Table 1.
Primary antibodies and sera used for virus in vitro-characterization.
Fig 1.
Construction of recombinant Newcastle disease virus (NDV) expressing rabies virus (RABV) glycoprotein (G).
The RABV transgene is inserted into the intergenic region (igr) of the NDV fusion protein (F) and the hemagglutinin-neuraminidase protein genes (HN). The open reading frame (orf) is flanked by NDV specific gene start (GS, grey triangle) and gene end sequences (GE, grey rectangle), and by the NDV HN specific non-coding regions (ncr, white rectangles).
Fig 2.
In vivo-replication in embryonated chicken eggs and in vitro replication in cell lines originated from different species.
Embryonated chicken eggs (ECE) (A), chicken embryo fibroblasts (CEF) (B), baby hamster kidney cells (BHK-21) (C), or bovine kidney cells (MDBK) (D) were infected with rNDV or rNDV_GRABV (moi 0.01). Allantoic fluids and cell culture supernatants were harvested at indicated time points after infection (p. i.). Viral titers (TCID50/mL) were determined after titration on quail muscle (QM9) cells and subsequent immunostaining. Bar charts depict mean viral titers standard deviation ((A) n = 4, four eggs from one experiment; (B, C, D) n = 4, two samples each from two independent experiments). Asterisks indicate significant differences of mean viral titer (α = 0.05).
Fig 3.
In vitro-analysis of viral protein expression and virion composition.
Chicken embryo fibroblasts (DF-1) (A), bovine kidney cells (MDBK) and baby hamster kidney cells (BHK-21) (B) were infected with rNDV or rNDV_GRABV (multiplicity of infection (moi) 5) and harvested 24 h post infection (p. i.). Cell lysates and lysates of purified virions (10 μg per lane) (C) were subjected to SDS-PAGE and subsequently to Western blot analysis. Viral proteins were visualized by immunostaining with respective antibodies. Β-Actin was detected on every cell lysate blot as loading control. Blots of cell lysates are representative of three biological replicates. Blot of purified virions were done once. Uncompartmentalized illustrations can be found in the supporting information (S2 Fig).
Fig 4.
In vitro-analysis of viral protein expression.
Quail muscle cells (QM9) were infected with rNDV or rNDV_GRABV (moi 0.1), fixed 24 h p. i., optionally permeabilized with 0.1% Triton X-100, and immunostained with respective antibodies. Cellular distribution was analyzed by confocal microscopy.
Table 2.
Pathotyping of rNDV and rNDV_GRABV according to pathogenicity indices in vivo.
Fig 5.
Analysis of virus replication and shedding after oral vaccination.
Goats and foxes were directly orally vaccinated with either parental rNDV (n = 3) or RABV G expressing rNDV_GRABV (n = 6). Nasal, oral and rectal swabs were taken from all animals at indicated days after vaccination (dpv) and analyzed by quantitative real-time RT-PCR (RT-qPCR) for the presence of NDV NP specific RNA. Red dots represent rNDV inoculated animals and black dots represent rNDV_GRABV inoculated animals respectively.
Fig 6.
Analysis of serum antibodies against NDV after oral vaccination.
Serum was taken from all animals at indicated days after vaccination (dpv) and analyzed for antibodies specific to NDV by a competitive ELISA (cELISA; seropositivity: inhibition ≥ 40%) (A), the hemagglutination inhibition (HI) assay (seropositivity: log2 ≥ 3) (B) and the virus neutralization assay (VNA; seropositivity: log2 ≥ 3) (C). Dotted lines indicate the respective thresholds. Floating bars depict the mean titers and individual values.
Fig 7.
Analysis of serum antibodies against RABV G after oral vaccination.
Serum was taken from all animals at indicated days after vaccination (dpv) and analyzed for RABV G specific antibodies by a competitive ELISA (cELISA; seropositivity: inhibition ≥ 40%) (A) and the rabies virus fluorescent focus inhibition test (RFFIT; seropositivity: IU/mL ≥ 0.5%) (B). Dotted lines indicate the respective thresholds. Floating bars depict the mean titers and individual samples.
Table 3.
Individual outcome of NDV and RABV serological assays of goat (G) and fox (F) sera 28 days after oral vaccination (dpv) with either rNDV or rNDV_GRABV.
Fig 8.
Analysis of T-cell specific IFN-γ production of pharyngeal lymphocytes from goats and foxes after oral vaccination.
Pharyngeal lymph nodes from all animals were removed post mortem (28 days after vaccination). Lymphocytes were isolated and their specific interferon γ (IFN-γ) response was measured by ELISpot assays. (A) Representative images of cavities showing IFN-γ spot forming units (SFU) of Concanavalin A (ConA, positive control), medium (negative control) and NDV or RABV antigen-stimulated fox and goat lymphocytes for rNDV and rNDV_GRABV vaccinated animals. (B) Graph shows antigen specific SFU per 106 cells, corrected for mock control.