Genetic Diversity of Artybash Virus in the Laxmann's Shrew ( Sorex caecutiens )

Abstract

Although based on very limited M and L segment sequences, Artybash virus (ARTV) was proposed previously as a unique hantavirus harbored by the Laxmann's shrew (Sorex caecutiens). To verify this conjecture, lung tissues from 68 Laxmann's shrews, captured during 2006 to 2014 in eastern Siberia, Russia, and Hokkaido, Japan, were analyzed for ARTV RNA using reverse transcription polymerase chain reaction (RT-PCR). ARTV RNA was detected in six Laxmann's shrews. Pairwise alignment and comparison of partial- and full-length S, M, and L segment sequences from these Laxmann's shrews, as well as phylogenetic analyses, using maximum likelihood and Bayesian methods indicated that ARTV was distinct from other soricine shrew-borne hantaviruses and representative hantaviruses harbored by rodents, moles, and bats. Taxonomic identity of the ARTV-infected Laxmann's shrews was confirmed by full-length cytochrome b mitochondrial DNA sequence analysis. Our data indicate that the hantavirus previously known as Amga virus (MGAV) represents genetic variants of ARTV. Thus, the previously proposed designation of ARTV/MGAV should be replaced by ARTV.

Introduction

Recent discovery of genetically distinct hantaviruses (family Bunyaviridae and genus Hantavirus) in multiple species of soricine and crocidurine shrews (order Eulipotyphla, family Soricidae, and subfamilies Soricinae and Crocidurinae), as well as in moles (family Talpidae and subfamilies Talpinae and Scalopinae) and insectivorous bats (order Chiroptera), challenges the conventional view that rodents (order Rodentia and families Muridae and Cricetidae) are the principal reservoir hosts. Broad taxonomic and geographic distribution of hosts indicates that small mammals other than rodents played a significant role in the evolutionary history of hantaviruses and points to the urgent need to understand the diversity, distribution, and phylogeography of hantaviruses worldwide (Bennett et al. 2014, Yanagihara et al. 2014).

Seewis virus (SWSV), originally detected in the Eurasian common shrew (Sorex araneus) in Switzerland (Song et al. 2007), has now been found across the vast distribution of its soricine reservoir host in the Czech Republic (Schlegel et al. 2012), Finland (Kang et al. 2009a, Ling et al. 2014), Germany (Schlegel et al. 2012), Hungary (Kang et al. 2009a), Poland (Gu et al. 2014), Russia (Yashina et al. 2010), Slovakia (Schlegel et al. 2012), and Slovenia (Korva et al. 2013; Resman et al. 2013).

Genetically distinct hantaviruses have also been detected in other Sorex species, including Ash River virus in the North American masked shrew (Sorex cinereus) and Jemez Springs virus in the dusky shrew (Sorex monticolus) (Arai et al. 2008a), Kenkeme virus in the Asian flat-skulled shrew (Sorex roboratus) (Kang et al. 2010), Asikkala virus in the Eurasian pygmy shrew (Sorex minutus) (Radosa et al. 2013), Yakeshi virus in the taiga shrew (Sorex isodon) (Guo et al. 2013), Qian Hu Shan virus in the stripe-back shrew (Sorex cylindricauda) (Zuo et al. 2014), and Sarufutsu virus in the long-clawed shrew (Sorex unguiculatus) (Arai et al. unpublished data).

In addition, based on very limited M and L segment sequences detected in a Laxmann's shrew (Sorex caecutiens) captured near Teletskoye Lake in the Altai Republic of western Siberia, a new hantavirus, named Artybash virus (ARTV), has been proposed. In an attempt to validate its reservoir host species and explore the genetic diversity of ARTV, we analyzed lung tissues collected from Laxmann's shrews trapped in eastern Siberia, Russia, and Hokkaido, Japan. Genetic and phylogenetic analyses, based on partial- and full-length genomes of a hantavirus formerly called Amga virus (MGAV), show that it represents genetic variants of ARTV. Thus, instead of referring to this hantavirus as ARTV/MGAV, as previously proposed (Bennett et al. 2014), ARTV should be the preferred designation.

