Genetic Interaction between Dobrava and Saaremaa Hantaviruses: Now or Millions of Years Ago?

In their paper (3), Klempa and coauthors reported results on (phylo)genetic analyses of hantaviruses occurring in two species of Apodemus mice, Dobrava virus (DOBV)-Af (from DOBV associated with A. flavicollis) and DOBV-Aa (associated with A. agrarius), and concluded that “DOBV-Af and DOBV-Aa are distinct but also subject to genetic exchanges that affect their evolutionary trajectories.” From these grounds, they then criticize our view that DOBV-Aa is a distinct hantavirus species, Saaremaa virus (SAAV) (1, 7, 9). (When we first discovered SAAV, it was designated a genetic lineage of DOBV [5, 8], and it was only with accumulating data that we came to the conclusion that it represents a distinct, new hantavirus.) We feel that the claim of Klempa et al. for currently occurring genetic exchanges between DOBV and SAAV is not well grounded and their view lacks an evolutionary dimension.

Although DOBV and SAAV are closely related, there are three most important differences. (i) While DOBV causes severe hemorrhagic fever with renal syndrome (fatality up to 12%), SAAV causes a milder form of the disease, similar to nephropathia epidemica (references 2 and 4 and our unpublished data). (ii) DOBV and SAAV are clearly differentiated by classical serology (reference 1 and our unpublished data). (iii) DOBV is lethal to suckling mice, while SAAV is not (J. Klingström and Å. Lundkvist, unpublished data).

In fact, in answering the crucial question of whether DOBV and SAAV are distinct entities or not, Klempa et al. came up with some controversy themselves: on the one hand, the two types are distinct; on the other, they are subject to genetic interactions (reassortment and recombination). Following this logic, DOBV and SAAV should, at the same time, be reproductively isolated and capable of “breeding”.

In our opinion, we are dealing here with a case of host switching, which occurred in the evolution of these hantaviruses (7, 9, 11), and we most recently obtained the crucial piece of evidence to support our view (6). Phylogenetic analysis revealed a discrepancy in the relationships of DOBV, SAAV, and Hantaan virus and their respective rodent hosts. This discrepancy is consistent with the transmission of (pre)DOBV/SAAV between A. flavicollis and A. agrarius, which resulted in the ecological and reproductive isolation of the two hantaviruses. Crucially, the time point of the host switching, 2.7 to 4.0 million years ago, was closer to the present than the estimated time of split between the two Apodemus species (<6.5 million years ago).

We would also like to address two specific points. (i) The claim for the reassortment between DOBV and SAAV is based on the lack of monophyly of all of the SAAV S segment sequences.

Klempa et al., using a TREE-PUZZLE program, showed for their set of the S segment sequences that the Estonian lineage of SAAV is not monophyletic with the Russian-Slovakian lineage (see Fig. 1 in reference 3). We shared this opinion until a year ago, when the set of S sequences in our analyses was smaller. However, when more strains became available (e.g., DOBV strains from Greece and Russia and an SAAV strain from Denmark), all SAAV S sequences turned monophyletic and, therefore, there was no longer any contradiction in the S- and M-segment-based phylogenies. This conclusion was still valid when the new DOBV strain from East Slovakia, Esl/400Af, was added to the data set. For these calculations we used the distant matrix approach (Fitch-Margoliash method). TREE-PUZZLE gave either monophyly of all SAAV strains (HKY model of nucleotide substitutions) or multifurcation of DOBV and SAAV lineages (TN model). Thus, the “reassortment claim” is not supported by the phylogenies seen with the use of larger data sets.

(ii) The claim for the recombination between DOBV and SAAV.

Recombination points suggested for the M segment of one of the SAAV strains (see Fig. 3 in reference 3) are not sharp, as one would expect for a recent recombination (10), but “diffuse” (which would be a logical result of genetic drift with the time passing). The idea of currently occurring recombination also demands a better explanation of the fact that the recombination seems to occur not only between the sympatric DOBV and SAAV from the same location in East Slovakia but between the Slovakian and Estonian viruses as well.

The host-switching hypothesis does not totally exclude the possibility of genetic exchange (in the past!) between the newly established ancestral SAAV in A. agrarius and the still very closely related (if not almost identical) ancestral DOBV in A. flavicollis—in biotopes where the two pairs of hosts and viruses coexisted. However, as in every case of origination of a new species, the crucial prerequisite is its reproductive isolation. Consequently, all of the genetic exchanges between the two diversifying hantaviruses should have stopped; otherwise, SAAV in A. agrarius (DOBV-Aa) would never have become distinct.

This reasoning leads us to the conclusion that Klempa et al. could have been right were they not two to four million years late.