
/ph
arm
acok
inet
ics
Neu
ram
inid
ase
inhi
bito
rsA
dult/
elde
rly
volu
ntee
rsSt
eady
-sta
te p
lasm
a co
ncen
trat
ions
The
tole
rabi
lity
and
phar
mac
okin
etic
s of
Ro
64-0
802,
a p
oten
t, se
lect
ive
inhi
bito
r of
influ
enza
neu
ram
inid
ase,
and
ifs
oral
pro
drug
ose
ltam
ivir
wer
e in
vest
igat
ed in
thre
e do
uble
-blin
d, p
lace
bo-c
ontr
olle
d st
udie
s. T
wo
stud
ies
invo
lved
he
alth
y ad
ult v
olun
teer
s (1
8–55
yea
rs) (
n =
48) w
ho re
ceiv
ed s
ingl
e (2
0–10
00 m
g) o
r bi
d do
ses
(50–
500
mg)
(n =
32)
of
ose
ltam
ivir
or
plac
ebo
for
7 da
ys. H
ealth
y el
derl
y vo
lunt
eers
(gre
ater
than
or
equa
l to
65 y
ears
) (n
= 24
) rec
eive
d os
elta
miv
ir 1
00 to
200
mg
bid
or p
lace
bo fo
r 7
days
in a
thir
d st
udy.
Mea
sura
ble
plas
ma
conc
entr
atio
ns o
f the
act
ive
met
abol
ite a
ppea
red
rapi
dly
in p
lasm
a an
d w
ere
sign
ifica
ntly
hig
her
and
long
er la
stin
g th
an th
ose
of o
selta
miv
ir.
Phar
mac
okin
etic
s of
bot
h co
mpo
unds
wer
e lin
ear.
Mul
tiple
-dos
e ex
posu
re w
as p
redi
ctab
le fr
om s
ingl
e-do
se d
ata,
and
st
eady
-sta
te p
lasm
a co
ncen
trat
ions
wer
e ac
hiev
ed w
ithin
3 d
ays
of b
id d
rug
adm
inis
trat
ion.
Ose
ltam
ivir
was
wel
l tol
er-
ated
at s
ingl
e do
ses
of u
p to
100
0 m
g an
d tw
ice-
daily
dos
es o
f up
to 5
00 m
g. A
dver
se e
vent
s w
ere
mild
in in
tens
ity.
Expo
sure
to b
oth
prod
rug
and
activ
e m
etab
olite
was
incr
ease
d in
eld
erly
pat
ient
s by
app
roxi
mat
ely
25%
. How
ever
, due
to
the
wid
e sa
fety
mar
gin
of b
oth
com
poun
ds, n
o do
se a
djus
tmen
t is
nece
ssar
y fo
r el
derl
y pa
tient
s.
Mas
sare
lla e
t al.
2000
Tabl
e 4.
1. In
hibi
tors
of h
aem
aggl
utin
in
Hae
mag
glut
inin
X-r
ay c
ryst
alog
raph
yRe
cept
or b
indi
ng
Hem
aggl
utin
in (H
A) i
s th
e re
cept
or-b
indi
ng a
nd m
embr
ane
fusi
on g
lyco
prot
ein
of in
fluen
za v
irus
and
the
targ
et fo
r in
fect
ivity
-neu
tral
izin
g an
tibod
ies.
The
str
uctu
res
of th
ree
conf
orm
atio
ns o
f the
ect
odom
ain
of th
e 19
68 H
ong
Kon
g in
fluen
za v
irus
HA
hav
e be
en d
eter
min
ed b
y X
-ray
cry
stal
logr
aphy
: the
sin
gle-
chai
n pr
ecur
sor,
HA
0; th
e m
etas
tabl
e ne
utra
l-pH
con
form
atio
n fo
und
on v
irus
, and
the
fusi
on p
H-i
nduc
ed c
onfo
rmat
ion.
The
se s
truc
ture
s pr
ovid
e a
fram
e-w
ork
for
desi
gnin
g an
d in
terp
retin
g th
e re
sults
of e
xper
imen
ts o
n th
e ac
tivity
of H
A in
rece
ptor
bin
ding
, the
gen
era-
tion
of e
mer
ging
and
reem
ergi
ng e
pide
mic
s, a
nd m
embr
ane
fusi
on d
urin
g vi
ral e
ntry
.
Skeh
el a
nd W
iley
2000
Sial
ylgl
ycan
sM
olec
ular
dyn
amic
s si
mul
atio
nH
aem
aggl
utin
in-r
ecep
tor
inte
ract
ion
Reco
gniti
on o
f cel
l-su
rfac
e si
alyl
disa
ccha
ride
s by
influ
enza
A h
emag
glut
inin
(HA
) tri
gger
s th
e in
fect
ion
proc
ess
of
influ
enza
. The
cha
nges
in g
lyco
sidi
c to
rsio
nal l
inka
ge a
nd th
e re
cept
or c
onfo
rmat
ions
may
alte
r th
e bi
ndin
g sp
ecifi
city
of
HA
s to
the
sial
ylgl
ycan
s. In
this
stu
dy, 1
0-ns
mol
ecul
ar d
ynam
ics
sim
ulat
ions
wer
e ca
rrie
d ou
t to
exam
ine
the
stru
c-tu
ral a
nd d
ynam
ic b
ehav
ior
of th
e H
As
boun
d w
ith s
ialy
ldis
acch
arid
es N
eu5A
c al
pha(
2-3)
Gal
(N23
G) a
nd N
eu5A
c al
pha(
2-6)
Gal
(N26
G).
The
ana
lysi
s of
the
glyc
osid
ic to
rsio
nal a
ngle
s an
d th
e pa
ir in
tera
ctio
n en
ergy
bet
wee
n th
e re
cept
or a
nd th
e in
tera
ctin
g re
sidu
es o
f the
bin
ding
site
reve
al th
at N
23G
has
two
bind
ing
mod
es fo
r H
1 an
d H
5 an
d a
sing
le b
indi
ng m
ode
for
H3
and
H9.
For
N26
G, H
1 an
d H
3 ha
s tw
o bi
ndin
g m
odes
, and
H5
and
H9
has
a si
ngle
bin
d-in
g m
ode.
The
dir
ect a
nd w
ater
-med
iate
d hy
drog
en b
ondi
ng in
tera
ctio
ns b
etw
een
the
rece
ptor
s an
d H
As
play
dom
i-na
nt ro
les
in th
e st
ruct
ural
sta
biliz
atio
n of
the
com
plex
es. I
t is
conc
lude
d fr
om p
air
inte
ract
ion
ener
gy a
nd M
olec
ular
M
echa
nic-
Pois
son-
Boltz
-man
n Su
rfac
e A
rea
calc
ulat
ions
that
N26
G is
a b
ette
r re
cept
or fo
r H
1 w
hen
com
pare
d w
ith
N23
G. N
23G
is a
bet
ter
rece
ptor
for
H5
whe
n co
mpa
red
with
N26
G. H
owev
er, H
3 an
d H
9 ca
n re
cogn
ize
N23
G a
nd
N26
G in
equ
al b
indi
ng s
peci
ficity
due
to th
e m
argi
nal e
nerg
y di
ffere
nce
(app
roxi
mat
e to
2.5
kca
l/m
ol).
The
ord
er o
f bi
ndin
g sp
ecifi
city
of N
23G
is H
3 >
H5
> H
9 >
H1
and
N26
G is
H1
> H
3 >
H5
> H
9, re
spec
tivel
y. T
he p
ropo
sed
con-
form
atio
nal m
odel
s w
ill b
e he
lpfu
l in
desi
gnin
g in
hibi
tors
for
influ
enza
vir
us.
Priy
adar
zini
et a
l. 20
12
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
141
Sial
ylla
ctos
e-co
ntai
ning
gl
ycop
olym
ers
Che
moe
nzym
atic
sy
nthe
sis
Gly
co-b
iose
nsor
Gly
coar
ray
We
repo
rt a
che
moe
nzym
atic
syn
thes
is o
f cha
in-e
nd fu
nctio
naliz
ed s
ialy
llact
ose-
cont
aini
ng g
lyco
poly
mer
s w
ith d
iffer
-en
t lin
kage
s an
d th
eir
orie
nted
imm
obili
zatio
n fo
r gl
ycoa
rray
and
SPR
-bas
ed g
lyco
-bio
sens
or a
pplic
atio
ns. S
peci
fical
ly,
O-c
yana
te c
hain
-end
func
tiona
lized
sia
lylla
ctos
e-co
ntai
ning
gly
copo
lym
ers
wer
e sy
nthe
size
d by
enz
ymat
ic a
2,3-
and
al
pha
2,6-
sial
ylat
ion
of a
lact
ose-
cont
aini
ng g
lyco
poly
mer
that
was
syn
thes
ized
by
cyan
oxyl
-med
iate
d fr
ee r
adic
al
poly
mer
izat
ion.
H-1
NM
R sh
owed
alm
ost q
uant
itativ
e al
pha
2,3-
and
alp
ha 2
,6-s
ialy
latio
n. T
he O
-cya
nate
cha
in-e
nd
func
tiona
lized
sia
lylla
ctos
e-co
ntai
ning
gly
copo
lym
ers
wer
e pr
inte
d on
to a
min
e-fu
nctio
naliz
ed g
lass
slid
es v
ia is
oure
a bo
nd fo
rmat
ion
for
glyc
oarr
ay fo
rmat
ion.
Spe
cific
pro
tein
bin
ding
act
ivity
of t
he a
rray
s w
as c
onfir
med
with
alp
ha 2
,3-
and
alph
a 2,
6-si
alyl
spe
cific
bin
ding
lect
ins
toge
ther
with
inhi
bitio
n as
says
. Fur
ther
, im
mob
ilizi
ng O
-cya
nate
cha
in-
end
func
tiona
lized
sia
lylla
ctos
e-co
ntai
ning
gly
copo
lym
ers
onto
am
ine-
mod
ified
SPR
chi
p vi
a is
oure
a bo
nd fo
rmat
ion
affo
rded
SPR
-bas
ed g
lyco
-bio
sens
or, w
hich
sho
wed
spe
cific
bin
ding
act
ivity
for
lect
ins
and
influ
enza
vir
al h
emag
glu-
tinin
s (H
A).
The
se s
ialy
lolig
o-m
acro
ligan
d de
rive
d gl
ycoa
rray
and
SPR
-bas
ed g
lyco
-bio
sens
or a
re c
lose
ly to
mim
ic 3
D
natu
re p
rese
ntat
ion
of s
ialy
lolig
osac
char
ides
and
will
pro
vide
impo
rtan
t hig
h-th
roug
hput
tool
s fo
r vi
rus
diag
nosi
s an
d po
tent
ial a
ntiv
iral
dru
g ca
ndid
ates
scr
eeni
ng a
pplic
atio
ns.
Nar
la a
nd S
un
2012
Sial
ylla
ctos
eSt
ruct
ural
ana
lysi
sC
ompu
ter s
imul
atio
n
We
desi
gned
and
syn
thes
ized
nov
el tr
ival
ent a
nti-
influ
enza
reag
ents
. Sia
lylla
ctos
e w
as lo
cate
d at
the
term
inal
of e
ach
vale
nce
whi
ch a
imed
to b
lock
eac
h re
cept
or-b
indi
ng s
ite o
f the
hem
aggl
utin
in (H
A) t
rim
er o
n th
e su
rfac
e of
the
viru
s.
Stru
ctur
al a
naly
ses
wer
e ca
rrie
d ou
t with
a m
odel
whi
ch w
as c
onst
ruct
ed w
ith a
com
pute
r si
mul
atio
n. A
pre
viou
sly
repo
rted
cyc
lic g
lyco
pept
ide
bloc
ker
boun
d to
the
HA
in th
e m
odel
. The
ana
lyse
s su
gges
t tha
t the
glu
tam
ine
resi
due
in th
e cy
clic
pep
tide
bear
ing
Neu
5A a
lpha
2,3
Gal
bet
a 1,
4Glc
tris
acch
arid
e vi
a a
linke
r in
tera
cts
with
the
Gln
189
in
HA
thro
ugh
hydr
ogen
bon
ding
. The
pre
sent
ant
i-in
fluen
za re
agen
ts li
kely
inte
ract
with
a g
luta
min
e re
sidu
e in
clud
ed
in th
e vi
cini
ty o
f Gln
189.
A p
lagu
e re
duct
ion
assa
y of
the
influ
enza
vir
us, A
/PR/
8/19
34 (H
1N1)
, was
per
form
ed in
M
DC
K c
ells
to e
valu
ate
for
the
synt
hesi
zed
com
poun
ds to
inhi
bit v
iral
repl
icat
ion.
One
of t
he c
ompo
unds
sho
wed
ap
prox
imat
ely
85%
inhi
bitio
n at
the
conc
entr
atio
n of
400
mu
M a
t 4 d
egre
es °C
.
Feng
et a
l. 20
10
Sial
ic a
cid
anal
ogue
sC
2-, C
4- a
nd
C5-
subs
titut
ions
M
olec
ular
doc
king
Aut
oDoc
k3.0
5
A m
olec
ular
doc
king
tool
of A
utoD
ock3
.05
was
eva
luat
ed fo
r its
abi
lity
to re
prod
uce
expe
rim
enta
lly d
eter
min
ed a
ffin
i-tie
s of
var
ious
sia
lic a
cid
anal
ogue
s to
war
d he
mag
glut
inin
of i
nflu
enza
A v
irus
. With
the
exce
ptio
n of
thos
e w
ith a
C
6-m
odifi
ed g
lyce
rol s
ide
chai
n, th
e ex
peri
men
tal b
indi
ng a
ffin
ities
of m
ost s
ialic
aci
d an
alog
ues
(C2,
C4
and
C5-
sub-
stitu
ted)
det
erm
ined
by
vira
l hem
adso
rptio
n in
hibi
tion
assa
y, h
emag
glut
inat
ion
inhi
bitio
n as
say
and
nucl
ear
mag
netic
re
sona
nce
corr
elat
ed w
ell w
ith th
e co
mpu
tatio
nally
est
imat
ed fr
ee e
nerg
y of
bin
ding
. Sia
lic a
cid
anal
ogue
s w
ith m
odi-
fied
glyc
erol
sid
e ch
ains
sho
wed
onl
y po
or c
orre
latio
n be
twee
n th
e ex
peri
men
tally
det
erm
ined
hem
aggl
utin
in in
hibi
-to
r af
finiti
es a
nd A
utoD
ock3
.05
scor
es, s
ugge
stin
g hi
gh m
obili
ty o
f the
glu
tam
ic a
cid
side
cha
in a
t the
gly
cero
l bin
ding
po
cket
, whi
ch is
diff
icul
t to
sim
ulat
e us
ing
a fle
xi-r
igid
mol
ecul
ar d
ocki
ng a
ppro
ach.
In c
oncl
usio
n, e
xcep
t for
som
e gl
ycer
ol-s
ubst
itute
d si
alic
aci
d an
alog
ues,
the
resu
lts s
how
ed th
e ef
fect
iven
ess
of A
utoD
ock3
.05
sear
chin
g an
d sc
orin
g fu
nctio
ns in
est
imat
ing
affin
ities
of s
ialic
aci
d an
alog
ues
tow
ard
influ
enza
A h
emag
glut
inin
, mak
ing
it a
relia
ble
tool
fo
r sc
reen
ing
a da
taba
se o
f vir
tual
ly d
esig
ned
sial
ic a
cid
anal
ogue
s fo
r he
mag
glut
inin
inhi
bito
rs.
Al-q
atta
n an
d M
ordi
201
0a
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
142
Sial
ic a
cid
anal
ogue
sC
2-, C
5- a
nd
C6-
subs
titut
ions
Fr
agm
ent-
base
d dr
ug
desi
gn
In th
is st
udy
frag
men
t-ba
sed
drug
des
ign
is c
ombi
ned
with
mol
ecul
ar d
ocki
ng si
mul
atio
n te
chni
que,
to d
esig
n da
taba
ses
of v
irtu
al si
alic
aci
d (S
A) a
nalo
gues
with
new
subs
titut
ions
at C
2, C
5 an
d C
6 po
sitio
ns o
f SA
scaff
old.
Usi
ng sp
aces
occ
u-pi
ed b
y C
2, C
5 an
d C
6 na
tura
l moi
etie
s of S
A w
hen
boun
d to
hem
aggl
utin
in (H
A) c
ryst
allo
grap
hic
stru
ctur
e, n
ew fr
ag-
men
ts th
at a
re c
omm
erci
ally
ava
ilabl
e w
ere
dock
ed in
depe
nden
tly in
all
the
pock
ets.
The
orie
nted
frag
men
ts w
ere
then
co
nnec
ted
to th
e SA
scaff
old
with
or w
ithou
t inc
orpo
ratio
n of
link
er m
olec
ules
. The
com
plet
ed a
nalo
gues
wer
e do
cked
to
the
who
le S
A b
indi
ng si
te to
est
imat
e th
eir b
indi
ng c
onfo
rmat
ions
and
affi
nitie
s, g
ener
atin
g th
ree
data
base
s of H
A-b
ound
SA
ana
logu
es. S
elec
ted
new
ana
logu
es sh
owed
hig
her e
stim
ated
affi
nitie
s tha
n th
e na
tura
l SA
whe
n te
sted
aga
inst
H3N
2,
H5N
1 an
d H
1N1
subt
ypes
of i
nflue
nza
A. A
n im
prov
emen
t in
the
bind
ing
ener
gies
indi
cate
s tha
t fra
gmen
t-ba
sed
drug
de
sign
whe
n co
mbi
ned
with
mol
ecul
ar d
ocki
ng si
mul
atio
n is
cap
able
to p
rodu
ce v
irtu
al a
nalo
gues
that
can
bec
ome
lead
co
mpo
und
cand
idat
es fo
r ant
i-flu
drug
dis
cove
ry p
rogr
am.
Al-q
atta
n an
d M
ordi
201
0b
Sial
ic a
cid
anal
ogue
sPo
lyac
ryla
mid
e co
njug
ate
Poly
vale
nt in
hibi
tor
An
ELIS
A a
ssay
is d
escr
ibed
for m
easu
ring
the
bind
ing
of in
fluen
za v
irus
A-X
31 to
alp
ha-s
ialo
side
gro
ups t
hat a
re li
nked
to
bio
tin-la
bele
d po
lyac
ryla
mid
es. Th
e effi
cacy
of t
hese
pol
ymer
s in
inhi
bitin
g th
e ad
hesi
on o
f infl
uenz
a vi
rus t
o er
ythr
o-cy
tes (
as m
easu
red
by a
hem
aggl
utin
atio
n as
say)
was
show
n to
be
dire
ctly
rela
ted
to th
e bi
ndin
g affi
nity
of t
he p
olym
ers
for t
he v
iral
surf
ace:
the
diffe
renc
es in
inhi
bito
ry e
ffica
cy a
mon
g th
e po
lym
eric
inhi
bito
rs a
nd m
onom
eric
alp
ha-m
ethy
l si
alos
ide,
am
ong
frac
tions
of a
pol
ymer
ic, p
olyv
alen
t inh
ibito
r with
nar
row
mol
ecul
ar w
eigh
t ran
ges,
and
am
ong
poly
-m
eric
inhi
bito
rs p
repa
red
by c
opol
ymer
izat
ion
or m
odifi
catio
n of
a p
refo
rmed
pol
ymer
cha
in, a
ll co
rrel
ated
with
diff
er-
ence
s in
the
affini
ty o
f the
inhi
bito
rs fo
r the
surf
ace
of th
e vi
rus,
The
poly
mer
ic in
hibi
tors
stud
ied
had
affini
ties f
or th
e vi
ral s
urfa
ce th
at ra
nged
bet
wee
n 10
3 and
> 1
06 gre
ater
than
alp
ha-m
ethy
l sia
losi
de, o
n th
e ba
sis o
f tot
al si
alic
aci
d gr
oups
in
solu
tion,
The
role
of s
teri
c st
abili
zatio
n in
the
mec
hani
sm b
y w
hich
thes
e po
lym
ers i
nhib
it he
mag
glut
inat
ion
was
in
vest
igat
ed, Th
e ab
ility
of t
he p
olym
eric
, pol
yval
ent i
nhib
itors
to in
hibi
t the
bin
ding
of a
pol
yclo
nal a
ntib
ody
to th
e vi
ral
surf
ace
sugg
ests
that
ster
ic st
abili
zatio
n m
ay a
lso
be a
n im
port
ant e
ffect
in th
is sy
stem
.
Siga
l et a
l. 19
96
Qui
none
mol
ecul
esBi
cycl
ic q
uino
nes
Poly
mer
ic in
hibi
tors
Influ
enza
A v
irus
Usi
ng th
e pl
aque
redu
ctio
n as
say,
rela
tivel
y si
mpl
e bi
cycl
ic q
uino
ne m
olec
ules
, as w
ell a
s mul
tiple
cop
ies t
here
of c
ova-
lent
ly a
ttac
hed
to a
long
pol
yglu
tam
ate-
base
d po
lym
eric
cha
in, w
ere
exam
ined
as n
ew in
hibi
tors
of v
ario
us n
atur
ally
oc
curr
ing
stra
ins o
f infl
uenz
a A
vir
us. Th
e po
lym
er-c
onju
gate
d in
hibi
tors
wer
e fo
und
to h
ave
a fa
r gre
ater
pot
ency
(for
so
me
as h
igh
as tw
o or
ders
of m
agni
tude
whe
n a
long
spac
er a
rm w
as e
mpl
oyed
) tha
n th
eir c
orre
spon
ding
par
ent m
ol-
ecul
es a
gain
st th
e hu
man
Wuh
an in
fluen
za st
rain
. How
ever
, suc
h po
lym
eric
inhi
bito
rs fa
iled
to e
xhib
it hi
gher
pot
ency
co
mpa
red
with
thei
r sm
all m
olec
ule
pred
eces
sors
aga
inst
the
hum
an P
uert
o Ri
co a
nd a
vian
turk
ey in
fluen
za st
rain
s. Th
ese
obse
rvat
ions
, fur
ther
exp
lore
d by
mea
ns o
f mol
ecul
ar m
odel
ing,
reve
al th
e pr
evio
usly
unr
ecog
nize
d un
pred
icta
bil-
ity o
f the
ben
efits
of m
ultiv
alen
cy, p
ossi
bly
beca
use
of p
oor a
cces
sibi
lity
of th
e vi
ral t
arge
ts to
pol
ymer
ic a
gent
s.
Lars
on e
t al.
2012
Ben
zoqu
inon
es,
hydr
oqui
none
sC
onfo
rmat
iona
l cha
nge
of
haem
aggl
utin
inV
irus
-cel
l mem
bran
e fu
sion
Past
effo
rts t
o em
ploy
a st
ruct
ure-
base
d ap
proa
ch to
des
ign
an in
hibi
tor o
f the
fusi
on-i
nduc
ing
conf
orm
atio
nal c
hang
e in
th
e in
fluen
za v
irus
hem
aggl
utin
in (H
A) y
ield
ed a
fam
ily o
f sm
all b
enzo
quin
ones
and
hyd
roqu
inon
es, Th
e m
ost p
oten
t of
thes
e, te
rt-b
utyl
hyd
roqu
inon
e (T
BHQ
), in
hibi
ts b
oth
the
conf
orm
atio
nal c
hang
e in
HA
from
stra
in X
:31
influ
enza
vir
us
and
vira
l inf
ectiv
ity in
tiss
ue c
ultu
re c
ells
with
50%
inhi
bito
ry c
once
ntra
tions
in th
e m
icro
mol
ar ra
nge.
A n
ew st
ruct
ure-
base
d in
hibi
tor d
esig
n se
arch
was
beg
un w
hich
invo
lved
the
rece
ntly
refin
ed c
ryst
al st
ruct
ure
(2.1
-Ang
stro
m re
solu
-tio
n) o
f the
HA
ect
odom
ain,
new
insi
ghts
into
the
conf
orm
atio
nal c
hang
e, a
nd im
prov
emen
ts in
the
mol
ecul
ar d
ocki
ng
prog
ram
, DO
CK
. As a
resu
lt, w
e id
entifi
ed n
ew in
hibi
tors
of H
A-m
edia
ted
mem
bran
e fu
sion
. Lik
e T
BHQ
, mos
t of t
hese
m
olec
ules
inhi
bit t
he c
onfo
rmat
iona
l cha
nge,
One
of t
he n
ew c
ompo
unds
, how
ever
, fac
ilita
tes r
athe
r tha
n in
hibi
ts th
e H
A c
onfo
rmat
iona
l cha
nge,
Non
ethe
less
, the
faci
litat
or, d
iiodo
fluor
esce
in, i
nhib
its H
A-m
edia
ted
mem
bran
e fu
sion
and
, ir
reve
rsib
ly, i
nfec
tivity
. We
furt
her c
hara
cter
ized
the
effec
ts o
f inh
ibito
rs fr
om b
oth
sear
ches
on
the
conf
orm
atio
nal
chan
ge a
nd m
embr
ane
fusi
on a
ctiv
ity o
f HA
as w
ell a
s on
vira
l inf
ectiv
ity. W
e al
so is
olat
ed a
nd c
hara
cter
ized
seve
ral
mut
ants
resi
stan
t to
each
cla
ss o
f inh
ibito
r. Th
e im
plic
atio
ns o
f our
resu
lts fo
r HA
-med
iate
d m
embr
ane
fusi
on, a
nti-
influ
enza
vir
us th
erap
y, an
d st
ruct
ure-
base
d in
hibi
tor d
esig
n ar
e di
scus
sed.
Hoff
man
et a
l. 19
97
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
143
new
hae
mag
glut
inin
in
hibi
tors
We
repo
rted
pre
viou
sly
that
a s
mal
l mol
ecul
e na
med
CL-
3853
19 c
ould
inhi
bit H
5N1
influ
enza
vir
us in
fect
ion
by
targ
etin
g he
mag
glut
inin
, the
env
elop
e pr
otei
n m
edia
ting
viru
s en
try.
In th
e pr
esen
t stu
dy, a
nov
el s
erie
s of
der
iva-
tives
focu
sed
on th
e st
ruct
ural
var
iatio
n of
CL-
3853
19 w
ere
synt
hesi
zed
as s
peci
fic in
hibi
tors
aga
inst
the
H5
subt
ype
of in
fluen
za A
vir
uses
. The
se s
mal
l mol
ecul
es in
hibi
ted
the
low
pH
-ind
uced
con
form
atio
nal c
hang
e of
hem
aggl
u-tin
in, t
here
by b
lock
ing
vira
l ent
ry in
to h
ost c
ells
. Com
poun
d 11
was
the
mos
t act
ive
inhi
bito
r in
this
ser
ies
with
an
IC50
of 0
.22
µM. T
he s
truc
ture
act
ivity
rela
tions
hips
ana
lysi
s of
thes
e co
mpo
unds
sho
wed
that
the
3-flu
oro-
5-(t
riflu
orom
ethy
l)ben
zam
ide
moi
ety
was
ver
y im
port
ant f
or a
ctiv
ity, a
nd th
e -F
gro
up w
as a
bet
ter
subs
titue
nt g
roup
th
an -C
F3 g
roup
in th
e ph
enyl
rin
g. T
he in
hibi
tory
act
ivity
was
sen
sitiv
e to
the
benz
amid
e be
caus
e th
e ox
ygen
and
hy
drog
en o
f the
am
ide
serv
ed a
s H
-bon
d ac
cept
or a
nd d
onor
, res
pect
ivel
y.
Zhu
et a
l. 20
12
Stac
hyfli
nC
onfo
rmat
iona
l cha
nge
of
haem
aggl
utin
in
Stac
hyfli
n is
a n
ovel
com
poun
d ha
ving
H1
and
H2
subt
ype-
spec
ific
anti-
influ
enza
A v
irus
act
ivity
. Sta
chyf
lin h
as n
o in
hibi
tion
on H
3 su
btyp
e in
fluen
za A
or
influ
enza
B v
irus
es. T
he s
usce
ptib
ility
of t
he re
asso
rtan
t vir
uses
bet
wee
n H
1 an
d H
3 su
btyp
e in
fluen
za A
vir
uses
to S
tach
yflin
indi
cate
d th
at it
s ta
rget
was
vir
us-e
ncod
ed h
emag
glut
inin
(HA
). T
he
resu
lts o
f the
tim
ing
of S
tach
yflin
add
ition
aga
inst
in v
itro
viru
s in
fect
ion
and
viru
s-m
edia
ted
hem
olys
is a
ssay
sug
-ge
sted
that
the
drug
inhi
bite
d th
e H
A-m
edia
ted
viru
s-ce
ll fu
sion
pro
cess
. Mor
e di
rect
ly, S
tach
yflin
inte
rfer
ed w
ith
HA
con
form
atio
nal c
hang
e in
duce
d by
low
pH
or
heat
trea
tmen
t. T
he e
ffect
of S
tach
yflin
cou
ld n
ot b
e el
imin
ated
by
was
hing
of t
he S
tach
yflin
-tre
ated
vir
us, w
hich
cau
sed
very
spe
cific
vir
ucid
al e
ffect
. Thi
s is
a re
mar
kabl
e pr
oper
ty
amon
g sm
all m
olec
ules
whi
ch in
hibi
t low
-pH
indu
ced
HA
con
form
atio
nal c
hang
e. F
rom
thes
e fin
ding
s, w
e co
nclu
ded
that
the
mec
hani
sm o
f Sta
chyf
lin a
ctio
n is
to in
hibi
t HA
con
form
atio
nal c
hang
e w
hich
is n
eces
sary
for
viru
s-ce
ll m
em-
bran
e fu
sion
. Sta
chyf
lin m
ay b
e us
ed a
s a
tool
for
a st
udy
of m
olec
ular
mec
hani
sm o
f low
-pH
indu
ced
HA
con
form
a-tio
nal c
hang
e, a
nd o
ffers
pot
entia
l as
a ph
arm
aceu
tical
age
nt.
Yosh
imot
o et
al.
1999
(+)-
stac
hyfli
nEn
antio
sele
ctiv
e sy
nthe
sis
A n
ovel
and
pot
ent h
emag
glut
inin
inhi
bito
r, (+
)-st
achy
flin,
was
eff
icie
ntly
syn
thes
ized
in a
n en
antio
sele
ctiv
e m
anne
r st
artin
g fr
om th
e (+
)-5-
met
hyl-W
iela
nd-M
iesc
her
keto
ne. T
he s
ynth
etic
met
hod
feat
ures
a B
F(3)
cent
er d
ot E
t(2)
O-i
nduc
ed c
asca
de e
poxi
de-o
peni
ng/r
earr
ange
men
t/cy
cliz
atio
n re
actio
n to
ste
reos
elec
tivel
y co
nstr
uct t
he re
quis
ite
pent
acyc
lic r
ing
syst
em in
one
ste
p. In
ord
er to
rat
iona
lize
the
mec
hani
sm o
f the
cas
cade
reac
tion,
qua
ntum
che
mic
al
calc
ulat
ions
of t
he p
ossi
ble
inte
rmed
iary
car
boca
tions
and
tran
sitio
n st
ates
in th
e m
odel
syn
thes
is w
ere
carr
ied
out.
An
alte
rnat
ive
appr
oach
to s
ynth
esiz
e (+
)-st
achy
flin
by e
mpl
oyin
g a
sim
ilar
casc
ade
reac
tion
was
als
o de
scri
bed.
Saku
rai e
t al.
2011
Ben
zam
id d
eriv
ativ
esV
irus
-cel
l mem
bran
e fu
sion
Sequ
ence
ana
lyse
sH
aem
aggl
utin
in g
ene
Dru
g re
sist
ance
In th
e in
itial
sta
ges
of in
fluen
za v
irus
infe
ctio
n, th
e he
mag
glut
inin
(HA
) pro
tein
of i
nflu
enza
vir
us m
edia
tes
both
ad
sorp
tion
and
pene
trat
ion
of th
e vi
rus
into
the
host
cel
l. Re
cent
ly, w
e id
entif
ied
and
char
acte
rize
d BM
Y-27
709
as
an in
hibi
tor
of th
e H
1 an
d H
2 su
btyp
es o
f inf
luen
za A
vir
us th
at s
peci
fical
ly in
hibi
ts th
e H
A fu
nctio
n ne
cess
ary
for
viru
s-ce
ll m
embr
ane
fusi
on. S
tudi
es p
rese
nted
her
ein
show
that
the
inhi
bitio
n is
med
iate
d th
roug
h sp
ecifi
c in
tera
ctio
n w
ith th
e H
A p
rote
in. T
his
bind
ing
repr
esse
s th
e lo
w-p
H-i
nduc
ed c
onfo
rmat
iona
l cha
nge
of th
e H
A p
rote
in w
hich
is
a pr
ereq
uisi
te fo
r m
embr
ane
fusi
on. I
n an
att
empt
to d
efin
e th
e bi
ndin
g po
cket
with
in th
e H
A m
olec
ule,
a n
umbe
r of
dr
ug-r
esis
tant
vir
uses
hav
e be
en is
olat
ed a
nd c
hara
cter
ized
. Seq
uenc
e an
alys
es o
f the
HA
gen
e of
thes
e dr
ug-r
esis
tant
vi
ruse
s m
appe
d am
ino
acid
cha
nges
resp
onsi
ble
for
drug
resi
stan
ce to
a re
gion
loca
ted
near
the
amin
o te
rmin
us o
f H
A2.
In a
dditi
on, w
e ha
ve id
entif
ied
inac
tive
anal
ogs
of B
RY-2
7709
whi
ch a
re a
ble
to c
ompe
te o
ut th
e in
hibi
tory
act
iv-
ity o
f BM
Y-27
709.
Thi
s fin
ding
sug
gest
s th
at in
hibi
tion
of th
e H
A-m
edia
ted
mem
bran
e fu
sion
by
this
cla
ss o
f com
-po
unds
is n
ot s
olel
y th
e re
sult
of b
indi
ng w
ithin
the
HA
mol
ecul
e bu
t req
uire
s sp
ecifi
c in
tera
ctio
ns.
Luo
et a
l. 19
97
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
144
Podo
carp
ic a
cid
deri
vati
ves
Sequ
ence
ana
lyse
sH
aem
aggl
utin
in g
ene
Dru
g re
sist
ance
Entr
y of
influ
enza
vir
us in
to th
e ho
st c
ell i
s de
pend
ent o
n th
e fu
sion
of t
he v
iral
env
elop
e w
ith th
e en
doso
mal
mem
-br
ane
and
is m
edia
ted
by a
low
-pH
-ind
uced
cha
nge
of th
e vi
ral h
emag
glut
inin
(HA
) to
a co
nfor
mat
ion
that
is fu
so-
geni
c. A
com
poun
d re
late
d to
pod
ocar
pic
acid
(180
299]
was
iden
tifie
d th
at in
hibi
ts m
ultic
ycle
repl
icat
ion
of in
fluen
za
A/K
awas
aki/
86 (H
1N1)
Vir
us in
cul
ture
. Tre
atm
ent o
f Mad
in-D
arby
can
ine
kidn
ey (M
DC
K) c
ells
with
180
299
at 1
h
befo
re in
fect
ion
resu
lted
in th
e in
hibi
tion
of v
iral
pro
tein
syn
thes
is. A
dditi
on o
f 20
µg o
f 180
299/
ml a
t 1 h
p.i.
had
no
effe
ct, i
ndic
atin
g th
at 1
8029
9 af
fect
s an
ear
ly s
tep
of th
e in
fluen
za v
iral
repl
icat
ion
cycl
e. G
enet
ic a
naly
sis
of re
as-
sort
ants
bet
wee
n se
nsiti
ve a
nd re
sist
ant v
irus
es d
emon
stra
ted
that
hem
aggl
utin
in (H
A) c
onfe
rred
the
1802
99-r
esis
t-an
t (18
0299
(r))
phe
noty
pe. T
wel
ve in
depe
nden
t iso
late
s of
influ
enza
A/K
awas
aki/
86 w
ere
sele
cted
for
resi
stan
ce
to 1
8029
9, a
nd s
eque
nce
anal
ysis
reve
aled
that
eac
h of
thes
e vi
ruse
s co
ntai
ned
amin
o ac
id s
ubst
itutio
ns in
the
HA
. T
hese
mut
atio
ns a
re d
ispe
rsed
thro
ugho
ut th
e H
A p
rim
ary
amin
o ac
id s
eque
nce
and
clus
ter
in o
ne o
f tw
o re
gion
s: th
e in
terf
ace
betw
een
HA
(1) a
nd H
A(2
) and
in a
regi
on n
ear
the
fusi
on d
omai
n of
HA
(2).
Whe
n co
mpa
red
with
the
pare
nt
viru
s, th
e pH
-of-
inac
tivat
ion
of th
e re
sist
ant m
utan
ts w
as in
crea
sed
by 0
.3 to
0.6
pH
uni
t, su
gges
ting
that
the
mut
ant
HA
s un
derg
o th
e co
nfor
mat
iona
l cha
nge
at a
n el
evat
ed p
H. F
usio
n of
hum
an e
ryth
rocy
tes
to M
DC
K c
ells
infe
cted
w
ith p
aren
t inf
luen
za A
/Kaw
asak
i/86
was
inhi
bite
d by
180
299
(0.1
–10
µg/m
l) in
a c
once
ntra
tion-
depe
nden
t: m
anne
r, w
here
as fu
sion
of e
ryth
rocy
tes
to M
DC
K c
ells
infe
cted
with
180
299(
r) m
utan
ts w
as n
ot: a
ffect
ed. T
hese
resu
lts s
ug-
gest
that
180
299
inte
ract
s w
ith th
e ne
utra
l pH
con
form
atio
n of
influ
enza
A H
A a
nd p
reve
nts
the
low
-pH
-ind
uced
ch
ange
of H
A to
its
fuso
geni
c co
nfor
mat
ion.
Stas
chke
et a
l. 19
98
dA
S181
Sial
idas
e fu
sion
pro
tein
Influ
enza
A a
nd B
Mou
se/f
erre
t mod
el
Influ
enza
is a
hig
hly
infe
ctio
us d
isea
se c
hara
cter
ized
by
recu
rren
t ann
ual e
pide
mic
s an
d un
pred
icta
ble
maj
or w
orld
-w
ide
pand
emic
s. R
apid
spr
ead
of th
e hi
ghly
pat
hoge
nic
avia
n H
5N1
stra
in a
nd e
scal
atin
g hu
man
infe
ctio
ns b
y th
e vi
rus
have
set
off
the
alar
m fo
r a
glob
al p
ande
mic
. To
prov
ide
an u
rgen
tly n
eede
d al
tern
ativ
e tr
eatm
ent m
odal
ity fo
r in
fluen
za, w
e ha
ve g
ener
ated
a re
com
bina
nt fu
sion
pro
tein
com
pose
d of
a s
ialid
ase
cata
lytic
dom
ain
deri
ved
from
A
ctin
omyc
es v
isco
sus
fuse
d w
ith a
cel
l sur
face
-anc
hori
ng s
eque
nce.
The
sia
lidas
e fu
sion
pro
tein
is to
be
appl
ied
topi
-ca
lly a
s an
inha
lant
to re
mov
e th
e in
fluen
za v
iral
rece
ptor
s, s
ialic
aci
ds, f
rom
the
airw
ay e
pith
eliu
m. W
e de
mon
stra
te
that
a s
ialid
ase
fusi
on c
onst
ruct
, DA
S181
, effe
ctiv
ely
clea
ves
sial
ic a
cid
rece
ptor
s us
ed b
y bo
th h
uman
and
avi
an in
flu-
enza
vir
uses
. The
trea
tmen
t pro
vide
s lo
ng-l
astin
g ef
fect
and
is n
onto
xic
to th
e ce
lls. D
AS1
81 d
emon
stra
ted
pote
nt
antiv
iral
and
cel
l pro
tect
ive
effic
acie
s ag
ains
t a p
anel
of l
abor
ator
y st
rain
s an
d cl
inic
al is
olat
es o
f IFV
A a
nd IF
V B
, w
ith v
irus
repl
icat
ion
inhi
bitio
n 50
% e
ffect
ive
conc
entr
atio
ns in
the
rang
e of
0.0
4 to
0.9
nM
. Mou
se a
nd fe
rret
stu
dies
co
nfir
med
sig
nific
ant i
n vi
vo e
ffic
acy
of th
e si
alid
ase
fusi
on in
bot
h pr
ophy
lact
ic a
nd tr
eatm
ent m
odes
.
Mal
akho
v et
al.
2006
dA
S181
Phas
e II
clin
ical
tria
lBa
ckgr
ound
. DA
S181
, a n
ovel
hos
t-di
rect
ed a
ntiv
iral
in d
evel
opm
ent f
or in
fluen
za tr
eatm
ent,
was
ass
esse
d in
this
pha
se
II c
linic
al tr
ial.
Met
hods
. This
stud
y w
as a
dou
ble-
blin
d, p
lace
bo-c
ontr
olle
d ph
ase
II c
linic
al tr
ial a
sses
sing
influ
enza
vi
ral l
oad
and
patie
nt sa
fety
in o
ther
wis
e he
alth
y in
fluen
za-in
fect
ed p
artic
ipan
ts. P
artic
ipan
ts w
ere
rand
omiz
ed to
a
sing
le-d
ose,
mul
tiple
-dos
e, o
r pla
cebo
gro
up a
nd w
ere
follo
wed
for s
afet
y an
d vi
rolo
gic
outc
omes
. Res
ults
. A to
tal o
f 177
la
bora
tory
-con
firm
ed in
fluen
za-in
fect
ed p
artic
ipan
ts w
ere
enro
lled
in th
e tr
ial,
whi
ch e
ncom
pass
ed 3
influ
enza
seas
ons
from
200
9–20
11 in
bot
h th
e N
orth
ern
and
Sout
hern
Hem
isph
eres
. Thir
ty-s
even
per
cent
of p
artic
ipan
ts h
ad c
onfir
med
in
fect
ion
with
influ
enza
B, 3
3% w
ith se
ason
al H
3N2,
29%
with
pan
dem
ic 2
009
H1N
1, a
nd 1
par
ticip
ant w
as p
ositi
ve
for b
oth
influ
enza
B a
nd p
ande
mic
200
9 H
1N1.
Sig
nific
ant e
ffect
s wer
e ob
serv
ed in
rega
rd to
dec
reas
ed c
hang
e fr
om
base
line
vira
l loa
d an
d vi
ral s
hedd
ing
in th
e m
ultip
le-d
ose
grou
p co
mpa
red
with
pla
cebo
as m
easu
red
by q
uant
itativ
e po
lym
eras
e ch
ain
reac
tion
(P <
0.0
5). N
o in
stan
ces o
f H27
4Y w
ere
obse
rved
am
ong
vira
l iso
late
s fro
m th
is tr
ial.
Ove
rall,
th
e dr
ug w
as g
ener
ally
wel
l tol
erat
ed. C
oncl
usio
ns. D
AS1
81 si
gnifi
cant
ly re
duce
d vi
ral l
oad
in p
artic
ipan
ts in
fect
ed w
ith
influ
enza
, thu
s war
rant
ing
futu
re c
linic
al d
evel
opm
ent o
f thi
s nov
el h
ost-
dire
cted
ther
apy.
Mos
s et a
l. 20
12
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
145
Fusi
on-i
nhib
itor
y pe
ptid
esC
hole
ster
ol-c
onju
gate
d co
mpo
unds
We
prev
ious
ly d
escr
ibed
fusi
on-in
hibi
tory
pep
tides
that
are
targ
eted
to th
e ce
ll m
embr
ane
by c
hole
ster
ol c
onju
gatio
n an
d po
tent
ly in
hibi
t env
elop
ed v
irus
es th
at fu
se a
t the
cel
l sur
face
, inc
ludi
ng H
IV, p
arai
nflue
nza,
and
hen
ipav
irus
es.
How
ever
, for
vir
uses
that
fuse
insi
de o
f int
race
llula
r com
part
men
ts, f
usio
n-in
hibi
tory
pep
tides
hav
e ex
hibi
ted
very
low
an
tivir
al a
ctiv
ity. W
e pr
opos
e th
at fo
r the
se v
irus
es, t
oo, m
embr
ane
targ
etin
g vi
a ch
oles
tero
l con
juga
tion
may
yie
ld
pote
nt c
ompo
unds
. Her
e w
e co
mpa
re th
e ac
tivity
of f
usio
n-in
hibi
tory
pep
tides
der
ived
from
the
influ
enza
hem
aggl
utin
in
(HA
) and
show
that
alth
ough
the
unco
njug
ated
pep
tides
are
inac
tive,
the
chol
este
rol-c
onju
gate
d co
mpo
unds
are
effe
ctiv
e in
hibi
tors
of i
nfec
tivity
and
mem
bran
e fu
sion
. We
hypo
thes
ize
that
the
chol
este
rol m
oiet
y, by
loca
lizin
g th
e pe
ptid
es to
th
e ta
rget
cel
l mem
bran
e, a
llow
s the
pep
tides
to fo
llow
the
viru
s to
the
intr
acel
lula
r site
of f
usio
n. Th
e ch
oles
tero
l-con
ju-
gate
d pe
ptid
es tr
ap H
A in
a tr
ansi
ent i
nter
med
iate
stat
e af
ter f
usio
n is
trig
gere
d bu
t bef
ore
com
plet
ion
of th
e re
fold
ing
step
s tha
t dri
ve th
e m
ergi
ng o
f the
vir
al a
nd c
ellu
lar m
embr
anes
. Thes
e re
sults
pro
vide
pro
of o
f con
cept
for a
n an
tivir
al
stra
tegy
that
is a
pplic
able
to in
trac
ellu
larl
y fu
sing
vir
uses
, inc
ludi
ng k
now
n an
d em
ergi
ng v
iral
pat
hoge
ns.
Lee
et a
l. 20
11
Rn
A a
ptam
ers
SELE
X te
chno
logy
Hae
mag
glut
inat
ion
inhi
bitio
n as
say
In th
is s
tudy
, we
inve
stig
ated
the
effic
acy
of u
sing
hem
aggl
utin
in (H
A) a
s a
targ
et fo
r an
tivir
al th
erap
y th
roug
h nu
clei
c ac
id a
ptam
ers.
Aft
er p
urifi
catio
n of
the
rece
ptor
bin
ding
dom
ain
(HA
1) o
f HA
pro
tein
, act
ivity
of r
ecom
bina
nt H
A1
was
con
firm
ed b
y us
ing
hem
aggl
utin
atio
n as
say.
We
sele
cted
RN
A a
ptam
er c
andi
date
s af
ter
15 ro
unds
of i
tera
tive
Syst
emat
ic E
volu
tion
of L
igan
ds b
y EX
pone
ntia
l enr
ichm
ent (
SELE
X) t
arge
ting
the
biol
ogic
ally
act
ive
HA
pro
tein
. T
he s
elec
ted
RNA
apt
amer
HA
S15-
5, w
hich
spe
cific
ally
bin
ds to
HA
1, e
xhib
ited
sign
ifica
nt a
ntiv
iral
eff
icac
y ac
cord
-in
g to
the
resu
lts o
f a h
emag
glut
inat
ion
inhi
bitio
n as
say
usin
g eg
g al
lant
oic
fluid
s ha
rbor
ing
the
viru
s. T
hus,
the
RNA
ap
tam
er H
AS1
5-5,
whi
ch a
cts
by b
lock
ing
and
inhi
bitin
g th
e re
cept
or-b
indi
ng d
omai
n of
vir
al H
A, c
an b
e de
velo
ped
as a
nov
el a
ntiv
iral
age
nt a
gain
st ty
pe H
5 av
ian
influ
enza
vir
us.
Park
et a
l. 20
11
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
146
Tabl
e 4.
