Recombinant Newcastle Disease Virus (NDV) Expressing Sigma C Protein of Avian Reovirus (ARV) Protects against Both ARV and NDV in Chickens

Abstract

Newcastle disease (ND) and avian reovirus (ARV) infections are a serious threat to the poultry industry, which causes heavy economic losses. The mesogenic NDV strain R2B is commonly used as a booster vaccine in many Asian countries to control the disease. In this seminal work, a recombinant NDV strain R2B expressing the sigma C (σC) gene of ARV (rNDV-R2B-σC) was generated by reverse genetics, characterized in vitro and tested as a bivalent vaccine candidate in chickens. The recombinant rNDV-R2B-σC virus was attenuated as compared to the parent rNDV-R2B virus as revealed by standard pathogenicity assays. The generated vaccine candidate, rNDV-R2B-σC, could induce both humoral and cell mediated immune responses in birds and gave complete protection against virulent NDV and ARV challenges. Post-challenge virus shedding analysis revealed a drastic reduction in NDV shed, as compared to unvaccinated birds.

Keywords: avian reovirus-σC protein; bivalent vaccine candidate; humoral and cell mediated immune responses; newcastle disease virus; protection efficacy.

Figure 1

Generation of a full-length infectious clone of NDV-R2B containing the σC gene of avian reovirus. The σC gene cassette containing the NDV gene start and gene end signals was engineered to be incorporated between the P and M genes of NDV-R2B genome flanked by the restriction enzymes PacI and AvrII. A Kozak sequence was incorporated preceding the σC gene coding sequence and one extra stop codon was artificially introduced at the end of σC coding sequence to maintain the “rule of six”. P-UTR and M-UTR contains the regulatory sequences of NDV, whereas P-CDS and M-CDS represent the complete ORF of P and M genes, respectively.

Figure 2

(A) immunofluorescence analysis of rNDV-R2B-σC virus expressing sigma C protein ×60. (a) DAPI stained cells; (b) cells expressing NDV proteins (Anti-NDV/Alexa); (c) cells expressing σC protein (Anti-ARV/FITC); (d) merged; (B) multistep growth kinetics of recombinant virus rNDV-R2B-σC in Vero cells. Monolayer of Vero cells were infected with rNDV-R2B-σC virus at MOI of 0.01 and culture supernatant was collected at every 12 h interval until 72 h, and viral titres were determined by limiting dilution assay and calculated as TCID₅₀ by the Reed and Muench method.

Figure 3

Schematic diagram showing the experimental design. NDV: Newcastle diseases virus, ELISA: enzyme-linked immunosorbent assay, HI: haemagglutination inhibition test, LTT: lymphocyte transformation test, dpc: days post challenge.

Figure 4

Assessment of NDV and ARV specific serum antibodies in experimental chickens by ELISA and HI. (A) birds of three groups were immunized at seven days of age with rNDV-R2B-FPCS and live LaSota vaccine as a primary vaccine. The control group birds were injected with phosphate buffered saline (PBS). Booster dose was given at 42 days of age with rNDV-R2B-σC and rNDV-R2B viruses to the corresponding groups and the control group was again injected with PBS. Serum samples were collected from the immunized and control group of birds at regular intervals and tested for NDV specific antibodies. The antibody titres higher than 200 was considered positive for NDV specific antibody; (B) one group was vaccinated with ARV inactivated vaccine at 6^th week of age. Serum samples were collected from the immunized and control group of birds at regular intervals and tested for anti- σC antibody. The O.D. value > 0.1779 (mean O.D. of the control birds + 3 S.D.) were considered positive for ARV antibodies. Bars (mean ± SE) indicate the representative data of a single experiment. Data with different capital letters superscript indicates the time effect (p < 0.01) and small letters superscript indicates the treatment effect (p < 0.05); (C) assessment of NDV specific serum antibodies in response to vaccination as determined by an HI test. Serum samples were collected at 14,21,28,35,42,49 and 56 days of age from all the birds. All HI titres were expressed as mean reciprocal log2 titre + SEM (standard error of the mean) (n = 10). Statistical differences were calculated by one-way ANOVA with p < 0.01 and Waller–Duncan as a post hoc test.

Figure 5

Assessment of antigen specific lymphocyte proliferative response in chickens at 49 and 56 days of age. Chicken PBMCs from immunized and control groups (n = 6) were stimulated with (A) NDV and (B) recombinant σC antigen expressed in E. coli. Bars (mean ± SE) indicate the representative data of a single experiment. Lymphocyte proliferative response was measured and expressed as stimulation index. Level not connected by same letter is significantly different (p < 0.05). (C) mRNA expression of different cytokine genes in response to vaccination as assessed by quantitative real time PCR and normalized to β-actin gene. Relative expression was determined by using the 2−^ΔΔCt method and relative fold change of each cytokine expression between vaccinated and control groups are presented. All of the data are represented as mean value ± standard errors. Data with different small letter superscript indicates the treatment effect (p < 0.05).

Figure 6

Survival curve for birds immunized with recombinant and vaccine viruses. Birds were challenged with 10⁵ ELD₅₀/mL virulent NDV at 62 days of their age and survivability was assessed up to 10 dpc. All of the control birds died by 4 dpc.

Figure 7

Reduction of virus shed due to vaccination in comparison to unvaccinated control group. (A) oropharyngeal and (B) cloacal swabs were collected on 2 and 4 dpc from all the birds for virus isolation. By day 4, all the control birds died due to an NDV challenge, whereas the viral shed from all the vaccinated groups reduced drastically by 4 dpc as compared to the control. Data with different small letter superscript indicates the treatment effect (p < 0.01) and capital letter superscript indicates the time effect (p < 0.01).

Figure 8

Gross lesions in control and vaccinated birds at 10 days post ARV challenge. (a) control birds had swelling and edema of footpads, swollen hock joints with edematous digital flexor tendons; (b) vaccinated birds had apparently normal footpads and hock joints.

Figure 9

Histopathological changes in various organs following virulent NDV challenge (a–c) caecal tonsil with normal histoarchitecture; (d) caecal tonsil with necrosis and lymphoid depletion; (e–g) normal histoarchitecture of spleen; (h) spleen with follicular necrosis and lymphoid depletion; (i) bursa with normal histoarchitecture; (j,k) section of bursa with mild lymphoid depletion and mild peri-follicular edema; (l) bursa with severe lymphoid depletion both in the cortex and medulla with mononuclear infiltration. Scale bar—100 µM. Normal histology of the organs is denoted by red arrows and histopathological changes by black arrows.

Figure 10

Histopathological changes in various organs following virulent ARV challenge (a) the footpad shows hyperkeratosis and necrotic lesions in epidermis and dermis; (b,c) footpad with mild mononuclear cell infiltration; (d) fibrin exudation, edema and mononuclear cell infiltration in the foot pad; (e) the spleen shows hyperplasia of reticular cells in Schweigger–Seidel sheath; (f,g) spleen with normal histoarchitecture; (h) spleen with mild lymphoid depletion; (i) caecal tonsils show severe lymphoid depletion; (j,l) caecal tonsils with normal histoarchitecture; (k) caecal tonsils show severe necrosis of the lymphoid follicles; (m) bursa shows severe lymphoid depletion; (n,p) bursa shows mild lymphoid depletion; (o) bursa shows moderate to severe lymphoid depletion. Scale bar—100 µM. Normal histology of the organs is denoted by red arrows and histopathological changes by black arrows.