Asian Zika Virus Strains Research Articles

Genetic diversity and phylogenetic analyses of Asian lineage Zika virus whole genome sequences derived from Culex quinquefasciatus mosquitoes and urine of patients during the 2020 epidemic in Thailand

Zika virus (ZIKV), a mosquito-borne flavivirus, has been continually emerging and re-emerging since 2010, with sporadic cases reported annually in Thailand, peaking at over 1000 confirmed positive cases in 2016. Leveraging high-throughput sequencing technologies, specifically whole genome sequencing (WGS), has facilitated rapid pathogen genome sequencing. In this study, we used multiplex amplicon sequencing on the Illumina Miseq instrument to describe ZIKV WGS. Six ZIKV WGS were derived from three samples of field-caught Culex quinquefasciatus mosquitoes (two males and one female) and three urine samples collected from patients in three different provinces of Thailand. Additionally, successful isolation of a ZIKV isolate occurred from a female Cx. quinquefasciatus. The WGS analysis revealed a correlation between the 2020 outbreak and the acquisition of five amino acid changes in the Asian lineage ZIKV strains from Thailand (2006), Cambodia (2010 and 2019), and the Philippines (2012). These changes, including C-T106A, prM-V1A, E-V473M, NS1-A188V, and NS5-M872V, were identified in all seven WGS, previously linked to significantly higher mortality rates. Furthermore, phylogenetic analysis indicated that the seven ZIKV sequences belonged to the Asian lineage. Notably, the genomic region of the E gene showed the highest nucleotide diversity (0.7–1.3%). This data holds significance in informing the development of molecular tools that enhance our understanding of virus patterns and evolution. Moreover, it may identify targets for improved methods to prevent and control future ZIKV outbreaks.

Viral Protein Accumulation of Zika Virus Variants Links with Regulation of Innate Immunity for Differential Control of Viral Replication, Spread, and Response to Interferon.

Asian lineage Zika virus (ZIKV) strains emerged globally, causing outbreaks linked with critical clinical disease outcomes unless the virus is effectively restricted by host immunity. We have previously shown that retinoic acid-inducible gene-I (RIG-I) senses ZIKV to trigger innate immunity to direct interferon (IFN) production and antiviral responses that can control ZIKV infection. However, ZIKV proteins have been demonstrated to antagonize IFN. Here, we conducted in vitro analyses to assess how divergent prototypic ZIKV variants differ in virologic properties, innate immune regulation, and infection outcome. We comparatively assessed African lineage ZIKV/Dakar/1984/ArD41519 (ZIKV/Dakar) and Asian lineage ZIKV/Malaysia/1966/P6740 (ZIKV/Malaysia) in a human epithelial cell infection model. De novo viral sequence determination identified amino acid changes within the ZIKV/Dakar genome compared to ZIKV/Malaysia. Viral growth analyses revealed that ZIKV/Malaysia accumulated viral proteins and genome copies earlier and to higher levels than ZIKV/Dakar. Both ZIKV strains activated RIG-I/IFN regulatory factor (IRF3) and NF-κB pathways to induce inflammatory cytokine expression and types I and III IFNs. However, ZIKV/Malaysia, but not ZIKV/Dakar, potently blocked downstream IFN signaling. Remarkably, ZIKV/Dakar protein accumulation and genome replication were rescued in RIG-I knockout (KO) cells late in acute infection, resulting in ZIKV/Dakar-mediated blockade of IFN signaling. We found that RIG-I signaling specifically restricts viral protein accumulation late in acute infection where early accumulation of viral proteins in infected cells confers enhanced ability to limit IFN signaling, promoting viral replication and spread. Our results demonstrate that RIG-I-mediated innate immune signaling imparts restriction of ZIKV protein accumulation, which permits IFN signaling and antiviral actions controlling ZIKV infection. IMPORTANCE ZIKV isolates are classified under African or Asian lineages. Infection with emerging Asian lineage-derived ZIKV strains is associated with increased incidence of neurological symptoms that were not previously reported during infection with African or preemergent Asian lineage viruses. In this study, we utilized in vitro models to compare the virologic properties of and innate immune responses to two prototypic ZIKV strains from distinct lineages: African lineage ZIKV/Dakar and Asian lineage ZIKV/Malaysia. Compared to ZIKV/Dakar, ZIKV/Malaysia accumulates viral proteins earlier, replicates to higher levels, and robustly blocks IFN signaling during acute infection. Early accumulation of ZIKV/Malaysia NS5 protein confers enhanced ability to antagonize IFN signaling, dampening innate immune responses to promote viral spread. Our data identify the kinetics of viral protein accumulation as a major regulator of host innate immunity, influencing host-mediated control of ZIKV replication and spread. Importantly, these findings provide a novel framework for evaluating the virulence of emerging variants.

