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Published in final edited form as: Infect Genet Evol. 2013 Aug 27;0:10.1016/j.meegid.2013.08.016. doi: 10.1016/j.meegid.2013.08.016

Complete genome sequence and molecular phylogeny of a newfound hantavirus harbored by the Doucet’s musk shrew (Crocidura douceti) in Guinea

Se Hun Gu a, Violaine Nicolas b, Aude Lalis b, Nuankanya Sathirapongsasuti a, Richard Yanagihara a,*
PMCID: PMC3844077  NIHMSID: NIHMS521834  PMID: 23994121

Abstract

Elucidation of the molecular phylogeny of shrew-borne hantaviruses in sub-Saharan Africa has been hampered by the lack of full-length viral genomes. In this report, we present the complete genome analysis of a newfound hantavirus, designated Bowé virus, detected in ethanol-fixed intercostal muscle of a Doucet’s musk shrew (Crocidura douceti), captured in southwestern Guinea in February 2012. Full-length amino acid sequence comparison of the S-, M- and L-segment gene products revealed that Bowé virus differed by 24.1–53.4%, 17.0–59.9% and 14.6–39.7%, respectively, from all other representative rodent-, shrew- and mole-borne hantaviruses. Phylogenetic analysis, using maximum-likelihood and Bayesian methods, under the GTR+I+Γ model of evolution, showed that Bowé virus shared a common ancestry with Tanganya virus, a hantavirus detected in the Therese’s shrew (Crocidura theresae) in Guinea. Whole genome analysis of many more hantaviruses from sub-Saharan Africa are needed to better clarify how the radiation of African shrews might have contributed to the phylogeography of hantaviruses.

Keywords: Hantavirus, Crocidura, Shrew, Phylogeny, Guinea

1. Introduction

Hantaviruses (genus Hantavirus), the etiologic agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), possess a single-stranded, negative-sense RNA genome, consisting of three segments, designated S, M and L, which encode the nucleocapsid protein, envelope glycoproteins and RNA-dependent RNA polymerase, respectively (Bi et al., 2008; Schmaljohn and Hjelle, 1997). Unlike other members of the Bunyaviridae family, hantaviruses have no arthropod host and have been long known to be harbored by rodents of multiple species in Europe, Asia and the Americas (Jonsson et al., 2010). Until recently, no hantavirus had been reported from Africa. This impasse was broken with the discovery of Sangassou virus (SANGV) in the African wood mouse (Hylomyscus simus) (Klempa et al., 2006) and Tanganya virus (TGNV) in the Therese’s shrew (Crocidura theresae), captured in Guinea (Klempa et al., 2007). More recently, another genetically distinct hantavirus, Azagny virus (AZGV), was detected in the West African pygmy shrew (Crocidura obscurior) in neighboring Côte d’Ivoire (Kang et al., 2011). However, the molecular phylogeny of crocidurine shrew-borne hantaviruses from Africa has been hampered because the genomes of TGNV and AZGV have not been fully sequenced.

In an attempt to obtain the complete genomes of TGNV and AZGV, ethanol-fixed, archival intercostal muscle tissues, collected from 39 West African pygmy shrews and 22 Therese’s shrews, as well as 11 Doucet’s musk shrew (Crocidura douceti) and 11 large-headed shrews (Crocidura grandiceps), which were captured as part of an ecological survey of small mammals in Bowé, Gbaolé, Hermakono and Zogota in Guinea (Fig. 1A), during May 2011 to February 2012, were analyzed for hantavirus RNA by reverse transcription polymerase chain reaction (RT-PCR), using previously described methods (Arai et al., 2008, 2012; Kang et al., 2009, 2011). In this report, we present the molecular phylogeny and complete genome sequence of a previously unrecognized hantavirus, designated Bowé virus (BOWV), harbored by the Doucet’s musk shrew.

Fig. 1.

Fig. 1

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(A) Map of Guinea, showing the locations of the four trap sites. A single Doucet’s musk shrew captured in Bowé was found to be infected with a previously unrecognized hantavirus. (B) Schematic comparison of consensus secondary structures of the full-length N protein of TPMV, MJNV, JJUV and BOWV, with the partial N protein of AZGV and TGNV. N protein structures for the crocidurine shrew-borne hantaviruses from Africa and Asia were predicted using several methods, including HNN, DSC, PHD, PREDATOR and MLRC, available at the NPS@ structure server (Combet et al., 2000). Alpha helices are represented by purple bars, beta-strand by yellow-green bars, and random coil and unclassified structure by red and gray bars, respectively.

