Pathogenesis of Aquatic Bird Bornavirus 1 in Domestic Chickens

doi:10.21203/rs.3.rs-1451335/v1

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Pathogenesis of Aquatic Bird Bornavirus 1 in Domestic Chickens

https://doi.org/10.21203/rs.3.rs-1451335/v1

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Aquatic bird bornavirus 1 (ABBV-1), classified in the Orthobornavirus genus, is a neurotropic virus that infects wild waterfowl causing persistent infection of the nervous system. Given the conspicuous presence of wild waterfowl in urban areas and farmlands, spillover of this virus into domesticated poultry species is a concern. The goal of this study was to test the ability of ABBV-1 to infect and cause disease in chickens. Two-day-old, White Leghorn chickens (n, 176) were inoculated with ABBV-1 through the oral, intramuscular, or intracranial routes, and sampled at 1, 4, 8, and 12-weeks post infection (wpi) to assess virus replication and lesion development. Chickens became infected only through the intracranial and intramuscular routes, developing earliest infection in the brain by 1 wpi (intracranial group), and spinal cord by 8 wpi (intramuscular group). Except for the kidney of one bird in the intracranial group, no other tissues (including choanal and cloacal swabs) tested positive for the virus. Therefore, while the virus reached the central nervous tissue (CNS) from the muscle (centripetal spread), it inefficiently reached peripheral sites after replication in the CNS (centrifugal spread). Inflammation in the CNS was observed in the intracranial and intramuscular groups starting at 8 and 12 wpi, respectively, and consisted of mononuclear perivascular cuffing.

This is the first study to document the pathogenesis of ABBV-1 in chickens, and indicates that this species is permissive to ABBV-1 infection, although less extensively than what is observed in waterfowl. This suggests that ABBV-1 replication is partially restricted in gallinaceous birds.

Virology

Pathology

ABBV-1

gallinaceous birds

encephalitis

persistent infection

host restriction

ABBV-1 can infect chickens through the intracranial and intramuscular routes.
Virus replication was identified almost exclusively in the nervous system.
Only one of 128 chickens inoculated with ABBV-1 developed clinical signs.
Less than half of tested chickens seroconverted by the end of the experiment.
No choanal or cloacal ABBV-1 shedding was identified.

Avian viruses in the Orthobornavirus genus, family Bornaviridae, have been collectively referred to as avian bornaviruses (ABVs). These viruses have been classified into 5 species (Passeriform orthobornavirus 1 and 2; Psittaciform orthobornavirus 1 and 2; and Waterbird 1 orthobornavirus ), based on the degree of genetic divergence and natural establishment in specific avian reservoirs (Amarasinghe et al., 2019). Viruses in the Psittaciform orthobornavirus species (i.e., parrot bornaviruses, PaBVs) are the causative agent of proventricular dilatation disease (PDD), a chronic disease of psittacine birds characterized by inflammation of the intestinal nervous ganglia and variable degrees of concurrent encephalitis and myelitis (Honkavuori et al., 2008; Kistler et al., 2008). Aquatic bird bornavirus 1 (ABBV-1), in the Waterbird 1 orthobornavirus species, causes a similar disease in wild waterfowl, characterized by inflammation of the central and peripheral nervous systems, distention of the proventriculus, and wasting. Disease associated with ABBV-1 infection was initially documented in Canada geese, trumpeter swans, and mute swans in a retrospective case series of wild waterfowl postmortem cases from Ontario, Canada (Delnatte et al., 2013). Subsequent surveillance studies have shown that ABBV-1 (as detected by molecular tests) is widely distributed in wild waterfowl in North America, with seroprevalence of up to 50% in certain waterfowl populations (Susan Payne et al., 2011; Guo et al., 2012; Delnatte et al., 2014).

Recently, our laboratory established an animal model of ABBV-1 infection in day-old Muscovy ducks (Cairina moschata), selected as a representative waterfowl species (Iverson et al., 2021). We have shown that ducklings can be infected with ABBV-1 through both intracranial and intramuscular inoculation routes, with high-titer virus replication in tissues, and both centripetal (from peripheral tissues to the central nervous tissue) and centrifugal (from the central nervous tissue to several peripheral sites) virus spread, including intermittent shedding through the choanal and cloacal secretions. Additionally, infected ducklings showed encephalitis and myelitis, recapitulating some of the microscopic findings observed in natural infection (Delnatte et al., 2013). This was the first study to demonstrate the infectious potential of ABBV-1 for domestic poultry species, and aligns with a growing body of evidence indicating that ABVs have the ability to spill over and infect a multitude of avian taxa, suggesting a broad host range (Delnatte et al., 2011; Guo et al., 2012; Payne et al., 2012b). For instance, ABBV-1 has been shown to infect and cause disease in gulls, raptors, and an emu, which are all birds taxonomically distant from waterfowl (Payne et al., 2012a; Guo et al., 2015; Nielsen et al., 2018). Phylogenetic analysis of several ABV isolates indicates that these viruses spread horizontally between multiple avian taxa, and it has been speculated that parrot bornaviruses may have originated from strains circulating in waterfowl species (Payne et al., 2012a, b).

