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Subrat Kumar Panda

Publications and source records attributed to Subrat Kumar Panda.

11 recordsLinked to original sources
PubMed2005

Replication of a recombinant hepatitis E virus genome tagged with reporter genes and generation of a short-term cell line producing viral RNA and proteins.

Hepatitis E virus (HEV) replication has been demonstrated in HepG2 cells transfected with full-length in vitro transcripts of an infectious cDNA clone. This cDNA clone was modified to generate several subgenomic HEV replicons with fused reporter genes. In vitro-transcribed capped RNAs generated from these were transfected into HepG2 cells. Negative-strand RNA was detected, indicating the occurrence of replication. The replicon containing an in-frame fusion of HEV ORF2 with enhanced green fluorescent protein (EGFP) was positive for fluorescence, whereas no signal was observed when the replicase domain was deleted. An HEV ORF3-EGFP in-frame fusion did not yield fluorescence. Deletions introduced into ORF2 did not affect the replication competency of the viral RNA. To explore the possibility of using a reporter-gene assay to monitor the synthesis of plus- and minus-strand RNA, the EGFP gene fused to the encephalomyocarditis virus internal ribosome entry site (IRES) was inserted into partially deleted ORF2 of HEV, in both the sense [HEV-IRES-EGFP(+)] and antisense [HEV-IRES-EGFP(-)] orientations. HepG2 cells transfected with HEV-IRES-EGFP(+) and HEV-IRES-EGFP(-) vectors were positive for EGFP fluorescence. To quantify HEV replication, EGFP was replaced with Renilla luciferase (RLuc). HEV-IRES-RLuc(+) showed approximately 10-fold higher luminescence than HEV-IRES-RLuc(-). There was complete loss of activity when the helicase-replicase domain in HEV-IRES-RLuc(-) was deleted. A short-term HepG2 cell line containing the full-length viral genome in the pcDNA3 vector was established. Viral RNA and proteins (RdRp, pORF2 and pORF3) could be detected in the geneticin-resistant cells, even after the seventh passage. In the absence of a reliable cell-culture system to study HEV biology, these reporter replicons, as well as the cell line, bestow immense utility.

Cell Line, Tumor↗
PubMed2004

Development and evaluation of a quantitative competitive reverse transcription polymerase chain reaction (RT-PCR) for hepatitis C virus RNA in serum using transcribed thio-RNA as internal control.

A method for quantitation of hepatitis C virus (HCV) RNA was developed based on competitive reverse transcription polymerase chain reaction (RT-PCR) using in vitro transcribed mutated thio-RNA as a competitor template. The thio-RNA is more resistant to RNAse and is stable over a year. This assay was compared with the commercially available Roche Amplicor HCV Monitor assay V 2.0 and real time PCR using SYBR green 1 dye method. A total of 18 pre-therapy serum samples from chronic hepatitis C cases were tested in parallel by the three assays. All samples could be quantitated using the in-house competitive RT-PCR and real time PCR and there was a significant correlation in the virus titer (P<0.05). However, 8 (44%) samples could not be quantified by Amplicor HCV Monitor assay, which has a lower detection range (10(2) to 10(5.5) copies/ml). The in-house method of competitive RT-PCR showed a detection range of 10(3) to 10(10) copies/ml. In the patients the mean viral titer was found to be (9.66+/-9.3)x10(6) copies/ml. Ten (55%) of the samples, assessed by the Amplicor HCV Monitor assay showed a mean viral titre of (1.13+/-0.75)x10(6) copies/ml, which was lower than the other two tests. The competitive PCR method and real time PCR could amplify all prevalent genotypes. This in-house quantitative competitive RT-PCR method is simple, cheap, reproducible and useful for estimation of HCV RNA load.

Genotype↗
PubMed2004

Occult hepatitis B virus infection in chronic liver disease: full-length genome and analysis of mutant surface promoter.

