Systemic inflammation, innate immunity and pathogenesis after Zika virus infection in cynomolgus macaques are modulated by strain-specificity within the Asian lineage.

Zika virus (ZIKV) is an emerging arbovirus with recent global expansion. Historically, ZIKV infections with Asian lineages have been associated with mild disease such as rash and fever. However, recent Asian sub-lineages have caused outbreaks in the South Pacific and Latin America with increased prevalence of neurological disorders in infants and adults. Asian sub-lineage differences may partially explain the range of disease severity observed. However, the effect of Asian sub-lineage differences on pathogenesis remains poorly characterized. Current study conducts a head-to-head comparison of three Asian sub-lineages that are representative of the circulating ancestral mild Asian strain (ZIKV-SG), the 2007 epidemic French Polynesian strain (ZIKV-FP), and the 2013 epidemic Brazil strain (ZIKV-Brazil) in adult Cynomolgus macaques. Animals infected intervenously or subcutaneously with either of the three clinical isolates showed sub-lineage-specific differences in viral pathogenesis, early innate immune responses and systemic inflammation. Despite the lack of neurological symptoms in infected animals, the epidemiologically neurotropic ZIKV sub-lineages (ZIKV-Brazil and/or ZIKV-FP) were associated with more sustained viral replication, higher systemic inflammation (i.e. higher levels of TNFα, MCP-1, IL15 and G-CSF) and greater percentage of CD14+ monocytes and dendritic cells in blood. Multidimensional analysis showed clustering of ZIKV-SG away from ZIKV-Brazil and ZIKV-FP, further confirming sub-lineage differences in the measured parameters. These findings highlight greater systemic inflammation and monocyte recruitment as possible risk factors of adult ZIKV disease observed during the 2007 FP and 2013 Brazil epidemics. Future studies should explore the use of anti-inflammatory therapeutics as early treatment to prevent ZIKV-associated disease in adults.

Viral metagenomics reveals the presence of novel Zika virus variants in Aedes mosquitoes from Barbados.

BACKGROUND: The Zika virus (ZIKV) epidemic of 2015/2016 spread throughout numerous countries. It emerged in mainland Latin America and spread to neighboring islands, including the Caribbean island of Barbados. Recent studies have indicated that the virus must have already been circulating in local mosquito populations in Brazil for almost 2 years before it was identified by the World Health Organization in 2015. Metagenomic detection assays have the potential to detect emerging pathogens without prior knowledge of their genomic nucleic acid sequence. Yet their applicability as vector surveillance tools has been widely limited by the complexity of DNA populations from field-collected mosquito preparations. The aim of this study was to investigate local vector biology and characterize metagenomic arbovirus diversity in Aedes mosquitoes during the ongoing 2015/2016 ZIKV epidemic. METHODS: We performed a short-term vector screening study on the island of Barbados during the ongoing 2015/2016 ZIKV epidemic, where we sampled local Aedes mosquitoes. We reanalyzed mosquito viral microbiome data derived from standard Illumina MiSeq sequencing to detect arbovirus sequences. Additionally, we employed deep sequencing techniques (Illumina HiSeq) and designed a novel bait capture enrichment assay to increase sequencing efficiency for arbovirus sequences from complex DNA samples. RESULTS: We found that Aedes aegypti seemed to be the most likely vector of ZIKV, although it prevailed at a low density during the observed time period. The number of detected viruses increased with sequencing depth. Arbovirus sequence enrichment of metagenomic DNA preparations allowed the detection of arbovirus sequences of two different ZIKV genotypes, including a novel one. To our knowledge, this is the first report of the S3116W mutation in the NS5 gene region of ZIKV polyprotein. CONCLUSIONS: The metagenomic arbovirus detection approach presented here may serve as a useful tool for the identification of epidemic-causing arboviruses with the additional benefit of enabling the collection of phylogenetic information on the source. Apart from detecting more than 88 viruses using this approach, we also found evidence of novel ZIKV variants circulating in the local mosquito population during the observed time period.

Identification of differentially expressed miRNAs in human cells infected with different Zika virus strains.

