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Biomedical subjects

S K Panda

Publications and source records attributed to S K Panda.

At least 19 recordsLinked to original sources
PubMed2001

The 3' end of hepatitis E virus (HEV) genome binds specifically to the viral RNA-dependent RNA polymerase (RdRp).

Hepatitis E virus (HEV) is the major cause of acute epidemic and sporadic hepatitis in the developing world. It is a positive-strand RNA virus with a genome length of about 7.2 kb. The replication mechanism of this virus is virtually unexplored. Identification of the regulatory elements involved in initiation of replication may help in designing specific inhibitors for therapy. In the positive-stranded RNA viruses the initiation of replication requires interaction of the 3' end of genome with its RNA-dependent RNA polymerase (RdRp) and possibly host-derived cofactors for synthesis of the minus-strand replicative intermediate. Secondary structure prediction of the conserved 3' end of the infectious HEV genome was carried out to identify possible stem-loop structures necessary for RNA-protein interaction and the model was confirmed by structure probing experiments. Electrophoretic mobility-shift assays showed specific binding of purified and refolded recombinant HEV RdRp protein to the 3' end of its RNA genome containing the poly(A) stretch. Mutations at the 3' end, in which the stem-loop structures were partially or completely destroyed or recreated revealed that the two stem-loop structures SL1 and SL2 at the 3' end and the poly(A) stretch are necessary for this binding. The interacting nucleotides in such an interaction were further identified by generating footprints of the complex by Pb(II)-induced hydrolysis. This specific binding of viral RdRp to the 3' end of HEV RNA directs the synthesis of complementary-strand RNA and thus such a binding domain might assume the role of a possible cis-acting element as a potential site for the initiation of replication.

3' Untranslated Regions↗
PubMed2000

Acute hepatic failure in India: a perspective from the East.

Acute hepatic failure (AHF) in India almost always presents with encephalopathy within 4 weeks of the onset of acute hepatitis. Further subclassification of AHF into hyperacute, acute and subacute forms may not be necessary in this geographical area, where the rapidity of onset of encephalopathy does not seem to influence survival. Viral hepatitis is the cause in approximately 95-100% of patients, who therefore constitute a more homogeneous population than AHF patients in the West. In India, hepatitis E (HEV) and hepatitis B (HBV) viruses are the most important causes of AHF; approximately 60% of cases are caused by to these viruses. Hepatitis B virus core mutants are very important agents in cases where hepatitis B results in AHF in this country. Half of the patients with AHF admitted to our centre are female, one-quarter of whom are pregnant. Therefore, pregnant females who contract viral hepatitis constitute a high-risk group for the development of AHF. However, the outcome of AHF in this group is similar to that in non-pregnant women and men. No association with any particular virus has been identified among sporadic cases of AHF. In our centre, approximately one-third of AHF patients survive with aggressive conservative therapy, whereas two-thirds of deaths occur within 72 h of hospitalization. Cerebral oedema and sepsis are the major fatal complications. Both fungal and gram-negative bacteria are major causes of sepsis. Among patients with AHF, despite the presence of sepsis, its overt clinical features (i.e. fever, leucocytosis) may be absent and objective documentation of the presence of sepsis in such patients is achieved by repeated culture of various body fluids. It should be possible to develop simple, clinical prognostic markers for AHF in this geographical region, in order to identify patients suitable for liver transplantation.

Adult↗
PubMed2000

The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious.

Hepatitis E virus (HEV) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the Indian subcontinent and prevalent in most of the developing parts of the world. The infection is often associated with acute liver failure and high mortality, particularly in pregnant women. In order to develop methods of intervention, it is essential to understand the biology of the virus. This is particularly important as no reliable in vitro culture system is available. We have constructed a cDNA clone encompassing the complete HEV genome from independently characterized subgenomic fragments of an Indian epidemic isolate. Transfection studies were carried out with HepG2 cells using in vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of negative-sense RNA, indicative of viral replication, was demonstrated in the transfected cells by strand-specific reverse transcription-PCR and slot blot hybridization. The viral proteins pORF2 and pORF3 and processed components of the pORF1 polyprotein (putative methyltransferase, helicase, and RNA-dependent RNA polymerase) were identified in the transfected cells by metabolic pulse-labeling with [(35)S]methionine-cysteine, followed by immunoprecipitation with respective antibodies. The expression of viral proteins in the transfected cells was also demonstrated by immunofluorescence microscopy. Viral replication was detected in the transfected cells up to 33 days posttransfection (six passages). The culture supernatant from the transfected cells was able to produce HEV infection in a rhesus monkey (Macaca mulatta) following intravenous injection, indicating the generation of viable HEV particles following transfection of cells with in vitro-synthesized genomic RNA. This transient cell culture model using in vitro-transcribed RNA should facilitate our understanding of HEV biology.

