Molecular Evolution Of P And HN Genes In Relation To Pathogenicity Of Field Newcastle Disease Viruses

Newcastle disease (ND), caused by Newcastle disease virus(NDV), is one of the most serious infectious diseases in poultry and even result in 100% morbidity and mortality in chicken flocks. In spite of constrol measures, including vaccination applies since the 1950s, ND still is a serious problem of the poultry industry all over the world which must be reported to OIE and Listed I pathogens in China. The new epidemic situations have been puzzling scientists.NDV is classified as a member of the family Paramyxoviridae in the order Mononegavirales, belonging to the new genus Avulavirus of subfamily Paramyxovirinae in the family. NDV, sole member of serotype 1 of avian paramyxovirus-1(APMV-1), contains a single-stranded, negative-sense, non- segmented RNA genome. The genomic RNA is approxmaitely 15.2kb nucleotides in length and contains six genes that codes at least seven proteins. P gene is unique in that it encodes several proteins. The unique genetic structure of NDV is one of major cause why it was reclassified into the new genus. To explore the nature of pathogenicty on NDV, too many studies have fouced on the glycoproteins existing at the surface of virons, especial F protein. However, recent tests demonstrated that the determinte for virlence is not sole F gene and but multigenes, including the V protein that is derived from the P gene. These provide the new ideas for the difficultly controlling and prevetion of ND. In our study, 14 NDV field strains were isolated in China from 1997 to 2007. Nucleotide sequence was determined for the P and HN genes of all strains were cloned and sequenced, and the pathogenicity of these isolates was evaluated. The HN and P genes were analyzed by the mothed of biological information based on the epidemical and biological data for the decade. The study was focused on the following areas:1. Isolation, identification and plaque-purification of NDV field isolates and comparisons of pathogenicity by different NDV strains.14 NDV field isolates were isolated from the poultry populations in China between 1997～ 2007.The viruses were identified by HI test using NDV and AIV (H5 and H9) positive serum.At the same time, all isolates were purified by plaque- purification test. Based on the value of MDT, ICPI and IVPI of the 14 NDV field isolates, all of the field isolates were designated as velogenic isolates.2. Pathogenicity in relation to molecular evolution of P gene from the filed strains.The nucleotide sequence was determined for the P gene from 14 field isolates of NDV. The P gene sequences of 41 NDV strains representing all defined pathotypes with different chronological and geographic origins in GenBank were also used in this study. The amino acid homology of the Chinese field NDV strains was lower compared with LaSota, Clone30 and F48E9, and foreign NDV strains.The unique residues in the amino acid sequence were found in the Chinese isolates. The C-terminal domain (CTD) of V protein played a role in host range and virulence. Phylogenetic analysis of P gene sequences among NDV isolates demonstrates there are two clades of these viruses. One group includes viruses isolated in the US prior to 1970, while a second cluster includes virulent viruses circulating worldwide.3. Phylogenetic and molecular evolution analysis of HN gene from the filed strains.HN gene sequences for 14 field isolates and 29 strains of older reference NDV were examined to determine phylogenetic relationships and pathogenicity among these viruses. The analysis of glycosylation site and antigen index showed that absence of one potential glycosylation site and two changing antigen epitopes occurred in current strains compared with traditional velogenic strains. The amino acid homology of the Chinese field NDV strains was lower compared with foreign NDV strains. The unique residues in the amino acid sequence were found in the Chinese isolates. Phylogenetic analysis revealed that two major separations occurred, Class I and ClassⅡ. Of that, ClassⅡis divided into two lineages. One includes viruses isolated in the US prior to 1970 and another cluster includes virulent viruses circulating worldwide.4. Molecular evolution and correlation of HN and P genes among the field Newcastle disease virusesThe nucleotide sequence and deduced amino acid sequence were analyzed for the Hemagglutinin-neuramindase (HN) and Phosphoprotein (P) gene from field isolates of Newcastle disease virus (NDV). The HN and P gene sequences of fifteen NDV reference strains from GenBank were also used in this study. The molecular evolution distance of nucleotides and amino acids were calculated by MEGA4.0 software, and analysis of variance and correlations were analyzed by SPSS11.0 software among different length sequences of the HN gene or P gene. The nucleotide and amino acids correlation of HN and P gene were analyzed respectively. The results indicated that there were significantly difference and good correlation of nucleotide and amino acid among different length sequences of the HN gene or P gene. These results revealed that the HN and P gene of NDV have the different response to selective pressure to adopt to landscape and the closely relationship on heredity mutations. In addition, the changing of HN and P genes have relationship with time of isolate.5. Molecular evolution analysis on the P and V genes of Newcastle disease virusPresented here is an anlysis of the molecular evolution of the P gene among 55 representative sequences of NDV. The homology of amino acid for the P and V proteins were analyzed.The nucleotide architecture that underlies the amino acid variation was determined in order to evaluate the role of selection in the evolution of the P gene overlapping reading frames. The significant diffierence and the different nucleotide substitutions occurred in the same nucleotides under the the overlapping reading frame The result show that evolution of highly conserved RNA editing and multicoding sequence is a response by virus to selective force. This is unique P gene distribution in sequence space..The experimentally determined functional region were found to be conserved, showing that important regions for function were conserved during evolution.