Vidya A. Arankalle

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. 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 (TCID(50)) and indirect immunofluorescence assay (IFA). 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. 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.

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. 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. 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. 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.

Phylogenetic analysis of the sequences of the 5′ noncoding regions (5′NCR) of 149 samples from hepatitis C virus (HCV) RNA-positive chronic carriers representing northern, southern, eastern, and western India showed that type 3 and type 1 are the predominant genotypes circulating in India, with an overall prevalence of 53.69 and 38.25%, respectively. Type 4 viruses (6.04%) were seen only in southern India. Sequence analysis of the core region of 51 of the above isolates enabled us to classify them further into subtypes as 1b (number of isolates [ n ] = 10), 1a ( n = 6), 3a ( n = 9), 3g ( n = 14), 3f ( n = 1), and 4d ( n = 3). Three new subtypes were identified for the first time and designated as 3i ( n = 5), 3j ( n = 2), and 6l ( n = 1). Sequencing the 5′NCR could differentiate HCV types, whereas classification at the level of subtype was possible with sequence analysis of the core region.

Summary. To determine the association of precore (Pre‐C)/basal core promoter (BCP) mutants with clinical outcome of hepatitis B in Western India, 192 hepatitis B virus (HBV) infected individuals were investigated. HBV‐DNA PCR positivity among asymptomatic hepatitis B surface antigen (HBsAg) positive carriers (61/100) was lower (P < 0.0001) than chronic hepatitis B (CHB), acute (P = 0.0001), and fulminant hepatitis B patients (P = 0.047). Pre‐C status was based on restriction fragment length polymorphism (RFLP, n = 153) and sequencing (n = 118). Prevalence of Pre‐C mutants was higher among carriers (23/61) than CHB (10/62, P = 0.0071) or acute (3/22; P = 0.037) patients. Children from carrier and CHB categories showed significantly higher circulation of Pre‐C‐wild than mutant HBV. Clinical manifestations were independent of BCP mutations (1762/64‐T/A). Hepatitis B e antigen (HBeAg) negative CHB patients [62.5% (15/24)] were circulating wild HBV. Higher HBV‐DNA levels were associated with chronic hepatitis and HBeAg positivity, whilst Pre‐C mutant positives had lower levels. BCP mutations did not affect HBV‐DNA levels. Multivariate regression analysis identified HBeAg (OR = 4.3) and Pre‐C mutants (OR = 3.1) to be associated with chronic hepatitis and carriers respectively. In a separate sub‐set analysis (n = 59), HBV‐DNA level was identified as the only variable. In conclusion, chronic or fulminant hepatitis B was not associated with Pre‐C or BCP mutants and switching over to Pre‐C mutant was beneficial for the infected individual in maintaining disease free status for extended periods.

Summary. Though a potent vaccine represents a powerful preventive tool, the policy of its use is governed by epidemiological and economical factors. Hepatitis A, an enterically trasmitted disease shows distinct association with socio‐economic status, populations with improvement experiencing lower exposure to the virus. With the availability of vaccine, it is pertinent to consider its use in the effective control of the disease. However, with the varied epidemiological patterns and economical constraints in different countries it does not seem to be possible to evolve universal policy for immunization. Though, universal immunization may be the most effective way of control, the same is not practical for many countries. It is proposed that irrespective of endemicity of hepatitis A, high‐risk groups such as travelers to endemic areas, patients suffering from chronic liver diseases, HBV and HCV carriers, tribal communities with high HBV carrier rates, food handlers, sewage workers, recipients of blood products, troops, and children from day‐care centers should be immunized with hepatitis A vaccine. In addition, for populations with intermediate prevalence, infants, children from affordable families may be immunized. As coupling the vaccine with EPI schedule would be beneficial, use of combined A & B or A, B & E vaccine may be an attractive alternative.

To determine hepatitis B virus genotype and subtype distribution among HBV infected individuals with different clinical manifestations in western India, serum samples from 19 asymptomatic hepatitis B surface antigen carriers, 30 chronic hepatitis B patients, 8 acute hepatitis B patients, 5 fulminant hepatitis B patients, and with circulating HBV DNA were genotyped and subtyped on the basis of the nucleotide sequence analysis of S region of the HBV genome. Genotype D was the predominant genotype circulating in western India (57/62; 91.93%). All 19 asymptomatic hepatitis B surface antigen carriers, 8 acute hepatitis B patients, 5 fulminant hepatic failure patients and 25/30 chronic hepatitis B patients were circulating genotype D and ayw3/ayw2 subtypes. HBV genotype A was prevalent in 8% (5/62) of the total number of patients and all belonged to chronic hepatitis B category. Subtyping analysis showed that all genotype A isolates were of subtype adw2. As most of the patients from different clinical categories were infected with HBV genotype D, it is concluded that this genotype did not influence the outcome of HBV infection. J. Med. Virol. 69:324–330, 2003.