Isolation of measles virus from cerebrospinal fluid of children with acute encephalopathy without rash.

OBJECTIVE: To determine the viral agent involved in cases of acute encephalopathy in children during an outbreak in Northern India. DESIGN: Virological and serological studies using serum and cerebrospinal fluid specimens from patients. METHODS: Serum and CSF specimens were tested by IgM ELISA for IgM antibodies to variety of viruses like Japanese encephalitis, West Nile, Dengue and Measles. The specimens were inoculated into Vero cell monolayer for virus isolation. The viral strains isolated were identified by indirect immunofluorescence test and qualitative in-vitro neutralization test using polyclonal and monoclonal antibodies to measles. Identity of the isolates was reconfirmed using RT-PCR method. RESULTS: Of the 28 specimens tested, 17 had IgM antibodies to measles. Commercial IgM ELISA kits confirmed the serological findings. Vero cell cultures yielded 4 isolates from CSF and 2 from serum specimens of six different patients. Cytopathic effect was typical of measles. Indirect imunofluorescence using polyclonal and monoclonal antibodies to measles HA protein, confirmed the measles etiology. Neutralization tests reconfirmed the measles strain isolation. RT-PCR amplified product was confirmed as measles. CONCLUSION: The isolation of measles virus from CSF and serum of children with acute encephalopathy without rash proved the etiological role of measles virus in this outbreak.

Loss of virus specific epitopes on JE virus 'E' glycoprotein by acetone treatment.

BACKGROUND & OBJECTIVES: Some Japanese encephalitis (JE) virus strains have been placed in group II based on the loss of reactivity against Hs (H = HI positive; s = JE virus specific) group of monoclonal antibodies (MAbs) in haemagglutination-inhibition (HI) test employing sucrose acetone (SA) extracted antigens. Also acetone-fixation of cells infected with some of the virus strains results in the loss of immunofluorescence (IF) against virus specific MAbs. The present study was undertaken to elucidate the effect of acetone on virus specific haemagglutination (HA) epitopes expressed on 'E' glycoprotein of group II strains of JE virus. METHODS: Porcine kidney (PS) cells were infected with JE virus strains (2 group I Indian strains, 5 group II strains and one neutralization-escape variant of 733913 group I strain). HI and complement fixation (CF) tests were carried out employing both polyethylene glycol (PEG) precipitated and SA extracted antigens of JE virus. RESULTS: Employing PEG precipitated antigens, Indian strain G9473 showed titres ranging from 1:40 to 1:160 against all the four virus specific HsMAbs and strain 641686 (1:160) with one of the four MAbs (Hs-1) by HI test whereas their SA extracted antigens did not react at all. In contrast, CF was positive employing both SA and PEG antigens in the presence of all four HsMAbs. The reactivity shown by PEG antigens in the HI test was confirmed by blocking the HA activity with the respective MAb. SA antigens, though negative in the HI test, were positive by the blocking assay. Interestingly, some of the non-HI MAbs which were negative against SA antigens, showed positive HI reaction with PEG antigens. Also, additional epitopes on Japanese (Yoken), Sri Lankan (691004) and two Indian (755468 and 641686) JE virus strains were detected either by blocking HA or surface IF. INTERPRETATION & CONCLUSIONS: It seems that the acetone treatment might result in HA property of the antigen which is no longer inhibited by an antibody in the HI test. The characterization of such labile and conformation-dependent epitopes is currently been undertaken to elucidate their role either in protection or immunopathogenesis of JE.

Preliminary observations on lymphocyte subpopulations in HIV seropositive & HIV seronegative tuberculosis patients in Pune, India.

CD4 and CD8 lymphocyte counts were determined in 59 HIV seropositive and 41 HIV seronegative newly diagnosed tuberculosis patients in Pune. There were significant differences in the CD4 counts and CD4/CD8 ratios between HIV seropositive and HIV seronegative tuberculosis patients. Majority of the HIV seropositive patients had a CD4 count less than 500 cells/cu.mm, whereas among the HIV seronegative patients, majority had a CD4 count more than 500 cells/cu.mm. In HIV seropositive patients with extrapulmonary and pulmonary tuberculosis, the CD4 counts were lower than in those who had only pulmonary or extrapulmonary tuberculosis. There was no significant differences in the CD8 counts between HIV seropositive and HIV seronegative tuberculosis patients, except for patients with pulmonary cavity, where the CD8 counts were significantly higher in HIV seropositive tuberculosis patients. In HIV seropositive individuals with pulmonary tuberculosis, the CD8 counts in those with pulmonary cavity were higher than in those without any pulmonary cavity. Absence of cavitation and presence of pulmonary with extrapulmonary tuberculosis occurred when immune activation was at a lower level.

