Shared Ancestry Between a Newfound Mole-Borne Hantavirus and Hantaviruses Harbored by ...

33 Pages·2011·3.76 MB·English

JVI Accepts, published online ahead of print on 1 June 2011
J. Virol. doi:10.1128/JVI.02450-10
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Kang et al. 1
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Shared Ancestry Between a Newfound Mole-Borne Hantavirus and Hantaviruses
Harbored by Cricetid Rodents

[Running title: Novel Talpid-Borne Hantavirus]
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Hae Ji Kang1, Shannon N. Bennett1, Andrew G. Hope2, Joseph A. Cook2, Richard Yanagihara1 n
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1Departments of Pediatrics and Tropical Medicine, Medical Microbiology and Pharmacology, fr
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John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96813; h
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2Department of Biology and Museum of Southwestern Biology, University of New Mexico, :/
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Albuquerque, New Mexico 87131 a
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*Corresponding author: Richard Yanagihara, M.D., Pacific Center for Emerging Infectious
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Diseases Research, John A. Burns School of Medicine, University of Hawaii at Manoa, 651 Ilalo N
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Street, BSB320L, Honolulu, HI 96813, USA; Telephone: [phone]; Fax: [phone]; E- m
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mail: [email protected] r

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Kang et al. 2
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ABSTRACT

Discovery of genetically distinct hantaviruses in multiple species of shrews (Order
Soricomorpha, Family Soricidae) and moles (Family Talpidae) contests the conventional
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view that rodents (Order Rodentia, Family Muridae and Cricetidae) are the principal
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reservoir hosts and suggests that the evolutionary history of hantaviruses is far more
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complex than previously hypothesized. We now report on Rockport virus (RKPV), a
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hantavirus identified in archival tissues of the eastern mole (Scalopus aquaticus), collected r
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in Rockport, Texas, in 1986. Pair-wise comparison of the full-length S-, M- and L-genomic h
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segments indicated moderately low sequence similarity between RKPV and other :/
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soricomorph-borne hantaviruses. Phylogenetic analyses, using maximum-likelihood and a
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Bayesian methods, showed that RKPV shared a most recent common ancestor with cricetid .o
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rodent-borne hantaviruses. Distributed widely across the eastern United States, the
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fossorial eastern mole is sympatric and syntopic with cricetid rodents known to harbor
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hantaviruses, raising the possibility of host-switching events in the distant past. Our m
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findings warrant more-detailed investigations on the dynamics of spillover and cross- r

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species transmission of present-day hantaviruses within communities of rodents and moles. ,

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Keywords: hantavirus, soricomorph, mole, evolution, host switching, North America
Abstract word count: 170
Text pages (including references, tables and figure legends): 28
Tables: 1
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Figures: 4
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GenBank accession numbers:
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RKPV S segment (HM015218, HM015223, HM015224); RKPV M segment (HM015219); r
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RKPV L segment (HM015220, HM015221, HM015222); Scalopus aquaticus cytochrome b gene h
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(HM461914, HM461915, HM461916, HM461917) :/
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INTRODUCTION

Hantaviruses (genus Hantavirus), like other Bunyaviridae genera (Orthobunyavirus,
Phlebovirus, Nairovirus and Tospovirus), possess a negative-sense, single-stranded, tripartite
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RNA genome, consisting of large (L), medium (M) and small (S) segments, which encode an
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RNA-dependent RNA polymerase (RdRP), two envelope glycoproteins (Gn, Gc) and a n
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nucleocapsid protein (NP), respectively (29, 37, 43, 44). However, hantaviruses are unique in
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that they have no known insect or arthropod host and instead are harbored by rodents (Order r
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Rodentia, Family Muridae and Cricetidae) (36, 56). Hantaviruses hosted by rodents in the h
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Subfamily Murinae and Arvicolinae cause hemorrhagic fever with renal syndrome in Eurasia (30, :/
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55, 56), while those carried by rodents in the Subfamily Neotominae and Sigmodontinae cause a
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hantavirus cardiopulmonary syndrome in the Americas (10, 35). .o
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The segregation of hantaviruses into clades that parallel the molecular phylogeny of
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rodents in the Murinae, Arvicolinae, Neotominae and Sigmodontinae subfamilies has suggested N
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that hantaviruses have co-evolved with their reservoir rodent hosts (20, 22, 37). Recently, this m
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premise has been strenuously challenged, on the basis of the disjunction between evolutionary r

