Construction And Biological Characteristics Of Duck Plague Virus US10-deletion Strain

Duck plague virus US10 gene encodes a tegument protein,we have investigated the function of US10 by generating deletion and revertant mutants,the main results are described as follows:1.The generation of DPV US10 deletion and revertant mutantsDPV CHv-BAC-G-?US10 and DPV CHv-BAC-G-US10FRT were generated by reverse genetics system based on BAC.PCR,sequencing and WB were performed and the results showed that the deletion and revertant mutants were generated successfully.2.Replication characteristics of recombinant viruses in DEFsIn multistep viral replication kinetics analyse,at 48 h.p.i,titer of DPV CHv-BAC-G and DPV CHv-BAC-G-US10FRT were 10^6.3 and10^6.18 TCID₅₀/0.1mL respectively,titer of DPV CHv-BAC-G-?US10 was 10^4.39 TCID₅₀/0.1mL,which was 100-fold lower than those in DPV CHv-BAC-G and DPV CHv-BAC-G-US10FRT.Genome copies of DPV CHv-BAC-G and DPV CHv-BAC-G-US10FRT were 10⁷.73.73 and 10^7.28 copies/0.1mL respectively,copies of DPV CHv-BAC-G-?US10 was 10^6.91 copies/0.1mL,which was 4-fold lower than those in DPV CHv-BAC-G and DPV CHv-BAC-G-US10FRT.In one-step viral replication kinetics analyse,at 24 h.p.i,titer of A to D groups were10^6.17,10^4.70,10^5.72 and 10⁵.03.03 TCID₅₀/0.1mL.Copies were 10^8.20,10^7.77,10⁷.67.67 and 10^8.06copies/0.1mL.These results indicated that deletion of US10 caused no impact on genome copies but strongly impaired the viral titer.3.The role of US10 in viral cell-to-cell spreadThe results detected by plaque assays showed that DPV CHv-BAC-G-?US10 and DPV CHv-BAC-G formed plaque in similar size,indicating that the deletion of the US10 gene cause no impact on cell-to-cell spread.4.The impact of US10 on immune-related genes transcription in DEFsDEFs in 12-well plates were infected with DPV CHv-BAC-G,DPV CHv-BAC-G-?US10 or mock infected at a MOI of 2.At the early stage of infection,transcription level of IL-4,IL-6,IL-10,Mx and OASL in DPV CHv-BAC-G-?US10 infected DEFs were significantly upregulated compared to those in DPV CHv-BAC-G infected DEFs.To summarize,DPV US10 is non-essencial in viral replication,it plays a role in virus assembly and maturation.Transcription level of IL-4,IL-6,IL-10,Mx and OASL in DPV CHv-BAC-G-?US10 infected DEFs were significantly upregulated.