Bat Coronaviruses, Ghana | CDC EID

Volume 15, Number 9–September 2009
Research
Distant Relatives of Severe Acute Respiratory Syndrome Coronavirus and Close Relatives of Human Coronavirus 229E in Bats, Ghana
Susanne Pfefferle, Samuel Oppong, Jan Felix Drexler, Florian Gloza-Rausch, Anne Ipsen, Antje Seebens, Marcel A. Müller, Augustina Annan, Peter Vallo, Yaw Adu-Sarkodie, Thomas F. Kruppa, and Christian Drosten
Author affiliations: Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany (S. Pfefferle, J.F. Drexler, T.F. Kruppa); University of Bonn Medical Centre, Bonn, Germany (S. Pfefferle, J.F. Drexler, M.A. Müller, C. Drosten); Kwame Nkrumah University of Science and Technology, Kumasi, Ghana (S. Oppong, Y. Adu-Sarkodie); Noctalis, Centre for Bat Protection and Information, Bad Segeberg, Germany (F. Floza-Rausch, A. Ipsen, A. Seebens); Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi (A. Annan, T.F. Kruppa); and Academy of Sciences of the Czech Republic, Brno, Czech Republic (P. Vallo)

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Abstract
We tested 12 bat species in Ghana for coronavirus (CoV) RNA. The virus prevalence in insectivorous bats (n = 123) was 9.76%. CoV was not detected in 212 fecal samples from Eidolon helvum fruit bats. Leaf-nosed bats pertaining to Hipposideros ruber by morphology had group 1 and group 2 CoVs. Virus concentrations were <45,000 copies/100 mg of bat feces. The diversified group 1 CoV shared a common ancestor with the human common cold virus hCoV-229E but not with hCoV-NL63, disputing hypotheses of common human descent. The most recent common ancestor of hCoV-229E and GhanaBt-CoVGrp1 existed in ≈1686–1800 ad. The GhanaBt-CoVGrp2 shared an old ancestor (≈2,400 years) with the severe acute respiratory syndrome–like group of CoV.

Coronaviruses (CoVs) (order Nidovirales, family Coronaviridae, genus Coronavirus) are enveloped viruses with plus-stranded RNA genomes of 26–32 kb, the largest contiguous RNA genomes in nature (1). They are classified into 3 groups, which contain viruses pathogenic for mammals (groups 1 and 2) and poultry (group 3) (1). Human CoVs (hCoVs)-229E, -NL63, -OC43, and -HKU1 are endemic worldwide and cause mainly respiratory infections in children and adults. The severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is a novel zoonotic coronavirus that caused an international epidemic in 2002–2003. Fortunately, efficient public health management interrupted this epidemic (2). Studies conducted in China in the aftermath of the SARS epidemic have identified CoVs in bats (Chiroptera) and implicated this speciose mammalian order as the most likely reservoir of all known coronaviruses (3–7). Among the most urgent concerns prompted by the SARS epidemic is the likelihood of similar future events. Thus, it seems highly relevant to study the ecology of bat CoVs in terms of diversity, host restriction, virus prevalence, risk of exposure, and the circumstances of past host transition events.

The genetic diversity of bat-borne CoVs is currently unclear. Preliminary data suggest that CoVs may be adapted in a stricter sense to a specific host species rather than to specific regions (5,6,8–12). A variety of pathogenic CoVs occur in other mammals or poultry. However, the genetic range within these animals is considerably less than that observed in even single bat species or subfamilies (7,8).

Estimates indicate that there are >100 bat species in sub-Sahran Africa. This finding is in contrast to ≈50 species in the entire Western Palaearctic region (Europe, Middle East, North Africa) (13,14). African bats have been shown to harbor pathogens that are occasionally transmitted to humans. This transmission may result in severe disease outbreaks, e.g., Ebola and Marburg viruses (15). Because bats are a part of the human diet in wide areas of Africa (16), it appears highly relevant to study CoVs in African bats.

We have demonstrated by serologic studies that African bats have antibodies against CoVs (10). Antibodies reactive with SARS-CoV antigen were detected in 47 (6.7%) of 705 bat serum specimens from 26 species (10). Recently, Tong et al. detected sequences of CoVs in bats from Kenya (17). We describe the results of studies on bats in Ghana obtained by using noninvasive sampling of frugivorous and insectivorous bats at 2 caves, a lake habitat of diverse insectivorous bats, and a large urban roosting site of frugivorous bats. Bayesian inference of diversification dates gave implications on the recency of the introduction of hCoV-229E into the human population, irrespective of its original source.