Infection with distinct Zika virus (ZIKV) strains in in vitro and in vivo models has demonstrated that the host's response to infection is strain-dependent. There has been no analysis of the impact of infection with different ZIKV strains on miRNA expression in human cells. We investigated miRNA expression in PNT1A cells upon infection with an African ZIKV strain (MR766) and a Brazilian ZIKV strain (ZIKVBR) using PCR array. Sixteen miRNAs were modulated in PNT1A cells: six miRNAs were modulated by both strains, while a set of ten miRNAs were modulated exclusively by ZIKVBR infection. In silico analysis showed that nine significant KEGG pathways and eight significant GO terms were predicted to be enriched upon ZIKVBR infection, and these pathways were related to cancer, environmental information processing, metabolism, and extracellular matrix. Differential modulation of miRNA expression suggests that distinct strains of ZIKV can differentially modulate the host response through the action of miRNAs.

Zika Virus-Like Particles Bearing a Covalent Dimer of Envelope Protein Protect Mice from Lethal Challenge.

Zika virus (ZIKV) envelope (E) protein is the major target of neutralizing antibodies in infected hosts and thus represents a candidate of interest for vaccine design. However, a major concern in the development of vaccines against ZIKV and the related dengue virus is the induction of cross-reactive poorly neutralizing antibodies that can cause antibody-dependent enhancement (ADE) of infection. This risk necessitates particular care in vaccine design. Specifically, the engineered immunogens should have their cross-reactive epitopes masked, and they should be optimized for eliciting virus-specific strongly neutralizing antibodies upon vaccination. Here, we developed ZIKV subunit- and virus-like particle (VLP)-based vaccines displaying E in its wild-type form or E locked in a covalently linked dimeric (cvD) conformation to enhance the exposure of E dimers to the immune system. Compared with their wild-type derivatives, cvD immunogens elicited antibodies with a higher capacity to neutralize virus infection in cultured cells. More importantly, these immunogens protected animals from lethal challenge with both the African and Asian lineages of ZIKV, impairing virus dissemination to brain and sexual organs. Moreover, the locked conformation of E reduced the exposure of epitopes recognized by cross-reactive antibodies and therefore showed a lower potential to induce ADE in vitro Our data demonstrated a higher efficacy of the VLPs in comparison with that of the soluble dimer and support VLP-cvD as a promising ZIKV vaccine.IMPORTANCE Infection with Zika virus (ZIKV) leads to the production by the host of antibodies that target the viral surface envelope (E) protein. A subset of these antibodies can inhibit virus infection, thus making E a suitable candidate for the development of vaccine against the virus. However, the anti-ZIKV E antibodies can cross-react with the E protein of the related dengue virus on account of the high level of similarity exhibited by the two viral proteins. Such a scenario may lead to severe dengue disease. Therefore, the design of a ZIKV vaccine requires particular care. Here, we tested two candidate vaccines containing a recombinant form of the ZIKV E protein that is forced in a covalently stable dimeric conformation (cvD). They were generated with an explicit aim to reduce the exposure of the cross-reactive epitopes. One vaccine is composed of a soluble form of the E protein (sE-cvD), the other is a more complex virus-like particle (VLP-cvD). We used the two candidate vaccines to immunize mice and later infected them with ZIKV. The animals produced a high level of inhibitory antibodies and were protected from the infection. The VLP-cvD was the most effective, and we believe it represents a promising ZIKV vaccine candidate.

Asian and African lineage Zika viruses show differential replication and innate immune responses in human dendritic cells and macrophages.

