Intrahost genetic diversity was analysed in naturally infected mosquitoes and birds to determine whether West Nile virus (WNV) exists in nature as a quasispecies and to quantify selective pressures within and between hosts. WNV was sampled from ten infected birds and ten infected mosquito pools collected on Long Island, NY, USA, during the peak of the 2003 WNV transmission season. A 1938 nt fragment comprising the 39 1159 nt of the WNV envelope (E) coding region and the 59 779 nt of the non-structural protein 1 (NS1) coding region was amplified and cloned and 20 clones per specimen were sequenced. Results from this analysis demonstrate that WNV infections are derived from a genetically diverse population of genomes in nature. The mean nucleotide diversity was 0?016 % within individual specimens and the mean percentage of clones that differed from the consensus sequence was 19?5 %. WNV sequences in mosquitoes were significantly more genetically diverse than WNV in birds. No host-dependent bias for particular types of mutations was observed and estimates of genetic diversity did not differ significantly between E and NS1 coding sequences. Non-consensus clones obtained from two avian specimens had highly similar genetic signatures, providing preliminary evidence that WNV genetic diversity may be maintained throughout the enzootic transmission cycle, rather than arising independently during each infection. Evidence of purifying selection was obtained from both intra-and interhost WNV populations. Combined, these data support the observation that WNV populations may be structured as a quasispecies and document strong purifying natural selection in WNV populations. INTRODUCTIONWest Nile virus (WNV) (family Flaviviridae, genus Flavivirus) was first detected in North America in 1999 in the New York City area, where it caused an avian and equine epizootic and 68 cases of human disease (CDC, 1999;Lanciotti et al., 1999). It subsequently spread throughout the United States and into Canada, Mexico and the Caribbean (Blitvich et al., 2003;Dupuis et al., 2003;Austin et al., 2004). Recently, several studies have examined the course of WNV evolution since its introduction and have concluded that WNV remains a relatively homogeneous virus population, with the most divergent strains containing only a few nucleotide and/or amino acid substitutions (Anderson et al., 2001;Ebel et al., 2001aEbel et al., , 2004Lanciotti et al., 2002;Beasley et al., 2003;Davis et al., 2003). However, a single WNV genotype that differs from the introduced strain has arisen since 1999 and has become dominant, largely displacing previously circulating strains throughout North America Ebel et al., 2004). Therefore, although WNV remains relatively genetically homogeneous, it appears to be undergoing a process of adaptation to local transmission cycles. To date, no published studies have quantitatively examined the role of positive and/or purifying selection in WNV since its introduction into North America.The mechanisms that lead to population-level genet...

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.

In nature, arthropod-borne viruses (arboviruses) perpetuate through alternating replication in vertebrate and invertebrate hosts. The trade-off hypothesis proposes that these viruses maintain adequate replicative fitness in two disparate hosts in exchange for superior fitness in one host. Releasing the virus from the constraints of a two-host cycle should thus facilitate adaptation to a single host. This theory has been addressed in a variety of systems, but remains poorly understood. We sought to determine the fitness implications of alternating host replication for West Nile virus (WNV) using an in vivo model system. Previously, WNV was serially or alternately passed 20 times in vivo in chicks or mosquitoes and resulting viruses were characterized genetically. In this study, these test viruses were competed in vivo in fitness assays against an unpassed marked reference virus. Fitness was assayed in chicks and in two important WNV vectors, Culex pipiens and Culex quinquefasciatus. Chick-specialized virus displayed clear fitness gains in chicks and in Cx. pipiens but not in Cx. quinquefasciatus. Cx. pipiens-specialized virus experienced reduced fitness in chicks and little change in either mosquito species. These data suggest that when fitness is measured in birds the trade-off hypothesis is supported; but in mosquitoes it is not. Overall, these results suggest that WNV evolution is driven by alternate cycles of genetic expansion in mosquitoes, where purifying selection is weak and genetic diversity generated, and restriction in birds, where purifying selection is strong.