Avian influenza: virology, diagnosis and surveillance

10.2217/FMB.13.81 © 2013 Future Medicine Ltd ISSN 1746-0913Future Microbiol. (2013) 8(9), 1209–1227

part of

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Avian influenza (AI) is an acute avian respiratory disease caused by AI viruses (AIVs) that affect many species of domestic and wild birds and can also infect other animal species. In poultry, AI is characterized by marked variation in mor-bidity and mortality. The notifiable form of AI (NAI) describes an outbreak of AI in poultry due to any H5 or H7 subtype of influenza A virus (IAV) or an infection by any AIV resulting in an intravenous pathogenicity index >1.2 or caus-ing at least 75% mortality [1]. Highly pathogenic NAI is a ‘list A’ notifiable disease (Box 1) of the World Organization for Animal Health (OIE), which describes serious transmissible diseases that can potentially spread very rapidly between countries and are of major significance for the international trade of animals and animal prod-ucts, and so have severe socioeconomic or public health costs [1]. Low pathogenic NAI includes all IAVs of H5 and H7 subtype that are not highly pathogenic NAI viruses. Low-pathogenicity AI (LPAI) refers to all infections caused by AIVs that are not NAI viruses and include H1-H4, H6 and H8-H16 LPAI [2,301].

The high-pathogenicity AI (HPAI) viruses are restricted to subtypes H5 and H7, although not all H5 and H7 viruses cause HPAI. The risk from AI is generally low in most people, as AIVs do not usually infect humans. However, there have been several cases of conjunctivitis in

humans related to LPAI. AIV has been trans-mitted to humans (e.g., H1N1 in 1976, 1986 and 1988; H3N2 in 1993; H7N7 in 1996; and H9N2 in 1998, 1999 and 2003) inducing mild, severe or fatal infections [3,4]. While it has been suggested that aerosolized droplets or direct contact with virus in poultry feces can lead to infections in humans [5], poultry-to-human transmission of IAVs is unlikely to cause pan-demic influenza. This requires the emergence of viral subtypes with human-to-human trans-missibility arising from mutations, genetic rear-rangement of viral RNA segments or genetic reassortment between different strains of virus [6,7] in a permissive host [8], with pigs being the probable ‘mixing vessel’ [9].

Several examples of human-adapted IAVs exist that have initiated a number of pandemics in the last century [10]. The emergence of the Asian-based HPAI H5N1 virus, in particular, high-lights the continuing public health concerns over human fatalities and its potential emergence as a future pandemic virus. This implicates AI as one of the most threatening transboundary diseases facing humans [11], highlights the potential risk IAV poses to human health and economies [12–14] and strongly emphasizes the urgent requirement for continued high levels of awareness and sur-veillance programs to monitor for emerging AIVs [15]. Accordingly, the WHO has complemented

Avian influenza: virology, diagnosis and surveillance

Mohamed E El Zowalaty*1,2,3, Stephen A Bustin1, Mohamed I Husseiny3,4 & Hossam M Ashour*5,6

1Postgraduate Medical Institute, Faculty of Health, Social Care & Education, Anglia Ruskin University, Chelmsford, Essex, UK 2Faculty of Public Health & Tropical Medicine, Jazan University, Jazan, Saudi Arabia 3Department of Microbiology & Immunology, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt 4Beckman Research Institute at City of Hope, Duarte, CA, USA 5Department of Pharmacy Practice, Eugene Applebaum College of Pharmacy & Health Sciences, Wayne State University, Detroit, MI, USA 6Department of Microbiology & Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt�*Authors for correspondence: [email protected] n [email protected]

Avian influenza virus (AIV) is the causative agent of a zoonotic disease that affects populations worldwide with often devastating economic and health consequences. Most AIV subtypes cause little or no disease in waterfowl, but outbreaks in poultry can be associated with high mortality. Although transmission of AIV to humans occurs rarely and is strain dependent, the virus has the ability to mutate or reassort into a form that triggers a life-threatening infection. The constant emergence of new influenza strains makes it particularly challenging to predict the behavior, spread, virulence or potential for human-to-human transmission. Because it is difficult to anticipate which viral strain or what location will initiate the next pandemic, it is difficult to prepare for that event. However, rigorous implementation of biosecurity, vaccination and education programs can minimize the threat of AIV. Global surveillance programs help record and identify newly evolving and potentially pandemic strains harbored by the reservoir host.

Keywords

n avian influenza n diagnosis n real-time PCR n surveillance n vaccines n virus isolation n waterfowl

Revie

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Future Microbiol. (2013) 8(9)1210 future science group

Review El Zowalaty, Bustin, Husseiny & Ashour

its human surveillance network with a program focusing on the ecology and surveillance of influenza viruses in wild animals [16,17].

Human influenza pandemicsThe three worldwide influenza outbreaks (pan-demics) in the 20th century occurred in 1918, 1957 and 1968 [18]. Each pandemic caused significant social and economic upheaval. The rapid spread, combined with high morbidity and mortality, gave rise to intense levels of anxiety and fear among the public. The ‘Spanish flu’ (1918–1919) was caused by direct transmission of the H1N1 virus from birds [19] and is considered to be the ‘mother of all pandemics’ [20]. It spread across the entire world within 3 months, infect-ing 50% of the world’s population and resulting in an estimated 30–50 million deaths [21]. The ‘Asian flu’ (1957–1958) was caused by genetic reassortment between human H1N1 and avian H2N2 influenza viruses. This pandemic spread across the world in approximately 6 months and, together with a second wave of infections, affected approximately 40–50% of the world’s population, killing approximately 1 million people [22]. The ‘Hong Kong flu’ (1968–1969) pandemic was caused by a H3N2 virus, which resulted from reassortment of circulating human H2N2 and avian H3 virus in China, spreading to Hong Kong and the rest of the world, and causing the deaths of a few million individuals [22].

