Antibody Responses to Zika Virus Infections in Environments of Flavivirus Endemicity.
ABSTRACT: Zika virus (ZIKV) infections occur in areas where dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), and other viruses of the genus Flavivirus cocirculate. The envelope (E) proteins of these closely related flaviviruses induce specific long-term immunity, yet subsequent infections are associated with cross-reactive antibody responses that may enhance disease susceptibility and severity. To gain a better understanding of ZIKV infections against a background of similar viral diseases, we examined serological immune responses to ZIKV, WNV, DENV, and YFV infections of humans and nonhuman primates (NHPs). Using printed microarrays, we detected very specific antibody responses to primary infections with probes of recombinant E proteins from 15 species and lineages of flaviviruses pathogenic to humans, while high cross-reactivity between ZIKV and DENV was observed with 11 printed native viruses. Notably, antibodies from human primary ZIKV or secondary DENV infections that occurred in areas where flavivirus is endemic broadly recognized E proteins from many flaviviruses, especially DENV, indicating a strong influence of infection history on immune responses. A predictive algorithm was used to tentatively identify previous encounters with specific flaviviruses based on serum antibody interactions with the multispecies panel of E proteins. These results illustrate the potential impact of exposure to related viruses on the outcome of ZIKV infection and offer considerations for development of vaccines and diagnostics.
Project description:Even in countries that are currently not facing a flavivirus epidemic, the spread of mosquito-borne flaviviruses presents an increasing public threat, owing to climate change, international travel, and other factors. Many of these countries lack the resources (viral strains, clinical specimens, etc.) needed for the research that could help cope with the threat imposed by flaviviruses, and therefore, an alternative approach is needed. Using an in silico approach to global databases, we aimed to design and develop flavivirus NS1 recombinant proteins with due consideration towards antigenic variation. NS1 genes analyzed in this study included a total of 6,823 sequences, from Dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV), and Yellow fever virus (YKV). We extracted and analyzed 316 DENV NS1 sequence types (STs), 59 JEV STs, 75 WNV STs, 30 YFV STs, and 43 ZIKV STs using a simple algorithm based on phylogenetic analysis. STs were reclassified according to the variation of the major epitope by MHC II binding. 78 DENV epitope type (EpT), 29 JEV EpTs, 29 WNV EpTs, 12 YFV EpTs, and 5 ZIKV EpTs were extracted according to their major epitopes. Also, frequency results showed that there were dominant EpTs in all flavivirus. Fifteen STs were selected and purified for the expression of recombinant antigen in Escherichia coli by sodium dodecyl sulfate extraction. Our study details a novel in silico approach for the development of flavivirus diagnostics, including a simple way to screen the important peptide regions.
Project description:Flaviviruses, such as dengue (DENV), West Nile (WNV), yellow fever (YFV) and Zika (ZIKV) viruses, are mosquito-borne pathogens that present a major risk to global public health. To identify host factors that promote flavivirus replication, we performed a genome-wide gain-of-function cDNA screen for human genes that enhance the replication of flavivirus reporter particles in human cells. The screen recovered seventeen potential host proteins that promote viral replication, including the previously known dolichyl-diphosphooligosaccharide--protein glycosyltransferase non-catalytic subunit (DDOST). Using silencing approaches, we validated the role of four candidates in YFV and WNV replication: ribosomal protein L19 (RPL19), ribosomal protein S3 (RPS3), DDOST and importin 9 (IPO9). Applying a panel of virological, biochemical and microscopic methods, we validated further the role of RPL19 and DDOST as host factors required for optimal replication of YFV, WNV and ZIKV. The genome-wide gain-of-function screen is thus a valid approach to advance our understanding of flavivirus replication.
Project description:Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1-4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the (98)DRXW(101) motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1-4, YFV, and WNV and confers protection from lethal challenge with DENV 1-4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.
