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.

Administration of E2 and NS1 siRNAs inhibit chikungunya virus replication in vitro and protects mice infected with the virus.

BACKGROUND: Chikungunya virus (CHIKV) has reemerged as a life threatening pathogen and caused large epidemics in several countries. So far, no licensed vaccine or effective antivirals are available and the treatment remains symptomatic. In this context, development of effective and safe prophylactics and therapeutics assumes priority. METHODS: We evaluated the efficacy of the siRNAs against ns1 and E2 genes of CHIKV both in vitro and in vivo. Four siRNAs each, targeting the E2 (Chik-1 to Chik-4) and ns1 (Chik-5 to Chik-8) genes were designed and evaluated for efficiency in inhibiting CHIKV growth in vitro and in vivo. Chik-1 and Chik-5 siRNAs were effective in controlling CHIKV replication in vitro as assessed by real time PCR, IFA and plaque assay. CONCLUSIONS: CHIKV replication was completely inhibited in the virus-infected mice when administered 72 hours post infection. The combination of Chik-1 and Chik-5 siRNAs exhibited additive effect leading to early and complete inhibition of virus replication. These findings suggest that RNAi capable of inhibiting CHIKV growth might constitute a new therapeutic strategy for controlling CHIKV infection and transmission.

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.

Survival of hepatitis A and E viruses in soil samples.

Survival of hepatitis A virus (HAV) and hepatitis E virus (HEV) in soil samples spiked with respective viruses was analysed using real-time PCR. Virus-spiked soil samples were incubated at environmental temperature (ET) and 37°C and processed weekly. Both HAV and HEV were less stable at fluctuating ET than at 37°C. Of the 403 soil samples collected in the vicinity of Mutha river, India, 19.1% and 4.9% were found to be contaminated with HAV and HEV, respectively.

Development of an inactivated candidate vaccine against Chandipura virus (Rhabdoviridae: Vesiculovirus).

A Vero cell based vaccine candidate against Chandipura (CHP) virus (Rhabdoviridae: Vesiculovirus), was developed and evaluated for immunogenicity in mice. Virus was purified by ultracentrifugation on 30% glycerol cushion followed by differential centrifugation on 10-60% sucrose gradient and inactivated with ß-propio lactone at a concentration of 1:3500. The inactivated product was blended with aluminium phosphate (3%) and immunized 4-week-old Swiss albino mice. Neutralizing antibodies in the range of 1:10 to 160 and 1:80 to 1:320 was detected with 85% and 100% sero-conversion after 2nd and 3rd dose, respectively. All the immunized mice with antibody titer above 1:20 survived live virus challenge. The vaccine candidate has potential to be an efficient vaccine against CHP virus.

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.

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.

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.

Complete genome sequences of hepatitis C virus subtype 3i and 3a subtype isolates from India.

AIM: Hepatitis C virus (HCV), a major causative agent of chronic hepatitis, is classified into six major genotypes. Genotype 3 HCV infection is more sensitive to interferon therapy. In India, genotype 3, particularly subtype 3a, HCV infections are common. Three novel HCV subtypes i.e., 3g, 3j, and 3i were identified from India based on partial genomic sequences. This report provides full genome sequences of one isolate each of subtypes 3i and 3a. METHODS: Serum samples positive for subtype 3i and 3a HCV RNA based on core region genomic sequences were studied. Complete HCV genomes were amplified as 11 overlapping PCR fragments and sequenced. RESULTS: The complete genomic sequence of Indian HCV 3i isolate clustered with other genotype 3 sequences, and was closer to subtypes 3b and 3a (80.5% and 79.1% [SD 0.4%] nucleotide identity). Nucleotide similarities were the highest in the core region (86.1-88.7%), and the least in the E2 region (69.4-70.7%). Phylogenetic tree analysis confirmed the existence of a separate subtype 3i. The Indian HCV 3a isolate's complete genomic sequences clustered with previously known genotype 3a sequences with a nucleotide similarity of 91.1% (SD 0.2%). Neither isolates showed evidence of recombination of different HCV genotypes. CONCLUSION: The information on complete genomic sequences of the genotype 3 HCV isolates should be helpful in future studies on HCV evolution and classification, and for development of newer therapeutic and preventive strategies against this infection.

Utility of pandemic H1N1 2009 influenza virus recombinant hemagglutinin protein-based enzyme-linked immunosorbent assay for serosurveillance.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against the pandemic H1N1 2009 influenza A virus, employing a recombinant hemagglutinin protein of the virus, was compared to the hemagglutination inhibition (HI) test using 783 serum samples. The results showed a concordance of 98.4%, suggesting the utility of the ELISA in serosurveillance. Two hundred sixty-nine (100%) serum samples with an HI titer of > or =20 were ELISA reactive.

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.

Evaluation of the immunogenicity of a recombinant glycoprotein-based Chandipura vaccine in combination with commercially available DPT vaccine.

