Screening And Identification Of Membrane Fusion Activity-related Sites Of Newcastle Disease Virus C22 Virulent Strain

Newcastle Disease(ND)is a highly contagious and lethal disease caused by a virulent strain of Newcastle Disease Virus(NDV)to a variety of poultry.According to the degree of pathogenicity to chickens,NDV is divided into virulent strains,moderately virulent strains and weak virulent strains,among which the virulent strains can sometimes lead to the death of all poultry.It has been reported that the membrane fusion activity and virulence of NDV are directly related to fusion(F)protein and hemagglutinin-neuraminidase(HN)protein,which are the determinants of syncytial lesions caused by membrane fusion in virus-infected cells.The laboratory isolated a Newcastle disease virus from a chicken farm in Tibet in the early stage.Whole gene sequencing and comparison analysis showed that the virus was of Class II gene VIId subtype,and named it as the C22 strain.The results of the challenge experiment showed that the strain could cause severe respiratory and gastrointestinal symptoms in chickens and had a high lethality rate.However,the strain showed weak membrane fusion ability after infecting cells.Generally,the stronger the membrane fusion ability,the stronger the virulence.However,some strains also showed strong membrane fusion ability,but showed weak toxicity.At present,there are no reports of NDV isolates with weak membrane fusion ability and strong virulence at the animal level.This characteristic of the C22 strain obviously broke the previous cognition of NDV.Based on this characteristic,relevant studies on the membrane fusion activity and pathogenicity of the virus were carried out.1.Association analysis of NDV pathogenicity and membrane fusion abilityIn this study,the NDV F48E9 strain preserved in the laboratory and the rescued r La Sota/Fmut strain were combined to determine the MDT,ICPI of the strain,and its pathogenicity to four-week-old low-antibody chickens.The results showed that F48E9 and C22 were virulent strains,but r La Sota/Fmut was lentogenic.After infecting BHK-21 cells with the three strains at a dose of 0.01 MOI,the size of the syncytia formed at different time points was observed,and the average size of the syncytia was measured by Image J software,and the differences in the membrane fusion activity of each strain were compared.NP protein,and indirect immunofluorescence observation of syncytia formed by virus-infected cells showed that the membrane fusion ability of C22 strain was significantly weaker than that of r La Sota/Fmut and F48E9 strains.These results indicated that C22 strain had weak membrane fusion ability and strong pathogenicity,r La Sota/Fmut strain had strong membrane fusion ability and weak pathogenicity,and F48E9 strain had strong membrane fusion ability and strong pathogenicity.Therefore,the pathogenicity of NDV is not always related to viral membrane fusion.2.Screening of key sites for membrane fusion activity of NDV C22 isolatesThe eukaryotic plasmids expressing the F and HN proteins of the F48E9 and C22 strains were constructed.After transfecting the cells,it was found that the ability of the F and HN proteins of the C22 strain to form syncytia was significantly weaker than that of F48E9,and the membrane fusion activity of the C22 strain did not follow enhanced with the increase in protein expression.In order to further determine the key sites,based on the crystal structure of the protein,the functional domain of the F protein of the C22 strain and F48E9 strain was replaced,and it was found that the DI region had the greatest impact on the membrane fusion activity of the C22 strain.Sequence analysis showed that in the DI region of F protein,C22 and F48E9 were not identical at amino acid residues 52,314,339 and 341.After point mutation of these sites and transfection of cells,the size of syncytia was counted,and it was found that the 339 th amino acid site had the greatest influence on the membrane fusion activity of C22 strain.Using a similar method,the key sites of HN protein were explored,and it was found that the 116 th amino acid site had the greatest impact on the membrane fusion activity of the C22 strain.Co-transfection of cells with F protein mutated to isoleucine F48E9 at position 339 of C22 strain and HN protein mutated to histidine 116 of F48E9 of C22 strain can significantly reduce the membrane fusion caused by F and HN proteins of F48E9 strain active.These results suggest that amino acid 339 of the F protein of the C22 strain and amino acid 116 of the HN protein may be the key sites that determine its weak membrane fusion activity.3.Validation of key sites for membrane fusion activity of NDV C22 isolatesUsing the reverse genetic system of the C22 strain constructed in the early stage of the laboratory,based on the screened key sites of the F protein and HN protein,the point mutant strain was rescued,and the qualitative and quantitative observation of the syncytia was performed after infection of the cells.It was found that the mutation of amino acid 339 of the F protein of C22 strain to the corresponding amino acid of F48E9 strain could significantly improve the membrane fusion activity of C22 strain.Similarly,mutation of amino acid 116 of HN protein can also improve the membrane fusion activity of C22 strain.Secondly,the related genes of C22 were replaced by the F and HN genes of F48E9,and the membrane fusion activity of the rescued chimeric strains was significantly improved.Based on this chimeric strain,the 339 amino acid of F protein and the 116 amino acid of HN protein of F48E9 strain were mutated to the corresponding sites of C22 strain,and the ability of the obtained mutant strain to form syncytia was significantly reduced after infecting cells.These findings confirmed that at the viral infection level,amino acids at position 339 of the F protein and 116 of the HN protein affected the membrane fusion activity of the C22 strain.Pathogenicity tests showed that the virulence of these strains did not change significantly compared with the C22 strain.The above results indicated that the pathogenicity of NDV C22 strain was independent of its membrane fusion activity.In summary,this study screened out two key amino acid sites that affect the membrane fusion activity of the NDV C22 virulent strain,which are located at the I339 site of the F protein and the G116 site of the HN protein.The substituted mutant strains further verified the effect of key sites on membrane fusion activity and virus virulence at the whole virus level,and found that the virulence of NDV was not always correlated with the membrane fusion activity of the virus.These research results enrich the biological characteristics of NDV virulent strains and provide more theoretical support for the determination of NDV virus virulence.