Single dose of recombinant baculovirus vaccine expressing sigma B and sigma C genes provides good protection against novel duck reovirus challenge in ducks

Abstract

The novel duck reovirus (NDRV) disease causes high economic losses, resulting in substantial economic losses in waterfowl industry. However, currently, no commercial vaccines are available to alleviate NDRV infection throughout the world. Here, we developed two subunit vaccine candidates for NDRV based on the insect cell-baculovirus expression system (IC-BEVS). Two recombinant viruses, namely rBac-σB and rBac-σC, were successfully generated based on the consensus sequence of NDRV. Then, the σB and σC subunit vaccine candidates were prepared by directly inactivating the recombinant virus infected-Sf9 cell suspension. The double antibody-sandwich ELISA was used for quantitative of σB or σC protein in the inactivated crude antigen. Protective efficacy results revealed that, compared with the whole virus inactivated vaccine, a single dose of 160 ng σB or σC protein showed advantages in inducing serum antibodies, elevating weight, alleviating liver and spleen injury, restraining viral shedding and viral replication in ducklings. To be noted, the subunit σC or the combination of subunit σB and σC vaccine candidates had better protective efficacies, especially the combined σB and σC vaccine group. Therefore, our study provides useful information for developing effective vaccine against NDRV infection.

Keywords: Ducks; IC-BEVS; NDRV; Subunit vaccine; σB; σC.

Fig. 1

Characterization of the recombinant baculovirus. (A) The rescued virus rBac-σB and rBac-σC were successfully identified using an immunofluorescence assay (IFA). The empty baculovirus was used as a mock control, and no specific immune fluorescence was detected in the mock cells. (B) The passage 2 rBac-σB virus (rBac-σB-P2) and the passage 3 rBac-σB virus (rBac-σB-P3) were confirmed by detecting the expression of σB by western blotting. Cells infected with an empty baculovirus were used as a mock control. (C) The passage 2 rBac-σC virus (rBac-σC-P2) and the passage 3 rBac-σC virus (rBac-σC-P3) were confirmed by detecting the expression of σC by western blotting. Cells infected with an empty baculovirus were used as a mock control. (D) The standard curve of the double antibody-sandwich ELISA method based on the σB protein. (E) The standard curve of the double antibody-sandwich ELISA method based on the σC protein.

Fig. 2

Serum antibodies induced by different vaccine candidates. (A) Time points of the duck experiments. (B) Grouping and the immune dose of the duck vaccination experiments. (C) The σB antibodies at day 7 post vaccination determined by the indirect-ELISA using the σB as the capture protein. (D) The σB antibodies at day 14 post vaccination determined by the indirect-ELISA using the σB as the capture protein. (E) The σC antibodies at day 7 post vaccination determined by the indirect-ELISA using the σC as the capture protein. (F) The σC antibodies at day 14 post vaccination determined by the indirect-ELISA using the σC as the capture protein. For all analysis, P < 0.05 (a, *), P < 0.01 (b, **), P < 0.001 (c, ***) or P < 0.0001 (d, ****) is considered as statistically significant between the indicated groups.

Fig. 3

Immune efficacy of the vaccine candidates. (A) VN antibodies of the duck serum collected on day 14 post vaccination. (B) Survival rates of the birds at day 10 post virus challenge. (C) The weight change of the birds for a period of 10 days after virus challenge. (D) The statistical analysis of the results from panel C. Statistical analyses were performed using One-way ANOVA analysis of variance with the nonparametric test based on Prism version. 9. All data were recorded as means ± SD. For all analysis, P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) or P < 0.0001 (****) is considered as statistically significant between the indicated groups.

Fig. 4

The gross lesions in the liver and spleen of the ducklings after virus challenge. On day 10 post challenge, 9 birds of each group were euthanatized to death for observation of lesion on liver and spleen. Three represented pictures of liver and spleen from each group were presented. For the liver and spleen, the most stable pathological changes are hemorrhagic-necrotic lesions. Mock indicates the unchallenged PBS control. PBS stands for the challenged PBS control.

Fig. 5

The pathological changes in the liver and spleen of the ducklings after virus challenge. On day 10 post challenge, 9 birds of each group were euthanatized to death for observation of pathological changes on liver and spleen by H&E staining. One represented picture of liver and spleen from each group were presented. , vascular stasis in the liver and mild congestion in the hepatic sinus; , a large number of severe necrotic hepatocytes were observed, and with small focal infiltration of peripheral lymphocytes around these necrotic hepatocytes; , ballooning degeneration and cytosolic vacuolation of the hepatocytes; , lymphocyte infiltration in the liver; , hyperplasia of the hepatic duct; , vacuolar degeneration of the hepatocytes; , congestion and dilatation of the medullary sinus in the spleen; , hyperplasia of the connective tissue in the spleen; , lymphocytes along with multinucleated giant cell infiltration in spleen; , large focal necrosis and nuclear fragmentation of the spleen cells; , heterophilic granulocytic infiltration in the spleen.

Fig. 6

Viral replication in liver and spleen of the infected birds. On day 3 and 5 post challenge, three ducks of each group were euthanatized to death for collection of liver and spleen for viral replication assay. (A) Viral replication in the liver of the infected birds at day 3 post challenge. (B) Viral replication in the spleen of the infected birds at day 3 post challenge. (C) Viral replication in the liver of the infected birds at day 5 post challenge. (D) Viral replication in the spleen of the infected birds at day 5 post challenge. Statistical analyses were performed using One-way ANOVA analysis of variance with the nonparametric test based on Prism version. All data were recorded as means ± SD. For all analysis, P < 0.01 (b, **), or P < 0.001 (c, ***), or P < 0.0001 (d, ****) is considered as statistically significant between the indicated groups.

Fig. 7

Viral shedding in throat and cloaca swabs of the infected birds. On day 3 and 7 post challenge, 8 ducks of each group were used for collection of throat and cloaca swabs for viral shedding assay. (A) Viral shedding in throat swabs of the infected birds at day 3 post challenge. (B) Viral shedding in cloaca swabs of the infected birds at day 3 post challenge. (C) Viral shedding in throat swabs of the infected birds at day 7 post challenge. (D) Viral shedding in cloaca swabs of the infected birds at day 7 post challenge. Statistical analyses were performed using One-way ANOVA analysis of variance with the nonparametric test based on Prism version. All data were recorded as means ± SD. For all analysis, P < 0.05 (a, *), P < 0.01 (b, **), P < 0.001 (c, ***) or P < 0.0001 (d, ****) is considered as statistically significant between the indicated groups.