Isolation And Identification Of PRRSV And Cloning And Sequence Analysis Of ORF5 Genes Of PRRSV Strain HN-Y

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. In order to investigate the causative agent of these cases , PRRSV was isolated and identificated.Several 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. 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. 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 HN-Ystrain.The complete cDNA encoding envelope protein (E or GP5) of PRRSV field strain HN-Y was amplified by one-tube reverse transcription polymerase chain reaction (RT-PCR). The cDNA fragment was further cloned and the complete sequences were determined. The results indicate that the cDNA of ORF5 encodes 200aa residues. Compared with the ORF5 gene of typical European strain (LV strain), North American strain (VR-2332 strain) and the first PRRSV isolate (CH-1a strain) in China, it was found that there was 58%, 90% and 95% aa identity respectively. The analysis of the phylogenetic tree also showed that PRRSV strain HN-Y is more closely related to the North American isolates than it is to the European isolates. So we deduced that PRRSV strain HN-Y blongs to North American type. But there exist differences in the potential N-glycosylation sites and antigen sites between PRRSV fideld strain HN-Y and other North American isolates.