Isolation,identification And Biological Characteristics Study Of Pseudorabies Virus Henan Isolates

Pseudorabies is an acute infectious disease caused by Pseudorabies virus(PRV),with fever,tickle(except pig)and encephalomyelitis of varieties of livestock,poultry and wild animals as main symptoms.Pigs are the natural host and the main infectious source f of the disease.Different ages of pigs can be infected by PRV,which main causes pregnancy sow's abortion,stillbirth and mummy foetus;Lactation piglet neurological symptoms,failure of death,and the death rate is almost as high as 100%.Since Liu Yongchun isolate PRV from pig in 1947,the infection of PRV had been reported in more than 20 provinces and cities in our country.In practical manufacture,to prevention of PR,it mainly relies on vaccines especially genetic deficiency engineering vaccine,to make sure that the disease effectively controled.However,since the spring of 2012,in many areas such as Henan,Jilin,Heilongjiang provinces where suspected PR occurred in piggeries which pigs were vaccinated by PRV genetic deficiency engineering vaccine.Nine epidemic strains of PRV in Henan province were identified in this study,the whole genome of gE,gC were sequenced,the related biological characteristics were identified,and the immunogenicity of the isolates of NY was explored.Polymerase Chain Reaction(PCR)was used for detection of suspected PRV epidemic materials acquired from different cities in Henan province,9 strains of the virus were successfully isolated by inoculating the positive pathologic material on the swine testicular(ST)cells,which respectively named NY,ZK,M5,JY,GY,MZ1,MZ2,LGX,ZM.Stable CPE appear relatively on ST cells with the nine strains' proliferation,and the specificity of gE gene(429 bp)can be amplified.The virulence titer of the strains basically reached to 10-5.6-10-9 at seventh generation.The 9 isolates were highly pathogenic in mice,causing skin inflammation and leading to death in all experimentally-infected mice at 48-120 h after challenge.According to NY strain,it found that the strain is sensitive to chloroform and trypsin;can tolerat acid,alkali,hot;and is not sensitive to ultraviolet.The virus was observed to be visible round with a diameter of 110-150 nm by electron microscopy,posterior capsule has radiate out in the surface of the fiber.All these confirmed that the nine strains of virus were PRV wild strains.Reference the gE gene sequence(AF171937)published in GenBank,one pair of primers were designed by Primer5.0,gE genes of the nine strains PRV wild strains were amplificated and determined respectively,the sequences and the derived amino acid sequences were analysised.The results show that the nucleotide homology between the nine strains 99.9%?99.9%,and the the nine strains have close kinship with the domestic isolates which were identified In recent years;The amino acid sequences derived from the gE genes were all inserted a aspartic acid at 48th and 494th locus,amino acid substitutions occurred in 448th and 510th locus(V448I,G510S),amino acid substitutions(T385M)only occur in NY strain,while amino acid substitutions(S530G)occur only in ZK strain.Reference the gC gene sequence published in GenBank,one pair of primers were designed by Primer5.0,gC genes of the nine strains PRV wild strains were amplificated and determined respectively,the sequences and the derived amino acid sequences were analysised.The results show that the nucleotide homology between the nine strains is 99.9%?100%,and after the 63th locus of the amino acid sequences seven amino acids(AASTPA)successive were inserted or deleted.NY isolate of pseudorabies virus was proliferated on ST cells,and the culture was inactivated with formaldehyde.Freund's adjuvant inactivated oil emulsion was prepared by mixing inactivated PRV NY with Freund's adjuvant.Female Kunming mice of 6 weeks were immunized by NY strains of inactivated oil emulsion,Bartha-K61 vaccine and Hubei 98 vaccine respectively.All groups were vaccinated intramuscularly,with a 2-week interval.After 28 days,the blood sera were collected for serum neutralization test,and mice were challenged with PRV NY isolate.The results showed that mice vaccinated with NY inactivated oil emulsion were fully protected,while mice vaccinated with Bartha-K61 and Hubei 98 were partially protected.Bartha-K61 and Hubei 98 have certain crossing antigens with PRV NY strain,but the antigen has lot of differences.