«Viruses 2014, 6, 1897-1910; doi:10.3390/v6051897 OPEN ACCESS viruses ISSN 1999-4915 Short Communication Molecular ...»
Viruses 2014, 6, 1897-1910; doi:10.3390/v6051897
Molecular Phylogeny of Hantaviruses Harbored by
Insectivorous Bats in Côte d’Ivoire and Vietnam
Se Hun Gu 1, Burton K. Lim 2, Blaise Kadjo 3, Satoru Arai 4, Jeong-Ah Kim 5, Violaine Nicolas 6,
Aude Lalis 6, Christiane Denys 6, Joseph A. Cook 7, Samuel R. Dominguez 8,
Kathryn V. Holmes 8, Lela Urushadze 9,10, Ketevan Sidamonidze 9, Davit Putkaradze 9, Ivan V. Kuzmin 11, Michael Y. Kosoy 12, Jin-Won Song 5 and Richard Yanagihara 1,* Pacific Center for Emerging Infectious Diseases Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA; E-Mail: firstname.lastname@example.org Department of Natural History, Royal Ontario Museum, Toronto, ON M5S 2C6, Canada;
E-Mail: email@example.com Department of Biology, Universitéde Cocody, Abidjan 22, Côte d’Ivoire;
E-Mail: firstname.lastname@example.org Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Tokyo 162-8640, Japan; E-Mail: email@example.com Department of Microbiology, College of Medicine, Korea University, Seoul 136-705, Korea;
E-Mails: firstname.lastname@example.org (J.-A.K.); email@example.com (J.-W.S.) Departement Systematique et Evolution, UMR CNRS 7205, Muséum National d’Histoire Naturelle, Paris 75005, France; E-Mails: firstname.lastname@example.org (V.N.); email@example.com (A.L.);
firstname.lastname@example.org (C.D.) Department of Biology, Museum of Southwestern Biology, University of New Mexico, Albuquerque, NM 87131, USA; E-Mail: email@example.com Department of Pediatrics, School of Medicine, University of Colorado, Aurora, CO 80045, USA;
E-Mails: firstname.lastname@example.org (S.R.D.); Kathryn.Holmes@ucdenver.edu (K.V.H.) National Center for Disease Control and Public Health, Tbilisi 0177, Georgia;
E-Mails: email@example.com (L.U.); firstname.lastname@example.org (K.S.);
email@example.com (D.P.) Institute of Chemical Biology, Ilia State University, Tbilisi 0162, Georgia;
E-Mail: firstname.lastname@example.org Global Alliance for Rabies Control, Manhattan, KS 66502, USA;
E-Mail: email@example.com Division of Vector Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521, USA; E-Mail: firstname.lastname@example.org * Author to whom correspondence should be addressed; E-Mail: email@example.com;
Tel.: +1-808-692-1610; Fax: +1-808-692-1976.
Viruses 2014, 6 1898 Received: 24 February 2014; in revised form: 4 April 2014 / Accepted: 8 April 2014 / Published: 29 April 2014 Abstract: 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 batborne 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.
Keywords: hantavirus; Chiroptera; evolution
1. Introduction Hantaviruses (genus Hantavirus, family Bunyaviridae) possess a negative-sense, single-stranded, tripartite segmented RNA genome, consisting of large (L), medium (M) and small (S) segments, encoding an RNA-dependent RNA polymerase (RdRp), envelope glycoproteins (Gn and Gc) and a nucleocapsid (N) protein, respectively . To date, 23 hantaviruses, hosted by reservoir rodent species, have been recognized as distinct species by the International Committee on Taxonomy of Viruses .
Several of these rodent-borne hantaviruses cause acute, febrile diseases of varying clinical severity and lethality in humans, known as hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome . Though once believed to be restricted to rodents (order Rodentia, family Muridae and Cricetidae), the reservoir host range of hantaviruses is far more expansive, as evidenced by the detection of divergent lineages of hantaviruses in multiple species of shrews and moles (order Soricomorpha, family Soricidae and Talpidae) throughout Asia, Europe, Africa and North America [4–19].
Despite their phylogenetic relatedness to the European mole (Talpa europaea) within the Laurasiatheria [20,21], as well as their rich genetic diversity, vast geographic range and ability to host many disease-causing viruses [22–24], bats (order Chiroptera) have not been extensively studied as potential reservoirs of hantaviruses. Although serological evidence of hantavirus infection was reported in the common serotine (Eptesicus serotinus) and greater horseshoe bat (Rhinolophus ferrumequinum) captured in Korea , genetic analysis of hantavirus isolates from these bat species suggested laboratory contamination .