Development of a TaqMan minor groove binding probe-based quantitative reverse transcription polymerase chain reaction for the detection and quantification of Zika virus.

Zika virus (ZIKV) infection has emerged as a global health concern following epidemic outbreaks of severe neurological disorders reported in Pacific and Americas since 2016. Therefore, a rapid, sensitive and specific diagnostic test for ZIKV infection is critical for the appropriate patient management and the control of disease spread. A TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the conserved sequence regions of 463 ZIKV NS2B genes. The designed ZIKV qRT-PCR assay was evaluated for its detection limit, strain coverage and cross-reactivity. We further assessed the clinical applicability of qRT-PCR assay for ZIKV RNA detection using a total 18 simulated clinical specimens. The detection limit of the qRT-PCR assay was 11.276 ZIKV RNA copies at the 95% probability level (probit analysis, p<= 0.05). Both Asian and African ZIKV strains were detected by the qRT-PCR assay without cross-reacting with DENV-1, DENV-2, DENV-3, DENV-4, CHIKV, JEV, LGTV, GETV and SINV. The qRT-PCR assay demonstrated a perfect agreement (k = 1.000, P < 0.001) with the reference assay; the sensitivity and specificity of the qRT-PCR assay were 100% (95% CI= 79.6-100) and 100% (95% CI= 43.9-100) respectively. The qRT-PCR assay developed in this study is a useful diagnostic tool for the broad coverage detection and quantification of both the Asian and African ZIKV strains.

Influence of Zika virus 3′-end sequence and nonstructural protein evolution on the viral replication competence and virulence

ABSTRACT Zika virus (ZIKV) has been circulating in human networks over 70 years since its first appearance in Africa, yet little is known about whether the viral 3′-terminal sequence and nonstructural (NS) protein diverged genetically from ancient ZIKV have different effects on viral replication and virulence in currently prevailing Asian lineage ZIKV. Here we show, by a reverse genetics approach using an infectious cDNA clone for a consensus sequence (Con1) of ZIKV, which represents Asian ZIKV strains, and another clone derived from the MR766 strain isolated in Uganda, Africa in 1947, that the 3′-end sequence –UUUCU-3′ homogeneously present in MR766 genome and the –GUCU-3′ sequence strictly conserved in Asian ZIKV isolates are functionally equivalent in viral replication and gene expression. By gene swapping experiments using the two infectious cDNA clones, we show that the NS1–5 proteins of MR766 enhance replication competence of ZIKV Con1. The Con1, which was less virulent than MR766, acquired severe bilateral hindlimb paralysis when its NS1–5 genes were replaced by the counterparts of MR766 in type I interferon receptor (IFNAR1)-deficient A129 mice. Moreover, MR766 NS5 RNA-dependent RNA polymerase (RdRp) alone also rendered the Con1 virulent, despite there being no difference in RdRp activity between MR766 and Con1 NS5 proteins. By contrast, the Con1 derivatives expressing MR766 Nsps, like Con1, did not develop severe disease in wild-type mice treated with an IFNAR1 blocking antibody. Together, our findings uncover an unprecedented role for ZIKV NS proteins in determining viral pathogenicity in immunocompromised hosts.

Human Neutrophils Present Mild Activation by Zika Virus But Reduce the Infection of Susceptible Cells.

