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Macrophages encounter flaviviruses early after injection by arthropod vectors. Using in vivo imaging of mice inoculated with firefly luciferase-expressing single-cycle flavivirus particles (FLUC-SCFV), we examined the initial dissemination of virus particles in the presence or absence of lymph node (LN)-resident macrophages. Higher luciferase activity, indicating higher SCFV gene expression, was detected in the footpad of macrophage-depleted mice after 24 h post infection (hpi). Moreover, FLUC-SCFV particles disseminated to the spleen within 14 hpi in macrophage-depleted, but not control mice. Although macrophages presented SCFV to naive T cells in vitro , depletion of subcapsular sinus (SCS) macrophages did not alter the magnitude or effector function of the WNV-specific CD8 + T cell response. Together, these results indicate that SCS macrophages play a role in limiting the dissemination of SCFV early in infection but are not required for the generation of a polyfunctional WNV-specific CD8 + T cell response in the draining LN.

We previously described a single-cycle dengue vaccine (RepliVAX D2) engineered from a capsid (C) gene-deleted West Nile virus (WNV) expressing dengue virus serotype 2 (DENV2) prM/E genes in place of the corresponding WNV genes. That work demonstrated that adaptation of RepliVAX D2 to grow in WNV C-expressing cells resulted in acquisition of non-synonymous mutations in the DENV2 prM/E and WNV NS2A/NS3 genes. Here we demonstrate that the prM/E mutations increase the specific infectivity of chimeric virions and the NS2A/NS3 mutations independently enhance packaging. Studies with the NS2A mutant demonstrated that it was unable to produce a larger form of NS1 (NS1'), suggesting that the mutation had been selected to eliminate a ribosomal frame-shift “slippage site” in NS2A. Evaluation of a synonymous mutation at this slippage site confirmed that genomes that failed to make NS1' were packaged more efficiently than WT genomes supporting a role for NS1/NS1' in orchestrating virion assembly.

Older adults exhibit higher morbidity and mortality from infectious diseases compared with those of the general population. The introduction and rapid spread of West Nile virus (WNV) throughout the continental United States since 1999 has highlighted the challenge of protecting older adults against emerging pathogens: to this day there is no therapy or vaccine approved for human use against West Nile encephalitis. In this study, we describe the characterization of T and B cell responses in old mice after vaccination with RepliVAX WN, a novel West Nile encephalitis vaccine based on single-cycle flavivirus particles. In adult mice, RepliVAX WN induced robust and long-lasting CD4+ and CD8+ T cell and Ab (B cell) responses against natural WNV epitopes, similar to those elicited by primary WNV infection. Primary and memory T and B cell responses in old mice against RepliVAX WN vaccination were significantly lower than those seen in younger mice, similar to the response of old mice to infection with WNV. Surprisingly, both the quality and the quantity of the recall Ab and T cell responses in vaccinated old mice were improved to equal or exceed those in adult animals. Moreover, these responses together (but not individually) were sufficient to protect both old and adult mice from severe WNV disease upon challenge. Therefore, at least two cycles of in vivo restimulation are needed for selection and expansion of protective lymphocytes in older populations, and live, single-cycle virus vaccines that stimulate both cellular and humoral immunity can protect older individuals against severe viral disease.

Flaviviruses transmitted by arthropods represent a tremendous disease burden for humans, causing millions of infections annually. All vector-borne flaviviruses studied to date suppress host innate responses to infection by inhibiting alpha/beta interferon (IFN-α/β)-mediated JAK-STAT signal transduction. The viral nonstructural protein NS5 of some flaviviruses functions as the major IFN antagonist, associated with inhibition of IFN-dependent STAT1 phosphorylation (pY-STAT1) or with STAT2 degradation. West Nile virus (WNV) infection prevents pY-STAT1 although a role for WNV NS5 in IFN antagonism has not been fully explored. Here, we report that NS5 from the virulent NY99 strain of WNV prevented pY-STAT1 accumulation, suppressed IFN-dependent gene expression, and rescued the growth of a highly IFN-sensitive virus (Newcastle disease virus) in the presence of IFN, suggesting that this protein can function as an efficient IFN antagonist. In contrast, NS5 from Kunjin virus (KUN), a naturally attenuated subtype of WNV, was a poor suppressor of pY-STAT1. Mutation of a single residue in KUN NS5 to the analogous residue in WNV-NY99 NS5 (S653F) rendered KUN NS5 an efficient inhibitor of pY-STAT1. Incorporation of this mutation into recombinant KUN resulted in 30-fold greater inhibition of JAK-STAT signaling than with the wild-type virus and enhanced KUN replication in the presence of IFN. Thus, a naturally occurring mutation is associated with the function of NS5 in IFN antagonism and may influence virulence of WNV field isolates.

Dengue viruses (DENV) cause the most common arboviral disease afflicting men. Clinical manifestations range from asymptomatic to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The mechanisms involved in the disease pathogenesis are not fully understood. The severity of the disease seems to be influenced by both viral and host factors. Subgenomic replicons of DENV can be used to study viral replication mechanisms and evaluate the effects of antiviral drugs on viral replication. The objective was to generate and characterize biologically a replicon from a clinical isolate of DENV-3, as part of our studies to understand how this new isolate interacts with cells. To obtain this replicon several RT-PCR fragments encoding the non-structural proteins genes were cloned in high-copy vectors, and used to assemble the replicon in a BAC plasmid vector containing a synthetic DNA molecule encoding the 5' and 3' ends of a viral cDNA with a T7 DNA-dependent RNA polymerase promoter and a ribozyme. In vitro transcribed RNA recovered from this BAC plasmid was transfected into C6/36 mosquito cells, and dengue virus protein expression was assessed by indirect immunofluorescence using polyclonal antibodies. The results showed that the replicon was replicated efficiently in cells, demonstrating successful assembly of a DENV-3 replicon.

During acute infection, West Nile virus (WNV) has been reported to infect a variety of cell types in various tissues of both experimentally and naturally infected hosts. Virus infects epithelial cells in the skin, kidney, intestine, and testes, although the importance of these findings is unclear. In the current study, we have observed that WNV infection of kidney tubules in mice coincides with the loss of expression of several members of the claudin family. Proteins of this family are often involved in epithelial barrier formation and function. WNV infection of epithelial cells in culture resulted in a decrease in the transepithelial electrical resistance, an increase in the efflux of mannitol across the monolayer, and a loss of intracellular levels of claudin-1 to -4. WNV capsid alone was sufficient for the degradation event, which was mediated through lysosomal proteases. Since epithelial cells are frequent sites of WNV infection, these observations imply a potential mechanism for virus dissemination and extraneural pathogenesis.

