A total of 326 clinical samples (185 blood and 141 cerebrospinal fluid [CSF]) were collected from the 326 patients who had a diagnosis of encephalitis. Two sets of blood samples, with and without anticoagulant, were collected for virus isolation and serologic tests. All serum and CSF samples were screened for JEV-specific immunoglobulin M (IgM) and IgG by using an in-house dipstick ELISA that incorporated nitrocellulose as the solid phase. Purified viral antigen was obtained from culture supernatant of infected C6/36
cultures by sucrose density gradient ultra centrifugation (4–6
). Results were confirmed by using an in-house IgM capture ELISA (7
JE-specific RNA was detected by using the Access quick one-step reverse transcription (RT)–PCR kit (Promega, Madison, WI, USA) with the primer pairs JED3S: ATG CGC GGA TCC GAC AAA CTG GCC CTG AA (1839–1867) and JED3C: GGG GAA GCT TCG TGC TTC CAG CTT TGT CC (2193–2165) on the basis of the sequence in domain III of the E gene of strain JaOArS982 (8
Virus isolation was attempted in C6/36
) from RT-PCR– and IgM-positive serum and CSF samples according to standard protocol (5
). Double-stranded sequencing of domain III of the E gene of JEV was performed on an ABI 310 sequencer (Applied Biosystems, Foster City, CA, USA) with the BigDye Terminator cycle sequencing ready reaction kit. The phylogenetic tree was constructed with the neighbor-joining method with bootstrap analysis of 1,000 replicates with the MEGA version 2.1 program (9
Rural populations between the ages of 3 months and 15 years were affected; almost 50% of children 6–10 years of age were affected, and 35% of children <5 years of age were affected. The epidemic affected boys and girls at a ratio of 1.9 to 1. The overall case-fatality ratio was 23%. Children dominated the case load because most adults in the area are immune to the virus. The trend of the epidemic showed that most cases were reported from the first to third weeks of October. Clinical history showed that all patients had fever (temperatures 38.5°C–40°C); prominent symptoms included severe headache, convulsions, and vomiting, leading to paralysis, coma, and death.
Analysis indicated an overall positivity of 50% of serum samples and 30% of CSF samples. The antibody profile of the serum samples showed 23% IgM, 19% IgG, and 7% both IgM and IgG positivity, compared with 26% IgM, 4% IgG, and 1% both IgM and IgG positivity in CSF samples. A total of 9% of CSF samples were positive for JEV-specific RNA (355-bp amplicon) as determined by RT-PCR. All these RT-PCR–positive CSF samples were also positive for IgM. None of the serum samples were positive by RT-PCR for viral RNA. Adding RT-PCR– and IgM-positive samples to C6/36 cells yielded 7 JEV isolates from IgM-positive CSF samples only, as confirmed by ELISA and RT-PCR. The antibody profile of the RT-PCR– and isolation-positive samples is depicted in .
Antibody profile of RT-PCR– and virus isolation–positive samples, 2005 Japanese encephalitis virus outbreak in India*
Further analysis of a 355-nucleotide sequence in domain III of the E gene of these isolates showed >95% homology with JEV on BLAST search. On comparison with 24 other geographically diverse JEV isolates (), all JEV isolates sequenced in this study were closely related (>99% homology). The isolates from this outbreak showed a nucleotide sequence identity of 95.6% and 94.6% with prototype JEV (Nakayama strain) and the first Indian JEV (isolated from Vellore in 1956), respectively. The dendrogram showed that the JEV isolates responsible for the 2005 Gorakhpur epidemic belong to genogroup 3 (G3) but form a cluster separate from earlier Indian isolates ().
Japanese encephalitis viruses compared for sequence analysis*
Figure Sequence phylogeny based on partial E gene sequence of Japanese encephalitis virus isolates from the Gorakhpur epidemic, with reference to other Southeast Asian isolates. The tree was generated by neighbor-joining method. Each strain is abbreviated with (more ...)