Animals↗
PubMed2000

Cloning, sequencing, and expression of the hepatitis E virus (HEV) nonstructural open reading frame 1 (ORF1).

Hepatitis E virus (HEV) causes enterically transmitted epidemic and sporadic viral hepatitis affecting millions of people in the developing world. Different geographical isolates of HEV show a high degree of homology at the nucleotide and amino acid levels. The approximately 7.2 kb RNA genome has three open reading frames of which ORF1 is predicted to code for the viral nonstructural polyprotein. The expression, processing and properties of the nonstructural ORF1 polyprotein have not been reported so far. In this study, the complete HEV ORF1 was reconstructed from overlapping fragments amplified by polymerase chain reaction (PCR) of total RNA isolated from the bile fluid of a rhesus monkey experimentally infected with HEV isolate from an epidemic. The complete assembled ORF1 was sequenced using HEV specific primers. The ORF1 polyprotein was expressed in E. coli, in a cell free translation system and in HepG2 cells, and was characterized by western blotting and immunoprecipitation using acute phase patient serum as well as polyclonal antibodies raised against defined parts of the ORF1 polyprotein. The nonstructural polyprotein of HEV was expressed as a 186 kDa protein. No processing was observed into discrete units, either in-vitro based on a kinetic analysis, or in HepG2 cells based on immunoprecipitation.

Animals↗
PubMed1999

Fecal excretion pattern of bile acids in rats fed high fat diets and neomycin in induced colon tumorigenesis.

Neomycin augments colon tumorigenesis in 1,2 - dimethylhydrazine treated rats fed polyunsaturated fat diet and decreases fecal cholic acid excretion, while it inhibits tumorigenesis with increased cholic acid and decreased deoxycholic acid excretions in rats fed high cholesterol diet. Participation of other fecal bile acids seems to be insignificant in relation to colon carcinogenesis.

1,2-Dimethylhydrazine↗
PubMed1999

Correlation of neomycin, faecal neutral and acid sterols with colon carcinogenesis in rats.

High fat diets have been implicated in incidence of colon cancer both in epidemiological and animal studies. Present investigation deals with the incidence, location and numbers of large and small bowel tumours induced by 1,2-dimethyl hydrazine (DMH) in rats fed high fat diets and neomycin. Neomycin was used to modify the faecal sterol metabolism and the relationship of the high fat diet and faecal neutral and acid sterols to the large bowel tumorigenesis was evaluated. DMH administered rats were fed with (a) 20% safflower oil; (b) 20% safflower oil and neomycin; (c) 20% safflower oil, cholesterol and cholic acid; and (d) 20% safflower oil, cholesterol, cholic acid and neomycin. Neomycin was found to be associated with both increase and decrease of tumour numbers. The faecal sterols lithocholic and deoxycholic acids were found to have no participation, while cholesterol and cholic acid were found to decrease with increase in tumour numbers. However, faecal coprostanol has been found to have a significant positive correlation with tumorigenesis in all dietary groups. Therefore coprostanol might possibly be associated with colon carcinogenesis in DMH-fed rats and cholesterol metabolism in gut appears to be related to the development of tumours.

1,2-Dimethylhydrazine↗
PubMed1999

Hepatitis E infection in children: study of an outbreak.

BACKGROUND: Hepatitis E virus (HEV) is responsible for most of the hepatitis epidemics in the developing world and it frequently affects young adults. Therefore, common perception is that it does not affect children. METHODS: A group of 20 school children (13 years old) were possibly exposed to hepatitis E virus infection during a 2 day trekking trip. Epidemiological and clinical information was correlated to the presence of the hepatitis E virus genome and antibodies to HEV structural and non-structural proteins found in the blood of the children, using polymerase chain reaction and line immunoassay techniques. RESULTS: Ten children developed icteric hepatitis, seven prodrome-like illness without jaundice while three remained asymptomatic. Immunoglobulin M (IgM) antibodies to open reading frame (ORF)2 protein (pORF2) were detected in all 19 children tested, whereas 11 and 10 of the children were positive for IgM antibodies against ORF1 (pORF1) and ORF3 (pORF3) proteins, respectively. The rate of HEV infection was found to be 85%. Viraemia was observed in 11 children and was present in four of the seven anicteric patients (55%) compared with six of the nine (66%) icteric patients. One child without any symptom also had viraemia. CONCLUSIONS: The data obtained indicate a high susceptibility of children for HEV infection and a frequently prolonged viraemia in those infected.

