Construction And Identification Of Infectious Clone Of NADC30-like Porcine Reproductive And Respiratory Syndrome Virus Strain HNyc15

Porcine reproductive and respiratory syndrome(PRRS)is a viral infectious disease leading to serious harm to the global pig industry caused by Porcine reproductive and respiratory syndrome virus(PRRSV).Since 2012,several field isolates of PRRSV were isolated at pig farms in China which showed the highest nucleotide similarity to a group represented by NADC30,a type 2 PRRSV that has been isolated in Unite States of America in 2008.Accordingly,these PRRSV isolates were designated as NADC30-like PRRSV.The strains had moderate pathogenicity,and current commercial PRRSV vaccine failed to protect NADC30-like PRRSV infection.So far,the disease has been reported to be widely spread in several provinces and led to huge amount of economic losses in China.It is of great scientific significance to investigate the genetic evolution of the NADC30-like PRRSV and associated molecular mechanisms with its pathogenicity.In the present study,a NADC30-like PRRSV strain HNyc15 was analyzed and the full-length genomes were sequenced in order to explore the evolution of NADC30-like PRRSV.By using revere genetic manipulation,the infectious c DNA clone of the NADC30-like PRRSV strain HNyc15 was constructed which could provide scientific evidences for elucidating the molecular pathogenesis of the NADC30-like PRRSV.In this study,PRRSV was detected in serum samples from Yichuan pig farm in Henan Province.The results showed that PRRSV was positive.The preliminary differentiating PCR results show that it was a NADC30-like PRRSV.The porcine alveolar macrophages(PAM)were used for the separation of virus.Typical cytopathic effect was observed in PAM cells 48 h post infection.After 96 hours,the cultures were harvested.The Nsp2 gene was amplified by RT-PCR and sequenced,Alignments of Nsp2 gene sequencing showed that there had the 131 amino acid deletion in the nonstructural protein 2(nsp2)region as other reported NADC30-like PRRSV strains.Therefore,this isolated virus was identified as a NADC30-like PRRSV and designated HNyc15 strain.In this study,twelve pairs of primers were synthesized accrording to the reported NADC30-like genome sequences at NCBI and RT-PCR method was used for amplification of the complete genome of HNyc15 strain.The results of each gene sequence were spliced and aligned.Genetic evolution analysis and recombination analysis was performed with the whole genome sequence of the reported strains.The results showed that the genome length of HNyc15 strain was 15049 bp.Genetic analyses demonstrated that HNyc15 exhibits 60.4%,85.2%,and 93.8% nucleotide identity with PRRSV strains Lelystad virus(LV),VR2332,and NADC30,respectively.The evolutionary analysis showed that HNyc15,HENAN-XINX,HNjz15,HENAN-HEB and NADC30 belonged to the same subgroup,and the subgroup was composed of NADC30 isolates,The results of recombination analysis showed that HNyc15 strainhad recombination with VR-2332 and CH-1a between ORF2 and ORF4.It is inferred that the NADC30-like PRRSV may be mutated by the NADC30 strain and type 2 PRRSV.In this study,the genome of NADC30-like PRRSV strain HNyc15 was divided into A,B,C,D,E and F six fragments according to the appropriate restriction sites.The amplified products were inserted into the p EASY-Blunt simple vector.The CMV eukaryotic promoter was inserted in the multiple cloning sites(MCS)of the p Bluescript II SK(+)vector,HDV and BGH sequences were inserted in the backward position.The modified vector was named p Blue-CMV-HB.Six fragments were ligated into the modified vector p Blue-CMV-HB to construct full-length c DNA clones p Blue-CMV-HNyc15.The C at position 7186 was mutated to T by PCR to create a restriction enzyme site Xba I as the genetic marker.The full-length c DNA plasmid was transfected into BHK-21 cells.After 48 hours,the cultures were harvested and inoculated in PAM cell.The cytopathic effects were observed at 48 h post infection.Immunoperoxidase monolayer assay(IPMA)and identification of genetic marker showed that virus was rescued successfully.The rescued virus exhibited a similar growth pattern to its parental virus in PAM cells with peak titers at 36 h post-infection.In conclusion,we rescued virus from an infectious c DNA clone of HNyc15 strain.The development of reverse genetics systems for NADC30-like PRRSV will be helpful to us for better understanding the moleculer mechanism of the virus.