Isolation And Identification Of Porcine Pseudorabies Virus Anhui Strains And Their Pathogenicity In Mice

Pseudorabies(PR)is an acute heat infectious disease caused by a variety of livestock and wildlife caused by Pseudorabies virus(PRV).Pigs are the main natural host and disseminator of PRV.Pigs in each stage of growth can feel PR.Clinically,neonatal piglets develop neurological symptoms and diarrhea.Sows have reproductive disorders,abortion,stillbirth and respiratory symptoms.Adult pigs It is a recessive infection,growth retardation,and long-term poisoning.Since the 1990s,with the introduction and widespread use of the PR gene deletion vaccine(Bartha-K61 strain),PR has been effectively controlled in most parts of China.However,from 2011 to the end of 2015,except for a few regions,there have been reports of PR outbreaks in various parts of China,causing serious economic losses to the pig industry.Studies have shown that the PRVs that are currently prevalent in China have undergone variability,not only in the enhancement of the virulence of the strains,but also in the immune protection provided by the Bartha-K61 attenuated vaccine.Therefore,understanding and mastering the characteristics(pathogenicity,antigenicity,etc.)of PRV isolates in different regions is significant for effective prevention and control of PR.In this study,PCR,cell inoculation test,virus plaque purification test,transmission electron microscopy,indirect immunofluorescence test(IFA),rabbit inoculation test,virus titer determination(TCID50)and other methods were used to treat pigs in different areas of Anhui Province.In the field,pigs with suspected PRV infection such as high fever,respiratory symptoms,neurological symptoms,reproductive disorders,and fatal encephalitis were collected,pathogens were detected for pathogen detection and PRV isolation and identification,and mice were used as experimental animal models for PRV Anhui isolation.LD50 measurement,histopathological observation,and viral load detection in each organ tissue.The aim is to obtain PRV epidemic strains in Anhui,to understand the infection of PRV in Anhui pig herds and the virulence characteristics of PRV Anhui strains,so as to lay a foundation for further study of the antigenicity of PRV Anhui isolates,and to effectively prevent and control PR in Anhui.Provide scientific evidence.The results showed that 13 of the diseased tissues from the affected farms in different areas of Anhui Province were only positive for PRV nucleic acid detection.The treated material tissue was inoculated with Vero cells.After the fifth generation,13 strains of virus could make the cells at 12h.Stable cytopathic changes such as aggregation,constriction,fusion,and shedding.The isolated virus was subjected to plaque purification,and the cells were cultured for about 1 week to obtain an obvious,near-circular plaque with an apparent diameter of about 5 mm and an undyed color by crystal violet staining.Purification of concentrated virus solution has a spherical structure,a particle diameter of about 180 nm,and a particle structure similar to herpesvirus under a transmission electron microscope.After 13 strains of virus infected Vero cells,obvious green fluorescence reaction appeared under the fluorescence microscope.The virus solution was inoculated into rabbits,and the rabbits died after typical PR clinical symptoms such as itching,local hair loss,and Angle bow.The 13 isolates of the virus were all PRV.The virus titer was detected by Reed-Muench method,and the TCID50 of 13 PRVs was between 10-3.12/0.1 mL?10-6 459/0.1 mL.The LD50 of 13 strains of PRV to mice was 102 569TCID50?105.167TCID50;the LD50 of strains 10(AH1802)and 11(AH1803)were 102.569TCIDso,102.833TCID50,3rd strain(AH1603)and 7th(AH1703),respectively.The LD50 of the strains was 105.167TCID50.Clinical necropsy showed that the liver,spleen,lung,kidney,brain and other parts of the challenge group had different degrees of hemorrhage and swelling,and there was no abnormality in the control group.Histopathology showed that 13 strains of PRV infected mice caused the tissues to be dirty.The lesions vary according to different strains.Among them,the 10th strain(AH1802 J group)and the 11th(AH1803 K group)cause brain,lung,liver,spleen and kidney lesions are more serious than other strains,and the virus is contained.The amount was also higher,and the difference was significant(p<0.05).The results showed that 13 strains of PRV were successfully isolated and identified in this study.Two strains of PRV from Chuzhou AH1802 and Suzhou AH1803 have the strongest virulence.The virulence of PRV isolates varies from region to region,which may be related to the variation of PRV isolates themselves.