1.Construction And Application Of A Pseudorabies Virus Fosmid Library 2.Screening And Identification Of Interferon-stimulated Genes That Regulate The Replication Of Pseudorabies Virus

Conventional genetic engineering of pseudorabies virus(PRV)is essentially based on homologous recombination or bacterial artificial chromosome.However,these techniques require multiple plaque purification,which is labor-intensive and time-consuming.The aim of the present study was to develop an efficient,direct and flexible genetic manipulation platform for PRV.To this end,the PRV genomic DNA was extracted from purified PRV virions and sheared into approximately 30-45-kb DNA fragments.After end-blunting and phosphorylation,the DNA fragments were separated by pulsed field gel electrophoresis,the recovered DNA fragments were inserted into the cloning-ready fosmids.The fosmids were then transformed into Escherichia coli and selected clones were end-sequenced for full-length genome assembly.Overlapping fosmid combinations that cover the complete genome of PRV were directly transfected into Vero cells and PRV was rescued.The morphology and one-step growth curve of the rescued virus were indistinguishable from those of the parent virus.VP26 is one of the first herpesvirus proteins to be fused with a fluorescent protein.Capsid-tagged virus mutants have been used to study capsid transport,intra-nuclear capsid dynamics,and nuclear egress.Therefore,in this study,the EGFP gene was inserted between the second and third codons of the VP26 gene by Counter Selection BAC Modification Kit according to the manufacturer's instructions.In the first step,the Red/ET expression plasmid(pRed/ET)and the fosmid were co-transformed into competent E.coli DH10B cells by electroporation.In the next step,the antibiotic selectable cassette(rpsL-neo)flanked by the homology arms was generated by PCR amplification with specific primers in Table 1 and inserted into the target site of the fosmid by the Red/ET-mediated recombination.To fuse the EGFP gene with the VP26 gene,the electro-component cells were prepared from the cells containing modified fosmid carrying a rpsL-neo cassette.In advance,the linear DNA fragment of the EGFP gene with homology arms was amplified with specific primers.The EGFP gene flanked by two oligonucleotide homology arms was transformed into the prepared electro-component cells to replace the rpsL-neo cassette by the Red/ET-mediated recombination.The modified VP26 ORFs were amplified and sequenced.Finally,the modified fosmid plus the other fosmids were transfected into Vero cells to rescue the virus.The recombinant virus expressing the VP26-EGFP fusion protein was rescued and named as rPRV-VP26-EGFP.The soma side of the microfluidic device was infected with the rPRV-VP26-EGFP,and the EGFP signals were imaged at 12 hpi,the EGFP-tagged capsid transported from the soma side to the axons that in the connecting region of soma and axonal side.The results indicate that rPRV-VP26-EGFP allows visualization of the EGFP-tagged capsid transport between neuron bodies and axons.Interferon-stimulated genes(ISGs)play critical roles in defense and clearance of exogenous pathogens.In this study,we screened the potential anti-PRV ISGs,and successfully screened 5 ISGs that significantly inhibited the of replication report virus,we demonstrated that ectopically expressed IFIT3 could restrict the replication of PRV.Further study showed that IFIT3 was up-regulated at the transcription level in PRV-infected PAM Cells and PRV-infected pigs.In summary,in this study,we constructed the PRV fosmid libiary.Based on this libiary,a recombinant PRV expressing EGFP fused with the VP26 gene was generated,and this recombinant virus can be used to observe the capsid transport in axons.In addition,the potential anti-PRV ISGs were screened by high content screening system.These studies will lay a foundation for the pathogenesis and vaccine research of PRV variant strains,and provide new ideas and means for PR control.