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

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.