Zika Virus In-Depth study into what exactly

Zika virus is or is not

Previous ECDC rapid risk assessments on Zika virus outbreaks:

Zika virus infection outbreak, French Polynesia’, 14 February 2014 Zika virus infection outbreak, Brazil and the Pacific region’, 25 May 2015 ‘Microcephaly in Brazil potentially linked to the Zika virus epidemic’, 24

November 2015

Zika virus was first isolated from a sentinel rhesus monkey placed in the Zika Forest near Lake Victoria, Uganda in April 1947; a second isolation from the

mosquito Aedes Africanus followed at the same site in January 1948 [2]

Although it is thought that enzootic ZIKV is maintained primarily in a monkey/mosquito transmission cycle, antibodies have been detected in numerous other

animal species including water buffalo, elephants, goats, hippos, impala, kongoni, lions, sheep, rodents, wildebeest, and zebras

[2]. Dick GW, Kitchen SF, Haddow AJ (1952) Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg 46: 509–520.

Yap Island, Federated States of Micronesia in 2007, and a 2010 pediatric case in Cambodia

[1] “Zika virus, has a positive-sense, single-stranded RNA genome”

Source [1] Genetic Characterization of Zika Virus Strains: Geographic Expansion of the Asian Lineage, Page 2

Zika Virus strain NS5 protein

Recombinant proteins and c DNA (cloned DNA)

Spondweni Virus SM-6-V1 polyprotein gene

Another emerging pathogen – Zika virusInstitute of Epidemiology, Military Medical Academy, Belgrade, Serbia

Zika virus (ZIKV) disease is caused by an emerging mosquito-borne virus from the Flavivirus genus, Flaviviridae family, from the Spondweni group. It was first

isolated in 1947 in rhesus monkeys through a monitoring network of sylvatic yellow fever, in the Zika forest Uganda, then in mosquitoes (Aedes africanus)

in the same forest in 1948, and in a human in Nigeria in 1952.

There are two ZIKV lineages:the African lineage and the Asian lineage which has recently emerged in the

Pacific and the Americas

The incubation period of disease is not clear, but is likely to be 3 to 12 days after the bite of an infected mosquito. Most of the infections remain

asymptomatic (between 60% to 80%). The symptoms are similar to other arbovirus infections such as dengue. Signs and symptoms are usually mild and

the disease is usually characterised by a short-lasting self-limiting febrile illness of 4–7 days duration. without severe complications, with no

associated fatalities and a low hospitalisation rate. The main symptoms are macular or papular rash, fever, arthralgia, non-purulent conjunctivitis/

conjunctival hyperaemia, myalgia and headache.

1 Biological Transmission of Arboviruses: Re-examination of and NewInsights into Components, Mechanisms, and Unique Traits as Well as

Their Evolutionary Trends

1 Goro Kuno* and Gwong-Jen J. Chang

The term arbovirus that derived from laboratory jargon used in early 1940s among the investigators in California refers to an animal virus that is

transmitted to vertebrate hosts by blood-sucking arthropod vectors. The basic requirements for arboviruses defined by the World Health Organization were viral replication in both phyla of hosts and viral transmission by blood-

sucking arthropod on vertebrate host demonstrating viremia.

In the original definition in microbiology, the word vector is used for all organisms, including vertebrates, that function as carriers of infectious

agents to another organisms. However, in this review it is used exclusively for hematophagous arthropods involved in biological transmission of arboviruses and is not used for the vertebrate hosts involved in direct

transmission between vertebrates.

Among the major modes of animal virus transmission, unquestionablydirect transmission is shared among all virus groups and considered the

fundamental mechanism in all animal virus groups, Thus, it is not surprising that this mode of transmission is widespread among all major

arbovirus groups

The common routes are intranasal, oral, venereal, and exposure of skin with abrasion, cornea, reproductive tissue, or any mucous tissue. The

common sources of contaminants include food, aerosol, bodily secretions,urine, fecal matter, saliva, milk, hair, feathers, and skin

The animal behaviours conducive to this mode of transmission include eating (including cannibalism), drinking, sniffing, licking, preening, nuzzling, and

any aggressive behaviours resulting in injury. Some viruses, such as Venezuelan equine encephalitis virus (VEE) are readily transmissible by

aerosol. Under special circumstances of high virus concentration, however, many arboviruses classified at a lower biohazard level (such as Dengue

virus [DENV]) also can infect vertebrates by aerosol

Insectivorous animals, such as bats, become infected by ingesting mosquitoes infected with viruses such as Rift Valley fever virus, Yellow fever virus (YFV), and

Japanese encephalitis virus (JEV)

But, it’s NOT the Aedes Aegypti - that is the AFRICAN strain. The strain that is present in South

America is ASIAN lineage

1 An additional interest in insectivorous bats is their potential role in the evolution of arboviruses originating from strictly insect viruses through ingestion of infected

insects. Recently, at least under laboratory conditions, an indirect mode of oral transmission was proposed to be possible for Vesicular stomatitis virus (VSV) New

Jersey serotype. According to the proposal, the chain of events is initiated first by grasshoppers cannibalizing infected grasshoppers and then by cattle accidentally ingesting infected grasshoppers in the pasture. Contact transmission of this virus

between livestock also has been recognized

1 Biological Transmission of Arboviruses: Re-examination of and New Insights into Components, Mechanisms, and Unique Traits as Well as Their Evolutionary Trends - Goro Kuno* and Gwong-Jen J. Chang

Some blood-sucking arthropods may be able to complete a gonotrophic cycle (a physiological cycle of egg maturation in the female vector) once after emergence

without engaging in blood feeding and ovipositing. This phenomenon, called autogeny, is recognized in about 60 species and is controlled by multiple genes. It is not clear

when this phenomenon evolved in some blood-sucking dipterans, such as mosquitoes (family Culicidae), biting midges (family Ceratopogonidae), sand flies (family

Psychodidae), black flies (family Simuliidae), and other flies.

Autogeny is clearly advantageous for survival when blood meal sources are either only seasonally available or when blood feeding is suppressed for some reasons. Variation in

autogeny within a species of vector is well documented. Many geographic strains of Aedes aegypti in Africa are autogenous; however, this phenomenon is not generally

observed in most laboratory colonies

Senior author, Dr Luciano Moreira, of the Oswaldo Cruz Foundation in Rio de Janerio, which is preparing to host the Olympics this summer, said: 'The idea

has been to release Aedes mosquitoes with Wolbachia over a period of a few months, so they mate with Aedes mosquitoes ... and over time, replace the

mosquito population.'

In the Zika study, the team infected field mosquitoes and Wolbachia-infected mosquitoes with two strains of Zika currently circulating in Brazil.

Interestingly, molecular clock studies of the antihemostatic genes of a diverse group of organisms ranging from blood-feeding insects and leeches to mammals (such as

vampire bats) revealed that the development of antihemostatic mechanisms in ticks coincided with the evolution of blood coagulation in vertebrates

Although genetic and ecological factors set the limits within which a vector selects vertebrate hosts, opportunistic behaviour of vectors often modifies actual host

selection, depending on the densities of available vertebrate hosts in a given location. As an example, Culex fatigans females collected from houses in Pakistan fed more often

on humans than cattle in sheds or in agricultural fields, but the same mosquitoes resting in cattle sheds in winter fed mostly on birds and bovids. However, they changed

to humans and bovids during the spring and then back to humans and birds during summer

Vector-Enhanced Transmission

Vectors facilitate virus replication by injecting a variety of substances in saliva. Some of these substances promote viral replication. Others are 1immunosuppressant, including antihemostatic (acting to help bleeding or a type of anticoagulant to halt blot clotting)

substances. Injection of the latter substances results in modified physiologic conditions or behaviours of vertebrate hosts or shortening of blood vessel probing time (and hence

enhanced feeding activity) for vectors

1. being highly resistant to a disease because of the formation of humoral antibodies or the development of immunologically competent cells, or both, or as a result of some other mechanism, as interferon activities in viral infections.

Both viral persistence and vertical transmission in vector are important not only for viral survival in nature but also for their role in the establishment of

biological transmission. However, a mathematical study revealed that viruses could not be maintained in nature with vertical mode of transmission alone

no matter how high the rate of vertical transmission was, thus requiring occasional horizontal transmission

1Regarding the stimulatory effect of the symptoms of viral infection, one of the questions raised for haemorrhagic viral infections (such as dengue) was if

increased vascular permeability as a result of thrombocytopenia increases the probability of transmission to biting mosquitoes. The effects for increased

transmission were also investigated with respect to increased level of odour in vertebrate hosts infected with Tick-Borne Encephalitis Virus (TBEV). It was found that the viral infection increased the blood level of testosterone, which, in turn, rendered infected male rodents to be more aggressive and attractive to

estrous females, thus enhancing viral dissemination

1 Biological Transmission of Arboviruses: Re-examination of and New Insights into Components, Mechanisms, and Unique Traits as Well as Their Evolutionary Trends - Goro Kuno* and Gwong-Jen J. Chang, page 11

Cellular and humoral immunity.