2. Materials and methods

Total RNA was extracted, using the PureLink Micro-to-Midi total RNA purification kit (Invitrogen, San Diego, CA, USA), from shrew tissues, and cDNA was prepared using the SuperScript III First-Strand Synthesis System (Invitrogen) and random hexamers and/or an oligonucleotide primer (OSM55: 5′-TAGTAGTAGACTCC-3′) designed from the conserved 5′-end of the S, M and L segments of hantaviruses. PCR was performed as described previously, with each 20-μL reaction containing 250 μM dNTP, 2 mM MgCl2, 1 U of AmpliTaq polymerase (Roche, Basel, Switzerland) and 0.25 μM of oligonucleotide primers, with modified cycling conditions. Innumerable trial-and-error testing of primers was necessary before ultimately obtaining the complete genome sequence of a newfound hantavirus (Table 1). Initial denaturation at 94°C for 5 min was followed by two cycles each of denaturation at 94°C for 40 sec, two-degree step-down annealing from 48°C to 38°C for 40 sec, and elongation at 72°C for 1 min, then 32 cycles of denaturation at 94°C for 40 sec, annealing at 42°C for 40 sec, and elongation at 72°C for 1 min, in a GeneAmp PCR 9700 thermal cycler (Perkin-Elmer, Waltham, MA). Amplicons were purified using the QIAQuick Gel Extraction Kit (Qiagen, Hilden, Germany), and DNA sequencing was performed using an ABI Prism 377XL Genetic Analyzer (Applied Biosystems, Foster City, CA).

Table 1.

Oligonucleotide primers used to amplify and sequence BOWV.

Segment Primer Name Position Primer Sequence (5′ to 3′)
S OSM55 1 TAG TAG TAG ACT CC
Shrew-S764R 680–697 CCA TNA CWG GRC TNA TCA
Han-S604F 604–624 GCH GAD GAR HTN ACA CCN GG
SO-S1220R 1216–1235 TCR TCA CCN AGR TGG AAR TG
Shrew-S1174F 1108–1127 TCM TCA TTY TAY CAA TCA TA
Shrew-end-EcoRI 1731 CTC GAA TTC TAG TAG T
M OSM55 1 TAG TAG TAG ACT CC
BOWV-M1594R 1563–1581 CTA CAA GTA TGC ACC ATG T
HTN-M1520F 1537–1555 TTG GNT GGN TNY TAA THC C
HTN-M2355R 2352–2374 CCW GGR CAA TCH VGA GGR TTA CA
BOWV-M2318F 2295–2312 TGG CAA GCA GCA GCA TGT
BOWV-M2856R 2806–2825 ATC TAT AGT AGC CAT AAG CT
Shrew-end-EcoRI 3597 CTC GAA TTC TAG TAG T
L OSM55 1 TAG TAG TAG ACT CC
BOWV-L1981R 1913–1931 CAG ATG GTA TTA AAT ATC T
Han-L1900F 1883–1905 ATG AAR NTN TGT GCN ATN TTT GA
Han-L2970R 2948–2970 CCN GGN GAC CAY TTN GTD GCA TC
Han-L-F1 2933–2952 ATG TAY GTB AGT GCW GAT GC
Han-L5239R 5162–5181 TGN AYR CAR TAW GCA TCA TA
BOWV-L4954F 4954–4978 AGC CAA TGA TGC ACC ATA TAT AGA C
Shrew-3′endR 6551 TAG TAG TAK RCT CCY TRA A
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In addition to traditional Sanger sequencing, BOWV RT-PCR products were sequenced using Ion Torrent PGM (Life Technologies, Inc., Carlsbad, CA) to validate the complete BOWV genome. Amplicons were size selected on 0.8% agarose gels, then purified with QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The purified DNA was quantified with Quant-iT™ Technology (Life Technologies, Inc.) using Quant-iT™ dsDNA Broad-Range Assay Kit. Libraries were then constructed using NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torrent™ (New England Biolabs, Inc., Ipswich, MA), and the Agilent Bioanalyzer™ with the Agilent High Sensitivity DNA Kit (Agilent Technologies, Inc., Santa Clara, CA) was used to analyze library sizes and molar concentrations. Template preparation and sequencing reactions were performed with Ion PGM™ 200 Xpress™ Template Kit and Ion PGM™ 200 Sequencing Kit (Life Technologies, Inc.), according to the manufacturer’s instructions.

