Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A reverse transcriptase (RT)-polymerase chain reaction (PCR)-oligoprobe (OP), or RT-PCR-OP, method was developed for the detection of the Norwalk virus, which causes acute, epidemic gastroenteritis, in stool specimens. The Norwalk virus genome regions encoding the following two proteins were amplified by RT-PCR: the RNA polymerase (260-bp product) and a putative immunogenic protein (224-bp product). The resulting DNA fragments (amplicons) were hybridized to a digoxigenin-labeled internal OP specific to each amplicon. The detection limit of Norwalk virus, as determined by the endpoint of RT-PCR amplification for serially diluted, positive stool specimens, was similar to the actual virion titer as estimated by electron microscopy and at least 100-fold greater than the titer determined by radioimmunoassay (RIA). The RT-PCR-OP assay was specific for Norwalk virus and negative for other enteric viruses, including human and animal caliciviruses, hepatitis E virus, Snow Mountain agent, astroviruses, 16 human enteroviruses, and 5 human rotaviruses. Components of fecal specimens that interfere with RT-PCR were removed successfully by Sephadex G-200 gel chromatography. Of 20 stool specimens from human volunteers that were positive for Norwalk virus by RIA, a specific RT-PCR-OP result was obtained in 95% (19 of 20) of the samples by using the immunogenic protein primers and 75% (15 of 20) by using the polymerase primers. Twenty-six stool specimens from asymptomatic children and adults were negative by the Norwalk virus RT-PCR-OP. RT-PCR-OP detected Norwalk virus in the 4 of 21 coded fecal specimens that were also positive by enzyme immunoassay. Two samples that were positive by RIA or enzyme immunoassay were negative by RT-PCR, perhaps because viral RNA was not present or RT-PCR inhibitors were not adequately removed.
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PMID:Detection of Norwalk virus in stool specimens by reverse transcriptase-polymerase chain reaction and nonradioactive oligoprobes. 128 Jun 49

The recently characterized fecal-orally transmitted agent of hepatitis E (formerly known as enterically transmitted non-A, non-B hepatitis) has been determined to be a new type of positive strand RNA virus. The complete sequencing of four different geographic isolates of the hepatitis E virus (HEV) has confirmed a similar genetic organization not previously recognized in nonenveloped positive strand RNA viruses. The approximately 7.5 kb RNA genome (including polyA tail) has nonstructural genes located at the 5' end and structural genes at the 3' end. Expression of these viral genes occurs in at least 3 different forward open reading frames. The largest open reading frame begins 27 nucleotides (nt) downstream of the apparent noncoding 5' end and extends 5,079 nt. Multiple nonstructural gene motifs/domains have been recognized in this 5' ORF1 including a methyltransferase, a papain-like protease, a helicase and the RNA-dependent, RNA polymerase. The second major ORF2 begins 37nt downstream of ORF1 and extends 1980 nt before terminating 65 nt upstream of the polyadenylation site. A third ORF of only 369 nt was identified by immunoscreening experiments as encoding an immunogenic epitope of the virus. Expression of the downstream ORF2 may occur through internal subgenomic RNA initiation at a sequence element found to have homology to internal RNA initiation sequences in Sindbis virus. This element in the HEV genome maps near the apparent 5' end of one of two identified subgenomic messages. The genomic organization and expression of HEV will be discussed and a hypothesis presented regarding the viral replication strategy.
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PMID:Molecular organization and replication of hepatitis E virus (HEV). 821 99

Partial genomic sequences representing 420 nucleotides of a nonstructional region, 480 nucleotides of the putative RNA polymerase region, and 540 nucleotides of the structural region of epidemic-associated Chinese strains of hepatitis E virus (HEV) were obtained by direct sequencing of PCR-amplified DNA. Comparison with previously published HEV sequences showed a clear relatedness of all Chinese strains to each other and to a Pakistani strain (Sar-55). All eight Chinese strains examined had very similar sequences (98.5-99.8% homology) in the regions examined and were much closer to the Pakistani strain (Sar-55) (97.9-98.4% homology) than to the Burmese strain (92.5-93.3% homology). Sequence comparisons of the three genomic regions in the Chinese strains indicated that the RNA polymerase region was much more conserved than the other nonstructural region or the structural region. HEV isolates from three remote geographic regions of China had sequences closely related to each other.
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PMID:Partial sequence comparison of eight new Chinese strains of hepatitis E virus suggests the genome sequence is relatively stable. 826 4

