The recent discovery of genetically distinct shrew- and mole-borne viruses belonging to the newly defined family Hantaviridae (order Bunyavirales) has spurred an extended search for hantaviruses in RNAlater®-preserved lung tissues from 215 bats (order Chiroptera) representing five families (Hipposideridae, Megadermatidae, Pteropodidae, Rhinolophidae and Vespertilionidae), collected in Vietnam during 2012 to 2014. A newly identified hantavirus, designated Đakrông virus (DKGV), was detected in one of two Stoliczka’s Asian trident bats (Aselliscus stoliczkanus), from Đakrông Nature Reserve in Quảng Trị Province. Using maximum-likelihood and Bayesian methods, phylogenetic trees based on the full-length S, M and L segments showed that DKGV occupied a basal position with other mobatviruses, suggesting that primordial hantaviruses may have been hosted by ancestral bats.
The long-standing consensus that hantaviruses are harbored exclusively by rodents has been disrupted by the discovery of distinct lineages of hantaviruses in shrews and moles of multiple species (order Eulipotyphla, families Soricidae and Talpidae) in Asia, Europe, Africa and North America1,2. Not surprisingly, bats (order Chiroptera, suborders Yangochiroptera and Yinpterochiroptera), by virtue of their phylogenetic relatedness to shrews and moles and other placental mammals within the superorder Laurasiatheria3,4, have also been shown to harbor hantaviruses1,2. Subsequently, based on phylogenetic analysis of the full-length S- and M-genomic segments, members of the genus Hantavirus (formerly family Bunyaviridae) have been reclassified into four newly defined genera (Loanvirus, Mobatvirus, Orthohantavirus and Thottimvirus) within a new virus family, designated Hantaviridae5,6.
All rodent-borne hantaviruses, as well as nearly all newfound hantaviruses hosted by shrews and moles, belong to the genus Orthohantavirus6. By contrast, bat-borne hantaviruses have been assigned to the Loanvirus and Mobatvirus genera. To date, bat-borne loanviruses include Mouyassué virus (MOYV) in the banana pipistrelle (Neoromicia nanus) from Côte d’Ivoire7 and in the cape serotine (Neoromicia capensis) from Ethiopia8, Magboi virus (MGBV) in the hairy slit-faced bat (Nycteris hispida) from Sierra Leone9, Huángpí virus (HUPV) in the Japanese house bat (Pipistrellus abramus) from China10, Lóngquán virus (LQUV) in the Chinese horseshoe bat (Rhinolophus sinicus), Formosan lesser horseshoe bat (Rhinolophus monoceros) and intermediate horseshoe bat (Rhinolophus affinis) from China10, and Brno virus (BRNV) in the common noctule (Nyctalus noctula) from the Czech Republic11.
Mobatviruses include Xuân Sơn virus (XSV) in the Pomona roundleaf bat (Hipposideros pomona) from Vietnam12,13, Láibīn virus (LAIV) in the black-bearded tomb bat (Taphozous melanopogon) from China14, Makokou virus (MAKV) in the Noack’s roundleaf bat (Hipposideros ruber) from Gabon15, and Quezon virus (QZNV) in the Geoffroy’s rousette (Rousettus amplexicaudatus) from the Philippines16. QZNV is the only hantavirus reported hitherto in a frugivorous bat species (family Pteropodidae).
Several orthohantaviruses hosted by murid and cricetid rodents cause either hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia17,18,19 or hantavirus cardiopulmonary syndrome (HCPS) in the Americas20,21. HFRS varies in clinical severity from mild to life threatening, with mortality ranging from <1% to ≥15%22,23, whereas HCPS is generally severe, and despite intensive care treatment, mortality rates are 25% or higher24,25. Humans are typically infected with rodent-borne orthohantaviruses by the respiratory route via inhalation of aerosolized excretions or secretions26,27. Transmission of hantaviruses from person-to-person has been reported only with Andes virus in Argentina28 and Chile29,30.
