Short Report: Phylogenetically Distinct Hantaviruses in the Masked Shrew (Sorex cinereus) and Dusky Shrew (Sorex monticolus) in the United States

A limited search for hantaviruses in lung and liver tissues of Sorex shrews (family Soricidae, subfamily Soricinae) revealed phylogenetically distinct hantaviruses in the masked shrew (Sorex cinereus) from Minnesota and in the dusky shrew (Sorex monticolus) from New Mexico and Colorado. The discovery of these shrew-borne hantaviruses, named Ash River virus and Jemez Springs virus, respectively, challenges the long-held dogma that rodents are the sole reservoir hosts and forces a re-examination of their co-evolutionary history. Also, studies now underway are aimed at clarifying the epizootiology and pathogenicity of these new members of the genus Hantavirus. Based on phylogenetic analyses of full-length viral genomic sequences and host mitochondrial DNA (mtDNA) sequences, hantaviruses segregate into clades that parallel the evolution of murinae, arvicolinae, neotominae, and sigmodontinae rodents. Whether insectivores (or soricomorphs), which are sympatric with rodents, are involved in the evolutionary origins of hantaviruses has not been systematically studied, despite previous reports of hantavirus antigens in tissues of the Eurasian common shrew (Sorex araneus), alpine shrew (Sorex alpinus), Eurasian water shrew (Neomys fodiens), and common mole (Talpa europea) captured in Russia and the former Yugoslavia. Also, the isolation of Thottapalayam virus (TPMV), from the Asian house shrew (Suncus murinus) in India, would suggest that soricids might serve as legitimate reservoir hosts of hantaviruses. Armed with the newly acquired full genome of TPMV and emboldened by our recent discovery of genetically distinct hantaviruses in soricine shrews, including Camp Ripley virus (RPLV) in the northern short-tailed shrew (Blarina brevicauda) in the United States and Cao Bang virus (CBNV) in the Chinese mole shrew (Anourosorex squamipes) in Vietnam, we launched a small-scale search for soricid-borne hantaviruses by accessing the archival tissue collection of Sorex (family Soricidae, subfamily Soricinae), housed in the Museum of Southwestern Biology at the University of New Mexico. RNA extracts, from 100 mg each of frozen lung or liver tissues from the masked shrew (S. cinereus), dusky or montane shrew (S. monticolus), dwarf shrew (S. nanus), northern water shrew (S. palustris), Trowbridge shrew (S. trowbridgii), tundra shrew (S. tundrensis), and vagrant shrew (S. vagrans), captured in the United States between 1983 and 2005 (Table 1), were analyzed for hantavirus sequences by reverse transcription-polymerase chain reaction (RT-PCR). The remarkably divergent genomes of soricid-borne hantaviruses presented challenges in designing suitable primers, but we eventually succeeded in amplifying regions of the S (outer OSM55: 5 -TAGTAGTAGACTCC-3 and HTN-S6: 5 -AGCTCIGGATCCATITCATC-3 ; inner Cro2R: 5 -AIGAYTGRTARAAIGAIGAYTTYTT-3 and PHS-5F: 5 -TAGTAGTAGA CTCCTTRAARAGC-3 ) and L (outer HAN-L-F1: 5 -ATGTAYGTBAGTGCWGATGC-3 and HAN-L-R1: 5 -AACCADTCWGTYCCRTCATC-3 ; inner HAN-L-F2: 5 -TGCWGATGCHACIAARTGGTC-3 and HAN-L-R2: 5 -GCRTCRTCWGARTGRTGDGCA A-3 ) segments. Firstand second-round PCR were performed in 20L reaction mixtures, containing 250 M dNTP, 2 mM MgCl2, 1 U of AmpliTaq polymerase (Roche, Basel, Switzerland), and 0.25 M of each primer. Initial denaturation at 94°C for 2 min was followed by two cycles each of denaturation at 94°C for 30 sec, two-degree step-down annealing from 46°C to 38°C for 40 sec, and elongation at 72°C for 1 min, then 30 cycles of denaturation at 94°C for 30 sec, annealing at 42°C for 40 sec, and elongation at 72°C for 1 min, in a GeneAmp PCR 9700 thermal cycler (Perkin-Elmer, Waltham, MA). PCR products were separated by agarose gel electrophoresis and purified using the Qiaex Gel Extraction Kit (Qiagen, Hilden, Germany). DNA was sequenced directly using an ABI Prism 377XL Genetic Analyzer (Applied Biosystems, Foster City, CA). Sequences were then processed using the Genetyx version 6 software (Genetyx Corporation, Tokyo, Japan) and aligned using Clustal W and transAlign. For phylogenetic analysis, maximum-likelihood consensus trees were generated by the Bayesian Metropolis–Hastings Markov Chain Monte Carlo (MCMC) tree-sampling methods as implemented by Mr. Bayes using a GTR+I+G model of evolution, as selected by Modeltest v.3.7, partitioned by codon position. Of the 54 Sorex shrews studied, hantavirus Sand L-segment sequences were detected in S. cinereus, captured near the Ash River Station in Voyageur’s National Park (48°26 N, 92°00 W) in St. Louis County, Minnesota, in August 1994, and in Chippewa National Forest in Cass County, Minnesota, in July 1983, as well as in S. monticolus, captured near Jemez Springs (35°48 N, 106°30 W) in Sandoval County, New Mexico, in September 1996, September 1998 and September 2000, and along Trapline ECJ (4027 N, 10600 W) in Jackson County, Colorado, in July 1994 (Table 1). The newly identified hantavirus sequences were designated Ash River virus (ARRV) and Jemez Springs virus (JMSV), respectively. Host identification was verified by morphologic assessment of voucher specimens and by mtDNA sequence analysis of a * Address correspondence to Richard Yanagihara, Pacific Center for Emerging Infectious Diseases Research, John A. Burns School of Medicine, University of Hawai‘i at Manoa, 651 Ilalo Street, BSB320L, Honolulu, HI 96813. E-mail: [email protected] Am. J. Trop. Med. Hyg., 78(2), 2008, pp. 348–351 Copyright © 2008 by The American Society of Tropical Medicine and Hygiene