Chandipura virus growth kinetics in vertebrate cell lines, insect cell lines & embryonated eggs.

Background & objectives: Since not much information on Chandipura virus is available, an attempt was made to study the growth kinetics of the virus in certain vertebrate, invertebrate cell lines and embryonated chicken eggs. Methods: Comparative study of Chandipura virus (CHPV) growth kinetics in three vertebrate cell lines [Vero E6, Rhabdo myosarcoma (RD), Porcine stable kidney (PS) cell lines], two insect cell lines [Aedes aegypti (AA) and Phlebotomus papatasi (PP-9) cell lines] and embryonated pathogen free chicken eggs was conducted, by tissue culture infective dose 50 per cent (TCID50) and indirect immunofluorescence assay (IFA). Results: All the cell lines and embryonated egg supported the growth of CHPV and yielded high virus titre. The vertebrate cell lines showed distinct cytopathic effect (CPE) within 4-6 h post infection (PI), while no CPE was observed in insect cell lines. PP-9 cell line was the most sensitive system to CHPV as viral antigen could be detected at 1 h PI by IFA. Interpretation & conclusions: Our results demonstrated that all the systems were susceptible to CHPV and achieved high yield of virus. However, the PP-9 cell line had an edge over the others due to its high sensitivity to the virus which might be useful for detection and isolation of the virus during epidemics.

Complete genome sequences of hepatitis C virus subtype 3i and 3a subtype isolates from India.

Aim Hepatitis C virus (HCV), a major causative agent of chronic hepatitis, is classified into six major genotypes. Genotype 3 HCV infection is more sensitive to interferon therapy. In India, genotype 3, particularly subtype 3a, HCV infections are common. Three novel HCV subtypes i.e., 3g, 3j, and 3i were identified from India based on partial genomic sequences. This report provides full genome sequences of one isolate each of subtypes 3i and 3a. Methods Serum samples positive for subtype 3i and 3a HCV RNA based on core region genomic sequences were studied. Complete HCV genomes were amplified as 11 overlapping PCR fragments and sequenced. Results The complete genomic sequence of Indian HCV 3i isolate clustered with other genotype 3 sequences, and was closer to subtypes 3b and 3a (80.5% and 79.1% [SD 0.4%] nucleotide identity). Nucleotide similarities were the highest in the core region (86.1–88.7%), and the least in the E2 region (69.4–70.7%). Phylogenetic tree analysis confirmed the existence of a separate subtype 3i. The Indian HCV 3a isolate’s complete genomic sequences clustered with previously known genotype 3a sequences with a nucleotide similarity of 91.1% (SD 0.2%). Neither isolates showed evidence of recombination of different HCV genotypes. Conclusion The information on complete genomic sequences of the genotype 3 HCV isolates should be helpful in future studies on HCV evolution and classification, and for development of newer therapeutic and preventive strategies against this infection.

Investigation of a hepatitis A outbreak from Shimla Himachal Pradesh.

Background & objective: Hepatitis A is an enterically transmitted viral disease, highly prevalent in India and mainly presents as a paediatric sporadic disease. This study investigated an outbreak of viral hepatitis at Shimla, Himachal Pradesh, India, during January-March 2007. Methods: Eighty seven blood samples, 3 water samples and 2 sewage samples were collected. Serum samples were tested for IgM and IgG anti HAV and IgM and IgG anti HEV antibodies. Serum, sewage and water samples were tested for HAV-RNA by nested RT- PCR. Nearly complete full genome (excluding extreme 5’ end) was amplified from one serum sample. Results: The hepatitis cases were mainly seen among children and young adults and 63.2 per cent (55/88) were positive for anti-HAV IgM. These cases were reported from the areas getting water supply from Ashwani Khud water supply system. This water purification system received water from a natural stream in which treated sewage water was let into 4 km upstream the collection point since one year. HAV-RNA present in serum, sewage and water samples showed 100 per cent sequence homology. Phylogenetic analysis based on 5’ non coding (5’ NC) and nearly complete genome showed the evidence of HAV genotype IIIA in all the samples. Interpretation and conclusion: The aetiological agent of the present outbreak was hepatitis A virus which is emerging in an outbreak form in India, emphasizing a definite need for formulating vaccination / control strategies.

