A synthetic peptide to the E glycoprotein of Murray Valley encephalitis virus defines multiple virus-reactive T- and B-cell epitopes.

Synthetic peptides from the envelope glycoprotein sequence of Murray Valley encephalitis (MVE) virus were previously evaluated in various strains of mice for both the induction of antibody and the in vitro proliferation of peptide-primed T-helper (Th) cells. MVE peptide 6 (amino acids 230 to 251) elicited reciprocal Th- and B-cell reactivity with native MVE virus after primary inoculation of C57BL/6 mice. In this study, we prepared overlapping subunit peptides of MVE peptide 6 and evaluated their immunogenicity. Analysis of these peptides delineated at least two B-cell epitopes that induced antibody reactive with MVE and other Japanese encephalitis serocomplex viruses. This antibody at low titer neutralized MVE virus. Genetic restriction of the antibody response to various T-cell elements within peptide 6 was observed in C3H, BALB/c, C57BL/6, and B10 congenic mice. One element demonstrable after primary immunization, located in the carboxy terminus, associated only with major histocompatibility complex class II IAb and IAbiEk glycoproteins. Functional stimulation with the peptides in association with IAkIEk and IAdIEd molecules was observed only after in vivo secondary stimulation. Peptide 6-1 (amino acids 230 to 241) was nonimmunogenic but could be recognized by Th cells from peptide 6-immunized mice. Further association of peptide 6 with the IAkIEk and IAdIEd subregions was demonstrated by the finding that T cells from MVE peptide 6-inoculated C3H and BALB/c mice primed for an antibody response to MVE virus. These results suggest that the peptide 6 sequence, which is relatively conserved among a number of flaviviruses, should be given consideration when synthetic immunogens for vaccine purposes are designed.     

Mechanism of virulence attenuation of glycosaminoglycan-binding variants of Japanese encephalitis virus and Murray Valley encephalitis virus

The in vivo mechanism for virulence attenuation of laboratory-derived variants of two flaviviruses in the Japanese encephalitis virus (JEV) serocomplex is described. Host cell adaptation of JEV and Murray Valley encephalitis virus (MVE) by serial passage in adenocarcinoma cells selected for variants characterized by (i) a small plaque phenotype, (ii) increased affinity to heparin-Sepharose, (iii) enhanced susceptibility to inhibition of infectivity by heparin, and (iv) loss of neuroinvasiveness in a mouse model for flaviviral encephalitis. We previously suggested that virulence attenuation of the host cell-adapted variants of MVE is a consequence of their increased dependence on cell surface glycosaminoglycans (GAGs) for attachment and entry (E. Lee and M. Lobigs, J. Virol. 74:8867-8875, 2000). In support of this proposition, we find that GAG-binding variants of JEV and MVE were rapidly removed from the bloodstream and failed to spread from extraneural sites of replication into the brain. Thus, the enhanced affinity of the attenuated variants for GAGs ubiquitously present on cells and extracellular matrices most likely prevented viremia of sufficient magnitude and/or duration required for virus entry into the brain parenchyma. This mechanism may also account, in part, for the attenuation of the JEV SA14-14-2 vaccine, given the sensitivity of the virus to heparin inhibition. A pronounced loss of the capacity of the GAG-binding variants to produce disease was also noted in mice defective in the alpha/beta interferon response, a mouse strain shown here to be highly susceptible to infection with JEV serocomplex flaviviruses. Despite the close genetic relatedness of JEV and MVE, the variants selected for the two viruses were altered at different residues in the envelope (E) protein, viz., Glu(306) and Asp(390) for JEV and MVE, respectively. In both cases the substitutions gave the protein an increased net positive charge. The close spatial proximity of amino acids 306 and 390 in the predicted E protein structure strongly suggests that the two residues define a receptor-binding domain involved in virus attachment to sulfated proteoglycans.     

