Complete Genome Sequence Of Newcastle Disease Virus (CH2000 Strain) And It's Pathogenicity Experiment To Waterfowl

Newcastle disease (ND), one of the most important diseases of poultry, caused by Newcastle disease virus (NDV), also called avian paramyxovirus type 1, is classified as a List A disease by the World Animal Health Organization ( Office International des Epizooties, OIE) because it is highly contagious and can causes severe disease and high mortalities in susceptible birds. NDV has been placed in the genus Avulavirus, sub-family Paramyxovirinae, family Paramyxoviridae, order Mononegavirales in the current taxonomy, which has a negative-sense, single-stranded RNA genome of 15 kb that contains 6 genes in the order of 3'-NP-P-M-F-HN-L-5', encoding six proteins (nucleoprotein, phosphorprotein, matrix protein, fusion protein, hemagglutinin-neuraminidase protein and large protein respectively).In this study, four NDV isolates, named CH2000, qd/06-74, d/06-87 and qd/07-12, which obtained from outbreaks of chicken in recent years in China were characterized both pathotypically and genotypically. The mean death time (MDT) and intracerebral pathogenicity index (ICPI) were used to evaluate the virulence of the isolates. The ICPI was proved to be 1.83, 1.92, 1.86 and 1.86 respectively, and the MDT was 60h, 67.2h, 62.4h and 52.8h. Pathogenicity tests showed that all ten isolates were velogenic strains. The main functional region of the F gene comprising 535 nucleotides was amplified by RT-PCR and sequenced. The amino-acid sequence of the fusion protein cleavage site in all four isolates was 112RRQKRF117, which is a typical sequence of velogenic strains and is in agreement with the results of in-vivo pathogenicity tests. For genotyping, a phylogenetic tree based on nucleotides 47–435 of the F gene was constructed. Phylogenetic analysis showed that all the isolates were of the genotype VII virus, which has became the predominant genotype of NDV responsible for most of outbreaks of ND in China in recent years. In order to obtain the complete genome sequence of NDV CH2000 isolate, 9 pairs of primers were designed to amplify the specific gene fragment by RT-PCR and sequenced. The complete genome sequence was analysised by SeqMan. The genomic sequence consists of 15192 nt, which is six nt longer than the published full length genome of the NDV strains La Sota. Phylogenetic tree was constructed to analysis the evolution origin among other representive NDV isolates which sequence obtained from GenBank. Phylogenetic analysis revealed that CH2000 isolate was similar to the Goose paramyxovirus SF02 and ZJ1.In order to analysis the pathogencity of different NDV isolates in waterfowl, CH2000 and the reference strain F48E8 were selected to perform virus challenge test on 15-day-old WuLong goose.The results revealed when using the dose of 105ELD50/ml, the morbidity and the mortality of the i.m. group of the CH2000 were 100%, and the p.o. group of the CH2000 were 93%; the morbidity and the mortality of the i.m. group of the F48E8 were separately 100% and 60%,the p.o. group of the F48E8 were 100% and 53% respectively.The latency period of the artificial infected goose was 50-80h, most of them wre died in the 4-5 days.In this study four NDV isolates obtained in recent years in China were characterized both pathotypically and genotypically. The result revealed that all the four isolates were proved to be Genotype VII virus and showed velogenic characterization according to the pathogenetic test and molecular motif of the Fusion protein. Complete genome sequence of CH2000 isolate were characterized in this paper, and it provided foundation to make research on reverse genetics. Natural infection test of two representive isolates in waterfowl were performed to analysis the pathogenic difference between different genotypes.