The pathogenicity of the newfound shrew-, mole- and bat-borne hantaviruses is unknown. That is, despite a report of IgG antibodies against recombinant nucleocapsid proteins of shrew-borne hantaviruses in humans from Côte d’Ivoire and Gabon31, there is no definitive proof that any of the recently reported orthohantaviruses, thottimviruses, loanviruses and mobatviruses, harbored by shrews, moles and bats, cause clinically identifiable diseases or syndromes in humans2.
This multi-institutional study represents an extended search for novel bat-borne loanviruses and mobatviruses to better understand their geographic distribution and host diversification. Our data indicate that a newly identified mobatvirus in the Stoliczka’s Asian trident bat (Aselliscus stoliczkanus) is genetically distinct and phylogenetically related to other bat-borne mobatviruses, suggesting that primordial hantaviruses may have been hosted by ancestral bats.
Repeated attempts to detect hantavirus RNA were successful in only one of the 215 lung specimens (Supplemental Table 1), despite using PCR protocols that led to the discovery of other bat-borne hantaviruses. Sequence analysis of the full-length S-, M- and L-genomic segments showed a novel hantavirus, designated Đakrông virus (DKGV), in one of two Stoliczka’s Asian trident bats (Fig. 1A), captured in Đakrông Nature Reserve (16.6091N, 106.8778E) in Quảng Trị Province (Fig. 1B), in August 2013.
The overall genomic organization of DKGV was similar to that of other hantaviruses. A nucleocapsid (N) protein of 427 amino acids was encoded by the 1,746-nucleotide S segment, beginning at position 58, and with a 405-nucleotide 3′-noncoding region (NCR). The hypothetical NSs open reading frame was not found.
A glycoprotein complex (GPC) of 1,127 amino acids was encoded by the 3,622-nucleotide M segment, starting at position 21, and with a 218-nucleotide 3′-NCR. Two potential N-linked glycosylation sites were found in the Gn at amino acid positions 344 and 396 and one in the Gc at position 924. Another possible site was present at amino acid position 133 of the Gn. For comparison, the amino acid positions of N-linked glycosylation sites of other representative hantaviruses are summarized in Table 1. Also, the highly conserved WAASA amino-acid motif was found at amino acid positions 641–645. The full-length Gn/Gc amino acid sequence similarity was highest between DKGV and LAIV (73.0%) (Table 2).
Analysis of the 6,535-nucleotide L segment, which encoded a 2,145-amino acid RNA-dependent RNA polymerase (RdRP), showed the highly conserved A, B, C, D and E motifs, The functional constraints on the RdRP were evidenced by the overall high nucleotide and amino acid sequence similarity of 60% or more in the L segment between DKGV and other hantaviruses (Table 2).
Comparison of the full-length S, M and L segments indicated amino acid sequence similarities ranging from 45.6% (BRNV GPC) to 81.2% (LAIV RdRP) between DKGV and representative bat-borne hantaviruses (Table 2), and showed that DKGV differed at the amino acid level by 30% or more from nearly all rodent- and shrew-borne orthohantaviruses.
Nucleocapsid and glycoprotein secondary structures
The DKGV N protein secondary structure, comprising 53.16% α-helices, 10.07% β-sheets and 35.13% random coils, resembled that of other hantavirus N proteins. A two-domain, primarily α-helical structure joined by a central β-pleated sheet, was observed. Although the N-terminal domain length was nearly the same, structural changes were evident in the central β-pleated sheet and adjoining C-terminal α-helical domain, according to the phylogenetic relationships of the N proteins.
That is, the N protein comprised two α-helical domains and a central β-pleated sheet (Fig. 2) irrespective of the low amino acid sequence similarity among the orthohantaviruses and thottimviruses. However, the central β-pleated sheet motif and RNA-binding region (amino acid positions 175 to 217) of DKGV differed from that of other hantaviruses, which resembled that of murid rodent-borne orthohantaviruses (Fig. 2).
The DKGV glycoprotein secondary structure, comprising 3.82% α-helices, 40.20% β-sheets and 55.99% random coils, resembled that of other hantavirus glycoproteins (Fig. 3). Also, the four transmembrane helices of the DKGV glycoprotein resembled that of other hantaviruses (Fig. 4), and the putative fusion loop (WGCNPVD) and zinc finger domain (CVVCTRECSCTEELKAHNEHCIQGSCPY CMRDLHPSQHVLTEHYKTC) were observed at residues 760–766 and 542–588, respectively.
