Studies On Molecular Genetic Variation And Vaccine Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS), also named"Blue-eared disease", is one of the most economically important infectious diseases of pigs throughout the world, defined by severe respiratory disorders in piglets and reproductive failure of gestating sows and gilts. In the present study, we conducted and discussed serological investigation, molecular genetic variation, development of inactivated vaccine, coinfection and application of PRRSV infectious cDNA clone.1. Serological investigation and virus isolationTo investigate the serological status of porcine reproductive and respiratory syndrome (PRRS) in Shandong Province of China, a total of 1,332 blood samples were collected from pigs that were not vaccinated for PRRS during 2004 and 2006. 59.5% of the samples were found to be sero-positive. For small-scale pig farms in the remote countryside, only 32.5% of the samples appeared to be positive. In contrast, large-scale pig farms were positive for 83.1%. Porcine reproductive and respiratory syndrome virus (PRRSV) was isolated from samples that were RT-PCR positive, and the virus induced typical cytopathic effect (CPE) in cell culture. Different types of CPE and virulence were observed from different isolates of PRRSV. The results indicated that the prevalence of PRRS was associated with the scale of pig farms and the breeding density of pig populations, and phenotypically different PRRSV isolates exist in the herds in this area.2. Molecular genetic variation of PRRSV Shandong isolatesOn the basis of serological survey of PRRS in Shandong area, five PRRSV strains were isolated and identified. ORF5, ORF6, ORF7 and Nsp2 gene were amplified by RT-PCR and PCR products were cloned into pMD-18T vector and sequenced respectively. Sequence analysis data showed that the nucleotide identity of ORF5, ORF6 and ORF7 gene among the five PRRSV isolates were 88.2%-99.2%, 94.9%-99.8% and 93.5%-100%, respectively. The two regular PRRSV isolates shared high nucleotide identity with VR2332, however, the three PRRSV variant isolates with 30 amino acids deletion shared low nuclotide identity with VR2332. The results showed the infection of PRRSV regular isolates and variant isolates coexisted in pig herds in Shandong province, and genetic variation analysis indicated that dominant PRRSVs showed a tendency to evovle from regular North American isolates to variant isolates in this area in recent years.3. Development of PRRS inactivated vaccine (SD1 strain)On the basis of biological study of PRRSV-SD1 strain, the production engineering of PRRS inactivated vaccine (SD1 strain) were evaluated in this study, including virus propagation, protocol of inactivation and emulsification, test of safety and efficacy and so on. PRRSV-SD1 strain culture fluid with the titer of over 107.0TCID50/mL was inactivated for 20 h in 37℃with 0.1% (v/v) of Formaldehyde, and heat-inactivated SD1 strain culture fluid was emulsified with the proportion of 66% of aqueous phase and 33% of oil phase. The properties of the kind of vaccine was water-in-oil emulsion and there is no side-effect or ill-effect to sows and piglets confirmed by vaccine safety test. When serum neutralization titer was more than 1:10, pigs showed no clinical signs after challenged by PRRSV virus. Period of immune validiy of the vaccine can persist 6 months and its storage life can maintain at least 12 months at 4℃. Field test and territorial test showed that reproductive failures was decreased by 8% in vaccinated sows and average survival can improved by 10% in vaccinated piglets, and the positive rate of serum neutralization (≥1:10) was over 80% in tested pig herds.4. Detection of PCV2 from retrospective cases associated with PRRSThe objective of this study was to determine the prevalence of porcine circovirus type 2 (PCV2) in retrospective cases of pigs associated with porcine reproductive and respiratory syndrome (PRRS). A total of 156 samples were collected during the period between March 2005 and August 2007 in this study, of which 102 samples were diagnosed positive for PRRS virus (PRRSV). A total of 46 (29.5%) of 156 pigs appeared to be positive for PCV2 in the collected organ pools of lung, liver, spleen, kidney, and inguinal lymph node. However, 32 (20.5%) pigs were positive for PCV2 in the serum samples determined by PCR. The positive rate for PCV2 was much higher in PRRSV-positive pigs than in PRRSV-negative pigs as determined by PCR from both sera and organ homogenate pools. Phylogenetic tree analysis based on the open reading frame 2 of PCV2 showed that all the PCV2 isolates in the present study shared higher nucleotide identity (98.3%~100%), and appeared to be much closer to one of PCV2 isolates of France than others. This result indicated that PCV2 was highly prevalent in the retrospective cases of pigs in the examined areas, and the prevalence of PCV2 infection varied between PRRSV-positive pigs and PRRSV-negative pigs (P<0.05).5. Construction of recombinant PRRSV-PCV2 using a PRRSV cDNA cloneThe recent development of infectious clones for PRRSV allows us to manipulate the viral genome using reverse genetics and generate altered PRRSVs with new genctic marker In Vitro. In the present study, PRRSV-mediated foreign gene expression was explored. Since porcine circovirus type 2 (PCV2) is an etiologic agent for post-weaning multisystemic wasting syndrome in pigs, the PCV2 capsid protein gene was cloned and inserted in PRRSV to generate PRRSV-PCV2. Expression of the PCV2 capsid protein was confirmed by PCR and immunofluorescence in virus-infected cells. To assess the immunogenicity of recombinant PRRSVs, three groups of five pigs were immunized twice intramuscularly with wild-type PRRSV, PRRSV-PCV2, or Marc-145 cell culture fluid, and blood samples were collected weekly for five weeks. PCV2 antibody increased until three weeks post-infection and then maintained a constant level thereafter. Our results demonstrate that a PRRSV-based vector is able to induce antibody responses in pigs to the products of inserted genes, and show a potential for PRRSV as a multivalent swine vaccine.