Vidya A. Arankalle

Chikungunya outbreaks caused by African genotype, India.

Chikungunya fever is reported in India after 32 years. Immunoglobulin M antibodies and virus isolation confirmed the cause. Phylogenic analysis based on partial sequences of NS4 and E1 genes showed that all earlier isolates (1963–1973) were Asian genotype, whereas the current and Yawat (2000) isolates were African genotype.

Thermal stability of hepatitis E virus

The thermal stability of virulent hepatitis E virus (HEV) and hepatitis A virus (HAV) was compared. Fecal suspensions of virus were heated to temperatures between 45 degrees C and 70 degrees C, and residual infectivity was determined in a cell culture system that was permissive for both viruses. Although HEV was less stable than was HAV, some HEV would most likely survive the internal temperatures of rare-cooked meat.

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.

AETIOLOGICAL ASSOCIATION OF A VIRUS-LIKE PARTICLE WITH ENTERICALLY TRANSMITTED NON-A, NON-B HEPATITIS

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.

Systemic involvements and fatalities during Chikungunya epidemic in India, 2006.

BACKGROUND In addition to classical manifestations of Chikungunya infection, severe infections requiring hospitalization were reported during outbreaks in India in 2006. OBJECTIVES To describe the systemic syndromes and risk groups of severe Chikungunya infections. STUDY DESIGN We prospectively investigated suspected Chikungunya cases hospitalized in Ahmedabad, Gujarat during September-October 2006, and retrospectively investigated laboratory-confirmed Chikungunya cases hospitalized with neurologic syndromes in Pune, Maharashtra. Hospital records were reviewed for demographic, comorbidity, clinical and laboratory information. Sera and/or cerebrospinal fluid were screened by one or more methods, including virus-specific IgM antibodies, viral RNA and virus isolation. RESULTS Among 90 laboratory-confirmed Chikungunya cases hospitalized in Ahmedabad, classical Chikungunya was noted in 25 cases and severe Chikungunya was noted in 65 cases, including non-neurologic (25) and neurologic (40) manifestations. Non-neurologic systemic syndromes in the 65 severe Chikungunya cases included renal (45), hepatic (23), respiratory (21), cardiac (10), and hematologic manifestations (8). Males (50) and those aged >or=60 years (50) were commonly affected with severe Chikungunya, and age >or=60 years represented a significant risk. Comorbidities were seen in 21 cases with multiple comorbidities in 7 cases. Among 18 deaths, 14 were males, 15 were aged >or=60 years and 5 had comorbidities. In Pune, 59 laboratory-confirmed Chikungunya cases with neurologic syndromes were investigated. Neurologic syndromes in 99 cases from Ahmedabad and Pune included encephalitis (57), encephalopathy (42), and myelopathy (14) or myeloneuropathy (12). CONCLUSIONS Chikungunya infection can cause systemic complications and probably deaths, especially in elderly adults.

Retrospective Analysis of Blood Transfusion Recipients: Evidence for Post–Transfusion Hepatitis E

Background and Objectives: Of 200 volunteer blood donors we had screened earlier for hepatitis E virus (HEV) RNA, using reverse–transcription polymerase chain reaction, 3 were positive, raising the possibility of transfusion–associated hepatitis E in areas endemic for this virus. This retrospective study was to reassess the extent of post–transfusion hepatitis E among transfusion recipients, investigated in 1982. Materials and Methods: We re–evaluated 56 recipients followed biweekly for 3 months after transfusion. The controls were 51 normal, healthy persons who gave blood at a 2–month interval, as well as 412 blood donors from whom blood was taken once in 1982. Results: Of the 56 transfusion recipients, 19 were positive for IgG antibodies against HEV (anti–HEV) in the pretransfusion sample. Two of the 37 IgG anti–HEV–negative recipients seroconverted to IgM and IgG anti–HEV 5 and 4 weeks after transfusion, 1 with raised serum alanine aminotransferase levels. None showed symptoms of hepatitis. Attempts to detect HEV RNA in transfused blood, from aliquot units stored at –20°C for over 17 years, were not successful. Of the controls, 17 out of 51 were IgG anti–HEV positive in the initial sample itself. None of the 34 IgG anti–HEV–negative controls seroconverted during the 2–month follow–up. Of the blood donors, 154 out of 412 were IgG anti–HEV positive. None of the 412 donors had circulating IgM anti–HEV antibodies. A significantly higher (p<0.03) proportion of susceptible transfusion recipients were IgM anti–HEV positive as compared with susceptible blood donors. Conclusion: The results suggest that, in countries where HEV is endemic, the transmission of hepatitis E may be associated with blood transfusion.

Phylogenetic analysis of hepatitis E virus isolates from India (1976-1993)

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.

Polymerase chain reaction-based prevalence of hepatitis A, hepatitis E and TT viruses in sewage from an endemic area

BACKGROUND/AIMS Hepatitis A and E viruses (HAV, HEV) are transmitted enterically and are highly endemic in India. This study aims to evaluate prevalence of these and TT virus (TTV) in the sewage. METHODS Influent and effluent samples from a sewage treatment plant from Pune, India were collected twice a week for 1 year and subjected to nested polymerase chain reaction (PCR) for the detection of HAV RNA, HEV RNA and TTV DNA. HAV and HEV PCR products were sequenced. Effluent samples were not collected for 5 months as the plant was non-functional. RESULTS The overall prevalence was 24.42% (21/86, HAV), 10.98% (9/82, HEV) and 12.7% (8/63, TTV). Prevalence of HAV was significantly higher than HEV (P=0.023). During summer months, significantly higher HAV RNA positivity was noted (P<0.01). A substantial reduction in HAV RNA positivity (15/48 vs. 2/48, P=0.0008) was recorded for treated sewage samples. However, HEV RNA or TTV DNA positivity did not reduce significantly. Of the 17 HAV and HEV RNA negative sewage samples concentrated using ultracentrifugation, 13 and none were positive for HAV and HEV RNA, respectively. Phylogenetic analyses grouped these viruses in IB and Ia, respectively, the genotypes most prevalent in India. CONCLUSIONS Sewage may play an important role in maintaining hyper-endemicity of these infections. Sustained efforts are obligatory to render sewage less/non-infectious.

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.