Given the widespread distribution of wild waterfowl in urban areas and farmlands, and the relatively high prevalence of infection of ABBV-1 in wild waterfowl populations, the introduction and establishment of ABBV-1 in domestic poultry flocks is a concern. We hypothesized that ABBV-1 can infect and cause disease in the chicken, selected as a representative gallinaceous bird (taxonomically distant from waterfowl) and the most relevant domestic poultry species worldwide. Pursuant to this hypothesis, we tested the ability of ABBV-1 to infect, cause clinical signs and lesions, and induce seroconversion in groups of two-day-old White Leghorn chickens (Gallus gallus domesticus) inoculated through three routes of administration.

Virus inoculum

A suspension of cell-free virus was produced from immortalized duck embryo fibroblasts (American Type Culture Collection CCL-141) that were persistently infected with ABBV-1, as previously described (Iverson et al., 2021). Briefly, confluent 100-150 mm dishes of persistently infected CCL-141 cells were lysed with deionized water (osmotic shock) and one cycle of freeze/thaw in a ultra-cold freezer (-80 °C). Cell debris and supernatant were then collected, cleared by centrifugation, and the cell-free virus was concentrated by precipitation with PEG-8000 solution (30 % w/v) and resuspended in phosphate-buffered saline (PBS) with 1 % fetal bovine serum (FBS) to a final volume ratio of 100:1. The virus stock was titered by the tissue culture infectious dose 50 % method (TCID₅₀, limiting dilution) in CCL-141 cells, coupled with immunofluorescence (IFA) to detect infected wells, using a monospecific antibody (Pacific Immunology) against the ABBV-1 nucleocapsid (N) protein (Leacy et al., 2020; Pham et al., 2020; Iverson et al., 2021). The final titer was calculated by the Spearman-Karber formula and reported as focus forming units (FFU)/ mL (Iverson et al., 2021). Inoculum for control birds was prepared in the same way from non-infected cells.

Experimental design

All experimental procedures involving animals were approved by the University of Guelph Animal Care and Use Committee (Animal Utilization Protocol 3978), and conducted in accordance with the relevant regulations. The experimental outline is similar to what is reported in detail elsewhere (Iverson et al., 2021). A brief description follows.

A total of 176, day-old specific pathogen free (SPF) White Leghorn chickens were sourced from the Canadian Food Inspection Agency (Ottawa, ON, Canada) and delivered to the University of Guelph Central Animal Facility Research Isolation Unit (Guelph, ON, Canada). Birds were randomly assigned to four experimental groups (40 birds / room plus 3-4 birds for attrition), neck tagged, placed on the floor with soft wood shavings, and provided food and water ad libitum. Infrared lamps, to aid in temperature regulation, were provided at floor level until 3 weeks of age. Artificial light provided a photoperiod of 12:12 hours of light:dark.

The day after placement, chicks from each room were inoculated with ABBV-1 through 1 of 3 routes (one route / room), as described previously (Iverson et al., 2021): intracranial (IC, 50 µL injected into the subdural space of the right cerebral hemisphere, corresponding to 6.5 x 10⁴ FFU / bird), intramuscular (IM, 100 µL injected into the right gastrocnemius muscle, 1.3 x 10⁵ FFU / bird), or oral (PO, 100 µL delivered into the oral cavity, 1.3 x 10⁵ FFU / bird). Sham-inoculated (control) birds (CO) were given a lysate from non-infected CCL-141 cells delivered through all 3 inoculation routes, using the same volumes (Iverson et al., 2021). Chickens were checked daily for clinical signs. Birds that died or were euthanized unexpectedly were sampled regardless of schedule (see below), and results were included in the following sampling group.

At 1, 4, and 8-weeks post infection (wpi), 10 chickens were randomly selected from each experimental group to be euthanized. During the last time point, extra birds included for attrition were also sampled. Immediately before euthanasia, swabs (choanal and cloacal) and blood for serum were collected. Birds up to 4 weeks of age were euthanized by carbon dioxide (CO₂) inhalation, after being anesthetized with isoflurane in a 7 L vented induction chamber (VetEquip). Older birds (8 and 12 wpi) were euthanized with 100 mg / kg of pentobarbital delivered intravenously.

All euthanized birds underwent complete postmortem analysis. At each time point, 3 birds from each experimental group (total, 48 birds) were sampled to obtain an extensive list of tissues to be processed for histopathology and immunohistochemistry, as detailed below. From the remaining birds (range 7-10), 5 tissues (brain, lumbar spinal cord, kidney, proventriculus and ventriculus) were sampled for both virus titration by reverse transcription quantitative PCR (RT-qPCR) and histopathology.

Histopathology

From the birds selected for extensive tissue sampling, the following organs were fixed in 10 % neutral buffered formalin and routinely processed for histology (hematoxylin and eosin, H&E): brain, spinal cord (cervical, thoracic, lumbar), ischiatic nerves, brachial plexuses, kidneys, gonads, proventriculus, ventriculus, heart, pancreas, adrenal glands, thyroid/parathyroid glands, segment of small intestine (at the level of the Meckel’s diverticulum), colon, lung, liver, spleen, trachea, esophagus, bursa of Fabricius, thymus, and the right gastrocnemius muscle (only for IM and CO groups, as this was the site of inoculation). Sectioning of the central nervous tissue (CNS) was done as reported previously (Iverson et al., 2021). Briefly, for birds sampled at 1 wpi, the brain and spinal cord were sectioned in situ within the skull and vertebral column, after brief decalcification. For older birds, brain and spinal cord were extracted from the skull and vertebral column before sectioning. Regardless of age, coronal sections of cerebrum, optic lobe, brainstem, and cerebellum were obtained. Two cross sections were taken from each segment of the spinal cord.