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.
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PMID:Phylogenetic analysis of hepatitis E virus isolates from India (1976-1993). 1042 37

Hepatitis E virus (HEV) is the major cause of enterically transmitted non-A, non-B hepatitis in many developing countries and is also endemic in many industrialized countries. Due to the lack of an effective cell culture system and a practical animal model, the mechanisms of HEV pathogenesis and replication are poorly understood. Our recent identification of swine HEV from pigs affords us an opportunity to systematically study HEV replication and pathogenesis in a swine model. In an early study, we experimentally infected specific-pathogen-free pigs with two strains of HEV: swine HEV and the US-2 strain of human HEV. Eighteen pigs (group 1) were inoculated intravenously with swine HEV, 19 pigs (group 2) were inoculated with the US-2 strain of human HEV, and 17 pigs (group 3) were used as uninoculated controls. The clinical and pathological findings have been previously reported. In this expanded study, we aim to identify the potential extrahepatic sites of HEV replication using the swine model. Two pigs from each group were necropsied at 3, 7, 14, 20, 27, and 55 days postinoculation (DPI). Thirteen different types of tissues and organs were collected from each necropsied animal. Reverse transcriptase PCR (RT-PCR) was used to detect the presence of positive-strand HEV RNA in each tissue collected during necropsy at different DPI. A negative-strand-specific RT-PCR was standardized and used to detect the replicative, negative strand of HEV RNA from tissues that tested positive for the positive-strand RNA. As expected, positive-strand HEV RNA was detected in almost every type of tissue at some time point during the viremic period between 3 and 27 DPI. Positive-strand HEV RNA was still detectable in some tissues in the absence of serum HEV RNA from both swine HEV- and human HEV-inoculated pigs. However, replicative, negative-strand HEV RNA was detected primarily in the small intestines, lymph nodes, colons, and livers. Our results indicate that HEV replicates in tissues other than the liver. The data from this study may have important implications for HEV pathogenesis, xenotransplantation, and the development of an in vitro cell culture system for HEV.
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PMID:Evidence of extrahepatic sites of replication of the hepatitis E virus in a swine model. 1152 25

Samples of serum, tissue and faeces from two pig herds in England were examined for hepatitis E virus by reverse-transcriptase PCR (RT-PCR), and a virus strain from each herd was partially sequenced. Eleven of 42 faecal samples and 16 of 21 tissue samples from two pigs were positive for the virus by RT-PCR. Analysis of two unique but closely related nucleotide sequences obtained from the two herds showed that the viruses clustered in genotype III (6) with a human strain of the virus from an autochthonously acquired case of acute hepatitis in the UK. An ELISA based on recombinant open reading frame 2 (ORF-2) was used to detect antibodies to hepatitis E virus in 256 pig sera from the UK; 85.5 per cent of the samples were positive, compared with 58 per cent of similar samples from Swedish pigs and 23.5 per cent of samples from Dutch pigs.
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PMID:Evidence for the presence of hepatitis E virus in pigs in the United Kingdom. 1500 46

The ancestor(s) of apparently Japan-indigenous strains of Hepatitis E virus (HEV) was probably of foreign origin, but it remains unclear when and from where it made inroads. In this study, 24 genotype 3 and 24 genotype 4 HEV strains recovered in Japan each showed a significant cluster, clearly distinct from those of foreign strains, in the phylogenetic tree constructed from an 821 nt RNA polymerase gene fragment. The evolutionary rate, approximately 0.8 x 10(-3) nucleotide substitutions per site per year, enabled tracing of the demographic history of HEV and suggested that the ancestors of Japan-indigenous HEV had made inroads around 1900, when several kinds of Yorkshire pig were imported from the UK to Japan. Interestingly, the evolutionary growth of genotype 3 in Japan has been slow since the 1920s, whereas genotype 4 has spread rapidly since the 1980s. In conclusion, these data suggest that the indigenization and spread of HEV in Japan were associated with the popularization of eating pork.
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PMID:Molecular tracing of Japan-indigenous hepatitis E viruses. 1652 44