High clonality of virus-specific T lymphocytes defined by TCR usage in the brains of mice infected with West Nile virus

It has been reported that brain-infiltrating T lymphocytes play critical roles in the clearance of West Nile virus (WNV) from the brains of mice. We characterized brain-infiltrating T lymphocytes by analyzing the TCR α- and β-chain repertoires, T cell clonality, and CDR3 sequences. CD3(+)CD8(+) T cells were localized in the WNV-infected brains. The expression of CD3, CD8, CD25, CD69, perforin, and granzymes positively correlated with viral RNA levels, and high levels of expression of IFN-γ, TNF-α, and IL-2 were detected in the brains, suggesting that Th1-like cytotoxic CD8(+) T cells are expanded in the brains in response to WNV infection. The brain-infiltrating T lymphocytes dominantly used TCR genes, VA1-1, VA2-1, VB5-2, and VB8-2, and exhibited a highly oligoclonal TCR repertoire. Interestingly, the brain-infiltrating T lymphocytes had different patterns of TCR repertoire usages among WNV-, Japanese encephalitis virus-, and tick-borne encephalitis virus-infected mice. Moreover, CD8(+) T cells isolated from the brains of WNV-infected mice produced IFN-γ and TNF-α after in vitro stimulation with peritoneal cells infected with WNV, but not with Japanese encephalitis virus. The results suggest that the infiltrating CD8(+) T cells were WNV-specific, but not cross-reactive among flaviviruses. T cells from the WNV-infected brains exhibited identical or similar CDR3 sequences in TCRα among tested mice, but somewhat diverse sequences in TCRβ. The results indicate that WNV-specific CD3(+)CD8(+) T cells expanding in the infected brains are highly oligoclonal, and they suggest that TCR α-chains play a dominant and critical role in Ag specificity of WNV-specific T cells.     

Serological evidence of inter-epidemic infection of feral pigs in New South Wales with Murray Valley encephalitis virus.

The sera of 617 feral pigs, collected from three widely separated areas of northern and central New South Wales, were examined for antibody to Murray Valley encephalitis (MVE) virus and to Ross River virus. Haemagglutination-inhibition (HI) antibody was detected to MVE in 58% of sera and to Ross River virus in 15% of sera. Neutralization tests suggested that the MVE HI antibody resulted from infection with MVE virus in the summers of 1971-1972 and 1972-1973 when the virus was not known to be active in New South Wales. These same tests suggested that more than one flavivirus infected the feral pigs in the summer of 1973-1974 and that Kunjin virus was active in the summer of 1975-1976.     

Antiviral cytotoxic T cells cross-reactively recognize disparate peptide determinants from related viruses but ignore more similar self- and foreign determinants

We have investigated the reactivities of cytotoxic T (Tc) cells against the two immunodominant, H-2K(k)-restricted determinants from the FLAVIVIRUS: Murray Valley encephalitis virus (MVE), MVE(1785) (REHSGNEI) and MVE(1971) (DEGEGRVI). The respective Tc cell populations cross-reactively lysed target cells pulsed with determinants from the MVE(1785)- and MVE(1971)-corresponding positions of six other flaviviruses, despite low sequence homology in some cases. Notably, anti-MVE(1785) Tc cells recognized a determinant (TDGEERVI) that shares with the determinant used for stimulation only the carboxyl-terminal amino acid residue, one of two H-2K(k) anchor residues. These reactivity patterns were also observed in peptide-dependent IFN-gamma production and the requirements for in vitro restimulation of memory Tc cells. However, the broad cross-reactivity appeared to be limited to flavivirus-derived determinants, as none of a range of determinants from endogenous mouse-derived sequences, similar to the MVE-determinants, were recognized. Neither were cells infected with a number of unrelated viruses recognized. These results raise the paradox that virus-immune Tc cell responses, which are mostly directed against only a few "immunodominant" viral determinants, are remarkably peptide cross-reactive.     