Ion Torrent base sequence quality below 20 were trimmed and reads shorter than 30 bases were discarded before further analysis. Sequence alignment was achieved using Bowtie 2–2.0.0 (Langmead and Salzberg, 2012), and Velvet 1.2.07 de novo assembler (Zerbino and Birney, 2008), with varying stringencies, was used to generate sequence contigs. Sequence comparison analysis for Ion Torrent sequencing was performed using BLAST (National Center for Biotechnology Information, Bethesda, MD), and ClustalW (DNASTAR, Inc., Madison, WI) (Thompson et al., 1994) and transAlign (Bininda-Emonds, 2005) were used for Sanger sequencing.

Phylogenetic trees were generated by maximum likelihood and Bayesian methods, implemented in PAUP* (Phylogenetic Analysis Using Parsimony, 4.0b10) (Swofford 2003), RAxML Blackbox webserver (Stamatakis et al., 2008) and MrBayes 3.1 (Ronquist and Huelsenbeck, 2003), under the best-fit GTR+I+Γ model of evolution selected by hierarchical likelihood-ratio test in MrModeltest v2.3 (Posada and Crandall, 1998) and jModelTest version 0.1 (Posada, 2008). Two replicate Bayesian Metropolis–Hastings Markov Chain Monte Carlo runs, each consisting of six chains of 10 million generations sampled every 100 generations with a burn-in of 25,000 (25%), resulted in 150,000 trees overall. Each genomic segment (S, M and L) was treated separately in phylogenetic analyses. Topologies were evaluated by bootstrap analysis of 1,000 iterations, and posterior node probabilities were based on 2 million generations and estimated sample sizes over 100 (implemented in MrBayes).

3. Results and discussion

Disappointingly, TGNV and AZGV were not detected in any of the Therese’s shrews and West African pygmy shrews, respectively, despite using TGNV- and AZGV-specific primers. As such, we were unable to definitively establish the host designation of TGNV and AZGV. The failure to detect TGNV and AZGV may be attributed to the ethanol fixation and the less than optimal tissue (intercostal muscle), as well as the small sample size and the likely low virus titers. Nevertheless, BOWV was identified in a Doucet’s musk shrew, captured on February 20, 2012 in a regenerating forested area in Bowé (8°5′60″N, 8°49′60″W), located in the Prefecture de Nzerekore region of Guinea (Fig. 1A). The taxonomic identity of the BOWV-infected Doucet’s musk shrew was confirmed by mitochondrial DNA sequence analysis of the 1,140-nucleotide full-length cytochrome b gene (GenBank accession no. KC684929).

The poor-quality fragmented RNA, extracted from the ethanol-fixed intercostal muscle, thwarted virus-specific RNA enrichment or host-sequence depletion efforts for RNA sequencing. Therefore, we performed deep sequencing of viral RT-PCR amplicons. Sequencing of the BOWV genome by Ion Torrent generated 2,777,993 reads with a mean read length of 113 bases. The sequence reads were trimmed and filtered for quality before being aligned to Sanger sequencing accomplished sequences and de novo assembled. Mapping sequence reads revealed 1,499,052 hits (55% of the quality reads) with mean coverage alignment of 13,319X; 244,726 reads (16.3%) mapped to the S segment, 501,888 reads (33.5%) to the M segment, and 752,438 reads (50.2%) to the L segment. Ultimately, the consensus sequences were accomplished from the alignment, which covered 73.4% of the BOWV genome. Comparison of the assembled contigs, consensus sequences by both Sanger and next-generation sequencing revealed more than 99% similarity for each of the segments. GenBank accession numbers for the S, M and L segments of BOWV were KC631782, KC631783 and KC631784, respectively.