Zika virus (ZIKV) infections in humans are considered to be mild or subclinical. However, during the recent epidemics in the Pacific Islands and the Americas, the infection was associated with Quillain-Barré syndrome and congenital infections with fetal brain abnormalities, including microcephaly. Thus, more detailed understanding of ZIKV-host cell interactions and regulation of innate immune responses by strains of differential evolutionary origin is required. Here, we characterized the infection and immune responses triggered by two epidemic Asian/American lineage viruses, including an isolate from fetal brains, and a historical, low passage 1947 African lineage virus in human monocyte-derived dendritic cells (DCs) and macrophages. The epidemic Asian/American ZIKV replicated well and induced relatively good antiviral responses in human DCs whereas the African strain replicated less efficiently and induced weaker immune responses. In macrophages both the African and Asian strains showed limited replication and relatively weak cytokine gene expression. Interestingly, in macrophages we observed host protein degradation, especially IRF3 and STAT2, at early phases of infection with both lineage viruses, suggesting an early proteasomal activation in phagocytic cells. Our data indicates that ZIKV evolution has led to significant phenotypic differences in the replication characteristics leading to differential regulation of host innate immune responses.

Prolonged Zika Virus RNA Detection in Semen of Immunosuppressed Patient.

Zika virus RNA has been detected in semen samples collected <370 days after symptom onset. We report unusual persistence of Zika virus RNA in semen, confirmed by sequencing at 515 days after symptom onset and detectable for >900 days, in a patient with immunosuppression.

Sustained Low-Level Transmission of Zika and Chikungunya Viruses after Emergence in the Fiji Islands.

Zika and chikungunya viruses were first detected in Fiji in 2015. Examining surveillance and phylogenetic and serologic data, we found evidence of low-level transmission of Zika and chikungunya viruses during 2013-2017, in contrast to the major outbreaks caused by closely related virus strains in other Pacific Island countries.

Diagnosing Zika virus infection against a background of other flaviviruses: Studies in high resolution serological analysis.

Zika virus (ZIKV) is a mosquito-borne flavivirus. Homologous proteins of different flaviviruses display high degrees of sequence identity, especially within subgroups. This leads to extensive immunological cross-reactivity and corresponding problems for developing a ZIKV-specific serological assay. In this study, peptide microarrays were employed to identify individual ZIKV antibody targets with promise in differential diagnosis. A total of 1643 overlapping oligopeptides were synthesized and printed onto glass slides. Together, they encompass the full amino acid sequences of ZIKV proteomes of African, Brazilian, USA, and French Polynesian origins. The resulting ZIKV scanning microarray chips were used to screen three pools of sera from recent Zika outbreaks in Senegal and Cape Verde, in Brazil, and from overseas travelers returning to the EU. Together with a mixed pool of well characterized, archived sera of patients suffering from infections by dengue, yellow fever, tick-borne encephalitis, and West Nile viruses, a total of 42 sera went into the study. Sixty-eight antibody target regions were identified. Most of which were hitherto unknown. Alignments and sequence comparisons revealed 13 of which could be classified as bona fide ZIKV-specific. These identified antibody target regions constitute a founding set of analytical tools for serological discrimination of ZIKV from other flaviviruses.

The Roles of prM-E Proteins in Historical and Epidemic Zika Virus-mediated Infection and Neurocytotoxicity.

The Zika virus (ZIKV) was first isolated in Africa in 1947. It was shown to be a mild virus that had limited threat to humans. However, the resurgence of the ZIKV in the most recent Brazil outbreak surprised us because it causes severe human congenital and neurologic disorders including microcephaly in newborns and Guillain-Barré syndrome in adults. Studies showed that the epidemic ZIKV strains are phenotypically different from the historic strains, suggesting that the epidemic ZIKV has acquired mutations associated with the altered viral pathogenicity. However, what genetic changes are responsible for the changed viral pathogenicity remains largely unknown. One of our early studies suggested that the ZIKV structural proteins contribute in part to the observed virologic differences. The objectives of this study were to compare the historic African MR766 ZIKV strain with two epidemic Brazilian strains (BR15 and ICD) for their abilities to initiate viral infection and to confer neurocytopathic effects in the human brain's SNB-19 glial cells, and further to determine which part of the ZIKV structural proteins are responsible for the observed differences. Our results show that the historic African (MR766) and epidemic Brazilian (BR15 and ICD) ZIKV strains are different in viral attachment to host neuronal cells, viral permissiveness and replication, as well as in the induction of cytopathic effects. The analysis of chimeric viruses, generated between the MR766 and BR15 molecular clones, suggests that the ZIKV E protein correlates with the viral attachment, and the C-prM region contributes to the permissiveness and ZIKV-induced cytopathic effects. The expression of adenoviruses, expressing prM and its processed protein products, shows that the prM protein and its cleaved Pr product, but not the mature M protein, induces apoptotic cell death in the SNB-19 cells. We found that the Pr region, which resides on the N-terminal side of prM protein, is responsible for prM-induced apoptotic cell death. Mutational analysis further identified four amino-acid residues that have an impact on the ability of prM to induce apoptosis. Together, the results of this study show that the difference of ZIKV-mediated viral pathogenicity, between the historic and epidemic strains, contributed in part the functions of the structural prM-E proteins.

