Patrick Barrière

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). Figure Maximum likelihood phylogenetic analysis of hantaviruses showing the phylogenetic placement of Tan826 (Tanganya virus, indicated by arrow) based on partial L segment nucleotide (A) and amino acid (B) sequences and partial S segment amino acid sequences ... 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°00′02″N, 10°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). 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). 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 (≈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.

Identification of Ebola virus sequences present as RNA or DNA in organs of terrestrial small mammals of the Central African Republic.

The life cycle of the Ebola (EBO) virus remains enigmatic. We tested for EBO virus in the organs of 242 small mammals captured during ecological studies in the Central African Republic. EBO virus glycoprotein or polymerase gene sequences were detected by reverse transcription PCR in RNA extracts of the organs of seven animals and by PCR in DNA extract of one animal. Neither live virus nor virus antigen was detected in any organ sample. Direct sequencing of amplicons identified the virus as being of the Zaire/Gabon subtype. Virus-like nucleocapsids were observed by electron microscopy in the cytoplasm of the spleen cells of one animal. The animals belonged to two genera of rodents (Muridae; Mus setulosus, Praomys sp1 and P. sp2) and one species of shrew (Soricidae; Sylvisorex ollula). These preliminary results provide evidence that common terrestrial small mammals living in peripheral forest areas have been in contact with the EBO virus and demonstrate the persistence of EBO virus RNA and DNA in the organs of the animals. Our findings should lead to better targeting of research into the life cycle of the EBO virus.

Biogeographic origin and radiation of the Old World crocidurine shrews (Mammalia: Soricidae) inferred from mitochondrial and nuclear genes

The crocidurine shrews include the most speciose genus of mammals, Crocidura. The origin and evolution of their radiation is, however, poorly understood because of very scant fossil records and a rather conservative external morphology between species. Here, we use an alignment of 3560 base pairs of mitochondrial and nuclear DNA to generate a phylogenetic hypothesis for the evolution of Old World shrews of the subfamily Crocidurinae. These molecular data confirm the monophyly of the speciose African and Eurasian Crocidura, which also includes the fossorial, monotypic genus Diplomesodon. The phylogenetic reconstructions give further credit to a paraphyletic position of Suncus shrews, which are placed into at least two independent clades (one in Africa and sister to Sylvisorex and one in Eurasia), at the base of the Crocidura radiation. Therefore, we recommend restricting the genus Suncus to the Palaearctic and Oriental taxa, and to consider all the African Suncus as Sylvisorex. Using molecular dating and biogeographic reconstruction analyses, we suggest a Palaearctic-Oriental origin for Crocidura dating back to the Upper Miocene (6.8 million years ago) and several subsequent colonisations of the Afrotropical region by independent lineages of Crocidura.

Terrestrial small mammals as reservoirs of Mycobacterium ulcerans in Benin

ABSTRACT Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is considered an environmental pathogen. Different mycobacteria were detected in 68 (12%) out of 565 small mammals collected in areas in Benin where BU is endemic. Although M. ulcerans was not found, we suggest that more research on M. ulcerans in African (small) mammals is needed.

Impact of removal pitfall trapping on the community of shrews (Mammalia : Soricidae) in two African tropical forest sites.

Removal trapping is still used for the study of tropical African shrews biodiversity and ecology, because shrew species identification requires cranio-dental analyses due to the existence of sibling species. Pitfall trapping has been found to be the most effective protocol to collect shrews. However, the impact of removal pitfall-trapping on density and diversity of shrew species is still unknown. In this paper, we test this impact on two African tropical forest shrew communities, by comparing the results of two trapping sessions conducted in two consecutive years. Our results support the view that removal trapping, with conditions described in this paper, does not adversely affect local population numbers and shrew species richness.

Species composition, abundance and vertical stratification of a bat community (Megachiroptera: Pteropodidae) in a West African rain forest

A pteropodid bat community was surveyed in a West African rain forest using mist-nets set for 3 mo in 2 consecutive years (1996-97). The captures were carried out in the understorey and in the canopy for a total of 5054 mist-net hours and were analysed to answer three main questions: (1) to what extent does the bat assemblage vary along the vertical axis, (2) does the observed vertical stratification depend on wingspan (with largest bats preferring canopy openings to the cluttered understorey) and (3) was the vertical stratification repeatable from one year to the next? Nine bat species were reported, among which two were captured exclusively in the canopy where the total capture rate tended to be higher. The community structure did not differ between the four canopy stations. Four species significantly favoured canopy, two significantly favoured understorey and two were opportunistic regarding vertical stratification. Wing size and canopy-preference index were not significantly correlated and except for Eidolon helvum , index values for the 2 sampling years were correlated. Important inter-annual and seasonal variations of species richness and capture rate were observed and these are discussed in relation to rainfall patterns and migratory behaviour of the species concerned.

