Suppressive activity and altered conventional phenotype markers/mediators of regulatory T cells in patients with self-limiting hepatitis E.

Hepatitis E virus (HEV) is a major cause of self-limiting acute viral hepatitis in several developing countries. Elevated levels of peripheral CD4(+) CD25(+) Foxp3(+) , CD4(+) CD25(-) Foxp3(+) and rise in IL-10 in hepatitis E have been associated with the involvement of regulatory T cells (Treg). The functional role of the same is yet elusive. In the current study, we have assessed (i) Foxp3 expression by real-time PCR and by flow cytometry, (ii) the levels of antigen-specific IL-10 and TGF-ß by ELISA, (iii) functional analysis of Treg cells and (iv) expression of Treg-associated conventional phenotypes by flow cytometry in 54 acute patients, 44 recovered individuals from hepatitis E and in 33 healthy controls. Foxp3 mRNA elevation in the acute compared with recovered group and elevation in Foxp3(+) cells in both patient groups were significantly elevated. The levels of IL-10 and TGF-ß in the acute patients and TGF-ß in the recovered individuals were elevated. Significantly higher expression of CTLA-4, PD1, GITR, CD95, CD103 and CD73 on Treg and T effector (Teff) cells was detected in the patient groups. Treg cells of acute patients and recovered individuals exhibited suppressive activity indicating that the Treg cells of hepatitis E patients are functional. The suppressive capacity of Treg cells in acute hepatitis E patients was significantly higher compared with the recovered individuals. Based on our findings, the suppressive functionality of these key markers associated with hepatitis E Treg function need further exploration to get a better understanding of the mechanisms of Treg-mediated suppression.

Peripheral T regulatory cells and cytokines in hepatitis E infection.

This study addresses the involvement of regulatory T cells in hepatitis E (HE) infection. The study population comprised 77 acute viral HE patients, 52 recovered individuals (overall, 129 individuals with HE) and 53 healthy controls. Peripheral CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)Foxp3(+) frequencies by flow cytometry and HE-specific cytokines/chemokines quantitation were carried out. The median percentage of CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)Foxp3(+) T cells in acute patients were significantly higher compared to controls and recovered individuals. Both of the T regulatory (Treg) subset populations in overall HE were significantly elevated compared to controls. Comparisons of cytokines/chemokines revealed that the levels of IL-10 were elevated in: (a) acute viral hepatitis E (AVH-E) versus recovered individuals and controls, and (b) HE versus controls. Overall, the elevation of CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)Foxp3(+) frequencies and the rise in IL-10 suggest that Treg cells might be playing a pivotal role in hepatitis E virus (HEV) infection.

Epidemic of hepatitis B with high mortality in India: association of fulminant disease with lack of CCL4 and natural killer T cells.

An explosive outbreak of Hepatitis B with high mortality was reported in 2009, in Modasa, Gujarat, India. Mortality was associated with basal core promoter and precore mutant hepatitis B virus (HBV). The current study addresses the role of immunological parameters in the progression to fulminant hepatitis. The study population comprised of 22 acute HBV patients, 13 fulminant HBV liver failure patients and 54 healthy controls. Hepatitis B surface antigen-induced CTL responses by enzyme-linked immunosorbent spot (ELISPOT), cytokine and chemokine quantitation by Bioplex assay, peripheral NK, natural killer T (NKT), CD4 and CD8 T-cell frequencies by flow cytometry were carried out. The median percentage of NK cells in the lymphocytes of the acute and fulminant liver failure patients were significantly lower compared to controls. Acute and fulminant liver failure patients had significantly high and comparable NKT cells compared to controls, respectively. Importantly, NKT cells were significantly lower in fulminant HBV liver failure than acute HBV patients. Circulating peripheral CD4/CD8 T-cell subsets among the patient categories and controls were comparable. In acute HBV patients, a significant increase in IFN-γ release was recorded (ELISPOT) by the unstimulated, antigen-stimulated and mitogen-stimulated cells when compared to controls. Comparisons of cytokines and chemokines among the disease categories revealed significantly lower levels of CCL4 in fulminant liver failure patients. NKT cells and CCL4 might be playing a pivotal role in limiting HBV infection among the patients investigated.

An outbreak of hepatitis B with high mortality in India: association with precore, basal core promoter mutants and improperly sterilized syringes.