Materials and Methods

Trapping and sample collection

Laxmann's shrews, which are widely distributed from northern Fennoscandia through Siberia and across northern Japan (Fig. 1A), were captured at three sites in the Sakha Republic, Russia, during July and August 2006 (Fig. 1B and Table 1) and five sites in Hokkaido, Japan, between 2008 and 2014 (Fig. 1C and Table 1). Protocols for trapping, euthanasia, and tissue processing were performed according to well-established guidelines (Animal Care and Use Committee 1998).

FIG. 1.

(A) Map showing the geographic distribution of the Laxmann's shrew (Sorex caecutiens) across Eurasia, where it inhabits broad-leaved forests on lowland and mountainous areas. (B) Map showing trap sites of Laxmann's shrews along the Lena River (N61°45′14.399 E129°31′30.0), Amga River (N61°35′34.810 E132°56′24.053), and Kenkeme River (N62°04′11.996 E128°56′16.821) in the Sakha Republic of Russia. (C) Map showing trap sites of Laxmann's shrews in Koshimizu (N43°56′10.615 E144°26′31.750), Shibetsu (N43°37′43.589 E145°11′34.594), Mukawa (N42°50′30.073 E142°10′01.675), Shari (N43°55′23.998, E144°50′39.800), and Tobetsu (N43°12′11.084 E141°26′58.999) in Hokkaido, Japan. Red circles indicate trap sites of ARTV-positive shrews.

Table 1.

Capture Sites of Laxmann's Shrews Tested for Hantavirus RNA

				Total		Positive
Country	Republic/prefecture	Location	Year	Male	Female	Male	Female
Russia	Sakha	Lena River	2006	4	1	0	0
		Amga River	2006	7	7	4	1
		Kenkeme River	2006	15	5	0	0
Japan	Hokkaido	Koshimizu	2008	1	2	0	0
		Shibetsu	2008	0	1	0	0
		Mukawa	2010	3	1	1	0
		Shari	2011	0	1	0	0
		Tobetsu	2013	2	2	0	0
			2014	9	7	0	0
Total				41	27	5	1

RNA extraction and reverse transcription polymerase chain reaction

Total RNA was extracted from lung tissues using the PureLink Micro-to-Midi Total RNA Purification Kit (Invitrogen, San Diego, CA) or MagDEA^® RNA 100 Kit (Precision System Science; PSS, Matstudo, Japan). cDNA was synthesized using SuperScript III First-Strand Synthesis System (Invitrogen) or PrimeScript™ II 1st strand cDNA Synthesis Kit (Takara Bio, Otsu, Japan) and an oligonucleotide primer (OSM55: 5′-TAGTAGTAGACTCC-3′) designed from the genus-specific conserved 3′-end of the S, M, and L segments of all hantaviruses. For initial screening by RT-PCR, primers were based on highly conserved regions of shrew-borne hantavirus genomes: S (outer: OSM55F, HTN-S6: 5′-AGCTCNGGATCCATNTCATC-3′; inner: Cro2F: 5′-AGYCCNGTNATGRGWGTNRTYGG-3′ and Cro2R: 5′-ANGAYTGRTARAANGANGAYTTYTT-3′) and L (outer: Han-L1880F: 5′-ATGAARNTNTGTGCNATNTTTGA-3′ and Han-L3000R: 5′-GCNGARTTRTCNCCNGGNGACCA-3′; inner: Han-L2520F: 5′-ATNWGHYTDAARGGNATGTCNGG-3′ and Han-L2970R: 5′-CCNGGNGACCAYTTNGTDGCATC-3′). Oligonucleotide primers designed for amplification and sequencing of the full genome of ARTV are shown in Table 2. Nested PCR cycling conditions and methods for DNA sequencing have been previously described (Arai et al. 2008a, 2008b, Kang et al. 2009b).

