Viral vector vaccines expressing nucleoprotein and phosphoprotein genes of avian bornaviruses ameliorate homologous challenge infections in cockatiels and common canaries

Avian bornaviruses are causative agents of proventricular dilatation disease (PDD), an often fatal disease of parrots and related species (order Psittaciformes) which is widely distributed in captive psittacine populations and may affect endangered species. Here, we established a vaccination strategy employing two different well described viral vectors, namely recombinant Newcastle disease virus (NDV) and modified vaccinia virus Ankara (MVA) that were engineered to express the phosphoprotein and nucleoprotein genes of two avian bornaviruses, parrot bornavirus 4 (PaBV-4) and canary bornavirus 2 (CnBV-2). When combined in a heterologous prime/boost vaccination regime, NDV and MVA vaccine viruses established self-limiting infections and induced a bornavirus-specific humoral immune response in cockatiels (Nymphicus hollandicus) and common canaries (Serinus canaria forma domestica). After challenge infection with a homologous bornavirus, shedding of bornavirus RNA and viral loads in tissue samples were significantly reduced in immunized birds, indicating that vaccination markedly delayed the course of infection. However, cockatiels still developed signs of PDD if the vaccine failed to prevent viral persistence. Our work demonstrates that avian bornavirus infections can be repressed by vaccine-induced immunity. It represents a first crucial step towards a protective vaccination strategy to combat PDD in psittacine birds.

. Bornavirus protein expression is retained after passaging of vaccine viruses in culture systems. rNDV (A) and rMVA (B) constructs were passaged five times in either CEF cultures or embryonated chicken eggs, respectively. Following the fifth passage, CEF cultures were infected and bornavirus N or P protein expression was visualized by immunofluorescence staining with the indicated antibodies as described for Supplementary figure S1.
Combined pharyngeal and cloacal swabs were collected at the indicated time points. (B, C) NDV was detected by conventional RT-PCR 3 . (D) Vaccine-derived PaBV-4 P DNA originating from rMVA/PaBV-4/P was quantified by qPCR. In experiment 1 no PaBV-4 P DNA was detectable (data not shown). The position of the X axis indicates the detection limits of the test.

Supplementary Fig. S4. Infectious bornavirus titers in the brains of cockatiels and canaries after challenge infection (experiments 1 & 3).
Brain samples were collected during necropsy at 17 or 15 weeks after challenge of cockatiels with PaBV-4 (A; experiment 1) or canaries with CnBV-2 (B; experiment 3), respectively. Ten-fold dilution series of ultrasonicated tissue homogenates were incubated with CEC-32 quail fibroblasts (A) or QM7 quail muscle cells (B) for four days before virus-positive cell foci were visualized by immunofluorescence staining and foci-forming units (ffu) were calculated. The procedure has been described in more detail elsewhere [4][5][6] . The position of the X axis indicates the detection limit of the test. P values < 0.05 indicate significant differences between the groups (Wilcoxon rank sum test).
Seventeen weeks after challenge infection all birds were euthanized and necropsied. Bird A6 (red box) remained free of detectable challenge virus whereas all other birds were persistently infected. Birds A2 and A4 (blue boxes) of the vaccine group showed a moderate dilatation of the proventriculus.

Group
Mononuclear infiltration score Bird cerebellum spleen liver kidney duodenum vaccine group A1 - Uninfected cockatiels originating from the same flock as the experimental birds served as controls.
Supplementary Fig. S6. Detection of NDV-specific HAI antibodies in vaccinated canaries (experiment 3). Two groups of 13 canaries each were vaccinated either with a mixture of rNDV/CnBV-2/N and rNDV/CnBV-2/P (vaccine group) or with the parental strain rNDV-wt (control group). Plasma samples were collected at the indicated time points after vaccination and tested for the presence of NDV-specific antibodies by HAI test. Due to limited sample volumes not all birds were tested at both time points. The position of the X axis indicates the detection limit of the test.

Group
CnBV - Birds vaccinated with rNDV and rMVA viruses expressing bornavirus proteins (vaccine group) or with rNDV-wt and MVA-wt (control group) were challenged with CnBV-2 #15864 by parenteral injection and necropsied at 15 weeks after challenge. Birds A10, B1, B8 and B11 had died or been euthanized prior to parental challenge. b Animal A6 was found dead during week 10 after parental challenge infection.