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rates of the host and virus species (39, 40). That is, rather than co-divergence, host switching and ,

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local species-specific adaptation have been proposed to account for the similarities between host
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and virus phylogenies. Since some sympatric and syntopic rodent species occasionally serve as
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reservoirs for the same hantavirus, host switching or cross-species transmission have clearly
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occurred during the evolution of hantaviruses (33). Topografov virus in the Siberian lemming
(Lemmus sibiricus) is an often-cited example (53). On the other hand, full-genome analysis of
Thottapalayam virus (TPMV), a hantavirus isolated from the Asian house shrew (Suncus
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murinus) more than 40 years ago (6, 58), shows an early evolutionary divergence from rodent-
associated hantaviruses (46, 54).
Moreover, the recent discovery of genetically diverse hantaviruses in shrews of multiple
species (Order Soricomorpha, Family Soricidae), including Tanganya virus in the Therese’s
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shrew (Crocidura theresae) (27), Imjin virus in the Ussuri white-toothed shrew (Crocidura
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lasiura) (49), Camp Ripley virus in the northern short-tailed shrew (Blarina brevicauda) (4), Cao n
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Bang virus in the Chinese mole shrew (Anourosorex squamipes) (48), Seewis virus in the
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Eurasian common shrew (Sorex araneus) (47), Ash River virus in the masked shrew (Sorex r
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cinereus) (2), Jemez Springs virus in the dusky shrew (Sorex monticolus) (2) and Kenkeme virus h
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in the flat-skulled shrews (Sorex roboratus) (25), as well as in moles (Family Talpidae), :/
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including Asama virus (ASAV) in the Japanese shrew mole (Urotrichus talpoides) (3), Oxbow a
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virus (OXBV) in the American shrew mole (Neurotrichus gibbsii) (23) and Nova virus (NVAV) .o
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in the European common mole (Talpa europaea) (24), suggests that the evolutionary history of
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hantaviruses is more complex than previously conjectured. N
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In particular, the highly divergent hantavirus in the European common mole (24) would m
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predict the existence of additional talpid-borne hantaviruses. High on the list of candidate talpid r

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hosts has been the eastern mole (Scalopus aquaticus) (Subfamily Scalopinae), which is widely ,

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distributed across the eastern United States (57). Here, we report on the molecular phylogeny of
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Rockport virus (RKPV), a newfound hantavirus in the eastern mole. The unexpected finding of a
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shared ancestry between RKPV and cricetid rodent-borne hantaviruses is consistent with cross-
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species virus transmission in the distant past, but leaves unanswered questions about which
mammalian lineage served as the original host of primordial hantaviruses.
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MATERIALS AND METHODS

Tissues. Frozen livers from 60 eastern moles, archived in the Museum of Southwestern
Biology at the University of New Mexico in Albuquerque, were analyzed. Moles were collected
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between 1984 and 1992 from the eastern United States (Fig. 1A), including Florida, Kansas,
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South Carolina, Tennessee and Texas (Table 1). n
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RNA extraction and RT-PCR analysis. Total RNA was extracted from tissues, using
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the PureLink Micro-to-Midi total RNA purification kit (Invitrogen, San Diego, CA), then reverse r
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transcribed, using the SuperScript III First-Strand Synthesis System (Invitrogen) and h
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oligonucleotide primer (OSM55, 5’-TAGTAGTAGACTCC-3’), designed from the conserved 3’- :/
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end of the S, M and L segments of hantaviruses. For amplification of hantavirus genes, a two- a
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step PCR was performed in 20-µL reaction mixtures, containing 250 µM dNTP, 2 mM MgCl2, 1 .o
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U of AmpliTaq polymerase (Roche, Basel, Switzerland) and 0.25 µM of each primer. Initial o
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denaturation at 94ºC for 5 min was followed by two cycles each of denaturation at 94ºC for 40 o
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sec, two-degree step-down annealing from 48ºC to 38ºC for 40 sec, and elongation at 72ºC for 1 m
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min, then 32 cycles of denaturation at 94ºC for 40 sec, annealing at 42ºC for 40 sec, and r

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elongation at 72ºC for 1 min, in a GeneAmp PCR 9700 thermal cycler (Perkin-Elmer, Waltham,
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MA). Amplicons were separated by electrophoresis on 1.5% agarose gels and purified using the 8