First results on the feeding ecology of sympatric shrews (Insectivora: Soricidae) in the Tai National Park, Ivory Coast

The feeding ecology of a multi-species community of shrews inhabiting secondary forest and cacao-coffee plantations in the Tai National Park (Ivory Coast) was investigated. A total of 553 shrews were captured and 194 alimentary tracts were examined. Ten species were found, includingSylvisorex megalura and nine species ofCrocidura, forming a series with respect to body size. New ecological data on these little known African species are presented. All species of shrews ate a wide diversity of arthropods, with Coleoptera, Araneae, Formicidae and Diplopoda making the largest dietary contributions. Lumbricidae were eaten by two species.C. obscurior had an exceptionally long intestine for its size but there was no evidence of dietary specialisation in this or other shrew species. All species investigated ate predominantly small prey and there was no correlation between size of prey items consumed and body mass of shrew species. There was little evidence of resource partitioning amongst the shrews, despite differences in body size.

MORPHOMETRIC VARIATION IN HYLOMYSCUS ALLENI AND H. STELLA (RODENTIA: MURIDAE), AND DESCRIPTION OF A NEW SPECIES

Abstract We compared 7 populations of woodmice, Hylomyscus stella (Thomas, 1911), from west-central, east-central, and east Africa using traditional morphometric data of the cranium. Our results are congruent with previous molecular and cytogenetic data, and demonstrate that specimens previously identified as H. stella represent 2 cryptic species: H. stella from east-central and east Africa, and Hylomyscus sp. nov. from west-central Africa. According to current knowledge, the new species of Hylomuscus is a forest-dwelling species inhabiting the region between the Sanaga River and the Oubangui and Congo rivers in Cameroon, Central African Republic, Gabon, and Republic of the Congo. It is sympatric, and even syntopic, with the morphologically closely related species H. alleni. These 2 cryptic species can be distinguished by traditional morphometric analysis of the cranium and by examination of molecular data.

A new species of Sylvisorex (Mammalia: Soricidae) from lowland forests north of Kisangani, Democratic Republic of Congo

Abstract We describe a new species of Sylvisorex (Soricidae) from the northern part of the Congo Basin. Specimens were recently found in three lowland forests on the right bank of the Congo River: Masako, Yelenge and Baliko. The new species, Sylvisorex akaibei n. sp., is unusual in its external morphology, having a short tail of which the basal part is covered by long bristle hairs. The skull is a small version of that of Sylvisorex lunaris, a species endemic to the mountains of the Albertine Rift; however, the two species are not closely related. The new discovery shows that the small mammal fauna of the lowland forests of the Congo Basin is more diverse than previously expected.

Microgeographical distribution of shrews (Mammalia, Soricidae) in the Congo River basin (Kisangani, D.R. Congo)

Abstract Research on the biodiversity of shrews was conducted in eight forest blocks at eight sampling localities: Djabir, Maiko, Masako, Yoko, Yelenge, Baliko, Bomane-1 and Bomane-2. We used pitfall traps combined with Sherman LFA traps placed on transects. We collected 724 shrews from primary forests, secondary forests, old fallow lands and old palm plantation for a total of 18,900 trap-nights. Shrews collected represent 5 genera and at least 19 species: Scutisorex (1 species), Crocidura (12 species), Paracrocidura (2 species), Sylvisorex (3 species) and Suncus (1 species). Sylvisorex nov. sp. is currently being described as a new species for the Kisangani region. Primary forests (16 species), secondary forests (17 species) and old fallow lands (16 species) are the habitats with higher species diversity. In an old palm plantation, only five species have been collected. Crocidura olivieri was caught in all types of prospected habitats. C. cf muricauda was only caught in old palm plantation and C. grassei only in old fallow land. Sylvisorex johnstoni was caught mainly in primary forest. Species composition is different for the different forest blocks studied, possibly indicating some influence of the larger rivers separating our sampling localities as natural barriers.