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Divergent lineage of a novel hantavirus in the banana pipistrelle (Neoromicia nanus) in Côte d'Ivoire
© Sumibcay et al; licensee BioMed Central Ltd. 2012
Received: 29 December 2011
Accepted: 26 January 2012
Published: 26 January 2012
Recently identified hantaviruses harbored by shrews and moles (order Soricomorpha) suggest that other mammals having shared ancestry may serve as reservoirs. To investigate this possibility, archival tissues from 213 insectivorous bats (order Chiroptera) were analyzed for hantavirus RNA by RT-PCR. Following numerous failed attempts, hantavirus RNA was detected in ethanol-fixed liver tissue from two banana pipistrelles (Neoromicia nanus), captured near Mouyassué village in Côte d'Ivoire, West Africa, in June 2011. Phylogenetic analysis of partial L-segment sequences using maximum-likelihood and Bayesian methods revealed that the newfound hantavirus, designated Mouyassué virus (MOUV), was highly divergent and basal to all other rodent- and soricomorph-borne hantaviruses, except for Nova virus in the European common mole (Talpa europaea). Full genome sequencing of MOUV and further surveys of other bat species for hantaviruses, now underway, will provide critical insights into the evolution and diversification of hantaviruses.
Discovery of phylogenetically divergent hantaviruses in shrews and moles (order Soricomorpha, family Soricidae and Talpidae) [1–13] raises the possibility that rodents (order Rodentia, family Muridae and Cricetidae) may not be the principal or primordial reservoirs. Moreover, newfound hantaviruses harbored by soricomorphs of multiple species, distributed in widely separated geographic regions across four continents, suggest that their host diversity may be far more expansive than previously assumed. Specifically, other mammals having shared ancestry or ecosystems with soricomorphs may serve as reservoirs and may be important in the evolutionary history and diversification of hantaviruses. In particular, bats (order Chiroptera) may be potential reservoirs by virtue of their rich diversity and vast geographical range, as well as their demonstrated ability to host myriad medically important, disease-causing viruses [14–18]. Surprisingly little attention, however, has been paid to this possibility.
As in our previous investigations on the spatial and temporal distribution of hantaviruses in soricomorphs [2–13], we relied on the availability of archival tissues. Using the PureLink Micro-to-Midi total RNA purification kit (Invitrogen, San Diego, CA), total RNA was extracted from 168 frozen and 45 ethanol-fixed liver and other visceral tissues of 213 insectivorous bats (representing 13 genera), collected during May 1981 to June 2011 in Asia, Africa and the Americas (Table 1). cDNA was then prepared with the SuperScript III First-Strand Synthesis System (Invitrogen) using random hexamers, and PCR was performed as described previously, using an extensive panel of oligonucleotide primers, designed on conserved genomic sequences of rodent- and soricomorph-borne hantaviruses [2–13, 19, 20]. Each reaction mixture contained 250 μ dNTP, 2 mM MgCl2, 1 U AmpliTaq polymerase (Roche, Basel, Switzerland) and 0.25 μ oligonucleotide primers. Initial denaturation at 94°C for 5 min was followed by two cycles each of denaturation at 94°C for 40 s, two-degree step-down annealing from 48°C to 38°C for 40 s, and elongation at 72°C for 1 min or 1 min 20 s, then 32 cycles of denaturation at 94°C for 40 s, annealing at 42°C for 40 s, 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).
Detection of hantavirus RNA in tissues of insectivorous bats by RT-PCR
A 423-nucleotide region of the RNA-dependent RNA polymerase-encoding L segment, amplified using a hemi-nested primer set (outer: 5'-GAAAGGGCATTNMGATGGGCNTCA GG-3', 5'-AACCADTCWGTYCCRTCATC-3'; inner: 5'-GNAAAYTNATGTATGTNAGT GC-3', 5'-AACCADTCWGTYCCRTCATC-3'), was aligned and compared with hantavirus sequences available in GenBank, using ClustalW (DNASTAR, Inc., Madison, WI)  and transAlign . The newfound hantavirus, designated Mouyassué virus (MOUV), exhibited low nucleotide and amino acid sequence similarity of less than 69% to all representative soricomorph- and rodent-associated hantaviruses, except for the 76.3% sequence similarity with Nova virus (NVAV), previously reported in the European common mole (Talpa europaea) . Interestingly, MOUV sequences were identical in the two banana pipistrelles (KB576 and KB577), a male-female pair captured simultaneously and presumed to be a mating couple, suggesting horizontal virus transmission or common-source infection.
