Coronaviruses (CoV; order
Nidovirales, family
Coronaviridae, subfamily
Coronavirinae) are enveloped viruses with unusually large plus-strand RNA genomes of 26 to 32 kb (
1-
3). According to a current proposal to the International Committee of Taxonomy of Viruses (ICTV) (
8), they are classified into 3 genera containing viruses pathogenic for mammals (
Alpha- and
Betacoronavirus) and, foremost, birds (
Gammacoronavirus). Alpha-, beta-, and gammacoronaviruses are also referred to as CoV groups 1, 2, and 3 (
4,
21,
34). In humans, there are four prototypic human CoV (hCoV) that cause endemic and epidemic respiratory disease, including the human alphacoronaviruses 229E and NL63 and the human betacoronaviruses OC43 and HKU1 (
25,
27,
55,
56). The severe acute respiratory syndrome (SARS) CoV is an additional human betacoronavirus that circulated temporarily in the human population during 2002 to 2003, resulting in an epidemic of SARS, with a 10% case fatality rate (
14,
17,
38,
49). Other important CoV include the transmissible gastroenteritis virus of swine (TGEV) and the feline infectious peritonitis virus (FIPV), which together define the species
Alphacoronavirus 1 in the genus
Alphacoronavirus. This genus currently contains seven more species that include the porcine epidemic diarrhea virus (PEDV) species, as well as hCoV 229E and NL63, each of which defines an independent
Alphacoronavirus species. The genus
Betacoronavirus includes the species
Betacoronavirus 1, defined by the bovine CoV, hCoV OC43, and the species murine hepatitis virus (MHV), as well as five more species, including one founded by the SARS-related CoV. The genus
Gammacoronavirus contains a species defined by the avian infectious bronchitis virus (IBV), one defined by a Munia bird CoV, and a single beluga whale CoV. The current proposal for the classification of independent CoV species demands an amino acid identity of less than 90% in all of seven defined functional domains in the seven nonstructural proteins (NSP), NSP3, -5, and -12 to -16, encoded in CoV open reading frame 1ab (ORF1ab).
Within the spectrum of potential zoonotic viruses, CoV may be regarded as particularly significant because of their potential to be transmitted via respiratory or fecal-oral routes, resulting in a proven ability to cause major epidemics after host transition. Studies undertaken in search of the natural host of SARS CoV identified related CoV in rhinolophid bats (genus
Rhinolophus, family
Rhinolophidae, order
Chiroptera) in China (
28,
31). Further investigations identified members of the majority of known mammalian CoV species in bats, as well as several additional species that exclusively occurred in bats (
20,
28,
31,
40,
41,
53,
58). While no gammacoronaviruses have been found in bats, bats may be natural reservoirs at least of most mammalian CoV, i.e., alpha- and betacoronaviruses. Anecdotally, bat CoV (BtCoV) appear to be linked to specific bats, with closely related CoV sequences being associated with the same bat species (or genus) in considerably distant geographic regions and with considerably distant CoV cooccurring in the same habitat in different bat species (
20,
60). Unfortunately, the currently proposed criteria for a CoV species classification are difficult to meet when only field samples are available. Because BtCoV have never been isolated in cell culture and bat feces contribute a strong background of substances that inhibit reverse transcription PCR (RT-PCR), it is rather difficult to sequence full BtCoV genomes and to analyze seven different genes from the CoV ORF1ab, as formally required, to assign CoV species (
8). Consequently, it has been difficult in the past to determine the significance and genealogies of novel CoV from bats. According to the concept that occupation of novel hosts and subsequent restriction to these hosts might result in CoV speciation, even a tentative and provisional delineation of species in field-collected BtCoV could greatly facilitate the prediction of associations between virus and host.
Due to recent implications that bats are infected with a number of highly pathogenic viruses, including viruses of the genera
Lyssavirus and
Henipavirus as well as the family
Filoviridae (
3,
12,
24,
29,
36,
51,
62), it is highly important to learn more about the ecology of bat-borne viruses. In contrast to most of the aforementioned viruses, which are carried by tropical fruit bats (
Megachiroptera), the predominant hosts of mammalian CoV are insect- and blood-eating
Microchiroptera, which are not limited to tropical climates. One paramount question therefore is that of the geographic extent of CoV reservoirs.
