Isolation And Identification Of A Mutated Pseudorabies Virus

Pseudorabies(PR)is a highly lethal acute infectious disease caused by Pseudorabies Virus(PRV).It is susceptible to a variety of livestock and wildlife and is mainly characterized by fever and itching(except pigs).And reproductive disorders.Classified by the World Organisation for Animal Health(OIE)as a Category B animal disease,China has also classified it as a Class II animal disease.Since 2011,a number of pigs in China that have been immunized with the PRV gE gene-deleted vaccine have developed PR-like symptoms,which have caused huge economic losses to farmers.In November 2016,in a small-scale pig farm in Shuangyang District,Changchun City,Jilin Province,there was a miscarriage and stillbirth in sows,and there were no deaths in sows.Piglets have diarrhoea,vomiting,elevated body temperature,lack of energy,loss of appetite,and mild neurological symptoms.The incidence of neonatal piglets is extremely high,with a mortality rate of 100%.The farm has never been vaccinated with PRV vaccine.According to the epidemic characteristics and clinical symptoms,it was initially identified as pseudorabies,porcine circle virus and porcine parvovirus.In order to verify the hypothesis,this study was conducted from pathological and etiological perspectives through pathogenesis investigations,histopathological observations,PCR and RT-PCR techniques,bacteriological examinations,electron microscopy observations,inoculation of experimental animals,and indirect immunofluorescence tests.The disease was analyzed and studied.1 pathological diagnosisRoutine pathological necropsy was performed on the pigs sent for examination,and necropsy found that the organs of the pigs examined had varying degrees of pathological changes: heart failure,more obvious left heart than right heart;congestion and edema of the lungs;mesenteric lymph nodes Large swollen,small intestine with dendritic hemorrhage;a large number of needle-like grayish white necrotic spots and necrotic foci on the liver and spleen,this pathological change is characteristic of swine pseudorabies.Subsequently,paraffin sections were made.Microscopic examination revealed focal necrosis of the liver and spleen.In the liver necrosis,inflammatory cells aggregated,erythrocytes in the hepatic sinusoids around the necrotic lesions were deposited,spleen necrosis was irregular in shape,central structure was unclear,and peripheral lymphocytes in the sheath artery were necrotic.This is consistent with the characteristic pathological changes of swine pseudorabies.2 etiological diagnosisThe DNA was extracted from the grinding material and specific primers for PRV were designed for PCR amplification,and fragments of the same size as the target were amplified.Subsequently,specific primers for porcine parvovirus and porcine circle virus were designed for differential diagnosis.PCR and RT-PCR specific amplification results were negative,excluding porcine parvovirus and Japanese encephalitis.The possibility.Bacteriological examinations by means of contact patch staining ruled out the possibility of bacterial infection.The well-conditioned supernatant with a double antibody was used to inoculate BHK-21 cells for virus isolation.There was no cytopathic effect(CPE)at 72 h after inoculation.We then performed blind transmission and blinded transmission.By the 8th generation,cells showed rounding,increased refractive index,and typical CPE phenomena such as syncytia.At this point,the cytopathic fluid was collected and observed by electron microscopy.The TCID50 was measured using the Muench method and the titer of the isolated strain was 10-5.4/0.1 mL.The cytopathic fluid was repeatedly frozen and thawed three times.The rabbits were vaccinated by centrifugation to inoculate the rabbits.After 3 days,hyperactivity,itching,and mild neurological symptoms occurred.The skin was scratched and bitten,and all died within 6 days.PCR was performed on rabbit tissues to amplify bands that were consistent with the size of the rabbit.Anatomical examination revealed meningeal hyperemia with a large amount of cerebrospinal fluid;heart congestion,a large effusion in the pericardium;and congestion and edema of the lungs.There were no obvious pathological changes in other organs.Microscopic examination revealed inflammatory cell infiltration in the lungs,congested capillaries in the alveolar walls;degeneration of the myocardium,widening of the stroma,and extensive inflammatory cell infiltration;narrowing of lymphoid follicles in the spleen,widening of the red pulp stroma,and localized red pulp stroma More fibroblasts are seen inside.Through IFA identification of PRV-infected cells and challenged rabbits,it was found that red fluorescence appeared in the cytoplasm and nucleus of the virus-inoculated BHK-21 cells and in the challenged rabbit brain tissue,which further confirmed that the isolated strain was Porcine pseudorabies virus.After isolation of PRV,the isolate gE gene was cloned and sequenced,and variability analysis,homology analysis,and phylogenetic tree analysis were performed.Through the analysis of the variability,it was determined that the isolate was a mutant strain;by homology analysis,it was found that the nucleotide and amino acid homology of the gE gene of the HeN1 strain isolated from the isolate was the highest,which was 99.9% and 99.8%,respectively.According to evolutionary tree analysis,the gE gene is mainly divided into two major branches,and the isolate has the closest genetic relationship with HeN1 strain isolated from Henan.This study has enriched the genetic evolution of PRV variants and laid a solid foundation for the research and prevention and control of PRV.