The genetic diversity of newfound hantaviruses recently detected in insectivorous bats preclude any possibility of contamination: Mouyassuévirus (MOYV) in the banana pipistrelle (Neoromicia nanus) from Côte d’Ivoire ; Magboi virus (MGBV) in the hairy slit-faced bat (Nycteris hispida) from Viruses 2014, 6 1899 Sierra Leone ; Xuan Son virus (XSV) in the Pomona roundleaf bat (Hipposideros pomona) from Vietnam ; Huangpi virus (HUPV) in the Japanese house bat (Pipistrellus abramus) and Longquan virus (LQUV) in the Chinese horseshoe bat (Rhinolophus sinicus), Formosan lesser horseshoe bat (Rhinolophus monoceros) and intermediate horseshoe bat (Rhinolophus affinis) from China . The primary goal of this multi-national collaborative study was to extend the search for hantaviruses in bats and to obtain more of the MOYV and XSV genomes. Our data indicate that bat-borne hantaviruses and Nova virus, a hantavirus hosted by the European mole, comprise a highly divergent phylogenetic lineage, suggesting that ancestral bats and/or soricomorphs, rather than rodents, may have served as the early reservoir hosts of primordial hantaviruses.
2. Results and Discussion
2.1. Hantavirus Detection and Sequence Analysis Exhaustive attempts to detect hantaviruses were unsuccessful in nearly all of the 454 bat tissue samples (Table 1 and Figure 1), despite employing oligonucleotide primers and PCR cycling conditions used to find MOYV  and XSV . In addition, hantavirus RNA was not detected in any of the 79 rectal swab and fecal samples. Because LQUV was previously found in four species of horseshoe bats in China , we expected to find the same or a similar hantavirus in the greater horseshoe bat, captured on Jeju Island in Korea. However, this was not the case, in spite of using LQUVspecific primers. Nevertheless, we did manage to obtain more of the MOYV and XSV genomes. That is, the original report of MOYV in the banana pipistrelle (Figure 2A,B) was based on a 423-nucleotide region of the L segment . Through repeated trial-and-error efforts, suitable primers were designed to obtain an additional 1268 nucleotides of the L segment (Table 2).
In addition, Arai and colleagues previously reported a novel hantavirus, designated XSV, in one of five Pomona roundleaf bats, captured during July 2012 in Xuan Son National Park in Phu Tho province in northern Vietnam . In analyzing archival kidney tissues from 44 Pomona roundleaf bats trapped in Tuyê Quang and Quang Nam provinces, hantavirus L-segment sequences were detected n in five animals (Figure 2C,D). Although a 15.7%–19.2% difference was found at the nucleotide level with prototype XSV, the high amino acid sequence similarity was consistent with these sequences representing genetic variants of XSV. Pair-wise alignment and comparison of the full-length S segment of XSV, amplified and sequenced from four bats (Table 2), indicated sequence similarity of 58.9%–60.3% at the amino acid level with LQUV, the only other bat-borne hantavirus for which the entire S segment has been sequenced. And sequence analysis of a 663-nucleotide (221 amino acid) region of the Gc envelope glycoprotein-encoding M segment showed that XSV differed by 45% from representative hantaviruses harbored by rodents and most soricomorphs. Collectively, the high level of sequence divergence in the N protein and Gc glycoprotein between XSV and other hantaviruses suggests that it might represent a new hantavirus species, using the guidelines proposed by Maes and co-workers .
However, the definitive taxonomic classification of XSV and other bat-borne hantaviruses must await their isolation in cell culture.
Viruses 2014, 6 1900
Figure 1. Geographic origin of 533 specimens from bats, belonging to seven families, were analyzed for hantavirus RNA, using RT-PCR.
The number of samples and genera and species of bats are shown for each country.
Figure 2. (A) Banana pipistrelle (Neoromicia nanus); (B) Map of Cote d’Ivoire, showing site where Mouyassuévirus-infected banana pipistrelles were captured during June 2011;
inset shows geographic distribution of banana pipistrelle; (C) Pomona roundleaf bat (Hipposideros pomona); (D) Map of Vietnam, showing Phu Tho, where Xuan Son virus (XSV) was first discovered, and Tuyê Quang and Quang Nam, where Pomona roundleaf n bats were captured in May 1997 and March 1999, respectively; (E) Comparison of the consensus secondary structures of the nucleocapsid protein of XSV, Longquan virus (LQUV), Nova virus (NVAV), Thottapalayam virus (TPMV), Imjin virus (MJNV), Hantaan virus (HTNV), Dobrava virus (DOBV), Seoul virus (SEOV), Puumala virus (PUUV), Sin Nombre virus (SNV) and Andes virus (ANDV), as predicted using methods available on the NPS@ structure server . Alpha helices are represented by blue bars, beta strands by red bars, and random coils and unclassified structures by magenta and gray bars, respectively.