In February 2009, a new swine-origin influ-enza A (H1N1) virus emerged in Mexico from multiple reassortment events involving a unique combination of gene segments from human, swine and avian type A viruses [23–27], result-ing in the first influenza pandemic of the 21st century [28,29]. By mid-April, it had reached the USA via human-to-human transmission, leading the WHO to raise its pandemic alert successively to phases 4 (27 April), 5 (29 April) and 6 (11 June) [24]. Although now in the post-pandemic

phase, the H1N1 (2009) virus is one of the seasonal influenza viruses in global circulation and continues to be a major challenge to public health.

History of HPAIIn 1878, a contagious disease of poultry asso-ciated with high mortality was first described in Italy and named ‘fowl plague’. In 1955, the causative agent was identified as an IAV, based on the presence of A type-specific ribonucleo-protein. This resulted in the term fowl plague being replaced by the more suitable term HPAI [17, 30–32]. In 1961, a high proportion of deaths in common terns (Sterna hirundo) in South Africa was attributed to the highHP A/H5N3 virus, which was the first demonstration of a HPAI virus in wild birds before the high-pathogenicity (HP) A/H5N1 [33]. The 1997 outbreak of res-piratory disease in humans in Hong Kong caused by H5N1, which was previously known to infect only avian species, raised serious concerns about the potential for another pandemic in humans [34,35]. The H5N1 virus seems to have emerged in southeast China during 1996 [36], where it reassorted with viruses circulating in other avian species and emerged as a new H5N1 virus in Hong Kong. After an initial major outbreak of HPAI in chickens and other birds, it was unex-pectedly transmitted to humans [37], and in May 1997 a 3-year-old boy became the first human to die of a respiratory illness related to an H5N1 virus infection [38]. An additional 17 cases were diagnosed in November and December of the same year [17]. A few years later, the virus spread to several Asian countries, infecting humans for the second time in February 2003 [39].

Since late 2003 until the present day, the world has witnessed the deadliest outbreak in the history of HPAI in birds. In addition, the transmission of A/H5N1 virus to humans is on the increase in Asia, southeast Asia, Africa and the Middle East. Between 1997 and April 2013, HPAI A/H5N1 infection has been identified in 628 human cases, with 374 deaths confirmed in 15 countries, resulting in a fatality rate of 59.55%; the highest numbers of cases/deaths were reported in Indonesia, Egypt, Vietnam and China, respectively [302]. Although A/H5N1 originated in China, it is still unclear why the number of cases is not the highest in its originat-ing location. So far, there has been no evidence of sustained human-to-human transmission of AIV. However, if the virus mutates or rearranges deliberately or spontaneously into a form that is more easily transmissible between humans,

Box 1. Notifiable avian influenza.

�n List A notifiable diseases of the World Organization for Animal Health are defined as transmissible diseases that have the potential for very serious and rapid spread, irrespective of national borders, are of serious socioeconomic or public health consequence and are of major importance in the international trade of animals and animal products.

�n Notifiable avian influenza (AI) is defined as an infection of poultry caused by any influenza A virus of the H5 or H7 subtypes or by any AI virus with an intravenous pathogenicity index in chickens of greater than 1.2 (or, as an alternative, at least 75% mortality). AI can be categorized into highly pathogenic notifiable AI, including high-pathogenicity H5 and H7 subtypes, low-pathogenicity AI (LPAI), which includes all H5 and H7 LPAI subtypes, and all other LPAIs, which are not notifiable to the World Organization for Animal Health, including H1–H4, H6 and H8–H16 LPAIs.

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Avian influenza: virology, diagnosis & surveillance Review

it will have the potential to kill millions in a pandemic.

The concurrent A/H5N1 virus possesses three of the four properties necessary to cause a pan-demic: it infects people, most people are immu-nologically naive and it is highly lethal. However, it lacks the capacity for sustained human-to-human transmission [18,40]. Although that capac-ity may require only a single genetic reassort-ment or mutation, up until now, most infected people have been infected through direct con-tact with infected poultry [41]. While there have been extensive efforts by the WHO to report all cases, the numbers might not reflect the real picture, especially in developing countries, since not all cases are being admitted to hospitals or reported by governments. In developed coun-tries, the strict implementation of biosecurity and preventive procedures in poultry farms helps guard against the spread of AIV. Annual surveil-lance programs among waterfowl have also been implemented in the USA and Europe.

In the spring of 2013, another ongoing out-break of novel avian A/H7N9 virus that origi-nated from multiple reassortment events has again emerged in China, where H7N9 has so far caused infection in 131 laboratory-confirmed human cases, including 36 deaths [42,43]. The recent A/H7N9 strain could potentially pro-duce a human pandemic since the virus is more humanized in traits and could increase human-to-human transmission, if transmitted among poultry or pigs, raising public health concerns of a looming pandemic influenza in humans [44,45].

In addition to AIV, other influenza viruses exist and can cause influenza epizootics among various animal species [5]. In some species, the disease mimics human influenza, while in others, there are no signs of disease. Since the demonstration of swine influenza (Hsw1N1) in 1930 and later in 1955, and with the identifica-tion of influenza viruses in horses and ducks, transmission to other animals has become more evident and has attracted the attention of influ-enza researchers. Influenza A/H3N2 viruses dis-covered in horses were also found in pigs, cattle, chickens, dogs and other species throughout the world [46]. An outbreak of severe respiratory disease in a pack of English foxhounds in the UK in September 2002 was caused by an equine A/H3N8 virus [47]. H3N8 and H3N2 canine influenza viruses have been reported in racing canine populations in the USA [48].