Mosquito saliva causes enhancement of West Nile virus infection in mice

West Nile virus (WNV) is transmitted to vertebrate hosts primarily by infected Culex mosquitoes. Transmission of arboviruses by the bite of infected mosquitoes can potentiate infection in hosts compared to viral infection by needle inoculation. Here we examined the effect of mosquito transmission on WNV infection and systematically investigated multiple factors that differ between mosquito infection and needle inoculation of WNV. We found that mice infected with WNV through the bite of a single infected Culex tarsalis mosquito exhibited 5- to 10-fold-higher viremia and tissue titers at 24 and 48 h postinoculation and faster neuroinvasion than mice given a median mosquito-inoculated dose of WNV (10(5) PFU) by needle. Mosquito-induced enhancement was not due to differences in inoculation location, because additional intravenous inoculation of WNV did not enhance viremia or tissue titers. Inoculation of WNV into a location where uninfected mosquitoes had fed resulted in enhanced viremia and tissue titers in mice similar to those in mice infected by a single infected mosquito bite, suggesting that differences in where virus is deposited in the skin and in the virus particle itself were not responsible for the enhanced early infection in mosquito-infected mice. In addition, inoculation of mice with WNV mixed with salivary gland extract (SGE) led to higher viremia, demonstrating that mosquito saliva is the major cause of mosquito-induced enhancement. Enhanced viremia was not observed when SGE was inoculated at a distal site, suggesting that SGE enhances WNV replication by exerting a local effect. Furthermore, enhancement of WNV infection still occurred in mice with antibodies against mosquito saliva. In conclusion, saliva from C. tarsalis is responsible for enhancement of early WNV infection in vertebrate hosts.     

Substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry

The flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (FG loop) in the envelope (E) protein. In some flaviviruses this domain harbors the integrin-binding motif Arg-Gly-Asp (RGD). One of us has shown earlier that host cell adaptation of Murray Valley encephalitis virus (MVE) can result in the selection of attenuated variants altered at E protein residue Asp(390), which is part of an RGD motif. Here, a full-length, infectious cDNA clone of MVE was constructed and employed to systematically investigate the impact of single amino acid changes at Asp(390) on cell tropism, virus entry, and virulence. Each of 10 different E protein 390 mutants was viable. Three mutants (Gly(390), Ala(390), and His(390)) showed pronounced differences from an infectious clone-derived control virus in growth in mammalian and mosquito cells. The altered cell tropism correlated with (i) a difference in entry kinetics, (ii) an increased dependence on glycosaminoglycans (determined by inhibition of virus infectivity by heparin) for attachment of the three mutants to different mammalian cells, and (iii) the loss of virulence in mice. These results confirm a functional role of the FG loop in the flavivirus E protein in virus entry and suggest that encephalitic flaviviruses can enter cells via attachment to glycosaminoglycans. However, it appears that additional cell surface molecules are also used as receptors by natural isolates of MVE and that the increased dependence on glycosaminoglycans for entry results in the loss of neuroinvasiveness.     

Lack of both Fas ligand and perforin protects from flavivirus-mediated encephalitis in mice

The mechanism by which encephalitic flaviviruses enter the brain to inflict a life-threatening encephalomyelitis in a small percentage of infected individuals is obscure. We investigated this issue in a mouse model for flavivirus encephalitis in which the virus was administered to 6-week-old animals by the intravenous route, analogous to the portal of entry in natural infections, using a virus dose in the range experienced following the bite of an infectious mosquito. In this model, infection with 0.1 to 10(5) PFU of virus gave mortality in approximately 50% of animals despite low or undetectable virus growth in extraneural tissues. We show that the cytolytic effector functions play a crucial role in invasion of the encephalitic flavivirus into the brain. Mice deficient in either the granule exocytosis- or Fas-mediated pathway of cytotoxicity showed delayed and reduced mortality. Mice deficient in both cytotoxic effector functions were resistant to a low-dose peripheral infection with the neurotropic virus.     

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.     

Antiviral peptides targeting the west nile virus envelope protein

West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 muM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 muM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses.     

T-helper cell and associated antibody response to synthetic peptides of the E glycoprotein of Murray Valley encephalitis virus.

