Synergistic Tumorigenesis Mechanism Of Avian Leukosis Virus Subgroup J And Marek's Disease Virus

Viral co-infection is prevalent in nature.During co-infection,viral and cellular biological properties such as viral replication,infection rate,pathogenicity,cell phenotype,tissue tropism and host range are altered.Avian leukosis virus subgroup J(ALV-J),an oncogenic retrovirus of poultry,which can cause immune suppression and oncogenesis in infected chickens,mainly inducing myeloma.No effective vaccine has been developed for prevention and control,and the spread of the virus is mainly controlled by elimination,which has caused serious economic losses to the poultry industry due to its unique ability to spread and pathogenicity.Marek's disease virus(MDV)is a highly oncogenic herpesvirus that also causes immunosuppression and oncogenesis in infected chickens,mainly inducing lymphocytoma.Vaccination has been the main strategy for prevention and control of Marek's disease(MD),but continued viral evolution has led to increasing virulence of the strain,posing a challenge to existing control strategies for MDV,making MDV still a threat to the poultry industry.However,on the one hand,co-infection of ALV-J and MD strains occurred frequently in the vaccinated chickens,due to the frequent contamination of MD vaccine by ALV-J and the increasing virulence of MDV under the pressure of vaccination;on the other hand,as the infection of ALV-J impairs the immune response of sick chickens to MD vaccine,the probability of secondary infection of MDV after infection with ALV-J increases,resulting in the increasingly serious co-infection of ALV-J and MDV.ALV-J and MDV co-infection have significant synergistic effects,leading to accelerated virus evolution,enhanced oncogenicity and expanded tumor spectrum,resulting in increased mortality,enhanced immunosuppression,immune response failure and frequent secondary infections in diseased chickens,posing serious challenges to the prevention and control of the two diseases,seriously affecting the healthy and sustainable development of the poultry industry.Tumor development and formation induced by ALV-J and MDV coinfection are the key factors causing death in diseased chickens,but their synergistic oncogenic mechanisms remain obscure.In this study,we constructed a co-infection model of ALV-J and MDV using response surface methodology,and verified the synergistic replication and oncogenesis of ALV-J and MDV in vitro and in vivo using pathology,molecular biology and bioinformatics technology.We screened and identified its co-activating host key molecule,doublecortin-like kinase 1(DCLK1),and revealed that DCLK1 induces epithelialmesenchymal transition(EMT)through activation of Wnt/?-catenin signaling pathway to promote tumorigenesis,finally elucidating the molecular mechanism of synergistic tumorigenesis of ALV-J and MDV.To clarify the synergistic replication and pathogenicity of ALV-J and MDV in vitro and in vivo,an in vitro cellular co-infection model was established,and it was observed by electron microscopy that ALV-J and MDV could co-infect the same cell.Based on the co-infection time,ALV-J TCID50 and MDV PFU as independent variables,and the copy number of ALV-J and MDV viruses as experimental indexes,the response surface methodology(RSM)of ALV-J and MDV co-infection was constructed,which visually reflected the trend of virus copy number changes with the independent variables during the co-infection process and determined the optimal time point for virus co-replication effect at 72 h post co-infection.Further confirmed by qPCR and WB,in co-infected cells,ALV-J and MDV synergistically promoted the replication of the two viruses,in which the Gag protein of ALV-J promoted MDV expression by 1.29-fold with the most obvious effect,and the Env protein promoted MDV expression by 1.15-fold,proving that the structural protein of ALV-J had a significant promotion effect on MDV replication.The release of inflammatory factors from infected cells is a fundamental condition for tumor development.In view of this,the dynamic expression of inflammatory factors IL-6,IL-10 and TGF-?,which are associated with tumor development,was detected by ELISA,indicating that ALV-J synergistically promotes the secretion of inflammatory factors with MDV.To further clarify the synergistic pathogenicity of ALV-J and MDV,a co-infected animal model was established.Mortality statistics showed that the mortality rate of chickens in the co-infected group was 45%,which was significantly higher than the mortality rate of 20%in the MDV group and 5%in the ALV-J group;blood smear observation showed that the number of lymphocytes and monocytes in the co-infected group was significantly higher than that in the single-infected group,which proved that the two viruses synergistically aggravated the inflammatory response of the organism.Histopathological observations revealed obvious mixed tumor foci of myelocytoma and lymphocytoma in the liver,kidney and heart of chickens in the co-infected group,while only inflammatory reactions were observed in the ALV-J and MDV single-infected groups.Viral load