2. M
2 io
n ch
anne
l blo
cker
s
Stru
ctur
e an
d fu
ncti
on o
f M
2 pr
otei
nC
ondu
ctan
ce o
f pro
tons
Hae
mag
glut
inin
-med
iate
d m
embr
ane
fusi
onM
2-ri
man
tadi
n co
mpl
ex
The
inte
gral
mem
bran
e pr
otei
n M
2 of
influ
enza
vir
us fo
rms
pH-g
ated
pro
ton
chan
nels
in th
e vi
ral l
ipid
env
elop
e (1
). T
he lo
w p
H o
f an
endo
som
e ac
tivat
es th
e M
2 ch
anne
l bef
ore
haem
aggl
utin
in-m
edia
ted
fusi
on. C
ondu
ctan
ce o
f pr
oton
s ac
idifi
es th
e vi
ral i
nter
ior
and
ther
eby
faci
litat
es d
isso
ciat
ion
of th
e m
atri
x pr
otei
n fr
om th
e vi
ral n
ucle
o-pr
otei
ns -
a re
quir
ed p
roce
ss fo
r un
pack
ing
of th
e vi
ral g
enom
e (2
). In
add
ition
to it
s ro
le in
rele
ase
of v
iral
nuc
leo-
prot
eins
, M2
in th
e tr
ans-
Gol
gi n
etw
ork
(TG
N) m
embr
ane
prev
ents
pre
mat
ure
conf
orm
atio
nal r
earr
ange
men
t of
new
ly s
ynth
esiz
ed h
aem
aggl
utin
in d
urin
g tr
ansp
ort t
o th
e ce
ll su
rfac
e by
equ
ilibr
atin
g th
e pH
of t
he T
GN
with
that
of
the
host
cel
l cyt
opla
sm (3
). In
hibi
ting
the
prot
on c
ondu
ctan
ce o
f M2
usin
g th
e an
ti- v
iral
dru
g am
anta
dine
or
rim
anta
dine
inhi
bits
vir
al re
plic
atio
n(4–
7). H
ere
we
pres
ent t
he s
truc
ture
of t
he te
tram
eric
M2
chan
nel i
n co
mpl
ex
with
rim
anta
dine
, det
erm
ined
by
NM
R. I
n th
e cl
osed
sta
te, f
our
tight
ly p
acke
d tr
ansm
embr
ane
helic
es d
efin
e a
narr
ow c
hann
el, i
n w
hich
a ‘t
rypt
opha
n ga
te’ i
s lo
cked
by
inte
rmol
ecul
ar in
tera
ctio
ns w
ith a
spar
tic a
cid.
A c
arbo
xy-
term
inal
, am
phip
athi
c he
lix o
rien
ted
near
ly p
erpe
ndic
ular
to th
e tr
ansm
embr
ane
helix
form
s an
inw
ard-
faci
ng
base
. Low
erin
g th
e pH
des
tabi
lizes
the
tran
smem
bran
e he
lical
pac
king
and
unl
ocks
the
gate
, adm
ittin
g w
ater
to
cond
uct p
roto
ns, w
here
as th
e C
-ter
min
al b
ase
rem
ains
inta
ct, p
reve
ntin
g di
ssoc
iatio
n of
the
tetr
amer
. Rim
anta
dine
bi
nds
at fo
ur e
quiv
alen
t site
s ne
ar th
e ga
te o
n th
e lip
id- f
acin
g si
de o
f the
cha
nnel
and
sta
biliz
es th
e cl
osed
con
for-
mat
ion
of th
e po
re. D
rug-
resi
stan
ce m
utat
ions
are
pre
dict
ed to
cou
nter
the
effe
ct o
f dru
g bi
ndin
g by
eith
er in
crea
s-in
g th
e hy
drop
hilic
ity o
f the
por
e or
wea
keni
ng h
elix
– h
elix
pac
king
, thu
s fa
cilit
atin
g ch
anne
l ope
ning
.
Schn
ell a
nd C
hou
2008
Stru
ctur
e of
M2
prot
ein
Chi
mer
ic M
2 ch
anne
lD
rug-
bind
ing
site
sD
rug
resi
stan
ce
The
M2
chan
nel o
f infl
uenz
a A
is a
targ
et o
f the
ada
man
tane
fam
ily a
ntiv
iral
dru
gs. T
wo
diffe
rent
dru
g-bi
ndin
g si
tes
have
bee
n re
port
ed: o
ne in
side
the
pore
, and
the
othe
r is a
lipi
d-fa
cing
poc
ket.
A p
revi
ous s
tudy
show
ed th
at a
chi
mer
a of
M2
vari
ants
from
influ
enza
A a
nd B
that
con
tain
s onl
y th
e po
re-b
indi
ng si
te is
sens
itive
to a
man
tadi
ne in
hibi
tion,
su
gges
ting
that
the
prim
ary
site
of i
nhib
ition
is in
side
the
pore
. To
obta
in a
tom
ic d
etai
ls o
f cha
nnel
-dru
g in
tera
ctio
n,
we
dete
rmin
ed th
e st
ruct
ures
of t
he c
him
eric
cha
nnel
with
and
with
out r
iman
tadi
ne. I
nsid
e th
e ch
anne
l and
nea
r the
N
-ter
min
al e
nd, m
ethy
l gro
ups o
f Val
27 a
nd A
la30
from
four
subu
nits
form
a h
ydro
phob
ic p
ocke
t aro
und
the
adam
an-
tane
, and
the
drug
am
ino
grou
p ap
pear
s to
be in
pol
ar c
onta
ct w
ith th
e ba
ckbo
ne o
xyge
n of
Ala
30. Th
e st
ruct
ures
als
o re
veal
diff
eren
ces b
etw
een
the
drug
-bou
nd a
nd -u
nbou
nd st
ates
of t
he c
hann
el th
at c
an e
xpla
in d
rug
resi
stan
ce.
Piel
ak e
t al.
2011
Affi
nity
stu
dyA
man
tadi
ne/r
iman
tadi
ne
bind
ing
to M
2Su
rfac
e pl
asm
on re
sona
nce
Lipo
som
es
The
influ
enza
A v
irus
con
tain
s a
prot
on-s
elec
tive
ion
chan
nel (
M2)
that
is th
e ta
rget
of t
he a
dam
anta
ne fa
mily
of
drug
inhi
bito
rs. T
wo
rece
ntly
pub
lishe
d st
udie
s re
latin
g to
ada
man
tane
bin
ding
of t
he M
2 io
n ch
anne
l usi
ng X
-ray
cr
ysta
llogr
aphy
and
sol
utio
n N
MR
have
re-i
gnite
d in
tere
st in
the
pote
ntia
l use
of a
dam
anta
nes
in c
omba
ting
the
spre
ad o
f inf
luen
za A
. How
ever
, the
se tw
o st
udie
s pr
opos
e di
ffere
nt b
indi
ng s
ites
for
the
adam
anta
ne d
rugs
with
th
e X
-ray
M2/
aman
tadi
ne s
truc
ture
favo
ring
an
ion
chan
nel p
ore-
bind
ing
mod
el a
nd th
e so
lutio
n N
MR
M2/
rim
an-
tadi
ne s
truc
ture
sug
gest
ing
the
exis
tenc
e of
a li
pid-
faci
ng b
indi
ng p
ocke
t. W
e co
nduc
ted
a se
ries
of s
urfa
ce p
lasm
on
reso
nanc
e (S
PR) e
xper
imen
ts d
esig
ned
to a
ccur
atel
y m
easu
re th
e af
finity
of a
man
tadi
ne a
nd r
iman
tadi
ne to
M2
ion
chan
nels
em
bedd
ed in
1,2
-dim
yris
toyl
-sn-
glyc
ero-
phos
phoc
holin
e (D
MPC
) lip
osom
es. W
e fin
d th
at th
is c
lass
of
drug
is c
apab
le o
f bin
ding
M2
with
two
diffe
rent
aff
initi
es in
the
orde
r of
10–4
and
10–7
M, s
ugge
stin
g th
at b
oth
pro-
pose
d bi
ndin
g si
tes
are
feas
ible
. Fur
ther
mor
e, b
y ex
amin
ing
drug
bin
ding
to M
2 m
utan
t con
stru
cts
(V27
A, S
31N
, an
d D
44A
), it
was
pos
sibl
e to
pro
be th
e lo
catio
n of
the
two
bind
ing
site
s. W
e sh
ow th
at a
hig
h-af
finity
bin
ding
site
co
rres
pond
s to
the
M2
ion
chan
nel p
ore
whe
reas
the
seco
ndar
y, lo
w-a
ffin
ity b
indi
ng s
ite c
an b
e at
trib
uted
to th
e lip
id fa
ce o
f the
por
e. T
hese
SPR
resu
lts a
re in
exc
elle
nt a
gree
men
t with
the
mos
t rec
ent s
olid
-sta
te N
MR
stud
y of
am
anta
dine
-bou
nd M
2 in
lipi
d bi
laye
rs a
nd p
rovi
de in
depe
nden
t sup
port
that
the
ion
chan
nel p
ore-
bind
ing
site
is
resp
onsi
ble
for
the
phar
mac
olog
ical
act
ivity
elic
ited
by th
e ad
aman
tane
dru
gs.
Rose
nber
g an
d C
asar
otto
201
0
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
147
M2
ion
chan
nel i
nhib
itio
nRi
man
tadi
ne
Mol
ecul
ar d
ynam
ics
sim
ulat
ions
In o
rder
to u
nder
stan
d ho
w r
iman
tadi
ne (R
MT
) inh
ibits
the
prot
on c
ondu
ctan
ce in
the
influ
enza
A M
2 ch
anne
l via
th
e re
cent
ly p
ropo
sed
“allo
ster
ic m
echa
nism
”, m
olec
ular
dyn
amic
s si
mul
atio
ns w
ere
appl
ied
to th
e M
2-te
tram
eric
pr
otei
n w
ith fo
ur R
MTs
bou
nd o
utsi
de th
e ch
anne
l at t
he th
ree
prot
onat
ion
stat
es: t
he 0
H-c
lose
d, 1
H-i
nter
med
iate
an
d 3H
-ope
n si
tuat
ions
. In
the
0H-c
lose
d st
ate,
a n
arro
w c
hann
el w
ith th
e R
MT-
Asp
44-T
rp41
H-b
ond
netw
ork
was
fo
rmed
, the
refo
re th
e w
ater
pen
etra
tion
thro
ugh
the
chan
nel w
as c
ompl
etel
y bl
ocke
d. T
he T
rp41
-Asp
44 in
tera
c-tio
n w
as a
bsen
t in
the
1H-i
nter
med
iate
sta
te, w
hils
t the
bin
ding
of R
MT
to A
sp44
rem
aine
d, w
hich
resu
lted
in a
w
eake
ned
helix
-hel
ix p
acki
ng, t
here
fore
the
chan
nel w
as p
artia
lly p
reve
nted
. In
the
3H-o
pen
stat
e it
was
foun
d th
at
the
elec
tros
tatic
repu
lsio
n fr
om th
e th
ree
char
ged
His
37 re
sidu
es a
llow
ed th
e Tr
p41
gate
to o
pen,
per
mitt
ing
wat
er
to p
enet
rate
thro
ugh
the
chan
nel.
Thi
s ag
reed
wel
l with
the
pote
ntia
l of t
he m
eans
forc
e w
hich
is in
the
follo
win
g or
der:
0H
> 1
H >
3H
.
Inth
arat
hep
et a
l. 20
11
dis
cove
ry o
f am
anta
dine
Ev
alua
tion
of v
irus
inhi
bitio
nI-
Ada
man
tana
min
e (a
man
tadi
ne) c
ause
s a se
lect
ive,
repr
oduc
ible
, dos
e-re
late
d in
hibi
tion
of in
fluen
za in
fect
ions
in
tissu
e cu
lture
, chi
ck e
mbr
yos,
and
mic
e. Th
e co
mpo
und
is n
ot v
iruc
idal
and
app
ears
to a
ct b
y in
terf
erin
g w
ith th
e pe
n-et
ratio
n of
the
host
cel
l by
the
viru
s. In
influ
enza
infe
ctio
ns o
f mic
e, g
reat
est e
ffica
cy o
ccur
s with
trea
tmen
t at t
he ti
me
of in
fect
ion;
how
ever
, the
re is
sign
ifica
nt a
ntiv
iral
act
ivity
with
trea
tmen
t del
ayed
up
to 7
2 ho
urs a
fter
infe
ctio
n. V
irus
in
hibi
tion
is n
ot c
ompl
ete
and
surv
ivor
s are
imm
une
to a
cha
lleng
e in
fect
ion
with
the
orig
inal
infe
ctin
g vi
rus.
Dav
ies e
t al.
1964
Ada
man
tane
der
ivat
ives
3-(2
-ada
man
tyl)p
yrro
lidin
esSy
nthe
sis a
nd e
valu
atio
n
The
3-(2
-ada
man
tyl)p
yrro
lidin
es 8
a-g.
14
wer
e sy
nthe
size
d an
d ev
alua
ted
for a
ctiv
ity a
gain
st in
fluen
za A
vir
us. Th
e pa
rent
N-H
com
poun
d 14
was
seve
ral t
imes
mor
e ac
tive
than
am
anta
dine
aga
inst
H2N
2 an
d H
3N2
influ
enza
A v
irus
. Th
e co
mbi
ned
use
of N
MR
spec
tros
copy
and
com
puta
tiona
l che
mis
try
show
ed th
at th
e co
nfor
mat
ion
arou
nd th
e py
rrol
idin
e-ad
aman
tyl c
arbo
n-ca
rbon
bon
d is
tran
s and
the
pyrr
olid
ine
hete
rocy
cle
has a
n en
velo
pe c
onfo
rmat
ion
with
C-2
out
of t
he p
lane
of t
he o
ther
ring
ato
ms.
N-D
ialk
ylam
inoe
thyl
subs
titut
ion
of c
ompo
und
14 re
sulte
d in
the
pote
nt d
iam
ine
anal
ogue
s 8e,
f,g. I
nter
estin
gly,
thei
r lac
tam
am
ine
prec
urso
rs w
ere
also
act
ive.
Com
poun
ds 8
e,f,g
are
th
e fir
st a
dam
anta
ne d
eriv
ativ
es, b
eari
ng tw
o am
ine
grou
ps, r
epor
ted
to b
e ac
tive
agai
nst i
nflue
nza
A v
irus
.
Stam
atio
u et
al.
2001
Ada
man
tane
der
ivat
ives
Pyrr
olid
ine
Aze
tidin
esA
ziri
dine
s
E2-(
1-ad
aman
tyl)-
2-m
ethy
l-py
rrol
idin
es 3
and
4, 2
-(1-
adam
atity
l)-2-
met
hyl-
azet
idin
es 5
and
6, a
nd
2-(1
-ada
man
tyl)-
2-m
ethy
l-az
irid
ines
7 a
nd 8
wer
e sy
nthe
size
d an
d te
sted
for
thei
r an
tivir
al a
ctiv
ity a
gain
st in
flu-
enza
A. P
aren
t mol
ecul
es 3
, 5, a
nd 7
con
tain
the
alph
a-m
ethy
l-1-
adim
anta
n-m
etha
nam
ine
2 ph
arm
acop
hori
c m
oiet
y (r
iman
tadi
ne).
The
rin
g si
ze e
ffect
on
anti-
influ
enza
A a
ctiv
ity w
as in
vest
igat
ed. P
yrro
lidin
e 3
was
the
mos
t po
tent
ant
i-in
fluen
za v
irus
A c
ompo
und,
9-f
old
mor
e po
tent
than
rim
anta
dine
2, 2
7-fo
ld m
ore
pote
nt th
an a
man
-ta
dine
1, a
nd 2
2-fo
ld m
ore
pote
nt th
an r
ibav
irin
. Aze
tidin
es 5
and
6 w
ere
both
mar
kedl
y ac
tive
agai
nst i
nflu
enza
A
H2N
2 vi
rus,
10-
to 2
0-fo
ld m
ore
pote
nt th
an a
man
tadi
ne. A
ziri
dine
7 w
as a
lmos
t dev
oid
of a
ny a
ctiv
ity a
gain
st
H2N
2 vi
rus
but e
xhib
ited
bord
erlin
e ac
tivity
aga
inst
H3N
2 in
fluen
za A
str
ain.
Thu
s, it
app
ears
that
cha
ngin
g th
e fiv
e-, t
o fo
ur- t
o a
thre
e-m
embe
red
ring
resu
lts in
a d
rop
of a
ctiv
ity a
gain
st in
fluen
za A
vir
us.
Zoi
dis e
t al.
2006
Ada
man
tane
der
ivat
ives
Azo
lo-a
dam
anta
nes
Rim
anta
dine
-res
ista
nt st
rain
s
Che
mot
hera
py a
nd c
hem
opro
phyl
axis
of i
nflu
enza
is o
ne o
f the
mos
t im
port
ant d
irec
tions
of h
ealth
pro
tect
ion
activ
ity. D
ue to
the
high
rat
e of
dru
g-re
sist
ant s
trai
ns o
f inf
luen
za v
irus
, the
re is
a n
eed
for
the
sear
ch a
nd fu
rthe
r de
velo
pmen
t of n
ew p
oten
t ant
ivir
als
agai
nst i
nflu
enza
with
a b
road
spe
ctru
m o
f act
ivity
. In
the
pres
ent s
tudy
, a
set o
f di-
, tri
- and
tetr
azol
e de
riva
tives
of a
dam
anta
ne w
as e
ffic
ient
ly p
repa
red
and
thei
r an
ti-in
fluen
za a
ctiv
ities
ev
alua
ted
agai
nst r
iman
tadi
ne-r
esis
tant
str
ain
A/P
uert
o Ri
co/8
/34.
In g
ener
al, d
eriv
ativ
es o
f tet
razo
le p
osse
ssed
the
high
est v
irus
-inh
ibiti
ng a
ctiv
ity. W
e de
mon
stra
ted
that
sev
eral
com
poun
ds o
f thi
s se
t exh
ibite
d m
uch
high
er a
ctiv
-ity
than
the
curr
ently
use
d an
tivir
al r
iman
tadi
ne, a
com
poun
d of
rela
ted
stru
ctur
e. M
oreo
ver,
we
show
ed th
at th
ese
azol
o- w
ere
sign
ifica
ntly
less
toxi
c. T
his
stud
y de
mon
stra
tes
that
influ
enza
vir
uses
can
be
inhi
bite
d by
ada
man
tyla
-zo
les
and
thus
hav
e po
tent
ial f
or d
evel
opin
g an
tivir
al a
gent
s w
ith a
n al
tern
ate
mec
hani
sm o
f act
ion.
Zar
ubae
v et
al.
2010
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
148
new
M2
ion
chan
nel
inhi
bito
rsT
he A
/M2
prot
on c
hann
el o
f inf
luen
za A
vir
us is
a ta
rget
for
the
anti-
influ
enza
dru
gs a
man
tadi
ne a
nd r
iman
ta-
dine
, who
se e
ffect
iven
ess
was
dim
inis
hed
by th
e ap
pear
ance
of n
atur
ally
occ
urri
ng p
oint
mut
ants
in th
e A
/M2
chan
nel p
ore,
am
ong
whi
ch th
e m
ost c
omm
on a
re S
31N
, V27
A, a
nd L
26F.
We
have
syn
thes
ized
and
cha
ract
eriz
ed
the
prop
ertie
s of
a s
erie
s of
com
poun
ds, o
rigi
nally
der
ived
from
the
A/M
2 in
hibi
tor
BL-1
743.
A le
ad c
ompo
und
emer
ging
from
thes
e in
vest
igat
ions
, spi
ro[5
.5]u
ndec
an-3
-am
ine,
is a
n ef
fect
ive
inhi
bito
r of
wild
-typ
e A
/M2
chan
-ne
ls a
nd L
26F
and
V27
A m
utan
t ion
cha
nnel
s in
vitr
o an
d al
so in
hibi
ts re
plic
atio
n of
reco
mbi
nant
mut
ant v
irus
es
bear
ing
thes
e m
utat
ions
in p
laqu
e re
duct
ion
assa
ys. D
iffer
ence
s in
the
inhi
bitio
n ki
netic
s be
twee
n BL
-174
3, k
now
n to
bin
d in
side
the
A/M
2 ch
anne
l por
e, a
nd a
man
tadi
ne w
ere
expl
oite
d to
dem
onst
rate
com
petit
ion
betw
een
thes
e co
mpo
unds
, con
sist
ent w
ith th
e co
nclu
sion
that
am
anta
dine
bin
ds in
side
the
chan
nel p
ore.
Inhi
bitio
n by
all
of th
ese
com
poun
ds w
as s
how
n to
be
volta
ge-i
ndep
ende
nt, s
ugge
stin
g th
at th
eir
char
ged
grou
ps a
re w
ithin
the
N-t
erm
inal
ha
lf of
the
pore
, pri
or to
the
sele
ctiv
ity fi
lter
that
def
ines
the
regi
on o
ver
whi
ch th
e tr
ansm
embr
ane
pote
ntia
l oc
curs
. The
se fi
ndin
gs n
ot o
nly
help
to d
efin
e th
e lo
catio
n an
d m
echa
nism
of b
indi
ng o
f M2
chan
nel-
bloc
king
dr
ugs
but a
lso
dem
onst
rate
the
feas
ibili
ty o
f dis
cove
ring
new
inhi
bito
rs th
at ta
rget
this
bin
ding
site
in a
num
ber
of
aman
tadi
ne-r
esis
tant
mut
ants
.
Bala
nnik
et a
l. 20
09
Synt
heti
c st
rate
gies
Mic
row
ave-
assi
sted
thre
e-co
mpo
nent
one
-pot
cy
cloc
onde
nsat
ion
met
hod
Ada
man
tyl m
oiet
y
A m
icro
wav
e-as
sist
ed th
ree-
com
pone
nt o
ne-p
ot c
yclo
cond
ensa
tion
met
hod
was
app
lied
for
the
synt
hesi
s of
nov
el
N-(
1-th
ia-4
-aza
spir
o[4.
5] d
ecan
-4-y
l)car
boxa
mid
e co
mpo
unds
car
ryin
g an
ada
man
tyl m
oiet
y. T
he s
truc
ture
s of
the
com
poun
ds w
ere
conf
irm
ed b
y sp
ectr
al a
nd e
lem
enta
l ana
lysi
s. A
ll co
mpo
unds
wer
e ev
alua
ted
for
antiv
iral
act
ivity
ag
ains
t inf
luen
za A
(H1N
1 an
d H
3N2)
and
influ
enza
B v
irus
in M
DC
K c
ell c
ultu
res.
The
com
poun
ds d
ispl
ayed
a
conf
ined
str
uctu
re-a
ctiv
ity re
latio
nshi
p. T
he N
-(2,
8-di
met
hyl-
3-ox
o-1-
thia
-4-a
zasp
iro[
4.5]
dec
-4-y
l)ada
man
tane
-1-
carb
oxam
ide
3b w
as th
e m
ost p
oten
t inh
ibito
r [an
tivir
al E
C50
: 1.4
µM
aga
inst
influ
enza
A/H
3N2
viru
s]. I
ts s
tron
g in
hibi
tory
effe
ct in
a v
irus
hem
olys
is a
ssay
sup
port
s th
at 3
b ac
ts a
s an
influ
enza
vir
us fu
sion
inhi
bito
r by
pre
vent
ing
the
conf
orm
atio
nal c
hang
e of
the
influ
enza
vir
us h
emag
glut
inin
at l
ow p
H.
Gok
tas e
t al.
2012
Enca
psul
atio
n of
ad
aman
tane
s in
lip
osom
es
The
aim
of t
he p
rese
nt s
tudy
was
to e
ncap
sula
te m
anno
syla
ted
1-am
inoa
dam
anta
ne a
nd m
anno
syla
ted
adam
an-
tyltr
ipep
tides
, nam
ely
[(2R
)-N
-(ad
aman
t-1-
yl)-
3-(a
lpha
,bet
a-D
-man
nopy
rano
sylo
xy)-
2-m
ethy
lpro
pana
mid
e an
d (2
R)-N
[3-(
alph
a-D
-man
nopy
rano
sylo
xy)-
2-m
ethy
lpro
pano
yl]-
D,L
-(ad
aman
t-2-
yl)g
lycy
l-L-
alan
yl-D
-iso
glut
amin
e]
in li
poso
mes
. The
cha
ract
eriz
atio
n of
lipo
som
es, s
ize
and
surf
ace
mor
phol
ogy
was
per
form
ed u
sing
dyn
amic
ligh
t sc
atte
ring
(DLS
) and
ato
mic
forc
e m
icro
scop
y (A
FM).
The
resu
lts h
ave
reve
aled
that
the
enca
psul
atio
n of
exa
min
ed
com
poun
ds c
hang
es th
e si
ze a
nd s
urfa
ce o
f lip
osom
es. A
fter
the
conc
anav
alin
A (C
onA
) was
add
ed to
the
lipos
ome
prep
arat
ion,
incr
ease
in li
poso
me
size
and
thei
r ag
greg
atio
n ha
s be
en o
bser
ved.
The
enl
arge
men
t of L
ipos
omes
w
as a
scri
bed
to th
e sp
ecifi
c bi
ndin
g of
the
Con
A to
the
man
nose
pre
sent
on
the
surf
ace
of th
e pr
epar
ed li
poso
mes
. T
hus,
it h
as b
een
show
n th
at th
e ad
aman
tyl m
oiet
y fr
om m
anno
syla
ted
1-am
inoa
dam
anta
ne a
nd m
anno
syla
ted
adam
anty
ltrip
eptid
es c
an b
e us
ed a
s an
anc
hor
in th
e lip
id b
ilaye
r fo
r ca
rboh
ydra
te m
oiet
y ex
pose
d on
the
lipos
ome
surf
ace.
Stim
ac e
t al.
2012
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
149
Effec
tive
ness
and
saf
ety
Am
anta
dine
Ri
man
tadi
neC
hild
ren/
elde
rly
Back
grou
nd T
he e
ffect
iven
ess
and
safe
ty o
f am
anta
dine
(AM
T) a
nd r
iman
tadi
ne (R
MT
) for
pre
vent
ing
and
trea
ting
influ
enza
A in
adu
lts h
as b
een
syst
emat
ical
ly re
view
ed. H
owev
er, l
ittle
is k
now
n ab
out t
hese
trea
tmen
ts in
chi
ldre
n an
d th
e el
derl
y. O
bjec
tives
To
syst
emat
ical
ly re
view
the
effe
ctiv
enes
s an
d sa
fety
of A
MT
and
RM
T in
pre
vent
ing
and
trea
ting
influ
enza
A in
chi
ldre
n an
d th
e el
derl
y. S
earc
h m
etho
ds W
e se
arch
ed th
e C
ochr
ane
Cen
tral
Reg
iste
r of
C
ontr
olle
d Tr
ials
(CEN
TR
AL)
(The
Coc
hran
e Li
brar
y 20
11, I
ssue
2) w
hich
con
tain
s th
e C
ochr
ane
Acu
te R
espi
ra-
tory
Infe
ctio
ns (A
RI) G
roup
’s Sp
ecia
lised
Reg
iste
r, M
EDLI
NE
(196
6 to
June
wee
k 3,
201
1) a
nd E
MBA
SE (1
980
to
June
201
1). S
elec
tion
crite
ria
Rand
omis
ed c
ontr
olle
d tr
ials
(RC
Ts) o
r qu
asi-
RC
Ts c
ompa
ring
AM
T a
nd/ o
r R
MT
w
ith p
lace
bo, c
ontr
ol, o
ther
ant
ivir
als
or d
iffer
ent d
oses
or
sche
dule
s of
AM
T o
r R
MT,
or
both
, or
no in
terv
en-
tion,
in c
hild
ren
and
the
elde
rly.
Dat
a co
llect
ion
and
anal
ysis
Tw
o re
view
aut
hors
inde
pend
ently
sel
ecte
d tr
ials
for
incl
usio
n an
d as
sess
ed m
etho
dolo
gica
l qua
lity.
We
reso
lved
dis
agre
emen
ts b
y co
nsen
sus.
In a
ll co
mpa
riso
ns e
xcep
t fo
r on
e, w
e se
para
tely
ana
lyse
d th
e tr
ials
in c
hild
ren
and
the
elde
rly
usin
g Re
view
Man
ager
sof
twar
e. M
ain
resu
lts A
to
tal o
f 12
stud
ies
invo
lvin
g 24
94 p
artic
ipan
ts (1
586
child
ren
and
adol
esce
nts
and
908
elde
rly)
com
pare
d A
MT
and
R
MT
with
pla
cebo
, par
acet
amol
(one
tria
l; 69
chi
ldre
n) o
r za
nam
ivir
(tw
o tr
ials
; 545
sen
iors
). A
ll st
udie
s w
ere
RCTs
bu
t mos
t wer
e st
ill s
usce
ptib
le to
bia
s. T
wo
tria
ls in
the
elde
rly
had
a hi
gh r
isk
of b
ias
beca
use
of in
com
plet
e ou
t-co
me
data
. In
one
of th
ose
tria
ls th
ere
was
als
o a
lack
of o
utco
me
asse
ssm
ent b
lindi
ng. R
isk
of b
ias
was
unc
lear
in
10 s
tudi
es d
ue to
unc
lear
ran
dom
seq
uenc
e ge
nera
tion
and
allo
catio
n co
ncea
lmen
t. O
nly
two
tria
ls in
chi
ldre
n w
ere
cons
ider
ed to
hav
e a
low
ris
k of
bia
s. A
MT
was
effe
ctiv
e in
pre
vent
ing
influ
enza
A in
chi
ldre
n. A
tota
l of 7
73 p
artic
-ip
ants
wer
e in
clud
ed in
this
out
com
e (r
isk
ratio
(RR)
0.1
1; 9
5% c
onfid
ence
inte
rval
(CI)
0.0
4 to
0.3
0). T
he a
ssum
ed
risk
of i
nflu
enza
in th
e co
ntro
l gro
up w
as 1
0 pe
r 10
0 an
d th
e co
rres
pond
ing
risk
in th
e R
MT
gro
up w
as o
ne p
er
100
(95%
CI 0
to 3
). T
he q
ualit
y of
the
evid
ence
was
con
side
red
low
. For
trea
tmen
t pur
pose
s, R
MT
was
ben
efi-
cial
for
abat
ing
feve
r on
day
thre
e of
trea
tmen
t. Fo
r th
is p
urpo
se o
ne s
tudy
was
sel
ecte
d w
ith lo
w r
isk
of b
ias
and
incl
uded
69
child
ren
(RR
0.36
; 95%
CI 0
.14
to 0
.91)
. The
ass
umed
ris
k w
as 3
8 pe
r 10
0 an
d th
e co
rres
pond
ing
risk
in
the
RM
T g
roup
was
14
per
100,
95%
CI 5
to 3
4. T
he q
ualit
y of
the
evid
ence
was
mod
erat
e. R
MT
did
not
sho
w a
pr
ophy
lact
ic e
ffect
aga
inst
influ
enza
in th
e el
derl
y, b
ut th
e qu
ality
of e
vide
nce
was
con
side
red
very
low
. The
re w
ere
103
part
icip
ants
(RR
0.45
; 95%
CI 0
.14
to 1
.41,
for
an a
ssum
ed r
isk
of 1
7 pe
r 10
0 an
d a
corr
espo
ndin
g ri
sk in
the
RM
T g
roup
of 7
per
100
, 95%
CI 2
to 2
3). W
e di
d no
t ide
ntify
any
AM
T tr
ials
in th
e el
derl
y th
at m
et o
ur in
clus
ion
crite
ria.
The
re w
as n
o ev
iden
ce o
f adv
erse
effe
cts
of A
MT
and
RM
T in
chi
ldre
n or
an
adve
rse
effe
ct o
f RM
T in
the
elde
rly.
We
did
not i
dent
ify a
ny A
MT
tria
ls in
the
elde
rly
that
met
our
incl
usio
n cr
iteri
a. A
utho
rs’ c
oncl
usio
ns A
MT
is
effe
ctiv
e in
pre
vent
ing
influ
enza
A in
chi
ldre
n bu
t the
NN
TB
is h
igh
(NN
TB:
12
(95%
CI 9
to 1
7). R
MT
pro
babl
y he
lps
the
abat
emen
t of f
ever
on
day
thre
e of
trea
tmen
t, bu
t the
qua
lity
of th
e ev
iden
ce is
poo
r. D
ue to
the
smal
l nu
mbe
r of
ava
ilabl
e st
udie
s, w
e co
uld
not r
each
a d
efin
itive
con
clus
ion
on th
e sa
fety
of A
MT
or
the
effe
ctiv
enes
s of
R
MT
in p
reve
ntin
g in
fluen
za in
chi
ldre
n an
d th
e el
derl
y.
Gal
vao
et a
l. 20
12
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
150
Res
ista
nce
to a
dam
anta
nes
281
influ
enza
A is
olat
esA
ustr
alia
Euro
peA
sia
The
ada
man
tane
s (a
man
tadi
ne a
nd r
iman
tadi
ne) w
ere
the
initi
al a
ntiv
iral
s lic
ense
d fo
r us
e ag
ains
t inf
luen
za A
vi
ruse
s an
d ha
ve b
een
used
in s
ome
coun
trie
s to
con
trol
sea
sona
l inf
luen
za a
nd h
ave
also
bee
n st
ockp
iled
for
pote
ntia
l pan
dem
ic u
se. W
hile
hig
h ra
tes
of re
sist
ance
hav
e be
en o
bser
ved
in re
cent
yea
rs w
ith A
(H3)
vir
uses
, the
ra
tes
of re
sist
ance
with
A(H
1) v
irus
es h
as v
arie
d w
idel
y. In
this
stu
dy w
e an
alys
ed 2
81 h
uman
influ
enza
A v
irus
es
isol
ated
in 2
007
that
wer
e re
ferr
ed to
the
WH
O C
olla
bora
ting
Cen
tre
for
Refe
renc
e an
d Re
sear
ch in
Mel
bour
ne,
mai
nly
from
Aus
tral
ia a
nd th
e su
rrou
ndin
g re
gion
s, fo
r ev
iden
ce o
f res
ista
nce
to a
dam
anta
nes
and
a su
bset
of
thes
e w
as e
xam
ined
for
resi
stan
ce to
the
neur
amin
idas
e in
hibi
tors
(NIs
). W
e fo
und
that
the
rate
s of
acd
aman
tane
re
sist
ance
in A
(H3)
vir
uses
con
tinue
d to
incr
ease
in m
ost c
ount
ries
in 2
007
but a
dis
tinct
var
iatio
n w
as s
een
with
A
(H1)
resi
stan
ce le
vels
. A(H
1) v
irus
es fr
om A
ustr
alia
, New
Zea
land
and
Eur
ope
had
low
rat
es o
f res
ista
nce
(2–9
%)
whe
reas
vir
uses
from
a n
umbe
r of
Sou
th E
ast (
SE) A
sian
cou
ntri
es h
ad h
igh
rate
s of
resi
stan
ce (3
3–10
0%).
Thi
s di
ffere
nce
can
be a
ttri
bute
d to
the
spre
ad o
f A/B
risb
ane/
59/2
007-
like
viru
ses
to m
any
part
s of
the
wor
ld w
ith th
e ex
cept
ion
of S
E A
sia
whe
re A
/Hon
g K
ong/
2652
/200
6-lik
e vi
ruse
s co
ntin
ue to
pre
dom
inat
e. W
hen
thes
e tw
o A
(H1)
su
bgro
ups
wer
e co
mpa
red
for
thei
r in
vitr
o se
nsiti
vity
to th
e ot
her
clas
s of
influ
enza
ant
ivir
al d
rugs
, the
neu
ram
ini-
dase
inhi
bito
rs, n
o di
ffere
nce
was
see
n be
twee
n th
e gr
oups
with
bot
h sh
owin
g no
rmal
leve
ls o
f sen
sitiv
ity to
thes
e dr
ugs,
The
find
ing
of re
duci
ng A
(H1)
resi
stan
ce r
ates
in A
ustr
alia
and
ris
ing
leve
ls in
SE
Asi
a in
200
7, re
vers
es th
e tr
end
seen
in 2
006
whe
n A
(H1)
resi
stan
ce le
vels
wer
e ri
sing
in A
ustr
alia
and
els
ewhe
re b
ut re
mai
ned
low
in m
ost o
f SE
Asi
a.
Barr
et a
l. 20
08
Res
ista
nce
to a
dam
anta
nes
Pyro
sequ
enci
ngC
onfir
mat
ory
sequ
ence
an
alys
isPh
enot
ypic
test
ing
Back
grou
nd a
dam
anta
nes
have
bee
n us
ed to
trea
t inf
luen
za A
vir
us in
fect
ions
for
man
y ye
ars.
Stu
dies
hav
e sh
own
a lo
w in
cide
nce
of re
sist
ance
to th
ese
drug
s am
ong
circ
ulat
ing
influ
enza
vir
uses
; how
ever
, the
ir u
se is
ris
ing
wor
ldw
ide
and
drug
resi
stan
ce h
as b
een
repo
rted
am
ong
influ
enza
A (H
5N1)
vir
uses
isol
ated
from
pou
ltry
and
hum
an b
eing
s in
Asi
a. W
e so
ught
to a
sses
s ad
aman
tane
resi
stan
ce a
mon
g in
fluen
za A
vir
uses
isol
ated
dur
ing
the
past
dec
ade
from
cou
ntri
es p
artic
ipat
ing
in W
HO
’s gl
obal
influ
enza
sur
veill
ance
net
wor
k. M
etho
ds w
e an
alys
ed
data
for
influ
enza
fiel
d is
olat
es th
at w
ere
obta
ined
wor
ldw
ide
and
subm
itted
to th
e W
HO
Col
labo
ratin
g C
ente
r fo
r In
fluen
za a
t the
US
Cen
ters
for
Dis
ease
Con
trol
and
Pre
vent
ion
betw
een
Oct
1, 1
994,
and
Mar
31,
200
5. W
e us
ed
pyro
sequ
enci
ng, c
onfir
mat
ory
sequ
ence
ana
lysi
s, a
nd p
heno
typi
c te
stin
g to
det
ect d
rug
resi
stan
ce a
mon
g ci
rcul
at-
ing
influ
enza
A H
3N2
(n =
652
4), H
1N1
(n =
589
), an
d H
1N2
(n =
83)
vir
uses
. Fin
ding
s M
ore
than
700
0 in
fluen
za
A fi
eld
isol
ates
wer
e sc
reen
ed fo
r sp
ecifi
c am
inoa
cid
subs
titut
ions
in th
e M
2 ge
ne k
now
n to
con
fer
drug
resi
stan
ce.
Dur
ing
the
deca
de o
f sur
veill
ance
a s
igni
fican
t inc
reas
e in
dru
g re
sist
ance
was
not
ed, f
rom
0.4
% in
199
4-19
95 to
12
.3%
in 2
003–
2004
. Thi
s in
crea
se in
the
prop
ortio
n of
resi
stan
t vir
uses
was
wei
ghte
d he
avily
by
thos
e ob
tain
ed
from
Asi
a w
ith 6
1% o
f res
ista
nt v
irus
es is
olat
ed s
ince
200
3 be
ing
from
peo
ple
in A
sia.
Inte
rpre
tatio
n ou
r da
ta r
aise
co
ncer
ns a
bout
the
appr
opri
ate
use
of a
dam
anta
nes
and
draw
att
entio
n to
the
impo
rtan
ce o
f tra
ckin
g th
e em
er-
genc
e an
d sp
read
of d
rug-
resi
stan
t inf
luen
za A
vir
uses
.
Brig
ht e
t al.
2005
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
151
Tabl
e 4.
3. In
hibi
tors
of v
iral
Rn
A p
olym
eras
e
Poly
mer
ase
basi
c
prot
ein
2C
ap-b
indi
ng d
omai
nC
ap a
nalo
gs
Influ
enza
vir
uses
cau
se a
sig
nific
ant l
evel
of m
orbi
dity
and
mor
talit
y in
the
popu
latio
n ev
ery
year
. The
ir re
sist
ance
to
curr
ent a
nti-
influ
enza
dru
gs in
crea
ses
the
diff
icul
ty o
f flu
trea
tmen
t. T
hus,
dev
elop
men
t of n
ew a
nti-
influ
enza
dru
gs
is n
eces
sary
in re
gard
s of
pre
vent
the
trag
edy
of in
fluen
za p
ande
mic
. The
Pol
ymer
ase
basi
c pr
otei
n 2
(PB2
) sub
unit
of in
fluen
za v
irus
RN
A p
olym
eras
e is
one
of p
oten
tial t
arge
ts fo
r ne
w d
rugs
bec
ause
the
bind
ing
of P
B2 w
ith th
e 5’
ca
p of
the
host
pre
-mRN
As
is th
e in
itial
ste
p of
the
viru
s’ pr
otei
n sy
nthe
sis.
In th
is s
tudy
, we
com
pare
d th
e bi
ndin
g po
tenc
y of
PB2
cap
bin
ding
dom
ain
with
two
smal
l mol
ecul
es, i
.e.,
RO a
nd P
PT28
, with
that
of P
B2 w
ith c
ap a
nalo
g m
7GT
P. T
he c
alcu
late
d bi
ndin
g en
ergi
es s
how
ed th
at R
O a
nd P
PT28
had
hig
her
bind
ing
affin
ity w
ith P
B2. F
urth
er
inte
ract
ion
anal
ysis
sho
wed
that
the
impo
rtan
t par
ts fo
r bi
ndin
g w
ere
the
five-
and
six-
mem
ber
hete
rocy
clic
rin
gs (t
he
6/5-
mem
ber
ring
s) o
f sm
all m
olec
ules
, as
wel
l as
the
hydr
opho
bic
part
s of
RO
and
PPT
28 w
hich
had
goo
d in
tera
c-tio
ns w
ith th
e hy
drop
hobi
c re
sidu
es in
the
bind
ing
cavi
ty. T
hus,
RO
and
PPT
28 a
re tw
o po
tent
ial a
nti-
influ
enza
dru
gs
targ
eted
PB2
, whi
ch m
ay in
hibi
t the
gro
wth
of i
nflu
enza
vir
us b
y co
mpe
titiv
ely
bind
ing
with
the
cap
stru
ctur
e bi
ndin
g do
mai
n of
PB2
.
Lv e
t al.
2011
Res
ista
nce
to a
dam
anta
nes
USA
S31N
subs
titut
ion
Influ
enza
A a
nd B
Thi
s re
port
des
crib
es n
ew fi
ndin
gs re
gard
ing
the
resi
stan
ce to
ada
man
tane
s of
influ
enza
A v
irus
es c
urre
ntly
ci
rcul
atin
g in
the
Uni
ted
Stat
es a
nd p
rovi
des
inte
rim
reco
mm
enda
tions
that
thes
e dr
ugs
not b
e us
ed d
urin
g th
e re
mai
nder
of t
he 2
005–
2006
influ
enza
sea
son.
Am
anta
dine
als
o is
use
d to
trea
t sym
ptom
s of
Par
kins
on d
isea
se a
nd
may
con
tinue
to b
e us
ed fo
r th
is in
dica
tion.
Res
ista
nce
of in
fluen
za A
vir
uses
to a
dam
anta
nes
can
occu
r sp
onta
ne-
ousl
y or
em
erge
rap
idly
dur
ing
trea
tmen
t. A
sin
gle
poin
t mut
atio
n in
the
codo
ns fo
r am
ino
acid
s at
pos
ition
s 26
, 27,
30
, 31,
or
34 o
f the
M2
prot
ein
can
conf
er c
ross
-res
ista
nce
to b
oth
aman
tadi
ne a
nd r
iman
tadi
ne. N
eith
er re
plic
a-tio
n, tr
ansm
issi
on, n
or v
irul
ence
of a
dam
anta
ne-r
esis
tant
influ
enza
A v
irus
es a
re im
pair
ed b
y th
e po
int m
utat
ions
co
nfer
ring
resi
stan
ce. A
rece
nt re
port
on
the
glob
al p
reva
lenc
e of
ada
man
tane
-res
ista
nt in
fluen
za A
vir
uses
indi
-ca
ted
a si
gnifi
cant
incr
ease
of d
rug
resi
stan
ce, f
rom
1.8
% d
urin
g th
e 20
01–2
002
influ
enza
sea
son
to 1
2.3%
dur
ing
the
2003
–200
4 se
ason
. In
the
Uni
ted
Stat
es, t
he fr
eque
ncy
of a
dam
anta
ne re
sist
ance
incr
ease
d fr
om 1
.9%
dur
ing
the
2003
–200
4 in
fluen
za s
easo
n to
11%
dur
ing
the
2004
-05
seas
on (C
DC
, unp
ublis
hed
data
, 200
5). I
n co
ntra
st to
ad
aman
tane
resi
stan
ce, n
eura
min
idas
e in
hibi
tor
resi
stan
ce re
mai
ns r
are
wor
ldw
ide.
Sin
ce th
e be
ginn
ing
of th
e 20
05–2
006
influ
enza
sur
veill
ance
sea
son,
WH
O a
nd N
REV
SS la
bora
tori
es h
ave
test
ed a
tota
l of 3
8,93
2 sp
ecim
ens
for
influ
enza
vir
uses
; 155
7 (4
.0%
) tes
ted
posi
tive.
Am
ong
the
1557
influ
enza
vir
uses
, 149
9 (9
6.3%
) wer
e in
fluen
za
A v
irus
es, a
nd 5
8 (3
.7%
) wer
e in
fluen
za B
vir
uses
. A to
tal o
f 765
(51.
0%) o
f the
149
9 in
fluen
za A
vir
uses
hav
e be
en
subt
yped
; 760
(99.
3%) w
ere
influ
enza
A (H
3N2)
vir
uses
, and
five
(0.7
%) w
ere
influ
enza
A (H
1N1)
vir
uses
. Dur
ing
Oct
ober
1, 2
005-
Janu
ary
14, 2
006,
a to
tal o
f 123
influ
enza
A v
irus
es c
olle
cted
from
23
stat
es w
ere
test
ed a
t CD
C
for
adam
anta
ne re
sist
ance
. Am
ong
the
120
influ
enza
A (H
3N2)
vir
uses
test
ed, 1
09 (9
1%) d
emon
stra
ted
the
S31N
su
bstit
utio
n in
the
M2
prot
ein
that
con
fers
resi
stan
ce to
am
anta
dine
and
rim
anta
dine
. Con
vent
iona
l seq
uenc
ing
on
a su
bset
of 2
0 vi
ruse
s co
nfir
med
this
sub
stitu
tion.
Am
ong
the
thre
e in
fluen
za A
(H1N
1) v
irus
es te
sted
, non
e co
n-ta
ined
any
mut
atio
ns a
ssoc
iate
d w
ith re
sist
ance
. As
of Ja
nuar
y 14
, all
U.S
. inf
luen
za v
irus
es s
cree
ned
for
antiv
iral
re
sist
ance
at C
DC
had
dem
onst
rate
d su
scep
tibili
ty to
neu
ram
inid
ase
inhi
bito
rs. P
roce
dure
s fo
r vi
rus
prop
agat
ion,
RN
A e
xtra
ctio
n, a
nd p
yros
eque
ncin
g fo
r ad
aman
tane
resi
stan
ce h
ave
been
des
crib
ed p
revi
ousl
y.
Brig
ht e
t al.
2006
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
152
Poly
mer
ase/
endo
nucl
ease
Cap
-bin
ding
Hos
t ada
ptat
ion
mec
hani
sms
The
het
erot
rim
eric
RN
A-d
epen
dent
RN
A p
olym
eras
e of
influ
enza
vir
uses
cat
alyz
es R
NA
repl
icat
ion
and
tran
scri
ptio
n ac
tiviti
es in
infe
cted
cel
l nuc
lei.
The
nuc
leot
ide
poly
mer
izat
ion
activ
ity is
com
mon
to b
oth
repl
icat
ion
and
tran
scri
p-tio
n pr
oces
ses,
with
an
addi
tiona
l cap
-sna
tchi
ng fu
nctio
n be
ing
empl
oyed
dur
ing
tran
scri
ptio
n to
ste
al s
hort
5’-c
appe
d RN
A p
rim
ers
from
hos
t mRN
As.
Cap
-bin
ding
, end
onuc
leas
e, a
nd p
olym
eras
e ac
tiviti
es h
ave
long
bee
n st
udie
d bi
o-ch
emic
ally
, but
str
uctu
ral s
tudi
es o
n th
e po
lym
eras
e an
d its
sub
units
hav
e be
en h
inde
red
by d
iffic
ultie
s in
pro
duci
ng
suff
icie
nt q
uant
ities
of m
ater
ial.