Adolescent↗
PubMed1999

Mutational analysis of glycosylation, membrane translocation, and cell surface expression of the hepatitis E virus ORF2 protein.

Hepatitis E virus (HEV) is the etiological agent for viral hepatitis type E, which is a major problem in the developing world. Because HEV cannot be cultured in vitro, very little information exists on the mechanisms of HEV gene expression and genome replication. HEV is a positive-strand RNA virus with three potential open reading frames (ORFs), one of which (ORF2) is postulated to encode the major viral capsid protein (pORF2). We earlier showed (S. Jameel, M. Zafrullah, M. H. Ozdener, and S. K. Panda, J. Virol. 70:207-216, 1996) pORF2 to be a approximately 88-kDa glycoprotein, carrying N-linked glycans and a potential endoplasmic reticulum (ER)-directing signal at its N terminus. Treatment with the drugs brefeldin A and monensin suggest that the protein may accumulate within the ER. Based on mutational analysis, we demonstrate Asn-310 to be the major site of N-glycan addition. In COS-1 cell expression and in vitro translation experiments, we confirm the ER-translocating nature of the pORF2 N-terminal hydrophobic sequence and show that the protein is cotranslationally, but not posttranslationally, translocated across the ER membrane. Earlier, we had also demonstrated cell surface localization of a fraction of the COS-1 cell-expressed pORF2. Using glycosylation- and translocation-defective mutants of pORF2, we now show that while transit of pORF2 into the ER is necessary for its cell surface expression, glycosylation of the protein is not required for such localization. These results may offer clues to the mechanisms of gene expression and capsid assembly in HEV.

Amino Acid Sequence↗
PubMed1998

A monoclonal antibody against the X protein of hepatitis B virus: fine mapping of its epitope and application in a quantitative ELISA of the X protein in sera of hepatitis B patients.

A HBx-specific mouse monoclonal antibody was developed and its epitope mapped to a hydrophilic segment 94HKRTLGL100 using the multipin peptide synthesis technique. A sensitive ELISA with a threshold of 5 to 10 ng was developed to identify the HBx-positive hepatitis B cases and measure the levels of HBx in sera. The same patient sera were also analyzed for the presence of anti-HBx using the purified recombinant antigen. HBx was present in 23% of the cases (15/65) whereas only 14% of the cases (9/65) were positive for anti-HBx. The mean value of HBx in acute hepatitis sera was higher (522 ng/ml) than in cirrhosis cases (48 ng/ml). PCR amplification of the S gene showed that all 15 HBx-positive cases were also positive for the viral DNA.

Animals↗
PubMed1998

Hepatitis G virus in multitransfused thalassaemics from India.

Hepatitis G virus (HGV)/GB virus-C (GBV-C) has been identified as a blood-borne agent with disputed pathogenicity. This virus belongs to the flaviviridae with a distant relationship to hepatitis C virus (HCV). Genetically divergent HGV isolates have been reported from different parts of the world. This study describes the prevalence of HGV in multitransfused thalassaemic children in India and genomic sequence variations in 11 HGV isolates from the same geographical location. Hepatitis G virus RNA was detected in 39.7% multitransfused thalassaemic children. The seroprevalence of hepatitis B virus (HBV) and HCV was 23.8% and 17.1%, respectively, and 11.4% had dual infection. The nucleotide sequence of a 166 bp HGV genomic segment from the putative capsid-envelope region (nucleotide; nt 578-743) from 11 Indian isolates was compared to the sequences available in the nucleotide databases. The isolates from India were 81.3-94.5% homologous to the isolates from other parts of the world. On phylogenetic analysis, it was observed that HGV isolates from India may belong to two genetically divergent types.

Amino Acid Sequence↗
PubMed1997

The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton.

Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information exists on the basic biology of the virus. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. We expressed pORF3 in transiently transfected COS-1 and Huh-7 cells and showed that it is a phosphoprotein which is modified at a serine residue(s). Deletion and site-directed mutants were created to establish Ser-80 as the phosphorylation site. This residue is present within a conserved primary sequence that showed consensus sites for phosphorylation by p34cdc2 kinase (cdc2K) and mitogen-activated protein kinase (MAPK). In vitro experiments with hexahistidine-tagged pORF3 expressed either in Escherichia coli or in COS-1 cells showed efficient phosphorylation with exogenously added MAPK. The pORF3 mutants also exhibited an in vitro phosphorylation profile with MAPK which was identical to that observed in vivo. In its primary sequence, pORF3 possesses two highly hydrophobic N-terminal domains. On subcellular fractionation, pORF3 was found to partition with the cytoskeletal fraction, and this association with the cytoskeleton was lost on deletion of hydrophobic domain I (amino acid residues 1 to 32). These results suggest that HEV pORF3 is a cytoskeleton-associated phosphoprotein and are discussed in terms of a possible function for pORF3 within the HEV replicative cycle.

Animals↗
PubMed1997

Chlamydia trachomatis in hydrocele fluid.

OBJECTIVE: To determine the presence of Chlamydia trachomatis in hydrocele fluid. METHODS: 90 male patients with hydrocele of tunica vaginalis from an endemic area for bancroftian filariasis were investigated for the presence of Chlamydia trachomatis in their hydrocele fluids. C trachomatis antigen detection tests-a direct immunofluorescence assay and an enzyme immunoassay along with polymerase chain reaction assay for amplification of a 517 bp fragment of C trachomatis endogenous plasmid-were used in this study. The patients were also tested for the presence of microfilaria in their hydrocele fluids and night blood. Histopathological examination was carried out to detect adult filarial worm in tunica vaginalis testes. RESULTS: Eight (8.88%) patients had chlamydia antigen in the hydrocele fluids; C trachomatis plasmid sequences could be amplified from five of these. Seven (7.77%) patients had microfilaria in the hydrocele fluids, three of them having adult worm in tunica vaginalis. CONCLUSION: C trachomatis infection might be associated with hydrocele in some of these patients.

Adult↗
PubMed1997

Magnitude of hepatitis C virus infection in India: prevalence in healthy blood donors, acute and chronic liver diseases.

An enzyme immunoassay (EIA) was developed in-house for the detection of anti-hepatitis C virus (HCV) antibody against the prevailing genotypes in India. The specific reactivity of the test was compared with commercial second and third-generation EIAs and reverse transcription nested polymerase chain reaction (RT-nested PCR). Fifteen thousand nine hundred twenty-two healthy blood donors at the All India Institute of Medical Sciences (AIIMS), New Delhi, India, were screened for anti-HCV antibody. Two hundred ninety-five (1.85%) of these donors were positive. The screening was also used to determine how many patients with acute hepatitis and chronic liver diseases were positive for anti-HCV antibody. Five hundred sixty-four chronic liver disease patients were screened for anti-HCV antibody and 78 (13.83%) were found positive. Two hundred forty-seven sporadic acute viral hepatitis patients were screened for viral infection markers. Hepatitis B and E viruses (HBV and HEV) were the major etiologic agents. HCV was associated with 9% of the acute cases. Anti-HCV core IgM with HCV RNA detection were found to be helpful for the diagnosis of acute HCV infection.

Acute Disease↗
PubMed1996

Genotype determination of hepatitis C virus from northern India: identification of a new subtype.

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68-79% overall sequence homology. This has led to problems in diagnosis of HCV using commercial immunoassays. Based on clustering of homologous sequences, various genotypes and subtypes of HCV have been described from different geographical regions. In the present study, 11 isolates from India were genotyped using sequence comparison for part of the non-structural (NS5) and structural (core) regions. Parts of the genome covering 451 bp (nt 9-459) of the core gene and a 249 bp fragment (nt 7959-8207) of the NS5 gene were reverse transcribed and amplified using nested polymerase chain reaction (RT-PCR). The amplified fragments were cloned and sequenced. The classification into genotypes was done on the basis of phylogenetic analysis. Four isolates showed sequence homology to type 1b. Two of the isolates were classified as type 3a. One isolate was classified as type 3b and the remaining four isolates were found to be variants of type 3 but did not belong to any designated subtype. On the basis of phylogenetic analysis two of the unclassified isolates were put into a new subtype of 3 named as 3g. In one of these variants, parts of a 5'-noncoding (5' NCR; 204 bp), envelope-E1 (435 bp), and NS3 (502 bp) regions were also amplified, cloned, and sequenced. This study demonstrates the type 3 variants including a new subtype (3g) to be the major cause of HCV infection in India.

Amino Acid Sequence↗