Study On The Mechanism Of Chicken Cell Surface Receptors Nectin4 And SLAMF1 Promoting NDV Proliferation

Newcastle disease(ND)is a highly contagious infectious disease caused by Newcastle disease virus(NDV),whichis widely spread around the world and can lead to a variety of poultry diseases.The incidence rate and mortality of the disease can reach100%,bringing huge economic losses to the global poultry industry.Signaling lymphocyte activation molecule(SLAM or CD150)and poliovirus receptor related protein 4(PVRL4 or Nectin4)are the main cellular receptors of Morbillivirus.Previous studies in our laboratory have found that SLAMF1 and Nectin4 are also involved in the process of NDV infection in host cells.In order to verify the role of chicken derived Nectin4 and SLAMF1 in NDV infection,this study used Lentivirus packaging method to introduce chNectin4 and chSLAMF1 genes into BHK21 cells,and constructed BHK21 cell lines stably expressing chNectin4 and chSLAMF1.Western blot and IFA results showed that the constructed BHK21 recombinant cell lines could stably expressed chNectin4 and chSLAMF1 proteins.The results of infection experiments showed that the NP m RNA level of BHK21 cell lines stably expressing chNectin4 and chSLAMF1 was significantly higher than that of their parent cells 12 and 24 hours after inoculation with NDV La Sota strain,indicating that chNectin4 and chSLAMF1 were helpful in promoting the proliferation of NDV in BHK21 cell lines,and the effect of chNectin4 on NDV proliferation was more obvious.Using chicken derived fibroblast line DF-1 cells as the researchobject,the role of chNectin4 and chSLAMF1 in NDV infected host cells was studied.The exogenous expression of chNectin4 and chSLAMF1 was detected in DF-1cells,respectively.After inoculation with NDV,the adsorption and proliferation efficiency of NDV were detected using RT-q PCR and Western blot methods.The results showed that exogenous expression of chSLAMF1 could promote the adsorption and proliferation efficiency of NDV;chNectin4 has no significant promoting effect on the adsorption and proliferation efficiency of NDV.In this study,lentivirus was obtained from 293 T cells using a three plasmid packaging system.The chSLAMF1 gene was introduced into DF-1 cells,and a DF-1 cell line stably expressing chSLAMF1 was obtained.After inoculation with NDV,the adsorption and proliferation efficien cy of NDV in DF-1 cells stably expressing chSLAMF1 was significantly increased compared to wild type DF-1 cells.The endogenous expression of chSLAMF1 in DF-1 cells can promote NDV adsorption and replication in DF-1 cells.Bioinformatics software was used to predict and analyze the key sites of the interaction between chNectin4,chSLAMF1 and NDV-HN protein,and point mutation plasmids were constructed.Western blot and q PCR methods were used to preliminarily screen the key sites of the interaction between chNectin4,chSLAMF1 and NDV-HN protein.The DLRH and FPLG sites of chNectin4 affect the promotive effect of chNectin4 on NDV infection in host cells;The E66 A,Y76A,Y106 A,and E116 A loci of chSLAMF1 affect the promotive effect of chSLAMF1 on NDV infection in host cells.In summary,this study has successfully constructed cell lines that stably express chNectin4 and chSLAMF1,respectively,demonstrating that chNectin4 and chSLAMF1 can promote NDV infection of host cells,and preliminary identified of key sites of chNectin4 and chSLAMF1 affecting NDV infection in host cells,laying a foundation for further researchon the role of chicken Nectin4 and SLAMF1 in NDV infection and the study of NDV cell vaccine.