DKGV was distinct from other hantaviruses in phylogenetic trees, based on S-, M- and L-segment sequences using the Markov chain Monte Carlo (MCMC) Bayesian methods (Fig. 5). In all analyses, DKGV and LAIV shared a common ancestry. The basal position of mobatviruses and loanviruses in phylogenetic trees suggested that primordial hantaviruses may have been hosted by ancestral bats (Fig. 5).
Taxonomic classification of the bats was confirmed by PCR amplification and sequencing of the cytochrome b (Cyt b) and cytochrome c oxidase I (COI) genes. Morphologically indistinguishable Aselliscus stoliczkanus and Aselliscus dongbacana were identified by Cyt b (KU161558–KU161575 and MG524933–MG524935) and COI (LC406430–LC406448) gene sequence analysis. Phylogenetic analysis of bats belonging to the suborders Yinpterochiroptera and Yangochiroptera resembled that of DKGV and other bat-borne hantaviruses (Fig. 6).
Co-phylogeny of hantavirus and host
Segregation of hantaviruses according to the subfamily of their reservoir hosts was demonstrated by co-phylogeny mapping, using consensus trees based on the Gn/Gc glycoprotein and RdRP protein amino acid sequences (Fig. 7). The phylogenetic positions of DKGV and other mobatviruses (XSV and MAKV) mirrored the phylogenetic relationships of their Hipposideridae hosts. By contrast, the phylogenetic positions of LAIV from Taphozous melanopogon, QZNV from Rousettus amplexicaudatus and LQUV from Rhinolophus affinis were mismatched between virus and host species tanglegram.
The Stoliczka’s Asian trident bat, one of three species in the genus Aselliscus, is found throughout Southeast Asia. The Dong Bac’s trident bat (Aselliscus dongbacana), a closely related species, overlaps in body size, distribution, echolocation and habitat32. However, we failed to detect hantavirus RNA in this latter species. As in previous studies, in which only one or two individual bats were found to be infected7,12,13,16, hantavirus RNA was detected in a single Stoliczka’s Asian trident bat. Technical issues (such as primer mismatches, suboptimal cycling conditions, limited tissues and degraded RNA), as well as the restricted transmission and/or immune-mediated clearance of hantavirus infection in bats, are possible contributing factors.
By virtue of the N protein and Gc glycoprotein sequence divergence between DKGV and other hantaviruses, as well as the unique host species, DKGV likely represents a new hantavirus species. A three-dimensional, bi-lobed protein architecture for RNA binding was suggested by the DKGV N protein secondary structure and that of other bat-borne hantaviruses. The fusion loop and zinc finger domain of the glycoprotein suggested that DKGV and other bat-borne loanvirus and mobatviruses have similar mechanisms of protein modification with rodent-borne orthohantaviruses33,34. On the other hand, the envelope GPC is implicated in virus attachment and cell entry. Further insights for receptor and receptor binding sites of hantaviruses harbored by shrews, moles and bats will contribute to a deeper understanding about their host specificities.
Recently, far greater genetic diversity than those in rodents have been detected in orthohantaviruses harbored by shrews and moles of multiple species, belonging to five subfamilies (Soricinae, Crocidurinae, Myosoricinae, Talpinae and Scalopinae) within the order Eulipotyphla, in Europe, Asia, Africa and/or North America. Similarly, hantavirus RNA has been detected in tissues of several bat species belonging to the families Emballonuridae, Nycteridae and Vespertilionidae (suborder Yangochiroptera) and the families Rhinolophidae, Hipposideridae and Pteropodidae (suborder Yinpterochiroptera). It is unclear to what extent spillover or host switching is responsible for the overall low prevalence of hantavirus infection in only 14 bat species to date, and the inability to detect hantavirus RNA in nearly 100 other bat species analyzed to date7,8,9,10,11,12,13,14,15,16.