Additional tissues (brain, spinal cord, kidney, proventriculus, and ventriculus), which were derived from birds sampled for virus titration in the IC and IM groups at 8 and 12 wpi, were also processed for histology. These birds (n, 32) were chosen to increase the details of the histological scoring in those groups and time points that showed microscopic lesions. Since these tissues were also used for RNA extraction and RT-qPCR, sections for histopathology were opportunistically collected, and not all brain and spinal cord sections were available for every bird.

Microscopic pathology and lesion scoring in the nervous system

Presence of microscopic lesions was recorded by routine evaluation of the slides. The severity of mononuclear inflammation in the brain and spinal cord was further assessed using a semi-quantitative grading scheme developed for a similar study in Muscovy ducks (Iverson et al., 2021). Briefly, the score was calculated by adding the intensity of inflammation (thickness of perivascular cuffs; no inflammation, score 0; 1-2 layers, score 1; 3-4 layers, score 2; ≥ 5 layers, score 3) and the extent of the inflammation (number of cuffed vessels; no inflammation, score 0; 1-10 vessels, score 1; 11-20 vessels, score 2; ≥ 21 vessels, score 3). Both parameters were assessed in up to 10, randomly selected 100X fields for each brain area (i.e., cerebrum, cerebellum, brainstem, optic lobe). For the spinal cord, scores were tallied on the entire available areas, and averaged by the number of available segments (score range, 0-6). A brain score was determined by adding the sub-scores of each brain area and dividing by the total number of available areas (score range, 0-6). Other lesions in the nervous system (meningitis, peripheral neuritis, gliosis) were tallied nominally, but were not included as part of the final score. Histological assessment and scoring were carried out by one member of the investigative team (M. Iverson).

Immunohistochemistry

Immunohistochemistry (IHC) for ABBV-1 was carried out on all tissues from three birds that underwent extensive tissue collection in the IC group at 12 wpi, as well as the bird that developed neurological signs and was euthanized (bird #136). This group had the highest number of positive birds and magnitude of ABBV-1 genome copy number in the central nervous system (CNS), and therefore was selected to evaluate ABBV-1 tissue distribution by IHC. The IHC for ABBV-1 was carried out using the same monospecific antibody used for IFA, as detailed elsewhere (Iverson et al., 2021). An age-matched control bird (12 wpi) was used as a negative control, and the brain tissue from a Canada goose (Branta canadensis) naturally infected with ABBV-1 was the positive control for the technique (Briese et al., 1994). Distribution of IHC reactivity was scored based on a semi-quantitative scale (no reactivity, score 0; 1-7 positive cells per 400X field, score 1; 8-14 positive cells per 400X field, score 2; ≥15 positive cells per 400X field, score 3), as assessed in up to 10, 400X fields of each brain area and spinal cord (Iverson et al., 2021).

On the CNS tissue of the same three birds used for ABBV-1 IHC, the relative contribution of T and B lymphocytes to the inflammatory population was assessed using IHC for CD3 (rabbit polyclonal antibody raised against the human homologue, Dako) and Pax5 (Pax5, mouse monoclonal antibody raised against the human homologue, clone 24, BD Biosciences), as previously described (Gibson et al., 2021; Iverson et al., 2021).

Quantification of virus genome copy number in tissues

For each experimental group and each time point, at least 7 birds (plus extra for attrition at 12 wpi) were sampled to collect tissues for RNA extraction and RT-qPCR. From each bird, brain, lumbar spinal cord, proventriculus, kidneys, and choanal and cloacal swabs (swabs were sampled from all necropsied birds) were collected into sterile screw cap tubes containing 1.0 mL of preserving solution (20 mM ethylenediaminetetraacetic acid [EDTA], 25 mM sodium citrate, and 70 % (w/v) ammonium sulfate with a pH of 5.2), and stored at -80 °C until RNA extraction. Total RNA was extracted, reverse transcribed, and the virus RNA was detected by RT-qPCR with primers and fluorescent probe targeting the ABBV-1 N gene, using a Roche LC (Light cycler)-480 model, as detailed previously (Iverson et al., 2021). Cycle threshold (Ct) less than 35 was considered to be positive. For each reaction, the number of genome copies per 150 ng of RNA (tissues) or 84 uL of swab fluid was interpolated based on a standard curve made by using limiting dilutions of a gBlock containing a 500 bp fragment of the ABBV-1 N gene (Integrated DNA Technologies). A dilution of the standard curve was run in parallel for each plate (Iverson et al., 2021).