Rece
ntly
, bec
ause
of h
eigh
tene
d ef
fort
and
adv
ance
s in
exp
ress
ion
and
crys
talli
zatio
n te
chno
logi
es, a
ser
ies
of h
igh
reso
lutio
n st
ruct
ures
of i
ndiv
idua
l dom
ains
hav
e be
en d
eter
min
ed. T
hese
she
d lig
ht o
n in
trin
sic
activ
ities
of t
he p
olym
eras
e, in
clud
ing
cap
snat
chin
g, s
ubun
it as
soci
atio
n, a
nd n
ucle
ocyt
opla
smic
tran
spor
t, an
d op
en u
p th
e po
ssib
ility
of s
truc
ture
-gui
ded
deve
lopm
ent o
f new
pol
ymer
ase
inhi
bito
rs. F
urth
erm
ore,
the
activ
ity
of in
fluen
za p
olym
eras
e is
hig
hly
host
- and
cel
l typ
e-sp
ecifi
c, b
eing
dep
ende
nt o
n th
e id
entit
y of
a fe
w k
ey a
min
o ac
id
posi
tions
in th
e di
ffere
nt s
ubun
its, e
spec
ially
in th
e C
-ter
min
al re
gion
of P
B2. N
ew s
truc
ture
s de
mon
stra
te th
e su
rfac
e ex
posu
re o
f the
se re
sidu
es, c
onsi
sten
t with
idea
s th
at th
ey m
ight
mod
ulat
e in
tera
ctio
ns w
ith h
ost-
spe
cific
fact
ors
that
en
hanc
e or
rest
rict
act
ivity
. Rec
ent p
rote
omic
and
gen
ome-
wid
e in
tera
ctom
e an
d RN
A in
terf
eren
ce s
cree
ns h
ave
sug-
gest
ed th
e id
entit
ies
of s
ome
of th
ese
pote
ntia
l reg
ulat
ors
of p
olym
eras
e fu
nctio
n.
Boiv
in e
t al.
2010
Ada
ptiv
e ev
olut
ion
Hos
t ada
ptat
ion
mec
hani
sms
Ada
ptiv
e ev
olut
ion
is c
hara
cter
ized
by
posi
tive
and
para
llel,
or re
peat
ed s
elec
tion
of m
utat
ions
. Mou
se a
dapt
atio
n of
in
fluen
za A
vir
us (I
AV) p
rodu
ces
viru
lent
mut
ants
that
dem
onst
rate
pos
itive
and
par
alle
l evo
lutio
n of
mut
atio
ns in
th
e he
mag
glut
inin
(HA
) rec
epto
r an
d no
n-st
ruct
ural
pro
tein
1 (N
S1) i
nter
fero
n an
tago
nist
gen
es. W
e no
w p
rese
nt a
ge
nom
ic a
naly
sis
of a
ll 11
gen
es o
f 39
mou
se a
dapt
ed IA
V v
aria
nts
from
10
repl
icat
e ad
apta
tion
expe
rim
ents
. Mut
a-tio
ns w
ere
map
ped
on th
e pr
imar
y an
d st
ruct
ural
map
s of
eac
h pr
otei
n an
d sp
ecifi
c m
utat
ions
wer
e va
lidat
ed w
ith
resp
ect t
o vi
rule
nce,
repl
icat
ion,
and
RN
A p
olym
eras
e ac
tivity
. Mou
se a
dapt
ed (M
A) v
aria
nts
obta
ined
aft
er 1
2 or
20
–21
seri
al in
fect
ions
acq
uire
d on
ave
rage
5.8
and
7.9
non
syno
nym
ous
mut
atio
ns p
er g
enom
e of
11
gene
s, re
spec
-tiv
ely.
Am
ong
a to
tal o
f 115
non
syno
nym
ous
mut
atio
ns, 5
1 de
mon
stra
ted
prop
ertie
s of
nat
ural
sel
ectio
n in
clud
ing
27 p
aral
lel m
utat
ions
. The
gre
ates
t deg
ree
of p
aral
lel e
volu
tion
occu
rred
in th
e H
A re
cept
or a
nd r
ibon
ucle
ocap
sid
com
pone
nts,
pol
ymer
ase
subu
nits
(PB1
, PB2
, PA
) and
NP.
Mut
atio
ns o
ccur
red
in h
ost n
ucle
ar tr
affic
king
fact
or
bind
ing
site
s as
wel
l as
site
s of
vir
us-v
irus
pro
tein
sub
unit
inte
ract
ion
for
NP,
NS1
, HA
and
NA
pro
tein
s. A
dapt
ive
regi
ons
incl
uded
cap
bin
ding
and
end
onuc
leas
e do
mai
ns in
the
PB2
and
PA p
olym
eras
e su
buni
ts. F
our
mut
atio
ns in
N
S1 re
sulte
d in
loss
of b
indi
ng to
the
host
cle
avag
e an
d po
lyad
enyl
atio
n sp
ecifi
city
fact
or (C
PSF3
0) s
ugge
stin
g th
at a
re
duct
ion
in in
hibi
tion
of h
ost g
ene
expr
essi
on w
as b
eing
sel
ecte
d. T
he m
ost p
reva
lent
mut
atio
ns in
PB2
and
NP
wer
e sh
own
to in
crea
se v
irul
ence
but
diff
ered
in th
eir
abili
ty to
enh
ance
repl
icat
ion
and
dem
onst
rate
d ep
ista
tic e
ffect
s. S
ev-
eral
pos
itive
ly s
elec
ted
RNA
pol
ymer
ase
mut
atio
ns d
emon
stra
ted
incr
ease
d vi
rule
nce
asso
ciat
ed w
ith >
300%
enh
ance
d po
lym
eras
e ac
tivity
. Ada
ptiv
e m
utat
ions
that
con
trol
hos
t ran
ge a
nd v
irul
ence
wer
e id
entif
ied
by th
eir
repe
ated
sel
ec-
tion
to c
ompr
ise
a de
fined
mod
el fo
r st
udyi
ng IA
V e
volu
tion
to in
crea
sed
viru
lenc
e in
the
mou
se.
Ping
et a
l. 20
11
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
153
Poly
mer
ase
PB2
subu
nit
Tem
pera
ture
-dep
ende
nt
poly
mer
ase
activ
ity
Mos
t avi
an in
fluen
za A
vir
uses
, whi
ch p
refe
rent
ially
repl
icat
e at
the
high
tem
pera
ture
s fo
und
in th
e di
gest
ive
trac
t of
bir
ds, h
ave
a gl
utam
ic a
cid
at re
sidu
e 62
7 of
the
vira
l RN
A p
olym
eras
e PB
2 su
buni
t (G
lu-6
27),
whe
reas
the
hum
an
viru
ses,
whi
ch o
ptim
ally
repl
icat
e at
the
low
tem
pera
ture
s ob
serv
ed in
the
hum
an re
spir
ator
y tr
act,
have
a ly
sine
(L
ys-6
27).
The
mec
hani
sm o
f act
ion
for
this
mut
atio
n is
stil
l not
und
erst
ood,
alth
ough
inte
ract
ion
with
hos
t fac
tors
ha
s be
en p
ropo
sed
to p
lay
a m
ajor
role
. In
this
stu
dy, w
e ex
plor
ed a
n al
tern
ativ
e, y
et re
late
d, h
ypot
hesi
s th
at th
is P
B2
mut
atio
n m
ay a
lter
the
tem
pera
ture
-dep
ende
nt e
nzym
atic
pol
ymer
ase
activ
ity o
f the
vir
al p
olym
eras
e. F
irst
, the
avi
an
poly
mer
ase
prot
ein,
whi
ch w
as p
urifi
ed fr
om b
acul
ovir
us e
xpre
ssio
n sy
stem
, ind
eed
rem
aine
d si
gnifi
cant
ly a
ctiv
e at
hi
gher
tem
pera
ture
s (i.
e. 3
7 an
d 42
°C),
whe
reas
the
hum
an E
627K
mut
ant d
rast
ical
ly lo
st a
ctiv
ity a
t the
se h
igh
tem
-pe
ratu
res.
Sec
ond,
our
ste
ady-
stat
e ki
netic
s da
ta re
veal
ed th
at th
e hu
man
E62
7K m
utan
t pol
ymer
ase
is c
atal
ytic
ally
m
ore
activ
e th
an th
e av
ian
Glu
-627
pol
ymer
ase
at 3
4 °C
. Im
port
antly
, the
E62
7K m
utat
ion
elev
ates
app
aren
t K(c
at) a
t lo
w te
mpe
ratu
res
with
litt
le e
ffect
on
Km
, sug
gest
ing
that
the
E627
K m
utat
ion
alte
rs th
e bi
oche
mic
al s
teps
invo
lved
in
enzy
me
cata
lysi
s ra
ther
than
the
inte
ract
ion
with
the
inco
min
g N
TP.
Thi
rd, t
his
tem
pera
ture
-dep
ende
nt k
inet
ic im
pact
of
the
hum
an E
627K
mut
atio
n w
as a
lso
obse
rved
with
diff
eren
t RN
A te
mpl
ates
, with
diff
eren
t pri
mer
s an
d al
so in
the
pres
ence
of n
ucle
opro
tein
. In
conc
lusi
on, o
ur s
tudy
sug
gest
s th
at th
e am
ino
acid
seq
uenc
e va
riat
ions
at r
esid
ue 6
27 o
f PB
2 su
buni
t can
dir
ectly
alte
r th
e en
zym
e ki
netic
s of
influ
enza
pol
ymer
ase.
Agg
arw
al e
t al.
2011
Poly
mer
ase
PB2
subu
nit
Hum
an-t
o-hu
man
tr
ansm
issi
on
Back
grou
nd: T
he id
entif
icat
ion
of m
utat
ions
that
con
fer
uniq
ue p
rope
rtie
s to
a p
atho
gen,
suc
h as
hos
t ran
ge, i
s of
fun-
dam
enta
l im
port
ance
in th
e fig
ht a
gain
st d
isea
se. T
his
pape
r de
scri
bes
a no
vel m
etho
d fo
r id
entif
ying
am
ino
acid
site
s th
at d
istin
guis
h sp
ecifi
c se
ts o
f pro
tein
seq
uenc
es, b
y co
mpa
rativ
e an
alys
is o
f mat
ched
alig
nmen
ts. T
he u
se o
f mut
ual
info
rmat
ion
to id
entif
y di
stin
ctiv
e re
sidu
es re
spon
sibl
e fo
r fu
nctio
nal v
aria
nts
mak
es th
is a
ppro
ach
high
ly s
uita
ble
for
anal
yzin
g la
rge
sets
of s
eque
nces
. To
supp
ort m
utua
l inf
orm
atio
n an
alys
is, w
e de
velo
ped
the
AVA
NA
sof
twar
e, w
hich
ut
ilize
s se
quen
ce a
nnot
atio
ns to
sel
ect s
ets
for
com
pari
son,
acc
ordi
ng to
use
r-sp
ecifi
ed c
rite
ria.
The
met
hod
pres
ente
d w
as a
pplie
d to
an
anal
ysis
of i
nflu
enza
A P
B2 p
rote
in s
eque
nces
, with
the
obje
ctiv
e of
iden
tifyi
ng th
e co
mpo
nent
s of
ad
apta
tion
to h
uman
-to-
hum
an tr
ansm
issi
on, a
nd re
cons
truc
ting
the
mut
atio
n hi
stor
y of
thes
e co
mpo
nent
s. R
esul
ts:
We
com
pare
d ov
er 3
,000
PB2
pro
tein
seq
uenc
es o
f hum
an-t
rans
mis
sibl
e an
d av
ian
isol
ates
, to
prod
uce
a ca
talo
gue
of
site
s in
volv
ed in
ada
ptat
ion
to h
uman
-to-
hum
an tr
ansm
issi
on. T
his
anal
ysis
iden
tifie
d 17
cha
ract
eris
tic s
ites,
five
of
whi
ch h
ave
been
pre
sent
in h
uman
-tra
nsm
issi
ble
stra
ins
sinc
e th
e 19
18 S
pani
sh fl
u pa
ndem
ic. S
ixte
en o
f the
se s
ites
are
loca
ted
in fu
nctio
nal d
omai
ns, s
ugge
stin
g th
ey m
ay p
lay
func
tiona
l rol
es in
hos
t-ra
nge
spec
ifici
ty. T
he c
atal
ogue
of
cha
ract
eris
tic s
ites
was
use
d to
der
ive
sequ
ence
sig
natu
res
from
his
tori
cal i
sola
tes.
The
se s
igna
ture
s, a
rran
ged
in
chro
nolo
gica
l ord
er, r
evea
l an
evol
utio
nary
tim
elin
e fo
r th
e ad
apta
tion
of th
e PB
2 pr
otei
n to
hum
an h
osts
. Con
clus
ion:
By
pro
vidi
ng th
e m
ost c
ompl
ete
eluc
idat
ion
to d
ate
of th
e fu
nctio
nal c
ompo
nent
s pa
rtic
ipat
ing
in P
B2 p
rote
in a
dapt
a-tio
n to
hum
ans,
this
stu
dy d
emon
stra
tes
that
mut
ual i
nfor
mat
ion
is a
pow
erfu
l too
l for
com
para
tive
char
acte
riza
tion
of
sequ
ence
set
s. In
add
ition
to c
onfir
min
g pr
evio
usly
repo
rted
find
ings
, sev
eral
nov
el c
hara
cter
istic
site
s w
ithin
PB2
are
re
port
ed. S
eque
nce
sign
atur
es g
ener
ated
usi
ng th
e ch
arac
teri
stic
site
s ca
talo
gue
char
acte
rize
con
cise
ly th
e ad
apta
tion
char
acte
rist
ics
of in
divi
dual
isol
ates
. Evo
lutio
nary
tim
elin
es d
eriv
ed fr
om s
igna
ture
s of
ear
ly h
uman
influ
enza
isol
ates
su
gges
t tha
t cha
ract
eris
tic v
aria
nts
emer
ged
rapi
dly,
and
rem
aine
d re
mar
kabl
y st
able
thro
ugh
subs
eque
nt p
ande
mic
s.
In a
dditi
on, t
he s
igna
ture
s of
hum
an-i
nfec
ting
H5N
1 is
olat
es s
ugge
st th
at th
is a
vian
sub
type
has
low
pan
dem
ic p
oten
-tia
l at p
rese
nt, a
lthou
gh it
pre
sent
s m
ore
hum
an a
dapt
atio
n co
mpo
nent
s th
an m
ost a
vian
sub
type
s
Mio
tto
et a
l. 20
08
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
154
Inhi
bito
r of
pol
ymer
ase
PA(C
) sub
unit
Chl
orog
enic
aci
d
The
avi
an in
fluen
za (H
5N1)
vir
al R
NA
pol
ymer
ase
prot
ein
PA(C
) was
use
d as
a ta
rget
to s
cree
n ni
ne c
hlor
ogen
ic
acid
der
ivat
ives
for
thei
r po
lym
eras
e in
hibi
tor
activ
ity. A
mon
g th
em, s
even
com
poun
ds w
ere
PAC
liga
nds,
and
fo
ur in
hibi
ted
influ
enza
RN
A p
olym
eras
e ac
tivity
. The
se re
sults
aid
in th
e de
sign
of a
nti-
influ
enza
age
nts
base
d on
ca
ffeoy
lqui
nic
acid
.
Li e
t al.
2012
b
Inhi
bito
r of
pol
ymer
ase
PA(C
) sub
unit
Lico
rice
-der
ived
co
mpo
unds
PA(C
) sub
unit
from
avi
an in
fluen
za (H
5N1)
vir
al R
NA
pol
ymer
ase
was
use
d in
this
wor
k as
a ta
rget
in th
e sc
reen
ing
for
anti-
influ
enza
age
nts
from
lico
rice
-der
ived
com
poun
ds. A
s a
resu
lt, 1
8 be
ta-g
lycy
rrhe
tinic
aci
d w
as s
ugge
sted
to b
e PA
(C) l
igan
d by
flex
ible
doc
king
, and
was
then
con
firm
ed b
y re
laxa
tion-
edite
d N
MR
. The
resu
lt of
ApG
pri
mer
ext
en-
sion
ass
ay in
dica
ted
that
this
PA
(C) l
igan
d ca
n in
hibi
t the
pol
ymer
ase
activ
ity, a
nd th
us m
ay p
oten
tially
be
valu
able
as
anti-
influ
enza
lead
com
poun
d. T
his
wor
k va
lidat
ed th
e po
ssib
ility
of s
cree
ning
pol
ymer
ase
inhi
bito
rs b
y us
ing
PAc
as a
ta
rget
, and
pro
vide
d a
star
ting
poin
t for
the
furt
her
disc
over
y of
new
ant
i-in
fluen
za d
rugs
.
Li e
t al.
2012
a
nuc
leos
ide
anal
ogue
T-70
5A
ntiv
iral
act
ivity
ev
alua
tion
T-70
5 (6
-flu
oro-
3-hy
drox
y-2-
pyra
zine
carb
oxam
ide)
has
bee
n fo
und
to h
ave
pote
nt a
nd s
elec
tive
inhi
bito
ry a
ctiv
ity
agai
nst i
nflu
enza
vir
us. I
n an
in v
itro
plaq
ue re
duct
ion
assa
y, T
-705
sho
wed
pot
ent i
nhib
itory
act
ivity
aga
inst
influ
enza
A
, B, a
nd C
vir
uses
, with
50%
inhi
bito
ry c
once
ntra
tions
(IC
(50)
s) o
f 0.0
13 to
0.4
8 µg
/ml,
whi
le it
sho
wed
no
cyto
-to
xici
ty a
t con
cent
ratio
ns u
p to
100
0 µg
/ml i
n M
adin
-Dar
by c
anin
e ki
dney
cel
ls. T
he s
elec
tivity
inde
x fo
r in
fluen
za
viru
s w
as m
ore
than
200
0. It
was
als
o ac
tive
agai
nst a
neu
ram
inid
ase
inhi
bito
r-re
sist
ant v
irus
and
som
e am
anta
dine
-re
sist
ant v
irus
es. T
-705
sho
wed
wea
k ac
tivity
aga
inst
non
-inf
luen
za v
irus
RN
A v
irus
es, w
ith th
e IC
(50)
s be
ing
high
er
for
non-
influ
enza
vir
us R
NA
vir
uses
than
for
influ
enza
vir
us, a
nd it
had
no
activ
ity a
gain
st D
NA
vir
uses
. Ora
lly
adm
inis
tere
d T-
705
at 1
00 m
g/kg
of b
ody
wei
ght/
day
(fou
r tim
es a
day
) for
5 d
ays
sign
ifica
ntly
redu
ced
the
mea
n pu
lmon
ary
viru
s yi
elds
and
the
rate
of m
orta
lity
in m
ice
infe
cted
with
influ
enza
vir
us A
/PR/
8/34
(3 ×
102 P
FU).
The
se
resu
lts s
ugge
st th
at T
-705
may
be
a co
mpo
und
that
is u
sefu
l and
hig
hly
sele
ctiv
e ag
ains
t inf
luen
za v
irus
infe
ctio
ns a
nd
that
has
a m
ode
of a
ctio
n di
ffere
nt fr
om th
ose
of c
omm
erci
ally
ava
ilabl
e dr
ugs,
suc
h as
am
anta
dine
, rim
anta
dine
, and
ne
uram
inid
ase
inhi
bito
rs.
Furu
ta e
t al.
2002
nuc
leos
ide
anal
ogue
T-70
5D
ose-
depe
nden
t effe
ct
GT
P-co
mpe
titio
n
T-70
5, a
sub
stitu
ted
pyra
zine
com
poun
d, h
as b
een
foun
d to
exh
ibit
pote
nt a
nti-
influ
enza
vir
us a
ctiv
ity in
vitr
o an
d in
vi
vo. I
n a
time-
of-a
dditi
on s
tudy
, it w
as in
dica
ted
that
T-7
05 ta
rget
ed a
n ea
rly
to m
iddl
e st
age
of th
e vi
ral r
eplic
atio
n cy
cle
but h
ad n
o ef
fect
on
the
adso
rptio
n or
rele
ase
stag
e. T
he a
nti-
influ
enza
vir
us a
ctiv
ity o
f T-7
05 w
as a
tten
uate
d by
add
ition
of p
urin
es a
nd p
urin
e nu
cleo
side
s, in
clud
ing
aden
osin
e, g
uano
sine
, ino
sine
, and
hyp
oxan
thin
e, w
here
as
pyri
mid
ines
did
not
affe
ct it
s ac
tivity
. T-7
05-4
-rib
ofur
anos
yl-5
’-tri
phos
phat
e (T
-705
RTP)
and
T-7
05-4
-rib
ofur
anos
yl-
5’-m
onop
hosp
hate
(T-7
05R
MP)
wer
e de
tect
ed in
MD
CK
cel
ls tr
eate
d w
ith T
-705
. T-7
05RT
P in
hibi
ted
influ
enza
vir
us
RNA
pol
ymer
ase
activ
ity in
a d
ose-
depe
nden
t and
a G
TP-
com
petit
ive
man
ner.
Unl
ike
riba
viri
n, T
-705
did
not
hav
e an
influ
ence
on
cellu
lar
DN
A o
r RN
A s
ynth
esis
. Inh
ibiti
on o
f cel
lula
r IM
P de
hydr
ogen
ase
by T
-705
RM
P w
as a
bout
15
0-fo
ld w
eake
r th
an th
at b
y ri
bavi
rin
mon
opho
spha
te, i
ndic
atin
g th
e sp
ecifi
city
of t
he a
nti-
influ
enza
vir
us a
ctiv
ity
and
low
er le
vel o
f cyt
otox
icity
of T
-705
. The
se re
sults
sug
gest
that
T-7
05RT
P, w
hich
is g
ener
ated
in in
fect
ed c
ells
, may
fu
nctio
n as
a s
peci
fic in
hibi
tor
of in
fluen
za v
irus
RN
A p
olym
eras
e an
d co
ntri
bute
s to
the
sele
ctiv
e an
ti-in
fluen
za v
irus
ac
tivity
of T
-705
.
Furu
ta e
t al.
2005
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
155
nuc
leos
ide
anal
ogue
sT-
705
Pera
miv
irA
ntiv
iral
act
ivity
ev
alua
tion
Mou
se m
odel
Favi
pira
vir
(T-7
05),
an in
fluen
za v
irus
RN
A p
olym
eras
e in
hibi
tor,
and
pera
miv
ir, a
n in
fluen
za v
irus
neu
ram
inid
ase
inhi
bito
r, w
ere
eval
uate
d al
one
and
in c
ombi
natio
n ag
ains
t pan
dem
ic in
fluen
za A
/Cal
iforn
ia/0
4/20
09 (H
1N1)
vir
us
infe
ctio
ns in
mic
e. In
fect
ed m
ice
wer
e tr
eate
d tw
ice
daily
for
5 da
ys s
tart
ing
4 h
afte
r vi
rus
chal
leng
e. F
avip
irav
ir w
as
40%
, 70%
, and
100
% p
rote
ctiv
e at
20,
40,
and
100
mg/
kg/d
ay. P
eram
ivir
was
30%
pro
tect
ive
at 0
.5 m
g/kg
/day
, but
inef
-fe
ctiv
e at
low
er d
oses
whe
n us
ed a
s m
onot
hera
py. C
ombi
natio
ns o
f fav
ipir
avir
and
per
amiv
ir in
crea
sed
the
num
bers
of
sur
vivo
rs b
y 10
–50%
whe
n th
e 0.
025,
0.0
5, a
nd 0
.1 m
g/kg
/day
dos
es o
f per
amiv
ir w
ere
com
bine
d w
ith 2
0 m
g/kg
/day
fa
vipi
ravi
r an
d w
hen
all d
oses
of p
eram
ivir
wer
e co
mbi
ned
with
40
mg/
kg/d
ay fa
vipi
ravi
r. T
hree
-dim
ensi
onal
ana
lysi
s of
dru
g in
tera
ctio
ns u
sing
the
Mac
Syne
rgy
met
hod
indi
cate
s st
rong
syn
ergy
for
thes
e dr
ug c
ombi
natio
ns. I
n ad
ditio
n,
an in
crea
se in
life
span
for
grou
ps o
f mic
e tr
eate
d w
ith d
rug
com
bina
tions
, com
pare
d to
the
mos
t effe
ctiv
e m
ono-
ther
apy
grou
p, w
as o
bser
ved
for
the
0.02
5, 0
.05,
and
0.1
mg/
kg/d
ay d
oses
of p
eram
ivir
com
bine
d w
ith fa
vipi
ravi
r at
the
20 m
g do
se le
vel.
The
refo
re, t
he 2
0 m
g/kg
/d d
ose
of fa
vipi
ravi
r w
as s
elec
ted
for
furt
her
com
bina
tion
stud
ies.
Incr
ease
d su
rviv
al w
as e
xhib
ited
whe
n th
is d
ose
was
com
bine
d w
ith p
eram
ivir
dos
es o
f 0.1
, 0.2
5 an
d 0.
5 m
g/kg
/day
(1 m
g/kg
/day
of
per
amiv
ir a
lone
was
100
% p
rote
ctiv
e in
this
exp
erim
ent)
. Im
prov
ed b
ody
wei
ght r
elat
ive
to e
ither
com
poun
d al
one
was
evi
dent
usi
ng 0
.25,
0.5
, and
1 m
g/kg
/day
of p
eram
ivir.
Sig
nific
ant r
educ
tions
in lu
ng h
emor
rhag
e sc
ore
and
lung
w
eigh
t wer
e ev
iden
t on
day
6 po
st-i
nfec
tion.
In a
dditi
on, v
irus
tite
rs w
ere
redu
ced
sign
ifica
ntly
on
day
4 po
st-i
nfec
tion
by c
ombi
natio
n th
erap
y co
ntai
ning
favi
pira
vir
com
bine
d w
ith p
eram
ivir
at 0
.25
and
0.5
mg/
kg/d
ay. T
hese
dat
a de
mon
-st
rate
that
com
bina
tions
of f
avip
irav
ir a
nd p
eram
ivir
per
form
bet
ter
than
sub
optim
al d
oses
of e
ach
com
poun
d al
one
for
the
trea
tmen
t of i
nflu
enza
vir
us in
fect
ions
in m
ice.
Tarb
et e
t al.
2012
nuc
leos
ide
anal
ogue
sPe
ram
ivir
Phas
e I o
f clin
ical
rese
arch
Pera
miv
ir is
a n
ovel
influ
enza
neu
ram
inid
ase
inhi
bito
r. In
this
art
icle
, hyd
roph
ilic
inte
ract
ion
chro
mat
ogra
phy
coup
led
with
tand
em m
ass s
pect
rom
etry
was
dev
elop
ed to
det
erm
ine
pera
miv
ir in
hum
an p
lasm
a. Th
e po
sitiv
e io
n M
RM m
ode
was
per
form
ed a
nd th
e pr
ecur
sor t
o th
e pr
oduc
t ion
tran
sitio
ns o
f m/z
329
≥ 1
00 a
nd 2
85 ≥
138
wer
e us
ed to
mea
sure
pe
ram
ivir
and
Ro
64-0
802
(I.S
.). C
hrom
atog
raph
ic se
para
tion
was
per
form
ed o
n an
Am
ide-
80 c
olum
n w
ith a
ceto
nitr
ile-
wat
er-f
orm
ic a
cid
(70:
30:0
.1, v
/v/v
, 0.5
ml/m
in).
The
met
hod
was
line
ar o
ver a
con
cent
ratio
n ra
nge
of 1
0–10
000
ng/
ml.
The
aver
age
inte
r-da
y/in
tra-
day
prec
isio
n va
lues
wer
e 3.
7 ±
1.8%
and
4.3
± 1
.8%
, res
pect
ivel
y, w
hile
the
accu
racy
val
ues
wer
e 97
.0 ±
4.8
%. T
his
met
hod
has
been
Suc
cess
fully
app
lied
to P
hase
I of
clin
ical
rese
arch
of p
eram
ivir.
Li e
t al.
2009
nuc
leos
ide
anal
ogue
sV
iram
idin
eRi
bavi
rin
Ant
ivir
al a
ctiv
ity
eval
uatio
nM
ouse
mod
el
Vir
amid
ine,
the
3-ca
rbox
amid
ine
deri
vativ
e of
rib
avir
in, w
as e
ffect
ive
agai
nst a
spe
ctru
m o
f inf
luen
za A
(H1N
1, H
3N2
and
H5N
1) a
nd B
vir
uses
in v
itro,
with
the
50%
effe
ctiv
e co
ncen
trat
ion
(EC
50) r
angi
ng fr
om 2
to 3
2 µg
/ml.
The
mea
n 50
% c
ytot
oxic
con
cent
ratio
n (C
C50
) in
the
MD
CK
cel
ls u
sed
in th
ese
expe
rim
ents
was
760
µg/
ml.
Riba
viri
n, r
un in
pa
ralle
l, ha
d a
sim
ilar
antiv
iral
spe
ctru
m, w
ith E
C50
val
ues
rang
ing
from
0.6
to 5
.5 µ
g/m
l; th
e m
ean
CC
50 fo
r ri
bavi
-ri
n w
as 5
60 µ
g/m
l. O
ral g
avag
e ad
min
istr
atio
ns o
f vir
amid
ine
or r
ibav
irin
to m
ice
infe
cted
with
influ
enza
A/N
WS/
33
(H1N
1), A
/Vic
tori
a/3/
75 (H
3N2)
, B[H
ong
Kon
g/5/
72 o
r B/
Sich
uan/
379/
99 v
irus
es w
ere
high
ly e
ffect
ive
in p
reve
ntin
g de
ath,
less
enin
g de
clin
e in
art
eria
l oxy
gen
satu
ratio
n, in
hibi
tion
Of l
ung
cons
olid
atio
n an
d re
duci
ng lu
ng v
irus
tite
rs.
The
min
imum
effe
ctiv
e do
se o
f vir
amid
ine
in th
ese
stud
ies
rang
ed fr
om 1
5 to
31
mg/
kg/d
ay, d
epen
ding
upo
n th
e vi
rus
infe
ctio
n, w
hen
adm
inis
tere
d tw
ice
daily
for
5 da
ys b
egin
ning
4 h
pre
-vir
us e
xpos
ure.
The
LD
50 o
f the
com
poun
d w
as
610
mg/
kg/d
ay. R
ibav
irin
’s m
inim
um e
ffect
ive
dose
var
ied
betw
een
18 a
nd 3
7.5
mg/
kg/d
ay w
ith th
e LD
50 d
eter
min
ed
to b
e 22
0 m
g/kg
/day
. Vir
amid
ine’s
eff
icac
y w
as a
lso
seen
aga
inst
an
influ
enza
A/N
WS/
33 (H
IN I)
vir
us in
fect
ion
in
mic
e, w
hen
the
com
poun
d w
as a
dmin
iste
red
in th
e dr
inki
ng w
ater
, the
min
imum
effe
ctiv
e do
se b
eing
50
dose
of e
ach,
w
as p
rote
ctiv
e 10
0 m
g/kg
/day
. Del
ay o
f the
initi
atio
n of
eith
er v
iram
idin
e or
rib
avir
in th
erap
y, u
sing
the
appr
oxim
ate
1/3
LD50
dos
e of
eac
h, w
as p
rote
ctiv
e as
late
as
48 h
aft
er e
xpos
ure
to th
e A
/NW
S/33
vir
us. W
hile
bot
h co
mpo
unds
ap
pear
to h
ave
sim
ilar
effic
acy
agai
nst i
nflu
enza
vir
us in
fect
ions
, whe
n on
e co
nsid
ers
the
less
er to
xici
ty, v
iram
idin
e m
ay w
an-a
nt fu
rthe
r ev
alua
tion
as a
pos
sibl
e th
erap
y fo
r in
fluen
za.
Sidw
ell e
t al.
2005
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
156
nuc
leos
ide
anal
ogue
2’-d
eoxy
-2’-
fluor
ogua
nosi
neA
ntiv
iral
act
ivity
ev
alua
tion
Chi
cken
em
bryo
mod
el
The
nuc
leos
ide
anal
og 2
’-deo
xy-2
’-flu
orog
uano
sine
(2’-f
luor
odG
uo) i
s ph
osph
oryl
ated
by
cellu
lar
enzy
mes
and
reve
rs-
ibly
inhi
bits
influ
enza
vir
us re
plic
atio
n in
chi
ck e
mbr
yo c
ells
with
in th
e fir
st 4
h o
f inf
ectio
n, R
NA
hyb
ridi
zatio
n st
ud-
ies
reve
aled
that
pri
mar
y an
d se
cond
ary
tran
scri
ptio
n of
influ
enza
vir
us R
NA
wer
e bl
ocke
d at
a c
ompo
und
conc
entr
a-tio
n of
10
µM, b
ut n
o in
hibi
tion
of c
ell p
rote
in s
ynth
esis
was
see
n ev
en a
t hig
h co
mpo
und
conc
entr
atio
ns (2
00 µ
M).
In v
itro,
the
trip
hosp
hate
of 2
’-flu
orod
Guo
is a
com
petit
ive
inhi
bito
r of
influ
enza
vir
us tr
ansc
ript
ase
activ
ity fr
om
disr
upte
d vi
rus,
with
a K
-i o
f 1.0
µM
. The
cel
lula
r po
lym
eras
es D
NA
pol
ymer
ase
alph
a an
d RN
A p
olym
eras
e II
wer
e on
ly w
eakl
y in
hibi
ted
or w
ere
insu
scep
tible
to 2
’-flu
orod
GT
P. In
kin
etic
stu
dies
with
the
influ
enza
vir
us tr
ansc
ript
ase,
2’
-flu
orod
GT
P, in
the
abse
nce
of G
TP,
blo
cked
elo
ngat
ion
of th
e vi
rus
RNA
cha
in. S
imila
rly,
by
usin
g pu
rifie
d ri
bonu
-cl
eopr
otei
n co
mpl
exes
it w
as fo
und
that
the
addi
tion
of a
sin
gle
nucl
eotid
e of
2’-f
luor
odG
TP
to th
e vi
rus
RNA
cau
sed
chai
n te
rmin
atio
n, w
hich
resu
lted
in th
e bl
ocka
ge o
f fur
ther
vir
us tr
ansc
ript
ion,
Fur
ther
mor
e, th
e sp
ecifi
city
for
influ
-en
za v
irus
tran
scri
ptas
e w
as c
onfir
med
whe
n th
e tr
ansc
ript
ase
from
par
tially
resi
stan
t vir
us w
as fo
und
to b
e 10
-fol
d le
ss s
usce
ptib
le to
2’-f
luor
odG
TP
(K-i
= 1
3.1
µM).
Tisd
ale
et a
l. 19
95
Endo
nucl
ease
inhi
bito
rs33
diff
eren
t typ
es o
f ph
ytoc
hem
ical
sM
arch
antin
sPl
agio
chin
APe
rrot
tetin
FD
ocki
ng si
mul
atio
n
Influ
enza
A p
osse
sses
an
endo
nucl
ease
with
in it
s RN
A p
olym
eras
e w
hich
com
pris
es P
A, P
B1 a
nd P
B2 s
ubun
its. T
o id
entif
y po
tent
ial n
ew a
nti-
influ
enza
com
poun
ds in
our
cur
rent
stu
dy, w
e sc
reen
ed 3
3 di
ffere
nt ty
pes
of p
hyto
chem
i-ca
ls u
sing
a P
A e
ndon
ucle
ase
inhi
bitio
n as
say
in v
itro
and
an a
nti-
influ
enza
A v
irus
ass
ay. T
he m
arch
antin
s ar
e m
ac-
rocy
clic
bis
bibe
nzyl
s fo
und
in li
verw
orts
, and
pla
gioc
hin
A a
nd p
erro
ttet
in F
are
mar
chan
tin-r
elat
ed p
hyto
chem
ical
s.
We
foun
d fr
om o
ur s
cree
n th
at m
arch
antin
A, B
, E, p
lagi
ochi
n A
and
per
rott
etin
F in
hibi
t inf
luen
za P
A e
ndon
ucle
ase
activ
ity in
vitr
o. T
hese
com
poun
ds h
ave
a 3,
4-di
hydr
oxyp
hene
thyl
gro
up in
com
mon
, ind
icat
ing
the
impo
rtan
ce o
f thi
s m
oiet
y fo
r th
e in
hibi
tion
of P
A e
ndon
ucle
ase.
Doc
king
sim
ulat
ions
of m
arch
antin
E w
ith P
A e
ndon
ucle
ase
sugg
est a
pu
tativ
e “f
ittin
g an
d ch
elat
ing
mod
el” a
s th
e m
echa
nism
und
erly
ing
PA e
ndon
ucle
ase
inhi
bitio
n. T
he d
ocki
ng a
min
o ac
ids
are
wel
l con
serv
ed b
etw
een
influ
enza
A a
nd B
. In
a cu
lture
d ce
ll sy
stem
, mar
chan
tin E
was
furt
her
foun
d to
in
hibi
t the
gro
wth
of b
oth
H3N
2 an
d H
1N1
influ
enza
A v
irus
es, a
nd m
arch
antin
A, E
and
per
rote
in F
sho
wed
inhi
bi-
tory
pro
pert
ies
tow
ards
the
grow
th o
f inf
luen
za B
. The
se m
arch
antin
s al
so d
ecre
ased
the
vira
l inf
ectiv
ity ti
ter,
with
m
arch
antin
E s
how
ing
the
stro
nges
t act
ivity
in th
is a
ssay
. We
addi
tiona
lly id
entif
ied
a ch
emic
al g
roup
that
is c
onse
rved
am
ong
diffe
rent
ant
i-in
fluen
za c
hem
ical
s in
clud
ing
mar
chan
tins,
gre
en te
a ca
tech
ins
and
dihy
drox
y ph
enet
hylp
heny
l-ph
thal
imid
es. O
ur p
rese
nt re
sults
indi
cate
that
mar
chan
tins
are
cand
idat
e an
ti-in
fluen
za d
rugs
and
dem
onst
rate
the
utili
ty o
f the
PA
end
onuc
leas
e as
say
in th
e sc
reen
ing
of p
hyto
chem
ical
s fo
r an
ti-in
fluen
za c
hara
cter
istic
s.
Iwai
et a
l. 20
11
Endo
nucl
ease
and
cap
-bi
ndin
g in
hibi
tors
Co-
crys
tal s
truc
ture
sC
ap-s
natc
hing
mec
hani
smD
iket
o co
mpo
unds
Gre
en te
a ca
tech
in
The
vir
al p
olym
eras
e, w
hich
per
form
s tr
ansc
ript
ion
and
repl
icat
ion
of th
e RN
A g
enom
e, is
an
attr
activ
e ta
rget
for
antiv
iral
dru
gs s
ince
pot
ent p
olym
eras
e in
hibi
tors
cou
ld d
irec
tly s
top
vira
l rep
licat
ion
at a
n ea
rly
stag
e. R
ecen
t str
uc-
tura
l stu
dies
on
func
tiona
l dom
ains
of t
he h
eter
otri
mer
ic p
olym
eras
e, w
hich
com
pris
es s
ubun
its P
A, P
B1 a
nd P
B2,
open
the
way
to a
str
uctu
re b
ased
app
roac
h to
opt
imis
e in
hibi
tors
of v
iral
repl
icat
ion.
In p
artic
ular
, the
uni
que
cap-
snat
chin
g m
echa
nism
of v
iral
tran
scri
ptio
n ca
n be
inhi
bite
d by
targ
etin
g ei
ther
the
PB2
cap-
bind
ing
or P
A e
ndon
ucle
-as
e do
mai
ns. H
ere
we
desc
ribe
hig
h re
solu
tion
X-r
ay c
o-cr
ysta
l str
uctu
res
of th
e 20
09 p
ande
mic
H1N
1 (p
H1N
1) P
A
endo
nucl
ease
dom
ain
with
a s
erie
s of
spe
cific
inhi
bito
rs, i
nclu
ding
four
dik
eto
com
poun
ds a
nd a
gre
en te
a ca
tech
in, a
ll of
whi
ch c
hela
te th
e tw
o cr
itica
l man
gane
se io
ns in
the
activ
e si
te o
f the
enz
yme.
Com
pari
son
of th
e bi
ndin
g m
ode
of
the
diffe
rent
com
poun
ds a
nd th
at o
f a m
onon
ucle
otid
e ph
osph
ate
high
light
s, fi
rstly
, how
diff
eren
t sub
stitu
ent g
roup
s on
the
basi
c m
etal
bin
ding
sca
ffold
can
be
orie
ntat
ed to
bin
d in
dis
tinct
sub
-poc
kets
with
in th
e ac
tive
site
cav
ity, a
nd
seco
ndly
, the
pla
stic
ity o
f cer
tain
str
uctu
ral e
lem
ents
of t
he a
ctiv
e si
te c
avity
, whi
ch re
sult
in in
duce
d fit
bin
ding
. The
se
resu
lts w
ill b
e im
port
ant i
n op
timis
ing
the
desi
gn o
f mor
e po
tent
inhi
bito
rs ta
rget
ing
the
cap-
snat
chin
g en
donu
clea
se
activ
ity o
f inf
luen
za v
irus
pol
ymer
ase.
Kow
alin
ski e
t al.
2012
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
157
Tran
scri
ptas
e an
d en
donu
clea
se
inhi
bito
rsFl
utim
ide
3-su
bstit
uted
2,
4-di
oxob
utan
oic
acid
s
A n
ovel
ant
i-in
fluen
za v
irus
com
poun
d, fl
utim
ide,
was
iden
tifie
d in
ext
ract
s of
a re
cent
ly id
entif
ied
fung
al s
peci
es,
Del
itsch
ia c
onfe
rtas
pora
. The
com
poun
d, a
sub
stitu
ted
2,6-
dike
topi
pera
zine
, sel
ectiv
ely
inhi
bite
d th
e ca
p-de
pend
ent
tran
scri
ptas
e of
influ
enza
A a
nd B
vir
uses
and
had
no
effe
ct o
n th
e ac
tiviti
es o
f oth
er p
olym
eras
es. S
imila
r to
the
3-su
bstit
uted
2,4
-dio
xobu
tano
ic a
cids
, a s
erie
s of
tran
scri
ptas
e in
hibi
tors
whi
ch w
e de
scri
bed
prev
ious
ly, t
his
inhi
bito
r, w
hich
is a
nat
ural
pro
duct
, affe
cted
nei
ther
the
initi
atio
n no
r th
e el
onga
tion
of in
fluen
za v
irus
mRN
A s
ynth
esis
, but
it
spec
ifica
lly ta
rget
ed th
e ca
p-de
pend
ent e
ndon
ucle
ase
of th
e tr
ansc
ript
ase.
Add
ition
ally
, the
com
poun
d w
as in
hibi
-to
ry to
the
repl
icat
ion
of in
fluen
za A
and
B v
irus
es in
cel
l cul
ture
. The
sel
ectiv
e an
tivir
al p
rope
rtie
s of
this
com
poun
d fu
rthe
r de
mon
stra
te th
e ut
ility
of i
nflu
enza
vir
us e
ndon
ucle
ase
as a
targ
et o
f ant
ivir
al a
gent
s.
Tom
assi
ni e
t al.
1996
Phos
phor
othi
oate
ol
igon
ucle
otid
esIn
a p
revi
ous
stud
y a
15-m
er p
hosp
horo
thio
ate
olig
onuc
leot
ide
(S-O
N) d
eriv
ed fr
om th
e pa
ckag
ing
sign
al in
the
5’ e
nd
of s
egm
ent 1
(PB2
) of i
nflu
enza
A v
irus
(des
igna
ted
5-15
b) p
rove
d m
arke
dly
inhi
bito
ry to
vir
us re
plic
atio
n. H
ere
we
inve
stig
ated
whe
ther
ana
logo
us in
hibi
tory
S-O
Ns
targ
etin
g th
e 5’
end
of s
egm
ents
2 (P
B1) a
nd 3
(PA
) cou
ld b
e id
enti-
fied
and
whe
ther
vir
al re
sist
ance
to S
-ON
s ca
n be
dev
elop
ed. S
imila
r to
our
ear
lier
resu
lt, 2
0-m
er S
-ON
s re
prod
ucin
g th
e 5’
end
s of
seg
men
ts 2
or
3 (c
ompl
emen
tary
to th
e 3’
-cod
ing
regi
ons
of P
B1 a
nd P
A, r
espe
ctiv
ely)
exe
rted
a p
ower
ful
antiv
iral
act
ivity
aga
inst
a v
arie
ty o
f inf
luen
za A
vir
us s
ubty
pes
in M
OC
K c
ells
. Ser
ial p
assa
ge o
f the
A/T
aiw
an/1
/86
H1N
1 st
rain
in th
e pr
esen
ce o
f S-O
N 5
-15b
or
its a
ntis
ense
as5
-15b
ana
logu
e sh
owed
that
mut
ant v
irus
es w
ith re
duce
d su
scep
tibili
ty to
the
S-O
N c
ould
inde
ed b
e ge
nera
ted,
alth
ough
the
resi
stan
t vir
uses
dis
play
ed re
duce
d re
plic
ativ
e fit
ness
. Seq
uenc
ing
the
resi
stan
t vir
uses
iden
tifie
d m
utat
ions
in th
e PB
1, P
B2, P
A a
nd M
1 ge
nes.
Intr
oduc
tion
of
thes
e ch
ange
s in
to th
e A
/PR/
8/34
H1N
1 st
rain
by
reve
rse
gene
tics,
sug
gest
ed th
at a
ltera
tions
to R
NA
func
tion
in th
e pa
ckag
ing
regi
ons
of s
egm
ents
2 a
nd 3
wer
e im
port
ant i
n de
velo
ping
resi
stan
ce to
S-O
N in
hibi
tion.
How
ever
, man
y of
th
e ot
her
sequ
ence
cha
nges
indu
ced
by S
-ON
trea
tmen
t wer
e m
arke
dly
dele
teri
ous
to v
irus
fitn
ess.
We
conc
lude
that
pa
ckag
ing
sign
als
in th
e in
fluen
za A
vir
us p
olym
eras
e se
gmen
ts p
rovi
de fe
asib
le ta
rget
s fo
r nu
clei
c ac
id-b
ased
ant
ivi-
rals
that
may
be
diff
icul
t for
the
viru
s to
eva
de th
roug
h re
sist
ance
mut
atio
ns.
Gia
nnec
chin
i et
al. 2
011
5’-c
appe
d sh
ort
phos
phor
othi
oate
R
nA
frag
men
tsC
ap d
ecoy
Lipo
som
e en
caps
ulat
ion
We
have
sho
wn
prev
ious
ly th
at th
e 5’
-cap
ped
shor
t pho
spho
dies
ter
RNA
frag
men
ts, C
ap d
ecoy
, (G
m 1
2 nt
) are
pot
ent
inhi
bito
rs o
f inf
luen
za v
irus
RN
A p
olym
eras
e ge
ne e
xpre
ssio
n. H
ere
we
inve
stig
ate
the
mod
ified
cap
ped
RNA
der
iva-
tive
cont
aini
ng p
hosp
horo
thio
ate
olig
onuc
leot
ides
(Cap
dec
oy) a
s a
pote
ntia
l inf
luen
za v
irus
RN
A p
olym
eras
e in
hibi
-to
r. T
he m
odifi
ed 5
’-cap
ped
shor
t pho
spho
roth
ioat
e RN
A fr
agm
ents
(Gm
s 12
–15
nt) w
ith th
e 5’
-cap
ped
stru
ctur
e (m
7Gpp
pGm
) wer
e sy
nthe
size
d by
T7
RNA
pol
ymer
ase.
The
5’-c
appe
d sh
ort R
NA
frag
men
ts (G
ms
12–1
5 nt
) wer
e en
caps
ulat
ed in
lipo
som
e pa
rtic
ulat
es a
nd te
sted
for
thei
r in
hibi
tory
effe
cts
on in
fluen
za v
irus
RN
A p
olym
eras
e ge
ne
expr
essi
on in
the
clon
e 76
cel
ls. T
he 1
2–15
nt l
ong
Gm
s RN
A fr
agm
ents
sho
wed
hig
hly
inhi
bito
ry e
ffect
s. B
y co
ntra
st,
the
inhi
bito
ry e
ffect
s of
the
13 n
t lon
g sh
ort R
NA
frag
men
ts (G
m 1
3 nt
) wer
e co
nsid
erab
ly le
ss in
com
pari
son
with
the
5’-c
appe
d sh
ort p
hosp
horo
thio
ate
RNA
frag
men
ts (G
ms
12–1
5 nt
). In
par
ticul
ar, t
he v
ario
us G
ms
RNA
cha
in le
ngth
s sh
owed
no
sign
ifica
nt d
iffer
ence
s in
the
inhi
bitio
n of
influ
enza
vir
us R
NA
pol
ymer
ase
gene
exp
ress
ion.
Fur
ther
mor
e,
the
capp
ed R
NA
with
a p
hosp
horo
thio
ate
back
bone
was
resi
stan
t to
nucl
ease
act
ivity
. The
se p
hosp
horo
thio
ate
RNA
fr
agm
ents
exh
ibite
d hi
gher
inhi
bito
ry a
ctiv
ity th
an th
e 5’
-cap
ped
shor
t RN
A fr
agm
ents
(Gm
12
nt).
The
se d
ecoy
s m
ay
prov
e to
be
usef
ul in
ant
i-in
fluen
za v
irus
ther
apeu
tics.