Previously, the overall congruence between gene phylogenies of rodent-borne orthohantaviruses and their hosts led to the conjecture that orthohantaviruses had co-evolved with their reservoir hosts4. However, recent studies, based on co-phylogenetic reconciliation and estimation of evolutionary rates and divergence times, conclude that local host-specific adaptation and preferential host switching account for the phylogenetic similarities between hantaviruses and their mammalian hosts35,36. Although our co-phylogenetic analysis also indicates that bat-borne loanvirus and mobatviruses and their host species have not co-diverged, the availability of whole genome sequences for only four of the 10 bat-borne hantaviruses presented a significant limitation. Thus, future efforts must focus on obtaining full-length genomes of newfound hantaviruses, particularly those harbored by bats in southeast Asia and Africa, to gain additional insights into the phylogeography and evolutionary origins of viruses in the family Hantaviridae.
Materials and Methods
Field procedures and protocols for trapping, euthanasia and tissue processing, conforming to the guidelines of the American Society of Mammalogists37,38, were approved by the Ministry of Agriculture and Rural Development in Vietnam. Moreover, permission for this study was obtained from the Vietnam Administration of Forest, belonging to the Ministry of Agriculture and Rural Development, before collecting bat specimens (permission numbers: 1492/TCLN-BTTN; 701/TCLN-BTTN; 389/TCLN-BTTN; 767/TCLN-BTTN). Also, the Institutional Animal Care and Use Committee, of the National Institute of Infectious Diseases, reviewed and approved the field protocols and experimental procedures (permission number: 112152).
Mist nets and harp traps were used to trap bats, during December 2012 to June 2014, in Xuân Lien Nature Reserve (19.8736N, 105.2156E) in Thanh Hóa Province; Vĩnh Cửu Nature Reserve, recently renamed Đồng Nai Culture and Nature Reserve (11.3808N, 107.0622E), in Đồng Nai Province; Khau Ca Nature Reserve (22.8381N, 105.1161E) in Hà Giang Province; Bắc Hướng Hóa Nature Reserve and Đakrông Nature Reserve in Quảng Trị Province; Nà Hang Nature Reserve (22.3532N, 105.4197E) in Tuyên Quang Province; and near Hoàng Liên National Park (22.2833N, 103.9219E) in Lào Cai Province. Live-caught bats were euthanized and lung tissues were preserved in RNAlater® (Qiagen) until testing by RT-PCR.
Using the MagDEA RNA 100 Kit (Precision System Science, Matsudo, Japan)39, total RNA was extracted from lung tissues of 215 bats, representing 15 genera and 46 species in five families (Hipposideridae, Megadermatidae, Pteropodidae, Rhinolophidae and Vespertilionidae) (Supplemental Table 1). RNA was reverse transcribed to cDNA was synthesized using the PrimeScript II 1st strand cDNA Synthesis Kit (Takara bio, Otsu, Japan) and an oligonucleotide primer (OSM55F, 5′–TAGTAGTAGACTCC–3′) designed from conserved 5′–end of the S, M and L segments of hantaviruses39.
RT-PCR and sequencing
Oligonucleotide primers previously used to detect hantaviruses12,13,16,39,40,41,42,43 were employed to amplify S, M and L segments (Supplemental Table 2). First-round PCR was performed in 20-μL reaction mixtures, containing 250 μM dNTP, 2.5 mM MgCl2, 1 U of Takara LA Taq polymerase Host Start version (Takara Bio) and 0.25 μM of each primer16. Second-round PCR was performed in 20-μL reaction mixtures, containing 200 μM dNTP, 2.5 mM MgCl2, 1 U of AmpliTaq 360 Gold polymerase (Life Technologies, Foster City, CA, USA) and 0.25 μM of each primer16. Initial denaturation at 95 °C for 1 min for first PCR or at 95 °C for 10 min for second PCR were followed by two cycles each of denaturation at 95 °C for 20 s, two-degree step-down annealing from 46 °C to 38 °C for 40 s, and elongation at 68 °C for 1 min, then 30 cycles of denaturation at 94 °C for 20 s, annealing at 42 °C for 40 s, and elongation at 68 °C for 1 min, in a Veriti thermal cycler (Life Technologies)7,12,13,16,39. PCR products, treated with ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instruction, were sequenced directly using an ABI 3730xl DNA Analyzer (Life Technologies)16.
