| Newcastle disease(ND)caused by Newcastle disease virus(NDV)is regarded as one of the most important viral infectious diseases,which is acute and highly contagious to susceptible birds.At present,the disease is a serious threat to healthy development of the poultry industry as one of the main diseases in our country.Therefore,studies of NDV isolation and identification,genetic variation,new and rapid diagnosis technique of pathogen will provide materials and theoretical basis for molecular epidemiology,disease diagnosis and prevention with great significance to the healthy development of the poultry industry.Three strains of virus was isolated from clinical samples suspected for Newcastle disease(ND)in Guangxi in 2011～2013.The isolates named as GXC110083,GXGF12001 and GXC201308 were proved to be NDVs by HA test,HI test and RT-PCR detection.The virulence evaluated by the classical pathogenicity tests e.g.MDT and ICPI for NDV,animal regression test and sequence analysis based on a portion of nucleotide sequences of F gene were also done.The results showed that MDT and ICPI of the NDV isolates GXC110083,GXGF12001 and GXC201308 were 62.6h,67.6 h,69.2h and 1.56,1.50,1.30 respectively.Obvious clinical signs of susceptible chicks of 30-day-old infected by the isolates was found at 2 days post inoculation and all the birds were dead in 5 days post inoculation.The NDV isolates was proved to be virulent strains.The cleavage site of F protein of the virus was 112R-R-R-K-R-F117,112R-R-Q-K-R-F117 and 112R-R-R-K-R-F117,with the motif characteristic of the virulent NDV strain.The result of phylogenetic analysis revealed that the NDV isolate GXGF12001 belonged to genotype Ⅷ,GXGF12001 and GXC201308 belonged to subgenotype Ⅶh.NDV strains GXC110083 and GXGF12001 were purified by three rounds of plaque purification on the BHK-21 cells.Primers for the amplification of the complete genome sequence of purified strains GXC110083 and GXGF 12001 were designed and synthesized,then the complete genome sequences were sequenced.The nucleotide homology and genetic variation of two strains were analyzed compared with other reference strains of NDV by the relevant molecular biological software.The results showed that the genome of two purified strains was 15192nt in length,in line with the "six bases rule".The GXC110083 purified strain belonged to genotype Ⅶ,sharing the highest nucleotide homology and the same genotype with Guangxi egret strain WBE-10.The GXGF 12001 purified strain belonged to genotype Ⅷ,sharing the higher nucleotide homology and the same genotype with Qinghai strains QH1 and QH4.The real time fluorescence quantitative RT-PCR assay with SYBR Green I was developed for rapid detection of Newcastle disease virus.The method had a good linear relationship with a correlation coefficient of 0.9948.The qRT-PCR assay had a detection limit of 5.03 copies·μL-1 of the virus nucleic acid and did not cross react with other viruses including AIV,IBV and IBDV.The variation coefficients of the assay were below 5%,which indicated good reproducibility.The method was used to detect the main organs including cecal tonsil,spleen,lung,trachea,brain of 40-day-old non-immune chicken infected with NDV.The results showed that the virus was first detected in the cecal tonsil at 48 hours post inoculation,then virus could be detected in all the other main organs at 144 hours post inoculation and the viral load had no apparent difference in detected organs.In addition to brain,the other five organs showed apparent lesions.Dynamic changes of NDV replication could be revealed by the test,which conformed that different lesions and clinical manifestations exhibited a certain correlation to viral load.The development of qRT-PCR method laid the technical foundation for the diagnosis of NDV and study of sustained virus infections. |
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