A survey of 158 rodents caught in the Czech Republic identified Dobrava virus sequences closely related to that of the Dobrava virus type strain in Apodemus sylvaticus and Mus musculus rodents. The identity of A. sylvaticus was unequivocally confirmed by random amplified polymorphic DNA analysis. The data seem to indicate hantavirus spillover from Apodemus flavicollis to other rodents.Hantaviruses are subdivided into the Eurasian and the American groups. The distinction manifests itself in serological and genetic differences as well as in the clinical picture of the diseases elicited in humans. The up to 20 individual hantavirus types have been observed to be strictly coevolving with the distinct rodent species that carry them. Prominent hantaviruses in western and central Europe are Dobrava virus (DOBV), Puumala virus (PUUV), and Tula virus (TULV) (6,16,21).DOBV was originally isolated in Slovenia from the rodent Apodemus flavicollis (DOBV-Af) (3) and is a Eurasian hantavirus that predominantly causes disease in humans in southeastern Europe. DOBV-Af has also been isolated in Greece, Albania, and Bosnia-Herzegovina (1,10,14). Serological studies have indicated the presence of DOBV-Af beyond the geographical limits of the Balkans in Slovakia (22) and recently in the Czech Republic (15,25). The isolation of a Dobrava-like virus from the rodent Apodemus agrarius (DOBV-Aa) was reported in Estonia (11), Russia (17), and Slovakia (23), supplemented by a report on genetic evidence of this DOBV-Aa type from rodent samples in Hungary (20). Whether this new type is a type of its own, as comparative serological and genetic analyses suggest, is still a matter of debate (7,12,18). The distribution of the two rodent host species overlaps in Europe, but rodent carriers of both DOBV types coexist only in the border region of southeastern and central Europe (2, 23). In order to investigate the occurrence of hantaviruses in a rodent population from the Czech Republic, we analyzed the kidneys, lungs, and spleens of 157 rodents and 1 shrew (A. flavicollis, n ϭ 77; A. sylvaticus, n ϭ 34; Mus musculus, n ϭ 2; Clethrionomys glareolus, n ϭ 41; Microtus arvalis, n ϭ 2; Microtus agrestis, n ϭ 1; Sorex araneus, n ϭ 1) caught at the military training area of Boletice, close to Ceské Budejovice in the south of the Czech Republic, from May to November 1999. MATERIALS AND METHODSA total of 10 to 50 mg of tissue of each organ was homogenized in a FastPrep machine with the FastRNA Green kit (Qbiogene, Hilden, Germany) to perform total RNA extraction. This closed system avoids the generation of contaminating aerosols. We screened the extracted RNA for DOBV-Af RNA by a one-step TaqMan reverse transcription (RT)-PCR and for hantavirus RNA with the degenerate primers for nested PCR published by Scharninghausen et al. (20). For the DOBV TaqMan RT-PCR, we used 500 nM concentrations of primers DOBFP (5Ј-TGGCTTGACCTCCCGTG-3Ј) and DOBRP (5Ј-CAAGCGCTCCT TGTCTTTGA-3Ј) and 200 nM probe DOBP (5Ј-ATCTCCAACGTCTTTGAC CAAAGGCCC-3Ј) tagged with 6-carboxyfluores...