Aquatic birds have been revealed as the source of influenza viruses in sea mammals, such as seals (H4N5, H7N7) and whales (H13N2, H13N9

and H1N3) [5]. In this context, it is worth noting the concerns expressed about the use of ferrets as a model. Ferrets are considered to be the best model for IAV because of their high suscepti-bility and the fact that IAV infection in ferrets closely resembles that in humans with respect to clinical signs, pathogenesis and immunity. Indeed, recent studies in ferrets have shown that IAV can acquire the capacity for airborne trans-mission between mammals without recombina-tion in an intermediate host, and therefore con-stitute a high risk for human pandemic influenza [49,50]. The ability to produce such an infection raises both biosecurity and biosafety concerns and could even lead to a human pandemic [51].

Genome & virology of AIVInfluenza viruses are members of the family Orthomyxoviridae (orthos, Greek for straight; myxa, Greek for mucus), which belongs to group V (negative-polarity ssRNA) of the Baltimore system of virus classification [52]. There are five genera: influenza virus A, B and C, Isavirus and Thogotovirus [53,54]. Each genus includes only one species of IAV, and influenza B and C viruses. Influenza A and C viruses infect multiple species, while influenza B viruses almost exclusively infect humans [55]. IAVs include avian, swine, equine and canine influenza viruses, as well as human IAVs.

The structure of IAVs has been extensively studied and reported [56]. The genome of IAVs comprises eight negative-sense, viral ssRNA seg-ments that are numbered in order of decreas-ing length, as summarized in TaBle 1. The two main surface glycoproteins of influenza viruses as shown in Figure 1 are the hemagglutinin (HA) and neuraminidase (NA) proteins, which function in the attachment and release of the virion to and from cells. They are major antigenic determi-nants and so are major targets for the immune response to which neutralizing antibodies are made [57]. Theoretically, different combinations from the 17 HA and ten NA subtypes [58,59] can be found, and each subtype may contain sev-eral further subtypes. Mutations can introduce additional variability, and so the number of AIV strains is indefinite.

In 1972, the WHO recommended a new sys-tem for influenza nomenclature and classification of influenza viruses into genetically distinct sub-types based on their NA and HA antigens [60,61]. The nomenclature consists of two parts: a strain designation and a description of the NA and HA antigens [60,62]. To date, only three HA (H1, H2 and H3) and two NA (N1 and N2) subtypes


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have caused human epidemics, as defined by sus-tained and widespread person-to-person trans-mission [8]. Nevertheless, the recent discovery of the highly divergent IAV subtype H17N10 in bats from Guatemala in South America rein-forces the importance of surveillance for moni-toring the evolution of influenza viruses among different animal populations. Thus far, this new distinct H17N10 virus is the only subtype that is not found in an avian species [58,59].

Epidemiology of AI The evolutionary success of IAV is a prototypic example of the ability of microbes to adapt to their many hosts. Influenza is fundamentally a recurring background disease that re-emerges slightly differently each year due to the con-tinuously evolving nature of their surface gly-coproteins, referred to as antigenic drift [63]. At unpredictable time periods, influenza presents a newly emerging disease caused by viruses with completely different surface antigens, termed

antigenic shift (Box 2), infecting humans with little or no immunity against these strains [57]. This antigenic variability of AIVs is due to their highly error-prone replication process [64] that, together with viral genome assortment, allows influenza viruses to adapt to their host, thus acquiring a pandemic potential [65].

The ecology and epidemiology of AI have changed substantially in the last two decades [66]. AI infections due to low-pathogenicity A/H9N2 subtype have become widespread in Asia, whereas the HP A/H5N1 subtype has been the causative agent of widespread infections in poultry across other areas of the world, result-ing in a modified ecoepidemiology and zoonotic potential [67].

As shown in Figure 2, the primary difference between LPAI and HPAI virus is local versus systemic replication, respectively. One of the key determinants of virulence is the ability of the host to proteolytically cleave the HA pre-cursor HA0 into HA1 and HA2 subunits, an

Hemagglutinin

Neuraminidase

M2 ion channel

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Figure 1. Molecular structure of influenza A virus showing two surface glycoproteins (hemagglutinin and neuraminidase), the major antigenic determinants of the virus. The hemagglutinin protein is responsible for binding to sialic acid receptors on host cells. Human influenza A viruses preferentially bind to sialic acids in an a2,6 conformation, while those from avian species bind mostly to sialic acids in an a2,3 conformation. RNP: Ribonucleoprotein. Image was reproduced with permission from the CDC, GA, USA [306].



essential requirement for binding to the cell sur-face receptors and fusion of the viral envelope with the endosomal membrane. An analysis of HA subtypes that circulate in aquatic birds, as well as HAs representative of the subtypes that have infected the human population, indicates that the cleavage efficiency can vary significantly for different HA subtypes and some display stringent selectivity for specific proteases [68]. The HA of mammalian viruses contains only a single arginine and, rarely, a single lysine at the cleavage site and is cleaved extracellularly, limit-ing their spread in mammalian hosts to tissues that express the appropriate proteases. Similarly, LPAI viruses have an HA cleavage site with one basic arginine or lysine residue that is cleaved only by proteases with monobasic specificity. As a result, LPAI infection in birds is restricted to the digestive or respiratory tracts and results in milder illness or no disease at all. By contrast, HPAI viruses are invariably HA subtypes H5 or H7 with a polybasic cleavage site, introduced either as a result of insertion or substitution [69–71]. This polybasic cleavage site is susceptible to the protease furin, which is ubiquitous in cells [72]. The factors that cause mutation from LPAI to HPAI, which occurs only in birds, are not known, but it seems that the wider the circula-tion of LPAI in poultry, the higher the chance that mutation into HPAI will occur [73]. It is important to remember, however, that acquisi-tion of a polybasic HA cleavage site is not the only necessary step for the evolution of LPAI strains into HPAI viruses, since not all H5 or H7 subtypes are hypervirulent. There are addi-tional virulence determinants within the HA itself [74,75] and in other viral proteins, suggesting that virulence is under polygenic control [73,76,77].