Chandipura virus (CHPV) belongs to family Rhabdovoridae and has emerged as an encephalitis causing pathogen with high mortality among pediatric population from three Indian states. The recombinant glycoprotein (rGp) was shown to be an excellent vaccine candidate as evaluated in a murine model. As the disease is predominantly rural, to ensure maximum coverage for Chandipura vaccine, an attempt was made to evaluate combination of rGp and a commercially available DPT vaccine (CHP-DPT). When CHP-DPT was used for immunization of mice, 90% seroconversion against rGp with high antibody titers (1:1200 by ELISA and 1:320 by neutralization test) was observed and did not differ from mice immunized with rGp alone (P>0.05). Similarly seroconversions and antibody titers against DPT were comparable in mice immunized with DPT alone or in combination with rGp. Seroconversions and antibody titers ranged from 90 to 100% and 1:1200 to 1:2400 respectively. Intracerebral challenge with homologous CHPV strain resulted in 90% survival in rGp alone and CHP-DPT groups. Lymphocyte proliferative responses were also comparable. Thus, neither components of the candidate combination vaccine inhibited immune response to the other component. Substantial decrease of CHPV RNA and absence of histopathological changes in the brains of surviving immunized mice after challenge than the unimmunized controls further confirm efficacy of the vaccine even after intracerebral challenge. In conclusion, a combination vaccine seems feasible for use in a restricted area where the disease is endemic and should be subjected to additional studies required for future use in humans.

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.

Full length genomes of genotype IIIA Hepatitis A Virus strains (1995-2008) from India and estimates of the evolutionary rates and ages.

With the changing epidemiology, outbreaks of Hepatitis A Virus (HAV) have been reported from different parts of India. To characterize HAV strains circulating in India (1995-2008), 6 full genome sequences of the predominant genotype, IIIA, were determined. Further, applying the Bayesian Markov Chain Monte Carlo (MCMC) framework to the full genomes of Indian HAV strains as well as other global strains (human as well as simian), we derived the mean nucleotide substitution rate and evolutionary timescales with emphasis on the age of genotype III and IIIA strains. The genomic length of all the 6 HAV isolates was 7464 nt excluding the poly A tract. Phylogenetic analysis confirmed that all the Indian isolates were close to Nor-21 (AJ299464) and HMH (AY644337) of subgenotype IIIA. The ORF of the isolates when compared within genotype III at amino acid level showed a highly conserved pattern. Under the best fit expansion population relaxed molecular clock model, the estimated mean substitution rate of the HAV full genomes (human and simian strains) was 1.73 x 10(-4) substitutions/site/year based on which the earliest transmission of HAV from simian to humans is estimated to have occurred about 3564 years ago. The mean substitution rate within human HAV full genomes under the same model was estimated to be 1.99 x 10(-4) substitutions/site/year. With this the mean age of genotype III strains was estimated to be 592 years while that of genotype IIIA was estimated to be 202 years. The time to the most common recent ancestor (tMRCA) of the Indian genotype IIIA isolates was calculated to be 116 years.

Recombinant glycoprotein based vaccine for Chandipura virus infection.

Chandipura virus (CHPV) has emerged as an important pediatric encephalitis-causing pathogen with very high mortality in India. No specific vaccine or treatment is available till date. We attempted to prepare a candidate vaccine employing recombinant CHPV Glycoprotein (rGp). The Glycoprotein gene (G-gene) of CHPV was expressed using Baculovirus expression system. The rGp was purified by HPLC and used for mice immunization, 3 doses, and 4 weeks apart. One microgram rGp was found to be optimum. Sero-conversion was observed as early as 2nd week by detecting anti-CHPV IgG antibodies. Antibody titres were immunogen-concentration dependent. Intracerebral challenge of the immunized mice with 100 LD(50) of the homologous strain demonstrated 90% protection. In in vitro neutralization, antibodies from the immunized mice were able to neutralize heterologous viruses. There was 60% T cell proliferation observed against rGp in immunized mice. The study shows that rGp induces both arms of immune response and represents an ideal vaccine candidate for further evaluations.

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.

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.

Outbreaks of hepatitis A among children in western India.

Hepatitis A in most developing countries is a sporadic childhood disease, but lately focal outbreaks have been observed among children in India. During 2004, we investigated a large-scale outbreak of hepatitis among children living in a residential colony in Daund Taluka of District Pune in the western region of India. In total, 123 overt and 56 sub-clinical cases were detected. All the patients were reactive for IgM antibodies against hepatitis A virus (IgM anti-HAV) and were negative for IgM anti-hepatitis E virus, confirming HAV to be the etiological agent of the outbreak. Serum samples, feces and sewage samples were tested for HAV RNA and molecular characterization of the positives showed the presence of genotype IIIA. Further, IgM anti-HAV-positive sera from eight focal outbreaks were analyzed. The causative HAV in all these small-scale outbreaks also belonged to genotype IIIA, indicating the predominance of genotype IIIA in this region. This report of a large-scale, explosive outbreak of hepatitis A in Indian children once again emphasizes the need to evolve proper public health strategies, especially for vaccination, in countries in the transitional phase from hyperendemicity to intermediate endemicity.