Tado
et a
l. 20
01
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
158
Shor
t cap
ped
olig
onuc
leot
ides
The
RN
A-d
epen
dent
RN
A p
olym
eras
e of
influ
enza
vir
us tr
ansc
ribe
s m
esse
nger
RN
A th
roug
h a
uniq
ue c
ap s
cave
ngin
g m
echa
nism
. Vir
al e
nzym
e bi
nds
to th
e ca
p st
ruct
ure
of h
ost m
RNA
, cle
aves
the
mol
ecul
e 9-
15 b
ases
dow
nstr
eam
of
the
cap,
and
use
s th
e sh
ort c
appe
d ol
igon
ucle
otid
e as
a p
rim
er fo
r m
RNA
syn
thes
is. P
revi
ousl
y, w
e ha
ve s
how
n th
at th
e vi
ral p
olym
eras
e ca
n ef
ficie
ntly
bin
d ca
pped
RN
As
shor
ter
than
9 n
ucle
otid
es in
leng
th, b
ut th
e vi
ral e
nzym
e ca
nnot
ut
ilize
thes
e RN
As
as p
rim
ers.
For
this
reas
on, t
hese
sho
rt c
appe
d ol
igon
ucle
otid
es a
re p
oten
t inh
ibito
rs o
f inf
luen
za
viru
s tr
ansc
ript
ion.
In th
ese
stud
ies,
it is
now
sho
wn
that
sho
rt c
appe
d ol
igom
ers
inhi
bit c
appe
d-RN
A d
epen
dent
tr
ansc
ript
ion
at th
e in
itial
ste
p of
cap
bin
ding
. In
cont
rast
, low
con
cent
ratio
ns o
f the
se s
hort
cap
ped
RNA
s ca
n ac
tu-
ally
stim
ulat
e vi
ral t
rans
crip
tion
prim
ed w
ith h
igh
conc
entr
atio
ns o
f the
din
ucle
otid
e A
pG. A
noth
er c
appe
d RN
A
deri
vativ
e co
ntai
ning
pho
spho
roth
ioat
e ol
igon
ucle
otid
es w
as a
lso
inve
stig
ated
as
a po
tent
ial p
olym
eras
e in
hibi
tor.
Thi
s lo
nger
cap
ped
RNA
was
abl
e to
bin
d to
the
poly
mer
ase,
but
cou
ld n
ot b
e cl
eave
d to
pri
mer
leng
th b
y th
e en
zym
e as
soci
ated
end
onuc
leas
e. T
hus,
the
capp
ed p
hosp
horo
thio
ate
RNA
inhi
bite
d ca
p-pr
imed
tran
scri
ptio
n at
the
step
of
cap
bind
ing.
How
ever
, in
cont
rast
to th
e sh
ort c
appe
d ol
igon
ucle
otid
e, it
als
o in
hibi
ted
ApG
pri
med
vir
al tr
ansc
ript
ion.
Cia
nci e
t al.
1997
Endo
nucl
ease
inhi
bito
rs4-
subs
titut
ed
2,4-
diox
obut
anoi
c ac
ids
We
prev
ious
ly id
entifi
ed a
seri
es o
f com
poun
ds w
hich
spec
ifica
lly in
hibi
ted
the
tran
scri
ptio
n of
influ
enza
A a
nd B
vir
uses
. Th
e co
mpo
unds
, 4-s
ubst
itute
d 2,
4-di
oxob
utan
oic
acid
s, se
lect
ivel
y ta
rget
ed th
e ca
p-de
pend
ent e
ndon
ucle
ase
activ
ity o
f th
e tr
ansc
ript
ase
com
plex
. Add
ition
ally
, sev
eral
of t
hese
com
poun
ds e
ffect
ivel
y in
hibi
ted
the
repl
icat
ion
of in
fluen
za v
irus
bu
t not
oth
er v
irus
es in
cel
l cul
ture
ass
ays.
Her
e, w
e re
port
on
the
anti-
influ
enza
vir
us a
ctiv
ities
of o
ther
pot
ent d
eriv
a-tiv
es o
f the
seri
es e
valu
ated
in b
oth
in v
itro
and
in v
ivo
infe
ctiv
ity a
ssay
s. Th
ese
com
poun
ds in
hibi
ted
the
repl
icat
ion
of
influ
enza
vir
us in
yie
ld re
duct
ion
assa
ys, w
ith 5
0% in
hibi
tory
con
cent
ratio
ns ra
ngin
g fr
om 0
.18
to 0
.71
µM. Th
ese
50%
in
hibi
tory
con
cent
ratio
ns w
ere
sim
ilar t
o th
ose
obse
rved
for i
nhib
ition
of i
n vi
tro
tran
scri
ptio
n (0
.32
to 0
.54
µM).
One
se
lect
ed c
ompo
und
also
elic
ited
a do
se-d
epen
dent
inhi
bitio
n of
influ
enza
vir
us re
plic
atio
n in
mic
e fo
llow
ing
an u
pper
re
spir
ator
y tr
act c
halle
nge.
Thes
e st
udie
s dem
onst
rate
the
antiv
iral
effi
cacy
of t
his i
nhib
itor c
lass
and
ther
eby
esta
blis
h th
e ut
ility
of i
nflue
nza
viru
s end
onuc
leas
e as
a c
hem
othe
rape
utic
targ
et.
Has
tings
et a
l. 19
96
Rep
licat
ion/
tr
ansc
ript
ion
inhi
bito
rsC
ompu
tatio
nal a
nd
expe
rim
enta
l scr
eeni
ngC
ompo
und
libra
ry
In th
is st
udy,
com
puta
tiona
l and
exp
erim
enta
l scr
eeni
ng o
f an
exte
nsiv
e co
mpo
und
libra
ry id
entifi
ed T
HC
19, w
hich
w
as a
ble
to su
ppre
ss in
fluen
za v
irus
repl
icat
ion.
This
com
poun
d ha
d no
cyt
otox
ic e
ffect
s and
did
not
dis
rupt
cel
l cyc
le
prog
ress
ion
or in
duce
apo
ptos
is in
MD
CK
cel
ls a
s con
firm
ed b
y W
ST-1
ass
ays,
flow
cyt
omet
ry a
naly
sis,
and
cas
pase
-3
assa
ys. T
ime-
of-a
dditi
on e
xper
imen
ts sh
owed
that
TH
C19
act
s at a
rela
tivel
y ea
rly
stag
e of
the
vira
l life
cycl
e. S
ubse
-qu
ent m
ini-
geno
me
assa
ys re
veal
ed th
at T
HC
19 in
hibi
ted
vira
l gen
ome
repl
icat
ion
and/
or tr
ansc
ript
ion,
sugg
estin
g th
at
it in
terf
eres
with
one
or m
ore
of th
e vi
ral c
ompo
nent
s tha
t for
m th
e ri
bonu
cleo
prot
ein
com
plex
es, n
amel
y po
lym
eras
e ba
sic
2 (P
B2),
poly
mer
ase
basi
c 1
(PB1
), po
lym
eras
e ac
idic
(PA
), nu
cleo
prot
ein
(NP)
and
vir
al R
NA
. Fin
ally
, min
i-ge
nom
e as
says
whe
re P
B2, P
B1, P
A o
r NP
from
A/W
SN/3
3 (H
1N1)
vir
us w
ere
repl
aced
with
thos
e fr
om A
/Udo
rn/3
07/1
972
(H3N
2) v
irus
effe
ctiv
ely
dem
onst
rate
d th
at T
HC
19 in
hibi
ted
vira
l mul
tiplic
atio
n in
a m
anne
r dep
ende
nt u
pon
the
PA
subu
nit.
Take
n to
geth
er, t
hese
resu
lts su
gges
t tha
t infl
uenz
a vi
rus P
A p
rote
in is
a p
oten
tial t
arge
t for
, and
may
aid
the
deve
lopm
ent o
f, no
vel c
ompo
unds
that
inhi
bit i
nflue
nza
A v
irus
repl
icat
ion.
Yam
ada
et a
l. 20
12
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
159
Shor
t int
erfe
ring
Rn
ARN
A p
olym
eras
e of
influ
enza
vir
us is
a sp
ecifi
c en
zym
e ne
cess
ary
for t
he v
iral
repl
icat
ion.
A si
RNA
aga
inst
the
RNA
po
lym
eras
e an
d th
e RN
A p
olym
eras
e in
hibi
tor L
-742
,001
redu
ced
accu
mul
atio
n of
vir
al R
NA
s in
the
infe
cted
cel
ls.
L-74
2,00
1 st
rong
ly in
hibi
ted
viru
s re-
grow
th a
fter
rem
oval
of t
he a
gent
from
the
cultu
re, w
here
as th
e ne
uram
inid
ase
inhi
bito
r zan
amiv
ir d
id n
ot. L
-742
,001
-res
ista
nt m
utan
ts sh
owed
a Th
r-20
to A
la su
bstit
utio
n in
the
PA su
buni
t of R
NA
po
lym
eras
e. Th
e dr
ug-r
esis
tant
vir
us sh
owed
a sl
ight
redu
ctio
n in
the
susc
eptib
ility
to L
-742
,001
in b
oth
the
plaq
ue a
ssay
(t
hree
fold
redu
ctio
n) a
nd e
nzym
e as
say
(tw
o- to
thre
e-fo
ld re
duct
ion)
. The
resi
stan
ce le
vels
wer
e lo
wer
than
thos
e of
za
nam
ivir-
resi
stan
t mut
ants
in th
e pl
aque
ass
ay. A
gain
st z
anam
ivir-
resi
stan
t mut
ants
, L-7
42,0
01 re
tain
ed th
e sa
me
anti-
vira
l act
ivity
as a
gain
st th
e w
ild-t
ype
stra
in. Th
ese
resu
lts in
dica
te th
at L
-742
,001
is m
ost l
ikel
y to
act
at t
he P
A su
buni
t, an
d po
sses
ses a
uni
que
profi
le. I
t is s
ugge
sted
that
PA
subu
nit o
f RN
A p
olym
eras
e is
a p
rom
isin
g ta
rget
for a
nti-
influ
enza
vi
rus a
gent
s.
Nak
azaw
a et
al.
2008
Tabl
e 4.
4. In
hibi
tors
of n
eura
min
idas
e
neu
ram
inid
ase
Rele
ase
and
spre
ad o
f pr
ogen
y vi
rion
sVi
rus e
ntry
into
cel
l
Influ
enza
vir
us n
eura
min
idas
e (N
A) p
lays
an
esse
ntia
l rol
e in
rele
ase
and
spre
ad o
f pro
geny
vir
ions
, fol
low
ing
the
intr
acel
lula
r vi
ral r
eplic
atio
n cy
cle.
To
test
whe
ther
NA
cou
ld a
lso
faci
litat
e vi
rus
entr
y in
to c
ell,
we
infe
cted
cul
ture
s of
hum
an a
irw
ay e
pith
eliu
m w
ith h
uman
and
avi
an in
fluen
za v
irus
es in
the
pres
ence
of t
he N
A in
hibi
tor
osel
tam
ivir
ca
rbox
ylat
e. T
wen
ty- t
o 50
0-fo
ld le
ss c
ells
bec
ame
infe
cted
in d
rug-
trea
ted
vers
us n
ontr
eate
d cu
lture
s (P
< 0
.000
1) 7
h
afte
r vi
rus
appl
icat
ion,
indi
catin
g th
at th
e dr
ug s
uppr
esse
d th
e in
itiat
ion
of in
fect
ion.
The
se d
ata
dem
onst
rate
that
vi
ral N
A p
lays
a ro
le e
arly
in in
fect
ion,
and
they
pro
vide
furt
her
ratio
nale
for
the
prop
hyla
ctic
use
of N
A in
hibi
tors
.
Mat
roso
vich
et
al. 2
004
neu
ram
inid
ase
Enzy
mat
ic a
ctiv
ityEn
docy
tic p
athw
ays
Vir
us re
plic
atio
n
N2
neur
amin
idas
e (N
A) g
enes
of t
he 1
957
and
1968
pan
dem
ic in
fluen
za v
irus
str
ains
pos
sess
ed a
vian
-lik
e lo
w-p
H
stab
ility
of s
ialid
ase
activ
ity, u
nlik
e m
ost e
pide
mic
str
ains
. We
gene
rate
d fo
ur re
vers
e-ge
netic
s vi
ruse
s fr
om a
gen
etic
ba
ckgr
ound
of A
/WSN
/33
(H1N
1) th
at in
clud
ed p
aren
tal N
2 N
As
of 1
968
pand
emic
(H3N
2) a
nd e
pide
mic
(H2N
2)
stra
ins
or th
eir
coun
terp
art N
2 N
As
in w
hich
the
low
-pH
sta
bilit
y of
the
sial
idas
e ac
tivity
was
cha
nged
by
subs
titu-
tions
of o
ne o
r tw
o am
ino
acid
resi
dues
. We
foun
d th
at th
e tr
ansf
ecta
nt v
irus
es b
eari
ng lo
w-p
H-s
tabl
e si
alid
ase
(WSN
/Sta
ble-
NA
s) s
how
ed 2
5- to
80-
times
-gre
ater
abi
lity
to re
plic
ate
in M
adin
-Dar
by c
anin
e ki
dney
(MD
CK
) cel
ls
than
did
the
tran
sfec
tant
vir
uses
bea
ring
low
-pH
-uns
tabl
e si
alid
ase
(WSN
/ Uns
tabl
e-N
As)
. Enz
ymat
ic a
ctiv
ities
of
WSN
/Sta
ble-
NA
s w
ere
dete
cted
in e
ndos
omes
of M
DC
K c
ells
aft
er 9
0 m
in o
f vir
us in
tern
aliz
atio
n by
in s
itu fl
uore
s-ce
nt d
etec
tion
with
5-b
rom
o-4-
chlo
ro-i
ndol
e-3-
yl-c
t-N
-ace
tyin
eura
min
ic a
cid
and
Fast
Red
Vio
let L
B. In
hibi
tion
of
sial
idas
e ac
tivity
of W
SN/S
tabl
e-N
As
on th
e en
docy
tic p
athw
ay b
y pr
etre
atm
ent w
ith 4
-gua
nidi
no-2
,4-d
ideo
xy-N
-ac
etyl
neur
amin
ic a
cid
(zan
amiv
ir) r
esul
ted
in a
sig
nific
ant d
ecre
ase
in p
roge
ny v
irus
es. I
n co
ntra
st, t
he e
nzym
atic
ac
tiviti
es o
f WSN
/Uns
tabl
e-N
As,
the
repl
icat
ion
of w
hich
had
no
effe
ct o
n pr
etre
atm
ent w
ith z
anam
ivir,
wer
e un
de-
tect
able
in c
ells
und
er th
e sa
me
cond
ition
s. H
emad
sorp
tion
assa
ys o
f tra
nsfe
ctan
t-vi
rus-
infe
cted
cel
ls re
veal
ed th
at
the
low
-pH
sta
bilit
y of
the
sial
idas
e ha
d no
effe
ct o
n th
e pr
oces
s of
rem
oval
of s
ialic
aci
d fr
om h
emag
glut
inin
in th
e G
olgi
regi
ons.
Mor
eove
r, hi
gh ti
ters
of v
irus
es w
ere
reco
vere
d fr
om th
e lu
ngs
of m
ice
infe
cted
with
WSN
/Sta
ble-
NA
s on
day
3 a
fter
intr
anas
al in
ocul
atio
n, b
ut W
SN/U
nsta
ble-
NA
s w
ere
clea
red
from
the
lung
s of
the
mic
e. T
hese
resu
lts
indi
cate
that
sia
lidas
e ac
tivity
in la
te e
ndos
ome/
lyso
som
e tr
affic
enh
ance
s in
fluen
za A
vir
us re
plic
atio
n.
Suzu
ki e
t al.
2005
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
160
FAn
AA
ntiv
iral
act
ivity
ev
alua
tion
Susc
eptib
ility
to th
e ne
uram
inid
ase-
inhi
bitin
g eff
ects
of a
synt
hetic
ana
log
of n
eura
min
ic a
cid,
2-d
eoxy
-2,3
-deh
ydro
-N-
trifl
uoro
acet
ylne
uram
inic
aci
d (F
AN
A) w
as fo
und
to v
ary
amon
g di
ffere
nt st
rain
s of i
nflue
nza
A v
irus
acc
ordi
ng to
the
neur
amin
idas
e th
ey c
onta
in. I
n pa
rtic
ular
, NW
S (H
0N1)
vir
us a
nd re
com
bina
nt st
rain
s whi
ch d
eriv
e th
eir n
eura
min
idas
e fr
om N
WS
are
espe
cial
ly su
scep
tible
to F
AN
A a
s mea
sure
d by
the
conc
entr
atio
ns o
f inh
ibito
r whi
ch re
duce
rate
s of e
lu-
tion
from
red
cells
, in
vitr
o ne
uram
inid
ase
activ
ity u
sing
fetu
in a
s a su
bstr
ate,
and
vir
us re
plic
atio
n in
cel
l cul
ture
und
er
agar
. Gro
wth
in ti
ssue
cul
ture
of X
-7 (H
0N2)
vir
us a
nd a
ll re
com
bina
nt v
irus
es c
onta
inin
g N
2 ne
uram
inid
ase
is a
ppro
xi-
mat
ely
50–2
00 ti
mes
less
susc
eptib
le to
inhi
bitio
n by
FA
NA
.Thes
e re
sults
pro
vide
furt
her e
vide
nce
that
the
inhi
bito
ry
effec
ts o
f FA
NA
on
the
repl
icat
ion
of in
fluen
za v
irus
es a
re m
edia
ted
by it
s spe
cific
neu
ram
inid
ase-
inhi
bito
ry a
ctiv
ity
and
conf
irm
that
neu
ram
inid
ase
activ
ity is
nec
essa
ry fo
r th
e re
plic
atio
n of
influ
enza
vir
uses
.
Schu
lman
and
Pa
lese
197
5
Zan
amiv
ird
An
A4-
amin
o-d
An
A a
ctiv
ity
Neu
ram
inid
ase
aciti
vity
Rece
ptor
bin
ding
Cel
l fus
ion
4-G
U-D
AN
A (z
anam
ivir)
(as w
ell a
s DA
NA
and
4-A
M-D
AN
A) w
as fo
und
to in
hibi
t the
neu
ram
inid
ase
activ
ity o
f hum
an
para
influ
enza
vir
us ty
pe 3
(HPF
3). Th
e vi
ral n
eura
min
idas
e ac
tivity
is a
ttri
buta
ble
to h
emag
glut
inin
-neu
ram
inid
ase
(HN
), an
env
elop
e pr
otei
n es
sent
ial f
or v
iral a
ttac
hmen
t and
for f
usio
n m
edia
ted
by th
e ot
her e
nvel
ope
prot
ein,
F. W
hile
ther
e is
no
evid
ence
that
HN
’s ne
uram
inid
ase
activ
ity is
ess
entia
l for
rece
ptor
bin
ding
and
sync
ytiu
m fo
rmat
ion,
we
foun
d th
at
4-G
U-D
AN
A p
reve
nted
hem
adso
rptio
n an
d fu
sion
of p
ersi
sten
tly in
fect
ed c
ells
with
uni
nfec
ted
cells
. In
plaq
ue a
ssay
s, 4-
GU
-DA
NA
redu
ced
the
num
ber (
but n
ot th
e ar
ea) o
f pla
ques
if p
rese
nt o
nly
duri
ng th
e ad
sorp
tion
peri
od a
nd re
duce
d pl
aque
are
a (b
ut n
ot n
umbe
r) if
add
ed o
nly
afte
r the
90-
min
ads
orpt
ion
peri
od. 4
-GU
-DA
NA
als
o re
duce
d th
e ar
ea o
f pl
aque
s for
med
by
a ne
uram
inid
ase-
defic
ient
var
iant
, con
firm
ing
that
its i
nter
fere
nce
with
cel
l-cel
l fus
ion
is u
nrel
ated
to
inhi
bitio
n of
neu
ram
inid
ase
activ
ity. Th
e or
der-
of-m
agni
tude
low
er 5
0% in
hibi
tory
con
cent
ratio
ns o
f 4-G
U-D
AN
A (a
nd
also
DA
NA
and
4-A
M-D
AN
A) f
or p
laqu
e ar
ea re
duct
ion
and
for i
nhib
ition
in th
e fu
sion
ass
ay th
an fo
r red
ucin
g pl
aque
nu
mbe
r or b
lock
ing
hem
adso
rptio
n in
dica
te th
e pa
rtic
ular
effi
cacy
of t
hese
sial
ic a
cid
anal
ogs i
n in
terf
erin
g w
ith c
ell-c
ell
fusi
on. I
n ce
ll lin
es e
xpre
ssin
g in
fluen
za v
irus
hem
aggl
utin
in (R
A) a
s the
onl
y vi
ral p
rote
in, w
e fo
und
that
4-G
U-D
AN
A
had
no e
ffect
on
hem
adso
rptio
n bu
t did
inhi
bit H
A2b
-red
blo
od c
ell f
usio
n, a
s jud
ged
by b
oth
lipid
mix
ing
and
cont
ent
mix
ing.
Thus
, 4-G
U-D
AN
A c
an in
terf
ere
with
bot
h in
fluen
za v
irus
- and
HPF
3-m
edia
ted
fusi
on. Th
e re
sults
indi
cate
that
(i)
in H
PF3,
4-G
U-D
AN
A a
nd it
s ana
logs
hav
e an
affi
nity
not
onl
y fo
r the
neu
ram
inid
ase
activ
e si
te o
f HN
but
als
o fo
r site
s im
port
ant f
or re
cept
or b
indi
ng a
nd c
ell f
usio
n an
d (ii
) sia
lic a
cid-
base
d in
hibi
tors
of i
nflue
nza
viru
s neu
ram
inid
ase
can
also
exe
rt a
dir
ect,
nega
tive
effe
ct o
n th
e fu
soge
nic
func
tion
of th
e ot
her
enve
lope
pro
tein
, HA
.
Gre
enga
rd e
t al.
2000
Zan
amiv
irM
ultic
ente
r clin
ical
stud
yA
ntiv
iral
act
ivity
Safe
tyTo
lera
bilit
yC
hina
2010
–201
1A
dole
scen
ts/a
dults
Back
grou
nd It
is th
e fir
st m
ultic
ente
r cl
inic
al s
tudy
in C
hina
to in
vest
igat
e za
nam
ivir
use
am
ong
Chi
nese
ado
lesc
ents
an
d ad
ults
with
influ
enza
-lik
e ill
ness
(ILI
) sin
ce 2
009,
whe
n in
hale
d za
nam
ivir
(REL
ENZ
A) w
as m
arke
ted
in C
hina
. M
etho
ds A
n un
cont
rolle
d op
en-l
abel
, mul
ticen
tre
stud
y to
eva
luat
e th
e an
tivir
al a
ctiv
ity, a
nd s
afet
y of
inha
led
zan-
amiv
ir (a
s Ro
tadi
sk v
ia D
iskh
aler
dev
ice)
; 10
mg
adm
inis
tere
d tw
ice
daily
for
5 da
ys in
sub
ject
s ≥
12 y
ears
old
with
ILI.
Patie
nts
wer
e en
rolle
d w
ithin
48
hour
s of
ons
et a
nd fo
llow
ed fo
r ei
ght d
ays.
Pat
ient
s w
ere
defin
ed a
s be
ing
influ
enza
-po
sitiv
e if
the
real
-tim
e re
vers
e tr
ansc
ript
ase-
poly
mer
ase
chai
n re
actio
n (r
RT-P
CR)
test
had
pos
itive
resu
lts. R
esul
ts
A to
tal o
f 400
pat
ient
s ≥
12 y
ears
old
wer
e sc
reen
ed fr
om 1
1 ce
nter
s in
sev
en p
rovi
nces
from
Mar
ch 2
010
to Ja
nuar
y 20
11. T
hree
hun
dred
and
nin
ety-
two
patie
nts
who
took
at l
east
one
dos
e of
zan
amiv
ir w
ere
ente
red
into
the
safe
ty
anal
ysis
. The
mea
n ag
e w
as 3
3.8
year
s an
d 50
% w
ere
mal
e. C
ardi
ovas
cula
r di
seas
es a
nd d
iabe
tes
wer
e th
e m
ost
com
mon
com
orbi
ditie
s. A
ll th
e re
port
ed a
dver
se e
vent
s, s
uch
as r
ash,
nas
al a
che,
mus
cle
ache
, nau
sea,
dia
rrhe
a, h
ead-
ache
, occ
urre
d in
less
than
1%
of s
ubje
cts.
Mild
sin
us b
rady
cadi
a or
arr
hyth
mia
occ
urre
d in
four
sub
ject
s (1
%).
Mos
t of
the
adve
rse
even
ts w
ere
mild
and
did
not
requ
ire
any
chan
ge o
f tre
atm
ent.
No
seve
re a
dver
se e
vent
s (S
AE)
or
fata
l ca
ses
wer
e re
port
ed. B
ronc
hosp
asm
was
foun
d in
a 3
8 ye
ars
old
wom
an w
hose
sym
ptom
s di
sapp
eare
d af
ter
stop
ping
za
nam
ivir
and
with
out a
dditi
onal
trea
tmen
t. A
ll th
e 61
influ
enza
vir
us is
olat
es (4
3 be
fore
enr
ollm
ent,
18 d
urin
g tr
eat-
men
t) p
rove
d to
be
sens
itive
to z
anam
ivir.
Con
clus
ions
Zan
amiv
ir is
wel
l tol
erat
ed b
y C
hine
se a
dole
scen
ts a
nd a
dults
w
ith IL
Is. T
here
is n
o ev
iden
ce fo
r th
e em
erge
nce
of d
rug-
resi
stan
t iso
late
s du
ring
trea
tmen
t with
zan
amiv
ir.
Cao
et a
l. 20
12
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
161
ose
ltam
ivir
Avi
an in
fluen
zaN
6 su
btyp
eSu
scep
tibili
ty te
stin
g
Avi
an in
fluen
za v
irus
es a
re a
sou
rce
of g
enet
ic m
ater
ial t
hat c
an b
e tr
ansm
itted
to h
uman
s th
roug
h di
rect
intr
oduc
-tio
n or
rea
ssor
tmen
t. A
lthou
gh th
ere
is a
wea
lth o
f inf
orm
atio
n co
ncer
ning
glo
bal m
onito
ring
for
antiv
iral
res
ista
nce
amon
g hu
man
vir
uses
of t
he N
1 an
d N
2 ne
uram
inid
ase
(NA
) sub
type
s, in
form
atio
n co
ncer
ning
avi
an v
irus
es o
f the
se
and
othe
r N
A s
ubty
pes
is li
mite
d. W
e un
dert
ook
a su
rvei
llanc
e st
udy
to in
vest
igat
e th
e an
tivir
al s
usce
ptib
ility
of
avia
n in
fluen
za N
6 N
A v
irus
es, t
he p
redo
min
ant s
ubty
pe a
mon
g w
ild w
ater
fow
l. W
e ev
alua
ted
73 v
irus
es fr
om N
orth
A
mer
ican
duc
ks a
nd s
hore
bird
s fo
r su
scep
tibili
ty to
the
NA
inhi
bito
r os
elta
miv
ir in
a fl
uore
scen
ce-b
ased
NA
enz
yme
inhi
bitio
n as
say.
Mos
t (90
%) h
ad m
ean
IC50
val
ues
rang
ing
from
< 0
.01
to 5
.0 n
M; 1
0% w
ere
from
5.1
to 5
0.0
nM;
and
none
wer
e >
50.0
nM
. Sus
cept
ibili
ty to
ose
ltam
ivir
rem
aine
d st
able
am
ong
all i
sola
tes
colle
cted
ove
r ap
prox
i-m
atel
y th
ree
deca
des
(P ≤
0.7
4). T
wo
isol
ates
with
122
2V N
A s
ubst
itutio
n ha
d m
oder
atel
y re
duce
d su
scep
tibili
ty to
os
elta
miv
ir in
vitr
o (I
C50
, 30.
0 an
d 40
.0 n
M).
One
fiel
d sa
mpl
e w
as a
mix
ed p
opul
atio
n co
ntai
ning
an
avia
n pa
ra-
myx
ovir
us (A
PMV
) and
H4N
6 in
fluen
za v
irus
, as
reve
aled
by
elec
tron
mic
rosc
opy
and
hem
aggl
utin
atio
n in
hibi
tion
assa
ys w
ith a
pan
el o
f ant
i-A
PMV
ant
iser
a. T
his
high
light
s th
e im
port
ance
of a
war
enes
s an
d ca
refu
l exa
min
atio
n of
no
n-in
fluen
za p
atho
gens
in fi
eld
sam
ples
from
avi
an s
ourc
es. T
his
stud
y sh
owed
that
ose
ltam
ivir
-res
ista
nt N
6 N
A
avia
n in
fluen
za v
irus
es a
re r
are,
and
mus
t be
test
ed b
oth
phen
otyp
ical
ly a
nd g
enot
ypic
ally
to c
onfir
m r
esis
tanc
e.
Ston
er e
t al.
2012
ose
ltam
ivir
car
boxy
late
Zan
amiv
irPr
oduc
tion
of v
iral
pa
rtic
les
Prot
ein
synt
hesi
sLo
ng tu
bula
r vir
us-li
ke
stru
ctur
es
Back
grou
nd: N
eura
min
idas
e (N
A) i
nhib
itors
use
d fo
r in
fluen
za th
erap
y ar
e be
lieve
d to
pre
vent
the
rele
ase
of
prog
eny
viru
s fr
om th
e su
rfac
e of
an
infe
cted
cel
l. In
this
stu
dy, w
e fo
und
that
NA
inhi
bito
rs h
ave
a no
vel a
ntiv
iral
fu
nctio
n ag
ains
t an
avia
n in
fluen
za v
irus
. Res
ults
: Mad
in-D
arby
can
ine
kidn
ey c
ells
, com
mon
ly u
sed
for
the
isol
a-tio
n an
d pr
opag
atio
n of
the
influ
enza
vir
us, w
ere
infe
cted
with
an
avia
n in
fluen
za v
iral
str
ain
A/c
hick
en/G
erm
an/
N/4
9(H
10N
7) (H
10/c
hick
en) o
r a
hum
an in
fluen
za v
iral
str
ain
A/O
saka
/981
/98(
H3N
2) (H
3/O
saka
) vir
us. C
ells
wer
e in
cuba
ted
in a
med
ium
with
out o
r w
ith a
NA
inhi
bito
r, os
elta
miv
ir c
arbo
xyla
te (G
S407
1), f
rom
1 to
13
h po
st in
fec-
tion
(p.i.
). In
fect
ed c
ells
wer
e w
ashe
d 12
h p
.i. to
rem
ove
GS4
071,
incu
bate
d fo
r 1
h w
ithou
t GS4
071,
and
ass
ayed
fo
r vi
rus
prod
uctio
n. In
cuba
tion
with
GS4
071
decr
ease
d th
e pr
oduc
tion
of in
fect
ious
vir
uses
. Whe
n H
10/c
hick
en
viru
s-in
fect
ed c
ells
wer
e in
cuba
ted
with
GS4
071
from
12
to 1
3 h
p.i.
(i.e.
, 1 h
bef
ore
the
viru
s pr
oduc
tion
assa
y),
the
inhi
bito
ry e
ffect
was
cle
arly
obs
erve
d, h
owev
er, t
he s
ame
was
not
evi
dent
for
H3/
Osa
ka v
irus
-inf
ecte
d ce
lls.
Furt
herm
ore,
vir
al p
rote
in s
ynth
esis
in in
fect
ed c
ells
was
not
affe
cted
by
GS4
071.
Usi
ng a
sca
nnin
g el
ectr
on m
icro
-sc
ope,
man
y si
ngle
sph
eric
al b
uds
wer
e ob
serv
ed o
n th
e su
rfac
e of
H3/
Osa
ka v
irus
-inf
ecte
d ce
lls in
cuba
ted
with
out
GS4
071,
whe
reas
man
y ag
greg
ated
par
ticle
s w
ere
obse
rved
on
the
surf
ace
of c
ells
incu
bate
d w
ith G
S407
1. H
owev
er,
man
y lo
ng tu
bula
r vi
rus-
like
stru
ctur
es, w
ith n
o ag
greg
ated
par
ticle
s, w
ere
obse
rved
on
the
surf
ace
of H
10/c
hick
en
viru
s-in
fect
ed c
ells
incu
bate
d w
ith G
S407
1. T
he s
ame
resu
lts w
ere
obta
ined
whe
n an
othe
r N
A in
hibi
tor,
zana
miv
ir,
was
use
d. C
oncl
usio
ns: T
hese
res
ults
indi
cate
that
NA
inhi
bito
rs in
terf
ered
with
vir
us p
artic
le fo
rmat
ion
in th
e H
10/
chic
ken
viru
s-in
fect
ed c
ells
, in
whi
ch th
e in
hibi
tor
caus
ed th
e fo
rmat
ion
of lo
ng tu
bula
r vi
rus-
like
stru
ctur
es in
stea
d of
sph
eric
al v
irus
par
ticle
s.
Ush
irog
awa
and
Ohu
chi 2
011
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
162
Pera
miv
irEv
alua
tion
of a
ntiv
iral
ac
tivity
Mou
se m
odel
New
and
em
ergi
ng in
fluen
za v
irus
stra
ins,
such
as t
he p
ande
mic
influ
enza
A (H
1N1)
vir
us re
quire
con
stan
t vig
ilanc
e fo
r ant
ivira
l dru
g se
nsiti
vity
and
resi
stan
ce. E
ffica
cy o
f int
ram
uscu
larly
(IM
) adm
inis
tere
d ne
uram
inid
ase
(NA
) inh
ibi-
tor,
pera
miv
ir, w
as e
valu
ated
in m
ice
infe
cted
with
rece
ntly
isol
ated
pan
dem
ic A
/Cal
iforn
ia/0
4/20
09 (H
1N1,
swin
e or
igin
, m
ouse
ada
pted
) infl
uenz
a vi
rus.
A si
ngle
IM in
ject
ion
of p
eram
ivir
(four
dos
e gr
oups
), gi
ven
1 h
prio
r to
inoc
ulat
ion,
si
gnifi
cant
ly re
duce
d w
eigh
t los
s (P
< 0.
001)
and
mor
talit
y (P
< 0
.05)
in m
ice
infe
cted
with
LD
(90)
dos
e of
pan
dem
ic A
/C
alifo
rnia
/04/
2009
(H1N
1) in
fluen
za v
irus
com
pare
d to
veh
icle
gro
up. Th
ere
was
20%
surv
ival
in th
e ve
hicl
e-tr
eate
d gr
oup,
w
here
as in
the
pera
miv
ir-tr
eate
d gr
oups
, sur
viva
l inc
reas
ed in
a d
ose-
depe
nden
t man
ner w
ith 6
0, 6
0, 9
0 an
d 10
0% su
rviv
ors
for t
he 1
, 3, 1
0, a
nd 3
0 m
g/kg
dos
es, r
espe
ctiv
ely.
Wei
ght l
oss o
n da
y 4
in th
e ve
hicl
e-tr
eate
d gr
oup
was
3.4
gm
, and
in th
e pe
ram
ivir-
trea
ted
grou
ps w
as 2
.1, 1
.5, 1
.8 a
nd 1
.8 g
for t
he 1
,3, 1
0 an
d 30
mg/
kg d
ose
grou
ps, r
espe
ctiv
ely.
In th
e tr
eatm
ent
mod
el, p
eram
ivir
give
n 24
h a
fter i
nfec
tion
as a
sing
le IM
inje
ctio
n at
50
mg/
kg d
ose,
show
ed si
gnifi
cant
pro
tect
ion
agai
nst
leth
ality
and
wei
ght l
oss.
Ther
e w
as 1
3% su
rviv
al in
the
vehi
cle-
trea
ted
grou
p w
hile
in th
e pe
ram
ivir-
trea
ted
grou
p at
24,
48,
an
d 72
h p
ost i
nfec
tion,
surv
ival
was
100
, 40,
and
50%
, res
pect
ivel
y. Su
rviv
al in
the
osel
tam
ivir
grou
ps (1
0 m
g/kg
/day
twic
e a
day,
oral
ly fo
r 5 d
ays)
was
90,
30
and
20%
at 2
4, 4
8 an
d 72
h, re
spec
tivel
y. Th
ese
data
dem
onst
rate
effi
cacy
of p
aren
tera
lly
adm
inis
tere
d pe
ram
ivir
agai
nst t
he re
cent
ly is
olat
ed p
ande
mic
influ
enza
vir
us in
mur
ine
infe
ctio
n m
odel
s.
Bant
ia e
t al.
2010
Pera
miv
iro
selt
amiv
irZ
anam
ivir
Effica
cy o
f a si
ngle
in
tram
uscu
lar i
njec
tion
Mou
se m
odel
In th
e ev
ent o
f an
influ
enza
out
brea
k, a
ntiv
iral
s inc
ludi
ng th
e ne
uram
inic
lase
(NA
) inh
ibito
rs, p
eram
ivir,
ose
ltam
ivir,
and
za
nam
ivir
may
pro
vide
val
uabl
e be
nefit
whe
n va
ccin
e pr
oduc
tion
is d
elay
ed, l
imite
d, o
r can
not b
e us
ed. H
ere
we
dem
-on
stra
te th
e effi
cacy
of a
sing
le in
tram
uscu
lar i
njec
tion
of p
eram
ivir
in th
e m
ouse
influ
enza
mod
el. P
eram
ivir
pot
ently
in
hibi
ts th
e ne
uram
inid
ase
enzy
me
N9
from
H1N
9 vi
rus i
n vi
tro
with
a 5
0% in
hibi
tory
con
cent
ratio
n (I
C50
) of 1
.3 ±
0.4
nM
. On-
site
dis
soci
atio
n st
udie
s ind
icat
e th
at p
eram
ivir
rem
ains
tigh
tly b
ound
to N
9 N
A (t
1/2 >
24
h), w
here
as, z
an-
amiv
ir a
nd o
selta
miv
ir c
arbo
xyla
te d
isso
ciat
ed ra
pidl
y fr
om th
e en
zym
e (t
1/2 =
1.2
5 h)
. A si
ngle
intr
amus
cula
r inj
ectio
n of
per
amiv
ir (1
0mg/
kg) s
igni
fican
tly re
duce
s wei
ght l
oss a
nd m
orta
lity
in m
ice
infe
cted
with
influ
enza
A/H
1N1,
whi
le
osel
tam
ivir
dem
onst
rate
s no
effica
cy b
y th
e sa
me
trea
tmen
t reg
imen
. This
may
be
due
to ti
ght b
indi
ng o
f per
amiv
ir to
th
e N
I NA
enz
ymes
sim
ilar t
o th
at o
bser
ved
for N
9 en
zym
e. A
dditi
onal
effi
cacy
stud
ies i
ndic
ate
that
a si
ngle
inje
ctio
n of
pe
ram
ivir
(2–2
0 m
g/kg
) was
com
para
ble
to a
q.d
. × 5
day
cou
rse
of o
rally
adm
inis
tere
d os
elta
miv
ir (2
–20m
g/kg
/day
) in
prev
entin
g le
thal
ity in
H3N
2 an
d H
1N1
influ
enza
mod
els.
A si
ngle
intr
amus
cula
r inj
ectio
n of
per
amiv
ir m
ay su
cces
sful
ly
trea
t infl
uenz
a in
fect
ions
and
pro
vide
an
alte
rnat
e op
tion
to o
selta
miv
ir d
urin
g an
influ
enza
out
brea
k.
Bant
ia e
t al.
2006
Lani
nam
ivir
Eval
uatio
n of
ant
ivir
al
activ
ityM
ouse
/fer
ret m
odel
We
have
repo
rted
on
lani
nam
ivir
(cod
e na
me,
R-1
2548
9), a
nov
el n
eura
min
idas
e in
hibi
tor,
and
have
dis
cove
red
that
th
e la
nina
miv
ir p
rodr
ug C
S-89
58 w
orke
d as
a lo
ng-a
ctin
g ne
uram
inid
ase
inhi
bito
r in
a m
ouse
influ
enza
vir
us in
fect
ion
mod
el w
hen
it is
intr
anas
ally
adm
inis
tere
d. In
this
stud
y, C
S-89
58 w
as a
dmin
iste
red
just
onc
e 7
days
bef
ore
infe
ctio
n an
d sh
owed
sign
ifica
nt e
ffica
cy in
viv
o. Th
e effi
cacy
of a
sing
le a
dmin
istr
atio
n of
CS-
8958
aft
er v
iral
infe
ctio
n w
as th
en
com
pare
d w
ith th
at o
f rep
eate
d ad
min
istr
atio
ns o
f ose
ltam
ivir
pho
spha
te o
r zan
amiv
ir in
mic
e an
d fe
rret
s. C
S-89
58
show
ed e
ffica
cy su
peri
or o
r sim
ilar t
o th
e effi
caci
es o
f the
two
licen
sed
NA
inhi
bito
rs. C
S-89
58 a
lso
sign
ifica
ntly
re
duce
d th
e tit
ers o
f an
osel
tam
ivir-
resi
stan
t H1N
1 vi
rus w
ith a
neu
ram
inid
ase
H27
4Y su
bstit
utio
n in
a m
ouse
infe
c-tio
n m
odel
. Thes
e re
sults
sugg
est t
hat s
ince
CS-
8958
is c
hara
cter
istic
ally
long
last
ing
in th
e lu
ngs,
it m
ay b
e id
eal f
or
the
prop
hyla
xis a
nd tr
eatm
ent o
f infl
uenz
a.
Kub
o et
al.
2010
Zan
amiv
irM
ultim
eric
con
juga
tes
A se
t of t
rim
eric
and
tetr
amer
ic d
eriv
ativ
es 6
-11
of th
e in
fluen
za v
irus
neu
ram
inid
ase
inhi
bito
r zan
amiv
ir 1
have
bee
n sy
nthe
size
d by
cou
plin
g a
com
mon
mon
omer
ic z
anam
ivir
deri
vativ
e 3
onto
var
ious
mul
timer
ic c
arbo
xylic
aci
d co
re g
roup
s. Th
ese
disc
rete
mul
timer
ic c
ompo
unds
are
all
sign
ifica
ntly
mor
e an
tivira
l tha
n za
nam
ivir
and
also
show
out
stan
ding
long
-la
stin
g pr
otec
tive
activ
ity w
hen
test
ed in
mou
se in
fluen
za in
fect
ivity
exp
erim
ents
. (C
) 200
4 El
sevi
er L
td. A
ll ri
ghts
rese
rved
.
Wat
son
et a
l. 20
04
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
163
Poly
vale
nt z
anam
ivir
co
njug
ates
Synt
hesi
sA
ntiv
iral
effe
ct
Poly
vale
nt s
ialid
ase
inhi
bito
rs b
eari
ng 4
-gua
nidi
no-N
eu5A
c2en
der
ivat
ives
on
a po
ly-L
-glu
tam
ine
back
bone
are
de
scri
bed.
Aim
ing
for
a lo
nger
rete
ntio
n tim
e of
4-g
uani
dino
-Neu
5Ac2
en (z
anam
ivir
) in
bron
chi a
nd lu
ngs,
we
focu
sed
on s
uper
mol
ecul
es b
eari
ng 4
-gua
nidi
no-N
eu5A
c2en
der
ivat
ives
bou
nd a
t the
ir C
-7 p
ositi
on th
roug
h no
n-cl
eava
ble
alky
l eth
er li
nkag
es. W
e fir
st fo
und
that
alk
ylat
ion
of th
e 7-
hydr
oxyl
gro
up o
f sia
lic a
cid
deri
vativ
e 8
pro-
ceed
ed s
moo
thly
, and
pro
duce
d 7-
O-a
lkyl
-4-g
uani
dino
-Neu
5Ac2
en d
eriv
ativ
es 1
3, w
hich
exh
ibite
d eq
uipo
tent
inhi
bi-
tory
act
ivity
aga
inst
not
onl
y in
fluen
za A
vir
us s
ialid
ase
but a
lso
influ
enza
A v
irus
in th
e ce
ll cu
lture
. Nex
t, w
e sy
n-th
esiz
ed p
oly-
L-gl
utam
ine
bear
ing
7-O
-alk
yl-4
-gua
nidi
no-N
eu5A
c2en
der
ivat
ives
link
ed b
y am
ide
bond
s, 2
6, w
hich
sh
owed
enh
ance
d an
tivir
al a
ctiv
ity a
gain
st in
fluen
za A
vir
us a
nd m
ore
pote
nt e
ffic
acy
in v
ivo
rela
tive
to a
mon
omer
ic
sial
idas
e in
hibi
tor.
Mas
uda
et a
l. 20
03
dua
l-ta
rget
ed
bifu
ncti
onal
ant
i-in
fluen
za d
rugs
Caff
eic
acid
-zan
amiv
ir
conj
ugat
eSu
ppre
ssio
n of
pro
-in
flam
ator
y cy
toki
nes
Influ
enza
ther
apy
with
a s
ingl
e ta
rget
ed c
ompo
und
is o
ften
lim
ited
in e
ffic
acy
due
to th
e ra
pidl
y de
velo
ped
drug
re
sist
ance
. Mor
eove
r, th
e un
cont
rolle
d vi
rus-
indu
ced
cyto
kine
s co
uld
caus
e th
e hi
gh m
orta
lity
of h
uman
infe
cted
by
H5N
1 av
ian
influ
enza
vir
us. I
n th
is s
tudy
, we
expl
ored
the
nove
l dua
l-ta
rget
ed b
ifunc
tiona
l ant
i-in
fluen
za d
rugs
fo
rmed
by
conj
ugat
ion
with
ant
i-in
flam
mat
ory
agen
ts. I
n pa
rtic
ular
, the
caf
feic
aci
d (C
A)-
bear
ing
zana
miv
ir (Z
A)
conj
ugat
es Z
A-7
-CA
(1) a
nd Z
A-7
-CA
-am
ide
(7) s
how
ed s
imul
tane
ous
inhi
bitio
n of
influ
enza
vir
us n
eura
min
idas
e an
d su
ppre
ssio
n of
pro
-inf
lam
mat
ory
cyto
kine
s. T
hese
ZA
con
juga
tes
prov
ided
rem
arka
ble
prot
ectio
n of
cel
ls a
nd
mic
e ag
ains
t inf
luen
za in
fect
ions
. Int
rana
sal a
dmin
istr
atio
n of
low
dos
age
(< 1
.2 µ
mol
/kg/
day)
of Z
A c
onju
gate
s ex
hibi
ted
muc
h gr
eate
r ef
fect
than
the
com
bina
tion
ther
apy
with
ZA
and
the
anti-
infla
mm
ator
y ag
ents
in p
rote
ctio
n of
the
leth
ally
infe
cted
mic
e by
H1N
1 or
H5N
1 in
fluen
za v
irus
es.
Liu
et a
l. 20
12
Thea
flavi
nsN
eura
min
idas
e ac
tivity
as
say
Hae
mag
glut
inat
ion
inhi
bitio
n as
say
Real
-tim
e qu
antit
ativ
e PC
R A
nti-i
nflam
mat
ory
prop
ertie
s
The
thea
flavi
ns fr
actio
n (T
F80%
, with
a p
urity
of 8
0%) a
nd th
ree
thea
flavi
n (T
F) d
eriv
ativ
es fr
om b
lack
tea
have
bee
n fo
und
to e
xhib
it po
tent
inhi
bito
ry e
ffect
s ag
ains
t inf
luen
za v
irus
in v
itro.