The full-length 1,731-nucleotide S-genomic segment of BOWV contained a single open reading frame (ORF), encoding a 431-amino acid nucleocapsid (N) protein (nucleotide positions, 28 to 1,320), and 3′- and 5′-noncoding regions (NCR) of 27- and 408-nucleotides, respectively. The hypothetical NSs ORF, typically found in hantaviruses hosted by arvicolid, neotomine and sigmodontine rodents, was absent. Employing prediction software available in the NPS@structure server (Combet et al., 2000), the BOWV N protein secondary structure generally resembled that of other rodent- and soricomorph-borne hantaviruses, comprising two α-helical domains packed against a central β-pleated sheet. Direct comparison of the N protein secondary structure demonstrated that BOWV most closely resembled AZGV, TGNV and Jeju virus (JJUV), and differed from Thottapalayam virus (TPMV) (Carey et al., 1971) and Imjin virus (MJNV) (Song et al., 2009), two crocidurine shrew-borne hantaviruses from Asia (Fig. 1B).

The complete M-genomic segment of BOWV was 3,597 nucleotides, with a predicted glycoprotein of 1,145 amino acids, starting at nucleotide position 36, and a 124-nucleotide 5′-NCR. Like other hantaviruses, the BOWV glycoprotein precursor had the highly conserved WAASA amino-acid motif (amino acid positions 652–656). Glycosylation sites, as predicted using NetNlyc 1.0 and Predictprotein (Gavel and von Heijne, 1990), showed five potential N-linked glycosylation sites (four in Gn at amino acid positions 142, 355, 407 and 526; and one in Gc at position 936).

The full-length 6,551-nucleotide L-genomic segment of BOWV encoded a predicted RNA-dependent RNA polymerase of 2,158 amino acids. The six major conserved motifs (designated as premotif A and motifs A, B, C, D and E) for RNA virus polymerases, which are shared by all hantaviruses, were also found in BOWV. Premotif A had a conserved lysine and two arginine residues. Motifs A, B and D had conserved aspartate, glycine and lysine, respectively. In motif C, there were two conserved aspartic acid residues. The XDD motif, essential for catalytic activity, and motif E, containing the E(F/Y)XS site, were also present.

Pairwise alignment and comparison of the S-segment coding sequences of BOWV and other non-African soricomorph-borne hantaviruses showed moderately low sequence similarities, ranging from 48.2–71.3% and 46.6–75.9% at the nucleotide and amino acid levels, respectively (Table 2). Amino acid sequence identity of the RNA-dependent RNA polymerase was highest (83.1% and 85.4%) between BOWV and AZGV and TGNV, two other African crocidurine shrew-borne hantaviruses (Table 2). This overall high sequence similarity was consistent with the presumed functional constraints on the RNA-dependent RNA polymerase.

Table 2.

Nucleotide and amino acid sequence identity (%) between BOWV strain VN1512 and representative rodent-, shrew- and mole-borne hantaviruses.

Virus strain S segment
M segment
L segment
1,293 nt 431 aa 3,435 nt 1145 aa 6,474 nt 2158 aa
HTNV 76-118 66.4 64.3 62.9 58.1 69.7 73.4
SEOV 80-39 63.9 63.6 61.2 56.9 69.1 72.3
SANGV SA14 65.3 63.2 60.7 56.7 68.4 72.0
SOOV SOO-1 64.0 63.6 63.3 58.2 70.2 73.2
DOBV Greece 65.0 62.2 62.6 56.8 69.9 73.5
PHV PH-1 63.3 58.0 59.2 50.7 66.4 65.4
PUUV Sotkamo 63.3 61.5 60.5 51.1 67.1 66.6
TULV 5302v 63.2 60.4 59.2 51.8 66.5 65.3
ANDV Chile9717869 65.0 61.4 59.6 51.7 67.2 66.5
SNV NMH10 61.4 59.1 59.3 51.8 66.2 66.7
CBNV CBN-3 69.0 74.5 66.5 66.2 72.2 78.2
ARRV MSB73418 67.3 65.6 - - 71.1 73.9
JMSV MSB144475 69.3 71.8 70.2 71.7 72.1 78.8
SWSV mp70 66.9 67.1 71.5 73.5 68.8 74.2
QDLV YN05-284 67.1 67.6 68.3 68.9 74.8 79.9
KKMV MSB148794 65.7 67.6 70.7 68.6 71.3 77.8
TGNV Tan826 72.2 73.5 - - 76.2 85.4
AZGV KBM15 65.0 65.6 73.1 83.0 74.2 83.1
JJUV 10-11 71.3 75.9 69.7 71.7 73.6 80.6
MJNV Cl05-11 49.1 47.6 41.5 41.1 64.4 60.3
TPMV VRC66412 48.2 46.6 40.3 40.1 63.5 60.5
RKPV MSB57412 64.3 61.2 62.1 51.3 67.8 65.8
OXBV Ng1453 68.1 70.3 64.7 61.6 70.4 75.7
ASAV N10 68.5 69.6 67.9 64.4 72.1 77.0
NVAV MSB95703 48.6 48.8 55.1 42.4 64.0 60.4
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Abbreviations: ANDV, Andes virus; ARRV, Ash River virus; ASAV, Asama virus; AZGV, Azagny virus; CBNV, Cao Bang virus; DOBV, Dobrava virus; HTNV, Hantaan virus; JMSV, Jemez Spring virus; JJUV, Jeju virus; KKMV, Kenkeme virus; MJNV, Imjin virus; NVAV, Nova virus; OXBV, Oxbow virus; PHV, Prospect Hill virus; PUUV, Puumala virus; QDLV, Qiandao Lake virus; RKPV, Rockport virus; SANGV, Sangassou virus; SEOV, Seoul virus; SNV, Sin Nombre virus; SOOV, Soochong virus; SWSV, Seewis virus; TGNV, Tanganya virus; TPMV, Thottapalayam virus; TULV, Tula virus. nt, nucleotides; aa, amino acids.

