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The lineage I strain of West Nile virus (WNV) frequently causes human epidemics, including the recent outbreak in North America (Lanciotti et al., 1999, Science 286:2333–2337). As an initial step in studying the replication and pathogenesis of WNV, we constructed several cDNA clones of a WNV replicon derived from an epidemic strain (lineage I) isolated from the epicenter of New York City in the year 2000. Replicon RNAs were in vitro transcribed from cDNA plasmids and transfected into BHK-21 cells. RNA replication in transfected cells was monitored by immunofluorescence analysis (IFA) and 5′ nuclease real-time RT-PCR (TaqMan). The replicon RNAs contained large in-frame deletions (greater than 92%) of the C-prM-E structural region yet still replicated efficiently in BHK-21 cells. 5′ nuclease real-time RT-PCR showed that a great excess of plus-sense replicon RNA over the minus-sense RNA was synthesized in transfected cells. Replication efficiency decreased upon insertion of a green fluorescent protein (GFP) reporter gene driven by an internal ribosomal entry site (IRES) in the upstream end of the 3′ untranslated region of the replicon. Strong GFP expression was detected in cells transfected with a replicon containing IRES-GFP positioned in the plus-sense orientation. IFA showed that GFP and viral proteins were exclusively coexpressed in transfected cells. In contrast, no GFP fluorescence was observed in cells transfected with a replicon containing IRES-GFP positioned in the minus-sense orientation, despite high levels of synthesis of viral proteins and RNA in the cells. Substitution of the GFP gene in the plus-sense GFP replicon with the neomycin phosphotransferase gene allowed selection of geneticin-resistant cells in which WNV replicons persistently replicated without apparent cytopathic effect. These results suggest that WNV replicons may serve as a noncytopathic RNA virus expression system and should provide a valuable tool to study WNV replication. [Copyright &y& Elsevier]

Abstract: Genome cyclization is essential for flavivirus replication. We used RNases to probe the structures formed by the 5′-terminal 190 nucleotides and the 3′-terminal 111 nucleotides of the West Nile virus (WNV) genomic RNA. When analyzed individually, the two RNAs adopt stem-loop structures as predicted by the thermodynamic-folding program. However, when mixed together, the two RNAs form a duplex that is mediated through base-pairings of two sets of RNA elements (5′CS/3′CSI and 5′UAR/3′UAR). Formation of the RNA duplex facilitates a conformational change that leaves the 3′-terminal nucleotides of the genome (position −8 to −16) to be single-stranded. Viral NS5 binds specifically to the 5′-terminal stem-loop (SL1) of the genomic RNA. The 5′SL1 RNA structure is essential for WNV replication. The study has provided further evidence to suggest that flavivirus genome cyclization and NS5/5′SL1 RNA interaction facilitate NS5 binding to the 3′ end of the genome for the initiation of viral minus-strand RNA synthesis. [Copyright &y& Elsevier]

Molecular epidemiologic studies of North American (NA) West Nile virus (WNV; Flaviviridae, Flavivirus) have documented the displacement of the introduced NY99 genotype with WN02. In addition, these studies have shown that particular substitutions are under positive selection. One occurs in the C-terminus of the NS4A coding sequence and results in a valine to methionine substitution at position nine of the 2K peptide. 2K-V9M confers the ability to overcome superinfection exclusion in vitro . We hypothesized that WNV strains bearing 2K-V9M have higher fitness than wildtype in Culex quinquefasciatus mosquitoes. Although infection rates and viral titers were not significantly different, virus dissemination rates were significantly higher with WNV 2K-V9M. As a super-infecting virus, WNV 2K-V9M was more successful than wildtype, however, in a mixed infection, 2K-V9M was not. These data support observations that 2K-V9M confers a context-specific selective advantage in mosquitoes and provides an in vivo mechanism for its positive selection. [ABSTRACT FROM AUTHOR]

Abstract: Most RNA viruses exist in their hosts as a heterogeneous population of related variants. Due to error prone replication, mutants are constantly generated which may differ in individual fitness from the population as a whole. Here we characterize three WNV isolates that contain, along with full-length genomes, mutants with large internal deletions to structural and nonstructural protein-coding regions. The isolates were all obtained from lorikeets that died from WNV at the Rio Grande Zoo in Albuquerque, NM between 2005 and 2007. The deletions are approximately 2kb, in frame, and result in the elimination of the complete envelope, and portions of the prM and NS-1 proteins. In Vero cell culture, these internally deleted WNV genomes function as defective interfering particles, reducing the production of full-length virus when introduced at high multiplicities of infection. In mosquitoes, the shortened WNV genomes reduced infection and dissemination rates, and virus titers overall, and were not detected in legs or salivary secretions at 14 or 21days post-infection. In mice, inoculation with internally deleted genomes did not attenuate pathogenesis relative to full-length or infectious clone derived virus, and shortened genomes were not detected in mice at the time of death. These observations provide evidence that large deletions may occur within flavivirus populations more frequently than has generally been appreciated and suggest that they impact population phenotype minimally. Additionally, our findings suggest that highly similar mutants may frequently occur in particular vertebrate hosts. [Copyright &y& Elsevier]

