Isolation,Identification And Biological Characteristics Analysis Of Pseudorabies Virus HB1 Strain Isolated From Fox

Pseudorabies?PR?is an acute infectious disease,caused by pseudorabies virus?PRV?,in varieties of livestock and wild animals.PRV is taxonomically classified in species Suid herpesvirus 1?SuHV-1?,the genus Varicellovirus,family Herpesviridae.PRV have a very broad host infection spectrum,and can infect the vast majority of mammal animals,including pigs,cattle,sheep,dogs,cats,rabbits,foxes,mink,raccoon and other animals.The new-emerging PRV mutant strains have been widely prevalent in the central and eastern regions of China since 2011,bringing huge economic losses to China's breeding and livestock industry.PRV is one of the most important viral pathogens that affecting the China's livestock industry.The main clinical symptoms of PR in fox are characterized by symptoms of the nervous system and respiratory symptoms.Currently there is no fur-specific PRV vaccine,and its prevention and control situation is serious.In our research,we investigated the new epidemic PRV mutant strain isolated from fox,and studied the biological characteristics of the isolate.Our research revealed the occurrence and epidemic characteristics of PR in fox,laying a foundation for its effective prevention and control.In this research,we collected samples of death foxes,which were suspected to be infected with PRV in Hebei region.The virus DNA was extracted after each tissue sample was homogenized for the PCR detection of PRV,and the PCR results were positive for PRV.The PK15 cell was inoculated with the tissue homogenate for PRV proliferation,and the CPE caused by PRV were observed in PK15 cells.By plaque purification,we obtained the PRV strain isolated from fox.The indirect immunofluorescence identification?IFA?showed that the PRV isolate could react specifically with the positive sera of PRV Bartha K-61 strain and PRV HeN1 strain to produce specific fluorescence,and the isolate was named PRV HB1 strain.The HB1 virus particles is approximately spheroidal and had a diameter of about150-180 nm under the transmission electron microscopy,which was consistent with the morphology of the herpes virus.The HB1 isolate can infect PK15 cell and other 12 kinds of cells,and the CPE and proliferation characteristics of HB1 isolate on different cell is different.The titer of HB1 strain on PK15 cell was10^7.67 TCID₅₀/mL by micromethod.One step growth curve showed that HB1 strain had similar growth kinetics with Bartha K-61 strain and SC strain,and the HB1 strain had the highest titer on PK15 cell.The neutralization test showed that the maximum dilution of positive sera against Bartha K-61 strain and HeN1 strain to neutralize HB1 strain were 8 times and 32 times respectively.The antiserum of the HeN1 mutant was able to completely neutralize the HB1 strain,but the antiserum of the Bartha K-61vaccine strain did not completely neutralize the HB1 strain.A total of 52 genes were obtained by sequencing the genome of HB1 with 96 pairs of primers,and the phylogenetic trees were constructed based on the 52 genes.The phylogenetic tree analysis showed that,HB1 is belong to the PRV gene type?.The gE gene of HB1 have the lowest homology,97.9%,with Becker and Kaplan strains,but the homology up to 99%with domestic isolates,such as HBCL and TJ strains.Compared with the reference strains in Europe and America,the amino acid sequences encoded by gE gene of HB1 had 19substitutions and 2 insertions.The gC gene of HB1 have the lowest homology,96.2%,with Bartha,Becker,Kaplan and SC strains,but the homology up to 99.5%with HBCL,HLJ8 and JS-2012 strains.Compared with the reference strains in Europe and America,the amino acid sequences encoded by gC gene of HB1 had 27 substitutions,and a continuous 7 amino acid insertion of AAASTPA in position of63 to 69,which were consistent with the PRV mutant strains prevalent in the past five years in China.BALB/c mice were inoculated with 10^4.67,10^5.67,10^6.67,and 10^7.67 TCID₅₀/mL HB1 isolate respectively,and mice can show itching and dyspnea and other typical clinical symptoms and oligodendrocytes hyperplasia in brain tissue and alveolar wall thickening and other pathological changes.10^7.67 TCID₅₀/mL infected group,5/5 mice died.10^6.67 TCID₅₀/mL infection group,4/5 mice died.10^5.67 TCID₅₀/mL infection group,3/5 mice died.10^7.67 TCID₅₀/mL infection Group,5 mice were not dead.The LD₅₀ of the HB1 isolate to Balb/C mice was 1×10^5.02 TCID₅₀/mL calculated according to the Reed-Muench method.