Genetic stability of A103G nucleotide substitution after ten serial passages in the absence of NH4Cl.

Mutant WNVs (Res population or A103G virus recovered from infectious clone pFLWNV-A103G) were subjected to 10 serial passages on Vero cells. Viral RNA was extracted and amplified by RT-PCR and the nucleotide sequence of the genomic region encoding the structural proteins was determined. Nucleotide replacements found on the genomic sequence of either Res or A103G subjected to 10 serial passages in the absence of NH4Cl were compared to WNV strain NY99-flamingo 382–99 [10] and to WNV recovered from plasmid pFLWNV [42] respectively. Amino acid replacements are shown in bold within parentheses. The presence of nucleotide substitution A103G was analyzed by nucleotide sequencing on different biological clones from each population subjected to 10 serial passages in the absence of NH4Cl. Each biological clone corresponded to an individual lysis plaque produced by infection in semisolid agar medium. N.D. Not done.