Dahonggou Creek virus, a divergent lineage of hantavirus harbored by the long-tailed mole (Scaptonyx fusicaudus)
© The Author(s) 2016
Received: 28 February 2016
Accepted: 13 May 2016
Published: 20 June 2016
Novel hantaviruses, recently detected in moles (order Eulipotyphla, family Talpidae) from Europe, Asia, and North America would predict a broader host range and wider ecological diversity. Employing RT-PCR, archival frozen tissues from the Chinese shrew mole (Uropsilus soricipes), broad-footed mole (Scapanus latimanus), coast mole (Scapanus orarius), Townsend’s mole (Scapanus townsendii), and long-tailed mole (Scaptonyx fusicaudus) were analyzed for hantavirus RNA. Following multiple attempts, a previously unrecognized hantavirus, designated Dahonggou Creek virus (DHCV), was detected in a long-tailed mole, captured in Shimian County, Sichuan Province, People’s Republic of China, in August 1989. Analyses of a 1058-nucleotide region of the RNA-dependent RNA polymerase-encoding L segment indicated that DHCV was genetically distinct from other rodent-, shrew-, mole-, and bat-borne hantaviruses. Phylogenetic trees, using maximum likelihood and Bayesian methods, showed that DHCV represented a divergent lineage comprising crocidurine and myosoricine shrew-borne hantaviruses. Although efforts to obtain the S- and M-genomic segments failed, the L-segment sequence analysis, reported here, expands the genetic database of non-rodent-borne hantaviruses. Also, by further mining natural history collections of archival specimens, the genetic diversity of hantaviruses will elucidate their evolutionary origins.
Hantaviruses are members of the family Bunyaviridae, all of whom possess a negative-sense, single-stranded tripartite RNA genome, consisting of large (L), medium (M), and small (S) segments, which encode an RNA-dependent RNA polymerase (RdRp), envelope glycoproteins (Gn and Gc), and a nucleocapsid (N) protein, respectively . However, unlike other members of this large virus family which are carried by insects or arthropods, hantaviruses are hosted by small mammals, notably rodents (order Rodentia, families Muridae and Cricetidae), as well as shrews (order Eulipotyphla, family Soricidae), belonging to three subfamilies (Soricinae, Crocidurinae, and Myosoricinae), and moles (family Talpidae) of the Talpinae and Scalopinae subfamilies [2, 3]. Recently, insectivorous bats (order Chiroptera) have also been shown to serve as reservoirs of divergent lineages of hantaviruses [4–9]. To date, the pathogenic potential of the newfound shrew-, mole-, and bat-borne hantaviruses is unknown, whereas selected rodent-borne hantaviruses have been associated with acute-onset febrile diseases of varying clinical severity, known as hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome .
Among the newfound mole-borne hantaviruses are Asama virus (ASAV) in the Japanese shrew mole (Urotrichus talpoides) , Oxbow virus (OXBV) in the American shrew mole (Neurotrichus gibbsii) , and Rockport virus (RKPV) in the eastern mole (Scalopus aquaticus) . Also, a divergent lineage of hantavirus, designated Nova virus (NVAV), has been identified in the widely distributed European mole (Talpa europaea) , in which very high prevalences of NVAV infection have been found in France  and Poland .
In testing archival frozen tissues from a natural history collection of moles, trapped in the People’s Republic of China and the USA, we now report the detection of a novel hantavirus, named Dahonggou Creek virus (DHCV), in the long-tailed mole (Scaptonyx fusicaudus).