The emergence of the Zika virus (ZIKV) has highlighted the need for a deeper understanding of virus-host interactions in order to pave the way for the development of antiviral therapies. The present work aimed to address the response of neutrophils during ZIKV infection. Neutrophils are important effector cells in innate immunity implicated in the host’s response to neurotropic arboviruses. Our results indicate that human neutrophils were not permissive to Asian or African ZIKV strain replication. In fact, after stimulation with ZIKV, neutrophils were mild primed against the virus as evaluated through CD11b and CD62L modulation, secretion of inflammatory cytokines and granule content, production of reactive oxygen species, and neutrophil extracellular traps formation. Overall, neutrophils did not affect ZIKV infectivity. Moreover, in vitro ZIKV infection of primary innate immune cells did not trigger neutrophil migration. However, neutrophils co-cultured with ZIKV susceptible cell lineages resulted in lower cell infection frequencies, possibly due to cell-to-cell contact. In vivo, neutrophil depletion in immunocompetent mice did not affect ZIKV spreading to the draining lymph nodes. The data suggest that human neutrophils do not play an antiviral role against ZIKV per se, but these cells might participate in an infected environment shaping the ZIKV infection in other target cells.

Characterization of m6 A modifications in the contemporary Zika virus genome and host cellular transcripts.

Zika virus (ZIKV) suddenly evolved from a neglected arthropod-borne flavivirus into a pandemic pathogen during 2015-2016. A panel of amino acid mutations has been shown to be responsible for the enhanced neurovirulence and transmissibility of ZIKV. Recent studies have demonstrated that ZIKV genomic RNA is modified by host N6-methyladenosine (m6 A) machinery during viral replication in host cells, and the m6 A profiles vary among different isolates and different host cells. In the present study, using a contemporary Asian ZIKV strain isolated in 2019 (SZ1901) as a model, we profiled m6 A modifications on both the viral genome RNA and cellular transcripts from the ZIKV-infected human hepatocarcinoma cell line Huh7. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) identified a unique m6 A map in the genome of ZIKV strain SZ1901 that is different from all previous isolates. Meanwhile, ZIKV infection induced m6 A upregulation in the CDS regions but downregulation in the 3' untranslated region of host RNA transcripts. The m6 A peak intensity in the majority of host genes was downregulated, including ISG-related genes. Overall, our study describes unique viral and host m6 A profiles in contemporary ZIKV-infected Huh7 cells, highlighting the complexity and importance of m6 A modification during viral infection.

Recombination, and Then What? How Zika Virus Hybrids Improve Their Fitness

Zika virus (ZIKV) is an emerging flavivirus primarily transmitted through the bite of Aedesmosquitoes. It spread explosively around the tropics in the 21st century, and still presents a threat to human health. There is a need to better understand the drivers of emergence of ZIKV and likely consequences of its future evolution. RNA recombination is a process of genetic exchange which occurs in positive-sense RNA viruses and contributes to virus evolution. We have developed a recombinant-specific PCR assay for detection of ZIKV recombinants in mammalian and insect cell co-infections. Using this system, we demonstrated the ability of distinct geographic and genetic isolates to recombine and tested their viability in tissue culture. Initial ZIKV recombinants produce plaques with a significantly smaller diameter than either parental strain. Recombinant large-plaque variants were isolated through serial passage and plaque purification, and the genome analysed for adaptive mutations. A single nucleotide coding mutation in NS1 was detected in several independent large-plaque variants. The role of this mutation in virus replication was investigated through reverse genetics to assess whether it gave the recombinant a replicative advantage. Understanding the genetic changes that arise during, and following recombination is important in the context of virus evolution. It is particularly relevant for ZIKV, as co-circulation of the African and Asian strains of ZIKV in nature has already occurred in parts of Latin America and Africa. Current and future studies will focus on the phenotype of recombinant ZIKV, their replication in mammalian and insect cell lines, and in mosquitoes.

Astrocyte Control of Zika Infection Is Independent of Interferon Type I and Type III Expression.

