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The pathogenicity of 13 field isolates of infectious bronchitis virus (IBV) isolated from Georgia broiler farms from 1989 to 1992 was evaluated using the IBV and Escherichia coli mixed-infection model. Based on the clinical signs, mortality, and lesions, the isolates were classified as high, intermediate, and low in pathogenicity. The in vivo classification was compared with the serotype classification results obtained by reverse transcriptase-polymerase chain reaction-restriction fragment length polymorphism analysis. The high-pathogenicity group was composed of five isolates representing three serotypes: Arkansas, Georgia variant (GAV), and Massachusetts. Isolates in the intermediate- and low-pathogenicity groups were all representatives of the Connecticut serotype, except for one isolate, which belonged to the Massachusetts serotype.
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PMID:In vivo evaluation of the pathogenicity of field isolates of infectious bronchitis virus. 783 13

Previously, we developed a rapid serotype identification test for infectious bronchitis virus (IBV) that utilizes the reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism analysis. The RT-PCR is used to amplify the S1 gene from RNA extracted from the virus grown in eggs. Restriction enzyme digestion and electrophoresis of that PCR product is used to determine the serotype of the virus. The purpose of this study was threefold. First, using a modified 5' PCR primer, we altered the procedures of our rapid serotype identification test and amplified the S1 gene of IBV in tracheal swabs collected from specific-pathogen-free leghorn chickens experimentally inoculated with the Arkansas or Mass 41 serotypes of IBV. Direct amplification of IBV in tracheal swabs eliminates the need to isolate the virus in eggs. Second, we attempted to amplify inactivated IBV in allantoic fluid, possibly allowing us to obtain and determine the serotype of isolates originating from outside the U.S.A. Virus inactivated by formalin (0.1% final concentration) could not be amplified by the RT-PCR procedure, but heat-inactivated IBV (56 C for 15 min) was successfully amplified. Third, we developed an internal control for the RT-PCR test by synthesizing RNA runoff transcripts of a cloned truncated S1 gene. The truncated S1 RNA transcripts were added to the RT-PCR reaction and a 1031-bp product was amplified, which could be distinguished from the coamplified S1 gene from viral RNA. The internal RNA control reduces the possibility of obtaining false-negative results in the RT-PCR test.
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PMID:Further development and use of a molecular serotype identification test for infectious bronchitis virus. 908 26

The S-1 peplomer gene sequences of 31 strains of avian coronavirus infectious bronchitis virus (IBV) from North America, Europe, and Australia were compared to identify common and unique regions for possible diagnostic applications. S-1 sequences that were conserved among serotypes and sequences that were variable between serotypes were identified. Based on conserved S-1 gene sequences, "general" degenerate oligonucleotide primers were designed that amplified IBV genomic RNA by the reverse transcriptase polymerase chain reaction (RT-PCR) procedure regardless of serotype. Primers specific for IBV serotypes Massachusetts, Connecticut, Arkansas, JMK, Delaware (DE/072/92), and California (CA/633/85) were designed from regions of the S-1 gene exhibiting extensive sequence hypervariability. The ability to identify these six serotypes of IBV by RT-PCR was demonstrated by testing the serotype-specific primers on a panel of unknown samples that included 30 reference strains and field isolates previously characterized by virus neutralization (VN). The use of serotype-specific primers in RT-PCR provides a rapid and accurate means of identifying IBV.
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PMID:Serotype identification of avian infectious bronchitis virus by RT-PCR of the peplomer (S-1) gene. 964 18

A reverse transcriptase, polymerase chain reaction (RT-PCR) procedure was used to amplify a segment of the genome of turkey coronavirus (TCV) spanning portions of the matrix and nucleocapsid (MN) protein genes (approximately 1.1 kb). The MN gene region of three epidemiologically distinct TCV strains (Minnesota, NC95, Indiana) was amplified, cloned into pUC19, and sequenced. TCV MN gene sequences were compared with published sequences of other avian and mammalian coronaviruses. A high degree of similarity (>90%) was observed between the nucleotide, matrix protein, and nucleocapsid protein sequences of TCV strains and published sequences of infectious bronchitis virus (IBV). The matrix and nucleocapsid protein sequences of TCV had limited homology (<30%) with MN sequences of mammalian coronaviruses. These results demonstrate a close genetic relationship between the avian coronaviruses, IBV and TCV.
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PMID:Sequence analysis of the matrix/nucleocapsid gene region of turkey coronavirus. 1039

An enteric disease of young turkeys, referred to as stunting syndrome (SS), causes reduced growth and impaired feed efficiency. A recently isolated virus, stunting syndrome agent, (SSA) has been found to be the etiologic agent of SS. The objective of the present study was to determine relatedness of the SSA with other viral agents. Serologic (viral neutralization and enzyme-linked immunosorbent assay [ELISA]) assays and a reverse transcriptase-polymerase chain reaction (RT-PCR) were used. The antisera against turkey enteric coronavirus (bluecomb agent), bovine coronavirus (BCV), bovine Breda-1 virus, bovine Breda-2 virus, avian infectious bronchitis virus (IBV), avian influenza virus, Newcastle disease virus (NDV), and transmissible gastroenteritis virus (TGEV) of swine were evaluated by dot-immunobinding avidin-biotin-enhanced ELISA and did not react with SSA. The homologous (anti-SSA) antiserum was positive by ELISA. Similarly, anti-SSA antiserum did not react when NDV, IBV, BCV, or TGEV was used as antigen but did react with the homologous (SSA) virus. The virus neutralization assay was performed by inoculating 24-to-25-day-old turkey embryos via the amniotic route and by assessing the embryo infectivity on the basis of gross intestinal lesions and intestinal maltase activity at 72 hr postinoculation. None of the aforementioned antisera neutralized SSA infectivity in embryos except for the homologous anti-SSA antiserum. A RT-PCR was performed with known primers specific for NDV, IBV, BCV, and TGEV. The known primers failed to amplify SSA genome but amplified their respective viral genomes. We concluded that the SSA was distinct from the viral agents that were evaluated.
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PMID:Characterization of the stunting syndrome agent: relatedness to known viruses. 1073 43

