Culture adaptation of serotype G6 human rotavirus strains from hospitalized diarrhea patients in India.

During serotyping of fecal specimens positive for rotavirus from hospitalized diarrhea patients in Pune, India, about 10% showed multireactivity in enzyme immunoassay with monoclonal antibodies specific for serotypes G1-4, 6, 8, and 10. In order to characterize some of these, three fecal specimens from children and one from adult were culture adapted. All the isolates showed long RNA pattern, but three out of four isolates belonged to subgroup I and II and one, to subgroup I. The isolates were confirmed as G6 by neutralization assay and polymerase chain reaction. Nucleotide sequences of cDNA derived from the gene encoding the outer capsid protein, VP7 of two strains indicated >94% identity with G6, the serotype, generally associated with cattle. The isolates were more close to G6 RF strain, which is a bovine rotavirus, reported from France. This is a first report of isolation of bovine serotype, G6 from children as well as adults from India.

Group B rotaviruses similar to strain CAL-1, have been circulating in Western India since 1993.

Generally, group A rotaviruses are the most common cause of paediatric diarrhoea. However, group B rotavirus, adult diarrhoea rotavirus (ADRV), was found to be involved in epidemics of severe gastroenteritis in several areas of China during 1982-1983 and had resulted in more than one million cases among adults as well as older children. Human group B rotavirus has been rarely reported outside China, but has been detected first from five adults with diarrhoea in Kolkata, India during 1997-1998 (strain CAL-1). During epidemiological studies at the National Institute of Virology (NIV) on hospitalized diarrhoea patients at Pune, India, faecal specimens from patients of >5 years age, which were negative for group A rotavirus by ELISA were tested by polyacrylamide gel electrophoresis (PAGE). We detected rotavirus RNA migration patterns similar to that of group B rotavirus in three faecal specimens from adults, two from the specimens collected in 1993 and one in 1998 from sporadic diarrhoea cases. RT-PCR was carried out using primers derived from gene 8 which codes for the NS2 protein, followed by nested PCR, which confirmed the presence of group B rotavirus in all three specimens. The sequences of the PCR products of NIV specimens were compared with that of CAL-1, ADRV and IDIR (infectious diarrhoea of infant rat) belonging to group B rotaviruses. The sequence analysis of the PCR products showed the highest identity with CAL-1, which was reported from Kolkata, India during 1997--1998. The finding suggests that human group B rotaviruses have been circulating in Pune. India, since 1993. This emerging virus may lead to more severe disease among adults in India. There is a need for surveillance of group B rotavirus infections, especially in adult diarrhoea cases and seroepidemiological studies on group B rotavirus are required among humans and animals of Western Maharashtra, India.

Rapid ELISA for the diagnosis of rotavirus.

BACKGROUND & OBJECTIVES: Rotavirus is the major cause of gastroenteritis in infants and young children all over the world. The objective of the study was to develop a rapid ELISA for the diagnosis of rotavirus infection in children hospitalised with diarrhoea. METHODS: Immune serum was raised in rabbits by inoculating semipurified rotavirus, SA-11 strain. Immunoglobulins were conjugated to horse radish peroxidase and a rapid ELISA for rotavirus diagnosis was developed. The rapid ELISA was compared with routine ELISA, developed earlier at NIV. RESULTS: Of the 155 faecal samples from patients with diarrhoea, 96 were positive by rapid ELISA and 95 in routine NIV ELISA. OD values were higher in rapid ELISA. The rapid ELISA takes only 4 h to complete. INTERPRETATION & CONCLUSION: Rotavirus diagnosis by rapid ELISA is simple and easy to perform. This may lead to a significant reduction in the unnecessary usage of antibiotics, which cannot control infection due to rotavirus. This technology is being commercialized.

An epidemic of rotavirus diarrhoea in Jawhar Taluk, Thane district, Maharashtra, India, December 2000-January 2001.

An epidemic of diarrhoea in Jawhar, a tribal area of Thane district, Maharashtra, India was investigated. Within a period of approximately 2 months 490 cases of acute diarrhoea were reported among children under 5 years of age, with a case fatality rate of 0.40%. Twenty-seven out of 39 (69.23%) rectal swabs/faecal specimens obtained from hospitalized paediatric patients up to 2 years of age from Jawhar were positive by ELISA for rotavirus. Of these, seven were in the age group of < or = 6 months. Seven ELISA-positive faecal specimens were positive for serotype G3 by RT PCR. Out of 15 serum samples collected from these patients, 12 showed the presence of rotavirus-specific IgM. Rotavirus appears to be the aetiological agent of this widespread outbreak in Jawhar, Thane district, Maharashtra state, India.

Prevalence of rotavirus diarrhea among outpatients and hospitalized patients: a comparison.