Simple SummaryZika virus (ZIKV) is a mosquito-borne virus first isolated from the Zika forest, Uganda, in 1947, which has been spreading across continents since then. We now know ZIKV causes both microencephaly in newborns and neurological complications in adults; however, no effective treatment options have yet been found. A more complete understanding of Zika-infection-mediated pathogenesis and host responses is required to enable the development of novel treatment strategies. In this study, efforts were made to elucidate the host responses following Zika virus infection using several astrocyte cell models, as astrocytes are a major cell type within the central nervous system (CNS) with significant antiviral ability. Our data suggest that astrocytes can resist ZIKV both in an interferon type I- and III-independent manner and suggest that an early and more diverse antiviral response may be more effective in controlling Zika infection. This study also identifies astrocyte cellular models that appear to display differential abilities in the control of viral infection, which may assist in the study of alternate neurotropic virus infections. Overall, this work adds to the growing body of knowledge surrounding ZIKV-mediated cellular host interactions and will contribute to a better understanding of ZIKV-mediated pathogenesis.Zika virus (ZIKV) is a pathogenic neurotropic virus that infects the central nervous system (CNS) and results in various neurological complications. Astrocytes are the dominant CNS cell producer of the antiviral cytokine IFN-β, however little is known about the factors involved in their ability to mediate viral infection control. Recent studies have displayed differential responses in astrocytes to ZIKV infection, and this study sought to elucidate astrocyte cell-specific responses to ZIKV using a variety of cell models infected with either the African (MR766) or Asian (PRVABC59) ZIKV strains. Expression levels of pro-inflammatory (TNF-α and IL-1β) and inflammatory (IL-8) cytokines following viral infection were low and mostly comparable within the ZIKV-resistant and ZIKV-susceptible astrocyte models, with better control of proinflammatory cytokines displayed in resistant astrocyte cells, synchronising with the viral infection level at specific timepoints. Astrocyte cell lines displaying ZIKV-resistance also demonstrated early upregulation of multiple antiviral genes compared with susceptible astrocytes. Interestingly, pre-stimulation of ZIKV-susceptible astrocytes with either poly(I:C) or poly(dA:dT) showed efficient protection against ZIKV compared with pre-stimulation with either recombinant IFN-β or IFN-λ, perhaps indicating that a more diverse antiviral gene expression is necessary for astrocyte control of ZIKV, and this is driven in part through interferon-independent mechanisms.

Replication Variance of African and Asian Lineage Zika Virus Strains in Different Cell Lines, Mosquitoes and Mice.

Since the epidemic in 2007, studies on vector competence for Zika virus (ZIKV) have intensified, showing that the transmission efficiency varies depending on the vector population, ZIKV strain, and dose of the infectious blood meal. In this study, we aimed to investigate the replication of African and Asian ZIKV strains in vitro and in vivo in order to reveal their phenotypic differences. In addition, we investigated the vector competence of Cambodian Aedes aegypti (Ae. aegypti) mosquitoes (urban and rural) for these ZIKV strains. We observed a significantly higher pathogenicity of the African ZIKV strain in vitro (in mosquito and mammalian cells), and in vivo in both Ae. aegypti and mice. Both mosquito populations were competent to transmit ZIKV as early as 7 days p.i., depending on the population and the ZIKV strain. Ae. aegypti from rural habitats showed significant higher transmission and survival rates than those from urban. We observed the highest transmission efficiency for the African ZIKV isolate (93.3% 14 days p.i.) and for the Cambodian ZIKV isolate (80% 14 days p.i.). Overall, our results highlight the phenotypic differences of the ZIKV lineages and the potential risk of ZIKV transmission by Ae. aegypti mosquitoes. Further investigations of Cambodian mosquito species and ZIKV specific surveillance in humans is necessary in order to improve the local risk assessment.

Experimental infections with Zika virus strains reveal high vector competence of Aedes albopictus and Aedes aegypti populations from Gabon (Central Africa) for the African virus lineage

ABSTRACT The two main Zika virus (ZIKV) vectors, Aedes albopictus and Aedes aegypti (invasive and native species, respectively), are present in Gabon (Central Africa). The aim of this study was to determine the entomological ZIKV risk associated with these mosquito species in Gabon by evaluating their vector competence for an African (i.e. representative of the endemic strains circulating in sub-Saharan Africa) and two Asian (i.e. representatives of exogenous epidemic strains that could be introduced) ZIKV strains. The transmission efficiency of one Ae. aegypti and two Ae. albopictus field-collected populations from Libreville and Franceville was assayed at day 7, 14 and 21 after experimental oral infection. The two mosquito species could transmit all three ZIKV strains already at day 7 post-infection, but transmission efficiency was higher for the African strain than the non-African strains (>60% versus <14%; incubation period of 14–21 days). The two mosquito species exhibited comparable vector competence for ZIKV, although the amount of viral particles (African strain) in saliva was significantly higher in Ae. albopictus than Ae. aegypti at day 14 post-infection. These findings suggest that overall, ZIKV risk in Gabon is mainly related to virus strains that circulate endemically across sub-Saharan Africa, although the transmission of non-African strains remain possible in case of introduction. Due to its high infestation indexes and ecological/geographical ranges, this risk appears mainly associated with Ae. albopictus. Vector surveillance and control methods against this invasive mosquito must be strengthened in the region to limit the risk of future outbreaks.