Molecular characterization of hepatitis A virus from a large outbreak from Kerala, India.

BACKGROUND & OBJECTIVES: Hepatitis A is highly prevalent in India and mainly presents as a sporadic disease. This study investigated an outbreak of viral hepatitis at Medical College Hospital area, Kottayam, Kerala state, India during January 2005. METHODS: Blood (133), faecal (1), sewage (4), and water samples (13) were collected. Sera were tested for IgG- and IgM-anti-HAV and IgM antibodies against hepatitis E (IgM-anti-HEV). Sewage, faeces and water samples were tested for HAV RNA in nested RT-PCR and HAV RNA positive samples were further processed for RNA quantitation using Real Time PCR. RESULTS: Of the 1180 total cases, 540 were reported from Medical college area. Two deaths were reported among doctors. Patients from the community gave a previous history of visit to medical college hospital area. The sewage treatment plant at the campus was non-functional since 1990 and the untreated sewage was constantly overflowing and getting mixed with a canal. At the time of the study, all the water sources were superchlorinated. HAV RNA was present in the faeces of hepatitis A patient (1.36 x 10(7) copies/ml), sewage tank (2.57 x 10(3) copies/ml and the canal (<100 copies/ml). None of the 13 water samples concentrated 10,000-fold and the soil sample showed presence of HAV RNA. Phylogenetic analysis based on 5'-non-coding and P2 regions showed HAV-genotype IIIA in all samples. INTERPRETATION & CONCLUSION: The aetiological agent of the present outbreak was found to be HAV. Epidemic hepatitis A (genotype-IIIA) is emerging in Indian adults, emphasizing the need for definite policy for control.

Vertical transmission of hepatitis A.

Two hospital delivered full term newborn babies were detected to have cholestatic jaundice in the first week of life. They had raised liver enzyme levels, which gradually declined over a period of one month. Both babies were anti HAV IgM positive on 6th day of life in Case 1 and on 7th day of life in Case 2 respectively. Both the mothers had jaundice 20 and 26 days before delivery and had anti HAV IgM positivity two and three weeks prior to delivery in Case 1 and 2 respectively. Hepatitis A virus is not transmitted vertically from mother to baby. However, there are 3 such case reports in literature stating vertical transmission of HAV infection. We are reporting it in two neonates for the first time in India.

Hepatitis G virus infection in India: prevalence and phylogenetic analysis based on 5' non-coding region.

OBJECTIVES: To determine the prevalence of hepatitis G virus (HGV) infection in western India and to carry out phylogenetic analysis of HGV isolates. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect HGV RNA in serum samples obtained from paid plasma donors, patients with hemophilia and voluntary blood donors. Nine Indian and one Kenyan HGV RNA-positive samples were sequenced in the 5' non-coding region (5'-NCR). Phylogenetic analysis based on the comparison of a 101 nucleotide fragment from a large number of HGV isolates from 22 countries (including Indian and Kenyan sequences obtained during the present study) was carried out. RESULTS: HGV RNA positivity rates among paid plasma donors from a commercial plasmapheresis unit (7/43, 16.3%) and patients with hemophilia (5/44, 11.4%) were significantly higher than that in voluntary blood donors (0/51; p=0.003 and 0.019, respectively). Among patients with acute non-A to E hepatitis and fulminant hepatic failure, 1 of 50 and 1 of 28 were HGV RNA-positive, whereas 6 of 49 (12%) patients with chronic liver disease had circulating HGV RNA. All Indian isolates belonged to genotype 2, whereas the Kenyan isolate formed a distinct branch within genotype 1 consisting of African isolates. CONCLUSION: Our results suggest existence of parenteral transmission of HGV in the Indian population. HGV was not an important cause of acute non-A to E hepatitis or fulminant hepatic failure among the patients investigated. Genotype 2 seems to be the most prevalent genotype in western India.

Ten-year serological follow up of hepatitis B vaccine recipients.

