Recombinant Duck Enteritis Virus-Vectored Bivalent Vaccine Effectively Protects Against Duck Hepatitis A Virus Infection in Ducks

Abstract

Duck enteritis virus (DEV) and duck hepatitis A virus (DHAV) are prevalent duck pathogens, causing significant economic losses in the duck industry annually. Using a fosmid-based rescue system, we generated two DEV recombinants, rDEV-UL26/27-P13C and rDEV-US7/8-P13C, in which the P1 and 3C genes from DHAV type 3 (DHAV-3) were inserted into the DEV genome between genes UL26 and UL27 or genes US7 and US8. We inserted a self-cleaving 2A-element between P1 and 3C, allowing the production of both proteins from a single open reading frame. P1 and 3C were simultaneously expressed in infected chicken embryo fibroblasts, with no difference in growth kinetics between cells infected with the recombinant viruses and those infected with the parent DEV. Both recombinant viruses induced neutralizing antibodies against DHAV-3 and DEV in ducks. A single dose of the recombinant viruses induced solid protection against lethal DEV challenge and completely prevented DHAV-3 infection as early as 7 days post-vaccination. These recombinant P1- and 3C-expressing DEVs provide potential bivalent vaccines against DEV and DHAV-3 infection in ducks.

Keywords: 3C protein; P1 protein; bivalent vaccine; duck enteritis virus; duck hepatitis A virus.

FIGURE 1

Construction of fosmids containing the P1 and 3C genes of DHAV-3. (A) The five fosmid combinations used for the generation of the DEV attenuated vaccine strain. Numbers represent the location of each fosmid fragment in the DEV vaccine strain genome. (B) The schematic diagrams of the recombinant fosmid with the P1 and 3C genes inserted between UL26 and UL27 in the DEV genome. (C) The schematic diagrams of the recombinant fosmid with the P1 and 3C genes inserted between US7 and US8 in the DEV genome.

FIGURE 2

Generation of recombinant DEVs with the P1 and 3C genes insertion. (A) The cytopathic effects induced by the recombinant DEVs containing the DHAV-3 P1 and 3C genes in CEFs. (B) Electron microscopy detection of the recombinant DEVs in CEFs. Bar length, 1 μm. Arrows represent the DEV virions detected in infected cells.

FIGURE 3

Detection of P1 and 3C expression by recombinant DEVs in infected CEFs. (A) PCR amplification of the P1-P2A-3C cassette from the recombinant viruses. (B) Detection of P1 and 3C expression by the recombinant viruses in indirect immunofluorescence assay with anti-DHAV3-VP1 and anti-DHAV3-3C antibodies.

FIGURE 4

Growth kinetics and genetic stability of the recombinant DEVs in CEFs. (A) Comparison of the replication kinetics of the recombinant DEVs and the parental virus (rDEV-WT) in CEFs. (B) Detection of the P1 and 3C genes inserted in the recombinant DEVs passaged 5 (F05), 10 (F10), 15 (F15), and 20 (F20) times in CEFs. (C) Confirmation of P1 and 3C expression by the recombinant DEVs passaged 20 times in CEFs with immunofluorescence assay.

FIGURE 5

Antibody responses induced by the recombinant DEVs in ducks. Groups of twenty ducks were inoculated with 1000 ELD₅₀ of the recombinant DEVs, and the sera samples were collected after 7 days of vaccination to detect neutralizing antibodies against DHAV-3 (A) and DEV (B). The neutralizing antibody titers for ducks are expressed as log₂. Data presented are the means ± standard deviations from twenty ducks per group. ns, no significant difference; *P < 0.05.

FIGURE 6

Protective efficacy of the recombinant DEVs against lethal DHAV-3 and DEV challenge in ducks. Ducks were inoculated with 1000 ELD₅₀ of the recombinant DEVs and then challenged with 100 ELD₅₀ of the virulent DHAV-3 A3 strain or 1000 minimum lethal doses of the virulent DEV CSC strain after 7 days of vaccination. (A) The survival rate of ducks challenged with DHAV-3 within an observation period of 14 days. (B) The survival rate of ducks challenged with DEV within an observation period of 14 days.