Mots-Clés: Talpa europaea, Talpa occidentalis, Hantavirus, NVAV, phylogénétique, phylogéographie. - PDF


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DIVERSITÉ GÉNÉTIQUE DE TALPA EUROPAEA ET DE L HANTAVIRUS NOVA (NVAV) EN FRANCE GENETIC DIVERSITY OF TALPA EUROPAEA AND NOVA HANTAVIRUS (NVAV) IN FRANCE Par Jean-Pierre HUGOT (1), Se Hun GU (2), Carlos FELIU (3), Jacint VENTURA (4), Alexis RIBAS (5), Jérôme DORMION (6), Richard YANAGIHARA (2) and Violaine NICOLAS (1) Communication présentée le 22 mai 2014 RÉSUMÉ L Hantavirus Nova (NVAV) a été identifié chez un spécimen de Talpa europaea capturé en Hongrie. L analyse de 94 spécimens de taupes capturés en France a révélé la présence de NVAV chez 50% des individus. Une étude populationnelle des taupes montre que les individus collectés entre Poitiers et Bordeaux sont génétiquement proches de l espèce voisine T. occidentalis, jusqu ici supposée être strictement endémique dans la péninsule ibérique. Plusieurs hypothèses permettant d expliquer ces observations sont discutées : 1) la présence jusqu ici ignorée de T. occidentalis dans le sud-ouest de la France ; 2) l existence d un ancien phénomène d introgression mitochondriale entre les deux espèces, ; 3) la présence d une zone d hybridation entre les deux espèces, produisant un phénotype particulier chez certains hybrides ; 4) l existence d une espèce nouvelle. NVAV n ayant été détecté chez aucun des spécimens du Sud-Ouest, la question de l existence d un Hantavirus particulier dans cette population et chez la taupe ibérique est posée. Mots-Clés: Talpa europaea, Talpa occidentalis, Hantavirus, NVAV, phylogénétique, phylogéographie. SUMMARY Nova hantavirus (NVAV) was first identified in a captured European mole (Talpa europaea) in Hungary. Analysis of lung tissues from 94 moles captured in France revealed NVAV in 50% of the animals. Based on the genetic diversity of the cytochrome b mtdna, moles collected in Poitiers and Bordeaux were more closely related to the Iberian mole (T. occidentalis), a species previously assumed to be restricted to the Iberian Peninsula. Several hypotheses are discussed to explain these observations: 1) the presence of hitherto unnoticed T. occidentalis in southwestern France; 2) the existence of an ancient mitochondrial introgression phenomenon between the two Talpa species, producing a particular phenotype in some hybrids; 3) the existence of a hybrid zone between the two species; and 4) the existence of a new Talpa species. NVAV was not detected in the southwestern moles, which raises the question of the possible presence of a particular Hantavirus species in this population and/or in the Iberian moles. Key-Words: Talpa europaea, Talpa occidentalis, Hantavirus, NVAV, phylogenetics, phylogeography. (1) UMR CNRS 7205, MNHN, 51, rue Buffon, Paris cedex, France. (2) Pacific Center for Emerging Infectious Diseases Research, John A. Burns School of Medicine, UNIVERSITY OF HAWAII AT MANOA, Honolulu, Hawaii, USA. (3) UNIV-BARCELONA, Facultad de Farmacia-Parasitología, Avda. Diagonal s/n 8028 Barcelona, Espagne. (4) UNIVERSITAT AUTÒNOMA DE BARCELONA, Unitat de Zoologia, Departament de Biologia Animal, Biologia Vegetal i Ecologia, Facultat de Biociències, Cerdanyola del Vallès, Espagne. (5) Biodiversity Research Group, Faculty of Science, UDON THANI RAJABHAT UNIVERSITY, Udon thani Thailand. (6) TAUP GREEN France, BP , Neuilly sur Seine, France. Bull. Acad. Vét. France Tome N INTRODUCTION Hantaviruses (family Bunyaviridae), long known to be harbored by rodents (rats, mice, voles, etc.) of the Muridae and Cricetidae families, have been detected in multiple species of shrews (Soricidae) and moles (Talpidae) (Yanagihara et al. 2014). More recently, highly divergent lineages of hantaviruses have also been discovered in insectivorous bats (Gu et al. 2014b; Guo et al. 2013; Yanagihara et al. 2014; table 1). In-depth studies of non-rodent hosts, which are heavily represented among micro-mammals all around the world, may provide a better