We established a countrywide alert network that included several physicians and diagnostic laboratories. This network enabled assessment of serum samples from patients during the early clinical phase of infection.

A total of 491 serum samples were tested for antibodies to hantavirus. Of these samples, 377 were positive for IgG against PUUV, of which 330 were positive for IgM against PUUV by an in-house ELISA (11) and immunoblot assay (recomLine Bunyavirus; Mikrogen, Neuried, Germany). These acute-phase antibody-positive samples were then screened by using a nested pan hantavirus reverse transcription PCR (12) specific for a conserved region within the polymerase gene (large genomic segment). Using this approach, we identified 102/330 IgM- and IgG-positive samples that were PCR positive. Sequencing of this large segment region confirmed infection by PUUV.

To differentiate virus strains within the PUUV species, a higher variable nucleotide sequence was targeted by amplification of a 504-nt fragment of the genomic S segment encoding the nucleocapsid protein (13). Using this approach, we found that 38 of 102 PUUV-positive samples were S segment positive. Nucleotide sequences of these 38 samples were determined and used for molecular phylogenetic analysis. The dataset also included sequences obtained from human and M. glareolus vole samples collected in previous years and from voles captured during the 2010 outbreak. Rodent trapping was performed after permission was obtained from Federal State authorities (permit no. NRW 20.09.210, BW 35-9185.82/0261).

Figure 1

Figure 1. . . . Distribution of investigated Puumala virus infections in Germany. Black dots indicate sequences obtained from patient samples in 2010; gray dots indicate sequences obtained from patient samples in 2007;...

Figure 2

Figure 2. . . . Neighbor-joining phylogenetic tree (TN93 evolutionary model) of Puumala virus (PUUV) strains constructed on the basis of partial sequences of the small segment (504-nt sequence, nt positions 392–894). Bootstrap...

Residences of patients in Germany from whom viral sequences were obtained and corresponding rodent trapping sites are shown in Figure 1. Results of molecular phylogenetic analysis of these strains are shown in Figure 2. In addition to 4 molecular clades (Swabian Jura, clade 1; Bavarian Forest, clade 2; Spessart Forest, clade 3; and Münsterland, clade 6) found during the 2007 outbreak, 2 novel clades (North East Hesse, clade 4; and Teutoburg Forest, clade 5) were defined. All 6 clades comprised human-derived and vole-derived sequences.

Identification of new PUUV clades in 2010 was associated with a higher incidence of human disease than in 2007. For example, in the Federal State of Hesse, incidence increased from 0.35 cases/100,000 persons in 2007 to 2.80 cases/100,000 persons in 2010. This increase in cases enabled us to collect blood specimens from patients shortly after clinical onset of disease, characterize these specimens, and define a new PUUV clade (North East Hesse, clade 4).

To analyze diversity within and between different clades, we aligned each clade and calculated mean pairwise amino acid and nucleotide identities. In addition, we identified the consensus sequence of each clade and compared each with those of other clades (Table). Sequences within 1 clade show pairwise amino acid identities >97% and nucleotide identities >96%. Identities between clades range from 90.3% (Swabian Jura vs. Spessart Forest) to 97.7% (Münsterland vs. Teutoburg Forest) on the amino acid level and 81.3% (Teutoburg Forest vs. Swabian Jura) to 89.0% (Münsterland vs. Teutoburg Forest) on the nucleotide sequence level.

Within a particular molecular clade, virus strains from localities near each other could be differentiated. For example, within PUUV strains from the Bavarian Forest, which has been known as an outbreak region since 2004 (13,14), newly characterized strains 10 Mu Eb51 and 10 Mu Eb14 (Bavarian Forest; Figure 1, Figure 2) originated from localities only 25 km apart. Because migration distances >1 km for bank voles are efficiently prevented by natural and artificial barriers (15), local vole populations are likely associated with specific PUUV strains.

Three years after a large 2007 PUUV outbreak, a subsequent epidemic with >2,000 human cases occurred in Germany. On the basis of beech mast coverage in 2009, growth of the reservoir rodent population in 2010 was expected. Model monitoring studies in July 2010 found mean ± SD bank vole densities of <141 ± 13 voles/hectare in investigated outbreak regions. PUUV seroprevalence in these bank vole populations was >40%. Bank vole densities in the same regions decreased to <15 voles/hectare in April 2011 (D. Reil, U.M. Rosenfeld, unpub. data).

Phylogenetic analysis of the involved PUUV strains identified 6 clades comprising virus strains of human and vole origin. Each clade clearly corresponds to a different outbreak region. High molecular similarities of human- and rodent-derived PUUV sequences from the same geographic origin and their clear molecular distinction from viruses of neighboring regions indicate spatial evolution of each virus clade. Thus, we conclude that the 2010 epidemic was not caused by countrywide spread of the same virus but resulted from multiple local outbreaks associated with simultaneous increases in densities and infection rates of bank voles in the different geographic regions.

At least for the investigated 504-nt S segment region, no evidence was found that viruses had undergone changes over time or as a result of rodent-to-human transmission. Lack of human-to-human transmission, local distribution of bank voles, and a high degree of identity between viruses of human and rodent origin enabled us to allocate certain virus strains to defined geographic regions. Future investigations may enable generation of risk maps with higher resolution and establishment of more sophisticated preventive measures in high-risk areas.