It has become increasingly apparent that the important groups of molecules used by mammalian hosts for surveillance to detect invasion of exogenous

viruses are Toll-like receptors (TLR). Each TLR recognizes a distinctive pathogen-associated molecular pattern and invokes various antimicrobial

innate immunity. The TLR3 recognizes double-stranded RNA, the replicative by-product during RNA virus replication. Some vertebrate cells involved in adaptive immune system (such as B and T cells) are endowed with innate

sensing of viruses by means of Toll-like receptors. The Toll-like receptors identified for Vesicular Stomatitis Virus (VSV) were TLR7 and TLR8. However, not all TLR functions are protective. TLR3 was speculated to facilitate West

Nile Virus (WNV) entry into the brain causing lethal encephalitis

Furthermore, in arboviruses, substitution rates were found to be higher during replication in vertebrates than during replication in vectors

1 Biological Transmission of Arboviruses: Re-examination of and New Insights into Components, Mechanisms, and Unique Traits as Well as Their Evolutionary Trends - Goro Kuno* and Gwong-Jen J. Chang, page 14/15

Apparently, some arboviruses have evolved genes to suppress development of apoptosis to facilitate unimpeded viral replication. As an example, African swine fever virus has two

genes encoding for two proteins that inhibit development of apoptosis in the hosts

Marine and other aquatic environments.

Nearly all arboviruses infect only terrestrial animals, and larval stage of manyvectors is spent in aquatic conditions. However, very little has been generally known about arboviral association with other aquatic environments. In fact, arboviruses are not entirely dissociated from marine, riverine, and other aquatic environments. Migratory seabirds are also recognized to be frequently infested with ticks carrying a variety of tick-borne arboviruses, and their role in long-distance dissemination of viruses has been known

As mentioned earlier in the section of direct transmission, water voles in the lakes in western Siberia were reported to be infected directly by the Omsk hemorrhagic fever virus released

into water from the corpses of diseased muskrats. Normal muskrats were, in turn, infected while wading in water previously virus contaminated by the water voles that

shed the virus in urine and feces. Interestingly, it was determined that the virus survived in water for up to 2 weeks in the summer and up to 3 months in the winter in Siberia. those

reports raised a possible mechanism by which virus transfer could occur between vertebrates and arthropods by direct transmission in such aquatic environments.

1 Biological Transmission of Arboviruses: Re-examination of and New Insights into Components, Mechanisms, and Unique Traits as Well as Their Evolutionary Trends - Goro Kuno* and Gwong-Jen J. Chang, page 17

Most experts agree that Aedes aegypti similarly originated from a sylvatic ancestor in Africa and became domesticated later through association with human activities

1In fact, in many parts of Africa, sylvatic Aedes aegypti populations are stillzoophilic, rarely biting humans, and play little or no role in DENV and YFV

transmission. In parts of Asia, subpopulations of mosquitoes normally considered outdoor species, such as Culex tritaeniorhynchus, have become endophilic(indoor living), and in the Reunion Island (Indian Ocean), two dengue vectors, Aedes aegypti and Aedes albopictus reversed the their typical habitats, the

former becoming more exophilic (outdoor living) and not 2anthropophilic and the latter becoming more endophilic and anthropophilic

2Human-seeking or human-preferring, especially with reference to: bloodsucking arthropods, denoting the preference of a parasite for the human host as a source of blood or tissues over an animal host

1Biological Transmission of Arboviruses: Re-examination of and New Insights into Components, Mechanisms, and Unique Traits as Well as Their Evolutionary Trends - Goro Kuno* and Gwong-Jen J. Chang, page 17-19

A survey of emerging infectious diseases clearly demonstrated that of the 26 viruses known to infect both birds and mammals all were RNA viruses,

despite statistical projection that predicts DNA viruses to represent as much as 24.6% of emerging viruses. In addition, the pathogens infecting more than one

order of animals were found generally more likely to emerge as zoonotic agents than those infecting only one order

1Although the mean longevity of adult Aedes aegypti mosquitoes under natural conditions in the tropics has been determined to be only about 8.5 days (283), under laboratory conditions, YFV-infected and DENV-infected females of this mosquito could transmit virus as long as 101 days and 75

days, respectively

283. Sheppard, P. M., W. M. Macdonald, R. J. Tonn, and B. Grab. 1969. The dynamics of an adult population of Aedes aegypti in relation to dengue haemorrhagic fever in Bangkok. J. Anim. Ecol. 38:661–697.

On the other hand, for DENV, once the virus became established in urbanenvironments, the genetic linkage with sylvatic viral populations was

completely severed; and the viral populations in urban areas have been independently perpetuated between domesticated mosquito and human.

Interestingly, both Aedes aegypti and Aedes albopictus, the vectors of urban dengue, were found to be susceptible to infection by the DENV-2 strains

isolated in urban outbreaks but not by the strains isolated in sylvatic environments

1Biological Transmission of Arboviruses: Re-examination of and New Insights into Components, Mechanisms, and Unique Traits as Well as Their Evolutionary Trends - Goro Kuno* and Gwong-Jen J. Chang, page 23

Autoimmune, neurological and neurodevelopmental conditions such as Guillain-Barré syndrome and microcephaly in fetuses and newborns from mothers possibly exposed

to ZIKV in the two first trimesters of the pregnancy were notified during recent Zika disease outbreaks in French Polynesia and Brazil, in 2013 and 2015, respectively.

Further evidence is needed to establish a causal link between these neurological/neurodevelopmental impairments and infections with ZIKV

Additional modes of transmission also have been identified. Perinatal transmission can occur most probably by trans-placental transmission or during delivery when the

mother is infected. Sexual transmission was reported in two case reports. 1There is a potential risk of ZIKV transfusion-derived transmission. ZIKV RNA has been detected

in urine up to 10 days after onset of the disease. From the day five post-onset of fever, serological investigations can be conducted by detection of Zika-specific IgM

antibodies

Diagnosis by serology can be difficult as the virus can cross-react with other flaviviruses, therefore serological results should be interpreted according to the vaccination status and

previous exposure to other flaviviral infections. There is currently no vaccine available. Treatment with acetylsalicylic acid (aspirin) and non-steroidal anti-inflammatory drugs was discouraged because of a potential increased risk of haemorrhagic syndrome reported with other flaviviruses as well as the risk of Reye's syndrome after viral infection in

children and teenagers.

1 Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau VM. Potential sexual transmission of Zika virus. Emerg InfectDis 2015; 21(2): 359−61.

Phylogenetic Evolutionary Tree

Zika Virus, French Polynesia, South Pacific, 2013

http://wwwnc.cdc.gov/eid/article/20/6/14-0138_articleDOI: http://dx.doi.org/10.3201/eid2006.140138

The phylogenetic tree shows that the ZIKV that recently emerged in French Polynesia is similar to Cambodia 2010 and Yap State 2007

strains, which corroborates previous findings of an expansion of ZIKV Asian lineage

This ZIKV outbreak is the largest documented and the first known to be caused by an arbovirus other than DENV in French Polynesia.

“Millions of people in Latin America have been infected with the mosquito-borne bug which has been linked to serious brain defects in newborns. At least 4,000 Brazilian babies have been born with the

condition – called microcephaly – since October.”TheMailonline, March 27, 2016

How Many Cases? 4,000? Where is the media getting this number in March 2016?

Feb 24, 2016

Image Source: http://www.huffingtonpost.com/entry/guide-to-zika-virus_us_56a272afe4b076aadcc675b6

Zika virus (ZIKV) is a member of the Spondweni serocomplex within the genus Flavivirus, family Flaviviridae. Other mosquito-borne flaviviruses of public health importance include yellow fever, dengue, St. Louis encephalitis, West Nile and

Japanese encephalitis viruses.

Zika virus was first isolated from a sentinel rhesus monkey (No. 766) placed in the Zika Forest near Lake Victoria, Uganda in April 1947; a second isolation from the mosquito Aedes africanus followed at the same site in January 1948

Genetic Characterization of Zika Virus Strains:Geographic Expansion of the Asian Lineage

1Although it is thought that enzootic ZIKV is maintained primarily in a monkey/mosquito transmission cycle, antibodies have been detected in

numerous other animal species including water buffalo, elephants, goats, hippos, impala, kongoni, lions, sheep, rodents, wildebeest, and zebras

1Darwish MA, Hoogstraal H, Roberts TJ, Ahmed IP, Omar F (1983) A seroepidemiological survey for certain arboviruses (Togaviridae) in Pakistan. Trans R Soc Trop Med Hyg 77: 442–445.

