African Zika Virus Strains Research Articles

The NS1 protein of contemporary West African Zika virus potentiates viral replication and reduces innate immune activation.

Mosquito-borne Zika virus (ZIKV) from sub-Saharan Africa has recently gained attention due to its epidemic potential and its capacity to be highly teratogenic. To improve our knowledge on currently circulating strains of African ZIKV, we conducted protein sequence alignment and identified contemporary West Africa NS1 (NS1CWA) protein as a highly conserved viral protein. Comparison of NS1CWA with the NS1 of the historical African ZIKV strain MR766 (NS1MR766), revealed seven amino acid substitutions. The effects of NS1 mutations on protein expression, virus replication, and innate immune activation were assessed in human cells using recombinant NS1 proteins and a chimeric viral clone MR766 with NS1CWA replacing NS1MR766. Our data indicated higher secretion efficiency of NS1CWA compared to NS1MR766 associated with a change in subcellular distribution. A chimeric MR766 virus with NS1CWA instead of authentic protein displayed a greater viral replication efficiency, leading to more pronounced cell death compared to parental virus. Enhanced viral growth was associated with reduced activation of innate immunity. Our data raise questions of the importance of NS1 protein in the pathogenicity of contemporary ZIKV from sub-Saharan Africa and point to differences within viral strains of African lineage.

Replication properties of a contemporary Zika virus from West Africa.

Zika virus (ZIKV) has become a global health problem over the past decade due to the extension of the geographic distribution of the Asian/American genotype. Recent epidemics of Asian/American ZIKV have been associated with developmental disorders in humans. There is mounting evidence that African ZIKV may be associated with increased fetal pathogenicity necessitating to pay a greater attention towards currently circulating viral strains in sub-Saharan Africa. Here, we generated an infectious molecular clone GUINEA-18 of a recently transmitted human ZIKV isolate from West Africa, ZIKV-15555. The available infectious molecular clone MR766MC of historical African ZIKV strain MR766-NIID was used for a molecular clone-based comparative study. Viral clones GUINEA-18 and MR766MC were compared for their ability to replicate in VeroE6, A549 and HCM3 cell lines. There was a lower replication rate for GUINEA-18 associated with weaker cytotoxicity and reduced innate immune system activation compared with MR766MC. Analysis of chimeric viruses between viral clones stressed the importance of NS1 to NS4B proteins, with a particular focus of NS4B on GUINEA-18 replicative properties. ZIKV has developed strategies to prevent cytoplasmic stress granule formation which occurs in response to virus infection. GUINEA-18 was greatly efficient in inhibiting stress granule assembly in A549 cells subjected to a physiological stressor, with NS1 to NS4B proteins also being critical in this process. The impact of these GUINEA-18 proteins on viral replicative abilities and host-cell responses to viral infection raises the question of the role of nonstructural proteins in the pathogenicity of currently circulating ZIKV in sub-Saharan Africa.

Infection of the maternal-fetal interface and vertical transmission following low-dose inoculation of pregnant rhesus macaques (Macaca mulatta) with an African-lineage Zika virus.

