Isolation, Identification Of Newcastle Disease Virus And Cloning And Sequencing Of Its HN Gene

Two isolates of virus which could agglutinate chicken red blood cell were isolated from two disease-infected poultry farms in Baoding of Hebei. These agglutinations were inhibited and neutralized by Newcastle disease virus standard antisera. These isolates were confirmed to be Newcastle disease virus based on HA and HI test and were named by HBB and HBW. Pathogenicity of the twe isolates was identificated. Mean death time (MDT) of chicken embryos of these isolates were 40.8, 53.7h separately. Intracerebral pathogenicity index (ICPI) of these isolates in l-day-old SPF chickens were 1.93, 1.86 , and Intracerebral pathogenicity index (IVPI) of these isolates in 6-week-old SPF chickens were 2.89 and 2.72 separately. The results demonstrated that the two isolates of virus were high virulent Newcastle disease virus.10 12-day chickens, which were not immuned by NDV, were challenged in the experiment. All were infected after 24 hours, of which challenged by HBB isolate caused a high mortality by 100%;chichens challenged by HBW had a high mortality by 80%;the other had a nerval symptom which were not recaver. Chickens with HI above 61og2 challenged by HBB isolate were all died during 192 hours;4 chickens with HI above 61og2 challenged by HBW isolate were died during 192 hours. The results indicated: The two isolates have a high virulent to chicken, and chickens above 61og2 were even not protected. The other result is that vaccine used now were not available.Ribonucleic acid were extracted by TRNzol from virions of Newcastle disease virus after propagated and condensed. Two forward primers were used for RT-PCR step to amplify hemagglutinin-neuraminidase(HN) gene of the two NDV strains;the PCR products checked by agrose gel electrophoresis were purified dy agrose gel fraction method, and cloned into PBS-T-easy vector. Cloned plasmids were transformed into E.coli JM109, and positive plasmids were selected to sequencing. According to the strains of the NDV reported home and abroad, the phylogenetic tree of the NDV and the analysis of genetic variation were obtaind by DNAStar software. The results demonstrated that: 897bp of the HN gene was amplified successfully, the homologous rate of HN gene nucleotides sequences was 99.7%, and of the deduced amino acid sequences was 99.3%. The HN genesequences from two isolates shared a high identity between 82.8%~93.2% with that of related sequences reported home and abroad.The amino acid residues predicted, shared an identity between 90.0%~96.0%with that of other strains. The phylogenetic tree showed that the two isolates and the epidemic strainsVII demestic were closely related.Study showed that: The isolates of HBB and HBW were all belong to high virulent strains of Newcastle Disease Virus. With high identity of nucleotide between the two isolates, the HBB strain have a higher virulence than the HBW strain.