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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two genetically distinct bovine enteric caliciviruses (BECs) have been identified: the norovirus (NLV) Jena and Newbury Agent-2 (NA-2) BECs, which are genetically related to human noroviruses, and the Nebraska (NB) BECs, which is related to sapoviruses and lagoviruses but may also represent a new calicivirus genus. The prevalence of these two BEC genotypes in cattle is unknown. Although reverse transcription-PCR (RT-PCR) primers for human NLV recognize NLV-BECs, the genetic relationships between NLV from humans and the NLV-BECs commonly circulating in cattle is undefined. In the present study, veal calf fecal samples were assayed for enteric caliciviruses by using six RT-PCR primer sets designed for the detection of human NLVs or BECs. Caliciviruses genetically related to the NLV-BEC Jena and NA-2 strains or to the recently characterized NB BEC strain were identified in three of four and four of four sampled veal herds, respectively. Extended 3'-terminal genome sequences of two NLV-BECs, designated CV95-OH and CV186-OH, encoding the RNA-dependent RNA polymerase (RdRp; open reading frame 1 [ORF-1]), VP1 (ORF-2), and VP2 (ORF-3) genes were determined. Phylogenetic and sequence identity analyses of each genome region demonstrated these viruses to be most closely related to the NLV-BEC Jena and NA-2 strains. In initial testing, the human P289-P290 (P289/290) primer set was found to be the most sensitive for calicivirus detection. However, its failure to identify all positive fecal pools (as determined by other assays) led us to design two new primer sets, CBECU-F/R and NBU-F/R, for the sensitive and specific detection of NLV-BEC (NLV-BEC Jena and NA-2) and BEC-NB-like viruses, respectively. The RT-PCR assays with the new primers were compared against other primer sets, including P289/290. Composite results of the tests completed by using the new assays identified 72% (54 of 75) of veal calf fecal samples as positive, with 21 of 21 sequenced reaction products specific for the target RdRp gene. The same design strategy used for the new BEC assays may also be applicable to the design of similar assays for the detection of human caliciviruses (HuCVs). Our data support the genetic relationship between NLV-BECs and NLV-HuCVs but with the NLV-BECs comprising two clusters within a third NLV genogroup.
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PMID:Reverse transcription-PCR assays for detection of bovine enteric caliciviruses (BEC) and analysis of the genetic relationships among BEC and human caliciviruses. 1284 48

Rotaviruses, members of family Reoviridae, are a major cause of acute gastroenteritis of infants and young children. The rotavirus genome consists of 11 segments of double-stranded (ds)RNA and the virion is an icosahedron composed of multiple layers of protein. The virion core is formed by a layer of VP2 and contains multiple copies of the RNA-dependent RNA polymerase VP1 and the mRNA-capping enzyme VP3. Double-layered particles (DLPs), representing cores surrounded by a layer of VP6, direct the synthesis of viral mRNAs. Rotavirus core- and DLP-like replication intermediates (RIs) catalyze the synthesis of dsRNA from viral template mRNAs coincidentally with the packaging of the mRNAs into the pre-capsid structures of RIs. In addition to structural proteins, the nonstructural proteins NSP2 and NSP5 are components of RIs with replicase activity. NSP2 self assembles into octameric structures that have affinity for ssRNA and NTPase and helix-destabilizing activites. Its interaction with nucleotides induces a conformational shift in the octamer to a more condensed form. Phosphate residues generated by the NTPase activity are believed to be transferred from NSP2 to NSP5, leading to the hyperphosphorylation of the latter protein. It is suspected that the transfer of the phosphate group to NSP5 allows NSP2 to return to its noncondensed state and, thus, to accept another NTP molecule. The NSP5-mediated cycling of NSP2 from condensed to noncondensed combined with its RNA binding and helix-destabilizing activities are consistent with NSP2 functioning as a molecular motor to facilitate the packaging of template mRNAs into the pre-capsid structures of RIs. Similarities with the bluetongue virus protein NS2 and the reovirus proteins sigmaNS and micro2 suggest that they may be functional homologs of rotavirus NSP2 and NSP5.
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PMID:Nonstructural proteins involved in genome packaging and replication of rotaviruses and other members of the Reoviridae. 1501 Feb 17

