Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A rapid and simple method was developed to detect enteroviruses and hepatitis A virus (HAV) in sewage and ocean water. Sewage samples were concentrated by Centriprep-100 and Centricon-100 at 1,000 x g. Samples collected from estuary and near-shore surf zone ocean water in Southern California were concentrated by vortex flow filtration and microconcentration. Reverse transcriptase-polymerase chain reaction (RT-PCR), with enterovirus primers or HAV capsid-specific primers, was used to detect enteroviruses or HAV in all concentrated samples. A nonradioactive internal probe was used to confirm the amplified products. Results of seeding experiments indicated that at 4 degrees C, HAV was more persistent than poliovirus in seawater and both HAV and poliovirus persisted longer at 4 degrees C than at 25 degrees C. RT-PCR was at least 500-fold more sensitive than cell culture. Results were obtained within 5 h by RT-PCR, in contrast with the 5 days to 3 weeks required for cell culture.
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PMID:Simple method of concentrating enteroviruses and hepatitis A virus from sewage and ocean water for rapid detection by reverse transcriptase-polymerase chain reaction. 750 33

PCR was used to introduce a 63-bp deletion into the putative RNA replicase coding sequence of hepatitis A virus. RNA was synthesized in vitro from the deletion mutant cloned into a transcription vector. Upon amplification by PCR, cDNA made from the competitor RNA generated an amplified fragment that could be easily distinguished from the product generated from wild-type hepatitis A virus genomic RNA by gel electrophoresis, when the same primers were used, without further manipulation. The competitor RNA was used as a positive control in PCR-based detection of very low copy numbers of hepatitis A virus genomic RNA in the presence of unrelated hard-shell clam RNA. When the competitor RNA was used for competitive PCR to quantitate wild-type RNA, the presence of one template at a 10-fold to 100-fold higher level almost completely inhibited product formation from the underrepresented template. The competitor RNA should be useful as a control for reverse transcription and PCRs to determine hepatitis A virus genome RNA when accidental contamination of test samples by a wild-type positive control template would compromise the results.
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PMID:Competitor template RNA for detection and quantitation of hepatitis A virus by PCR. 813 24

Genomic probes were used to investigate hepatitis A virus (HAV) and enterovirus RNAs in two types of shellfish from natural beds (Atlantic coast, France). After elution concentration, nucleic acid extracted by proteinase K and purified by phenol-chloroform and ethanol precipitation was assayed by dot blot hybridization. The probes used were a specific HAV probe corresponding to the 3' end (3D polymerase coding region) and an enterovirus probe corresponding to the 5' noncoding region. The method was first tested under experimental conditions by using virus-spiked shellfish before being applied under field conditions. Our results show that shellfish were highly contaminated: enterovirus and HAV RNAs were found in 63 and 67%, respectively, of samples examined with the riboprobes. On the same site, viral (HAV and enterovirus) RNAs were found in a larger fraction of cockles than mussels. Statistical tests of dependence showed no relationship between viral contamination and bacterial contamination (evaluated by fecal coliform counts).
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PMID:Use of genomic probes to detect hepatitis A virus and enterovirus RNAs in wild shellfish and relationship of viral contamination to bacterial contamination. 828

Hepatitis A virus (HAV) cDNAs encoding the P3 region proteins were expressed in vivo and in vitro to characterize the HAV 3D protein and to identify the cleavage site between 3C and 3D. Protein coding sequences were placed under control of a T7 promoter and an EMCV translational initiation signal. T7 RNA polymerase was provided by simultaneous infection of transfected BS-C-1 cells with a recombinant vaccinia virus vTF7-3 (T. R. Fuerst et al., Proc. Natl. Acad. Sci. USA 83, 8122-8126, 1986). Efficient synthesis and processing of P3 proteins occurred to yield 3CD (78 kDa), 3D (54 kDa), 3ABC (33 kDa), 3BC (25 kDa), and 3C (23 kDa). Similar products were produced by translation of T7 transcripts in a rabbit reticulocyte lysate in vitro. The 3C/D cleavage site was mapped by comparing the mobility of 3D in SDS-PAGE with 3D proteins engineered to begin at each of the two proposed cleavage sites; in addition, direct N-terminal sequencing of radiolabeled 3D protein from translation in vitro was performed. The results showed that 3D was formed by cleavage at the glutamine-arginine (Q/R) pair at position 1738 and 1739 of the HAV polyprotein. HAV 3D protein produced by autocatalytic cleavage of P3 precursor proteins in BS-C-1 cells is virtually completely insoluble and sediments after low-speed centrifugation. This is in contrast to the poliovirus 3D protein, produced from a similar construct, a significant portion of which remains soluble. Extracts containing the poliovirus 3D protein manifested high levels of RNA-dependent RNA polymerase activity, whereas those containing the HAV 3D protein showed no detectable activity by the same assay.
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PMID:Expression of hepatitis A virus precursor protein P3 in vivo and in vitro: polyprotein processing of the 3CD cleavage site. 829 Dec 34