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QIAQuick Gel Extraction Kit (Qiagen, Hilden, Germany). DNA was sequenced directly using an
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ABI Prism 377XL Genetic Analyzer (Applied Biosystems, Foster City, CA). s
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Genetic analysis. Complete S-, M- and L-genomic nucleotide and amino acid sequences
of RKPV were aligned with representative rodent- and soricomorph-borne hantavirus sequences,
using the ClustalW method (TranslatorX server and BioEdit 7.0.5) (1, 15, 52). Nucleotide
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sequences were also analyzed using multiple recombination-detection methods within the RDP3
Beta34 software. The NP secondary structure was predicted from the entire amino acid sequence
of the RKPV S segment using five methods: DSC (26), HNN (26), MLRC (14), PHD (42) and
PREDATOR (12), at the NPS@ structure server (9). COILS (31) was also used to scan the NP
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for coiled-coil regions. To determine the glycosylation and transmembrane sites for the RKPV
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Gn and Gc, NetNlyc 1.0 and Predictprotein (13) and TMHMM version 2.0 (28) were used, n
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respectively.
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Phylogenetic analysis. To determine the phylogenetic relationship of RKPV with well- r
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characterized hantaviruses, phylogenetic trees were generated, based on the entire coding regions h
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of the S, M and L segments, using the maximum likelihood (ML) method implemented in :/
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PAUP* (Phylogenetic Analysis Using Parsimony, 4.0b10) (51) and RAxML Blackbox a
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webserver (50), as well as a Bayesian approach (19) using MrBayes 3.1 (41). The optimal .o
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evolutionary model was estimated as the GTR+I+Γ model of evolution, as selected by
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jModelTest version 0.1 (38). ML topologies were evaluated by bootstrap analysis of 1,000 N
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neighbor-joining iterations (in PAUP*) or 1,000 ML iterations (in RAxML). Bayesian analysis m
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consisted of two million Markov Chain Monte Carlo (MCMC) generations sampled every 100 r

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generations to ensure convergence across two runs of four chains each, with average standard ,

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deviations of split frequencies less than 0.01 and effective sample sizes over 100, resulting in
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consensus trees supported by posterior-node probabilities. Phylogenetic trees were readdressed
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to construct a tanglegram of host and associated hantaviruses in TreeMap 2.0b (7, 8, 23).
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Mitochondrial DNA (mtDNA) host phylogeny. Genomic DNA was extracted from
tissues using the QIAamp DNA Mini Kit (Qiagen) to verify the taxonomic identity of the
hantavirus-infected eastern moles and to study their phylogenetic relationships. The complete
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1,140-nucleotide cytochrome b gene was amplified by PCR using well-tested primers (forward:
5’-CGAAGCTTGATATGAAAAACCATCGTTG- 3’; and reverse: 5’-
CTGGTTTACAAGACCAGAGTAAT-3’) (21). Host phylogenies based on mtDNA cytochrome
b sequences, along with published sequences for shrews and moles for this gene region, were
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generated, using ML and Bayesian methods described previously (2-4, 23, 24). The tree was
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based on 3,000,000 MCMC generations, sampled every 100 generation and burn-in after 10,000 n
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trees.
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RESULTS h
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RT-PCR detection of hantavirus. Of the 60 eastern moles studied, hantavirus RNA was a
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detected in four of five Scalopus aquaticus captured in Aransas National Wildlife Refuge in .o
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Rockport (latitude 28.042° N; longitude 97.052° W), Texas, in October 1986 (Fig. 1B). Despite
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using a series of oligonucleotide primers that proved useful for the amplification of RKPV and N
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other soricomorph-borne hantaviruses, three or more separate attempts to detect hantavirus RNA m
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in each of the remaining 55 eastern moles captured elsewhere in Texas and in Florida, Kansas, r

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South Carolina and Tennessee failed (Table 1). ,

