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.Keywords: Hantavirus, Bat, Phylogeny, Côte d'Ivoire, Africa FindingsDiscovery of phylogenetically divergent hantaviruses in shrews and moles (order Soricomorpha, family Soricidae and Talpidae) [1][2][3][4][5][6][7][8][9][10][11][12][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][15][16][17][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 FirstStrand 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 rodentand soricomorph-borne hantaviruses [2][3][4][5][6][7][8][9][10][11][12][13]19,20]. Each reaction mixture contained 250 μ dNTP, 2 mM MgCl 2 , 1 U Ampl...

BackgroundTanganya virus (TGNV), the only shrew-associated hantavirus reported to date from sub-Saharan Africa, is harbored by the Therese's shrew (Crocidura theresae), and is phylogenetically distinct from Thottapalayam virus (TPMV) in the Asian house shrew (Suncus murinus) and Imjin virus (MJNV) in the Ussuri white-toothed shrew (Crocidura lasiura). The existence of myriad soricid-borne hantaviruses in Eurasia and North America would predict the presence of additional hantaviruses in sub-Saharan Africa, where multiple shrew lineages have evolved and diversified.MethodsLung tissues, collected in RNAlater®, from 39 Buettikofer's shrews (Crocidura buettikoferi), 5 Jouvenet's shrews (Crocidura jouvenetae), 9 West African pygmy shrews (Crocidura obscurior) and 21 African giant shrews (Crocidura olivieri) captured in Côte d'Ivoire during 2009, were systematically examined for hantavirus RNA by RT-PCR.ResultsA genetically distinct hantavirus, designated Azagny virus (AZGV), was detected in the West African pygmy shrew. Phylogenetic analysis of the S, M and L segments, using maximum-likelihood and Bayesian methods, under the GTR+I+Γ model of evolution, showed that AZGV shared a common ancestry with TGNV and was more closely related to hantaviruses harbored by soricine shrews than to TPMV and MJNV. That is, AZGV in the West African pygmy shrew, like TGNV in the Therese's shrew, did not form a monophyletic group with TPMV and MJNV, which were deeply divergent and basal to other rodent- and soricomorph-borne hantaviruses. Ancestral distributions of each hantavirus lineage, reconstructed using Mesquite 2.74, suggested that the common ancestor of all hantaviruses was most likely of Eurasian, not African, origin.ConclusionsGenome-wide analysis of many more hantaviruses from sub-Saharan Africa are required to better understand how the biogeographic origin and radiation of African shrews might have contributed to, or have resulted from, the evolution of hantaviruses.

No abstract

The hypothesis of Pleistocene forest refugia was tested using comparative phylogeography of Scotonycterini, a fruit bat tribe endemic to Africa containing four species: Scotonycteris zenkeri, Casinycteris argynnis, C. campomaanensis, and C. ophiodon. Patterns of genetic structure were assessed using 105 Scotonycterini (including material from three holotypes) collected at 37 localities, and DNA sequences from the mitochondrial cytochrome b gene (1140 nt) and 12 nuclear introns (9641 nt). Phylogenetic trees and molecular dating were inferred by Bayesian methods. Multilocus analyses were performed using supermatrix, SuperTRI, and *BEAST approaches. Mitochondrial analyses reveal strong phylogeographical structure in Scotonycteris, with four divergent haplogroups (4.9-8.7%), from Upper Guinea, Cameroon, western Equatorial Africa, and eastern Democratic Republic of the Congo (DRC). In C. argynnis, we identify two mtDNA haplogroups corresponding to western and eastern Equatorial Africa (1.4-2.1%). In C. ophiodon, the mtDNA haplotypes from Cameroon and Ivory Coast differ by only 1.3%. Nuclear analyses confirm the validity of the recently described C. campomaanensis and indicate that western and eastern populations of C. argynnis are not fully isolated. All mtDNA clusters detected in Scotonycteris are found to be monophyletic based on the nuclear dataset, except in eastern DRC. In the nuclear tree, the clade from western Equatorial Africa is closely related to individuals from eastern DRC, whereas in the mitochondrial tree it appears to be the sister-group of the Cameroon clade. Migrate-n analyses support gene flow from western Equatorial Africa to eastern DRC. Molecular dating indicates that Pleistocene forest refugia have played an important role in shaping the evolution of Scotonycterini, with two phases of allopatric speciation at approximately 2.7 and 1.6 Mya, resulting from isolation in three main forest areas corresponding to Upper Guinea, Cameroon, and Equatorial Africa. Two cryptic species and two subspecies are described herein in the genus Scotonycteris. Female philopatry and male biased dispersal are supported for the smallest taxa, i.e., the three species of Scotonycteris and C. argynnis. The Congo, Ntem, and Sanaga rivers are identified as biogeographic barriers to the dispersal of Scotonycteris during interglacial periods. A greater capacity for long-distance dispersal is inferred for the largest species, C. ophiodon.

The recent discovery of genetically distinct hantaviruses in multiple species of shrews and moles prompted a further exploration of their host diversification by analyzing frozen, ethanol-fixed and RNAlater®-preserved archival tissues and fecal samples from 533 bats (representing seven families, 28 genera and 53 species in the order Chiroptera), captured in Asia, Africa and the Americas in 1981–2012, using RT-PCR. Hantavirus RNA was detected in Pomona roundleaf bats (Hipposideros pomona) (family Hipposideridae), captured in Vietnam in 1997 and 1999, and in banana pipistrelles (Neoromicia nanus) (family Vespertilionidae), captured in Côte d’Ivoire in 2011. Phylogenetic analysis, based on the full-length S- and partial M- and L-segment sequences using maximum likelihood and Bayesian methods, demonstrated that the newfound hantaviruses formed highly divergent lineages, comprising other recently recognized bat-borne hantaviruses in Sierra Leone and China. The detection of bat-associated hantaviruses opens a new era in hantavirology and provides insights into their evolutionary origins.

