Dobrava-Belgrade Virus Spillover Infections, Germany

We present the molecular identification of Apodemus agrarius (striped field mouse) as reservoir host of the Dobrava-Belgrade virus (DOBV) lineage DOBV-Aa in 3 federal states of Germany. Phylogenetic analyses provided evidence for multiple spillover of DOBV-Aa to A. flavicollis, a crucial prerequisite for host switch and genetic reassortment.

We present the molecular identifi cation of Apodemus agrarius (striped fi eld mouse) as reservoir host of the Dobrava-Belgrade virus (DOBV) lineage DOBV-Aa in 3 federal states of Germany. Phylogenetic analyses provided evidence for multiple spillover of DOBV-Aa to A. fl avicollis, a crucial prerequisite for host switch and genetic reassortment.
E uropean hantaviruses are emerging viruses that can cause hemorrhagic fever with renal syndrome (HFRS) of differing severities. Dobrava-Belgrade virus (DOBV) is a hantavirus that appears in 3 distinct lineages hosted by different Apodemus species. The DOBV-Af lineage associated with the yellow-necked mouse (A. fl avicollis) has caused serious HFRS in southeast Europe with a case-fatality rate <12% (1,2). Human infections with Caucasian wood mouse (A. ponticus)-associated DOBV-Ap have resulted in more moderate than severe HFRS in the southern part of European Russia (3). Mild-to-moderate human DOBV disease in central and eastern Europe has been connected with infection by DOBV-Aa lineage carried by the striped fi eld mouse (A. agrarius) (3)(4)(5). Other A. agariusassociated strains, found in Estonia and called Saaremaa virus, have been proposed to form a distinct hantavirus species (6). In Germany, human DOBV cases with mild to moderate clinical outcomes have been detected by sero-logic investigations (4,7) but only 1 short DOBV-Aa small (S) segment sequence derived from a patient in northern Germany has been identifi ed (8). The natural host and the geographic distribution of DOBV in its reservoir host has remained unknown in Germany. spillover infections (Figure 2, panel A; online Technical Appendix).
Morphologic species determination for all DOBVseroreactive and RT-PCR-positive rodents was confi rmed by a mitochondrial cytochrome b gene-specifi c PCR (9,10), sequence determination, and comparison with available GenBank sequences from A. agrarius and A. fl avicollis (online Technical Appendix).

Conclusions
Based on a large panel of the entire N-and GPC-encoding DOBV sequences, we report direct molecular evidence that DOBV in Germany is represented by a genetic lineage associated with A. agrarius (DOBV-Aa). In contrast, we found no evidence for the occurrence of DOBV-Af in A. fl avicollis or other Apodemus species from Germany. Consistent with the geographic distribution of A. agrarius (11) and the report of human DOBV disease exclusively in northern and northeastern Germany, this fi nding may confi rm DOBV-Aa as the sole causative agent of DOBV infections in Germany (4; Robert Koch-Institut, SurvStat, www.rki.de).
Previously A. agrarius-associated Saaremaa virus was experimentally shown to be able to infect A. agrarius and A. fl avicollis mice (12). We report multiple natural spillover infections of A. fl avicollis by a DOBV strain originally hosted by A. agrarius. The observed spillover infections represent a crucial prerequisite for genetic reassortment. This observation is in contrast to other reports from Slovenia  is exclusively carrying the DOBV-Af and A. agrarius the DOBV-Aa lineage (4,13). In contrast to our observations, single DOBV-Af spillover infections of A. sylvaticus and Mus musculus have been reported previously (14). The phylogenetic analyses demonstrated 2 well-separated clusters within the DOBV-Aa lineage. These rodentderived DOBV sequences in Germany represent a major contribution to the DOBV genomics and phylogenetics. Future investigations should help to identify specifi c features of these DOBV-Aa strains resulting in its frequent spillover to A. fl avicollis and to prove a putative adaptation of DOBV-Aa on A. fl avicollis after spillover, as well as possible reassortment processes. . Before tree construction, automated screening for recombination between the S segment sequences was performed using program RDP3 (15), which used 6 recombination detection programs: Bootscan, Chimeric, GENECONV, MaxChi, RDP, and SiScan with their default parameters. No putative recombinant regions could be conclusively detected by >3 programs and subsequently verifi ed by phylogenetic trees. programs and subsequently verified by phylogenetic trees.