Viruses 2014, 6 1902
2.2. Nucleocapsid Secondary Structure In employing software available on the @NPS structure server , the overall predicted secondary structures of the N proteins were similar. That is, despite the relatively low amino acid sequence similarity among the rodent-, shrew-, mole- and bat-borne hantaviruses, the N protein comprised two major α-helical domains packed against a central β-pleated sheet (Figure 2E). However, the central β-pleated sheet motif of XSV, including the RNA-binding region (amino acid positions 175 to 217), was unlike that of other hantaviruses, even that of LQUV, which more closely resembled murid rodent-borne hantaviruses, such as Hantaan virus (HTNV 76-118), Dobrava virus (DOBV Greece) and Seoul virus (SEOV 80-39) (Figure 2E). The distinctive α-helix motif between two β-strands of the Viruses 2014, 6 1903 RNA-binding region, observed in the prototype mole-borne hantavirus, Nova virus (NVAV MSB95703), as well as HTNV and SEOV, but not in LQUV, may have a significant effect on binding specificity.
2.3. Phylogenetic Analysis Phylogenetic analyses, based on S-, M- and L-genomic sequences, indicated that XSV and MOYV shared a common ancestry with other bat-borne hantaviruses (Figure 3). In all analyses, NVAV from the European mole segregated with the bat-associated hantaviruses, which was reminiscent of trees based on the complete mitochondrial genomes of the European mole and bats [20,21]. The basal position of chiropteran-borne hantaviruses and selected soricomorph-borne hantaviruses, such as Nova virus in the European mole, Thottapalayam virus in the Asian house shrew and Imjin virus in the Ussuri white-toothed shrew, in phylogenetic trees based on the S- and L-genomic sequences suggests that soricomorphs and/or chiropterans, rather than rodents, may have been the primordial mammalian hosts of ancestral hantaviruses (Figure 3). Geographic-specific clustering was evidenced by the close phylogenetic relationship between prototype XSV VN1982 from Phu Tho province and XSV F42640 and XSV F42682 from neighboring Tuyê Quang province in northern Vietnam. On the other hand, n XSV F44583, XSV 44601 and XSV 44580 from Quang Nam province in central Vietnam clustered together. Although limited differences were present in phylogenetic trees based on each segment, tree topologies were generally congruent and supported by significant bootstrap values (70%) and posterior node probabilities (0.70).
Figure 3. Phylogenetic trees were generated by maximum-likelihood and Bayesian methods, using the GTR+I+Γ model of evolution, based on the S-, M- and L-genomic sequences of hantavirus strains.
Because tree topologies were nearly identical using RAxML and MrBayes programs, the trees generated by MrBayes were displayed.
The evolutionary relationships between Xuan Son virus (XSV), Mouyassuévirus (MOYV) and other bat-borne hantaviruses, including Magboi virus (MGBV), Longquan virus (LQUV) and Huangpi virus (HUPV), are shown, as are representative soricomorph-borne hantaviruses, including Nova virus (NVAV MSB95703, S: FJ539168; M: HQ840957; L: FJ593498), Thottapalayam virus (TPMV VRC66412, S: AY526097; M: EU001329; L: EU001330), Imjin virus (MJNV Cl05-11, S: EF641804; M: EF641798; L: EF641806), Seewis virus (SWSV mp70, S: EF636024; M: EF636025; L: EF636026), Kenkeme virus (KKMV MSB148794, S: GQ306148, M: GQ306149; L: GQ306150), Lianghe virus (LHEV As217, M: JX465406), Boginia virus (BOGV 2074, M: JX990966), Cao Bang virus (CBNV CBN-3,
S: EF543524; M: EF543526; L: EF543525), Ash River virus (ARRV MSB 73418, S:
EF650086; L: EF619961), Jemez Springs virus (JMSV MSB144475, S: FJ593499; M:
FJ593500; L: FJ593501), Qian Hu Shan virus (QHSV YN05-284, S: GU566023; M:
GU566022; L: GU566021), Tanganya virus (TGNV Tan826, S: EF050455; L: EF050454), Azagny virus (AZGV KBM15, S: JF276226; M: JF276227; L: JF276228), Jeju virus (JJUV 10-11, S: HQ834695; M: HQ834696; L: HQ834697), Bowévirus (BOWV VN1512,
M: KC631783; L: KC631784), Asama virus (ASAV N10, S: EU929072; M: EU929075; L: