Detection and identification of avian, duck, and goose reoviruses by RT-PCR: goose and duck reoviruses are part of the same genogroup in the genus Orthoreovirus.
A reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of avian, duck, and goose reovirus (ARV, DRV, and GRV) RNA from cell culture supernatant and clinical samples was established. Based on multiple sequence alignment, a pair of degenerate primers was selected and synthesized. The amplified, cloned, and sequenced 598-base-pair products from the sigmaA-encoding gene fragment from 16 isolates (ranging over 30 years) indicated that the primer regions were well conserved. The sensitivity of this method was determined to be 10(-2) PFU. The specificity of the RT-PCR method was determined by testing specimens containing avian influenza A viruses, Newcastle disease virus, and infectious bronchitis virus, all of which yielded negative results with no discernible background. The efficiency of the system for detection of ARV, DRV, and GRV directly in 71/83 clinical samples was confirmed. The nucleotide sequence analysis indicated that DRV and GRV isolated from China in different locales and years were closely related, showing 97.4-100% homology to each other, but with only 86.7-88.5% identity to DRV 89026. The nucleotide and amino acid sequence identities in the amplified sigmaA-encoding gene were 74.2-78.4% and 86.9-92.0%, respectively, between duck/goose and chicken species. Phylogenetic analysis indicated that GRV and DRV aggregated into the same specified genogroup within subgroup II of the genus Orthoreovirus and are more closely related to ARV than to Nelson Bay virus. Overall, this study developed a sensitive and specific technique for the identification ARV, DRV, and GRV, and sequencing analysis has enhanced our understanding of the evolutionary relationship between ARV, DRV, and GRV.
Zhang
Y
Y
National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, P.R. China. yunzhang03@yahoo.com
Liu
M
M
Shuidong
O
O
Hu
Q L
QL
Guo
D C
DC
Chen
H Y
HY
Han
Z
Z
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens / Q000821:virology; D004372:Ducks / Q000821:virology*; D005347:Fibroblasts / Q000821:virology; D005777:Geese / Q000821:virology*; D005838:Genotype; D008969:Molecular Sequence Data; D030101:Orthoreovirus, Avian / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D010802:Phylogeny; D011201:Poultry Diseases / Q000821:virology; D016601:RNA-Binding Proteins / Q000235:genetics; D012088:Reoviridae Infections / Q000662:veterinary* / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014758:Viral Core Proteins / Q000235:genetics
D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; C403292:sigmaA protein, avian reovirus