Because of limited volume of plasma specimens available, viruses were isolated from Vero cell cultures for sequence analysis. We assessed the potential of the viral isolation procedure to cause mutations in the viral genome by comparison of sequences of the structural region of 6 specimens and their respective isolates after 3 passages in Vero cells. There were no changes in the RNA sequences obtained in each original plasma sample and the RNA sequences obtained in each of the passages in culture.
We investigated the genetic variability of 30 WNV isolates collected from plasma specimens from nucleic acid amplification testing–positive blood donors in 13 states during 2002–2005 (). Isolates were generated by cultivation in Vero cells. The fragment that encompasses the 5′-UTR, all structural genes, and part of NS1 (bp 1–2,685) from all 30 specimens were subjected to genomic sequencing. Eight of 30 isolates were fully sequenced.
The Appendix Table
compares conserved nucleotide mutations and deduced amino acid substitutions identified in structural regions of all 30 isolates with the WN-NY99 isolate (AF196835). Approximately 80% of the nucleotide changes in structural regions were transitions (T ↔ C) and 75% were silent mutations. All mutations in the preM and membrane (M) regions were silent, and 16 isolates shared the transition 660 C → T. Several WNV strains from Europe and Asia, as well as Kunjin virus isolates, also had T at position 660, but both the prototype WN-NY99 and the IS-98 (AF481864) isolates contain 660 C. Twenty-nine of 30 isolates shared 2 conserved nucleotide mutations in the env
gene: 1442 T → C (Val 159 → Ala) and 2466 C → T that differentiates the new dominant genotype, WN02, from the preceding genotype WN99.
Construction of a phylogenetic tree by the maximum parsimony method () showed the degree of divergence of isolates from WN-NY99. The average nucleotide divergence for structural genes has increased from 0.18% in 2002 to 0.37% in 2005.
Figure 1 Phylogenetic analyses based on maximum parsimony comparing the 2,685-bp nucleotide sequence, including the complete structural and the 5′-untranslated region of prototype West Nile virus (WNV) strain WN-NY99 with 30 WNV isolates collected during (more ...)
The env protein has several biologic roles, which include viral entry, virion assembly and release, agglutination of erythrocytes, and induction of B- and T-cell responses that are associated with protective immunity. Thus, this protein may be involved in WNV evolution. The phylogenetic tree shown in was constructed by maximum parsimony analysis with env gene sequences from US isolates from 1999–2006 in GenBank and sequences of human isolates in our study. The isolates clustered in 2 clades correlated with the parsimony-revealing mutation sites at positions 1442 and 2466.
Figure 2 Distance analysis of envelope glycoprotein of West Nile virus isolates collected during 1999–2006 epidemics in the United States. Phylogram is based on maximum parsimony analysis of complete nucleotide sequences of the envelope gene. Diamonds (more ...)
Although preM, M, and env sequences show adequate phylogenetic representation, we also analyzed 8 complete genomes of WNV isolates for stronger evidence of evolutionary relationships between isolates and additional mutations that may have implications in phenotypic properties of these isolates. Nucleotide changes and deduced amino acid substitutions of complete genomic sequences from 8 isolates are shown in and , respectively. When compared with WN-NY99 sequences, these sequences showed an increased number of nucleotide mutations. FDA/Hu-02 isolated in 2002 showed 20 nt mutations plus 1 insertion at position 10497, and 5 mutations resulted in amino acid substitutions on the basis of deduced sequence of viral polyprotein. ARC10–02 isolated in 2002 had 22 mutations, 3 of which resulted in amino acid substitutions. These 2 isolates showed ≈0.2% nucleotide divergence.
Deduced amino acid substitutions in 8 completely sequenced West Nile virus isolates compared with isolate WN-NY99*
Nucleotide mutations conserved in fully sequenced West Nile virus isolates from 2002– 2005 epidemics in the United States compared with isolate WN-NY99*
The 2003 isolate BSL5–03 showed 39 mutations (nucleotide divergence 0.35%), 7 of which were associated with predicted amino acid substitutions. These 3 isolates from 2002 and 2003 had 11 common mutations: 2 were in env (1442 T → C resulting in Val 449 → Ala and 2466 C → T, a silent mutation); 8 were in the NS regions (4146 A → G in NS2A; 2 C → T transitions at positions 4803 and 6138 in NS3; 6996C → T and 7015T → C in NS4B; T → C transitions at positions 7938 and 8811 and 9352 C → T at NS5); and 1 in the 3-′UTR (10851 A → G).
The 2004 isolate BSL5–04 had 42 mutations with 7 aa substitutions. The isolates from 2005 were as follows: GCTX1-05 had 56 mutations with 17 aa substitutions; GCTX2-05 had 41 mutations with 3 aa substitutions; BSL2–05 had 44 mutations with 10 aa substitutions plus a deletion of 14 nt (10480 to 10493) in the 3′-UTR; and BSL13–05 had 48 mutations with 8 aa substitutions. Isolates from 2004 and 2005 shared a nucleotide mutation 6721 G → A, which resulted in amino acid substitution Ala2209Thr in the NS4A. Four isolates from 3 consecutive years (BSL5–03 from 2003, BSL5–04 from 2004, GCTX1 and BSL13–05 from 2005), shared an amino acid substitution (Lys2842Arg) in NS5. Three isolates from 2005 (GCTX1–05, BSL2–05 and BSL13–05) plus 1 isolate from 2004 (BSL5–04) also shared a silent mutation at position 8550 C → T.
The overall nucleotide divergence from the WN-NY99 isolates from 2003, 2004, and 2005 showed a steady but small increase (BSL5–03, 035%; BSL5–04, 0.38%; BSL2–05, 0.39%; BSL13–05, 0.43%; GCTX1–05, 0.5%; and GCTX2–05, 0.37%). These findings suggest relative stasis in WNV divergence. The 8 completely sequenced isolates shared conserved nucleotide mutations in the preM, M, env, NS2A, NS3, NS4B, and NS5 genes and the 3′-UTR (). The largest number of conserved mutations was in the NS3 and NS5 genes. No conserved mutations were observed in the core, M, NS1, and NS2B genes or the 5′-UTR.