BACKGROUND AND AIMS: Genome sequence of hepatitis B virus (HBV) from occult chronic infection is scarce. Fifty-six (9.4%) of 591 patients seronegative for hepatitis B surface antigen (HBsAg) with chronic liver disease were positive for HBV DNA. The complete HBV genome from 9 of these patients (S1-S9) and 5 controls positive for HBsAg (SWT.1-SWT.5) were analyzed. METHODS: Overlapping genome fragment amplification, cloning, and sequencing was performed on these cases. Functional analysis of surface promoter was conducted using fusion construct. RESULTS: All patients with occult infection except one (S8) had a low viral titer. Eight patients had infection with genotype A (S1-S5, SWT.1-2, SWT.5) and 6 had infection with genotype D (S6-S9, SWT.3-4). S4 and S5.1 of genotype A had the characteristic nucleotide deletions in core and pre-S1 region seen in genotype D. The major observations in patients with occult HBV infection were as follows: frequent quasispecies variation, deletions in pre-S2/S region affecting the surface promoters (nt 3025-54) and pre-S protein (S3, S5, S6, S8), truncated precore (S6, S8, S7.1) and core (S9) owing to stop signal, alternate start codon for the Polymerase gene (S3, S9), and YMDD mutation (S1, S4, S9) in patients not on antiviral therapy. HBsAg and core proteins could be shown immunohistochemically in 3 of 5 liver biopsy specimens available. The mutant surface promoters (pre-S2 and S) on functional analysis showed alterations in HBsAg expression. CONCLUSIONS: These changes in the regulatory region with possible alterations in the ratio of large and small surface proteins along with other mutations in the genome may decrease the circulating HBsAg level synergistically, making the immunodetection in serum negative.

Adolescent↗
PubMed2004

Treatment of hepatitis C virus infection in patients of northern India.

BACKGROUND AND AIM: The purpose of the present study was to evaluate the therapeutic response of north Indian patients with chronic hepatitis C (CH-C) to two different treatment regimens of interferon and ribavirin. METHODS: Consecutive patients with a diagnosis of CH-C attending the Liver Clinic at the All India Institute of Medical Sciences, New Delhi between April 1999 and April 2002 were included in the study. A competitive reverse transcription-polymerase chain reaction (RT-PCR) method developed in the authors' laboratory was used for quantification of hepatitis C virus (HCV)-RNA. Genotyping of HCV was also determined. The clinical, biochemical, virological and histological parameters were used to assess the therapeutic response among a clinical cohort of patients with chronic hepatitis C. They were treated with two different protocols (interferon [IFN]-alpha-2b, 3 million units daily and ribavirin 10.6 mg/kg daily in two divided doses for 6 months or IFN-alpha-2b, 3 million units thrice weekly and ribavirin 10.6 mg/kg daily for 6 months). RESULTS: Sixty-five patients with CH-C were included in the study. Blood transfusion (n = 28, 43%) and community-acquired (n = 23, 35%) HCV infections were the commonest. The mean HCV load was high (24.14 +/- 12.5 x 10(8) copies/mL). Genotype 2 and 3 were prevalent in 80% (41/51) of the patients. Forty-five patients received 3 million units of IFN thrice weekly and 20 received the same dose daily. All received the same dose of ribavirin. A sustained virological response (SVR) of 95% (19/20) was achieved among patients receiving daily IFN, whereas 64.4% (29/45) of those who received IFN thrice weekly had SVR. The virological relapse was significantly lower among patients who received daily IFN than in those treated with thrice weekly IFN (n = 1/20, 5% vs 10/39, 25.6%; P = 0.015). The proportion of patients receiving daily IFN among those achieving SVR (19/48, 40%) was significantly higher than the proportion of patients receiving similar therapy among patients without SVR (1/17, 6%; P = 0.02). CONCLUSIONS: Transfusion and community-acquired HCV infection were the major causes of CH-C. Genotype 2 and 3 HCV were most prevalent among these patients. Despite high viral load, these patients responded well to a combination of daily IFN-alpha-2b and ribavirin.