Human rhinoviruses (HRVs; family Picornaviridae), the most frequent causative agents of respiratory infections, comprise more than 100 distinct serotypes. According to previous phylogenetic analysis of the VP4/VP2-coding sequences, all but one of the HRV prototype strains distribute between the two established species, Human rhinovirus A (HRV-A) and Human rhinovirus B (HRV-B). Here, partial sequences of the RNA-dependent RNA polymerase (3D polymerase)-coding gene of 48 HRV prototype strains and 12 field isolates were analysed. The designated division of the HRV strains into the species HRV-A and HRV-B was also seen in the 3D-coding region. Phylogenetically, HRV-B clustered closer to human enterovirus (HEV) species HEV-B, HEV-C and poliovirus than to HRV-A. Intraspecies variation within both HRV-A and HRV-B was greater in the 3D-coding region than in the VP4/VP2-coding region, with the difference maxima reaching 48 % at the nucleotide level and 36 % at the amino acid level in HRV-A and 53 and 35 %, respectively, in HRV-B. Within both species, a few strains formed a separate cluster differing from the majority of strains as much as HEV-B from HEV-C. Furthermore, the tree topology within HRV-A differed from that for VP4/VP2, suggesting possible recombination events in the evolutionary history of the strains. However, all 12 field isolates clustered similarly, as in the capsid region. These results showed that the within-species variation in the 3D region is greater in HRV than in HEV. Furthermore, HRV variation in the 3D region exceeds that in the capsid-coding region.
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PMID:Sequence analysis of human rhinoviruses in the RNA-dependent RNA polymerase coding region reveals large within-species variation. 1526 68

Ten antibody escape mutants of coxsackievirus B3 (CVB3) were used to identify nucleotide substitutions that determine viral virulence for the heart and pancreas. The P1 region, encoding the structural genes of each mutant, was sequenced to identify mutations associated with the lack of neutralization. Eight mutants were found to have a lysine-to arginine mutation in the puff region of VP2, while two had a glutamate-to-glycine substitution in the knob of VP3. Two mutants, EM1 and EM10, representing each of these mutations, were further analyzed, initially by determining their entire sequence. In addition to the mutations in P1, EM1 was found to have two mutations in the 3D polymerase, while EM10 had a mutation in stem-loop II of the 5' nontranslated region (5'NTR). The pathogenesis of the mutants relative to that of CVB3 strain RK [CVB3(RK)] then was examined in A/J mice. Both mutants were found to be less cardiotropic than the parental strain, with a 40-fold (EM1) or a 100- to 1,000-fold (EM10) reduction in viral titers in the heart relative to the titers of CVB3(RK). The mutations in VP2, VP3, and the 5'NTR were introduced independently into the RK infectious clone, and the phenotypes of the progeny viruses were determined. The results substantiated that the VP2 and VP3 mutations reduced cardiovirulence, while the 5'NTR mutation in EM10 was associated with a more virulent phenotype when expressed on its own. Stereographic imaging of the two mutations in the capsomer showed that they lie in close proximity on either side of a narrow cleft between the puff and the knob, forming a conformational epitope that is part of the putative binding site for coreceptor DAF.
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PMID:Attenuating mutations in coxsackievirus B3 map to a conformational epitope that comprises the puff region of VP2 and the knob of VP3. 1556 6