Persistence of AIV in the environmentLPAI viruses are ubiquitous [16] and approxi-mately 90 species from some 12 of the 50 orders of birds carry all strains of influenza, with birds inhabiting wetland and aquatic environments showing the highest rates of influenza infec-tion [78]. Mixed infections with different influ-enza subtypes among waterfowl are also com-mon [12,13,79]. Carriers are mainly of the orders Anseriformes (especially the families Anatidae [ducks, geese and swans], Charadriiformes [terns and waders] and Procellariiformes [shorebirds, gulls and seabirds]) and thus act as a reservoir for the virus [80]. The migratory nature of many of these species and the persistence of influenza in these populations facilitates the dissemina-tion of influenza viruses worldwide [81]. Influenza

viruses infect different species and have become very successful parasites in their avian hosts, causing mostly silent or asymptomatic infections while creating the opportunity to infect other immunologically naive hosts [5].

Interspecies barriers and the host species speci-ficity mechanisms, such as receptor preference and host factors interacting with HA, NA, viral polymerase and other internal genes, are impor-tant molecular constraints and determinants for their transmissibility of AIVs among different species [82,83]. IAV particles bind their target cells through interaction between their HA molecules and the sialic acid-containing cell-surface recep-tors on the host cells. Human influenza viruses bind preferentially to N-acetylneuraminic acid (sialic acid), which is attached to a galactose molecule by an a2,6 linkage (SAa2,6Gal), while AIVs mostly bind to silalic acid with an a2,3 linkage [84]. In human tracheal epithelial cells, a2,6 linkages predominate, while a2,3-linkages are more common in duck gut epi-thelial cells [83]. Recently, it was indicated that epithelial cells of the human lower respiratory tract contain both SAa2,3Gal and SAa2,6Gal but with different distributions [85]. In humans, a2,3 linkages are also present in respiratory epi-thelial cells, but their presence is less abundant than a2,6 linkages [86]. This explains the low infectivity but high pathogenicity of some AI strains in mammalian species [56]. AIVs do not generally replicate well in humans and vice versa [87,88], so it is possible that reassortment takes place in an intermediate host. Pig populations have been proposed as a potential mixing vessel where reassortment takes place, since both avian and human strains can infect and replicate in pigs [89]. This is attributed to the fact that tra-cheal cells of pigs, where influenza replication occurs, contain a2,3-linked sialic acid receptors preferred by avian viruses, as well as those with the a2,6-linkage favored by human strains [89].

Box 2. Antigenic drift versus antigenic shift.

�n Antigenic drift is defined as the process whereby small changes or mutations accumulate in the virus, mainly in the surface glycoproteins (predominantly hemagglutinin [HA]), which happen continually over time. The new virus strains thus produced have different antigenic profiles and may not be recognized by antibodies against the older strains, thereby making the individual susceptible to infection with the new strains.

�n Antigenic shift is an abrupt, major change in the HA and/or neuraminidase (NA) proteins of the virus that occurs when surface HA and NA segments reassort between different viruses, resulting in the emergence of a new subtype. Alternatively, the new HA and NA combination may have emerged from an animal population that is so different from the same subtype in humans that most people do not have immunity to the new virus.



The mechanisms by which influenza viruses pass from one bird to another and cause infec-tion are not fully understood, but may be due to combinations of factors that include strain of virus, species of bird and environmental factors. In wild ducks, for example, influenza viruses replicate preferentially in the cells lin-ing the intestinal tract and are excreted in high concentrations in the feces. Contamination of water supplies by infective feces and fomites that contain concentrations as high as 107 infectious particles/g is one obvious route of infection [5,90]. This has led to the assessment of ducks being the ‘Trojan horses’ of H5N1 in their sur-reptitious spread of viruses because they do not show symptoms after HP H5N1 infection [91]. Influenza viruses have coevolved with ducks over a very long period of time, allowing the establishment of equilibria between hosts and viruses so that neither suffers a significant loss of biological fitness. Hence, it is important to determine whether antigenic diversity is driven naturally in ducks or whether it is it the conse-quence of vaccine usage. Furthermore, it will be necessary to determine the genomic character-istics of ducks that are associated with natural resistance in some species and ascertain what dose of vaccine antigen is required to prevent

transmissible levels of virus excretion by ducks of different species [91].

Clearly, the natural reservoir of IAVs could be the source of the next human influenza pan-demic [92] and so the routine surveillance and early detection of these viruses [93,94], their hosts and their migratory patterns [95], as well as the study of the impact of environmental and social factors on their interactions [96], are key to the control, management and eradication of the disease [97]. One consequence of this has been the formulation in the USA of an interagency strategic plan by a group of federal and state resources, as well as science agencies, to conduct surveillance in wild birds in order to determine the possible pathways of entry [98].