Materials and Methods
Capturing and Sampling
Figure 1

Figure 1. Location of Kwamang caves near the village of Kwamang, (6°58´N, 1°16´W), 50 km northeast of Kumasi, Ashanti region, Ghana...


Figure 2 (please, see the complete note)

Figure 2. Two morphotypes of Hipposideros caffer ruber bats held by one of the authors (F.G.-R.), who was wearing a leather glove...


Figure 3 (please, see the complete note)

Figure 3. A) Phylogeny of coronaviruses (CoVs) in the RNA-dependent RNA polymerase gene (RdRp, 817-bp fragment) with root point dates derived from Bayesian inference...

In the locations identified in Figure 1, mist netting and sampling were conducted as described (11). In Kumasi Zoo, fecal samples were collected with plastic foil under trees occupied by Eidolon helvum bats (estimated colony size 300,000). For all capturing and sampling, permission was obtained from the Wildlife Division of the Ministry of Lands, Forestry, and Mines in Ghana. Research samples were exported under a state agreement between the Republic of Ghana and the Federal Republic of Germany, represented by the City of Hamburg. Additional export permission was obtained from the Veterinary Services of the Ghana Ministry of Food and Agriculture.

Processing and Analysis of Samples
Samples (1–4 fecal pellets or swabs suspended in RNA stabilization solution [RNAlater Tissue Collection; Applied Biosystems, Foster City, CA, USA]) were tested at the Kumasi Centre for Collaborative Research in Tropical Medicine as described (11,18). After initial sequencing, specific primers were designed for each group of CoV found. Nested reverse transcription–PCR (RT-PCR) primer sets used for sequencing of longer fragments of representative viruses are available upon request. The following sequences were derived from this study and were submitted to GenBank under the listed accession numbers: RNA-dependent RNA polymerase (RdRp) sequences: BtCoV/Hip/GhanaBoo/348/2008, FJ710043; BtCoV/Hip/GhanaBoo/344/2008, FJ710044; BtCoV/Hip/GhanaKwam/8/2008, FJ710045; BtCoV/Hip/GhanaKwam/19/2008, FJ710046; BtCoV/Hip/GhanaKwam/20/2008, FJ710047; BtCoV/Hip/GhanaKwam/13/2008, FJ710048; BtCoV/Hip/GhanaKwam/31/2008, FJ710049; BtCoV/Hip/GhanaKwam/27/2008, FJ710050; BtCoV/Hip/GhanaKwam/26/2008, FJ710051; BtCoV/Hip/GhanaKwam/24/2008, FJ710052; BtCoV/Hip/GhanaKwam/10/2008, FJ710053; BtCoV/Hip/GhanaKwam/22/2008, FJ710054/nucleocapsid sequences; BtCoV/Hip.sp/GhanaBoo/344/2008, FJ710055; BtCoV/Hip.sp/GhanaKwam/19/2008, FJ710056.

Phylogenetic Analysis
Nucleic acid alignments were conducted based on amino acid code by using the ClustalW algorithm (www.ebi.ac.uk/clustalw) in the Molecular Evolutionary Genetics Analysis version 4.0 software package (www.megasoftware.net) (19). Two gap-free nucleotide alignments (817 bp and 1,221 bp) were generated. Tree topologies were determined on both datasets by using MrBayes version 3.1 (20). The analysis used a general time reversible (GTR) substitution model with 6 rate categories to approximate a gamma-shaped rate distribution across sites and an invariant site assumption (GTR + Γ6 + I). Markov chain Monte Carlo (MCMC) chains of 107 iterations were sampled every 500 generations, resulting in 20,000 sampled trees. Two Metropolis-coupled chains (1 cold and 3 heated chains each) were run in parallel, compared, and pooled. Convergence of chains was confirmed by the potential scale reduction factor statistic in MrBayes (21) and by visual inspection of each cold chain using the TRACER program (22). Phylogenetic dating was conducted by using Bayesian evolutionary analysis sampling trees (BEAST) (22). Chain lengths in BEAST were at least 20,000,000 generations with sampling every 500 generations. Convergence of the model was checked visually and by the effective sample size statistic with TRACER.