The Crocidura obscurior or West African pygmy shrew complex is endemic to West African forests from south‐eastern Guinea, eastern Liberia, southern Côte d'Ivoire and south‐western Ghana. We explore the genetic and morphometric diversity of 239 individuals of the C. obscurior complex from 17 localities across its geographical range. Using genetic data from three mitochondrial (16S, cytochrome b and COI) and four nuclear markers (BRCA1, STAT5A, HDAC2 and RIOK3) and skull geometric morphometrics, we show that this complex is composed of two cryptic and sympatric species, C. obscurior and C. eburnea. We then test several hypotheses to infer their evolutionary history. The observed phylogeographical pattern based on cytochrome b and COI sequences fits the forest refuge theory: during arid phases of the Plio‐Pleistocene, around 3.5, 2.1, 1 and 0.5 Mya, a small number of populations survived in isolated forest patches and diverged allopatrically. During wetter climatic periods, forests expanded, leading to secondary contacts between previously isolated populations. Our results also suggest the possible contribution of episodes of isolation in subrefuges. Historical variation of the West African hydrographic network could also have contributed to the observed patterns of genetic differentiation. Rivers such as the Volta and Sassandra may act as past and/or current barriers to gene flow. Although these two species have sympatric distributions, their phylogeographical histories are somewhat dissimilar due to small differences in their dispersal abilities and ecological requirements.

Both Ebolavirus and Marburgvirus were detected in several fruit bat species of the family Pteropodidae, suggesting that this taxon plays a key role in the life cycle of filoviruses. After four decades of Zaire Ebolavirus (ZEBOV) outbreaks in Central Africa, the virus was detected for the first time in West Africa in 2014. To better understand the role of fruit bats as potential reservoirs and circulating hosts between Central and West Africa, we examine here the phylogeny and comparative phylogeography of Pteropodidae. Our phylogenetic results confirm the existence of four independent lineages of African fruit bats: the genera Eidolon and Rousettus, and the tribes Epomophorini and Scotonycterini, and indicate that the three species suspected to represent ZEBOV reservoir hosts (Epomops franqueti, Hypsignathus monstrosus, and Myonycteris torquata) belong to an African clade that diversified rapidly around 8-7 Mya. To test for phylogeographic structure and for recent gene flow from Central to West Africa, we analysed the nucleotide variation of 675 cytochrome b gene (Cytb) sequences, representing eight fruit bat species collected in 48 geographic localities. Within Epomophorina, our mitochondrial data do not support the monophyly of two genera (Epomops and Epomophorus) and four species (Epomophorus gambianus, Epomops franqueti, Epomops buettikoferi, and Micropteropus pusillus). In Epomops, however, we found two geographic haplogroups corresponding to the Congo Basin and Upper Guinea forests, respectively. By contrast, we found no genetic differentiation between Central and West African populations for all species known to make seasonal movements, Eidolon helvum, E. gambianus, H. monstrosus, M. pusillus, Nanonycteris veldkampii, and Rousettus aegyptiacus. Our results suggest that only three fruit bat species were able to disperse directly ZEBOV from the Congo Basin to Upper Guinea: E. helvum, H. monstrosus, and R. aegyptiacus.

Members of the family Pteropodidae, also known as Old World fruit bats, are represented in Africa by 14 genera and 44 species. Here, we sequenced 67 complete mitochondrial genomes from African and Asian pteropodids to better understand the evolutionary history of the subfamily Rousettinae, which includes most of the African species. An increased frequency of guanine to adenine transitions is detected in the mtDNA genomes of Macroglossus sobrinus and all species of Casinycteris and Scotonycteris. Our phylogenetic and molecular dating analyses based on 126 taxa and 15,448 characters indicate a low signal for deep relationships within the family, suggesting a rapid diversification during the Late Oligocene period of “warming.” Within the subfamily Rousettinae, most nodes are highly supported by our different analyses (all nucleotide sites, SuperTRI analyses of a sliding window, transversions only, coding genes only, and amino acid sequences). The results indicate the existence of four tribes: Rousettini—distributed from Africa through Mediterranean region and South Asia to South‐East Asia; Eonycterini—found in Asia; and Epomophorini and Scotonycterini—restricted to sub‐Saharan Africa. Although most interspecies relationships are highly supported, three parts of the Rousettinae mitochondrial tree are still unresolved, suggesting rapid diversification: (a) among the three subtribes Epomophorina (Epomophorus sensu lato, i.e., including Micropteropus, Epomops, Hypsignathus, Nanonycteris), Plerotina (Plerotes), and Myonycterina (Myonycteris, Megaloglossus) in the Late Miocene; (b) among Epomops, Hypsignathus, and other species of Epomophorina at the Pliocene–Pleistocene boundary; and (c) among Myonycteris species in the Early Pleistocene. Within the Epomophorini, Stenonycteris lanosus emerged first, suggesting that lingual echolocation may have appeared in the common ancestor of Epomophorini and Rousettini. Our analyses suggest that multiple events of mtDNA introgression occurred within the Epomophorus species complex during the Pleistocene.