In addition to carrying mixed populations of AIVs, waterfowl can host additional viruses, especially the avian paramyxoviruses (APMVs) [99,100]. Several studies have demonstrated the cir-culation of influenza virus with APMV-1 (also known as Newcastle disease virus [NDV]), APMV-2, APMV-4 and APMV-6 [101–105]. The coexistence of NDV with AIV in field samples might present a diagnostic problem when it is desired to isolate and characterize only AIVs for surveillance and epidemiological purposes. The overwhelming growth of NDV may inhibit AIV

Avian strains isolated from human H5N1 (1997 HK)

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Avian strains isolated from human H7N7 (2003 NL)

Avian strains isolated from human H5N1 (2004 Asia)

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Figure 2. Protease cleavage site in influenza A viruses hemagglutinin. HA0 is cleaved into disulfide linked subunits HA1 and HA2 at a specific cleavage site shown in red (single letter amino acid code is used to identify the amino acid sequence at the hemagglutinin cleavage site). The hemagglutinins of LPAI viruses are cleaved by proteases that are localized in respiratory and intestinal organs, resulting in mild localized infections, whereas the hemagglutinins of HPAI viruses have multiple basic amino acids, which are cleaved by ubiquitous proteases in a wide range of organs, resulting in lethal systemic infection. Most human isolates of avian strains also possess polybasic cleavage sites. The TM domain is shown in orange. HK: Hong Kong; HPAI: High-pathogenicity avian influenza; LPAI: Low-pathogenicity avian influenza; NL: The Netherlands; TM: Transmembrane.



growth, which in turn will decrease the chance of detection of AI subtypes. In several AIV surveil-lance studies, if a sample is found to be positive for NDV, it is not processed for AIV detection; only if a sample is found to be negative for NDV is it screened for AIV [106]. In a recent report, it was found that treatment of the sample with NDV polyclonal antiserum facilitates the isola-tion of AI from samples containing both AIVs and NDV [102].

Diagnosis of AI An effective strategy for understanding the ecology and epidemiology of the virus, and thus controlling the spread of influenza viruses, depends on the availability of reliable laboratory techniques permitting accurate and sensitive detection of AIVs in waterfowl [107,108]. Clinical diagnosis of AIV in birds is performed by the isolation and characterization of the virus, which varies with species and type of infection [109] and can be either presumptive or definitive. Clinical symptoms can be a valuable tool for presump-tive diagnosis of HPAI, whereas LPAI infection is asymptomatic [110]. Definitive diagnosis of AIV depends on specific laboratory methods, including the indirect evidence of infection by serological methods, which detect anti-influenza antibodies, and direct detection methods for live virus, viral antigen or viral nucleic acid [111].

Specimen collectionClinical specimens for AIV diagnosis in birds can be obtained from either live or dead birds. Wild birds in surveillance programs of migrating waterfowl are usually trapped using night light-ing, rocket netting and hunter harvesting tech-niques [303,304]. Samples from live birds should include both tracheal and cloacal swabs, since it has been shown that the analysis of both oro-pharyngeal and cloacal swabs provides an accu-rate snapshot of AIV status in birds [79,112–114]. If this is not possible, the collection of fresh feces may also serve as an alternative [115].

Samples should be placed in an isotonic phos-phate-buffered saline or viral transfer medium consisting of brain–heart infusion medium containing several antibiotics to eliminate any bacteria that may interfere with subsequent diagnostic procedures [116]. Although diagnos-tic samples can be stored temporarily at 4°C, it is recommended that all samples be kept at -80°C until tested, especially if prolonged stor-age is anticipated [1]. In addition, field samples should be maintained under strict cold chain conditions from the moment of acquisition and

should not be subjected to frequent freeze–thaw cycles, as this results in significantly decreased virus titers [79]. In addition, technical details such as the use of dry or wet swabs, the pH of viral transfer medium and the time taken to transport samples to the testing laboratory play a crucial role in the successful isolation of AIVs from field samples [114].

Serological methodsSerological methods for the diagnosis of AI in birds play an important role in monitoring the disease in populations and can provide an accurate method for the detection of influenza infection [117]. Serological diagnosis is important in case of LPAI to ensure freedom of infection for commercial purposes. Conversely, in cases of HPAI, serological methods are of little value since birds die before producing antibodies [111]. Serological methods are thus useful epidemio-logically for strain surveillance, but cannot be used for rapid diagnosis, which would allow therapeutic intervention [118]. Common serologic tests for AIV include hemagglutination inhibi-tion (HI), NA inhibition, agar gel precipitation (also known as agar gel immunodiffusion), virus neutralization, complement fixation (CF), enzyme immunoassay and indirect immunofluo-rescence [119,120]. These tests are based on the presence of influenza-specific antibodies that first appear approximately 2 weeks after initial infection and peak at 4–7 weeks after initial infection [120]. HI and NA inhibition assays are labor-intensive and time-consuming and require several controls for standardization. However, they are inexpensive, use readily available rea-gents and display high specificity in identifying strains [121]. HI is more specific in differenti-ating between HA subtypes [122]. The agar gel immunodiffusion test detects IgM and some IgG and is type-specific [123]. Although CF tests have been used for the identification of influ-enza isolates [118], the Canadian Public Health Laboratories stopped offering this test in 2009 because influenza CF serology does not provide a reliable indicator of immunity or response to vaccination and is not recommended for the diagnosis of acute influenza infection [305].

Virus isolationInfluenza virus was first isolated in 1933, by inoculation of specimens into the amniotic cavity of 10–12-day-old embryonating chicken eggs (ECEs) [124], where permissive infection occurs in the cells lining the amniotic and allantoic cavities [118]. This method, using either



specific pathogen-free chicken eggs or specific antibody-negative eggs [125], continues to be the gold-standard technique for AIV diagnosis and remains the most sensitive method for generat-ing very high titers of all but one AIV [119]. The exception is the recently discovered bat virus A/H17N10; to date, all attempts to propagate this virus in cell cultures or chicken embryos have failed [59]. Although commercial non-specific pathogen-free eggs have been used for AIV propagation and isolation, their vaccination history should be carefully checked to confirm the absence of other pathogens that might affect AIV isolation [13].