Table 2.

Oligonucleotide Primers for the Amplification of the S, M, and L Genomic Segments of Artybash Virus

Segment	Primer	Sequence (5′→3′)	Length	Polarity
S	OSM55	TAG TAG TAG ACT CC	14	+
	CAS-381F	CAN GTG GNC ARA CWG CWG AYT GG	23	+
	Han-S604F	GCH GAD GAR HTN ACA CCN GG	20	+
	Cro2F	AGY CCN GTN ATG RGW GTN RTY GG	24	+
	Han-S974R	TCN GGN GCH CHN GCA AAN AHC CA	23	−
	Cro2R	ANG AYT GRT ARA ANG ANG AYT TYT T	25	−
	HTN-S6	AGC TCN GGA TCC ATN TCA TC	20	−
	MKWS-1612R	AGA GTG TTT GAG GTA GTG GAG TG	23	−
	Han-S3R1	TAG TAG TAN NCT CCN	15	−
	PHS-3endR	TAG TAG TAT ACT CCT TGA AAA GC	23	−
M	OSM55	TAG TAG TAG ACT CC	14	+
	JAM-241F	GTT CAT GYA GYA TGG ATG TRC A	22	+
	MKWM-433R	TNC GCA TCT TGTA RGC CTG YTC	22	−
	HTM-1490F	TGT GTN CCW GGN TTY CAT GGN T	22	+
	CAM-1685F	ACN AAG GGY TCW ATG GTN TGT GA	23	+
	SHM-1706R	CAT ACA TCA CAN ACC ATW GAA CC	23	−
	SHM-2371F	TGY AAC CCN GTN GAT TGY CCW GG	23	+
	MKWM-2458R	ATA GGC AGT CCC GAC AGC CTT	21	−
	MKWM-2631R	CAT GAT RTC NCC AGG RTC NCC	21	−
	TM-2957R	GAA CCC CAD GCC CCNTCY AT	20	−
	AM-3280F	CCA TAT CTA TGA TGA TGG TGC	21	+
	PHM-3endR	TAG TAG TAG ACT CCG CAA GAA	21	−
L	OSM55	TAG TAG TAG ACT CC	14	+
	PHL-173F	GAT WAA GCA TGA YTG GTC TGA	21	+
	CAL-539F	TAT NTC MAC ACA RTG GCC WAG T	22	+
	HTL-577R	CMC CNK CAT THC KYC TAC TNG GC	23	−
	SL-761F	CCT ATT TNA GTA YTG TAA GGW NTG G	25	+
	Han-L1880F	CAR AAR ATG AAR NTN TGT GC	20	+
	CS-L-2319R	CTT CYT CAT TYA CAT TMC CAT G	22	−
	Han-L2520F	ATN WGH YTD AAR GGN ATG TCN GG	23	+
	Tal-L2855F	GAA AGG GCA TTN MGA TGG GCN TCA GG	26	+
	Tal-L2928F	GNA AAY TNA TGT ATG TNA GTG C	22	+
	HAN-L-F1	ATG TAY GTB AGT GCW GAT GC	20	+
	HAN-L-F2	TGC WGA TGC HAC NAA RTG GTC	21	+
	Han-L3000R	GCN GAR TTR TCN CCN GGN GAC CA	23	−
	HAN-L-R2	GCR TCR TCW GAR TGR TGD GCA A	22	−
	HAN-L-R1	AAC CAD TCW GTY CCR TCA TC	20	−
	MKWL-4020F	YAA YCA TGA RAG GTT GGG NGA G	22	+
	MKWL-4132F	GAG ACT TGG AGT GAN CAA CAY CC	23	+
	SL-4321R	AAYGTTACCCAYTCACCAYCCA	22	−
	PHL-5167R	CAT AYT GYT THC CTG AAT AWG C	22	−
	CAL-6375F	GKR TWG TKA ARG GYT GGG GWG A	22	−
	HNL-6388R	CTC WGT YAA RTC ATA WGG ATC	21	−
	HNL-3R	TAG TAG TAK GCT CCG	15	−

Mixed bases: B = G, T, C; D = G, A, T; H = A, T, C; K = G, T; M = A, C; N = A, T, G, C; R = A, G; W = A, T; and Y = C, T.

