Isolation,Identification And Complete Genome Analysis Of A Racing Pigeon-Origin ?k Sub-Genotype Newcastle Disease Virus

Newcastle disease?ND?,caused by Newcastle disease virus?NDV?,is a kind of infectious diseases,which causes the highly mortality to poultry or avian including pigeons.Pigeon-origin NDV,also known as Pigeon Paramyxovirus type 1?PPMV-1?,has a negative-sense and single-stranded RNA genome,which contains six structural proteins and two nonstructural proteins.The coding genes are listed as 3'-NP-P-M-F-HN-L-5'in the viral genome,and the length of viral genome followed the‘rule of six'.According to the pathogenicity of NDV to chickens that it can be divided into three types:velogenic strain,mesogenic strain and lentogenic strain.However,pigeon-origin NDV has highly pathogenicity to pigeons and mainly causes typical neurological symptom.According to the genome size and phylogenic analysis that NDV strains can be separated into Class?and Class?.Nowadays,Class?viruses were classified into 9 genotypes and Class?viruses were classified into 18 genotypes.Generally,pigeon-origin NDV strains were classified into genotype?in Class?.In this study,the tissue samples from infected racing pigeon including their liver,spleen,kidney,brain and so on were collected in Xi'an city,Shaanxi Province.Isolation of virus used 9-day old SPF chicken embryos,and identified by HA and HI,and then a NDV strain named NDV/pigeon/China/Shaanxi-06/2018 was isolated.The F gene of NDV/pigeon/China/Shaanxi-06/2018 isolated was amplified by RT-PCR,and the phylogenic analysis results showed that the isolated belonged to the Class?sub-genotype?k.Nucleotide sequence analysis revealed that NDV/pigeon/China/Shaanxi-06/2018 strain was more similarly with Shanghai strains Pigeon/Shanghai/215/2011,Pi/China/SH/2013/163and Pi/China/SH/2013/168.And protein sequence analysis revealed that NDV/pigeon/China/Shaanxi-06/2018 strain was more similarly with Shandong strain Pigeon/China/SD069/2012 and Belgium strain PPMV-1/Belgium/11-09620/2011.According to the standard of OIE,the results of the MDT?110 h?and ICPI?0.31?,which showed it's a lentogenic strains.But,the fusion protein cleavage site is ¹¹²R-R-Q-K-R-F¹¹⁷,which is conform to the molecular biological standard of velogenic strain of OIE.Based on the Pigeon/Shanghai/215/2011 whole genome sequence to design 6 pairs of primers with overlapping regions and covering the whole genome of NDV/pigeon/China/Shaanxi-06/2018 strain.The method of RT-PCR,molecular cloning and sequence analysis were performed and acquire the whole genome of NDV/pigeon/China/Shaanxi-06/2018 strain,which is length into 15192 nt.The phylogenic and sequence analysis based on the whole genome indicated that the Shanghai strain Pigeon/Shanghai/215/2011,Shandong strain Pigeon/China/SDLC/2011,Yunnan strain Pigeon/Yunnan/1111/2013 and Anhui strain Pigeon/Anhui/2369/2012 was more similarly with NDV/pigeon/China/Shaanxi-06/2018 strain.Besides,the vaccine strains,genotype?NDV strains and Class?NDV strains was less similarly with isolated strain.The phylogenic and sequence analysis results of each structural gene is the same as genome-wide analysis.The analysis of the whole genome sequence of NDV/pigeon/China/Shaanxi-06/2018 isolated will provide a theoretical basis for the genetic and variation of pigeon-origin NDV.Furthermore,the spread situation of the sub-genotype?k NDV prompt that it is necessary to pay close attention to pigeons and carry out the ND epidemiological investigation of pigeon.