Cloning and nucleotide sequencing of the S4 genome segment of avian reovirus S1133.

Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.
Archives of Virology (Impact Factor: 2.28). 02/1997; 142(12):2515-20. DOI: 10.1007/s007050050258
Source: PubMed

ABSTRACT The sequence of RNA genome segment S4 of the avian reovirus (ARV) strain S1133 was determined. S4 RNA is 1185 base pairs long and contains one open reading frame encoding a protein of 367 amino acid residues (40.6 kDa), the similar size as the known S4 gene product (sigma NS), with a net charge of -1 at neutral pH. The S4 RNA sequence possesses a pentanucleotide sequence UCAUC at the 3'-terminus of its plus strand like in ARV S1 and S3 segments and ten segments of mammalian reovirus (MRV). The predicted amino acid sequence comparison revealed that the homology is 44.02%, 45.71%, and 42.33% for ARV sigma NS and three serotypes of MRV sigma NS, respectively. The relatively high content of alpha-helix structure in the C-terminal portion of ARV sigma NS suggests that this protein may functionally relate to MRV sigma NS. Northern blot hybridization showed that a 32P-labeled cDNA insert S4-49 from ARV S4 RNA cross-hybridized with the corresponding RNA segments of all seven strains of ARV tested.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The genome segment S2 of p6ian reovirus (ARV) S1133 was cloned and sequenced. The entire S2 nucleotide sequence is 1325 bp long with one long open reading frame that encodes a protein of 415 amino acids, corresponding to varsigmaA, a major core protein of ARV. S2 possesses a pentanucleotide, TCATC, at the 3'-terminus of its plus strand, common to other known genome segments of ARV and to 10 genome segments of mammalian reovirus. Amino acid sequence analysis revealed that varsigmaA contains a carboxy-terminal region (one-fourth of the protein) that is formed from alpha-helices and beta-turns, and the remainder (three-fourths of the protein) is formed predominantly from beta-strands and beta-turns. Analysis of binding activity to poly(rI)-poly(rC)-agarose suggested that ARV protein A present in total virus-infected chicken embryo fibroblasts (CEF) had dsRNA-binding activity. To further characterize the binding activity, protein varsigmaA was subsequently expressed in Escherichia coli BL21(DE3) cells as a fusion protein and isolated by metal chelate affinity chromatography. The expressed protein evarsigmaA was further purified through a Superdex 75 HR 10/30 column after digestion of the purified fusion peptide with enterokinase. The expressed protein evarsigmaA has the same molecular weight as virion protein varsigmaA purified from ARV-infected CEF and is indistinguishable from virion protein varsigmaA by immunoblot analysis. The evarsigmaA binds cooperatively alpha (32)P-labeled dsRNA probe produced by run-off transcription of clone pGEM-3Zf(+)S4. The binding reaction is blocked by homologous ARV dsRNA or heterologous infectious bursal disease virus dsRNA and poly(rI)-poly(rC), but not by salmon sperm DNA. The results indicate that the expressed protein evarsigmaA has dsRNA-binding activity similar to that of varsigmaA obtained from infected cells, and its binding is sequence-independent.
    Virology 01/2000; 266(1):33-41. DOI:10.1006/viro.1999.0020 · 3.28 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND: Genotype analyses of avian reoviruses isolated from organ samples collected from chickens with suspicious clinical symptoms, between 1997--2008, was based on sequences for both sigmaC and sigmaB genes and aligned with those published in the Genbank, making it possible to carry out studies of molecular classification and relationships. METHODS: The full length of the known variable protein sigmaC and part of the sigmaB encoding genes, were amplified with RT-PCR, using conserved primers. PCR products were sequenced and the sequences were analyzed and aligned with avian reovirus sequences from the Genbank database. RESULTS: The sequences of sigmaC-encoding genes of all the isolated strains indicated their close relationship with the American, Chinese and Indian strains. Taking the American strain S1133 as a reference, the two Tunisian isolates 97.1 and 97.2 showed some nucleotide substitutions. For isolate 97.1, the substitution was silent whereas for strain 97.2 the mutation was at the first position of the corresponding codon and induced the substitution of the amino acid encoded. For the sigmaB-encoding gene, the sequences of the Tunisian strains showed mutations at positions two or three of the corresponding codons, inducing substitutions of amino acids at these positions. The phylogenic trees based on sigmaC and sigmaB encoding genes indicated closer relationship between Tunisian, American and Taiwanese isolates of genotype I. CONCLUSION: Our study describes the genotype of avian reoviruses that are not yet well characterized genetically. The characterization and classification of these viruses might be significant for understanding the epidemiology of malabsorption syndrome and viral arthritis, and improving our knowledge of the genotype of strains circulating in Tunisian flocks. Furthermore, the study of their variable pathogenicity could be extremely important in the choice of the appropriate vaccine strain to control disease.
    Virology Journal 01/2013; 10(1):12. DOI:10.1186/1743-422X-10-12 · 2.09 Impact Factor
  • Source


1 Download
Available from