understanding about their evolutionary origins. Since some hantaviruses may infect and cause diseases in humans, such studies may also interest epidemiologists (Pontier & Fouchet, 2013; Reynes, 2013; Tordo et al. 2013). Between October 2012 and March 2013, we sampled European moles, Talpa europaea, in various regions of France. The purpose of this investigation was: 1) to verify the presence of Nova hantavirus (NVAV), originally described in a single specimen of T. europaea from Hungary (Kang et al. 2009); 2) to assess the prevalence and genetic variability of NVAV; and 3) to compare NVAV genetic diversity with mole population genetics. The study revealed the presence of NVAV in France and the existence of a genetic variability of the virus linked to the localities where the moles were captured (Gu et al. 2014a). Analysis of the cytochrome b mtdna produced an unexpected finding: that is, although only one mole species (T. europaea) was considered to be present in France, some moles collected in the southwestern part of the country were genetically more closely related to T. occidentalis, a different species previously believed to be endemic to the Iberian Peninsula. Details of these results are presented and discussed. Research of hantavirus A subset of 94 moles captured either in the Golf of Ozoir-la-Ferrière ( N, E), or in the Beauvais agreement park called «Le Plan d'eau du Canada» ( N, E), were studied. We also analyzed 24 mole specimens collected between Bordeaux and Poitiers and genetically different from T. europaea. After capture, moles were frozen at -20 C; for tissue dissection, moles were partially thawed and lung tissues were removed using ethanol-cleaned instruments and placed in RNAlater RNA Stabilization Reagent (Qiagen Inc., Valencia, CA). Hantavirus detection and sequencing Total RNA was extracted from mole lung tissues, using the PureLink Micro-to-Midi total RNA purification kit (Invitrogen, San Diego, CA), and cdna was prepared using the SuperScript III First-Strand Synthesis System (Invitrogen) and random hexamers. PCR was performed as described previously using oligonucleotide primers designed from NVAV and other soricomorph-borne hantaviruses (Song et al. 2007; Arai et al. 2008) and DNA sequencing was performed using an ABI Prism 377XL Genetic Analyzer (Applied Biosystems, Foster City, CA). MATERIAL AND METHODS Collecting moles Approximately 350 moles were captured in different localities in France (figure 1) from October 2012 to March 2013, using Putange traps and following a protocol approved by French professional mole catchers (Dormion, 2012). Figure 1. Map of collection localities. Map of France, showing localities where European moles were captured during October 2012 to March The results presented in this manuscript were obtained from specimens collected in the localities indicated in red (Blanquefort, St-Loubès, Sadirac, Ambarès, Arcins and Izon are small cities situated on either side of the Gironde estuary, in the vicinity of Bordeaux). 278 Bull. Acad. Vét. France Tome N 3 Virus name Logo Host species Host family Biorealm Country Collection year Publication year Genbank 1 Thottapalayam TPMV Suncus murinus Soricidae Oriental India AY Tanganya TGNV Crocidura theresae Soricidae Ethiopian Guinea EF Cao Bang CBNV Anourosorex squamipes Soricidae Oriental Vietnam EF Ash River ARRV Sorex cinereus Soricidae Nearctic USA EF Amga MGAV Sorex caecutiens Soricidae Palearctic Russia Jemez Spring JMSV Sorex monticolus Soricidae Nearctic USA FJ Kenkeme KKMV Sorex roboratus Soricidae Palearctic Russia GQ Kathmandu TPMV Suncus murinus Soricidae Oriental Nepal HQ Imjin MJNV Crocidura lasiura Soricidae Palearctic South Korea EF Qian Hu Shan QHSV Sorex cylindricauda Soricidae Palearctic China GU Asagny AZGV Crocidura obscurior Soricidae Ethiopian Côte d'ivoire JF Longwan TPMV Suncus murinus Soricidae Palearctic China JF Uluguru ULUV Myosorex geata Soricidae Ethiopian Tanzania JX Kilimanjaro KMJV Myosorex zinki Soricidae Ethiopian Tanzania JX Seewis