We have previously described a novel flavivirus vaccine technology based on a single-cycle, capsid (C) gene-deleted flavivirus called RepliVAX. RepliVAX can be propagated in cells that express high levels of C but undergoes only a single cycle of infection in vaccinated hosts. Here we report that we have adapted our RepliVAX technology to produce a dengue vaccine by replacing the prM/E genes of RepliVAX WN (a West Nile virus [WNV] RepliVAX) with the same genes of dengue virus type 2 (DENV2). Our first RepliVAX construct for dengue virus (RepliVAX D2) replicated poorly in WNV C-expressing cells. However, addition of mutations in prM and E that were selected during blind passage of a RepliVAX D2 derivative was used to produce a second-generation RepliVAX D2 (designated D2.2) that displayed acceptable growth in WNV C-expressing cells. RepliVAX D2.2 grew better in DENV2 C-expressing cells than WNV C-expressing cells, but after several passages in DENV2 C-expressing cells it acquired further mutations that permitted efficient growth in WNV C-expressing cells. We tested the potency and efficacy of RepliVAX D2.2 in a well-described immunodeficient mouse model for dengue (strain AG129; lacking the receptors for both type I and type II interferons). These mice produced dose-dependent DENV2-neutralizing antibody responses when vaccinated with RepliVAX D2.2. When challenged with 240 50% lethal doses of DENV2, mice given a single inoculation of RepliVAX D2.2 survived significantly longer than sham-vaccinated animals, although some of these severely immunocompromised mice eventually died from the challenge. Taken together these studies indicate that the RepliVAX technology shows promise for use in the development of vaccines that can be used to prevent dengue.

Safer vaccines are needed to prevent flavivirus diseases. To help develop these products we have produced a pseudoinfectious West Nile virus (WNV) lacking a functional C gene which we have named RepliVAX WN. Here we demonstrate that RepliVAX WN can be safely propagated at high titer in BHK cells and vaccine-certified Vero cells engineered to stably express the C protein needed to trans-complement RepliVAX WN growth. Using these BHK cells we selected a better growing mutant RepliVAX WN population and used this to generate a second-generation RepliVAX WN (RepliVAX WN.2). RepliVAX WN.2 grown in these C-expressing cell lines safely elicit strong protective immunity against WNV disease in mice and hamsters. Taken together, these results indicate the clinical utility of RepliVAX WN.2 as a vaccine candidate against West Nile encephalitis.

Before 1990, West Nile virus (WNV) was considered to be one of many arthropod-borne viruses that caused mild febrile illness in man. However, in the 1990s, the virus was associated with severe CNS disease that produced mortality in horses and man in Europe. In 1999, WNV was identified as the etiologic agent of an outbreak of human and avian encephalitis in New York City (NY, USA). Like many other Flaviviridae family members, WNV is generally considered to cause acute infections, however, persistent WNV infections have been observed in laboratory-infected animals and in human patients. These persistent infections could be facilitated by changes to the viral genome that allow the virus to evade detection by the host cell, a property that has been studied in cell culture. This review highlights our current knowledge of persistent WNV infections in vitro and in vivo, and speculates on how persistence could influence virus transmission.

The effect of long-term West Nile virus (WNV) infection on Culex salivary gland morphology and viability was evaluated by transmission electron microscopy during a four week period post-blood feeding. These studies showed that apoptosis and other cytopathologic changes occurred more frequently in WNV-infected mosquitoes compared with uninfected controls. The effect of long-term infection on WNV transmission was evaluated by titering virus in saliva over the same time period. Although the mean titer of WNV in mosquito saliva did not change significantly over time, the percentage of saliva samples containing WNV decreased. Because of the importance of saliva in blood meal acquisition and virus delivery, salivary gland pathology has the potential to affect mosquito feeding behavior and virus transmission. Results from this study add to a growing body of evidence that arbovirus infections in mosquito vectors can be cytopathic, and offer a potential mechanism for virus-induced cell death in mosquitoes.

A stable cell system for high-efficiency packaging of West Nile virus (WNV) subgenomic replicons into virus-like particles (VLPs) was developed. VLPs could be propagated on these packaging cells and produced infectious foci similar to foci produced by WNV. Focus size correlated with the replicative capacity of WNV replicons, indicating that genome copy number, rather than amount of trans-complementing structural proteins, was rate-limiting in packaging of VLPs. Comparison of VLP production from replicon genomes encoding partial or complete C genes indicated that portions of C downstream of the cyclization sequence could improve genome replication or that cis expression of C could enhance packaging. Interestingly, a rapid loss of replicon-encoded reporter gene activity was detected within two serial passages of reporter gene-containing VLPs. The loss of reporter activity correlated with gene deletion and better VLP growth, indicating a powerful selection pressure for WNV genomes lacking reporter genes.

West Nile virus (WNV) is a positive-sense, single-stranded RNA virus of the family Flaviviridae. WNV persistently infects insect cells, but can causes acute cytopathic infection of mammalian cells and is an etiologic agent of viral encephalitis in humans. By using a cell line expressing a WNV subgenomic replicon [Rossi, S.L., Zhao, Q., O'Donnell, V.K., Mason, P.W., 2005. Adaptation of West Nile virus replicons to cells in culture and use of replicon-bearing cells to probe antiviral action. Virology 331 (2), 457-470], we developed a high-throughput assay and used it to screen a library of small molecule compounds for inhibitors of WNV replication in the absence of live virus. Here we report the identification of novel small molecule inhibitors for WNV replicon replication. We demonstrate that the compounds inhibited WNV replication-dependent luciferase expression in the replicon cells and reduced WNV viral protein accumulation and viral RNA copy number in the replicon cells. Two classes of compounds with multiple hits, parazolotrahydrothophenes and pyrozolopyrimidines, showed preliminary structure-activity relationships. In WNV infection assays, one pyrozolopyrimidine compound was confirmed to have antiviral activity. These compounds should be valuable for developing anti-WNV therapeutic drugs as well as research tools to study the mechanism of WNV replication.

The T lymphocytes play an important role in prevention and recovery from viral infections. To characterize T lymphocyte responses to Japanese encephalitis (JE) virus infections, we analyzed JE virus-specific T lymphocytes in peripheral blood mononuclear cells (PBMC) obtained from seven JE patients and 10 vaccinees who had received a formalin-inactivated, purified JE virus vaccine (Biken vaccine). These PBMC were examined for proliferative responses against live JE virus, a glutaraldehyde-fixed lysate of cells infected with JE virus, and extracellular particles (EPs; subviral membrane vesicles released from cells infected with recombinant vaccinia viruses encoding the JE virus premembrane and envelope proteins). Japanese encephalitis virus-specific T cell proliferation was demonstrated with PBMC from both patients and vaccinees after stimulation with infectious JE virus or the lysate of JE virus-infected cells. Proliferating PBMC included CD4+ T lymphocytes and CD8+ T lymphocytes in responses to either form of JE viral antigens. Responses to EPs were observed only with PBMC from some American vaccinees whose PBMC also responded to the virus and lysate. These results indicate that JE virus infection and immunization with an inactivated JE vaccine induce JE virus-specific CD4+ and CD8+ T memory lymphocytes that can be induced to proliferate by infectious JE virus and noninfectious JE antigens.

