Epidemiological Investigation Of Porcine Reproductive And Respiratory Syndrome In Some Areas Of China From 2019 To 2020 And Construction Of PRRSV Chimeric Virus PMLV+NADC30

Porcine reproductive and respiratory syndrome(PRRS)is an acute and highly infectious disease caused by the porcine reproductive and respiratory syndrome virus(PRRSV).In 2014,NADC30-like PRRSV strain was isolated from southern China.In the following years,the prevalence rate of this strain increased so fast that it has become a new dominant PRRSV strain in China.The key measure of PRRS prevention and control is to develop specific and efficient vaccine against PRRSV epidemic strains.In order to understand the epidemic situation and genetic evolution of PRRSV in some areas of China in recent years and to provide a technical platform for the research of new vaccine candidate strains,this study carried out etiological detection and genetic evolution analysis of PRRSV epidemic strains in some areas of China from 2019 to 2020,and completed the construction of infectious clone of PRRSV classic strains and chimeric strains through reverse genetic operation technology and rescued the viruses.The main results of this study are as follows:1.Research on epidemic strains of PRRSV in China from 2019 to 2020.The dominant epidemic strains in some areas of China in recent two years were MLV-like strains(subgroup 1)and NADC30-like strains(subgroup 5).A total of 4,238 samples were collected from 2019 to 2020 and used RT-PCR method for pathogen detection and the positive rates were 7.02% and 10.57% respectively.The positive rates of PRRSV were high in Northeast(10.00%)and East(10.78%)of China in 2019,while it was South(13.43%)and Northwest(27.17%)of China in 2020.ORF5 gene sequencing and genetic evolution analysis showed that MLV-like(subgroup 1)and NADC30-like strains(subgroup 5)were the main epidemic strains in recent two years.In particular,NADC30-like strains accounted for 67.61% and 45.95% in 2019 and 2020 respectively.2.Construction of the PRRSV chimeric virus pMLV+NADC30.Successfully constructed the full length infectious clone of classical virus pMLV and the chimeric virus pMLV+NADC30 and obtained the rescued viruses.Using the genome of MLV-like virus strain as template,the full-length plasmid pMLV was constructed by dividing it into four fragments and ligating them into p WSK29-CMV vector in turn.It’s infectious clone was rescued by transfection into Marc-145 cells.Furthermore,the structural proteins(ORF2～7)of NADC30-like strain was substituted into the corresponding coding region of pMLV to construct the chimeric plasmid pMLV+NADC30.The live virus was successfully rescued after transfection.The biological characteristics of the two rescued viruses showed that the growth curves of pMLV and pMLV+NADC30 were similar with their parental strain MLV.The rescued viruses could be passaged on Marc-145 cells stably.3.Detection of the stability of PRRSV chimeric virus strain pMLV+NADC30.Chimeric virus could stably pass on Marc-145 cells and no recombination was found when the chimeric virus was co-infected with three epidemic strains in different subgroups at the cellular level.The sequences of Nsp2,ORF3 and ORF5 of chimeric plasmid and its rescued viruses F5 and F10 were compared.The results showed that the chimeric virus could stably pass on Marc-145 cells and no mutation was found.The chimeric virus pMLV+NADC30was co-infected into Marc-145 cell in different proportions with MLV-like,NADC30-like and HP-PRRSV strains,respectively.After the co-infected viruses were passed to F10,the viral plaque purification test was carried out and aligned the sequence of Nsp2,ORF3 and ORF5.The results showed that no recombination was found when the chimeric virus was co-infected with three strains from different subgroups,while the replication ability of the chimeric virus pMLV+NADC30 was stronger than MLV-like strain and HP-PRRSV strain,but weaker than NADC30-like strain.In summary,molecular epidemiology found that MLV-like strains and NADC30-like strains were the main domestic epidemic strains in the past two years,while NADC30-like strains accounted for the highest proportion.The rescued viruses pMLV and pMLV+NADC30 could be stably passed on Marc-145 cells and had a similar growth kinetic with the parental MLV strain.The chimeric virus pMLV+NADC30 was relatively stable after co-infection with the epidemic strains from three subgroups at the cellular level,no recombination between strains was found and the chimeric virus pMLV+NADC30 had stronger replication ability compared with MLV-like strain and HP-PRRSV strain,but weaker than NADC30-like strain.