Project description:The Flavivirus genus of the Flaviviridae family encompasses numerous enveloped plus-strand RNA viruses. Dengue virus (DENV), a flavivirus, is the leading cause of serious arthropod-borne disease globally. The genomes of DENV, like the genomes of yellow fever virus (YFV), West Nile fever virus (WNV), or Zika virus (ZIKV), control their translation by a 5'-terminal capping group. Three other genera of Flaviviridae are remarkable because their viruses use internal ribosomal entry sites (IRESs) to control translation, and they are not arthropod transmitted. In 2006, E. Harris' group published work suggesting that DENV RNA does not stringently need a cap for translation. They proposed that instead DENV translation is controlled by an interplay between 5' and 3' termini. Here we present evidence that the DENV or ZIKV 5' untranslated regions (5'-UTRs) alone have IRES competence. This conclusion is based, first, on the observation that uncapped monocistronic mRNAs 5' terminated with the DENV or ZIKV 5'-UTRs can efficiently direct translation of a reporter gene in BHK and C6/36 cells and second, that either 5'-UTR placed between two reporter genes can efficiently induce expression of the downstream gene in BHK cells but not in C6/36 cells. These experiments followed observations that uncapped DENV/ZIKV genomic transcripts, 5' terminated with pppAN… or GpppAN…, can initiate infections of mammalian (BHK) or mosquito (C6/36) cells. IRES competence of the 5'-UTRs of DENV/ZIKV raises many open questions regarding the biology and control, as well as the evolution, of insect-borne flaviviruses.IMPORTANCE Members of the genus Flavivirus of Flaviviridae are important human pathogens of great concern because they cause serious diseases, sometimes death, in human populations living in tropical, subtropical (dengue virus [DENV], Zika virus [ZIKV], and yellow fever virus), or moderate climates (West Nile virus). Flaviviruses are known to control their translation by a cap-dependent mechanism. We have observed, however, that the uncapped genomes of DENV or ZIKV can initiate infection of mammalian and insect cells. We provide evidence that the short 5' untranslated region (5'-UTR) of DENV or ZIKV genomes can fulfill the function of an internal ribosomal entry site (IRES). This strategy frees these organisms from the cap-dependent mechanism of gene expression at an as yet unknown stage of proliferation. The data raise new questions about the biology and evolution of flaviviruses, possibly leading to new controls of flavivirus disease.
Project description:Many flaviviruses, such as Zika virus (ZIKV), Dengue virus (DENV1-4) and yellow fever virus (YFV), are significant human pathogens. Infection with ZIKV, an emerging mosquito-borne flavivirus, is associated with increased risk of microcephaly in newborns and Guillain-Barré syndrome and other complications in adults. Currently, specific therapy does not exist for any flavivirus infections. In this study, we found that erythrosin B, an FDA-approved food additive, is a potent inhibitor for flaviviruses, including ZIKV and DENV2. Erythrosin B was found to inhibit the DENV2 and ZIKV NS2B-NS3 proteases with IC50 in low micromolar range, via a non-competitive mechanism. Erythrosin B can significantly reduce titers of representative flaviviruses, DENV2, ZIKV, YFV, JEV, and WNV, with micromolar potency and with excellent cytotoxicity profile. Erythrosin B can also inhibit ZIKV replication in ZIKV-relevant human placental and neural progenitor cells. As a pregnancy category B food additive, erythrosin B may represent a promising and easily developed therapy for management of infections by ZIKV and other flaviviruses.