In 2009, an outbreak of hepatitis B with high mortality was observed in Sabarkantha district, Gujarat state, India with 456 cases and 89 deaths. Hospitalized patients with self-limiting disease (152, AVH)) and fulminant hepatic failure (39, FHF including 27 fatal and 12 survivals) were investigated. These were screened for diagnostic markers for hepatitis viruses, hepatitis B virus (HBV) genotyping and mutant analysis. Complete HBV genomes from 22 FHF and 17 AVH cases were sequenced. Serosurveys were carried out in the most and least affected blocks for the prevalence of HBV and identification of mutants. History of injection from a physician was associated with FHF and AVH cases. Co-infection with other hepatitis viruses or higher HBV DNA load was not responsible for mortality. Four blocks contributed to 85.7% (391/456) of the cases and 95.5% (85/89) mortality while two adjacent blocks had negligible mortality. Sequence analysis showed the presence of pre-core and basal core promoter mutants and 4 amino acid substitutions exclusively among FHF cases. None of the self-limiting patients exhibited these dual mutations. Genotype D was predominant, D1 being present in all FHF cases while D2 was most prevalent in AVH cases. Probably due to violation of accepted infection control procedures by the qualified medical practitioners, HBV prevalence was higher in the affected blocks before the outbreak. Gross and continued use of HBV contaminated (mutant and wild viruses) injection devices led to an explosive outbreak with high mortality with a striking association with pre-C/BCP mutants and D1 genotype.

Investigation of a Chikungunya-like illness in Tirunelveli district, Tamil Nadu, India 2009-2010.

OBJECTIVE: To identify the aetiological agent/s of an outbreak of chikungunya-like illness with high morbidity and several fatalities in Tamil Nadu, India, 2009-2010. METHODS: Two hundred and seventeen serum samples were collected from the affected areas and screened for chikungunya virus (CHIKV), dengue virus (DENV) and Japanese encephalitis virus (JEV) IgM antibodies using MAC-ELISA kits. A few selected samples were also tested for Ross River, Sindbis, and Murrey Valley viruses by RT-PCR and Hantan virus by serology. Twelve acute serum and mosquito samples were processed for virus isolation in C6/36 cells. CHIKV isolate was characterised by RT-PCR and sequencing. RESULTS: Diagnostic levels of IgM antibodies were detected in 107 (49.3%) CHIKV samples and 22 (10.1%) DENV samples. IgM antibodies against JEV were not detected (n=46). Characterisation of the CHIKV isolate at genetic level demonstrated it as ECSA (E1: 226A). Thirty-six selected samples were also negative for Ross River, Sindbis, Murrey Valley and Hantan viruses. CONCLUSION: High prevalence of CHIKV IgM antibody positivity, clinical symptoms, virus isolation and the presence of vector mosquitoes clearly suggest CHIKV as the aetiological agent responsible for the outbreak.

A novel HBV recombinant (genotype I) similar to Vietnam/Laos in a primitive tribe in eastern India.

Genotyping of 20 strains of Hepatitis B virus (HBV) from the Idu Mishmi primitive tribe of northeast India identified multiple genotypes and the presence of a unique cluster grouping with strains from Vietnam and Laos identified as novel recombinants/genotype I. Sequence analysis (similarity and bootscan plots) of three complete HBV genomes from the tribe provided evidence of recombination. Phylogenetic analyses supported recombination between genotypes A, G and C. The Pre-S gene between nt 2943 and 397 was clearly of genotype A origin, whereas nt 397-1397 represented genotype G and nt 1397-2943 represented genotype C. Percentage divergence from genotypes B, D, E, F, G and H varied from 9.2 +/- 0.45% to 13.8 +/- 0.53%, whereas genotype A and C differed by 7.9 +/- 0.42% and 7.4 +/- 0.39% respectively. The identification of similar recombinant viruses in three countries, especially in a primitive tribe with no contact with the outside world suggests that these viruses do not represent recent recombination events, but circulation of closely related viruses highly divergent from known HBV genotypes and should be classified as members of genotype 'I'.

Hepatitis E virus-based evaluation of a virion concentration method and detection of enteric viruses in environmental samples by multiplex nested RT-PCR.

