Investigation of buffalopox outbreaks in Maharashtra State during 1992-1996.

During 1992-96, outbreaks of buffalopox zoonosis were reported from different villages in Jalgaon, Dhule and Beed districts of Maharashtra State. In humans, pox lesions were observed on the hands whereas in affected buffaloes and cows the lesions were noticed mainly on the teats and udder. Twenty two virus strains were isolated from the skin scabs collected from infected humans and milch animals. Neutralizing antibodies were detected not only in the sera of affected humans but also in their contacts. Detection of antibodies in young individuals from endemic area, who were neither vaccinated for smallpox nor had any contact with buffaloes or history of any poxvirus disease, is suggestive of occurrence of subclinical infection. A few children who had no contact with infected animals also showed clinical manifestations with disseminated lesions on the face, arm and buttocks, and thus suspected to have acquired infection through their infected parents or other family members indicating a possible man to man transmission. Therefore, in the light of discontinuation of smallpox vaccination, buffalopox outbreaks need to be monitored carefully as this may emerge as a serious zoonotic disease in India.

Susceptibility of laboratory-bred rodents to the experimental infection with dengue virus type 2.

Various strains of laboratory-bred rodents viz. mice [Swiss, C57BL/6, C3H/Hej, DBA/2, BALB/c, NMRI (nu/nu) and BL6 (nu/nu) and their heterozygous siblings (nu/+)], Mastomys natalensis, Wistar rat, golden hamster and Indian desert gerbil were inoculated intracerebrally (ic) with mouse-adapted dengue virus type 2 (DV-2). The inoculated animals were observed daily for dullness, anorexia, occult blood in faeces, patechial haemorrhages, lacrymation, paralysis, cachexia, death. Necropsied animals were examined for gastrointestinal haemorrhages and lymphadenopathy. The severity of clinical symptoms in various rodents declined as follows: (i) BL6 (nu/nu) mice exhibited most severe manifestation of all the aforementioned symptoms followed by (ii) NMRI (nu/nu), (iii) BL6 (nu/+) (iv) NMRI (nu/+) and C57BL6, (v) DBA, C3H/Hej and BALB/c, and (vi) Swiss. These results indicate that adaptation of DV-2 to the mouse may be an important factor in exaltation of virulence. Interstrain variation in manifestation of symptoms in mice indicates that the susceptibility to DV-2 may be determined by host genetic factors.

Antibodies against hepatitis E virus in Old World monkeys.

To examine whether Indian monkeys are infected with hepatitis E virus (HEV) in nature, serum samples from wild rhesus (Macaca mullata), bonnet (M. radiata) and langur (Presbytes entellus) monkeys were screened for anti-HEV IgG antibodies in recombinant antigen-based ELISA assays. The positivity rates were 36.7%, 19.1% and 2% respectively. The protection of such antibodies against human HEV was studied in four rhesus monkeys. Of the two rhesus monkeys with anti-HEV titres of 100 and 1000 respectively which were inoculated with the KOL-91 strain of HEV, the former demonstrated a 10-fold rise in anti-HEV titres. Anti-HEV titre in the second rhesus monkey remained unchanged. Neither of the monkeys showed any rise in serum alanine transaminase (ALT) or presence of virus in the faeces, as tested by polymerase chain reaction (PCR). Two other rhesus monkeys with anti-HEV titres of 10,000 and 100 respectively were inoculated with the AKL-90 strain of HEV. Serum ALT levels and anti-HEV titres remained unchanged in the first monkey. Excretion of virus in faeces was not noted (PCR). The second monkey developed a typical HEV infection. HEV infection could be produced in anti-HEV negative control monkeys inoculated with both strains of HEV. These results show that either human or simian HEV, or a closely related agent, is circulating among Indian macaques. Titre-dependent protection of naturally occurring anti-HEV antibodies supports this view.

Susceptibility of BL6 nude (congenitally athymic) mice to Japanese encephalitis virus by the peripheral route.

Studies on the susceptibility of adult BL6 nude (congenitally athymic, nu/nu) mice, their euthymic littermates (+/nu) and Swiss mice to Japanese encephalitis (JE) virus inoculated subcutaneously were carried out. The mice were observed over a period of 60 days p.i. for sickness and/or death, which was noticed only in nu/nu mice. However, the onset and the duration of sickness varied and no definite pattern was observed. Thirty four of 53 nu/nu, 28 of 42 +/nu and 30 of 52 Swiss mice bled during the first 5 days p.i. exhibited viraemia. Interestingly, only nu/nu mice had secondary viraemia during the period of sickness. The cause of sickness and death in nu/nu mice was confirmed by recovering the virus from the blood, the brain and other organs. Antibodies were detected in the sera of +/nu and Swiss mice from the 10th day p.i. onwards but not in nu/nu mice. These findings indicate the important role of functional T cells both in induction of active immunity and protection against JE virus infection in mice.

Two-way cross-protection between West Nile and Japanese encephalitis viruses in bonnet macaques.

