Monocytes and B cells support active replication of Chandipura virus.

BACKGROUND: Interaction between immune system and Chandipura virus (CHPV) during different stages of its life cycle remain poorly understood. The exact route of virus entry into the blood and CNS invasion has not been clearly defined. The present study was undertaken to assess the population in PBMC that supports the growth of virus and to detect active virus replication in PBMC as well as its subsets. METHODS: PBMC subsets viz.: CD3(+), CD14(+), CD19(+), CD56(+)cells were separated and infected with CHPV. The infected cells were then assessed for transcription (N gene primer) and replication (NP gene primer) of CHPV by PCR. The supernatant collected from infected cells were titrated in Baby Hamster Kidney (BHK) cells to assess virus release. The cytokine and chemokine expression was quantified by flow cytometry. RESULTS: Amplification of N and NP gene was detected in CD14(+) (monocyte) and CD19(+) (B cell), significant increase in virus titre was also observed in these subsets. It was observed that, although the levels of IL-6 and IL-10 were elevated in CD14(+) cells as compared to CD19(+)cells, the differences were not significant. However the levels of TNFα and IL-8 were significantly elevated in CD14(+) cells than in CD19(+)cells. The levels of chemokine (CXCL9, CCL5, CCL2, CXCL10) were significantly elevated in CHPV infected PBMC as compared to uninfected cells. CCL2 and CXCL9 were significantly increased in CHPV infected CD14(+)cells as compared to CD19(+) cells. CONCLUSION: CD14(+)and CD19(+)cells support active replication of CHPV. High viral load was detected in CD14(+) cells infected with CHPV hence it might be the primary target cells for active replication of CHPV. An elevated levels of cytokines and chemokines observed in CD14(+) cells may help in predicting the pathogenecity of CHPV and possible entry into the central nervous system.

Development and characterization of reverse genetics system for the Indian West Nile virus lineage 1 strain 68856.

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.

Genetic characterization of Bagaza virus (BAGV) isolated in India and evidence of anti-BAGV antibodies in sera collected from encephalitis patients.

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.

Enteroviruses in patients with acute encephalitis, uttar pradesh, India.

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.

Detection and isolation of Japanese encephalitis virus from blood clots collected during the acute phase of infection.

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.