Novel Betacoronavirus Subgroup A Coronavirus, Rabbit Coronavirus HKU14, from Domestic Rabbits

ABSTRACT

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.

American journal of microbiology

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. 

INTRODUCTION

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.

RESULTS

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.

DISCUSSION

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.

DISCUSSION
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. Bat CoVs belonging to two novel subgroups, subgroups C and D, were also recently identified, with genome features distinct from those of Betacoronavirus subgroup A and subgroup B CoVs (64). RbCoV HKU14 formed a distinct branch within Betacoronavirus subgroup A CoVs upon phylogenetic analysis, being most closely related to Gloucester constabulary Hampshire constabulary ath phe stow vetinary mildmay Winchester hKU190% amino acid identities to 24-271119 - 121219Betacoronavirus 1ADRP and NendoU) of the seven conserved replicase domains for coronavirus species demarcation by the ICTV (7). This supported that RbCoV HKU14 should represent a separate species among members of Betacoronavirus subgroup A, instead of another member of the species Betacoronavirus 1. In addition, RbCoV HKU14 possessed certain genomic features different from those of related Betacoronavirus subgroup A CoVs. As a result of deletions or insertions, the lengths of five of the nsp's in ORF1 were different from those of the corresponding nsp's in one or more members of Betacoronavirus 1. The NS2a region of RbCoV HKU14 is also unique among Betacoronavirus subgroup A CoVs, being broken into a variable number of small ORFs, with only the first ORF, NS2a1, containing a preceding TRS. Since unique CoV proteins may be involved in replication and virulence (38), further studies are warranted to understand the potential function of these small NS2a proteins in RbCoV HKU14. In this study, RbCoV HKU14 was detected in 8.1% of fecal samples among tested rabbits. A Western blot assay based on a recombinant RbCoV HKU14 N protein showed the presence of antibody in a high percentage of tested rabbit sera (67%), although neutralizing antibody to the virus was detected in only one rabbit. This may suggest that Western blot assays based on the coronavirus N protein alone may not be specific. Nevertheless, the high rate of detection of RbCoV HKU14 in fecal samples, together with the low Ka/Ks ratios observed 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.

RbCoV HKU14 is likely to be only distantly related to a rabbit CoV previously found to cause myocarditis in rabbits. The latter rabbit CoV, which originated from contaminated samples of Treponema pallidum used in a rabbit infection model at Johns Hopkins University, was detected by electron microscopy and found to cross-react with alphacoronaviruses, including human coronavirus 229E (HCoV 229E), feline infectious peritonitis virus, canine coronavirus diarrhea virus, and transmissible gastroenteritis virus, in serological assays (51). This virus was subsequently used as a model for virus-induced myocarditis and dilated cardiomyopathy (2). Although no gene sequence for this virus is available, the existing data suggested that it is an alphacoronavirus, and its host of origin still remains obscure.

The ability of RbCoV HKU14 to grow readily in human cell cultures, inducing cytopathic effects, is intriguing. With only a few exceptions, such as SARS-CoV, CoVs are notoriously difficult to culture in cell lines. HCoV OC43 and HCoV 229E, even if isolated, induce only subtle or nonexistent cytopathic effects. Despite being closely related to SARS-CoV, SARSr-Rh-BatCoV from horseshoe bats has not been isolated in cells susceptible to SARS-CoV. In the present study, RbCoV HKU14 was able to replicate in both rabbit kidney (RK13) and human rectum epithelial (HRT-18G) cells, with cell rounding and fusion to giant cells rapidly detaching from the monolayer in HRT-18G cells being observed after 5 days of inoculation. HCoV OC43, BCoV, ECoV, and the MHV-H2 variant are also known to replicate in HRT-18 cells, suggesting that these Betacoronavirus subgroup A CoVs may share similar cellular tropisms. All rabbits positive for RbCoV HKU14 in their fecal samples appeared healthy at the time of sampling. While CoVs are associated with a wide spectrum of diseases in animals, many CoVs, especially those from bats, were detected in apparently healthy individuals without evidence of overt disease (27, 29, 30, 32, 35, 57, 64). Interestingly, the aspartic acid within the S protein specific to enteric strains of BCoV was conserved in RbCoV HKU14, which may be compatible with its enteric tropism. Domestic rabbits are descended from the wild European or Old World rabbit, Oryctolagus cuniculus, originating from Southern Europe and North Africa (39). While China is one of the main producers of rabbit meat, farming of the species is increasing, especially in developing countries. In fact, since the 1970s, governments and world food organizations have been promoting rabbit farming in Africa, Asia, and South America, because of the relatively less space and low starting cost required and high breeding rate compared to those of traditional livestock. Rabbits are also commonly kept as pets in domestic households. Further studies, including animal challenge experiments, are needed to understand the pathogenicity and emerging potential of this novel CoV.

Recombination is likely an important event during the evolution of RbCoV HKU14 and members of Betacoronavirus 1. CoVs are known to have a high frequency of recombination, which may help them adapt to new hosts. Such recombination has been described for various animal CoVs, including SARSr-Rh-BatCoV and other bat CoVs, MHV, BCoV, infectious bronchitis virus, feline CoV, and canine CoV (17, 22, 23, 27, 32, 33, 52). Natural recombination leading to the generation of different genotypes in HCoV HKU1 has also been described (67, 68). We have also recently found natural recombination among different genotypes of HCoV OC43, giving rise to an emerging genotype D, associated with pneumonia (31). PHEV, BCoV, and HCoV OC43 are genetically and antigenically related betacoronaviruses that have been shown to have originated from a relatively recent common ancestor dating back to the end of the 19th century to the beginning of the 20th century, which is also supported by the present data (Fig. 7) (61, 62). CoVs closely related to BCoV were recently isolated from a sable antelope and a giraffe during an outbreak of winter dysentery in an Ohio wild-animal habitat (1, 16). These CoVs were so closely related to BCoV that no specific genomic markers can allow discrimination between them (1), suggesting that they could well represent spillovers of BCoV to other mammalian hosts. The present study demonstrated recombination events during the evolution of RbCoV HKU14 and members of Betacoronavirus 1, which may have arisen during cross-species transmission. However,

