Rapid characterization of avian reoviruses using phylogenetic analysis, reverse transcription-polymerase chain reaction and restriction enzyme fragment length polymorphism.
A reverse transcription-polymerase chain reaction is described, which amplified the full-length sigmaC-encoding and sigmaNS-encoding genes of avian reovirus (ARV). DNA fragments of 1022 and 1152 base pairs were amplified among ARV isolates, respectively, indicating that there were no apparent deletions or insertions in these regions. Fragments amplified from vaccine strains and field isolates were digested with five different restriction enzymes Bcn I, Hae III, Taq I, Dde I, and Hinc II, respectively. Restriction fragment profiles observed on polyacrylamide gels showed heterogeneity between vaccine and Taiwanese isolates. All ARV isolates tested showed different restriction enzyme cleavage patterns and could be clearly distinguished. The strain-typing based on the cleavage sites in the sigmaC-encoding gene of ARV showed that viruses could be classified into four distinct groups. A phylogenetic tree based on the nucleotide sequences of the sigmaC-encoding gene revealed that Taiwanese ARV isolates were classified into four distinct groups, indicating that the genotyping is consistent with typing based on restriction enzyme fragment length polymorphism of the sigmaC-encoding gene of ARV. The results suggested that polymerase chain reaction followed by restriction enzyme analysis provided a simple and rapid approach for characterization of ARV isolates. Also, it is possible to determine whether a new variant strain has been introduced into a flock or a given virus strain has spread from one flock to another.
Liu
Hung J
HJ
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan. hjliu@mail.npust.edu.tw
Lee
Long H
LH
Shih
Wen L
WL
Li
Ying J
YJ
Su
Hung Y
HY
D000818:Animals; D002522:Chlorocebus aethiops; D005814:Genes, Viral / Q000235:genetics; D030101:Orthoreovirus, Avian / Q000235:genetics*; D010802:Phylogeny; D012150:Polymorphism, Restriction Fragment Length; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics
D014764:Viral Proteins