A virus-type specific serological diagnosis of flavivirus infection using virus-like particles

Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members, plaque reduction neutralization test (PRNT) is the only available assay to determine the infecting flavivirus type.Since PRNT requires culturing raw viruses, it must be performed in biosafety level-3 or level-4 containment for many flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed flavivirus viral-like particles (VLPs) that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps: (ⅰ) VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h; (ⅱ)the neutralized VLPs are used to infect Vero cells; and (ⅲ) the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen (as quantified by the luciferase activities) is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the "gold standard" of the classic PRNT; importantly, it shortens the assay time from ＞10 days to ＜1 day, and can be performed in biosafety level-2 facility.     

Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections

Dengue virus (DENV) is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV), Yellow Fever virus (YFV) or Tick-borne encephalitis virus (TBEV). Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL) domain in the viral envelope (E) protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity.     

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.     

Vector Competence in West African Aedes aegypti Is Flavivirus Species and Genotype Dependent

Background

Vector competence of Aedes aegypti mosquitoes is a quantitative genetic trait that varies among geographic locations and among different flavivirus species and genotypes within species. The subspecies Ae. aegypti formosus, found mostly in sub-Saharan Africa, is considered to be refractory to both dengue (DENV) and yellow fever viruses (YFV) compared to the more globally distributed Ae. aegypti aegypti. Within Senegal, vector competence varies with collection site and DENV-2 viral isolate, but knowledge about the interaction of West African Ae. aegypti with different flaviviruses is lacking. The current study utilizes low passage isolates of dengue-2 (DENV-2-75505 sylvatic genotype) and yellow fever (YFV BA-55 -West African Genotype I, or YFV DAK 1279-West African Genotype II) from West Africa and field derived Ae. aegypti collected throughout Senegal to determine whether vector competence is flavivirus or virus genotype dependent.

Methodology/Principal Findings

Eight collections of 20–30 mosquitoes from different sites were fed a bloodmeal containing either DENV-2 or either isolate of YFV. Midgut and disseminated infection phenotypes were determined 14 days post infection. Collections varied significantly in the rate and intensity of midgut and disseminated infection among the three viruses.

Conclusions/Significance

Overall, vector competence was dependent upon both viral and vector strains. Importantly, contrary to previous studies, sylvatic collections of Ae. aegypti showed high levels of disseminated infection for local isolates of both DENV-2 and YFV.     