Despite the overall success of our brute-force RT-PCR approach at identifying previously unrecognized hantaviruses in frozen tissues [2, 3, 5–7, 10–13] and tissues preserved in RNAlater® RNA Stabilization Reagent [4, 8], designing universal primers for the amplification of soricomorph-borne hantaviruses has presented continuing challenges. Thus, while it is likely that many more hantaviruses await discovery, overcoming technical barriers is essential to facilitating their detection. Viewed in this context, the failure to detect hantavirus RNA in all but one bat species was not altogether unexpected and may be attributed simply to suboptimal primer design and imperfect cycling conditions. Also, low RNA yields and poor RNA preservation in tissues fixed in ethanol under field conditions may have thwarted our efforts at obtaining more of the MOUV genome. That said, the successful amplification of hantavirus RNA from ethanol-fixed tissues is highly instructive and augments the pool of archival tissues for future exploratory studies of hantaviruses in bats, and possibly other insectivorous small mammals that share ancestral lineages with soricomorphs, such as hedgehogs (order Erinaceomorpha, family Erinaceidae).
Dating to the seminal discovery of Hantaan virus in lung tissue of the striped field mouse (Apodemus agrarius) , lung has been the preferred tissue in studies aimed at finding new hantaviruses [28–30]. However, lung is not the only tissue in which hantaviruses can be detected [27, 31]. In our search of genetically distinct hantaviruses in long-stored archival tissues from shrews and moles, lung tissue was frequently unavailable. Instead, liver tissue was more often accessible and proved to be quite suitable [4, 5, 12, 13]. Similarly, liver tissues were more often available in the present study. As in reservoir rodents and soricomorphs, hantavirus RNA is likely to be present in many tissues of persistently infected bats. Real-time quantitative RT-PCR analysis of lung, liver and other viscera will clarify the tissue distribution of MOUV in newly captured banana pipistrelles from Mouyassué.
Having their fossil origins in the Eocene epoch, approximately 50 million years before present, bats occur on every continent except Antarctica and are among the most speciose orders of mammals, with more than 1,100 extant species . The banana pipistrelle, which is distributed widely in forests and savannas across sub-Saharan Africa (Figure 1C, inset), is one of 13 species in the genus Neoromicia of the family Vespertilionidae and subfamily Vespertilioninae. Like other vesper bats, the banana pipistrelle is insectivorous. Unlike large fruit bats, such as the straw-colored fruit bat (Eidolon helvum) and hammer-headed bat (Hypsignathus monstrosus), which are sold as bush meat, the banana pipistrelle, weighing approximately 3 g, is not consumed as food. However, because banana pipistrelles occasionally roost within houses or reside near human habitation, rare human encounters raise the possibility of hantavirus exposure.
Previously, serological evidence of hantavirus infection was reported in the common serotine (Eptesicus serotinus) and greater horseshoe bat (Rhinolophus ferrumequinum) captured in Korea , but genetic analysis of hantaviral isolates from these insectivorous bat species proved to be indistinguishable from prototype Hantaan virus , suggesting laboratory contamination. In the present study, the strikingly divergent lineage of MOUV precluded any possibility of contamination and lends support to our earlier conjecture that the ancient origins of hantaviruses may have involved insect-borne viruses [7, 10], with subsequent adaptation to and host switching between early soricomorph and chiropteran ancestral hosts in the mammalian superorder Laurasiatheria. However, since the biological and evolutionary implications of bats as reservoirs of hantaviruses are considerable, studies are underway to establish that the banana pipistrelle is the natural host of MOUV. Moreover, high-throughput sequencing technology is being applied to obtain the full genome of MOUV and to ascertain the geographic range and genetic diversity of hantaviruses harbored by bats.
We thank Melissa S. Nagata, Nelson I.B. Lazaga, Moti L. Chapagain and Pakieli H. Kaufusi for technical assistance. We also thank Shaobin Hou and the staff of the Advanced Studies in Genomics, Proteomics and Bioinformatics for DNA sequencing. This work was supported in part by 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.