Detection and serotyping of dengue viruses in febrile patients consulting at the New-Bell District Hospital in Douala, Cameroon.

Arboviruses are a major public health problem worldwide and are predominantly present in intertropical areas. Chikungunya, dengue and zika viruses have been implicated in recent epidemics in Asia, America and Africa. In Cameroon, data on these viruses are fragmentary. The purpose of this study was to determine the frequency of detection of these three viruses in febrile patients in Douala, Cameroon. A cross-sectional and descriptive study was conducted from March to April 2017 at the New-Bell District Hospital in Douala. Blood samples were collected from febrile patients and tested for malaria infections using Rapid Diagnostic test. Plasma harvested was later analyzed for the presence of chikungunya, dengue and zika viruses by a Trioplex real-time RT-PCR at Centre Pasteur of Cameroon. A total of 114 participants were included, of which 63.2% were females, reflecting a sex ratio (female/male) of 1.7. The median age was 26 years, range [0.25-81]. Eight (7%) of the 114 participants were infected with Dengue virus (DENV) among which 5 were identified as serotype 1. No cases of infection by either Zika virus or Chikungunya virus were detected. Three cases of dengue-malaria co-infection (13%) were recorded. No association was found between socio-demographic factors and dengue infection. The phylogenetic analysis of the partial envelope E gene showed that all the five DENV serotype 1 samples belonged to subtype V, similarly to strains from West African countries, particularly those from Nigeria, Senegal and Côte d'Ivoire. This study showed the circulation of DENV serotype 1 in febrile patients and raises the alarm for the establishment of a sustained surveillance system to detect cases and prevent potential outbreaks in Cameroon. The existence of dengue-malaria co-infections suggests that surveillance of arboviruses should not be limited to febrile, non-malarial cases.

An observational clinical case of Zika virus-associated neurological disease is associated with primary IgG response and enhanced TNF levels.

Descriptive clinical data help to reveal factors that may provoke Zika virus (ZIKV) neuropathology. The case of a 24-year-old female with a ZIKV-associated severe acute neurological disorder was studied. The levels of ZIKV in the cerebrospinal fluid (CSF) were 50 times higher than the levels in other compartments. An acute anti-flavivirus IgG, together with enhanced TNF-alpha levels, may have contributed to ZIKV invasion in the CSF, whereas the unbiased genome sequencing [obtained by next-generation sequencing (NGS)] of the CSF revealed that no virus mutations were associated with the anatomic compartments (CSF, serum, saliva and urine).

Zika detection: comparison of methodologies

ABSTRACT Many countries in the Americas have detected local transmission of multiple arboviruses that cause febrile illnesses. Therefore, laboratory testing has become an important tool for confirming the etiology of these diseases. The present study aimed to compare the sensitivity and specificity of three different Zika virus detection assays. One hundred serum samples from patients presenting with acute febrile symptoms were tested using a previously reported TaqMan® RT-qPCR assay. We used a SYBR® Green RT-qPCR and a conventional PCR methodologies to compare the results. Of the samples that were determined to be negative by the TaqMan® RT-qPCR assay, 100% (Kappa = 0.670) were also found to be negative by SYBR® Green RT-qPCR based on Tm comparison; however, 14% (Kappa = 0.035) were found to be positive by conventional PCR followed by agarose gel electrophoresis. The differences between the ZIKV strains circulating worldwide and the low viremia period can compromise diagnostic accuracy and thereby the accuracy of outbreak data. Therefore, improved assays are required to improve the diagnosis and surveillance of arbovirus.