Protein Kinase G Phosphorylates Mosquito-Borne Flavivirus NS5

Serine/threonine phosphorylation of the nonstructural protein 5 (NS5) is a conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to compare the phosphorylation sites of the NS5 proteins of yellow fever virus (YFV) and dengue virus (DENV), two flaviviruses transmitted by mosquitoes. Seven DENV phosphopeptides were identified, but only one conserved phosphoacceptor site (threonine 449 in DENV) was identified in both viruses. This site is predicted to be a protein kinase G (PKG) recognition site and is a strictly conserved serine/threonine phosphoacceptor site in mosquito-borne flaviviruses. In contrast, in tick-borne flaviviruses, this residue is typically a histidine. A DENV replicon engineered to have the tick-specific histidine residue at this position is replication defective. We show that DENV NS5 purified from Escherichia coli is a substrate for PKG in vitro and facilitates the autophosphorylation of PKG as seen with cellular substrates. Phosphorylation in vitro by PKG also occurs at threonine 449. Activators and inhibitors of PKG modulate DENV replication in cell culture but not replication of the tick-borne langat virus. Collectively, these data argue that PKG mediates a conserved serine/threonine phosphorylation event specifically for flaviviruses spread by mosquitoes.The flavivirus genus contains many medically important species, including dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV). More than 2 billion people are at risk of infection by DENV alone, leading to an estimated 50 million cases annually, which may increase further as the range of the mosquito vector expands with urbanization (24). While disease from mosquito-borne flaviviruses is particularly common, there are other flaviviral human pathogens that exist with transmission cycles that do not involve mosquitoes. Tick-borne transmission is the other well-described route, but non-arthropod-borne routes also exist (for example, bats). It is likely that each transmission route has genetic adaptations that facilitate that route, but such changes are not yet understood (7).Serine/threonine phosphorylation is a conserved feature across all three genera of the family Flaviviridae, including the genus flavivirus (the others genera being pestivirus and hepacivirus). Among the features of Flaviviridae, the most-studied examples are the multiple phosphorylations of nonstructural protein 5A (NS5A) of hepatitis C virus, which exists in both basal (termed p56) and hyperphosphorylated (termed p58) states mediated by multiple kinases that both are necessary for and limit replication (14, 18, 23). Phosphorylation of NS5B, the RNA-dependent RNA polymerase (RdRP), has also been shown to affect replicon activity (10). In the genus flavivirus, several mosquito-borne viruses (DENV, WNV, and YFV) and at least one tick-borne encephalitis virus are known to have phosphorylated forms of nonstructural protein NS5 (2, 9, 11, 13, 19). In the genus flavivirus, NS5 is central to viral replication, as it possesses both RdRP and methyltransferase activities. DENV phosphorylation of NS5 correlates with the loss of NS5 interactions with the viral helicase NS3. A hyperphosphorylated form of NS5 was found to localize to the nucleus, away from the cytoplasmic sites of viral replication (6, 9). A nuclear localization sequence is present in DENV NS5 and is phosphorylated in vitro by host CKII, but the relationship between phosphorylation and nuclear localization has yet to be fully elucidated (17). Multiple different serine/threonine phosphorylation events likely occur in the flaviviral life cycle, potentially affecting various functions of NS5 (2), but the role of these events and identity of the kinase(s) responsible are largely unknown.In this report, we used mass spectrometry to identify serine/threonine phosphorylation sites in DENV. A single phosphoacceptor site, previously identified in YFV, is conserved specifically in the mosquito-borne flaviviruses but not the tick-borne flaviviruses. Furthermore, in vitro studies reveal that this site is phosphorylated by a cyclic-nucleotide-dependent kinase, protein kinase G (PKG), and a phosphoacceptor threonine/serine is required for replication. Taken together, these data implicate the PKG pathway in flaviviral replication for the first time and suggest a host cell pathway that could be targeted by antiviral therapy.     

Noncoding Flavivirus RNA Displays RNA Interference Suppressor Activity in Insect and Mammalian Cells

West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3′-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses.     

Appraising the roles of CBLL1 and the ubiquitin/proteasome system for flavivirus entry and replication

The ubiquitin ligase CBLL1 (also known as HAKAI) has been proposed to be a critical cellular factor exploited by West Nile virus (WNV) for productive infection. CBLL1 has emerged as a major hit in a recent RNA interference screen designed to identify cellular factors required for the early stages of the WNV life cycle. Follow-up experiments showed that HeLa cells knocked down for CBLL1 by a small interfering RNA (siRNA) failed to internalize WNV particles and resisted infection. Furthermore, depletion of a free-ubiquitin pool by the proteasome inhibitor MG132 abolished WNV endocytosis, suggesting that CBLL1 acts in concert with the ubiquitin proteasome system to mediate virus internalization. Here, we examined the effect of CBLL1 knockdown and proteasome inhibitors on infection by WNV and other flaviviruses. We identified new siRNAs that repress the CBLL1 protein and strongly inhibit the endocytosis of Listeria monocytogenes, a bacterial pathogen known to require CBLL1 to invade host cells. Strikingly, however, we detected efficient WNV, dengue virus, and yellow fever virus infection of human cells, despite potent downregulation of CBLL1 by RNA interference. In addition, we found that the proteasome inhibitors MG132 and lactacystin did not affect WNV internalization but strongly repressed flavivirus RNA translation and replication. Together, these data do not support a requirement for CBLL1 during flavivirus entry and rather suggest an essential role of the ubiquitin/proteasome pathway for flavivirus genome amplification.     

A broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of E protein

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.     

Bicluster pattern of codon context usages between flavivirus and vector mosquito Aedes aegypti: relevance to infection and transcriptional response of mosquito genes