Cross-protection between Japanese encephalitis (JE) and West Nile (WN) viruses was tested in bonnet macaques (Macaca radiata) immunized either with JE virus (JEV) or WN virus (WNV). JEV immunized monkeys were challenged by intranasal (i.n.) route with WNV and vice versa. Four control unimmunized monkeys were similarly infected either with WNV or JEV. Two of three control monkeys infected with WNV, developed paralysis followed by death. Virus was recovered from the central nervous system (CNS) of the both dead control monkeys and the histopathological examination of CNS revealed changes suggestive of viral encephalitis. The control monkey infected with JEV developed encephalitis and the virus was recovered from the blood and CNS. All the 3 JEV-immunized monkeys withstood WNV challenge, whereas only 2 of the 5 WNV immunized monkeys withstood the challenge with JEV. Out of 3 WNV-immunized monkeys surviving challenge with JEV, 2 revealed symptoms suggestive of mild encephalitis followed by complete recovery. The third monkey died on the 60th day post-infection (p.i.) without any symptoms and virus was recovered only from the olfactory lobe. These studies indicate that the immunization with JEV protects the bonnet macaques against WNV, whereas the WNV immunization only reduces the severity of the disease due to JEV.

Characterization of & induction of immune response to anti-idiotypic antibodies for JE virus.

Anti-idiotypic antibodies (anti-Ids, Ab2s) were prepared by immunizing rabbits with two murine monoclonal antibodies (Ab1) having specificities for two independent haemagglutinin (HA) epitopes on JE virus [viz., Hs-1, monoclonal antibody (MAb) specific for Japanese encephalitis virus (JEV) and Hx-1, MAb common to flaviviruses]. Anti-Hs-1 (S-Ab2) and Anti-Hx-1 (X-Ab2) reacted specifically with the immunizing Ab1. In addition, they could react with other MAbs whose reactivity was similar to their immunizing homologous Ab1. The paratope inhibition assay indicated that both anti-idiotypes recognized paratope related idiotopes on their respective Ab1 and could therefore be designated as Ab2 beta. Experimental animals (Swiss mice, Balb/c mice and guineapigs) immunized with S-Ab2 or X-Ab2 produced anti-JE virus antibodies (Ab3) which could be detected by enzyme linked immunosorbent assay, immunofluorescence, haemagglutination inhibition and neutralization tests. The anti-idiotypes were also found to stimulate a cellular immune response in vitro as assessed by 3H thymidine incorporation by lymphocytes from JE vaccinated individuals and experimentally immunized Balb/c mice. The findings of the present study suggest that both the anti-Id antibodies are homobodies which may act as surrogate antigens to manipulate the immune response against JEV.

Susceptibility of four species of mosquitoes to Chandipura virus and its detection by immunofluorescence.

Susceptibility of Culex tritaeniorhynchus, Cx. Bitaeniorhynchus, Cx. quinquefasciatus, and Aedes aegypti to Chandipura (CHP) virus was compared after parental inoculation of the mosquitoes. Virus detection was done by indirect immunofluorescence (IF). CHP antigen in head squashes of all the four species was seen at 24 hr post infection (p.i.). The mosquitoes supported the virus growth and transmission by bite to 2 days old suckling Swiss albino mice. Ae. aegypti which was found the most susceptible mosquito species for CHP virus can be used as a substitute for laboratory mice.

Protective effect of 6-MFA, a fungal interferon inducer against Japanese encephalitis virus in bonnet macaques.

6-MFA, an extract from the fungus Aspergillus ochraceus was administered to 8 bonnet macaques. An equal number of monkeys matched for age, sex and weight received placebo and served as controls. Twenty hours after the administration of the 6-MFA/placebo the monkeys were challenged with an Indian strain of Japanese encephalitis virus by the intranasal route. Signs and symptoms of the disease such as fever, tremors, loss of appetite, dehydration, flaccid paraplegia or quadriplegia were pronounced in all the control monkeys, while in the 6-MFA treated group only two developed symptoms. Virus could be isolated from only one of the 6-MFA treated monkeys on day 6, and from four control monkeys; one each from CSF, spinal cord, blood and from both nasal swab and blood of the fourth monkey. The appearance of HI and N antibodies in 6-MFA treated group was either delayed or completely suppressed. The results indicate that 6-MFA is a potential antiviral agent which can be used to reduce the morbidity and mortality in bonnet macaques (Macaca radiata) experimentally infected with Japanese encephalitis virus.

Replication of Japanese encephalitis virus in monkey, pig and chick leucocyte cultures.

Japanese encephalitis (JE) virus replicated in monkey, pig and day-old chick leucocyte cultures. The titres obtained on days 3 to 5 after infection in monkey, pig and chick leucocyte cultures were comparable. Treatment of monkey leucocyte cultures with the mitogens phytohaemagglutinin P, pokeweed mitogen (PWM), formalinized Staphylococcus aureus (Cowan I) or concanavalin A and pig leucocytes with PWM did not significantly affect their ability to support replication of JE virus. No relationship was observed between the amount of [3H]thymidine incorporated in untreated or mitogen treated monkey or pig leucocyte cultures and the titres of JE virus in such cultures. The ability of monkey, pig and chick leucocyte cultures to support JE virus replication was abrogated following silica treatment. These findings suggest that monocytes may serve as one of the important sites of JE virus replication.