Pathological And Ultrastructural Analysis Of PRRSV-SCQ Isolate, CDNA Cloning And Bioinformatics Analysis Of It's Structure Proteins

Porcine Reproductive and Respiratory Syndrome Virus(PRRSV),a member of Arterivirus Group, represents the causative agent of a new porcine disease which characterized the symptoms of fever, anorexia, abortion or premature delivery, mummied fetuses and stillbirths in the herds of the productive sows, high mortality and dyspnoea of piglets,and stunt of grower-finisher pigs. Since the disease broke out first in America in 1987,great damages have took place in pig industry.The genome of PRRSV is 15kb in length, encoding 8 ORFs. ORF1a and ORF1b, the longest ORF, encode the polymerase of PRRSV.ORF2-ORF7 encode the structural gene of PRRSV respectively.The item covered with some pig farms in which some sows and piglets had productive failures and respiratory problems in Sichuan district. Investigated by ELISA,sera samples of some sows,piglets and grouwer-finisher pigs were checked.Based on the test,we got at least two PRRSV isolates, one named PRRSV-SCQ strain.Then some molecular biological characteristics of the PRRSV-SCQ and bioinformatics of the main structural proteins were analysed. Finally we constructed the expression recombinants of ORF2-ORF7 respectively. The paper disclosed the following contents.1. 873 sera samples were collected from different pig farms in Sichuan district,and were checked by ELISA kit(offered by IDEXX company). The results showed that 326 sera samples were antibody positive,and the positive ratio was 37.34%(326/873).Among these samples, antibody positive ratio of sows including nursery pigs, piglets including weaning piglets and boars were 38.29%(219/572),36.84%(98/266) and 25.72%(9/35),respectively.2. The obvious CPE could not be seen until the fourth passage when Mark-145 cells were vaccinated with PRRSV positive serum, and 70%-80% cells showed CPE in the sixth passage in only 2-3 days, indicating that PRRSV titres could increase. Localization of PRRSV in vivo and the pathology of PRRSV-infected porcine have been studiedextensively, however, no exact criterion has been achieved.3. This study used SCQ to inoculate 5 piglets. The result indicates that the patient had the symptoms of pulmonary collapse and wide microcirculation malfunction, which may be used as an index for the diagnosis of PRRS. With One Step RT-PCR virus was detected in different tissue, such as spleen, lmyph nodes and lung,and virions stayed in macrophages and vaSCQlar endothelial cells were determined by electric microscope.4. From PRRSV-SCQ, cDNA fragments of the different structural genes of ORF2-ORF7 were acquired respectively by RT-PCR after designing different pairs of primers which were based on the published sequences from Genebank and optimizing the amplifying conditions. In order to clarify the source and genetic background of PRRSV from different parts of China and from abroad, the sequences of the structural genes of PRRSV-SCQ were analyzed and compared with those of VR-2332, LV, CH-1a, BJ-4.HN1,HB-1,HB-2,RespVaccine strain and SPVaccine strain.The results showed as following description. The base composition was basically the same among different PRRSV strains in China. Among those nucleosides that changed, more than 80% were transformed between A-G and T-C. The results also showed that all PRRSV strains in China are closely related to the strain VR-2332. Among different isolates in China, ORF5 displayed the most significant changes, while the ORF6 and ORF7 were the most conservative. Also,the paper analysed codon bias of ORF2-ORF7,the hydrophobic ity and the prediction of transmembrane helices of structural proteins.5. Expression recombinant plasmids expressing PRRSV ORF2-ORF7 under the control of powerful double promoters of PrPL and temperature-sensitivity gene cI857 were constructed, respectively. Ecoli.JM109 strain were used as host bacteria to express these genes.In summary, this experiment finished the analysis of structural genes of PRRSV from different areas of China. Homology analysis showed all isolates belong to American strain.The results set biological foundation for vaccine research,molecular diagnosis methods and measures of prevention.