Construction Of Type ? Bovine Herpes Virus Expressing Rabies Virus G Protein Based On CRISPR-Cas9 Technology

Rabies virus(RABV)is one of zoonotic diseases,with approximately 59,000 deaths from rabies reported each year.At present,stray dogs have become one of the main transmission routes of rabies virus(RABV).Wild carnivores such as bats,foxes,wolves,ferret badgers and other wild carnivores are the natural reservoirs of RABV.These rabies virus-carrying animals pose potential public health risks to humans and domestic animals.In order to control the spread of the virus from the source,these wild animals need to be vaccinated.However,most of the current rabies vaccination uses intramuscular injections.This type of vaccination is costly and has not been widely used in many developing countries.On the other hand,the current rabies vaccine immunization titer still needs to be improved.Therefore,it is necessary to develop a safe,effective and cost-effective rabies vaccine.Bovine herpes virus type ?(BHV-1)is a double-stranded DNA virus,in which g E glycoprotein is a non-essential protein for viral replication,and deletion of g E gene does not affect viral replication and immunogenicity.At present,BHV-1 vaccine with g E gene deletion has been widely used in Europe.Therefore,BHV-1 with g E gene deletion is safe to be used as the vector to construct recombinant rabies virus.Based on CRISPR-Cas9 and homologous recombination technology,this study used Bovine herpes virus type ?(BHV-1)as vector.Recombinant virus(BHV-1?g E/EGFP~+/G~+)with deletion of g E gene and expression of enhanced green fluorescent protein gene(EGFP)and G gene of rabies virus was constructed.The EGFP gene in the BHV-1?g E/EGFP~+/G~+viral genome was knocked out by small guide RNA(sg RNA),and the recombinant virus BHV-1 was finally obtained.Sequencing and Western blot results showed that the recombinant virus was successfully constructed and could stably express the G protein of rabies virus.In vitro biological characteristics analysis showed that the growth characteristics of BHV-1?g E/G~+and BHV-1 were similar.However,in the comparison of plaque morphology,the plaque of BHV-1?g E/G~+was significantly larger than that of BHV-1,and the apoptosis experiment proved that the recombinant virus BHV-1?g E/G~+could promote cell apoptosis.Flow cytometry was used to detect the changes of the number of DC and B cells in the submandibular lymph nodes of mice after oral administration of the recombinant virus.The results showed that the number of DC and B cells increased,and the recombinant virus could produce a good immune response.Finally,the mice were immunized with the recombinant virus BHV-1?g E/G~+by intramuscular injection and oral administration,and then challenged with the(CVS-11)strain.The results showed that the survival rate of mice by intramuscular inoculation and oral inoculation was as high as 71%,and the recombinant virus could produce effective neutralizing antibodies by oral inoculation.The above research results show that the successfully constructed recombinant virus BHV-1?g E/G~+is an excellent candidate for a new generation of oral recombinant rabies vaccine for humans and animals,and BHV-1 is an ideal carrier for oral vaccines.