Isolation,Identification And Genetic Characteristics Of Porcine Circovirus Type 2 From Tibetan Pigs

Porcine circovirus type 2(Porcine circovirus type 2,PCV2)plays an important role in causing a Porcine Circovirus Associated Disease(PCVAD),and is the smallest mammalian virus.At present,there are few reports about porcine circovirus type 2 in Tibet.This study aims to understand the prevalence of PCV2 in Linzhi,Tibet,and establish a rapid and quantitative PCV2 genotyping method for in-depth research on the significance and effect of genetic variation of Tibetan pig PCV2 isolates through epidemiological investigations,establishment of multiple fluorescent quantitative PCV2 genotyping methods,and infectious clone construction and genetic characteristics research.1.In order to understand the molecular epidemiology of porcine circovirus type 2 in Linzhi,Tibet and the genetic variation of the virus strain,the polymerase chain reaction analysis method was used to analyze 95 samples collected from different pig farms in Linzhi,Tibet from 2018 to 2019.The samples were tested for PCV2,and the whole genome sequence of positive samples was amplified and cloned and sequenced,and 9 PCV2 full-length genome sequences were obtained.The positive Tibetan pigs were inoculated with PK-15 cells after aseptic treatment.After PCR detection and sequencing,a PCV2 strain from Tibetan pigs was successfully isolated and named as ZZ2 strain.The genetic variation analysis of the whole genome sequence of the positive samples showed that among the 9 PCV2 positive strains,there were 6PCV2 b subtypes and 3 PCV2 d subtypes.Among them,2 Tibetan pig isolates were PCV2 b subtypes,but the total length was only 1756 bp,there is a 11 bp deletion of the nucleotide sequence in the replication start region.2.To establish a multiple real-time fluorescent quantitative PCR method for detecting porcine circovirus type 2,which can detect and type three genotypes PCV2 a,PCV2b,and PCV2 d.Universal primers was designed based on the conservative sequence region of the ORF2 gene of PCV2,and Taqman probes was designed according to different genotypes.The three probes are labeled with different fluorophores,and the reaction system of multiple fluorescence quantitative PCR method is established and optimized to detect the sensitivity,repeatability and specificity.The results showed that the minimum number of copies detected by the established multiplex fluorescence quantitative PCR method was PCV2 a 100 copies/?L,PCV2 b 57 copies/?L and PCV2d100 copies/?L.The method has good sensitivity and specificity based on testing.227 positive samples including 9 positive samples from Tibet were detected by this method.The results showed that the mixed infection of PCV2 a,PCV2b,and PCV2 d was more serious,with a detection rate of 55.95%,and the second rate was the rate of co-infection of PCV2 b and PCV2 d with the value of 17.62%.Part of the samples were selected for sequencing and identification,and the results showed that the sequencing results were highly consistent with the results of multiple fluorescent quantitative PCR typing.The 9 samples from Tibet that tested positive were all PCV2 monotype infections and the typing results were consistent.3.In order to further study whether the isolated 11 bp nucleotide sequence of the isolated ZZ2 strain has an effect on the replication ability and pathogenicity of PCV2,the reverse genetic technology was used to construct a full-length infectious clone plasmid of ZZ2 with nucleotide deletion and the full-length infectious cloning plasmid of normal sequence 5123 strain with its genotype being PCV2 b.Besides,gene recombination technology was used to construct a full-length infectious cloning plasmid ZZ2(1767 bp)after nucleotide replenishment and the cloned plasmid of 5123(1756 bp)nucleotide deletion after nucleotide knockout.infectivity,nucleotide-replenishing and nucleotide-knockout sequences are the corresponding deleted nucleotide fragments on the isolated Tibetan pig strains.After the constructed four infectious cloning plasmids were transfected into PK-15 cells,the PCV2ZZ2 strain,ZZ2 nucleotide replenishment strain,5123 strain and 5123 nucleotide knockout virus were detected by PCR,q PCR,IFA and Western blot methods.The strain of virus was successfully rescued.The proliferation of the virus tended to be stable until the 10 th generation,but the copy number of the rescued strain of ZZ2 was lower than that of the other three strains.By detecting the transcription levels of related immune factors,autophagy marker factors,and apoptosis factors,it was found that nucleotide-deleted strains inhibit IFN-?1 transcription,promote IL-8 expression in the late infection stage,inhibit autophagy in the early stage of infection,and has effects on limiting apoptosis.