The Effect Of Animo Acid Residue 347 Of Hemagglutinin-Neuraminidase Protein On The Antigenicity Of Class ? Genotype ? Newcastle Disease Virus

Newcastle disease?Newcastle diseases,ND?is an acute and highly contacting infectious disease with worldwide distribution for domestic and wild birds,posing a great threat to poultry industry.Genotype VII NDVs have been the predominant circulating strains in China since 1985.Researchers found that many genotype VII NDVs harbor amino acid substitutions in and near a linear epitope of HN protein?amino acid residues 346 to 353?,espeacially the substitution at position 347?E to K?could increase antigenic differences between NDV isolates and vaccine strains.Previous research of our laboratory has shown that genotype II virus have similar substitutions,however,it remains unclear that whether the substitution could effect the antigenicity of genotype II NDVs or not.Go/CH/LHLJ/2/06 strain was isolated from non-immunized healthy geese by our laboratory in2006.Phylogenetic analysis indicated that this isolates belonged to Class II genotype II and the amino acid at position 347 of HN protein was lysine K,however,the amino acid at that position of genotype II NDV was glutamate E traditionally.The full-length cDNA clone of go/CH/LHLJ/2/06 strain was cloned into the transcription plasmid pOLTV5 to construct the resulting plasmid named pNDVgo,then we rescued virus vNDVgo using reverse genetics technique.Morever,two more mutant viruses were rescued with E347K or K347E substitution by introduction of A1039G or G1039A mutation into the HN gene of go/CH/LHLJ/2/06 and La Sota respectively.The HI and VN assay were performed to determine whether amino acid substitution was successfully achieved,and the results showed that viruses with E347?La Sota and go/CH/LHLJ/2/06 347E?in the HN protein could be recognized by HN mAb,while viruses with K347?La Sota 347K and go/CH/LHLJ/2/06?escaped recognition by HN mAb,which indicated that amino acid substitution mutant strains of La Sota and go/CH/LHLJ/2/06 were generated.These two rescued viruses,go/CH/LHLJ/2/06 347E and La Sota 347K,showed similar low virulence and multicycle growth kinetics characteristics with parental viruses,replicating effectively in chicken eggs with high titers as the parental viruses.Subsequently,to explore the effect of the 347th amino acid of HN protein on the biological properties of Class II genotype II NDVs and HN protein,the following experiment was carried out respectively:?1?The binding capacity to host cells was assessed by ELISA employing chicken antiserum against HN protein and chicken antiserum against NDVs,the result of the ELISA test showed that these four NDV strains could be recognized by chicken antiserum against HN protein or chicken antiserum against NDVs at the same level?p>0.05?,indicating that the 347th amino acid of HN protein had no effect on the polyclonal antibody binding capacity of genotype II NDVs.?2?Hemagglutination test,which was carried out with the same amount of viruses(10^8.5 EID₅₀),showed that the 347th amino acid of HN protein had no significant effect on the titer of genotype II NDVs agglutinating red blood cells?p>0.05?;After infecting DF-1 cells with four NDV strains for 1 h,the cells were harvested and submitted to total viral RNA extraction and quantification of viral load via real-time RT-PCR,the results showed that there was no difference between viruses harboring E347 or K347 on the adsorption ability to host cells?p>0.05?,indicating that amino acid at site 347 of NDV HN protein had no effect on genotype II NDVs in terms of the adsorption ability to host cells;?3?DF-1 cells were infected with La Sota,La Sota 347K,go/CH/LHLJ/2/06,and go/CH/LHLJ/2/06,after being properly treated,cells were subjected to flow cytometry analysis and the fluorescence of 10,000 cells was measured to evaluate the cell surface expression of HN protein.The results showed that there was no significant difference between viruses that harbored E or K at position 347 on cell surface expression of HN protein?p>0.05?,indicating that the 347th amino acid of HN protein had no effect on synthesis and transportation of HN protein of genotype II NDVs.Finally,the cross-HI and-VN assay were performed to determine the antigenic relationships among these four NDV viruses,however,R-values were between 0.67 and 1.5 in terms of any combination,revealing that there was no antigenic difference among them?p>0.05?and residue 347 had nothing to do with antigenicity of genotype II NDVs.The results above demonstrated that the 347th amino acid of HN protein had no effect on replication ability,virulence and receptor binding activity of genotype II NDVs.Moreover,unlike genotype VII NDVs,the 347th amino acid of HN protein had no effect on antigenicity of Class II Genotype II NDVs.