Isolation And Identification Of Porcine Reproductive And Respiratory Syndrome Virus And Pseudorabies Virus

Porcine reproductive and respiratory syndrome (PRRS) is a new and highly infectious swine disease. The disease was characterized by inappetence, fever and respiratory disease in pigs of any age , abortion or increased proportions of stillbirn and infirm piglets in pregnant sows and high mortality in pre-weaning piglets. Although porcine reproductive and respiratory syndrome (PRRS) was first reported in swine herds in the United States in 1987, it spread rapidly and became endemic in most of the major swine-producing areas throughout the world during the 1980, bringing an enormous loss in swine production in the world. Today, it is one of the most economically important diseases of the global pork-producing community. Pseudorabies is an acutely infected disease caused by a swine herpesvirus belonging to the sub-family Alphaherpesvirinae. Typical clinical signs of pseudorabies virus(PRV) infection in susceptible animals include abortion,dead fetuses and mummified fetuses in pregnant sows, fearing cold, quivering, having loose bowes and vomitting in shortly born piglets and 100% mortality in pre-15-day pigs. In recent years, most of cases from many farms indicated respiratory problems in piglets and reproductive failaure in pregnant sows . In order to investigate the causative agent of these cases, PRRSV and PRV was isolated and identificated.1. Isolation and identification of PRRSVSeveral doubted PRRSV srains were isolated from visceral organs of dead fetuses and piglets from the farms in Hubei, Hunan, Jiangxi, Henan and Zhejiang ect. One of the strains was identified. After the isolate was inocaluted on MARC-145 for several genenrations, the result of its'TCID50 test was 10-4.39/0.1ml.The isolate was sensitive to chloroform, completely inactivated when exposed to 56 for one hour, and could not be inhibited by IUDR when it replicated on MARC-145 cells. The diameter of virus particle was about 50~65nm. The isolated virus was spherical and has envelope. Virus neutralization test showed that the isolate was neutralized by the serum to American genotype of PRRSV, not by the sera separately agaist HCV PPV PRV JEV. The fragment of about 500bp ORF6 gene of PRRSV was amplified from the isolated virus by RT-PCR. However this 500bp fragment was not amplified from the negative control. According to the above results, the isolated virus was identificated as American geotype PRRSV and named PRRSV HA strain.2. Cloning and sequence analysis of ORF5 and ORFTgenes ofPRRSV strain HAAccording to Nucleotide acids sequence of ORF5 and ORF7 genes of porcine reproductive and respiratory syndrome virus (PRRSV) strain VR-2332, two pairs of primers were designed , which contain a complete encoding region of ORF5 and ORF7 gene, respectively. Using the pairs of special primers,the ORF5 and ORF7 of PRRSV strain HA were amplified by RT-PCR. After agarose gel electrophoresis,the fragments of about 600bp and that of 400bp were collected , respectively ,then inserted into pMD18-T,building pMD-ORF5 and pMD-ORF7. After recombinant plasmids, pMD-ORF5 and pMD-ORF7,were cloned largely in E.coli DH5a,the ORF5 gene and ORF7 gene in two recombinant plasmid was sequenced respectively.The nucleotide and deduced amino acids sequences indicated that there are 603bp and 200 amino acids in the ORF5 gene of PRRSV strain HA , When compared with PRRSV VR-2332,PRRSV strain Bj-4,and PRRSV strain LV , the homology at nucleotide acid level were 98% 99% 70%; the homology at deduced amino acid level were 94% 94%, 57%,respectively . The sequenced result of ORF7 was 372bp. When the sequence of ORF7 was used to compare with the above PRRSV strains , the homology values were 99% 99% 93%,respectively.3. Isolation and identification of PRVIn a large-scale farm, located in YiCheng city of Hubei province, reproductive failure was observed in 90% of pregnant sows, showing abortion, dead futuses, ect. We isolated a strain of PRV from visceral organs of dead fetuses, and carried out preliminary identification . Cuturing the virus on BHK-21 cells, PK-15 cells, IBRS-2...