Better knowledge of taxonomic associations between bats and CoV could enable predictions of CoV geographic ranges, since habitats of bats are well characterized in many regions. In China, members of the genus Rhinolophus have been specifically associated with SARS-related CoV. We investigated in this study whether SARS-related CoV might also occur in Europe in bats of the same genus.
Five different species of rhinolophid bats inhabit large parts of Europe, including Spain, France, and the United Kingdom, as well as most countries of southeastern Europe (Fig.
1). All five European species share a small area of overlapping occurrences (sympatry) on the Balkan Peninsula and in the eastern Mediterranean, including in Turkey, Bulgaria, and Greece (
7). In this study, we examined European rhinolophids in their area of sympatry for their CoV genetic range, prevalence, and seasonality. A surrogate classification criterion for tentative CoV species based on sequences of RNA-dependent RNA polymerases (RdRp) was developed, and the entire genome of a SARS-related CoV from European rhinolophids was determined. The resulting taxonomic associations between bats and CoV were used to derive predictions of potential geographic ranges of novel CoV. Finally, the complete spike protein reading frames of human SARS CoV, SARS-related BtCoV/Rp3, and the European SARS-related CoV were expressed on the surfaces of BHK-21 cells and stained with a set of anti-SARS CoV sera in order to determine antigenic relatedness within the extended SARS-related CoV species.
MATERIALS AND METHODS
Sample collection and processing.
No bats were killed for this study. For all capturing and sampling of bats, permission was obtained from the Bulgarian Ministry of Environment and Water. Geographic coordinates of all seven sampling sites in Bulgaria are given in Table
1. Sampling was performed in the spring and autumn of 2008 (months of April and September). Bats were identified on site by trained field biologists. Additionally, mitochondrial DNA in representative fecal samples was amplified and sequenced for species confirmation as described previously (
57). Bats were caught with mist nets within 2 to 5 meters' distance from the entrances of caves and tunnels. Captured bats were freed from nets immediately and put into cotton bags for 2 to 15 min to allow them to calm down before examination. Species, sex, reproductive status, forearm length, and body mass were determined. While being kept in bags, bats produced fecal pellets that were collected with clean tweezers and spiked into RNAlater RNA stabilization solution (Qiagen, Hilden, Germany) for sample processing. Duplicate sampling of individual bats was prevented by marking the toes of captured bats with nail polish upon first capture. Approximately 100 mg of bat feces was suspended in 500 μl of RNAlater (Qiagen, Hilden, Germany) and homogenized by vortexing. Of the suspensions, 50 μl was suspended into 560 μl of buffer AVL from the Qiagen viral RNA minikit and processed further according to the instructions of the manufacturer. The elution volume was 50 μl.
Analysis of samples by RT-PCR.
Reverse transcription-PCR (RT-PCR) covering the family
Coronaviridae was done as previously described, yielding amplicons of 455 bp located in the highly conserved motifs in the RdRp that encode LMGWDYPKCD and MMILSDDAV (
10). Following nucleotide sequencing of initial amplicons, nested or real-time RT-PCR assays were designed for the same amplicon region (oligonucleotide sequences are available on request). The above-described 455-bp amplicons were extended toward the 5′ end of the genome by using downstream primers within the fragment and upstream primers based on alignments of phylogenetically related CoV from GenBank. The final size of sequences used for phylogenetic analyses was 816 bp. 5′ primer sequences for the different CoV lineages were SP3080, 5′-CTTCTTCTTTGCTCAGGATGGCAATGCTGC-3′; SP3195, 5′-ATACTTTGATTGTTACGATGGTGGCTG-3′; SP3374, 5′-CTATAACTCAAATGAATCTTAAGTATGC-3′; GrISP1, 5′-TTCTTTGCACAGAAGGGTGATGC-3′; and GrISP2, 5′-CTTTGCACAAAAAGGTGATGCWGC-3′.
In vitro-transcribed RNA standards.
Photometrically quantified
in vitro RNA transcripts of all CoV lineages described in this study were generated as described previously (
13). Briefly, the 455-bp amplicon from the initial screening assay was TA cloned, and plasmids were purified and reamplified with vector-specific oligonucleotides and finally
in vitro transcribed using a T7 promoter-based Megascript kit (Applied Biosystems, Darmstadt, Germany). All six described real-time RT-PCR assays showed comparable lower limits of detection in the single-copy range. The
in vitro-transcribed RNAs were used as calibration standards for virus quantification in bat fecal samples, as described previously (
40).
Whole-genome sequencing.
