Construction Of A Recombinant Newcastle Disease Virus Expressing Codon Optimized H5N1Subtypes Avian Influenza Virus HA Protein

Avian influenza virus (AIV) induced serious syndrome including respiratory symptoms, neurological complications, gastrointestinal symptoms and critical generalized disorder, even death in infected birds. Epidemic outbreaks of highly pathogenic avian influenza (HPAI) caused by viruses of the subtype H5N1emerged in2003in several countries of Southeast Asia. Most of human infection and death cases were traced to direct intense contact with domestic poultry infected with HPAFV H5N1. Traditionally, people often cull the whole infected flocks for controlling AIV epidemic. However, because of the wide spread of HPAI, culling may not be effective to eliminate the virus. The development and application of vaccine are still the most effective means against HPAIV. Current vaccines include subunit vaccines, DNA vaccines, inactivated vaccines and live vector vaccines, and each has limitations.Like Avian influenza virus, Newcastle diseases Virus (NDV) can be widely spread in poultry and caused high mortality rate of Disease. NDV is a single-strain, negative sense and non-segmented RNA virus, which is a member of paramyxovirus family and have genome approximately15Kb. The full length genome encode viral phosphor protein(P), the RNA dependent RNA polymerase (L), matrix protein (M), nucleoprotein (NP), hemagglutinin-neuraminidase protein (HN) and fusion protein (F).In many countries, the immune prevention and control of Newcastle disease is compulsory. NDV as a live vaccine vector has its unique advantages. First of all, it can be very convenient to test the immune and none-immune animals via serological detection. Secondly, the synthesis can be easily administrated to large number of animals by drinking water, aerosol, or eye-drop. Thus, it is very suitable for mass immunization of poultry.In order to enhance the efficacy of protection, a variety of new-novel vaccines have been successfully developed. Codon-optimization as a useful approach of enhancing the protection efficiency has already been developed. In this study, two recombinant Newcastle disease viruses (rNDVs) expressing the H5HA gene without cleavage site (HAmut), and codon-optimized H5HA (optiH5HA) gene of the strain A/Duck/Guangdong/1/1322/H5N1(GD/1/1322) have been generated as rL-H5HAmut and rL-optiH5HA, respectively. Those recombinant viruses can expressed HA protein in BHK and CEF cells stably, which were confirmed by western blot and indirect immunofluorescence (IF) assay. Serum samples collected before and after vaccination were assessed by hemagglutination inhibition (HI) assay. Significant HI titers were observed in both rL-optiH5HA vaccination group and the rL-H5HAmut group at one week post-prime, reached a peak at three weeks and decreased slowly at four weeks post inoculation. However, all HI titers were significantly higher in the rL-optiH5HA-vaccinated group compared with the group given rL-H5HAmut from one week to four weeks post-prime. A subsequent boost immunization confirmed that the HI titers in chickens vaccinated with rL-optiH5HA were still significantly higher than those in chicken vaccinated with rL-H5HAmut in same dose. These results showed that codon optimization improved immunogenic efficacy and recombinant virus rL-optiH5HA has great potential for application in market.