Detection And Epidemiological Investigation Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is a worldwide recognized disease with important economic impact on swine industry. The disease is characterized by reproductive problems such as abortions, farrowing of dead or weak piglets, high preweaning mortality and mortality in sows. The PRRS virus (PRRSV) is an enveloped positive strand RNA virus classified in the Arteriviridae family. There are two serotypes of PRRSV, American type and European type. American type isolates are genetically highly variable particularly at the level of the E glycoprotein. PRRSV has spread in all over country since it has been confirmed in 1996, while little is known about the epidemiology and genomic variability of PRRSV.This study included as following:1. According to the genome sequence of Porcine reproductive and respiratory syndrome virus (PRRSV), three pairs of primers (N₁/N₂ AdGP5.1/AdGP5.2, RFLP5.1/RFLP5.2) were designed and the RT-PCR methods were developed to amplify the corresponding gene fragments. Their sensitivity specificity and positive rate of samples were detected and compared. And one step RT-PCR was also constructed for the detection of PRRSV. The results showed that: (1)Three pairs of primers could be used to amplify specifily the gene fragments of PRRSV. (2)The RT-PCR using primers N₁/N2 was able to detect at least 7.9 TCID₅0 PRRSV, while the RT-PCR using primers AdGP5.1/AdGP5.2 or primers RFLP5.1/RFLP5.2 only 79 TCID50 PRRSV. (3)Among forty-eight clinical samples, twenty-eight samples were identified as positive to PRRSV by RT-PCR using primers N₁/N₂, and twenty-seven were positive by using primers AdGP5.1/AdGP5.2 and twenty-five in using primers RFLP5.1/RFLP5.2. Furthermore, the expected 374bp fragment was also amplified by one step RT-PCR method with the primer N₁/N2. It indicated that the RT-PCR with the primers N₁/N2had higher sensitivity for PRRSV than that with the other two pairs of primers, and it is suitable to be used to detect PRRSV in clinical samples.2. Two hundred seventy-one field samples originating from one hundred twenty-nine pig farms in eight provinces (Jiangsu, Shanghai, Zhejiang, Anhui,Shandong, Jiangxi, Guangxi and Henan) were collected and tested with RT-PCR using primers N₁/N2 for the presence of PRRSV during the period from May 2002 to February 2005. The results showed that PRRSV were detected from 166 field samples originating from 89 pig farms. Positive rate was 61.2% in all samples, especially it was higher in Anhui, Jiangsu and Shanghai than that in other provinces. From 2002 to 2005, positive rate was 74.4%, 65.5%, 57.0% and 38.1% respectively. Clinical and epidemiological data were collected through a questionnaire for each of the submitted field samples. Data indicated that about 70% of the samples were submitted during autumn and winter. Over 90% of field cases were submitted for respiratory problems or reproductive- respiratory problems, only 6.6% for reproductive problems. Most striking macroscopic lesions observed were lymph node hemorrhage and/or edema(100%) and diffuse interstitial pneumonia(88%), in a low proportion of samples, intestine thin-walled, kidneys hemorrhage, spleen hemorrhage and pleuritis were observed. In 2002, morbidity of preweaning piglets was higher than that of nursery pigs, while the case was just the reverse in following years. In 24% of the samples the animals submitted had been vaccinated with RespPRRS MLV.3. One hundred sixty-six samples identified with porcine reproductive and respiratory syndrome virus (PRRSV) were collected, and ORF5 gene of PRRSV was amplified by RT-PCR with the primers AdGP5.1/AdGP5.2, and the restriction fragment length polymorphism (RFLP) patterns for enzymes Mlu, Hinc II and Sac II were determined on ORF5 gene sequences. A total of 3 RFLP patterns were obtained, which named as, 2-2-1, 1-1-1, 1-3-1. The main RFLP patterns obtained were 2-2-1(91.0%), and the other two patterns (1-1-1 and 1-3-1) were determined for the only 1.8% and 7.2% remaining strains, respectively. Furthermore fifty-four PRRSV isolates were...