We describe the isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14 (Rb. CoV HKU14), from domestic rabbits. The virus was detected in 11 (8.1%) of 136 rabbit fecal samples by reverse transcriptase PCR (RT-PCR), with a viral load of up to 108 copies/ml. Rb. CoV HKU14 was able to replicate in HRT-18G and RK13 cells with cytopathic effects. Northern blotting confirmed the production of subgenomic mRNAs coding for the HE, S, NS5a, E, M, and N proteins. Subgenomic mRNA analysis revealed a transcription regulatory sequence, 5′-UCUAAAC-3′. Phylogenetic analysis showed that Rb. CoV HKU14 formed a distinct branch among Betacoronavirus subgroup A coronaviruses, being most closely related to but separate from the species Betacoronavirus 1. A comparison of the conserved replicase domains showed that Rb-CoV HKU14 possessed <90% amino acid identities to most members of Betacoronavirus 1 in ADP-ribose 1"-phosphatase (ADRP) and nidoviral uridylate-specific endoribonuclease (NendoU), indicating that Rb. CoV HKU14 should represent a separate species. Rb. CoV HKU14 also possessed genomic features distinct from those of other Betacoronavirus subgroup A coronaviruses, including a unique NS2a region with a variable number of small open reading frames (ORFs). Recombination analysis revealed possible recombination events during the evolution of Rb. CoV HKU14 and members of Betacoronavirus 1, which may have occurred during cross-species transmission. Molecular clock analysis using RNA-dependent RNA polymerase (Rd. Rp) genes dated the most recent common ancestor of Rb. CoV HKU14 to around 2002, suggesting that this virus has emerged relatively recently. Antibody against Rb. CoV was detected in 20 (67%) of 30 rabbit sera tested by an N-protein-based Western blot assay, whereas neutralizing antibody was detected in 1 of these 20 rabbits. © 2012, American Society for Microbiology.
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