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We describe the isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14 (RbCoV 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. RbCoV HKU14 was able to replicate in HRT-18G and RK13 cells with cytopathic effects.
Recombination analysis revealed possible recombination events during the evolution of RbCoV HKU14 and members of Betacoronavirus 1, which may have occurred during cross-species transmission.
Coronaviruses (CoVs) are found in a wide variety of animals, in which they can cause respiratory, enteric, hepatic, and neurological diseases of various severities. Based on genotypic and serological characterizations, CoVs were traditionally classified into three distinct groups (5, 25). Recently, the Coronavirus Study Group of the International Committee on Taxonomy of Viruses (ICTV) has revised the nomenclature and taxonomy to classify coronaviruses into three genera, Alphacoronavirus, Betacoronavirus, and Gammacoronavirus, replacing the traditional group 1, 2, and 3 CoVs (7). Historically, alphacoronaviruses and betacoronaviruses were found in mammals, while gammacoronaviruses were found in birds, although recent findings also suggested the presence of gammacoronaviruses in mammals (21, 37, 70). Novel CoVs, which represented a novel genus, Deltacoronavirus, have also been identified in birds and pigs (69, 70, 74). As a result of the unique mechanism of viral replication, CoVs have a high frequency of recombination, which, coupled with high mutation rates, may allow them to adapt to new hosts and ecological niches (17, 25, 32, 67).
The discovery of the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) as the causative agent of the SARS epidemic and the identification of SARS-CoV-like viruses from palm civets and horseshoe bats in China have boosted interests in the discovery of novel CoVs in both humans and animals (15, 27, 29, 35, 36, 42, 46, 71). During the post-SARS era, two novel human CoVs, both associated with respiratory tract infections, have been discovered. Human coronavirus NL63 (HCoV NL63), an alphacoronavirus, was reported independently by two groups in the Netherlands in 2004 (12, 60), whereas human coronavirus HKU1 (HCoV HKU1), a betacoronavirus, was identified in patients from Hong Kong in 2005 (28, 68, 72). As for animal CoVs, a previously unknown diversity of CoVs was described for various bat species in China and subsequently in other countries (9, 13, 30, 32, 43, 57, 59, 64, 73). In addition, a number of novel CoVs have been identified in other animals (10, 16, 21, 37, 70, 77), suggesting that our understanding of the diversity and evolution of CoVs in animals is still far from complete (74).addition, a number of novel CoVs have been identified in other animals (10, 16, 21, 37, 70, 77), suggesting that our understanding of the diversity and evolution of CoVs in animals is still far from complete (74).
Despite the identification of horseshoe bats in China as the natural reservoir of SARS-CoV-like viruses, it is still unknown if these animals are the direct origin of SARS-CoV in civet and human (27, 29, 35). In particular, the spike protein of SARS-related Rhinolophus bat coronavirus (SARSr-Rh-BatCoV) showed only ∼80% amino acid identity to that of civet SARS-CoV, with significant differences from the receptor binding domain of SARS-CoV (29, 35, 44). Since bats are commonly found and served in wild-animal markets and restaurants in Guangdong, which often house a variety of animals (65), we attempted to study other animals in Guangdong wet markets, which may have served as intermediate hosts for interspecies transmission or may harbor CoVs that could have recombined with SARSr-Rh-BatCoV to generate a SARS-CoV capable of infecting civet. During the investigations, a previously undescribed Betacoronavirus subgroup A CoV, rabbit coronavirus HKU14 (RbCoV HKU14), was detected in domestic rabbits. In this study, we describe the discovery and characterization of RbCoV HKU14, which was successfully isolated from HRT-18G and RK13 cell cultures. Complete genome analyses of four RbCoV HKU14 strains were carried out to study the genome features and molecular evolution in relation to those of other Betacoronavirus subgroup A CoVs.
MATERIALS AND METHODS
Sample collection.All specimens were collected from live food animal markets in Guangzhou, China, from March 2006 to June 2009. A total of 165 animal or environmental samples from markets with a diversity of food animals and, subsequently, 136 fecal and 30 serum samples from domestic rabbits (Oryctolagus cuniculus) were collected by using procedures described previously (29, 70). All samples were placed into viral transport medium before transportation to the laboratory for nucleic acid extraction.
Identification of a novel CoV from rabbits.Of 165 various animal/environmental samples from markets, RT-PCR for a 440-bp fragment in the RdRp gene of CoVs was positive for a potentially novel CoV in two samples, both from domestic rabbits (one rabbit anal swab sample and one rabbit cage swab sample). Sequencing results suggested that the potential novel virus was most closely related to members of the species Betacoronavirus 1, which includes bovine coronavirus (BCoV), equine coronavirus (ECoV), porcine hemagglutinating encephalomyelitis virus (PHEV), and HCoV OC43, with ≤91.6% nucleotide identities. In view of these preliminary results, fecal samples from 136 domestic rabbits were subsequently collected.
Moreover, human sera from a patient with HCoV OC43 infection, 28 of 32 healthy blood donors, and 32 of 33 SARS patients were positive for antibody against a recombinant RbCoV HKU14 N protein by a Western blot assay. 4 to detect neutralizing antibodies in rabbit and human sera. One of the 20 rabbit sera positive for antibody against the recombinant RbCoV HKU14 N protein and human sera from a patient with HCoV OC43 infection, 5 of 10 healthy blood donors, and 4 of 10 SARS patients were found to possess neutralizing antibody to RbCoV HKU14 with a titer of ≥1:8.
We isolated and characterized a novel Betacoronavirus subgroup A CoV, RbCoV HKU14, from domestic rabbits in wet markets in Guangzhou, China. Betacoronavirus subgroup A CoVs include the traditional “group 2 CoVs,” including MHV, HCoV HKU1, HCoV OC43, BCoV, and PHEV, whereas SARS-CoV-like viruses were classified under Betacoronavirus subgroup B CoVs. for most of its ORFs, including the S gene, suggested that rabbits are likely the natural reservoir of RbCoV HKU14. Interestingly, anti-RbCoV HKU14 N antibodies and neutralizing antibody to RbCoV HKU14 were also detected in a significant proportion of healthy blood donors and SARS patients. This may due to the presence of cross-reacting antibodies due to past infection by human betacoronaviruses such as HCoV OC43, in line with our previous findings on cross-reactivity between HCoV OC43 and SARS-CoV (66). Further studies are required to understand the cross-reactivity among the different betacoronaviruses.