Research On The Dynamics Of Viral Replication After Chicken Embryonic Fibroblasts Co-infection With Newcastle Disease Virus And H9N2 Subtype Avian Influenza Virus

Newcastle disease(ND)is an acute and highly contagious disease of chickens,turkeys and many birds caused by Newcastle disease virus(NDV),which seriously harms poultry.H9N2 is a low-pathogenic avian influenza,but it can cause severe immune suppression in poultry and increase the possibility of secondary pathogens.Newcastle disease and H9N2 subtype avian influenza are more common in the clinic and have more significant harm.The mixed infection of the two diseases will increase the condition of the diseased poultry and bring more enormous losses to the poultry industry.The research on NDV and H9N2 subtype AIV co-infection are limited,and people only initially explored the interaction between the two,and the process and pathogenic mechanism of co-infection are still unclear.The purpose of this study was to establish an in vitro cell model of NDV and H9N2 subtype AIV co-infection.After studying and analyzing the co-infection of chicken embryo fibroblasts(CEF)with the two viruses,RNA levels,virus titer,and the correlation between the single infection group and the co-infection group observed.Changes in immune factors,etc.,lay a foundation for studying the immune response and resistance mechanism of the host system to the virus under co-infection conditions and provide new ideas and new directions for clinical prevention and control of ND and H9N2 mixed infection.It divides into four parts:1.In this study,four pairs of primers were designed and synthesized by referring to the M gene sequences of NDV and H9N2 subtype AIV published in GenBank and related literature.The M genes were amplified using the cDNA in the extracted allantoic fluid as a template.It was ligated to the pMD19-T vector and the standard positive plasmid for the M gene was selected and a standard curve was established using the q-PCR method.2.Design the co-infection experiments of NDV and H9N2 subtype AIV on chicken embryo fibroblasts and use the standard curve to detect the copy number of NDV and H9N2 in the experimental group.The results showed that at 6 h and 12 h,the copy number of M gene in the cell culture of the single infection group was higher than that of the co-infection,and the virus replication was slower;at 24 h,36 h,and 48 h,the single infection cell culture M The copy number of the gene was lower than that of the co-infection group,and the virus replicates faster.It was initially concluded that,within the entire virus replication,NDV and H9N2 subtype AIV were inhibited in the co-infected group 24 hours before,and after 24 hours,the two promoted each other's replication in CEF cells.3.Cellular immunohistochemical(ICC)technique was used to determine the proliferation curves of NDV and H9N2 subtype AIV in experimental groups,respectively.The results showed that the viral titer of the single infection NDV group was higher than that of the mixed infection group,and the viral titer of the single infection H9N2 group was lower than that of the co-infection group,indicating that H9N2 subtype AIV inhibited NDV in CEF cells in the event of co-infection.Intracellular replication,while NDV promotes the replication of H9N2 subtype AIV in CEF cell.4.Q-PCR determined the expression of six intrinsic immune-related factors IFN-?,IFN-?,IL18,IL-1?,TLR4,and NLRC5,and this result shows an upward adjustment.The early stage expression of IFN-? and IFN-? gene was inhibited,and both of them showed up-regulation at the late stage of co-infection.IL-1? gene was up-regulated during the entire co-infection phase;IL18,TLR4 and NLRC5 genes did not show significant changes in the early stage,and they all showed up-regulation at the late stage of co-infection.The results showed that after co-infection of CEF cells with NDV and H9N2 subtype AIV for 24 h,the two promote each other's replication and act together on CEF cells,aggravating the infectivity of CEF cells.