Adverse birth outcomes associated with Zika virus exposure during pregnancy in São José do Rio Preto, Brazil.

OBJECTIVES: We aimed to report the first 54 cases of pregnant women infected by Zika virus (ZIKV) and their virologic and clinical outcomes, as well as their newborns' outcomes, in 2016, after the emergence of ZIKV in dengue-endemic areas of São Paulo, Brazil. METHODS: This descriptive study was performed from February to October 2016 on 54 quantitative real-time PCR ZIKV-positive pregnant women identified by the public health authority of São José do Rio Preto, São Paulo, Brazil. The women were followed and had clinical and epidemiologic data collected before and after birth. Adverse outcomes in newborns were analysed and reported. Urine or blood samples from newborns were collected to identify ZIKV infection by reverse transcription PCR (RT-PCR). RESULTS: A total of 216 acute Zika-suspected pregnant women were identified, and 54 had the diagnosis confirmed by RT-PCR. None of the 54 women miscarried. Among the 54 newborns, 15 exhibited adverse outcomes at birth. The highest number of ZIKV infections occurred during the second and third trimesters. No cases of microcephaly were reported, though a broad clinical spectrum of outcomes, including lenticulostriate vasculopathy, subependymal cysts, and auditory and ophthalmologic disorders, were identified. ZIKV RNA was detected in 18 of 51 newborns tested and in eight of 15 newborns with adverse outcomes. CONCLUSIONS: Although other studies have associated many newborn outcomes to ZIKV infection during pregnancy, these same adverse outcomes were rare or nonexistent in this study. The clinical presentation the newborns we studied was mild compared to other reports, suggesting that there is significant heterogeneity in congenital Zika infection.

Zika virus infection dysregulates human neural stem cell growth and inhibits differentiation into neuroprogenitor cells.

The current outbreak of Zika virus-associated diseases in South America and its threat to spread to other parts of the world has emerged as a global health emergency. A strong link between Zika virus and microcephaly exists, and the potential mechanisms associated with microcephaly are under intense investigation. In this study, we evaluated the effect of Zika virus infection of Asian and African lineages (PRVABC59 and MR766) in human neural stem cells (hNSCs). These two Zika virus strains displayed distinct infection pattern and growth rates in hNSCs. Zika virus MR766 strain increased serine 139 phosphorylation of histone H2AX (γH2AX), a known early cellular response proteins to DNA damage. On the other hand, PRVABC59 strain upregulated serine 15 phosphorylation of p53, p21 and PUMA expression. MR766-infected cells displayed poly (ADP-ribose) polymerase (PARP) and caspase-3 cleavage. Interestingly, infection of hNSCs by both strains of Zika virus for 24 h, followed by incubation in astrocyte differentiation medium, induced rounding and cell death. However, astrocytes generated from hNSCs by incubation in differentiation medium when infected with Zika virus displayed minimal cytopathic effect at an early time point. Infected hNSCs incubated in astrocyte differentiating medium displayed PARP cleavage within 24-36 h. Together, these results showed that two distinct strains of Zika virus potentiate hNSC growth inhibition by different mechanisms, but both viruses strongly induce death in early differentiating neuroprogenitor cells even at a very low multiplicity of infection. Our observations demonstrate further mechanistic insights for impaired neuronal homeostasis during active Zika virus infection.

Differential Neurovirulence of African and Asian Genotype Zika Virus Isolates in Outbred Immunocompetent Mice.