One bat fecal sample showing the highest virus concentration of all European SARS-related CoV samples was used for whole-genome sequencing. Based on related CoV genome sequences available in GenBank, sets of oligonucleotides that yielded small amplicons ranging from 150 to 300 bp at approximately every 5,000 bases along the SARS CoV genome were generated. Following nucleotide sequencing of amplicons, specific oligonucleotide primers were designed and long-range PCR was performed with the Expand high-fidelity (Roche, Penzberg, Germany) and Phusion DNA (New England Biolabs, Frankfurt, Germany) polymerase kits. Amplicons ranging from 4 to 6 kb were sequenced using primer walking. The 3′ end of the genome was determined using a 3′-RACE (rapid amplification of cDNA ends) strategy, based on gene-specific forward primers and inclusion of reverse primers by an oligo(dT)-driven reverse transcription step.
Phylogenetic analysis.
Sanger sequencing of PCR products was done using dye terminator chemistry (Applied Biosystems, Darmstadt, Germany). Nucleic acid alignments were done based on amino acid codes by the BLOSUM algorithm in the MEGA 4 software package (
52). A gap-free nucleotide alignment of 816 bp containing the novel viruses as well as CoV reference strains was generated (see Tables S1 and S2 in the supplemental material). Tree topologies were determined with MrBayes v3.1 (
45). The analysis used a substitution model with GTR plus gamma plus an invariant site. Metropolis-coupled Markov chains (MCMC) of 6×10E6 iterations were sampled every 100 generations, resulting in 60,000 sampled trees. Two MCMC Monte Carlo (MCMCMC) analyses (1 cold chain and 3 heated chains each) were run in parallel, and results were compared and pooled. Convergence of chains was confirmed by the potential scale reduction factor statistic in MrBayes (
19), as well as by visual inspection of each cold chain using TRACER (
15).
Construction of expression plasmids.
The construction of a protein gene of SARS CoV spike (CUHK-W1) in pCG1 was described previously (
46). The spike gene of BtCoV/Rp3/
Rhinolophus pearsoni [Rhi pea]/China/2004 was recloned from the plasmid pcDNA3.1-Rp3S by inserting the BamHI/XbaI-digested insert into the pCG1 vector (kindly provided by Roberto Cattaneo, Mayo Clinic Rochester, Rochester, MN), to give pCG1-Rp3-S. The full-length S gene of European SARS-related BtCoV/BM48-31/
Rhinolophus blasii [Rhi bla]/Bulgaria/2008 was cloned by PCR from cDNA prepared from original fecal material. Primers contained BamHI and XbaI restriction sites (forward, 5′-TTGGATCCATGAAATTTTTGGCTTTTCTCTGTCTTCTTGGC-3′, reverse, 5′-TTTCTAGATTATGTGTAATGTAACTTCACTCCAG-3′). The PCR product was purified using a PCR purification kit (Qiagen, Hilden, Germany) and digested overnight with BamHI and XbaI, and the digest was inserted into the vector pCG1 to yield pCG1-BtCoV/BM48-31-S. The open reading frame was deposited in GenBank (accession no. GU190215). For construction of a carboxy-terminal FLAG-tagged BtCoV/BM48-31/Rhi bla/Bulgaria/2008 spike protein, the reverse primer 5′-TTTCTAGATTACTTGTCGTCGTCGTCCTTGTAGTCTGTGTAATGTAACTTCACTCCAGTAAGCAC-3′ was used instead.
Immunofluorescence.
BHK-21 cells grown on 12-mm-diameter coverslips were transfected with 1 μg of the respective plasmid DNA and 2 μl of Lipofectamine 2000 reagent (Invitrogen, Karlsruhe, Germany), followed by incubation at 37°C for 24 h. The cells were fixed with 3% paraformaldehyde for 20 min at room temperature and permeabilized with 0.2% Triton X-100 -phosphate-buffered saline for 5 min. The spike proteins were detected with the antibodies indicated in the legend to Fig. 6 in phosphate-buffered saline containing 1% bovine serum albumin, followed by incubation with a fluorescein isothiocyanate-conjugated secondary antibody. Fluorescence was visualized with a Zeiss Axioplan 2 microscope.
Virus isolation attempts.
Based on prior findings (
18,
54), isolation of virus was attempted from feces suspended in RNAlater solution. Vero and CaCo2 cells were used, as well as primary cells from colon, lung, and kidney of
Myotis nattereri,
Eidolon helvum, and
Rousettus aegyptiacus bats (unpublished data). No cytopathic effect was observed, and no virus growth was seen by RT-PCR, despite repeated trials (data not shown).