Virus isolation from brain

Isolation of ABBV-1 was attempted from the brain of 2 chickens in the IC group, which had tested positive by RT-qPCR, as described before (Iverson et al., 2021). Briefly, brain homogenates (Precellys 24 homogenizer, Bertin Instruments) were incubated with primary duck embryo fibroblasts (DEF) overnight (approximately 100 µL of homogenate per confluent well of a 6-well plate). Cells were then washed, and after the third consecutive passage, ABBV-1 infection was detected by IFA using the same monospecific antibody against the N protein that was used for IHC (Leacy et al., 2020; Pham et al., 2020; Iverson et al., 2021). As a negative control, brains from two CO chickens were homogenized and used for virus isolation using the same method.

Western blot and serology

Blood was collected from all euthanized birds and allowed to coagulate for up to 8 h, followed by serum separation by centrifugation (2000 g for 10 min). Sera were used as the primary antibody to blot protein lysates of Vero cells either transfected or not-transfected with a eukaryotic expression vector (pCA; Addgene) encoding for the open reading frame of the ABBV-1 N gene (pCA-N). Briefly, Vero cells were seeded into 10-cm dishes and transfected with 5 µg of pCA-N using GeneJuice™ Transfection Reagent (Millipore Sigma). Two days post transfection, cells were scraped off the plate, washed three times in PBS, and protein was extracted using radio immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Nonidet P-40 [NP-40], 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate). Six birds per two time points (1 and 12 wpi) per each inoculation group were tested (total, 48). Briefly, cells lysates (20 μg of protein, as determined by bicinchoninic acid [BCA] assay) from transfected and not-transfected Vero cells were loaded onto 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, resolved, and transferred onto polyvinylidene difluoride (PVDF) membranes (25 V for 30 min; semi-dry transfer, BioRad). After being blocked with 5 % skim milk or 5 % fish gelatin in PBS-T (PBS supplemented with 20 % Tween20) at 4 °C overnight, the membranes were incubated with test sera (1:1000 dilution) diluted in 5 % skim milk overnight at 4 °C. Before incubation on membranes, serum samples (8 μL) were cross-absorbed with untransfected Vero cells (450 μg cell lysate in 500 to 1000 μL NP-40 buffer [20 mM Tris HCl pH 8, 137 mM NaCl, 10 % glycerol, 1 % Nonidet P-40 (NP-40), 2 mM EDTA]) for 3 h at RT or overnight at 4 °C . Antibody binding was detected using a horseradish peroxidase secondary goat antibody against pan-avian IgY-heavy and light chain at 1:5000 dilution (A140-110P-Bethyl Laboratories Inc.; Cedarlane) for 1 h, coupled with the SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher) using a ChemiDoc MP Imaging System and Image Lab 6.0.1. software (Bio Rad). A positive control was run separately using a monospecific rabbit antibody against the N protein (used also for IHC) instead of the test sera. All blots were exposed up to 1 minute to assess band presence.

Statistical analysis

Differences in proportions of nervous tissue samples with lesions, and differences in proportions of tissue samples positive by RT-qPCR were assessed by Chi-square test. The severity of nervous microscopic lesions between groups, time points, and different segments of the CNS were tested using the Kruskal-Wallis test with the Benjamini, Krieger, and Yekutieli correction for false discovery rate. The Kruskal-Wallis test with Dunn’s test for multiple comparison was used to assess differences in IHC reactivity between segments of the CNS. Differences of the average concentration of genome copy number in the tissues of infected birds was tested by a two-way ANOVA, with inoculation route and time point as the independent variables, followed by multiple comparisons between groups (Tukey’s test). In this calculation, tissues with a Ct value ³ 35 were considered negative and tallied as 0 in the calculation of the group averages. Statistical analysis was carried out using GraphPad Prism for iOS, version 9 (GraphPad Software, La Jolla, USA); for all tests, significance was set at < 0.05.

Clinical disease and gross pathology

Two chicks in the IC group died immediately after intracranial inoculation due to peracute cerebral hemorrhage (these were excluded from the study). Clinical signs were identified in two chickens that were euthanized. One chicken in the control group (#416) was euthanized at 16 days post infection (dpi) due to progressing lameness of the right leg; the bird was otherwise bright and alert. Gross and histologic examination did not identify a cause for the lameness, and the bird tested negative for ABBV-1 by RT-qPCR, however the intramuscular injection into the right gastrocnemius muscle was suspected as the possible cause of lameness. At 68 dpi, one bird in the IC group (#136) exhibited severe torticollis and was euthanized. Postmortem analysis revealed no gross musculoskeletal abnormalities. This bird tested positive for ABBV-1 infection by IHC and histologically had prominent lymphoplasmacytic perivascular cuffs in the central nervous tissue (see below). This bird was processed for extensive tissue sampling for pathology and IHC (not for RT-qPCR), and results were included in the cohort for the next sampling time (12 wpi).

No clinical signs or gross lesions were identified in any of the other birds. Based on the postmortem results, the experimental groups had the following sex distribution: 23 males, 18 females and 3 undetermined in the IC group (n, 44); 15 males, 28 females in the IM group (n, 43); 24 males, 19 females in the PO group (n, 43); and 19 males, 23 females and 1 undetermined in the CO group (n, 43).

Histopathology and severity of inflammation

An extensive set of tissues for histopathological evaluation was collected from a total of 48 birds (3 birds per time point per experimental group). In addition, partial sections of brain, spinal cord, kidney, proventriculus, and ventriculus were evaluated histologically from an additional cohort of birds at 8 and 12 wpi from the IC and IM groups (total, 32 additional birds), which were also sampled for virus titration by RT-qPCR. For bird #136, extensive tissue sampling for histology was carried out, and results were included in the 12 wpi group.

