Development and evaluation of immunogenicity and protective efficacy of two recombinant attenuated newcastle disease viruses expressing the VP2 protein of infectious bursal disease virus

Abstract

Newcastle disease (ND) and Infectious bursal disease (IBD) are highly contagious and economically significant viral diseases affecting poultry worldwide. Despite widespread vaccination, current vaccines often require multiple doses and provide limited protection against emerging strains. This study developed and evaluated bivalent vaccines based on recombinant Newcastle disease virus (NDV) expressing the VP2 protein of a novel variant Infectious bursal disease virus (IBDV), SHG19 strain. To enhance immunogenicity and antigenic compatibility, two recombinant NDVs were generated: rHV, using a modified genotype VII HEB strain backbone, and rLHV, using the lentogenic LaSota backbone incorporating HEB F and HN genes. Protective efficacy was evaluated in specific pathogen-free (SPF) chickens through immunization followed by challenges with both NDV and IBDV. Results demonstrated robust humoral immune responses, significant reductions in viral shedding, and effective dual protection against both pathogens. These findings highlight the potential of NDV-based bivalent vaccines to streamline vaccination protocols and offer enhanced protection against circulating NDV and IBDV strains.

Keywords: Infectious bursal disease virus; Newcastle disease virus; VP2 protein; immunogenicity; recombinant attenuated virus.

Fig. 1

Construction and validation of recombinant plasmids. (A) Schematic diagrams illustrating the genomic organization of the final recombinant NDV full-length plasmids pCI-aH-VP2 and pCI-L-H(aF/HN)-VP2, showing the insertion of the IBDV VP2 gene between the P and M genes and indicating key backbone modifications (F gene mutation or F/HN gene replacement). (B and C) Agarose gel electrophoresis confirming the integrity of constructed helper plasmids following Xba I digestion. Lanes M: DNA marker; Lane 1: Xba I digested plasmid. (B) HEB-derived helper plasmids: (a) pCI-H-NP; (b) pCI-H-P; (c) pCI-H-L. (C) LaSota-derived helper plasmids: (a) pCI-L-NP; (b) pCI-L-P; (c) pCI-L-L.

Fig. 2

Confirmation of recombinant virus rescue by hemagglutination (HA) assay following propagation in embryonated eggs. Allantoic fluid, harvested after three serial passages in 9-day-old SPF embryonated chicken eggs, was subjected to HA assay. (A) rHV exhibited a titer of 2⁶ HA units. (B) rLHV exhibited a titer of 2⁷ HA units.

Fig. 3

Confirmation of VP2 protein expression by Western blot and immunofluorescence assay. (A) Western blot analysis of BHK-T7 cell lysates using a mouse anti-VP2 polyclonal antibody. Lane M: protein marker. Lane 1 shows lysate from infected cells; Lane 2 shows lysate from uninfected control cells for: (a) rHV infection; (b) rLHV infection. (B) IFA of BHK-T7 cells infected with rHV (top row), rLHV (middle row), or mock-infected (Control, bottom row) at 24 h post-infection. Cells were co-stained with chicken anti-NDV serum (detected by FITC, green) and mouse anti-VP2 antibody (detected by TRITC, red). Nuclei were counterstained with DAPI (blue). Merged images are shown in the right column. Scale bars = 100 μm.

Fig. 4

Humoral antibody responses in chickens following immunization. Serum samples collected at 7, 14, and 21 days post-immunization (dpi) were analyzed. (A) NDV-specific antibody titers determined by hemagglutination inhibition (HI) assay, expressed as log₂ values. (B) IBDV VP2-specific antibody titers determined by ELISA, expressed as ng/L. Data points represent the mean ± SEM.

Fig. 5

Survival curves of chickens following NDV challenge. The graph displays the percentage survival over time (days post-challenge, dpc) for each experimental group.

Fig. 6

Gross pathological changes in kidneys and proventriculus following NDV challenge. Representative images display kidneys (top row) and proventriculus (bottom row) from chickens in the indicated experimental groups at 3 days post-challenge (dpc). Labels indicate the treatment/challenge group.

Fig. 7

Gross pathological changes in the bursa of Fabricius following IBDV challenge. Representative images show bursae collected from chickens in the indicated experimental groups at 3 days post-challenge following challenge with either IBDV GF6 (top row of challenge groups) or IBDV BC6/85 (bottom row of challenge groups). Arrows indicate representative lesions such as petechial (black arrow) or extensive (red arrow) hemorrhages observed primarily in control groups.

Fig. 8

Histopathological changes in tissues following NDV challenge. Representative images of hematoxylin and eosin (H&E)-stained tissue sections (trachea, spleen, lung, kidney, duodenum) from chickens in the indicated experimental groups at 3 days post-challenge. Scale bar = 100 μm.

Fig. 9

Histopathological changes in the bursa of Fabricius following IBDV challenge. Representative images of hematoxylin and eosin (H&E)-stained bursa sections from chickens in the indicated experimental groups at 3 days post-challenge. The top row shows groups challenged with IBDV GF6; the bottom row shows groups challenged with IBDV BC6/85. Scale bar = 50 μm.

Fig. 10

Viral loads were determined by RT-qPCR and expressed as log viral RNA copies per 0.1 g of tissue. (A) NDV viral loads in various tissues (heart, liver, spleen, lung, kidney, thymus, bursa of Fabricius, trachea, duodenum) of chickens challenged with NDV (HEB strain). (B) IBDV viral loads in the bursa of Fabricius of chickens challenged with the IBDV GF6 strain. (C) IBDV viral loads in the bursa of Fabricius of chickens challenged with the IBDV BC6/85 strain. Data are presented as mean ± SEM. Asterisks indicate statistically significant differences compared to the respective unvaccinated control group (***P < 0.001).