Human case reports of clinically diagnosed ZIKV infections include self-limiting acute febrile illnesses with fever, headache, myalgia and rash, similar to that caused by many

other arboviruses found throughout the tropics. This clinical picture could easily be mistaken for dengue (DEN) or chikungunya (CHIK) fevers, two common arboviral

infections which both produce similar clinical presentations.

Zika virus, has a positive-sense, single-stranded RNA genome approximately 11 kilobases in length. A previous genetic study using nucleotide sequences derived from the

NS5 gene indicated three ZIKV lineages (East African, West African and Asian)

The Spondweni virus strain SM-6 V-1 was used as the outgroup for all phylogenies, as Spondweni virus is the most closely related flavivirus (antigenically and genetically) to

ZIKV

1Some viruses, such as Venezuelan equine encephalitis virus, are readily transmissible by aerosol. Under special circumstances of high virus concentration, however, many arboviruses classified at a lower biohazard level (such as Dengue virus [DENV]) also

can infect vertebrates by aerosol.

Omsk haemorrhagic fever virus is also spread among muskrats by direct contact, as the virus is shed in urine and faeces.

1 Biological Transmission of Arboviruses: Re-examination of and New Insights into Components, Mechanisms, and Unique Traits as Well as Their Evolutionary Trends

Recently, at least under laboratory conditions, an indirect mode of oral transmission was proposed to be possible for Vesicular stomatitis virus (VSV) New Jersey serotype.

According to the proposal, the chain of events is initiated first by grasshoppers cannibalizing infected grasshoppers and then by cattle accidentally ingesting infected grasshoppers in the pasture. Contact transmission of this virus between livestock also

has been recognized

However, direct transmission of arboviruses to mosquitoes occurs in aquatic environments as well. In fact, YFV, JEV, Western equine encephalitis virus, and Rift Valley fever virus in contaminated water, could be transmitted orally to mosquito larvae through feeding activity. Under such conditions, viruses from the infected

animals released into water as a result of cannibalism or death due to infection could be ingested by mosquito larvae.

Autogeny - a hypothetical organic phenomenon by which living organisms are created from non-living matter (or without a blood meal to create eggs in female mosquitos)Vertical Transmission - A vertically transmitted infection is an infection caused by bacteria, viruses, or in rare cases, parasites transmitted directly from the mother to an embryo, fetus, or baby during pregnancy or childbirth

Interestingly, vertical transmission of St. Louis encephalitis virus through eggs laid by autogenous Aedes mosquitoes was also proven in a laboratory experiment

It has been reported that wind-blown mosquitoes can travel distances of several hundred (700Km) kilometers over the open ocean [25,26]. However,

due to the great distances involved, it seems likely that the virus was introduced as a result of travel or trade activities whereby either a viremic person, enzootic host species, and/or an infected and subsequently infective mosquito (adult or immature) was transported to the island as suggested by Duffey et al. [9].

This hypothesis is further supported by the fact that no monkeys were present on Yap Island during the 2007 epidemic

25. Asahina S (1970) Transoceanic flight of mosquitoes on the Northwest Pacific Jpn J Med Sci Biol 23: 255–258.26. Curry DP (1939) A documented record of a long flight of Ae. sollicitans. Proc New Jersey Mosq Exterm Ass 26: 36–399. Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, et al. (2009) Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med 360: 2536–2543..

1The E protein (<53 kDa) is the major virion surface protein. E is involved in various aspects of the viral cycle, mediating binding and membrane fusion

1. Molecular Evolution of Zika Virus during Its Emergence in the 20th Century

Molecular Evolution of Zika Virus during Its Emergencein the 20th Century (February, 2014)

1 This virus received little attention until its sudden emergence in Yap Island (Micronesia) in 2007, which involved about 5000 persons, revealing its epidemic capacity. Patients develop

a mild dengue-like syndrome, including fever, headache, macular or papular rash, periarticular edema (fluid retention and swelling of joints), arthralgia (pain in the joints);

sometimes asthenia (weakness, lacking bodily strength; vomiting;, diarrhea and abdominal pain if another virus is present) and non-purulent conjunctivitis. This clinical similarity

with other, more commonly diagnosed arboviral infections such as chikungunya (CHIKV) and dengue (DENV), might delay the diagnosis and/or lead to underestimation of

ZIKV infections.

1 Zika Virus in Gabon (Central Africa) – 2007: A New Threat from Aedes albopictus (Asian tiger mosquito)?

While some mosquito species (including Aedes aegypti commonly known as Yellow Fever mosquito) previously found to be associated with ZIKV, were captured and tested here, only Aedes albopictus pools were positive for this virus. Moreover, this species largely

outnumbered Aedes aegypti in the suburbs of Libreville where human cases were detected, suggesting that Aedes albopictus played a major role in ZIKV transmission in

Libreville.

1Now is the time for the Zika virus – Expected arrival in Columbia, 2015 (note: Zika Forest Virus is neurotropic virus that is capable of infecting nerve cells)

1The ZIKV is a flavivirus and the same family of viruses of dengue, yellow fever and West Nile. The Zika is a RNA virus closely related to the Spondweni, Ilheus, Rocio

and St. Louis encephalitis viruses.

The ZIKV apparently does not trigger haemorrhagic disorders such as dengue and chikungunya. Hospitalization is generally not required and no mortality has been reported yet. Moreover, an important and peculiar epidemiological aspect of ZIKV is that it also

can be transmitted through sexual contact

But perhaps, from the clinical and public health point of view, most disturbing it is that the three viruses cause similar clinical symptoms and that the country would not have

sufficient resources to make a differential diagnosis of the virus

1 This event coincided with the first report of autochthonous transmission of ZIKV in Brazil and with the confirmation of the Asiatic lineage of the circulating strain. Currently, Brazil is the country with the highest number of ZIKV-positive cases in Latin America with

circulation of the virus in almost the entire national territory (see next 2 corresponding slides for images)

1 Overview of Zika virus (ZIKV) infection in regards to the Brazilian epidemic: Brazilian Journal of Medical and Biological Research (2016) 49(5): e5420, http://dx.doi.org/10.1590/1414-431X20165420

The genome is a single-stranded, positive-sense RNA of approximately 11 kb. The genomic organization follows that of the flaviviruses with two flanking non-coding regions

(NCR), i.e. 59 and 39, and a long open reading frame encoding a single polyprotein: (NCR59)-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-(NCR39). (See Slide 7 for non-

structural protein NS5)

Worldwide dissemination of ZIKV is thought to be due to loss of the N154 glycosylation site of the E protein, thus permitting viral adaptation to a broader range of mosquito

vectors

A recent finding demonstrated that pools of Aedes albopictus collected in the capital city of Gabon, Libreville, are positive for the ZIKV African lineage, strongly suggesting the

participation of Aedes albopictus mosquitoes in the ZIKV urban cycle in Africa

It is unknown how the urban transmission occurs. Probably during heavy rainfalls, the sylvatic mosquito population can grow up progressively and disseminate the virus to nearby villages, and from there to larger urban centers, thus the urban cycle may occur with human-to-human transmission (A). Another possible route for human-to-human ZIKV infection is the direct human invasion of the forest habitats, where the infection can be transmitted to human hosts from forest dwelling mosquitoes (B).

Other modes of ZIKV transmission, including vertical, sexual and parenteral have also been proposed. The detection of ZIKV RNA in semen from a Tahiti man complaining from

hematospermia during the ZIKV outbreak in French Polynesia (1), and the probable sexual transmission of ZIKV from a man to woman with sexual contact a few days

before the onset of the man’s symptoms (2) suggests that the ZIKV could be transmitted sexually.

(1). Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau VM. Potential sexual transmission of Zika virus. Emerg/Infect Dis 2015; 21: 359–361, doi: 10.3201/eid2102.141363.(2). Foy BD, Kobylinski KC, Chilson Foy JL, Blitvich BJ, Travassos da RA, Haddow AD, et al. Probable non-vectorborne transmission of Zika virus, Colorado, USA. EmergInfect Dis 2011; 17: 880–882, doi: 10.3201/eid1705.101939

Another relationship suggesting ZIKV vertical transmission is the observed unusual increase of microcephaly cases in the Northeast part of Brazil. The Brazilian health authorities are suspicious of a connection between the microcephaly and ZIKV, as the increase is associated spatio-temporally to the ZIKV outbreak in the country. Detection of

ZIKV sequences in fetuses with malformations (microcephaly, hidrancephaly, hydropsy) strongly suggests ZIKV transplacental passage and capacity to cause congenital

infection with neuronal damage. However, the temporal association between ZIKV and microcephaly is not sufficient to establish a ‘‘cause-effect’’ relationship

Even if it is supposed that ZIKV causes intrauterine infections, more studies are needed in order to define the pathogenic effect of ZIKV on the neuronal tissues

Moreover, an increased prevalence of microcephaly could be due to multiple causes including other viruses and parasites, irradiation,

toxic substances, intrauterine growth retardation and chromosomal disorders.

