Genetic Variation Of NADC30-like PRRSV And Development Of A Detection Method Based On CRISPR-Cas13a For PRRSV

Porcine reproductive and respiratory syndrome virus(PRRSV)has been prevalent in the world for more than 30 years since its discovery.PRRSV is an important pathogen that seriously harms the pig industry.PRRSV causes porcine reproductive and respiratory syndrome(PRRS)in pigs,which is characterized by reproductive disorders such as abortion in pregnant sows and respiratory symptoms and death in pigs of all ages.PRRSV has harmed China's pig industry for more than 20 years,which has brought huge economic losses to the pig industry.PRRSV is a positive-sense single-stranded RNA virus encapsulated by envelope.PRRSV is divided into PRRSV-1 and PRRSV-2.And PRRSV-2 is divided into 9 lineages.The mainly prevalent lineages in China are lineages 1,3,5 and 8 of PRRSV-2 and NADC30-like strains in lineage 1 have gradually become dominant strains,bringing great resistance to the prevention and control of PRRS.In this study,genetic variation of NADC30-like PRRSV was analyzed and a detection method based on CRISPR-Casl3a for PRRSV was developed,which may provide support for the prevention and control of PRRS.In order to understand genetic variation of NADC30-like PRRSV,the complete genome sequences of 10 NADC30-like strains were obtained by sequencing.And genome annotation,nucleotide sequence similarity analysis of genome and ORFs,amino acid sequence similarity analysis of structural proteins and non-structural proteins,deletion characteristic analysis of NSP2 protein,construction of phylogenetic trees respectively based on ORF5,NSP2,and whole genome,recombination analysis of genome,antigenic epitopes analysis of glycosylated proteins and N-glycosylation sites analysis of GP5 protein.The genomic length of the 10 strains range from 15004-15020bp.The similarity between the whole genome of 10 strains and LV(representative strain of PRRSV-1)are 59.8-60.1%,the similarity with VR2332(representative strain of PRRSV-2)are 84.7-86.7%,the similarity with NADC30 are 90.5-94.3%,NSP2 proteins of 10 strains all have 131 discontinuous amino acid deletions consistent with NADC30,indicating that all 10 strains are NADC30-like PRRSV.The amino acid sequence similarity analysis showed that amino acid sequences of each protein of 10 strains had different degrees of variation.The phylogenetic tree respectively based on NSP2 and whole genome showed that all 10 strains were in NADC30-like PRRSV branch of lineage 1,while the phylogenetic tree based on ORF5 showed that 7 strains were in NADC3 0-like PRRSV branch and 3 strains were in other branches,indicating that the genetic diversity of NADC30-like PRRSV was complex.Recombination analysis of genome showed that 8 out of 10 strains had recombination,and the recombination patterns were diverse,indicating that NADC30-like PRRSV had extensive recombination.Antigenic epitopes analysis of glycosylated proteins showed that 91?105aa epitope in GP3 and epitope in GP4 of 10 strains had larger variation.N-glycosylation sites analysis of GP5 protein showed that N-glycosylation patterns of GP5 in 10 strains were diverse.In addition,the adaption on MARC-145 cells of 15 NADC3 0-like PRRSV strains was preliminarily evaluated,and the results showed that most of NADC30-like PRRSV could not adapt well to MARC-145 cells.Detection and identification of PRRSV are the premise for the study and prevention and control of PRRS.However,methods such as RT-PCR and RT-qPCR commonly used to detect PRRSV nucleic acids rely on sophisticated equipment and have certain limitation for being used in field or in grass-root laboratories.Therefore,in this study,a visual,sensitive and specific nucleic acid detection method based on CRISPR-Casl3a was developed for PRRSV.The novel method was an isothermal detection at 37?,and the detection can be used for real-time analysis or visual readout.The detection limit of the novel method was 172 copies/?L,and there were no cross-reactions with porcine circovirus 2,porcine parvovirus,classical swine fever virus and pseudorabies virus.And,the enhanced Casl3a detection can work well in clinical samples.