Epizootiological Investigation Of Avian Reovirus Infections And The Sequencing, Cloning Of S1 Genes Of Guangxi Isolates

Avian reovirus (ARV) infection is ubiquitous in the poultry industry around the world. Arthritis/tenosynovitis of chickens is the most important disease caused by ARV. ARV infected birds are also characterized by diarrhea, disorders of enteric function, low conversion rate of feed, growth tardiness etc. And it is also one of the immunosuppressive diseases and subsequently results in more susceptive to the infection of other pathogens, and causes serious economy loss for poultry industry. It is one of the hot topics for the industry and the researchers in recent years.The rapid diagnosis technique based on the reverse transcription polymerase chain reaction (RT-PCR), the isolation and identification of field viruses and the sequencing and cloning of S1 gene, and epizootiology of ARV infections in Guangxi were studied in the thesis.Two sets of primers, AR1-AR2-AR3 and S1A-S1H for the RT-PCR to amplify ORF1 and ORF2 and ORF3 of S1 gene respectively were designed on the basis of nucleotide sequence published of ARV reference strain S1133. A fragment of 534 base pairs (bps) was amplified by the primary RT-PCR using primers AR1-AR2, a fragment of 393 bps was amplified by a hemi-nested PCR using primers AR1-AR3, and both fragments span the majority of ORF1 and ORF2. A fragment of 1022 bps was amplified using primers S1A-S1H, it covers ORF3 of S1 gene and encodes the important protein 3 of ARV. At the same time however, the amplifications applied to other pathogens were all negative.14 out of 102 field samples collected from Guangxi, Anhui and Shandong were positively detected by using the developed RT-PCR based rapid diagnosis technique with a percentage of 13.73%.Samples of the positive birds were collected and suspended as the regular method, and inoculated into 7-day-old chicken embryos of specific-pathogen-free (SPF) via allantoid cavity and yolk sac respectively. Four local isolates were successfully obtained from Guangxi chicken. And it is proved that inoculation via yolk sac is more effective than via allantoid cavity for the isolation of ARV.The amplified 534-bp fragments of S1 gene from the isolates were sequenced,cloned. The comparison of the nucleotide sequences among the isolates and the reference strains indicated that isolate SH004 was 99.6% homologous in the nucleotide level to strain SI 133, while isolate JD2 was only 79.1%~89.3% homologous to isolate SH004 and other reference strains based on the sequences published in GenBank. At the same time,the sequences of p10-encoding gene and p17-encoding gene of isolates SH004 and JD2 were aligned and analysed. 1022bp fragments of S 1 gene amplified from isolates NN263 and NN344 were successfully cloned into plasmid vector pMD18-T for further study.