analysis of liver,spleen and bursa of chickens in each group revealed that the viral load of each tissue in the co-infected group was significantly higher than that in the single-infected group,which proved that ALV-J and MDV synergistically promoted virus replication in both groups in vivo.The above results demonstrated that ALV-J and MDV have synergistic replication,synergistic inflammation and synergistic tumorigenesis.EMT is an important indication of tumorigenesis and transformation.To clarify the effect of ALV-J and MDV on the EMT process,the expression of EMT marker molecules in ALV-J and MDV co-infected cells and tissues was detected.In vitro assays,qPCR and WB demonstrated that ALV-J synergistically upregulated the expression of EMT mesenchymal markers N-cadherin,Vimentin and transcription factor Snail,synergistically downregulated the expression and distribution of epithelial marker E-cadherin in cells with MDV;In vivo assay,the expression and distribution of E-cadherin in liver,spleen,kidney and duodenum were downregulated by ALV-J in synergy with MDV as demonstrated by tissue immunofluorescence assay.These results suggest that ALV-J and MDV synergistically promote EMT process.To clarify the key host molecules and signaling pathways associated with the synergistic activation of EMT by ALV-J and MDV,co-infected cell samples at 72 h post co-infection with optimal synergistic replication effect were selected for iTRAQ proteomic assay based on response surface model optimization conditions,and cells in the single-infected and Mock groups were used as controls.A total of 15 differentially expressed proteins were found,and the tumor-related and significantly up-regulated DCLK1 and Wnt-induced proteins(WISP2)were screened out by functional analysis.To clarify the synergistic activation of DCLK1 expression by ALV-J and MDV,the expression of DCLK1 was first detected by qPCR and WB,and the results showed that DCLK1 expression was significantly upregulated in the co-infected group of cells.The expression of DCLK1 in tissues was further detected by IHC,and the results showed that ALV-J and MDV synergistically promoted the expression of DCLK1 in the duodenal intestinal crypts,confirming that ALV-J and MDV synergistically promoted the expression of DCLK1 in vitro and in vivo.Overexpression and knockdown of DCLK1 results showed that DCLK1 significantly promoted replication and cell proliferation of ALV-J with MDV.To clarify the effect of DCLK1 on EMT process,DCLK1 was overexpressed in the cell system co-infected with ALV-J and MDV,and the expression of EMT marker molecules was detected by qPCR and WB,which showed that E-cadherin expression was down-regulated and N-cadherin,Vimentin and Snail expression were up-regulated,indicating that DCLK1 promoted EMT process.The above results demonstrate that ALV-J and MDV promote the EMT process by synergistically activating DCLK1.To reveal the pathway of DCLK1-promoted EMT,the expression of marker proteins in the classical Wnt/?-catenin signaling pathway activated by WISP2 was examined.The results indicated that co-infection of ALV-J with MDV enhanced the protein expression of Wnt and ?catenin in cells and promoted the transfer of ?-catenin from the cytoplasm to the nucleus.In vivo studies showed that ALV-J co-infection with MDV promotes ?-catenin expression in the liver,spleen,kidney and duodenum.Overexpression of DCLK1 revealed that DCLK1 promoted Wnt/?-catenin protein expression,whereas knockdown of DCLK1 inhibited its protein expression while attenuating the nuclear allosteric site of ?-catenin;The Wnt/?-catenin pathway activator CHIR-99021 was used to rescue the expression of ?-catenin and its downstream molecule C-myc and EMT markers induced by DCLK1 knockdown.The fluorescence overlap between DCLK1 and ?-catenin was demonstrated by in vivo immunofluorescence assay,indicating that DCLK1 and ?-catenin promoted the formation of ?catenin complex and transcription of downstream target gene C-myc through interaction,thereby inducing the EMT process.The above results reveal that DCLK1 regulates the EMT process by targeting the Wnt/?-catenin signaling pathway.In conclusion,this study elucidated the mechanism of tumorigenesis induced by the synergistic effect of simple retrovirus ALV-J and herpesvirus MDV.Through in vitro and in vivo co-infection models,it was verified that ALV-J and MDV synergistically promote the replication of the two viruses and synergistically promote tumorigenesis and EMT processes.To clarify the molecular mechanism of its tumorigenesis and EMT activation,DCLK1,a key host molecule for the synergistic activation of EMT by ALV-J and MDV,was screened by proteomics.DCLK1 promotes viral replication and cell proliferation and mediates the synergistic activation of Wnt/?-catenin signaling pathway by ALV-J and MDV to induce EMT process,thus synergistically promoting tumor formation.This study provides a theoretical basis and technical support for the efficient combined prevention and control of the two viruses,as well as a reference for the study of the synergistic tumorigenic mechanism of retrovirus and herpesvirus.