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Several H-2 defined cell lines were examined for their ability to support infection and replication of Japanese encephalitis virus (JEV) before their use in in vitro and in vivo stimulation protocols for generating cytotoxic T lymphocytes (CTLs) against JEV. Among II different cell lines tested, two H-2(d) macrophage tumour lines (P388D1, RAW 264.7), an H-2(d) hybridoma (Sp2/0), an H-2K(k)D(d) neuroblastoma (Neuro 2a), and H-2(k) fibroblast cell line (L929) were found to support JEV infection and replication. These cell lines were used to generate anti-JEV CTLs by using in vivo immunization followed by in vitro stimulation of BALB/c mice. We observed that not only syngeneic and allogeneic infected cells but also JEV-infected xenogeneic cells could prime BALB/c mice for the generation of JEV-specific CTLs upon subsequent in vitro stimulation of splenocytes with JEV-infected syngeneic cells. Although infected xenogeneic cells were used for immunization, the anti-JEV effecters that were generated lysed infected syngeneic targets but not JEV-infected xenogeneic or allogeneic target cells in a 5h Cr-51 release assay. These anti-JEV effecters recognized syngeneic target cells infected with West Nile virus to a lesser extent and were shown to be Lyt-2.2(+) T cells. The results of unlabelled cold target competition studies suggested alterations in the cell surface expression of viral antigenic determinants recognized by these CTLs. We further demonstrate that the JEV-specific CTLs generated could virtually block the release of infectious virus particles from infected P388D1 and Neuro 2a cells in vitro.

SummaryObjectivesCurrently three tests are approved for the estimation of neutralizing antibodies after rabies vaccination: the mouse neutralization test (MNT), the rapid fluorescent focus inhibition test (RFFIT), and the fluorescent antibody virus neutralization (FAVN) test. Performance of these tests requires a lot of expertise and is generally carried out in reference laboratories and, hence, they are not available to many people. The aim of the present study was to develop and evaluate a competitive ELISA (C-ELISA) for estimation of neutralizing antibodies in order to make this testing more widely available.MethodsThe C-ELISA was designed based on competition between a murine neutralizing monoclonal antibody (Mab) and the antibodies in serum of vaccinated people. The test was initially standardized using known negative and known positive serum samples for determining the optimal dilution of the Mab as well as the cut-off value (%) for ascertaining the level of inhibition. Nine hundred and ninety serum samples were tested from 250 people who had been administered purified chick embryo cell vaccine (PCECV). Serum samples were collected on days 0, 14, 30 and 90 post-vaccination, and were tested by C-ELISA.ResultsAll the serum samples that were positive by RFFIT were also positive by C-ELISA. The titers obtained with C-ELISA were marginally higher than the RFFIT titers, but a significant correlation was noted between the two tests (r=0.897). None of the negative controls were detected to be positive for rabies antibodies by either of these tests. Therefore the C-ELISA was found to be 100% specific and sensitive in comparison to RFFIT. Further, the initial rise and fall of antibody titers on different days post-vaccination was comparable for both tests.ConclusionsThe C-ELISA described herein can be used to quantify rabies neutralizing antibody levels after vaccination. This test is simple and can be conveniently used under field conditions for monitoring seroconversion after post-exposure rabies vaccination. Moreover it does not require handling of infectious virus by the end user.

Detection of virus, viral antigen, and class-specific antibody was carried out in cerebrospinal fluid (CSF) and sera of 27 children with Japanese encephalitis. The diagnosis could be confirmed in 78.57% of cases (22/27) by demonstration of virus-specific IgM in CSF (15/22), viral antigen in CSF (5/22), or by virus isolation (2/22). Absence of virus specific IgM in CSF was associated with a fatal outcome (P = 0.05).

To investigate the prognostic role of tumour necrosis factor (TNF) in Japanese encephalitis virus (JEV) infection, we measured the immunoreactive forms of TNF concentrations in the serum and cerebrospinal fluid (CSF) of 47 laboratory-confirmed cases of JE. It was observed that TNF levels were elevated (>15 pgm/ml) in all the 47 serum samples (range 19.4–923.8 pg/ml), while in 46/47 CSF samples TNF was elevated (range 10.8–376 pg/ml). The mean (SD) TNF levels in the serum of fatal cases was 234.34 pg/ml (304.40) as compared to the mean of 85.31 pg/ml (SD 153.92) in nonfatal cases. Similar observations were also made with respect to the TNF levels in the CSF; the mean of fatal cases was 69.39 pg/ml (SD 39.00) in contrast to the mean of 62.41 pg/ml (SD 75.25) of nonfatal cases. The increase in TNF levels did not show any correlation to the duration of illness. It was further observed that the mortality rate increased with increasing concentrations of TNF in the serum and CSF. Correlation of laboratory parameters to final outcome revealed that TNF concentrations above 50 pg/ml in serum correlated significantly (P = .05) with a fatal outcome, whilst high levels of JEV-IgM antibodies (>500 units) in the CSF correlated with a nonfatal outcome (P = .03). These results suggest that TNF can be used as a possible prognosticator of a fatal outcome in JEV infection. J Med Virol 51:132–136, 1997. © 1997 Wiley-Liss, Inc.

We characterized the molecular nature of a large number of primary HIV-1 isolates in the four southern states of India. In addition to confirming a predominance of subtype C infection, for the first time we identified three B/C recombinant viruses in a subset of 115 samples. Unexpectedly, env sequences of two of the three B/C recombinants phylogenetically clustered with subtype B strains of the USA. The determination of the real incidence of the recombinant viruses is of importance.