The
y w
ere
eval
uate
d w
ith a
neu
ram
inid
ase
(NA
) act
ivity
ass
ay, a
hem
aggl
utin
atio
n (H
A) i
nhib
ition
ass
ay, a
real
-tim
e qu
antit
ativ
e PC
R (q
PCR)
ass
ay fo
r ge
ne
expr
essi
on o
f hem
aggl
utin
in (H
A) a
nd a
cyt
opat
hic
effe
ct (C
PE) r
educ
tion
assa
y. T
he e
xper
imen
tal r
esul
ts s
how
ed
that
they
all
exer
ted
sign
ifica
nt in
hibi
tory
effe
cts
on th
e N
A o
f thr
ee d
iffer
ent s
ubty
pes
of in
fluen
za v
irus
str
ains
[A/
PR/8
/34(
H1N
1), A
/Syd
ney/
5/97
( H3N
2) a
nd 8
/Jia
ngsu
/10/
2003
] with
50%
inhi
bito
ry c
once
ntra
tion
(IC
50) v
alue
s ra
ngin
g fr
om 9
.27
to 3
6.55
µg/
ml,
and
they
als
o di
spla
yed
an in
hibi
tory
effe
ct o
n H
A; t
hese
inhi
bito
ry e
ffect
s m
ight
co
nstit
ute
two
maj
or m
echa
nism
s of
thei
r an
tivir
al a
ctiv
ity. T
ime-
of-a
dditi
on s
tudi
es d
emon
stra
ted
that
TF
deri
vativ
es
mig
ht h
ave
a di
rect
effe
ct o
n vi
ral p
artic
le in
fect
ivity
, whi
ch w
as c
onsi
sten
t with
the
inhi
bito
ry e
ffect
on
HA
. Sub
se-
quen
tly, t
he in
hibi
tory
effe
ct o
f TF
deri
vativ
es o
n th
e re
plic
atio
n of
the
vira
l HA
gen
e as
ass
ayed
by
qPC
R an
d on
the
nucl
ear
loca
lizat
ion
of th
e in
fluen
za v
irus
vRN
P fu
rthe
r de
mon
stra
ted
that
they
may
pri
mar
ily a
ct d
urin
g th
e ea
rly
stag
e of
infe
ctio
n. In
tere
stin
gly,
bes
ides
the
activ
ity a
gain
st fu
nctio
nal v
iral
pro
tein
s, T
F de
riva
tives
als
o de
crea
sed
the
expr
essi
on le
vel o
f the
infla
mm
ator
y cy
toki
ne IL
-6 d
urin
g vi
ral i
nfec
tion,
exp
ress
ion
of w
hich
may
resu
lt in
ser
ious
tis
sue
inju
ry a
nd a
popt
osis
. Our
resu
lts in
dica
ted
that
TF
deri
vativ
es a
re p
oten
tial c
ompo
unds
with
ant
i-in
fluen
za
vira
l rep
licat
ion
and
anti-
infla
mm
ator
y pr
oper
ties.
The
se fi
ndin
gs w
ill p
rovi
de im
port
ant i
nfor
mat
ion
for
new
dru
g de
sign
and
dev
elop
men
t for
the
trea
tmen
t of i
nflu
enza
vir
us in
fect
ion.
Zu e
t al.
2012
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
164
Trad
itio
nal C
hine
se
med
icin
esW
ater
ext
ract
s fro
m
plan
tsM
ouse
mod
el
Etno
phar
mac
olog
ical
rele
vanc
e: N
eura
min
idas
e (N
A) i
nhib
itors
are
cur
rent
ly th
e m
ost e
ffect
ive
drug
s to
trea
t inf
lu-
enza
A v
irus
es in
fect
ion.
Man
y tr
aditi
onal
Chi
nese
med
icin
es (T
CM
s) h
ave
been
use
d in
the
clin
ics
to tr
eat i
nflu
-en
za. T
he a
nti-
vira
l mec
hani
sms
of th
ese
TC
Ms
and
thei
r in
hibi
tory
effe
cts
tow
ards
NA
nee
d to
be
syst
emat
ical
ly
test
ed. A
im o
f the
stu
dy: T
o ev
alua
te th
e an
ti-N
A a
ctiv
ity o
f the
TC
Ms
and
the
anti-
influ
enza
A v
irus
effe
cts
of th
e N
A in
hibi
tory
TC
Ms
in v
itro
and
in v
ivo.
Mat
eria
l and
met
hods
: We
test
ed th
e in
hibi
tory
act
ivity
of w
ater
ext
ract
s fr
om 4
39 T
CM
s to
war
ds N
A. T
he in
vitr
o an
ti-in
fluen
za v
irus
act
iviti
es o
f the
5 T
CM
s w
ere
eval
uate
d us
ing
the
stra
in A
/Cal
iforn
ia/7
/200
9 (H
1N1)
NYM
C X
-179
A o
f inf
luen
za A
vir
us. A
ran
dom
ly s
elec
ted
TC
M w
ith N
A in
hibi
-to
ry a
ctiv
ity. M
elia
toos
enda
n ex
trac
t, w
as fu
rthe
r ev
alua
ted
usin
g a
mou
se m
odel
infe
cted
with
influ
enza
A v
irus
. Re
sults
: Fiv
e T
CM
s, D
uche
snea
indi
ca, F
ocke
(Fra
gari
a in
dica
), Li
quid
amba
r fo
rmos
ana,
Lith
ospe
rmum
ery
thro
rhi-
zon,
Mel
ia to
osen
dan,
and
Pru
nella
vul
gari
s, e
xert
ed p
oten
t inh
ibito
ry a
ctiv
ity to
war
ds N
A. T
hese
TC
Ms
in th
e ra
nge
of 2
5–25
0 µg
/ml h
ad th
e ab
ility
to re
duce
vir
us-i
nduc
ed c
ytop
athi
c ef
fect
(CPE
) and
the
viru
s yi
eld
in M
DC
K c
ells
. M
elia
toos
enda
n si
gnifi
cant
ly re
duce
d de
ath
rate
and
pro
long
ed m
ean
day
to d
eath
(MD
D) o
f the
vir
al in
fect
ed m
ice.
C
oncl
usio
ns: T
his
stud
y de
scri
bes
five
TC
Ms
exer
ted
stro
ng in
hibi
tory
act
iviti
es to
war
ds N
A, a
nd e
xhib
ited
antiv
i-ra
l effe
ct a
gain
st in
fluen
za A
vir
us b
y re
duci
ng v
iral
repr
oduc
tion
and
redu
ced
CPE
of t
he v
iral
infe
cted
cel
ls. M
elia
to
osen
dan,
sig
nific
antly
redu
ced
deat
h ra
te a
nd p
rolo
nged
sur
viva
l of t
he H
1N1
vira
l inf
ecte
d m
ice.
Tian
et a
l. 20
11
Plan
t flav
onol
sG
ossy
petin
Kae
mpf
erol
Eval
uatio
n of
ant
ivir
al
activ
ity
Five
flav
onol
s (3
, 5, a
nd 9
–11)
wer
e is
olat
ed fr
om R
hodi
ola
rose
a, a
nd c
ompa
red
with
com
mer
cial
ly a
vaila
ble
fla-
vono
ids
(1, 2
, 4, 6
-8, a
nd 1
2–14
) to
faci
litat
e an
alys
is o
f the
ir s
truc
ture
-act
ivity
rela
tions
hip
(SA
R). A
ll co
mpo
unds
(1
–14)
sho
wed
neu
ram
inid
ase
inhi
bito
ry a
ctiv
ities
with
IC50
val
ues
rang
ing
from
0.8
to 5
6.9
µM. T
he in
vitr
o an
ti-in
fluen
za v
irus
act
iviti
es o
f fla
vono
ids
1-6,
8–1
2, a
nd 1
4 w
ere
eval
uate
d us
ing
two
influ
enza
vir
al s
trai
ns, H
1N1
(A/
PR/8
/34)
and
H9N
2 (A
/Chi
cken
/Kor
ea/M
S96/
96),
test
ing
thei
r ab
ility
to re
duce
vir
us-i
nduc
ed c
ytop
athi
c ef
fect
(C
PE) i
n M
DC
K c
ells
. We
foun
d th
at th
e ac
tivity
of t
hese
com
poun
ds r
ange
d fr
om 3
0.2
to 9
9.1
µM a
gain
st H
1N1-
and
18
.5 to
133
.6 µ
M a
gain
st H
9N2-
indu
ced
CPE
. Of c
ompo
unds
1–1
4, g
ossy
petin
(6) e
xhib
ited
the
mos
t pot
ent i
nhib
i-to
ry a
ctiv
ity, w
ith IC
50 v
alue
s of
0.8
and
2.6
µM
on
neur
amin
idas
es fr
om C
lost
ridi
um p
erfr
inge
ns a
nd re
com
bina
nt
influ
enza
vir
us A
(rvH
1N1)
, res
pect
ivel
y. In
con
tras
t, ka
empf
erol
(3) e
xhib
ited
the
high
est a
ctiv
ity a
gain
st tw
o in
flu-
enza
vir
uses
, H1N
1 an
d H
9N2
with
EC
50 v
alue
s of
30.
2 an
d 18
.5 µ
M, r
espe
ctiv
ely.
Act
ivity
dep
ende
d on
the
posi
tion
and
num
ber
of h
ydro
xy g
roup
s on
the
flavo
noid
s ba
ckbo
ne. I
n ki
netic
stu
dies
, all
isol
ated
com
poun
ds b
ehav
ed a
s no
ncom
petit
ive
inhi
bito
rs.
Jeon
g et
al.
2009
1,3-
diar
ylpr
op-2
-en-
1-on
e de
riva
tive
sSy
nthe
sis
Eval
uatio
n
A s
erie
s of
1,3
-dia
rylp
rop-
2-en
-1-o
ne d
eriv
ativ
es 3
a-v
have
bee
n sy
nthe
size
d an
d ev
alua
ted
for
thei
r ab
ility
to in
hibi
t ne
uram
inid
ase
(NA
). A
mon
g th
e pr
epar
ed c
ompo
unds
, the
less
lipo
phili
c de
riva
tive
3k s
how
ed th
e m
ost p
oten
t in
vitr
o in
hibi
tory
act
ivity
aga
inst
NA
with
an
IC50
val
ue o
f 1.5
µ M
.
Kin
ger e
t al.
2012
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
165
Cyc
lope
ntan
e de
riva
tive
sA
nov
el se
ries
of c
yclo
pent
ane
deri
vativ
es h
ave
been
foun
d to
exh
ibit
pote
nt a
nd se
lect
ive
inhi
bito
ry e
ffect
s on
influ
enza
vi
rus n
eura
min
idas
e, Th
ese
com
poun
ds, d
esig
nate
d RW
J-27
0201
, BC
X-1
827,
BC
X-1
898,
and
BC
X-1
923,
wer
e te
sted
in
par
alle
l with
zan
amiv
ir a
nd o
selta
miv
ir c
arbo
xyla
te a
gain
st a
spec
trum
of i
nflue
nza
A (H
1N1,
H3N
2, a
nd H
5N1)
an
d in
fluen
za B
vir
uses
in M
DC
K c
ells
, inh
ibiti
on o
f vir
al c
ytop
athi
c eff
ect a
scer
tain
ed v
isua
lly a
nd b
y ne
utra
l red
dye
up
take
was
use
d, w
ith 5
0% e
ffect
ive
(vir
us-in
hibi
tory
) con
cent
ratio
ns (E
C50
) det
erm
ined
. Aga
inst
the
H1N
1 vi
ruse
s A/
Baye
rn/0
7/95
, A/B
eijin
g/26
2/95
, A/P
R/8/
34, a
nd A
/Tex
as/3
6/91
, EC
(50)
s (de
term
ined
by
neut
ral r
ed a
ssay
) of t
he n
ovel
co
mpo
unds
wer
e le
ss th
an o
r equ
al to
1.5
µM
. Tw
elve
stra
ins o
f H3N
2 an
d tw
o st
rain
s of a
vian
H5N
1 vi
ruse
s wer
e in
hib-
ited
at <
0.3
µM
Influ
enza
B/B
eijin
g/18
4/93
and
B/H
arbi
n/07
/94
viru
ses w
ere
inhi
bite
d at
< 0
.2 µ
M, w
ith th
ree
othe
r B
viru
s str
ains
inhi
bite
d at
0.8
to 8
µM
, The
nove
l inh
ibito
rs w
ere
com
para
ble
in p
oten
cy to
(or s
light
ly m
ore
pote
nt th
an)
zana
miv
ir a
nd o
selta
miv
ir c
arbo
xyla
te. N
o cy
toto
xici
ty w
as se
en w
ith th
e co
mpo
unds
at c
once
ntra
tions
of l
ess t
han
or
equa
l to
1 m
M in
cel
l pro
lifer
atio
n as
says
. The
antiv
iral
act
ivity
of R
WJ-
2702
01, c
hose
n fo
r clin
ical
dev
elop
men
t, w
as
stud
ied
in g
reat
er d
etai
l. It
s pot
ency
and
that
of o
selta
miv
ir c
arbo
xyla
te d
ecre
ased
with
incr
easi
ng m
ultip
licity
of v
irus
in
fect
ion.
Tim
e-of
-add
ition
stud
ies i
ndic
ated
that
trea
tmen
t with
eith
er c
ompo
und
need
ed to
beg
in 0
to 1
2 h
afte
r vir
us
expo
sure
for o
ptim
al a
ctiv
ity. E
xpos
ure
of c
ells
to R
WJ-
2702
01 c
ause
d m
ost o
f the
vir
us to
rem
ain
cell
asso
ciat
ed, w
ith
extr
acel
lula
r vir
us d
ecre
asin
g in
a c
once
ntra
tion-
depe
nden
t man
ner.
This
is c
onsi
sten
t with
its e
ffect
as a
neu
ram
inid
ase
inhi
bito
r. RW
J-27
0201
show
s pro
mis
e in
the
trea
tmen
t of h
uman
influ
enza
vir
us in
fect
ions
.
Smee
et a
l. 20
01
Res
ista
nce
to
neur
amin
idas
e in
hibi
tors
Con
serv
ed re
sidu
esV
iral
fitn
ess
Neu
ram
inid
ase
inhi
bito
rs (N
AIs
) are
ant
ivir
als d
esig
ned
to ta
rget
con
serv
ed re
sidu
es a
t the
neu
ram
inid
ase
(NA
) enz
yme
activ
e si
te in
influ
enza
A a
nd B
vir
uses
. The
cons
erve
d re
sidu
es th
at in
tera
ct w
ith N
AIs
are
und
er se
lect
ive
pres
sure
, but
on
ly a
few
hav
e be
en li
nked
to re
sist
ance
. In
the
A/W
uhan
/359
/95
(H3N
2) re
com
bina
nt v
irus
bac
kgro
und,
we
char
ac-
teri
zed
seve
n ch
arge
d, c
onse
rved
NA
resi
dues
(111
18, R
371,
E22
7, R
152,
R22
4, E
276,
and
D15
1) th
at d
irec
tly in
tera
ct
with
the
NA
ls b
ut h
ave
not b
een
repo
rted
to c
onfe
r res
ista
nce
to N
AIs
. Thes
e N
A re
sidu
es w
ere
repl
aced
with
am
ino
acid
s tha
t pos
sess
side
cha
ins h
avin
g si
mila
r pro
pert
ies t
o m
aint
ain
thei
r ori
gina
l cha
rge.
The
NA
mut
atio
ns w
e in
tro-
duce
d si
gnifi
cant
ly d
ecre
ased
NA
act
ivity
com
pare
d to
that
of t
he A
/Wuh
an/3
59/9
5 re
com
bina
nt w
ild-t
ype
and
R292
K
(an
NA
mut
atio
n fr
eque
ntly
repo
rted
to c
onfe
r res
ista
nce)
vir
uses
, whi
ch w
ere
anal
yzed
for c
ompa
riso
n. H
owev
er, t
he
reco
mbi
nant
vir
uses
diff
ered
in re
plic
atio
n effi
cien
cy w
hen
we
seri
ally
pas
sage
d th
em in
vitr
o; th
e gr
owth
of t
he R
118K
an
d E2
27D
vir
uses
was
mos
t im
pair
ed. Th
e R2
24K
E27
6D, a
nd R
371K
mut
atio
ns c
onfe
rred
resi
stan
ce to
bot
h za
nam
ivir
an
d os
elta
miv
ir, w
hile
the
D15
1E m
utat
ion
redu
ced
susc
eptib
ility
to o
selta
miv
ir o
nly
(sim
ilar t
o 10
-fol
d) a
nd th
e R1
52K
m
utat
ion
did
not a
lter s
usce
ptib
ility
to e
ither
dru
g. B
ecau
se th
e R2
24K
mut
atio
n w
as g
enet
ical
ly u
nsta
ble
and
the
emer
-ge
nce
of th
e R3
71K
mut
atio
n in
the
N2
subt
ype
is st
atis
tical
ly u
nlik
ely,
our r
esul
ts su
gges
t tha
t onl
y th
e E2
76D
mut
atio
n is
like
ly to
em
erge
und
er se
lect
ive
pres
sure
. The
resu
lts o
f our
stud
y m
ay h
elp
to o
ptim
ize
the
desi
gn o
f NA
Is.
Yen
et a
l. 20
06
Res
ista
nce
to
adam
anta
nes
Cen
tral
/Sou
th A
mer
ica
Back
grou
nd R
ecen
t inf
luen
za a
ntiv
iral
resi
stan
ce s
tudi
es re
veal
an
alar
min
g in
crea
se in
bot
h ad
aman
tane
s an
d ne
uram
inid
ase
inhi
bito
rs (N
AIs
) res
ista
nt v
iral
str
ains
wor
ldw
ide,
par
ticul
arly
in A
sia,
Eur
ope
and
the
Uni
ted
Stat
es.
Obj
ectiv
es In
this
stu
dy, w
e ha
ve e
valu
ated
influ
enza
vir
us re
sist
ance
in C
entr
al a
nd S
outh
Am
eric
a. M
etho
ds In
flu-
enza
vir
uses
, iso
late
d fr
om s
ympt
omat
ic p
atie
nts
thro
ugho
ut C
entr
al a
nd S
outh
Am
eric
a in
200
5-20
08 w
ere
anal
yzed
fo
r in
hibi
tor
resi
stan
ce. T
he M
2 an
d N
A g
enes
of i
nflu
enza
vir
uses
wer
e se
quen
ced
and
resi
stan
ce w
as in
ferr
ed b
y co
mpa
riso
n w
ith p
ublis
hed
sequ
ence
s an
d kn
own
resi
stan
t mut
atio
ns. R
esul
ts O
ur re
sults
indi
cate
that
: (i)
resi
stan
ce
to a
dam
anta
nes
was
see
n in
the
maj
ority
(95.
5%) o
f the
influ
enza
A/H
3N2
isol
ates
but
onl
y in
one
isol
ate
of th
e in
flu-
enza
A/H
1N1
viru
ses;
(ii)
resi
stan
ce to
NA
Is b
egan
to b
e de
tect
ed in
A/H
1N1
isol
ates
from
Cen
tral
Am
eric
a in
200
8;
and
(iii)
none
of t
he in
fluen
za B
vir
uses
ana
lyze
d w
ere
resi
stan
t to
NA
Is. C
oncl
usio
ns T
hese
find
ings
sug
gest
a li
mite
d ef
fect
iven
ess
of in
fluen
za in
hibi
tors
due
to th
e de
tect
ion
of re
sist
ance
am
ong
A/H
1 an
d A
/H3
viru
ses.
Gar
cia
et a
l. 20
09
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
166
Phar
mac
okin
etic
stu
dies
Ose
ltam
ivir
Bioa
vaila
bilit
yRe
nal c
lear
ance
Stea
dy-s
tate
pla
sma
conc
entr
atio
n
Ose
ltam
ivir
is a
n et
hyl e
ster
pro
drug
of P
o 64
-080
2, a
sele
ctiv
e in
hibi
tor o
f infl
uenz
a vi
rus n
eura
min
idas
e. O
ral a
dmin
is-tr
atio
n of
ose
ltam
ivir
deliv
ers t
he a
ctiv
e an
tivira
l Po
64-0
802
to th
e bl
oods
trea
m, a
nd th
us a
ll si
tes o
f infl
uenz
a in
fect
ion
(lung
, nas
al m
ucos
a, m
iddl
e ea
r) a
re a
cces
sibl
e. Th
e ph
arm
acok
inet
ic p
rofil
e of
ose
ltam
ivir
is si
mpl
e an
d pr
edic
tabl
e, a
nd
twic
e da
ily tr
eatm
ent r
esul
ts in
effe
ctiv
e an
tivira
l pla
sma
conc
entr
atio
ns o
ver t
he e
ntire
adm
inis
trat
ion
inte
rval
. Afte
r or
al a
dmin
istr
atio
n, o
selta
miv
ir is
read
ily a
bsor
bed
from
the
gast
roin
test
inal
trac
t and
ext
ensi
vely
con
vert
ed to
the
activ
e m
etab
olite
. The
abso
lute
bio
avai
labi
lity
of th
e ac
tive
met
abol
ite fr
om o
rally
adm
inis
tere
d os
elta
miv
ir is
80%
. The
activ
e m
etab
olite
is d
etec
tabl
e in
pla
sma
with
in 3
0 m
inut
es a
nd re
ache
s max
imal
con
cent
ratio
ns a
fter 3
to 4
hou
rs. A
fter p
eak
plas
ma
conc
entr
atio
ns a
re a
ttai
ned,
the
conc
entr
atio
n of
the
activ
e m
etab
olite
dec
lines
with
an
appa
rent
hal
f-lif
e of
6 to
10
hou
rs. O
selta
miv
ir is
elim
inat
ed p
rim
arily
by
conv
ersi
on to
and
rena
l exc
retio
n of
the
activ
e m
etab
olite
. Ren
al c
lear
ance
of
bot
h co
mpo
unds
exc
eeds
glo
mer
ular
filtr
atio
n ra
te, i
ndic
atin
g th
at re
nal t
ubul
ar se
cret
ion
cont
ribu
tes t
o th
eir e
limin
a-tio
n vi
a th
e an
ioni
c pa
thw
ay. N
eith
er c
ompo
und
inte
ract
s with
cyt
ochr
ome
P450
mix
ed-f
unct
ion
oxid
ases
or g
lucu
rono
-sy
ltran
sfer
ases
. The
phar
mac
okin
etic
pro
file
of th
e ac
tive
met
abol
ite is
line
ar a
nd d
ose-
prop
ortio
nal,
with
less
than
2-f
old
accu
mul
atio
n ov
er a
dos
age
rang
e of
ose
ltam
ivir
50 to
500
mg
twic
e da
ily. S
tead
y-st
ate
plas
ma
conc
entr
atio
ns a
re a
chie
ved
with
in 3
day
s of t
wic
e da
ily a
dmin
istr
atio
n, a
nd a
t a d
osag
e of
75
mg
twic
e da
ily th
e st
eady
-sta
te p
lasm
a tr
ough
con
cent
ra-
tions
of a
ctiv
e m
etab
olite
rem
ain
abov
e th
e m
inim
um in
hibi
tory
con
cent
ratio
n fo
r all
influ
enza
stra
ins t
este
d. E
xpos
ure
to
the
activ
e m
etab
olite
at s
tead
y st
ate
is a
ppro
xim
atel
y 25
% h
ighe
r in
elde
rly c
ompa
red
with
you
ng in
divi
dual
s; ho
wev
er, n
o do
sage
adj
ustm
ent i
s nec
essa
ry. I
n pa
tient
s with
rena
l im
pair
men
t, m
etab
olite
cle
aran
ce d
ecre
ases
line
arly
with
cre
atin
ine
clea
ranc
e. A
dos
age
redu
ctio
n to
75
mg
once
dai
ly is
reco
mm
ende
d fb
r pat
ient
s with
cre
atin
ine
clea
ranc
e <
30 m
l/min
(1
.8 l/
h). Th
e ph
arm
acok
inet
ics i
n pa
tient
s with
influ
enza
are
qua
litat
ivel
y si
mila
r to
thos
e in
hea
lthy
youn
g ad
ults
. In
vitr
o an
d in
viv
o st
udie
s ind
icat
e no
clin
ical
ly si
gnifi
cant
dru
g in
tera
ctio
ns. N
eith
er p
arac
etam
ol (a
ceta
min
ophe
n) n
or c
imet
idin
e al
tere
d th
e ph
arm
acok
inet
ics o
f Ro
64-0
802.
Coa
dmin
istr
atio
n of
pro
bene
cid
resu
lted
in a
2.5
-fol
d in
crea
se in
exp
osur
e to
Ro
64-
0802
, how
ever
, thi
s com
petit
ion
is u
nlik
ely
to re
sult
in c
linic
ally
rele
vant
effe
cts.
Thes
e pr
oper
ties m
ake
osel
tam
ivir
a su
itabl
e ca
ndid
ate
for u
se in
the
prev
entio
n an
d tr
eatm
ent o
f infl
uenz
a.
He
et a
l. 19
99
Tabl
e 4.
5. H
ost c
ell f
acto
r ta
rget
ing
Sign
allin
g ca
scad
e bl
ocke
rsC
-Jun
kin
ase
Bloc
king
of
tran
scri
ptio
n an
d RN
A sy
nthe
sis
C-J
un N
-ter
min
al k
inas
es (
JNK
) are
act
ivat
ed in
cou
rse
of m
any
vira
l inf
ectio
ns. H
ere
we
anal
yzed
the
activ
ity o
f JN
K
inhi
bito
rs o
n in
fluen
za A
vir
us (I
AV) a
mpl
ifica
tion.
Hum
an lu
ng e
pith
elia
l cel
ls w
ere
infe
cted
with
eith
er th
e hi
ghly
pa
thog
enic
avi
an v
irus
str
ain
A/F
PV/B
ratis
lava
/79
(H7N
7) o
r th
e pa
ndem
ic s
win
e-or
igin
influ
enza
vir
us A
/Ham
-bu
rg/4
/09
(H1N
1v).
The
app
licat
ion
of th
e JN
K in
hibi
tors
SP6
0012
5 an
d A
S601
245
redu
ced
IAV
am
plifi
catio
n by
sup
-pr
essi
ng v
iral
pro
tein
and
RN
A s
ynth
esis
. Alth
ough
AS6
0124
5 ap
pear
ed to
gen
eral
ly b
lock
the
tran
scri
ptio
n of
new
ly
intr
oduc
ed g
enes
, SP6
0012
5 sp
ecifi
cally
affe
cted
vir
al R
NA
syn
thes
is. O
vere
xpre
ssio
n of
a d
omin
ant n
egat
ive
mut
ant
of S
EK/M
KK
4 an
d si
RNA
-med
iate
d su
ppre
ssio
n of
JNK
2 ex
pres
sion
con
firm
ed th
at s
peci
fic m
anip
ulat
ion
of th
e JN
K
path
way
att
enua
tes
viru
s pr
opag
atio
n. A
n IA
V m
inig
enom
e re
plic
atio
n as
say
reve
aled
that
SP6
0012
5 di
d no
t dir
ectly
af
fect
the
activ
ity o
f the
vir
al R
NA
pol
ymer
ase
com
plex
but
see
ms
to s
uppr
ess
an a
nti-
influ
enza
non
stru
ctur
al p
rote
in
1-m
edia
ted
viru
s su
ppor
tive
func
tion.
Fin
ally
, whe
n H
7N7-
or H
1N1v
-inf
ecte
d m
ice
wer
e tr
eate
d w
ith S
P600
125,
the
vira
l loa
d is
redu
ced
in lu
ngs
of tr
eate
d co
mpa
red
with
unt
reat
ed m
ice.
Our
dat
a su
gges
t tha
t thi
s cl
ass
of A
TP
com
peti-
tive
inhi
bito
rs o
nce
optim
ized
for
antiv
iral
act
ion
pote
ntia
lly re
pres
ent n
ovel
dru
gs fo
r an
tivir
al in
terv
entio
n.
Nac
ken
et a
l. 20
12
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
167
Sign
allin
g ca
scad
e bl
ocke
rsRa
f/M
EK/E
RK
sign
allin
g pa
thw
ayO
selta
miv
ir re
sist
ant
stra
ins
Mou
se m
odel
The
em
erge
nce
of th
e 20
09 H
1N1
pand
emic
sw
ine
influ
enza
A v
irus
is a
goo
d ex
ampl
e of
how
this
vir
al in
fect
ion
can
impa
ct h
ealth
sys
tem
s ar
ound
the
wor
ld in
a v
ery
shor
t tim
e. T
he c
ontin
uous
zoo
notic
cir
cula
tion
and
reas
sort
men
t po
tent
ial o
f inf
luen
za A
vir
uses
(IAV
) in
natu
re re
pres
ents
an
enor
mou
s pu
blic
hea
lth th
reat
to h
uman
s. B
esid
e va
ccin
a-tio
n an
tivir
als
are
need
ed to
eff
icie
ntly
con
trol
spr
eadi
ng o
f the
dis
ease
. In
the
pres
ent w
ork
we
inve
stig
ated
whe
ther
the
MEK
inhi
bito
r U
0126
, tar
getin
g th
e in
trac
ellu
lar
Raf/
MEK
/ERK
sig
nalin
g pa
thw
ay, i
s ab
le to
sup
pres
s pr
opag
atio
n of
th
e 20
09 p
ande
mic
IV H
1N1v
(v =
var
iant
) as
wel
l as
high
ly p
atho
geni
c av
ian
influ
enza
vir
uses
(HPA
IV) i
n ce
ll cu
lture
an
d al
so in
viv
o in
the
mou
se lu
ng. U
0126
sho
wed
ant
ivir
al a
ctiv
ity in
cel
l cul
ture
aga
inst
all
test
ed IA
V s
trai
ns in
clud
-in
g Ra
f/M
EK/E
RK s
igna
ling
path
way
. Fur
ther
mor
e, w
e w
ere
able
to d
emon
stra
te th
at tr
eatm
ent o
f mic
e w
ith U
0126
via
th
e ae
roso
l rou
te le
d to
inhi
bitio
n of
MEK
act
ivat
ion
in th
e lu
ng re
duct
ion
of p
roge
ny IA
V ti
ters
com
pare
d to
unt
reat
ed
cont
rols
pro
tect
ion
of IA
V in
fect
ed m
ice
agai
nst a
100
× le
thal
vir
al c
halle
nge.
Mor
eove
r, no
adv
erse
effe
cts
of U
0126
w
ere
foun
d in
cel
l cul
ture
or
in th
e m
ouse
. Thu
s, w
e co
nclu
de th
at U
0126
, by
inhi
bitin
g th
e ce
llula
r ta
rget
MEK
, has
an
antiv
iral
pot
entia
l not
onl
y in
vitr
o in
cel
l cul
ture
, but
als
o in
viv
o in
the
mou
se m
odel
.
Dro
ebne
r et a
l. 20
11
Cat
helic
idin
pep
tide
sD
ecre
ase
of p
ro-
infla
mat
ory
cyto
kine
sPr
otec
tion
agai
nst
influ
enza
The
ext
ensi
ve w
orld
-wid
e m
orbi
dity
and
mor
talit
y ca
used
by
influ
enza
A v
irus
es h
ighl
ight
s th
e ne
ed fo
r ne
w in
sigh
ts
into
the
host
imm
une
resp
onse
and
nov
el tr
eatm
ent a
ppro
ache
s. C
atio
nic
Hos
t Def
ense
Pep
tides
(CH
DP,
als
o kn
own
as a
ntim
icro
bial
pep
tides
), w
hich
incl
ude
cath
elic
idin
s an
d de
fens
ins,
are
key
com
pone
nts
of th
e in
nate
imm
une
syst
em
that
are
upr
egul
ated
dur
ing
infe
ctio
n an
d in
flam
mat
ion.
Cat
helic
idin
s ha
ve im
mun
omod
ulat
ory
and
anti-
vira
l effe
cts,
bu
t the
ir im
pact
on
influ
enza
vir
us in
fect
ion
has
not b
een
prev
ious
ly a
sses
sed.
We
ther
efor
e ev
alua
ted
the
effe
ct o
f ca
thel
icid
in p
eptid
es o
n di
seas
e ca
used
by
influ
enza
A v
irus
in m
ice.
The
hum
an c
athe
licid
in, L
L-37
, and
the
mur
ine
cath
elic
idin
, mC
RA
MP,
dem
onst
rate
d si
gnifi
cant
ant
i-vi
ral a
ctiv
ity in
viv
o, re
duci
ng d
isea
se s
ever
ity a
nd v
iral
repl
ica-
tion
in in
fect
ed m
ice
to a
sim
ilar
exte
nt a
s th
e w
ell-
char
acte
rize
d in
fluen
za v
irus
-spe
cific
ant
ivir
al d
rug
zana
miv
ir. In
vi
tro
and
in v
ivo
expe
rim
ents
sug
gest
ed th
at th
e pe
ptid
es m
ay a
ct d
irec
tly o
n th
e in
fluen
za v
irio
n ra
ther
than
via
rece
p-to
r-ba
sed
mec
hani
sms.
Influ
enza
vir
us-i
nfec
ted
mic
e tr
eate
d w
ith L
L-37
had
low
er c
once
ntra
tions
of p
ro-i
nfla
mm
ator
y cy
toki
nes
in th
e lu
ng th
an d
id in
fect
ed a
nim
als
that
had
not
bee
n tr
eate
d w
ith c
athe
licid
in p
eptid
es. T
hese
dat
a su
gges
t th
at tr
eatm
ent o
f inf
luen
za-i
nfec
ted
indi
vidu
als
with
cat
helic
idin
-der
ived
ther
apeu
tics,
or
mod
ulat
ion
of e
ndog
enou
s ca
thel
icid
in p
rodu
ctio
n m
ay p
rovi
de s
igni
fican
t pro
tect
ion
agai
nst d
isea
se.
Barl
ow e
t al.
2011
Vacu
olar
pro
ton-
AT
Pase
inhi
bito
rC
onca
nam
ycin
A
The
sel
ectiv
e in
hibi
tor
of th
e va
cuol
ar p
roto
n-A
TPa
se, c
onca
nam
ycin
A, p
ower
fully
blo
cks
influ
enza
vir
us e
ntry
into
ce
lls, i
f pre
sent
dur
ing
the
initi
al ti
mes
of v
irus
infe
ctio
n. A
ttac
hmen
t of v
irus
par
ticle
s to
cel
ls is
not
pre
vent
ed b
y co
n-ca
nam
ycin
A, r
athe
r th
e ex
it of
influ
enza
vir
us fr
om e
ndos
omes
is th
e st
ep b
lock
ed b
y th
is m
acro
lide
antib
iotic
. Inh
ibi-
tion
of in
fluen
za v
irus
ent
ry in
to c
ells
by
conc
anam
ycin
A o
r by
nig
eric
in ta
kes
plac
e un
der
acid
ic c
ondi
tions
. Mor
eove
r, if
the
pH g
radi
ent i
s ab
olis
hed
by p
re-i
ncub
atio
n of
cel
ls in
aci
dic
pH, i
nflu
enza
vir
us e
ntry
doe
s no
t occ
ur e
ven
in th
e ab
senc
e of
any
inhi
bito
rs. T
hese
resu
lts in
dica
te th
at a
cidi
c co
nditi
ons
per
se a
re n
ot s
uffic
ient
to p
rom
ote
viru
s en
try
into
cel
ls; r
athe
r th
is s
tep
of v
irus
infe
ctio
n re
quir
es a
pH
gra
dien
t.
Gui
nea
and
Car
-ra
sco
1994
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
168
Inhi
bito
r of
vac
uola
r-ty
pe p
roto
n pu
mp
Bafil
omyc
in A
lEn
doso
me
acid
ifica
tion
We
stud
ied
the
effe
ct o
f baf
ilom
ycin
Al (
Baf-
A1)
, a n
ovel
and
hig
hly
spec
ific
inhi
bito
r fo
r va
cuol
ar-t
ype
prot
on (V
-H+)
pu
mp,
on
the
grow
th o
f inf
luen
za A
and
B v
irus
es in
Mad
in-D
arby
can
ine
kidn
ey c
ells
. Vita
l flu
ores
cenc
e m
icro
scop
ic
stud
y sh
owed
that
Baf
-A1
indu
ced
the
com
plet
e di
sapp
eara
nce
of a
cidi
fied
com
part
men
ts s
uch
as e
ndos
omes
and
ly
soso
mes
bot
h in
infe
cted
and
uni
nfec
ted
cells
by
the
trea
tmen
t with
0.1
mu
M in
hibi
tor
for
1 h
at 3
7 °C
. In
addi
tion,
vi
rus
grow
th w
as in
hibi
ted
whe
n Ba
f-A
1 w
as p
rese
nt fr
om 1
h b
efor
e in
fect
ion
to th
e en
d of
incu
batio
n, o
r ad
ded
with
in
as e
arly
as
5–10
min
aft
er in
fect
ion.
Con
vers
ely,
the
viru
s gr
owth
was
reco
vere
d in
cor
rela
tion
with
the
reap
pear
ance
of
aci
difie
d co
mpa
rtm
ents
aft
er re
mov
al o
f Baf
-A1.
The
se d
ata
sugg
est t
hat B
af-A
1-se
nsiti
ve V
-H+
pum
ps a
re s
olel
y re
spon
sibl
e fo
r th
e ac
idifi
catio
n of
end
osom
es a
nd ly
soso
mes
, and
thus
Baf
-A1
inhi
bits
the
grow
th o
f inf
luen
za A
and
B
viru
ses
by a
ffect
ing
the
acid
ified
com
part
men
ts in
whi
ch lo
w p
H is
ess
entia
l for
the
unco
atin
g pr
oces
s of
influ
enza
vir
us
grow
th a
t an
earl
y st
age
of in
fect
ion.
Och
iai e
t al.
1995
Prot
easo
me
inhi
bito
rRe
cent
ly it
has
bee
n sh
own
that
the
proi
nfla
mm
ator
y N
F-ka
ppa
B pa
thw
ay p
rom
otes
eff
icie
nt in
fluen
za v
irus
pro
paga
-tio
n. B
ased
on
thes
e fin
ding
s, it
was
sug
gest
ed th
at N
F-ka
ppa
B bl
ocka
de m
ay b
e a
prom
isin
g ap
proa
ch fo
r an
tivir
al
inte
rven
tion.
The
cla
ssic
al v
irus
-ind
uced
act
ivat
ion
of th
e N
F-ka
ppa
B pa
thw
ay re
quir
es p
rote
asom
al d
egra
datio
n of
the
inhi
bito
r of
NF-
kapp
a B,
I ka
ppa
B. T
here
fore
, we
hypo
thes
ized
that
inhi
bitio
n of
pro
teas
omal
I ka
ppa
B de
grad
atio
n sh
ould
impa
ir in
fluen
za A
vir
us (I
AV) r
eplic
atio
n. W
e ch
ose
the
spec
ific
prot
easo
me
inhi
bito
r PS
-341
, whi
ch is
a c
lini-
cally
app
rove
d an
tican
cer
drug
als
o kn
own
as B
orte
zom
ib o
r Ve
lcad
e. A
s ex
pect
ed, P
S-34
1 tr
eatm
ent o
f inf
ecte
d A
549
cells
in a
con
cent
ratio
n ra
nge
that
was
not
toxi
c re
sulte
d in
a s
igni
fican
t red
uctio
n of
pro
geny
vir
us ti
ters
. How
ever
, we
coul
d no
t obs
erve
the
prop
osed
sup
pres
sion
of N
F-ka
ppa
B-si
gnal
ing
in v
itro.
Rat
her,
PS-3
41 tr
eatm
ent r
esul
ted
in a
n in
duct
ion
of I
kapp
a B
degr
adat
ion
and
activ
atio
n of
NF-
kapp
a B
as w
ell a
s th
e JN
K/A
P-1
path
way
. Thi
s co
inci
des
with
en
hanc
ed e
xpre
ssio
n of
ant
ivir
al g
enes
, suc
h as
inte
rleu
kin-
6 an
d, m
ost i
mpo
rtan
tly, M
xA, w
hich
is a
str
ong
inte
rfer
on
(IFN
)-in
duce
d su
ppre
ssor
of i
nflu
enza
vir
us re
plic
atio
n. T
his
sugg
ests
that
PS-
341
may
act
as
an a
ntiv
iral
age
nt v
ia
indu
ctio
n of
the
type
I IF
N re
spon
se. A
ccor
ding
ly, P
S-34
1 di
d no
t affe
ct v
irus
tite
rs in
Ver
o ce
lls, w
hich
lack
type
I IF
N
gene
s, b
ut s
tron
gly
inhi
bite
d re
plic
atio
n of
ves
icul
ar s
tom
atiti
s vi
rus
(VSV
), a
high
ly IF
N-s
ensi
tive
path
ogen
. Thu
s,
we
conc
lude
that
PS-
341
bloc
ks IA
V a
nd V
SV re
plic
atio
n by
indu
cing
an
antiv
iral
sta
te m
edia
ted
by th
e N
F-ka
ppa
B-de
pend
ent e
xpre
ssio
n of
ant
ivir
us-a
ctin
g ge
ne p
rodu
cts.
Dud
ek e
t al.
2010
Prot
easo
me
inhi
bito
rRe
duct
ion
of c
ytok
ine
rele
ase
In th
e pr
esen
t stu
dy w
e sh
ow th
at th
e pr
otea
som
e in
hibi
tor
VL-
01 le
ads
to re
duct
ion
of in
fluen
za v
irus
repl
icat
ion
in
hum
an lu
ng a
deno
carc
inom
a ep
ithel
ial c
ells
(A54
9) a
s de
mon
stra
ted
with
thre
e di
ffere
nt in
fluen
za v
irus
str
ains
, A
/Pue
rto
Rico
/8/3
4 (H
1N1)
(EC
50 v
alue
of 1
.7 µ
M),
A/R
egen
sbur
g/D
6/09
(H1N
1v) (
EC50
val
ue o
f 2.4
µM
) and
A/M
al-
lard
/Bav
aria
/1/2
006
(H5N
1) (E
C50
val
ue o
f 0.8
µM
). In
in v
ivo
expe
rim
ents
we
coul
d de
mon
stra
te th
at V
L-01
-aer
osol
-tr
eatm
ent o
f BA
LB/c
mic
e w
ith 1
4.1
mg/
kg re
sults
in n
o to
xic
side
effe
cts,
redu
ced
prog
eny
viru
s tit
ers
in th
e lu
ng
(1.1
± 0
.3 lo
g 10 p
fu) a
nd e
nhan
ced
surv
ival
of m
ice
afte
r in
fect
ion
with
a 5
-fol
d M
LD50
of t
he h
uman
influ
enza
A v
irus
st
rain
A/P
uert
o Ri
co/8
/34
(H1N
1) u
p to
50%
. Fur
ther
mor
e, tr
eatm
ent o
f mic
e w
ith V
L-01
redu
ced
the
cyto
kine
rele
ase
of IL
-alp
ha/b
eta,
IL-6
, MIP
-1 b
eta,
RA
NT
ES a
nd T
NF-
alph
a in
duce
d by
LPS
or
high
ly p
atho
gen
avia
n H
5N1
influ
enza
A
vir
us. T
he p
rese
nt d
ata
dem
onst
rate
s an
ant
ivir
al e
ffect
of V
L-01
in v
itro
and
in v
ivo
and
the
abili
ty to
redu
ce in
flu-
enza
vir
us in
duce
d cy
toki
nes
and
chem
okin
es.
Haa
sbac
h et
al.
2011
nF-
kapp
a B
inhi
bito
rPy
rone
poly
ene
C-g
luco
side
A n
ew p
yron
epol
yene
C-g
luco
side
, nam
ed is
o-D
8646
-2-6
(1) t
oget
her
with
the
know
n re
late
d co
mpo
und
D86
46-2
-6 (2
), w
as is
olat
ed fr
om th
e sp
onge
-ass
ocia
ted
fung
us E
pico
ccum
sp.
JJY4
0. T
hey
show
ed N
F-ka
ppa
B in
hibi
tory
and
ant
i-in
fluen
za A
vir
al (H
1N1)
act
iviti
es.
Peng
et a
l. 20
12
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
169
Prot
ease
inhi
bito
rsA
prot
inin
Leup
eptin
Cam
osta
t
Effo
rts
to d
evel
op n
ew a
ntiv
iral
che
mot
hera
peut
ic a
ppro
ache
s ar
e fo
cusi
ng o
n co
mpo
unds
that
targ
et e
ither
influ
enza
vi
rus
repl
icat
ion
itsel
f or
host
fact
or(s
) tha
t are
cri
tical
to in
fluen
za re
plic
atio
n. H
ost p
rote
ase
med
iate
d in
fluen
za
hem
aggl
utin
in (H
A) c
leav
age
is c
ritic
al fo
r ac
tivat
ion
of v
irus
infe
ctiv
ity a
nd a
s su
ch is
a c
hem
othe
rape
utic
targ
et. I
nflu
-en
za p
atho
gene
sis
invo
lves
a “v
icio
us c
ycle
” in
whi
ch h
ost p
rote
ases
act
ivat
e pr
ogen
y vi
rus
whi
ch in
turn
am
plifi
es re
p-lic
atio
n an
d st
imul
ates
furt
her
prot
ease
act
iviti
es w
hich
may
be
detr
imen
tal t
o th
e in
fect
ed h
ost.
Apr
otin
in, a
58
amin
o ac
id p
olyp
eptid
e pu
rifie
d fr
om b
ovin
e lu
ng th
at is
one
of a
fam
ily o
f hos
t-ta
rget
ed a
ntiv
iral
s th
at in
hibi
t ser
ine
prot
ease
s re
spon
sibl
e fo
r in
fluen
za v
irus
act
ivat
ion.
Thi
s dr
ug a
nd s
imila
r ag
ents
, suc
h as
leup
eptin
and
cam
osta
t, su
ppre
ss v
irus
H
A c
leav
age
and
limit
repr
oduc
tion
of h
uman
and
avi
an in
fluen
za v
irus
es w
ith a
sin
gle
argi
nine
in th
e H
A c
leav
age
site
. Si
te-d
irec
ted
stru
ctur
al m
odifi
catio
ns o
f apr
otin
in a
re p
ossi
ble
to in
crea
se it
s in
trac
ellu
lar
targ
etin
g of
cle
avag
e of
hig
hly
viru
lent
H5
and
H7
hem
aggl
utin
ins
poss
essi
ng m
ulti-
argi
nine
/lys
ine
clea
vage
site
. An
addi
tiona
l mec
hani
sm o
f act
ion
for
seri
ne p
rote
ase
inhi
bito
rs is
to ta
rget
a n
umbe
r of
hos
t med
iato
rs o
f inf
lam
mat
ion
and
dow
n re
gula
te th
eir
leve
ls in
vi
rus-
infe
cted
hos
ts. A
prot
inin
is a
gen
eric
dru
g ap
prov
ed fo
r in
trav
enou
s us
e in
hum
ans
to tr
eat p
ancr
eatit
is a
nd li
mit
post
-ope
rativ
e bl
eedi
ng. A
s an
ant
iinflu
enza
l com
poun
d, a
prot
inin
mig
ht b
e de
liver
ed b
y tw
o ro
utes
: (i)
a sm
all-
part
icle
ae
roso
l has
bee
n ap
prov
ed in
Rus
sia
for
loca
l res
pira
tory
app
licat
ion
in m
ild-t
o-m
oder
ate
influ
enza
and
(ii)
a pr
opos
ed
intr
aven
ous
adm
inis
trat
ion
for
seve
re in
fluen
za to
pro
vide
bot
h an
ant
ivir
al e
ffect
and
a d
ecre
ase
in s
yste
mic
pat
holo
gy
and
infla
mm
atio
n.
Zhi
rnov
et a
l. 20
11
Prot
ease
inhi
bito
rsC
amos
tat m
esila
teN
afam
osta
t mes
ilate
Six
nucl
eosi
de a
nalo
gues
, tw
o su
lfate
d po
lysa
ccha
ride
s, a
nd fo
ur p
rote
ase
inhi
bito
rs w
ere
eval
uate
d in
vitr
o as
inhi
bito
rs
of in
fluen
za v
irus
repl
icat
ion.
Fou
r gu
anos
ine
anal
ogue
s (m
izor
ibin
e, r
ibav
irin
, pyr
azof
urin
, and
5-e
thyn
yl-1
-bet
a-D
-ri
bofu
rano
sylim
idaz
ole-
4-ca
rbox
amid
e), t
he s
ulfa
ted
poly
sacc
hari
de d
extr
an s
ulfa
te (m
olec
ular
wei
ght 5
00 0
00),
and
two
prot
ease
inhi
bito
rs (c
amos
tat m
esila
te a
nd n
afam
osta
t mes
ilate
) wer
e in
hibi
tory
to th
e re
plic
atio
n of
str
ains
of i
nflu
-en
za v
irus
type
s A
and
B a
t con
cent
ratio
ns d
own
to 0
.3 µ
g/m
l. O
f the
se s
even
com
poun
ds, r
ibav
irin
, cam
osta
t mes
ilate
, an
d na
fam
osta
t mes
ilate
wer
e ef
ficac
ious
in b
oth
redu
cing
the
viru
s tit
er a
nd in
crea
sing
the
surv
ival
rat
e of
influ
enza
vi
rus-
infe
cted
chi
ck e
mbr
yos.
For
cam
osta
t mes
ilate
, the
ED
50 (r
equi
red
to im
prov
e th
e su
rviv
al r
ate
of in
fluen
za
viru
s-in
fect
ed c
hick
em
bryo
s by
50%
) was
0.8
0 µg
/g, a
nd it
s se
lect
ivity
inde
x, b
ased
on
the
ratio
of t
he 5
0% to
xic
dose
(r
equi
red
to re
duce
the
viab
ility
of c
hick
em
bryo
s by
50%
) to
ED50
, was
280
. Cam
osta
t mes
ilate
des
erve
s fu
rthe
r ex
plo-
ratio
n fo
r its
pot
entia
l in
the
trea
tmen
t of i
nflu
enza
vir
us in
fect
ion.