All of these factors have to be discarded in order to establish a vertical transmission of ZIKV and potential for induction of birth

defects or stillbirths.

1 Martin DA, Muth DA, Brown T, Johnson AJ, Karabatsos N,Roehrig JT. Standardization of immunoglobulin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections. J Clin Microbiol 2000; 38: 1823–1826.

Up to this day, there is no officially approved commercial kit for ZIKV serological diagnosis. During the Yap ZIKV epidemic IgM-serological investigation on the collected samples was performed at the Arboviral Diagnostic and Reference Laboratory, Centers for Disease Control and Prevention (Atlanta, GA, USA) following a routine protocol for anti-

arboviral 1IgM detection

?

?

Really

Really

1 'We feel it's time to move from precautionary language to more forceful language to get people to take action,' said Dr Bruce Aylward, who is

leading WHO's Zika response.

The virus causes only a mild and brief illness, at worst, in most people. But in the last year, infections in pregnant women have been strongly linked to fetal deaths

and devastating birth defects, mostly in Brazil, where the Health Ministry said Tuesday that 1,113 cases of microcephaly have been confirmed since

October.1 TheMailonline, April 13, 2016

“Millions of people in Latin America have been infected with the mosquito-borne bug which has been linked to serious brain defects in newborns. At least 4,000 Brazilian babies have been born with the

condition – called microcephaly – since October.”TheMailonline, March 27, 2016

Now, compare the propagandising headline 3 weeks prior to this on March 27th concerning how many people were allegedly infected

below

As the microcephaly cases rose in Latin America, a number of alternative theories circulated through the public. Some

claimed the cause was a vaccine given to pregnant women. Some suspected a mosquito-killing larvicide, and others

wondered whether genetically modified mosquitoes were to blame.

They still don't have some of the evidence they hope for. So far, for example, there have been no published studies demonstrating Zika causes such birth defects in animals.

'The purist will say that all the evidence isn't in yet, and they're right but this is public health and we need to act,'

the WHO's Aylward said.(TheMailonline, April 13, 2016)

No relationship between myelitis and ZIKV infection has been established. However, the neurotropism of some flaviviruses such as DENV, Japanese

encephalitis, and West Nile causing encephalitis and myelitis is well documented, therefore, it is highly probable that ZIKV can also be involved as a

causing agent in inflammatory neurological disease.

There are different causes for microcephaly development, such as exposure to environmental factors (heavy metals, smoking, alcohol, radiation), genetic

causes, maternal diabetes, and pathogens (cytomegalovirus, toxoplasmosis, rubella, herpes simplex, syphilis, HIV, and West Nile virus)

1 Mlakar J, Korva M, Tul N, Popovic M, Poljsak-Prijatelj M,Mraz J, et al. Zika Virus Associated with Microcephaly, N EnglJ Med 2016; 374: 951–958, doi: 10.1056/NEJMoa1600651.

Similar results were obtained by the study of 1Mlakar et al, where ZIKV positive fetus with microcephaly had almost complete agyria (silver poisoning), internal hydrocephalus, and calcifications in the cortex and subcortical white matter

in the frontal, parietal, and occipital lobes. Almost all of the case-reports demonstrate fetal abnormalities apart from microcephaly including multifocal

intracranial and placental calcifications, ventriculomegaly, intrauterine growth restriction, brain atrophy, and other less frequent abnormalities

A report from Paraiba, Brazil related two fetuses with microcephaly by prenatal ultrasound from two pregnant women who had ZIKV symptoms but were ZIKV RNA negative in blood, and ZIKV positive

in amniotic fluid… neonate was born with severe ventriculomegaly, 1microphtalmia, cataract (clouding of the lens of the eye) and severe arthrogryposis

(Greek, literally meaning "curving of joints“ of the legs and arms - stock image below)

1 https://ghr.nlm.nih.gov/condition/microphthalmia- Microphthalmia is an eye abnormality that arises before birth. In this condition, one or both eyeballs are abnormally small. Microphthalmia may or may not result in

significant vision loss. May also have a condition called coloboma. Colobomas are missing pieces of tissue in structures that form the eye. They may appear as notches or gaps in the coloured part of the eye

called the iris; the retina, which is the specialized light-sensitive tissue that lines the back of the eye; the blood vessel layer under the retina called the choroid; or in the optic nerves, which carry information from

the eyes to the brain. Also can be caused by West Nile virus infection, toxoplasmosis, cytomegalovirus, rubella, herpes simplex virus and syphilis

Fetal deaths and miscarriages have also been reported in ZIKV-positive pregnant women. 1,2The reported miscarriages occurred during the first trimester (11th and 13th weeks).

Women who presented fetal deaths were infected with ZIKV during the second and third trimester, and 2presented at least maculopapular rash during the acute phase of infection. 1Newborn lethality within the first 20 hrs after birth from ZIKV infected mothers has also

been reported.

1Martines RB, Bhatnagar J, Keating MK, Silva-Flannery L,Muehlenbachs A, Gary J, et al. Notes from the field: evidence of zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses – Brazil, 2015. MMWR Morb Mortal Wkly Rep 2016; 65: 159–160. Available at: http://www.ncbi.nlm.nih.gov/pubmed/26890059, doi: 10.15585/mmwr.mm6506e1.2Brasil P, Pereira JP Jr, Raja GC, Damasceno L, Wakimoto M, Ribeiro Nogueira RM, et al. Zika virus infection in pregnant women in Rio de Janeiro – Preliminary Report. N Engl J Med 2016. Available at: http://www.ncbi.nlm.nih.gov/pubmed/26943629.

Currently, Brazil is experiencing the largest ZIKV epidemic among the countries of Latin America. Preliminary data announced on December 2015, by the European Centre for

Disease Prevention and Control, estimate that between 440,000 and 1.3 million cases of autochthonous transmission of ZIKV have occurred in Brazil, but these data are largely underestimated and the real magnitude of the Brazilian ZIKV epidemic may be much

higher3Microcephaly is either primary (anomaly of development during the first 7 months of

gestation) or secondary to an insult incurred during the last 2 months of gestation or during the perinatal period

3Zika Virus Infection and Microcephaly, J.G.Millichap, Division of Neurology, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL; and Departments of Pediatrics and Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL

Source: http://time.com/4202262/zika-brazil-doctors-recife-investigation-outbreak/ - February 1, 2016

Please note: From all the slides so far, and the slides to come, you the viewer will notice that ALL the studies confirm that The Americas and Latin America are specifically dealing with the ASIAN lineage of the Virus. Aedes Aegypti may be a vector, but all the genetic markers are conclusively expressing the Asian Tiger Mosquito and the ASIAN branch

According to an epidemiological report for monitoring cases of microcephaly in Brazil, until February 6, 2016, 75.8% of the cases reported (3,852/5,079)

remained under investigation, of which 62.5% (n=3,174) were reported in 2015 and 37.5% (n=1,905) in 2016.

According to the final classification, 24.2% of the total cases were investigated and classified and, of these, 15.1% (n=765) were discarded for the presence of

microcephaly and/or changes in the central nervous system suggestive of congenital infection. Of the remaining, 9.1% (462/5,079) were confirmed

according to specific protocol definitions and, of these, only 8.9%, i.e., 41 out of 462, were confirmed as associated with infection with Zika virus by clinical and laboratory testing following the criteria for diagnostic confirmation . As for cases progressing to miscarriage or postpartum death, 1.8% of the total

(91/5,079) were recorded.

Zika virus infection and pregnancy: http://dx.doi.org/10.1590/1806-9282.62.02.108 

In the primary databases consulted, there is only one case report occurred in French Polynesia in which guillain-barré syndrome was diagnosed in a patient

infected with Zika virus. The report showed the first case of GBS manifested seven days after febrile illness characterized as Zika virus infection based on

serological results

1,2 Information about laboratory abnormalities during Zika virus infection are scarce in the literature, but leukopenia, thrombocytopenia, elevation of serum lactate dehydrogenase, and elevated markers of inflammatory activity such as C-reactive protein are reported. To date (March 2016), there are no commercial tests allowing serological diagnosis of Zika virus

infection. The diagnosis of acute infection can be done by RT-PCR within a week from the onset of symptoms.

