An outbreak of viral encephalitis occurred in northern India in 2006. Attempts to identify an etiologic agent in cerebrospinal fluid by using reverse transcription–PCR showed positivity to enterovirus (EV) in 66 (21.6%) of 306 patients. Sequencing and phylogenetic analyses of PCR products from 59 (89.3%) of 66 specimens showed similarity with EV-89 and EV-76 sequences.

During investigations into the outbreak of encephalitis in 1996 in the Kerala state in India, an arbovirus was isolated from a Culex tritaeniorhynchus mosquito pool. It was characterized as a Japanese encephalitis and West Nile virus cross-reactive arbovirus by complement fixation test. A plaque reduction-neutralization test was performed using hyperimmune sera raised against the plaque-purified arbovirus isolate. The sera did not show reactivity with Japanese encephalitis virus and were weakly reactive with West Nile virus. Complete open reading frame sequence analysis characterized the arbovirus as Bagaza virus (BAGV), with 94.80 % nucleotide identity with African BAGV strain DakAr B209. Sera collected from the encephalitic patients during the acute phase of illness showed 15 % (8/53) positivity for anti-BAGV neutralizing antibodies. This is the first report of the isolation of BAGV from India. The presence of anti-BAGV neutralizing antibodies suggests that the human population has been exposed to BAGV.An outbreak of Japanese encephalitis (JE) was reported from the Allapuzza, Thiruvanthapuram and Kottayam districts of Kerala state, India, during 1996. Only 33 % (50/ 150) of the sera collected from hospitalized cases were confirmed as JE by immunoglobulin M (IgM) ELISA. Other clinical specimens were not available for further investigations. Entomological investigations during the outbreak were carried out and 184 mosquito pools collected from the affected area were processed for isolation in 2-day-old Swiss mice by the intra-cranial route (Rodrigues et al., 1980;George et al., 1984). One pool from Culex tritaeniorhynchus showed sickness in inoculated mice. Brains from sick mice were harvested and suspended in 10 % bovalbumin phosphate saline. The suspensions were stored at 270 u C and designated as the arbovirus isolate (96363). The isolate showed cross-reactivity with anti-JE virus (JEV) and anti-West Nile virus (WNV) immune sera in a complement fixation (CF) test (Pavri & Ghosh, 1969; Rodrigues et al., 1980;Damle et al., 1998).The isolate did not react with immune sera raised against other circulating arboviruses, including Chandipura (Rhabdoviridae), Sindbis (Togaviridae), Chikungunya (Togaviridae), Kyasanur forest disease (Flaviviridae), Batai (Bunyaviridae) and Dengue (Flaviviridae) viruses (Paul et al., 1970; Rodrigues et al., 1980;George et al., 1984).In this study, we present the genetic characterization of the arbovirus isolate and serological analysis of available sera collected from encephalitis patients during 1996. The Institutional Animal Ethical Committee approved this work and ethical guidelines were strictly followed according to their recommendations. The arbovirus isolate was plaque-purified to rule out the possibility of isolation of both JEV and WNV from the mosquito pool. The mouse brain stock of the arbovirus isolate was passaged twice in porcine stable kidney (PS) cells to amplify the virus. A single plaque was selected from the first PS cell passage and then subjected to two sequential ...

Clinical specimens from an encephalitis outbreak in the Lakhimpur area of Uttar Pradesh, India, were investigated for identification and characterization of the etiologic agent. IgM capture ELISA showed recent Japanese encephalitis virus (JEV) infection. JEV isolation was attempted from white blood cells (WBCs) separated from blood clots of 12 patients (9 IgM positive and 3 negative) by serial co-culturing with phytohemagglutinin P-stimulated peripheral blood mononuclear leukocytes (PBMCs) obtained from pre-screened JEV sero-negative healthy individuals. JEV was isolated from two IgM-positive blood clots. Isolate 014178 was detected in WBCs and in the first passage of PBMCs by ELISA and reverse transcriptase-polymerase chain reaction. Isolate 014173 was detectable only after a second passage in PBMC co-culture. Sequence analysis of 346 nt of the C-prM region showed homology with JEV strain GP78. This is the first report on isolation of JEV from patient blood clots. Our study shows that the co-cultures of PBMCs separated from patient blood clots provide an additional source for JEV isolation.

Japanese encephalitis is an acute infection of the central nervous system caused by Japanese encephalitis virus (JEV). The importance of an effective humoral response in preventing JEV infection has already been established, although the contribution of cellular immunity remains unclear. This study used an experimental murine model to understand the protective effects of cell-mediated immunity in JEV infection. Fourteen-day-old mice adoptively transferred with JEV-immune splenocytes were found to be protected from peripheral JEV challenge. The survival rate was reduced when transferred cells were depleted of their CD4 + T-cell population.Correspondingly, increased protection was observed when JEV-primed isolated CD4 + T cells were transferred compared with isolated CD8 + T cells. Mice protected from JEV infection by the adoptive transfer of JEV-immune splenocytes had higher levels of immunomodulatory cytokines and decreased expression of pro-inflammatory cytokines. Concurrent with the increase in Th2 cytokines, JEV-specific IgM and IgG1 antibody titres were found to be elevated in protected mice. Taken together, these data indicate a definite role for CD4 + T cells in protection from lethal JEV infection in naïve 14-day-old mice. Induction of a Th2 cytokine response and IgG1 antibody probably achieves an immunomodulatory effect that results in the enhanced survival of these animals.

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