Newfound Hantavirus in Chinese Mole Shrew, Vietnam

Sequence analysis of the full-length medium segment and the partial small and large segments of a hantavirus, detected by reverse transcription–PCR in lung tissues of the Chinese mole shrew (Anourosorex squamipes) captured in Cao Bang Province, Vietnam, in December 2006, indicated that it is genetically distinct from rodentborne hantaviruses.

I nsectivores (or soricomorphs) have been largely ignored as being important in the evolutionary dynamics of hantaviruses, despite the isolation of Thottapalayam virus (TPMV) from the Asian house shrew (Suncus murinus) (1,2) and the detection of hantavirus antigens in tissues of the Eurasian common shrew (Sorex araneus), alpine shrew (S. alpinus), Eurasian water shrew (Neomys fodiens), and common mole (Talpa europea) (3). Recently, genetically distinct hantavirus sequences have been found by reverse transcription-PCR in the Therese shrew (Crocidura theresae) in Guinea (4) and the northern short-tailed shrew (Blarina brevicauda) in the United States (5). In addition, a phylogenetically distinct hantavirus has been isolated from lung tissues of the Ussuri shrew (C. lasiura), captured along the Imjin River near the demilitarized zone in South Korea (J.-W. Song and R. Yanagihara, unpub. data).
Gene-amplifi cation reactions were performed in 50-μL reaction mixtures, containing 200 μmol deoxyribonucleoside triphosphate, 0.5 U of super-therm polymerase (PureTech Co., Ltd, Seoul, South Korea), 1 μg of cDNA, and 10 pmol of each primer. Initial denaturation, at 94°C for 5 min, was followed by touchdown cycling with denaturation at 94°C for 40 s, annealing from 50°C to 37°C for 40 s, elongation at 68°C for 1 min 20 s, then 25 cycles of denaturation at 94°C for 40 s, annealing at 40°C for 40 s, and elongation at 68°C for 1 min 20 s in a Mastercycler ep gradient S (Eppendorf AG, Hamburg, Germany). PCR products were purifi ed by the Wizard PCR Preps DNA Purifi cation System (Promega). DNA sequencing of at least 3 clones of each amplicon was performed in both directions with the dye primer cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, USA) on an automated sequencer (Model 377, Perkin Elmer Co., Waltham, MA, USA) (6).
Phylogenetic trees based on sequences of the fulllength M segment and partial S and L segments, generated by the maximum likelihood and neighbor-joining methods using the GTR+I+G model of evolution, showed similar topologies supported by bootstrap analysis, in which CBNV was relatively distinct from rodentborne and other shrewborne hantaviruses (Figure). A strong association with TGNV was observed on the basis of the S segment (1,185 bases), however. Further sequence information will clarify the relationship between CBNV and other soricidborne hantaviruses and whether these form a monophyletic group in parallel with the evolution of Soricinae and Crocidurinae shrews. If one judges by the distant evolutionary relationship between shrews and rodents, future sequences of other soricidborne hantaviruses will provide considerable insights into their evolutionary origins.

Conclusions
Designing suitable primers for the amplifi cation of CBNV presented unanticipated challenges. Ironically, the recently acquired full genome of TPMV (J.-W. Song and R. Yanagihara, unpub. data) was not particularly helpful, since CBNV was genetically more divergent from TPMV than from well-characterized rodentborne hantaviruses (78% bootstrap support, S segment). Also, because the tissues were collected in RNAlater, virus isolation attempts could not be performed. As such, progress in obtaining the full-length sequence of CBNV has been slow.
A forest-dwelling soricine typically residing at elevations of 1,500-3,000 m, the Chinese mole shrew (family Soricidae, subfamily Soricinae) has a vast geographic range, extending from western and central People's Republic of China, northern Myanmar, northern Thailand, Assam, Bhutan, northern Vietnam, Taiwan, and possibly Lao People's Democratic Republic. That a hantavirus has been identifi ed in the Chinese mole shrew was not completely unexpected, in view of the isolation of a HTNV-like virus from this species in Sichuan Province in 1986 (7). However, those authors may have prematurely concluded that their hantavirus isolate was closely related to HTNV, since no genetic analysis was performed.
Viewed within the context of newly identifi ed, genetically distinct hantaviruses in the northern short-tailed shrew (B. brevicauda), Eliot's short-tailed shrew (B. hylophaga),  OSM55  TAG TAG TAG ACT CC  +  OSM47  GGC CAG ACA GCA GAT TGG  +  CBS1063F  ATK GCA TCH AAR ACA GTN GGN A  +  CBS1016F  GGA GRA CWC AAT CAA TGG GT  +  CBS1195F  GCN TGG GGN AAR GAG GCW GT  +  CBS593R  GAC TGG GCA TTN GGC ATN GA  -CBS506R  ATH CTT GTC CCY TTR TTA TC  -S6  ACG TCI GGA TCC ATI TCA TC  -CBS-3′endR  TAG TAG TAK RCT CCY TRA   shrews captured in Taiwan (S. Arai and R. Yanagihara, unpub. data). Also, investigations on the genetic diversity of CBNV and other newly identifi ed members of the Hantavirus genus will provide additional insights into the phylogeography and co-evolution of hantaviruses and their soricid reservoir hosts. One or more of these newfound shrewborne viruses may yield valuable clues about the molecular determinants of hantavirus pathogenicity.