Project description:Members of the flavivirus genus share a high level of sequence similarity and often circulate in the same geographical regions. However, whether T cells induced by one viral species cross-react with other related flaviviruses has not been globally addressed. In this study, we tested pools of epitopes derived from dengue (DENV), Zika (ZIKV), Japanese encephalitis (JEV), West Nile (WNV), and yellow fever (YFV) viruses by intracellular cytokine staining (ICS) using peripheral blood mononuclear cells (PBMCs) of individuals naturally exposed to DENV or immunized with DENV (TV005) or YF17D vaccine. CD8 T cell responses recognized epitopes from multiple flaviviruses; however, the magnitude of cross-reactive responses was consistently severalfold lower than those to the autologous epitope pools and was associated with lower expression of activation markers such as CD40L, CD69, and CD137. Next, we characterized the antigen sensitivity of short-term T cell lines (TCL) representing 29 different individual epitope/donor combinations. TCL derived from DENV monovalent vaccinees induced CD8 and CD4 T cells that cross-reacted within the DENV serocomplex but were consistently associated with >100-fold-lower antigen sensitivity for most other flaviviruses, with no cross-recognition of YFV-derived peptides. CD8 and CD4 TCL from YF17D vaccinees were associated with very limited cross-reactivity with any other flaviviruses and in five out of eight cases >1,000-fold-lower antigen sensitivity. Overall, our data suggest limited cross-reactivity for both CD4 and CD8 T cell responses between flaviviruses and have implications for understanding immunity elicited by natural infection and strategies to develop live attenuated vaccines against flaviviral species.IMPORTANCE The envelope (E) protein is the dominant target of neutralizing antibodies for dengue virus (DENV) and yellow fever virus (YFV). Accordingly, several DENV vaccine constructs use the E protein in a live attenuated vaccine format, utilizing a backbone derived from a heterologous flavivirus (such as YF) as a delivery vector. This backbone comprises the nonstructural (NS) and capsid (C) antigens, which are dominant targets of T cell responses. Here, we demonstrate that cross-reactivity at the level of T cell responses among different flaviviruses is very limited, despite high levels of sequence homology. Thus, the use of heterologous flavivirus species as a live attenuated vaccine vector is not likely to generate optimal T cell responses and might thus impair vaccine performance.
Project description:Dengue viruses (DENV) cause countless human deaths each year, whilst West Nile virus (WNV) has re-emerged as an important human pathogen. There are currently no WNV or DENV vaccines licensed for human use, yet vaccines exist against other flaviviruses. To investigate flavivirus cross-reactivity, sera from a human cohort with a history of vaccination against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV) were tested for antibodies by plaque reduction neutralization test. Neutralization of louping ill virus (LIV) occurred, but no significant neutralization of Murray Valley encephalitis virus was observed. Sera from some individuals vaccinated against TBEV and JEV neutralized WNV, which was enhanced by YFV vaccination in some recipients. Similarly, some individuals neutralized DENV-2, but this was not significantly influenced by YFV vaccination. Antigenic cartography techniques were used to generate a geometric illustration of the neutralization titres of selected sera against WNV, TBEV, JEV, LIV, YFV and DENV-2. This demonstrated the individual variation in antibody responses. Most sera had detectable titres against LIV and some had titres against WNV and DENV-2. Generally, LIV titres were similar to titres against TBEV, confirming the close antigenic relationship between TBEV and LIV. JEV was also antigenically closer to TBEV than WNV, using these sera. The use of sera from individuals vaccinated against multiple pathogens is unique relative to previous applications of antigenic cartography techniques. It is evident from these data that notable differences exist between amino acid sequence identity and mapped antigenic relationships within the family Flaviviridae.
Project description:The explosive spread of Zika virus (ZIKV) and associated complications in flavivirus-endemic regions underscore the need for sensitive and specific serodiagnostic tests to distinguish ZIKV, dengue virus (DENV) and other flavivirus infections. Compared with traditional envelope protein-based assays, several nonstructural protein 1 (NS1)-based assays showed improved specificity, however, none can detect and discriminate three flaviviruses in a single assay. Moreover, secondary DENV infection and ZIKV infection with previous DENV infection, both common in endemic regions, cannot be discriminated. In this study, we developed a high-throughput and multiplex IgG microsphere immunoassay (MIA) using the NS1 proteins of DENV1-DENV4, ZIKV and West Nile virus (WNV) to test samples from reverse-transcription-polymerase-chain reaction-confirmed cases, including primary DENV1, DENV2, DENV3, WNV and ZIKV infections, secondary DENV infection, and ZIKV infection with previous DENV infection. Combination of four DENV NS1 IgG MIAs revealed a sensitivity of 94.3% and specificity of 97.2% to detect DENV infection. The ZIKV and WNV NS1 IgG MIAs had a sensitivity/specificity of 100%/87.9% and 86.1%/78.4%, respectively. A positive correlation was found between the readouts of enzyme-linked immunosorbent assay and MIA for different NS1 tested. Based on the ratio of relative median fluorescence intensity of ZIKV NS1 to DENV1 NS1, the IgG MIA can distinguish ZIKV infection with previous DENV infection and secondary DENV infection with a sensitivity of 88.9-90.0% and specificity of 91.7-100.0%. The multiplex and high-throughput assay could be applied to serodiagnosis and serosurveillance of DENV, ZIKV and WNV infections in endemic regions.