A rapid-plate hemagglutination (HA) test to detect infectious bronchitis virus (IBV) in allantoic fluid of embryonated eggs was introduced into routine procedures for IBV identification. This system was tested in 468 diagnostic cases received by the Poultry Diagnostic and Research Center at the University of Georgia. Allantoic fluids from inoculated embryos were harvested and treated with commercially available neuraminidase enzyme. IBV in neuraminidase-treated allantoic fluid was identified by clear and consistent HA of chicken red blood cells within 1 min of incubation. The specificity of the neuraminidase rapid-plate HA assay was examined with other avian viruses in individual and dual embryonic infections. Sensitivity of this test was compared with embryo lesions and reverse transcriptase-polymerase chain reaction (RT-PCR). The rapid-plate HA assay of neuraminidase-treated allantoic fluid correlated with the RT-PCR during the early stages of IBV detection, identification, and isolation in embryonated eggs.
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PMID:A rapid-plate hemagglutination assay for the detection of infectious bronchitis virus. 1073 49

Diagnosis of the DE072 strain of infectious bronchitis virus (IBV) by the reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) serotype identification test was not possible because the primer used in the RT-PCR did not amplify the S1 gene of the DE072 strain. The 3' end of the polymerase gene and the 5' end of the S2 gene of the DE072 strain were sequenced and compared with the forward and reverse RT-PCR primers, respectively. A 2-bp mismatch at the 3' end of the reverse primer was found. On the basis of these data, a degenerate primer that could amplify the S1 gene of the DE072 strain as well as eight other serotypes of the virus was synthesized. In addition, we were able to differentiate the DE072 strain from all of the other IBV strains examined by RFLP analysis of the RT-PCR product.
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PMID:Redesign of primer and application of the reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism test to the DE072 strain of infectious bronchitis virus. 1100 14

Ten infectious bronchitis virus (IBV) isolates were recovered from broiler chickens in the states of Queretaro and Guanajuato in Mexico. The viruses were isolated from trachea, lung, kidney, and cecal tonsils of birds that showed respiratory signs in spite of vaccination with Massachusetts (Mass) and Connecticut strains of IBV. Each isolate was identified by an accession number from 1 to 10. Six of the isolates were neutralized by Mass monoclonal antibodies, whereas the other four were not. In addition, these four isolates did not produce lesions in embryos in the first five to seven passes. These four isolates were further characterized by the reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism techniques. The electrophoretic patterns for the four isolates were identical but were different from other known IBV isolates.
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PMID:Characterization of Mexican strains of avian infectious bronchitis isolated during 1997. 1119 51

The nucleocapsid (N) gene of turkey coronavirus (TCV) was amplified by reverse transcriptase-polymerase chain reaction, cloned, and expressed in the baculovirus expression system. A recombinant baculovirus containing the TCV N gene (rBTCV/N) was identified by polymerase chain reaction and expression of TCV N protein as determined by western immunoblot analysis. Two TCV-specific proteins, 52 and 43 kDa, were expressed by rBTCV/N; one of these proteins, p52, was comparable in size to native TCV N protein. Baculovirus-expressed N proteins were used as antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for detection of TCV-specific antibodies. The ELISA detected antibodies specific for TCV and infectious bronchitis virus, a closely related avian coronavirus, but did not detect antibodies specific for other avian viruses (avian influenza, avian reovirus, avian paramyxovirus 3, avian adenovirus 1, or Newcastle disease virus). These findings indicate that baculovirus-expressed TCV N protein is a suitable source of antigen for ELISA-based detection of TCV-specific antibodies in turkeys.
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PMID:Baculovirus expression of turkey coronavirus nucleocapsid protein. 1133 74

Eleven infectious bronchitis virus (IBV) isolates foreign to the United States were analyzed by using reverse transcriptase (RT)-polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) and S1 glycoprotein gene sequencing. Two of the isolates generated RFLP patterns that resembled the Mass 41 strain. Seven novel RFLP patterns were detected among the other nine foreign IBV isolates. Five of the foreign isolates were further analyzed by S1 glycoprotein gene sequencing in our laboratory. Phylogenetic analysis of S1 glycoprotein-deduced amino acid sequences for 4/91 pathogenic, 4/91 attenuated, and Variant 1 were greater than 90% similar to viruses belonging to the 793/B serogroup and, therefore, are possibly serologically related. Variant 2 was only 81.0% similar to viruses belonging to the European serogroup B, and, therefore, predicting its serotype is difficult. Isolates 98-07484 and 97-8123 were genotypically unique and therefore might be serologically unique. With the RFLP patterns and the deduced S1 amino acid sequence data as a reference, none of the IBV isolates foreign to the United States have been detected in the United States.
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PMID:Molecular characterization of infectious bronchitis virus isolates foreign to the United States and comparison with United States isolates. 1141 34


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