Statistical analyses.
All analyses were performed with EpiInfo 3.5.1 (CDC, Atlanta, GA) and with SPSS 17 (SPSS, Munich, Germany).
Nucleotide sequence accession numbers.
All sequences were submitted to GenBank under accession numbers GU190215 to GU190248 (see Table S2 in the supplemental material). GU190215 represents the full-genome sequence of the Bulgarian SARS-related bat coronavirus BtCoV/BM48-31/Rhi bla/Bulgaria/2008.
RESULTS
To obtain a comprehensive sample of European rhinolophid species, bats were studied in Strandja Park Sanctuary, Bulgaria, located in the area of sympatry of all five European rhinolophid species (Fig.
1) (
2,
7). Bat feces were first tested by CoV broad-range RT-PCR (
10) and sequenced. Detected viruses pertained to five major alphacoronavirus clades and one betacoronavirus. For each lineage, BLAST searches were done with default BLASTn settings to obtain alignments with closest phylogenetic neighbors. Redundant sequences were eliminated, and alignments were used to design lineage-specific RT-PCRs in order to increase detection sensitivity. Retesting increased the number of positive samples in the collection approximately 6-fold (202 RT-PCR products from 499 animals) (Table
1).
To obtain reliable resolution in phylogenetic analyses, we found earlier that it was insufficient especially for the betacoronaviruses to compare sequences of just the ∼440-bp universal CoV RT-PCR fragment that is generally used in ecological investigations (
10,
40,
59). Lineage-specific combinations of nested RT-PCR primers were therefore designed, and an 816-bp fragment of the 5′-terminal RdRp (NSP12) gene was successfully generated for representatives of all CoVs found in this study. Results of a Bayesian phylogenetic analysis based on 816-bp RdRp fragments are shown in Fig.
2.
All viruses with completed 816-bp RdRp gene sequences are listed along with their hosts in Table S2 in the supplemental material. In summary, three
Alphacoronavirus clades occurred exclusively in
Miniopterus bats, one related to BtCoV/HKU7/1/
Miniopterus tristis [Min tri]/China/2005, one related to BtCoV/HKU8/
Miniopterus pusillus [Min pus]/China/2008, and one related to
Miniopterus BtCoV-1A (Fig.
2A). Thirteen of these 38
Miniopterus bats were either doubly or triply infected. One unique alphacoronavirus (BtCoV/BNM98-30/
Nyctalus leisleri [Nyc lei]/Bulgaria/2008) was detected in
Nyctalus leisleri. Two
Alphacoronavirus clades were detected in bats of the genus
Rhinolophus, one related to BtCoV/HKU2/HK46/
Rhinolophus sinicus [Rhi sin]/China/2006 and one unique lineage represented by BtCoV/BB98-15/Rhi bla/Bulgaria/2008.
SARS-related CoV, divided into two monophyletic clades, occurred exclusively in
Rhinolophus, one of which preferentially occurs in Mediterranean horseshoe bats (
Rhinolophus euryale), the other in
Rhinolophus blasii,
Rhinolophus ferrumequinum, and
Rhinolophus mehelyi (Fig.
2A). Table
1 summarizes detection rates for all viruses. It should be mentioned that SARS-related CoV were detected at a significantly higher frequency than any alphacoronavirus (26% of 389
Rhinolophus bats,
P < 0.001, χ
2 = 84.48). Double infections occurred in 24 of 389 rhinolophids (6.2%), while two rhinolophids were triply infected (0.5%). All multiple infections included the SARS-related CoV.
In order to determine virus concentrations in fecal samples, specific real-time RT-PCRs with
in vitro-transcribed RNA quantification standards were established for each of the above-described CoV clades. As shown in Fig.
3, there were no discernible differences in virus concentrations between clades as well as between sampling seasons (spring and autumn). Maximal concentrations were observed for the SARS-related CoV, reaching up to 2.4 × 10
8 copies per gram of feces.
Classification of CoV.
Except for the SARS-related CoV described below, it was impossible to sequence larger genome portions necessary for species classification (
8). To obtain a surrogate estimation of genetic taxonomical limits, distance matrices covering the 816-bp RdRp gene fragment were calculated for all available CoVs and summarized as shown in Fig.