A summary of histological findings in the nervous system is shown in Table 1. Microscopic lesions were identified exclusively in the central nervous system of birds in the IC and IM groups, and consisted of non-heterophilic inflammation with prominent perivascular cuffs of lymphocytes, which often spilled into the adjacent neuropil. Overall, inflammation did not appear to target a specific area of the central nervous system. In the IC group, encephalitis (inflammation of any section of the cerebrum, optic lobes, brainstem or cerebellum) was identified in 100 % of birds at both 8 and 12 wpi (Figures 1A-B), while myelitis was seen in 29 % and 33 % of birds at these time points (Figures 1C), respectively. In this group, gliosis affecting less than 10 % of the tissue in section was identified in 40 % and 83 % of birds at 8 and 12 wpi, respectively. Gliosis was characterized by randomly distributed glial cells arranged in variably sized clusters or scattered hypertrophied and euchromatic glial cells (Figure 1D). In the IM group, encephalitis, myelitis, and spinal meningitis were seen at 12 wpi only in one bird out of 13 (the same bird was affected, #210). In birds from the IM group, no gliosis was observed.

In the IC group, the total number of birds with encephalitis was significantly higher than those with myelitis (chi-square test, p < 0.001). At 8 wpi, the inflammatory lesions in the spinal cord were significantly less severe compared to the inflammation of the cerebrum; and at 12 wpi, lesions in the spinal cord were less severe compared to the cerebrum, optic lobe, and brainstem, and lesions in the cerebrum were significantly more severe compared to the cerebellum. While there was an average increase in the severity of histological lesions between 8 and 12 wpi, no differences were significant between the same segments of the CNS across these two time points (Figure 2A). There were no differences between frequency and scores of inflammation between segments of the central nervous tissue in birds from the IM group (data not shown).

Taking into consideration the overall brain scores, averaged over all the brain segments, inflammation was more severe in birds from the IC group at 12 compared to 8 wpi, although this difference was not significant; the inflammatory score in the IM group at 12 wpi was significantly lower compared to the scores in the IC group at both 8 and 12 wpi (Figure 2B).

Immunohistochemistry

Immunohistochemistry for CD3 (Figure 1E) and Pax-5 (Figure 1F) revealed that the majority of inflammatory cells forming the perivascular cuffs were formed by T lymphocytes, with only a minority of B cells. As expected, CD3 showed a finely granular, cytoplasmic to membranous reactivity, and Pax-5 showed nuclear reactivity (Gibson et al., 2021).

The distribution of ABBV-1 N protein was evaluated by IHC from 3 chickens in IC group at 12 wpi, plus the bird with severe clinical signs that was euthanized at 68 dpi. Reactivity for ABBV-1 N protein was detected in all 3 birds exclusively in the brain and spinal cord; none of the other tissues evaluated (n, 17) tested positive, including the gonads, peripheral nerves, and the plexuses in the wall of the gastrointestinal tract (Table 2). Signal in the brain was multifocal and randomly distributed, without clear predilection for specific sites. Reactivity was observed mainly in neurons (Figure 3A-C) and rarely in glial cells, and presented as finely granular, predominately nuclear though occasionally cytoplasmic. The bird that presented clinical signs (#136) showed immunoreactivity exclusively in the cerebrum and optic lobes, with no reactivity in the brainstem and spinal cord, despite presence of inflammation in these areas. The extent of IHC reactivity was scored (Table 2) for separate areas of the central nervous system, and no differences were identified between brain areas (Kruskal-Wallis test with Dunn’s multiple comparisons, adjusted p > 0.07).

Virus replication in tissues

The genome copy number of ABBV-1 was interpolated based on a standard curve with serial dilution of a gBlock fragment of the N gene. No virus genetic material was detected in the tissues from the CO and PO groups. In the IC group, ABBV-1 RNA was first detected in the brain at 1 wpi in 5/7 birds (71 %), and in the subsequent time points was detected in the brain of all tested birds (100 %) with increasing magnitude up until 12 wpi (Table 3). In the spinal cord, virus RNA was detected starting at 4 wpi in 2/7 birds (29 %), and increased gradually at 8 and 12 wpi in both frequency (> 75 % of birds) and magnitude. When all time points were considered, the differences in proportions of ABBV-1-positive brains and spinal cords were not significant (Chi-square, p < 0.001). The magnitude of genome copy number in the brain at 12 wpi was highest, and significantly different when compared to the titer in the brain at previous time points and the spinal cord at 12 wpi (Figure 4A). Only one bird had viral RNA in the renal tissue at 4 wpi, albeit at very low titer. No other tissues or swabs were positive for ABBV-1 RNA in the IC group.

In the IM group, virus RNA was first detected in the spinal cord of 3/7 birds (43 %) at 8 wpi, with an increase in magnitude of copy number at 12 wpi (Table 3). In the brain, ABBV-1 RNA was detected exclusively in 2/10 birds (20 %) at 12 wpi, averaging to less than 100 genome copies per 150 ng of total RNA. No differences were significant in the IM group, when comparing the total proportion of positive brains and spinal cords (Chi-square, p = 0.12), or the average virus copy number between different organs or time points (Figure 4B). No other tissues or swabs were positive for ABBV-1 RNA in the IM group.