AIMS: The prevalence of enteric viruses in drinking and river water samples collected from Pune, India was assessed. During an outbreak of HEV in a small town near pune, water samples were screened for enteric viruses. METHODS AND RESULTS: The water samples were subjected to adsorption-elution-based virus concentration protocol followed by multiplex nested PCR. Among 64 Mutha river samples, 49 (76.56%) were positive for Hepatitis A Virus, 36 (56.25%) were positive for Rotavirus, 33 (51.56%) were positive for Enterovirus and 16 (25%) were positive for Hepatitis E Virus RNA. Only enterovirus RNA was detected in 2/662 (0.3%) drinking water samples, and the samples from the city's water reservoir tested negative for all four viruses. HEV RNA was detected in three out of four river water samples during HEV outbreak and partial sequences from patients and water sample were identical. CONCLUSIONS: The study suggests absence of enteric viruses both in the source and in the purified water samples from Pune city, not allowing evaluation of the purification system and documents high prevalence of enteric viruses in river water, posing threat to the community. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid, sensitive and relatively inexpensive protocol developed for virological evaluation of water seems extremely useful and should be adapted for evaluating viral contamination of water for human consumption. This will lead to development of adequate control measures thereby reducing disease burden because of enteric viruses.

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 (TCID(50)) 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.

Simian hepatitis A virus derived from a captive rhesus monkey in India is similar to the strain isolated from wild African green monkeys in Kenya.

SUMMARY: A simian hepatitis A virus (HAV) was identified retrospectively in a faecal sample from a rhesus monkey in India, inoculated in 1995 with a faecal suspension from a suspected patient of non-A to E hepatitis. The monkey was in captivity for 2 years in one of the experimental primate facilities in western India before being moved to the National Institute of Virology, Pune for experimentation. Phylogenetic analysis based on a partial sequence of the 5' noncoding region placed this virus in genotype V, the only other member being the AGM-27 strain recovered in 1986 from African green monkeys in Kenya. The source of infection of the monkey remains unclear. The full genome was amplified in nine fragments and sequenced. The genome of the Indian simian HAV (IND-SHAV) is 7425 nucleotides long including the poly-A tail of 14 nucleotides at the 3' end. At the nucleotide and amino acid levels, IND-SHAV was 99.8 and 100% identical with AGM27, respectively.

Viral load, antibody titers and recombinant open reading frame 2 protein-induced TH1/TH2 cytokines and cellular immune responses in self-limiting and fulminant hepatitis e.

OBJECTIVES: Hepatitis E virus (HEV) is the predominant cause of water-borne epidemics, sporadic acute viral hepatitis (AVH) in adults and fulminant hepatic failure (FHF) among pregnant women and other adults in India. This preliminary study was designed to examine the association of viral load and certain host immune responses with uneventful recovery or progression to FHF. METHODS: Viral load, anti-HEV antibody titers, rORF2p-induced Th1/Th2 cytokines levels and cellular immune responses were assessed in 47 patients with self-limiting hepatitis E and 14 FHF-E cases. The controls included 16 anti-HEV-IgM and IgG-negative healthy individuals. RESULTS: In AVH category, the viral load was 2.4 x 10(4) +/- 1.92 x 10(4) copies/ml while except for one, all FHF patients were negative for HEV RNA; anti-HEV-IgM and IgG titers were higher in the FHF group. Lymphocyte proliferative response to rORF2p was comparable in both groups. As compared to AVH, significantly higher levels of both Th1 (IFN-gamma, IL-2 and TNF-alpha) and Th2 (IL-10) cytokines were recorded in FHF patients. Analysis of sequential samples differentiated FHF recovered and fatal patients with respect to IFN-gamma and IL-12. CONCLUSION: The results document increased Th1/Th2 responses and anti-HEV titers in FHF patients that warrant in-depth immunological studies.

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 & 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.

The detection and characterization of hepatitis E virus in pig livers from retail markets of India.

The zoonotic transmission of Hepatitis E virus (HEV) is a well-established fact and pigs are known reservoirs of the virus. The human and swine HEV from countries such as the USA, Japan and Taiwan, show a remarkable sequence identity. Swine liver samples from markets in Japan and US were shown to be HEV RNA positive. In contrast, in India, viruses belonging to different genotypes, that is, genotypes 1 and 4 circulate in humans and pigs respectively, at least for the last 20 years. To assess possible exposure of the Indian population to swine HEV, 240 pig liver samples were collected from retail markets of Pune, western India. Two (0.83%) samples were found positive for HEV RNA by nested RT-PCR. Nucleotide sequence-based phylogenetic analysis showed the presence of genotype 4 HEV with 90-91% similarity and clustering with Indian swine HEV sequences generated earlier. The data suggest the possibility of HEV infection in persons consuming infected pig liver. So far, it has not been possible to detect any type 4 HEV infection in humans. The absence of type 4 infections in humans may be attributed to cooking leading to the inactivation of the virus.