Allantoic fluids negative for virus can be pas-saged into ECEs for up to three passages and retested for HA before being recorded as nega-tive. However, AIV isolation in ECEs requires a readily available continuous supply of ferti-lized chicken eggs and special incubators [126]. It can take days to weeks to achieve high titers, the method is labor- and resource-intensive and there is a mandatory requirement of a high level of biosecurity facilities (generally biosafety level 3) if HPAI is suspected. Hence, it is not widely used for the routine diagnosis of influ-enza infection. On the other hand, egg isolation provides high quantities of live infectious virus and reference laboratories therefore utilize this culture system to ensure high sensitivity and for biological characterization, full-length sequence analysis and antiviral resistance studies [111,127]. This method also enables the production of virus stocks for epidemiological monitoring and vac-cine updating, which is a critical requirement for the diagnosis of AI in the index cases.

The technique of conventional culture of influ-enza viruses in a cell culture was introduced in the 1940s [120] and provides a useful method for the primary isolation of some human or swine influenza isolates that do not grow well in ECEs [117,128]. Since viral culture amplifies the inocu-lum, it is more sensitive than direct methods of detection [13]. Cell cultures can be monitored for the development of cytopathic effects, the mani-festation of hemadsorption after the addition of erythrocytes or for the presence of influenza anti-gens [120]. Various mammalian and bird cell lines have been used to isolate influenza viruses. The most commonly used cells are: primary rhesus monkey kidney and rhesus monkey kidney (LLC MK2) cells, African green monkey kidney cells, mink lung epithelial cell line (Mv1Lu), buffalo green monkey kidney, Madin–Darby canine kid-ney, green monkey continuous cell line (Vero), human lung embryonating cells (MRC-5),

CACO-2 cell lines, CCL-141 (duck embryo) cells, CCL-169 (goose embryonic kidney) cells, duck embryo fibroblast cells and chicken embryo fibroblast cells [129–134].

Alternatively, since viruses are released slowly from the cell surface of virus-infected cells, hemadsorption results in erythrocytes adher-ing directly to these infected cells, which can be observed microscopically [118]. In addition to ECEs, Madin–Darby canine kidney cells are now considered a valuable system for the isola-tion of influenza viruses. However, since AIVs have different host and in vitro growth proper-ties depending on the strain [135,136] and not all host cells are universally permissive to all AIV subtypes, virus isolation is effective only when the cell culture is sensitive to the inoculated virus [137]. In addition, this method requires the rapid transport of specimens to the laboratory, since delays may lead to inactivation of the virus and hence failure to isolate any infectious agent [138].

Although conventional cell culture takes up to 2 weeks to generate results, it is very sensi-tive. Cytopathic effects such as intracytoplasmic basophilic inclusion bodies are observed. The presence of influenza virus can be ascertained using either hemadsorption with guinea pig red blood cells [108] or immunofluorescence on cul-tured cells. Immunofluorescence has the twin advantages of higher sensitivity in the detection of positive cultures and can also be used to type the isolated virus.

Another useful technique for the culture of AIVs is the rapid shell vial culture, which uses single or mixed cell lines using monolayers of two different cell types in a single vial [120]. This tech-nique has the advantage of enhancing sensitivity and shortening the time to detection through enhancing the viral infectivity of the cells by cen-trifugation (time to diagnosis: 24 h vs 13 days postinoculation) [139,140]. Sensitivity is equiva-lent to that of conventional tube cultures and greater than that of direct fluorescent-antibody tests [141].

Molecular methods Molecular methods, also known as nucleic acid tests, detect viral nucleic acid (i.e., RNA) and promise to shorten the turnaround time for the laboratory diagnosis of AIV. There is a range of different types of molecular diagnostic tests. Until recently, the most commonly used was reverse-transcription PCR (RT-PCR), where purified RNA of the virus, isolated from chicken eggs, cell cultures or from clinical specimens, is reverse transcribed into cDNA, which is then



exponentially amplified using AIV-specific oli-gonucleotide primers [142–147]. Conventional RT-PCR is an end point assay, where the PCR product is analyzed on agarose gel after ethidium bromide staining to separate the PCR product according to molecular weight, which allows presumptive identification [127,145,148]. Its main disadvantage is the ease with which the assay can be contaminated and the tedious amount of work involved in running and visualizing gels.

Other nucleic acid test methods include the use of RT-PCR with detection by ELISA [149], nucleic acid sequence-based amplification [150] and H5 reverse transcription loop-mediated isothermal amplification [151]. However, none of these can compete with quantitative reverse tran-scription real-time PCR (RT-qPCR), which is a homogeneous assay requiring minimal hands-on time and no post-PCR processing [152]. RT-qPCR typically uses a fluorescently labeled probe to detect the increase in PCR product while the test is being performed and the results are reported in real time. All these methods have the potential to provide rapid and sensitive diagnostic results.

RNA is most commonly extracted from AIVs using phenol–guanidinium thiocyanate [153] or one of the recently developed commercially available kits that depend on the adsorption of RNA to silica gel-based columns. These methods for the detection of influenza are highly sensitive, specific and versatile [154], with the column-based methods having the advantage in terms of the reliable generation of intact RNA [155]. Once the viral RNA is extracted, it can be used not only to identify the isolate as AIV, but also to further determine the subtype and even the strain by sequence ana lysis utilizing an array of several influenza-specific primers. The viral genotype can be determined by sequencing some or all of the AIV genes, although some level of virus amplification in ECEs or cell culture is required for full-length sequence ana lysis [120]. Molecular methods are rapid, of acceptable sensitivity and provide results within a relatively short time for targeted treatment and limitation of the spread of infection. This is crucial in situations in which rapid diagnosis is necessary, such as in epidemics and in patients with specific medical conditions [119,156].

