| Newcastle disease (ND) is an acute, contagious severely and fatal infection caused by Newcastle disease virus (NDV). NDV has a special tropism for mucous membrane and infects through respiratory tract and digestive tract easily. Mucous membrane is the first line of defence against NDV, improve the defensive ability of mucosal immune systems can effectively restrain the adsorption and invasion of NDV.Currently, the vaccine with at present commonly used vaccinate can be divided into2kinds:conventional attenuated live vaccine and inactivated vaccine, while several limitations are found in the practice application, how to improve the immune effectiveness of vaccine is urgently needed to conquer in vaccine research field. Transforming the existing vaccine formulations and delivery methods is one possible ways to facilitate this work. Nowdays, nanopartciles mucosa immunity delivery system seems to be one of the most hopeful ways to solve the problem.N-2-hydroxypropyltrimethyl ammonium chloride chitosan (N-2-HACC) has permanent positive charge and water solubility in physiological environment, and can effectively reduce transepithelial electrical resistance, opening the tight junction of epithelial cells, and enhance the transfer of large hydrophilic molecular with Paracellular route, increase the drug uptake rate. In this study, biodegradable material N-2-HACC was chosen as drug carriers, Newcastle disease virus was selected as the model drug, sodium tripolyphosphate as the crosslinking agent, NDV-N-2-HACC-NPs were obtained, which was prepared using a ionic gelation method. In this study, the preparation procedure has been optimized, the physicochemical characteristics, release characteristics in vitro, storage stability and virus content of NDV-N-2-HACC-NPs were systematic studied; The cytotoxicity test and safety test were used to determine the safety of NDV-N-2-HACC-NPs mucosa immunity delivery system. NDV-N-2-HACC-NPs were administrated through intranasal or oral routes and the humoral and cellular immune responses in SPF chicken induced by NDV-N-2-HACC-NPs were observed. The protection rate was investigated by challenge test. The results of study demonstrate that the NDV-N-2-HACC-NPs produced by the optimal formulation have been produced with suitable size, morphous regulation, extremely spherical shape, regular and well-distributed. The mean size of NDV-N-2-HACC-NPs was (303.88±49.84) nm, Zata Potential was+(45.77±0.75)mV, entrapment efficiency was (94.26±4.03)%(n=5) and loading capacity was (54.06±7.37)%(n=5). Releasing tests in vitro has shown that NDV-N-2-HACC-NPs could make NDV sustained release; The cytotoxicity of NDV-N-2-HACC-NPs was lower and N-2-HACC nanopartciles mucosa immunity delivery system has higher safety. The prapared NDV-N-2-HACC-NPs administrated through intranasal or oral routes could induce effective humoral immune responses and local mucosal immune responses in SPF chicken through intranasal or oral vaccination, and protective efficacy was superior to general ND live vaccine.In addition, a viral strain which has heagglutination activity was isolated. The heagglutination activity could be inhibited by Newcastle disease virus La Sota strain standard positive serum. The isolate was confirmed to be Newcastle disease virus based on HA and HI test and was named by JL-DH/10strain. Plaque purification was carried out on Chicken embryo fiber (CEF) cell, and intracerebral pathogenicity index (ICPI) of the JL-DH/10strain in one-day-old SPF chickens were1.86; F gene full-length was cloned followed with sequence analysis and homogeneity analysis, the result showed that the JL-DH/10strain was NDV virulent strain. Phylogenetic tree was drawed by Meg Align software, the result showed that JL-DH/10was NDV Ⅶ virulent strain.Besides, JL-DH/10strain inactivated oil emulsion vaccine, La Sota strain N-2-HACC nanoparticles inactivated oil emulsion vaccine, JL-DH/10strain N-2-HACC nanoparticles inactivated oil emulsion vaccine was developed, the inspection of the semifinished products and finished products were carried out and immune effect to SPF chicken was evaluated. The result showed that the prepared ND inactivated vaccine all were water in oil type and milk white emulsions, with low viscosity, well stability, and the immunity duration was superior to conventional inactivated vaccine.N-2-HACC nanopartciles mucosa immunity delivery system had been constructed successfully allows vaccine to release the antigen slowly, which could prolong immunity duration, decrease the inoculation times, protect the antigen from damage, simplify inoculation procedure, decrease the vaccination methods, increase the transport efficiency in cell, enhance the immunological effect, which possessed both scientific significance and application values and had potential prospect in applications.
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