Tissues, stored frozen at −80 °C at the Museum of Southwestern Biology in Albuquerque, New Mexico, had been collected from five Chinese shrew moles (Uropsilus soricipes) and two long-tailed moles, captured in Shimian Xian, Sichuan Province, in the People’s Republic of China in 2005 and 1989, respectively, and from two broad-footed moles (Scapanus latimanus), two coast moles (Scapanus orarius), and two Townsend’s moles (Scapanus townsendii), in California (Sonoma) and Washington (Walla Walla and Gray’s Harbor) in 1984, according to well-established protocols, approved by the Institutional Animal Care and Use Committee of the University of New Mexico. Tissues were analyzed for hantavirus RNA by RT-PCR, using newly designed and previously employed oligonucleotide primers [12–16]. Briefly, total RNA was extracted from tissues using the PureLink Micro-to-Midi total RNA purification kit (Invitrogen, San Diego, CA), and cDNA was synthesized using the SuperScript III First-Strand Synthesis Systems (Invitrogen) with a highly conserved primer and/or random hexamers by two-step RT-PCR cycles. First- and second-round PCR were performed in 20-μL reaction mixtures, containing 250 μM dNTP, 2.5 mM MgCl2, 1 U of Takara LA Taq polymerase (Takara, Shiga, Japan), and 0.25 μM of each primer. Initial denaturation at 94 °C for 2 min was followed by two cycles each of denaturation at 94 °C for 30 s, two-degree step-down annealing from 46 to 38 °C for 40 s, and elongation at 72 °C for 1 min, then 30 cycles of denaturation at 94 °C for 30 s, annealing at 42 °C for 40 s, and elongation at 72 °C for 1 min, in a GeneAmp PCR 9700 thermal cycler (PerkinElmer, Waltham, MA). PCR products were separated, using MobiSpin S-400 spin columns (MoBiTec, Goettingen, Germany), and amplicons were sequenced directly using an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Phylogenetic trees were generated using maximum likelihood and Bayesian methods, implemented in the RAxML Blackbox webserver  and MrBayes 3.1 , under the best-fit GTR+I+Γ model of evolution selected by hierarchical likelihood-ratio test in MrModeltest v2.3  and jModelTest version 0.1 . Two replicate Bayesian Metropolis-Hastings Markov chain Monte Carlo 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 overall. Topologies were evaluated by bootstrap analysis of 1000 iterations (implemented in RAxML Blackbox), and posterior node probabilities were based on 2 million generations and estimated sample sizes over 100 (implemented in MrBayes).
Results and discussion
The whole genomes of many hantaviruses, previously described and recently discovered, are unavailable. A 1-kb sequence from a single specimen is far from optimal and full-length genomes from multiple long-tailed moles would be required to gain a more definitive conclusion about the molecular phylogeny and genetic diversity of DHCV. However, while admittedly incomplete, the sequence analysis presented in this report would facilitate and guide future studies by other investigators, seeking to expand the genetic database of non-rodent-borne hantaviruses. Also, completion of the DHCV genome from tissues of more recently collected long-tailed moles, using next-generation sequencing technology, is warranted.
Nucleotide and amino acid sequence similarity (%) between DHCV strain MSB281632 and representative rodent-, shrew-, mole-, and bat-borne hantaviruses
The fossorial long-tailed mole, which closely resembles the American shrew mole and Japanese shrew mole in size and appearance, as well as ecological habits , is restricted to high altitudes (2000–4100 m) in montane coniferous forests in central and southern China, extending to northern Myanmar and northern Vietnam. It represents the only species within the Scaptonyx genus. Because the long-tailed mole is sympatric with Uropsilus moles, the latter might also serve as a potential reservoir host of hantaviruses. Thus, efforts to test tissues from additional Uropsilus moles are also warranted.
Museum curators and field mammalogists, who willingly granted access to their priceless archival tissue collections, have contributed greatly to the acquisition of new knowledge about the geospatial distribution and temporal dynamics, host range, and genetic diversity of hantaviruses in shrews, moles, and insectivorous bats [2, 3]. The availability of these specimens, which were not originally intended for our studies, provide compelling justification for the continued expansion and long-term maintenance of natural history tissue repositories for future investigations, including virus-discovery efforts to better catalog the vast environmental microbiome. In so doing, important improvements could be made in preparedness and response to new and emerging zoonotic infectious diseases .
This work was supported in part by the US Public Health Service grants R01AI075057, P20GM103516, and P30GM114737 from the National Institutes of Health. The funding agency had no role in the study design, data collection and analysis, manuscript preparation, and/or decision to publish.
JAC and RY conceived and designed the project. HJK and SHG performed the experiments. HJK, SHG, and RY analyzed the data. All authors prepared and reviewed the manuscript.
The authors declare that they have no competing interests.
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