Molecular phylogeny of a newfound hantavirus in the Japanese shrew mole (Urotrichus talpoides)

Abstract

Recent molecular evidence of genetically distinct hantaviruses in shrews, captured in widely separated geographical regions, corroborates decades-old reports of hantavirus antigens in shrew tissues. Apart from challenging the conventional view that rodents are the principal reservoir hosts, the recently identified soricid-borne hantaviruses raise the possibility that other soricomorphs, notably talpids, similarly harbor hantaviruses. In analyzing RNA extracts from lung tissues of the Japanese shrew mole (Urotrichus talpoides), captured in Japan between February and April 2008, a hantavirus genome, designated Asama virus (ASAV), was detected by RT-PCR. Pairwise alignment and comparison of the S-, M-, and L-segment nucleotide and amino acid sequences indicated that ASAV was genetically more similar to hantaviruses harbored by shrews than by rodents. However, the predicted secondary structure of the ASAV nucleocapsid protein was similar to that of rodent- and shrew-borne hantaviruses, exhibiting the same coiled-coil helix at the amino terminus. Phylogenetic analyses, using the maximum-likelihood method and other algorithms, consistently placed ASAV with recently identified soricine shrew-borne hantaviruses, suggesting a possible host-switching event in the distant past. The discovery of a mole-borne hantavirus enlarges our concepts about the complex evolutionary history of hantaviruses.

Fig. 1.

Japanese shrew mole (Urotrichus talpoides) (family Talpidae, subfamily Talpinae), one of two endemic shrew mole species found only in Japan.

Fig. 2.

Consensus secondary structure of N protein of ASAV and representative rodent- and soricid-borne hantaviruses, predicted using a high-performance method implemented on the NPS@ structure server (47). As shown, the ASAV N protein was very similar to that of other hantaviruses, characterized by the same coiled-coil helix at the amino terminal end and similar secondary structure motifs at their carboxyl terminals. The predicted structures were represented by colored bars to visualize the schematic architecture: α-helix, blue; β-sheet, red; coil, magenta; unclassified, gray. For simplicity, turns and other less frequently occurring secondary structural elements were omitted. All sequences are numbered from Met-1.

Fig. 3.

Phylogenetic trees generated by the ML method, using the GTR+I+G model of evolution as estimated from the data, based on the alignment of the coding regions of the full-length (A) 1,302-nucleotide S and (B) 3,423-nucleotide M segments, and partial (C) 6,126-nucleotide L-genomic segment of ASAV. The phylogenetic positions of ASAV strains H4, N9, and N10 are shown in relationship to representative murinae rodent-borne hantaviruses, including Hantaan virus (HTNV 76–118, NC_005218, NC_005219, NC_005222), Soochong virus (SOOV SOO-1, AY675349, AY675353, DQ056292), Dobrava virus (DOBV AP99, NC_005233, NC_005234, NC_005235), and Seoul virus (SEOV HR80–39, NC_005236, NC_005237, NC_005238); arvicolinae rodent-borne hantaviruses, including Tula virus (TULV M5302v, NC_005227, NC_005228, NC_005226), Puumala virus (PUUV Sotkamo, NC_005224, NC_005223, NC_005225), and Prospect Hill virus (PHV PH-1, Z49098, X55129, EF646763); and a neotominae rodent-borne hantavirus, Sin Nombre virus (SNV NMH10, NC_005216, NC_005215, NC_005217). Also shown are Thottapalayam virus (TPMV VRC, AY526097, EU001329, EU001330) from the Asian house shrew (Suncus murinus); Imjin virus (MJNV 05–11, EF641804, EF641798, EF641806) from the Ussuri white-toothed shrew (Crocidura lasiura); Cao Bang virus (CBNV CBN-3, EF543524, EF543526, EF543525) from the Chinese mole shrew (Anourosorex squamipes); Ash River virus (ARRV MSB 73418, EF650086, EF619961) from the masked shrew (Sorex cinereus); Jemez Springs virus (JMSV MSB89332, EF619962, EF619960) from the dusky shrew (Sorex monticolus); and Seewis virus (SWSV mp70, EF636024, EF636025, EF636026) from the Eurasian common shrew (Sorex araneus). The numbers at each node are posterior node probabilities based on 30,000 trees: two replicate MCMC runs consisting of six chains of 3 million generations each sampled every 1,000 generations with a burn-in of 7,500 (25%). The scale bar indicates nucleotide substitutions per site. GenBank accession numbers: ASAV S segment (H4, EU929070; N9, EU929071; N10, EU929072); ASAV M segment (H4, EU929073; N9, EU929074; N10, EU929075); and ASAV L segment (H4, EU929076; N9, EU929077; N10, EU929078).

Fig. 4.

Confirmation of host identification of ASAV-infected Urotrichus talpoides by mtDNA sequencing. Phylogenetic tree, based on the 1,140-nucleotide cytochrome b (cyt b) gene, was generated by the ML method. The phylogenetic positions of Urotrichus talpoides H4 (EU918369), N9 (EU918370), and N10 (EU918371) are shown in relationship to other Urotrichus talpoides cyt b sequences from GenBank (Ut76835: AB076835; Ut76834: AB076834; Ut76833: AB076833; Ut76832: AB076832), as well as other talpids, including Desmana moschata (AB076836), Talpa altaica (AB037602), Talpa europea (AB076829), Mogera imaizumii (AB037616), Dymecodon pilirostris (AB076830), and Bos frontalis (EF061237). Also shown are representative murinae rodents, including Apodemus agrarius (AB303226), Apodemus flavicollis (AB032853), and Rattus norvegicus (DQ439844); arvicolinae rodents, including Microtus arvalis (EU439459), Myodes glareolus (DQ090761), and Microtus pennsylvanicus (AF119279); and a neotominae rodent, Peromyscus maniculatus (AF119261), as well as crocidurinae shrews, including Suncus murinus (DQ630386), Crocidura lasiura (AB077071), and Crocidura dsinezumi (AB076837); and soricinae shrews, including Anourosorex squamipes (AB175091), Blarina brevicauda (DQ630416), Sorex cinereus (EU088305), Sorex monticolus (AB100273), Sorex araneus (DQ417719), Sorex caecutiens (AB028563), Sorex unguiculatus (AB028525), and Sorex gracillimus (AB175131). The numbers at each node are posterior node probabilities based on 30,000 trees: two replicate MCMC runs consisting of six chains of 3 million generations each sampled every 1,000 generations with a burn-in of 7,500 (25%). The scale bar indicates nucleotide substitutions per site.