AIM: To determine long-term persistence of antibodies to hepatitis B surface antigen (anti-HBs) after vaccination against hepatitis B. METHODS: Thirty-four laboratory workers received hepatitis B vaccine in 1989 in a 0-1-6 month vaccination schedule. Group A (n = 16) received a booster at 3 years after vaccination whereas Group B (n = 18) did not. Anti-HBs was quantitated at 1 month and 1, 2, 3, 5, 6 and 8 years post-vaccination. RESULTS: At eight-year follow up, 10 of 15 subjects in Group A and 3 of 16 in Group B had protective levels of anti-HBs; in addition, two and four subjects, respectively, had detectable anti-HBs though below protective levels. At ten years, 9/15 and 3/16 were anti-HBs positive in Groups A and B, respectively. One subject in each group had rise in anti-HBs titer at 6-year follow up but both of them tested negative for IgG antibodies to hepatitis B core antigen (anti-HBc). A booster dose at 10 years to anti-HBs negative subjects led to an anamestic response in 3/4 and 8/10 persons in Groups A and B, respectively. CONCLUSION: Immunological memory after vaccination against hepatitis B is maintained for at least 10 years.

Prevalence of hepatitis B infection among the primitive tribes of Andaman & Nicobar Islands.

BACKGROUND & OBJECTIVES: Andaman and Nicobar Islands, is the home of six primitive tribes. No information is available on the prevalence of hepatitis B virus (HBV) infection among them. Hence a study was undertaken with the objective of assessing the sero-prevalence of HBV infection among the four accessible tribes of these islands. METHODS: A total of 1266 serum samples were collected from four tribes i.e., Nicobarese, Shompens, Onges and Great Andamanese and tested for the presence of HBsAg and anti-HBs. Information about different risk factors associated with HBV infection was also collected from the Nicobarese tribe. RESULTS: The overall seropositivity rate of HBsAg among the Nicobarese was 23.3 per cent (95% C.I. 21.0-25.9). Hepatitis B was also found to be an important health problem among the Shompens and Onges with HBsAg positivity of 37.8 and 31.0 per cent respectively. The age-wise distribution of these serological markers among Nicobarese tribe indicate that the infection is very common in all the age groups. The sero-prevalence was also found to be very high among the children. CONCLUSIONS & INTERPRETATION: The findings of the present study indicate that hepatitis B infection is hyper endemic among the primitive tribes of these islands. Though none of the risk factors studied in the Nicobarese was found to be significantly associated with HBV infection, the fact that almost 20 per cent of the women in the reproductive age group were positive for HBsAg indicates the possibility of vertical transmission among the Nicobarese. Further studies are required to find out other modes of transmission.

Epidemic hepatitis E: serological evidence for lack of intrafamilial spread.

INTRODUCTION: Hepatitis E presents as epidemic as well as sporadic disease. Fecal contamination of drinking water results in epidemics of hepatitis E. The extent of intrafamilial spread needs to be assessed employing serological assays. AIMS: To understand the dynamics of intrafamilial spread of the disease. METHODS: The study was conducted using blood samples collected during the 1988 and 1989 epidemics of viral hepatitis in Kudal and Atit villages of Maharashtra state; the epidemics were subsequently shown to be due to hepatitis E virus (HEV). The one-time collection carried out at the end of the Kudal epidemic was from 184 apparently healthy individuals irrespective of family history of jaundice during the epidemic. In the Atit epidemic, 153 family contacts of 49 IgM anti-HEV positive patients were bled. An additional 151 blood samples were collected from apparently healthy individuals irrespective of family history of jaundice during the epidemic. One month later, blood samples were collected from 64 of the 153 family contacts. Relevant history was recorded each time. All serum samples were tested for ALT levels and for IgM and IgG antibodies to hepatitis E virus employing ELISA. RESULTS: IgM anti-HEV positivity among persons with family history of jaundice was not different from those without such a history (8/62 [12.9%] and 11/122 [9%] at Kudal; 9/57 [15.8%] and 22/94 [23.4%] at Atit; p > 0.1). Excluding IgG anti-HEV positive samples from the analysis also yielded non-significant results. Of the 32 follow-up samples collected from family contacts without IgG or IgM antibodies to HEV in the initial blood sample, 31 remained IgM and IgG anti-HEV negative at the end of 1 month. One of the family contacts was found to be IgG anti-HEV positive in the second blood sample. The disease was not related to the index case. CONCLUSION: Intrafamilial spread of HEV is negligible.