Genetic and phylogenetic analysis

Pairwise alignment and comparison of partial- and full-length S, M, and L segment sequences of hantaviruses from Laxmann's shrews, as well as other representative rodent-, shrew-, mole-, and bat-borne hantaviruses, were performed using the ClustalW method (Thompson et al. 1994). Phylogenetic analyses were conducted using maximum likelihood and Bayesian methods, implemented in MrBayes 3.1 (Ronquist and Huelsenbeck 2003) and RAxML (Stamatakis et al. 2008), under the best-fit GTR + I + Γ model of evolution using MrModeltest 2.3 (Posada 2008). 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 100 iterations, and posterior node probabilities were based on 10 million generations and estimated sample sizes more than 100 (implemented in MrBayes).

Host species identification

Because shrews are inherently difficult to identify by morphological features alone, host verification of ARTV-infected shrews was confirmed by analyzing the entire 1140-base pair cytochrome b gene of mitochondrial DNA (mtDNA), amplified by PCR, using modified universal primers (Cy-14726F: 5′-GACYARTRRCATGAAAAAYCAYCGTTGT-3′ and Cy-15909R: 5′-CYYCWTYIYTGGTTTACAAGACYAG-3′) (Arai et al. 2008b). A Bayesian approach, as described above, with midpoint rooting was used to determine the phylogenetic relationship between Laxmann's shrews and other shrew and mole species known to harbor distinct hantaviruses.

Results and Discussion

ARTV RNA was detected by RT-PCR in five of 39 (12.8%) and one of 29 (3.4%) Laxmann's shrews captured in Russia and Japan, respectively (Table 1). All but one of the six ARTV-infected shrews were males.

Analysis of the full-length nucleotide and amino acid sequences of the S, M, and L segments (GenBank accession numbers: KF974360, KF974359, and KF974361, respectively) of ARTV strain Mukawa AH301 showed considerable divergence from representative hantaviruses harbored by shrews in the Soricinae subfamily in Eurasia (Table 3): S, 22.9–27.3% and 13.3–16.0%; M, 20.9–23.8% and 8.4–12.7%; and L, 20.2–22.1% and 5.7–8.7%, respectively. In contrast, alignment and comparison between ARTV strain Mukawa AH301 and the very limited S, M, and L segments of ARTV strains ART502, Galkino2712, and Parnaya1205 showed higher sequence similarity at the amino acid level (Table 3).

Table 3.

Sequence Identities of the Full-Length S, M, and L Segments of ARTV Strain Mukawa AH301 from Sorex caecutiens in Japan to Other Representative Hantaviruses