A battery of 16 synthetic peptides, selected primarily by computer analysis for predicted B- and T-cell epitopes, was prepared from the deduced amino acid sequence of the envelope (E) glycoprotein of Murray Valley encephalitis (MVE) virus. We examined all of the peptides for T-helper (Th)-cell recognition and antibody induction in three strains of mice: C57BL/6, BALB/c, and C3H. Lymphoproliferative and interleukin-2 assays were performed on splenic T cells from mice inoculated with peptides in Freund's incomplete adjuvant or with MVE virus. Several peptides found to contain predicted T-cell epitopes elicited a Th-cell response in at least one strain of mice, usually with a concomitant antibody response. Peptides 145 (amino acids 145 to 169) and 17 (amino acids 356 to 376) were strongly recognized by T cells from all three inbred strains of mice. Peptide 06 (amino acids 230 to 251) primed C57BL/6 mice for Th- and B-cell reactivity with native MVE virus, and T cells from virus-immune mice were stimulated by this peptide. Peptide 06 was recognized by several Th-cell clones prepared from mice immunized with MVE, West Nile, or Kunjin virus. These results indicate that it may be feasible to design synthetic flavivirus peptides that define T-cell epitopes capable of generating a helper cell response for B-cell epitopes involved in protective immunity.     

Serological Evidence of Widespread Circulation of West Nile Virus and Other Flaviviruses in Equines of the Pantanal,Brazil

A recent study reported neutralizing antibodies to West Nile virus (WNV) in horses from four ranches of southern Pantanal. To extend that study, a serosurvey for WNV and 11 Brazilian flaviviruses was conducted with 760 equines, 238 sheep and 61 caimans from 17 local cattle ranches. Among the tested equines, 32 were collected from a ranch where a neurologic disorder outbreak had been recently reported. The sera were initially screened by using a blocking ELISA and then titrated by 90% plaque-reduction neutralization test (PRNT₉₀) for 12 flaviviruses. Employing the criterion of 4-fold greater titer, 78 (10.3%) equines were seropositive for Ilheus virus, 59 (7.8%) for Saint Louis encephalitis virus, 24 (3.2%) for WNV, two (0.3%) for Cacipacore virus and one (0.1%) for Rocio virus. No serological evidence was found linking the neurological disease that affected local equines to WNV. All caimans and sheep were negative by blocking ELISA for flaviviruses. There were no seropositive equines for Bussuquara, Iguape, Yellow fever and all four Dengue virus serotypes. The detection of WNV-seropositive equines in ten ranches and ILHV and SLEV-seropositive equines in fourteen ranches of two different sub-regions of Pantanal is strong evidence of widespread circulation of these flaviviruses in the region.     

Reconciling West Nile virus with the autophagic pathway

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus responsible for recurrent outbreaks of meningitis and encephalitis. Several studies analyzing the interactions of this pathogen with the autophagic pathway have reported opposite results with evidence for and against the upregulation of autophagy in infected cells. In this regard, we have recently reported that minimal genetic changes (single amino acid substitutions) in nonstructural proteins of WNV can modify the ability of the virus to induce autophagic features such as LC3 modification and aggregation in infected cells. We think that these results could help explain some of the previously reported discrepancies. These findings could also aid in deciphering the interactions of this pathogen with the autophagic pathway at the molecular level aimed to develop feasible antiviral strategies to combat this pathogen, and other related flaviviruses.     

Infection and injury of neurons by West Nile encephalitis virus

West Nile virus (WNV) infects neurons and leads to encephalitis, paralysis, and death in humans, animals, and birds. We investigated the mechanism by which neuronal injury occurs after WNV infection. Neurons in the anterior horn of the spinal cords of paralyzed mice exhibited a high degree of WNV infection, leukocyte infiltration, and degeneration. Because it was difficult to distinguish whether neuronal injury was caused by viral infection or by the immune system response, a novel tissue culture model for WNV infection was established in neurons derived from embryonic stem (ES) cells. Undifferentiated ES cells were relatively resistant to WNV infection. After differentiation, ES cells expressed neural antigens, acquired a neuronal phenotype, and became permissive for WNV infection. Within 48 h of exposure to an exceedingly low multiplicity of infection (5 x 10(-4)), 50% of ES cell-derived neurons became infected, producing nearly 10(7) PFU of infectious virus per ml, and began to die by an apoptotic mechanism. The establishment of a tractable virus infection model in ES cell-derived neurons facilitates the study of the molecular basis of neurotropism and the mechanisms of viral and immune-mediated neuronal injury after infection by WNV or other neurotropic pathogens.     

