A phylogenetic approach to following West Nile virus in Connecticut

Abstract

The 1999 outbreak of West Nile (WN) virus in the northeastern United States was the first known natural occurrence of this flavivirus in the Western Hemisphere. In 1999 and 2000, 82 independent Connecticut WN virus isolates were cultured from nine species of birds, five species of mosquitoes, and one striped skunk. Nucleotide sequences obtained from these isolates identified 30 genetic changes, compared with WN-NY99, in a 921-nt region of the viral genome beginning at nucleotide position 205 and ending at 1125. This region encodes portions of the nucleocapsid and envelope proteins and includes the entire coding regions for the premembrane and membrane proteins. Amino acid changes occurred at seven loci in six isolates relative to the WN-NY99 strain. Although 34 of the isolates showed sequences identical to the WN-NY99 isolate, we were able to show geographical-based clusters of mutations. In particular, 26 isolates were characterized by mutation of C to T at position 858. This group apparently originated in Stamford, CT and disseminated to sites located as far as 54 miles from Stamford. Sequences of WN virus isolated from both brain and heart tissues from the same avian host were identical in all 14 tested individual birds, suggesting that the mutations we have documented are real and not caused by culture, RNA extraction, or PCR procedures. We conclude that this portion of the viral genome will enable us to follow the geographical and temporal movement of variant WN virus strains as they adapt to North America.

Figure 1

Unrooted phylogram based on maximum parsimony analysis (heuristic search) comparing a reverse transcriptase–PCR of a 921-nt sequence of the WN-NY99 isolate (GenBank accession no. AF196835) with 82 WN isolates from Connecticut. Branch lengths represent 0, 1, 2, or 3 nucleotide changes. Neighbor-joining analysis gave identical results. Each mutation is identified above the branch as a nucleotide of the WN virus genome. Amino acid changes resulting from nucleotide changes also are identified above the line. Isolation number, host, Connecticut town where host was collected, and WN strain identification number are provided for each isolate. GenBank accession numbers are provided for each isolate in the data deposition footnote.

Figure 2

Map of Connecticut showing county boundaries and the distribution of WN virus isolates with sequences identical to WN-NY99 (□) and isolates containing the C to T mutation at genome position 858 shown in the majority of the isolates from Stamford (●).