SWSV Sorex araneus Soricidae Palearctic Switzerland EF Jeju JJUV Crocidura shantungensis Soricidae Palearctic South Korea HQ Bowé BOWV Crocidura douceti Soricidae Ethiopian Guinea KC Lianghe CBNV Anourosorex squamipes Soricidae Palearctic China JX Yakeshi YKSV Sorex isodon Soricidae Palearctic China JX Asikkala ASIV Sorex minutus Soricidae Palearctic Germany KC Boginia BOGV Neomys fodiens Soricidae Palearctic Poland Asama ASAV Urotrichus talpoides Talpidae Palearctic Japan EU Nova NVAV Talpa europaea Talpidae Palearctic Hungary FJ Oxbow OXBV Neurotrichus gibbsii Talpidae Nearctic USA FJ Dahonggou Creek DHCV Scaptonyx fusicaudus Talpidae Palearctic China HQ Rockport RKPV Scalopus aquaticus Talpidae Nearctic USA HM Magboi MGBV Nycteris hispida Nycteridae Ethiopian Sierra Leone Mouyassué MOYV Neoromicia nanus Vespertilionidae Ethiopian Côte d'ivoire Xuan Son XSV Hipposideros pomona Hipposideridae Oriental Vietnam KF Longquan LQUV Rhinolophus spp Rhinolophidae Palearctic China JX Huangpi HUPV Pipistrellus abramus Vespertilionidae Palearctic China JX Table 1: Checklist of the hantaviruses hosted by Soricomorpha and Chiroptera. Since the report of Thottapalayam virus in 1971, more than 30 other hantavirus strains have been detected either from Soricomorpha (Soricidae, Talpidae) or Chiroptera, from: the Palearctic (19), Nearctic (5), Oriental (3) or Ethiopian (7) Biorealms, respectively. Accession numbers in GenBank are given when the short gene sequence (S) of the corresponding hantavirus strain is available. Bull. Acad. Vét. France Tome N Mitochondrial DNA sequencing DNA was extracted from ethanol-preserved tissues by the CTAB method (Winnepenninckx et al., 1993). The cytochrome b gene was amplified from 40 mole specimens using polymerase chain reaction (PCR) primers L14723 and H15915 (Ducroz et al. 2001; Nicolas et al. 2008). Sequence alignment Alignment of the coding region of the hantavirus S segment, or the cytochrome b of the moles was performed using CLUS- TAL-X automatic procedure (Thompson et al. 1994) then improved manually using SEAL v2.0a11 (Rambaut, 1996) and validated using the amino acid translation. Phylogenetic analyses Evolutionary relationships among sequences were estimated by constructing phylogenetic trees using maximum parsimony (MP) and Bayesian Markov chain Monte Carlo phylogenetic analyses (MCMC). MP analysis was performed with PAUP 4b10 (Swofford, 2001) and Bayesian analysis with MrBayes (Huelsenbeck and Ronquist, 2001). The MP analysis was performed with tree-bisection-reconnection (TBR) branch swapping option and 10 random addition replicates. We estimated the robustness of internal nodes by 1,000 bootstrapping replicates (each with a single replication of random addition of taxa). An equal weighting of character-state transformations was applied. In all MCMC analyses three heated chains and one single cold chain were employed, and runs were initiated with random trees. Two independent MCMC runs were conducted with six million generations per run; trees (and parameters) sampled every 100 generations. Stationarity was assessed by: examining the average standard deviation of split frequencies and, the Potential Scale Reduction Factor (Ronquist et al. 2005). For each run, the first 25% of sampled trees were discarded as burn-in. A consensus tree was computed using the halfcompat option, equivalent to the 50% majority rule and rooted using the midpoint root option. Proportion values of posterior probability of bipartition were used for evaluation of robustness of the nodes. RESULTS Prevalence of NVAV in moles Hantavirus RNA was detected in 47 moles (50%) by RT-PCR. Of 36 and 28 moles captured in Ozoir-la-Ferrière on October 18, 2012 and February 21, 2013, 15 and 14, respectively, were positive, while 18 of 30 moles captured in Beauvais on March 3, 2013 were positive (Gu et al. 2014a). NVAV was not detected in the 24 southwestern moles. Phylogenetic analysis of hantavirus found in micro-mammals Phylogenetic analysis, using Bayesian methods (figure 2) showed that: 1) NVAV from France and prototype NVAV from Hungary grouped together in a particular lineage; 2) NVAV strains from France segregated along geographic-specific lineages; 3) NVAV clade was strongly associated with another clade, comprising hantaviruses detected in insectivorous bats; 4) other hantaviruses hosted by soricomorphs were distributed into two separate clades: i) the most divergent included seven strains from shrews captured in India, South Korea, China and Tanzania; ii) the second clade included strains from moles and shrews captured in North America, China, Japan and Europe, and associated with hantaviruses hosted by Murinae rodents; 5) two other clades grouping hantaviruses hosted by Sigmodontinae-Neotominae and Arvicolinae rodents, respectively; 6) a single mole-borne hantavirus, Rockport virus from the eastern mole in North America, was situated between the Sigmodontinae and Arvicolinae clades. All the basal nodes of the cladogram had the maximum posterior probability of bipartition value (= 1). Genetic affinities within the Family Talpidae Phylogenetic analysis, using Bayesian methods, of all available sequences from Talpidae species (figure 3) showed that: 1) the genus Talpa may be considered a monophyletic group, in which each different species is recognized as a distinct clade; 2) T. europaea and T. occidentalis are sister taxa; 3) specimens captured in Brittany, Northern France and Saôneet-Loire clustered with strains identified as T. europaea and collected from different European countries (Sweden, Denmark, Switzerland, Hungary, Germany, Italy and Greece); 4) specimens captured in the vicinity of Bordeaux, or Poitiers, clustered with specimens captured either from Spain or Portugal and identified as T. occidentalis, the Iberian mole. All the nodes of the cladogram corresponding to a particular mole species had the maximum posterior probability of bipartition value (= 1). DISCUSSION Mole infection Prevalence of anti-viral antibodies and/or viral antigens in rodent species known to harbor disease-causing hantaviruses generally varies from less than 1% to more than 25%, depending on seasonal factors, reservoir population density, geographical area and ecological diversity (Lee et al.1978; Dobly et 280 Bull. Acad. Vét. France Tome N 3 COMMUNICATION Figure 2: Phylogeny of hantaviruses hosted by Chiroptera, Soricomorpha or Rodents. Cladogram resulting of Bayesian (MCMC) analysis (GTR+I+G model), based on the entire coding region of the S gene (nucleotides ). Hantaviruses hosted by different host groups are highlighted in: red (SigmodontinaeNeotominae), green (Arvicolinae), blue (Murinae), pink (Soricidae), light brown (Talpidae) and yellow (Chiroptera). The analysis used 1) an extent data set of the S gene coding region available on GenBank, including most of the hantavirus strains detected either in rodents or non-rodent mammals (Soricomorpha and Chiroptera), and 2) 11 complete S-segment sequences from moles captured either in Ozoir (4) or Bauvais (7). GenBank accession numbers: FJ539168, NVAV from Hungary; KF KF010576, NVAV strains from Ozoir-la-Ferrière; KF KF010571, NVAV strains from Beauvais. For Chiroptera and Soricomorpha hosts, see Table 1; for rodent hosts, see Herbreteau et al., 2007, Table Figure 3. Phylogenetic analysis of Talpa spp. and closely related taxa Bayesian (MCMC) analysis (GTR+I+G model) based on the entire coding region of the cytochrome b gene. The analysis was rooted using as outgroups several taxa included within the Talpidae: Urotrichus, Dymecodon, Mogera, Euroscaptor, Uropsilus, Desmana and Galemys. All the sequences available in GenBank for these taxa or for Talpa spp. were included. The tip labels indicate the GeneBank accession number and the name of the species (for the Talpa spp.), the name of the genus for the other taxa. Our own mole samples are labeled using the field collection number + the name of the locality of capture and highlighted in red. Bull. Acad. Vét. France Tome N