Congenital Zika virus (ZIKV) infection can result in birth defects, including malformations in the fetal brain and visual system. There are two distinct genetic lineages of ZIKV: African and Asian. Asian-lineage ZIKVs have been associated with adverse pregnancy outcomes in humans; however, recent evidence from experimental models suggests that African-lineage viruses can also be vertically transmitted and cause fetal harm. To evaluate the pathway of vertical transmission of African-lineage ZIKV, we inoculated nine pregnant rhesus macaques (Macaca mulatta) subcutaneously with 44 plaque-forming units of a ZIKV strain from Senegal, (ZIKV-DAK). Dams were inoculated either at gestational day 30 or 45. Following maternal inoculation, pregnancies were surgically terminated seven or 14 days later and fetal and maternal-fetal interface tissues were collected and evaluated. Infection in the dams was evaluated via plasma viremia and neutralizing antibody titers pre- and post- ZIKV inoculation. All dams became productively infected and developed strong neutralizing antibody responses. ZIKV RNA was detected in maternal-fetal interface tissues (placenta, decidua, and fetal membranes) by RT-qPCR and in situ hybridization. In situ hybridization detected ZIKV predominantly in the decidua and revealed that the fetal membranes may play a role in ZIKV vertical transmission. Infectious ZIKV was detected in the amniotic fluid of three pregnancies and one fetus had ZIKV RNA detected in multiple tissues. No significant pathology was observed in any fetus; and ZIKV did not have a substantial effect on the placenta. This study demonstrates that a very low dose of African-lineage ZIKV can be vertically transmitted to the macaque fetus during pregnancy. The low inoculating dose used in this study suggests a low minimal infectious dose for rhesus macaques. Vertical transmission with a low dose in macaques further supports the high epidemic potential of African ZIKV strains.

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.

Pathogenicity and Structural Basis of Zika Variants with Glycan Loop Deletions in the Envelope Protein.

The glycan loop of Zika virus (ZIKV) envelope protein (E) contains the glycosylation site and has been well documented to be important for viral pathogenesis and transmission. In the present study, we report that deletions in the E glycan loop, which were recorded in African ZIKV strains previously, have re-emerged in their contemporary Asian lineages. Here, we generated recombinant ZIKV containing specific deletions in the E glycan loop by reverse genetics. Extensive in vitro and in vivo characterization of these deletion mutants demonstrated an attenuated phenotype in an adult A129 mouse model and reduced oral infections in mosquitoes. Surprisingly, these glycan loop deletion mutants exhibited an enhanced neurovirulence phenotype, and resulted in a more severe microcephalic brain in neonatal mouse models. Crystal structures of the ZIKV E protein and a deletion mutant at 2.5 and 2.6 Å, respectively, revealed that deletion of the glycan loop induces encephalitic flavivirus-like conformational alterations, including the appearance of perforations on the surface and a clear change in the topology of the loops. Overall, our results demonstrate that the E glycan loop deletions represent neonatal mouse neurovirulence markers of ZIKV. IMPORTANCE Zika virus (ZIKV) has been identified as a cause of microcephaly and acquired evolutionary mutations since its discovery. Previously deletions in the E glycan loop were recorded in African ZIKV strains, which have re-emerged in the contemporary Asian lineages recently. The glycan loop deletion mutants are not glycosylated, which are attenuated in adult A129 mouse model and reduced oral infections in mosquitoes. More importantly, the glycan loop deletion mutants induce an encephalitic flavivirus-like conformational alteration in the E homodimer, resulting in a significant enhancement of neonatal mouse neurovirulence. This study underscores the critical role of glycan loop deletion mutants in ZIKV pathogenesis, highlighting a need for global virological surveillance for such ZIKV variants.

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.

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.

A methanol extract and N,N-dimethyltryptamine from Psychotria viridis Ruiz & Pav. inhibit Zika virus infection in vitro.

Zika virus (ZIKV) is a public health problem due to its association with serious fetal and neurological complications and the lack of antiviral agents and licensed vaccines against this virus. Surveillance studies have alerted about the potential occurrence of a new South American epidemic episode due to the recent circulation of an African ZIKV strain detected in Brazil. Therefore, it is essential to discover antiviral agents, including natural substances, that are capable of neutralizing the action of ZIKV. Several Psychotria species have antimicrobial and anti-inflammatory properties. Thus, a methanol extract and dimethyltryptamine from Psychotria viridis were evaluated for their ability to inhibit ZIKV infection in vitro by measuring the effective concentration that protects 50% of cells and investigating their possible mechanisms of action. The tested samples showed antiviral activity against ZIKV. The extract showed virucidal activity, affecting viral and non-cellular elements, inactivating the virus before infection or when it becomes extracellular after the second cycle of infection. It was also observed that both extract and dimethyltryptamine could inhibit the virus at intracellular stages of the viral cycle. In addition to dimethyltryptamine, it is believed that other compounds also contribute to the promising virucidal effect observed for the methanol extract. To our knowledge, this is the first report of the activity of a methanolic extract and dimethyltryptamine from Psychotria viridis against cellular ZIKV infection. These two samples, extracted from natural sources, are potential candidates for use as antiviral drugs to inhibit ZIKV infections.