4. In the nucleotide-based analysis, distances within and between potential taxonomic units were not clearly separated (Fig.
4A and B). At the amino acid level, however, distances always exceeded 32% between established CoV genera (Fig.
4C and D). Established
Alphacoronavirus species were at least 5.9% distant from each other (Table
2). This also included BtCoV/HKU6, which has not been fully sequenced and is therefore not formally accepted as a CoV species (
20,
60).
Betacoronavirus species were always at least 6.3% distant from each other (Table
2).
In order to classify those CoV described here as well as in our recent studies on CoV in African and northern German bats (
20,
40), amino acid distances of the novel viruses from related reference species and phylogenetic neighbors were calculated as listed in Table
3. In a first approach, the above-mentioned amino acid distance criteria were used to classify viruses into RdRp grouping units (RGU) according to distances between established species (Table
2). However, it became clear that the two prototype viruses defining the HKU8 species, i.e., BtCoV/HKU8/Min pus/China/2008 and BtCoV/HKU7/1/Min tri/China/2005 (
6), were each represented by closely related European viruses that in turn were separated from each other by the same distance as that between BtCoV/HKU8 and BtCoV/HKU7. This independent relationship was strongly supported by Bayesian-likelihood-based phylogeny (Fig.
2A) and by distance-based phylogeny using the neighbor-joining method (Fig.
2B). Because of the paraphyly of both the European and Chinese representatives of the HKU8 species, the separation criterion for RGU within the genus
Alphacoronavirus was reduced to a 4.8% amino acid distance. This corresponded to the separation limit between BtCoV/HKU8 and BtCoV/HKU7, yielding two independent RGU within the formally defined BtCoV/HKU8 species. The following criteria for defining separate RGU in mammalian CoV were thus adopted: a >4.8% amino acid distance in the analyzed 816-bp fragment for alphacoronaviruses and a >6.3% amino acid distance for betacoronaviruses.
Using these criteria, two novel RGU were defined in the genus
Alphacoronavirus, represented by BtCoV/BB98-15/Rhi bla/Bulgaria/2008 and BtCoV/BNM98-30/Nyc lei/Bulgaria/2008 (refer to the right-hand column in Fig.
2A for RGU designations). Another novel RGU was defined for a virus in direct phylogenetic relationship to SARS CoV represented by BtCoV/GhanaKwam20/
Hipposideros sp. [Hip spe]/Ghana/2008, which was described previously to occur in Ghanaian
Hipposideros bats by our group (
40).
Six more viruses were determined to be extensions of six existing RGU, as shown in Fig.
2A (related to HKU8, HKU7,
Miniopterus BtCoV1A, HKU6, HKU2, and SARS). Of note, within all six RGU, the respective Chinese and European members were carried by corresponding bat genera (Fig.
2A and Table S2 in the supplemental material). To test the hypothesis that associations between CoV RGU and their hosting bat genus are valid across wide geographies,
Nyctalus leisleri bats were sampled in Munster, Germany, 2,000 km from Strandja Park. A virus of 97.7% nucleotide and 100% amino acid identity with the Bulgarian
Nyctalus virus BtCoV/BNM98-30/Nyc lei/Bulgaria/2008 was retrieved in one of eight bats. No other CoV were found.
Full genome analysis and distinctive features of European SARS-related CoV.
Virus isolation was attempted without success from Vero cells and CaCo2 cells, as well as from several primary bat cell cultures (data not shown). However, a comprehensive design of nested RT-PCR primer sets allowed for a determination of the entire sequence of BtCoV/BM48-31/Rhi bla/Bulgaria/2008 (here referred to as BM48-31), except the 5′-most 72 nucleotides (nt), directly from fecal material of a single Blasius's horseshoe bat (
Rhinolophus blasii). As shown in Fig. S1 in the supplemental material, the phylogenetic position of BM48-31 in relation to prototype alpha- and betacoronaviruses was preserved along all analyzed genome portions, including those for NSP1, NSP3, NSP5 (3C-like protease), and NSP13 (helicase), as well as the structural S, E, M, and N proteins. No further attempts were therefore made to identify evidence of recombination. Pairwise amino acid identities with fully sequenced SARS-related CoV are listed by protein domain in Table
4. According to criteria of the ICTV proposal (
8), BM48-31 was a member of the SARS-related CoV species, confirming RGU classification. Three proteins showed the most significant deviations from SARS CoV.
Low conservation of open reading frame 3b.
