| Pseudorabies(PR)is an infectious disease that can cause a variety of animals disease,pigs are the only natural host.Porcine pseudorabies can cause boars-reproductive dysfunction,Breeding pigs of miscarriage,newborn piglets with neurological symptoms and high mortality.Once infected with the disease,pigs are carrying the virus lifelong.Since 2012,A number of domestic pig farms those using Bartha-K61 immunized vaccine,have erupted PRV.To investigate the prevalence and genetic variation of PRV in Guangxi province,enriching the complete sequence genome-database of PRV and exploring the differences in pathogenicity from biological characteristics,this thesis offers the experimental conclusion for the Prevention and controlling of the disease,and helps to develop more effective and safer vaccine.We have isolated 34 strains pseudorabies virus since 2013 to 2016.By analyzing the five virulence genes of PRV,we found one PRV isolated which is from Laibin named GXLB-2015 strains that five virulence genes were in two different branches of genetic evolution.And a PRV deletion of a TK gene were found in one PRV isolated from Guigang named GXGG-2016 strains.In this study,PRV GXLB-2015 and GXGG-2016 strains were analyzed by complete genome sequencing.The sequence of the PRV genome was compared with the published genome in NCBI and analyzed with Simplot program.The results showed that GXLB-2015 had full length of 143656bp,and were the homologous recombination of the foreign vaccine strains Bartha and the domestic mutants.The full length of GXGG-2016 was 142264bp,and 69 amino acids were deleted on the TK gene.The deletion site was consistent with the artificial deletion site of HB-98.In the genetic evolution tree,GXLB-2015 is located among the foreign strains and domestic mutants,GXGG-2016 is located in the traditional branch of the domestic.Compared with foreign and domestic strains,The GXLB-2015 and GXGG-2016 Nucleotides homology was 95.48-98.83%and 94.76-99.54%respectively.GXLB-2015 are highly consistent with Bartha in the UL region.However,the US gene of the virus is consistent with the domestic mutant strain,and in the same genetic phylogenetic tree branch.GXGG-2016 in the whole sequence genes are consistent with the domestic traditional strains.In order to analyse the biological characteristics of the two PRV with others,the mutant strain GXNN-2014 and the traditional strain GXLB-2013 were compared with the vaccine Bartha and HB-98.The Multi-step growth curve showed that the virus could be detected after 4h,and the virus titer peak reached after 48h,GXGG-2016 were detected virus titer increasing 10 times faster than others.Plaque experiments showed that the plaques produced by different PRV were significantly different.The mutant strain GXNN-2014 strain produced the largest plaque,followed by the traditional strain GXLB-2013 and GXLB-2015,Indicating that the mutant strain's infection capacity was the strongest,and GXGG-2016 with TK deleted was significantly smaller than other strains.The results of pathogenicity test in mice showed that GXLB-2015 had the strongest pathogenicity to mice with LD50 was 103.5TCID50,and the mutant GXN-2014 was the second,while GXGG-2016 had no pathogenicity and no obvious neurological symptoms.Detection of PRV in brain tissue of mice,PRV was only detectable from the brain tissue of diseased mice.The results showed that the recombinant strain GXLB-2015 had the strongest pathogenicity and the most serious symptoms.While the GXGG-2016 with 69 amino acids deletion is not pathogenic to mice.This study enriches the domestic database of PRV virus,which provides an important scientific basis for our understanding and controlling of PRV. |
PDF Full Text Request