Banerjee K

Antibodies against hepatitis E virus in old world monkeys

SUMMARY. 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 10000 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.

Role of immune serum globulins in pregnant women during an epidemic of hepatitis E

The efficacy of an Indian preparation of immune serum globulins (ISG) was evaluated among pregnant women during an epidemic of hepatitis E in Karad, Western India from January to March 1993. Ten of 55 women receiving ISG developed immunoglobulin M (IgM) antibodies to hepatitis E virus (anti‐HEV) during the 1 month of follow‐up compared with 18 out of 53 control subjects. Although the total number of recent HEV infections was significantly less in the ISG‐treated group, no significant difference could be shown in the proportion of clinical hepatitis E cases because of the very small numbers of patients who developed clinical disease. The observed marginal beneficial effect of ISG might be the result of a low immunoglobulin G (IgG) anti‐HEV IgG titre (1:500) of the ISG preparation used. Preparation and testing of high‐titred ISG should be a high priority for protecting pregnant women during epidemics of hepatitis E.

Contribution of HEV and HCV in causing fulminant non-A, non-B hepatitis in western India

Summary. During 1990, 38 patients with fulminant non‐A, non‐B hepatitis (NANB) died in Government Medical College Hospital, Aurangabad. Serum samples from these patients were tested for antibodies to hepatitis C virus (anti‐HCV) and IgM antibodies to hepatitis E virus (IgM‐anti‐HEV). All samples were also subjected to polymerase chain reaction (PCR) for the detection of HBV DNA, HCV RNA and HEV RNA. None of the patients had circulating anti‐HCV antibodies; three had HCV RNA. Based on anti‐HEV‐IgM positivity 14 patients (37%) could be diagnosed as suffering from hepatitis E. None was positive for HEV RNA. In the absence of serological markers, HBV DNA was present in three cases. None of the HBV DNA positive patients had anti‐δ antibodies. Dual infections (HBV with HEV, and HBV with HCV) were seen in two cases. The aetiology of half of the NANB cases could not be assigned to the known hepatitis viruses using current techniques.

A high prevalence of antibodies to hepatitis C virus among commercial plasma donors from Western India.

Summary. A very high prevalence of anti‐hepatic C virus (anti‐HCV) antibodies (63/73, 86.3%) was noted among commercial plasma donors of an organization manufacturing blood products, studied in 1989. Retrospective serological analysis of these donors revealed continued high prevalence of anti‐HCV, i.e. 35/40 (87.5%) in 1988, 28/31 (90.3%) in 1987 and 86/94 (91.4%) in 1986. HCV RNA detection by polymerase chain reaction (PCR) demonstrated 15/33 (45.45%) plasma donors to be positive in 1989.

Detection of HEV RNA in faeces, by RT‐PCR, during the epidemics of hepatitis E in India (1976–1995)

SUMMARY. Out of the 15 hepatitis E (HEV) epidemics that occurred during the years 1976–1995 in the Gujarat and Maharashtra states of India, 45.78% (76/166) stool samples showed the presence of HEV RNA. HEV RNA was found significantly more often in samples that were transported in liquid nitrogen (50.9%) compared with samples that were transported in wet ice (37.0%) (P < 0.05). Stool samples collected within 7 days after the onset of the disease (59.2%) were more often positive for HEV RNA when compared with samples that were collected 7–20 days after the onset of the disease (28.5%) (P < 0.01). It has been observed in experimentally infected Rhesus monkeys that they excrete HEV throughout the incubation period and for a variable length of time after the elevation of serum ALT levels. A similar situation is found in humans.