A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

BackgroundEarly detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.MethodsIn the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.ResultsThe detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6–98.2) and 100% (95% CI = 78.5–100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P < 0.001).ConclusionThe RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

Reversion to ancestral Zika virus NS1 residues increases competence of Aedes albopictus.

Both mosquito species-specific differences and virus strain -specific differences impact vector competence. Previous results in our laboratory with individual populations of N. American mosquitoes support studies suggesting Aedes aegypti are more competent than Ae. albopictus for American Zika virus (ZIKV) strains and demonstrate that U.S. Ae. albopictus have higher competence for an ancestral Asian ZIKV strain. A982V, an amino acid substitution in the NS1 gene acquired prior to the American outbreak, has been shown to increase competence in Ae. aegypti. We hypothesized that variability in the NS1 could therefore contribute to species-specific differences and developed a reverse genetics system based on a 2016 ZIKV isolate from Honduras (ZIKV-WTic) to evaluate the phenotypic correlates of individual amino acid substitutions. In addition to A982V, we evaluated G894A, which was acquired during circulation in the Americas. Reversion of 982 and 894 to ancestral residues increased infectivity, transmissibility and viral loads in Ae. albopictus but had no effect on competence or replication in Ae. aegypti. In addition, while host cell-specific differences in NS1 secretion were measured, with significantly higher secretion in mammalian cells relative to mosquito cells, strain-specific differences in secretion were not detected, despite previous reports. These results demonstrate that individual mutations in NS1 can influence competence in a species-specific manner independent of differences in NS1 secretion and further indicate that ancestral NS1 residues confer increased competence in Ae. albopictus. Lastly, experimental infections of Ifnar1-/- mice demonstrated that these NS1 substitutions can influence viral replication in the host and, specifically, that G894A could represent a compensatory change following a fitness loss from A982V with some viral genetic backgrounds. Together these data suggest a possible role for epistatic interactions in ZIKV fitness in invertebrate and vertebrate hosts and demonstrate that strains with increased transmission potential in U.S. Ae. albopictus could emerge.

Cutaneous vaccination ameliorates Zika virus-induced neuro-ocular pathology via reduction of anti-ganglioside antibodies

ABSTRACT Zika virus (ZIKV) causes moderate to severe neuro-ocular sequelae, with symptoms ranging from conjunctivitis to Guillain-Barré Syndrome (GBS). Despite the international threat ZIKV poses, no licensed vaccine exists. As ZIKV and DENV are closely related, antibodies against one virus have demonstrated the ability to enhance the other. To examine if vaccination can confer robust, long-term protection against ZIKV, preventing neuro-ocular pathology and long-term inflammation in immune-privileged compartments, BALB/c mice received two doses of unadjuvanted inactivated whole ZIKV vaccine (ZVIP) intramuscularly (IM) or cutaneously with dissolving microneedle patches (MNP). MNP immunization induced significantly higher B and T cell responses compared to IM vaccination, resulting in increased antibody titers with greater avidity for ZPIV as well as increased numbers of IFN-γ, TNF-α, IL- and IL-4 secreting T cells. When compared to IM vaccination, antibodies generated by cutaneous vaccination demonstrated greater neutralization activity, increased cross-reactivity with Asian and African lineage ZIKV strains (PRVABC59, FLR, and MR766) and Dengue virus (DENV) serotypes, limited ADE, and lower reactivity to GBS-associated gangliosides. MNP vaccination effectively controlled viremia and inflammation, preventing neuro-ocular pathology. Conversely, IM vaccination exacerbated ocular pathology, resulting in uncontrolled, long-term inflammation. Importantly, neuro-ocular pathology correlated with anti-ganglioside antibodies implicated in demyelination and GBS. This study highlights the importance of longevity studies in ZIKV immunization, and the need of exploring alternative vaccination platforms to improve the quality of vaccine-induced immune responses.

