title: Development and characterization of reverse genetics system for the Indian West Nile virus lineage 1 strain 68856.
abstract: Our previous studies on West Nile virus (WNV) strains isolated from human patients in India suggested substantial variation at the genetic level reflecting their variable pathogenesis. This study describes the development of reverse genetics system for a neurovirulent WNV isolate 68856 and its characterization. Full length viral cDNA was cloned into bacterial artificial chromosome (BAC) under the transcription control of T7 promoter. The RNA transcripts obtained by in vitro transcription were infectious in mammalian cells upon transfection. Cytopathic effect caused by synthetic RNA transcripts in mammalian cells, detection of cell associated viral protein after transfection and recovery of genetic markers in the progeny virus genome marked the successful development of reverse genetics system for WNV. Replication potential and plaque morphology of newly expressed virus along with its antigenic cross reactivity with the parental virus suggests synthesis of biologically identical replicative virus. Comparative neuropathogenesis studies in murine model indicated that the three genetic changes occurred in the recombinant virus during in vitro transcription has no impact on viral pathogenesis. The stable infectious cDNA clone generated from the neurovirulent Indian WNV strain will serve as a valuable experimental tool to study the viral factors contributing towards pathogenesis, host-virus interaction and immune evasion.
authors.affiliation: Encephalitis Group, National Institute of Virology, Microbial Containment Complex, Pashan, Pune 411021, India. Electronic address: dayapavitrakar@gmail.com.
authors.forename: Daya V
authors.initials: DV
authors.lastname: Pavitrakar
authors.affiliation: Encephalitis Group, National Institute of Virology, Microbial Containment Complex, Pashan, Pune 411021, India. Electronic address: vmayachit@gmail.com.
authors.forename: Vijay M
authors.initials: VM
authors.lastname: Ayachit
authors.affiliation: Encephalitis Group, National Institute of Virology, Microbial Containment Complex, Pashan, Pune 411021, India. Electronic address: sonal.mundhra@gmail.com.
authors.forename: Sonal
authors.initials: S
authors.lastname: Mundhra
authors.affiliation: Encephalitis Group, National Institute of Virology, Microbial Containment Complex, Pashan, Pune 411021, India. Electronic address: vpbondre@gmail.com.
authors.forename: Vijay P
authors.initials: VP
authors.lastname: Bondre
mesh_terms: D000818:Animals; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D006801:Humans; D007194:India; D051379:Mice; D023421:Models, Animal; D059386:Reverse Genetics / Q000379:methods*; D014902:West Nile virus / Q000235:genetics* / Q000276:immunology
chemical_list: D018076:DNA, Complementary
keywords: Bacmid vector; Encephalitis; Flavivirus; Infectious cDNA clone; Reverse genetics system; West Nile virus