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.

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.

Recent African strains of Zika virus display higher transmissibility and fetal pathogenicity than Asian strains

The global emergence of Zika virus (ZIKV) revealed the unprecedented ability for a mosquito-borne virus to cause congenital birth defects. A puzzling aspect of ZIKV emergence is that all human outbreaks and birth defects to date have been exclusively associated with the Asian ZIKV lineage, despite a growing body of laboratory evidence pointing towards higher transmissibility and pathogenicity of the African ZIKV lineage. Whether this apparent paradox reflects the use of relatively old African ZIKV strains in most laboratory studies is unclear. Here, we experimentally compare seven low-passage ZIKV strains representing the recently circulating viral genetic diversity. We find that recent African ZIKV strains display higher transmissibility in mosquitoes and higher lethality in both adult and fetal mice than their Asian counterparts. We emphasize the high epidemic potential of African ZIKV strains and suggest that they could more easily go unnoticed by public health surveillance systems than Asian strains due to their propensity to cause fetal loss rather than birth defects.

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.

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.

Recombination between African and Asian lineages of Zika virus in vitro and its consequences for viral phenotype

Recombination is a process of extensive genetic exchange that is known to contribute to virus evolution and has been frequently observed in positive-sense RNA viruses. Zika virus (ZIKV) is an emerging arbovirus of the family Flaviviridae with two distinct lineages – African and Asian. While some phylogenetic evidence suggests that recombination in the envelope-encoding region of the ZIKV genome has occurred during evolution, there has been no experimental evidence for ZIKV recombination to date. We conducted co-infections of mammalian and insect cells, using the prototype African ZIKV strain (MR766) and an Asian isolate from the 2015-16 ZIKV outbreak in Brazil (BeH819015), and used a recombinant-specific PCR assay to detect recombinant sequences from total cell RNA extracts. In brief, a 564bp fragment spanning the boundary between the structural and the non-structural genes of the viral genome was amplified using a primer pair consisting of an Asian-specific and an African-specific primer. A total of 24 individual sequences were screened. All were in-frame recombinants and they formed 10 unique junctions. Several of the detected recombinant sequences were chosen for construction of full-length infectious clones to test the viability and phenotype of the recombinant viruses. This study represents the first isolation of recombinant ZIKV sequences from co-infected cultured cells and demonstrates the capacity of ZIKV to recombine in an experimental system. Further investigation is required to better understand the evolutionary potential of this mechanism and its putative role in the emergence of ZIKV.

Benzimidazole Derivatives as Novel Zika Virus Inhibitors.

We have synthesized 50 benzimidazole (BMZ) derivatives with 1,2-phenylenediamines and aromatic aldehydes under mild oxidation conditions by using inexpensive, nontoxic inorganic salt sodium metabisulfite in a one-pot condensation reaction and screened their ability to interfere with Zika virus (ZIKV) infection utilizing a cell-based phenotypic assay. Seven BMZs inhibited an African ZIKV strain with a selectivity index (SI=CC50 /EC50 ) of 9-37. Structure-activity relationship analysis demonstrated that substitution at the C-2, N-1, and C-5 positions of the BMZ ring were important for anti-ZIKV activity. The hybrid structure of BMZ and naphthalene rings was a structural feature responsible for the high anti-ZIKV activity. Importantly, BMZs inhibited ZIKV in human neural stem cells, a physiologically relevant system considering the severe congenital anomalies, like microcephaly, caused by ZIKV infection. Compound 39 displayed the highest antiviral efficacy against the African ZIKV strain in Huh-7 (SI>37) and neural stem cells (SI=12). Compound 35 possessed the highest activity in Vero cells (SI=115). Together, our data indicate that BMZs derivatives have to be considered for the development of ZIKV therapeutic interventions.

