Construction And Purification Of Recombinant MDV Expressing The HA Gene Of Subtype H5 HPAIV

The highly pathogenic avian influenza (HPAI) is a notifiable disease according to the Office International des Epizooties (World Organization for Animal Health). Specifically, the highly pathogenic H5N1 avian influenza virus have been circulating among poultry in China in recent years. Killing infected birds is the main measure for the control of HPAI taken by the developed contries. However, for economic and practical reasons, inoculation of inactivated vaccine is common in China instead of using slaughter policy, but this kind of vaccine could not induce good cellular and mucosal immunity, and was interfered by maternal antibodies. Thererfore, novel vaccines are needed to control this disease.Several virus vectors that express foreign antigens of other avian pathogens have been developed, and these viruses showed significant vaccine efficacy against a variety of avian diseases. Among them, MD live vaccine viruses are known to infect persistently within chickens in spite of the presence of neutralizing antibodies in sera and induce a high titer of antibody against MDV. In addition, MDV has a natural host range limited to avian, and therefore, there is no rearrangement and variation of AIV genome during the use of recombinant MDV.In this study, we constructed recombinant MD viruses expressing AIV-H5 hemagglutinin. Two pairs of primers were designed according to the published complete genomic sequence of MDV to amplify the flanking regions of 2 nonessential regions (IRS-US and US9-US10). The HA gene, gB promoter and CMV promoter were amplified and cloned into pCR2.1 vector. Plasmids pCR2.1-CMV-HA-GFP and pCR2.1-gB-HA-GFP were constructed and exogenous gene lac/smGFP was inserted between duplex loxp sequences as a marker. Eight transfer vectors were obtained by the ligation of flanking regions, HA genes and promoters with different combinations.Transfer vectors were transfected into the CEF cells with genomic DNA of MDV Rispense CVI 988 strains. A total of 8 recombinant MDVs were successfully generated, and the recombinant MDVs were the vaccine candidates for the control of HPAI and MD diseases.