Virus isolation from brain

ABBV-1 was successfully isolated in DEF from the brain of 2 chickens sampled at 12 wpi from the IC group (data not shown). The isolation procedure from birds in the CO group did not yield infectious virus.

Serology

Presence of antibodies against the N protein of ABBV-1 was tested in the serum of infected birds at 1 and 12 wpi (6 from each group), using protein blots of Vero cells transfected with a plasmid expressing the coding region of the ABBV-1 N gene. A rabbit monospecific antibody (positive control) showed that the lysate from transfected Vero cells expressed large amounts of ABBV-1 N protein. None of the birds in the CO and PO groups had reactive sera. In the IC and IM groups, no birds had reactive sera at 1 wpi, while there were 4/6 and 1/6 birds seropositive at 12 wpi, respectively, highlighting a band between 35 and 40 kDa (Figure 5).

The present study documents that chickens are susceptible to infection with ABBV-1, when inoculated through the IC and IM routes. This is consistent with an increasing body of literature, which shows that experimental administration of ABVs through the oral route is highly inefficient (Piepenbring et al., 2012, 2016; Iverson et al., 2021). To our knowledge, this is the first successful experimental infection of a gallinaceous species with ABBV-1. Prior to our study, Ludwig and colleagues inoculated one day-old chicks intracranially with the brain homogenate from Borna disease virus (BoDV)-infected rabbits (Ludwig et al., 1973). All chicks (100%) in the experimental group developed neurologic signs including paralysis of legs and wings, and birds examined histologically had characteristic BoDV intranuclear Joest-Degen inclusion bodies. A more recent report documented another gallinaceous bird (Himalayan monal, a type of pheasant) naturally infected with psittacine bornavirus 4 (PaBV-4), as confirmed by RT-qPCR and IHC of brain tissue. This bird presented with neurological signs, crop stasis, and wasting (Bourque et al., 2015).

In the present study, intracranial inoculation of White Leghorn chickens with ABBV-1 led to establishment of persistent infection almost exclusively in the CNS, as shown by RT-qPCR and IHC signal limited to neurons and glial cells of the brain and spinal cord. These results differ from our previous study in Muscovy ducks with ABBV-1, as well as numerous infection trials of psittacine birds with PaBVs, which showed that intracranial inoculation leads to infection of both the CNS and several visceral tissues, suggesting a centrifugal virus spread (i.e., from the central nervous system to peripheral tissues) (Piepenbring et al., 2012, 2016; Iverson et al., 2021). This is also consistent with reports of natural ABBV-1 infection in waterfowl, which is associated with a wide tissue distribution of the virus (Delnatte et al., 2013). On the other hand, inoculation of chicks in the gastrocnemius muscle led to CNS infection in less than 20% of birds, indicating only a moderate ability of the virus to spread centripetally from the periphery to the CNS. In Muscovy ducks inoculated in a similar way, however, spread from the gastrocnemius muscle to the CNS was more efficient, leading to CNS infection in more than 70% of inoculated birds (Iverson et al., 2021). Taken together, these data suggest that, although chickens can become infected with ABBV-1, virus distribution in tissues is limited compared to ducks naturally or experimentally infected with ABBV-1, as well as psittacine birds infected with PaBVs. Furthermore, lack of ABBV-1 in the choanal and cloacal swabs suggests that chickens may not shed ABBV-1 in the environment and transmit the virus. These results are consistent with the current understanding that wild waterfowl are the natural reservoir of ABBV-1 (Payne et al., 2012a; Delnatte et al., 2013), and indicate partial host restriction of ABBV-1 in chickens. This conclusion is also supported by our earlier work showing that ABBV-1 grows poorly in chicken and primary immortalized cell lines (Leacy et al., 2020; Pham et al., 2020). It remains to be evaluated if passaging of ABBV-1 in chickens could promote host adaptation and ultimately increase its replicative efficacy in this species.

Microscopic lesions, characterized by lymphocytic perivascular inflammation within the nervous tissue, were present almost exclusively in the IC chicken group. At 8 and 12 wpi, 100% of IC chickens had encephalitis, with the highest overall inflammation scores occurring at 12 wpi. In contrast, only one IM chicken developed a mild encephalitis at 12 wpi, despite some brains (2/10) and spinal cords (3/9) from the same timepoint testing positive by RT-qPCR. Differences in inflammation between the CNS of chickens in the IC and IM groups are likely the consequence of timing and magnitude of infection. No inflammation in the IC group was observed before 8 wpi, as virus titers before this time point remained relatively low. Similarly, ABBV-1 RNA in the brain of IM chickens was almost at the limit of detection even at 12 wpi, consistent with lack of inflammation in this group except for one bird. These findings agree with our previous experiment in Muscovy ducks, which showed that inflammation temporally followed ABBV-1 infection in tissues, with lesions appearing first in the brain or spinal cord of birds infected intracranially or intramuscularly, respectively (Iverson et al., 2021). Gliosis was observed exclusively in IC chickens at 8 and 12 wpi, and likely was associated with the increased severity of inflammation.