Results
Virus Detection
During February 2008, bats were sampled in the described locations around Kumasi, Ghana. Initially, 7 fecal samples tested positive by pan-CoV PCR. Products (440 bp, RdRp gene) were sequenced and aligned with prototype CoV. Neighbor-joining phylogenies indicated 2 distinct groups of sequences that belonged to CoV group 1 (n = 4) and group 2 (n = 3), respectively. Specific primer pairs for the group 1 and group 2 sequences were designed and applied again to all samples. Five additional viruses were found, resulting in a total CoV prevalence of 9.76% in insect-eating bats (n = 123). No virus was found in any oral swab. All virus findings in fecal samples are listed by capture site in Table 1.

Notably, all CoV findings were in insect-eating leaf-nosed bats of the genus Hipposideros. Within the genus, the species H. abae could be discriminated unambiguously by morphology (Table 1). The remaining Hipposideros species were assigned to the complex of forms related to currently recognized species H. caffer and H. ruber. Because 2 morphotypes were present (Figure 2), the mitochondrial cytochrome b gene was sequenced as described (23). Both morphotypes belonged to phylogenetic lineages distinct from H. caffer and possibly represented 2 distinct species (P. Vallo, personal ongoing investigation). Both are collectively referred to as H. caffer (cf.) ruber in this study. A fraction of 15.4 % of H. cf. ruber specimens yielded CoV, without a difference between sexes (14%/19%, n = 57/21 [M/F], respectively). Only adult males and nonlactating adult females, but no lactating females, juveniles, and subadults of H. cf. ruber were encountered.

Virus Concentrations
To estimate the quantity of CoV genomes in bat feces, we did end-point dilution experiments with the nested pan-CoV RT-PCR (18). The previously determined sensitivity limit of the PCR assay was 5–45 copies/PCR (18). In the assay, the equivalent of 1 mg feces was tested per PCR tube (100 mg feces collected, 1:10 dilution extracted, 1:10 dilution tested). The highest dilution factor that still yielded an amplification signal in any of the samples was 1:10, which suggested a maximal concentration of 50 to 450 CoV RNA copies/mg of feces.

Virus Classification
Group 1 CoV
In H. cf. ruber bats in the Kwamang and Booyem caves, a diverse group 1 CoV was found. Further analysis was complicated by the low RNA content in samples. Based on alignments of prototype group 1 viruses, 5 different nested RT-PCRs were designed and the RdRp fragment could finally be extended by 441 bp to the 5´ end, providing an 817-nt fragment for phylogenetic analysis. All methods of phylogenetic inference placed this virus next to a common ancestor with human coronavirus 229E, which circulates worldwide in humans (Figure 3). Bootstrap support of the hCoV-229E/GhanaBt-CoVGrpI root point in neighbor-joining analysis was 100%. The corresponding Bayesian posterior probability was 1.0. The most closely related member of the GhanaBt-CoVGrp1 clade shared 91.90% nucleotide identity with hCoV-229E in the analyzed fragment. The most distant member was 86.50% identical. The next phylogenetic neighbor, the human CoV hCoV-NL63, was only 74.70%–78.60% identical in the analyzed fragment.

Group 2 CoV

With the pan-CoV screening assay, a group 2 CoV was initially found in the Kwamang cave. Sequences from 3 bats were identical. The secondary group-specific PCR identified 4 additional samples of this virus, 1 of them from Booyem Cave B and the remaining from Kwamang. Nucleotide identity among these sequences was 97.2%–100%. Phylogenetic analysis with different methods of inference (neighbor-joining nucleotide-based, neighbor-joining amino acid–based, Bayesian) yielded variable tree topologies suggesting basal associations with either the 2a, 2d, or 2b subgroups (data not shown) (24). Based on alignments of prototype group II viruses, 8 additional nested RT-PCR primer sets were designed and 2 of the samples could be amplified. Sequences could be extended 520 bp upstream and 383 bp downstream of the initial fragments, yielding 1,221-bp fragments for phylogenetic analysis. Bayesian phylogenetic inference with different substitution models and parallel analysis using Metropolis coupling now placed the virus reliably next to a common ancestor with the 2b group of CoV (SARS-like viruses, Figure 3). The Bayesian posterior probability of the CoV 2b/GhanaBt-CoVGrp2 clade being monoyphletic was 1.0. A maximum of 72.2% nucleotide identity was shared with SARS CoV.

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Bat Coronaviruses, Ghana | CDC EID