The objective of the present study was to detect hepatitis E virus (HEV) in different samples from naturally infected pigs and to characterise genetically the detected strains. Serum, bile, liver, lymph nodes and faeces of 69 animals from 1 week to 4 months of age with different pathological conditions were collected. Reverse transcriptase-polymerase chain reaction (RT-PCR) to detect HEV and histopathology of tissues was conducted. Positive RT-PCR samples were sequenced and phylogenetically analysed. HEV was detected in at least one sample in 26 out of 69 animals (37.7%). Bile was the most frequently positive sample, followed by mesenteric lymph nodes, liver, faeces and serum. HEV was detected in pigs of 1 (n = 7), 2 (n = 8) and 3 (n = 11) months of age. A total of 22 of 69 (31.9%) pigs had mild to moderate hepatitis and 15 of them were HEV RT-PCR positive in at least one of the tested samples. The highest sensitivity of viral detection was achieved using samples that cannot be obtained from live pigs, such as liver, mesenteric lymph node and bile. Phylogenetic analyses confirmed that all Spanish swine HEV strains detected belonged to genotype III. Therefore, genotype III strains are present in a relative high proportion of pigs between 1 and 3 months of age. Through this study, it cannot be ruled out if concomitant infections may influence the distribution of HEV in infected pigs.
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PMID:Detection of hepatitis E virus in liver, mesenteric lymph node, serum, bile and faeces of naturally infected pigs affected by different pathological conditions. 1699 12

In this study we tried to find the role of some waterborne viruses in repeated abortion of women. The study includes maternal blood serum and fetal tissue. The serum of full-term delivered women was taken as a control. All collected samples were inoculated on BGM and Hep2G cells to detect entero and Hepatitis E viruses. Enzyme-linked immunosorbent assay was also carried out for IgM and IgG antibodies against HEV in all serum samples. HEV-Ag was determined by dot-ELISA, which used also for enterovirus typing. Reverse transcriptase polymerase chain reaction was used for detection of entero and HE virus RNAs in the collected serum samples. To follow up the source of virus transmission, the wastewater treatment plant which serves the area of samples population was studied at the intake and the final effluent for the presence of hepatitis E virus and enteroviruses with special reference to coxsackieviruses. Wastewater samples were collected for 1 year and for enterovirus concentration the adsorption-elution on nitrocellulose membrane was used and for HEV, two methods of virus concentration were used, urea arginine phosphate buffer (U-APB) and PEG8000. The results of HEV investigation of aborted women sera was 22% for IgG, 3% for IgM, 20% HEV-Ag, and 16% of HEV-RNA by RT-PCR. For fetal tissue, HEV-Ag was detected in 5% of the collected samples. The detected enteroviruses were coxsackieviruses types 2, 3,4 and 5 in all serum samples and wastewater samples. The results showed also, that virus concentration by U-APB is much better than PEG-8000 but not highly efficient.
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PMID:Waterborne viruses associated with repeated abortion. 1721 39

Hepatitis E virus is a major cause of acute viral hepatitis. The diagnosis of acute viral hepatitis E is based essentially on antibodies and hepatitis E virus RNA. We describe herein a case of acute hepatitis E associated with a false-positive serology for Epstein-Barr virus (EBV). This case report suggests that anti-viral capsid antigen IgM must be interpreted with caution in acute E hepatitis. When positive, EBV RNA polymerase chain reaction can be useful as a false positivity of anti-viral capsid antigen IgM and can be misinterpreted as an acute infection. EBV false positivity was probably related to polyclonal stimulation of the immune system, favoured by hepatitis E.
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PMID:Acute hepatitis E infection associated with a false-positive serology against Epstein-Barr virus. 1977 53


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