Mechanism Research Of Superinfection Exclusion Of Newcastle Disease Virus

Superinfection Exclusion(SIE)is a phenomenon in which cells or hosts are prevented from re-infection by the same or similar pathogen after being infected.It is widely found among viruses and bacteria.With SIE,the host with low antibody can use the lentogen strain to block the infection of the velogenic strain,providing a new idea for clinical disease prevention and control.Newcastle Disease(ND)is an infectious disease caused by Newcastle disease virus(NDV),which can cause great harm to poultry.At present,the prevention and control measures mainly rely on vaccination.NDV SIE has been reported,and some studies suggested that NDV SIE may be related to hemagglutinin-neuraminidase protein(HN).HN plays an important role in the entire life cycle of NDV infection as a prerequisite for NDV to recognize,absorb host cells and release progeny viruses from cells.However,how HN is involved in NDV SIE has not been reported,and whether other factors contribute to the occurrence of NDV SIE is unknown.This study mainly focuses on the exploration of the occurrence mechanism of NDV SIE.The specific research contents and results are as follows:(1)Establishment of NDV SIE cellular model.In order to study the mechanism of NDV SIE,it is necessary to use NDV strains with different phenotypes to infect cells successively and observe the ability of cells to inhibit the re-infection of NDV after the first viral infection,so as to establish the SIE model of NDV.NDV strains expressing different fluorescent proteins were used to infect DF-1 and BHK-21 cells at different intervals.The ability of cells infected with NDV to reject the virus for re-infection was analyzed qualitatively and quantitatively at the cell,protein and RNA levels by fluorescence microscopy,flow cytometry,Q-PCR and WB tests.It was found that cells could reject the re-infection of NDV within 2 h after being infected with NDV,and the degree of rejection gradually became stronger with the extension of time between two viral infections.NDV SIE cell model was successfully established.Further studies showed that SIE was interspecific and could occur between lentogen and velogenic NDV viruses,but could not occur between NDV and Vesicular Stomatitis Virus(VSV),indicating that SIE of NDV is related to the virus itself,and has nothing to do with the natural immunity that induces broad spectrum antiviral of cells.(2)The mechanism of HN protein regulation of NDV SIE.In order to explore the molecular mechanism of HN protein regulating NDV SIE.First,the eukaryotic expression vector of NDV HN protein was constructed and the rescue of Peste des Petits Ruminants virus(PPRV)PPRV-HN-GFP expressing NDV HN protein.The successful expression of HN protein was confirmed by transfection with eukaryotic plasmid or recombinant PPRV virus infection,and the associated hemadsorption activity(HAd),neuraminidase activity(NA)and membrane fusion activity were detected.After plasmid transfection or infection with PPRV-HN-GFP strain,related cells were then infected with NDV strain and the infection level of NDV was detected.It was found that the pre-expressed HN protein had a repulsive effect on NDV infection at both plasmid level and virus level.Secondly,eukaryotic plasmids expressing HN protein mutants with lost,decreased or increased HAd and NA activity were obtained by point mutation,and PPRV strains chiming these HN protein mutants were rescued by reverse genetic techniques.After plasmid transfection or infection with PPRV-HN-GFP strain,related cells were then infected with NDV strain.The HN protein was found to regulate SIE through NA activity at cell,protein,and RNA levels,using the methods used in SIE cell model.However,the expression of HN protein and NA activity could not be detected 2～6 h after NDV infection,suggesting that SIE may be regulated by other components of NDV in the early stage of virus infection.(3)Mechanism of Nucleocapsid protein(NP)regulating NDV SIE.In order to explore whether other viral proteins besides HN protein are involved in NDV SIE.The eukaryotic expression plasmid of all the proteins of NDV was constructed.Through cell transfection,virus infection and infection monitoring,it was found that the NP protein of the virus in advance could also inhibit the infection of NDV.PPRV strain(PPRV-NP-GFP)expressing NP protein was rescued,and the expression of NP protein and its ability to drive NDV microreplicons(microgenome)were detected after the cells were infected.It was also confirmed that NP protein significantly inhibited the infection of NDV.In order to study the mechanism of NP induced NDV SIE,we constructed plasmids with truncated amino terminus(N-terminus)and carboxyl terminus(C-terminus)of NP protein,and infected NDV after transfection.The results showed that amino acid sequence 1～40 of N-terminus and amino acid sequence 353～489 of C-terminus were the key sequences of NP protein involved in NDV SIE.Recombinant PPRV strains PPRV-NPΔN41-GFP and PPRV-NPΔC352-GFP expressing NPtruncated protein were rescued.The cells were infected with NDV after expressing the recombinant strain.The results showed that compared with the wild type NP protein,the NP protein truncated by the deletion of amino acids 1-40 at the N-terminal lost the ability to inhibit NDV infection,and the NP protein with the deletion of amino acids 353 to 489 at the Cterminal of the NP protein reduced the ability to inhibit NDV infection.Further studies showed that this ability to regulate NDV SIE was positively correlated with the degree of NP protein self-aggregation.The self-aggregation ability of wild NP protein was significantly stronger than that of NPΔC352,and the self-aggregation of NPΔN41 truncated protein could hardly be detected.However,how NP protein self-aggregation regulates SIE in NDV remains to be further studied.In conclusion,from the protein level,NDV SIE is mainly regulated by viral HN protein and NP protein.This study for the first time found and confirmed that HN protein participated in SIE of NDV through NA activity and NP protein participated in SIE through self-assembly and aggregation,providing ideas for further understanding SIE of other viruses.