- Klempa B, Fichet-Calvet E, Lecompte E, Auste B, Aniskin V, Meisel H, Barrière P, Koivogui L, ter Meulen J, Krüger DH: Novel hantavirus sequences in shrew, Guinea. Emerg Infect Dis 2007, 13: 520-522.PubMedPubMed CentralView ArticleGoogle Scholar
- Song J-W, Gu SH, Bennett SN, Arai S, Puorger M, Hilbe M, Yanagihara R: Seewis virus, a genetically distinct hantavirus in the Eurasian common shrew ( Sorex araneus ). Virol J 2007, 4: 114. 10.1186/1743-422X-4-114PubMedPubMed CentralView ArticleGoogle Scholar
- Arai S, Song J-W, Sumibcay L, Bennett SN, Nerurkar VR, Parmenter C, Cook JA, Yates TL, Yanagihara R: Hantavirus in northern short-tailed shrew, United States. Emerg Infect Dis 2007, 13: 1420-1423.PubMedPubMed CentralView ArticleGoogle Scholar
- Song J-W, Kang HJ, Song KJ, Truong TT, Bennett SN, Arai S, Truong NU, Yanagihara R: Newfound hantavirus in Chinese mole shrew, Vietnam. Emerg Infect Dis 2007, 13: 1784-1787.PubMedPubMed CentralView ArticleGoogle Scholar
- Arai S, Bennett SN, Sumibcay L, Cook JA, Song J-W, Hope A, Parmenter C, Nerurkar VR, Yates TL, Yanagihara R: Phylogenetically distinct hantaviruses in the masked shrew ( Sorex cinereus ) and dusky shrew ( Sorex monticolus ) in the United States. Am J Trop Med Hyg 2008, 78: 348-351.PubMedPubMed CentralGoogle Scholar
- Song J-W, Kang HJ, Gu SH, Moon SS, Bennett SN, Song KJ, Baek LJ, Kim HC, O'Guinn ML, Chong ST, Klein TA, Yanagihara R: Characterization of Imjin virus, a newly isolated hantavirus from the Ussuri white-toothed shrew ( Crocidura lasiura ). J Virol 2009, 83: 6184-6191. 10.1128/JVI.00371-09PubMedPubMed CentralView ArticleGoogle Scholar
- Kang HJ, Arai S, Hope AG, Song J-W, Cook JA, Yanagihara R: Genetic diversity and phylogeography of Seewis virus in the Eurasian common shrew in Finland and Hungary. Virol J 2009, 6: 208. 10.1186/1743-422X-6-208PubMedPubMed CentralView ArticleGoogle Scholar
- Kang HJ, Kadjo B, Dubey S, Jacquet F, Yanagihara R: Molecular evolution of Azagny virus, a newfound hantavirus in the West African pygmy shrew ( Crocidura obscurior ) in Côte d'Ivoire. Virol J 2011, 8: 373. 10.1186/1743-422X-8-373PubMedPubMed CentralView ArticleGoogle Scholar
- Kang HJ, Kosoy MY, Shrestha SK, Shrestha MP, Pavlin JA, Gibbons RV, Yanagihara R: Genetic diversity of Thottopalayam virus, a hantavirus harbored by the Asian house shrew ( Suncus murinus ) in Nepal. Am J Trop Med Hyg 2011, 85: 540-545.PubMedPubMed CentralView ArticleGoogle Scholar
- Arai S, Ohdachi SD, Asakawa M, Kang HJ, Mocz G, Arikawa J, Okabe N, Yanagihara R: Molecular phylogeny of a newfound hantavirus in the Japanese shrew mole ( Urotrichus talpoides ). Proc Natl Acad Sci USA 2008, 105: 16296-16301. 10.1073/pnas.0808942105PubMedPubMed CentralView ArticleGoogle Scholar
- Kang HJ, Bennett SN, Dizney L, Sumibcay L, Arai S, Ruedas LA, Song J-W, Yanagihara R: Host switch during evolution of a genetically distinct hantavirus in the American shrew mole ( Neurotrichus gibbsii ). Virology 2009, 388: 8-14. 10.1016/j.virol.2009.03.019PubMedPubMed CentralView ArticleGoogle Scholar
- Kang HJ, Bennett SN, Sumibcay L, Arai S, Hope AG, Mocz G, Song J-W, Cook JA, Yanagihara R: Evolutionary insights from a genetically divergent hantavirus harbored by the European common mole ( Talpa europaea ). PLoS One 2009, 4: e6149. 10.1371/journal.pone.0006149PubMedPubMed CentralView ArticleGoogle Scholar
- Kang HJ, Bennett SN, Hope AG, Cook JA, Yanagihara R: Shared ancestry between a mole-borne hantavirus and hantaviruses harbored by cricetid rodents. J Virol 2011, 85: 7496-7503. 10.1128/JVI.02450-10PubMedPubMed CentralView ArticleGoogle Scholar
- Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T: Bats: important reservoir hosts of emerging viruses. Clin Microbiol Rev 2006, 19: 531-545. 10.1128/CMR.00017-06PubMedPubMed CentralView ArticleGoogle Scholar
- Johara M, Field H, Rashdi A, Morrissy C, vander Heide B, Rota P, Azri A, White J, Daniels P, Jamaluddin A, Ksiazek T: Serological evidence of infection with Nipah virus in bats (order Chiroptera) in Peninsular Malaysia. Emerg Infect Dis 2001, 7: 439-441.View ArticleGoogle Scholar