An avipox virus, canarypox (ALVAC), which is naturally host-range restricted, was used to construct recombinants encoding the Japanese encephalitis virus (JEV) prM, E and NS1 genes (vCP107) and prM and E genes (vCP140). Mice immunized with these recombinant viruses produced JEV neutralizing antibodies and were protected from lethal JEV challenge. Protection was also observed in mice immunized with a subunit vaccine candidate, consisting of extracellular particles (EPs; RNA-free subviral membrane vesicles containing prM/M and E proteins) derived from HeLa cell cultures infected with a JEV—vaccinia recombinant. Mice primed with vCP107 and boosted with EPs had higher antibody levels than mice immunized twice with EPs alone, although the levels were comparable to that obtained in mice immunized twice with the recombinant virus. Mice immunized with a mixture of recombinant virus (vCP107) plus EPs had neutralizing antibody titres higher than mice immunized with the recombinant virus or EPs alone.

Recently, we demonstrated that a single-cycle West Nile virus (WNV) named RepliVAX WN could be used to produce a chimeric Japanese encephalitis (JE) vaccine (RepliVAX JE) by replacing the WNV prM/E genes with those of JEV. Here, we tested if replacement of WNV NS1 gene in RepliVAX JE with that of JEV (producing TripliVAX JE) could produce a superior vaccine. TripliVAX JE elicited higher anti-E immunity and displayed better efficacy in mice than RepliVAX JE. Furthermore, TripliVAX JE displayed reduced immune interference caused by pre-existing anti-NS1 immunity. Thus, we propose prM/E/NS1 chimerization as a new strategy for flavivirus vaccine development.

We recently reported that immunization with RepliVAX WN, a single-cycle West Nile virus (WNV) vaccine, protected mice against WNV challenge. We have extended these studies by characterizing the RepliVAX WN-elicited antibody and T cell responses. WNV-specific IgG antibody responses comprised predominantly of IgG(2c) and IgG(2b) subclasses were detected 8 months after immunization. Vigorous WNV-specific CD4(+) and CD8(+) T cell responses directed at both structural and nonstructural WNV proteins were detected which were characterized by cytolytic activity and secretion of IFN-γ and TNF-α. Importantly, RepliVAX WN immunization resulted in vigorous CD8(+) memory T cell responses detected at 8 months after immunization.

West Nile virus (WNV) causes serious neurologic disease, but no licensed vaccines are available to prevent this disease in humans. We have developed RepliVAX WN, a single-cycle flavivirus with an expected safety profile superior to other types of live-attenuated viral vaccines. In this report we describe studies examining RepliVAX WN safety, potency, and efficacy in a non-human primate model of WNV infection. A single immunization of four rhesus macaques with RepliVAX WN was safe and elicited detectable neutralizing antibody titers and IgM and IgG responses, and IgG titers were increased in two animals that received a second immunization. After challenge with WNV, three of four immunized animals were completely protected from viremia, and the remaining animal showed minimal viremia on one day. In contrast, the unvaccinated animal developed viremia that lasted six days. These results demonstrate the efficacy and safety of RepliVAX WN in this primate model of WNV infection.

A highly attenuated strain of vaccinia virus (NYVAC) was engineered to express the Japanese encephalitis virus (JEV) prM, E, and NS1 genes or the prM and E genes. The recombinant viruses were tested as vaccine candidates in pigs, a natural host of JEV. JEV-neutralizing and hemagglutination-inhibiting antibodies appeared in swine sera 7 days after immunization with 10(8) PFU of the recombinant viruses and increased after a second dose at 28 days. The JEV levels detected in the serum after JEV challenge (d56) of the swine with 2 x 10(5) PFU of JEV were significantly reduced in animals inoculated with the recombinant viruses. These results demonstrate the ability of these NYVAC-vectored recombinants to protect pigs from JEV viremia.

The aminoglycoside, geneticin (G418), was recently shown to have antiviral activity against bovine viral diarrhea virus (BVDV). Since BVDV, dengue virus (DENV) and yellow fever virus (YFV) all belong to the Flaviviridae family, it seemed possible that a common step in their life cycle might be affected by this aminoglycoside. Here it is shown that geneticin prevented the cytopathic effect (CPE) resulting from DENV-2 infection of BHK cells, in a dose-dependent manner with an 50% effective concentration (EC(50)) value of 3+/-0.4microg/ml. Geneticin had no detectable effect on CPE caused by YFV in BHK cells. Geneticin also inhibited DENV-2 viral yield with an EC(50) value of 2+/-0.1microg/ml and an EC(90) value of 20+/-2microg/ml. With a CC(50) value of 165+/-5microg/ml, the selectivity index of anti-DENV activity of geneticin in BHK cells was established to be 66. Furthermore, 25microg/ml of geneticin nearly completely blocked plaque formation induced by DENV-2, but not YFV. In addition, geneticin, inhibited DENV-2 viral RNA replication and viral translation. Gentamicin, kanamycin, and the guanidinylated geneticin showed no anti-DENV activity. Neomycin and paromomycin demonstrated weak antiviral activity at high concentrations. Finally, aminoglycoside-3'-phosphotransferase activity of neomycin-resistant gene abolished antiviral activity of geneticin.

West Nile virus (WNV) causes significant disease, yet no vaccines exist to prevent WN disease in humans. We have previously reported that RepliVAX WN is a safe and efficacious vaccine in mouse and hamster models of WN disease. Here, we report that vaccination of hamsters with RepliVAX WN induces antibody responses that remain stable for at least 6 months. Furthermore, animals challenged with virulent WNV 6 months after vaccination were protected from disease as well as those challenged 2 months post-vaccination, with no vaccinated animals succumbing to WNV challenge. These results indicate that RepliVAX is capable of inducing durable protective immunity after a single dose.

Immunization with recombinant vaccinia viruses that specified the synthesis of Japanese encephalitis virus (JEV) glycoproteins protected mice from a lethal intraperitoneal challenge with JEV. Recombinants which coexpressed the genes for the structural glycoproteins, prM and E, elicited high levels of neutralizing (NEUT) and hemagglutination inhibiting (HAI) antibodies in mice and protected mice from a lethal challenge by JEV. Recombinants expressing only the gene for the nonstructural glycoprotein, NS1, induced antibodies to NS1 but provided low levels of protection from a similar challenge dose of JEV. Antibodies to the NS3 protein in postchallenge sera, representing the degree of infection with challenge virus, were inversely correlated to NEUT and HAI titers and levels of protection. These results indicate that although vaccinia recombinants expressing NS1 can provide some protection from lethal JEV infection, recombinants expressing prM and E elicited higher levels of protective immunity.