	S		M		L
Virus	1290 nt	429 aa	3420 nt	1139 aa	6456 nt	2151 aa
ARTV Amga MSB148558	1290 nt^a	429 aa	1088 nt	362 aa	4598 nt	1532 aa
	79.8%	93.5%	78.1%	95.0%	79.7%	96.0%
ARTV Amga MSB148559	—	—	—	—	1401 nt	467 aa
					79.8%	97.4%
ARTV Amga MSB148436	—	—	727 nt	242 aa	2454 nt	818 aa
			78.3%	97.1%	80.2%	97.3%
ARTV Amga MSB148457	—	—	683 nt	227 aa	2397 nt	799 aa
			78.2%	96.9%	80.0%	97.4%
ARTV Amga MSB148347	582 nt	194 aa	729 nt	243 aa	1302 nt	434 aa
	76.5%	88.7%	78.2%	97.5%	80.2%	97.2%
ARTV ART502	837 nt	279 aa	247 nt	82 aa	347 nt	115 aa
	76.9%	92.8%	79.8%	96.3%	80.1%	96.5%
ARTV Galkino2712	559 nt	186 aa	—	—	—	—
	75.9%	89.2%
ARTV Parnaya1205	767 nt	255 aa	—	—	347 nt	115 aa
	76.8%	92.6%			80.4%	95.7%
SWSV mp70	1290 nt^a	429 aa	250 nt	83 aa	3288 nt	1096 aa
	77.1%	86.7%	78.0%	91.6%	77.9%	94.3%
KKMV MSB148794	1290 nt^a	429 aa	1002 nt	334 aa	4295 nt	1432 aa
	74.6%	83.3%	79.1%	91.6%	77.9%	91.5%
QHSV YN05-284	1290 nt^a	429 aa	1430 nt	476 aa	450 nt	149 aa
	72.7%	83.5%	76.2%	89.9%	79.8%	93.3%
YKSV Si-210	1290 nt^a	429 aa	3420 nt^a	1139 aa	339 nt	113 aa
	74.3%	84.0%	76.8%	88.2%	79.4%	91.3%
ASIV Drahany	1290 nt^a	429 aa	3420 nt^a	1139 aa	1729 nt	576 aa
	73.2%	84.7%	76.8%	87.3%	79.3%	93.8%
ARRV MSB73418	1059 nt	353 aa	—	—	347 nt	115 aa
	62.5%	64.0%			75.8%	86.1%
JMSV MSB144475	1290 nt^a	429 aa	1412 nt	470 aa	4928 nt	1642 aa
	65.8%	69.1%	72.6%	79.8%	74.8%	85.7%
CBNV CBN-3	1287 nt^a	428 aa	3420 nt^a	1139 aa	412 nt	137 aa
	64.7%	69.0%	68.9%	73.9%	75.0%	84.9%
TGNV Tan826	442 nt	147 aa	—	—	412 nt	137 aa
	62.4%	61.2%			71.8%	77.4%
BOWV VN1512	1296 nt^a	431 aa	3438 nt^a	1145 aa	6477 nt^a	2158 aa
	63.6%	67.8%	67.0%	66.8%	70.9%	78.2%
AZGV KBM15	540 nt	180 aa	687 nt	229 aa	4556 nt	1518 aa
	60.7%	60.6%	71.0%	73.8%	71.4%	77.7%
JJUV SH42	1287 nt^a	428 aa	3408 nt	1135 aa	6474 nt^a	2157 aa
	60.8%	61.8%	66.2%	65.8%	71.3%	77.6%
MJNV Cl 05-11	1311 nt^a	436 aa	3363 nt^a	1120 aa	6450 nt^a	2149 aa
	53.8%	45.3%	51.7%	42.5%	62.3%	61.7%
TPMV VRC66412	1308 nt^a	435 aa	3366 nt^a	1121 aa	6450 nt^a	2149 aa
	50.9%	44.2%	52.3%	43.1%	61.6%	61.7%
ULUV FMNH158302	1187 nt	395 aa	1389 nt	463 aa	6459 nt^a	2152 aa
	52.8%	45.7%	52.8%	42.8%	61.9%	61.9%
KMJV FMNH174124	1269 nt^a	423 aa	1240 nt	413 aa	6447 nt^a	2148 aa
	53.7%	47.6%	53.8%	45.1%	63.0%	62.7%
RKPV MSB57412	1287 nt^a	428 aa	3411 nt	1136 aa	6462 nt^a	2153 aa
	60.1%	60.3%	57.7%	51.8%	65.3%	67.9%
OXBV Ng1453	1287 nt^a	428 aa	3426 nt	1141 aa	4345 nt	1448 aa
	64.4%	68.3%	66.9%	67.4%	72.4%	80.3%
ASAV N10	1302 nt^a	433 aa	3423 nt^a	1140 aa	6456 nt^a	2151 aa
	65.1%	70.0%	71.6%	77.6%	75.4%	85.0%
NVAV Te34	1287 nt^a	428 aa	3384 nt^a	1127 aa	6474 nt^a	2157 aa
	56.1%	49.7%	53.5%	44.8%	63.6%	62.1%

For virus names and hosts, refer to legend of Figure 2.

