The West Nile virus mutant spectrum is host-dependant and a determinant of mortality in mice

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.

Figure 1.

Mean titer of WNV during sequential passage in mosquitoes (A) and chickens (B). Each of three independent lineages (designated A–F) per host type is shown.

Figure 2.

Host‐dependence of the WNV mutant spectrum. WNV derived from an infectious cDNA clone was passaged 20 times in either chickens or mosquitoes, and the mutant spectrum was characterized at passages 1, 5, 10, 15 and 20. Panels A and C: proportion of nucleotides sequenced that differed from the specimen‐specific consensus sequence in chicken (A) and mosquito (C) passaged WNV. Open symbols indicate values for each of three replicate lineages, designated G, W and R for chickens and A, B, and C for mosquitoes. Solid line indicates background error rate as determined by analysis of clone‐derived WNV, dotted line indicates homologous field‐derived observation (Jerzak, Bernard et al., 2005). Panels B and D: proportion of clones that differed from the specimen‐specific consensus sequence and chicken (B) and mosquito (D) passaged WNV. Open symbols, solid and dotted lines are as above.

Figure 3.

Survival of mice infected with (A) chicken‐ and (B) mosquito‐passed WNV. Groups of eight six‐week‐old female C3H mice were inoculated subcutaneously in the left rear footpad with 10^2.5 PFU of WNV and survival was monitored. Mock‐inoculated mice (not shown) remained healthy for the duration of the study. Results were obtained from two independent trials, and the survival curve for mice inoculated with the parental (unpassed) WNV is the combined data from both trials. Lineages are designated R, G and W for chick‐passaged and A, B, and C for mosquito‐passaged lineages. (C): percentage mortality at the study conclusion was significantly negatively correlated with overall genetic diversity in the inoculum.