Detection of HIV-1, HBV and HCV Antibodies in Blood Donors from Surat, Western India

HIV continues to spread in India mainly through heterosexual intercourse, but also among homosexual men and through blood transfusion. The government of India has mandated since 1992 that donor blood from the larger cities be screened for hepatitis B (HBV) and HIV infections. It is expected that this policy will be extended to other cities. Surat is a town 250 km north of Bombay. Approximately 50% of blood is obtained from professional donors to meet the requirement of blood for transfusion. The authors investigated the extent to which blood from professional and voluntary blood donors was infected with HIV-1, HIV-2, HBV, and hepatitis C virus (HCV). They tested 85 blood samples from professional blood donors and 94 samples from voluntary donors from Surat and Pune, respectively, using ELISA and immunoassay. 56 of the professional blood donors were HIV-1-seropositive, 2 of whom were also seropositive for HIV-2. The voluntary donors were seronegative for both HIV-1 and HIV-2. With regard to infection with HBV, 9 samples from professional donors were reactive for HBsAg, 62 of the total 85 samples were reactive for anti-HBs, and 1 sample was borderline reactive. Among the voluntary donors, 6 were reactive for HBsAg, 3 were borderline reactive for HBsAg, and 5 were reactive for anti-HBs. 47 samples from professional donors were reactive for antibodies to HCV, while 13 samples were indeterminate. 4 voluntary donors were reactive for anti-HCV and 5 were indeterminate. The professional blood donors typically were of lower socioeconomic level, among whom awareness about HIV infection and AIDS is incomplete. The voluntary donors, however, were of relatively higher socioeconomic status, and donated blood to help society or as replacement donors. This latter group was more aware of HIV transmission and AIDS. Many blood banks in India still procure blood from professional donors, most likely the reason why parenterally-transmitted HIV infection still takes place in India.

A cell line from the gill tissues of Indian cyprinoid Labeo rohita.

Dear Editor: The fish gill is a multifunctional organ responsible for respiration, osmoregulation, acid base balance, nitrogen excretion, and metabolism of circulating hormones. Because of its anatomical position and being continuously bathed with water, it is the principal target organ for toxic compounds and infective agents. Many aquatic pollutants are assayed by their deleterious effect on the cellular ultrastructure of the gill epithelium (Matei, 1993), histopathological changes (Singh and Sahai, 1990), perturbation in carbohydrate metabolism (Reddy and Yellamma, 1991), nitrogen metabolism (Reddy et al., 1991), or protein metabolism (Suresh et al., 1991). However, these studies were carried out on intact gills in vivo. In vitro approaches have not been fully exploited because of lack of cell culture system and standardization of assay protocol. Recent literature survey (Fryer and Lannan, 1994) shows that only four cell lines BG/G (bluegill sunfish), G1B (walking catfish), ZG (zebrafish), and GI (common carp) have been developed so far from the gill tissues of fishes. A recent addition has been MG-3, a cell line developed by our group from the gills of Cirrhinus mrigala (Sathe et al., 1995). Our attempts to develop newer cell lines from indigenous fresh water fishes have resulted in establishment of yet another cell line, RG-1, from gill tissues of Labeo rohita (Fig. 1), commonly known in India as Rohu. We herein report some biochemical characteristics and ultrastructural details of the cell line. While attempting to develop cell lines from gill tissues of Rohu, two of the explant cultures (RG-1 and RG-2) set up on two different days showed outgrowth of cells from the explants and were capable of indefinite propagation by following the routine procedures of subculture. Both cell lines have been maintained in L-15 medium with 10% fetal calf serum (FCS) with optimal growth at 28 ° C. The meth-

Detection of human immunodeficiency virus antibody among homosexual men from Bombay