This paper describes 2 outbreaks of hepatitis A infection in Finland, a very low endemic area of hepatitis A infection, where a large proportion of the population is now susceptible to infection by hepatitis A virus (HAV). The first outbreak involved people attending several schools and day-care centres; the second employees of several bank branches in a different city. The initial investigation revealed that both were related to food distributed widely from separate central kitchens. Two separate case-control studies implicated imported salad food items as the most likely vehicle of infection. HAV was detected in the stool of cases from both outbreaks using reverse-transcriptase polymerase chain reaction; however, comparison of viral genome sequences proved that the viruses were of different origin and hence the outbreaks, although occurring simultaneously, were not linked. Foodborne outbreaks of HAV may represent an increasing problem in populations not immune to HAV.
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PMID:Foodborne outbreaks of hepatitis A in a low endemic country: an emerging problem? 952 18

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.
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PMID:Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine. 1048 33

The duration of viremia and time course for development of IgM antibodies were determined prospectively in natural and experimental hepatitis A virus (HAV) infection. Serial serum samples from HAV-infected men (n=13) and experimentally infected chimpanzees (n=5) were examined by nested reverse-transcriptase polymerase chain reaction analysis to detect HAV RNA and by ELISA to detect IgM antibodies to HAV. Among infected humans, HAV RNA was detected an average of 17 days before the alanine aminotransferase peak, and viremia persisted for an average of 79 days after the liver enzyme peak. The average duration of viremia was 95 days (range, 36-391 days). Results were similar in chimpanzees. In addition, HAV RNA was detected in serum of humans and chimpanzees several days before IgM antibodies to HAV were detected. These results indicate that adults with HAV infection are viremic for as long as 30 days before the onset of symptoms and that the duration of viremia may be longer than previously described.
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PMID:Duration of viremia in hepatitis A virus infection. 1088 76

The importance of leptospirosis in Southeast Asia was assessed in conjunction with other studies supported by the U.S. Naval Medical Research Unit No. 2 (US NAMRU-2), Jakarta, Republic of Indonesia. These included studies of hospital-based, acute clinical jaundice in Indonesia, Lao PDR, and Socialist Republic of Vietnam; nonmalarial fever in Indonesia; and hemorrhagic fever in Cambodia. Background prevalence estimates of leptospiral infection were obtained by a cross-sectional, community-based study in Lao PDR. Laboratory testing methods involved serology, microscopic agglutination test, and reverse-transcriptase polymerase chain reaction. Suggestive evidence of recent leptospiral infections was detected in 17%, 13%, and 3% of patients selected on the basis of non-hepatitis A through E jaundice, nonmalarial fever, and hemorrhagic fever (in the absence of acute, dengue viral infections). Leptospiral IgG antibody, reflective of prior infections, was detected in 37% of human sera, collected in Lao PDR. The predominant leptospiral serogroups identified from cases with clinical jaundice were Hurstbridge, Bataviae, and Icterohaemorrhagiae tonkini LT 96 69. Among the nonmalarial febrile cases, Bataviae was the most frequently recognized serogroup. Pyrogenes and Hurstbridge were the principal serogroups among the hemorrhagic fever case subjects. These findings further attest to the relative importance of clinical leptospirosis in Southeast Asia. The wide spectrum of clinical signs and symptoms associated with probable, acute, leptospiral infections contributes to the potential of significant underreporting.
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PMID:The importance of leptospirosis in Southeast Asia. 1240 67

Proteins interacting with RNA structures at the 3' non-translated region (3'NTR) of picornaviruses are probably important during viral RNA replication. We have shown previously that a dominant cellular cytoplasmic protein of 38 kDa (p38) interacts with the 3'NTR and upstream regions of the hepatitis A virus (HAV) RNA (Kusov et al., J Virol 70, 1890-1897, 1996). Immunological and biochemical analyses of p38 have indicated that it is identical to GAPDH, which has previously been described as modulating translational regulation of the HAV RNA by interacting with the 5'NTR (Schultz et al., J Biol Chem 271, 14134-14142, 1996). Three separate binding regions for GAPDH in the 3'NTR and in the upstream 3D polymerase-coding region were identified. Structural analysis of these RNA regions by computer modelling and direct enzymatic cleavage suggested the presence of several AU-rich stem-loop structures having the potential for tertiary interactions. Binding of GAPDH to these structures was confirmed by RNA footprint analysis and resulted in the loss of double-stranded RNA regions. A different panel of RNA binding proteins (p28, p41 and p65) was detected in the ribosomal fractions of several cell lines (BSC-1, FRhK-4 and HeLa), whereas RNA binding of the GAPDH that was also present in these fractions was only marginal or absent.
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PMID:Interaction of glyceraldehyde-3-phosphate dehydrogenase with secondary and tertiary RNA structural elements of the hepatitis A virus 3' translated and non-translated regions. 1256 May 73

Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells. The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems. Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season. Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs). In addition, cell culture was used to detect culturable enteroviruses. Companion water samples were collected for total coliforms, Escherichia coli, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses. Among the 50 wells, four (8%) were positive for viruses by RT-PCR. Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample. Contamination was transient, since none of the wells was virus positive for two sequential samples. Culturable enteroviruses were not detected in any of the wells. Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride. The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses.
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PMID:Incidence of enteric viruses in groundwater from household wells in Wisconsin. 1257 Oct 44


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