Generating West Nile Virus from an Infectious Clone

Abstract

WNV infectious clones are valuable tools for elucidating WNV biology. Nevertheless, relatively few infectious WNV clones have been generated because their construction is hampered by the instability of flaviviral genomes. More recently, advances in cloning techniques as well as the development of several two-plasmid WNV infectious clone systems have facilitated the generation of WNV infectious clones. Here we described a protocol for recovering WNV from a two-plasmid system. In this approach, large quantities of these constructs are digested with restriction enzymes to produce complementary restriction sites at the 3' end of the upstream fragment and the 5' end of the downstream fragment. These fragments are then annealed to produce linear template for in vitro transcription to synthesize infectious RNA. The resulting RNA is transfected into cells and after several days WNV is recovered in the culture supernatant. This method can be used to generate virus from infectious clones encoding high- and low-pathogenicity strains of WNV, as well as chimeric virues.

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 4 B), 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 4 B).…”

“…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 4 A).…”

“…In an attempt to isolate a pure WNV-NY/GLuc population, we repeated the electroporation of Vero cells with WNV-NY/GLuc RNA and collected culture supernatant at 4 and 7 days post-transfection. 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.…”