Hos
oya
et a
l. 19
93
Prot
ease
inhi
bito
rsN
afam
osta
t mes
ilate
Cam
osta
t mes
ilate
Gab
exat
e m
esila
teA
prot
inin
Chi
cken
em
bryo
s
We
stud
ied
the
effe
cts
of e
ight
pro
teas
e in
hibi
tors
on
the
mul
ticyc
le re
plic
atio
ns o
f var
ious
ort
hom
yxov
irus
es a
nd
para
myx
ovir
uses
. Am
ong
the
com
poun
ds, n
afam
osta
t mes
ilate
, cam
osta
t mes
ilate
, gab
exat
e m
esila
te, a
nd a
prot
inin
, w
hich
are
wid
ely
used
in th
e tr
eatm
ent o
f pan
crea
titis
, inh
ibite
d in
fluen
za v
irus
A a
nd B
repl
icat
ion
at c
once
ntra
tions
th
at w
ere
sign
ifica
ntly
low
er th
an th
eir
cyto
toxi
c th
resh
olds
in v
itro.
Non
e of
the
prot
ease
inhi
bito
rs h
ad a
ctiv
ity a
gain
st
resp
irat
ory
sync
ytia
l vir
us, m
easl
es v
irus
, or
para
influ
enza
vir
us ty
pe 3
at t
he h
ighe
st c
once
ntra
tions
test
ed. C
amos
tat
mes
ilate
was
foun
d to
be
the
mos
t sel
ectiv
e in
hibi
tor.
Its
50%
effe
ctiv
e co
ncen
trat
ion
for
influ
enza
vir
us A
repl
icat
ion
was
2.2
µg/
ml,
and
the
sele
ctiv
ity in
dex,
whi
ch w
as b
ased
on
the
ratio
of t
he 5
0% in
hibi
tory
con
cent
ratio
n fo
r ho
st
cell
prol
ifera
tion
to th
e 50
% e
ffect
ive
conc
entr
atio
n fo
r in
fluen
za v
irus
A re
plic
atio
n, w
as 6
80. W
hen
the
in o
vo a
nti-
vira
l act
ivity
of t
he c
ompo
unds
was
test
ed b
y us
ing
chic
ken
embr
yos,
cam
osta
t mes
ilate
at a
dos
e of
10
µg/g
mar
kedl
y re
duce
d th
e he
mag
glut
inin
tite
rs o
f inf
luen
za v
irus
es A
and
B.
Hos
oya
et a
l. 19
92
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
170
Hae
mag
glut
inin
fo
ldin
gG
luta
thio
ne d
eriv
ativ
e
Aim
: The
aim
of t
his
stud
y w
as to
det
erm
ine
whe
ther
GSH
-C4,
a h
ydro
phob
ic g
luta
thio
ne d
eriv
ativ
e, a
ffect
s in
vitr
o an
d in
viv
o in
fluen
za v
irus
infe
ctio
n by
inte
rfer
ing
with
redo
x-se
nsiti
ve in
trac
ellu
lar
path
way
s in
volv
ed in
the
mat
urat
ion
of v
iral
hem
aggl
utin
in (H
A).
Resu
lts: G
SH-C
4 st
rong
ly in
hibi
ted
influ
enza
A v
irus
repl
icat
ion
in c
ultu
red
cells
and
in
leth
ally
infe
cted
mic
e, w
here
it a
lso
redu
ced
lung
dam
age
and
mor
talit
y. In
cel
l-cu
lture
stu
dies
, GSH
-C4
arre
sted
vir
al
HA
fold
ing;
the
disu
lfide
-ric
h gl
ycop
rote
in re
mai
ned
in th
e en
dopl
asm
ic re
ticul
um a
s a
redu
ced
mon
omer
inst
ead
of
unde
rgoi
ng o
ligom
eriz
atio
n an
d ce
ll pl
asm
a-m
embr
ane
inse
rtio
n. H
A m
atur
atio
n de
pend
s on
the
host
-cel
l oxi
dore
duc-
tase
, pro
tein
dis
ulfid
e is
omer
ase
(PD
I), w
hose
act
ivity
in in
fect
ed c
ells
is p
roba
bly
faci
litat
ed b
y vi
rus-
indu
ced
glu-
tath
ione
dep
letio
n. B
y co
rrec
ting
this
def
icit
, GSH
-C4
incr
ease
d le
vels
of r
educ
ed P
DI a
nd in
hibi
ted
esse
ntia
l dis
ulfid
e bo
nd fo
rmat
ion
in H
A. H
ost-
cell
glyc
opro
tein
exp
ress
ion
in u
ninf
ecte
d ce
lls w
as u
naffe
cted
by
glut
athi
one,
whi
ch th
us
appe
ars
to a
ct e
xclu
sive
ly o
n gl
utat
hion
e-de
plet
ed c
ells
. Inn
ovat
ion:
All
curr
ently
app
rove
d an
ti-in
fluen
za d
rugs
targ
et
esse
ntia
l vir
al s
truc
ture
s, a
nd th
eir
effic
acy
is li
mite
d by
toxi
city
and
by
the
alm
ost i
nevi
tabl
e se
lect
ion
of d
rug-
resi
stan
t vi
ral m
utan
ts. G
SH-C
4 in
hibi
ts in
fluen
za v
irus
repl
icat
ion
by m
odul
atin
g re
dox-
sens
itive
pat
hway
s in
infe
cted
cel
ls,
with
out p
rodu
cing
toxi
city
in u
ninf
ecte
d ce
lls o
r an
imal
s. N
ovel
ant
i-in
fluen
za d
rugs
that
targ
et in
trac
ellu
lar
path
way
s es
sent
ial f
or v
iral
repl
icat
ion
(“ce
ll-ba
sed
appr
oach
”) o
ffer
two
impo
rtan
t pot
entia
l adv
anta
ges:
they
are
mor
e di
ffi-
cult
for
the
viru
s to
ada
pt to
and
thei
r ef
ficac
y sh
ould
not
be
depe
nden
t on
viru
s ty
pe, s
trai
n, o
r an
tigen
ic p
rope
rtie
s.
Con
clus
ion:
Red
ox-s
ensi
tive
host
-cel
l pat
hway
s ex
ploi
ted
for
vira
l rep
licat
ion
are
prom
isin
g ta
rget
s fo
r ef
fect
ive
anti-
influ
enza
str
ateg
ies.
Sgar
bant
i et a
l. 20
11
Prot
easo
me
inhi
bito
rsW
e ha
ve d
emon
stra
ted
that
influ
enza
A v
irus
(IAV
) RN
A s
ynth
esis
dep
ends
on
the
ubiq
uitin
-pro
teas
ome
syst
em. I
AV
repl
icat
ion
was
redu
ced
both
by
prot
easo
me
inhi
bito
rs a
nd in
E36
ts20
cel
ls, w
hich
con
tain
the
ther
mol
abile
ubi
quiti
n-ac
tivat
ing
enzy
me
E1. W
hile
vir
us e
ntry
was
not
affe
cted
in E
36ts
20 c
ells
, the
pro
teas
ome
inhi
bito
r M
G13
2 re
tain
ed
vira
l par
ticle
s in
the
cyto
plas
m. A
dditi
on-r
emov
al e
xper
imen
ts o
f MG
132
in c
ombi
natio
n w
ith b
afilo
myc
in A
1, a
wel
l-es
tabl
ishe
d in
hibi
tor
of IA
V e
ntry
and
fusi
on, s
how
ed th
at M
G13
2 af
fect
ed IA
V in
fect
ion
at a
pos
tfus
ion
step
. Thi
s w
as
conf
irm
ed b
y th
e la
ck o
f inh
ibiti
on o
f IAV
ent
ry b
y pr
otea
som
e in
hibi
tors
in a
vir
us-l
ike
part
icle
fusi
on a
ssay
.
Wid
jaja
et a
l. 20
10
Hos
t fac
tor
targ
etin
gG
enom
e-w
ide
RNA
in
terf
eren
ce sc
reen
ing
295
cellu
lar c
ofac
tors
Influ
enza
A v
irus
is a
n RN
A v
irus
that
enc
odes
up
to 1
1 pr
otei
ns a
nd th
is s
mal
l cod
ing
capa
city
dem
ands
that
the
viru
s us
e th
e ho
st c
ellu
lar
mac
hine
ry fo
r m
any
aspe
cts
of it
s lif
e cy
cle.
Kno
wle
dge
of th
ese
host
cel
l req
uire
men
ts n
ot o
nly
info
rms
us o
f the
mol
ecul
ar p
athw
ays
expl
oite
d by
the
viru
s bu
t als
o pr
ovid
es fu
rthe
r ta
rget
s th
at c
ould
be
purs
ued
for
antiv
iral
dru
g de
velo
pmen
t. H
ere
we
use
an in
tegr
ativ
e sy
stem
s ap
proa
ch, b
ased
on
geno
me-
wid
e RN
A in
terf
er-
ence
scr
eeni
ng, t
o id
entif
y 29
5 ce
llula
r co
fact
ors
requ
ired
for
earl
y-st
age
influ
enza
vir
us re
plic
atio
n. W
ithin
this
gro
up,
thos
e in
volv
ed in
kin
ase-
regu
late
d si
gnal
ling,
ubi
quiti
natio
n an
d ph
osph
atas
e ac
tivity
are
the
mos
t hig
hly
enri
ched
, an
d 18
1 fa
ctor
s as
sem
ble
into
a h
ighl
y si
gnifi
cant
hos
t-pa
thog
en in
tera
ctio
n ne
twor
k. M
oreo
ver,
219
of th
e 29
5 fa
ctor
s w
ere
conf
irm
ed to
be
requ
ired
for
effic
ient
wild
-typ
e in
fluen
za v
irus
gro
wth
, and
furt
her
anal
ysis
of a
sub
set o
f gen
es
show
ed 2
3 fa
ctor
s ne
cess
ary
for
vira
l ent
ry, i
nclu
ding
mem
bers
of t
he v
acuo
lar
AT
Pase
(vA
TPa
se) a
nd C
OPI
-pro
tein
fa
mili
es, f
ibro
blas
t gro
wth
fact
or re
cept
or (F
GFR
) pro
tein
s, a
nd g
lyco
gen
synt
hase
kin
ase
3 (G
SK3)
-bet
a. F
urth
erm
ore,
10
pro
tein
s w
ere
conf
irm
ed to
be
invo
lved
in p
ost-
entr
y st
eps
of in
fluen
za v
irus
repl
icat
ion.
The
se in
clud
e nu
clea
r im
port
com
pone
nts,
pro
teas
es, a
nd th
e ca
lciu
m/c
alm
odul
in-d
epen
dent
pro
tein
kin
ase
(CaM
kin
ase)
II b
eta
(CA
MK
2B).
Not
ably
, gro
wth
of s
win
e-or
igin
H1N
1 in
fluen
za v
irus
is a
lso
depe
nden
t on
the
iden
tifie
d ho
st fa
ctor
s, a
nd w
e sh
ow th
at
smal
l mol
ecul
e in
hibi
tors
of s
ever
al fa
ctor
s, in
clud
ing
vAT
Pase
and
CA
MK
2B, a
ntag
oniz
e in
fluen
za v
irus
repl
icat
ion.
Kon
ig e
t al.
2010
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
171
Tabl
e 4.
6. o
ther
ant
i-in
flue
nza
agen
ts
nuc
leop
rote
in
inhi
bito
rsN
ucle
ozin
Fluo
resc
ence
-que
nchi
ng
effec
t
Rece
nt s
tudi
es h
ave
show
n th
at N
P (n
ucle
opro
tein
), w
hich
pos
sess
es m
ultip
le fu
nctio
ns in
the
vira
l life
cyc
le, i
s a
new
po
tent
ial a
nti-
influ
enza
dru
g ta
rget
. NP
inhi
bito
rs re
liabl
y in
duce
con
form
atio
nal c
hang
es in
NPs
, and
thes
e ch
ange
s m
ay c
onfe
r in
hibi
tion
of th
e in
fluen
za v
irus
. The
six
con
serv
ed tr
ypto
phan
resi
dues
in N
P ca
n be
use
d as
an
intr
insi
c pr
obe
to m
onito
r th
e ch
ange
in fl
uore
scen
ce o
f the
tryp
toph
an re
sidu
es in
the
prot
ein
upon
bin
ding
to a
n N
P in
hibi
tor.
In th
e pr
esen
t stu
dy, w
e fo
und
that
the
fluor
esce
nce
of re
com
bina
nt N
P pr
otei
ns w
as q
uenc
hed
follo
win
g th
e bi
ndin
g of
ava
ilabl
e N
P in
hibi
tors
(suc
h as
nuc
leoz
in) i
n a
conc
entr
atio
n- a
nd ti
me-
depe
nden
t man
ner,
whi
ch s
ugge
sts
that
the
inhi
bito
r in
duce
d co
nfor
mat
iona
l cha
nges
in th
e N
Ps. T
he m
inim
al fl
uore
scen
ce-q
uenc
hing
effe
ct a
nd w
eak
bind
ing
cons
tant
of n
ucle
ozin
to th
e sw
ine-
orig
in in
fluen
za v
irus
H1N
1pdm
09 (S
OIV
) NP
reve
aled
that
the
SOIV
is re
sist
ant
to n
ucle
ozin
. We
have
use
d th
e flu
ores
cenc
e-qu
ench
ing
prop
erty
of t
rypt
opha
ns in
NPs
that
wer
e bo
und
to li
gand
s in
a
96-w
ell-
plat
e-ba
sed
drug
scr
een
to a
sses
s th
e ab
ility
of p
rom
isin
g sm
all m
olec
ules
to in
tera
ct w
ith N
Ps a
nd h
ave
iden
ti-fie
d on
e ne
w a
nti-
influ
enza
dru
g, C
SV0C
0010
18, w
ith a
hig
h SI
val
ue. T
his
conv
enie
nt m
etho
d fo
r dr
ug s
cree
ning
may
fa
cilit
ate
the
deve
lopm
ent o
f ant
ivir
al d
rugs
that
targ
et v
irus
es o
ther
than
the
influ
enza
vir
us, s
uch
as H
IV a
nd H
BV.
Hun
g et
al.
2012
nuc
leop
rote
in
inhi
bito
rsN
ucle
ozin
The
influ
enza
vir
us n
ucle
opro
tein
(NP)
is a
n em
ergi
ng ta
rget
for
anti-
influ
enza
dru
g de
velo
pmen
t. N
ucle
ozin
(1) a
nd
its c
lose
ly re
late
d de
riva
tives
had
bee
n id
entif
ied
as N
P in
hibi
tors
dis
play
ing
anti-
influ
enza
act
ivity
. Util
izin
g 1
as a
lead
m
olec
ule,
we
succ
essf
ully
des
igne
d an
d sy
nthe
size
d a
seri
es o
f 1H
-1,2
,3-t
riaz
ole-
4-ca
rbox
amid
e de
riva
tives
as
new
ant
i-in
fluen
za A
age
nts.
One
of t
he m
ost p
oten
t com
poun
ds, 3
b, in
hibi
ted
the
repl
icat
ion
of v
ario
us H
3N2
and
H1N
1 in
flu-
enza
A v
irus
str
ains
with
IC50
val
ues
rang
ing
from
0.5
to 4
.6 µ
M. C
ompo
und
3b a
lso
stro
ngly
inhi
bite
d th
e re
plic
atio
n of
H5N
1 (R
G14
), am
antid
ine-
resi
stan
t A/W
SN/3
3 (H
1N1)
, and
ose
ltam
ivir
-res
ista
nt A
/WSN
/193
3 (H
1N1,
274
Y) v
irus
st
rain
s w
ith IC
50 v
alue
s in
sub
-mu
M r
ange
s. F
urth
er c
ompu
tatio
nal s
tudi
es a
nd m
echa
nism
inve
stig
atio
n su
gges
ted
that
3b
mig
ht d
irec
tly ta
rget
influ
enza
vir
us A
nuc
leop
rote
in to
inhi
bit i
ts n
ucle
ar a
ccum
ulat
ion.
Che
ng e
t al.
2012
Inhi
bito
rs o
f non
-st
ruct
ural
pro
tein
The
inna
te im
mun
e sy
stem
gua
rds
agai
nst v
irus
infe
ctio
n th
roug
h a
vari
ety
of m
echa
nism
s in
clud
ing
mob
iliza
tion
of th
e ho
st in
terf
eron
sys
tem
, whi
ch a
ttac
ks v
iral
pro
duct
s m
ainl
y at
a p
ostt
rans
crip
tiona
l lev
el. T
he in
fluen
za v
irus
N
S1 p
rote
in is
a m
ultif
unct
iona
l fac
ilita
tor
of v
irus
repl
icat
ion,
one
of w
hose
act
ions
is to
ant
agon
ize
the
inte
rfer
on
resp
onse
. Sin
ce N
S1 is
requ
ired
for
effic
ient
vir
us re
plic
atio
n, it
was
reas
oned
that
che
mic
al in
hibi
tors
of t
his
prot
ein
coul
d be
use
d to
furt
her
unde
rsta
nd v
irus
-hos
t int
erac
tions
and
als
o se
rve
as p
oten
tial n
ew a
ntiv
iral
age
nts.
A y
east
-ba
sed
assa
y w
as d
evel
oped
to id
entif
y co
mpo
unds
that
phe
noty
pica
lly s
uppr
ess
NS1
func
tion.
Sev
eral
suc
h co
mpo
unds
ex
hibi
ted
sign
ifica
nt a
ctiv
ity s
peci
fical
ly a
gain
st in
fluen
za A
vir
us in
cel
l cul
ture
but
had
no
effe
ct o
n th
e re
plic
atio
n of
an
othe
r RN
A v
irus
, res
pira
tory
syn
cytia
l vir
us. I
nter
estin
gly,
cel
ls la
ckin
g an
inte
rfer
on re
spon
se w
ere
drug
resi
stan
t, su
gges
ting
that
the
com
poun
ds b
lock
inte
ract
ions
bet
wee
n N
S1 a
nd th
e in
terf
eron
sys
tem
. Acc
ordi
ngly
, the
com
poun
ds
reve
rsed
the
inhi
bitio
n of
bet
a in
terf
eron
mRN
A in
duct
ion
duri
ng in
fect
ion,
whi
ch is
kno
wn
to b
e ca
used
by
NS1
. In
addi
tion,
the
com
poun
ds b
lock
ed th
e ab
ility
of N
S1 p
rote
in to
inhi
bit d
oubl
e-st
rand
ed R
NA
-dep
ende
nt a
ctiv
atio
n of
a
tran
sfec
ted
beta
inte
rfer
on p
rom
oter
con
stru
ct. T
he e
ffect
s of
the
com
poun
ds w
ere
spec
ific
to N
S1, b
ecau
se th
ey
had
no e
ffect
on
the
abili
ty o
f the
sev
ere
acut
e re
spir
ator
y sy
ndro
me
coro
navi
rus
papa
inlik
e pr
otea
se p
rote
in to
blo
ck
beta
inte
rfer
on p
rom
oter
act
ivat
ion.
The
se d
ata
dem
onst
rate
that
the
func
tion
of N
S1 c
an b
e m
odul
ated
by
chem
ical
in
hibi
tors
and
that
suc
h in
hibi
tors
will
be
usef
ul a
s pr
obes
of b
iolo
gica
l fun
ctio
n an
d as
sta
rtin
g po
ints
for
clin
ical
dru
g de
velo
pmen
t.
Basu
et a
l. 20
09
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
172
Func
tion
of n
S1
prot
ein
Inac
tivat
ion
of N
F-ka
ppa
B Pr
even
tion
of in
terf
eron
in
duct
ion
The
alph
a/be
ta in
terf
eron
(IFN
-alp
ha/b
eta)
syst
em re
pres
ents
one
of t
he fi
rst l
ines
of d
efen
se a
gain
st v
irus
infe
ctio
ns. A
s a
resu
lt, m
ost v
irus
es e
ncod
e IF
N a
ntag
onis
tic fa
ctor
s whi
ch e
nhan
ce v
iral r
eplic
atio
n in
thei
r hos
ts. W
e ha
ve p
revi
ously
show
n th
at a
reco
mbi
nant
influ
enza
A v
irus
lack
ing
the
NS1
gen
e (d
elN
S1) o
nly
repl
icat
es e
ffici
ently
in IF
N-a
lpha
/bet
a -d
efici
ent
syst
ems.
Con
sist
ent w
ith th
is o
bser
vatio
n, w
e fo
und
that
infe
ctio
n of
tiss
ue c
ultu
re c
ells
with
del
NS1
vir
us, b
ut n
ot w
ith w
ild-
type
influ
enza
A v
irus
, ind
uced
hig
h le
vels
of m
RNA
synt
hesi
s fro
m IF
N-a
lpha
/bet
a ge
nes,
incl
udin
g IF
N-b
eta.
It is
kno
wn
that
tran
sact
ivat
ion
of th
e IF
N-b
eta
prom
oter
dep
ends
on
NF-
kapp
aB a
nd se
vera
l oth
er tr
ansc
ript
ion
fact
ors.
Inte
rest
ingl
y, ce
lls in
fect
ed w
ith d
elN
S1 v
irus
show
ed h
igh
leve
ls of
NF-
kapp
aB a
ctiv
atio
n co
mpa
red
with
thos
e in
fect
ed w
ith w
ild-t
ype
viru
s. Ex
pres
sion
of d
omin
ant-
nega
tive
inhi
bito
rs o
f the
NF-
kapp
aB p
athw
ay d
urin
g de
INS1
vir
us in
fect
ion
prev
ente
d th
e tr
ansa
ctiv
atio
n of
the
IFN
-bet
a pr
omot
er, d
emon
stra
ting
a fu
nctio
nal l
ink
betw
een
NF-
kapp
aB a
ctiv
atio
n an
d IF
N-a
lpha
/bet
a sy
nthe
sis i
n de
INS1
vir
us-in
fect
ed c
ells
. Mor
eove
r, ex
pres
sion
of t
he N
S1 p
rote
in p
reve
nted
vir
us- a
nd/o
r dou
ble-
stra
nded
RN
A (d
sRN
A)-
med
iate
d ac
tivat
ion
of th
e N
F-ka
ppaB
pat
hway
and
of I
FN-b
eta
synt
hesi
s. Th
is in
hibi
tory
pro
pert
y of
the
NS1
pro
tein
of i
nflue
nza
A v
irus
was
dep
ende
nt o
n its
abi
lity
to b
ind
dsRN
A, s
uppo
rtin
g a
mod
el in
whi
ch b
indi
ng o
f NS1
to
dsRN
A g
ener
ated
dur
ing
influ
enza
vir
us in
fect
ion
prev
ents
the
activ
atio
n of
the
IFN
syst
em. N
S1-m
edia
ted
inhi
bitio
n of
the
NF-
kapp
aB p
athw
ay m
ay th
us p
lay
a ke
y ro
le in
the
path
ogen
esis
of i
nflue
nza
A v
irus
.
Wan
g et
al.
2000
Rn
A in
terf
eren
ceSh
ort i
nter
feri
ng R
NA
sVi
ral m
RNA
deg
rada
tion
Hig
hly
path
ogen
ic a
vian
influ
enza
(HPA
I) H
5N1
viru
s is e
ndem
ic in
man
y re
gion
s aro
und
the
wor
ld a
nd re
mai
ns a
sign
ifi-
cant
pan
dem
ic th
reat
. To
date
H5N
1 ha
s cla
imed
alm
ost 3
00 h
uman
live
s wor
ldw
ide,
with
a m
orta
lity
rate
of 6
0% a
nd h
as
caus
ed th
e de
ath
or c
ullin
g of
hun
dred
s of m
illio
ns o
f pou
ltry
sinc
e its
initi
al o
utbr
eak
in 1
997.
We
have
des
igne
d m
ulti-
func
tiona
l RN
A in
terf
eren
ce (R
NA
i)-ba
sed
ther
apeu
tics t
arge
ting
H5N
1 th
at d
egra
de v
iral
mRN
A v
ia th
e RN
Ai p
athw
ay
whi
le a
t the
sam
e tim
e au
gmen
ting
the
host
ant
ivir
al re
spon
se b
y in
duci
ng h
ost t
ype
I int
erfe
ron
(IFN
) pro
duct
ion.
Mor
eo-
ver,
we
have
iden
tified
two
fact
ors c
ritic
al fo
r max
imis
ing
the
imm
unos
timul
ator
y pr
oper
ties o
f sho
rt in
terf
erin
g (s
i) RN
As
in c
hick
en c
ells
(i) m
ode
of sy
nthe
sis a
nd (i
i) nu
cleo
side
sequ
ence
to a
ugm
ent t
he re
spon
se to
vir
us. Th
e 5-
bp n
ucle
osid
e se
quen
ce 5
’-UG
UG
U-3
’ is a
key
det
erm
inan
t in
indu
cing
hig
h le
vels
of e
xpre
ssio
n of
IFN
-alp
ha, -
beta
, -la
mbd
a an
d in
terl
euki
n 1-
beta
in c
hick
en c
ells
. Pos
ition
ing
of th
is 5
’-UG
UG
U-3
’ mot
if at
the
5’- e
nd o
f the
sens
e st
rand
of s
iRN
As,
but
no
t the
3’-
end,
resu
lted
in a
rapi
d an
d en
hanc
ed in
duct
ion
of ty
pe I
IFN
. An
anti-
H5N
1 av
ian
influ
enza
siRN
A d
irec
ted
agai
nst t
he P
B1 g
ene
(PB1
-225
7) ta
gged
with
5’-U
GU
GU
-3’ i
nduc
ed ty
pe I
IFN
ear
lier a
nd to
a g
reat
er e
xten
t com
pare
d to
a
non-
tagg
ed P
B1-2
257.
Tes
ted
agai
nst H
5N1
in v
itro,
the
tagg
ed P
B1-2
257
was
mor
e eff
ectiv
e th
an n
on-t
agge
d PB
1-22
57.
Thes
e da
ta d
emon
stra
te th
e ab
ility
of a
n im
mun
ostim
ulat
ory
mot
if to
impr
ove
the
perf
orm
ance
of a
n RN
Ai-
base
d an
tivi-
ral,
a fin
ding
that
may
influ
ence
the
desi
gn o
f fut
ure
RNA
i-bas
ed a
nti-i
nflue
nza
ther
apeu
tics.
Stew
art e
t al.
2011
Rn
A in
terf
eren
ceSh
ort i
nter
feri
ng R
NA
sSi
lenc
e ge
ne e
xpre
ssio
nV
iral
mRN
A d
egra
datio
n
RNA
inte
rfer
ence
(RN
Ai)
is a
pow
erfu
l too
l to
sile
nce
gene
exp
ress
ion.
Sm
all i
nter
feri
ng R
NA
(siR
NA
)-in
duce
d RN
A d
egra
-da
tion
has b
een
rece
ntly
use
d as
an
antiv
irus
age
nt to
inhi
bit s
peci
fic v
irus
repl
icat
ion.
Her
e, w
e sh
owed
that
seve
ral s
iRN
As
spec
ific
for c
onse
rved
regi
ons o
f infl
uenz
a vi
rus m
atri
x (M
2) a
nd n
ucle
ocap
sid
prot
ein
(NP)
gen
es c
ould
effe
ctiv
ely
inhi
bit
expr
essi
on o
f the
cor
resp
ondi
ng v
iral p
rote
in. W
e al
so e
valu
ated
the
antiv
iral p
oten
tial o
f the
se si
RNA
s tar
getin
g M
2 an
d N
P of
H5N
1 av
ian
influ
enza
vir
us (A
IV),
whi
ch a
re e
ssen
tial t
o vi
ral r
eplic
atio
n. W
e in
vest
igat
ed th
e in
hibi
tory
effe
ct o
f M2-
spe-
cific
siRN
As a
nd N
P-sp
ecifi
c si
RNA
s on
influ
enza
A v
irus
(H5N
1, H
1N1
and
H9N
2) re
plic
atio
n in
Mad
in-D
arby
can
ine
kidn
ey (M
DC
K) c
ells
and
BALB
/c m
ice.
The
resu
lts sh
owed
that
trea
tmen
t with
thes
e si
RNA
s cou
ld sp
ecifi
cally
inhi
bit i
nflu-
enza
A v
irus
repl
icat
ion
in M
DC
K c
ells
(0.5
1–1.
63 T
CID
50 re
duct
ion
in v
irus
tite
rs),
and
deliv
ery
of p
S-M
48 a
nd p
S-N
P138
3 si
gnifi
cant
ly re
duce
d lu
ng v
irus
tite
rs in
the
infe
cted
mic
e (1
6–50
-fol
d re
duct
ion
in lu
ng v
irus
tite
rs) a
nd p
artia
lly p
rote
cted
th
e m
ice
from
leth
al in
fluen
za v
irus
cha
lleng
e (a
surv
ival
rate
of 4
/8 fo
r H1N
1 vi
rus-
infe
cted
mic
e an
d 2/
8 fo
r H5N
1 vi
rus
infe
cted
mic
e). M
oreo
ver,
the
trea
tmen
t of p
S-M
48 a
nd p
S-N
P138
3 co
uld
supp
ress
repl
icat
ion
of d
iffer
ent s
ubty
pes o
f infl
u-en
za A
vir
uses
, inc
ludi
ng a
H5N
1 hi
ghly
pat
hoge
nic
avia
n is
olat
e st
rain
. The
resu
lts p
rovi
ded
a ba
sis f
or fu
rthe
r dev
elop
men
t of
siRN
A fo
r pro
phyl
axis
and
ther
apy
of in
fluen
za v
irus
infe
ctio
n in
hum
ans a
nd a
nim
als.
Zho
u et
al.
2007
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
173
Rn
A in
terf
eren
ceSh
ort i
nter
feri
ng R
NA
ssi
RNA
-res
ista
nt v
irus
esH
1-sh
ort h
airp
in R
NA
RNA
inte
rfer
ence
(RN
Ai)
prov
ides
a p
ower
ful n
ew m
eans
to in
hibi
t vir
al in
fect
ion
spec
ifica
lly. H
owev
er, t
he s
elec
tion
of s
iRN
A-r
esis
tant
vir
uses
is a
maj
or c
once
rn in
the
use
of R
NA
i as
antiv
iral
ther
apeu
tics.
In th
is s
tudy
, we
cond
ucte
d a
lent
ivir
al v
ecto
r w
ith a
H1-
shor
t hai
rpin
RN
A (s
hRN
A) e
xpre
ssio
n ca
sset
te to
del
iver
sm
all i
nter
feri
ng R
NA
s (s
iRN
As)
in
to m
amm
alia
n ce
lls. U
sing
this
vec
tor
that
als
o ex
pres
ses
enha
nced
gre
en fl
uore
scen
ce p
rote
in (E
GFP
) as
surr
ogat
e m
arke
r, st
able
shR
NA
-exp
ress
ing
cell
lines
wer
e su
cces
sful
ly e
stab
lishe
d an
d th
e in
hibi
tion
effic
ienc
ies
of r
atio
nally
de
sign
ed s
iRN
As
targ
etin
g to
con
serv
ed re
gion
s of
influ
enza
A v
irus
gen
ome
wer
e as
sess
ed. T
he re
sults
sho
wed
that
a
siRN
A ta
rget
ing
influ
enza
M2
gene
(siM
2) p
oten
tly in
hibi
ted
vira
l rep
licat
ion.
The
siM
2 w
as n
ot o
nly
effe
ctiv
e fo
r H
1N1
viru
s bu
t als
o fo
r hi
ghly
pat
hoge
nic
avia
n in
fluen
za v
irus
H5N
1. In
add
ition
to it
s M
2 in
hibi
tion,
the
siM
2 al
so
inhi
bite
d N
P m
RNA
acc
umul
atio
n an
d pr
otei
n ex
pres
sion
. A lo
ng te
rm in
hibi
tion
effe
ct o
f the
siM
2 w
as d
emon
stra
ted
and
the
emer
genc
e of
siR
NA
-res
ista
nt m
utan
ts in
influ
enza
qua
sisp
ecie
s w
as n
ot o
bser
ved.
Tak
en to
geth
er, o
ur s
tudy
su
gges
ted
that
M2
gene
mig
ht b
e an
opt
imal
RN
Ai t
arge
t for
ant
ivir
al th
erap
y. T
hese
find
ings
pro
vide
use
ful i
nfor
ma-
tion
for
the
deve
lopm
ent o
f RN
Ai-
base
d pr
ophy
laxi
s an
d th
erap
y fo
r hu
man
influ
enza
vir
us in
fect
ion.
Sui e
t al.
2009
Alk
aloi
ds fr
om
Am
aryl
lidac
eae
Lyco
rine
Hae
man
tham
ine
Influ
enza
A v
irus
es o
ccas
iona
lly c
ause
larg
e ep
idem
ics
and
kill
thou
sand
s ev
ery
year
. Whi
le li
ttle
is k
now
n ab
out t
he
mec
hani
sm o
f cel
l fus
ion
in d
isea
ses,
esp
ecia
lly in
fluen
za v
irus
infe
cted
and
pro
tect
ion
from
Am
aryl
lidac
eae
Alk
aloi
ds.
The
two
com
poun
ds ly
cori
ne (A
A1)
and
hae
man
tham
ine
(AA
3) w
ere
obta
ined
from
bul
bs o
f L. r
adia
te, e
xhib
ited
anti-
influ
enza
act
ivity
aft
er in
fluen
za v
irus
ent
ry c
ells
. The
two
com
poun
ds w
ere
inve
stig
ated
for
in v
itro
disp
layi
ng d
iffer
-en
t lev
els
of re
sist
ance
to p
ro-a
popt
otic
stim
uli.
Seve
n in
fluen
za v
irus
es w
ere
used
, A/C
K/G
D/1
78/0
4 (H
5N, 1
78),
A/
DK
/GD
/212
/04
(H5N
1, 2
12),
A/s
win
e/G
D/1
66/0
6 (H
3N2,
166
), A
/CK
/HN
/170
/03
(H1N
1, 1
70),
A/P
uert
o Ri
co/8
/34
(H1N
1, P
R8),
A/C
k/G
D/4
00/0
7 (H
9N2,
400
), A
/Ck/
GD
/228
/04
(H9N
2, 2
28),
the
two
com
poun
ds e
xhib
ited
pote
ncy
in th
e si
ngle
-dig
it m
icro
mol
ar r
ange
. The
stu
dies
als
o sh
owed
that
AA
1 an
d A
A3
exer
ted
thei
r in
vitr
o an
ti-in
fluen
za
activ
ity th
roug
h cy
tost
atic
rat
her
than
cyt
otox
ic e
ffect
s. M
any
viru
ses
inte
ract
with
the
host
cel
ls to
cha
nge
thei
r ow
n gr
owth
whi
ch th
ey fa
vor
the
spee
d. T
he c
ells
infe
cted
with
vir
us, g
row
th o
f MD
CK
cel
ls w
as s
low
ed d
own
by a
rres
ting
cell
cycl
e at
G1/
S ph
ase.
With
the
com
poun
d tr
eate
d, th
e gr
owth
cyc
le w
as d
ecre
ased
in S
pha
se. W
ith H
5N1
influ
enza
vi
rus
trea
ted,
the
cyto
skel
eton
of c
ells
was
cha
nged
whi
le w
ith th
e co
mpo
und
trea
ted
the
prot
ectio
n of
cyt
oske
leto
n w
as
obvi
ousl
y pr
otec
ted.
The
dat
a sh
owed
diff
eren
ces
betw
een
drug
trea
ted
cells
and
vir
us in
fect
ed c
ells
, pro
vide
d a
basi
s to
fu
rthe
r ex
plor
e ce
ll fu
sion
and
ant
i-in
fluen
za m
echa
nism
of t
he tw
o co
mpo
unds
.
He
et a
l. 20
12
Poly
sacc
hari
des
from
se
awee
dsSu
lfate
d fu
cans
Het
erog
lycu
rona
ns
To e
xplo
re th
e po
lysa
ccha
ride
s fr
om s
elec
ted
seaw
eeds
of A
tlant
ic C
anad
a an
d to
eva
luat
e th
eir
pote
ntia
l ant
i-in
fluen
za
viru
s ac
tiviti
es, p
olys
acch
arid
es w
ere
isol
ated
from
sev
eral
Atla
ntic
Can
adia
n se
awee
ds, i
nclu
ding
thre
e re
d al
gae
(Pol
y-si
phon
ia la
nosa
, Fur
cella
ria
lum
bric
alis
, and
Pal
mar
ia p
alm
ata)
, tw
o br
own
alga
e (A
scop
hyllu
m n
odos
um a
nd F
ucus
ve
sicu
losu
s), a
nd o
ne g
reen
alg
a (U
lva
lact
uca)
by
sequ
entia
l ext
ract
ion
with
col
d w
ater
, hot
wat
er, a
nd a
lkal
i sol
utio
ns.
The
se p
olys
acch
arid
es w
ere
anal
yzed
for
mon
osac
char
ide
com
posi
tion
and
othe
r ge
nera
l che
mic
al p
rope
rtie
s, a
nd th
ey
wer
e ev
alua
ted
for
anti-
influ
enza
vir
us a
ctiv
ities
. Tot
al s
ugar
con
tent
s in
thes
e po
lysa
ccha
ride
s ra
nged
from
15.
4% (i
n U
. lac
tuca
) to
91.4
% (i
n F.
lum
bric
alis
); su
lfatio
n le
vel w
as a
s hi
gh a
s 17
.6%
in a
pol
ysac
char
ide
from
U. l
actu
ca, w
here
as
it co
uld
not b
e de
tect
ed in
an
alik
ali-
extr
act f
rom
P. p
alm
aria
. For
pol
ysac
char
ides
from
red
seaw
eeds
, the
mai
n su
gar
units
wer
e su
lfate
d ga
lact
ans
(aga
r or
car
rage
enan
) for
P. l
anos
a, F
. lum
bric
alis
, and
xyl
ans
for
P. p
alm
ata.
In b
row
n se
awee
ds, t
he p
olys
acch
arid
es la
rgel
y co
ntai
ned
sulfa
ted
fuca
ns, w
here
as th
e po
lysa
ccha
ride
s in
gre
en s
eaw
eed
wer
e m
ainl
y co
mpo
sed
of h
eter
ogly
curo
nans
. Scr
eeni
ng fo
r an
tivir
al a
ctiv
ity a
gain
st in
fluen
za A
/PR/
8/34
(H1N
1) v
irus
re
veal
ed th
at b
row
n al
gal p
olys
acch
arid
es w
ere
part
icul
arly
effe
ctiv
e. S
eaw
eeds
from
Atla
ntic
Can
ada
are
a go
od s
ourc
e of
mar
ine
poly
sacc
hari
des
with
pot
entia
l ant
ivir
al p
rope
rtie
s.
Jiao
et a
l. 20
12
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
174
Car
rage
enan
s fr
om r
ed
alga
eK
appa
-car
rage
enan
ol
igos
acch
arid
es
Car
rage
enan
s, th
e su
lpha
ted
gala
ctan
s der
ived
from
red
alga
e, a
re a
ttra
ctin
g in
crea
sing
inte
rest
in d
evel
opin
g po
tent
ial
anti-
vira
l dru
gs. I
n th
is st
udy,
low
mol
ecul
ar w
eigh
t kap
pa-c
arra
geen
an o
ligos
acch
arid
es (K
CO
) and
thei
r sul
phat
ed d
eriv
-at
ives
(KC
O-S
) wer
e pr
epar
ed, a
nd th
eir a
nti-i
nflue
nza
A v
irus
(IAV
) pro
pert
ies w
ere
inve
stig
ated
. The
resu
lts in
dica
ted
that
KC
O a
nd K
CO
-S c
ould
effe
ctiv
ely
inhi
bit I
AV m
ultip
licat
ion
in M
DC
K c
ells
in a
dos
e-de
pend
ent m
anne
r. Fu
rthe
r-m
ore,
a st
ruct
ure-
activ
ity re
latio
nshi
p st
udy
show
ed th
at th
e de
gree
of s
ulph
atio
n an
d m
olec
ular
wei
ght w
ere
the
mai
n fa
ctor
s tha
t infl
uenc
ed th
e an
ti-IA
V a
ctiv
ity o
f kap
pa-c
arra
geen
an o
ligos
acch
arid
e. Th
e m
ost a
ctiv
e ka
ppa-
carr
agee
nan
oli-
gosa
ccha
ride
had
a su
lpha
te c
onte
nt o
f 0.8
–1.0
mol
e/m
ole
of d
isac
char
ide
and
a m
olec
ular
wei
ght o
f 1–3
kD
a. In
add
ition
, K
CO
and
KC
O-S
cou
ld si
gnifi
cant
ly im
prov
e su
rviv
al a
nd d
ecre
ase
pulm
onar
y vi
ral t
itres
in IA
V-i
nfec
ted
mic
e. M
oreo
ver,
the
antiv
iral
effe
ct o
f KC
O a
nd K
CO
-S d
oes n
ot se
em to
be
depe
nden
t on
the
inte
rfer
on sy
stem
. In
conc
lusi
on, c
arra
-ge
enan
olig
osac
char
ide
and
its su
lpha
ted
deri
vativ
e ha
ve g
ood
inhi
bito
ry a
ctio
ns o
n IA
V re
plic
atio
n in
vitr
o an
d in
viv
o.
Wan
g et
al.
2012
Subs
tanc
es fr
om M
osla
di
anth
era
Phen
olic
sesq
uite
rpen
esA
rom
atic
com
poun
ds
Ethn
opha
rmac
olog
ical
rele
vanc
e: M
osla
dia
nthe
ra a
s an
arom
atic
her
b is
use
d in
folk
med
icin
e fo
r the
trea
tmen
t of c
ough
, co
lds,
feve
r, br
onch
itis,
nasa
l con
gest
ion
and
head
ache
. Aim
of t
he st
udy:
To
char
acte
rize
che
mic
al c
ompo
sitio
ns a
nd to
ev
alua
te th
e an
ti-in
fluen
za e
ffect
s of e
ssen
tial o
ils o
f M. d
iant
hera
(MD
EO) i
n in
fluen
za v
irus
A (I
VA) i
nfec
ted
mic
e. M
ater
i-al
s and
met
hods
: MD
EO w
as o
btai
ned
by h
ydro
dist
illat
ion
and
anal
ysed
by
gas c
hrom
atog
raph
y-m
ass s
pect
rom
etry
(G
C-M
S). I
CR
mic
e w
ere
trea
ted
with
MD
EO fo
r 5 c
onse
cutiv
e da
ys a
t dos
es o
f 90–
360
mg/
kg a
fter p
ost-
infe
cted
. Lev
els o
f Se
rum
IL-4
and
IFN
-gam
ma
wer
e as
saye
d by
ELI
SA. L
evel
s of M
OD
, SO
D, T
AO
C a
nd G
SH-P
x in
lung
tiss
ue w
ere
dete
r-m
ined
by
colo
rim
etri
c m
etho
d. R
esul
ts: G
C-M
S an
alys
is re
veal
ed th
e pr
esen
ce o
f 29
com
pone
nts t
hat a
ccou
nt fo
r 97.
74%
of
phen
olic
sesq
uite
rpen
es a
nd a
rom
atic
com
poun
ds. Th
e m
ajor
com
poun
ds w
ere
elem
icin
(16.
51%
), th
ymol
(14.
77%
), be
ta-
cary
ophy
llene
(14.
49%
), is
o-el
emic
in (9
.22%
), as
aron
e (6
.09%
) and
alp
ha-c
aryo
phyl
lene
(5.2
6%).
It ha
d si
gnifi
cant
effe
cts o
n de
crea
sing
lung
vira
l tite
rs, i
nhib
iting
pne
umon
ia, r
educ
ing
leve
ls of
seru
m IF
N-g
amm
a an
d IL
-4, a
nd e
nhan
cing
ant
ioxi
dant
ac
tivity
in th
e lu
ng ti
ssue
of I
VA in
fect
ed m
ice.
Con
clus
ions
: MPE
cou
ld e
xhib
it th
erap
eutic
al e
ffect
s in
IVA
infe
cted
mic
e as
a su
ppre
ssor
of I
VA re
plic
atio
n an
d in
flam
mat
ory
med
iato
rs a
nd a
pro
mot
er o
f ant
ioxi
dant
pot
entia
ls. Th
eref
ore,
MD
EO
coul
d pr
ovid
e a
safe
and
effe
ctiv
e th
erap
eutic
can
dida
te fo
r tre
atm
ent o
f infl
uenz
a an
d its
subs
eque
nt v
iral p
neum
onia
.
Wu
et a
l. 20
12
Inhi
bito
rs fr
om h
erba
l ex
trac
tBe
rber
ine
Obj
ectiv
e: T
o ex
plor
e th
e po
tent
ial e
ffect
s of b
erbe
rine
on in
fluen
za v
irus i
nfec
tion
both
in v
itro
and
in v
ivo.
Met
hods
: In
vitr
o an
ti-in
fluen
za v
irus a
ssay
s wer
e pe
rfor
med
by
cyto
path
ogen
ic e
ffect
and
neu
ram
inid
ase
assa
ys in
Mad
in D
arby
can
ine
kidn
ey
cells
. In
vivo
ant
i-infl
uenz
a vi
rus a
ssay
s wer
e pe
rfor
med
on
the
vira
l pne
umon
ia m
odel
of m
ice.
The
num
bers
of m
ice
that
die
d w
ithin
day
2 to
day
14
post
infe
ctio
n w
ere
reco
rded
to c
alcu
late
the
mor
talit
y. O
n da
ys 2
, 4, a
nd 6
, the
vira
l tite
rs in
the
lung
s w
ere
dete
rmin
ed b
y he
mag
glut
inat
ion
assa
y; h
emat
oxyl
in/e
osin
stai
ning
was
use
d to
ass
ess t
he p
atho
geni
c ch
ange
s of l
ung
tis-
sues
; the
con
cent
ratio
ns o
f tum
or n
ecro
sis fa
ctor
-alp
ha (T
NF-
alph
a) a
nd m
onoc
yte
spec
ific
chem
oattr
acta
nt m
olec
ule
(MC
P-1)
w
ere
mea
sure
d by
radi
o im
mun
oass
ay o
r enz
yme-
linke
d im
mun
osor
bent
ass
ay; t
he c
once
ntra
tions
of n
itric
oxi
de (N
O) a
nd
indu
cibl
e ni
tric
oxi
de sy
nthe
tase
(iN
OS)
wer
e de
tect
ed b
y co
lorim
etric
met
hod;
reve
rse
tran
scrip
tion
poly
mer
ase
chai
n re
actio
n w
as u
sed
to d
etec
t the
mRN
A le
vel o
f TN
F-al
pha
and
MC
P-1.
Res
ults
: Ber
berin
e sh
owed
inhi
bito
ry e
ffect
s on
cyto
path
ogen
ic
effec
ts a
nd n
eura
min
idas
e ac
tivity
of v
irus,
with
the
ther
apeu
tic in
dex
9.69
. In
vivo
, ber
berin
e de
crea
sed
mic
e m
orta
lity
from
90
% to
55%
, red
uced
viru
s tite
rs in
the
lung
s on
day
2 po
stin
fect
ion
(P <
0.0
5). Th
e lu
ng h
istol
ogy
scor
es w
ere
1.50
±0.
67, 4
.50
± 1.
00, a
nd 5
.50
± 1.
00 in
the
berb
erin
e gr
oup
on d
ays 2
, 4, a
nd 6
, res
pect
ivel
y, w
hich
wer
e sig
nific
antly
redu
ced
com
pare
d to
2.
17 ±
0.2
2, 6
.83
± 0.
44, a
nd 8
.50
± 0.
33 in
the
infe
cted
gro
up (P
< 0
.05)
. The
prod
uctio
ns o
f NO
and
iNO
S w
ere
repr
esse
d by
be
rber
ine
com
pare
d w
ith th
ose
in th
e in
fect
ed g
roup
(P <
0.0
1). Th
e tr
ansc
riptio
n an
d ex
pres
sion
of T
NF-
alp
ha w
ere
inhi
bite
d by
ber
berin
e on
day
4 (P
< 0
.01)
and
day
6 (P
< 0
.05)
, and
thos
e of
MC
P-1
wer
e in
hibi
ted
on d
ay 6
(P <
0.0
1) c
ompa
red
with
the
infe
cted
gro
up. C
oncl
usio
ns: B
erbe
rine
exhi
bite
d an
tivira
l effe
cts o
n th
e in
fluen
za v
irus b
oth
in v
itro
and
in v
ivo.