Background and Objectives In India, heterosexual transmission of HIV-infection is considered to be the major mode of transmission. However, no report is available on transmission of HIV-infection among homosexually active men. The prevalence of human immunodeficiency virus-1 (HIV-1) and human immunodeficiency virus-2 (HIV-2) infections among homosexual men from Bombay is discussed. Goal of the Study To determine the extent of presence of anti-HIV-1, anti-HIV-2 antibodies, or both anti-HIV-1 and anti-HIV-2 antibodies among homosexual men in India. Study Design Sixty-three blood samples were collected from two STD clinics of Bombay over a 6-month period from men with a history of homosexual behavior who were asymptomatic for HIV-infection. The mean age of the subjects was 31.6 years. For serological detection anti-HIV-1 antibody ELISA was used as the primary screening test followed by Western blot to confirm the results. For distinction between anti-HIV-1 and anti-HIV-2 antibody, line immunoassay was used. The sexually transmitted diseases (STDs) were diagnosed clinically, although Venereal Disease Research Laboratory (VDRL) tests were carried out as a routine test for screening STDs. For detection of gonorrhea, Gram stains of urethral smear were done routinely. Results From the 63 blood samples tested, 10 samples were reactive by ELISA for HIV-1 infection, and three samples were borderline reactive. These three samples were found to be reactive for anti-HIV-2 by the line immunoassay. The above 10 samples were also positive by Western blot for anti-HIV-1 antibody. Two blood samples were positive for both anti-HIV-1 and anti-HIV-2 antibodies. Using clinical diagnosis as the criteria, the different types of STD among the 63 subjects were as follows: condylomata (22), herpes (20), gonorrhea (15), candidiasis (3), and syphilis (3). However with VDRL, seven subjects were found to be reactive. Gram stains indicated gonorrhea in all the 15 subjects. Conclusions This study reports for the first time the homosexual transmission of both HIV-1 and HIV-2 infections in India, although heterosexual transmission still is the major mode of transmission of the infection. The associated incidence of STDs among these men and that a few of these subjects were bisexual make them at high risk for transmission of HIV infection.

Detection and cloning of potent transforming gene(s) from chewing tobacco-related human oral carcinomas.

High molecular weight DNA isolated from 14 primary tumour tissues of human oral carcinoma patients was analysed for transforming activity by NIH3T3 co-transfection assay using pSV2neo gene as a selectable marker, followed by nude mouse tumorigenicity assay. Ten of the patient tumour tissues demonstrated molecular lesions in myc, ras or/and EGF-R genes, whereas 4 patients did not show tumour associated aberrations in these oncogenes. The G418-resistant transfected cells from 12 of 14 individual patients demonstrated transforming potential by colony formation in soft agar and tumour induction in nude mice within 25-80 days. DNAs from the transfected cells, consequent nude mice tumours and corresponding cell lines, contained human Alu sequences. Southern blot hybridisation with ras, myc, EGF-R oncogenes demonstrated the presence of human H-ras oncogene in one of the 12 sets of nude mice tumours. In contrast, DNA from the other 11 sets of nude mice tumours indicated absence of c-myc, N-myc, L-myc, H-ras, K-ras, N-ras and EGF-R genes on Southern analysis. Further, DNAs from five first cycle tumorigenic transformants were subjected to a second cycle of transfection, and induced tumours in nude mice with a shorter latency period of 21-50 days. The secondary transformants contained discrete human Alu sequences; however, the DNA did not hybridise with myc/ras/EGF-R probes. A genomic library was constructed from a second cycle nude mice tumour, using EMBL-3 as the vector. Four human Alu sequence positive clones were isolated on screening 2 x 10(5) plaques, and one of the recombinant clones subjected to fine restriction mapping using 16 restriction enzymes. The lack of association of the nude mice tumour DNA with myc/ras/EGF-R showing aberrations in the primary human tumour, implies activation of an alternative potent transforming gene(s) in the chewing tobacco-related oral carcinomas in India.

Isolation of measles virus below 4 months of age during an outbreak in Pune, India

This article reports the isolation of a measles virus from infants under 4 months of age during an outbreak in an orphanage at Pune, India, in 1996. With a total population of 24 orphanages, 6 inmates showed the signs and symptoms of measles; 4 were under 4 months old and the other 2 were 7 and 11 months old, respectively. Bronchopneumonia and weight loss were observed as the main complications of the disease. Isolation of the measles virus was done by obtaining throat swabs, skin scrapings, and blood samples from the victims. Serum testing of the 6 cases showed a positive result for immunoglobulin M (IgM) antibodies. During the same period, mothers of 4 IgM-positive infants were screened for antibodies; 3 mothers were negative for measles-specific antibodies. Because of this, lack of maternal antibodies, malnutrition, and crowding were accounted for the outbreak of measles in the orphanage. These findings reflect a need to assess the prevalence of measles within this age group and develop prevention strategies for measles.