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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 © 2012 Elsevier Inc. All rights reserved.)

Abstract: To investigate differential evolutionary rates and selective forces of WNV in hosts and vectors, we measured the genetic diversity that arose during alternating passage in mosquitoes and birds. Within-host genetic diversity was monitored in each of three experimentally passed replicates, and the complete genome sequence of each WNV strain was determined after passage. The intrahost genetic diversity that arose during alternating passage was significantly greater than the diversity generated during chicken-only passage and similar to mosquito-only passage. d N /d S ratios suggested purifying selection similar to chick-passed virus, but not to mosquito-passed virus. Thus, the abundant genetic variation contributed to WNV populations through infection of mosquitoes and the strong purifying selection contributed by infection of birds may be maintained despite frequent host switching. [Copyright &y& Elsevier]

Abstract: A reporting replicon of West Nile virus (WN) was used to distinguish between the function of the 3′ untranslated region (UTR) in viral translation and RNA replication. Deletions of various regions of the 3′ UTR of the replicon did not significantly affect viral translation, but abolished RNA replication. A systematic mutagenesis showed that the flavivirus-conserved penta-nucleotide (5′-CACAG-3′ located at the top of the 3′ stem-loop of the genome) requires a specific sequence and structure for WN RNA synthesis, but not for viral translation. (i) Basepair structure and sequence at the 1st position of the penta-nucleotide are critical for RNA replication. (ii) The conserved nucleotides at the 2nd, 3rd, and 5th positions, but not at the 4th position of the penta-nucleotide, are essential for RNA synthesis. (iii) The nucleotide U (which is partially conserved in the genus Flavivirus) immediately downstream of the penta-nucleotide is not essential for viral replication. [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: West Nile virus is similar to most other RNA viruses in that it exists in nature as a genetically diverse population. However, the role of this genetic diversity within natural transmission cycles and its importance to virus perpetuation remain poorly understood. Therefore, we determined whether highly genetically diverse populations are more fit compared to less genetically diverse WNV populations. Specifically, we generated three WNV populations that varied in their genetic diversity and evaluated their fitness relative to genetically marked control WNV in vivo in Culex quinquefasciatus mosquitoes and chickens. Our results demonstrate that high genetic diversity leads to fitness gains in vector mosquitoes, but not chickens. [Copyright &y& Elsevier]

Abstract: West Nile virus (WNV) is a mosquito-borne pathogen. During replication, WNV acquires different carbohydrates and lipid membranes, depending on its mosquito or vertebrate hosts. Consequently, WNV derived from mosquito and vertebrate cell lines differ in their infectivity for dendritic cells (DCs) and induction of type I interferon (IFN-α/β) in vitro. We evaluated the pathogenesis of WNV derived from mosquito (WNVC6/36) and vertebrate (WNVBHK) cell lines in mice. The tissue tropism, infectivity, clinical disease, and mortality did not differ for mice inoculated with WNVC6/36 or WNVBHK, and there were only minor differences in viral load and serum levels of IFN-α/β. The replication kinetics of WNVC6/36 and WNVBHK were equivalent in primary DCs and skin cells although primary DCs were more susceptible to WNVC6/36 infection than to WNVBHK infection, suggesting that less virus is produced per infected cell for WNVC6/36. In conclusion, viral source has minimal effect on WNV pathogenesis in vivo. [Copyright &y& Elsevier]

Abstract: To define the impact of mosquitoes and birds on intrahost WNV population dynamics, the mutant spectra that arose as a result of 20 serial in vivo passages in Culex pipiens and young chickens were examined. Genetically homogeneous WNV was serially passaged 20 times in each host. Genetic diversity was greater in mosquito-passaged WNV compared to chicken-passaged WNV. Changes in the viral consensus sequence occurred in WNV passaged in mosquitoes earlier and more frequently than in chicken-passaged WNV. Analysis of synonymous and nonsynonymous variation suggested that purifying selection was relaxed during passage in mosquitoes. Mortality in mice was significantly negatively correlated with the size of the WNV mutant spectrum. These studies suggest that mosquitoes serve as sources for WNV genetic diversity, that birds are selective sieves, and that both the consensus sequence and the mutant spectrum contribute to WNV phenotype. [Copyright &y& Elsevier]