Inhibition Of The Reactivation Of Pseudorabies Virus By Using The CRISPR/Cas9 System

Pseudorabies(PR)is an acute infectious disease,characterized by fever,itching,encephalomyelitis,respiratory and nervous system disorders in many livestock and wild animals.PRV is a neurotropic virus that can infect pigs and establish latent infection in trigeminal ganglions of the host.Pseudorabies vaccines have played an important role in the prevention and control of the disease,but the vaccines cannot prevent the establishment of latent infection and reactivation.Pigs in the state of latent infection cannot be diagnosed by serological methods.The latent PRV can be reactivated under certain conditions,which will cause virus shedding and recurrence of the disease.Therefore,it is useful to develop a method to clear the latent PRV or block the reactivation of latent PRV.Firstly,we constructed a recombinant reporter virus rPRVTJ-US9-EGFP by homologous recombination,which identified by fluorescent,PCR and one-step growth curve.These results showed that rPRVTJ-US9-EGFP can express the green fluorescent protein stably,and the growth of rPRVTJ-US9-EGFP was similar to that of PRV TJ strain.We got sensory neurons from dorsal root ganglions(DRGs)of newborn mice,and identified it with the ??-tubulin antibody by indirect immunofluorescence.To purify sensory neurons,we inhibited the growth of non-neuron cells by cytosine arabinoside(Ara-C),and the concentration was screened.It turned out 5?M Ara-C is the most suitable concentration.At the same time,the toxicity of acyclovir(ACV)to nerve cells was studied,and the results showed that 0.1~0.7 mM ACV had no significant effect on the growth of neural cells.We established a model of PRV latent infection in sensory neurons,3 kinds of multiplicity of infection and 5 kinds of ACV treatment concentrations were tested with a checkerboard titration method.The parameters of latent infection were as followed: 0.001 MOI of rPRVTJ-US9-EGFP,processing 0.5 mM ACV for 7 days.Subsequently,we collected PRV latent and lytic samples,the copy numbers of virus genome,the virus titer in supernatant and the expression of virus genes were identified.The results showed that there were virus genome in the cell when the fluorescence disappeared,but the virus could not replicate,did not produce infectious virus particles,it was really in the latency.The latent PRV can be reactivated when stimulating with 10 ?M LY294002.Then we selected the genes UL9,UL42,UL54 and VP16 that have an effect on PRV replication and reactivation.Eukaryotic expression vectors containing target gene were constructed,and the editing effect of sgRNA was initially screened by protein expression.The results showed that UL9-sgRNA1,UL9-sgRNA3,UL54-sgRNA1,UL54-sgRNA2 and UL54-sgRNA3 could significantly reduce the protein expression of target gene.Then we constructed a series of recombinant lentiviruses containing Cas9 components and sgRNAs targeting genes affecting the reactivation of PRV.Finally,the effects of the recombinant lentiviruses inhibiting the reactivation of PRV were evaluated in the latent infection model.The recombinant lentiviruses containing Cas9 components and sgRNAs targeting UL9 gene effectively inhibited the reactivation of PRV.This study lays a foundation for the development of a pseudorabies vaccine that possesses the ability to clear or block latent PRV and will also provide reference for the study of latent infection of other herpesviruses.