Role of the mutant spectrum in adaptation and replication of West Nile virus

Abstract

West Nile virus (WNV) has successfully spread throughout the USA, Canada, Mexico, the Caribbean and parts of Central and South America since its 1999 introduction into North America. Despite infecting a broad range of both mosquito and avian species, the virus remains highly genetically conserved. This lack of evolutionary change over space and time is common with many arboviruses and is frequently attributed to the adaptive constraints resulting from the virus cycling between vertebrate hosts and invertebrate vectors. WNV, like most RNA viruses studied thus far, has been shown in nature to exist as a highly genetically diverse population of genotypes. Few studies have directly evaluated the role of these mutant spectra in viral fitness and adaptation. Using clonal analysis and reverse genetics experiments, this study evaluated genotype diversity and the importance of consensus change in producing the adaptive phenotype of WNV following sequential mosquito cell passage. The results indicated that increases in the replicative ability of WNV in mosquito cells correlate with increases in the size of the mutant spectrum, and that consensus change is not solely responsible for alterations in viral fitness and adaptation of WNV. These data provide evidence of the importance of quasispecies dynamics in the adaptation of a flavivirus to new and changing environments and hosts, with little evidence of significant genetic change.

Fig. 1

Genetic diversity of WNV generated by in vitro passage in C6/36 cells (CP), DF-1 cells (DP) and in C6/36 cells followed by DF-1 cells (AP1). Diversity values (%) were determined by clonal analyses of the region containing nt 1311–3248 (E/NS1). (a) Nucleotide diversity and amino acid diversity represent the percentage of changes per sequenced units. (b) Sequence diversities represent the percentage of genotypes different from the consensus sequence. Filled bars, nucleotide diversity; empty bars, amino acid diversity.

Fig. 2

Growth of unpassaged (◆), C6/36-passage 40 (○) and alternate-passages (▲) WNV in C6/36 and DF-1 cell cultures. AP1 refers to WNV passaged 39 times in C6/36 cells followed by a single passage in DF-1 cells. The m.o.i. for all growth curves was 0.01 p.f.u. per cell. Results are presented as the means ± sd of duplicate assays. (a) WNV growth in C6/36 cell culture. (b) WNV growth in DF-1 cell culture.

Fig. 3

Growth of unpassaged (◆) and DF-1-passaged (□, DP10; △, DP20) WNV in C6/36 and DF-1 cell culture. The m.o.i. for all growth curves was 0.01 p.f.u. per cell. Results are presented as the means ± sd of duplicate assays. (a) WNV growth in C6/36 cell culture. (b) WNV growth in DF-1 cell culture.

Fig. 4

Growth of full-length WNV infectious clone (–––) and WNV mutants (- - -) in C6/36, DF-1 and Vero cell cultures. Single mutants are WNV A1712G and WNV G6687T. WNV E/NS4 contains the mutations A1712G and G6687T. The m.o.i. for all growth curves was 0.01 p.f.u. per cell. Results are presented as the means ± sd of duplicate assays.