Based on previous data showing that JEV and DEN-1 fusion proteins can be used as antigens, we engineered analogous fusion proteins for the same regions of the E proteins of DEN-2, DEN-3, and DEN-4, using the polymerase chain reaction. The resulting fusion proteins were tested in a modified ELISA for their reactivity with mouse immune ascitic fluids (MIAF) generated against 10 different flaviviruses. The results showed that these recombinant antigens could be used as type-specific immunological reagents, and suggest that these antigens could serve as the basis for rapid, safe, and inexpensive flavivirus serosurveys.

Four recombinant vaccinia viruses were engineered for expression of different portions of the Japanese encephalitis virus (JEV) open reading frame. All four recombinant vaccinias contained the NS1 and NS2A genes, and each of these viruses specified the synthesis, glycosylation, and secretion of the nonstructural glycoprotein (NS1). All four recombinants also contained the E gene, and each virus correctly directed the synthesis and glycosylation of the envelope glycoprotein (E). Interestingly, two of these viruses (vP555 and vP650), which expressed the prM gene in addition to E and NS1, produced an extracellular hemagglutinin containing M and E that migrated in sucrose gradients similarly to the slowly-sedimenting hemagglutinin found in the culture fluid of JEV-infected cells. Immunization of 3-week-old mice with the recombinant viruses vP555 and vP658 resulted in immune responses to NS1, whereas only the virus that directed the synthesis of extracellular forms of E (vP555) induced an immune response to E. Both viruses provided protection against lethal challenge with JEV. Animals given two inoculations with vP555 were fully protected from greater than 10,000 LD50 of JEV. This high level of protection was correlated with the production of high titers of neutralizing and hemagglutination-inhibiting antibodies.

Flaviviruses are arthropod-borne pathogens that cause significant disease on all continents of the world except Antarctica. Flavivirus diseases are particularly important in tropical regions where arthropod vectors are abundant. Live-attenuated virus vaccines (LAVs) and inactivated virus vaccines (INVs) exist for some of these diseases. LAVs are economical to produce and potent, but are not suitable for use in the immunocompromised. INVs are safer, but are more expensive to produce and less potent. Despite the success of both classes of these first-generation flavivirus vaccines, problems associated with their use indicate a need for improved products. Furthermore, there are no suitable vaccines available for important emerging flavivirus diseases, notably dengue and West Nile encephalitis (WNE). To address these needs, new products, including LAVs, INVs, viral-vectored, genetically engineered LAVs, naked DNA, and subunit vaccines are in various stages of development. Here we describe the current state of these first- and second-generation vaccine candidates, and compare these products to our recently described single-cycle, encapsidation defective flavivirus vaccine: RepliVAX. RepliVAX can be propagated in C-expressing cells (or as a unique two-component virus) using methods similar to those used to produce today's economical and potent LAVs. However, due to deletion of most of the gene for the C protein, RepliVAX cannot spread between normal cells, and is unable to cause disease in vaccinated animals. Nevertheless, RepliVAX is potent and efficacious in animal models for WNE and Japanese encephalitis, demonstrating its utility as a third-generation flavivirus vaccine that should be potent, economical to produce, and safe in the immunocompromised.

Existing live-attenuated flavivirus vaccines (LAV) could be improved by reducing their potential to recombine with naturally circulating viruses in the field. Since the highly conserved cyclization sequences (CS) found in the termini of flavivirus genomes must be complementary to each other to support genome replication, we set out to identify paired mutant CS that could support the efficient replication of LAV but would be unable to support replication in recombinant viruses harboring one wild-type (WT) CS. By systematic evaluation of paired mutated CS encoded in West Nile virus (WNV) replicons, we identified variants having single and double mutations in the 5′- and 3′-CS components that could support genome replication at WT levels. Replicons containing only the double-mutated CS in the 5′ or the 3′ ends of the genome were incapable of replication, indicating that mutated CS could be useful for constructing safer LAV. Despite the identity of the central portion of the CS in all mosquito-borne flaviviruses, viruses carrying complementary the double mutations in both the 5′- and the 3′-CS were indistinguishable from WT WNV in their replication in insect and mammalian cell lines. In addition to the utility of our novel CS pair in constructing safer LAV, we demonstrated that introduction of these mutated CS into one component of a recently described two-component genome system (A. V. Shustov, P. W. Mason, and I. Frolov, J. Virol. 81:11737-11748, 2007) enabled us to engineer a safer single-cycle WNV vaccine candidate with reduced potential for recombination during its propagation.

West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem1. However, our understanding of the molecular interaction of WNV (and related flaviviruses) with mammalian host cells is limited1. WNV encodes only 10 proteins, implying that the virus may use many cellular proteins for infection1. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway1–3. RNA-interference (RNAi) presents a powerful forward genetics approach to dissect virus-host cell interactions4–6. Here we report the identification of 305 host proteins impacting WNV infection, using a human genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic reticulum-associated degradation (ERAD) pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides the first comprehensive molecular portrait of WNV-human cell interactions that forms a paradigm for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.

Flaviviruses are arthropod-borne pathogens that cause significant disease on all continents of the world except Antarctica. Flavivirus diseases are particularly important in tropical regions where arthropod vectors are abundant. Live-attenuated virus vaccines (LAVs) and inactivated virus vaccines (INVs) exist for some of these diseases. LAVs are economical to produce and potent, but are not suitable for use in the immunocompromised. INVs are safer, but are more expensive to produce and less potent. Despite the success of both classes of these first-generation flavivirus vaccines, problems associated with their use indicate a need for improved products. Furthermore, there are no suitable vaccines available for important emerging flavivirus diseases, notably dengue and West Nile encephalitis (WNE). To address these needs, new products, including LAVs, INVs, viral-vectored, genetically engineered LAVs, naked DNA, and subunit vaccines are in various stages of development. Here we describe the current state of these first- and second-generation vaccine candidates, and compare these products to our recently described single-cycle, encapsidation defective flavivirus vaccine: RepliVAX. RepliVAX can be propagated in C-expressing cells (or as a unique two-component virus) using methods similar to those used to produce today's economical and potent LAVs. However, due to deletion of most of the gene for the C protein, RepliVAX cannot spread between normal cells, and is unable to cause disease in vaccinated animals. Nevertheless, RepliVAX is potent and efficacious in animal models for WNE and Japanese encephalitis, demonstrating its utility as a third-generation flavivirus vaccine that should be potent, economical to produce, and safe in the immunocompromised.

