Isolation of Ontario aquatic bird bornavirus 1 and characterization of its replication in immortalized avian cell lines

Abstract

Background: Aquatic bird bornavirus 1 (ABBV-1) has been associated with neurological diseases in wild waterfowls. In Canada, presence of ABBV-1 was demonstrated by RT-qPCR and immunohistochemistry in tissues of waterfowls with history of neurological disease and inflammation of the central and peripheral nervous tissue, although causation has not been proven by pathogenesis experiments, yet. To date, in vitro characterization of ABBV-1 is limited to isolation in primary duck embryo fibroblasts. The objectives of this study were to describe isolation of ABBV-1 in primary duck embryonic fibroblasts (DEF), and characterize replication in DEF and three immortalized avian fibroblast cell lines (duck CCL-141, quail QT-35, chicken DF-1) in order to evaluate cellular permissivity and identify suitable cell lines for routine virus propagation.

Methods: The virus was sequenced, and phylogenetic analysis performed on a segment of the N gene coding region. Virus spread in cell cultures, viral RNA and protein production, and titres were evaluated at different passages using immunofluorescence, RT-qPCR, western blotting, and tissue culture dose 50% (TCID₅₀) assay, respectively.

Results: The isolated ABBV-1 showed 97 and 99% identity to European ABBV-1 isolate AF-168 and North American ABBV-1 isolates 062-CQ and CG-N1489, and could infect and replicate in DEF, CCL-141, QT-35 and DF-1 cultures. Viral RNA was detected in all four cultures with highest levels observed in DEF and CCL-141, moderate in QT-35, and lowest in DF-1. N protein was detected in western blots from infected DEF, CCL-141 and QT-35 at moderate to high levels, but minimally in infected DF-1. Infectious titre was highest in DEF (between approximately 10⁵ to 10⁶ FFU / 10⁶ cells). Regarding immortalized cell lines, CCL-141 showed the highest titre between approximately 10⁴ to 10⁵ FFU / 10⁶ cells. DF-1 produced minimal infectious titre.

Conclusions: This study confirms the presence of ABBV-1 among waterfowl in Canada and reported additional in vitro characterization of this virus in different avian cell lines. ABBV-1 replicated to highest titre in DEF, followed by CCL-141 and QT-35, and poorly in DF-1. Our results showed that CCL-141 can be used instead of DEF for routine ABBV-1 production, if a lower titre is an acceptable trade-off for the simplicity of using immortalized cell line over primary culture.

Keywords: ABBV-1 replication in avian cells; Aquatic bird bornavirus-1; Avian bornavirus; Immortalized avian cell lines, duck embryo fibroblasts; Persistent infection.

Fig. 1

Detection of ABBV-1 from persistently infected DEF using western blot, IFA, RT-qPCR and virus titration. For panels a), b) and c), DEF were incubated with either the brain homogenate from an ABBV-1 positive goose or control medium, and monitored for potential ABBV-1 replication using western blot and IFA. a Western blot showing detection of ABBV-1 N protein in lysates from infected DEF but not in uninfected control DEF culture. β-actin was detected in both infected and control DEF cultures. b and c IFA of control and infected DEF cultures, respectively, using a primary antibody against ABBV-1 N protein (green) and counterstained with DAPI (blue). Scale bar, 100 μm. For panels d, e and f, DEF were infected with cell-culture harvested ABBV-1 and monitored for ABBV-1 replication using RT-qPCR, western blot, and virus titration. D) RT-qPCR of DEF infected with ABBV-1 over the first seven passages. E) Western blot of ABBV-1-infected DEF at passages 1–3, 6–8 and 11–13. F) Virus titration of ABBV-1-infected DEF at passages 6–8 and 10–12

Fig. 2

Detection of ABBV-1 replication by IFA, in DF-1, CCL-141, and QT-35 at early passages. Representative pictures of IFA from ABBV-1-infected DF-1, CCL-141, and QT-35 at passages 1–3 post-infection. Blue fluorescence shows DAPI nuclear counterstain, and green fluorescence indicates ABBV-1 N protein. The fourth column shows a magnified area of the picture on the third column. Speckled or punctate signal in the nucleus is more readily observed in infected CCL-141 (fourth column) compared to the than the other cell lines. Scale bar, 100 μm

Fig. 3

Detection of ABBV-1 N gene, by RT-qPCR, in DF-1, CCL-141, and QT-35 at early passages. RT-qPCR performed on DF-1, CCL-141, and QT-35 infected with ABBV-1 at passages 2 to 7 post-infection in two independent experiments (panels a and b). The primers and probes targeted ABBV-1 N gene. Cycle threshold (Ct) less than 35 was considered positive, with lower Ct values indicating higher target concentrations. Viral N gene levels increased in CCL-141 and QT-35 with subsequent passages, but in DF-1 remained steady at levels close to the detection threshold

Fig. 4

N protein expression in infected DF-1, CCL-141, and QT-35. Western blot of N protein levels in cell lysates collected from cultures of infected DF-1, CCL-141, and QT-35 in two independent experiments. A) Western blot of passages 11–13 (infection 1) showing detection of intense N protein bands in infected CCL-141 and QT-35 cultures and faint N protein bands in infected DF-1 cultures. β-actin protein bands were detected with strong intensity in all samples. B) Western blot of passages 1–3, 6–8, and 11–13 (infection 2). N protein was detected in CCL-141 starting from passage 3, in QT-35 starting from passage 7 (faint band), and in DF-1 starting from passage 12 (faint band)