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.

A Novel Mechanism for Zika Virus Host-Cell Binding.

Zika virus (ZIKV) recently emerged in the Western Hemisphere with previously unrecognized or unreported clinical presentations. Here, we identify two putative binding mechanisms of ancestral and emergent ZIKV strains featuring the envelope (E) protein residue asparagine 154 (ASN154) and viral phosphatidylserine (PS). Synthetic peptides representing the region containing ASN154 from strains PRVABC59 (Puerto Rico 2015) and MR_766 (Uganda 1947) were exposed to neuronal cells and fibroblasts to model ZIKV E protein/cell interactions and bound MDCK or Vero cells and primary neurons significantly. Peptides significantly inhibited Vero cell infectivity by ZIKV strains MR_766 and PRVABC59, indicating that this region represents a putative binding mechanism of ancestral African ZIKV strains and emergent Western Hemisphere strains. Pretreatment of ZIKV strains MR_766 and PRVABC59 with the PS-binding protein annexin V significantly inhibited replication of PRVABC59 but not MR_766, suggesting that Western hemisphere strains may additionally be capable of utilizing PS-mediated entry to infect host cells. These data indicate that the region surrounding E protein ASN154 is capable of binding fibroblasts and primary neuronal cells and that PS-mediated entry may be a secondary mechanism for infectivity utilized by Western Hemisphere strains.

Comparative Analysis of African and Asian Lineage-Derived Zika Virus Strains Reveals Differences in Activation of and Sensitivity to Antiviral Innate Immunity.

In recent years, Asian lineage Zika virus (ZIKV) strains emerged to cause pandemic outbreaks associated with a high rate of congenital ZIKV syndrome (CZVS). The reasons for the enhanced spread and severe disease caused by newly emerging strains are not fully understood. Here we compared viral sequences, viral replication, and innate immune signaling induction of three different ZIKV strains derived from African and Asian lineages and West Nile virus, another flavivirus. We found pronounced differences in activation of innate immune signaling and inhibition of viral replication across ZIKV strains. The newly emerged Asian ZIKV strain Brazil Fortaleza 2015, which is associated with a higher rate of neurodevelopmental disorders like microcephaly, induced much weaker and delayed innate immune signaling in infected cells. However, superinfection studies to assess control of innate immune signaling induced by Sendai virus argue against an active block of IRF3 activation by the Brazilian strain of ZIKV and rather suggest an evasion of detection by host cell pattern recognition receptors. Compared to the Asian strain FSS13025 isolated in Cambodia, both ZIKV Uganda MR766 and ZIKV Brazil Fortaleza appear less sensitive to the interferon-induced antiviral response. ZIKV infection studies of cells lacking the different RIG-I-like receptors identified RIG-I as the major cytosolic pattern recognition receptor for detection of ZIKV.IMPORTANCE Zika Virus (ZIKV), discovered in 1947, is divided into African and Asian lineages. Pandemic outbreaks caused by currently emerging Asian lineage strains are accompanied by high rates of neurological disorders and exemplify the global health burden associated with this virus. Here we compared virological and innate immunological aspects of two ZIKV strains from the Asian lineage, an emerging Brazilian strain and a less-pathogenic Cambodian strain, and the prototypic African lineage ZIKV strain from Uganda. Compared to the replication of other ZIKV strains, the replication of ZIKV Brazil was less sensitive to the antiviral actions of interferon (IFN), while infection with this strain induced weaker and delayed innate immune responses in vitro Our data suggest that ZIKV Brazil directs a passive strategy of innate immune evasion that is reminiscent of a stealth virus. Such strain-specific properties likely contribute to differential pathogenesis and should be taken into consideration when choosing virus strains for future molecular studies.

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.

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.