A Zika virus envelope mutation preceding the 2015 epidemic enhances virulence and fitness for transmission

Arboviruses maintain high mutation rates due to lack of proofreading ability of their viral polymerases, in some cases facilitating adaptive evolution and emergence. Here we show that, just before its 2013 spread to the Americas, Zika virus (ZIKV) underwent an envelope protein V473M substitution (E-V473M) that increased neurovirulence, maternal-to-fetal transmission, and viremia to facilitate urban transmission. A preepidemic Asian ZIKV strain (FSS13025 isolated in Cambodia in 2010) engineered with the V473M substitution significantly increased neurovirulence in neonatal mice and produced higher viral loads in the placenta and fetal heads in pregnant mice. Conversely, an epidemic ZIKV strain (PRVABC59 isolated in Puerto Rico in 2015) engineered with the inverse M473V substitution reversed the pathogenic phenotypes. Although E-V473M did not affect oral infection of Aedes aegypti mosquitoes, competition experiments in cynomolgus macaques showed that this mutation increased its fitness for viremia generation, suggesting adaptive evolution for human viremia and hence transmission. Mechanistically, the V473M mutation, located at the second transmembrane helix of the E protein, enhances virion morphogenesis. Overall, our study revealed E-V473M as a critical determinant for enhanced ZIKV virulence, intrauterine transmission during pregnancy, and viremia to facilitate urban transmission.

Zika structural genes determine the virulence of African and Asian lineages

ABSTRACT The Asian lineage of Zika virus (ZIKV) is responsible for the recent epidemics in the Americas and severe disease, whereas the African lineage of ZIKV has not been reported to cause epidemics or severe disease. We constructed a cDNA infectious clone (IC) of an African ZIKV strain, which, together with our previously developed Asian ZIKV strain IC, allowed us to engineer chimeric viruses by swapping the structural and non-structural genes between the two lineages. Recombinant parental and chimeric viruses were analyzed in A129 and newborn CD1 mouse models. In the A129 mice, the African strain developed higher viremia, organ viral loading, and mortality rate. In CD1 mice, the African strain exhibited a higher neurovirulence than the Asian strain. A chimeric virus containing the structural genes from the African strain is more virulent than the Asian strain, whereas a chimeric virus containing the non-structural genes from the African strain exhibited a virulence comparable to the Asian strain. These results suggest that (i) African strain is more virulent than Asian strain and (ii) viral structural genes primarily determine the virulence difference between the two lineages in mouse models. Other factors may contribute to the discrepancy between the mouse and epidemic results.

Mechanism of differential Zika and dengue virus neutralization by a public antibody lineage targeting the DIII lateral ridge.

We previously generated a panel of human monoclonal antibodies (mAbs) against Zika virus (ZIKV) and identified one, ZIKV-116, that shares germline usage with mAbs identified in multiple donors. Here we show that ZIKV-116 interferes with ZIKV infection at a post-cellular attachment step by blocking viral fusion with host membranes. ZIKV-116 recognizes the lateral ridge of envelope protein domain III, with one critical residue varying between the Asian and African strains responsible for differential binding affinity and neutralization potency (E393D). ZIKV-116 also binds to and cross-neutralizes some dengue virus serotype 1 (DENV1) strains, with genotype-dependent inhibition explained by variation in a domain II residue (R204K) that potentially modulates exposure of the distally located, partially cryptic epitope. The V-J reverted germline configuration of ZIKV-116 preferentially binds to and neutralizes an Asian ZIKV strain, suggesting that this epitope may optimally induce related B cell clonotypes. Overall, these studies provide a structural and molecular mechanism for a cross-reactive mAb that uniquely neutralizes ZIKV and DENV1.

