| Porcine reproductive and respiratory syndrome (PRRS) is one of the most seriousdiseases affected the development of pig industry, which is caused by porcine reproductiveand respiratory syndrome virus (PRRSV). PRRSV can cause severe respiratory andreproductive disorders in pigs. Since the last century, people around the world in many fields,especially farmers, veterinary authorities and researchers have invested a lot of effort andachieved certain result in PRRS. But until now, many questions around PRRSV, such aspathogenesis and genetic variation are still not known. PRRS is still prevalent and causesenormous damage to global pig industry.For the carried out of PRRS prevention and control technology research, with aims tocontrol and reduce the disease incidence, it is very important to comprehensively developepathogenic and serological diagnostic methods of PRRSV, investigate the epidemicpandemic, isolate and identify the epidemic virus strains and analysis the genetic variationtrend of PRRSV strains, and so on. In this study, samples were collected from pig farms in9areas of Shandong, Henan and Hebei provinces. Using these samples, several PRRSVisolates were isolated and identified; RT-PCR detection methods for virus RNA andindirect-ELISA method for virus antibodies were found. Based on the establishment of PCRmethod and antibody diagnostic method, monoclonal antibodies were developed for studyand diagnostic uses; antibody and etiology epidemiological investigations during2007-2012were carried out; ORF5and partical nsp2gene squences of isolated virus and GenBankassesion strains were identified and analyzed to find the genetic variation trend.Using partical nsp2gene and ORF7gene as target genes, two pairs of primers weredesigned to establish differential detection RT-PCR diagnostic methods of PRRSV andconducted a preliminary optimized. The methods had specifically sensitivities to thedifferential diagnosis with classical strains and deficient strains, Americas strains andEuropean strains, which were able to detect the virus concentration of lower than0.1TCID50(EO subtype virus) and0.01TCID50(US subtype virus).Recombinant N protein, which was cloned, expressed, identified and purified by otherresearchers in the lab, was applied as the coating antigen to construct an indirect ELISAmethod, to detect the N protein-specific antibodies in pigs. The tests showed that the methodhad great sensivity, specificity, and stability, and was well coincided to IDEXX ELISA kit.An isolate of Highly pathogenic PRRSV was collected and purified by density gradientcentrifugation and immunized to BALB/c mice. Following immunication, cell fusion, cellselection and several identifications, three positive cell strains, named3F1,7F2and6A4,which could induce anti-PRRSV monoclonal antibodies, were obtained. During them, 7F2could specifically recognize some epitope on N protein, and6A4could recognize someepitope on GP5protein. By the ADE and antibody neutralization tests,6A4and3F1showedneutralization activity, and7F2showed no ADE activity or neutralization activity, but it canbe potential applicated in N protein detection by ELISA. All the three antibodies were higheffecency and specificity, and could be well used in PRRSV diagnology, immunologystudied.Using MARC-145cells as a research tool, representative five PRRSV isolates wereisolated and identified for biological characteristics. One of five isolates (SDDY2007) wassimilar to a vaccine strain (US subtype), and the other four were more similar to highpathogenic strains, in which an isolate of the four (SDBZ2006) was identified by pigexperiments infection, and another one (HN2010) showed a lower proliferative capacity inMARC-145cells.PRRS prevalence survey was carried out in Shandong Province and surrounding areasbased on established PCR and ELISA.957serum samples and272suspected tissues andblood samples were examined. The average positive rate of PRRSV antibody was49.8%.From2007to2012, though the antibody positive rate obviously fluctuated, a significantupward trend was found during2012. Antibody-positive rate of different regions are quitedifference. The average positive rate of PRRSV neutralizing antibody was25.1%(using cellneutralization test), with outstanding differences in different regions. PRRS pathgens in the272samples were detected by RT-PCR, which showed that73samples were virus positive(positive rate of26.84%), and several farms had co-infection in them. A sample of genetypeⅠ,6samples of classical genetype Ⅱ were found,66sample of nsp2deleted, and1samplewith both classical and deleted subtype viruses were detected. Virus isolation positive ratesvaried in different diseased tissues and organs. During2007-2012,15strains were isolatedand identified by MARC-145cells, the ORF5and partial nsp2gene were amplified, clonedand sequencing. The sequences were blasted with68ORF5sequences and41nsp2sequencein GenBank, showed that all the15isolates were belong to American genetype strain, ofwhich13strains showed high homology with JXA1strain(95.5%~97.5%), one (DY2007strain) was closed to the vaccine strain MLV and VR-2332strain, and one might be a straincoming from genetic recombination.The study established good PCR and ELISA methods and applicated well. Virusisolation, serological surveys, pathogen detection and genetic variation analysis showed thathigh pathogenic PRRSV is the advantage strain in Shandong and surrounding areas from2007to2012. High pathogenic PRRSV is more serious prevalent in the region and canco-infection with other viruses. There may be some recombinant strains, too. It seemed thatvaccine immunization has not been able to play an enough prevention acctions in PRRScontrol. Addition to high pathogenic strains, there are also classic US strains liked vaccinestrains and European strain in Shandong and the surrounding areas. Pandemic strains in the three provinces have some genetic differences, but no obvious regional characteristics.In clusion, the established diagnostic methods, antibodies, isolates and epidemiology,genetic diversity information provide us effective technical tools and reliable data referencefor PRRS prevention and control。... |
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