关键词: 鸭肠炎病毒, H9亚型禽流感病毒, HA, 重组病毒

Abstract:

【Background】The H9N2 avian influenza virus (AIV) pathogenicity and transmissibility have recently showed an increasing trend. Moreover, it donates partial or even whole cassette of internal genes to generate novel reassortants, which is serious threat to poultry industry and public health. Waterfowls are considered as the natural host and reservoirs of AIVs and play an important role in the spread and mutation of AIV. Therefore, successful control of the spread of H9N2 in waterfowls contributes significantly to poultry industry and public health. Duck enteritis virus (DEV) taxonomically belongs to family Herpesviridae and infects ducks, geese, and swans, which results in high mortality and decreased egg production in domestic and wild waterfowl. DEV may be a promising candidate viral vector for aquatic poultry vaccination because it has a large genome and good immunogenicity. 【Objective】 In this study, we constructed a recombinant DEV expressing the hemagglutinin (HA) gene of a H9N2 virus that was inserted into the deleted viral gE gene, and then its characterization to explore the feasibility of the recombinant DEV as a live vectored vaccine was studied.【Method】 The HA gene of H9N2 was cloned to construct the transfer vector pT-gE-HA. Plasmid pT-gE-HA and rDEV-△gE-GFP were co-transfected into CEF cells. After plaque-purification, we obtained a pure recombinant virus which expressed H9N2 AIV HA protein, and named as rDEV-△gE-HA; PCR and sequencing assay were used to identify the recombinant virus. The recombinant virus was passaged in primary CEF 20 times to evaluate the genetic stability of the foreign gene in the recombinant virus. Ducks were inoculated with 10 ³EID₅₀ rDEV-△gE-HA, then challenged with lethal DEV. Ducks were vaccinated intramuscularly with 10 ³-10 ⁶ TCID₅₀ of rDEV-△gE-HA. At 14, 21, and 28 days post-vaccination (d.p.v.), sera were obtained from all ducks to monitor HI antibody against H9N2 AIV. At 28 d.p.v. all ducks were challenged with 10 ⁸ EID₅₀ H9N2 (A/duck/GD/08) by intravenous injection. Oropharyngeal swabs were collected from H9N2 virus challenged ducks to detect viral shedding.【Result】The recombinant expression vector pT-gE-HA was constructed and transfected with rDEV-△gE-GFP in chicken embryo fibroblasts (CEF). After 3 rounds of plaque-purification, the purified rDEV-△gE-HA was obtained. The results of the PCR and sequencing indicated that the HA expression cassette had already successfully been inserted into the DEV. The HA gene were stably maintained after the recombinant was passaged 20 times in CEF. Ducks inoculated with 10 ³ TCID₅₀ of rDEV-△gE-HA were protected against lethal DEV. HI antibody was detected in all vaccinated ducks at 14 d.p.v. and slightly increased at 21 d.p.v.. Challenge with H9N2 at 28 d.p.v., ducks inoculated with 10 ³, 10 ⁴ and 10 ⁶TCID₅₀ were completely protected from challenge, as evidenced by the finding that no virus was recovered from collected oropharyngeal swabs, while 80% ducks (4/5) inoculated with 10 ⁵TCID₅₀ were protected.【Conclusion】In this research, we successfully constructed a stable recombinant DEV expressing the HA of H9N2 AIV. The recombinant DEV remained the protective efficacy of the parental virus against lethal DEV parental virus. Moreover, it could induce HI antibody in ducks and protect no less 80% ducks against H9N2 AIV challenge, although the titer of HI antibody was not too high. This study laid a foundation for developing bivalent vaccine controlling DEV and AIV infection.

Key words: duck enteritis virus, H9 subtype AIV, HA, recombinant virus