The
poss
ible
th
erap
eutic
mec
hani
sm o
f ber
berin
e on
influ
enza
-indu
ced
vira
l pne
umon
ia m
ight
be
inhi
bitin
g th
e vi
rus i
nfec
tion,
as w
ell a
s im
prov
ing
the
path
ogen
ic c
hang
es b
y re
pres
sing
infla
mm
ator
y su
bsta
nces
rele
ase.
Wu
et a
l. 20
11
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
175
nan
obod
ies
Targ
etin
g of
H
5N1h
emag
glut
inin
We
asse
ssed
the
prot
ectiv
e po
tent
ial o
f mon
oval
ent a
nd b
ival
ent N
anob
odie
s (A
blyn
x) a
gain
st c
halle
nge
with
this
vir
us.
Thes
e N
anob
odie
s wer
e de
rive
d fr
om ll
amas
and
targ
et H
5N1
hem
aggl
utin
in. I
ntra
nasa
l adm
inis
trat
ion
of N
anob
odie
s eff
ectiv
ely
cont
rolle
d ho
mol
ogou
s infl
uenz
a A
vir
us re
plic
atio
n. A
dmin
istr
atio
n of
Nan
obod
ies b
efor
e ch
alle
nge
stro
ngly
re
duce
d H
5N1
viru
s rep
licat
ion
in th
e lu
ngs a
nd p
rote
cted
mic
e fr
om m
orbi
dity
and
mor
talit
y af
ter a
leth
al c
halle
nge
with
H
5N1
viru
s. Th
e bi
vale
nt N
anob
ody
was
at l
east
60-
fold
mor
e eff
ectiv
e th
an th
e m
onov
alen
t Nan
obod
y in
con
trol
ling
viru
s re
plic
atio
n. In
add
ition
, Nan
obod
y th
erap
y af
ter c
halle
nge
stro
ngly
redu
ced
vira
l rep
licat
ion
and
sign
ifica
ntly
del
ayed
tim
e to
dea
th. E
pito
pe m
appi
ng re
veal
ed th
at th
e V
HH
Nan
obod
y bi
nds t
o an
tigen
ic si
te B
in H
5 he
mag
glut
inin
. Bec
ause
Nan
o-bo
dies
are
smal
l, st
able
, and
sim
ple
to p
rodu
ce, t
hey
are
a pr
omis
ing,
nov
el th
erap
eutic
age
nt a
gain
st in
fluen
za.
Iban
ez e
t al.
2011
nan
obod
ies
M2
prot
ein
targ
etin
gN
eutr
aliz
ing
antib
odie
s aga
inst
the
high
ly c
onse
rved
M2
ion
chan
nel i
s tho
ught
to o
ffer b
road
pro
tect
ion
agai
nst i
nflue
nza
A
viru
ses.
Her
e, w
e sc
reen
ed sy
nthe
tic C
amel
sing
le-d
omai
n an
tibod
y (V
HH
) lib
rari
es a
gain
st n
ativ
e M
2 io
n ch
anne
l pro
tein
. O
ne o
f the
isol
ated
VH
Hs,
M2-
7A, s
peci
fical
ly b
ound
to M
2-ex
pres
sed
cell
mem
bran
e as
wel
l as i
nflue
nza
A v
irio
n, in
hibi
ted
repl
icat
ion
of b
oth
aman
tadi
ne-s
ensi
tive
and
resi
stan
t infl
uenz
a A
vir
uses
in v
itro,
and
pro
tect
ed m
ice
from
a le
thal
influ
enza
vi
rus c
halle
nge.
Mor
eove
r, M
2-7A
show
ed b
lock
ing
activ
ity fo
r pro
ton
influ
x th
roug
h M
2 io
n ch
anne
l. Th
ese
piec
es o
f evi
-de
nce
colle
ctiv
ely
dem
onst
rate
for t
he fi
rst t
ime
that
a n
eutr
aliz
ing
antib
ody
agai
nst M
2 w
ith b
road
spec
ifici
ty is
ach
ieva
ble,
an
d M
2-7A
may
hav
e po
tent
ial f
or c
ross
pro
tect
ion
agai
nst a
num
ber o
f var
iant
s and
subt
ypes
of i
nflue
nza
A v
irus
es.
Wei
et a
l. 20
11
Hum
anis
ed m
onoc
lona
l an
tibo
dies
Hae
mag
glut
inin
ta
rget
ing
Hum
aniz
ed m
onoc
lona
l ant
ibod
ies (
mA
bs) t
hat n
eutr
aliz
e H
5N1
viru
s cou
ld b
e us
ed a
s pro
phyl
axis
and
trea
tmen
t to
aid
in th
e co
ntai
nmen
t of s
uch
a pa
ndem
ic. M
etho
ds: N
eutr
aliz
ing
mA
bs a
gain
st H
5 he
mag
glut
inin
wer
e hu
man
ized
and
in
trod
uced
into
C57
BL/6
mic
e (1
, 5, o
r 10
mg/
kg b
odyw
eigh
t) o
ne d
ay p
rior
to-,
one
day
post
- and
thre
e da
ys p
ost-
leth
al
chal
leng
e w
ith H
5N1
A/V
ietn
am/1
203/
04 v
irus
. Effi
cacy
was
det
erm
ined
by
obse
rvat
ion
of w
eigh
t los
s as w
ell a
s sur
viva
l. Re
sults
: Tw
o m
Abs
neu
tral
izin
g fo
r ant
igen
ical
ly v
aria
nt H
5N1
viru
ses,
A/V
ietn
am/1
203/
04 a
nd A
/Hon
g K
ong/
213/
03
wer
e id
entifi
ed a
nd h
uman
ized
with
out l
oss o
f spe
cific
ity. B
oth
antib
odie
s exh
ibite
d pr
ophy
lact
ic e
ffica
cy in
mic
e, h
ow-
ever
, VN
04-2
-huG
1 pe
rfor
med
bet
ter r
equi
ring
onl
y 1
mg/
kg b
odyw
eigh
t for
com
plet
e pr
otec
tion.
Whe
n us
ed to
trea
t in
fect
ion
VN
04-2
-huG
1 w
as a
lso
com
plet
ely
prot
ectiv
e, e
ven
whe
n in
trod
uced
thre
e da
ys p
ost i
nfec
tion,
alth
ough
hig
her
dose
of a
ntib
ody
was
requ
ired
. Con
clus
ion:
Pro
phyl
axis
and
trea
tmen
t usi
ng n
eutr
aliz
ing
hum
aniz
ed m
Abs
is e
ffica
ciou
s ag
ains
t let
hal c
halle
nge
with
A/V
ietn
am/1
203/
04, p
rovi
ding
pro
of o
f pri
ncip
le fo
r the
use
of p
assi
ve a
ntib
ody
ther
apy
as a
co
ntai
nmen
t opt
ion
in th
e ev
ent o
f pan
dem
ic in
fluen
za.
Han
son
et a
l. 20
06
Hum
an m
onoc
lona
l an
tibo
dies
Pre-
exis
ting
neut
raliz
ing
antib
ody
prov
ides
the
first
line
of d
efen
ce a
gain
st p
atho
gens
in g
ener
al. F
or in
fluen
za v
irus
, ann
ual
vacc
inat
ions
are
giv
en to
mai
ntai
n pr
otec
tive
leve
ls of
ant
ibod
y ag
ains
t the
cur
rent
ly c
ircul
atin
g st
rain
s. H
ere
we
repo
rt th
at
afte
r boo
ster
vac
cina
tion
ther
e w
as a
rapi
d an
d ro
bust
influ
enza
-spe
cific
IgG
+ ant
ibod
y- se
cret
ing
plas
ma
cell
(ASC
) res
pons
e th
at p
eake
d at
app
roxi
mat
ely
day
7 an
d ac
coun
ted
for u
p to
6%
of p
erip
hera
l blo
od B
cel
ls. Th
ese
ASC
s cou
ld b
e di
stin
guis
hed
from
influ
enza
- spe
cific
IgG
+ mem
ory
B ce
lls th
at p
eake
d 14
–21
days
afte
r vac
cina
tion
and
aver
aged
1%
of a
ll B
cells
. Im
por-
tant
ly, a
s muc
h as
80%
of A
SCs p
urifi
ed a
t the
pea
k of
the
resp
onse
wer
e in
fluen
za sp
ecifi
c. Th
is A
SC re
spon
se w
as c
hara
cter
-iz
ed b
y a
high
ly re
stri
cted
B-c
ell r
ecep
tor (
BC
R) re
pert
oire
that
in so
me
dono
rs w
as d
omin
ated
by
only
a fe
w B
-cel
l clo
nes.
This
pau
cicl
onal
resp
onse
, how
ever
, sho
wed
ext
ensi
ve in
trac
lona
l div
ersi
ficat
ion
from
acc
umul
ated
som
atic
mut
atio
ns. W
e us
ed th
e im
mun
oglo
bulin
var
iabl
e re
gion
s iso
late
d fr
om so
rted
sing
le A
SCs t
o pr
oduc
e ov
er 5
0 hu
man
mon
oclo
nal a
ntib
od-
ies (
mA
bs) t
hat b
ound
to th
e th
ree
influ
enza
vac
cine
stra
ins w
ith h
igh
affini
ty. Th
is st
rate
gy d
emon
stra
tes t
hat w
e ca
n ge
ner-
ate
mul
tiple
hig
h-affi
nity
mA
bs fr
om h
uman
s with
in a
mon
th a
fter v
acci
natio
n. Th
e pa
nel o
f infl
uenz
a-vi
rus-
spec
ific
hum
an
mA
bs a
llow
ed u
s to
addr
ess t
he is
sue
of o
rigi
nal a
ntig
enic
sin
(OA
S): t
he p
heno
men
on w
here
the
indu
ced
antib
ody
show
s hi
gher
affi
nity
to a
pre
viou
sly e
ncou
nter
ed in
fluen
za v
irus
stra
in c
ompa
red
with
the
viru
s str
ain
pres
ent i
n th
e va
ccin
e (1
). H
owev
er, w
e fo
und
that
mos
t of t
he in
fluen
za- v
irus
- spe
cific
mA
bs sh
owed
the
high
est a
ffini
ty fo
r the
cur
rent
vac
cine
stra
in.
Thus
, OA
S do
es n
ot se
em to
be
a co
mm
on o
ccur
renc
e in
nor
mal
, hea
lthy
adul
ts re
ceiv
ing
influ
enza
vac
cina
tion.
Wra
mm
ert e
t al.
2008
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
176
Tabl
e 5.
Com
bina
tion
ther
apy
of in
flue
nza
infe
ctio
ns
ose
ltam
ivir
wit
h am
anta
dine
Mou
se m
odel
In th
e pr
esen
t stu
dy, t
he e
ffect
of c
ombi
ning
ant
i-infl
uenz
a dr
ugs a
ctiv
e at
diff
eren
t ste
ps o
f the
influ
enza
vir
us re
plic
a-tio
n cy
cle,
ose
ltam
ivir
as a
neu
ram
inid
ase
(NA
) inh
ibito
r and
am
anta
dine
targ
etin
g M
2 pr
otei
n, w
as in
vest
igat
ed in
viv
o by
ora
l adm
inis
trat
ion
in a
mou
se m
odel
of a
eros
ol in
fluen
za v
irus
infe
ctio
n an
d in
vitr
o in
MD
CK
cel
ls. I
n m
ice,
dos
es
of o
selta
miv
ir a
nd a
man
tadi
ne p
rovi
ding
50–
60%
surv
ival
aga
inst
A/H
ongk
ong/
1/68
(H3N
2) o
r A/P
R/8/
34 (H
1N1)
wer
e ca
pabl
e of
con
ferr
ing
com
plet
e pr
otec
tion
whe
n us
ed si
mul
tane
ousl
y, su
gges
ting
that
incr
ease
d in
hibi
tion
of in
fluen
za
viru
s rep
licat
ion
by c
ombi
ning
ose
ltam
ivir
and
am
anta
dine
in v
itro
tran
slat
es in
to p
rote
ctio
n fr
om le
thal
infe
ctio
n of
m
ice.
The
com
bina
tion
of a
man
tadi
ne w
ith o
selta
miv
ir re
quir
ed 1
5-fo
ld le
ss o
selta
miv
ir th
an m
onot
hera
py to
con
fer
com
plet
e pr
otec
tion
agai
nst l
etha
l aer
osol
influ
enza
vir
us in
fect
ion.
Rem
arka
bly,
aman
tadi
ne-b
ased
com
bina
tion
chem
o-pr
ophy
laxi
s was
eve
n eff
ectiv
e ag
ains
t am
anta
dine
-res
ista
nt A
/PR/
8/34
influ
enza
vir
us. Th
us, c
ombi
natio
n ch
emot
hera
py
may
be
mor
e effi
caci
ous t
han
mon
othe
rapy
aga
inst
new
ly e
mer
ging
Influ
enza
A su
btyp
es.
Mas
ihi e
t al.
2007
Zan
amiv
ir w
ith
imm
uno-
mod
ulat
ors
Cel
ecox
ibG
emfib
rozi
lM
esal
azin
eM
ouse
mod
el
The
mor
talit
y of
hum
an in
fect
ion
by in
fluen
za A
/H5N
1 vi
rus c
an e
xcee
d 80
%. Th
e hi
gh m
orta
lity
and
its p
oor r
espo
nse
to
the
neur
amin
idas
e in
hibi
tor o
selta
miv
ir h
ave
been
att
ribu
ted
to u
ncon
trol
led
viru
s-in
duce
d cy
toki
ne st
orm
. We
chal
-le
nged
BA
LB/c
mic
e w
ith 1
000
LD50
of i
nflue
nza
A/V
ietn
am/1
194/
04. S
urvi
val,
body
wei
ght,
hist
opat
holo
gy, i
nflam
ma-
tory
mar
kers
, vir
al lo
ads,
T ly
mph
ocyt
e co
unts
, and
neu
tral
izin
g an
tibod
y re
spon
se w
ere
docu
men
ted
in in
fect
ed m
ice
trea
ted
indi
vidu
ally
or i
n co
mbi
natio
n w
ith z
anam
vir,
cele
coxi
b, g
emfib
rozi
l, an
d m
esal
azin
e. T
o im
itate
the
real
-life
sce-
nari
o, tr
eatm
ent w
as in
itiat
ed a
t 48
h af
ter v
iral
cha
lleng
e. Th
ere
wer
e si
gnifi
cant
impr
ovem
ents
in su
rviv
al ra
te (P
= 0
.02)
, su
rviv
al ti
me
(P <
0.0
2), a
nd in
flam
mat
ory
mar
kers
(P <
0.0
1) in
the
grou
p tr
eate
d-w
ith a
trip
le c
ombi
natio
n of
zan
amiv
ir,
cele
coxi
b, a
nd m
esal
azin
e w
hen
com
pare
d w
ith z
anam
ivir
alo
ne. Z
anam
ivir
with
or w
ithou
t im
mun
omod
ulat
ors r
educ
ed
vira
l loa
d to
a si
mila
r ext
ent.
Insi
gnifi
cant
pro
long
atio
n of
surv
ival
was
obs
erve
d w
hen
indi
vidu
al a
gent
s wer
e us
ed
alon
e. S
igni
fican
tly h
ighe
r lev
els o
f CD
4+ and
CD
8+ T ly
mph
ocyt
es a
nd le
ss p
ulm
onar
y in
flam
mat
ion
wer
e al
so fo
und
in
the
grou
p re
ceiv
ing
trip
le th
erap
y. Z
anam
ivir
alo
ne re
duce
d vi
ral l
oad
but n
ot in
flam
mat
ion
and
mor
talit
y. Th
e su
rviv
al
bene
fits o
f add
ing
cele
coxi
b an
d m
esal
azin
e to
zan
amiv
ir c
ould
be
caus
ed b
y th
eir s
yner
gist
ic e
ffect
s in
redu
cing
cyt
okin
e dy
sfun
ctio
n an
d pr
even
ting
apop
tosi
s. C
ombi
natio
ns o
f a n
eura
min
idas
e in
hibi
tor w
ith th
ese
imm
unom
odul
ator
s sho
uld
be c
onsi
dere
d in
rand
omiz
ed c
ontr
olle
d tr
eatm
ent t
rial
s of p
atie
nts s
uffer
ing
from
H5N
1 in
fect
ion.
Zhe
ng e
t al.
2008
ose
ltam
ivir
wit
h co
rtic
oste
roid
sM
ethy
lpre
dnis
olon
eH
ydro
cort
ison
eLu
ng in
jury
acu
te
resp
irat
ory
dist
ress
sy
ndro
me
PURP
OSE
: Dur
ing
the
2009
H1N
1 in
fluen
za A
vir
us p
ande
mic
, a m
inor
ity o
f pat
ient
s dev
elop
ed ra
pidl
y pr
ogre
ssiv
e pn
eum
onia
lead
ing
to a
cute
lung
inju
ry (A
LI)-
acut
e re
spir
ator
y di
stre
ss sy
ndro
me
(ARD
S). A
rece
nt m
eta-
anal
ysis
pro
-vi
des s
uppo
rt fo
r pro
long
ed c
ortic
oste
roid
trea
tmen
t in
ALI
-ARD
S. W
e pr
ospe
ctiv
ely
eval
uate
d th
e re
spon
se to
ose
ltam
i-vi
r and
pro
long
ed c
ortic
oste
roid
trea
tmen
t in
patie
nts w
ith A
LI-A
RDS
and
susp
ecte
d H
1N1
influ
enza
. MET
HO
DS:
Fr
om Ju
ne 2
4 th
roug
h 12
July
200
9, 1
3 pa
tient
s with
susp
ecte
d H
1N1
pneu
mon
ia a
nd A
LI-A
RDS
wer
e ad
mitt
ed to
the
inte
nsiv
e ca
re u
nit (
ICU
) of a
tert
iary
car
e ho
spita
l. H
1N1
influ
enza
was
con
firm
ed w
ith re
al-t
ime
reve
rse
tran
scri
ptas
e-po
lym
eras
e ch
ain
reac
tion
assa
y in
eig
ht p
atie
nts.
Ose
ltam
ivir
and
cor
ticos
tero
id tr
eatm
ent w
ere
initi
ated
con
com
itant
ly
at IC
U a
dmis
sion
; tho
se w
ith se
vere
ARD
S re
ceiv
ed m
ethy
lpre
dnis
olon
e (1
mg/
kg/d
ay),
and
othe
rs re
ceiv
ed h
ydro
cor-
tison
e (3
00 m
g/da
y) fo
r a d
urat
ion
of 2
1 ±
6 da
ys. R
ESU
LTS:
Pat
ient
s with
and
with
out c
onfir
med
H1N
1 in
fluen
za h
ad
sim
ilar d
isea
se se
veri
ty a
t pre
sent
atio
n an
d a
com
para
ble
resp
onse
to tr
eatm
ent.
By d
ay 7
of t
reat
men
t, pa
tient
s exp
eri-
ence
d a
sign
ifica
nt im
prov
emen
t in
lung
inju
ry a
nd m
ultip
le o
rgan
dys
func
tion
scor
es (P
< 0
.001
). Tw
elve
pat
ient
s (92
%)
impr
oved
lung
func
tion,
wer
e ex
tuba
ted,
and
dis
char
ged
aliv
e fr
om th
e IC
U. H
ospi
tal l
engt
h of
stay
and
mor
talit
y w
ere
18.7
± 9
.6 d
ays a
nd 1
5%, r
espe
ctiv
ely.
Surv
ivor
s wer
e di
scha
rged
hom
e w
ithou
t oxy
gen
supp
lem
enta
tion.
CO
NC
LUSI
ON
S:
In A
RDS
patie
nts,
with
and
with
out c
onfir
med
H1N
1 in
fluen
za, p
rolo
nged
low
-to-
mod
erat
e do
se c
ortic
oste
roid
trea
t-m
ent w
as w
ell t
oler
ated
and
ass
ocia
ted
with
sign
ifica
nt im
prov
emen
t in
lung
inju
ry a
nd m
ultip
le o
rgan
dys
func
tion
scor
es a
nd a
low
hos
pita
l mor
talit
y. Th
ese
findi
ngs p
rovi
de th
e ra
tiona
le fo
r dev
elop
ing
a ra
ndom
ized
tria
l.
Qui
spe-
Laim
e et
al
. 201
0
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
177
Lani
nam
ivir
oct
anoa
te
wit
h ar
tific
ial
surf
acta
nt
Mou
se m
odel
Back
grou
nd: P
atie
nts
with
influ
enza
vir
us in
fect
ion
can
deve
lop
seve
re p
neum
onia
and
acu
te re
spir
ator
y di
stre
ss
synd
rom
e (A
RDS)
whi
ch h
ave
a hi
gh m
orta
lity.
Influ
enza
vir
us in
fect
ion
is tr
eate
d w
orld
wid
e m
ainl
y by
neu
ram
inid
ase
inhi
bito
rs (N
AIs
). H
owev
er, m
onot
hera
py w
ith N
AIs
is in
suff
icie
nt fo
r se
vere
pne
umon
ia s
econ
dary
to in
fluen
za v
irus
in
fect
ion.
We
prev
ious
ly d
emon
stra
ted
that
mic
e in
fect
ed w
ith a
leth
al d
ose
of in
fluen
za v
irus
dev
elop
diff
use
alve
olar
da
mag
e (D
AD
) with
alv
eola
r co
llaps
e si
mila
r to
that
see
n in
ARD
S in
hum
ans.
Add
ition
ally
, pul
mon
ary
surf
acta
nt p
ro-
tein
s w
ere
grad
ually
incr
ease
d in
mou
se s
erum
, sug
gest
ing
a de
crea
se in
pul
mon
ary
surf
acta
nt in
the
lung
. The
refo
re,
the
pres
ent s
tudy
exa
min
ed w
heth
er c
ombi
natio
n th
erap
y of
NA
I with
exo
geno
us a
rtifi
cial
sur
fact
ant a
ffect
s m
orta
l-ity
of i
nflu
enza
vir
us-i
nfec
ted
mic
e. M
etho
dolo
gy/P
rinc
ipal
Fin
ding
s: B
ALB
/c m
ice
wer
e in
ocul
ated
with
sev
eral
vir
al
dose
s of
influ
enza
A/P
uert
o Ri
co/8
/34
(PR8
) vir
us (H
1N1)
. The
mic
e w
ere
addi
tiona
lly a
dmin
iste
red
exog
enou
s ar
tifi-
cial
sur
fact
ant i
n th
e pr
esen
ce o
r ab
senc
e of
a n
ew
up to
20
days
aft
er in
ocul
atio
n. V
iral
tite
r an
d cy
toki
ne/c
hem
okin
e le
vels
in th
e lu
ngs,
lung
wei
ght,
path
olog
ical
ana
lysi
s, a
nd b
lood
O2 a
nd C
O2 p
ress
ures
wer
e ev
alua
ted.
Infe
cted
mic
e tr
eate
d w
ith c
ombi
natio
n th
erap
y of
lani
nam
ivir
oct
anoa
te w
ith a
rtifi
cial
sur
fact
ant s
how
ed a
sig
nific
antly
hig
her
surv
ival
rat
e co
mpa
red
with
thos
e th
at re
ceiv
ed la
nina
miv
ir o
ctan
oate
mon
othe
rapy
(P =
0.0
03).
How
ever
, vir
us ti
ter,
lung
wei
ght a
nd c
ytok
ine/
chem
okin
e re
spon
ses
wer
e no
t diff
eren
t bet
wee
n th
e gr
oups
. His
topa
thol
ogic
al e
xam
inat
ion,
a
hydr
osta
tic lu
ng te
st a
nd b
lood
gas
ana
lysi
s sh
owed
pos
itive
resu
lts in
the
com
bina
tion
ther
apy
grou
p. C
oncl
usio
ns/
Sign
ifica
nce:
Com
bina
tion
ther
apy
of la
nina
miv
ir o
ctan
oate
with
art
ifici
al s
urfa
ctan
t red
uces
leth
ality
in m
ice
infe
cted
w
ith in
fluen
za v
irus
, and
eve
ntua
lly s
uppr
esse
s D
AD
form
atio
n an
d pr
eser
ves
lung
func
tion.
Thi
s co
mbi
natio
n co
uld
be e
ffect
ive
for
prev
entio
n of
sev
ere
pneu
mon
ia s
econ
dary
to in
fluen
za v
irus
infe
ctio
n in
hum
ans,
whi
ch is
not
im
prov
ed b
y N
AI m
onot
hera
py.
Fuku
shi e
t al.
2012
ose
ltam
ivir
wit
h ke
toti
fen
Mas
t cel
l deg
ranu
latio
n in
hibi
tor
In v
itro/
in v
ivo
test
s
Alth
ough
an
impo
rtan
t rol
e fo
r m
ast c
ells
in s
ever
al v
iral
infe
ctio
ns h
as b
een
dem
onst
rate
d, it
s ro
le in
the
inva
sion
of
hig
hly
path
ogen
ic H
5N1
influ
enza
vir
us is
unk
now
n. In
the
pres
ent s
tudy
, we
dem
onst
rate
that
mas
t cel
ls w
ere
activ
ated
sig
nific
antly
by
H5N
1 vi
rus
(A/c
hick
en/H
enan
/1/2
004)
infe
ctio
n bo
th in
viv
o an
d in
vitr
o. M
ast c
ells
cou
ld
poss
ibly
inte
nsify
the
lung
inju
ry th
at re
sults
from
H5N
1 in
fect
ion
by re
leas
ing
proi
nfla
mm
ator
y m
edia
tors
, inc
lud-
ing
hist
amin
e, tr
ypta
se, a
nd g
amm
a in
terf
eron
(IFN
-gam
ma)
. Lun
g le
sion
s an
d ap
opto
sis
indu
ced
by H
5N1
infe
ctio
n w
ere
redu
ced
dram
atic
ally
by
trea
tmen
t with
ket
otife
n, w
hich
is a
mas
t cel
l deg
ranu
latio
n in
hibi
tor.
A c
ombi
natio
n of
ke
totif
en a
nd th
e ne
uram
inid
ase
inhi
bito
r os
elta
miv
ir p
rote
cted
100
% o
f the
mic
e fr
om d
eath
pos
tinfe
ctio
n. In
con
-cl
usio
n, o
ur d
ata
sugg
est t
hat m
ast c
ells
pla
y a
cruc
ial r
ole
in th
e ea
rly
stag
es o
f H5N
1 in
fluen
za v
irus
infe
ctio
n an
d pr
ovid
e a
new
app
roac
h to
com
bat h
ighl
y pa
thog
enic
influ
enza
vir
us in
fect
ion.
Hu
et a
l. 20
12
dua
l-ta
rget
ed
bifu
ncti
onal
ant
i-in
fluen
za d
rugs
Ant
i-infl
amm
ator
y eff
ect
Caff
eic
acid
-zan
amiv
ir
conj
ugat
eIn
viv
o/in
vitr
o te
sts
Influ
enza
ther
apy
with
a s
ingl
e ta
rget
ed c
ompo
und
is o
ften
lim
ited
in e
ffic
acy
due
to th
e ra
pidl
y de
velo
ped
drug
resi
st-
ance
. Mor
eove
r, th
e un
cont
rolle
d vi
rus-
indu
ced
cyto
kine
s co
uld
caus
e th
e hi
gh m
orta
lity
of h
uman
infe
cted
by
H5N
1 av
ian
influ
enza
vir
us. I
n th
is s
tudy
, we
expl
ored
the
nove
l dua
l-ta
rget
ed b
ifunc
tiona
l ant
i-in
fluen
za d
rugs
form
ed b
y co
njug
atio
n w
ith a
nti-
infla
mm
ator
y ag
ents
. In
part
icul
ar, t
he c
affe
ic a
cid
(CA
)-be
arin
g za
nam
ivir
(ZA
) con
juga
tes
ZA
-7-C
A (1
) and
ZA
-7-C
A-a
mid
e (7
) sho
wed
sim
ulta
neou
s in
hibi
tion
of in
fluen
za v
irus
neu
ram
inid
ase
and
supp
res-
sion
of p
ro-i
nfla
mm
ator
y cy
toki
nes.
The
se Z
A c
onju
gate
s pr
ovid
ed re
mar
kabl
e pr
otec
tion
of c
ells
and
mic
e ag
ains
t in
fluen
za in
fect
ions
. Int
rana
sal a
dmin
istr
atio
n of
low
dos
age
(<1.
2 m
u m
ol/k
g/da
y) o
f ZA
con
juga
tes
exhi
bite
d m
uch
grea
ter
effe
ct th
an th
e co
mbi
natio
n th
erap
y w
ith Z
A a
nd th
e an
ti-in
flam
mat
ory
agen
ts in
pro
tect
ion
of th
e le
thal
ly
infe
cted
mic
e by
H1N
1 or
H5N
1 in
fluen
za v
irus
es.
Liu
et a
l. 20
12
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
178
neu
ram
inid
ase
inhi
bito
rs w
ith
rim
anta
dine
Add
itive
and
syne
rgis
tic
effec
tsIn
vitr
o te
sts
The
re is
insu
ffici
ent i
nfor
mat
ion
abou
t com
bina
tion
ther
apy
with
app
rove
d an
ti-in
fluen
za a
gent
s. W
e te
sted
com
bina
-tio
ns th
at p
aire
d a
neur
amin
idas
e (N
A) i
nhib
itor (
zana
miv
ir, o
selta
miv
ir c
arbo
xyla
te, o
r per
amiv
ir) w
ith ri
man
tadi
ne
agai
nst i
nfec
tion
of M
DC
K c
ells
with
H1N
1 an
d H
3N2
subt
ypes
of i
nflue
nza
A v
irus
and
cha
ract
eriz
ed th
eir m
ode
of
inte
ract
ion.
Whe
n re
duct
ion
of e
xtra
cellu
lar v
irus
was
ana
lyze
d by
indi
vidu
al re
gres
sion
mod
els a
nd th
ree-
dim
ensi
onal
re
pres
enta
tions
of t
he d
ata,
all
thre
e co
mbi
natio
ns sh
owed
add
itive
and
syne
rgis
tic e
ffect
s with
no
cyto
toxi
city
. Max
imum
sy
nerg
y ag
ains
t A/N
ew C
aled
onia
/20/
99 (H
1N1)
vir
us in
fect
ion
was
obs
erve
d w
ith <
2.5
µM
rim
anta
dine
pai
red
with
low
co
ncen
trat
ions
of N
A in
hibi
tors
. All
com
bina
tions
redu
ced
the
extr
acel
lula
r yie
ld o
f A/P
anam
a/20
07/9
9 (H
3N2)
influ
enza
vi
rus s
yner
gist
ical
ly. H
owev
er, o
ur fi
ndin
gs w
ere
diffe
rent
for t
he c
ell-a
ssoc
iate
d vi
rus y
ield
. At s
ome
drug
con
cent
ratio
ns,
the
yiel
d of
cel
l-ass
ocia
ted
viru
s was
inhi
bite
d an
tago
nist
ical
ly. Th
eref
ore,
the
met
hod
of a
naly
sis c
an b
e a
cruc
ial f
acto
r in
eval
uatin
g th
e in
tera
ctio
ns o
f dru
gs w
ith d
iffer
ent m
echa
nism
s. W
e hy
poth
esiz
e th
at a
ssay
s bas
ed o
n ce
ll-as
soci
ated
vir
us
yiel
d m
ay u
nder
estim
ate
the
effica
cies
of d
rug
com
bina
tions
that
incl
ude
an N
A in
hibi
tor.
Take
n to
geth
er, o
ur re
sults
su
gges
t tha
t reg
imen
s tha
t com
bine
NA
inhi
bito
rs a
nd ri
man
tadi
ne e
xert
syne
rgis
tic a
nti-
influ
enza
effe
cts i
n vi
tro.
Thes
e fin
ding
s pro
vide
bas
elin
e in
form
atio
n fo
r the
rape
utic
test
ing
of th
e dr
ug c
ombi
natio
ns in
viv
o.
Gov
orko
va e
t al.
2004
Trip
le-c
ombi
nati
on
anti
vira
l dru
g th
erap
yA
man
tadi
neRi
bavi
rin
Ose
ltam
ivir
Adu
lt pa
tient
s
A re
cent
in v
itro
stud
y sh
owed
that
the
thre
e co
mpo
unds
of a
ntiv
iral
dru
gs w
ith d
iffer
ent m
echa
nism
s of a
ctio
n (a
man
ta-
dine
, rib
avir
in, a
nd o
selta
miv
ir) c
ould
resu
lt in
syne
rgis
tic a
ntiv
iral
act
ivity
aga
inst
influ
enza
vir
us. H
owev
er, n
o cl
inic
al
stud
ies h
ave
eval
uate
d th
e effi
cacy
and
safe
ty o
f com
bina
tion
antiv
iral
ther
apy
in p
atie
nts w
ith se
vere
influ
enza
illn
ess.
A
tota
l of 2
45 a
dult
patie
nts w
ho w
ere
criti
cally
ill w
ith c
onfir
med
pan
dem
ic in
fluen
za A
/H1N
1 20
09 (p
H1N
1) v
irus
infe
c-tio
n an
d w
ere
adm
itted
to o
ne o
f the
inte
nsiv
e ca
re u
nits
of 2
8 ho
spita
ls in
Kor
ea w
ere
revi
ewed
. Pat
ient
s who
requ
ired
ve
ntila
tor s
uppo
rt a
nd re
ceiv
ed e
ither
trip
le-c
ombi
natio
n an
tivir
al d
rug
(TC
AD
) the
rapy
or o
selta
miv
ir m
onot
hera
py
wer
e an
alyz
ed. A
tota
l of 1
27 p
atie
nts w
ere
incl
uded
in o
ur a
naly
sis.
Am
ong
them
, 24
patie
nts r
ecei
ved
TCA
D th
erap
y, an
d 10
3 pa
tient
s rec
eive
d os
elta
miv
ir m
onot
hera
py. Th
e 14
-day
mor
talit
y w
as 1
7% in
the
TCA
D g
roup
and
35%
in th
e os
elta
miv
ir g
roup
(P =
0.0
8), a
nd th
e 90
-day
mor
talit
y w
as 4
6% in
the
TCA
D g
roup
and
59%
in th
e os
elta
miv
ir g
roup
(P
= 0
.23)
. Non
e of
the
toxi
citie
s att
ribu
tabl
e to
ant
ivir
al d
rugs
occ
urre
d in
eith
er g
roup
of o
ur st
udy,
incl
udin
g he
mol
ytic
an
emia
and
hep
atic
toxi
citie
s rel
ated
to th
e us
e of
riba
viri
n. L
ogis
tic re
gres
sion
ana
lysi
s ind
icat
ed th
at th
e od
ds ra
tio fo
r th
e as
soci
atio
n of
TC
AD
with
90-
day
mor
talit
y w
as 0
.58
(95%
con
fiden
ce in
terv
al, 0
.24
to 1
.42;
P =
0.2
4). A
lthou
gh th
is
stud
y w
as re
tros
pect
ive
and
did
not p
rovi
de v
irol
ogic
out
com
es, o
ur re
sults
sugg
est t
hat t
he tr
eatm
ent o
utco
me
of th
e tr
iple
com
bina
tion
of a
man
tadi
ne, r
ibav
irin
, and
ose
ltam
ivir
was
com
para
ble
to th
at o
f ose
ltam
ivir
mon
othe
rapy
.
Kim
et a
l. 20
11
Trip
le-c
ombi
nati
on
anti
vira
l dru
g th
erap
yA
man
tadi
neRi
bavi
rin
Ose
ltam
ivir
Dos
e-de
pend
ent
prot
ectio
nSy
nerg
istic
effe
ctBr
oad-
spec
trum
act
ivity
Mou
se m
odel
The
lim
ited
effic
acy
of e
xist
ing
antiv
iral
ther
apie
s fo
r in
fluen
za –
cou
pled
with
wid
espr
ead
base
line
antiv
iral
resi
stan
ce
– hi
ghlig
hts
the
urge
nt n
eed
for
mor
e ef
fect
ive
ther
apy.
We
desc
ribe
a tr
iple
com
bina
tion
antiv
iral
dru
g (T
CA
D) r
egi-
men
com
pose
d of
am
anta
dine
, ose
ltam
ivir,
and
rib
avir
in th
at is
hig
hly
effic
acio
us a
t red
ucin
g m
orta
lity
and
wei
ght l
oss
in m
ouse
mod
els
of in
fluen
za in
fect
ion.
TC
AD
ther
apy
was
sup
erio
r to
dua
l and
sin
gle
drug
regi
men
s in
mic
e in
fect
ed
with
dru
g-su
scep
tible
, low
pat
hoge
nic
A/H
5N1
(A/D
uck/
MN
/152
5/81
) and
am
anta
dine
-res
ista
nt 2
009
A/H
1N1
influ
-en
za (A
/Cal
iforn
ia/0
4/09
). Tr
eatm
ent w
ith T
CA
D a
fford
ed >
90%
sur
viva
l in
mic
e in
fect
ed w
ith b
oth
viru
ses,
whe
reas
tr
eatm
ent w
ith d
ual a
nd s
ingl
e dr
ug re
gim
ens
resu
lted
in 0
% to
60%
sur
viva
l. Im
port
antly
, am
anta
dine
had
no
activ
ity
as m
onot
hera
py a
gain
st th
e am
anta
dine
-res
ista
nt v
irus
, but
dem
onst
rate
d do
se-d
epen
dent
pro
tect
ion
in c
ombi
natio
n w
ith o
selta
miv
ir a
nd r
ibav
irin
, ind
icat
ive
that
am
anta
dine
’s ac
tivity
had
bee
n re
stor
ed in
the
cont
ext o
f TC
AD
ther
apy.
Furt
herm
ore,
TC
AD
ther
apy
prov
ided
sur
viva
l ben
efit
whe
n tr
eatm
ent w
as d
elay
ed u
ntil
72 h
ours
pos
t-in
fect
ion,
w
here
as o
selta
miv
ir m
onot
hera
py w
as n
ot p
rote
ctiv
e af
ter
24 h
ours
pos
t-in
fect
ion.
The
se fi
ndin
gs d
emon
stra
te in
viv
o ef
ficac
y of
TC
AD
ther
apy
and
conf
irm
pre
viou
s re
port
s of
the
syne
rgy
and
broa
d sp
ectr
um a
ctiv
ity o
f TC
AD
ther
apy
agai
nst s
usce
ptib
le a
nd re
sist
ant i
nflu
enza
str
ains
in v
itro.
Ngu
yen
et a
l. 20
12
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
179
Com
bina
tion
of
favi
pira
vir
and
osel
tam
ivir
Mou
se m
odel
Favi
pira
vir
(T-7
05 [6
-flu
oro-
3-hy
drox
y-2-
pyra
zine
carb
oxam
ide]
) and
ose
ltam
ivir
wer
e co
mbi
ned
to tr
eat i
nflu
enza
vi
rus
A/N
WS/
33 (H
1N1)
, A/V
icto
ria/
3/75
(H3N
2), a
nd A
/Duc
k/M
N/1
525/
81 (H
5N1)
infe
ctio
ns. T
-705
alo
ne in
hibi
ted
viru
ses
in c
ell c
ultu
re a
t 1.4
to 4
.3 µ
M. O
selta
miv
ir in
hibi
ted
thes
e th
ree
viru
ses
in c
ells
at 3
.7, 0
.02,
and
0.1
6 µM
and
in
neu
ram
inid
ase
assa
ys a
t 0.9
4, 0
.46,
and
2.3
1 nM
, res
pect
ivel
y. O
ral t
reat
men
ts w
ere
give
n tw
ice
daily
to m
ice
for
5 to
7
days
sta
rtin
g, g
ener
ally
, 24
h af
ter
infe
ctio
n. S
urvi
val r
esul
ting
from
5 d
ays
of o
selta
miv
ir tr
eatm
ent (
0.1
and
0.3
mg/
kg/d
ay) w
as s
igni
fican
tly b
ette
r in
com
bina
tion
with
20
mg/
kg o
f bod
y w
eigh
t/da
y of
T-7
05 a
gain
st th
e H
1N1
infe
ctio
n.
Trea
tmen
t of t
he H
3N2
infe
ctio
n re
quir
ed 5
0 m
g/kg
/day
of o
selta
miv
ir fo
r 7
days
to a
chie
ve 6
0% p
rote
ctio
n; 2
5 m
g/kg
/da
y w
as in
effe
ctiv
e. T
-705
was
≥ 7
0% p
rote
ctiv
e at
50
to 1
00 m
g/kg
/day
but
inac
tive
at 2
5 m
g/kg
/day
. The
com
bina
tion
of in
hibi
tors
(25
mg/
kg/d
ay e
ach)
incr
ease
d su
rviv
al to
90%
. The
H5N
1 in
fect
ion
was
not
ben
efite
d by
trea
tmen
t with
os
elta
miv
ir (≤
100
mg/
kg/d
ay fo
r 7
days
). T-
705
was
30
to 7
0% p
rote
ctiv
e at
25
to 1
00 m
g/kg
/day
. Sur
viva
l im
prov
ed
slig
htly
with
com
bina
tion
trea
tmen
ts. I
ncre
ased
act
ivity
was
see
n ag
ains
t H5N
1 in
fect
ion
by s
tart
ing
trea
tmen
ts 2
h
befo
re in
fect
ion.
Ose
ltam
ivir
was
inef
fect
ive
at ≤
40
mg/
kg/d
ay. T
-705
was
100
% p
rote
ctiv
e at
40
and
80 m
g/kg
/day
and
in
activ
e at
20
mg/
kg/d
ay. C
ombi
ning
inef
fect
ive
dose
s (2
0 m
g/kg
/day
of T
-705
and
10
to 4
0 m
g/kg
/day
of o
selta
miv
ir)
affo
rded
60
to 8
0% p
rote
ctio
n an
d im
prov
ed b
ody
wei
ghts
dur
ing
infe
ctio
n. T
hus,
syn
ergi
stic
resp
onse
s w
ere
achi
eved
w
ith lo
w d
oses
of T
-705
com
bine
d w
ith o
selta
miv
ir. T
hese
com
poun
ds m
ay b
e vi
able
can
dida
tes
for
com
bina
tion
trea
t-m
ent o
f hum
an in
fluen
za in
fect
ions
.
Smee
et a
l. 20
10
ose
ltam
ivir
wit
h za
nam
ivir
Tran
smis
sion
in
hous
ehol
ds
Back
grou
nd: T
he e
ffect
iven
ess
of n
eura
min
idas
e in
hibi
tors
to re
duce
tran
smis
sion
whe
n us
ed a
s tr
eatm
ent i
n in
flu-
enza
-inf
ecte
d pa
tient
s re
mai
ns d
ebat
ed. M
etho
ds: I
n a
pres
peci
fied
anal
ysis
of a
blin
ded
rand
omiz
ed c
ontr
olle
d tr
ial
on th
e ef
ficac
y of
ose
ltam
ivir
-zan
amiv
ir c
ombi
natio
n th
erap
y ve
rsus
ose
ltam
ivir
and
zan
amiv
ir m
onot
hera
py c
on-
duct
ed d
urin
g th
e 20
08–2
009
seas
onal
influ
enza
epi
dem
ic, w
e co
mpa
red
the
rate
of s
econ
dary
illn
ess
in h
ouse
hold
co
ntac
ts o
f inf
luen
za-p
ositi
ve in
dex
patie
nts
betw
een
arm
s. S
econ
dary
illn
ess
was
def
ined
as
occu
rren
ce in
con
tact
s of
feve
r pl
us c
ough
with
in 7
day
s fr
om r
ando
miz
atio
n of
inde
x pa
tient
s. A
naly
ses
wer
e co
nduc
ted
acco
rdin
g to
the
dela
y be
twee
n pa
tient
s’ on
set o
f sym
ptom
s an
d in
terv
entio
n. R
esul
ts: A
tota
l of 5
43 h
ouse
hold
con
tact
s of
267
inde
x pa
tient
s w
ere
incl
uded
, of w
hich
466
had
follo
w-u
p as
sess
men
t. A
sec
onda
ry il
lnes
s w
as re
port
ed in
58
(12.
5%) c
on-
tact
s w
ith n
o si
gnifi
cant
diff
eren
ce b
etw
een
arm
s ov
eral
l (P
= 0.
07).
Whe
n th
e an
alys
is w
as li
mite
d to
the
232
cont
acts
of
136
inde
x pa
tient
s w
ith fi
rst t
reat
men
t int
ake
with
in 2
4 h
of o
nset
of s
ympt
oms,
a lo
wer
rat
e of
sec
onda
ry il
lnes
s w
as re
port
ed in
the
com
bina
tion
ther
apy
arm
(2 o
f 56
[4%
]) th
an in
the
osel
tam
ivir
arm
(14
of 8
1 [1
7%];
P =
0.01
4) a
nd
the
zana
miv
ir a
rm (1
4 of
95
[15%
]; P
= 0.
031)
. Mul
tivar
iate
ana
lysi
s ac
coun
ting
for
intr
a-ho
useh
old
corr
elat
ion
con-
firm
ed th
ese
findi
ngs.
Con
clus
ions
: Our
ana
lysi
s su
gges
ts a
gre
ater
effe
ctiv
enes
s of
the
com
bina
tion
ther
apy
to re
duce
tr
ansm
issi
bilit
y w
hen
give
n to
the
inde
x pa
tient
with
in 2
4 h
of o
nset
of s
ympt
oms.
As
the
findi
ng w
as o
btai
ned
from
a
subg
roup
ana
lysi
s, it
sho
uld
be in
terp
rete
d w
ith c
autio
n.
Car
rat e
t al.
2012
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
180
Tabl
e 6.
Infl
uenz
a dr
ug r
esis
tanc
e
Mec
hani
sm o
f dru
g re
sist
ance
Neu
ram
inid
ase
mut
ants
Tam
iflu
Rele
nza
New
mut
ants
of h
uman
influ
enza
vir
us (A
/H1N
1) e
xhib
it re
sist
ance
to a
ntiv
iral
dru
gs. T
he m
echa
nism
whe
reby
they
de
velo
p in
sens
itivi
ty to
thes
e m
edic
atio
ns is
, how
ever
, not
yet
com
plet
ely
unde
rsto
od. A
cry
stal
logr
aphi
c st
ruct
ure
of
A/H
1N1
neur
amin
idas
e ha
s be
en p
ublis
hed
rece
ntly
. Usi
ng m
olec
ular
dyn
amic
sim
ulat
ions
, it i
s no
w p
ossi
ble
to c
har-
acte
rize
at t
he a
tom
ic le
vel t
he m
echa
nism
that
und
erlie
s th
e lo
ss o
f bin
ding
aff
inity
of t
he d
rugs
. In
this
stu
dy, f
ree-
ener
gy p
ertu
rbat
ion
was
use
d to
eva
luat
e th
e re
lativ
e bi
ndin
g fr
ee e
nerg
ies
of T
amifl
u an
d Re
lenz
a w
ith H
274Y
, N29
4S,
and
Y252
H n
eura
min
idas
e m
utan
ts. O
ur re
sults
dem
onst
rate
a re
mar
kabl
e co
rrel
atio
n be
twee
n th
eore
tical
and
exp
eri-
men
tal d
ata,
whi
ch q
uant
itativ
ely
conf
irm
s th
at th
e m
utan
ts a
re re
sist
ant t
o Ta
mifl
u bu
t are
stil
l str
ongl
y in
hibi
ted
by
Rele
nza.
The
sim
ulat
ions
furt
her
reve
al th
e ke
y in
tera
ctio
ns th
at g
over
n th
e af
finity
of t
he tw
o dr
ugs
for
each
mut
ant.
Thi
s in
form
atio
n is
env
isio
ned
to p
rove
use
ful f
or th
e de
sign
of n
ovel
neu
ram
inid
ase
inhi
bito
rs a
nd fo
r th
e ch
arac
teri
-za
tion
of n
ew p
oten
tial m
utan
ts.
Verg
ara-
Jaqu
e et
al
. 201
2
Res
ista
nce
to z
anam
ivir
an
d os
elta
miv
irM
olec
ular
mar
kers
of
resi
stan
cePy
rose
quen
cing
We
repo
rt h
ere
the
desi
gn o
f a p
yros
eque
ncin
g ap
proa
ch fo
r th
e de
tect
ion
of m
olec
ular
mar
kers
of r
esis
tanc
e to
the
neur
amin
idas
e in
hibi
tors
zan
amiv
ir a
nd o
selta
miv
ir in
influ
enza
vir
uses
of t
ype
B. P
rim
ers
wer
e de
sign
ed to
ana
lyze
the
sequ
ence
s at
eig
ht a
min
o ac
id p
ositi
ons
E119
, R15
2, D
198.