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Genetic analysis. The complete genome of RKPV, designated strain MSB57412, was
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amplified from one of the four hantavirus-positive eastern moles. Full-length S and L segment
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sequences were also obtained from RKPV strains MSB57411 and MSB57413.
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The full-length 1,830-nucleotide S-genomic segment of RKPV strains MSB57411,
MSB57412 and MSB57413 contained a single open reading frame (ORF), encoding a 428-amino
acid NP (nucleotide positions, 33 to 1,319), and 32- and 511-nucleotide 3’ and 5’ noncoding
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regions (NCR). The hypothetical NSs ORF was absent. Employing prediction software available
in the NPS@structure server, the RKPV NP secondary structure resembled that of other rodent-,
soricid- and talpid-borne hantaviruses, showing 48.7% α helices and 9.95% β sheets, and two
major α-helical domains with the characteristic coiled-coil domain in the N-terminal region
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(residues 1-35 and 51-68) and a central β-pleated sheet at the presumed RNA-binding domain
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(residues 175-217). n
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Despite technical difficulties, previously experienced in amplifying and sequencing other
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soricomorph-borne hantaviruses, the full-length RKPV M segment was obtained from one r
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eastern mole, and in another, a partial sequence of 500 nucleotides was obtained. The complete h
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M-genomic segment of RKPV strain MSB57412 was 3,647 nucleotides, with a predicted :/
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glycoprotein of 1,136 amino acids, starting at nucleotide position 57, and a 179-nucleotide 5’- a
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NCR. Like other rodent- and soricomorph-borne hantaviruses, the RKPV glycoprotein precursor .o
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had the highly conserved WAASA amino-acid motif (amino acid positions 632-636) and four
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potential N-linked glycosylation sites (three in Gn at amino acid positions 135, 401 and 577; and N
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one in Gc at position 929). m
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The full-length 6,558-nucleotide L-genomic segment of RKPV strains MSB57411, r

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MSB57412 and MSB57413 encoded a 2,153-amino acid RNA-dependent RNA polymerase ,

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(nucleotide positions, 44-6505), and exhibited six major conserved motifs (designated premotif
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A and motifs A, B, C, D and E), which have been reported for the RNA polymerase function in
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RNA viruses, including hantaviruses.
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Percent sequence similarities at the nucleotide and amino acid levels were assessed
between the S-, M-, and L-genomic segments of RKPV strain MSB57412 and representative
rodent- and soricomorph-borne hantaviruses. RKPV was highly divergent from other
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hantaviruses, ranging from 28.4-48.2% (nucleotide) and 20.8-57.9% (amino acid), respectively.
RKPV sequences were even more divergent from crocidurine shrew-derived hantaviruses, such
as TPMV strain VRC66412 and MJNV strain Cl05-11, differing overall by more than 36.7 %
(nucleotide) and 38.2 % (amino acid). On the other hand, RKPV exhibited a higher degree of
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sequence homology with cricetid rodent-borne hantaviruses at the nucleotide (S, 67.1-70.7%; M,
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63.4-65.4%; L, 70.1-71.6%) and amino acid (S, 72.2-79.2%; M, 61.6-63.1%; L, 76.0-77.9%) n
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levels. The degrees of sequence variation among RKPV strains MSB57411, MSB57412 and
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MSB57413 were 0.1-1.3% (nucleotide) and 0-0.2% (amino acid) for the S segment and 0.3-1.9% r
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(nucleotide) and 0.5-0.6% (amino acid) for the L segment. An exhaustive search for h
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recombination within the full-length S, M and L segments of RKPV, using multiple :/
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recombination-detection methods, revealed no convincing evidence of genetic recombination. a
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Phylogenetic analysis. Phylogenetic trees, based on the coding regions of the full-length .o
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S, M and L segments, revealed identical topologies with ML and Bayesian methods (Fig. 2).
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Consistently and unexpectedly, the newfound mole-borne hantavirus clustered with Andes virus N
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(ANDV) and Sin Nombre virus (SNV), two prototype hantaviruses harbored by sigmodontine m
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and neotomine rodents, in both the S- and L-genomic segment-based phylogenetic trees and with r

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Puumala virus (PUUV), Tula virus (TULV) and Prospect Hill virus (PHV), well-characterized ,

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arvicolid rodent-associated hantaviruses, in the M-genomic segment phylogenetic tree (Fig. 2).
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The Subfamily Sigmodontinae, Neotominae and Arvicolinae are all within the Family Cricetidae.
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Phylogenetic trees, based on the deduced amino acid sequences of the S, M and L segment-
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encoded proteins of RKPV and other representative hantaviruses, also revealed similar
topologies, with RKPV sharing an ancestral node with hantaviruses harbored by cricetid rodents.
Other shrew- and rodent-borne hantaviruses formed two well-defined groups according to their