latter rabbit CoV, which originated from contaminated samples of Treponema pallidum used in a rabbit infection model at Johns Hopkins University, was detected by electron microscopy and found to cross-react with alphacoronaviruses, including human coronavirus 229E (HCoV 229E), feline infectious peritonitis virus, canine coronavirus diarrhea virus, and transmissible gastroenteritis virus, in serological assays (51). This virus was subsequently used as a model for virus-induced myocarditis and dilated cardiomyopathy (2). Although no gene sequence for this virus is available, the existing data suggested that it is an alphacoronavirus, and its host of origin still remains obscure.

The ability of RbCoV HKU14 to grow readily in human cell cultures, inducing cytopathic effects, is intriguing. With only a few exceptions, such as SARS-CoV, CoVs are notoriously difficult to culture in cell lines. HCoV OC43 and HCoV 229E, even if isolated, induce only subtle or nonexistent cytopathic effects. Despite being closely related to SARS-CoV, SARSr-Rh-BatCoV from horseshoe bats has not been isolated in cells susceptible to SARS-CoV. In the present study, RbCoV HKU14 was able to replicate in both rabbit kidney (RK13) and human rectum epithelial (HRT-18G) cells, with cell rounding and fusion to giant cells rapidly detaching from the monolayer in HRT-18G cells being

specific to enteric strains of BCoV was conserved in RbCoV HKU14, which may be compatible with its enteric tropism. Domestic rabbits are descended from the wild European or Old World rabbit, Oryctolagus cuniculus, originating from Southern Europe and North Africa (39). While China is one of the main producers of rabbit meat, farming of the species is increasing, especially in developing countries. In fact, since the 1970s, governments and world food organizations have been promoting rabbit farming in Africa, Asia, and South America, because of the relatively less space and low starting cost required and high breeding rate compared to those of traditional livestock. Rabbits are also commonly kept as pets in domestic households. Further studies, including animal challenge experiments, are needed to understand the pathogenicity and emerging potential of this novel CoV.

Recombination is likely an important event during the evolution of RbCoV HKU14 and members of Betacoronavirus 1. CoVs are known to have a high frequency of recombination, which may help them adapt to new hosts. Such recombination has been described for various animal CoVs, including SARSr-Rh-BatCoV and other bat CoVs, MHV, BCoV, infectious bronchitis virus, feline CoV, and canine CoV (17, 22, 23, 27, 32, 33, 52). Natural recombination leading to the generation of different genotypes in HCoV HKU1 has also been described (67, 68). We have also recently found natural recombination among different genotypes of HCoV OC43, giving rise to an emerging genotype D, associated with pneumonia (31). PHEV, BCoV, and HCoV OC43 are genetically and antigenically related betacoronaviruses that have been shown to have originated from a relatively recent common ancestor dating back to the end of the 19th century to the beginning of the 20th century, which is also supported by the present data (Fig. 7) (61, 62). CoVs closely related to BCoV were recently isolated from a sable antelope and a giraffe during an outbreak of winter dysentery in an Ohio wild-animal habitat (1, 16). These CoVs were so closely related to BCoV that no specific genomic markers can allow discrimination between them (1), suggesting that they could well represent spillovers of BCoV to other mammalian hosts. The present study demonstrated recombination events during the evolution of RbCoV HKU14 and members of Betacoronavirus 1, which may have arisen during cross-species transmission. However, it remains to be determined if such recombination would have resulted in animal-to-human transmission and the emergence of HCoV OC43. Interestingly, the tMRCA of RbCoV HKU14 was estimated to be rather recent, at around 2002 (HPDs, 1997 to 2005). We speculate that this virus may have emerged as a result of interspecies transmission during the mixing of game food animals in markets during a period of economic boost in China, as in the case of SARS-CoV. Further studies are required to understand the possible existence and prevalence of RbCoV HKU14 in rabbits from other geographical regions. Continuous surveillance studies will also be important to monitor the genetic evolution of CoVs in various food animals.

Yeah, not reading all that. A summary would have been dandy....

a reply to: Nyiah

Domestic rabbit = likely reservoir source of sars2cv19

originally posted by: DoctorBluechip
a reply to: Nyiah

Domestic rabbit = likely reservoir source of sars2cv19

Which kinda blows, no bag limit, no tags on rabbit.

I have a cat awaiting burial in our freezer that died from the feline corona virus. It most often causes lung issues, but in this case it was the wet version which caused fluid to build up in it's abdomen, fluid created by the proteinase of the virus disolving muscle and cartilage. The research I did on that like three months ago was a primer to expand my knowledge of this virus. I have to build it a coffin pretty quick, the funeral will be in the spring in the pet cemetary on our property.

My question is what does this particular virus have to do with what we see now with SARS2Covid19? The treatment for the cat works with this virus too, but we started it too late, I was trying to disolve the virus's proteinase, but most of the cat's muscle was already wasted. It did get two more weeks of a decent less hurtful life, but it was too late already, I could only slow the progression of my daughters cats disease.

The Rabbit virus can infect human cells but it does not say our immune system won't kill it, It is more similar to the wet type cat coronavirus I suppose, destroying the organs but nothing is said about it destroying muscles and collagens in the animal like the cat coronavirus..

So how come there was never an order for dogs to be masked if out being walked by owners if this thing can jump from animal to human & vice versa ?