TECHNICAL FIELD

The present disclosure relates generally to molecular biology technology and microbiology, and in particular relates to a heat-resistant recombinant Newcastle Disease Virus (NDV) vaccine strain capable of expressing truncated Fiber 2 protein of Fowl Adenovirus serotype 4.

More specifically, the present disclosure also relates to a reverse genetic manipulation technology used to transform the NDV LaSota vaccine strain by HN gene replacement and truncated Fiber 2 gene insertion to obtain a new recombinant NDV vaccine strain rLS-tFib2-C, and application of the strain in preparation of heat-resistant live vaccine against Angraradisease and Newcastle disease.

BACKGROUND

Fowl Adenovirus (FAdV) is a worldwide infectious disease pathogen that frequently occurs in poultry and wild birds. FAdV is a member of the avian adenovirus genus of the adenovirus family. The viruses of this genus can be divided into three groups, namely group I, group II and group III. The relationship between group II, group III FAdV and disease is relatively clear, while group I FAdV has the most types of strains and the most complicated relationship with disease, and there are as many as 12 serotypes of group I FAdV. Fowl Adenovirus serotype 4 (FAdV-4) is one of the representative strains of FAdV in group I. The virus mainly causes Angraradisease, also known as hydropericardial-hepatitis syndrome. The disease was first discovered in the Ankara region of Pakistan, so it is called Angraradisease, and its fatality rate can reach 30%-70%. Since 2015, Angraradisease has spread and broke out in Henan, Anhui, Shandong and other places, causing huge economic losses to the poultry industry in China. At present, there is no vaccine against Angraradisease on the market, which has caused great difficulties in prevention and control of the disease. The development of a safe and efficient Fowl Adenovirus vaccine is an important means to control the virus epidemic.

FAdV is a double-stranded DNA virus with a molecular diameter of about 70-90 nm, no envelope structure, and with an icosahedral symmetric structure. The capsid protein of the virus is mainly composed of Hexon, Penton and Fiber. Fiber and Penton are respectively responsible for the two processes of virus adsorption and invasion during the virus infection cycle. Fiber protein has two independent Fiber encoding genes named Fiber1 and Fiber2. Penton and Fiber2 are antigenic and can induce virus neutralizing antibodies.

Newcastle Disease (ND) is an acute, highly lethal and highly infectious disease in chickens caused by Newcastle Disease Virus (NDV). The epidemic of ND has caused huge economic losses to the poultry industry and is listed as one of the two A poultry infectious diseases by the International Veterinary Administration. NDV has only one serotype, but the virulence of different strains is quite different. According to the virulence, the Newcastle disease virus can be divided into virulent strains, medium virulence and weak virulent strains.

In China, Newcastle disease is one of the animal diseases subject to national compulsory immunization. Newcastle disease vaccines are divided into two categories: live vaccines and inactivated vaccines. Live vaccines include lineage I vaccines (Mukteswar strain), lineage II vaccines (HB1), lineage III vaccines (F strain), lineage IV vaccines (LaSota strain), Clone-30 and V4 strain. Among them, lineage I vaccines are medium-virulence live vaccines, and the others are attenuated live vaccines. The inactivated vaccine is mostly an oil emulsion vaccine prepared by lineage IV vaccines or clone 30 after inactivation. LaSota strain vaccine is very popular among farmers for its low virulence, good immunogenicity and high immune protection rate. However, the LaSota strain virus has poor thermal stability, and the live vaccine prepared therefrom has high requirements on the cold chain, which is not conducive to the promotion and application in tropical and remote areas.

With the continuous advancement of reverse genetic manipulation technology, new multiple live vaccine based on NDV vectors have become a hot spot for new vaccine research and development. Many pathogenic immunogenic genes have been successfully expressed in NDV vectors, and have achieved better immune protection effects, such as VP2 protein of infectious bursal disease virus and HA protein of H5 subtype avian influenza virus. Such vaccines take advantages of inducing systemic immunity, high growth characteristics of chicken embryos, low production costs, and simple immunization methods.

However, most of current research reports are solely on the Angraradisease vaccine or Newcastle disease vaccine, and there is no research report on the dual live vaccine. Therefore, in view of the two important diseases of Angraradisease and Newcastle Disease, which seriously endanger China's poultry industry, there is an urgent need to develop a new dual live vaccine that is safe, efficient, stable, and simple in immunization.

