Novel hantavirus sequences in Shrew, Guinea.

To the Editor: Hantaviruses, family Bunyaviridae, have been known as causative agents of hemorrhagic fever with renal syndrome in Asia and Europe (1,2) and hantavirus cardiopulmonary syndrome in the Americas (3). Hantaviruses are spread by aerosolized rodent excreta and are strongly associated with their natural hosts, rodents of the family Muridae. Based on phylogenetic analyses, hantaviruses have been divided into 3 major groups that resemble 3 subfamilies of their natural hosts (Figure, panel A).

Recently, we found the first indigenous African hantavirus, Sangassou virus (SANGV), in an African wood mouse (Hylomyscus simus) collected in Guinea (5). Thottapalayam virus (TPMV), isolated from an Asian house shrew (Suncus murinus) in India (6), is the only known hantavirus to be hosted by a shrew instead of a rodent (7,8). We report the recovery of hantavirus RNA of a novel sequence from a shrew, collected in Guinea, West Africa.

During a study of rodentborne hemorrhagic fever viruses performed in Guinea in 2002-2004, 32 shrews of the genus Crocidura were collected and screened for hantavirus RNA by reverse transcription-PCR (5). An RNA sample designated Tan826 produced a PCR product of the expected size. The animal host was a male Crocidura theresae collected in the grassland savannah around the village Tanganya (10[degrees]00'02"N, 10[degrees]58"22"W) in January 2004. Species identification, following the taxonomic nomenclature (9), was performed on the basis of morpho-anatomical characteristics and was supported by molecular analyses.

Partial L segment sequence of 412 nt was determined by cloning and sequencing of the obtained PCR product. Nucleotide sequence comparisons between Tan826 and other representatives of the genus Hantavirus showed very low sequence identity values, ranging from 67.7% (Andes virus) to 72.3% (Puumala virus). Corresponding sequences of deduced viral RNA polymerase (137 aa) showed only slightly higher similarity values of 69.3% (Tula virus) to 76.6% (SANGV). In a maximum likelihood phylogenetic tree (Figure, panel A), Tan826 did not unambiguously cluster with any of the major groups (i.e., Murinae-, Arvicolinae-, Sigmodontinae-associated viruses) and showed equal relatedness to all 3 groups. This exceptional position of the Tan826 sequence within the tree is consistent with its detection in a shrew instead of a rodent host. Because the sequence is only distantly related to other hantaviruses, sequences from additional members of the Bunyaviridae family were analyzed. Despite use of a suboptimal dataset of very divergent and short sequences, the phylogenetic placement of Tan862 within the genus Hantavirus could be clearly demonstrated (Figure, panel B).

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Furthermore, a partial S segment sequence (442 nt, 147 aa of the putative nucleoprotein) was determined to compare Tan826 directly with the shrew-associated TPMV (for which only an S segment sequence was available in GenBank). Rather unexpectedly, the Tan826 sequence showed the lowest similarity to TPMV: 47.5% on nt level and 39.4% on aa level. The identity values to other Hantavirus members were also extremely low, 52.2% (Sin Nombre virus) to 62.1% (SANGV) on nt level and 50.6% (Andes virus) to 56.7% (Hantaan, Dobrava virus) on aa level. Corresponding aa sequences were then used for phylogenetic analysis to reduce problems derived from higher sequence diversities. In the resulting evolutionary tree, Tan826 and TPMV did not join any of the 3 major groups but also did not cluster together (Figure, panel C).

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Our attempts to obtain more sequence data were hampered by the unique nature of the Tan826 virus sequence, which makes it difficult to design additional effective PCR primers, as well as by the limited amount of available biological material from the shrew. Nevertheless, the sequence and phylogenetic analyses of the 2 partial sequences strongly indicate that they represent a novel hantavirus. The amino acid sequences are highly divergent ([approximately equal to] 25%-50%) from those of other hantaviruses and in phylogenetic trees; the Tan826 virus sequence appeared approximately equally related to those of all other hantaviruses. We propose to name the putative new species Tanganya virus (TGNV), after the locality where it was detected.

Detecting the virus in 1 of 32 Crocidura shrews, 15 of them C. theresae, is not sufficient to define C. theresae as a reservoir animal of this novel virus. However, the unique position of TGNV in evolutionary trees supports the idea that a shrew instead of a rodent is the natural host of TGNV. Therefore, it is rather surprising that TGNV did not form a monophyletic group with TPMV. Before this observation becomes either a challenge or support for the hantavirus-host coevolution concept, more extensive sequence data (for comprehensive phylogenetic analysis) and epizootiologic studies (to confirm the natural hosts of both viruses) are necessary.