al. 2012). Recently, a similarly high prevalence of NVAV infection, as evidenced by RT-PCR and confirmed by DNA sequencing, has been found in European moles captured in central Poland, suggesting that NVAV is widespread throughout the vast distribution of T. europaea (Gu et al. 2014a). The high prevalence of this hantavirus suggests that the mechanisms of transmission between individuals are very efficient. NVAV was not detected in the southwestern moles, but few specimens (24) were available and the RNA quality was suboptimal. Thus, future studies are warranted to ascertain if NVAV or NVAV-related hantaviruses are harbored by moles in southwestern France. These investigations will be extended to mole specimens collected from different parts of the Iberian Peninsula. Until now no human infection due to NVAV has been recorded. However, moles have a high propensity to occupy arable fields, deciduous woodland and permanent pastures, and hantavirus are known to be able to survive for prolonged periods (12-15 days) in external environment (Kallio et al. 2006; Hardestam et al. 2007). Any unusual clinical syndromes, recorded among individuals reporting contact with European moles, should be thoroughly studied by physicians and public health workers. Host and hantavirus coevolution Hantaviruses were traditionally considered to have codiverged (co-speciatiated) with their rodent hosts (Herbreteau et al. 2006, 2007; Hentonnen et al. 2008; Guo et al. 2013; Yanagihara et al. 2014) and some evidence for such codivergence is apparent here. In particular, rodent-borne hantaviruses clustered according to whether their hosts were members of the Murinae, Arvicolinae or Sigmodontinae-Neotominae subfamilies. However, codivergence alone cannot explain the clustering of hantaviruses hosted by Soricomorpha and Chiroptera, which exist in several paraphyletic clades with no correspondence between the association of the viruses, their geographic origin and/or host taxonomy (figure 2; Tordo et al. 2013). Presence of genetically distinct moles in France Identification of two genetically divergent mole lineages in France was unexpected. Until now, only T. europaea was considered to be present in France. In the Iberian Peninsula, the situation is quite different and two species have been recorded for a long time. T. europaea is present in Northeast Spain (Catalunya, Navarra and the Eastern part of Pais Vasco) while the closely related T. occidentalis is present in the Northwest and South of Spain and in Portugal (Palomo et al. 2007). To explain this presence of mitochondrial haplotypes related to T. occidentalis in the Southwest of France, the simplest hypothesis is to admit that T. occidentalis was able to cross the mountains, leaving Spain and partly colonizing a territory extending from the Pyrenees to the river Loire. However, this hypothesis doesn t fit well with the comparison of the morphology of the different populations. Table 2 shows that: i) if the measurements of the specimens clustering with the other European moles fit with the values attributed to T. europaea (Palomo et al. 2007; Aulagnier et al. 2008); conversely, ii) the comparison of the measurement of the southwestern mole specimens with the values attributed to T. occidentalis (Palomo et al. 2007; Aulagnier et al. 2008) are incompatible; Weight H & Body Tail Foot Head MAX 118,0 166,0 40,1 30,8 49,0 MIN 48,0 122,8 14,0 16,6 31,6 MOY 78,0 145,5 26,8 20,8 43,6 330 moles from different sites in France Talpa europaea MAX 130,0 165,0 51,0 25,0 38,0 MIN 36,0 100,0 20,0 16,0 30,0 MOY 83,0 132,5 35,5 20,5 34,0 Weight H & Body Tail Foot Head MAX 147,0 165,0 35,0 26,0 49,0 MIN 69,0 141,0 19,0 19,0 39,0 MOY 92,5 153,2 27,5 21,3 45,8 24 moles from Southwest of France Talpa occidentalis MAX 70,0 135, ,0 32,0 MIN 30,0 90, ,0 28,0 MOY 50,0 112,5 25,5 16,0 30,0 Table 2: Comparison of the measurements of the moles captured in France, following their genetic affinities The measure
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