Pathogenesis of West Nile Virus in Humans

Generation of West Nile Virus Infectious Clones Containing Amino Acid Insertions Between Capsid and Capsid Anchor

“…Moreover, the only virus detected by RT-PCR following a single passage of this population in Vero cells was the smaller KTG➔MGV virus (Figure 4B), suggesting that the GLuc sequence, like the TAT(1-67) sequence, was unstable. This instability was further confirmed by the observation that a single passage of the day 4 p0 virus, which had produced only small plaques, yielded a virus stock of mixed small- and large-plaque phenotypes [26] and RT-PCR products of variable lengths (Figure 4B). …”

“…Vero cells were electroporated with in vitro- transcribed WNV-NY/TAT RNA. Although no cytopathic effects (CPE) were observed, a relatively high titer viral stock (p0) (2.5 × 10 6 plaque-forming units (pfu)/mL), with a plaque phenotype indistinguishable from that of wild-type WNV-NY [26], was recovered at 7 days post-electroporation. Passage of this viral stock in Vero cells generated a 4.9 × 10 7 pfu/mL (p1) stock.…”

“…The plaque phenotype of the recovered virus was highly variable. Some plaques were indistinguishable from those of wild-type WNV-NY, while others were significantly smaller [26], suggesting the virus population was mixed. Indeed, when RNA isolated from the infected Vero cells was used as template for RT-PCR, two predominate PCR products were generated (Figure 4A).…”

“…While only 75 pfu/mL of virus was recovered on day 4, the plaques had a uniformly small plaque phenotype [26], indicative of a single virus population. In contrast, virus recovered on day 7 displayed both small- and large-plaque phenotypes, similar to those observed in the previous isolation.…”

“…Numerous GLuc-reactive bands were detected in WNV-NY/GLuc(ALIC➔APC)-pp1 or -pp2-infected cells [26], indicating that the GLuc reporter was translated. However, we did not detect GLuc activity in cell lysates or culture supernatants of Vero cells infected with WNV-NY/GLuc(ALIC➔APC)-pp1 or -pp2 (Figure S3), suggesting that the virus does not produce functional GLuc.…”