Venereal transmission of Chandipura virus by Phlebotomus papatasi (Scopoli).

Experiments were conducted in the laboratory on Phlebotomus papatasi to determine the possible role of males in maintaining or sustaining the Chandipura virus (CHPV) activity in nature. This study indicated that infected males are capable of passing on the virus to female sand flies while mating. The infection rate was found to be 12.5% in uninfected females when mated with infected males. The occurrence of venereal transmission of this virus may have epidemiologic importance in the natural cycle of CHPV.

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.

Hepatitis delta virus infection among the tribes of the Andaman and Nicobar Islands, India.

Hepatitis B virus infection is highly endemic among the tribes of Andaman and Nicobar Islands, India. We screened 223 hepatitis B surface antigen-positive members of these tribes for hepatitis delta virus infection (HDV). The infection was observed only among the Nicobarese. Considering the serious consequences of HDV infection, we suggest that the tribes of these islands should be monitored for HDV infection.

Detection of chandipura virus from sand flies in the genus Sergentomyia (Diptera: Phlebotomidae) at Karimnagar District, Andhra Pradesh, India.

A total of 191 adult sand flies belonging to the genus Sergentomyia were collected from seven villages in Karimnagar and Warangal districts of Andhra Pradesh State, India, after an outbreak of encephalitis due to Chandipura virus (CHPV). Fifteen pools, each containing two specimens, were tested by reverse transcriptase-polymerase chain reaction and sequencing. One pool of Sergentomyia from Kolanur village in Karimnagar District was positive for CHPV.

Fecal shedding of hepatitis A virus in Indian patients with hepatitis A and in experimentally infected Rhesus monkey.

Hepatitis A is highly endemic in India. The surveillance reports for the disease from this region are primarily based on the demonstration of hepatitis A virus (HAV) specific serum IgM and IgG antibodies. The present study was conducted to assess the presence and duration of fecal shedding of HAV in patients with hepatitis A and in an experimentally infected rhesus monkey. Nested reverse transcription polymerase chain reaction (RT-PCR) was applied to fecal specimens from 67 sporadic cases of hepatitis A. Recent infection with HAV in these cases was evidenced by the presence of serum anti-HAV IgM. Fecal HAV RNA positivity was observed in nearly 40% patients. The proportion of HAV RNA positivity in fecal specimens obtained within the first week (36.58%) was not different from those collected in 2-12 weeks post onset (42.42%) (P>0.05). A significant number of HAV RNA positive stool specimens showed presence of full virus particles by immune electron microscopy (IEM). Extended fecal shedding of HAV could be a major contributory factor for high circulation of virus thereby maintaining hyperendemicity of the disease. One of the IEM positive samples was inoculated into an anti-HAV negative rhesus monkey. Serum alanine amino transferase levels of the monkey remained within the normal limits. However, HAV RNA positivity in the feces was noted from 3 to 50 days post inoculation. The monkey seroconverted to anti-HAV IgM on day 31. This study records prolonged excretion of HAV in humans as well as in experimentally infected rhesus monkey.

Detection of IgM against hepatitis A virus using tissue culture derived antigen.

Sera from healthy donors and four groups of subjects with acute viral hepatitis were tested for anti-HAV IgM by ELISA with hepatitis A virus antigen grown in tissue culture. The results were compared with those obtained by formalin inactivated HAV from 'HAVAB-M' test kit (Abbott Laboratories, USA). With both preparations of antigens, sera from healthy donors and patients suffering from acute hepatitis B or non-A, non-B did not show anti-HAV IgM antibodies whereas acute phase sera from hepatitis A patients showed strong reactions indicating the presence of anti-HAV IgM antibodies. The results indicate similarity in the specificity of both preparations of HAV against anti-HAV antibodies and encourage the use of tissue culture derived HAV for serological diagnosis of hepatitis A.

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.

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.