Visualization Of The Replication And Spread Of Pseudorabies Virus In A Mouse Model

Pseudorabies（PR）is a devastating infectious disease of pigs caused by pseudorabies virus（PRV）.PRV can infect many species of livestock and wildlife.The natural host of PRV is pigs.PR has been well controlled owing to the utilization of the gE-deleted vaccines,even eradicated in America and partial European countries.Since 2011,PR outbreaks have occurred in many Bartha-K61-vaccinated pig farms in China.Previous studies indicated that the epidemic strains exhibited increased pathogenicity in pigs and mice.The phylogenetic analysis indicated that the PRV epidemic and classical strains belong to different branches.The new epidemic PRV strains were named as PRV variants.However,it is not clear about the mechanism of enhanced pathogenicity for the PRV.The emergence of variant PRV strains necessitates the ability to rapidly assess their pathogenicity.Herein,we constructed a recombinant PRV（rPRVTJ-NLuc）expressing NanoLuc luciferase（NLuc）by homologous recombination.The red fluorescent protein DsRed is fusion expressing with NLuc for screening and purification of the recombinant PRV.rPRVTJ-NLuc was passaged in PK-15 cells more than 20 generations and showed genetics stability.There was no significance difference in the replication between rPRVTJ-NLuc and the parent strain PRVTJ.rPRVTJ-NLuc can express robust luminescence stably in PK-15 cells,and there is a positive correlation between luminescence activity and virus titer.When mice infected with PRVTJ and rPRVTJ-NLuc,no significant difference was found in the clinical signs,weight changes,survival rates,pathological changes and viral tissue distribution between the mice infected with PRVTJ or rPRVTJ-NLuc.These results indicate that the insertion of the reporter genes has not influenced the pathogenicity of PRV in mice.To visualize the replication and spread of PRV,mice were infected with 10^3.0 TCID₅₀rPRVTJ-NLuc and in vivo imaging was performed daily.The bioluminescent signal was observed as early as 4 days post-infection（dpi）.The signal was observed at the site of the spinal cord at 6 dpi.The luminescence signal of the mice increased firstly and then declined,which indicates the initiation of PRV infection and final clearance.We confirmed the source of the signal by imaging of excised organs finally.In conclusion,we visualized the replication and spread of PRV by in vivo imaging with a recombinant PRV stably expressing NLuc luciferase.This study will provide new methods for the study of pathogenicity of PRV,assessment the efficacy of PRV vaccine candidates and new antiviral agents.