The biosynthesis of the flavivirus nonstructural glycoprotein, NS1, has important implications for (1) vaccine production, since NS1 immunity can protect animals from flavivirus infection; and (2) virion maturation, since NS1 is coretained with immature forms of the structural glycoprotein E in the endoplasmic reticulum (ER) of infected cells. To examine the molecular basis for NS1 retention within the ER we have expressed fragments of Japanese encephalitis virus (JEV) cDNA encoding NS1 in BHK cells. These transient expression studies showed that the JEV NS1 protein was faithfully processed when expressed in isolation, and have revealed that NS1 expressed in the absence of any other viral genes behaves like the NS1 protein found in JEV-infected cells with respect to retention in the ER, secretion, glycosylation, membrane association, and dimerization.

Multiple vaccines exist to control Japanese encephalitis (JE), but all suffer from problems. We have developed a new type of flavivirus vaccine, a pseudoinfectious virus (RepliVAX WN) that prevents West Nile virus (WNV)-induced disease. Here, we describe production of a chimeric RepliVAX (RepliVAX JE) that expresses the JE virus (JEV) prM and E proteins. Our prototype RepliVAX JE replicated poorly in cells, but blind passage produced a better-growing derivative, and analyses of this derivative allowed us to engineer a second-generation RepliVAX (RepliVAX JE.2) that grew to high titers. RepliVAX JE.2 elicited neutralizing antibodies in both mice and hamsters and provided 100% protection from a lethal challenge with JEV or WNV, respectively. These results demonstrate the utility our RepliVAX platform for producing a JE vaccine.

Dendritic cells (DCs) play a central role in innate immunity and antiviral responses. In this study, we investigated the production of alpha interferon (IFN-α) and inducible chemokines by human monocyte-derived dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) infected with West Nile virus (WNV), an emergent pathogen whose infection can lead to severe cases of encephalitis in the elderly, children, and immunocompromised individuals. Our experiments demonstrated that WNV grown in mammalian cells (WNVVero) was a potent inducer of IFN-α secretion in pDCs and, to a lesser degree, in mDCs. The ability of WNVVeroto induce IFN-α in pDCs did not require viral replication and was prevented by the treatment of cells with bafilomycin A1 and chloroquine, suggesting that it was dependent on endosomal Toll-like receptor recognition. On the other hand, IFN-α production in mDCs required viral replication and was associated with the nuclear translocation of IRF3 and viral antigen expression. Strikingly, pDCs failed to produce IFN-α when stimulated with WNV grown in mosquito cells (WNVC7/10), while mDCs responded similarly to WNVVeroor WNVC7/10. Moreover, the IFN-dependent chemokine IP-10 was produced in substantial amounts by pDCs in response to WNVVerobut not WNVC7/10, while interleukin-8 was produced in greater amounts by mDCs infected with WNVC7/10than in those infected with WNVVero. These findings suggest that cell-specific mechanisms of WNV recognition leading to the production of type I IFN and inflammatory chemokines by DCs may contribute to both the innate immune response and disease pathogenesis in human infections.

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.

West Nile virus (WNV) is a member of the Flavivirus genus of the family Flaviviridae. This genus contains a number of arthropod-borne human pathogens, including dengue virus (DV), Japanese encephalitis virus, yellow fever virus, and tick-borne encephalitis virus (26). WNV was associated with fever and infrequent encephalitis cases in humans in Africa, the Middle East, and Europe from its discovery in 1938 through the 1990s. In 1999, WNV was first detected in New York City, and from there it rapidly spread across the United States, some regions of Canada, Mexico, and Central America. The majority of WNV infections are asymptomatic; however, a portion of infections result in West Nile fever, and a subset of infections lead to viral invasion of the central nervous system that results in encephalitis, paralysis, and meningitis, outcomes which are especially prevalent in immunocompromised and aged individuals (4). The mechanisms by which WNV causes disease are not completely understood, but studies with a hamster model of the disease suggest that following a brief peripheral replication cycle, the virus crosses the blood-brain barrier, where it infects neurons, causing cell death (47). The direct effect of WNV on neurons or an immune response to their infection may also be responsible for encephalitis and meningoencephalitis in humans (13). Pretreatment of animals with type I interferons (IFN-α/IFN-β) has been shown to block flavivirus disease (3, 23), and animals defective in the IFN response have been shown to be more susceptible to flavivirus infection (19, 28, 39). Although the precise IFN-stimulated genes (ISGs) that are responsible for preventing or controlling flavivirus infections are unknown, a number of studies have demonstrated that flavivirus-infected cells prevent IFN-induced phosphorylation of STAT molecules, which carry the signal from the ligated IFN receptor to the nucleus to activate the transcription of ISGs (1, 11, 20, 24, 25, 32, 37, 44). Interestingly, the precise mechanism by which flaviviruses alter STAT phosphorylation appears to differ among members of the genus (1, 24, 32). IFN is produced by most eukaryotic cells in response to viral infection and/or recognition of virus-associated macromolecules (known as pathogen-associated molecular patterns [PAMPs]), such as single- or double-stranded RNA (ssRNA and dsRNA, respectively). In many mammalian cell types, ligation of Toll-like receptor 3 (TLR3) to extracellular dsRNA or recognition of intracellular dsRNA by the intracellular helicase mda5 or RIG-I induces the phosphorylation and activation of the constitutively expressed IFN regulatory factor 3 (IRF3), leading to nuclear translocation and activation of transcription of the genes for IFN-β and IFN-α subtype 4 (14). Recent studies have indicated that in cell cultures, a subset of viruses appear to activate the mda5 pathway, whereas others, including the flavivirus Japanese encephalitis virus, activate the RIG-I pathway (21). In some cases, IFN-α expression has been linked to protein kinase R activation via binding of intracellular dsRNA (9). Furthermore, multiple IFN-α subtypes can also be induced by PAMP-stimulated signal transduction pathways that lead to the phosphorylation of IRF7, which can be triggered by binding of ssRNA to TLR7/8. Interestingly, IRF7 is constitutively expressed in only a subset of cells, but the IRF7 gene is an ISG, so following IFN binding, many cells can produce IRF7, permitting them to amplify the IFN signal if stimulated by ssRNA (12). For many infections, a subset of cells known as plasmacytoid dendritic cells (pDCs), which express IRF7 constitutively, have been implicated as key IFN producers (16). These cells can produce extremely high levels of IFN in response to stimulation with infectious disease agents or components thereof. Activated pDCs also produce other cytokines, notably interleukin-12 (IL-12), tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and IL-3, and chemokines, such as CCL3, CCL4, CCL5, CCL22, CCL19, and CXCL13 (2, 5, 33). However, pDCs are not the only cell type that express IRF7 constitutively. Other lymphocytes, including monocytes, B cells, and dendritic cell precursors (pDC2) (18), also express IRF7 in the resting state and are hence able to induce synthesis of IFN-α through the IRF7 pathway (reviewed in reference 22). Virus-like particles (VLPs) have been used as tools to study RNA virus infection in vitro and in vivo. VLPs consist of subgenomic replicating genomes lacking structural protein genes (replicons) that have been encapsidated by the missing structural proteins provided in trans by packaging cells. VLPs are thus able to infect cells and initiate genome replication in a manner that mimics that of normal virus, but unlike infections with normal virus, VLP infections cannot spread in the absence of trans-expressed structural proteins. In the case of both alphaviruses (35) and flaviviruses (43), VLPs have been used to identify the first cells that are infected in insect vectors of these viruses. Furthermore, for the alphavirus Venezuelan equine encephalitis virus, VLPs have been used to identify the first cells that are infected in animal models (29), and recent studies with Venezuelan equine encephalitis virus VLPs have shown that these VLPs (referred to as VRPs in the previous study) can strongly stimulate antiviral responses (46). In the studies described in this paper, we used WNV VLPs to study the early events in WNV infection in mice, demonstrating that WNV VLPs induce a rapid IFN response, resulting in very high levels of IFN-α in the serum between 8 and 24 h after either intraperitoneal (i.p.) inoculation or subcutaneous inoculation in the footpad (f.p. inoculation). Since VLPs can undergo only a single round of infection, these studies have demonstrated that the very first cells infected in an animal are capable of stimulating the production of high levels of IFN. IFN production was dependent on the replicative capacity of VLPs, since mice inoculated with UV-inactivated VLPs did not produce IFN. Immunohistochemical (IHC) detection of WNV antigen-positive cells in the popliteal lymph nodes (pLN) after f.p. inoculation with VLPs and the demonstration of high levels of IFN mRNA in pLN collected from VLP-inoculated animals implicate this lymphoid organ as a prominent source of IFN production. Finally, the finding that IRF3−/− animals produced levels of IFN similar to those of wild-type animals suggests that neither the RIG-I/mda5 nor the TLR3 pathway, known to be important for IFN-β induction, is important for this early response to WNV infection.