There are several concerns with molecular diagnostic methods. Foremost is the techni-cal problem associated with the viral sequence that gives rise to both genetic drift and shift. The exquisite specificity of PCR can suddenly become a liability if the sequence change or reassortment includes primer binding sites and

the assay no longer amplifies the altered target sequences. Consequently, nucleic acid-based testing requires routine updating of the primers [13]. Another is the potential for obtaining false-negative results that may occur due to the pres-ence of inhibitors (which are concentrated along with pathogens during sample processing), low virus titers, degradation of target RNA before amplification and errors in setting up a reaction [157,158]. The problem of RNA quality, in par-ticular, is a critical issue, as these tests require RNA samples to be of good quality and the use of degraded RNA or inefficient PCR reac-tions can make these methods unreliable and may yield inaccurate results [13,107]. Hence, it is important to follow a rigid quality assessment program and, taking the example of RT-qPCR, there is a requirement to report the protocols accurately and comprehensively using the mini-mum information for publication of quantitative real-time PCR experiments (MIQE) guidelines [159]. Even then, the conversion of mRNA to cDNA is highly variable, and RT-qPCR results can vary considerably with the choice of reverse transcriptase.

mRNA molecules are not linear molecules, but instead form secondary structures through extensive intrastrand base pairing. This results in numerous stem-loop structures consisting of sin-gle-stranded loops and double-stranded hairpin structures of varying lengths. The efficiency of the RT step depends heavily on the primers used for cDNA synthesis not targeting the hairpin structures, since the intermolecular (i.e., primer to mRNA) hybridization kinetics are infavorable compared with intramolecular (i.e., mRNA to itself) hybridization. Hence, it is important to choose suitable RT primers that target the loops, rather than the stems. Similarly, it is important to ensure that the amplicon itself is free from secondary structures at the PCR primer binding sites, as extensive secondary structures there can give rise to significantly different results. It must be noted that assays only determine total patho-gen number and do not provide information as to whether a pathogen has the ability to establish an infection or not. Hence, a positive result may not necessarily pose a public health threat, and so there is a requirement for additional assays that can determine viability.

None of the diagnostic tests discussed so far are easily carried out in the field, and none can provide virtually instant results. Rapid point-of-care diagnosis is critical for the control and man-agement of influenza infection in humans, as it enables the fast administration of appropriate



antiviral therapy within the first 2 days of illness [160], reduces the length of in-patient hospital stay and minimizes the unnecessary use of antibi-otics [161]. Rapid antigen capture immunoassay tests can provide results within 30 min or less and are easy to perform [162]. They are based on an immunochromatographic reaction that uses a monoclonal antibody targeted to a specific antigen of influenza, usually the nucleoprotein. They can therefore detect any AIV [163–166] and include tests such as the Directigen™ Flu A and Directigen Flu A and B tests (Becton Dickin-son Diagnostic Systems, MD, USA) and the BinaxNow® influenza A and B test (Binax, Inc., ME, USA) [162].

VaccinesVaccination in poultry can minimize the threat of AI. Vaccines against AI are important tools for protecting the poultry industry and humans, since vaccines help increase resistance to infec-tion, prevent illness and death, reduce virus replication and reduce viral transmission to birds and mammals, including humans. DNA vaccination could provide protective immunity in animal models against different infectious diseases. The basic principle of DNA vaccina-tion is the induction of an immune response by intramuscular injection or the use of a ‘gene gun’ of naked DNA (plasmid) encoding the targeted gene into the host cells [167]. The restrictions of DNA vaccination are vaccine cost, problems with delivery into birds, low efficiency in birds and its requirement of large amounts of puri-fied plasmids [168]. The reason DNA vaccines are not suitable in the poultry industry include their time-consuming application, expenses and undue stress to the chickens.

DNA delivery technologies are effective and essential for inducing a strong and long-lasting immune response that is required to induce high and continued levels of antigen production at proper target sites. A variety of intracellular bac-teria have been utilized as live carriers for effi-cient delivery of either DNA vaccine constructs or vaccine antigens [169] directly into professional APCs [170]. This strategy can elicit humoral and cellular responses against the pathogens from which the target genes are derived [171–174].

Live oral attenuated Salmonella typhimurium is a well-known, useful carrier for the expres-sion and delivery of heterologous antigens to mucosal lymphoid tissue, including the HA of AIV [175]. The viable bacteria multiply inside the Salmonella-containing vacuole and deliver the recombinant antigen into the host cell

cytosol [176,177]. DNA vaccines delivered by atten-uated S. typhimurium SL7207 and boosted with a conventional killed vaccine confer protection on chickens against infection with the H9 sub-type of AIV [178]. Another approach is the utiliza-tion of SPI2-encoded type III secretion system [179] for antigen expression and delivery into the cytoplasm of APCs, which increases safety and efficacy. Such vaccines have been shown to be very effective in eliciting both CD8+ and CD4+ T-cell-mediated immune responses in models of infectious diseases and cancer [177,180–183].

Interestingly, the development of an immune response to HA glycoprotein is important for the protection of chickens against challenge with the AIV infection [184]. Accordingly, a wide variety of vaccines against AIV has been developed and tested in experimental conditions, but only inac-tivated whole AIV vaccines and live recombinant fowlpox virus vectors expressing HA vaccines have been licensed and widely used in various countries [185].