An IgM monoclonal antibody to Japanese encephalitis virus recognizing a cross-reactive epitope on nuclear histones.

An IgM class of monoclonal antibody (MAb) raised against 'envelope' (E) glycoprotein of Japanese encephalitis (JE) virus, cross reacted with nuclear histones, in addition to recognizing the viral antigen present in the cytoplasm of infected cells by indirect fluorescent antibody (FA) technique. The experiments on histone depletion by the acid treatment of uninfected PS (porcine kidney) cells, revealed the loss of nuclear immunofluorescence (IF) which was regained after the reconstitution of acid treated cells with histones, prior-to reacting with MAb NHA-2. The IgM MAb recognized specifically the viral antigens expressed on the surface of JE virus infected PS cells by a modified indirect FA. The adsorption of MAb NHA-2 with calf thymus histones (type II-AS) showed a comparative higher drop in the reactivity to JE virus (54.2% reduction) as compared to that against uncomplexed histones (33.3%) by ELISA, thus indicating a higher MAb affinity to the former. In contrast, the adsorption of MAb with chicken RBC nuclei resulted in comparatively more reduction in the reactivity to the uncomplexed histones (52.4% reduction) as against JE virus (37.5%), suggesting that DNA plays some role in modifying and presenting these epitopes. The cross-linkage of epitopes by glutaraldehyde treatment of JE virus antigen and histones showed a 2-fold and higher rise in the MAb reactivity as against those with unfixed or methanol fixed antigens (no cross-linkage), suggesting that the epitope is conformation dependent. Thus, histones seem to share a partial conformational homology with 'E' glycoprotein of JE virus and immune reaction with histones might lead to an autoimmune disorder.

Isolation & preliminary characterization of two HIV-2 strains from Pune, India.

Two HIV-2 strains were isolated from peripheral blood mononuclear cells of two HIV-2 seropositive patients with pulmonary tuberculosis by co-cultivating the cells with phytohaemagglutinin-P stimulated heterologous normal lymphocytes. Biological characterization of the isolates indicated that both isolates were syncytium inducing and induced cytopathic effect in the form of giant cells and syncytia formation in four T lymphoid cell lines. The isolates differed in their replication pattern. The isolates were confirmed as HIV-2 by nested PCR using HIV-1 and HIV-2 specific oligonucleotide primers from the env region and by supplementary tests like indirect immunofluorescence assay, syncytium inhibition assay using reference and HIV-2 reactive patients' sera, western blot and electron microscopy. Neutralization of one isolate (TB1) with two Senegal reference sera also indicated that the isolate may be related to the Senegal strain. To our knowledge, this is the first report of isolation of HIV-2 in India.

Cross-reactive HIV-1-specific CTL in recent seroconverters from Pune, India.

In the light of the diversity of HIV, matching the genotype of candidate HIV vaccines with the transmitted genotype may be required. Alternatively, matching the immunotype of HIV vaccines and transmitted subtypes may be the best option. Since studies of cross-subtype HIV-1 immunity are limited, subtype B specific cytolytic T lymphocyte (CTL) responses were measured in subtype C infected individuals. HIV-1 subtype B-specific CTLs, plasma viral load and absolute CD4 and CD8 lymphocyte numbers were measured in six HIV-1 subtype C infected individuals within a year of seroconversion. HIV-1 subtype B env. gag and nef-specific CTL precursor frequencies were measured by limiting dilution analysis. Three of the six subjects had demonstrable CTL directed at more than one HIV-1 subtype B antigens. One individual demonstrated CTL directed against all three HIV-1 subtype B antigens, while two individuals did not demonstrate CTL against HIV-1 subtype B antigens. The frequencies of CTL precursor were not associated with plasma viral load or absolute CD4 cell counts in peripheral blood. These findings suggest that some individuals recently infected with subtype C HIV-1 generate cross-reactive CTL that are directed against HIV-1 subtype B.

Transmission of genetically diverse strains of HIV-1 in Pune, India.