Fulminant hepatic failure: etiology, viral markers and outcome.

OBJECTIVE: To investigate the etiology and outcome of fulminant hepatic failure (FHF) in children. SETTING: Hospital based descriptive. METHODS: 36 children (22 males and 14 females) presenting with FHF over a period of one year were investigated. The ages ranged from 1.5 to 9 years. FHF was defined as occurrence of encephalopathy within eight weeks of onset of jaundice with no evidence of pre-existing liver disease. Detailed history, clinical examination, routine biochemical parameters and relevant diagnostic tests were carried out. Viral markers studied were anti HAV-IgM, HBsAg, anti HBc-IgM, anti-HCV and anti HEV-IgM. RESULTS: A viral etiology could be established in 22 children (61.1%). Hepatitis A (n = 12), Hepatitis B (n = 3), Hepatitis A and B (n = 2), and Hepatitis A and E (n = 4). Two children had enteric fever (1 with associated HEV), 2 children had Wilson's disease, 1 child had Indian Childhood Cirrhosis (ICC) and 2 children had drug induced hepatitis. Etiological diagnosis was not possible in 8 children (22%). Fourteen children (39%) died. Poor outcome was associated with spontaneous bleeding, raised prothrombin time, lower transaminases and higher bilirubin on admission. CONCLUSION: Viral hepatitis is the commonest cause of FHF in children. HAV alone or in combination is responsible for upto 50% of all FHF in children. Chronic liver disease can also present as FHF. Etiological diagnosis is not possible to upto one-fourth of all cases.

Prevalence of hepatitis A antibodies in western Indian population: changing pattern.

This report pertains to a retrospective study conducted between 1983 and 1995 at three time points to evaluate the prevalence of hepatitis A virus (HAV) infection in the population of Bhor Taluk, situated in western India. Serum samples from children and adults were tested for anti-HAV antibodies using blocking ELISA test. There was a significant decrease in anti-HAV prevalence among children aged 5-10 years in 1995 (87.36%) as compared to that of 1983 (97.58%) and 1987 (96.48%). All individuals >11 years of age were seropositive for anti-HAV antibodies. Anti-HAV prevalence was similar in the users of well water, but was significantly reduced in individuals supplied with piped water in 1995 (88.61%) compared with that in 1983 (98.77%). A significant decrease in anti-HAV positivity was noted in children from Bhor Taluk as compared to children from Pune bled in 1992. These results underline the need for periodic surveillance of seroepidemiology of hepatitis A to determine the measures for prevention and control of the disease.

Insignificant prevalence of antibodies to hepatitis C in a rural area of western Maharashtra.

OBJECTIVE: To determine the age-specific seroprevalence of hepatitis C virus (HCV) in a rural population in Maharashtra. METHODS: 1054 serum samples collected from apparently healthy persons were tested by recombinant immunoblot assay for antibodies against HCV (anti-HCV). Anti-HCV positive samples were tested for HCV-RNA by nested reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: One man tested positive for anti-HCV; his sample was also HCV-RNA positive. CONCLUSIONS: HCV infection is infrequent in this rural area in Maharashtra.

Exposure of Indian children to hepatitis A virus & vaccination age.

It is known that 90 per cent of children in India are exposed to hepatitis A virus (HAV) by the age of six years. The aim of the study was to determine when in early childhood maximum HAV infections take place and to deduce an appropriate age for vaccination against HAV. Blood samples of 499 children between the ages of three days and six years were collected and tested for the presence of antibodies against hepatitis A. A statistically significant negative correlation between IgG anti-HAV and age was observed (P < 0.01) up to 11.67 months when IgG anti-HAV positivity was found to be minimum (9.25%). Subsequently a significant positive correlation was noted (P < 0.01). Exposure to HAV was 28.9 per cent soon after the waning of maternal antibodies in the 13-15 month age group which increased to 52.5 per cent by two years of age and 90.9 per cent by 6 yr. It is concluded that in addition to other preventive measures, if children in India are to be vaccinated against hepatitis A they should be immunised against HAV by 9-10 months of age when the maternal antibodies disappear.