Complete coding region.

—, sequences unavailable; aa, amino acids; ARTV, Artybash virus; nt, nucleotides; SWSV, Seewis virus.

Phylogenetic analyses of ARTV strain Mukawa AH301, based on the full-length coding regions (comprising 1290-nucleotide S, 3420-nucleotide M, and 6456-nucleotide L segment sequences), showed similar topologies, with ARTV strains from the Sakha Republic and ARTV strains detected independently in Laxmann's shrews captured in the Altai Republic and Khabarovsk Krai of Russia forming a separate geographic-specific cluster (Fig. 2). Moreover, ARTV strains were distinct from recently detected hantaviruses harbored by other Sorex shrew species in Eurasia. In contrast, differences in the phylogenetic positions of some soricine shrew-borne hantaviruses may be attributed to the limited sequences, especially for the M and L segments.

FIG. 2.

Phylogenetic trees generated by Bayesian and maximum likelihood methods under the best-fit GTR + I + Γ model of evolution as estimated based on the entire coding regions of the full-length 1290-nucleotide S, 3420-nucleotide M, and 6456-nucleotide L genomic segments of Artybash virus (ARTV) strain Mukawa AH301. Phylogenetic trees show the positions of ARTV Mukawa AH301 (GenBank accession numbers: S: KF974360; M: KF974359; and L: KF974361), ARTV Amga MSB148558 (S: KM201411; M: KM201412; and L: KM201413), ARTV Amga MSB148559 (S: KM201414), ARTV Amga MSB148436 (M: KM201415 and L: KM201416), ARTV Amga MSB148457 (M: KM201417 and L: KM201418), ARTV Amga MSB148347 (S: KM201419; M: KM201420; and L: KM201421), ARTV ART502 (S: KM288698; M: EU424340; and L: EU424339), ARTV Galkino2712 (S: KM288699), and ARTV Parnaya1205 (S: KU253274 and L: KU253275) from Sorex caecutiens. Also shown are Ash River virus (ARRV MSB73418, S: EF650086 and L: EF619961) from Sorex cinereus, Jemez Springs virus (JMSV MSB144475, S: FJ593499; M: FJ593500; and L: FJ593501) from Sorex monticolus, Seewis virus (SWSV mp70, S: EF636024; M: EF636025; and L: EF636026) from Sorex araneus, Asikkala virus (ASIV Drahany, S: KC880342; M: KC880345; and L: KC880348) from Sorex minutus, Kenkeme virus (KKMV MSB148794, S: GQ306148; M: GQ306149; and L: GQ306150) from Sorex roboratus, Qian Hu Shan virus (QHSV YN05-284, S: GU566023; M: GU566022; and L: GU566021) from Sorex cylindricauda, Yakeshi virus (YKSV Si-210, S: JX465423; M: JX465403; and L: JX465389) from Sorex isodon, and Cao Bang virus (CBNV CBN-3, S: EF543524; M: EF543526; and L: EF543525) from Anourosorex squamipes, as well as Thottapalayam virus (TPMV VRC66412, S: AY526097 and L: EU001330) from Suncus murinus, Imjin virus (MJNV Cl 05-11, S: EF641804; M: EF641798; and L: EF641806) from Crocidura lasiura, Azagny virus (AZGV KBM15, S: JF276226; M: JF276227; and L: JF276228) from Crocidura obscurior, Tanganya virus (TGNV Tan826, S: EF050455 and L: EF050454) from Crocidura theresea, Bowé virus (BOWV VN1512, S: KC631782; M: KC631783; and L: KC631784) from Crocidura douceti, Jeju virus (JJUV SH42, S: HQ663933; M: HQ663934; and L: HQ663935) from Crocidura shantungensis, Uluguru virus (ULUV FMNH158302, S: JX193695; M: JX193696; and L: JX193697) from Myosorex geata, and Kilimanjaro virus (KMJV FMNH174124, S: JX193698; M: JX193699; and L: JX193700) from Myosorex zinki. Mole-borne hantaviruses include Asama virus (ASAV N10, S: EU929072; M: EU929075; and L: EU929078) from Urotrichus talpoides, Nova virus (NVAV Te34, S: KR072621; M: KR072622; and L: KR072623) from Talpa europaea, Oxbow virus (OXBV Ng1453, S: FJ5339166; M: FJ539167; and L: FJ593497) from Neurotrichus gibbsii, and Rockport virus (RKPV MSB57412, S: HM015223; M: HM015222; and L: HM015221) from Scalopus aquaticus. Bat-borne hantaviruses include Magboi virus (MGBV MGB1209, L: JN037851) from Nycteris hispida, Mouyassué virus (MOYV KB576, L: JQ287716) from Neoromicia nanus, Huangpi virus (HUPV Pa-1, S: JX473273 and L: JX465369) from Pipistrellus abramus, Longquan virus (LQUV Ra-25, S: JX465415; M: JX465397; and L: JX465381) from Rhinolophus sinicus, Laibin virus (LBV BT20, S: KM102247; M: KM102248; and L: KM102249) from Taphozous melanopogon, and Xuan Son virus (XSV VN1982B4, S: KC688335; M: KU976427; and L: JX912953) from Hipposideros pomona. Other taxa include Sin Nombre virus (SNV NMH10, S: NC_005216; M: NC_005215; and L: NC_005217), Andes virus (ANDV Chile9717869, S: AF291702; M: AF291703; and L: AF291704), Prospect Hill virus (PHV PH-1, S: Z49098; M: X55129; and L: EF646763), Tula virus (TULV M5302v, S: NC_005227; M: NC_005228; and L: NC_005226), Puumala virus (PUUV Sotkamo, S: NC_005224; M: NC_005223; and L: NC_005225), Dobrava virus/Belgrade virus (DOBV/BGDV Greece, S: NC_005233; M: NC_005234; and L: NC_005235), Hantaan virus (HTNV 76-118, S: NC_005218; M: NC_005219; and L: NC_005222), Soochong virus (SOOV SOO-1, S: AY675349; M: AY675353; and L: DQ056292), Sangassou virus (SANGV SA14, S: JQ082300; M: JQ082301; and L: JQ082302) and Seoul virus (SEOV 80-39, S: NC_005236; M: NC_005237; and L: NC_005238). The numbers at each node are posterior node probabilities (left) based on 150,000 trees: two replicate Markov chain Monte Carlo runs, consisting of six chains of 10 million generations each sampled every 100 generations with a burn-in of 25,000 (25%) and bootstrap values (right), based on 100 replicates executed on the RAxML BlackBox Web server.