Infectious pancreatic necrosis virus (IPNV), a prototype virus of the family Birnaviridae, exhibits a high degree of antigenic variability, pathogenicity and virulence in salmonid species. The Genomic Segment A encodes all the structural (VP2 and VP3) and nonstructural (NS) proteins, whereas Segment B encodes the viral RNA-dependent RNA polymerase (VP1). We tested 3 different IPNV isolates (Sp103, Sp116 and Sp122) isolated during field outbreaks in Norway for their ability to cause mortality in fry and post-smolt of Atlantic salmon Salmo salar L. The cumulative mortality following experimental challenge in fry was 29% for Sp122 followed by 19% for Sp116 and 15% for Sp103. In post-smolt, the corresponding mortality rates were 79, 46 and 16%, respectively. Comparisons of the deduced amino acid sequences of Segments A and B of all 3 Sp strains revealed substitutions of residues in 13 positions, of which 6 are in VP2, 2 in VP3, and 5 in VP1. Our results suggest that these residues, especially those in the outer capsid VP2, may be involved in the virulence of IPNV. Genome Segment A of the Sp serotype is 3097 nucleotides long and contains a major open reading frame (ORF) encoding a polyprotein of 972 amino acids, which initiates at the second in-frame start codon at Position 119. This was ascertained by making mutants of Segment A clone using site-directed mutagenesis, followed by in vitro transcription-coupled translation reaction and immunoprecipitation analyses. In addition, Segment A also encodes a 15 kDa arginine-rich non-structural protein from a small ORF, preceding and partially overlapping the polyprotein ORF, which is truncated to 12 kDa in the virulent Sp122 strain. Moreover, Segment A could encode a novel, putative 25 kDa protein from another ORF between VP2 and VP4 coding regions, which is only detected in the Sp serotype. Segment B is 2777 nucleotides long and encodes in a single large ORF (a polypeptide of 844 amino acid residues), VP1.
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PMID:Molecular characterization of Sp serotype strains of infectious pancreatic necrosis virus exhibiting differences in virulence. 1558 7

Deformed wing virus (DWV) of honeybees (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. The virus was purified from diseased insects, and its genome was cloned and sequenced. The genomic RNA of DWV is 10,140 nucleotides in length and contains a single large open reading frame encoding a 328-kDa polyprotein. The coding sequence is flanked by a 1,144-nucleotide 5' nontranslated leader sequence and a 317-nucleotide 3' nontranslated region, followed by a poly(A) tail. The three major structural proteins, VP1 (44 kDa), VP2 (32 kDa), and VP3 (28 kDa), were identified, and their genes were mapped to the N-terminal section of the polyprotein. The C-terminal part of the polyprotein contains sequence motifs typical of well-characterized picornavirus nonstructural proteins: an RNA helicase, a chymotrypsin-like 3C protease, and an RNA-dependent RNA polymerase. The genome organization, capsid morphology, and sequence comparison data indicate that DWV is a member of the recently established genus Iflavirus.
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PMID:Molecular and biological characterization of deformed wing virus of honeybees (Apis mellifera L.). 1664 Dec 91

Porcine enteroviruses (PEVs) and teschoviruses (PTVs) are described as causative agents of neurological disorders, fertility disorders and dermal lesions of swine. Difficulties in the serological detection of these viruses may lead to a significant underestimation of infections with clinical symptoms. With the recent availability of genome sequence data for all the serotypes, molecular diagnosis is a possibility. The present study describes a new approach to molecular 'serotyping' of PTVs and PEV-B viruses, involving the amplification and sequencing of a genomic fragment of the VP1 coding region. A molecular characterization of Italian entero-teschovirus isolates was performed using a set of previously published and newly designed polymerase chain reaction primers. A total of 33 porcine isolates and 10 reference strains were analysed. Porcine enterovirus-B samples were first diagnosed as positive for enterovirus by amplification of the 5'-non-translated region. Samples were then typed by amplification and sequencing of a portion of the VP1 coding region. Porcine enterovirus-A and PTVs were detected by a published assay in the 5'-NC region that allows them to be differentiated according to the size of amplification product, using the same set of primers. For serotype characterization of PTV, we evaluated four different regions: the N terminus of the capsid protein VP2, the region encoding for RNA-dependent RNA polymerase, and the capsid VP1 and VP4 regions. The newly designed primers in the VP1 region was proved to be broad in range and suitable for serotype assessment and therefore constitute a useful diagnostic tool for molecular diagnosis of porcine teschovirus/enterovirus strains and for the study of molecular epidemiology and evolution of these viruses.
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PMID:Validation of rt-PCR assays for molecular characterization of porcine teschoviruses and enteroviruses. 1690 56