Although first isolated almost 70 years ago, Zika virus (ZIKV; Flavivirus, Flaviviridae) has only recently been associated with significant outbreaks of disease in humans. Several severe ZIKV disease manifestations have also been recently documented, including fetal malformations, such as microcephaly, and Guillain-Barré syndrome in adults. Although principally transmitted by mosquitoes, sexual transmission of ZIKV has been documented. Recent publications of several interferon receptor knockout mouse models have demonstrated ZIKV-induced disease. Herein, outbred immunocompetent CD-1/ICR adult mice were assessed for susceptibility to disease by intracranial (i.c.) and intraperitoneal (i.p.) inoculation with the Ugandan prototype strain (MR766; African genotype), a low-passage Senegalese strain (DakAr41524; African genotype) and a recent ZIKV strain isolated from a traveler infected in Puerto Rico (PRVABC59; Asian genotype). Morbidity was not observed in mice inoculated by the i.p. route with either MR766 or PRVABC59 for doses up to 6 log10 PFU. In contrast, CD-1/ICR mice inoculated i.c. with the MR766 ZIKV strain exhibited an 80-100% mortality rate that was age independent. The DakAr41524 strain delivered by the i.c route caused 30% mortality, and the Puerto Rican ZIKV strain failed to elicit mortality but did induce a serum neutralizing immune response in 60% of mice. These data provide a potential animal model for assessing neurovirulence determinants of different ZIKV strains as well as a potential immunocompetent challenge model for assessing protective efficacy of vaccine candidates.

In Vitro Assembly and Stabilization of Dengue and Zika Virus Envelope Protein Homo-Dimers.

Zika virus (ZIKV) and the 4 dengue virus (DENV) serotypes are mosquito-borne Flaviviruses that are associated with severe neuronal and hemorrhagic syndromes. The mature flavivirus infectious virion has 90 envelope (E) protein homo-dimers that pack tightly to form a smooth protein coat with icosahedral symmetry. Human antibodies that strongly neutralize ZIKV and DENVs recognize complex quaternary structure epitopes displayed on E-homo-dimers and higher order structures. The ZIKV and DENV E protein expressed as a soluble protein is mainly a monomer that does not display quaternary epitopes, which may explain the modest success with soluble recombinant E (sRecE) as a vaccine and diagnostic antigen. New strategies are needed to design recombinant immunogens that display these critical immune targets. Here we present two novel methods for building or stabilizing in vitro E-protein homo-dimers that display quaternary epitopes. In the first approach we immobilize sRecE to enable subsequent dimer generation. As an alternate method, we describe the use of human mAbs to stabilize homo-dimers in solution. The ability to produce recombinant E protein dimers displaying quaternary structure epitopes is an important advance with applications in flavivirus diagnostics and vaccine development.

Identification of diagnostic peptide regions that distinguish Zika virus from related mosquito-borne Flaviviruses.

Zika virus (ZIKV) is a member of the Flavivirus genus of positive-sense single-stranded RNA viruses, which includes Dengue, West Nile, Yellow Fever, and other mosquito-borne arboviruses. Infection by ZIKV can be difficult to distinguish from infection by other mosquito-borne Flaviviruses due to high sequence similarity, serum antibody cross-reactivity, and virus co-circulation in endemic areas. Indeed, existing serological methods are not able to consistently differentiate ZIKV from other Flaviviruses, which makes it extremely difficult to accurately calculate the incidence rate of Zika-associated Guillain-Barre in adults, microcephaly in newborns, or asymptomatic infections within a geographical area. In order to identify Zika-specific peptide regions that could be used as serology reagents, we have applied comparative genomics and protein structure analyses to identify amino acid residues that distinguish each of 10 Flavivirus species and subtypes from each other by calculating the specificity, sensitivity, and surface exposure of each residue in relevant target proteins. For ZIKV we identified 104 and 116 15-mer peptides in the E glycoprotein and NS1 non-structural protein, respectively, that contain multiple diagnostic sites and are located in surface-exposed regions in the tertiary protein structure. These sensitive, specific, and surface-exposed peptide regions should serve as useful reagents for seroprevalence studies to better distinguish between prior infections with any of these mosquito-borne Flaviviruses. The development of better detection methods and diagnostic tools will enable clinicians and public health workers to more accurately estimate the true incidence rate of asymptomatic infections, neurological syndromes, and birth defects associated with ZIKV infection.

