The Fusion Protein Gene Cloning And Sequencing Of Pigeon Paramyxovirus Type Ⅰand Pathogenicity

M. S. Candidate: Yu BinAdvisor: Prof. Wang YongkunAnimal Science And Veterinary Medicine College. Yangzhou UniversityYangzhou, Jiangsu 225009P. R. ChinaABSTRACTAccording to the published Fusion(F) protein gene sequence of Newcastle disease virus(NDV), two pairs of oligonucleotide primers were designed and synthesised. The anticipated nucleotide sequence of F gene function domain with a length of about 500bps were amplified from F gene of pigeon origin paramyxovirus type I (PPMVI )isolates SZb9803.. NC970 1 NP9904~ PK99 10, and the complete gene encoding Fusion protein about 1.7kb were also amplified from F gene of PPMV- I isolates SZa98O3 ~. YZ9712~ PB9601 by reverse transcription-polymerase chain reaction (RTPCR).Four fragments with a length of about 500bps and three fragments with a length of about 1.7kb were all cloned into plasmid vector pGEMR~T respectively and further sequenced.Compared with those of NDV genotype IL. IlL. VL VII reference strains, the nucleotide sequences from nucleotide 1-389 in F gene encoding region and deduced amino acid were aligned and analysed by software Clustal W, the result indicate that those of six PPMV- I isolates and Zhaoqing isolate ZQ98-l of pigeon origin had 96%97%.. 96%-98% homology in nucleotide and amino acid level respectively, The six PPMV- I isolates above and the England reference strain 1168/84.. Wales reference strain 760/83 of PPMV- I had 93%-95%.. 91%-94% homology in nucleotide and amino acid level respectively. Also the six PPMV- I isolates above and strain Lasota of NDV genotype II -. strain F4~E~ of NDV genotype III-. isolate YG97 of NDV genotype VII had 82%-83%~. 84%-85%.. 88%-89% homology in nucleotide level and 85%-87%~.485%-87% ~. 89%-91% homology in amino acid level respectively. Based on the sequences from Genebank, the phylogenetic tree was constructed using software PHYLIP, and it showed that the cluster in NDV genotype VI including six isolates was formed, and isolate PK99 10 belonging to NDV genotype II, clearly distinct from all other PPMV- I isolates.According to the three incides of measuring the pathogenicity of NDV: the mean death time(MDT)~. the intracerebral pathogenicity index(ICPI) with 1-day-old SPF chickens the intravenous pathogenicity index(IVPI) with 6-week-old SPF chicken, they were measured for five PPMV- I isolates, and the results were that the MDT of five isolates was 87.6-88.8 hours, the ICPI was 1.38-1.53, the IVPI with 6-week-old SPF chicken was 1.00-1.50. The five isolates were also measured using five-week-old pigeon, and the IVPI was 1.78-2.07. The IVPIs of isolate PK9910 measured using chicken and pigeon all were 0. These results confirmed the five PPMV- I isolates measured were mesogenic NDV to chicken and velogenic NDV to pigeon.The difference of the PPMV- I isolate and strain Lasota compared by crossreaction of hemagglutination inhibition (HI)test, and showed that the PPMV- I isolates, except PK99 10, had some difference with strain Lasota, although they all had high level of cross-reaction to Lasota in HI test.