Characterization of Imjin virus, a newly isolated hantavirus from the Ussuri white-toothed shrew (Crocidura lasiura)

Abstract

Until recently, the single known exception to the rodent-hantavirus association was Thottapalayam virus (TPMV), a long-unclassified virus isolated from the Asian house shrew (Suncus murinus). Robust gene amplification techniques have now uncovered several genetically distinct hantaviruses from shrews in widely separated geographic regions. Here, we report the characterization of a newly identified hantavirus, designated Imjin virus (MJNV), isolated from the lung tissues of Ussuri white-toothed shrews of the species Crocidura lasiura (order Soricomorpha, family Soricidae, subfamily Crocidurinae) captured near the demilitarized zone in the Republic of Korea during 2004 and 2005. Seasonal trapping revealed the highest prevalence of MJNV infection during the autumn, with evidence of infected shrews' clustering in distinct foci. Also, marked male predominance among anti-MJNV immunoglobulin G antibody-positive Ussuri shrews was found, whereas the male-to-female ratio among seronegative Ussuri shrews was near 1. Plaque reduction neutralization tests showed no cross neutralization for MJNV and rodent-borne hantaviruses but one-way cross neutralization for MJNV and TPMV. The nucleotide and deduced amino acid sequences for the different MJNV genomic segments revealed nearly the same calculated distances from hantaviruses harbored by rodents in the subfamilies Murinae, Arvicolinae, Neotominae, and Sigmodontinae. Phylogenetic analyses of full-length S, M, and L segment sequences demonstrated that MJNV shared a common ancestry with TPMV and remained in a distinct out-group, suggesting early evolutionary divergence. Studies are in progress to determine if MJNV is pathogenic for humans.

FIG. 1.

Map of Paju City, Yeoncheon County, and Pocheon City near the DMZ, showing the locations of the 13 trap sites on U.S. Army installations. MJNV RT-PCR-positive Ussuri shrews (red boxes) were trapped at six sites (designated DN, F1, L1, L3, MP, and SR).

FIG. 2.

Detection of new hantavirus. (A) Crocidura lasiura (Ussuri white-toothed shrew) inhabits forests and fields, occasionally near human habitation. (B) The geographic range of Crocidura lasiura extends throughout Korea, southeastern Siberia, and northeastern China (shaded area). (C) Representative agarose gel showing 696-bp product (circled) amplified by RT-PCR from RNA extracted from the lung tissue of an Ussuri shrew. M, molecular size markers.

FIG. 3.

Thin-section electron micrograph of MJNV virion in Vero E6 cells.

FIG. 4.

Comparative seasonal prevalences of MJNV infection, as determined by IFA and RT-PCR tests, in Ussuri white-toothed shrews captured near the DMZ during the spring, summer, autumn, and winter of 2004 and 2005.

FIG. 5.

Phylogenetic trees generated by the ML method using the GTR+I+Γ model of evolution as estimated from the data based on the alignment of the entire coding regions of the 1,353-nucleotide S, 3,498-nucleotide M, and 6,480-nucleotide L genomic segments of MJNV. The phylogenetic positions of MJNV strains 05-11 and 04-55 are shown in relationship to those of representative murine rodent-borne hantaviruses, including HTNV strain 76-118 (GenBank accession no. NC_005218, NC_005219, and NC_005222), Sangassou virus strain SA14 (SANV; GenBank accession no. DQ268650, DQ268651, and DQ268652), Dobrava virus strain AP99 (DOBV; GenBank accession no. NC_005233, NC_005234, and NC_005235), and SEOV strain 80-39 (GenBank accession no. NC_005236, NC_005237, and NC_005238); arvicoline rodent-borne hantaviruses, including Tula virus strain M5302v (TULV; GenBank accession no. NC_005227, NC_005228, and NC_005226) and PUUV strain Sotkamo (GenBank accession no. NC_005224, NC_005223, and NC_005225); and sigmodontine and neotomine rodent-borne hantaviruses, including Andes virus strain Chile 9717869 (ANDV; GenBank accession no. NC_003466, NC_003467, and NC_003468) and Sin Nombre virus strain NMH10 (SNV; GenBank accession no. NC_005216, NC_005215, and NC_005217). The position of TPMV strain VRC66412 (GenBank accession no. AY526097, EU001329, and EU001330), from the Asian house shrew (Suncus murinus), is also shown. Host identification of Crocidura lasiura was confirmed by mtDNA sequencing (data not shown). The numbers at each node are bootstrap support values (expressed as the percentage of replicates in which the node was recovered), as determined for 100 ML iterations under the same model of evolution by the RAxML Web server (39). The scale bars indicate the numbers of nucleotide substitutions per site.