122
2, S
250,
H27
4, R
371,
and
G40
2 (u
nive
rsal
A/N
2 nu
mbe
r-in
g) in
the
neur
amin
idas
e (N
A) w
hich
hav
e be
en p
revi
ousl
y fo
und
to b
e as
soci
ated
with
resi
stan
ce o
r re
duce
d su
scep
ti-bi
lity
to o
selta
miv
ir a
nd/o
r za
nam
ivir
in th
e N
A in
hibi
tion
assa
y. In
add
ition
, the
des
igne
d pr
imer
s co
uld
be u
tiliz
ed to
th
e di
stin
guis
h be
twee
n th
e N
As
of in
fluen
za B
vir
uses
from
the
two
maj
or li
neag
es (V
icto
ria
and
Yam
agat
a) th
at h
ave
co-c
ircu
late
d gl
obal
ly in
rece
nt y
ears
, thu
s pr
ovid
ing
a va
luab
le to
ol fo
r vi
rus
stra
in s
urve
illan
ce.
Sheu
et a
l. 20
10
Res
ista
nce
to
osel
tam
ivir
Five
yea
rs o
f non
-pr
escr
iptio
n os
elta
miv
irN
ew Z
eala
nd
In 2
007
New
Zea
land
(NZ
) bec
ame
the
first
cou
ntry
to m
ake
osel
tam
ivir
(Tam
iflu)
ava
ilabl
e of
f-pr
escr
iptio
n. T
his
stud
y in
vest
igat
ed th
e ex
tent
of p
harm
acis
t sup
ply
of o
selta
miv
ir o
ver
5 ye
ars,
incl
udin
g du
ring
the
influ
enza
A(H
1N1)
pan
-de
mic
, and
the
impa
ct o
f pha
rmac
ist s
uppl
y of
ose
ltam
ivir
on
influ
enza
vir
us o
selta
miv
ir s
usce
ptib
ility
, per
sona
l sto
ck-
pilin
g an
d in
fluen
za v
acci
ne u
ptak
e. R
ando
mly
sel
ecte
d co
mm
unity
pha
rmac
ies
in N
Z re
port
ed o
selta
miv
ir p
rovi
sion
by
pres
crip
tion
and
thro
ugh
phar
mac
ist s
uppl
y fr
om 1
Janu
ary
2007
to 1
5 Se
ptem
ber
2011
. Ose
ltam
ivir
resi
stan
ce d
ata
on
influ
enza
vir
uses
isol
ated
dur
ing
influ
enza
sur
veill
ance
from
200
8 to
201
1 w
ere
obta
ined
, alo
ng w
ith in
fluen
za v
acci
ne
upta
ke d
ata
from
200
5 to
201
1 an
d in
fluen
za d
etec
tion
data
. Sev
enty
of 8
5 el
igib
le p
harm
acie
s co
mpl
eted
the
stud
y (8
2 re
spon
se r
ate)
. Mos
t sup
plie
s of
ose
ltam
ivir
thro
ugho
ut th
e 5
year
s w
ere
disp
ense
d ag
ains
t a p
resc
ript
ion
rath
er th
an
phar
mac
ist s
uppl
ied,
with
pha
rmac
ist s
uppl
y re
spon
sibl
e fo
r 11
of s
uppl
ies
duri
ng th
e pa
ndem
ic y
ears
(200
9–20
10)
vers
us 2
7 an
d 31
dur
ing
2007
and
200
8, re
spec
tivel
y. P
harm
acis
t-su
pplie
d os
elta
miv
ir d
id n
ot a
ppea
r to
be
asso
ciat
ed
with
the
deve
lopm
ent o
f res
ista
nce,
with
iden
tifie
d lik
ely
stoc
kpili
ng o
r w
ith a
dec
line
in in
fluen
za im
mun
izat
ion.
Pha
r-m
acis
t sup
plie
s la
rgel
y m
atch
ed th
e tim
ing
of in
fluen
za in
the
com
mun
ity a
nd p
eake
d in
June
200
9, a
s di
d pr
escr
iptio
n su
pplie
s. F
ive
year
s of
non
-pre
scri
ptio
n os
elta
miv
ir in
NZ
has
resu
lted
in n
o si
gnifi
cant
cha
nge
in th
e de
velo
pmen
t of
resi
stan
ce o
r ra
tes
of in
fluen
za im
mun
izat
ion.
Sup
plie
s re
mai
ned
mod
est a
nd s
igni
fican
t con
sum
er s
tock
pilin
g th
roug
h ph
arm
acis
t sup
ply
has
not o
ccur
red,
eve
n du
ring
the
influ
enza
A(H
1N1)
pdm
09 p
ande
mic
in 2
009
and
2010
. Pha
rma-
cist
s co
uld
be b
ette
r ut
ilize
d in
ens
urin
g fa
st d
istr
ibut
ion
of a
ntiv
iral
s to
influ
enza
suf
fere
rs d
urin
g a
pand
emic
Gau
ld e
t al.
2012
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
181
Res
ista
nce
to
aman
tadi
ne
Am
anta
dine
trea
ted
child
ren
Sequ
enci
ng
Clin
ical
sam
ples
from
15
aman
tadi
ne-t
reat
ed c
hild
ren
wer
e co
llect
ed s
eria
lly-b
efor
e, d
urin
g, a
nd/o
r af
ter
trea
tmen
t and
w
ere
stud
ied
to d
eter
min
e th
e ac
tual
pre
vale
nce,
tim
ing,
and
clin
ical
impl
icat
ions
of M
2 m
utat
iona
l eve
nts.
Aft
er v
iral
RN
A e
xtra
ctio
n an
d re
vers
e-tr
ansc
ript
ase
poly
mer
ase
chai
n re
actio
n am
plifi
catio
n of
the
vira
l RN
A e
ncod
ing
the
M2
prot
ein,
the
prod
ucts
wer
e cl
oned
into
pla
smid
s, a
nd th
eir
sequ
ence
s w
ere
dete
rmin
ed. F
ive
mut
atio
ns k
now
n to
con
fer
aman
tadi
ne re
sist
ance
in c
linic
al s
ampl
es w
ere
iden
tifie
d in
12
(80%
) of 1
5 ev
alua
ble
patie
nts,
and
9 p
atie
nts
had
> 1
(2–4
) mut
ant v
irus
. The
pat
tern
of e
mer
genc
e of
mut
ant s
trai
ns w
as c
lari
fied
from
the
stud
y of
6 p
atie
nts
with
at l
east
4
seri
al s
ampl
es. A
lthou
gh v
irus
es w
ith M
2 m
utat
ions
tend
ed to
bec
ome
the
dom
inan
t pop
ulat
ions
, in
2 ca
ses,
wild
-ty
pe v
irus
es b
ecam
e do
min
ant a
fter
dec
reas
ing
to lo
w le
vels
. The
se re
sults
sug
gest
that
resi
stan
t vir
uses
em
erge
in a
m
uch
high
er p
ropo
rtio
n of
am
anta
dine
-tre
ated
pat
ient
s th
an h
as b
een
sugg
este
d by
pre
viou
s st
udie
s.
Shir
aish
i et a
l. 20
03
Res
ista
nce
to
aman
tadi
ne
Am
ino
acid
subs
titut
ions
In tw
o in
fluen
za s
easo
ns d
urin
g w
hich
H1N
1 an
d H
3N2
coci
rcul
ated
, res
ista
nce
was
mor
e fr
eque
nt in
H3N
2 st
rain
s th
an in
H1N
1 st
rain
s af
ter
aman
tadi
ne tr
eatm
ent.
Pred
omin
ant a
min
o ac
id s
ubst
itutio
ns in
M2
prot
ein
occu
rred
at
posi
tion
31 (s
erin
e to
asp
arag
ine)
in H
3N2
stra
ins
and
at p
ositi
on 2
7 (v
alin
e to
ala
nine
) in
H1N
1 st
rain
s.
Saito
et a
l. 20
03
Res
ista
nce
to
adam
anta
nes
281
influ
enza
isol
ates
Aus
tral
iaEu
rope
Asi
a
The
ada
man
tane
s (a
man
tadi
ne a
nd r
iman
tadi
ne) w
ere
the
initi
al a
ntiv
iral
s lic
ense
d fo
r us
e ag
ains
t inf
luen
za A
vir
uses
an
d ha
ve b
een
used
in s
ome
coun
trie
s to
con
trol
sea
sona
l inf
luen
za a
nd h
ave
also
bee
n st
ockp
iled
for
pote
ntia
l pan
-de
mic
use
. Whi
le h
igh
rate
s of
resi
stan
ce h
ave
been
obs
erve
d in
rece
nt y
ears
with
A(H
3) v
irus
es, t
he r
ates
of r
esis
t-an
ce w
ith A
(H1)
vir
uses
has
var
ied
wid
ely.
In th
is s
tudy
we
anal
ysed
281
hum
an in
fluen
za A
vir
uses
isol
ated
in 2
007
that
wer
e re
ferr
ed to
the
WH
O C
olla
bora
ting
Cen
tre
for
Refe
renc
e an
d Re
sear
ch in
Mel
bour
ne, m
ainl
y fr
om A
us-
tral
ia a
nd th
e su
rrou
ndin
g re
gion
s, fo
r ev
iden
ce o
f res
ista
nce
to a
dam
anta
nes
and
a su
bset
of t
hese
was
exa
min
ed fo
r re
sist
ance
to th
e ne
uram
inid
ase
inhi
bito
rs (N
Is).
We
foun
d th
at th
e ra
tes
of a
cdam
anta
ne re
sist
ance
in A
(H3)
vir
uses
co
ntin
ued
to in
crea
se in
mos
t cou
ntri
es in
200
7 bu
t a d
istin
ct v
aria
tion
was
see
n w
ith A
(H1)
resi
stan
ce le
vels
. A(H
1)
viru
ses
from
Aus
tral
ia, N
ew Z
eala
nd a
nd E
urop
e ha
d lo
w r
ates
of r
esis
tanc
e (2
–9%
) whe
reas
vir
uses
from
a n
umbe
r of
Sou
th E
ast (
SE) A
sian
cou
ntri
es h
ad h
igh
rate
s of
resi
stan
ce (3
3–10
0%).
Thi
s di
ffere
nce
can
be a
ttri
bute
d to
the
spre
ad o
f A/B
risb
ane/
59/2
007-
like
viru
ses
to m
any
part
s of
the
wor
ld w
ith th
e ex
cept
ion
of S
E A
sia
whe
re A
/Hon
g K
ong/
2652
/200
6-lik
e vi
ruse
s co
ntin
ue to
pre
dom
inat
e. W
hen
thes
e tw
o A
(H1)
sub
grou
ps w
ere
com
pare
d fo
r th
eir
in
vitr
o se
nsiti
vity
to th
e ot
her
clas
s of
influ
enza
ant
ivir
al d
rugs
, the
neu
ram
inid
ase
inhi
bito
rs, n
o di
ffere
nce
was
see
n be
twee
n th
e gr
oups
with
bot
h sh
owin
g no
rmal
leve
ls o
f sen
sitiv
ity to
thes
e dr
ugs,
The
find
ing
of re
duci
ng A
(H1)
resi
st-
ance
rat
es in
Aus
tral
ia a
nd r
isin
g le
vels
in S
E A
sia
in 2
007,
reve
rses
the
tren
d se
en in
200
6 w
hen
A(H
1) re
sist
ance
leve
ls
wer
e ri
sing
in A
ustr
alia
and
els
ewhe
re b
ut re
mai
ned
low
in m
ost o
f SE
Asi
a.
Barr
et a
l. 20
08
Res
ista
nce
to
adam
anta
nes
and
neur
amin
idas
e in
hibi
tors
Cen
tral
/Sou
th A
mer
ica
Back
grou
nd R
ecen
t inf
luen
za a
ntiv
iral
resi
stan
ce s
tudi
es re
veal
an
alar
min
g in
crea
se in
bot
h ad
aman
tane
s an
d ne
u-ra
min
idas
e in
hibi
tors
(NA
Is) r
esis
tant
vir
al s
trai
ns w
orld
wid
e, p
artic
ular
ly in
Asi
a, E
urop
e an
d th
e U
nite
d St
ates
. O
bjec
tives
In th
is s
tudy
, we
have
eva
luat
ed in
fluen
za v
irus
resi
stan
ce in
Cen
tral
and
Sou
th A
mer
ica.
Met
hods
Influ
enza
vi
ruse
s, is
olat
ed fr
om s
ympt
omat
ic p
atie
nts
thro
ugho
ut C
entr
al a
nd S
outh
Am
eric
a in
200
5–20
08 w
ere
anal
yzed
for
inhi
bito
r re
sist
ance
. The
M2
and
NA
gen
es o
f inf
luen
za v
irus
es w
ere
sequ
ence
d an
d re
sist
ance
was
infe
rred
by
com
-pa
riso
n w
ith p
ublis
hed
sequ
ence
s an
d kn
own
resi
stan
t mut
atio
ns. R
esul
ts O
ur re
sults
indi
cate
that
: (i)
resi
stan
ce to
ad
aman
tane
s w
as s
een
in th
e m
ajor
ity (9
5.5%
) of t
he in
fluen
za A
/H3N
2 is
olat
es b
ut o
nly
in o
ne is
olat
e of
the
influ
enza
A
/H1N
1 vi
ruse
s; (i
i) re
sist
ance
to N
AIs
beg
an to
be
dete
cted
in A
/H1N
1 is
olat
es fr
om C
entr
al A
mer
ica
in 2
008;
and
(ii
i) no
ne o
f the
influ
enza
B v
irus
es a
naly
zed
wer
e re
sist
ant t
o N
AIs
. Con
clus
ions
The
se fi
ndin
gs s
ugge
st a
lim
ited
effe
c-tiv
enes
s of
influ
enza
inhi
bito
rs d
ue to
the
dete
ctio
n of
resi
stan
ce a
mon
g A
/H1
and
A/H
3 vi
ruse
s.
Gar
cia
et a
l. 20
09
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
182
Res
ista
nce
to
adam
anta
nes
Pyro
sequ
enci
ng
Back
grou
nd a
dam
anta
nes
have
bee
n us
ed to
trea
t inf
luen
za A
vir
us in
fect
ions
for
man
y ye
ars.
Stu
dies
hav
e sh
own
a lo
w
inci
denc
e of
resi
stan
ce to
thes
e dr
ugs
amon
g ci
rcul
atin
g in
fluen
za v
irus
es; h
owev
er, t
heir
use
is r
isin
g w
orld
wid
e an
d dr
ug re
sist
ance
has
bee
n re
port
ed a
mon
g in
fluen
za A
(H5N
1) v
irus
es is
olat
ed fr
om p
oultr
y an
d hu
man
bei
ngs
in A
sia.
W
e so
ught
to a
sses
s ad
aman
tane
resi
stan
ce a
mon
g in
fluen
za A
vir
uses
isol
ated
dur
ing
the
past
dec
ade
from
cou
ntri
es
part
icip
atin
g in
WH
O’s
glob
al in
fluen
za s
urve
illan
ce n
etw
ork.
Met
hods
We
anal
ysed
dat
a fo
r in
fluen
za fi
eld
isol
ates
th
at w
ere
obta
ined
wor
ldw
ide
and
subm
itted
to th
e W
HO
Col
labo
ratin
g C
ente
r fo
r In
fluen
za a
t the
US
Cen
ters
for
Dis
-ea
se C
ontr
ol a
nd P
reve
ntio
n be
twee
n O
ct 1
, 199
4, a
nd M
ar 3
1, 2
005.
We
used
pyr
oseq
uenc
ing,
con
firm
ator
y se
quen
ce
anal
ysis
, and
phe
noty
pic
test
ing
to d
etec
t dru
g re
sist
ance
am
ong
circ
ulat
ing
influ
enza
A H
3N2
(n =
652
4), H
1N1
(n =
58
9), a
nd H
1N2
(n =
83)
vir
uses
. Fin
ding
s M
ore
than
700
0 in
fluen
za A
fiel
d is
olat
es w
ere
scre
ened
for
spec
ific
ami-
noac
id s
ubst
itutio
ns in
the
M2
gene
kno
wn
to c
onfe
r dr
ug re
sist
ance
. Dur
ing
the
deca
de o
f sur
veill
ance
a s
igni
fican
t in
crea
se in
dru
g re
sist
ance
was
not
ed, f
rom
0.4
% in
199
4–19
95 to
12.
3% in
200
3–20
04. T
his
incr
ease
in th
e pr
opor
tion
of re
sist
ant v
irus
es w
as w
eigh
ted
heav
ily b
y th
ose
obta
ined
from
Asi
a w
ith 6
1% o
f res
ista
nt v
irus
es is
olat
ed s
ince
200
3 be
ing
from
peo
ple
in A
sia.
Inte
rpre
tatio
n O
ur d
ata
rais
e co
ncer
ns a
bout
the
appr
opri
ate
use
of a
dam
anta
nes
and
draw
at
tent
ion
to th
e im
port
ance
of t
rack
ing
the
emer
genc
e an
d sp
read
of d
rug-
resi
stan
t inf
luen
za A
vir
uses
.
Brig
ht e
t al.
2005
Vira
l fitn
ess
and
tran
smis
sibi
lity
Neu
ram
inid
ase
mut
atio
nsO
selta
miv
ir c
arbo
xyla
te
Lim
ited
antiv
iral
com
poun
ds a
re a
vaila
ble
for
the
cont
rol o
f inf
luen
za, a
nd th
e em
erge
nce
of re
sist
ant v
aria
nts
wou
ld
furt
her
narr
ow th
e op
tions
for
defe
nse.
The
H27
5Y n
eura
min
idas
e (N
A) m
utat
ion,
whi
ch c
onfe
rs re
sist
ance
to
osel
tam
ivir
car
boxy
late
, has
bee
n id
entif
ied
amon
g th
e se
ason
al H
1N1
and
2009
pan
dem
ic in
fluen
za v
irus
es; h
owev
er,
thos
e H
275Y
resi
stan
t var
iant
s de
mon
stra
ted
dist
inct
epi
dem
iolo
gica
l out
com
es in
hum
ans.
Spe
cific
ally
, dom
inan
ce
of th
e H
275Y
var
iant
ove
r th
e os
elta
miv
ir-s
ensi
tive
viru
ses
was
onl
y re
port
ed fo
r a
seas
onal
H1N
1 va
rian
t dur
ing
2008
–200
9. H
ere,
we
syst
emat
ical
ly a
naly
ze th
e ef
fect
of t
he H
275Y
NA
mut
atio
n on
vir
al fi
tnes
s an
d tr
ansm
issi
bil-
ity o
f A(H
1N1)
pdm
09 a
nd s
easo
nal H
1N1
influ
enza
vir
uses
. The
NA
gen
es fr
om A
(H1N
1)pd
m09
A/C
alifo
rnia
/04/
09
(CA
04),
seas
onal
H1N
1 A
/New
Cal
edon
ia/2
0/19
99 (N
ewC
al),
and
A/B
risb
ane/
59/2
007
(Bri
sban
e) w
ere
indi
vidu
-al
ly in
trod
uced
into
the
gene
tic b
ackg
roun
d of
CA
04. T
he H
275Y
mut
atio
n le
d to
redu
ced
NA
enz
yme
activ
ity, a
n in
crea
sed
Km
for
3’-s
ialy
lact
ose
or 6
’-sia
lyla
ctos
e, a
nd d
ecre
ased
infe
ctiv
ity in
muc
in-s
ecre
ting
hum
an a
irw
ay e
pith
elia
l ce
lls c
ompa
red
to th
e os
elta
miv
ir-s
ensi
tive
wild
-typ
e co
unte
rpar
ts. A
tten
uate
d pa
thog
enic
ity in
bot
h RG
-CA
04(N
A-
H27
5Y) a
nd R
G-C
A04
x B
risb
ane(
NA
-H27
5Y) v
irus
es w
as o
bser
ved
in fe
rret
s co
mpa
red
to R
G-C
A04
vir
us, a
lthou
gh
the
tran
smis
sibi
lity
was
min
imal
ly a
ffect
ed. I
n pa
ralle
l exp
erim
ents
usi
ng re
com
bina
nt B
risb
ane
viru
ses
diffe
ring
by
hem
aggl
utin
in a
nd N
A, c
ompa
rabl
e di
rect
con
tact
and
resp
irat
ory
drop
let t
rans
mis
sibi
litie
s w
ere
obse
rved
am
ong
RG-
New
Cal
(HA
,NA
), RG
-New
Cal
(HA
,NA
-H27
5Y),
RG-B
risb
ane(
HA
,NA
-H27
5Y),
and
RG-N
ewC
al(H
A) ×
Bri
sban
e(N
A-
H27
5Y) v
irus
es. O
ur re
sults
dem
onst
rate
that
, des
pite
the
H27
5Y m
utat
ion
lead
ing
to a
min
or re
duct
ion
in v
iral
fitn
ess,
th
e tr
ansm
issi
on p
oten
tials
of t
hree
diff
eren
t ant
igen
ic s
trai
ns c
arry
ing
this
mut
atio
n w
ere
com
para
ble
in th
e na
ive
ferr
et m
odel
.
Won
g et
al.
2012
Rim
anta
dine
-res
ista
nce
mut
atio
nsG
row
th c
hara
cter
istic
s an
d vi
rule
nce
The
influ
ence
of r
iman
tadi
ne-r
esis
tanc
e m
utat
ions
on
the
viru
lenc
e of
hum
an H
3N2
viru
ses
in fe
rret
s w
as e
xam
ined
. T
he s
imila
ritie
s in
vir
ulen
ce o
f the
dru
g-re
sist
ant m
utan
ts w
ith s
ingl
e am
ino
acid
sub
stitu
tions
at t
hree
diff
eren
t loc
a-tio
ns, 2
7, 3
0, a
nd 3
1, w
ithin
the
M2
sequ
ence
and
thei
r co
rres
pond
ing
sens
itive
wild
-typ
e is
olat
es c
ontr
aste
d w
ith d
if-fe
renc
es in
vir
ulen
ce b
etw
een
the
thre
e pa
irs
of v
irus
es. T
hese
dat
a pr
ovid
e fu
rthe
r ev
iden
ce th
at r
iman
tadi
ne-r
esis
tant
vi
ruse
s th
at e
mer
ge d
urin
g tr
eatm
ent o
f pat
ient
s w
ith th
e dr
ug a
re u
nalte
red
both
in th
eir
grow
th c
hara
cter
istic
s an
d vi
rule
nce.
Swee
t et a
l. 19
91
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
183
Res
ista
nce
to
neur
amin
idas
e in
hibi
tors
Con
serv
ed re
sidu
esVi
ral fi
tnes
sN
eura
min
idas
e ac
tivity
Neu
ram
inid
ase
inhi
bito
rs (N
AIs
) are
ant
ivir
als d
esig
ned
to ta
rget
con
serv
ed re
sidu
es a
t the
neu
ram
inid
ase
(NA
) enz
yme
activ
e si
te in
influ
enza
A a
nd B
vir
uses
. The
cons
erve
d re
sidu
es th
at in
tera
ct w
ith N
AIs
are
und
er se
lect
ive
pres
sure
, but
on
ly a
few
hav
e be
en li
nked
to re
sist
ance
. In
the
A/W
uhan
/359
/95
(H3N
2) re
com
bina
nt v
irus
bac
kgro
und,
we
char
acte
r-iz
ed se
ven
char
ged,
con
serv
ed N
A re
sidu
es (1
1118
, R37
1, E
227,
R15
2, R
224,
E27
6, a
nd D
151)
that
dir
ectly
inte
ract
with
th
e N
Als
but
hav
e no
t bee
n re
port
ed to
con
fer r
esis
tanc
e to
NA
Is. Th
ese
NA
resi
dues
wer
e re
plac
ed w
ith a
min
o ac
ids
that
pos
sess
side
cha
ins h
avin
g si
mila
r pro
pert
ies t
o m
aint
ain
thei
r ori
gina
l cha
rge.
The
NA
mut
atio
ns w
e in
trod
uced
si
gnifi
cant
ly d
ecre
ased
NA
act
ivity
com
pare
d to
that
of t
he A
/Wuh
an/3
59/9
5 re
com
bina
nt w
ild-t
ype
and
R292
K (a
n N
A
mut
atio
n fr
eque
ntly
repo
rted
to c
onfe
r res
ista
nce)
vir
uses
, whi
ch w
ere
anal
yzed
for c
ompa
riso
n. H
owev
er, t
he re
com
bi-
nant
vir
uses
diff
ered
in re
plic
atio
n effi
cien
cy w
hen
we
seri
ally
pas
sage
d th
em in
vitr
o; th
e gr
owth
of t
he R
118K
and
E22
7D
viru
ses w
as m
ost i
mpa
ired
. The
R224
K E
276D
, and
R37
1K m
utat
ions
con
ferr
ed re
sist
ance
to b
oth
zana
miv
ir a
nd o
selta
mi-
vir,
whi
le th
e D
151E
mut
atio
n re
duce
d su
scep
tibili
ty to
ose
ltam
ivir
onl
y (s
imila
r to
10-f
old)
and
the
R152
K m
utat
ion
did
not a
lter s
usce
ptib
ility
to e
ither
dru
g. B
ecau
se th
e R2
24K
mut
atio
n w
as g
enet
ical
ly u
nsta
ble
and
the
emer
genc
e of
the
R371
K m
utat
ion
in th
e N
2 su
btyp
e is
stat
istic
ally
unl
ikel
y, ou
r res
ults
sugg
est t
hat o
nly
the
E276
D m
utat
ion
is li
kely
to
emer
ge u
nder
sele
ctiv
e pr
essu
re. Th
e re
sults
of o
ur st
udy
may
hel
p to
opt
imiz
e th
e de
sign
of N
AIs
.
Yen
et a
l. 20
06
Viru
s tr
ansm
issi
onRe
sist
ance
to
neur
amin
idas
e in
hibi
tors
Thre
e ty
pe A
influ
enza
vir
uses
, eac
h of
whi
ch h
as a
dis
tinct
neu
ram
inid
ase-
gene
mut
atio
n an
d is
resi
stan
t to
the
neur
ami-
nida
se in
hibi
tor o
selta
miv
ir, h
ave
been
isol
ated
. Pre
viou
sly,
in th
e fe
rret
mod
el, a
n R2
92K
mut
ant o
f a ty
pe A
(H3N
2) v
irus
w
as n
ot tr
ansm
itted
und
er c
ondi
tions
in w
hich
the
wild
-typ
e vi
rus w
as tr
ansm
itted
. This
mod
el w
as u
sed
to in
vest
igat
e w
heth
er th
e E1
19V
mut
ant o
f a ty
pe A
(H3N
2) v
irus
and
the
H27
4Y m
utan
t of a
type
A (H
1N1)
vir
us w
ould
be
tran
smit-
ted
unde
r sim
ilar c
ircu
mst
ance
s. B
oth
mut
ant v
irus
es w
ere
tran
smitt
ed, a
lthou
gh th
e H
274Y
mut
ant r
equi
red
a 10
0-fo
ld-
high
er d
ose
for i
nfec
tion
of d
onor
ferr
ets a
nd w
as tr
ansm
itted
mor
e sl
owly
than
was
the
wild
type
. Bot
h th
e m
utan
t and
th
e w
ild-t
ype
viru
ses r
etai
ned
thei
r gen
otyp
ic c
hara
cter
istic
s.
Her
loch
er e
t al.
2004
Res
ista
nce
to
neur
amin
idas
e in
hibi
tors
and
ad
aman
tane
sC
ompu
tatio
nal 3
D
stru
ctur
esRe
sidu
e m
utat
ions
The
neur
amin
idas
e (N
A) a
nd M
2 pr
oton
cha
nnel
of i
nflue
nza
viru
s are
the
drug
-tar
getin
g pr
otei
ns, b
ased
on
whi
ch se
vera
l dr
ugs w
ere
deve
lope
d. H
owev
er th
ese
once
pow
erfu
l dru
gs e
ncou
nter
ed d
rug-
resi
stan
t pro
blem
to th
e H
5N1
and
H1N
1 flu
. To
addr
ess t
his p
robl
em, t
he c
ompu
tatio
nal 3
D st
ruct
ures
of N
A a
nd M
2 pr
otei
ns o
f 200
9-H
1N1
influ
enza
vir
us w
ere
built
usi
ng th
e m
olec
ular
mod
elin
g te
chni
que
and
com
puta
tiona
l che
mis
try
met
hod.
Bas
ed o
n th
e m
odel
s the
stru
ctur
e fe
atur
es o
f NA
and
M2
prot
eins
wer
e an
alyz
ed, t
he d
ocki
ng st
ruct
ures
of d
rug-
prot
ein
com
plex
es w
ere
com
pute
d, a
nd th
e re
sidu
e m
utat
ions
wer
e an
nota
ted.
The
resu
lts m
ay h
elp
to so
lve
the
drug
-res
ista
nt p
robl
em a
nd st
imul
ate
desi
gnin
g m
ore
effec
tive
drug
s aga
inst
200
9-H
1N1
influ
enza
pan
dem
ic.
Du
et a
l. 20
10
Mol
ecul
ar m
arke
rs o
f re
sist
ance
Pyro
sequ
enci
ngM
2 bl
ocke
rsN
eura
min
idas
e in
hibi
tors
In th
e pr
esen
t stu
dy, w
e de
scri
be h
ow a
pyr
oseq
uenc
ing
met
hod
can
be u
sed
to ra
pidl
y de
tect
est
ablis
hed
mol
ecul
ar m
ark-
ers o
f res
ista
nce
to M
2 bl
ocke
rs a
nd N
A in
hibi
tors
in in
fluen
za A
(H5N
1) v
irus
es. Th
e re
sidu
es L
26, V
27, A
30, S
31, a
nd
G34
in th
e M
2 pr
otei
n w
ere
targ
eted
for p
yros
eque
ncin
g. Th
e N
A re
sidu
es fo
r pyr
oseq
uenc
ing
anal
ysis
incl
uded
the
esta
b-lis
hed
mar
kers
of d
rug
resi
stan
ce (H
274
and
N29
4), a
s wel
l as r
esid
ues o
f les
s cer
tain
rele
vanc
e (V
116,
I117
, Q13
6, K
150,
an
d I2
22).
A si
ngle
pai
r of p
yro-
reve
rse
tran
scri
ptio
n (R
T)-
PCR
prim
ers w
as d
esig
ned
to a
llow
am
plifi
catio
n of
an
appr
oxi-
mat
ely
600-
nucl
eotid
e-lo
ng a
mpl
icon
of t
he N
A g
enes
of H
5N1
viru
ses f
rom
var
ious
cla
des/
subc
lade
s ass
ocia
ted
with
in
fect
ions
in h
uman
s. Th
e se
nsiti
vity
of t
he a
ssay
was
dem
onst
rate
d by
the
succ
essf
ul p
yros
eque
ncin
g of
RN
A e
xtra
cted
fr
om sa
mpl
es o
f ser
ially
dilu
ted
(10–5
to 1
0–7 v
irus
stoc
ks w
ith in
itial
con
cent
ratio
ns ra
ngin
g fr
om 1
05 to 1
08 PFU
/ml.
The
mar
kers
of r
esis
tanc
e w
ere
dete
cted
in sa
mpl
es w
ith th
resh
old
cycl
e va
lues
rang
ing
from
32
to 3
7, a
s det
erm
ined
by
real
-tim
e RT
-PC
R. Th
e py
rose
quen
cing
app
roac
h m
ay p
rovi
de a
val
uabl
e to
ol fo
r rap
id d
etec
tion
of m
arke
rs o
f dru
g re
sist
ance
in
H5N
1 vi
ruse
s and
faci
litat
e th
e el
ucid
atio
n of
the
role
of s
uch
chan
ges i
n na
tura
l and
acq
uire
d dr
ug re
sist
ance
.
Dey
de e
t al.
2009
Review Article Veterinarni Medicina, 58, 2013 (3): 113–185
184
Iden
tific
atio
n of
the
pote
ntia
l res
ista
nce
site
sC
ompu
ter-
aide
d m
etho
dO
selta
miv
irZ
anam
ivir
Mol
ecul
ar b
asis
of d
rug
resi
stan
ce
The
out
brea
k an
d hi
gh s
peed
glo
bal s
prea
d of
the
new
str
ain
of in
fluen
za A
(H1N
1) v
irus
in 2
009
pose
s a
seri
ous
thre
at
to th
e ge
nera
l pop
ulat
ion
and
gove
rnm
ents
. At p
rese
nt, t
he m
ost e
ffect
ive
drug
s fo
r th
e tr
eatm
ent o
f 200
9 in
fluen
za A
(H
1N1)
vir
us a
re n
eura
min
idas
e in
hibi
tors
: mai
nly
osel
tam
ivir
and
zan
amiv
ir. T
he u
se o
f the
se tw
o in
hibi
tors
will
un
doub
tedl
y in
crea
se, a
nd th
eref
ore
it is
mor
e lik
ely
that
dru
g-re
sist
ant i
nflu
enza
str
ains
will
ari
se. T
he id
entif
icat
ion
of th
e po
tent
ial r
esis
tanc
e si
tes
for
thes
e dr
ugs
in a
dvan
ce a
nd th
e un
ders
tand
ing
of c
orre
spon
ding
mol
ecul
ar b
asis
to
caus
e dr
ug re
sist
ance
are
no
doub
t ver
y im
port
ant t
o fig
ht a
gain
st th
e ne
w re
sist
ant i
nflu
enza
str
ains
. In
this
stu
dy,
first
, the
com
plex
es o
f neu
ram
inid
ase
with
the
subs
trat
e si
alic
aci
d an
d tw
o in
hibi
tors
ose
ltam
ivir
and
zan
amiv
ir w
ere
obta
ined
by
fittin
g th
em to
the
3D s
truc
ture
of 2
009
influ
enza
A (H
1N1)
neu
ram
inid
ase
obta
ined
by
hom
olog
y m
ode-
ling.
By
usin
g th
ese
com
plex
es a
s th
e in
itial
str
uctu
res,
mol
ecul
ar d
ynam
ics
sim
ulat
ion
and
mol
ecul
ar m
echa
nics
gen
er-
aliz
ed B
orn
surf
ace
area
(MM
-GBS
A) c
alcu
latio
ns w
ere
perf
orm
ed to
iden
tify
the
resi
dues
with
sig
nific
ant c
ontr
ibut
ion
to th
e bi
ndin
g of
sub
stra
te a
nd in
hibi
tors
. By
anal
yzin
g th
e di
ffere
nce
of in
tera
ctio
n pr
ofile
s of
sub
stra
te a
nd in
hibi
tors
, th
e po
tent
ial d
rug
resi
stan
ce s
ites
for
two
inhi
bito
rs w
ere
iden
tifie
d. P
arts
of t
he id
entif
ied
site
s ha
ve b
een
veri
fied
to
conf
er re
sist
ance
to o
selta
miv
ir a
nd z
anam
ivir
for
influ
enza
vir
us o
f the
pas
t flu
epi
dem
ic. T
he id
entif
ied
pote
ntia
l re
sist
ance
site
s in
this
stu
dy w
ill b
e us
eful
for
the
deve
lopm
ent o
f new
effe
ctiv
e dr
ugs
agai
nst t
he d
rug
resi
stan
ce a
nd
avoi
d th
e si
tuat
ion
of h
avin
g no
effe
ctiv
e dr
ugs
to tr
eat n
ew m
utan
t inf
luen
za s
trai
ns.
Liu
et a
l. 20
10
Prev
alen
ce o
f am
anta
dine
-res
ista
nt
viru
ses
M2
chan
nel m
utat
ions
Gen
ome
sequ
enci
ngJa
pan
Back
grou
nd: T
he p
reva
lenc
e of
am
anta
dine
-res
ista
nt in
fluen
za A
/H3N
2 vi
ruse
s (b
elon
ging
to th
e N
-lin
eage
), po
sses
s-in
g an
S31
N m
utat
ion
in th
e M
2 pr
otei
n an
d S1
93F
and
D22
5N s
ubst
itutio
ns in
thei
r H
A1
subu
nit,
has
sign
ifica
ntly
in
crea
sed
wor
ldw
ide
sinc
e 20
05. T
he a
im o
f thi
s st
udy
was
to c
lari
fy th
e ge
nom
ic e
vent
s co
ntri
butin
g to
the
evol
utio
n an
d co
ntin
uity
of t
he N
-lin
eage
am
anta
dine
-res
ista
nt v
irus
es. M
etho
ds: T
he fu
llgen
ome
sequ
ence
of A
/H3N
2 is
olat
es,
incl
udin
g bo
th a
man
tadi
ne-r
esis
tant
and
am
anta
dine
-sen
sitiv
e vi
ruse
s, c
olle
cted
in Ja
pan
betw
een
2006
and
200
8, w
as
dete
rmin
ed a
nd p
hylo
gene
tical
ly c
ompa
red
with
isol
ates
obt
aine
d fr
om th
e da
taba
se. R
esul
ts: O
n th
e ba
sis
of th
e fu
ll ge
nom
e se
quen
ce a
naly
sis,
the
N-l
inea
ge c
ould
be
furt
her
divi
ded
into
thre
e ge
netic
ally
rela
ted
clad
es: N
i (A
/Wis
cons
in/6
7/20
05-l
ike
aman
tadi
ne-r
esis
tant
vir
uses
from
yea
rs 2
005–
2007
), N
2 (a
man
tadi
ne-s
ensi
tive
viru
ses
from
200
7) a
nd N
3 (A
/Bri
sban
e/10
/200
7-lik
e am
anta
dine
-res
ista
nt v
irus
es fr
om 2
007
and
2008
). T
he 2
006/
2007
sea
son
show
ed c
ocir
cula
tion
of a
ntig
enic
var
iant
s of
am
anta
dine
-res
ista
nt v
irus
es o
f cla
des
Ni a
nd N
3 in
add
ition
to th
e N
2-se
nsiti
ve v
irus
es. I
n th
e 20
07/2
008
seas
on, t
he c
lade
N3
aman
tadi
ne-r
esis
tant
line
age
dom
inat
ed a
nd re
plac
ed o
ther
st
rain
s. P
hylo
gene
tic a
naly
sis
of e
ach
indi
vidu
al s
egm
ent s
ugge
sted
that
N2
and
N3
wer
e ge
nera
ted
from
two
inde
pend
-en
t rea
ssor
tmen
t eve
nts
invo
lvin
g cl
ade
Ni v
irus
es a
nd p
re-N
-lin
eage
str
ains
. Con
clus
ions
: Our
dat
a sh
ow th
at s
ever
al
reas
sort
men
t eve
nts
have
con
trib
uted
to th
e ev
olut
ion
of a
man
tadi
ne-r
esis
tant
A/H
3N2
stra
ins
and,
con
sequ
ently
, to
the
succ
essf
ul s
prea
d of
this
line
age.
Alth
ough
am
anta
dine
resi
stan
ce is
cau
sed
by s
ingl
e am
ino
acid
mut
atio
ns in
the
M2
prot
ein,
gen
ome-
wid
e ad
just
men
t inv
olvi
ng m
ultip
le g
enes
app
ears
to b
e ne
cess
ary
to o
btai
n ef
ficie
nt re
plic
atio
n an
d tr
ansm
issi
on o
f res
ista
nt v
irus
es. S
uch
adju
stm
ents
are
att
aina
ble
thro
ugh
reas
sort
men
t of s
egm
ents
am
ong
diffe
r-en
t vir
us li
neag
es.
Zar
aket
et a
l. 20
10
Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article
185
Prev
alen
ce o
f ant
ivir
al
resi
stan
ceO
selta
miv
irZ
anam
ivir
Am
anta
dine
Arg
entin
a20
05–2
008
Obj
ectiv
e. T
o de
scri
be th
e vi
rolo
gica
l cha
ract
eris
tics
of th
e in
fluen
za s
trai
ns c
ircu
latin
g in
Arg
entin
a in
200
5–20
08
and
to a
sses
s th
e pr
eval
ence
of a
ntiv
iral
resi
stan
ce. M
etho
ds. O
n th
e ba
sis
of th
eir
geog
raph
ical
spr
ead
and
prev
alen
ce,
influ
enza
Lam
bda
and
B is
olat
es g
row
n in
Mad
in-D
arby
can
ine
kidn
ey c
ells
wer
e se
lect
ed a
fter
ant
igen
ic a
nd g
enom
ic
char
acte
riza
tion
to b
e an
alyz
ed fo
r an
tivir
al re
sist
ance
by
enzy
mat
ic a
ssay
and
pyr
oseq
uenc
ing.
Am
anta
dine
sus
cep-
tibili
ty w
as e
valu
ated
by
pyro
sequ
enci
ng fo
r kn
own
resi
stan
ce m
arke
rs o
n 45
str
ains
of i
nflu
enza
A. S
usce
ptib
ility
to
osel
tam
ivir
and
zan
amiv
ir w
as e
valu
ated
by
enzy
mat
ic a
ssay
of 6
7 in
fluen
za L
ambd
a an
d 46
influ
enza
B s
trai
ns, s
ome
of w
hich
wer
e fu
rthe
r an
alyz
ed b
y se
quen
cing
the
neur
amin
idas
e ge
ne. R
esul
ts. R
esis
tanc
e to
am
anta
dine
was
obs
erve
d on
ly o
n A
(H3N
2) s
trai
ns (2
9/33
); al
l of t
hem
car
ried
the
mut
atio
n S3
1N in
thei
r M
2 se
quen
ce. O
selta
miv
ir re
sist
-an
ce w
as o
bser
ved
in 1
2 (3
4.3%
) of t
he 3
5 A
(H1N
1) s
trai
ns fr
om 2
008;
all
of th
em c
arri
ed th
e m
utat
ion
H27
5Y in
thei
r ne
uram
inid
ase
sequ
ence
. All
thes
e vi
ruse
s re
mai
ned
sens
itive
to z
anam
ivir.
Con
clus
ions
. Thi
s st
udy
desc
ribe
s a
high
in
cide
nce
of a
man
tadi
ne-r
esis
tant
influ
enza
A(H
3N2)
vir
uses
sin
ce 2
006
and
an u
npre
cede
nted
incr
ease
in o
selta
miv
ir
resi
stan
ce d
etec
ted
only
in in
fluen
za A
(H1N
1) v
irus
es is
olat
ed in
200
8. In
fluen
za A
and
B v
irus
es w
ere
mor
e se
nsiti
ve
to o
selta
miv
ir th
an to
zan
amiv
ir, a
nd in
fluen
za A
vir
uses
wer
e m
ore
sens
itive
to b
oth
neur
amin
idas
e in
hibi
tors
than
the
influ
enza
B v
irus
es. T
he n
atio
nal d
ata
gene
rate
d an
d an
alyz
ed in
this
stu
dy m
ay h
elp
incr
ease
kno
wle
dge
abou
t inf
lu-
enza
ant
ivir
al d
rug
resi
stan
ce, w
hich
is a
pro
blem
of g
loba
l con
cern
.
Pont
orie
ro e
t al.
2011
Res
ista
nce
to
haem
aggl
utin
in
inhi
bito
rsSt
achy
flin
In si
lico
scre
enin
g
In th
is s
tudy
we
perf
orm
ed m
olec
ular
dyn
amic
s si
mul
atio
ns o
n a
spik
e pr
otei
n on
the
vira
l env
elop
, hem
aggl
utin
in
for
the
wild
type
and
thre
e ki
nds
of m
utan
ts u
sing
a m
odel
sys
tem
con
sist
ing
of a
trim
eric
hem
aggl
utin
com
plex
, vir
al
lipid
mem
bran
e. s
olva
tion
wat
ers,
and
ions
. A
nat
ural
pro
duct
sta
chyf
lin, w
hich
sho
ws
a hi
gh le
vel o
f ant
ivir
al a
ctiv
ity
spec
ific
to s
ome
subt
ypes
of i
nflu
enza
vir
uses
, was
exa
min
ed o
n bi
ndin
g to
the
wild
-typ
e he
mag
glut
inin
was
cla
rifie
d.
Nex
t, 8
com
poun
ds w
ere
sele
cted
from
a c
hem
ical
dat
abas
e by
in s
ilico
scr
eeni
ng, c
onsi
deri
ng th
e fin
ding
s fr
om th
e si
mul
atio
ns. I
nhib
itory
act
iviti
es to
sup
pres
s th
e pr
olife
ratio
n of
influ
enza
vir
us w
ere
mea
sure
d by
cel
l-ba
sed
antiv
iral
as
says
, and
che
mic
al s
caffo
lds
wer
e fo
und
to b
e po
tent
for
an in
hibi
tor.
Mor
e th
an 3
0 de
riva
tives
bea
ring
eith
er o
f the
se
two
chem
ical
sca
ffold
s w
ere
synt
hesi
zed,
and
cel
l cul
ture
ass
ays
wer
e ca
rrie
d ou
t of e
valu
ate
the
com
poun
d po
tenc
y. Se
vera
l der
ivat
ives
dis
play
ed a
hig
h co
mpo
und
pote
ncy,
and
50%
effe
ctiv
e co
ncen
trat
ions
of t
wo
synt
hesi
zed
com
-po
unds
wer
e be
low
1 µ
M.
Yana
gita
et a
l. 20
12
Res
ista
nce
to
phos
phor
o-th
ioat
e ol
igon
ucle
otid
e
In a
pre
viou
s st
udy
a 15
-mer
pho
spho
roth
ioat
e ol
igon
ucle
otid
e (S
-ON
) der
ived
from
the
pack
agin
g si
gnal
in th
e 5’
end
of
seg
men
t 1 (P
B2) o
f inf
luen
za A
vir
us (d
esig
nate
d 5-
15b)
pro
ved
mar
kedl
y in
hibi
tory
to v
irus
repl
icat
ion.
Her
e w
e in
vest
igat
ed w
heth
er a
nalo
gous
inhi
bito
ry S
-ON
s ta
rget
ing
the
5’ e
nd o
f seg
men
ts 2
(PB1
) and
3 (P
A) c
ould
be
iden
ti-fie
d an
d w
heth
er v
iral
resi
stan
ce to
S-O
Ns
can
be d
evel
oped
. Sim
ilar
to o
ur e
arlie
r re
sult,
20-
mer
S-O
Ns
repr
oduc
ing
the
5’ e
nds
of s
egm
ents
2 o
r 3
(com
plem
enta
ry to
the
3’-c
odin
g re
gion
s of
PB1
and
PA
, res
pect
ivel
y) e
xert
ed a
pow
erfu
l an
tivir
al a
ctiv
ity a
gain
st a
var
iety
of i
nflu
enza
A v
irus
sub
type
s in
MO
CK
cel
ls. S
eria
l pas
sage
of t
he A
/Tai
wan
/1/8
6 H
1N1
stra
in in
the
pres
ence
of S
-ON
5-1
5b o
r its
ant
isen
se a
s 5-
15b
anal
ogue
sho
wed
that
mut
ant v
irus
es w
ith re
duce
d su
scep
tibili
ty to
the
S-O
N c
ould
inde
ed b
e ge
nera
ted,
alth
ough
the
resi
stan
t vir
uses
dis
play
ed re
duce
d re
plic
ativ
e fit
-ne
ss. S
eque
ncin
g th
e re
sist
ant v
irus
es id
entif
ied
mut
atio
ns in
the
PB1,
PB2
, PA
and
M1
gene
s. In
trod
uctio
n of
thes
e ch
ange
s in
to th
e A
/PR/
8/34
H1N
1 st
rain
by
reve
rse
gene
tics,
sug
gest
ed th
at a
ltera
tions
to R
NA
func
tion
in th
e pa
ck-
agin
g re
gion
s of
seg
men
ts 2
and
3 w
ere
impo
rtan
t in
deve
lopi
ng re
sist
ance
to S
-ON
inhi
bitio
n. H
owev
er, m
any
of
the
othe
r se
quen
ce c
hang
es in
duce
d by
S-O
N tr
eatm
ent w
ere
mar
kedl
y de
lete
riou
s to
vir
us fi
tnes
s. W
e co
nclu
de th
at
pack
agin
g si
gnal
s in
the
influ
enza
A v
irus
pol
ymer
ase
segm
ents
pro
vide
feas
ible
targ
ets
for
nucl
eic
acid
-bas
ed a
ntiv
iral
s th
at m
ay b
e di
ffic
ult f
or th
e vi
rus
to e
vade
thro
ugh
resi
stan
ce m
utat
ions
.
Gia
nnec
chin
i et
al. 2
011