The antiviral activity of iminocyclitol compounds with a deoxynojirimycin (DNJ) head group and either a straight chain alkyl or alkylcycloalkyl group attached to the nitrogen atom have been tested in vitro against multiple-enveloped viruses. Several of these analogues were superior to previously reported DNJ compounds. Iminocyclitols that inhibit the glycan-processing enzyme endoplasmic-reticular glucosidase have been shown to inhibit the morphogenesis of viruses that bud from the endoplasmic reticulum (ER) at non-cytotoxic concentrations. Bovine viral diarrhoea virus (BVDV) has been used as a surrogate system for study of the hepatitis C virus, which belong to the virus family ( Flaviviridae) as West Nile virus (WNV) and dengue virus (DV). N-Nonyl-DNJ (NNDNJ) was previously reported to have micromolar antiviral activity against BVDV, but a limiting toxicity profile. N-Butylcyclohexyl-DNJ (SP169) was shown to be as potent as NNDNJ in assays against BVDV and less toxic. However, it was inactive against hepatitis B virus (HBV). The present study reports efforts to improve the performance profiles of these compounds. Introduction of an oxygen atom into the N-alkyl side chain of DNJ, either as an ether or a hydroxyl functionality, reduced toxicity but sacrificed potency. Introduction of a hydroxyl group at the tertiary carbon junction of the cycloalkyl and linear alkyl group, as in N-pentyl-(1-hydroxycyclohexyl)-DNJ (OSL-95II), led to a structure that was as well tolerated as DNJ (CC50>500 µM), but retained micromolar antiviral activity against all ER morphogenesis budding viruses tested: BVDV, WNV, DV and HBV. The implication of this modification to the development of broad-spectrum antiviral agents is discussed.

The emergence of West Nile virus (WNV) in the Western Hemisphere is marked by the spread of pathogenic lineage I strains, which differ from typically avirulent lineage II strains. To begin to understand the virus-host interactions that may influence the phenotypic properties of divergent lineage I and II viruses, we compared the genetic, pathogenic, and alpha/beta interferon (IFN-α/β)-regulatory properties of a lineage II isolate from Madagascar (MAD78) with those of a new lineage I isolate from Texas (TX02). Full genome sequence analysis revealed that MAD78 clustered, albeit distantly, with other lineage II strains, while TX02 clustered with emergent North American isolates, more specifically with other Texas strains. Compared to TX02, MAD78 replicated at low levels in cultured human cells, was highly sensitive to the antiviral actions of IFN in vitro, and demonstrated a completely avirulent phenotype in wild-type mice. In contrast to TX02 and other pathogenic forms of WNV, MAD78 was defective in its ability to disrupt IFN-induced JAK-STAT signaling, including the activation of Tyk2 and downstream phosphorylation and nuclear translocation of STAT1 and STAT2. However, replication of MAD78 was rescued in cells with a nonfunctional IFN-α/β receptor (IFNAR). Consistent with this finding, the virulence of MAD78 was unmasked upon infection of mice lacking IFNAR. Thus, control of the innate host response and IFN actions is a key feature of WNV pathogenesis and replication fitness.

A trans -packaging system for West Nile virus (WNV) subgenomic replicon RNAs (repRNAs), deleted for the structural coding region, was developed. WNV repRNAs were efficiently encapsidated by the WNV C/prM/E structural proteins expressed in trans from replication-competent, noncytopathic Sindbis virus-derived RNAs. Infectious virus-like particles (VLPs) were produced in titers of up to 10 9 infectious units/ml. WNV VLPs established a single round of infection in a variety of different cell lines without production of progeny virions. The infectious properties of WNV and VLPs were indistinguishable when efficiencies of infection of a number of different cell lines and inhibition of infection by neutralizing antibodies were determined. To investigate the usefulness of VLPs to address biological questions in vivo, Culex pipiens quinquefasciatus mosquitoes were orally and parenterally infected with VLPs, and dissected tissues were analyzed for WNV antigen expression. Antigen-positive cells in midguts of orally infected mosquitoes were detected as early as 2 days postinfection and as late as 8 days. Intrathoracic inoculation of VLPs into mosquitoes demonstrated a dose-dependent pattern of infection of secondary tissues and identified fat body, salivary glands, tracheal cells, and midgut muscle as susceptible WNV VLP infection targets. These results demonstrate that VLPs can serve as a valuable tool for the investigation of tissue tropism during the early stages of infection, where virus spread and the need for biosafety level 3 containment complicate the use of wild-type virus.

