Isolation And Identification Of Pseudorabies Virus DL14/08 Strain From Mink And Immunogenicity Research

Pseudorabies virus（PRV）,a member of the family Herpesviridae,subfamily Alphaherpesvirinae,and genus Varicellovirus,is more commonly known as pseudorabies.The incidence of PRV infection among vaccinated pigs has been increasing on farms in northern China.This trend has been evident in thirteen provinces and municipalities,including Liaoning Province.An outbreak of severe PRV infection in farmed mink occurred in northern China in late years,causing significant economic losses in the local fur industry.There are no effective prevention and vaccines for fur animal,so the study on the vaccine of the fur-bearing animal against PR current epidemic strains is of great significance.We collected suspected samples of minks and foxes infected by PRV from Shandong,Hebei,and the three provinces in Northeast China from 2012 to 2014.The mink origin PRV strain named DL14/08was identified by PCR,proliferation,IFA,electron microscopy,and artificial replication experiment.We amplified the complete sequences of the PRV isolates from Liaoning Province using the Illumina HiSeq 4000 platform.The gene sequence of the DL14/08 strain was submitted to GenBank（accession no.KU360259）.The complete genome sequence of the DL14/08 strain was used for sequence alignments and phylogenetic analysis and was compared with other strains for which sequences are available in the GenBank database（e.g.,accession numbers KM061380,JF797217）.The genome was143,642 bp long,and the sequences were run far with 67%⁸2%similarity against vaccine strains,but had a highly similarity with 97%¹00%on JS-2012,ZJ01,HeN1,HNB,TJ strains.This study provide a theoretical basis for the mink origin PR genetic characteristics and swines PR and genetic relationships.In this study,we obtained a PRV from brain of the affected mink,and the inactivated vaccine was prepared with the virus in a titer of 10^7.10TCID₅₀/mL by the conventional methods consulted’The New Veterinary Biological Products Quality Standards’.The vaccine was inactivated by 0.2%formaldehyde solution,shaken 7 days with 5%Aluminum hydroxide gel adjuvants in 4℃.When the physical properties test,sterility test,and inactivation test results met the inactivated vaccine standard respectively,the safety inspections examined on minks and rabbits.Continuous monitoring of laboratory animals,and obtained safety mink origin PR inactivated vaccine.In order to compare inactived vaccine and commodity vaccine we designed three experiments:The minimum immune dose and immune effect of the inactivated vaccine were detected in mink and rabbit.The results showed that the MID is 5×10^6.10 TCID₅₀/mL and 5×10^6.10 TCID₅₀/mL in mink and rabbit respectively.Inactived vaccine and commodity vaccine of PRV was inoculated in healthy mink.The protective immunity test in mink show titer of inactived vaccine and commodity vaccine of PRV neutralizing antibody reached an average of 1:516 and 1:348 the protection rate reached 100%and 80%in mink when been attacked with 10 LD₅₀ PRV-DL14/10 strain in 21 days after the immunization.The results indicated that inactive vaccine antibody titer and protection rate were higher than the commodity vaccine.PRV-DL/14 strain inactivation by formalin and preparation inactivated vaccine could protect minks against the challenge of PRV virulent strain infection,it could be develop a novel vaccine candidate of PRV.