Molecular Characteristic Analysis Of F And HN Genes Of 12 Newcastle Disease Virus(NDV) And Biological Characteristic Analysis Of Duck Source NDV

Newcastle disease virus?NDV?is an important infectious disease with a typical respiratory and gastrointestinal mucosal hemorrhage in avians.NDV was mostly be considered as one of the important infectious diseases which caused huge enconocim damage to poultry industry worldwide.Currently,waterfowl infected by NDV with new characteristics were frequently found and reported.The new clinical symbols were not only showed as a low production capacity,but also morbidity,mortality was gradually increased.Went with such trend of NDV evaluation,significant economic losses was attacking and influencing worldwide poultry industry.In this research,samples were collected from different poultry with significant clinical symbol in Foshan,Zhaoqing,Yunfu,etc cities,Guangdong province from 1997 to 2016.The F gene and HN gene of 12 NDV isolates were amplified through RT-PCR.PCR products were cloned into 18-T vector and were followed by sequenced.The sequence results were analyzed by comparing with NDV reference strains published in GeneBank.The genetic evolution analysis of F gene showed that 9 of 12 NDV isolates belonged to genotype?and9 strains were isolated from chicks and waterfowl in various regions of Guangdong province from 1997 to 2006.The phylogenetic analysis results showed that genotype?NDV strains was considered as the virulent strains circulating in Guangdong province.In addition,CK/FS-SS/N/1997 and MDK/FS-SS/485/2014 belong to genotype?,which were isolated from Sanshui district of Foshan,and PG/GZ-HD/PN/2011 belong to genotype?.Thus genotype?NDV strains were threaten to poultry,but the spread of the epidemic is still relatively slow.The aligment and analysis results show that both of the F gene and HN gene had homology rates from 80.9%to 91.9%by comparing with those reference strains including the wide used vaccine canddidates,such as LaSota,B1,Mukteswar and Clone 30.Genotype?NDV strains in this study were showed with high homology?from 99.7%to99.6%?compared with standard virulent F48E8.The nucleotide sequence homology are higher than 97%between those five goose strains and one duck strain.However,the homology of other strains located in type?were only from 83.4%to 87.8%.There are genotypic differences between strains of the same genotype in Guangdong province.The results of amino acid sequence analysis of F protein and HN protein of 12 strains were in conformity with the velogen strain of NDV characteristics;and nine strains showed neutralization site variation.There was significant antigenic diversion happened between most strains and traditional vaccine strains.In 2007,the duck source strain,NDV MDK/FS-SS/E/2007,which isolated from a sick duck.The results of pathogenicity index measured show that the ELD₅₀?MDT?ICPI and IVPI were 10^-8.668/0.1 mL?75.2 h?1.725 and 2.48.Therefore,the results of the artificial infection test are shownd ducklings and goslings appear mental depression,paralysis and twisting neck and other neurological symptoms,and most of organs appear degeneration and necrosis,which was similar to the symptoms of NDV of chicken.Results of the immunize protective evaluation study suggested that this vaccine provide good immunization for ducklings.In addition,the open reading frame of F protein was expressed and the Western blot analysis showed that the purified protein can specific reaction with the monoclonal antibody of anti-His tag protein of antigenic sites about blood clotting properties and neutralizing activity and rabbit polyclonal,indicating that the recombinant protein has a good immunogenic.It lays the foundation of next further more preparation of monoclonal antibodies and in-depth study of its epitope preparations.