Isolation Of PRV Virulent Strains And Genetic Evolution Analysis Of GB And GD Genes

Pseudorabies?PR?,also known as Aujesky's disease,is an infectious disease caused by Pseudorabies virus?PRV?,which is characterized by fever and respiratory symptoms in fattening pigs,high death rate in newborn piglets.Pregnant sows infected with PRV show abortion,stillbirth,mummified symptoms.PRV has a wide range of hosts,among which pigs are the natural host and reservoir,and also are the most important source of infection and detoxifier in other animals,which has caused great losses in pig industry.In recent years,pseudorabies outbreaks occurred in pig herds which have been immunized with PRV vaccine like Bartha-K61.Some studies showed that the field isolates from died pigs were new variant strains of PRV,and also have very high infection rates of g E,which indicating that the traditional PRV attenuated vaccine like Bartha-K61 can not provide complete protection against these pseudorabies virus variants.Therefore,it is extremely urgent and necessary to find out the current variation and wild virus infection in prevalence of major PRV strains and develop new vaccines against these pseudorabies virus variants.The conventional serological detection method can't distinguish pigs immunized with PRV vaccine from pigs infected by PRV wild strains.The g E gene is a sign of wild PRV strains infection,and the g H gene is highly conservative,and it is also necessary for viral replication within PRV genes.Two pairs of primers were designed according to the PRV genome sequence in Gen Bank.After optimizing annealing temperature and other conditions,the duplex PCR method was established for the differentiation of attenuated and virulent strains of pseudorabies virus.The reacting condition test showed that the anneal temperature was 53?.In the duplex PCR assay,two fragments of 429 bp for g E gene and 355 bp for g H gene were simultaneously amplified from pseudorabies virus virulent strains DNA,only a 355-bp fragment for g H gene was amplified from pseudorabies virus vaccine strains DNA,whereas no PCR products were amplified from porcine circovirus type 2 and porcine parvovirus.Then,the detection limit of duplex PCR was estimated to be 1×10²TCID₅₀/0.1m L.348 clinical samples were collected from pigs with suspected PRV in Zhengzhou,Xuchang,Puyang,Jiaozuo,Luoyang and other cities in Henan Province,as well as Wen shui in Shanxi Province and Handan in Hebei province,and tested by the duplex PCR assay.The results showed the assay only required only 3 h?4 h.This method was special,sentive and stable,which can distinguish pigs immunized with gene deletion PRV vaccine from pigs infected by PRV virulent strain,and also can provide technical support for differentiation of wild-type pseudorabies virus and vaccine strains in clinical specimen.Five samples with suspected PRV were acquired from Xuchang,Wenshui,and Handan,and then inoculated them into ST cells after aseptic processing.The viral DNA was extracted from each generation cells harvested for PCR amplification,and the results showed all positive.The 7th generation virus was inoculated to 6-week-old mice,and the animals began to die after 48 h after vaccination,and all died after 62 h,while no morbidity and mortality were displayed for the control group after 168 h.The results showed that the five isolates were all PRV virulent strains,and named XC,SX-1,SX-2,HD1 and HD2.Taking XC strain as an example,the physicochemical properties of XC strain were tested,and the test results showed that it had the physicochemical properties of PRV,and the growth curve of XC strain on ST cells showed that the XC strain gradually increased at 4-12 h after infection and reached its peak at 36 h(TCID50/m L was 10^7.49),and then the viral titer began to decrease.This is consistent with the growth dynamics of PRV on ST cells.Therefore,5 PRV virulent strains were successfully isolated,which provided a theoretical basis for the isolation and identification of PRV and the control of the epidemic of PRV.Two pairs of primers were designed according to g B and g D gene sequences published in Gen Bank.g B and g D genes of 5 isolated pseudorabies strains were amplified and sequenced respectively,and the sequences were analyzed by homology and genetic evolution.The results showed that 5 PRV strains isolated from different areas had high homology of the nucleotide or amino acid.Compared with the strains in earlier years such as Ea,Fa,SC,there were a total of five common mutations of amino acids in the 5 isolated strains of g B gene,which are consistent with the domestic popular variants of amino acids in recent years.When compared with the foreign strains,about 20 several mutations were found in the domestic strains.Based on the deduced amino acid sequence of g D gene,foreign strains?except Becker and NIA3?and the domestic epidemic strains in recent years showed one amino acid deletion at position 278 compared with the domestic epidemic strains in early years,and there were eight common mutation sites in domestic strains including 5 isolates compared with the foreign strains?except Becker and NIA3?.The results showed that because of geographical isolation,5 isolated strains differed genetically from the foreign strains,and the different regions of the PRV infection and epidemic situation were not the same.The 5 isolated strains are far from the domestic epidemic strains isolated before 2011,indicating that the epidemic strains of PRV have changed since 2011,it can be inferred that the occurrence of PR epidemic in different regions in China after 2011 may be caused by the emergence of PRV variant strains.The results of genetic evolution tree showed that these isolates were far from the previous classical strains such as Bartha-K 61,which also explains why PRV infections still occur in pig farms that have been immunized throughout China in recent years.