A controlled, randomized, double-blind clinical trial evaluated whether two attenuated recombinant poxviruses with identical Japanese encephalitis virus (JEV) gene insertions, NYVAC-JEV and ALVAC-JEV, were safe and immunogenic in volunteers. Groups of 10 volunteers distinguished by vaccinia immune status received two doses of each vaccine. The vaccines appeared to be equally safe and well tolerated in volunteers, but more reactogenic than licensed formalin-inactivated JE and placebo vaccines given as controls. NYVAC-JEV and ALVAC-JEV vaccine recipients had frequent occurrence of local warmth, erythema, tenderness, and/or arm pain after vaccination. There was no apparent effect of vaccinia immune status on frequency or magnitude of local and systemic reactions. NYVAC-JEV elicited antibody responses to JEV antigens in recipients but ALVAC-JEV vaccine poorly induced antibody responses. However, NYVAC-JEV vaccine induced neutralizing antibody responses only in vaccinia-nonimmune recipients while vaccinia-immune volunteers failed to develop protective antibodies (5/5 vs. 0/5 seroconversion, p

A dengue DNA vaccine candidate was developed and evaluated for immunogenicity in mice. The vaccine, designated pcD2ME, is a pcDNA3-based plasmid encoding the signal sequence of premembrane (prM), prM and envelope (E) genes of the New Guinea C strain of dengue type 2 virus. CHO-K1 cells transfected with pcD2ME expressed prM and E as determined by immunochemical staining with monoclonal antibodies. BALB/c mice inoculated intramuscularly with 100 microg of pcD2ME two or three times at an interval of 2 weeks developed a low level of neutralizing antibody (1:10 at a 90% plaque reduction). Immunization twice with 10 microg or 1 microg of pcD2ME or three times with 100 microg of pcDNA3 did not induce detectable levels of neutralizing antibody. Mice immunized two or three times with 100 microg of pcD2ME raised neutralizing antibody titers to 1:40 or greater on days 4 and 8 after challenge with 3x10(5) plaque forming units (PFU) of the New Guinea C strain of dengue type 2 virus, showing strong anamnestic responses to the challenge. In contrast, mice immunized two or three times with 100 microg of pcDNA3 developed no detectable neutralizing antibody on days 4 and 8 after challenge. These results indicate that immunization with pcD2ME induces neutralizing antibody and dengue type 2 virus-responsive memory B cells in mice.

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA 14 -14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA 14 -14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10 6 PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.

Poxvirus-based recombinant Japanese encephalitis (JE) vaccine candidates, NYVAC-JEV and ALVAC-JEV, were examined for their ability to induce JE virus-specific cytotoxic T lymphocytes (CTLs) in a phase I clinical trial. These vaccine candidates encoded the JE virus premembrane (prM), envelope (E) and non-structural 1 (NS1) proteins. The volunteers received subcutaneous inoculations with each of these candidates on days 0 and 28, and blood was drawn 2 days before vaccination and on day 58. Anti-E and anti-NS1 antibodies were elicited in most vaccinees inoculated with NYVAC-JEV and in some vaccinees inoculated with ALVAC-JEV. Peripheral blood mononuclear cells (PBMCs) obtained from approximately one half of vaccines showed positive proliferation in response to stimulation with live JE virus. Cytotoxic assays demonstrated the presence of JE virus-specific CTLs in in vitro-stimulated PBMCs obtained from two NYVAC-JEV and two ALVAC-JEV vaccinees. Cell depletion tests using PBMCs from one NYVAC-JEV recipient indicated that the phenotype of CTLs was CD8+CD4-.

We previously reported that extracellular particles (EPs) composed of premembrane (prM) and envelope (E) proteins were released from cells infected with recombinant vaccinia viruses encoding Japanese encephalitis (JE) virus prM and E genes. In the present study, EPs were evaluated for induction of JE virus-specific antibody and specific T lymphocytes in mice. Six- to 8-week-old male Balb/c mice were inoculated intraperitoneally once or twice (at a 3-week interval) with purified EPs containing 1 microgram of E without adjuvant. Neutralizing antibody was detected and spleen cells proliferated against JE viral antigen 3 weeks after the second immunization with EPs. Neutralizing antibody and JE virus-specific T lymphocytes were also detected 10 months after immunization with EPs containing 2 micrograms of E. Spleen cells obtained from EP-immunized mice and stimulated in vitro with live JE virus, expressed JE virus-specific cytotoxic activity. The cytotoxic activity was reduced by treatment with anti-CD3 antibody and complement. These results indicate that immunization with EPs induces long-lasting specific antibody and memory T cells in mice.

Four recombinant vaccinia viruses expressing different portions of the dengue type 1 virus (DEN-1) genome (C-prM-E-NS1-NS2A-NS2B; prM-E; prM-E-NS1-NS2A-NS2B; or NS1-NS2A) were constructed in order to establish the most immunogenic configuration of DEN-1 proteins. Both recombinants producing prM and E in the absence of C induced the synthesis of extracellular forms of E in vitro. Mice inoculated with these two recombinants produced DEN-1 neutralizing (NEUT) and haemagglutination inhibiting (HAI) antibodies. The other two recombinant vaccinia viruses, which did not induce the production of extracellular forms of E, did not induce E-specific immune responses. These results support our previous studies on the design of flavivirus-vaccinia vaccine candidates by showing the importance of co-expressing prM and E in order to induce the synthesis of extracellular E and to elicit NEUT and HAI antibodies.

Extracellular subviral particles produced by HeLa cells infected with a recombinant vaccinia virus encoding the prM and E genes of Japanese encephalitis virus (JEV) were purified and characterized. These particles contained the JEV prM/M and E proteins embedded in a lipid bilayer, and RNA was not detected in particles using the polymerase chain reaction and primers recognizing a part of the JEV E gene. The particles were uniformly spherical with a 20-nm diameter and had 5-nm projections on their surface. Mice that received a single inoculation of the purified extracellular particles emulsified with Freund's complete adjuvant were fully protected against 4.9 × 105 LD50 of JEV. Comparison of the neutralizing and hemagglutination-inhibiting antibody titers and radioimmunoprecipitation data showed that immunization with the particles induced an immune response similar to that following inoculation with the recombinant vaccinia virus.

Recombinant Japanese encephalitis (JE) vaccine candidates based on a highly attenuated vaccinia virus (NYVAC-JEV) and a canarypox virus (ALVAC-JEV) were evaluated for their ability to induce specific antibodies and cytotoxic T lymphocytes (CTLs) in mice. Six- to eight-week-old male Balb/c mice that received one or two intraperitoneal inoculations with these JE vaccine candidates at a dose of 1 × 10 7 PFU per mouse produced neutralizing antibody and antibodies to the envelope (E) and nonstructural 1 (NS1) proteins as determined by radioimmunoprecipitation. Immunization with either of these vaccine candidates also induced JE virus-specific T lymphocytes that proliferated in response to stimulation with infectious virus and/or noninfectious viral antigens. Mice maintained detectable levels of neutralizing antibody and JE virus-specific memory T cells for at least 6 months after immunization with NYVAC-JEV and for 4 months after immunization with ALVAC-JEV. Cells induced to proliferate after stimulation with live virus contained specific CD8 + CTLs that lysed primary Balb/c mouse kidney cells infected with JE virus and P815 mastocytoma cells infected with a recombinant vaccinia virus expressing the premembrane (prM), E, and NS1 proteins. These CTLs also lysed P815 cells infected with vaccinia recombinants expressing prM and E, and those expressing E and NS1, but did not lyse P815 cells infected with a recombinant virus expressing only NS1, indicating that the CTLs mainly recognized E, but did not recognize NS1. These results demonstrate that both recombinant JE vaccines, NYVAC-JEV and ALVAC-JEV, induce JE virus-specific antibody and CTLs in mice.