Project description:Mosquito-borne flaviviruses, including yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV), profoundly affect human health. The successful transmission of these viruses to a human host depends on the pathogen's ability to overcome a potentially sterilizing immune response in the vector mosquito. Similar to other invertebrate animals and plants, the mosquito's RNA silencing pathway comprises its primary antiviral defense. Although a diverse range of plant and insect viruses has been found to encode suppressors of RNA silencing, the mechanisms by which flaviviruses antagonize antiviral small RNA pathways in disease vectors are unknown. Here we describe a viral suppressor of RNA silencing (VSR) encoded by the prototype flavivirus, YFV. We show that the YFV capsid (YFC) protein inhibits RNA silencing in the mosquito Aedes aegypti by interfering with Dicer. This VSR activity appears to be broadly conserved in the C proteins of other medically important flaviviruses, including that of ZIKV. These results suggest that a molecular "arms race" between vector and pathogen underlies the continued existence of flaviviruses in nature.
Project description:UNLABELLED:Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat due in part to its association with congenital abnormalities. Other globally relevant flaviviruses include dengue virus (DENV) and West Nile virus (WNV). High-resolution structures of ZIKV reveal many similarities to DENV and suggest some differences, including an extended glycan loop (D. Sirohi, Z. Chen, L. Sun, T. Klose, T. C. Pierson, et al., 352:467-470, 2016, http://dx.doi.org/10.1126/science.aaf5316) and unique interactions among envelope (E) protein residues that were proposed to confer increased virion stability and contribute mechanistically to the distinctive pathobiology of ZIKV (V. A. Kostyuchenko, E. X. Lim, S. Zhang, G. Fibriansah, T. S. Ng, et al., Nature 533:425-428, 2016, http://dx.doi.org/10.1038/nature17994). However, in the latter study, virus stability was inferred by measuring the loss of infectivity following a short incubation period. Here, we rigorously assessed the relative stability of ZIKV, DENV, and WNV by measuring changes in infectivity following prolonged incubation at physiological temperatures. At 37°C, the half-life of ZIKV was approximately twice as long as the half-life of DENV (11.8 and 5.2 h, respectively) but shorter than that of WNV (17.7 h). Incubation at 40°C accelerated the loss of ZIKV infectivity. Increasing virion maturation efficiency modestly increased ZIKV stability, as observed previously with WNV and DENV. Finally, mutations at E residues predicted to confer increased stability to ZIKV did not affect virion half-life. Our results demonstrate that ZIKV is not uniquely stable relative to other flaviviruses, suggesting that its unique pathobiology is explained by an alternative mechanism. IMPORTANCE:Zika virus (ZIKV) belongs to the Flavivirus genus, which includes other clinically relevant mosquito-borne pathogens such as dengue virus (DENV) and West Nile virus (WNV). Historically, ZIKV infection was characterized by a self-limiting, mild disease, but recent outbreaks have been associated with severe clinical complications, including Guillain-Barré syndrome and microcephaly, which are atypical of other flavivirus infections. Moreover, ZIKV has been detected in saliva, urine, and semen, and it may be sexually transmitted. Analysis of a high-resolution cryo-electron microscopic reconstruction of ZIKV hypothesized that the unusual stability of this virus contributes to its distinctive pathobiology. Here, we directly compared the stability of ZIKV to that of other flaviviruses following prolonged incubation in solution at physiological temperatures. We found that the stability of multiple ZIKV strains, including those from recent outbreaks, is intermediate between that of DENV and WNV, suggesting an alternative explanation for the unique clinical manifestations of ZIKV infection.