X-ray and electron microscopy analysis of Bluetongue virus (BTV), the type species of the Orbivirus genus within the family Reoviridae, have revealed various aspects of the organisation and structure of the proteins that form the viral capsid. Orbiviruses have a segmented dsRNA genome, which imposes constraints on their structure and life cycle. The atomic structure of the BTV core particle, the key viral component which transcribes the viral mRNA within the cell cytoplasm, revealed the architecture and assembly of the major core proteins VP7 and VP3. In addition, these studies formed the basis for a plausible model for the organisation of the dsRNA viral genome and the arrangement of the viral transcriptase complex (composed of the RNA-dependent RNA polymerase, the viral capping enzyme and RNA helicase) that resides within the core particle. Electron cryo-microscopy of the viral particle has shown how the two viral proteins VP2 and VP5 are arranged to form the outer capsid, with distinct packing arrangements between them and the core protein VP7. By comparison of the outer capsid proteins of orbiviruses with those of other nonturreted members of the family Reoviridae, we are able to propose a more detailed model of these structures and possible mechanisms for cell entry. Further structural results are also discussed including the atomic structure of an N-terminal domain of nonstructural protein NS2, a protein involved in virus genome assembly and morphogenesis.
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PMID:Structural studies on orbivirus proteins and particles. 1690 1

Infectious bursal disease virus (IBDV) is an important poultry pathogen and is distributed world wide that can cause immune suppression and lesions of the bursa of Fabricius. The main component of the virus, VP2, is not only responsible for the bird's immune response, but is important for the molecular identification of this virus as well. The nucleic acid of the virus must be adequately preserved to be analyzed by reverse-transcriptase PCR (RT-PCR) and sequenced for the molecular characterization of the field strain. Phenol inactivation has been the standard for IBDV tissue collection and international shipment; however, there have been some reports of interference with molecular detection capabilities when using phenol. Phenol is also a hazardous chemical and must be handled and shipped carefully. The ability to use the Flinders Technology Associates filter paper (FTA card) for inactivation of several avian pathogens has been proven previously, however no work has been published on its use in IBDV nucleic acid detection. Bursas from experimentally infected birds was imprinted on FTA cards, and then placed in phenol. Samples were evaluated and compared based on molecular detection capabilities between the two inactivation methods. The nucleic acid of the virus was detected in 85% of the FTA card inactivated samples compared to 71% in the phenol inactivated samples. Sequence analysis was performed on samples inactivated by both methods and no differences were found. When comparing the RNA stability at different temperatures, euthanized IBDV infected birds were held at two different temperatures before sampling. No differences were detected for FTA sampling; however, for tissues in phenol the nucleic acid was only detectable up to 2 h post-mortem in the tissues held at 4 degrees C prior to sampling. These findings indicate that the FTA card is an efficient and reliable alternative collection method for molecular detection and characterization of IBDV.
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PMID:Evaluation of FTA paper and phenol for storage, extraction and molecular characterization of infectious bursal disease virus. 1697 12

Infectious bursal disease virus (IBDV), a double-stranded RNA virus, is a member of the Birnaviridae family. Four pathotypes of IBDV, attenuated, virulent, antigenic variant, and very virulent (vvIBDV), have been identified. We isolated and characterized the genomic reassortant IBDV strain ZJ2000 from severe field outbreaks in commercial flocks. Full-length genomic sequence analysis showed that ZJ2000 is a natural genetic reassortant virus with segments A and B derived from attenuated and very virulent strains of IBDV, respectively. ZJ2000 exhibited delayed replication kinetics as compared to attenuated strains. However, ZJ2000 was pathogenic to specific pathogen free (SPF) chickens and chicken embryos. Similar to a standard virulent IBDV strain, ZJ2000 caused 26.7% mortality, 100% morbidity, and severe bursal lesions at both gross and histopathological levels. Taken together, our data provide direct evidence for genetic reassortment of IBDV in nature, which may play an important role in the evolution, virulence, and host range of IBDV. Our data also suggest that VP2 is not the sole determinant of IBDV virulence, and that the RNA-dependent RNA polymerase protein, VP1, may play an important role in IBDV virulence. The discovery of reassortant viruses in nature suggests an additional risk of using live IBDV vaccines, which could act as genetic donors for genome reassortment.
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PMID:Genetic reassortment of infectious bursal disease virus in nature. 1701 Sep 36


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