Isolation, Identification Of The Porcine Reproductive And Respiratory Syndrome Virus HN-HW Strain And Molecular Characteristics Analysis Of Complete Genome

In this study,a porcine reproductive and respiratory syndrome virus(PRRSV) strains was isolated from the samples collected from the infected pig in Ningxiang county of Hunan province by inoculating the samples into the Marc-145 cell,and the propagation in Marc-145 cell were identified by RT-PCR,FA and immune electron microscopy.All cDNA fragments of the strain,which was named HN-HW,were amplified and cloned by RT-PCR,5' Full RACE and 3' Full RACE,and the complete genomes were obtained by assembling these sequence.Sequence comparison suggested the strain belong to American type,then the molecular characteristics of HN-HW was analyed by bioinformatics.1.The samples,such as lung treated with grind and filtration,were inoculated on Marc-145 cell.A strain was obtained,and was designated as HN-HW.The obvious cytopathogenic effects(CPE) were appeared after 72 hours post infection,such as conspicuous cell clusters,pyknosis and defluxion.The virus propagation in Marc-145 cell was determined by RT-PCR.The specific fluorescence was observed in plasma by FA using the fluorescent antibody of PRRSV VR-2332 standard strain.Observation of ultramicrostructure with the immune electron microscope showed that the size of typical shape of PRRSV was 50 nm-60 ran.The TCID₅₀ of passage 5 of the virus in Marc-145 was l0^-5.76TCID₅₀/mL。2.The cDNA fragments covered full genomes of HN-HW were amplified from total RNA isolated from virus culture by 5' Full RACE method,3' Full RACE method and RT-PCR,and were cloned into pGEM-Teasy vector respectively.Then the complete genome sequence of the isolate was obtained by sequencing and assembling these fragments.3.Sequence comparison showed that the complete nucleotide sequence of HN-HW strain shared 85.3%-99.4%identity with American type isolates,but only 59.7%with European prototype LV, which suggested HN-HW belong to American type in genotype.Sequence comparison of Nsp2 gene indicated that the NH-HW strain shared a feature of 30 amino acids deletion.In order to well understand the genetic relations of American type isolates,a phylogenetic tree was generated based on complete gemomes.It indicated that HN-HW was a new variation strains and belonged to the new branch with JXA1,HEB-1,etc.