The SARS CoV ORF3b, which was shown to counteract type I interferon (IFN) induction in human cells (
26), was not conserved in BM48-31, which comprises only 115 instead of 154 amino acids (aa). ORF3b in Chinese SARS-related BtCoV/Rf1/
Rhinolophus ferrumequinum [Rhi fer]/China/2004 and BtCoV/273/Rhi fer/China/2005 also comprised only 114 aa, while the respective domains in all other known SARS-like bat viruses, including the most SARS-related virus, BtCoV/Rp3/Rhi pea/China/2004, were even shorter. In the 115-aa ORF3b of BM48-31, identity with SARS CoV was 44.3% and similarity was 57.4%. In contrast, the 114-aa protein putatively encoded by BtCoV/Rf1/Rhi fer/China/2004 was 91.2% identical and 93.0% similar to that of SARS CoV, respectively.
Loss of open reading frame 8.
The ORF8 domain that acquired a 29-nt deletion during host transition of the SARS CoV was missing entirely from the BM48-31 genome, unlike with all other SARS-related CoV (
5,
23,
28,
37,
43). In order to determine how recently the ORF8 region had been lost in the European SARS-related viruses, representatives of both European SARS-related CoV sublineages from all possible hosts (
Rhinolophus ferrumequinum,
R. blasii, and
R. mehelyi,
R. euryale) were amplified with primers flanking the deleted region (compared to sequences of Chinese SARS-related viruses). The deletion was present in all viruses, as shown in Fig. S2 in the supplemental material, suggesting a loss of the ORF8 region already in an ancestor common to all European SARS-related viruses. Nucleotide sequencing further confirmed almost 100% conservation in the amplified genomic region.
Spike protein.
The spike protein has been shown to mediate virus entry and to contain binding domains of virus-neutralizing antibodies that provide relevant protection against infection in a mouse model (
9). BtCoV/Rp3/Rhi pea/China/2004, which is most closely related to the human SARS CoV, was 78.0% identical and 86.8% similar to SARS CoV in the whole Spike protein. For comparison, the Spike protein of BM48-31 identified in this study was slightly more distant from that of SARS CoV, at 74.7% identity and 83.8% similarity. Because the binding of the SARS CoV spike protein to its human receptor, ACE-2, is determined by aa 319 to 518 (
22,
30), this region was analyzed as shown in Fig.
5. Whereas the region homologous to the SARS CoV receptor binding domain (RBD) showed two deletions in all Chinese SARS-related BtCoV, BM48-31 had deletions in only one region. With the receptor binding motif of SARS CoV (aa 424 to 495), the corresponding sequence in BM48-31 showed 67% similarity (BLOSUM62 similarity matrix). For comparison, similarities in this region between SARS CoV and the closest Chinese BtCoV, BtCoV/HKU3/1/Rhi sin/China/2005 and BtCoV/Rp3/Rhi pea/China/2004, were only 36 and 33%, respectively. The two most critical domains for interaction of SARS CoV spike with ACE-2, aa 472 and 487, were identical (aa 472) or highly similar (aa 487), as were their contexts, in BM48-31 (
22,
42). Phylogenetic analysis confirmed that the RBDs of SARS CoV and related civet CoV were closer in relation to corresponding sequences in BM48-31 than to those in Chinese SARS-related CoV from bats (Fig.
5C).
For a functional estimate of antigenic relatedness within the SARS-like group of CoV, the entire spike protein reading frames of SARS CoV, BtCoV/Rp3/Rhi pea/China/2004, and BM48-31, followed by C-terminal FLAG tags, were cloned into the expression vector pCG1 downstream of a cytomegalovirus promoter and transfected into BHK-21 cells. Transfected cells were confirmed to express spike proteins on their surfaces by anti-FLAG immunofluorescence assay (IFA) staining (Fig.
6B). Cells were then stained with a polyclonal rabbit serum raised against a recombinant SARS CoV spike protein or, alternatively, with sera of two different human SARS patients, one from China and one from Germany. As shown in Fig.
6A, the rabbit serum showed reactivity with SARS CoV and BtCoV/Rp3/Rhi pea/China/2004, while both human sera reacted only with the SARS CoV spike protein. The BM48-31 spike protein was recognized by none of the sera, including the rabbit serum.