The disparate phylogenetic positions of BOWV in trees based on the S- and on the M- and L-segment sequences could not be attributed to reassortment or recombination. Multiple recombination-detection methods, including GENECONV, Bootscan, Chimaera, 3SEQ, RDP, SiScan, MaxChi and HyPhy Single Recombinant Breakpoint (Pond et al., 2005), failed to disclose any evidence of genetic recombination in the BOWV genome.

Unrooted phylogenetic trees, based on the nucleotide sequences, showed four distinct, well-supported clades, comprising hantaviruses harbored by rodents of the Muridae family; rodents of the Cricetidae family; soricomorphs of the Soricidae and Talpidae families; and two shrew species (Crocidurinae subfamily) and one talpid mole species (Talpinae subfamily), which represent the most divergent hantaviruses found to date. The phylogenetic trees obtained for the M and L segments showed that the three African crocidurine shrew-borne hantaviruses (BOWV, TGNV and AZGV) clustered with JJUV (Arai et al., 2012), hosted by the Asian lesser white-toothed shrew (Crocidura shantungensis) in Korea (Fig. 2). This crocidurine shrew-borne hantavirus lineage was very well supported, and BOWV always occupied a basal position within this cluster. By contrast, as discussed previously, the divergent and basal position of MJNV and TPMV in the phylogeny of hantaviruses harbored by crocidurine shrews suggested that it may have arisen from a host-switching event (Arai et al., 2012).

Fig. 2.

Fig. 2

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Phylogenetic trees were generated by the maximum-likelihood and Bayesian methods, using the GTR+I+Γ model of evolution, based on the alignment of the S-, M- and L-segment sequences of BOWV strain VN1512. Since tree topologies were very similar using RAxML and MrBayes, the trees generated by MrBayes were displayed. The phylogenetic positions of BOWV are shown in relationship to crocidurine shrew-borne hantaviruses, including Tanganya virus (TGNV Tan826: EF050455, EF050454), Azagny virus (AZGV KBM15: JF276226, JF276227, JF276228), Jeju virus (JJUV 10–11: HQ834695, HQ834696, HQ834697), Thottapalayam virus (TPMV VRC66412: AY526097, EU001329, EU001330) and Imjin virus (MJNV Cl05–11: EF641804, EF641798, EF641806). Soricine shrew-borne hantaviruses included Cao Bang virus (CBNV CBN-3: EF543524, EF543526, EF543525), Ash River virus (ARRV MSB73418: EF650086, EF619961), Jemez Springs virus (JMSV MSB144475: FJ593499, FJ593500, FJ593501), Kenkeme virus (KKMV MSB148794: GQ306148, GQ306149, GQ306150), Qiandao Lake virus (QDLV YN05-284: GU566023, GU566022, GU566021) and Seewis virus (SWSV mp70: EF636024, EF636025, EF636026). Also shown are mole-borne hantaviruses, including Asama virus (ASAV N10: EU929072, EU929075, EU929078), Nova virus (NVAV MSB95703: FJ539168, HQ840957, FJ593498), Oxbow virus (OXBV Ng1453: FJ539166, FJ539167, FJ593497) and Rockport virus (RKPV MSB57412: HM015223, HM015219, HM015221). Rodent-borne hantaviruses included Hantaan virus (HTNV 76–118: NC_005218, Y00386, NC_005222), Soochong virus (SOOV SOO-1: AY675349, AY675353, DQ562292), Dobrava virus (DOBV Greece: NC_005233, NC_005234, NC_005235), Seoul virus (SEOV 80–39: NC_005236, NC_005237, NC_005238), Sangassou virus (SANGV SA14: JQ082300, JQ082301, JQ082302), Tula virus (TULV M5302v: NC_005227, NC_005228, NC_005226), Puumala virus (PUUV Sotkamo: NC_005224, NC_005223, NC_005225), Prospect Hill virus (PHV PH-1: Z49098, X55129, EF646763), Andes virus (ANDV Chile9717869: NC_003466, NC_003467, NC_003468) and Sin Nombre virus (SNV NMH10: NC_005216, NC_005215, NC_005217). The numbers at each node are posterior node probabilities based on 150,000 trees (left) and bootstrap values of 1,000 replicates executed on the RAxML BlackBox web server (right), respectively. The scale bars indicate nucleotide substitutions per site.

