Cloning and sequencing of F gene of avian paramyxovirus isolates

The whole sequence of F gene of avian paramyxovirus isolates was amplified by reverse transcription-polymerase chain reaction(RT-PCR),and further cloned into pMD18-T vector,and positive recombinant plasmids were obtained by polymerase chain reaction(PCR).The sequence analysis showed that all the nucleotide sequence of the F gene were 1 662 bp in length and had a single opening read frame(ORF),which encoded polypeptide of 533 animo acid,contained 6 potential asparagine-linked glycosylation sites,their amino acid sequence of cleavage site region residues were 112R-R-Q-K-R-F117,matched to the characteristics of virulent strain,the isolates possessed K101and V121,which were similar to the characteristic of genotype Ⅶ;WF00C strain contained 13 cysteine(Cys) residues,but WF00D and WF00G strains only had 12Cys residues,all isolates contained 973 nt and 1 249 nt RsaⅠrestriction endonuclease(RE)sites(nt334-1 682).Comparison of F gene nucleotide sequences of WF00D strain with 15 other published sequences from GenBank showed that the homology of the nucleotide was 85.0%-99.3%,the homology of the deduced amino acid sequences was 88.8%-98.9%.Three isolates from Anhui shared higher homonology each other,and higher homonology compared with ZJ1、SF02、NA_1 too,but lower homonology compared with F48E9、LaSota.The results of phylogenetic tree,restriction site analysis and genotype characterization showed that three strains WF00C,WF00D,WF00G belonged to novel subgenotype Ⅶd.