The identities of the six ARTV-infected shrews were confirmed as Laxmann's shrews (GenBank no. KF974362 for strain AH301; GU562415, GU562416, GU562417, GU562418, and GU562422 for strains MSB148558, MSB148559, MSB148436, MSB148457, and MSB148347, respectively). The cytochrome b mtDNA sequences of the ARTV-infected Laxmann's shrews from Japan and Russia differed by 4.0–6.4%. The ARTV-negative shrews from Japan were also identified as Laxmann's shrews by analysis of the 1140-nucleotide cytochrome b mtDNA gene (GenBank nos. KU760858 to KU760884). However, the full-length cytochrome b mtDNA sequence variation was 0–1.1% and 0.5–1.1% among Laxmann's shrews in Japan and Russia, respectively.

Host phylogenies based on mtDNA cytochrome b sequences of shrew and mole species, which harbor genetically distinct hantaviruses, showed two separate lineages of Laxmann's shrews (Fig. 3). This was consistent with previous studies suggesting at least two genetic races of Laxmann's shrews distributed in Eurasia and Japan (Ohdachi et al. 2003). That is, based on analysis of the full-length cytochrome b gene, Laxmann's shrews were separated into Hokkaido and Eurasian Continent–Sakhalin–Cheju clusters. Individuals in the latter cluster do not always reflect the geographical proximity of their capture locations, which is consistent with an ancestral isolation of the Hokkaido population, occurring ∼13,000 years before present (Tanabe et al. 2015), and recent rapid range expansion of the modern Eurasian Continent–Sakhalin–Cheju population (Ohdachi et al. 2003).

FIG. 3.

Bayesian phylogenetic tree based on 1140 nucleotides of the cytochrome b mtDNA of shrews and moles (order Eulipotyphla and families Talpidae and Soricidae). The tree was rooted using Elephantulus (order Macroscelidea, GenBank nos. DQ901019, DQ901206, and DQ901201) as the outgroup. Numbers at nodes indicate posterior probability values based on 150,000 trees: two replicate Markov chain Monte Carlo runs, consisting of six chains of 10 million generations each sampled every 100 generations with a burn-in of 25,000 (25%). GenBank numbers for all taxa are provided in the tree.

The phylogeographic variation in ARTV reflects the distinct evolutionary histories of these variants. Similar patterns of geographic variation have also been reported for rodent-borne hantaviruses. For example, geographic-specific genetic variants have been reported for Puumala virus in the bank vole (Myodes glareolus) (Garanina et al. 2009), Tula virus in the European common vole (Microtus arvalis) (Song et al. 2004), and Andes virus in the colilargo (Oligoryzomys longicaudus) (Torres-Perez et al. 2011). Both Muju virus in the royal vole (Myodes regulus) (Lee et al. 2014) and Hokkaido virus in the grey red-backed vole (Myodes rufocanus) and northern red-backed vole (Myodes rutilus) (Yashina et al. 2015) may represent genotypes of Puumala virus. These examples point to the urgency of studying the phylogeographic variation of viruses and their hosts to provide an essential foundation for understanding their evolutionary histories and as a prelude to forecasting their future emergence under changing environmental conditions (Hope et al. 2013, Campbell et al. 2015).

Although ARTV was detected in a single Laxmann's shrew captured in the Mukawa area in Japan, the evidence is compelling that this Sorex shrew species harbors ARTV across its broad geographic range. Our findings suggest that Laxmann's shrews, resident on Hokkaido Island before the geologic separation from the Eurasian continent, might have already been infected with ARTV. Renewed attempts to isolate ARTV from Laxmann's shrews captured in Eurasian Continent–Sakhalin–Cheju and Japan are warranted to better understand its evolutionary origins and phylogeography, as well as its pathogenic potential in humans.

Footnotes

Acknowledgments

We thank Masashi Hamada, Yu Ikeyama, and Keita Aoki for their technical assistance and Kimiyuki Tsuchiya, Hidenori Nishizawa, and Kyle R. Taylor for supporting field investigations. We also thank Nikolai Dokuchaev, Andrew G. Hope, Anson Koehler, Stephen O. MacDonald, Robert A. Nofchissey, and Albina Tsvetkova for assistance with field collections made in Russia through the National Science Foundation support (DEB0415668). In addition, this research was supported in part by a grant-in-aid for Research on Emerging and Re-emerging Infectious Diseases, Health Labour Sciences Research Grant in Japan (H22-Shinko-Ippan-006 and H25-Shinko-Ippan-008), a grant-in aid for Research Program on Emerging and Re-emerging Infectious Diseases, Japan Agency for Medical Research and Development (AMED), and a grant-in-aid from the Japan Society for the Promotion of Science (S13205 Invitation Fellowship for Research in Japan), as well as by the U.S. Public Health Service grants R01AI075057 from the National Institute of Allergy and Infectious Diseases and P20GM103516 and P30GM114737 (Centers of Biomedical Research Excellence) from the National Institute of General Medical Sciences, National Institutes of Health.

Author Disclosure Statement

No competing financial interests exist.

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