The mosquito Aedes aegypti is the primary vector of dengue virus (DENV) infection in most of the subtropical and tropical countries. Besides DENV, yellow fever virus (YFV) is also transmitted by A. aegypti. Susceptibility of A. aegypti to West Nile virus (WNV) has also been confirmed. Although studies have indicated correlation of codon bias between flaviviridae and their animal/insect hosts, it is not clear if codon sequences have any relation to susceptibility of A. aegypti to DENV, YFV and WNV. In the current study, usages of codon context sequences (codon pairs for neighboring amino acids) of the vector (A. aegypti) genome as well as the flaviviral genomes are investigated. We used bioinformatics methods to quantify codon context bias in a genome-wide manner of A. aegypti as well as DENV, WNV and YFV sequences. Mutual information statistics was applied to perform bicluster analysis of codon context bias between vector and flaviviral sequences. Functional relevance of the bicluster pattern was inferred from published microarray data. Our study shows that codon context bias of DENV, WNV and YFV sequences varies in a bicluster manner with that of specific sets of genes of A. aegypti. Many of these mosquito genes are known to be differentially expressed in response to flaviviral infection suggesting that codon context sequences of A. aegypti and the flaviviruses may play a role in the susceptible interaction between flaviviruses and this mosquito. The bias in usages of codon context sequences likely has a functional association with susceptibility of A. aegypti to flaviviral infection. The results from this study will allow us to conduct hypothesis-driven tests to examine the role of codon context bias in evolution of vector–virus interactions at the molecular level.     

Research advances in plant-made flavivirus antigens

Outbreaks of flaviviruses such as dengue (DV), yellow fever (YFV), Japanese encephalitis (JEV), tick-borne encephalitis (TBEV) and West Nile (WNV) affect numerous countries around the world. The fast spread of these viruses is the result of increases in the human population, rapid urbanisation and globalisation. While vector control is an important preventive measure against vector-borne diseases, it has failed to prevent the spread of these diseases, particularly in developing countries where the implementation of control measures is intermittent. As antiviral drugs against flaviviruses are not yet available, vaccination remains the most important tool for prevention. Although human vaccines for YFV, TBEV and JEV are available, on-going vaccination efforts are insufficient to prevent infection. No vaccines against DENV and WNV are available. Research advances have provided important tools for flavivirus vaccine development, such as the use of plants as a recombinant antigen production platform. This review summarises the research efforts in this area and highlights why a plant system is considered a necessary alternative production platform for high-tech subunit vaccines.     

The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production

Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter₁₈) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter₁₈ and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter₁₈, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization.     

An Integrated Systems Biology Approach Identifies the Proteasome as A Critical Host Machinery for ZIKV and DENV Replication

The Zika virus (ZIKV) and dengue virus (DENV) flaviviruses exhibit similar replicative processes but have distinct clinical outcomes. A systematic understanding of virus–host protein–pro-tein interaction networks can reveal cellular pathways critical to viral replication and disease patho-genesis. Here we employed three independent systems biology approaches toward this goal. First, protein array analysis of direct interactions between individual ZIKV/DENV viral proteins and 20,240 human proteins revealed multiple conserved cellular pathways and protein complexes, including proteasome complexes. Second, an RNAi screen of 10,415 druggable genes identified the host proteins required for ZIKV infection and uncovered that proteasome proteins were crucial in this process. Third, high-throughput screening of 6016 bioactive compounds for ZIKV inhibition yielded 134 effective compounds, including six proteasome inhibitors that suppress both ZIKV and DENV replication. Integrative analyses of these orthogonal datasets pinpoint proteasomes as crit-ical host machinery for ZIKV/DENV replication. Our study provides multi-omics datasets for fur-ther studies of flavivirus–host interactions, disease pathogenesis, and new drug targets.     

Stability of yellow fever virus under recombinatory pressure as compared with chikungunya virus

Recombination is a mechanism whereby positive sense single stranded RNA viruses exchange segments of genetic information. Recent phylogenetic analyses of naturally occurring recombinant flaviviruses have raised concerns regarding the potential for the emergence of virulent recombinants either post-vaccination or following co-infection with two distinct wild-type viruses. To characterize the conditions and sequences that favor RNA arthropod-borne virus recombination we constructed yellow fever virus (YFV) 17D recombinant crosses containing complementary deletions in the envelope protein coding sequence. These constructs were designed to strongly favor recombination, and the detection conditions were optimized to achieve high sensitivity recovery of putative recombinants. Full length recombinant YFV 17D virus was never detected under any of the experimental conditions examined, despite achieving estimated YFV replicon co-infection levels of ∼2.4×10⁶ in BHK-21 (vertebrate) cells and ∼1.05×10⁵ in C₇10 (arthropod) cells. Additionally YFV 17D superinfection resistance was observed in vertebrate and arthropod cells harboring a primary infection with wild-type YFV Asibi strain. Furthermore recombination potential was also evaluated using similarly designed chikungunya virus (CHIKV) replicons towards validation of this strategy for recombination detection. Non-homologus recombination was observed for CHIKV within the structural gene coding sequence resulting in an in-frame duplication of capsid and E3 gene. Based on these data, it is concluded that even in the unlikely event of a high level acute co-infection of two distinct YFV genomes in an arthropod or vertebrate host, the generation of viable flavivirus recombinants is extremely unlikely.     

