Molecular Identification And Variation Analysis Of PRRSV In Henan Province From 2014 To 2015 And Analysis Of Complete Genome Sequences Of HP-PRRSV Isolates

Porcine reproductive and respiratory syndrome(PRRS)is currently the most economically significant disease impacting the swine industry worldwide,caused by porcine reproductive and respiratory syndrome virus(PRRSV),and characterized by reproductive failure in sows,respiratory problems affecting pigs of all ages and increased morbidity and mortality in piglets.PRRSV possesses several properties related to viral pathogenesis,including cytopathic replication in macrophages,the capacity to establish a persistent infection and immunological suppression.Since the first outbreak of PRRS was found in 1995 in China,this disease has already accompanied with the Chinese swine industry over twenty years.Especially in 2006,a so-called porcine high-fever syndrome,caused by highly pathogenic PRRSV(HP-PRRSV),broke out in southern China and rapidly spread throughout the country,causing enormous economic losses.The PRRSV strain NADC30,containing a discontinuous 131-amino acids deletion in Nsp2 protein compared with VR-2332,was isolated in United States in 2008.Since2012,the PRRSV strains with the same deletion feature,sharing a high nucleotide identity with NADC30,have spread to 13 provinces in China and recombined with domestic PRRSV prevalent strains.In addition,the genome of PRRSV is characterized of its genetically extensive variation,and thus field isolates showed a variable genetic,antigenic and pathogenic diversity,which is regarded as one of the most important reasons for prevention and elimination of PRRS.In this study,the suspected PRRS clinical samples in Henan province were detected and analyzed based on the NSP2 and ORF5 genes to reveal the ultramodern molecular epidemiology and genetic variation of PRRSV.Then the PRRSV prevalent strains were isolated and identified,and subsequently the complete genome of these isolates were sequenced and analyzed to understand its partial biology,variation and evolution characteristics,expecting that it will provide scientific reference data for modification of diagnosis methods,design of novel vaccine and antiviral drugs,and the current prevention and control strategy of PRRS.1.Molecular identification and variation analysis of NSP2 and ORF5 genes of PRRSV in Henan province from 2014 to 2015NSP2 and ORF5 genes of the PRRSV positive samples were amplified,sequenced and analyzed,based on the detection of 414 clinical samples by RT-PCR,collected from165 farms during 2014-2015 in different regions of Henan province.The results showed that 123 samples were positive for PRRSV,and the positive rate was 29.7%.50 NSP2 genes and 64 ORF5 genes were obtained.The phylogenetic analysis revealed that all sequenced strains belonged to the North American genotype.Among the 64 positive samples,40 samples were highly homologous with NADC30 containing a discontinuous131-amino acids deletion in Nsp2 protein,which had been reported in recent years;another 21 strains were highly homologous with HP-PRRSV.This assay showed that the NADC30-Like strains has been the dominant strains in Henan at present,meanwhile both NSP2 and ORF5 genes had significant variations.Especially,compared with the results during 2012-2013,NADC30-Like strains accounted for higher percentage and most of HP-PRRSV strains were closely related to the vaccine strain JXA1-P80 during 2014-2015,suggesting that wide use of HP-PRRSV-derived live vaccines and increasing importation of boars are the reasons for the change of the dominant strains and the rapid spread of PRRSV.Thus,more attention should be continuously paid to monitor the epidemiology and genetic variation of PRRSV and more research should be conducted about the mechanism of pathogenicity and immunological suppression so as to provide a reference for the research and development of clinical diagnosis and prevention and control of PRRS.2.Isolation and identification of four HP-PRRSV strainsThe PRRSV positive specimens were passaged through a 0.22-?m filter and inoculated onto MARC-145 cells,and the viruses harvested from the supernatant were identified by RT-PCR and indirect immunofluorescence assay(IFA).The results revealed that four PRRSV strains were isolated from 52 positive specimens and generated stable cytopathic effect(CPE)on Marc-145 cells.These isolates were identified as HP-PRRSV strains and designated as HENZK-1,HENPDS-2,HENZMD-5 and HENXC-1,and viral titers of their fifth viral passage were determined as 105.77 TCID50 /m L,106.75 TCID50 /m L,104.80 TCID50 /m L and 104.36 TCID50 /m L,respectively.This assay provided reliable reference materials for the research about pathogenicity and evolution of PRRSV.3.Genomic sequence analysis of HP-PRRSV isolates HENZK-1 and HENPDS-2The complete genome sequences of HP-PRRSV isolates HENZK-1 and HENPDS-2were amplified,sequenced and analyzed.The results showed that the genomic sequences of HENZK-1 and HENPDS-2 were 15019 bp and 15020 bp in length,respectively,excluding the poly(A)tail.The genomic sequences alignment analysis revealed that both of them contained a discontinuous 30-amino acids deletion in Nsp2 protein compared with VR-2332,which was considered as the genetic marker of HP-PRRSVstrains,and meanwhile novel genomic variations existed.The genomic nucleotide identity and phylogenetic analysis revealed that the two isolates shared 99.3%~99.4% and99.6%~99.8% nucleotide identity with the JXA1 derivatives(JXA1-P10~P170)and three revertants of the vaccine strain JXA1-P80(NT1,NT2 and NT3),respectively,clustered into a main branch belonged to subgroup 4 represented by JXA1,together with the JXA1-P10~P170,NT1,NT2 and NT3,and were closely related to JXA1-P45 and NT3,respectively,implying that the two isolates are likely to be revertants of the vaccine strain JXA1-P80,and the preliminary results of animal experiments showed that infected piglets exhibited significant clinical signs and lung pathology.This assay provided scientific reference data not only for the study on the virulence and genomic evolution of HP-PRRSVstrains,but for the development and use of vaccine and the current prevention and control strategy of PRRS.4.Genomic sequence analysis of PRRSV NADC30-Like strain HENXX-1The complete genome sequences of NADC30-Like strain HENXX-1 were amplified,sequenced and analyzed.The results showed that the genomic sequences of HENXX-1were 15019 bp in length,excluding the poly(A)tail.The genomic nucleotide identity analysis revealed that HENXX-1 shared the highest nucleotide identity(96.0%)with NADC30,and shared 85.3%,84.6% and 84.0% nucleotide identity with VR-2332,CH-1a and JXA1,respectively.The genomic sequence alignment analysis revealed that HENXX-1 contained a discontinuous 131-amino acids deletion in Nsp2 protein compared with VR-2332,which was considered as the genetic marker of NADC30-Like strains.Phylogenetic analysis revealed that HENXX-1 clustered into a main branch belonged to novel subgroup represented by NADC30,closely related to the NADC30-Like strains reported in China in recent years.Recombination analysis showed that HENXX-1 was without recombination,implying that it possibly was a variants of NADC30 in China.This assay provided reliable reference data for the research about genetic variation and evolution of NADC30-Like strains in China.