The Synergistic Replication Mechanism Of Marek's Disease Virus And Reticuloendotheliosis Virus

Viral co-infection is a common phenomenon in nature and associated with more severe clinical consequences.Marek's disease virus(MDV)and reticuloendotheliosis virus(REV)cause Marek's disease(MD)and reticuloendotheliosis(RE),respectively.Co-infection with MDV and REV is common in chickens,causing synergistic pathogenic effects and serious losses to the poultry industry.However,whether there is synergism between the two viruses in viral replication and the roles of host factors in regulating MDV and REV co-infection remain elusive.It has been reported that the protective efficacy of existing MD vaccines was attenuated by co-infection of MDV and REV.Recombinant MDV increased horizontal transmission due to REV long terminal repeat.However,whether there is a synergism between the two viruses in viral replication and the roles of host factors in regulating MDV and REV coinfection remains elusive.The outcome of co-infection usually is viral interference.Besides interference,co-infections of viruses may also enhance viral replication and virulence.To verify the synergistic replication between MDV and REV,an in vitro co-infection model was established.The results of immunofluorescence showed that the two viruses could locate the same CEF cell.The detection of virus growth curve showed that the replication of MDV and REV was enhanced and the transcription level and protein expression level of MDV and REV were significantly increased in co-infected cells.The fold change between coinfection and single-infection of MDV and REV reached the peak at 48 hpi.In order to further verify the co-replication of MDV and REV in vivo,animal models of co-infection were established.The results showed that the mortality and tumor incidence of MDV and REVcoinfected chicken were higher than that of single infected chicken.Severe growth inhibition and obvious tissue lesions were occurred in coinfected chicken.The viral load of coinfected chickens was significantly higher than that of single infected chicken.These results indicated that MDV and REV replicated synergistically in vitro and in vivo.To further study the host factors and molecular mechanisms involved in replication and pathogenicity of MDV and REV under co-infection,we explore the host cell responses to MDV and REV coinfection using tandem mass tag(TMT)peptide labeling coupled with liquid chromatography-tandem mass spectrometry(LC-MS/MS).Compared with MDV/REV-infected cells,38 proteins increased(fold change>1.2)and 60 decreased(fold change<0.83)their abundance in MDV and REV coinfected cells.Differentially accumulated proteins(DAPs)were involved in important biological processes involved in the immune system process,cell adhesion and migration,cellular processes,and multicellular organismal systems,PPAR pathway.STRING analysis found that Akt and IRF7 may be associated with MDV and REV synergistic replication in chicken embryo fibroblasts.DNA and RNA viruses usually activate intracellular PI3K/Akt signaling to promote viral infection and replication.To verify whether the Akt pathway mediates the synergistic replication of MDV and REV,CEFs were transfected with expression plasmid or shRNA of Akt.The results showed that overexpression of Akt promoted synergistic replication of MDV and REV.Conversely,inhibition of Akt suppressed synergistic replication of MDV and REV.However,PI3K inhibition did not affect synergistic replication of MDV and REV,suggesting that the PI3K/Akt pathway is not involved in the synergism of MDV and REV.In addition,we revealed that RIOK3 was recruited to regulate Akt in MDV and REV co-infected cells.Moreover,wild-type RIOK3,but not kinase-dead RIOK3,mediated Akt phosphorylation and promoted synergistic replication of MDV and REV.Our results illustrate that MDV and REV activated a novel RIOK3-Akt signaling pathway to facilitate their synergistic replication.MDV and REV coinfection trigger a strong innate immune response.In this study,we identify the RIOK3 as a important immune modulator for the cytosolic nucleic acid-induced type I IFN production and for the antiviral response to virus.overexpression of RIOK3 markedly reduces viral RNA and DNA-triggered IFN-?production and promotes viral replication,while knockdown of RIOK3 enhances the IFN-? response.RIOK3 selectively inhibits IRF7 but not NF-?B activation.Furthermore,we found that RIOK3 but not K290A interacts and hyperphosphorylates with IRF7,thereby blocking its nuclear translocation,resulting in reduced IFN-? production.These results suggest that chRIOK3 is a negative regulator of the IRF7-mediated IFN response in chicken cells that probably serves to attenuate the establishment of an antiviral state during viral infection.This study described the synergistic replication of MDV and REV in vivo and in vitro,and revealed that the RiOK3-Akt signaling pathway and RIOK3-IRF7 pathway play an important role in the synergistic replication of MDV and REV.These findings provide theoretical basis for studying the co-infection of MDV and REV and the co-infection of herpes virus and retrovirus.