Inhibition of japanese encephalitis virus infection by flavivirus recombinant e protein domain III

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therapies for all of the flavivirus and only a few highly effective vaccines are licensed for human use. In this paper, the E protein domain III (DIII) of six heterologous flaviviruses (DENV1-4, WNV and JEV) was expressed in Escherichia coli successfully. The proteins were purified after a solubilization and refolding procedure, characterized by SDS-PAGE and Western blotting. Competitive inhibition showed that all recombinant flavivirus DIII proteins blocked the entry of JEV into BHK-21 cells. Further studies indicated that antibodies induced by the soluble recombinant flavivirus DIII partially protected mice against lethal JEV challenge. These results demonstrated that recombinant flavivirus DIII proteins could inhibit JEV infection competitively, and immunization with proper folding flavivirus DIII induced cross-protection against JEV infection in mice, implying a possible role of DIII for the cross-protection among flavivirus as well as its use in antigens for immunization in animal models.     

Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule.     

Seroprevalence of arboviruses in Ecuador: Implications for improved surveillance

Introduction:Arthropod-borne viruses (arboviruses) cause morbidity and mortality in humans and domestic animals worldwide. The percentage of population immunity or susceptibility to these viruses in Ecuador is unknown.Objectives:To investigate the proportion of Ecuadorian populations with IgG antibodies (Abs) (past exposure/immunity) and IgM Abs (current exposure) against flaviviruses and alphaviruses and to study the activity of these viruses in Ecuador.Materials and methods:During 2009-2011, we conducted a serosurvey for selected arboviruses in humans (n=1,842), equines (n=149), and sentinel hamsters (n=84) at two coastal locations and one in the Amazon basin (Eastern Ecuador) using enzyme-linked immunosorbent assay and hemagglutination inhibition test.Results:From 20.63% to 63.61% of humans showed IgG-antibodies for the flaviviruses: Dengue virus (DENV), yellow fever virus (YFV) Saint Louis encephalitis virus, and West Nile virus (WNV); from 4.67% to 8.63% showed IgG-Abs for the alphaviruses: Venezuelanequine encephalitis virus, eastern equine encephalitis virus, and western equine encephalitis virus. IgM-Abs were found for DENV and WNV. Equines and hamsters showed antibodies to alphaviruses in all locations; two hamsters seroconverted to YFV in the Amazonia.Conclusions:The results show a YFV vaccination history and suggest the activity of arboviruses not included in the current surveillance scheme. Enhanced arbovirus and mosquito surveillance, as well as continued YFV vaccination and evaluation of its coverage/ effectiveness, are recommended.     

Production of pseudoinfectious yellow fever virus with a two-component genome

Application of genetically modified, deficient-in-replication flaviviruses that are incapable of developing productive, spreading infection is a promising means of designing safe and effective vaccines. Here we describe a two-component genome yellow fever virus (YFV) replication system in which each of the genomes encodes complete sets of nonstructural proteins that form the replication complex but expresses either only capsid or prM/E instead of the entire structural polyprotein. Upon delivery to the same cell, these genomes produce together all of the viral structural proteins, and cells release a combination of virions with both types of genomes packaged into separate particles. In tissue culture, this modified YFV can be further passaged at an escalating scale by using a high multiplicity of infection (MOI). However, at a low MOI, only one of the genomes is delivered into the cells, and infection cannot spread. The replicating prM/E-encoding genome produces extracellular E protein in the form of secreted subviral particles that are known to be an effective immunogen. The presented strategy of developing viruses defective in replication might be applied to other flaviviruses, and these two-component genome viruses can be useful for diagnostic or vaccine applications, including the delivery and expression of heterologous genes. In addition, the achieved separation of the capsid-coding sequence and the cyclization signal in the YFV genome provides a new means for studying the mechanism of the flavivirus packaging process.