Isolation,identification And Epidemiological Investigation Of Porcine Pseudorabies Virus (PRV) In Liaoning Province And Research And Application Of Novel Detection Methods For PRV

Pseudorabies（PR）caused by Pseudorabies virus（PRV）is an important infectious disease that causes substantial economic losses to swine industry worldwide.Pigs are the only natural reservoir hosts for PRV and main transmission source of PRV.PRV can cause serious illnesses to pigs.Piglets mostly die from central nervous system disorders after infection,with morbidity and mortality can be as high as 100%.Adult pigs usually show respiratory symptoms after infection,and those that have survived from the acute infection become retarded or even stiffened,and carry the virus recessive lifelong,as the continuous transmission sources of the virus.Infected breeding pigs show reproductive failure.Since the late 2011,the disease had been broke out again in pigs of intensive pig farms in many provinces in China,including the pigs vaccinated with PRV vaccines,which caused huge economic losses to China’s pig industry.Recently,several cases of human infection with PRV have been reported successively,and these infected persons were pig breeders or closely contacted with pigs,so PRV has become a zoonosis with potential public health hazards,which should be highly concerned with further investigation and research needed.A total of 25 clinical samples were detected positive for wild-type PRV during 2015-2017,the positive rate was 0.55%.By inoculating Vero cells for virus isolation,four representative strains of PRV were obtained,named PRV-LN01,PRV-LN09,PRV-LN10 and PRV-LN11,respectively.And their TCID₅₀ were between 10^6.28 and 10^7.26/0.1m L.For these strains,the typical morphology of herpes virus were observed by electron microscopy and the inoculation test of sheep showed that the isolated virus strains could cause typical clinical symptoms in sheep and finally lead to death.The isolated strains had the same evolutionary origin as the HB/BD strain,HLJ8 strain,JS-2012 strain,FJ-N1 strain and other PRV epidemic variants isolated in China in recent years and the vaccine strains（SA215 and HB-98）,with the nucleotide homology of g D and g E were both above 99.3%.and they are belong to the same gene type II（GII）.Therefore,the isolated strains in this study were the current domestic epidemic PRV variant strains.The understanding of the genetic characteristics of the epidemic strains in Liaoning province will provide a scientific basis for the screening of targeted and effective PRV vaccines,and lay a foundation for the research and development of diagnostic reagents and new vaccines.In order to distinguish epidemic strains from the g E-deleted vaccine strains of PRV rapidly,sensitively and specifically,a novel real-time quantitative PCR（q PCR）assay were developed based on two specific sets of primers and Taq Man probes for g D and g E genes of PRV.The linear correlation coefficients（R²）of q PCR for g D and g E genes were 0.996 and 0.98,and the amplification efficiency were 99.96%and 98.02%,with the detection limits at 39.4 and 12.1copies/μL,respectively.The coefficients of variation（CV）of the intra and inter batches was between 0.98%and 2.07%.Upon the established q PCR method,a new detection method for identification and accurate quantitative droplet digital PCR（dd PCR）was established.The detection limits for PRV g D and g E genes were 3.9 copies/μL and 2.3 copies/μL,respectively,which were 10 times and 5 times higher than the established q PCR method,respectively.The intra and inter batch CV of dd PCR were between 2.06%and 3.88%.Comparing the virus isolation method with q PCR and dd PCR methods,45 clinical samples were tested.The results showed that the sensitivity of the dd PCR assays were the highest,then the virus isolation and q PCR,Kappa consistency test showed good consistency among the three methods（Kappa≥0.88）.Two suspicious samples（Ct value 30-37）detected by q PCR were positive for PRV wild strain by dd PCR,and the nucleic acid concentrations were 8.6 copies/μL and 11.2 copies/μL,respectively.The dd PCR showed more advantageous in the detection of low-copy clinical samples.Both q PCR and dd PCR can be used as effective,accurate and reliable detection methods for distinguishing PRV wild strains from vaccine strains.In addition,dd PCR is the"gold standard"method for the determination of PRV nucleic acid standard material.It provides a basis for the advancement of diagnostic standardization in the veterinary field.In order to systematically understand the prevalence of PR in pig farms in Liaoning Province during 2015 and 2020.A total of 12586 serum samples from 452 pig farms were tested for PRV g B antibody,12586 pig serum samples from 536 pig farms were tested for PRV g E infectious antibody,and 7490 tissue samples collected from pig farms,slaughterhouses and harmless treatment plants were detected for PRV nucleic acid.The test results showed that the average herd-level and individual-level g B positive rate were 61.3%and 70.2%,respectively;Chi-square test and binary logistic regression analysis showed that there exsited differences of g B antibody levels among different years,scale types,production stages,seasons and regions.The g B antibody level was the highest in 2019（83.1%）and the lowest in 2017（42.4%）;the small-sized farms were significantly higher than those in the large and medium-sized farms（P<0.01）;the finishing pigs were higher than producing sows higher than the gilts;In the winters were higher than other seasons（P<0.01）.In northern region were significantly higher than other three regions（P<0.01）.The herd apparent prevalence（HAP）and individual apparent prevalence（AP）of PRV g E antibodies were 40.3%and 20.8%,respectively.There also exsited significant differences of g E antibody levels among different years,scale types,production stages,seasons and regions.The g E antibody level was the highest in the year 2017 and the lowest in 2016,and set the 2016 as the reference,the infection risk（Odds ratio,OR）values in 2015,2017,2019 and 2020 were 2.7 times,3.2 times,2.1 times and 1.6 times,respectively;Small-sized farms was significantly higher than that of large and medium-sized farms（P<0.01）,and the OR was 1.8 times;Among different production stages,producing sows higher than the gilts higher than the stud boars higher than the finishing pigs,and the OR was 2.5 times,2.1 times and 1.5 times,respectively;In winters was significantly higher than that in the other three seasons（P<0.01）,and the OR was about 1.5 times.There were significant differences among different regions（P<0.01）,and the OR in southern,eastern and northern Liaoning were 2.0 times,3.2 times and 1.7 times of that in western region,respectively,showing obvious regional differences.A total of 56 clinicalsamples were detected as PRV wild virus positive and the positive rate was 0.75%,The most positive samples were detected in the breeding process,accounting for 66.1%,followed by the slaughter process,accounting for26.8%,and 7.1%in the harmless treatment plants.The results showed that PRV immune antibody level was not high and the virus infection was relatively serious in pig farms in Liaoning Province.In conclusion,four PRV epidemic variant strains were isolated and identified in Liaoning Province in this study.A Taq Man q PCR and a new dd PCR were developed which can process accurate quantitative,rapid,sensitive and specific differential diagnosis between the current endemic wild-type PRV strain and the widely used g E gene deleted vaccine strains.Through the systematic epidemiological investigation and research,the epidemic situation of PR infection in large-scale pig farms（especially breeding pig farms）in Liaoning Province has been basically mastered.Based on the investigation results,the integrated and standardized research on the comprehensive prevention,control and eradication technologies of PRV in large-scale pig farms has been processed,which can be applied in the whole province to provide scientific data and effective technical support for the precise prevention,control and eradication of PRV in large-scale pig farms in Liaoning Province.