- Li W, Shi Z, Yu M, Ren W, Smith C, Epstein JH, Wang H, Crameri G, Hu Z, Zhang H, Zhang J, McEachern J, Field H, Daszak P, Eaton BT, Zhang S, Wang LF: Bats are natural reservoirs of SARS-like coronaviruses. Science 2005, 310: 676-679. 10.1126/science.1118391PubMedView ArticleGoogle Scholar
- Leroy EM, Kumulungui B, Pourrut X, Rouquet P, Hassanin A, Yaba P, Délicat A, Paweska JT, Gonzalez JP, Swanepoel R: Fruit bats as reservoirs of Ebola virus. Nature 2005, 438: 575-576. 10.1038/438575aPubMedView ArticleGoogle Scholar
- Biek R, Walsh PD, Leroy EM, Real LA: Recent common ancestry of Ebola Zaire virus found in a bat reservoir. PLoS Pathog 2006, 2: e90. 10.1371/journal.ppat.0020090PubMedPubMed CentralView ArticleGoogle Scholar
- Klempa B, Fichet-Calvet E, Lecompte E, Auste B, Aniskin V, Meisel H, Denys C, Koivogui L, ter Meulen J, Krüger DH: Hantavirus in African wood mouse, Guinea. Emerg Infect Dis 2006, 12: 838-840. 10.3201/eid1205.051487PubMedPubMed CentralView ArticleGoogle Scholar
- Arthur RR, Lofts RS, Gomez J, Glass GE, LeDuc JW, Childs JE: Grouping of hantaviruses by small (S) genome segment polymerase chain reaction and amplification of viral RNA from wild-caught rats. Am J Trop Med Hyg 1992, 47: 210-224.PubMedGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22: 4673-4680. 10.1093/nar/22.22.4673PubMedPubMed CentralView ArticleGoogle Scholar
- Bininda-Emonds OR: transAlign: using amino acids to facilitate the multiple alignment of protein-coding DNA sequences. BMC Bioinformatics 2005, 6: 156. 10.1186/1471-2105-6-156PubMedPubMed CentralView ArticleGoogle Scholar
- Swofford D: PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods). Sunderland, Massachusetts: Sinauer Associates; 2003.Google Scholar
- Stamatakis A, Hoover P, Rougemont J: A rapid bootstrap algorithm for the RAxML web servers. Syst Biol 2008, 57: 758-771. 10.1080/10635150802429642PubMedView ArticleGoogle Scholar
- Ronquist F, Huelsenbeck JP: MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 2003, 19: 1572-1574. 10.1093/bioinformatics/btg180PubMedView ArticleGoogle Scholar
- Posada D: jModelTest: phylogenetic model averaging. Mol Biol Evol 2008, 25: 1253-1256. 10.1093/molbev/msn083PubMedView ArticleGoogle Scholar
- Lee HW, Lee P-W, Johnson KM: Isolation of the etiologic agent of Korean hemorrhagic fever. J Infect Dis 1978, 137: 298-308. 10.1093/infdis/137.3.298PubMedView ArticleGoogle Scholar
- Brummer-Korvenkontio M, Vaheri A, Hovi T, von Bonsdorff CH, Vuorimies J, Manni T, Penttinen K, Oker-Blom N, Lähdevirta J: Nephropathia epidemica: detection of antigen in bank voles and serologic diagnosis of human infection. J Infect Dis 1980, 141: 131-134. 10.1093/infdis/141.2.131PubMedView ArticleGoogle Scholar
- Lee HW, Baek LJ, Johnson KM: Isolation of Hantaan virus, the etiologic agent of Korean hemorrhagic fever, from wild urban rats. J Infect Dis 1982, 146: 638-644. 10.1093/infdis/146.5.638PubMedView ArticleGoogle Scholar
- Nichol ST, Spiropoulou CF, Morzunov S, Rollin PE, Ksiazek TG, Feldmann H, Sanchez A, Childs J, Zaki S, Peters CJ: Genetic identification of a hantavirus associated with an outbreak of acute respiratory illness. Science 1993, 262: 914-917. 10.1126/science.8235615PubMedView ArticleGoogle Scholar
- Lee HW, French GR, Lee PW, Baek LJ, Tsuchiya K, Foulke RS: Observations on natural and laboratory infection of rodents with the etiologic agent of Korean hemorrhagic fever. Am J Trop Med Hyg 1981, 30: 477-482.PubMedGoogle Scholar
- Wilson DE, Reeder DM: Mammal Species of the World: A Taxonomic and Geographic Reference. 3rd edition. Baltimore, Maryland: The Johns Hopkins University Press; 2005.Google Scholar
- Kim GR, Lee YT, Park CH: A new natural reservoir of hantavirus: isolation of hantaviruses from lung tissues of bats. Arch Virol 1994, 134: 85-95. 10.1007/BF01379109PubMedView ArticleGoogle Scholar
- Jung YT, Kim GR: Genomic characterization of M and S RNA segments of hantaviruses isolated from bats. Acta Virol 1995, 39: 231-233.PubMedGoogle Scholar
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