Quasispecies composition and evolution of a typical Zika virus clinical isolate from Suriname.

The arthropod-borne Zika virus (ZIKV) is currently causing a major international public health threat in the Americas. This study describes the isolation of ZIKV from the plasma of a 29-year-old female traveler that developed typical symptoms, like rash, fever and headache upon return from Suriname. The complete genome sequence including the 5' and 3' untranslated regions was determined and phylogenetic analysis showed the isolate clustering within the Asian lineage, close to other viruses that have recently been isolated in the Americas. In addition, the viral quasispecies composition was analyzed by single molecule real time sequencing, which suggested a mutation frequency of 1.4 × 10-4 for this ZIKV isolate. Continued passaging of the virus in cell culture led to the selection of variants with mutations in NS1 and the E protein. The latter might influence virus binding to cell surface heparan sulfate.

Viral and serological kinetics in Zika virus-infected patients in South Korea.

Zika virus is a mosquito-borne flavivirus that causes clinical symptoms similar to those observed in dengue and chikungunya virus infections. The Korea Centers for Disease Control and Prevention initiated laboratory testing using a real-time reverse transcription-polymerase chain reaction in January 2016. More than 1,000 suspected cases of infection were tested and nine were confirmed as imported cases of Zika virus infection from January to July 2016. The travel destinations of the infected individuals were Brazil, Philippines, Viet Nam, Guatemala, Puerto Rico, and the Dominican Republic. Phylogenetic analysis based on the partial envelope gene indicated that the viruses belonged to the Asian genotype circulating in South America. We further investigated the duration for which the viral RNA and virus-specific antibodies were detectable after the symptom onset. After the day of symptom onset, Zika virus was detectable until 6 days in serum, 14 days in urine and saliva, and 58 days in semen. Immunoglobulin M against Zika virus was detected as early as 2 days after the symptom onset and was maintained at these levels until 41 days, whereas Immunoglobulin G was detectable from 8 days after the symptom onset and was maintained until 52 days. These findings would help diagnostic laboratories improve their testing programs for Zika virus infection.

Comparative analysis of protein evolution in the genome of pre-epidemic and epidemic Zika virus.

Zika virus (ZIKV) causes microcephaly in congenital infection, neurological disorders, and poor pregnancy outcome and no vaccine is available for use in humans or approved. Although ZIKV was first discovered in 1947, the exact mechanism of virus replication and pathogenesis remains unknown. Recent outbreaks of Zika virus in the Americas clearly suggest a human-mosquito cycle or urban cycle of transmission. Understanding the conserved and adaptive features in the evolution of ZIKV genome will provide a hint on the mechanism of ZIKV adaptation to a new cycle of transmission. Here, we show comprehensive analysis of protein evolution of ZIKV strains including the current 2015-16 outbreak. To identify the constraints on ZIKV evolution, selection pressure at individual codons, immune epitopes and co-evolving sites were analyzed. Phylogenetic trees show that the ZIKV strains of the Asian genotype form distinct cluster and share a common ancestor with African genotype. The TMRCA (Time to the Most Recent Common Ancestor) for the Asian lineage and the subsequently evolved Asian human strains was calculated at 88 and 34years ago, respectively. The proteome of current 2015/16 epidemic ZIKV strains of Asian genotype was found to be genetically conserved due to genome-wide negative selection, with limited positive selection. We identified a total of 16 amino acid substitutions in the epidemic and pre-epidemic strains from human, mosquito, and monkey hosts. Negatively selected amino acid sites of Envelope protein (E-protein) (positions 69, 166, and 174) and NS5 (292, 345, and 587) were located in central dimerization domains and C-terminal RNA-directed RNA polymerase regions, respectively. The predicted 137 (92 CD4 TCEs; 45 CD8 TCEs) immunogenic peptide chains comprising negatively selected amino acid sites can be considered as suitable target for sub-unit vaccine development, as these sites are less likely to generate immune-escape variants due to strong functional constrains operating on them. The targeted changes at the amino acid level may contribute to better adaptation of ZIKV strains to human-mosquito cycle or urban cycle of transmission.