1 Zammarchi L, Stella G, Mantella A, Bartolozzi D, Tappe D, Günther S, et al. Zika virus infections imported to Italy: clinical, immunological and virological findings, and public health implications. J Clin Virol. 2015; 63:32-5. 2 Cao-Lormeau VM, Roche C, Teissier A, Robin E, Berry AL, Mallet HP, et al. Zika virus, French polynesia, South pacific, 2013. Emerg Infect Dis. 2014; 20(6):1085-6.

Zika virus infection and pregnancy: http://dx.doi.org/10.1590/1806-9282.62.02.108 

Immunoenzymatic test (ELISA) for detection of immunoglobulins (IgG and IgM) and plaque-reduction neutralization test (PRNT) can be used.

IgG antibodies are found in all body fluids. They are the smallest but most common antibody (75% to 80%) of all the antibodies in the body. IgG antibodies are very important in fighting

bacterial and viral infections. IgG antibodies are the only type of antibody that can cross the placenta in a pregnant woman to help protect her baby (fetus).

IgM antibodies are the largest antibody. They are found in blood and lymph fluid and are the first type of antibody made in response to an infection. They also cause other

immune system cells to destroy foreign substances. IgM antibodies are about 5% to 10% of all the antibodies in the body

Simultaneous outbreaks of dengue, chikungunya and Zika virus infections: diagnosis challenge in a returning traveller with nonspecific

febrile illness (Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious

Diseases - 11 February 2016)

We report the first Zika virus infection case imported to Switzerland. On 19 November 2015 a 29-year-old woman attended our clinic with a 4-day flulike

syndrome after a trip in Colombia from 30 October to 15 November. On serum collected on 20 November (5 days after onset of symptoms), chikungunya

IgM/IgG (immunofluorescence commercial test chikungunya) ..was negative, but real-time Zika reverse transcription (RT) PCR was positive.

Urine collected 12 days after symptom onset was positive for Zika by real-time RT-PCR with a higher virus load than in serum collected 7 days before.

At the last medical visit, 20 days after symptom onset, she described progressive relief from the arthralgia, and urine real-time RT-PCR was negative.

May 28 2016 - A disfiguring tropical disease that had been contained to Syria has now spread across the Middle East as millions are displaced from the war-

torn region.

Cutaneous leishmaniasis is a parasitic disease spread by bites from infected sand flies. It can lead to severe scarring,

often on the face, and regularly goes undiagnosed and untreated.

As well as severe scarring, the disease causes open sores on the skin,

breathing difficulty, nose bleeds and swallowing difficulties

A possible cause for Microencephaly – Vanadium Pentoxide

Vanadium pentoxide is used in ceramics and as a catalyst as well as in the production of superconductive magnets.

Populations in areas with high levels of residual fuel oil consumption may also be exposed to above-background levels of vanadium, from increased particulate deposition

upon food crops and soil in the vicinity of power plants. Vanadium concentrations in surface water can range from approximately 0.04 to 220 µg/L depending on

geographical location.

http://www.atsdr.cdc.gov/PHS/PHS.asp?id=274&tid=50 (Agency for Toxic Substances and Disease Registry)

We do not know whether vanadium can cause birth defects in people.

Studies in animals exposed during pregnancy have shown that vanadium can cause decreases in growth and increases in the occurrence of birth defects. These effects are usually observed at levels which cause effects in the

mother. Effects have also been observed at vanadium doses which did not cause effects in the mother.

Breathing air with vanadium pentoxide can result in coughing which can last a number of days after exposure.

Damage to the lungs, throat, and nose have been observed in rats and mice exposed to vanadium pentoxide

A number of effects have been found in rats and mice ingesting several vanadium compounds. The effects include: decreases in

number of red blood cells, increased blood pressure, mild neurological effects and developmental effects in animals

Lung cancer has been found in mice exposed to vanadium pentoxide. The International Agency for Research on Cancer (IARC) has

determined that vanadium is possibly carcinogenic to humans.

Guillain-Barré syndrome associated with the Zika virus outbreak in Brazil (DOI: 10.1590/0004-282X20160035)

In 2007, the first large outbreak of ZIKV occurred in the Yap Islands, an archipelago located within the Federated States of Micronesia. Forty-nine cases of ZIKV infection

were confirmed, with roughly 73% of the islanders aged 3 years or older being potentially infected with ZIKV according to a household survey. A larger outbreak

occurred in the end of 2013 in the French Polynesia. It is still unclear how ZIKV was introduced in Brazil. The lineage identified in serum samples from the first cases in

the State of Bahia (image below) resembles a previously identified Asian lineage

1 Guillain-Barre syndrome, an acute inflammatory demyelinating polyneuropathy which typically

occurs after minor viral and bacterial infections.

The syndrome usually begins with tingling and weak-ness in the feet and legs and spreads to the upperbody and arms, often evolving into paralysis over a 4-week period, with accompanying disturbances of sensation and cranial nerve function. The risk ofGuillain-Barre syndrome increases with age, with a

predilection for males

1 Pathogenesis of Zika Virus-Associated Embryopathy, BioResearch Open Access Volume 5.1, 2016 DOI: 10.1089/biores.2016.0004

A recent study showed that ZIKV portends similar cellular targets as dengue virus (DENV). Fibroblasts and keratinocytes seem to play a

major role in the pathogenesis of the disease, likely serving as a gateway to infection. Another

noticeable finding is that 1ZIKV promotes apoptosis, leading to decreased immune response, the same strategy used by DENV.

Finally, dendritic cells seem to be more susceptible to ZIKV infection. A German study using a mice model observed active replication in neurons, confirming an active role of ZIKV in the

destruction of nervous tissue. In addition, it was observed that the host´s immune response also contributes to the damage to the

central nervous system

1Hamel R, Dejarnac O, Wichit S, Ekchariyawat P, Neyret A, Luplertlop N et al. Biology of Zika Virus infection in human skin cells. J Virol. 2015;89(17):8880-96. doi:10.1128/JVI.00354-15

Teratogenic – an external agent that causes birth defects

Guillain-Barré syndrome associated with the Zika virus outbreak in Brazil(DOI: 10.1590/0004-282X20160035)

(Thrombocytopenia)

The Media still continue to insist that Aedes Aegypti is the cause, and not the ASIAN Lineage that all studies and phylogenetic trees conclude IS the correct lineage!

1Similarities to Hypervitiminosis A

Strong parallels between the manifestations of ZIKV infection and those of hypervitaminosis A can be seen in the RA (Retinoic Acid) syndrome associated with the treatment of acute promyelocytic leukemia with RA, and in the effects of vitamin A supplements or excess dietary intakes of

vitamin A-containing foods such as liver.

Symptoms of acute vitamin A poisoning include fever, arthralgia and arthritis, myalgia, headache, flu-like symptoms, conjunctival congestion, lymphadenopathy, pruritus, erythematous rash, weakness, anorexia, skin peeling and altered mental status.

Less common effects include hepatosplenomegaly, miscarriage, Guillain-Barre syndrome and thrombocytopenic purpura.

Likewise, bone pain, fatigue and headache are major symptoms of hypervitaminosis A

1 Pathogenesis of Zika Virus-Associated Embryopathy, BioResearch Open Access Volume 5.1, 2016 DOI: 10.1089/biores.2016.0004

After the suspicion was raised that the increase in birth defects could be attributed to the massive use of the larvicide pyriproxifen, Brazil’s health ministry quickly

posted a clarification on the use of pyriproxifen, noting that there is no scientific basis for a link between the use of the larvicide and microcephaly and that some cities that have not used it have also reported microcephaly cases.

1 Zika virus and microcephaly: is the correlation causal or coincidental?

1 February 2 2016 Giuseppina Liuzzi, Vincenzo Puro, Simone Lanini, Francesco Vairo, Emanuele Nicastri, Maria Rosaria Capobianchi, Antonino Di Caro, Mauro Piacentini, Alimuddin Zumla1, Giuseppe Ippolito

In fact, it is well known that the blockade of autophagy during embryogenesis leads to nervous system defects (Fimia et al., 2007). This provides some

evidence to support the involvement of ZIKV in the damage of cells from multiple lineages, including neural cells (Bell et al., 1971).

Besides perturbation of the autophagy pathway, a summary of pathogenetic mechanisms involved in the generation of microcephaly is reported in the

section “What is microcephaly?” Although the mechanisms of ZIKV pathogenesis appear to fall in line with the requirements for centrosome abnormalities, there is as yet no evidence to prove culpability. Future studies need to be

performed to definitively establish this link.

Zika Virus Outbreak in Haiti in 2014: Molecular and Clinical Data

Zika virus is currently spreading rapidly through the Americas, including the Caribbean, where it has emerged as a major public health problem due to the

linkage with birth defects, including microcephaly. We report the isolation of Zika virus from 3 children in rural Haiti (port-au-prince) in December, 2014, as part of a study of acute undifferentiated febrile illness that was being conducted by our research group; from one of these children, we also isolated dengue virus

serotype 1 (DENV-1).

