Leenata P. Chobe

Prevalence of anti-hepatitis E virus antibodies in different Indian animal species

Prevalence of IgG antibodies to hepatitis E virus (IgG‐anti‐HEV) was determined among different animal species from India. Seropositivity varied from 4.4% to 6.9% in cattle, 54.6–74.4% in pigs and 2.1–21.5% in rodents. Of the 44 dogs screened, 10 were positive (22.7%). None of the 250 goat sera tested were found to be anti‐HEV positive. Among rodents, over 50% serum samples collected in 1985 from Bandicota bengalensis were positive for anti‐HEV antibodies. No evidence of HEV infection was obtained following experimental inoculation of an Indian strain (AKL‐90) of HEV into anti‐HEV negative pigs and goats. The results document varied prevalence of anti‐HEV antibodies in different animal species from India and of inability of Indian pigs and goats to support replication of at least one human strain of HEV.

Human and swine hepatitis E viruses from Western India belong to different genotypes

BACKGROUND/AIMS Hepatitis E is endemic in India. Earlier, we showed prevalence of IgG antibodies to hepatitis E virus (IgG-anti-HEV) in different animal species and inability of at least one human hepatitis E virus (HEV) strain to infect pigs. In the US where hepatitis E is not endemic in humans, zoonotic spread of HEV was suspected as swine and human HEV were closely related and cross-species infection was documented. The present study attempts to identify and partially characterize swine HEV from India. METHODS Serum samples from 284 pigs were screened for the presence of HEV-RNA (nested polymerase chain reaction; PCR) and IgG-anti-HEV (enzyme-linked immunosorbent assay; ELISA). PCR products (Open Reading Frame-2 region) were sequenced and subjected to phylogenetic analysis. Two sero-negative pigs were inoculated with swine HEV-positive serum pool. RESULTS ELISA and PCR positivity were 42.9 and 4.6%, respectively. All Indian swine HEV sequences clustered with genotype IV. Pigs could be experimentally infected with swine HEV. CONCLUSIONS Swine HEV circulates in Indian pigs. In contrast to US and Taiwan wherein both human and swine HEV isolates belong to same genotype, Indian human HEV isolates belong to genotype I whereas genotype IV circulates in swine. Though experimental infection with Indian swine HEV was possible, at least one human HEV strain could not infect pigs.

Evaluation of human (genotype 1) and swine (genotype 4)-ORF2-based ELISAs for anti-HEV IgM and IgG detection in an endemic country and search for type 4 human HEV infections

Summary.  Open reading frame 2 proteins (ORF2) from swine (genotype 4, S‐ORF2) and human (genotype 1, H‐ORF2) hepatitis E virus (HEV) having 91.4% identity at amino acid level were expressed using baculovirus expression system. Comparison of ELISAs based on the two proteins yielded identical results when sequential serum samples from monkeys and pigs experimentally infected with genotypes 1 and 4 HEV, respectively, were tested. Samples from patients (n = 258) suffering from non‐A, non‐B hepatitis during outbreaks of the disease and 180 sera from apparently healthy children were screened by H‐ORF2‐, S‐ORF2‐based ELISAs and Genelabs ELISA, a widely used commercial test for HEV diagnosis. Specificity of all three tests in detecting IgM and IgG antibodies in healthy children was comparable. Excellent correlation was noted in detecting both IgM (98.7% concordance) and IgG (97.7% concordance) anti‐HEV antibodies when H‐ORF2 and S‐ORF2 ELISAs were compared. When compared with Genelabs ELISA, both H‐ORF2 and S‐ORF2 ELISAs identified 34 and 18 additional positives, respectively, in IgM and IgG anti‐HEV tests showing comparatively less sensitivity of the commercial assay. The concordance of Genelabs ELISA in IgM detection was 86.4% and 85.6%, respectively, with H‐ORF2 and S‐ORF2 ELISAs. The concordance between Genelabs ELISA and H‐ORF2 decreased further to 73.6% when 129 human samples from recent HEV epidemics (2002–2004) were tested for IgM. Similar results were obtained when sequential samples from 11 hepatitis E patients were examined. Screening of serum samples from 137 sporadic non‐A, non‐B hepatitis cases further confirmed the superiority of the H‐ORF2 and S‐ORF2 ELISAs. All 36/137 HEV‐RNA‐positive samples from sporadic cases belonged to genotype 1 confirming absence/rarity of type 4 human infections. H‐ORF2 and S‐ORF2 antigens were swappable in ELISAs for detecting both genotypes 1 and 4 HEV infections.

Type‐IV Indian swine HEV infects rhesus monkeys

Summary.  In contrast to countries reporting zoonotic spread of hepatitis E virus (HEV), distinct genotypes circulate in humans (genotype 1) and pigs (genotype 4) from India indicating rarity of such spread. Pigs were refractory to human HEV. As rhesus is an excellent animal model for human HEV, an attempt was made to infect rhesus monkeys with swine HEV. Experimental infection of both the rhesus monkeys with swine‐HEV as evidenced by seroconversion to anti‐HEV antibodies and presence of viraemia suggests possibility of human infections or differential susceptibility. Comparison of Open Reading Frame‐2 and hypervariable regions of HEV genomes showed identity of swine and monkey‐derived HEV.

Long-term serological follow up and cross-challenge studies in rhesus monkeys experimentally infected with hepatitis E virus

BACKGROUND/AIMS The aims of this study were to examine the decline of IgG anti-HEV antibodies over a period of 7 years in rhesus monkeys experimentally infected with hepatitis E virus, and to assess the protectivity of these antibodies by challenging the monkeys with a heterologous isolate of hepatitis E virus, 5 years after the primary inoculation. METHODS Nine rhesus monkeys (six non-pregnant and three pregnant at the time of hepatitis E virus inoculation) were followed serologically and biochemically for 7 years post-inoculation. Based on regression analysis, estimated time for IgG anti-HEV titers to reach 1:100 or 1:50 was calculated. Three of the monkeys inoculated initially with AKL-90 isolate and challenged 2 years later with PUN-85 isolate of hepatitis E virus were rechallenged with KOL-91 isolate of the virus, 5 years post-primary inoculation. Evidence of viral replication was assessed by measuring serum alanine aminotransferase levels, excretion of the virus in feces or bile (reverse-transcription polymerase chain reaction) and rise in IgG anti-HEV titers (ELISA). RESULTS None of the challenged monkeys showed evidence of disease. In contrast to extensive replication of the virus in anti-HEV-negative control monkeys, limited replication was noted in one of the challenged monkeys. The estimated time for the titers to reach 1:100 or 1:50 varied from 3.15 to 44.9 years (19.4+/-11.6 years) and 6.9 to 84.3 years (35.4+/-21.3 years), respectively. Decline in titers was independent of the pregnancy status at the time of infection or reexposure of the monkeys to HEV CONCLUSION: The results show persistence of IgG anti-HEV antibodies for a long time and protectivity of low titered antibodies against reinfection, leading to disease even after intravenous exposure to a heterologous isolate of hepatitis E virus.

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.