TGNV represents, after the recently described SANGV (5), a second hantavirus from Africa. Its low sequence similarity to other hantaviruses should make this virus serologically distinct from other hantaviruses, as shown for TPMV (10). Therefore, human infections by TGNV might be missed when using antibody detection assays based on antigens from conventional hantaviruses.

Acknowledgments

We thank Bernard Allali, Amadou Dore, Kekoura Koulemou, Fode Kourouma, Barre Soropogui, Thomas Strecker, and Oumar Sylla for help in small mammal trapping. We also thank Christiane Denys for her assistance. Trapping was authorized by the Ministry of Public Health, Republic of Guinea (permission no. 2003/PFHG/05/GUI).

This study was supported by Deutsche Forschungsgemeinschaft (grant KR 1293/2-4), the European Community (INCO-DEV grant ICA4-CT2002-10050, VIZIER project LSHG-CT-2004-511960, Marie Curie fellowship MEIF-CT2003506686), and the Howard Hughes Medical Institute.

References

(1.) Schmaljohn CS, Hjelle B. Hantavirnses: a global disease problem. Emerg Infect Dis. 1997;3:95-104.

(2.) Kruger DH, Ulrich R, Lundkvist A. Hantavirus infections and their prevention. Microbes Infect. 2001;3:1129-44.

(3.) Mertz GJ, Hjelle BL, Bryan RT. Hantavirus infection. Adv Intern Med. 1997;42: 369-421.

(4.) Schmidt HA, Strimmer K, Vingron M, von Haeseler A. TREE-PUZZLE: Maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics. 2002;18:502-4.

(5.) Klempa B, Fichet-Calvet E, Lecompte E, Auste B, Aniskin V, Meisel H, et al. Hantavirus in African wood mouse, Guinea. Emerg Infect Dis. 2006; 12:838-40.

(6.) Carey DE, Reuben R, Panicker KN, Shope RE, Myers RM. Thottapalayam virus: a presumptive arbovirus isolated from a shrew in India. Indian J Med Res. 1971;59:1758-60.

(7.) Zeller HG, Karabatsos N, Calisher CH, Digoutte JP, Cropp CB, Murphy FA, et al. Electron microscopic and antigenic studies of uncharacterized viruses. II. Evidence suggesting the placement of viruses in the family Bunyaviridae. Arch Virol. 1989;108:211-27.

(8.) Xiao SY, Leduc JW, Chu YK, Schmaljohn CS. Phylogenetic analyses of virus isolates in the genus Hantavirus, family Bunyaviridae. Virology. 1994; 198:205-17.

(9.) Hutterer R. Order Soricomorpha. In: Wilson DE, Reeder DM, editors. Mammal species of the world: A taxonomic and geographic reference, 3rd ed, vol. 1. Baltimore: Johns Hopkins University Press; 2005. p. 220-311.

(10.) Chu YK, Jennings G, Schmaljohn A, Elgh F, Hjelle B, Lee HW, et al. Cross-neutralization of hantaviruses with immune sera from experimentally infected animals and from hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome patients. J Infect Dis. 1995;172:1581-4.

[1] Current affiliation: Leiden University Medical Center, Leiden, the Netherlands ([section]) Philipps University, Marburg, Germany; ([paragraph]) Severtsov Institute of Ecology and Evolution, Moscow, Russia; # Universite de Rennes 1, Paimpont, France; and ** Viral Hemorrhagic Fever Project, Conakry, Guinea

Boris Klempa, * ([dagger]) Elisabeth Fichet-Calvet, ([double dagger]) Emilie Lecompte, ([section]) Brita Auste, * Vladimir Aniskin, ([paragraph]) Helga Meisel, * Patrick Barriere, # Lamine Koivogui, ** Jan ter Meulen, ([section]) [1] and Detlev H. Kruger *

* University Hospital Charite, Berlin, Germany; ([dagger]) Slovak Academy of Sciences, Bratislava, Slovak Republic; ([double dagger]) Museum National d'Histoire Naturelle, Paris, France;

Address for correspondence: Detlev H. Kruger, Institute of Virology, Helmut-Ruska-Haus, University Hospital Charite, Campus Charite Mitre, Chariteplatz 1, D-10117 Berlin, Germany; email: [e-mail]