On analysis of nucleotide sequence data from these and Zika strains from other locales, the South American/Haitian sequences cluster within the Asian clade and clearly branch out from a sequence circulating in Easter Island, which originated, in turn, from French Polynesia. On further analysis of one specific

gene sequence for which more data were available, there appeared to be slight separation of Haitian strains and the strains from Brazil, Suriname,

Puerto Rico and Guatemala, with molecular clock analysis suggesting that Zika virus was present in Haiti as early as mid-2013.

These findings raise questions about the origin of Zika virus in the Caribbean, and subsequent patterns of circulation of the virus within the

Americas.

If this case was Zika-Virus related 19 years ago, the phylogenetic tree would have already established this presumption. But this strain is of the ASIAN Lineage, not the Aedes

Aegypti which is the AFRICAN lineage

In the present study, we describe the entry receptors and cellular targets of the most recent ZIKV isolate, responsible for the recent epidemic in French

Polynesia, to provide general insights into the interaction between this virus and its human host.

The capacity of ZIKV to replicate ex vivo in human skin cells was also studied. Infection of human skin explants with ZIKV resulted in a gradual increase in

the viral copy number, with maximal levels at 5 days post-infection (dpi), pointing to a process of active viral replication

Immature dendritic cells (iDC) have been reported to be permissive for DENV infection, and as such, they are recognized as an important target for

propagation of this virus in the human skin. ZIKV infection was therefore also investigated on this cell type, using DENV as a control, by analyzing the

intracellular presence of the viral envelope protein by flow cytometry. Our results show that about 50% of human in vitro-generated immature dendritic cells

challenged with ZIKV at an MOI of 0.5 for 24 hpi expressed the viral envelope. This percentage is identical to that for cells infected with DENV under the

same experimental conditions.

These results indicate that immature dendritic cells are also permissiveto infection by this member of the flavivirus family.

1 RESULTS - Human skin cells are permissive for ZIKV infection and replication.Given the capacity of mosquitoes to inoculate ZIKV into the human skin during the blood-

feeding process, the potential target cells for infection with this virus are likely to be localized to the epidermis and dermis, which also constitute the first line of defense

1 Biology of Zika Virus Infection in Human Skin Cells, Hamel et al

1A susceptible mouse model for Zika virus infection

1 Dowall S.D., Graham V.A., Rayner E., Atkinson B, Hall G., Watson R., Bosworth A., Bonney L. Kitchen S, and Hewson R. Public Health England, Porton Down, Salisbury, Wiltshire, SP4 0JG. UK April 1st 2016

ZIKV (strain MP1751, isolated in 21962 from pools of Aedes africanus) was obtained from the National Collection of Pathogenic Viruses, UK. Virus was

cultivated in Vero cells (European Collection of Cell Cultures, UK) grown in with Dulbecco’s Modified Eagle Medium containing GlutaMAX (Invitrogen, UK) and

supplemented with 2% heat-inactivated foetal bovine serum (Sigma, UK).

2 Twelve Isolations of Zika Virus from Aedes (Stegomyla) africanus (Theobald) taken in andAbove a Uganda Forest - A. J.HADDOW, M. C.WILLIAMS, J.P.WOODALL, D.I.H.SIMPSON & L.K.H.GOMA

1Our report has studied the effect of ZIKV infection in type-I interferon receptor deficient (A129) mice and the parent strain (129Sv/Ev) after subcutaneous challenge in the lower leg to mimic a mosquito bite. A129 mice developed

severe symptoms with widespread viral RNA detection in the blood, brain, spleen, liver and ovaries. Histological changes were also striking in these animals. 129Sv/Ev mice developed no clinical symptoms or histological

changes, despite viral RNA being detectable in the blood, spleen and ovaries, albeit at lower levels than those seen in A129 mice. Our results identify A129

mice as being highly susceptible to ZIKV and thus A129 mice represent a suitable, and urgently required, small animal model for the testing of

vaccines and antivirals.

The most significant lesions referable to infection with ZIKV in the mice were those observed in the brain. Nuclear fragmentation, perivascular cuffing by

inflammatory cells, and degenerative cells in the hippocampus have been reported previously. Since the ZIKV field is still developing, there is currently no

“prototype” strain that researchers are using to allow comparison of results between laboratories. ZIKV is subdivided into two phylogenetically distinct

genotypes, African and Asian. This is the first report of a suitable murine model and can accelerate the testing of novel interventions against this pathogen declared

as public health emergency of international concern.

1 Dowall S.D., Graham V.A., Rayner E., Atkinson B, Hall G., Watson R., Bosworth A., Bonney L. Kitchen S, and Hewson R. Public Health England, Porton Down, Salisbury, Wiltshire, SP4 0JG. UK April 1st 2016

http://www.inovio.com/clinical-trials/infectious-disease-trials/

Using a technology called electroporation (EP) which delivers brief electrical pulses

It’s unfortunate to see some of the world’s worst nefarious corporations are all heavily involved in making a ‘’safe’’ vaccine for humans to take, such as DARPA, with the emerging new fields of Synthetic

biology and artificially-created un-natural Artificial DNA and Intelligence and genes, testing vaccines using mice, especially genetically-engineered knock-out mice, are assumed to work in the same way in a conscious human being? And using Green Monkey kidney cells are not human cells, even though

studies have been done on human fibroblasts and neuroblastomas in vitro and ex vivo

Summary so far – findings and problems

• Can be remain and be transferred via saliva, semen and urine• No Monkeys were on the Island for Yap Island (2007), suggesting it was not

of sylvatic cycles. Aedes Albopictus was the most prominent Mosquito found and not Aedes Aegypti, the mosquito named to be in the Americas

• Zika virus is of the Asian lineage, the species is cited to be Aedes Aegypti, but the procrastinator appears to be of Haitan subtype (Port-Au-Prince area) of the Asian lineage and transferred into Brasil via 2014 football tournament

• Has the ability to cross the placental barrier, increases pro-tumour cells, and targets specific cells and stem cells such as immature dendritic cells and epithelial cells

• Possible other causes is known as Hypervitaminosis A• Possible poisoning by Vanadium Pentoxide (patient has same symptoms)• Possible mercury and heavy metal toxicity acting as adjuvant• Quick infector between 3-7 days but doesn’t leave long IgM markers• Zika Forest Virus RNA is found in cells in amniotic fluid• Possible contaminant larvicide pyriproxifen• It’s well known that the blockade of autophagy during embryogenesis leads

to nervous system defects; perturbation of the autophagy pathway• mechanisms of ZIKV pathogenesis appear to fall in line with the

requirements for centrosome abnormalities

• Fibroblasts and keratinocytes seem to play a major role in the pathogenesis of the disease, likely serving as a gateway to infection

• it was observed that the host´s immune response also contributes to the damage to the central nervous system

• Can cause Guillian-Barre Syndrome and miscarriages/ abortions• Can be spread vertically, parenterally and via body fluids/sexually• Climate now enables Autochthonous transmission of ZIKV in Brazil• ZIKV is able to increase its replication via induction of autophagy in

the host cell.• Can be transmitted via blood transfusion

The availability of different entry receptors is likely to provide an evolutionary advantage for the virus, and as a result, the virus is able to infect a wide range of target cells and

invade the human host. We show here that ZIKV entry is mediated by DC-SIGN, AXL, Tyro3, and, to a lesser extent, TIM-1. Indeed, ZIKV infection strongly enhanced TLR3 expression,

in association with the production of Interferon (IFN) Type1 and IFN Type 2 in infected cells

The precise mechanism by which ZIKV induces autophagy still needs to be determined. Nevertheless, similar to the findings for DENV, the results from our study demonstrating the

co-localization of ZIKV with LC3 strongly suggest that autophagocytic vacuoles are the site of viral replication. However, the exact molecular mechanism(s) by which ZIKV highjacks

components of the autophagosome pathways remains to be determined.

The autophagy pathway is also used by plasmacytoid dendritic cells to recognize viral single-stranded RNA (ssRNA). Toll-like receptors (TLRs) are key molecules in innate immunity, which recognize various molecules derived from microbes. Some TLRs are present on the plasma membrane, while others localize to endosomes (TLR3, TLR7, TLR8, and TLR9). Among these, TLR7

recognizes viral ssRNA in endosomes and triggers immune responses, such as induction of inflammatory cytokines

interferon-α the major interferon produced by virus-induced leukocyte cultures; its primary producer cells are null CELLS, and its major activities are antiviral activity and activation of NK CELLS.

interferon-β the major interferon produced by double-stranded RNA-induced fibroblast cultures; the major producer cells are FIBROBLASTS, epithelial cells, and MACROPHAGES, and its major activity is antiviral.interferon-γ the major interferon produced by lymphocyte cultures that have been immunologically stimulated by mitogens or antigens; the major producer cells are T LYMPHOCYTES, and its major activity is IMMUNOREGULATION.

