Differences In Nerve Conduction And Virulence Between Variant And Classical Pseudorabies Virus Strains

Pseudorabies(PR),caused by pseudorabies virus(PRV),is an acute infectious animal disease.The genome of PRV is about 143 kb,which is difficult to be edited.In this study,the fosmid library of the PRV SC strain was constructed successfully,which contains 200 recombinant plasmids and covered the complete genome sequence of the PRV SC strain.Five combinations containing thirteen plasmids were selected and the recombinant viruses were rescued efficiently within 5～7 days.All the five recombinant viruses shared the same biological characteristics in vitro as parental PRV.Based on the fosmid library and the homologous recombination technology mediated by Red/ET,the PRV SC genome can be edited accurately.A recombinant virus rPRVSC-UL6-EGFP with the EGFP reporter gene inserted into the N-terminal of the UL36 gene of PRV SC was constructed by using Red/ET technology and fosmid library.There was no obvious difference between rPRVSC-UL6-EGFP and PRV SC in the biological characteristics and pathogenicity in mice.The recombinant virus can be used to trace the stages of invasion,transport,replication and release of a single PRV particle in the neuron effectively.Compared with the classical strain PRV SC,the variant strain PRV TJ is more pathogenic to mice with lower LD50 and more severe itching symptoms.It is confirmed that the invasion of PRV TJ into N2a cells was significantly higher than that of PRV SC.There were multiple amino acids differences in the gB,gC and gD proteins related to PRV invasion between the two strains,the fosmid library of PRV TJ and PRV SC strains were used to construct a series of gB,gC and gD gene replaced recombinant viruses.The recombinant viruses were compared in the adsorption and internalization efficiency on the N2a cells with parental viruses.As a result,the variations in the gB,gC,and gD proteins all improved the adsorption efficiency of PRV TJ.Moreover,the mutation in the gB and gD proteins caused the internalization efficiency of PRV TJ.Among them,the gD protein played a key role in internalization efficiency.gC did not participate in the internalization process directly,but affected the internalization efficiency.Finally,the pathogenicity of gB-,gC-,and gD-interchanged chimeric viruses was evaluated in mice.It was confirmed that the 50%lethal doses of recombinant TJ strain with the gB,gC and gD proteins of the PRV SC was consistent with the parental virus PRV SC,meanwhile,the LD50 of SC strain with the gB,gC and gD proteins of TJ was consistent with that of the parental virus PRV TJ,and the pruritus symptoms of mice changed correspondingly,these indicating that the mutation of the invasion-related protein changed the virulence of PRV.In summary,the PRV SC fosmid library constructed in this study can quickly and efficiently complete the construction of PRV recombinant virus;Microfluidic was used to culture primary neural cells,and show the process of PRV single virus particles moving in the axon,which provided technical support for study on neurotropism,nerve conduction of different PRV strains,and neuron axon transport mechanism.Finally,gB,gC and gD proteins of TJ displayed enhanced internalization to N2a cells and pathogenicity to mice,and gD played the mainly role in improving entry,which provided new ideas for PRV vaccines.