A Preliminary Study On The Effect Of Coinfection Of IBDV With Other Pathogens On Cellular Innate Immunity

Infectious bursal disease virus(IBDV)is the pathogen of infectious bursal disease(IBD).Chickens are susceptible to IBDV.It can not only cause apoptosis of B lymphocytes,but also inhibit innate immunity,damage immune organs such as bursa,thymus and spleen,resulting in permanent immune deficiency and even death of infected chickens.Immunosuppression caused by IBDV often increases the susceptibility to other pathogens.For example,the common clinical cases of mixed infection of viruses such as IBDV and Newcastle disease virus(NDV)or bacteria such as Escherichia coli have caused huge losses to the breeding industry.At present,there is still no relevant molecular mechanism on how to improve the ability of the body to infect other pathogens after IBDV infection.In this experiment,LPS was used to simulate bacterial endotoxin.DF-1 was infected with IBDV and NDV corporately or was acted with IBDV and LPS corporately.The molecular mechanism of mixed and secondary infection caused by IBDV was research from the point of innate immunity,which provides a basis for elucidating the molecular pathogenic mechanism of IBDV secondary infection.Firstly,cytopathic effect was observed when IBDV and NDV were co-infected,infect IBDV and NDV were infected successively,IBDV and LPS were treated at the same time,and IBDV and LPS were treated successively.Then quantitative Real-time PCR(QPCR)was used to detect the dynamic changes of virus replication in each group.The results showed that both IBDV and NDV could replicate in DF-1 cells at the same time,but the replication platform of IBDV was controlled by NDV,which advanced the replication platform of IBDV.In addition,when IBDV and NDV were co-infected,the replication between the two viruses did not interfere with each other before the virus replication platform.When IBDV and NDV were infected successively,IBDV interfered with the replication of NDV and NDV could interfere with the replication of IBDV after the virus replication platform.After IBDV replication,stimulation in LPS can interfere with IBDV replication.Secondly,in order to study the effect of IBDV and NDV co-infection on host innate immune antiviral pathway and the mechanism that affects the replication of these two viruses,we set up IBDV and NDV co-infection group and IBDV colonization replication reinfection NDV group.The changes of m RNA levels of genes related to congenital antiviral pathway were detected by QPCR,and the changes of IFN-? protein level were detected by Elisa.The results showed that in the study of co-infection of IBDV and NDV,the changes of factors of congenital antiviral pathway in the co-infection group were similar to those in the NDV infection group.Besides the expression of MDA5 and OAS in the co-infection group was similar to that in the NDV infection group,while the expression of MX and NF-?B was significantly up-regulated in the co-infection group compared with the single infection group.Subsequently,in the later stage of infection,the expression of TLR3,MDA5,interferon I and OAS was significantly inhibited by NDV when IBDV and NDV were co-infected,resulting in a stronger expression of IL8.In the infection experiment of IBDV and NDV successively,the congenital antiviral pathway in the successive infection group was always inhibited compared with the blank group in the early stage of infection,and the expression of TLR3 was more strongly inhibited,and then the congenital antiviral pathway was activated,especially IRF3,MX and OAS in the late infection group were significantly higher than those in the single infection group.The change trend of IFN-? protein level was consistent with that of m RNA in congenital antiviral pathway,and the IFN-? protein level in 40 hpi infection group was significantly higher than that in IBDV infection group,but there was no significant difference between NDV infection group and NDV infection group.At the same time,the expression of MDA5,IFN-? and OAS in the successive infection group was significantly higher than that in the NDV infection group,and the expression of type I interferon and interferon-induced gene expression in the subsequent infection group was always higher than that in the NDV infection group.In the early stage of infection,the m RNA level of IL8,TNF-? and IL1 ? in the successively infected group were significantly lower than those in the IBDV group,and then the expression began to up-regulate.In conclusion,the co-infection of IBDV and NDV inhibited the innate immune antiviral pathway of host cells and was beneficial to the coexistence of the two viruses;when IBDV and NDV were co-infected,NDV could interfere with the replication of IBDV through the congenital anti-disease pathway,while when IBDV and NDV were successively infected,IBDV could interfere with the replication of NDV through the congenital anti-disease pathway,and NDV up-regulated the expression of IFN-? in the later stage of infection to interfere with the replication of IBDV.In order to study the effect of IBDV and NDV co-infection on apoptosis pathway,and then affect the replication mechanism of the two viruses,the changes of m RNA level of genes related to apoptosis pathway in IBDV and NDV co-infection were detected by fluorescence quantitative PCR,and further verified by MTT and flow cytometry.The results showed that the ratio of Bax to Bcl-2 in the early stage of IBDV and NDV co-infection was lower than that of single infection,and it was found that NDV had a greater effect on the apoptotic pathway.When IBDV and NDV were infected successively,compared with the single infection group,the m RNA level of apoptosis pathway in the co-infection group was first inhibited and then enhanced,which was consistent with the results of cell viability detected by MTT method,and the specific effect caspase of successive infection group was significantly higher than that of NDV infection group at each time point.In summary,the co-infection of IBDV and NDV inhibited apoptosis in the early stage of infection,which was beneficial to the replication and coexistence of the two viruses,but promoted apoptosis and increased cell damage in the later stage of infection.NDV can interfere with IBDV replication through congenital disease resistance pathway and apoptosis pathway.When IBDV and NDV were infected successively,IBDV could interfere with NDV replication through apoptotic pathway.Finally,the mechanism of LPS interfering with IBDV replication was further studied.LPS was applied to DF-1 cells infected with IBDV,and the changes of congenital antiviral pathway were detected by fluorescence quantitative PCR.The results showed that LPS always activated the antiviral pathway related to type I interferon in IBDV infected DF-1 cells,which was the key for LPS to interfere with IBDV replication.To sum up,both IBDV and NDV co-infection can replicate in DF-1 cells at the same time,while IBDV and NDV co-infection can inhibit the innate antiviral pathway and apoptosis pathway of host cells in the early stage of infection,which is beneficial to the coexistence of the two viruses,promote apoptosis and increase cell damage in the later stage of infection.When IBDV and NDV are co-infected,NDV can interfere with IBDV replication through congenital disease resistance pathway and apoptosis pathway.When IBDV and NDV were infected successively,IBDV could interfere with NDV replication through congenital disease resistance pathway and apoptosis pathway,while NDV could up-regulate the expression of IFN-? and interfere with IBDV replication in the later stage of infection.In addition,the continuous upregulation of congenital antiviral pathway caused by LPS on DF-1 cells infected with IBDV can interfere with IBDV replication.