1Outside of the context of viral infection, deregulation of autophagy can lead to various pathologies including heart disease, neurodegeneration and cancer

(see slides concerning TP53)

1 Dengue Virus and Autophagy - Nicholas S. Heaton and Glenn Randall Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA, July 2011, page 3

AutophagyZIKV infection also induced an autophagy program, as demonstrated by the presence

of characteristic autophagosome-like vesicles in the infected fibroblasts.

Autophagy is a process characterized by the presence of double-membrane vesicles, known as autophagosomes, that recruit cytoplasmic material and subsequently fuse with

lysosomes for protein degradation. Autophagy not only participates in the degradation of proteins and damaged organelles in the cytoplasm to maintain homeostasis but also is involved in host immunity against pathogen infection. This is particularly illustrated by vesicular stomatitis virus, Sendai virus, and herpes simplex virus 1-infected cells, in

which autophagymediated degradation of viral proteins limits viral replication andpromotes cell survival.

In contrast, the autophagy process can be subverted by viruses. This is true for several arboviruses, including DENV, Chikungunya virus, and Japanese encephalitis virus, that

use components of the autophagy pathway to promote their replication and dissemination by clearing cells through multiple mechanisms.

1 Despite its antiviral functions, autophagy is subverted by some viruses to perform pro-viral roles. Poliovirus, coxsackievirus B3 virus, coronaviruses, hepatitis C virus, and dengue virus are among some of the best characterized examples of viruses that activate and require

some aspect of autophagy for robust viral replication

1 Dengue Virus and Autophagy - Nicholas S. Heaton and Glenn Randall Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA, July 2011, page 3

Autophagy: process and function

Noboru Mizushima - Department of Physiology and Cell Biology, Tokyo Medical and Dental University, Tokyo 113-8519, Japan; Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Tokyo 102-0075, Japan

Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome. Despite its simplicity, recent progress has

demonstrated that autophagy plays a wide variety of physiological and pathophysiological roles, which are sometimes complex.

Autophagy is mediated by a unique organelle called the autophagosome. As autophagosomes engulf a portion of cytoplasm, autophagy is generally thought to

be a non-selective degradation system. However, recent studies have clearly demonstrated that autophagy has a greater variety of physiological andpathophysiological roles than expected, such as starvation adaptation, intracellular protein and organelle clearance, development, anti-aging,

elimination of microorganisms, cell death, tumour suppression, and antigen presentation

Additionally, in some situations, the contribution of autophagy seems to bevery complicated

If both apoptosis and autophagy are suppressed, cell survival is severely impaired. Intriguingly, the resulting necrotic cell death promotes

tumorigenesis, which is probably mediated by the inflammatory respponse. Thus, tumorigenesis may be a secondary effect of autophagy suppression in

this context

It should be emphasized that excess production of amino acids by autophagy is an acute response or emergency action. Therefore, induction of

autophagy can support cell survival only for a short time.

The elimination of cytoplasmic contents by autophagy is so important that defects cause various cellular malfunctions. Two possible outcomes of

autophagy defects, neurodegeneration and tumorigenesis,

Another well-known pathogen persisting inside phagocytes is Mycobacterium tuberculosis. These bacteria inhibit phagosome maturation and survive in

premature phagosomes. However, when autophagy is stimulated by starvation, rapamycin, or Interferon, mycobacterial phagosomes are

enclosed, delivered to lysosomes, and acidified, which results in microbacterial death

1Detection of Zika virus in saliva

1Didier Mussoa, Claudine Rocheb, Tu-Xuan Nhana, Emilie Robina, Anita Teissier , Van-Mai Cao-Lormeaub

The overall number of RT-PCR positive ZIKV samples was 210 of 748 (28.1%) in blood and 182 of 319 (57.1%) in saliva. ZIKV RNA was more frequently

detected in saliva than in blood samples…From the 137 patients with only saliva samples, 95 samples tested were positive for ZIKV, of which 38 were

children less than 10 years old (including 7 neonates).

We found that the ability to detect ZIKV RNA in saliva was higher compared to blood; was neither found to be related to a particular clinical presentation nor to the

age of the patient. The use of saliva improved the ability to detect ZIKV RNA within the first week from symptoms onset but it did not increase the window of detection in contrast to what was reported for urine. However, as ZIKV RNA

detection can be negative in saliva while positive in blood and blood sample is required for other laboratory tests, saliva cannot replace blood samples.

For acute phase,

ZIKV fever diagnosis we recommend to collect both blood and saliva samples to increase the sensitivity of molecular detection of ZIKV, urine sample can

be associated at the late stages of the disease.

A complete ZIKV genome sequence (10,808 nucelotides) was recovered from brain tissue. Phylogenetic analysis showed the highest identity (99.7%) with the

ZIKV strain isolated from a patient from French Polynesia in 2013 (KJ776791) and ZIKV detected in Sao Paolo, Brazil, in 2015 (KU321639), followed by a strain isolated in Cambodia in 2010 (JN860885, with 98.3%

identity) and with a strain from the outbreak in Micronesia in 2007 (EU545988, with 98% identity). In the ZIKV polyprotein, 23 polymorphisms were detected

in comparison with the strain from Micronesia and 5 polymorphisms in comparison with the isolate from French Polynesia

1 Zika Virus Associated with Microcephaly

1 Jernej Mlakar, M.D.,

The complete genome sequence of ZIKV that was recovered in this study is consistent with the observation that the present strain in Brazil has

emerged from the Asian lineage. The presence of two major amino acid substitutions positioned in non-structural proteins NS1 and NS4B

probably represents an accidental event or indicates a process of eventual adaptation of the virus to a new environment.

1Complete coding sequence of Zika virus from Martinique outbreak in 2015

Maximum likelihood phylogenetic reconstruction (GTR+G+I model, determined from the data set using the MEGA6 program) indicates that it belongs to the

Asian lineage, sharing common ancestorship and c.99.9% nucleotide and amino acid identities with isolates circulating previously in French Polynesia and

currently in South America (accession no. KJ776791 in French Polynesia, 2013, and accession no. KU321639 in Brazil, 2015). The membrane gene is the

most divergent with reference to the French Polynesia 2013 and Brazil 2015 isolates.

The virus genome sequence described here has been deposited in the GenBank database under the accession no KU647676.

1G. Piorkowski1, P. Richard2, C. Baronti1, P. Gallian1,3, R. Charrel1, I. Leparc-Goffart4 and X. de Lamballerie1, March 8, 2016

Zika virus is an Aedes-borne Flavivirus causing fever, arthralgia, myalgia rash, associated with Guillain–Barré syndrome and suspected to induce microcephaly in the fetus. We report here the complete coding sequence of the first characterized

Caribbean Zika virus strain, isolated from a patient from Martinique in December, 2015

Zika Virus Infection and Stillbirths: A Case of Hydrops Fetalis, Hydranencephaly and Fetal Demise

Citation: Sarno M, Sacramento GA, Khouri R, do Rosário MS, Costa F, Archanjo G, et al. (2016) Zika Virus Infection and Stillbirths: A Case of Hydrops Fetalis, Hydranencephaly and Fetal Demise. PLoS Negl Trop Dis 10(2): e0004517. doi:10.1371/journal.pntd.0004517

We detected ZIKV RNA in the central nervous system and amniotic fluid andnot in heart, lung, liver or placenta, yet our findings were limited by the sampling

procedure and lack of histopathological analysis of tissues. The mechanism by which ZIKV may cause hydrops fetalis therefore remains speculative

We obtained ZIKV-specific RT-PCR amplification products from extracts of cerebral cortex, medulla oblongata and cerebrospinal and amniotic fluid.

Analysis of extracts of heart, lung, liver, vitreous body of the eye and placenta did not yield detectable products. Amplification products mapped within the NS5

gene of ZIKV strains belonging to the Asian lineage, with closest relationship to sequences from French Polynesian and Surinamese strains. We cannot extrapolate from this single case the overall risk for developing hydrops fetalis

and fetal demise among pregnant women exposed to the virus. The strain detected in this case of fetal demise appears to be the same as the epidemic

strain that has spread across the Americas and Caribbean

The Hamel et al study also showed that ZIKV infection triggered autophagy in the infected fibroblasts. Generally, autophagy-mediated degradation of viral proteins prevents the

replication of the virus; however, some viruses, including ZIKV, utilise certain components of the autophagy cycle, and promote viral replication and dissemination.

Electron microscopic studies also revealed membrane vesicles (70 – 100 nm) in close association with the ER; this indicates that ZIKV replication occurs intimately with the

host cell membrane.