Visceral tissues, such as lung and spleen, were unavailable from the hantavirus-infected Doucet’s musk shrew. However, the detection of hantavirus RNA in intercostal muscle was consistent with widespread persistent infection and strongly suggests a genuine hantavirus-reservoir host relationship. Moreover, although not ideal and far inferior to frozen tissues or tissues preserved in RNAlater®, ethanol-fixed archival tissues from small mammals hold promise in virus-discovery efforts, as has been demonstrated recently with the identification of a highly divergent hantavirus in ethanol-fixed liver tissue collected from banana pipistrelles (Neoromicia nanus) captured in Côte d’Ivoire (Sumibcay et al., 2012). That is, ethanol-fixed tissues, originally collected for other purposes, serve as precious archival research resources in microbiology, evolutionary biology and host genetics.

The Doucet’s musk shrew is a semi-arboreal species, which inhabits the forest-savanna border of Guinea and Côte d’Ivoire. Sparse populations of this species have been recorded in Adiopodoumé and the Taï National Park in Côte d’Ivoire, and in Sérédou and at the foot of Mount Nimba in upper Guinea. Of the 11 Doucet’s musk shrews from Guinea in the present study, six were trapped in Gbaolé, three in Zogota and one each in Hermakono and Bowé. Although not currently listed as a threatened or endangered species by the International Union for Conservation of Nature and Natural Resources, general deforestation is a presumed threat. More information is needed about the natural history and ecology of this little-known African crocidurine shrew species.

Genome-wide analyses of many more shrew-borne hantaviruses are necessary to ascertain their evolutionary origin and phylogeography. Viewed within the context that newly recognized hantaviruses detected in African shrews greatly outnumber those reported in rodents (Klempa et al., 2006; Meheretu et al., 2012), the recent identification of highly divergent lineages of hantaviruses in insectivorous bats in Côte d’Ivoire (Sumibcay et al., 2012) and Sierra Leone (Weiss et al., 2012), as well as in China (Guo et al., 2013) and Vietnam (Arai et al., 2013), further supports the emerging concept that rodents were not the primordial hosts of ancestral hantaviruses.

Highlights.

  • Detection of a previously unrecognized genetically distinct hantavirus in the Doucet’s musk shrew

  • First full genome analysis of an African shrew-borne hantavirus

  • Ethanol-fixed archival tissues hold promise in virus-discovery efforts

Acknowledgments

This work was supported in part by U.S. Public Health Service grants R01AI075057 from the National Institute of Allergy and Infectious Diseases and P20GM103516 from the National Institute of General Medical Sciences, National Institutes of Health. We also thank the Service de Systématique Moléculaire of the Muséum National d’Histoire Naturelle (CNRS - UMS 2700, Paris, France). Trapping of shrews was approved by the Ministère de l’environnement et du développement durable, République de Guinée (permit numbers 0003394, 0003329 and 0003374).

Footnotes

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