Susceptibility of mosquito and tick cell lines to infection with various flaviviruses

The genus Flavivirus consists of more than 70 virus species and subtypes, the majority of which are transmitted by mosquitoes or ticks, although some have no known vector (NKV). The ability of these viruses to infect cultured cells derived from mosquito or tick species offers a useful insight into the suitability of such vectors to harbour and replicate particular viruses. We undertook a comparative study of the susceptibility of mammalian Vero cells, a clonal mosquito cell line (C6/36) and recently developed cell lines derived from the ticks (Acari: Ixodidae) Ixodes ricinus (L.) (IRE/CTVM18), I. scapularis (Say) (ISE6), Rhipicephalus appendiculatus (Neumann) (RAE/CTVM1) and Amblyomma variegatum (Fabricius) (AVL/CTVM17) to infection with 13 flaviviruses (and one alphavirus) using immunofluorescence microscopy and plaque assay techniques. The C6/36 mosquito cell line was infected by all the mosquito-borne flaviviruses tested but not by NKV viruses or tick-borne viruses, with the exception of Langat virus (LGTV). The tick cell lines were susceptible to infection by all of the tick-borne viruses tested, as well as two mosquito-borne viruses, West Nile virus (WNV) and the alphavirus, Venezuelan equine encephalitis virus (VEEV), but not other mosquito-borne viruses or NKV viruses.     

Pivotal role of antibody and subsidiary contribution of CD8+ T cells to recovery from infection in a murine model of Japanese encephalitis

The immunological correlates for recovery from primary Japanese encephalitis virus (JEV) infection in humans and experimental animals remain poorly defined. To investigate the relative importance of the adaptive immune responses, we have established a mouse model for Japanese encephalitis in which a low-dose virus inoculum was administered into the footpads of adult C57BL/6 mice. In this model, ~60% of the mice developed a fatal encephalitis and a virus burden in the central nervous system (CNS). Using mice lacking B cells (μMT(-/-) mice) and immune B cell transfer to wild-type mice, we show a critically important role for humoral immunity in preventing virus spread to the CNS. T cell help played an essential part in the maintenance of an effective antibody response necessary to combat the infection, since mice lacking major histocompatibility complex class II showed truncated IgM and blunted IgG responses and uniformly high lethality. JEV infection resulted in extensive CD8(+) T cell activation, judged by upregulation of surface markers CD69 and CD25 and cytokine production after stimulation with a JEV NS4B protein-derived H-2D(b)-binding peptide and trafficking of virus-immune CD8(+) T cells into the CNS. However, no significant effect of CD8(+) T cells on the survival phenotype was found, which was corroborated in knockout mice lacking key effector molecules (Fas receptor, perforin, or granzymes) of cytolytic pathways triggered by T lymphocytes. Accordingly, CD8(+) T cells are mostly dispensable for recovery from infection with JEV. This finding highlights the conflicting role that CD8(+) T cells play in the pathogenesis of JEV and closely related encephalitic flaviviruses such as West Nile virus.     

Fas ligand interactions contribute to CD8+ T-cell-mediated control of West Nile virus infection in the central nervous system