West Nile virus (WNV) infections in vertebrates are generally acute but persistent infections have been observed. To investigate the ability of WNV to produce persistent infections, we forced subgenomic WNV replicons to replicate within a cell without causing cell death. Detailed analyses of these cell-adapted genomes revealed mutations within the nonstructural protein genes NS2A (D73H, M108K), NS3 (117Kins), NS4B (E249G) and NS5 (P528H). WNV replicons and WNVs harboring a subset of NS2A or NS3 mutations showed a reduction in genome replication, a reduction in antigen accumulation, a decrease in cytopathic effect, an increased ability to persist in cell culture and/or attenuation in vivo. Taken together, these data indicate that WNV with a defect in replication and an increased potential to persist within the host cell can be generated by point mutations at multiple independent loci, suggesting that persistent viruses could arise in nature.

Flaviviruses are emerging threats to public health worldwide. Recently, one flavivirus, West Nile virus (WNV), has caused the largest epidemic of viral encephalitis in US history. Like other flaviviruses, WNV is thought to cause a persistent infection in insect cells, but an acute cytopathic infection of mammalian cells. To study adaptation of WNV to persistently replicate in cell culture and generate a system capable of detecting antiviral compounds in the absence of live virus, we generated subgenomic replicons of WNV and adapted these to persistently replicate in mammalian cells. Here we report that adaptation of these replicons to cell culture results in a reduction of genome copy number, and demonstrate that hamster, monkey, and human cells that stably carry the replicons can be used as surrogates to detect the activity of anti-WNV compounds. Additionally, we have used these cells to investigate the interaction of WNV genomes with interferon (IFN). These studies demonstrated that IFN can cure cells of replicons and that replicon-bearing cells display lower responses to IFN than their IFN-cured derivatives.

Expression of antigenic fragments of the Japanese encephalitis virus envelope protein (E) in Escherichia coli has been used to define the boundaries of an antigenic domain that contains the binding sites for 10 anti-E monoclonal antibodies (MAbs). All of these antibodies neutralized the virus in vitro and some of them passively protected mice from a fatal virus challenge. We have shown previously that nine of these antibodies react with the antigenic determinants encoded by a 405 bp fragment of viral cDNA. To determine the amino acid sequences of specific determinants, truncated polypeptides were expressed as fusion proteins in E. coli following progressive Bal 31 exonuclease digestion of the 5' and 3' ends of the cDNA fragment. Examination of the immunoreactivity of these polypeptides revealed that the region from methionine 303 to tryptophan 396 was the shortest sequence capable of reacting with any of the 10 MAbs or with a polyclonal, antiviral hyperimmune mouse ascitic fluid. Biochemical tests showed that an intramolecular disulphide cross-linkage between cysteine 304 and cysteine 335 of the E protein sequence was required for presentation of the binding site(s) for these MAbs. Although this 95 amino acid antigenic domain appeared to be capable of forming several conformational neutralizing epitopes, it was not an effective immunogen for inducing neutralizing or protective antibodies in mice.

Sequence results are presented for a 3745-nucleotide region at the 5′ end of the dengue type-1 virus (DEN-1) genome. The strain characterized is a Western Pacific isolate from Nauru Island. The sequenced region contains the beginning of a continous open reading frame which specifies the capsid (C), membrane (M), and envelope (E) structural proteins and the nonstructural protein NS1. The sequences are compared with corresponding segments for seven other flaviviruses, including two of the three remaining dengue serotypes, DEN-2 and DEN-4. The results show the DEN-1 genome size and organization to be similar to those of other characterized flaviviruses and that major features of the individual proteins are conserved. It is of special interest that comparisons of the E glycoprotein sequences between the dengue serotypes (DEN-1, -2, -4) reveal only moderately greater sequence relatedness (63–68%) than occurs in comparisons of DEN-1 with five other flaviviruses (46–54%). For the other structural proteins, C and M, the relatedness values are 59–74% for comparisons between DEN-1 and the other dengue serotypes and 31–45% for comparisons between DEN-1 and the five other flaviviruses.

The expression of Japanese encephalitis virus (JE) cDNA in Escherichia coli has been used to study the functional organization of the viral genome. JE protein coding sequences were expressed in E. coli by subcloning random fragments of cloned cDNA (P. C. McAda, P. W. Mason, C. S. Schmaljohn, J. M. Dalrymple, T. L. Mason, and M. J. Fournier, 1987 , Virology 158, 348–360) into the bacteriophage λτ11 expression vector. Over 120 λτl1 recombinants expressing viral protein sequences as β-galactosidase fusion proteins were identified immunologically with monoclonal antibodies (MAbs) and polyclonal hyperimmune mouse ascites fluid (HMAF). This expression and immunological detection strategy has been used to (1) map viral protein coding sequences to the JE genome; (2) demonstrate that contiguous viral protein coding regions can be expressed as single polypeptides in E. coli , providing functional confirmation for a long viral open reading frame; (3) localize important antigenic domains within the envelope protein E; and (4) identify in JE-infected cells a form of the glycosylated nonstructural protein NS1 that contains a hydrophobic C-terminal extension encoded by portions of the “ns2a” region of the JE genome.

Approximately 10 kb of the estimated 10.9-kb genome of the Japanese encephalitis virus (JE; Nakayama strain) has been cloned as cDNA; the uncloned portion includes 430 bases at the 5′-terminus and 450 bases at the 3′-end. A map of the genome has been developed through nucleotide sequencing and in vivo expression with the Escherichia colt expression vector λτ11 and immunological identification. Sequence results for 4320 nucleotides suggest the JE genome organization is very similar to those of three other flaviviruses for which sequence information is available. Like the other flaviviruses, the JE proteins are encoded by a single open reading frame that continues uninterrupted throughout the region sequenced. Considerable homology exists between the JE RNA and protein sequences and those of the other characterized flaviviruses. Comparative nucleotide and (amino acid) homology values for the M-E-NS1-ns2 segment of JE are approximately MVE, 70% (80%), WN, 68% (76%), and YF, 50% (45%). Even greater homology is suggested when the protein hydrophobicity profiles are compared. The molecular relationships are consistent with the established serological relationships among JE, MVE, and WN viruses and argue that these flaviviruses may have been derived from a common evolutionary ancestor.