Evaluation of the Safety and Immunogenicity of Duck-Plague Virus gE Mutants

Abstract

Duck plague (DP) is an acute infectious disease in the duck industry. The duck plague virus (DPV) is the pathogen, a subfamily of alphaherpesvirinae. gE is a type I membrane protein that contains three parts: an extracellular domain, a transmembrane domain, and a cytoplasmic domain. gE is the major virulence determinant of α-herpesvirus. However, the functions of the gE extracellular and cytoplasmic domains have not been reported in DPV. In this study, a gE extracellular domain deletion mutant and a gE cytoplasmic domain deletion mutant were constructed from DPV. Virus replication kinetics showed that the growth titers of both the gE ectodomain-deleted mutant virus and the gE cytoplasmic domain-deleted virus in DEFs were lower than that of the parental virus CHv-50. DPV CHv-gEΔET and DPV CHv-gEΔCT were continuously passed to the 20th passage in DEFs and the 10th in ducklings. The mutant virus DNA after passage was extracted for identification. The results showed that the gE ectodomain and gE cytoplasmic domain deletion mutant viruses have good genetic stability. The ducklings in each group (n=10) were inoculated with the same titers of DPV CHv-gEΔET, DPV CHv-gEΔCT, DPV CHv-ΔgE, and parental CHv-50, respectively. Clinical symptoms and serum antibody levels were detected after inoculation. The results showed that the virulence of DPV CHv-gEΔCT to ducklings was reduced compared with parental CHv-50, while the virulence of DPV CHv-gEΔET to ducklings was significantly reduced. 105 TCID50 DPV CHv-gEΔET or DPV CHv-ΔgE can induce ducklings to produce DPV-specific antibodies, protect the ducklings from virulent CHv challenge. Therefore, DPV CHv-gEΔET may serve as a promising vaccine candidate to prevent and control duck plague.

Evaluation of safety and immunogenicity of duck-plague virus gC/gE double gene deletion

“…In practical production, duck embryonic attenuated vaccine and chicken embryo fibroblast vaccine are mainly used to control duck plague ( 14 , 28 , 29 ). Previous studies have shown that the deletion of viral genes or gene functional domains can reduce the virulence of herpes virus, have no pathogenicity or weaken pathogenicity to natural hosts, and can achieve protective effects, which can be used for further genetic engineering vaccine research ( 23 , 30 , 31 ). In this study, based on the constructed CHv-ΔgC single-gene deletion strain, the gE gene was deleted using the BAC gene editing system ( 24 ), and an attenuated CHv-ΔgC/gE strain with double gene deletion was obtained.…”

“…Duck embryo fibroblasts (DEFs) were prepared from 9-day-old duck embryos and cultured in DMEM medium (Gibco, Shanghai, China) containing 10% neonatal bovine serum (NBS, Gibco, Rockford, USA) according to conventional methods ( 23 ). Duck plague virus Chinese virulent strain (CHv) (GenBank: NO.…”

“…The plasmid with the correct sequence was transfected into DEFs, and plaque-purified to obtain the virus CHv-ΔgC/gE, which was identified by IFA and WB with gE polyclonal antibody. The primer sequences have been described elsewhere ( 23 , 25 , 26 ) ( Table 1 ).…”

“…In order to test the immunogenicity and the protective effect of immunized ducks against virulent challenge, 30 14-day-old ducklings were divided into 3 groups, the first group was inoculated with 1 mL CHv-ΔgC/gE (10 6 TCID 50 /mL), the second group was inoculated with a dose (10 7.7 copies/dose) of live duck plague vaccine CVCC AV1222 (AV1222, VAC, Yangzhou, China), the third group was the control group, inoculated with 1 mL of MEM. After 10 days of immunization, all ducks were challenged with 100 LD 50 CHv, the methods of immunization and challenge were intramuscular injection in the right leg ( 23 ). Body temperature, body weight and survival were recorded daily after challenge.…”

“…The level of neutralizing antibodies in the immunized ducks was determined by the fixed virus-diluted serum method. Briefly, 0.1 mL of 10-fold diluted serum (heat inactivated at 56° C. for 30 minutes) was mixed with an equal volume of 200 TCID 50 /0.1mL of DPV, and neutralizing antibodies were detected by conventional methods ( 23 ). NAbs titers were expressed as the maximum dilution at which 50% of the virus was neutralized.…”