scholarly journals Reverse Genetic Analysis of the Transcription Regulatory Sequence of the Coronavirus Transmissible Gastroenteritis Virus

2004 ◽  
Vol 78 (11) ◽  
pp. 6061-6066 ◽  
Author(s):  
Kristopher M. Curtis ◽  
Boyd Yount ◽  
Amy C. Sims ◽  
Ralph S. Baric
Keyword(s):  
Genetic Analysis ◽  
Full Length ◽  
Transmissible Gastroenteritis Virus ◽  
Regulatory Sequence ◽  
Reverse Genetic ◽  
Conserved Sequence ◽  
Gastroenteritis Virus ◽  
Flanking Sequences ◽  
Transmissible Gastroenteritis ◽  
Discontinuous Transcription

ABSTRACT Coronavirus discontinuous transcription uses a highly conserved sequence (CS) in the joining of leader and body RNAs. Using a full-length infectious construct of transmissable gastroenteritis virus, the present study demonstrates that subgenomic transcription is heavily influenced by upstream flanking sequences and supports a mechanism of transcription attenuation that is regulated in part by a larger domain composed of primarily upstream flanking sequences which select appropriately positioned CS elements for synthesis of subgenomic RNAs.

scholarly journals Strategy for Systematic Assembly of Large RNA and DNA Genomes: Transmissible Gastroenteritis Virus Model

2000 ◽  
Vol 74 (22) ◽  
pp. 10600-10611 ◽  
Author(s):  
Boyd Yount ◽  
Kristopher M. Curtis ◽  
Ralph S. Baric
Keyword(s):  
Full Length ◽  
Host Cells ◽  
Transmissible Gastroenteritis Virus ◽  
Plaque Morphology ◽  
Permissive Host ◽  
Gastroenteritis Virus ◽  
Novel Approach ◽  
Transmissible Gastroenteritis ◽  
Cdna Construct ◽  
Rna And Dna

ABSTRACT A systematic method was developed to assemble functional full-length genomes of large RNA and DNA viruses. Coronaviruses contain the largest single-stranded positive-polarity RNA genome in nature. The ∼30-kb genome, coupled with regions of genomic instability, has hindered the development of a full-length infectious cDNA construct. We have assembled a full-length infectious construct of transmissible gastroenteritis virus (TGEV), an important pathogen in swine. Using a novel approach, six adjoining cDNA subclones that span the entire TGEV genome were isolated. Each clone was engineered with unique flanking interconnecting junctions which determine a precise systematic assembly with only the adjacent cDNA subclones, resulting in an intact TGEV cDNA construct of ∼28.5 kb in length. Transcripts derived from the full-length TGEV construct were infectious, and progeny virions were serially passaged in permissive host cells. Viral antigen production and subgenomic mRNA synthesis were evident during infection and throughout passage. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar plaque morphology in permissive host cells. Host range phenotypes of the molecularly cloned and wild-type viruses were similar in cells of swine and feline origin. The recombinant viruses were sequenced across the unique interconnecting junctions, conclusively demonstrating the marker mutations and restriction sites that were engineered into the component clones. Full-length infectious constructs of TGEV will permit the precise genetic modification of the coronavirus genome. The method that we have designed to generate an infectious cDNA construct of TGEV could theoretically be used to precisely reconstruct microbial or eukaryotic genomes approaching several million base pairs in length.

scholarly journals The Application of Immunogold Silver Staining (IGSS) for the Detection of Transmissible Gastroenteritis Virus in Fixed Tissues

1993 ◽  
Vol 5 (1) ◽  
pp. 16-20 ◽  
Author(s):  
Renke Larochelle ◽  
Ronald Magar
Keyword(s):  
Silver Staining ◽  
Protein A ◽  
Immunogold Electron Microscopy ◽  
Transmissible Gastroenteritis Virus ◽  
Tissue Sections ◽  
Gastroenteritis Virus ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Transmissible Gastroenteritis ◽  
Immune Aggregates

Protein A-gold (PAG) and a primary porcine antiserum were used in immunogold silver staining (IGSS) for the detection of transmissible gastroenteritis virus (TGEV) in formalin-fixed paraffin-embedded tissue sections of small intestine originating from infected pigs. Immunogold electron microscopy was used to evaluate the reactivity of the prepared PAG marker with the specific porcine TGEV antiserum. Gold particles were closely associated with single virions and immune aggregates of TGEV. When IGSS, using PAG as the marker, was applied to tissue sections, dark staining of TGEV-infected villous enterocytes was observed. Background was low, allowing good visualization by light microscopy of the distribution of viral antigen. Two other gold conjugates, protein A/G-gold (PA/GG) and protein G-gold (PGG), were tested in IGSS. The labeling with PA/GG was comparable to that obtained with PAG. However, no staining was observed when PGG was used. The use of IGSS and PAG offers advantages and may represent a useful technique for the detection of other viral pathogens.

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