The prevalence of rotavirus diarrhea was compared in two settings, among children attending outpatient clinics and those hospitalized (inpatients) at Pune, India. A total of 489 and 628 fecal specimens were collected during October 1993 to September 1996 from outpatients and inpatients respectively. Overall occurrence of rotavirus diarrhea was more among hospitalized children. Using the stratification on the variable age, it is shown that age is indeed a confounding variable. The important finding of the study was, in < or = 6 months age group, it was observed that the occurrence of rotavirus diarrhea was more in the outpatients (30.26%) than among the inpatients (10.11%). Children of this age group are likely to be partially protected by maternal antibodies. The effect of seasonality and sex distribution did not differ in the two settings. It was found that G2 serotype was the major cause of diarrhea among the outpatients.

Circulation of group A rotavirus subgroups and serotypes in Pune, India, 1990-1997.

Group A rotavirus-positive stool specimens, collected from 432 hospitalized patients of all age groups with diarrhoea during 1990-1997 from Pune, India, were characterized for subgroups (SGs) and G serotypes (1, 2, 3, 4, 6, 8, and 10). ELISA for subgrouping was carried out by employing subgroup I and II-specific monoclonal antibodies (MAbs). For serotyping, MAbs against G1 (Ku), G2 (S2), G3 (Yo), and G4 (ST-3) were used. In addition, MAbs against G3 (RV-3), G8 (B37), G6 (bovine U.K.), and G10 (B223) were also employed. Of the 432 specimens, 174 (40.27%) belonged to subgroup I, 187 (43.29%) to subgroup II, 15 (3.47%) to subgroup I and II, and 56 (12.96%) did not react to MAbs specific to subgroup I and subgroup II MAbs. Of the 432 specimens, 111 (25.69%) reacted to one of the MAbs used. Thirty-five of the 111 specimens were serotyped as G1, 34 as G2, and 42 as G3, G4, G6, G8, and G10. Sixty-seven (21%) specimens gave dual reaction mainly to MAbs against G6, G10; G2, and G4, and in several other combinations. Forty-seven specimens (10.88%) showed multireactivities. A large number of specimens (47.92%) did not show any reactivity with MAbs employed in this study, and remained non-serotypeable. Subgroup I was found to be more common in Pune, and most specimens negative for subgroup I and II were non-serotypeable. The results implicate the need for characterization of unusual and non-typeable strains before undertaking any rotaviral vaccine studies in India.

Prevalence of rotavirus diarrhoea among hospitalized children in Pune, India.

Rotavirus was detected in 266 (28.15%) out of 945 faecal specimens collected between July 1992 and June 1996 from children < or = 5 yr of age. Statistical analysis using odds ratios and multivariate logistic regression analysis revealed that seasonality had a strong influence on the number of rotavirus diarrhoea cases admitted to the hospital. Maximum cases occurred in the winter and minimum in the rainy season. Age was strongly associated with the prevalence of rotavirus diarrhoea. The age group of 6-24 months was the most susceptible. This disease was more predominant in males.

Time series analysis of patients with rotavirus diarrhoea in Pune, India.

The effects of seasonality and other temporal patterns on the occurrence of rotavirus diarrhoea were studied among hospitalised cases at Pune, India from July 1992 to June 1996. The well-accepted Box-Jenkins methodology based on modelling was employed for the analysis. This is the first presentation of such analysis for rotavirus diarrhoea. The model suggests strong influence of climatic changes on the incidence of the disease. Further study of weather parameters not only confirms that daily minimum temperature is the principal factor but also reveals that easterly wave, a characteristic feature of tropical weather, is useful in predicting the peak of hospital admissions and the geographical sequence of outbreaks of the disease in tropical India.

Prevalence of human group A rotavirus serotypes in Pune, India (1990-1993).

A total of 205 specimens positive for rotaviruses obtained during 1990-93 surveillance studies of hospitalized patients with diarrhoea were tested by ELISA for G serotypes. Of these, 107 (52.19%) specimens could be serotyped. Seventy four specimens were assigned to one of the G1-G4 serotypes. Of the 74 specimens, 49 (66.2%) reacted with MAb against G2. Of 107 typeable specimens, 33 showed dual or multiple reactivity. Eighteen specimens reacted with MAb specific to G1 and G2 serotypes. Five specimens reacted with MAb specific to G2 and G4. RNA profile of some of the specimens showing dual reactivities did not show additional RNA band. The specimens showing reaction to G1 and G2, had long RNA pattern, those with G2 and G4, showed short RNA pattern. Ten (9.17%) specimens reacted to 3 or more rotavirus serotypes. Of these, three reacted to MAb raised against G6, a bovine rotavirus. Ninety eight specimens could not be serotyped.