Hepatitis A in day care centre.

A focal outbreak of hepatitis was detected in a day-care centre for children centrally located in Pune. The source of infection was suspected to be an 11-year-old child who probably got the infection from his school. Seven out of 15 children from day-care centre developed clinical hepatitis. Two cases of secondary infection were identified among the family contacts of infected children. Sera from all the nine sick children were positive for anti-hepatitis A virus-IgM antibodies. A stool sample from a case of secondary infection showed presence of HAV-RNA by RT-nested PCR. These findings proved that the outbreak was caused by hepatitis A virus.

Prevalence of anti-HCV antibodies in western India.

Nearly 2000 serum samples collected from different risk groups from Pune and Bombay metropolitan areas were tested for antibodies to hepatitis C virus (anti-HCV) by Recombinant Immunoblot Assay-3 (RIBA-3). Patients undergoing haemodialysis showed 24.5 per cent seropositivity whereas 5.7 and 5.3 per cent of multiply transfused patients (>2 units) and chronic liver disease patients respectively were anti-HCV positive. Leprosy patients had almost 0.7 per cent seropositivity. In other risk groups the positivity rate was nil. In normal population only one out of 830 persons had anti-HCV antibodies. It is therefore apparent that the prevalence of hepatitis C virus (HCV) in western India is not high. However, special care needs to be taken for dialysis patients. As none of the 430 pregnant women and 86 children below the age of 5 yr were anti-HCV positive, vertical mode of HCV transmission seems to be negligible.

Indigenous anti-hepatitis A virus IgM capture ELISA for the diagnosis of hepatitis A.

Anti-hepatitis A virus IgM capture ELISA was developed by using the reagents produced in the NIV laboratory. The major reagents of the assay were anti-human IgM antibody, hepatitis A virus (HAV) and anti-HAV IgG-horse radish peroxidase (HRP) conjugate. Of these, anti-human IgM antibodies were generated in rabbit against IgM secreted by human hybridoma clone(G3). HAV was derived from buffalo green money kidney cell line infected with HM-175 strain. Virus purified from the cell lysates was used for immunization of rabbits and guinea-pigs. There was very low anti-HAV response. A seropositive rhesus monkey was inoculated with monkey adapted strain of HAV to boost the anti-HAV antibody titre. Anti-HAV IgGs derived from hyperimmune sera of monkey and hepatitis A patient were conjugated with HRP. The preparations of conjugate--particularly human antibody--HRP conjugate yielded highly satisfactory results in anti-HAV capture ELISA. The assay appears to be specific, sensitive and quick and is useful in differentiating acute HAV infection from other acute infections caused by B, E and non-A non-B hepatitis viruses.

Hepatitis E virus infection in pregnant rhesus monkeys.

Ten non-pregnant female monkeys and four pregnant monkeys (all Macaca mulatta) in the last third of their gestation period were infected intravenously with the stool sample of a patient with hepatitis E virus infection (immuno-electronmicroscopy positive for hepatitis E virus). Four more non-pregnant monkeys were inoculated with a lower dose (less number of virus particles by IEM) of a stool sample collected on a different day from the same patient. The average incubation period as evidenced by the rise of serum alanine transferase in the non-pregnant monkeys, was 36.4 +/- 4.9 days. The dose of the virus did not affect the incubation period. Two of the pregnant monkeys had incubation periods of 9 and 13 days respectively. They delivered healthy babies on 40th and 53rd day respectively after inoculation. At the age of 11 months, both babies were negative for anti-HEV antibodies. One monkey which delivered a healthy baby on the 2nd day after inoculation had incubation period of 36 days. The baby of this monkey was anti-HEV positive at the age of 11 months. The incubation period was 41 days in the fourth monkey which delivered a macerated foetus on the 36th day after infection. No fatality was recorded in the infected monkeys. Bile samples collected from all monkeys showed strong signals in nested polymerase chain reaction (PCR). It seems that the incubation period in pregnant monkeys was determined by the state of pregnancy.