Construction Of A US7/US8/UL23/US3-deleted Recombinant Pseudorabies Virus And Evaluation Of Its Pathogenicity In Dogs

Pseudorabies?PR?is an acute infectious disease caused by pseudorabies virus?PRV?.Pigs are the main nature host of PRV,immunization with attenuated vaccines is one of the effective ways to prevent and control pseudorabies.The US7/US8-deleted and US7/US8/UL23-deleted attenuated vaccines have been widely used in the prevention and control of pseudorabies in pigs and achieved good immune effects.In recent years,it has been reported in Shandong,Hebei,Liaoning and Beijing that dogs,minks and other fur animals have been infected with PRV through contact with raw meat or offal of pigs inoculated with the attenuated vaccine.The dogs developed symptoms including severe neurological disorders,high fever,respiratory and digestive disorders.After inoculation with the above attenuated vaccines,it was found that the attenuated vaccines could spread horizontally in dogs and cause disease.Although the PRV attenuated vaccines is safe for pigs,but still highly pathogenic to dogs and other fur animals.At present,the widely used PRV vaccine has safety problems for dogs,which needs to be further improved to reduce its virulence.US3 gene is another important virulence gene of herpes virus,which also plays a key role in regulating cell apoptosis and host immune response.In this study,r PRV^{del US7/US8/UL23} was used as the parent strain to construct the US7/US8/UL23/US3-deleted using CRISPR/Cas9 technology.The pathogenicity of rPRV^{del US7/US8/UL23}and US7/US8/UL23/US3-deleted recombinant PRV strains were evaluated in dogs.In order to explore its safety to dogs,so as to provide a safe and effective vaccine for the prevention and control of pseudorabiesIn this study,PRV US3 gene was firstly cloned into a prokaryotic expression vector,and t he prokaryotic expression was followed by the preparation of US3 antiserum in immunized mice.Using CRISPR/Cas9 method to design a pair of sgRNAs,cloned it into the expression vector and transfection with r PRV^{del US7/US8/UL23} genome for 48 hours can see the typical cytopathic effect.After 72 hours,the supernatant of transfected cells was harvested and inoculate pk-15 cells to plaque purification and screening.Plaque was picked up under a fluorescence microscope.After 10 rounds of purification,PCR and Western blotting were used for identification,the obtained virus was named r PRV^{del US7/US8/UL23/US3}.The recombinant virus is genetically stable,and there are no missing back mutations.The growth capacity of r PRV^{del US7/US8/UL23/US3} was evaluated by replication kinetics test and cytopathic test.Compared with wild type and r PRV^{del US7/US8/UL23},r PRV^delUS7/US8/UL23/US3elUS7/US8/UL23/US3 has lower titer,but it still has good replication ability,and no cell fusion was observed after infection.Pathogenicity analysis by clinical observation,pathological changes and tissue viral load.Under the same challenge dose?2×10⁴?,r PRV^{del US7/US8/UL23} was lethal to dogs,while the r PRV^{del US7/US8/UL23/US3} challenged dogs were living,body temperature was normal,clinical symptoms were not obvious,viral load in each tissue was very low,and pathological changes were not significant.All these data demonstrated that,compared with wild type and r PRV^{delUS7/US8/UL23},the pathogenicity of rPRV^{del US7/US8/UL23/US3} was significantly reduced.This study will reveal for the first time that US3 gene is the virulence gene of PRV vaccine strain infection after infection in dogs,it provides a theoretical basis for PRV vaccine to further improve.