Secondary structure analysis
Full-length amino acid sequences were submitted to NPS@ structure server44 to predict secondary structures of the N protein and Gn/Gc glycoproteins36. Glycosylation and transmembrane sites were predicted at the NetNlyc 1.0 and Predictprotein45 and TMHMM version 2.046, respectively. The program COILS47 was used to scan the N protein for expected coiled-coil regions36.
Maximum likelihood and Bayesian methods, implemented in RAxML Blackbox webserver48 and MrBayes 3.149, under the best-fit GTR + I + Γ model of evolution and jModelTest version 2.1.650, were used to generate phylogenetic trees13. Two replicate Bayesian Metropolis–Hastings MCMC runs, each consisting of six chains of 10 million generations sampled every 100 generations with a burn-in of 25,000 (25%), resulted in 150,000 trees overall13,16. The S-, M- and L-genomic segments were treated separately in the phylogenetic analyses. Topologies were evaluated by bootstrap analysis of 1,000 iterations, and posterior node probabilities were based on 10 million generations and estimated sample sizes over 100 (implemented in MrBayes)16. With a robust phylogeny of shrew-, mole-, bat- and rodent-borne hantaviruses10, we readdressed the co-evolutionary relationship between hantaviruses and their hosts that formed the basis of our predictive paradigm for hantavirus discovery, by comparing the degree of concordance between reservoir host and hantavirus cladograms in TreeMap 3β124351.
Total DNA was extracted from lung tissues, using MagDEA DNA 200 Kit (Precision System Science), and PCR amplification of the 1,140-nucleotide Cyt b gene and 1,545-nucleotide COI gene52,53 was performed with newly designed primer sets: Cy-14724F (5′–GACYARTRRCATGAAAAAYCAYCGTTGT–3′)/Cy-15909R (5′–CYYCWTYIYTGGTTTACAAGACYAG–3′)16 and KOD multi enzyme (Toyobo, Osaka, Japan), and MammMt-5533F (5′–CYCTGTSYTTRRATTTACAGTYYAA–3′)/MammMt-7159R (5′–GRGGTTCRAWWCCTYCCTYTCTT–3′) and Phusion enzyme (New England Biolabs, Ipswitch, MA, USA), respectively. Initial denaturation at 95 °C for 2 min was followed by two cycles each of denaturation at 95 °C for 15 s, two-degree step-down annealing from 60 °C to 50 °C for 30 s, and elongation at 68 °C for 1 min 30 s, then 30 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 30 s, and elongation at 68 °C for 1 min 30 s, in a Veriti thermal cycler13. PCR products were purified by Mobispin S-400 (Molecular Biotechnology, Lotzzestrasse, Germany) and were sequenced directly.
GenBank accession numbers
MG663534, MG663535 and MG663536 for Đakrông virus; MG524933–MG524935 and LC406452–LC406456 for cytochrome b and LC406430–LC406448 for cytochrome c oxidase I of Aselliscus stoliczkanus and Aselliscus dongbacana.
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We thank Hitoshi Suzuki, Shinichiro Kawada, Satoshi D. Ohdachi and Kimiyuki Tsuchiya for supporting field investigations and for valuable advice. This work was supported in part by a grant-in-aid Research on Emerging and Re-emerging Infectious Diseases, Health Labour Sciences Research Grant in Japan (H25-Shinko-Ippan-008), grants-in-aid on Research Program on Emerging and Re-emerging Infectious Diseases, Japan Agency for Medical Research and Development (AMED) JP15fk0108005, JP16fk0108117, JP17fk0108217, JP18fk0108017 and JP19fk0108097, grant-in aid from the Japan Society for the Promotion of Science 24405045 (Scientific Research grant B), grant-in-aid on NAFOSTED (106-NN.05-2016.14), and VAST-JSPS (QTJP01.02/18-20).
The authors declare no competing interests.
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