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Seventeen Indian hepatitis E virus (HEV) isolates, representing epidemic and sporadic hepatitis E cases during 1976-1991, were sequenced in the RNA polymerase (RNAP) region. Five isolates were also sequenced in the non-structural hypervariable region of open reading frame 1. Open reading frames 2 and 3 were sequenced only for the prototype isolate. On the basis of the comparison of all the available sequences of the conserved RNAP region, the HEV isolates were divided into three genotypes, differing from each other by >15%. Genotype I included African and Asian isolates, whereas II and III were represented by Mexican and US isolates, respectively. Genotype I was further divided into four sub-genotypes. The majority of the Indian isolates (15/20), along with the Burmese and Nepali isolates, belonged to genotype IA. Genotype IB included HEV isolates from China, Pakistan and the former USSR and 2/20 Indian isolates, which represented the oldest (1976) HEV sequenced so far. Genotype IC included both the African isolates, whereas 3/20 Indian isolates formed genotype ID. Nucleotide sequence analysis of other regions of the HEV genome also placed isolates in the same genotypes. Both the Indian cities experiencing second HEV epidemics, after intervals of 8 and 10 years, showed shifts in the sub-genotypes found; from IB (Ahm-76) to IA (Ahm-84) and from IA (Kol-81) to ID (Kol-91). However, no major shift in the genotypes was noted. Overall, HEV genotypes appear to be segregated geographically.

Raw sewage samples from an area where hepatitis E is not endemic (Barcelona, Spain) were analyzed by reverse transcriptase-PCR followed by nested PCR. One of the 37 tested samples showed a positive result for hepatitis E virus (HEV). The detected strain was amplified by inoculation into rhesus monkeys, and the course of the infection was studied by analyzing serological and biochemical parameters and by monitoring the presence of HEV in serum and feces. Fecal suspensions from the rhesus monkeys were used as the source of viral particles for sequence analysis. Eighty percent of the genome of the isolated strain, named BCN, was sequenced and found to be phylogenetically related to Asian (Indian) strains, with a 98% nucleotide identity with an isolate from Madras, India. Since this was a single isolation we cannot conclude that HEV is regularly present in the sewage. However, the finding of viable HEV in sewage has implications for contamination of the environment and shellfish by HEV and must be considered in the diagnosis of viral hepatitis in regions of nonendemic hepatitis.

The age-specific seroprevalence of antibody to hepatitis A virus (HAV) and antibody to hepatitis E virus (HEV) were studied in persons in Pune, India, where both viruses are endemic. The data showed that HAV infected the majority of persons by age 3 years and virtually 100% by late childhood. In contrast, infection with HEV was rare in children and did not reach peak prevalence (33%-40%) until early adulthood. The reason for the differences in infection rates between HAV and HEV is not known. Age-specific antibody patterns in serum samples obtained 10 years apart show that neither HAV nor HEV has diminished in medical importance in this Indian community.

Many epidemics of water-borne hepatitis have occurred throughout India. These were thought to be epidemics of hepatitis A until 1980, when evidence for an enterically transmitted non-A, non-B hepatitis was first reported. Subsequently, hepatitis E virus was discovered and most recent epidemics of enterically transmitted non-A, non-B hepatitis have been attributed to hepatitis E virus infection. However, only a limited number of cases have been confirmed by immuno electron microscopy, polymerase chain reaction, or seroconversion. In the present study we have performed a retrospective seroepidemiologic study of 17 epidemics of water-borne hepatitis in India. We have confirmed that 16 of the 17 epidemics were caused at least in part by serologically closely related hepatitis E viruses. However, one epidemic, in the Andaman Islands, and possibly a significant minority of cases in other epidemics, appears to have been caused by a previously unrecognized hepatitis agent.

69 serum pairs from two common-source water-borne outbreaks and one series of endemic cases of hepatitis in three parts of India were tested for hepatitis A and hepatitis B virus infections. None of the patients had evidence of HAV infection and only 10.1% had evidence of HBV infection. A large proportion of hepatitis in India seems to be caused by previously unrecognised agents.

Waterborne non-A, non-B hepatitis (NANB) is responsible for outbreaks of hepatitis with a predilection for young adults. The disease is usually mild, except in pregnant women, who have a high case-fatality rate from fulminant hepatic failure. Diagnosis is largely based on the epidemiological findings of faecal contamination of drinking water and serological exclusion of hepatitis A and B virus infection. Histological features of liver biopsy specimens are characteristic and virus-like particles in the stool are aggregated by antibody present in acute and convalescent phase sera of the test subject. NANB is widespread in India and several countries of South-East Asia; it is increasingly recognised in Africa and may occur in Latin America. Control measures include provision of clean water supplies, safe disposal of human excreta, and sound personal and food hygiene practices. Passive immunisation with immunoglobulin derived from healthy donors resident in the countries affected by the disease may protect vulnerable groups.

Virus-like particles, approximately 27 nm in diameter, were identified in faeces from an Indian patient with enterically transmitted non-A, non-B (ENANB) hepatitis. They were serologically distinct from hepatitis A virus (HAV). Nucleic acid extracted from the particles did not hybridize with cDNA probes representing the genomes of HAV, enteroviruses, and cardioviruses. Chimpanzees were experimentally inoculated with faecal suspensions containing this 27 nm particle or with faeces from another case of ENANB hepatitis. Mild histological and biochemical hepatitis developed in these animals and there was serological evidence of infection with the virus-like particle as shown by immunoelectronmicroscopy (IEM). Serological analysis by IEM suggested that this agent or an antigenically similar virus was the aetiological agent of two epidemics and a sporadic case of ENANB hepatitis in India and of an epidemic of the illness in the USSR. Antibody to the particle was found in sera from patients with ENANB hepatitis from various geographic areas over a 30-year period.