Adult↗
PubMed2004

Role of polymerase chain reaction and liver biopsy in the evaluation of patients with asymptomatic transaminitis: implications in diagnostic approach.

BACKGROUND AND AIM: Detection of an asymptomatic rise in the hepatic aminotransferase (ARHA) value has become a distinct and frequent clinical problem. We evaluated a three-step diagnostic algorithm in such patients for maximum yield. METHODS: Consecutive patients with an ARHA value 1.5-fold the upper limit of normal for at least 4 weeks and who were apparently healthy were included in the study. Each patient underwent standard biochemical investigations and a stepwise investigative protocol. In the first step, serological markers for hepatitis viruses, serum ferritin, 24-h urinary copper, alpha-1-antitrypsin phenotyping, and autoimmune markers were carried out. In step two, patients who tested negative for all the above markers had polymerase chain reaction (PCR) analysis for hepatitis B virus (HBV)-DNA and hepatitis C virus (HCV)-RNA. Patients without a diagnosis despite the above investigations underwent a liver biopsy as part of step three. RESULTS: Of 105 patients with ARHA, 38 were excluded for various reasons and 67 were included for the final analysis. The mean age was 35.11 +/- 11.96 years and 56 patients were men. The mean body mass index was 24.17 +/- 3.2 kg/m(2). The stepwise diagnostic algorithm achieved a diagnosis in 65/67 (97%) patients. Non-alcoholic steatohepatitis (NASH) and chronic viral hepatitis were the most common diagnoses, in 24 (36%) patients each. Using the diagnostic algorithm a diagnosis was reached in 34% of patients with only serological and biochemical investigations, whereas PCR for HBV and HCV could further detect the presence of active HBV or HCV viremia in 21% (14/97) and a liver biopsy was necessary to establish the diagnosis in 28/67 (42%) patients. CONCLUSIONS: A stepwise diagnostic algorithm in patients with ARHA resulted in an optimal use of PCR and invasive tests such as liver biopsy. Cryptic HBV and HCV infection was frequent among these patients and PCR was necessary in such cases. NASH and chronic viral hepatitis were the most frequent causes of ARHA.

Adolescent↗
PubMed2003

Targeted cleavage of hepatitis E virus 3' end RNA mediated by hammerhead ribozymes inhibits viral RNA replication.

The 3' end of hepatitis E virus (HEV) contains cis-acting regulatory element, which plays an important role in viral replication. To develop specific replication inhibitor at the molecular level, mono- and di-hammerhead ribozymes (Rz) were designed and synthesized against the conserved 3' end sequences of HEV, which cleave at nucleotide positions 7125 and 7112/7125, respectively. Di-hammerhead ribozyme with two catalytic motifs in tandem was designed to cleave simultaneously at two sites spaced 13 nucleotides apart, which increases the overall cleavage efficiency and prevents the development of escape mutants. Specific cleavage products were obtained with both the ribozymes in vitro at physiological conditions. The inactive control ribozymes showed no cleavage. The ribozymes showed specific inhibition of HEV 3' end fused-luciferase reporter gene expression by approximately 37 and approximately 60%, respectively in HepG2 cells. These results demonstrate a feasible approach to inhibit the HEV replication to a limited extent by targeting the cis-acting 3' end of HEV with hammerhead ribozymes.

Base Sequence↗
PubMed2003

Hepatocellular carcinoma in a hepatitis B 'x' transgenic mouse model: A sequential pathological evaluation.