DISCUSSION
CoV are predominantly associated with
Microchiroptera, which are not limited to tropical climates. Their geographic range might therefore be considerably wider than that of other bat-borne viruses. In this study, we have demonstrated that SARS-related CoV exist in Europe. In addition to identifying these betacoronaviruses, we have identified novel alphacoronavirus lineages in rhinolophid and vespertilionid bats that might in part represent novel CoV species. To enable a taxonomic assignment of BtCoV from field data with reasonable effort, we have developed a surrogate criterion for defining tentative coronavirus species based on distance matrices involving an 816-bp fragment from the RdRp (RdRp grouping units [RGU]). The 816-bp fragment can easily be analyzed in samples showing evidence of CoV in generic screening assays (
9,
41) and is sufficient, as demonstrated in this study, to resolve all currently agreed upon CoV species. It is acknowledged that the CoV species classification as defined recently in a joint proposal to the ICTV has to be met before a CoV species can be formally declared. However, for most field-sampled CoV, this task is impossible to complete. Even if only surrogate criteria are available, it will be practically useful in ecological investigations to assign preliminary connections between specific taxonomic units of bats and specific CoV in order to assess potential geographic ranges of CoV reservoirs. With six different CoV RGU as examples, our study revealed that European and Chinese representatives were consistently associated with the same bat host genera. Moreover, the Ghana348-related RGU that constitutes the closest phylogenetic neighbor of the SARS-related RGU was detected in bats of the genus
Hipposideros, which in turn is the closest phylogenetic neighbor of the genus
Rhinolophus (
40). We can thus conclude that associations exist between CoV RGU and bat genera. If we assume that RGU are predictive of species, this suggests that CoV speciation may have frequently been caused by extensions of host ranges of ancestral CoV into novel bat genera. On the other hand, because host associations rarely extend to bat species, it appears that CoV may experience limited genetic barriers toward host switching within bat genera.
Virus-host associations can be used in a prospective manner to predict geographic distributions of reservoir-borne CoV. For example, we have identified in this study a novel alphacoronavirus RGU in Nyctalus leisleri. The concept of taxonomic association suggests that similar viruses might occur in other regions of the world harboring Nyctalus bats, including most of northern Europe and the boreal regions of the Asian continent. Indeed, such a virus could be confirmed in a specimen of the same genus from Germany, 2,000 km away. Similarly, there was one Rhinolophus-associated CoV that occurred as an independent RGU in Bulgaria but that has never been detected in China (represented by BtCoV/BB98-15). We may predict that this virus can also be found in Rhinolophus bats elsewhere in Europe and Asia.
The availability of a full genome sequence of the Bulgarian SARS-related CoV allows for an unequivocal classification of the virus as an extension of the recognized SARS CoV species. The existence of SARS-related CoV in a considerably larger geographic area than previously assumed demands a comprehensive approach to characterizing the relevance of this virus as a potential zoonotic agent. The complete genome sequence enables the comparison of several features that have been functionally characterized for SARS CoV, in order to derive hypotheses for future experimental work. For example, the important papain-like protease domain of NSP3 that has been shown to interact with IRF3 phosphorylation and nuclear translocation and thereby to prevent induction of type I interferon fell significantly below the general level of identity observed in the nonstructural proteins (>94%), at only 84.3 to 86.5% (
11). The Bulgarian virus might show a discernible difference from SARS CoV in its ability to interact with the human interferon system. Reverse genetics experiments are under way to investigate this important implication (
39). Another interesting domain whose differential functionality should be studied in comparison with that of SARS CoV is the ADRP domain in NSP3, with a level of identity to prototypic SARS-like CoVs of only 64.4 to 69.1%. This domain is conserved across the alphavirus-like superfamily but is of yet-unclear function in CoV replication (
16,
35,
61). Within the genome portion containing structural and accessory genes, ORF3b has been identified as an important interferon antagonist (
26). While among the structural proteins E, M, and N the level of identity is generally around 90%, a low degree of conservation in ORF3b among the now-extended range of SARS-related CoV speaks against a generally important role of the encoded protein for virus replication. This difference might identify one of the distinctive features causing the high pathogenicity of the epidemic 2002-2003 SARS CoV as opposed to the obviously mild courses following postepidemic zoonotic transitions of closely related viruses in 2003-2004 (
33,
50).