Zika Virus: Life cycle and pathogenicity in humans

First detection of autochthonous Zika virus transmission in a HIV-infected patient In Rio de Janeiro, Brazil

G.A. Calvet et al. / Journal of Clinical Virology 74 (2016) 1–3

Fig. 1. Maximum likelihood phylogenetic tree from partial NS5 gene of Zikavirus. Phylogenetic tree constructed By MEGA 6.06 software, with 1000 bootstrap replicates,(support values greater than 75% shown) by using Tamura-Nei 93 model With Gamma distribution and invariant sites correction (Chosen by AIC Criteria). The branch lengthis indicated below of branch. The square represents the sample analyzed

The MEGA 6.6 software package was used for choosing the best model of nucleotide substitution and the phylogenetic tree assembly. Phylogenetic

analysis of the ZIKV detected in patient sera clustered within the Asian clade (Fig. 1). The sequences obtained in this study were deposited in the GenBank

under accession number KT200609. Patient sera from day 1 and day 28 were tested for dengue and no detectable IgM/IgG antibodies was found. The CD4+

cell count and HIV viral load levels remained unchanged four weeks after Zika virus infection (CD4+ T cell count of 619 cells/mm3 and undetectable viral load).

To the best of our knowledge, this is the first case of Zika virus infection reported in a HIV-infected patient.

aPreviously reports show a transient suppression of HIV-1 replication during acute DENV infection

a G. Watt, P. Kantipong, K. Jongsakul, Decrease in human immunodeficiency virus type 1 load during acute dengue fever, Clin. Infect. Dis. 36 (8) (2003)1067–1069b J.H. McLinden, J.T. Stapleton, Q. Chang, J. Xiang, Expression of the dengue virustype 2 NS5 protein in a CD4(+) T cell line inhibits HIV replication, J. Infect. Dis.198 (6) (2008) 860–863.

bIn a case series of HIV and CHIKV co-infection there was an association between CHIKV co-infection and lymphopenia, lower CD4 cell count and

severe immunosuppression

The main non-specific laboratory findings are:

Similarly to other arboviruses, the use of aspirin is banned due to the risk of bleeding complications Normal leukocyte count or mild leukopenia (reduced white blood cells)

Platelet count (thrombocytopenia) normal to slightly decreased. Slight increase in aspartate aminotransferase (AST) transaminase; gamma-glutamyl

transferase (gama GT); lactic dehydrogenase (DHL) and C-reactive protein.

Brazilian researchers found recent Asian epidemic strains of Zika virus that have changes in NS1 and NS4 genes and are more adapted to 1humans.

These authors point out that the pandemic potential of Zika virus is maximized to also be transmitted by Aedes albopictus, a mosquito that occurs at high

latitudes. (Asian Tiger Mosquito)

1 Freire CCM, Iamarino A, Lima Neto DF, Sall AA, Zanotto PMA. Spread of the pandemic Zika virus lineage is associated with NS1 codon usage adaptation in humans. bioRxiv. 2015. Available from: <http://biorxiv.org/content/biorxiv/ early/2015/11/25/032839.full.pdf>.

Technical Opinion on Examination Request presented at the 171st Plenary Meeting of the National Technical Commission on Biosafety (CTNBio), held on

April 10th, 2014 Applicant: Oxitec do Brasil Participações Ltd.

The project is well informed and the three studies referred in Cayman, Malaysia and Brazil (Juazeiro, State of Bahia, during 2012 and 2013) present interesting

preliminary results, showing it to be a promising alternative in the fight against dengue.

It has to be stressed that all opinions that support the request for commercial release (including the consolidated one) consider the OX513A mosquito Risk Class I, when the applicant company understands the issue as distinct and

deserving of greater caution. On page 67 of the dossier presented by the applicant it can be read that “the risk classification of the Aedes aegypti OX513A was

evaluated and in accordance with Normative Resolution No. 2 of November 27th, 2006, it was established as Risk Class II: moderate individual risk and low

collectivity risk”.

This topic should be clarified before any decision. In its statement, the company affirmed that it works with Risk Class II events, and it benefited from a Quality

Certificate in Biosecurity Class NB-1 and developed the planned release into the environment based on CTNBio Normative Resolution No. 7, which is

restricted to genetically modified, Risk Class I organisms. If the transgenic mosquito is classified as Risk Class II, the LPMA then followed, at least, the

guidance of an “inadequate” Normative Resolution.

Risk assessment associated with the introduction of massive quantities of OX513A into the environment

The dossier presented by the applicant company presents a vast set of scientific data, complemented by a rich bibliographic review, covering aspects pertaining to

the biology of the A. aegypti, associated risks on environment including the OX513A in trophic chains and potential consequences of releasing

genetically modified females undesirably. However, the process lacks certain biosafety aspects

The occupation of the ecological niche of Aedes Aegypti by Aedes Albopictus has not received sufficient attention from the dossier and the

other evaluators

The large-scale release of OX513A, altering the reproductive performance of the Aedes aegypti, can trigger a population explosion of other vectors, with

implications for adaptive dengue virus mechanisms in epidemiological terms and consequences for public health. Therefore, it is important to check the possibility of alterations in hosts, vectors, or even infectious

profiles.

The alterations made by releasing hundreds of thousand transgenic mosquitoes with the characteristic of lethality passed down to Aedes

Aegypti descendants will benefit other insects. As local populations of Aedes Aegypti compete with local populations of Aedes Albopictus (species

that have invasive ecologic characteristics) wouldn’t the suppression of the first favour a population explosion of the second?

Available references suggest Aedes Albopictus is adapted to the peridomestic environment just as Aedes Aegypti, where it feeds from human

and animal blood, laying eggs in many natural and artificial water-accumulating containers. Scientific reports support the fact that up to the XVIII and XIX centuries, Aedes Albopictus was the most frequent daytime biting

species in the majority of the cities in Asia

The inclusion of Aedes Albopictus in the list of the world’s 100 most invasive species leaves no doubts as to its aggressiveness and potential to occupy that ecological niche. In other words: the almost complete suppression of

local populations of Aedes Aegypti by the OX513A will possibly cause migration flows in local populations of Aedes Albopictus, compromising the disease-reduction goal, for the simple fact that a new vector of the disease will occupy the ecological niche that was abruptly abandoned by the main

competitor.

3.2. The ecological imbalance caused by mass introduction of the OX513A into the environment can cause implications for the epidemiological profile of

the dengue virus, aside from transmitting other viral human and zoonotic diseases

Therefore, considering the hypothesis that mass releasing of the OX513 mosquito will cause mass occupation of the Aedes Aegypti’s ecological

niche by Aedes Albopictus, this could cause changes in the dengue virus’ epidemiologic profile, as well as in other viral diseases (human, animal and zoonotic). These are some of issues that were not examined in the dossier.

Mutations in dengue strains that would allow Aedes Albopictus to become a highly efficient transmission vector could occur, getting around the

immunological properties provided by the symbiote bacteria Wolbachia (as it was with the CHIKV). In that case, Aedes Albopictus would become the

dengue virus’ main epidemic vector.

In both cases, a new epidemic vector for the dengue virus would replace Aedes Aegypti, followed by new risks. In said conditions, the change in

vector would mean alterations in the infectivity mechanisms of the dengue virus itself, making its control by health agencies more complex.

Additionally, mass releases of the OX513A into urban zones could favour the entry of other human, animal and zoonotic viral diseases, which do not

occur today thanks to the occupation of the ecological niche by Aedes Aegypti, that is not a vector for these diseases. Considering that Aedes

Albopictus on its own, facing the current conditions, it is possible to speculate on risks involving the whole set of viral diseases, whether human,

animal or zoonotic which that species hosts.

It is a known fact that the Aedes Albopictus is susceptible to infection and is capable of transmitting most viruses that have been tested on it. The list

includes 8 alphaviruses, 8 flaviviruses and 4 bunyaviruses, representing the three main types of arbovirus that include human pathogens (revised in Paupy

et al., 2009).

In this sense, besides transmitting dengue, Aedes Albopictus also transmits yellow fever and the Chikungunya virus (Hochedez et al., 2006), as well as

other viral diseases. It is worth noting the recent Chikungunya epidemics in the Indian Ocean islands (especially La Réunion), in Central Africa (Gabon,

among other countries) and in Italy, derived from the Aedes Albopictus vector (Lambrechts et al., 2010).

The quoted authors also warn that Aedes Albopictus deserves special attention in the South and Central Americas, for it is a vector of yellow fever and Venezuelan Equine Encephalitis viruses. At this point, it is worth noticing that the Eastern Equine Encephalitis, Venezuelan Equine Encephalitis (VEE)

and WEE (Western Equine Encephalitis) viruses are present in Brazil

TP53 and Ascorbic Acid (Vitamine C)