By 12 wpi, only 4 of 6 IC and 1 of 6 IM chickens had seroconverted, while none of the birds from the other groups did. This corresponds with the higher frequency and magnitude of inflammation in the IC group, suggesting a heightened immune response. In comparison, 6/6 and 2/6 of Muscovy ducks infected with ABBV-1 via the IC and IM routes, respectively, showed seroconversion by 12 wpi (Iverson et al., 2021); a finding that is consistent with the relatively higher ABBV-1 replication and inflammation severity in tissues of ducks, as discussed.

Despite development of inflammatory lesions in the CNS of 23 birds, only one chicken developed neurological signs, and none displayed a dilated proventriculus. Lack of clinical signs were also observed in Muscovy ducks infected with ABBV-1, despite a higher frequency of microscopic lesions (Iverson et al., 2021). It is plausible that the 12-week duration of our experiment might not have afforded sufficient time for development of clinical signs. A long incubation period has been described in other animal models of ABVs, including experimental infection of PaBV-4 in adult cockatiels via a combination of IM and PO routes, which did not result in clinical signs until 92 dpi (S. Payne et al., 2011). Age may have also played a role, as infections at a very early age may have led to immune tolerance, limiting the development of a cytotoxic T cell response. In a recent study, mature cockatiels infected with PaBV-4 developed encephalitis and clinical signs, while younger birds developed only inflammatory lesions but no clinical signs (Gartner et al., 2021). However, it remains unclear how immunotolerant birds could seroconvert, albeit limitedly. Quantitative ways of assessing seroconversion, as opposed to Western blotting, may reveal a more direct relationship between immune response and lesion development. Lastly, it should be noted that natural cases of ABBV-1 infection have been identified in many healthy waterfowl (Susan Payne et al., 2011; Thomsen et al., 2018), indicating that there are certain factors able to trigger development of clinical signs in birds that had been persistently infected for a long time.

To confirm that the virus detected by RT-qPCR was infectious, we successfully isolated ABBV-1 from the brain of 2 chickens from the IC group. This is significant, as it enforces our results by indicating that there is replication of infectious virus in the brains of infected birds.

The SPF White Leghorn chickens used in this study were obtained from the Canadian Food Inspection Agency at one-day-old. While we did not have a method to confirm each chick was ABBV-1 negative prior to inoculation (PCR of the CNS being the best method for detection), we are confident in our results as at no point during the study were tissues from birds in the CO or PO groups RT-qPCR positive for ABBV-1.

We demonstrated that day-old White Leghorn chickens can become infected with ABBV-1 when inoculated via the intracranial and intramuscular, but not oral, routes. In infected birds, ABBV-1 replicated almost exclusively in the central nervous system, causing development of characteristic histological lesions, although neurological signs were observed in only one bird. The virus did not replicate significantly in visceral tissues, suggesting that ABBV-1 is partially host restricted in chickens compared to waterfowl. Further investigation into the development of clinical signs and potential for horizontal transmission is needed in order to better assess the risk that ABBV-1 poses to the commercial poultry industry

Author Contributions. MI wrote the manuscript and had critical input in the study design. LS devised and designed the experiment, co-wrote the manuscript, received funding and regulatory approval to conduct the study. MI, AL, PP, and LS carried out the live animal study. MI conducted microscopic analysis of sampled tissues, and devised and implemented a semi-quantitative score for histology and immunohistochemistry. AL and PP prepared the ABBV-1 virus stock, and conducted RNA extraction, RT-qPCR, Western blot analysis, and virus isolation form brain. EN provided the virus and primary cell culture systems, critically reviewed the experimental protocol, and edited the manuscript. MI and LS participated in data analysis, figure and graph production, and statistical analysis. BL and EB contributed to finalize the study design, data analysis, and critically reviewed the manuscript.

Competing interests. The authors declare no competing interests.

Declarations. Animal use and experimental procedures involving animals were approved by the University of Guelph Animal Care and Use Committee (Animal Utilization Protocol #3978).

Consent for publication. All authors reviewed the manuscript and approved it in its final form.

Availability of data and materials. Any additional data is submitted as Supplementary Material.

Funding. This work was funded by the Alliance program of the Ontario Ministry of Agriculture, Food and Rural Affairs (grant UofG2016-2577), and the Discovery program of the Natural Science and Engineering Research Council (grant 401127). The Doctor of Veterinary Science student fund of the Pathobiology Department provided Dr. Iverson’s stipend.

Acknowledgements. We thank Drs. Dale Smith and Pauline Delnatte for their work with ABBV-1 at the University of Guelph and providing the initial isolate. We thank Susan Lapos and Josepha Delay for the help with tissue processing and immunohistochemistry. Summer students from the Ontario Veterinary College (OVC), Elena Huong and Akshu Valecha, for their help with postmortem sessions.

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Table 1. Frequency and time distribution of chickens presenting inflammation and/or gliosis in different areas of the central nervous system upon inoculation with ABBV-1 by the intracranial (IC) and intramuscular (IM) routes.

