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Abstract: Background: Since its introduction into North America in 1999, West Nile virus (WNV) has spread rapidly across United States (US). Objective: To genetically analyze WNV isolates from US blood donors during 2002–2005. Study design: Full-length nucleotide (NT) sequences of WNV isolates from 23 US volunteer blood donors of different geographic areas from 2002 to 2005 were determined and analyzed. Results: Results indicated an overall lack of geographic pattern to WNV in US. Analyses of the viral genetic diversity demonstrated that the WNV evolved at approximately five NT substitutions and 0.8 amino acid (AA) mutations per genome per year. Comparison of the functional sequences of WNV genome showed a higher evolution rate in the coding region than in the non-coding region. Furthermore, a greater diversity was observed in the nonstructural proteins as compared to the structural proteins. Sequence alignment analysis revealed that the rapid spread of WNV in US was accompanied by the establishment of a dominant genetic variant with 11 conserved NT mutations. Conclusions: The establishment of a dominant genotype across US and the displacement and possible extinction of earlier progenitor genotypes appears to have resulted from the accumulation and fixation of 11 nucleotide mutations throughout the coding region of WNV genome. [Copyright &y& Elsevier]

Abstract: Background: Starting in 1999, the West Nile virus (WNV) epidemic represents the largest outbreak of arboviral encephalitis ever recorded in the U.S. The effective means to determine an infection are detection of viral nucleic acid and/or viral specific immunoglobulin, IgM and/or IgG. Objective: To develop a highly sensitive and specific TaqMan RT-PCR assay for the detection and quantification of WNV RNA of lineage 1 and lineage 2. Study design: A TaqMan RT-PCR primer–probe was designed to perfectly match target sequences of all sequenced WNV strains and isolates, which added a layer of protection against false-negative results due to strain variability. In addition, the inclusion of a low level RNA internal control (IC) in the assay increased the precision and accuracy of the assay. Results: By optimizing the RNA preparation procedure for increased WNV RNA recovery, together with optimizing the primer–probe and TaqMan conditions for improved amplification efficiency, we developed a highly sensitive assay with the detection limit of 10 copies/mL. To evaluate the assay, we tested plasma samples from 12 transfusion-transmitted implicated cases in 2002 and from 68 positive blood donors in 2003. All tested specimens were WNV positive. The viral load of 68 positive blood donor samples collected in 2003 ranged from 10 copies/mL to 67,000 copies/mL with a mean of 8100 copies/mL. Furthermore, high sensitivity of the assay was achieved without compromising specificity. All 100 routine donor samples tested negative. Conclusions: The assay results demonstrate that our in-house TaqMan RT-PCR procedure can detect and quantify WNV RNA of lineage1 and lineage 2 in human plasma with high sensitivity and specificity. [Copyright &y& Elsevier]