A Safe and Effective Bivalent Vaccine Candidate Against Duck Enteritis Virus and Duck‐Origin Novel Goose Parvovirus

FIGURE 1. Construction of recombinant pDEV‐NGPV VP2. (A) A mini‐F vector (pHA2) was inserted into the intergenic region between UL15B and UL18 of the DEV vaccine strain. (B) The pCMV promoter driving GFP expression in pDEV‐vac was replaced with the pEF1 gene. (C) Schematic diagram of the recombinant virus showing insertion of the pCMV‐NGPV VP2‐BGH‐pA expression cassette into the intergenic region between DEV US7 and US8 through two‐step Red‐mediated recombination (en passant). The procedures shown in the dotted box (A, B) were reported previously (Chen et al. 2013).

FIGURE 2. Identification of recombinant BAC clones by RFLP (A) and PCR (B). (A) Analysis of recombinant BAC clones by BamH I digestion. DNA marker: 15,000, 10,000, 7500, 5000, 2500 and 1000 bp. Lane 1, pE1; lane 2, pkan‐NGPV VP2; lane 3, pNGPV VP2. (B) Identification of BAC clones by PCR amplification. Lane 1, pE1 (641 bp); lane 2, pkan‐NGPV VP2 (4465 bp); lane 3, pNGPV VP2 (3408 bp).

FIGURE 3. Rescue of recombinant viruses (100×). (A) rE1. (B) rNGPV VP2. (C) rNGPV VP2‐Cre. (A) rE1, the parental strain was constructed and stored in our laboratory; (B) rNGPV VP2 was rescued by transfecting pNGPV VP2 into CEFs using the calcium phosphate precipitation method. (C) rNGPV VP2‐Cre was generated by excision of the mini‐F sequence from rNGPV VP2 through co‐transfection of pCAGGS‐NLS/Cre and pNGPV VP2 into CEFs, followed by selection of plaques lacking GFP fluorescence.

FIGURE 4. Plaque size comparison of recombinant virus rNGPV VP2 and parental virus rE1 in CEFs. Plaque areas of 100 plaques for each virus were measured using ImageJ 1.42 software. Statistical analysis was performed using a two‐tailed unpaired t‐test (n = 100 per group). ns, not significant.

FIGURE 5. Western blot detection of NGPV VP2 expression in recombinant virus‐infected CEFs. CEFs infected with rNGPV VP2, rNGPV VP2‐Cre or rE1 were collected for Western blot analysis using anti‐GPV VP2 mAb as the primary antibody, with DEV US3 protein as a viral protein control. M, EasySee Western marker (90, 75, 60, 40 kDa); lane 1, rE1; lane 2, rNGPV VP2; lane 3, rNGPV VP2‐Cre.

FIGURE 6. Detection of VP2 expression in rNGPV VP2‐Cre‐infected CEFs by IEM (A) and observation of VLP formation by TEM (B). Thin sections of CEF monolayers infected with rNGPV VP2‐Cre were examined by IEM and TEM. Immunogold staining using GPV VP2 mAb indicates VP2 protein expression. Representative micrographs are shown. Scale bars, 200 nm.

FIGURE 7. IFA analysis of NGPV VP2 expression and localisation in CEFs. CEFs infected with rNGPV VP2‐Cre or rE1 were fixed and analysed by IFA using anti‐GPV VP2 mAb as the primary antibody and Cy3‐labelled goat anti‐mouse IgG as the secondary antibody. Cell nuclei were stained with DAPI. Red fluorescence indicates VP2 protein localisation.

FIGURE 8. Schematic diagram of the vaccine efficacy experiment in ducklings.

FIGURE 9. Detection of virus shedding in swabs (A) and viral loads in tissues (B–D) by qPCR. Two‐day‐old ducklings were immunised with 1 × 105 TCID50 rNGPV VP2‐Cre (Group 1, labelled A), 1 × 104 TCID50 rNGPV VP2‐Cre (Group 2, labelled B) or culture medium (Group 3, labelled PBS). Group 4 remained untreated (labelled NC). Eight days after immunisation, ducklings in Groups 1–3 were challenged with the highly virulent NGPV ZJ strain. Viral DNA in anal swabs, throat swabs and tissues (heart, liver, spleen, lung, kidney and intestinal tract, including duodenum, jejunum, ileum, cecum, colon and rectum) was measured by qPCR at 1, 3, 5 and 7 d.p.c. Statistical analysis was performed using ordinary one‐way ANOVA followed by Tukey multiple comparisons test (n = 10). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

FIGURE 10. Histopathological analysis of the duck intestine. Duodenum samples were collected from immunised and control ducks at 5 d.p.c and examined by histopathological analysis after haematoxylin and eosin (H&E) staining. Scale bar, 100 μm. And the microscopic lesions were evaluated using a 5‐point severity scoring system. A: the untreated normal control group; B: the unimmunised challenged group; C: the high‐dose immunisation and challgenged group; D: the low‐dose immunisation and challgenged group.

FIGURE 11. Protective efficacy of rNGPV VP2‐Cre against lethal DEV challenge. Four groups of 2‐day‐old ducklings were vaccinated intramuscularly with 1 × 105 TCID50 rNGPV VP2‐Cre (Group 1, labelled A), 1 × 104 TCID50 rNGPV VP2‐Cre (Group 2, labelled B) or culture medium (Group 3, labelled PBS). Group 4 remained untreated (labelled NC). Eight days after immunisation, ducklings in Groups 1–3 were challenged with the highly virulent NGPV ZJ strain. At 47 d.p.c, ducklings in Groups 1 and 2 received booster immunisation with 1 × 105 TCID50 or 1 × 104 TCID50 rNGPV VP2‐Cre, respectively. Fourteen days after the booster immunisation, all ducks in Groups 1–3 were challenged intramuscularly with 100 × DLD50 of the highly virulent DEV CHv strain. Group 4 remained untreated, and clinical signs and mortality were monitored for 14 days after challenge. Statistical analysis was performed using ordinary one‐way ANOVA followed by Dunnett multiple comparisons test (n = 10). ns, not significant.

FIGURE 12. Detection of VP2‐specific antibody titers by iELISA (A) and virus neutralisation test (B). (A) Serum samples from ducklings were collected and analysed by iELISA using recombinant VP2 protein. NC (2 days), 2‐day‐old control ducklings; PBS (8 d.p.i), ducklings injected with DMEM and sampled at 8 d.p.i; A (8 d.p.i), ducklings immunised with 1 × 105 TCID50 rNGPV VP2‐Cre and sampled at 8 d.p.i; B (8 d.p.i), ducklings immunised with 1 × 104 TCID50 rNGPV VP2‐Cre and sampled at 8 d.p.i; PBS+NGPV (16 d.p.c), DMEM‐injected ducklings challenged with highly virulent NGPV and sampled at 16 d.p.c; A+NGPV (16 d.p.c), ducklings immunised with 1 × 105 TCID50 rNGPV VP2‐Cre, then challenged with NGPV and sampled at 16 d.p.c; B+NGPV (16 d.p.c), ducklings immunised with 1 × 104 TCID50 rNGPV VP2‐Cre, then challenged with NGPV and sampled at 16 d.p.c. (B) Serum samples were analysed by the virus neutralisation (VN) test. Statistical analysis was performed using ordinary one‐way ANOVA followed by Tukey multiple comparisons test (n = 10). ns, not significant.