PIP: Surveillance of the geographic and temporal distribution of rotavirus serotypes is essential to the development of vaccine strategies. In the present study, 205 specimens positive for group A rotaviruses obtained during 1990-93 surveillance studies of diarrhea inpatients at Naidu Infectious Disease Hospital in Pune, India, were tested by enzyme-linked immunosorbent assay for G serotypes. Although serotype G1 is reported most frequently on a worldwide basis, G2 predominated in this study. Of the 107 specimens (52.2%) that could be further serotyped, 15 (13.8%) were serotype G1, 49 (45.0%) were serotype G2, 9 (8.3%) were serotype G4, and 1 (0.09%) was serotype G3. 23 specimens (22.9%) reacted with 2 G serotypes (primarily G1 and G2) and 10 (9.2%) reacted to 3 or more G serotypes. The specimens reacting to G1 and G2 had a long RNA pattern, while those with G2 and G4 showed a short RNA pattern. Nontypeable strains pose a significant diagnostic challenge in countries such as India.

Assay of neutralizing antibodies to animal rotavirus strains and human rotavirus serotype G8 by a modified method in the residents of Pune, India.

In the western literature, four G serotypes (G1-G4) of human rotaviruses have been found to be of a major epidemiological importance. During the analysis of rotavirus serotypes from faecal samples in Pune, over 50% of specimens could not be serotyped with the available monoclonal antibodies against G1-G4 serotypes. The results prompted to look for the prevalence of neutralizing antibodies against serotypes other than the major human serotypes (G1-G4) in adults. Neutralizing antibodies against animal rotavirus serotype, viz. G3, G6 and G10, and human rotavirus serotype G8 were determined in adult sera, by a modified technique, which is ELISA-based and mechanized. The results showed that, of the 68 sera tested at 1:100 dilution, 65 (95.58%) were reactive for G3 (SA-11), 52 (76.47%) for G6 (Bovine Lincoln), 6 (8.82%) for G10 (B223), and 40 (58.82%) for G8 (M69) serotypes. It appears that the prevalence of rotaviruses in India may be quite different from that in the developed countries.

Development of indigenous ELISA for rotavirus diagnosis & its comparison with commercial kit.

Preparation of reagents for the diagnosis of rotavirus by enzyme linked immunosorbent assay (ELISA) was carried out at the National Institute of Virology (NIV), Pune. This is a double antibody sandwich ELISA test. The coating antibody was raised in guinea pig against SA-11 (Simian rotavirus), and is used at 1:20,000 dilution. The indicator antibody was also raised against SA-11 in rabbits. The rabbit preimmune serum is used as negative control serum. Both, pre- and post-immune rabbit antisera are used at 1:10,000 dilution in the test. Goat IgG-HRP conjugate against rabbit IgG was procured commercially (Sigma, USA). Results of ELISA test can be read visually. A total of 63 pretested faecal specimens and 38 specimens of rotavirus in different concentrations were compared simultaneously by the NIV ELISA and Dakopatts ELISA. Of the 63 specimens, 36 were positive in NIV ELISA and 33 positive by Dakopatts ELISA. Human and animal rotavirus strains also showed higher titers in NIV ELISA than Dakopatts ELISA. From our data we conclude that NIV ELISA is 100 per cent specific and more sensitive than Dakopatts ELISA test. It is much more economical than any other commercial kit available for the diagnosis of rotavirus. Since the reagents are in lyophilized form, they are suitable for use in developing countries.

Characterization of concanavalin A induced peritoneal exudate cells in infant mice.

Con A induced peritoneal exudate (PE) cells were characterized in 9-12 days old infant mice. It was observed that 100-200 micrograms of Con A induces two peaks viz. first at 48 hrs post inoculation (PI) and second on 8-10 days PI. Majority of the induced cell population was adherent macrophages (75-80%) and non adherent population was 20-25%. During 2nd peak of induction non adherent population increased upto 40%. The macrophage population was heterogenous. However, majority of them were 8-12 micron in size. The cells showed strong non specific esterase and acid phosphatase activities. Subsequently, there was vesicle formation in this population. Con A also induced vesiculation in PE cells markedly when compared with normal unstimulated mice.

In vivo opsonization of Japanese encephalitis virus by peritoneal macrophages in infant mice.

Anti-JE antibody in nonneutralizable concentration and Concanavalin A are synergistic in protecting 10-day-old mice from lethal JE virus challenge by i.p. route.

Macrophage-virus interaction during Con A-induced protection against Japanese encephalitis virus in infant mice.

Eight to ten-day-old mice when inoculated i.p. with Concanavalin A yielded a large number of cells in the peritoneal exudate (PE), the majority of which belonged to the macrophage series. Con A-treated and untreated (control) mice were infected i.p. with Japanese encephalitis (JE) virus. The PE cells of Con A-treated mice showed a higher percentage and greater intensity of virus specific immunofluorescent cells as compared with the cells from control mice. No live virus was detected in the Con A-induced cells. However, traces of live virus were found in the cells of control mice, 3-6 hr after infection. In vitro, peritoneal macrophages from Con A-treated mice showed a higher uptake of the virus (83.3%) within 18 hr as compared to the cells from paraffin-treated mice (34%). The protection of Con A-induced mice to JE virus challenge by i.p. route could be due to increased uptake and subsequent inactivation of the JE virus in the peritoneal exudate cells.