Abstract：Avain influenza (AI) and Infectious bursal disease (IBD) are harmful to poultry industry, and vaccination is one of the most effective method of controling these two diseases. In order to explore the feasibility of Duck enteritis virus (DEV) vector combined vaccine against AI and IBD that were used in chicken (Gallus gallus), two recombinant viruses that co-expressed hemagglutinin (HA) gene of H5 type Avian influenza virus (AIV) and virus protein 2 (VP2) gene of Infectious bursal disease virus (IBDV) were constructed, and experiments were done to evaluate the immune efficacy of the recombinant viruses. HA gene from A/Chicken/Guizhou/4/2013 (H5N1) (GZ/4) and VP2 gene from IBDV (HLJ-0504) were connected by internal ribosome entry site (IRES) sequence in different orders, HA-IRES-VP2 and VP2-IRES-HA. The two gene fragments were then inserted between unique short region 7 (US7) and US8 genes of DEV genome in fosmid T-us78Kan ccdB which is overlapping fosmid DNAs rescue system. Using this system, the target gene between the US7 and US8 genes of the DEV genome were successfully inserted, and the two recombinant viruses rDEV-HA/VP2 and rDEV-VP2/HA were successfully constructed. The in vitro experiments showed that the inserted gene could be stably inherited and express without affecting replication of virus vector in cells. In order to evaluate the protective efficacy induced by recombinant virus in specific pathogen free (SPF) chicken, groups of ten 3-week-old SPF chicken were immunized with 103 tissue culture infective dose (TCID50), 104 TCID50, 105 TCID50 recombinant virus, and challenged with 100 median lethal dose (LD50) GZ/4 or 100 LD50 very virulent IBDV (vvIBDV) at 14 days post vaccination. Recombinant DEV with only HA gene of GZ/4 inserting between US7 and US8 genes (rDEV H5-8) and commercial IBDV vaccine Gt strain were immunized as control. The result showed that chicken which were immunized with 103 TCID50, 104 TCID50, 105 TCID50 rDEV-HA/VP2 induced low level of hemagglutination inhibition (HI) antibody, and no IBD antibody could be detected. The protective efficiency against H5 AIV was lower than 70%, and the protective efficiency against vvIBDV was lower than 30%, respectively. While, chicken which were immunized with 103 TCID50, 104 TCID50, 105 TCID50 rDEV-VP2/HA could not induce HI or IBD antibody. The protective efficiency against IBDV was lower than 40%, and the protective efficiency against H5 AIV was 0. However, chicken which were immunized with rDEV H5-8 or Gt strain could induce 100% protection against H5 AIV or vvIBDV. In conclusion, the two recombinant viruses rDEV-HA/VP2 and rDEV-VP2/HA could not induce complete protection against AIV or IBDV, and the strategy of using IRES to connect HA and VP2 genes to construct the DEV vector vaccine against AI and IBD was not feasible. This study provides a reference for the construction of DEV vectored multi vaccine in chicken
[1]Neumann G, Chen H, Gao G F, et al.H5N1 influenza viruses：outbreaks and biological properties[J].Cell Research, 2010, 20(1):51-61
[2]Peng Y S, Li X D, Zhu H B, et al.Continual Antigenic Diversification in China Leads to Global Antigenic Complexity of Avian Influenza H5N1 Viruses[J]., 2017, 7:-
[3]祁小乐，高立，吴关，等.鸡传染性法氏囊病病毒基因突变对病毒体外复制的影响[J].畜牧兽医学报, 2011, 42(11):1577-1583
[4]Prandini F, Simon B, Jung A, et al.Comparison of infectious bursal disease live vaccines and a HVT-IBD vector vaccine and their effects on the immune system of commercial layer pullets[J].Avian Pathology, 2016, 45(1):114-125
[5]于建立，王雷，魏波，等.鸡传染性法氏囊病疫苗应用现状与研究进展[J].安徽农业科学, 2016, 44(1):91-92
[6]Li C, Bu Z, Chen H.Avian influenza vaccines against H5N1‘bird flu’[J].Trends in Biotechnology, 2014, 32(3):147-156
[7] Hermann M, Egbert M, Nicolas E, et al.Current status of vaccines against infectious bursal disease[J].Avian Pathology, 2012, 41(2):133-139
[8]陈柳，余斌，倪征，等.表达小鹅瘟病毒VP2蛋白重组鸭瘟病毒的构建及其生物学特性[J].中国农业科学, 2016, 14:2813-2821
[9]Liu J X, Chen P C, Jiang Y P, et al.A duck enteritis virus-vectored bivalent live vaccine provides fast and complete protection against H5N1 avian influenza virus infection in ducks[J].Journal of Virology, 2011, 85(21):10989-10998
[10]Liu J X, Chen P C, Jiang Y P, et al.Recombinant duck enteritis virus works as a single-dose vaccine in broilers providing rapid protection against H5N1 influenza infection[J].Antiviral Research, 2013, 97:329-333
[11]陈普成.2009.鸭瘟病毒部分基因组序列的克隆及重组病毒转移载体的构建[D].硕士学位论文，中国农业科学院，导师：田国彬.(Chen P C.2009.Cloning and analysis of partial genomic sequence of duck enteritis virus and construction of transfer vector for recombinant virus [D].Thesis for M.S, Chinese Academy of Agricultural Sciences, Supervisor: Tian G B.)
