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.
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"Recently, the ARV σA protein, encoded by the S2 gene , has been identified as a double-stranded RNA binding protein that may be involved in interferon resistance . Another viral protein, σNS encoded by the S4 gene, has been reported as having a single-stranded RNA binding activity [22,23]. "
[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 σC and σB genes and aligned with those published in the Genbank, making it possible to carry out studies of molecular classification and relationships.
The full length of the known variable protein σC and part of the σB 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.
The sequences of σC-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 σB-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 σC and σB encoding genes indicated closer relationship between Tunisian, American and Taiwanese isolates of genotype I.
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.
"In contrast, sequence information from ARV isolates is more limited. While the entire complement of S-class genome segments (for example, [14,15,35-39]) and M-class genome segments (for example, [40,41]) have been determined for some ARV clones, and sequence information is available for some ARV L1 and L3 genome segments [42,43], there is, at present, no sequence information for the ARV L2 genome segment. This segment is presumed to encode for the viral RNA-dependent RNA polymerase (RdRp) protein, an essential enzyme for RNA virus replication. "
[Show abstract][Hide abstract] ABSTRACT: The orthoreoviruses are infectious agents that possess a genome comprised of 10 double-stranded RNA segments encased in two concentric protein capsids. Like virtually all RNA viruses, an RNA-dependent RNA polymerase (RdRp) enzyme is required for viral propagation. RdRp sequences have been determined for the prototype mammalian orthoreoviruses and for several other closely-related reoviruses, including aquareoviruses, but have not yet been reported for any avian orthoreoviruses.
We determined the L2 genome segment nucleotide sequences, which encode the RdRp proteins, of two different avian reoviruses, strains ARV138 and ARV176 in order to define conserved and variable regions within reovirus RdRp proteins and to better delineate structure/function of this important enzyme. The ARV138 L2 genome segment was 3829 base pairs long, whereas the ARV176 L2 segment was 3830 nucleotides long. Both segments were predicted to encode lambdaB RdRp proteins 1259 amino acids in length. Alignments of these newly-determined ARV genome segments, and their corresponding proteins, were performed with all currently available homologous mammalian reovirus (MRV) and aquareovirus (AqRV) genome segment and protein sequences. There was approximately 55% amino acid identity between ARV lambdaB and MRV lambda3 proteins, making the RdRp protein the most highly conserved of currently known orthoreovirus proteins, and there was approximately 28% identity between ARV lambdaB and homologous MRV and AqRV RdRp proteins. Predictive structure/function mapping of identical and conserved residues within the known MRV lambda3 atomic structure indicated most identical amino acids and conservative substitutions were located near and within predicted catalytic domains and lining RdRp channels, whereas non-identical amino acids were generally located on the molecule's surfaces.
The ARV lambdaB and MRV lambda3 proteins showed the highest ARV:MRV identity values (approximately 55%) amongst all currently known ARV and MRV proteins. This implies significant evolutionary constraints are placed on dsRNA RdRp molecules, particularly in regions comprising the canonical polymerase motifs and residues thought to interact directly with template and nascent mRNA. This may point the way to improved design of anti-viral agents specifically targeting this enzyme.
"All ARV-encoded proteins, that include at least 10 structural proteins (A, B, C, A, B, BC, BN, C, A, and B), and four nonstructural proteins (NS, P10, P17, and (Gonzalez-Lopez et al., 2003). Another protein of ARV, NS, encoded by the genome segment S4 (Chiu and Lee, 1997), has been reported for its single-stranded RNA (ssRNA) binding activity (Yin and Lee, 1998; Touris-Otero et al., 2005). More recently, an epitope on the NS required for ssRNA binding has been mapped (Huang et al., 2005). "
[Show abstract][Hide abstract] ABSTRACT: A robust, ultrasensitive, and accurate quantitative assay was developed for avian reovirus (ARV) with the Light Cycler SYBR Green-based real-time reverse transcription-PCR (real-time LC RT-PCR). The assay exhibited high specificity as all negative controls and other avian pathogens, such as Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), avian influenza virus (AIV), and mycoplasma synovia (MS), failed to show any positive detection. A minimum of 39 copies/microl of ARV genomic RNA could be detected by the assay. By dilution analysis, the real-time LC RT-PCR developed in this study was 3-log more sensitive than the conventional RT-PCR for the detection of ARV. The vaccine and field isolates of ARV were detected by the real-time LC RT-PCR. As a result of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time LC RT-PCR will be useful as a routine assay for the clinical diagnosis of ARV infection.
Full-text · Article · May 2006 · Journal of Virological Methods