Construction of recombinant Marek's disease virus (MDV) lacking the meq oncogene and co-expressing AIV-H9N2 HA and NA genes under control of exogenous promoters.
To develop a recombinant Marek's disease virus (rMDV1) co-expressing the hemagglutinin gene (HA) and neuramidinase gene (NA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain and lacking the meq oncogene that shares homology with the Jun/Fos family of transcriptional factors, a wild strain of MDV GX0101 was used as parental virus, the HA and NA genes co-expression cassette under control of the CMV and SV40 early promoters was inserted at two meq sites of GX0101 to form a new meq knock-out mutant MDV (MZC12HA/NA) through homologous recombination. MZC12HA/NA was reconstituted by transfection of recombinant BAC-MDV DNA into the secondary chicken embryo fibroblast (CEF) cells. Highly purified MZC12HA/NA was obtained after four rounds of plaque purification and proliferation. In vitro growth properties of recombinant virus were also inspected and concluded that the MZC12HA/NA had the same growth kinetics in CEF cultures as its parental wild type virus GX0101. Southern blot indicated that co-expression cassette was successfully inserted at two copies sites of meq gene, so two meq genes were knocked-out completely. RT-qPCR showed transcription and expression levels of the HA and NA genes were both significantly higher than that of GX0101 own pp38 gene. Indirect fluorescence antibody (IFA) test, and Western blot analyses indicated that HA and NA genes were co-expressed simultaneously under control of the different promoters but meq genes were not. These results herald a new and effective recombinant meq-deleted MDV-based AIV-H9N2 vaccine may be useful in protecting chickens from very virulent MDV and H9N2 challenges.
College of Veterinary Medicine, Shandong Agricultural University, No. 61 Daizong Road, Taian 271018, Shandong, China.
Zhenjie
Z
Zhang
College of Veterinary Medicine, Shandong Agricultural University, No. 61 Daizong Road, Taian 271018, Shandong, China.
Wenqing
W
Chen
College of Veterinary Medicine, Shandong Agricultural University, No. 61 Daizong Road, Taian 271018, Shandong, China.
Chengtai
C
Ma
College of Veterinary Medicine, Shandong Agricultural University, No. 61 Daizong Road, Taian 271018, Shandong, China.
Peng
P
Zhao
College of Veterinary Medicine, Shandong Agricultural University, No. 61 Daizong Road, Taian 271018, Shandong, China.
Luntao
L
Duan
College of Veterinary Medicine, Shandong Agricultural University, No. 61 Daizong Road, Taian 271018, Shandong, China.
Fushou
F
Zhang
Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA.
Aijun
A
Sun
Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun Nandajie, Beijing 100083, China.
Yanpeng
Y
Li
College of Veterinary Medicine, Shandong Agricultural University, No. 61 Daizong Road, Taian 271018, Shandong, China.
Hongqin
H
Su
College of Veterinary Medicine, Shandong Agricultural University, No. 61 Daizong Road, Taian 271018, Shandong, China.
Sifei
S
Li
College of Veterinary Medicine, Shandong Agricultural University, No. 61 Daizong Road, Taian 271018, Shandong, China.
He
H
Cui
College of Veterinary Medicine, Shandong Agricultural University, No. 61 Daizong Road, Taian 271018, Shandong, China. Electronic address: zzcui@sdau.edu.cn.
Zhizhong
Z
Cui
D000818:Animals; D002642:Chick Embryo; D002645:Chickens / Q000235:genetics / Q000821:virology; D015967:Gene Expression Regulation, Viral; D006388:Hemagglutinins / Q000096:biosynthesis* / Q000235:genetics; D008381:Herpesvirus 2, Gallid / Q000235:genetics*; D053127:Influenza A Virus, H9N2 Subtype / Q000235:genetics; D008380:Marek Disease / Q000235:genetics* / Q000821:virology; D009439:Neuraminidase / Q000096:biosynthesis* / Q000235:genetics; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D011401:Promoter Regions, Genetic
C100369:Eco-Q protein, Gallid herpesvirus 2; D006388:Hemagglutinins; D009856:Oncogene Proteins, Viral; D009439:Neuraminidase
H9N2-HA gene; H9N2-NA gene; Homologous recombination; Marek's disease virus (MDV); meq oncogene