West Nile virus (WNV) is a neurotropic flavivirus that causes encephalitis, most frequently in elderly and immunocompromised humans. Previous studies demonstrated that CD8+ T cells utilize perforin-dependent cytolytic mechanisms to limit WNV infection. Nonetheless, the phenotype of perforin-deficient CD8+ T cells was not as severe as that of an absence of CD8+ T cells, suggesting additional effector control mechanisms. In this study, we evaluated the contribution of Fas-Fas ligand (FasL) interactions to CD8+ T-cell-mediated control of WNV infection. Notably, the cell death receptor Fas was strongly upregulated on neurons in culture and in vivo after WNV infection. gld mice that were functionally deficient in FasL expression showed increased susceptibility to lethal WNV infection. Although antigen-specific priming of CD8+ T cells in peripheral lymphoid tissues was normal in gld mice, increased central nervous system (CNS) viral burdens and delayed clearance were observed. Moreover, the adoptive transfer of WNV-primed wild-type but not gld CD8+ T cells to recipient CD8(-/-) or gld mice efficiently limited infection in the CNS and enhanced survival rates. Overall, our data suggest that CD8+ T cells also utilize FasL effector mechanisms to contain WNV infection in Fas-expressing neurons in the CNS.     

Attenuation of Tick-Borne Encephalitis Virus Using Large-Scale Random Codon Re-encoding

Large-scale codon re-encoding (i.e. introduction of a large number of synonymous mutations) is a novel method of generating attenuated viruses. Here, it was applied to the pathogenic flavivirus, tick-borne encephalitis virus (TBEV) which causes febrile illness and encephalitis in humans in forested regions of Europe and Asia. Using an infectious clone of the Oshima 5–10 strain ("wild-type virus"), a cassette of 1.4kb located in the NS5 coding region, was modified by randomly introducing 273 synonymous mutations ("re-encoded virus"). Whilst the in cellulo replicative fitness of the re-encoded virus was only slightly reduced, the re-encoded virus displayed an attenuated phenotype in a laboratory mouse model of non-lethal encephalitis. Following intra-peritoneal inoculation of either 2.10⁵ or 2.10⁶ TCID50 of virus, the frequency of viraemia, neurovirulence (measured using weight loss and appearance of symptoms) and neuroinvasiveness (detection of virus in the brain) were significantly decreased when compared with the wild-type virus. Mice infected by wild-type or re-encoded viruses produced comparable amounts of neutralising antibodies and results of challenge experiments demonstrated that mice previously infected with the re-encoded virus were protected against subsequent infection by the wild-type virus. This constitutes evidence that a mammalian species can be protected against infection by a virulent wild-type positive-stranded RNA virus following immunisation with a derived randomly re-encoded strain. Our results demonstrate that random codon re-encoding is potentially a simple and effective method of generating live-attenuated vaccine candidates against pathogenic flaviviruses.     

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.     

A thiopurine drug inhibits West Nile virus production in cell culture, but not in mice

Many viruses within the Flavivirus genus cause significant disease in humans; however, effective antivirals against these viruses are not currently available. We have previously shown that a thiopurine drug, 6-methylmercaptopurine riboside (6MMPr), inhibits replication of distantly related viruses within the Flaviviridae family in cell culture, including bovine viral diarrhea virus and hepatitis C virus replicon. Here we further examined the potential antiviral effect of 6MMPr on several diverse flaviviruses. In cell culture, 6MMPr inhibited virus production of yellow fever virus, dengue virus-2 (DENV-2) and West Nile virus (WNV) in a dose-dependent manner, and DENV-2 was significantly more sensitive to 6MMPr treatment than WNV. We then explored the use of 6MMPr as an antiviral against WNV in an immunocompetent mouse model. Once a day treatment of mice with 0.5 mg 6MMPr was just below the toxic dose in our mouse model, and this dose was used in subsequent studies. Mice were treated with 6MMPr immediately after subcutaneous inoculation with WNV for eight consecutive days. Treatment with 6MMPr exacerbated weight loss in WNV-inoculated mice and did not significantly affect mortality. We hypothesized that 6MMPr has low bioavailability in the central nervous system (CNS) and examined the effect of pre-treatment with 6MMPr on viral loads in the periphery and CNS. Pre-treatment with 6MMPr had no significant effect on viremia or viral titers in the periphery, but resulted in significantly higher viral loads in the brain, suggesting that the effect of 6MMPr is tissue-dependent. In conclusion, despite being a potent inhibitor of flaviviruses in cell culture, 6MMPr was not effective against West Nile disease in mice; however, further studies are warranted to reduce the toxicity and/or improve the bioavailability of this potential antiviral drug.