| Newcastle Disease Virus (NDV) belongs to the genus Avulavirus within the family Paramyxoviridae. Newcastle disease (ND), a extremely contagious destructive disease, is caused by NDV and identified as one out of two the most serious disease. Currently, immune prevention is the main method of prevention and control of Newcastle disease. Thermostable vaccine represented by NDV V4heat-resistant strains has the advantages of lower requirements for cold chain, easy to use, low toxicity, good immune effects, good cohabitation infection and so on, and has broad application prospects in China and other developing countries. So far there are some NDV heat-resistant strains, such as V4,I-2, HB92, B95, TS09and so on. TS09strain was obtained from V4strain through heat-resistant breeding and purification on chicken embryo by our team. If NDV heat-resistant strains is used as a heat-resistant carrier by reverse genetics technology, the key immune-derived genes of other poultry disease is inserted into it to develop polyvalent thermostable live vaccines for main diseases of poultry. That will generate far-reaching impact on the prevention and control of poultry disease. However, there has been no report on successful building of the reverse genetic operating system of NDV thermostable strain. Therefore, on the basis of TS09strain, this study carried out the construction of the reverse genetic operating system of NDV thermostable strain.1. Cell-adaptive culture of NDV thermostable strainExisting reverse genetics operating systems were mainly established on the cell, but the TS09strain, a chick embryo adapted strain, could not highly proliferate on cell. First of all, the work of adaptive culture and purification on BHK cell of TS09strain was carried out. The results of optimization of cell culture condition showed that0.2μg/mL TPCK trypsase concentration was maximum non-toxic concentrations. A new, purified and BHK cell adapted strain, named TS09-C strain, was gained after17continuous passages cultured and3times purification by limiting dilution. Determination results of biological characteristics showed that EID50value of TS09-C declined from the original108.6/mL to105.7/mL, but TCID50value increased from the original103.3/mL to107.9/mL. It indicated TS09-C strain was cell adapted strain and maintained the characteristics of non-toxic and heat-resistant of parent strains. So cell-adaptive strain of NDV thermostable strain was successfully established. Sequence analysis showed that TS09, TS09-C and V4still kept a close genetic relationship. 2. Whole genome sequencing and analysis of TS09-C strains10pairs PCR primers were designed for TS09-C strain genome sequencing and analysis. After sequence splicing of sequencing results, TS09-C complete genome sequence was obtained, which was15186nt. Sequence analyses indicated that TS09-C was on the branch of type Ⅰ, the same with1-2,1-2progenitor and Ulster-67, and encoded6structural proteins (NP, P, M, F, L and HN) and two non-structural proteins (V and W). Compared amino acid homology of every protein between TS09-C strain and V4strain, it was found that the M protein had the minimum homology which was92.3%, and F and HN protein had high homology, both greater than99%.The preliminary study on the mechanism of heat-resistant of TS09-C strain was carried out by Bioinformatics Technology. When amino acid sequence compared between heat-resistant strains and non-heat-resistant strains,23different amino acids were found, but most of the mutations of the conservative, and non-conservative mutations were found on the P and W proteins. Protein secondary structure comparative analysis showed that differences on the secondary structure between non-heat-resistant strains and heat-resistant strains were mainly in the alpha helical region of the HN protein. The discovery of specific sites (or structures) of these heat-resistant strains offered idea for our further study on mechanism of heat-resistant.3. Construction of infectious full length cDNA clone of TS09-C strainBased on TS09-C strain whole genome sequence, the restriction endonuclease sites analysis was carried out, and a construction strategy of infectious full length cDNA clone of TS09-C strain was designed. A total of8pairs of primers were designed for construction of full-length cDNA clones.8fragments (A, B, C, D, E, F, G1and G2) were amplified by high-fidelity RT-PCR, and G was gained by fusion PCR of G1and G2fragments. The T7RNA polymerase promoter was introduced in upstream of the A fragment, T7RNA polymerase termination and Delta hepatitis virus ribozyme sequence were introduced to the downstream of G fragment by fusion PCR.3intermediate plasmids (pBR-ABC, pBR-DE and pBR-FG) were built by enzyme-connection method. Fragment ABC and DE were inserted into plasmid pBR-FG by enzyme-connection method to generate plasmid pBR-TS09-C. Compared with TS09-C strain genome sequences, there were6mutations on the plasmid,4of which were synonymous mutations. And the other2mutations caused amino acid changes which existed in the other strain, so it not affect subsequent work of virus rescue recovery. 4. Establishment and application of fluorescent RT-PCR for NDV detectionIn order to provide a rapid and quantitative detection method for the subsequent NDV research, in this study a method of fluorescent RT-PCR for detection of NDV was established. A relatively conservative sequence was found in Newcastle disease virus F gene by alignmenting Newcastle disease virus sequences published in NCBI. A pair of primers and a probe was designed based on the relatively conservative sequence. A highly sensitive, specific and reproducible method of fluorescent RT-PCR for detecting Newcastle disease virus was established by a series of condition optimizations, specific experiment, sensitivity experiment and repeated experiment. The lowest concentration of the initial virus that could be detect by the method was102.2ELD50/mL, and in the range of108.2-102.2ELD50/mL, Ct value and the logarithm of the concentration of the virus (1gELD50/mL) showed a good linear relationship, R2of which was0.9975. The method had no cross-reaction with avian influenza virus, fowl pox virus, E. coli and other avian pathogenic nucleic. And inter-and intra-assay reproducibility was good. In clinical sample detection assays, compared with the method of virus isolation by chicken embryo, the agreement, diagnostic sensitivity and diagnostic specificity of fluorescent RT-PCR were98.5%,50%and98.4%, respectively. For this reason, the development of fluorescent RT-PCR method provided a rapid and effective method for Newcastle disease virus detection and laid a foundation for the study of Newcastle disease virus.To sum up, this study completed the works of establishment of cell-adaptive strain of NDV thermostable strain, sequencing and analysis of TS09-C strains whole genome and construction of infectious full length cDNA clone of TS09-C strain, which laid a good basis for establishment of reverse genetic operating system of NDV thermostable strain. Fluorescent RT-PCR method for detection of NDV provides a technology platform for follow-up research of NDV... |
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