West Nile virus (WNV) is a mosquito borne member of the Flaviviridae family and is distributed throughout Africa, Asia, part of Southern Europe, United States, southern Canada and has recently been isolated in Mexico. It is a major cause of human infection and is responsible for a high frequency of inapparent infection in children and sometimes causes encephalitis in elders. In India, WNV has been isolated from sporadic human cases of encephalitis, bats and certain mosquito species. Some isolates can cause fatal infection in mice even by peripheral route. Determinant(s) and factors responsible for Flavivirus pathogenicity is still unresolved. To understand some of the determinants of peripheral pathogenicity neutralization escape mutants IVC3F10 1.2 and IF1A71.1 of highly pathogenic WNV (68856) isolate from bat were selected. The loss of peripheral pathogenicty of WN 68856 was found to be associated with the mutations in the envelope (E) protein. To elucidate the mechanism of loss of pathogenicity in mutants, the kinetics of growth of WNV was examined in murine peritoneal exudate cell culture (PEC) and assayed in Pig kidney (PS) cells. Cytokine expression in virus infected murine peritoneal cultures was studied using ELISA (BD Biosciences) according to the manufacturer's instructions. The concentration of nitrites in the supernatant culture fluid was taken as measure of NO production. Virus replication kinetics in PEC of wild type and two mutant strains was studied at MOI of 1 and 0.1 at 24 hr interval. Peak titers of wild type and mutants were similar. Wild type and mutant IVC3F10 1.2 virus showed significant titers within 24hrs and attained peak by 48hrs, however, mutant IF1A71.1 showed 100-1000 fold lower titer exhibiting slower replication kinetics during early period of replication in PEC. Tumor necrosis factor (TNF) á and interleukin (IL)-1â in the cell culture supernatant was up-regulated along with replication of in case of parent and IVC3F10. Although the peak titers were same, TNF á did not increase above the base line in PEC infected with IF1A71.1. Interferon (IFN) â and IL-6 were up-regulated when the virus titers were decreasing in wild and both mutant viruses. Nitric oxide production followed similar trend as TNF á. In cultures infected with parent and IVC3F10 1.2 the production of NO was found to be 60 and 50μM respectively whereas, IF1A71.1 showed 5 μM NO production. However, no increased induction of the anti-inflammatory cytokine such as IL-10 was observed in any of the infected cultures. These results indicate that slower growth kinetics might be partially responsible for loss of pathogenicity. IFN â and IL-6 secretion did not alter in pathogenic and nonpathogenic virus infected cultures. In case of TNF á and IL-1â secretion, nonpathogenic virus showed lower secretion and it did not correlate with virus titer indicating a role in pathogenic potential.
