Analysis Of Genetic Variation And Isolation And Dentification Of PRV Strains In Some Areas Of Henan Province

Porcine pseudorabies(Pseudorabies,PR)is a highly contagious infectious disease caused by the pseudorabies virus(PRV)in a variety of domestic animals,poultry and wild animals with fever,itching and encephalomyelitis as the main symptoms.PRV can cause abortion,stillbirth and mummified fetuses in pregnant sows;suckling piglets have neurological symptoms,exhaustion and death,and the mortality rate is almost 100%;adult pigs are infected with PRV,but they can carry the virus for a long time;clinical symptoms appear tolerant pigs can also become carriers of the virus and become a source of infection,thereby increasing the difficulty of prevention and treatment of PRV.Since 2011,a suspected pseudorabies disease has broken out in largescale pig farms immunized with PRV gene-deficient live vaccines in many provinces in my country.The sows gave birth to weak piglets,stillbirths or abortions,and piglets showed neurological symptoms and death.Studies have confirmed that the disease is caused by a variant strain of PRV,which has caused huge economic losses to the pig industry in my country.This experiment uses the PRV double PCR detection method to detect,sequence,compare and analyze the nucleotide sequence of suspected PRV disease materials in some areas of Henan Province from 2018 to 2020,and explain the current epidemic strains from the perspective of genetic variation.The genetic relationship with the mutant strain in 2011 provides theoretical support for the purification of pseudorabies pigs in Henan Province.By designing primers to clone the gB and gE genes,a double PCR detection method for porcine pseudorabies virus was established.Using this double PCR detection method,PCV2,CSFV,PRRSV,and sterile double-distilled water could not amplify any bands,and the double PCR amplification was repeated three times.The test results were completely consistent.For pMD-18T-PRV-gE and The minimum detectable nucleic acid copy n?mber of pMD-18T-PRV-gB recombinant plasmid template is 1.04×103 copy/?L.It shows that the dual PCR detection method has the advantages of strong specificity,high sensitivity,good reproducibility,etc.and is simple to operate,short in time,and less pollution.It can distinguish infections of wild virus and vaccine strains,and is suitable for the detection of clinical samples.Using the above-mentioned double PCR method to detect 311 clinical samples in parts of Henan Province from 2018 to 2020,a total of 14 PRV positive samples were detected,with a positive rate of 4.5%,and the full gC and gE gene cloning and genetic evolution of PRV were performed.analysis.The analysis results showed that the nucleotide sequence homology of the whole gC and gE genes among the 14 cases of PRV in this experiment was 99.6%-100%and 98.7%-100%,which was the same as the nucleotide sequence of the domestic mutant strain HeN1-2012.The origin is 99.8%?100%and 98.7%?99.9%,and the nucleotide sequence homology with the classical foreign strains is 94.7%?95.8%and 96.6%?97.7%.The results showed that PRV and the mutant strain HeN1-2012 belong to the same genotype and the closest genetic relationship.It belongs to the same branch as the domestic classic strain Ea and other strains.It can be inferred that both are from the earliest domestic epidemic strains,and are similar to foreign classic strains.The genetic relationship of the strains is relatively distant.Cell proliferation and culture of the above 14 cases of positive disease materials were separated into one PRV,named JZ-45(BankIt:MT796654),after plaque purification,TCID50 determination,and indirect immunofluorescence to detect the cell line.At the same time,the pathogenicity and LD50 were determined by animal inoculation test.The results showed that the strain showed a relatively stable CPE phenomenon on ST cells.After continuous plaque purification,the TCID50 of the purified JZ-45 virus was 10-6.58/0.1mL.Indirect immunofluorescence showed a specific fluorescent label.The vaccinated Kunming mice began to show neurological symptoms on the second day,cramping and spasm,biting the injection site,and finally died on the third day.The LD50 measurement result was 1.5×106 TCID50,but the mice in the control group injected with the same dose of DMEM showed no abnormalities.which performed.