Downloaded from www.microbiologyresearch.org by IP: 54.211.192.98 On: Wed, 19 Apr 2017 06:27:42 Journal of General Virology (2000), 81, 1747–1755. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Genetic analysis of wild-type Dobrava hantavirus in Slovenia : co-existence of two distinct genetic lineages within the same natural focus Tatjana Avsic-Zupanc, 1 Kirill Nemirov, 3 Miro Petrovec, 1 Tomi Trilar, 2 Mario Poljak, 1 Antti Vaheri 3 and Alexander Plyusnin 3 1 Institute of Microbiology and Immunology, Medical Faculty of Ljubljana, Zaloska 4, 1000 Ljubljana, Slovenia 2 Slovenian Museum of Natural History, Ljubljana, Slovenia 3 Haartman Institute, University of Helsinki, Helsinki, Finland Genetic analysis was performed of wild-type (wt) Dobrava hantavirus (DOB) strains from Slovenia, the country where the virus was first discovered and where it was found to cause haemorrhagic fever with renal syndrome (HFRS), with a fatality rate of 12 %. Two hundred and sixty mice of the genus Apodemus, trapped in five natural foci of DOB-associated HFRS during 1990–1996, were screened for the presence of anti-hantavirus antibodies and 49 Apodemus flavicollis and four Apodemus agrarius were found to be positive. RT–PCR was used to recover partial sequences of the wt-DOB medium (M) and small (S) genome segments from nine A. flavicollis and one A. agrarius. Sequence comparison and phylogenetic analysis of the Slovenian wt-DOB strains revealed close relatedness of all A. flavicollis-derived virus sequences (nucleotide diversity up to 6 % for the M segment and 5 % for the S segment) and the geographical clustering of genetic variants. In contrast, the strain harboured by A. agrarius showed a high level of genetic diversity from other Slovenian DOB strains (14 %) and clustered together on phylogenetic trees with other DOB strains harboured by A. agrarius from Russia, Estonia and Slovakia. These findings suggest that the DOB variants carried by the two species of Apodemus in Europe represent two distinct genetic lineages. Introduction Hantaviruses comprise a genus of the family Bunyaviridae. They possess a tripartite, negative-sense RNA genome encoding a viral RNA polymerase, surface glycoproteins G1 and G2 and a nucleocapsid (N) protein (for reviews see Elliott et al., 1991 ; Schmaljohn & Hjelle, 1997). The number of known distinct hantavirus sero- and genotypes has grown significantly during recent years (for reviews see Plyusnin et al., 1996, 1999 b) and currently totals 22 (Elliott et al., 2000). Members of the genus Hantavirus are maintained in nature in persistently infected rodents and, unlike other bunyaviruses, are not transmitted by arthropod vectors. They are thought to be Author for correspondence : Tatjana Avsic-Zupanc. Fax 386 1 543 7401. e-mail tatjana.avsicmf.uni-lj.si The nucleotide sequence data reported in this paper have been deposited in the EMBL/GenBank/DDBJ databases under accession numbers AJ251996–AJ252009, AJ276305 and AJ276306. transmitted primarily via infectious aerosols generated by contaminated rodent urine, faeces and saliva (Lee & van der Groen, 1989). One of the main features of hantaviruses is the close association between the virus type and the host species. This results in the circulation of distinct hantaviruses in the Old and New Worlds and in geographical clusters of hantavirus genetic variants (Plyusnin et al., 1996). Many hantaviruses are pathogenic for humans. Hantaan (HTN), Seoul (SEO), Puumala (PUU) and Dobrava (DOB) viruses cause haemorrhagic fever with renal syndrome (HFRS) in Eurasia, whereas Sin Nombre (SN), New York (NY), Black Creek Canal (BCC), Bayou (BAY), Laguna Negra (LN) and Andes viruses are the causative agents of hantavirus pulmonary syndrome and occur endemically in the Americas (Schmaljohn & Hjelle, 1997). Two pathogenic hantaviruses that cause HFRS have so far been proven to circulate in Europe : PUU and DOB. DOB was isolated originally from the lungs of a yellow-necked mouse (Apodemus flavicollis) trapped in the Dolenjska region of Slovenia, where a number of severe HFRS cases had occurred 0001-6929  2000 SGM BHEH