A strikingly low identity with prototypic members of the SARS CoV species existed in protein 6 (48.4 to 53.1%), which has been determined to interfere with type I interferon-mediated signaling via inhibition of nuclear translocation of STAT1 (
26,
63). Again, it would be warranted to study differences that may exist between both viruses' proteins 6 in their ability to ablate interferon (IFN) signaling. Yet another interesting observation is that identities between ORF7b proteins were clearly higher than those between ORF7a proteins, suggesting that ORF7b, whose translation in cell culture is probably mediated by ribosomal leaky scanning, is differentially selected and, thus, probably expressed
in vivo in bats. Interestingly, the entire region encoded by subgenomic RNA 8 was not present in BM48-31. This includes ORF8, whose 29-nucleotide deletion in human SARS CoV as opposed to in SARS-like bat and civet viruses has been hypothesized to represent a major functional hallmark in the transition of SARS CoV to humans (
5,
23,
28,
43). The absence of ORF8 in both sublineages of European SARS-related CoV suggests that protein 8 may not be generally essential for the maintenance of SARS-related CoV in bats. Confirmation in a BtCoV animal model is warranted, yet such models are not available so far.
Finally, the CoV spike protein mediates entry of virus into host cells and acts as a functionally relevant virus neutralization domain targeted by virus-neutralizing antibodies. Interestingly, neither human sera nor a high-titer rabbit serum raised against a recombinant trimeric SARS CoV spike protein bound the BM48-31 spike protein, while at least some expectable degree of cross-reactivity was observed among SARS CoV and a close relative from Chinese bats, BtCoV/Rp3/Rhi pea/China/2004 (
28,
31,
43). Even in the absence of virus neutralization tests, we may therefore draw the preliminary conclusion that it would be of little value to use existing SARS CoV vaccine candidates for an immunization of the population in case of an epidemic caused by the European SARS-related virus.
Another highly relevant experimental task is to determine whether the spike protein of BM48-31 might mediate entry into human or domestic animal cells. It has been shown previously that members of the Chinese BtCoV lineage in the SARS-related species cannot utilize the human SARS CoV entry receptor, ACE-2 (
44). However, the RBD of BM48-31 is related considerably more closely to the SARS CoV RBD than that of any other BtCoV characterized so far. The most critical domains for receptor binding in SARS CoV, i.e., aa 472 and aa 487, as well as their contexts, are present and highly similar (
22,
32,
42). To reflect adaptive processes in nature, it would therefore be highly interesting to simulate the behavior of the BM48-31 spike protein (or RBD) in the context of a replicating SARS CoV. It has already been demonstrated that recombinant CoV can be pseudotyped with BtCoV spike proteins and rescued with replicative activity (
1,
47,
48). It should therefore be possible to study adaptive mechanisms of the BM48-31 RBD during transition to novel hosts by passaging of recombinant viruses in cells of other mammals. However, as a first prerequisite, such experiments would require susceptible cell cultures from bats, either from the genus
Rhinolophus or ultimately from the exact species yielding BM48-31,
R. blasii. Such cells are not available to us so far.
In summary, this study shows approaches to utilizing data from field investigations for ecological and experimental assessments of the zoonotic potential of bat-borne CoV. Bats are a major current focus of zoonotic risk assessment, due to their large population sizes, their ability to fly, and their relatedness with other mammals. Coronaviruses provide important models for studying ecological and molecular mechanisms of virus transition from bats to other mammals, including humans.
Acknowledgments
We thank Elena Tilova and Liubomir Iankov at Green Balkans NGO, Bianca Steimle at Noctalis, Justin Robarge and Milen Rashkov at DPP Strandja, Liubomir Jeliazkov of the Bulgarian Cave Rescue Team, Andreas Kiefer of the Nature Conservation Project Mayen, and the volunteers at the Bonn Consortium for Bat Conservation for field assistance. Nyctalus bat samples from Munster, Germany, were kindly provided by Lena Grosche, Frauke Meier, and Myriam Götz from Echolot GbR. The rabbit anti-SARS serum was kindly provided by Markus Eickmann at the Institute of Virology, University of Marburg. Helpful suggestions were provided by Stefan Zlatarov, director of the Strandja Nature Park and Boyan Petrov, National Museum of Natural History, Sofia, Bulgaria.
Victor Corman received a scholarship from a BONFOR intramural grant at the University of Bonn. This study was funded by the European Union FP7 projects EMPERIE (grant agreement 223498) and EVA (grant agreement 228292) and the German Federal Ministry of Education and Research (BMBF) via the project Ecology and Pathogenesis of SARS, an Archetypical Zoonosis (project code 01KIO701).