Abstract: The West Nile virus (WNV) genome contains a single RNA-dependent RNA polymerase (RdRp) gene, which is responsible for replication of the viral genome and, as such, is an important target for antiviral therapy. Viral RdRps are known to lack proofreading capabilities and as a result viruses such as WNV exist as a mixture of viral genotypes within an infection, enabling the virus to readily emerge and adapt to new host environments. To test the consequences of subtle structural alterations remote from the RdRp active-site, the following single point mutations were engineered in the WNV NS5 RdRp coding region: T363N, A365N, and T537I; these mutations were selected in an effort to stabilize the secondary structural elements near the rNTP binding pocket of the RdRp. Mutant viruses were tested in vitro on Vero, C6/36, Culex tarsalis and DF-1 cell types and in vivo in one day old chickens and Culex pipiens mosquitoes. Plaque morphology was affected by each mutation and growth and RNA replication kinetics were altered as well. Our results demonstrate that subtle alteration of the RdRp protein away from the active site can have a significant overall biological effect on WNV fitness, and that this effect can be host-dependent. [Copyright &y& Elsevier]

Abstract: We report a genetic interplay among three pairs of long-distance RNA interactions that are involved in West Nile virus (WNV) genome cyclization and replication: 5′CS/3′CSI (conserved sequence), 5′UAR/3′UAR (upstream AUG region), and 5′DAR/3′DAR (downstream AUG region). Deletion of the complete 3′CSI element is lethal for WNV replication, but the replication of the 3′CSI deletion virus could be rescued by second site mutations. Functional analysis, using a genome-length RNA and replicon, mapped the compensatory mutations to the 5′UAR/3′UAR and 5′DAR/3′DAR regions. Biochemical analysis showed that the 3′CSI deletion abolished the 5′ and 3′ RNA interaction of the genome; the compensatory mutations could partially restore the 5′ and 3′ genome cyclization. These results demonstrate, for the first time, that a flavivirus without 3′CSI could restore genome cyclization and viral replication through enhancement of the 5′UAR/3′UAR and 5′DAR/3′DAR interactions. [Copyright &y& Elsevier]

Abstract: Lycorine potently inhibits flaviviruses in cell culture. At 1.2-μM concentration, lycorine reduced viral titers of West Nile (WNV), dengue, and yellow fever viruses by 102- to 104-fold. However, the compound did not inhibit an alphavirus (Western equine encephalitis virus) or a rhabdovirus (vesicular stomatitis virus), indicating a selective antiviral spectrum. The compound exerts its antiviral activity mainly through suppression of viral RNA replication. A Val→Met substitution at the 9th amino acid position of the viral 2K peptide (spanning the endoplasmic reticulum membrane between NS4A and NS4B proteins) confers WNV resistance to lycorine, through enhancement of viral RNA replication. Initial chemistry synthesis demonstrated that modifications of the two hydroxyl groups of lycorine can increase the compound''s potency, while reducing its cytotoxicity. Taken together, the results have established lycorine as a flavivirus inhibitor for antiviral development. The lycorine-resistance results demonstrate a direct role of the 2K peptide in flavivirus RNA synthesis. [Copyright &y& Elsevier]

Abstract: To determine if West Nile virus (WNV) infection of insect cells induces a protective RNAi response, Drosophila melanogaster S2 and Aedes albopictus C6/36 cells were infected with WNV, and the production of WNV-homologous small RNAs was assayed as an indicator of RNAi induction. A distinct population of ~25 nt WNV-homologous small RNAs was detected in infected S2 cells but not C6/36 cells. RNAi knockdown of Argonaute 2 in S2 cells resulted in slightly increased susceptibility to WNV infection, suggesting that some WNV-homologous small RNAs produced in infected S2 cells are functional small interfering RNAs. WNV was shown to infect adult D. melanogaster, and adult flies containing mutations in each of four different RNAi genes (Argonaute 2, spindle-E, piwi, and Dicer-2) were significantly more susceptible to WNV infection than wildtype flies. These results combined with the analysis of WNV infection of S2 and C6/36 cells support the conclusion that WNV infection of D. melanogaster, but perhaps not Ae. albopictus, induces a protective RNAi response. [Copyright &y& Elsevier]