Surveillance of the different HIV-1 subtypes has important implications for developing candidate vaccines and understanding the dynamics of HIV transmission in various populations. In this study, HIV-1 viral subtypes were determined for homologies in the V3-V5 region by heteroduplex mobility assay (HMA) in 46 patients with sexually transmitted diseases (STD) in Pune, India. Proviral DNA from peripheral blood mononuclear cells (PBMCs) from 20 recent sero-coverters and 26 HIV seropositive individuals were analyzed. Of the 46 samples analyzed, 44 (96%) were HIV-1 subtype C and one each of subtypes A and B. Further analyses revealed that 29 (66%) of the C subtype samples had maximum homology to the C3-Indian reference strain, while 15 (34%) were most homologous to the C2-Zambian strain. The C3 genotype prevailed in the majority (80%) of the seropositive individuals. Most of the C3 (Indian) strains were closely homologous to each other, while more nucleotide sequence divergence was seen in C2 samples. A higher quasispecies complexity was observed in the samples collected from seropositive individuals. These findings may have important implications for the design and testing of effective candidate HIV-1 vaccines for India.

Increasing trend of HIV seroprevalence among pulmonary tuberculosis patients in Pune, India.

A total of 4618 tuberculosis patients attending the TB clinic at the Sassoon General Hospitals, Pune between 1991 and 1996 were screened for anti-HIV antibodies. Of these 694 were found reactive in enzyme immuno assay (EIA) and 624 were further confirmed by a second test, either rapid EIA or Western blot. HIV-1 reactivity was predominant among tuberculosis patients with HIV-2 reactivity appearing only in 1995. HIV-2 seroreactivity accounted for 0.54 and 1.02 per cent of all HIV reactive samples in 1995 and 1996. HIV-1 and HIV-2 dual reactivity accounted for 1.63 and 2.04 per cent of all infections in 1995 and 1996. The overall seroprevalence of HIV among newly diagnosed tuberculosis patients rose from 3.2 per cent in 1991 to 20.1 per cent in 1996.

Evidence for high prevalence & rapid transmission of HIV among individuals attending STD clinics in Pune, India.

Prevalence and incidence of HIV-1 infection among persons attending two STD clinics in Pune between May 1993 and October 1995 are reported. On screening 5321 persons, the overall prevalence of HIV-1 infection was found to be 21.2 per cent, being higher in females (32.3%) than in males (19.3%). Analysis of behavioural and biological factors showed that old age, sex work, lifetime number of sexual partners, receptive anal sex, lack of circumcision, genital diseases and lack of formal education were related to a higher HIV-1 seroprevalence. The observed incidence rate of 10.2 per cent per year was very high, much higher in women than in men (14.2% and 9.5% per year respectively) and over three times higher among the sex workers. Females in sex work, males having recent contacts with female sex workers (FSWs) and living away from the family and persons with previous or present genital diseases had a higher risk of seroconversion. Condom usage was shown to have a protective effect in seroprevalence and seroincidence analysis. With limited available resources and lack of a suitable vaccine or a drug, long-term prevention policy of creating awareness in the community must be supplemented by strengthening STD control measures and promotion of condom use and safe sex. Factors related to availability and utilization of condoms must be carefully investigated.

Characterization of temperature sensitive mutants of Japanese encephalitis virus isolated from persistently infected mammalian cells.

Four new temperature-sensitive (ts) mutants of Japanese encephalitis virus (JEV) isolated from porcine kidney cells persistently infected with JEV and seven previously isolated ts mutants were studied. Of the eight mutants tested, five mutants ts1, ts14, ts36, ts48 and ts71, were thermolabile. Analyses of virus induced intracellular polypeptides revealed that with majority of the ts mutants, when grown at restrictive temperature, the viral proteins were quantitatively affected. All the five ts mutants tested for mouse virulence showed attenuation in infant mice by the intracerebral route. Two ts mutants, ts36 and ts48, escaped neutralization with two anti-JEV envelope protein specific monoclonal antibodies, however, these escape mutants reacted efficiently with the same monoclonal antibodies in antigen capture ELISA.

Localization of arboviral antigen in brain tissues from patients with encephalitis.

Brain tissues from 38 patients with a clinical suspicion of encephalitis or encephalopathy were examined by two immunoenzymatic techniques for the detection of arboviral antigen. Group B arboviral antigen was identified in 23 of these tissues. This simple method could be used for the diagnosis of the causal agent of encephalitis.