Selection of a Representative Virus Clone and Construction of a Full-length cDNA Clone of La Sota Strain of Newcastle Disease Virus
Newcastle disease (ND) is a major animal infectious disease of poultry. At present, it is listed as notifiable disease reported to the OIE and class one animal disease ruled by Chinese government. Reverse genetic system for negative-strand RNA viruses is a very powerful tool to study the mechanism of pathogenesis or develop new vaccines on the basis of gene manipulation.First of all, La Sota strain of Newcastle disease virus (NDV) which has been widely used for prevention and control of ND was purified. Now that lentogenic strains hardly form plaques in chicken embryo fibroblast (CEF) cell cultures, but can produce typical cytopathic effects, we designed a method called cytopathogen assay to purify the La Sota virus. In essence, the method is the same as plaque assay. The unique feature of the method was to clone the virus by \'picking\' the cytopathogens-that is, literally excising a small plug of agar and infected cells from a cytopathogenic area using a Pasteur pipette. As a result, eleven virus clones were aquired after purification. Then partial genome sequencing including F gene 47～435 nucleotides of those virus clones indicated that most virus clones had shown more than 99% homology with La Sota strain. This just justified the reliability of the method, and that they were all lentogenic strains. But how different were between clones picked? If there was a significant difference in the biological properties between them, was it necessary to make a choice to select a better or representive one from them? In view of this, those virus clones were tested, analyzed and compared by hemagglutinin thermostability assay, virus growth kinetics and immunogenicity assessment. The main purpose for choosing a representive virus clone which ought to have good biological properties is to avoid eliminating the one which had worst biological properties. Finally, clone 4 that had shown best growth property in SPF chicken embryos and had induced relatively high antibody titres in SPF chicken, was chosen as the representive virus clone.Then eight primers were designed for the amplication of the whole genome of La Sota virus. Meanwhile, molecular tag inserted in the primer was used to identify the rescued virus and original parent virus. By means of RT-PCR method and bioinformatics tools (softwares for RNA secondary structure prediction), eight cDNA fragments covering the whole genome were obtained. Subsequently, they were cloned into pMD18-T vector and sequenced.Since Fusion (F) protein plays a vital role in the process of virus infection and is closely related to the virus virulence, after sequencing bioinformatics tools such as Emboss tools, DNAstar software, Swiss-Model Server, were used to predict and analyze the secondary structure, three-dimensional structure and antigen sites of F protein. Then the relationship between its structure and biological function was deeply discussed. Furthermore, genetic diversity of functionally important region in fusion gene of NDV was analyzed by Mega and DnaSP softwares. As a result, some evolutional information of NDV was gotten. It means that negative selection pressure is the main power to propel the evolution of the genome of NDV. However, some small regions or nucleotide sites in the genome are experiencing adaptive evolution. It just coincides with the epidemic situation of ND. Besides, after sequencing, we were capable of knowing the distribution of restriction enzyme sites in the genome. Subsequently, eight cDNA fragments were linked together after continual restriction enzyme digestion and ligation in sequence. Eventually a full-length cDNA clone of La Sota strain of NDV named pBR-NDV was successfully constructed. However, reverse genetic system of negative-strand RNA viruses which is a very useful platform can not be achieved in one day, so this research builds a solid foundation for the establishment of reverse genetic system of La Sota strain of NDV.