Future perspectiveThe recent outbreak in humans of a novel influenza A/H7N9 in China in March 2013 shed light on the importance of surveillance efforts to alleviate public health concerns of AI. Extensive global surveillance programs are urgently required to help predict the circulating AI strains that may produce potential pandem-ics in humans. Vaccines and molecular immu-nology approaches are currently the subject of extensive research efforts aimed at minimiz-ing the threat of AI. These are complemented by surveillance programs that are essential for monitoring the AI dynamics in their recognized reservoirs. The outcome of active surveillance for AIVs in different parts of the world, including Malaysia, will fill the gap in knowledge regard-ing the true disease status in Malaysia, Egypt and several countries in southeast Asia. Similar to many countries in southeast Asia, Malaysia is endemic for NDV, but its current AI disease status is unknown. Outbreaks of HP A/H5N1 were reported in 2004, 2006 and 2007 in poul-try, although none have been reported since. The country borders Indonesia, which is endemic for A/H5N1, and Thailand, which has also had outbreaks of HP A/H5N1 AI. In addition, in countries such as Thailand, Indonesia and China where pigs are not fully protected from acquir-ing AIVs, the generation of pandemic influenza strains is a possibility. It was recently reported that the unprotected transport of pigs and the breach of biosecurity measures could facilitate



the mixed infection of humans and pigs with avian viruses and the generation of newer strains of greater pandemic potential [186, 187]. This could also explain the emergence of influenza A strains of pandemic potential in China and southeast Asia. Because of the lack of active surveillance, particularly in duck populations, the true disease status in such regions remains undetermined.

Although the use of influenza vaccines to protect the poultry industry and humans is an effective procedure to control the disease, the current use of vaccination encounters several challenges [188]. A major hurdle is the increased chance of having asymptomatically infected poultry spreading the virus. In addition, the use of the current vaccines for AI in poultry is not efficient to elicit immune responses. This is the most common cause of vaccine failure in poul-try; therefore search for new strategies to produce a new delivery system for vaccination against AI

such as using live attenuated bacteria or viruses as vectors or using nanovaccines should be advo-cated to improve the outcome data (immuno-genicity, efficacy and effectiveness [efficiency]) of available AI vaccines. Another drawback of currently applied vaccines used in poultry is the low antigen stability in the vaccines and the short duration of immunity. Worryingly, it has been recently reported that independent recombina-tion events between distinct attenuated infec-tious laryngotracheitis virus vaccine strains can result in virulent recombinant viruses [189]. This prospect might arise in AIVs as well. Although the use of nanotechnology to deliver AI vaccines may improve vaccine efficacy and overcome the recombination challenges [190,191], for now, we advocate strict biosecurity procedures coupled with the implementation of worldwide intera-gency surveillance efforts modeled on those of the USA and Europe in high-risk regions, such

Executive summary

Background & history�n Avian influenza virus (AIV), which is closely related to human influenza virus, appeared nearly 150 years ago and has recently

re-emerged, causing grave concern among certain populations.�n Several pandemics of AIV have afflicted human populations with devastating impact.�n The recently emerged viruses, mainly A/H5N1 and A/H7N9, arose from several reassortment events in different hosts and pose a

significant threat to human public health.

Genome & virology of AIV�n Although the structure of AIV has been fully elucidated, it remains difficult to predict when the next pandemic will occur. �n Hemagglutinin and neuraminidase are the major antigenic determinants of influenza A viruses.�n Acquisition of a polybasic hemagglutinin cleavage site is not the only necessary step for the evolution of low-pathogenicity avian

influenza strains into high-pathogenicity AIVs. There are other factors that are also required.

Epidemiology of AIV�n AIVs are natural inhabitants of waterfowl, mainly migratory ducks.�n Several interspecies barriers determine the transmissibility of AIVs among different species and, finally, to humans.�n Surveillance is of importance in order to monitor the current status of avian influenza in populations and to update vaccine databases.�n Global programmed surveillance programs in Asia, a potential origin of the next pandemic, should be promptly initiated.

Diagnosis of AIV�n Diagnosis of avian influenza infection in waterfowl is different from diagnosis in humans.�n Suitable and reliable diagnostic techniques in real time will guard against severe infection in humans and allow monitoring of AIVs

spreading in their natural reservoirs.�n Virus isolation in embryonating chicken eggs should be advocated as the cornerstone diagnostic technique of isolation of live AIVs for

subsequent characterization and updating of vaccine databases.�n Nucleic acid-based tests are useful techniques for the screening of AIVs during surveillance studies.�n The specificity of quantitative PCR is subject to several factors, such as the primers, RNA extraction method, RNA integrity and presence

of inhibitors, among others.�n Virologic and serological techniques, along with rapid molecular methods (e.g., quantitative PCR), will provide information and suitable

intervention strategies to limit the pandemic potential of avian influenza in humans and poultry.

Vaccines�n Vaccines are useful tools to minimize the threat of avian influenza in poultry, but a concern that is associated with the use of vaccines in

poultry is that vaccines could increase the chance of having asymptomatically infected poultry spreading the virus.�n DNA vaccines and nanovaccines are promising tools to improve currently applied avian influenza vaccines in poultry.�n In addition, live-attenuated bacteria and viruses might be important tools as delivery systems for the production of avian influenza

vaccines in humans and poultry.



as in developing countries, to identify potentially pandemic strains [192–194]. Surveillance and diag-nosis of AI in humans and birds are not similar and involve different techniques. Thus, AIV surveillance in AIV’s natural reservoirs of wild birds is of high importance in order to monitor the silent circulating strains and protect poultry from being infected with LPAI strains, as well as HPAI. Biosecurity measures, environmental pro-tection and handling of poultry in at-risk farms or locations of endemic infections will limit and control the spread of the disease in humans and will minimize poultry-to-human transmission. Virologic and serological monitoring, along with the use of rapid molecular methods (e.g., quantitative PCR), will provide information in real time in order to enable suitable intervention

strategies to limit the AI pandemic and disease potential in humans and poultry. Finally, it is advisable to use complementary methods for the identification of AIV, since this approach is most likely to yield a reliable and comprehensive evaluation of circulating viruses [13,107].

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the sub-ject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

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Avian influenza: virology, diagnosis and surveillance

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