Affected Area or Disease Category

1 wpi^a

4 wpi

8 wpi

12 wpi^f

Total

1 wpi

4 wpi

8 wpi

12 wpi

Total

Cerebrum^c

0/3^b

0/3

10/10

12/12

22/28

0/3

0/10

0/12

0/28

Optic lobe

0/2

0/3

7/8

11/12

18/25

0/3

0/9

0/12

0/27

Brainstem

0/2

0/3

7/8

9/9

16/22

0/3

0/2

0/6

1/9

1/20

Cerebellum

0/3

7/9

8/11

15/26

0/3

0/2

0/8

0/13

0/26

Spinal cord

0/3

2/7

3/9

5/22

0/3

0/9

1/11

1/26

Brain meninges

0/3

7/10

9/11

16/27

0/3

0/10

0/12

0/28

Spinal meninges

0/3

0/7

5/9

5/22

0/3

0/8

1/11

1/25

Peripheral nerves

0/3

0/4

0/13

0/3

0/12

Encephalitis^d

0/3

10/10

12/12

22/28

0/3

0/10

1/13

1/29

Gliosis in brain^e

0/3

4/10

10/12

14/28

0/3

0/10

0/13

0/29

^aWpi, week post-infection. ^bIndicates birds with disease in that tissue over the number of birds tested at that time point, or over all the time points (total). ^cIndicates anatomical areas affected by inflammation. ^dEncephalitis was assigned if birds had inflammatory lesions in any section of the brain section (cerebrum, optic lobe, brainstem, cerebellum), excluding gliosis. ^eGliosis was assigned in birds with proliferation / hypertrophy of glial cells or glial nodules in any section of the brain (cerebrum, optic lobe, brainstem, cerebellum). ^fThe 12 wpi group includes the results of the only bird (#136) that presented clinical signs and was euthanized at 68 days post-infection.

Table 2. Reactivity for ABBV-1 N protein, as assessed by immunohistochemistry (IHC), in the tissues of four chickens experimentally infected through the IC route and sampled between 68 days post-infection (bird #136) and 12 weeks post-infection (birds #1-3).

Tissues	#1	#2	#3	#136	Total	Description of cells with IHC reactivity^f
Cerebrum	2^a	2	1	2	4/4^b	Neurons, glial cells, Purkinje cells
Optic lobe	2	1	2	1	4/4
Brainstem	1	0	1	0	2/4
Cerebellum	1	0	0	0	1/4
Spinal cord	1	0	1	0	2/4	Neurons, glial cells
Peripheral nerves	-^c	-	-	-	0/4	-
Kidney	-	-	-	-	0/4	-
Gonad	- (M)^d	- (F)	- (F)	- (F)	0/4	-
Proventriculus	-	-	-	-	0/4	-
Ventriculus	-	-	-	-	0/4	-
Heart	-	-	-	-	0/4	-
Pancreas	-	-	NA	-	0/3	-
Adrenal	-	-	-	-	0/4	-
Thyroid/parathyroid	-	-	-	-	0/4	-
Small intestine	-	-	-	-	0/4	-
Colon	-	-	-	-	0/4	-
Lung	-	-	-	-	0/4	-
Liver	-	-	-	-	0/4	-
Spleen	-	-	-	-	0/4	-
Trachea	-	-	-	-	0/4	-
Esophagus	-	NA^e	-	-	0/3	-
Bursa	-	-	NA	-	0/3	-

^aDistribution score was assessed only for the central nervous system (cerebrum, optic lobe, brainstem, cerebellum, and spinal cord). ^bIndicates the number of birds with immunohistochemical reactivity for that tissue, over the total birds available for examination. ^cPresence (+) or absence (-) of immunohistochemical reactivity in tissues for which distribution score was not tallied. ^dMale (M) or female (F) sex of birds. ^eNA, tissue was not available for analysis. ^fOnly cells with nuclear, with or without cytoplasmic reactivity, were considered reactive.

Table 3. Frequency and time distribution of select tissues that tested positive for the ABBV-1 N gene, sampled from chickens inoculated with ABBV-1 by the intracranial (IC) and intramuscular (IM) routes.

Samples

1 wpi^a

4 wpi

8 wpi

12 wpi

Total

1 wpi

4 wpi

8 wpi

12 wpi

Total

Brain

5/7^b

7/7

8/8

27/29

0/7

2/10

2/31

(2.07 x10²)^c

(7.62 x 10⁵)

(1.08 x 10⁶)

(5.62 x 10⁶)

(6.22 x 10¹)

Spinal cord

0/6

2/7

7/7

6/8

15/28

0/7

3/7

3/9

6/30

(4.22 x 10¹)

(3.73 x 10⁴)

(2.40 x 10⁶)

(4.14 x 10²)

(1.87 x 10⁶)

Proventriculus

0/7

0/8

0/29

0/7

0/10

0/31

Kidney

0/7

1/7

0/7

0/8

1/29

0/7

0/10

0/31

(9.72 x 10¹)

Cloacal swab

0/10

0/11

0/41

0/10

0/13

0/43

Choanal swab

0/10

0/11

0/41

0/10

0/13

0/43

^aWpi, week post-infection.^bIndicates birds with disease in that tissue over the number of birds tested at that time point, or over all the time points (total). ^cIn parenthesis is the average genome copy number, calculated from birds that tested positive.

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Pathogenesis of Aquatic Bird Bornavirus 1 in Domestic Chickens

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