Isolation And Identification Of PRRSV Henan-1 And Analysis Of Its Complete Genoms

Porcine reproductive and respiratory syndrome (PRRS),a kind of highly infectious disease emerged in late 1980s,can cause series breeding disorder syndromes of pregnant swine,such as abortion,fetal death and mummy embryo,and respiratory tract of various ages,especially piglet.The pathogen is Porcine reproductive and respiratory syndrome virus.The genome length of PRRSV is 15 kb,which containing eight ORFs.In this study,one strain of porcine reproductive and respiratory syndrome virus (PRRSV) was isolated from the samples collected from hogpen in Henan province by inoculating the samples into the Marc-145 cell.The strain was identified with RT-PCR,and its TCID50 was determined when propagated on Marc-145 cell.All cDNA fragments of the strain,which was named Henan-1,were amplified and cloned by RT-PCR,and the complete genomes were obtained by assembling these sequences.The genome of Henan-1 strain belonged to American type, when analyzed by bioinformatics.1.The samples,such as lung treated with polish and filtration,were inoculated on Marc-145 cell.After 72 hours,there appeared conspicuous cell clusters,then it showed some cell pathological changes,such as pyknosis and defluxion.The virus propagation in Marc-145 cell were determined by RT-PCR,and it was named Henan-1.The TCID50 of its third passage virus was 104.8 TCID50/ml.2.The cDNA fragments covered full genomes of Henan-1 were amplified from total RNA isolated from virus cultures by RT-PCR method,and were cloned into pMD18-T vector respectively.Then the complete genomes sequence of the isolate was obtained by sequencing and assembling these fragments.The complete genome cDNA fragments of Henan-1 PRRSV were amplified by RT-PCR and cloned on pMD18-T Vector.The positive recombined plasmid was sequenced,and then the complete genome of the Henan-1 strain was obtained.Results of sequence comparison showed that the complete nucleotide sequence of Henan-1 shared 88.8%～99.3% identity with American type isolates,but only 50.5%with European prototypr LV,which suggested Henan-1 belongs to American genotype.Nsp2 gene of Henan-1 has no 30 amino acids identity with the other American strains,ATCC VR2332,HB-1 etc.In order to well understand the genetic relations of American type isolates,a phylogenetic tree was generated based on Nsp2.It indicated that Henan-1 was a new variation strains and belongs to the new branch with JXA1,HUB2,etc. CH-1a belongs to the other branch with PA8,VR-2332,etc.