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jwk3029(金币+90, 翻译EPI+1): 2010-05-20 17:16:48
sirljz(金币+3):谢谢交流 2010-05-20 17:33:41
仅供LZ参考。
Chicken Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious, acute septic infectious disease. ND is among the most serious disease threats to the poultry industry worldwide. It’s included in the list A infectious disease by OIE. NDV, is a negative-sense single-stranded RNA virus, a member of rubulavirus, paramyxoviridae family, affects most avian species, and spread as the immigration of birds.
For a long time, ND had been well prevented by a comprehensive measure mainly by vaccination in China. But, ND has new features in recent years, such as more and more non-typical ND, even in chicken flocks have high antibodies concentration, which might induced by variant or highly virulent strains. Newly identified geese-derived NDV variant attracted researchers’ attention on the pathogenesis of inapparent ND.
In this study, we isolated a wild NDV strain (strain NDV ND-xx08) from layers with no apparent ND symptoms, in a hennery of Xinxiang. After basic assay on its biological characteristic, the F and HN gene were amplified, immune protection efficiency was evaluated, which would provide theoretical foundation for investigation on ND variation and prevention, provide important basis for formulation of ND prevention methods, and also be a useful reference for NDV molecular epidemiology in Henan, enrichment for ND molecular epidemiology features in China.
Firstly, biological characteristics of NDV ND-xx08 were examined. Cultured by inoculating SPF chicken embryo, this new strain was able of inhibiting blood coagulation, and was neutralized by NDV standard positive serum, can’t be inhibited by bird flu (subtype H5, H9) and EDS-76 standard positive serum. So we identified this isolation as NDV. It took average 50h (MDT) for minimum lethal dose of this virus to kill chick embryo. Pathogenicity index for 1-day SPF chicken inoculated the isolation in brain (ICPI) was 1.71, Pathogenicity index for 6-week chicken inoculated the virus through vein (IVPI) was 2.38. These data revealed NDV ND-xx08 was a highly virulent strain.
Secondly, the F and HN gene were amplified by RT-PCR from RNA (extracted by Trizol method) using primers we designed. After sequencing, we analyzed the nucleotide sequences and the protein sequences. DNAstar software was used to compare the NDVF gene 47nt-420nt region of this strain with other 46 typical strains published in China and other countries, draw the gene phylogenetic tree and analyze the genetic variation.
The open reading frame of NDV ND-xx08 strain F gene is 1662 bp, encodes 553 amino acids. The lysis site locates at 112R-R-Q-K-R-F117. Phylogenetic analysis shows this strain belongs to VIIe genotype. Comparison of the nucleotide sequence for this strain and other published strains, revealed 82.7%-97.8% homology, 87.5%-97.7% homology in amino acid sequence. A 91.5% amino acids sequence homology was found when compared to F48E9, 88.1%, 88.6% and 88.3% to Lasota, Clone30 and V4 respectively. This means NDV ND-xx08 is a highly virulent NDV strain has some variation in the molecular level.
The open reading frame of NDV ND-xx08 strain HN gene is 1716 bp, encodes 571 amino acids, belonging to C group. The nucleotide sequence of this strain has 80-98.4% homology to other 24 strains. High homology was found to Beijing strain (SRZ03) and Jiangsu strain (zj1), 98.1% and 98.4% respectively. On the gene phylogenetic tree, ND-xx08 strain is near to Beijing strain (SRZ03) and Jiangsu strain (zj1), they are basically in the same gene group; but far away from Lasota and Clone30, belonging to different groups. The amino acid sequence of ND-xx08 has 87.2%-98.2% homology with other 24 strains, a low homology of 88.2% and 89.3% to Lasota and Clone30 respectively.
At last, immune protection experiments were carried out using this strain. Inactivated oil emulsified vaccine were prepared by original amniotic fluid and amniotic fluid diluted 10, 100 and 1000 times according to traditional method. Chicken flocks were inoculated twice with these four oil-emulsified vaccines, following with examination of antibody concentrations. Subsequently, we evaluated the protection efficiency of chicken flocks to the challenge of the virus strain, found the vaccine with high efficiency and best dose.
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