Comparative analysis of viral entry for Asian and African lineages of Zika virus

Zika virus (ZIKV) is an emerging pathogen with global health and economic impacts. ZIKV circulates as two major lineages, Asian or African. The Asian lineage has recently been associated with significant disease in humans. Numerous studies have revealed differences between African and Asian ZIKV strains with respect to cellular infectivity, pathogenesis, and immune activation. Less is known about the mechanism of ZIKV entry and whether viral entry differs between strains. Here, we characterized ZIKV entry with two Asian and two African strains. All viruses exhibited a requirement for clathrin-mediated endocytosis and Rab5a function. Additionally, all ZIKV strains tested were sensitive to pH in the range of 6.5–6.1 and were reliant on endosomal acidification for infection. Finally, we provide direct evidence that ZIKV primarily fuses with late endosomes. These findings contribute new insight into the ZIKV entry process and suggest that divergent ZIKV strains enter cells in a highly conserved manner.

Efficiencies and kinetics of infection in different cell types/lines by African and Asian strains of Zika virus.

After recent outbreaks, Zika virus (ZIKV) was linked to severe neurological diseases including Guillain-Barré syndrome in adults and microcephaly in newborns. The severities of pathological manifestations have been associated with different ZIKV strains. To better understand the tropism of ZIKV, we infected 10 human and four nonhuman cell lines (types) with two African (IbH30656 and MR766) and two Asian (PRVABC59 and H/FP/2013) ZIKV strains. Cell susceptibility to ZIKV infection was determined by examining viral titers, synthesis of viral proteins, and replication of positive and negative strands of viral genome. Among nonhuman cell lines, only Vero cells were efficiently infected by ZIKV. Among human cell lines, all were permissive to ZIKV infection. However, 293T and HeLa cells showed differential susceptibility towards African strains. In 293T cells, the NS1 protein was expressed at the high level by African strains but was almost not expressed by Asian strains though there was no obvious difference in viral genome replication, suggesting that the differential susceptibility might be controlled at the stage of viral protein translation. This study provides comprehensive results of the permissiveness of different cell types to both African and Asian ZIKV strains, which might help clarify their different pathogenesis.

Differential transmission of Asian and African Zika virus lineages by Aedes aegypti from New Caledonia

Zika virus (ZIKV) is a Flavivirus that is transmitted to humans by Aedes mosquitoes. ZIKV is divided into two phylogenetic lineages, African and Asian. In the Asian lineage, Pacific and American clades have been linked to the recent worldwide outbreak of ZIKV. The aim of this study was to measure the vector competence of Aedes aegypti for seven ZIKV strains belonging to both lineages. We demonstrate that Ae. aegypti from New Caledonia (NC), South Pacific region, is a low-competence vector for Asian ZIKV (<10% transmission efficiency). No significant differences were observed in vector competence with respect to the sampling date and collection site of Asian ZIKV strains used (2014 and 2015 for New Caledonia, Pacific clade, and 2016 for French Guiana, American clade). The ability of the New Caledonian Ae. aegypti to transmit ZIKV is significantly greater for the earlier viral isolates belonging to the African lineage (>37% transmission efficiency after 9 days post-infection) compared to recent ZIKV isolates from African (10% transmission efficiency) and Asian lineages (<10% transmission efficiency). The results of this study demonstrate that Ae. aegypti from NC can become infected and replicate different ZIKV strains belonging to all lineages. Our data emphasize the importance of studying the interaction between vectors and their arboviruses according to each local geographic context. This approach will improve our understanding of arbovirus transmission to prevent their emergence and improve health surveillance.

ZIKV Infection Induces an Inflammatory Response but Fails to Activate Types I, II, and III IFN Response in Human PBMC.

The recent epidemic in the Americas caused by Zika virus (ZIKV), Asian lineage, spurred the research towards a better understanding of how ZIKV infection affects the host immune response. The aim of this study was to evaluate the effects of Asian and East African ZIKV strain infection on the induction of IFN and proinflammatory and Th2 cytokines in human PBMC. We reported a slight modulation of type II IFN in PBMC exposed to Asian strain, but not to African strain, and a complete lack of type I and III IFN induction by both strains, suggesting the ability of ZIKV to evade the IFN system not only inhibiting the antiviral IFN response but also IFN production. Moreover, we highlighted a polyfunctional immune activation only in PBMC exposed to Asian strain, due to the induction of an inflammatory profile (IL-6, IL-8) and of a Th9 (IL-9) response. Overall, our data show a different ability of the ZIKV Asian strain, with respect to the African strain, to activate host immune response that may have pathogenetic implications for virus spread in vivo, including mother-to-child transmission and induction of severe fetal complications, as birth defects and neurological disorders.