Characteristic Analysis Of Various Subgenotype PRRSV And Construction Of DIVA Vaccine Strain

Porcine reproductive and respiratory syndrome virus(PRRSV)is the causative agent of porcine reproductive and respiratory syndrome(PRRS).In the early 1990 s,Type I and Type II PRRSV appeared almost simultaneously in Europe and North America.In 1996,the PRRSV CH-1a strain was isolated for the first time in mainland China.After more than two decades of evolution,the diversity of PRRSV is more complicated.To study the prevalence and characteristics of new PRRSVs in different Sublineages,PRRSVs were detected and isolated from suspected PRRSV infection in 14 Provinces from 2014 to 2018.We sequenced 321 ORF5 genes and 50 whole genomes in the present study.Five sublineages PRRSV were identified,including NADC30-like PRRSV(Sublineage 1.8),NADC34-like PRRSV(1-7-4,Sublineage 1.5),QYYZ-like PRRSV(Sublineage 3.5),RespPRRS MLV like PRRSV(Sublineage 5.1)and Sublineag 8.7 PRRSV.Importantly,NADC34-like PRRSV was first discovered in China.There are 6 parts in this chapter.(1)We divided Lineage 1 PRRSV into 9 Sublineages(1.1-1.9)based on Shi et al.(2010).NADC30-like PRRSV was classified into Sublineage 1.8.Sublineage 1.8 PRRSV has faster variation and more frequent recombination,low levels of whole-genome similarity and a wide variety of recombination patterns.Interestingly,the recombination breakpoints mainly occurred in genes encoding nsps and/or minor structural proteins.(2)We isolated two novel recombinant PRRSV strains,named LNWK96 and LNWK130,which had 100-aa deletion in Nsp2 protein.Both strains had highest nucleotide similarity with NADC34 isolated from America,which called NADC34-like PRRSV or 1-7-4 PRRSV.The phylogenetic analysis showed that NADC34-like PRRSV were classified into Sublineage 1.5.We speculated that NADC34-like PRRSV originated from North America.(3)We divided Lineage 3 PRRSV into 5 Sublineages,including Sublineage 3.1-3.5.Sublineage 3.5 PRRSV had higher recombination frequency and distributed in south of China after recombining with local strains.Sublineage 3.5 PRRSV originated from Sublineage 3.2 PRRSV based on the phylogenetic analysis of intermediate strains FJ-1 and FJFS.(4)Lineage 5 PRRSV vaccine had been applicated in China for a long time.Phylogenetic analyze showed that all the Lineage 5 PRRSVs isolated from China were classified into Sublineage 5.1.Sublineage 5.1 PRRSV in China had lower variation,which had higher nucleotide similarity with ATCC VR2332 and RespPRRS MLV strain.We considered that Sublineage 5.1 PRRSVs isolated in China were closely related to application of RespPRRS MLV.(5)Phylogenetic analyze showed that Sublineage 8.7 PRRSV was classified into Sublineage 8.7.1(CH-1a like PRRSV),Sublineage 8.7.2(HP-PRRSV like PRRSV)and Sublineage 8.7.3(HP-PRRSV).In recent years,Sublineage 8.7.3 PRRSV had undergone great variation compared with HP-PRRSV isolated from 2006.However,the pathogenicity and the protection of commercial vaccines against these strains are unclear.(6)Sublineage 1.8 PRRSV is novel strain in China and become the main circulating strain in short time.The pathogenicity experiment and efficiency evaluation of commercial vaccine HuN4-F112 showed that Sublineage 1.8 PRRSV SD53-1603 had medium pathogenicity to piglets and commercial vaccine HuN4-F112 could significantly decrease the clinical signs and virus loads or viremia.In order to find the genetic evolution of PRRSV,this study conducted a four-year PRRSV monitoring of relatively closed large-scale breeding group.This study collected 338 suspected PRRSV-infected tissues or serum from different farms of the large-scale breeding group.The ORF5 sequence of 119 strains of PRRSV and the genome-wide sequence of 34 strains of PRRSV were identified and sequenced.Four types of PRRSV were identified in the large-scale breeding group,namely NADC30-like PRRSV(Sublineage 1.8),QYYZ-like PRRSV(Sublineage 3.5),RespPRRSV MLV like PRRSV(Sublineage 5.1)and Sublineag 8.7 PRRSV,respectively.It is explained that the current complex recombination model is gradually formed.The same strain can be recombined at different locations,increasing the diversity of recombination and providing more possibilities for the survival of the virus.RespPRRS MLV-like PRRSV are likely to be revertant of the attenuated vaccine RespPRRS MLV.Five aas related to virulence and four aas related to revertant ware determined.In addition,the distribution and transmission of PRRSV is closely related to transportation of pigs and the use of vaccine in large pig farm.Currently,attenuated vaccine is one of the main methods to prevent and control PRRS.However,one of the shortcomings of using PRRS live vaccine is that the antibody of the vaccine strain cannot be distinguished from the antibody of the wild strain,which is not conducive to the evaluation of vaccine immunity and eradication of PRRSV.To solve this problem,our laboratory has established a blocking ELISA for 1C8 epitope of M protein.Then we constructed the infectious clone of NADC30-like PRRSV SD53-1603-M40 and rescued the virus successfully.Based on this infectious clone,we constructed 12 different clones with aa deletion or substitution against 1C8 epitope.Fortunately,we rescued 5 PRRSVs.These 5 PRRSVs were passaged to 65 th generation on Marc-145 cells after IFA and sequencing.Experimental results of vaccine immunization showed that there was a significant difference between rSD53-1603-913S914S-M65 and rSD53-1603-M65 in producing specific antibody against the 1C8 epitope.The above results laid foundation for developing DIVA vaccine of PRRSV.