Infectious cDNA Clone of the Epidemic West Nile Virus from New York City

Abstract

We report the first full-length infectious clone of the current epidemic, lineage I, strain of West Nile virus (WNV). The full-length cDNA was constructed from reverse transcription-PCR products of viral RNA from an isolate collected during the year 2000 outbreak in New York City. It was cloned into plasmid pBR322 under the control of a T7 promoter and stably amplified in Escherichia coli HB101. RNA transcribed from the full-length cDNA clone was highly infectious upon transfection into BHK-21 cells, resulting in progeny virus with titers of 1 ؋ 10 9 to 5 ؋ 10 9 PFU/ml. The cDNA clone was engineered to contain three silent nucleotide changes to create a StyI site (C to A and A to G at nucleotides [nt] 8859 and 8862, respectively) and to knock out an EcoRI site (A to G at nt 8880). These genetic markers were retained in the recovered progeny virus. Deletion of the 3-terminal 199 nt of the cDNA transcript abolished the infectivity of the RNA. The plaque morphology, in vitro growth characteristics in mammalian and insect cells, and virulence in adult mice were indistinguishable for the parental and recombinant viruses. The stable infectious cDNA clone of the epidemic lineage I strain will provide a valuable experimental system to study the pathogenesis and replication of WNV.

Differential Replication of Pathogenic and Nonpathogenic Strains of West Nile Virus within Astrocytes

“…Working stocks of WNV-NY strain 3356 were generated from an infectious clone, pFL-WNV (19). Briefly, infectious particles were recovered as previously described (19), passaged once in 293 cells at a low multiplicity of infection (MOI), and subsequently passaged in Vero cells.…”

“…Briefly, infectious particles were recovered as previously described (19), passaged once in 293 cells at a low multiplicity of infection (MOI), and subsequently passaged in Vero cells. WNV-MAD78 was obtained from the World Reference Center of Emerging Viruses and Arboviruses (Galveston, TX).…”

Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

“…There is a common difficulty in the construction of full-length clones of flaviviruses, because the plasmids containing a full-length cDNA of these viruses are often unstable during propagation in E. coli. Therefore, by using low-copy-number plasmids and specific bacterial hosts, stable full-length infectious clones have been developed for several flaviviruses (Gritsun and Gould, 1998;Hayasaka et al, 2004;Kinney et al, 1997;Shi et al, 2002;Yamshchikov et al, 2001;Yun et al, 2003). In this study, the low-copy-number plasmid pACNR was used for the construction of the full-length OHFV cDNA.…”

IS-98-ST1 West Nile virus derived from an infectious cDNA clone retains neuroinvasiveness and neurovirulence properties of the original virus

“…WNV is maintained in endemic cyles in Africa, West Asia, Russia, India and Europe, and more recently in North America [3]. Phylogenetic analysis has permitted identification of lineages 1 (divided into clades 1a to 1b) to 5 [12,13].…”

“…Phylogenetic analysis has permitted identification of lineages 1 (divided into clades 1a to 1b) to 5 [12,13]. Lineage 1 WNV strains have been reported in many regions, including Africa, Europe, the Middle East, Russia, India, and North America since 1999 [3]. WNV subsequently spread to South America [14].…”