BACKGROUND: The introduction of transgenic technology has made it possible to study the steps of carcinogenesis and directly establish the link between viral subgenomic fragments and specific types of cancer. Research directed at hepatitis B virus (HBV)-related carcinogenesis has benefited from this technology. We present a detailed pathological evaluation of the sequential steps of hepatocarcinogenesis in a hepatitis B 'x' (HBx) transgenic mouse model. In this model, the transgene incorporates the region encoding amino acids 58-154 of the HBV X protein and the murine c-myc gene. This model demonstrated changes in the liver from birth with foci of multicentric dysplasia evolving into nodules and overt hepatocellular carcinoma between 20 and 28 weeks. METHODS AND RESULTS: The hepatocytes were mitotically active and showed increased proliferative capacity soon after birth, with exponential increase thereafter. This was accompanied by a high rate of apoptosis, which later declined as the tumors developed. Other functional and immunophenotypic characteristics included a high c-myc expression in the neoplastic lesions, no alteration in p53 expression, and no alteration in the expression of hepatic enzymes except for diffuse expression of succinic dehydrogenase. CONCLUSION: The entire process illustrates the disturbances of cell growth and death because of the collaborative influence of HBx and c-myc genes that result in the development of hepatocellular carcinoma after a prolonged latent period.

Animals↗
PubMed2003

Evaluation of serologic screening of blood donors in India reveals a lack of correlation between anti-HBc titer and PCR-amplified HBV DNA.

BACKGROUND: Transfusion associated-HBV (TAHBV) is estimated at approximately 1.5 percent in postsurgical recipients and 50 percent or more in multiple-transfusion recipients in India. Not transfusing blood with high-titer anti-HBc, which reportedly correlates with the presence of HBV DNA, helped reduce TAHBV in Japan. This study tested anti-HBc-reactive donors for PCR-amplified HBV DNA and its correlation with anti-HBc titers. STUDY DESIGN AND METHODS: In total, 30,853 donors from Cohort 1 (24,694 in 2001) and Cohort 2 (6159 in 2000) were screened for anti-HBc and anti-HBs. Amplification of HBV DNA was performed on an unselected subset of 147 out of 3304 anti-HBc-only sera from Cohort 1 and 230 out of 6159 from Cohort 2. Correlation of anti-HBc titers in DNA positive (n = 48), DNA negative (n = 40), anti-HBs reactive (n = 44), and HBsAg reactive (n = 45) donors was by Fisher's exact test. RESULTS: In Cohort 1, 2673 (10.82%) donors were reactive for anti-HBc, of whom 1038 (4.20%) were anti-HBc only. HBV DNA was detected in 40 out of 147 (27.21%) and 48 out of 230 (20.87%) donors with anti-HBc only from the two cohorts. Anti-HBc titers detected no significant difference between the first three groups. CONCLUSION: Cryptic HBV infection was observed in approximately 25 percent of anti-HBc-only donors. No correlation was established between HBc titers and presence of HBV DNA.

Blood Donors↗
PubMed2002

Vaccination against hepatitis A virus may not be required for schoolchildren in northern India: results of a seroepidemiological survey.

OBJECTIVES: To evaluate the current seroprevalence of antibodies against hepatitis A virus (HAV) in a sample of schoolchildren above 10 years of age and to determine the prevalence of HAV-induced hepatitis in adults at a tertiary care hospital in northern India between January 1992 and December 2000. METHODS: Sera from 276 male and 224 female schoolchildren aged 10-17 years were tested for anti-HAV antibodies by enzyme-linked immunosorbent assay. Consecutive patients with a diagnosis of acute viral hepatitis who attended a liver clinic were tested for the serological markers of HAV, hepatitis B Virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus. FINDINGS: Of the male and female children, 96.3% and 98.2%, respectively had anti-HAV antibodies in their sera. The prevalence of these antibodies in the age groups 10-12, 13- 14, and 15-17 years were 98.6%, 94.8%, and 98.3% respectively. The frequency of HAV- induced acute viral hepatitis (69/870, 8%) in adults did not show an increasing trend. CONCLUSION: Mass HAV vaccination may be unnecessary in northern India because the seroprevalence of protective antibodies against HAV in schoolchildren aged over 10 years remains above 95% and there has been no apparent increase in HAV-induced acute viral hepatitis in adults.

Adolescent↗
PubMedDate not supplied

Musings on HEV.

Explore the source record for details and available documents.

Hepatitis E↗