Saaremaa Virus Research Articles

Optimization of ELISA and immunoblot methods for the detection of IgG antibodies against old world hantaviruses in wild rodents

Hantaviruses infect humans via inhalation of viral particles in infected rodents' secretions such as saliva, urine and faeces or via direct contact with infected rodents. The rodent species that are known as the carriers of Dobrava (DOBV), Puumala (PUUV), Saaremaa (SAAV), Tula (TULV) and Seoul (SEOV) viruses are found in our country. The presence of specific antibodies against hantaviruses have been demonstrated in rodents collected from Black Sea and Aegean Regions of Turkey in 2004 for the first time. The first hantavirus-related hemorrhagic fever with renal syndrome (HFRS) cases were reported in Black Sea region in 2009. The determination of the hantavirus prevalence in wild life and rodent populations in the field is crucial for the information about hantavirus-related cases and to clarify the state of risk. There is no commercial product optimized for the screening of rodent serum samples in terms of HFRS agents like DOBV and PUUV that are widely seen in Eurasia as well as Turkey. In this study, the antigens belonging to the commercial enzyme-linked immunoassay (ELISA) and immunoblot tests that are produced for the screening of human sera were used for the development of antibody screening tests against hantavirus in rodent sera and were optimized. The most appropriate serum and conjugate dilutions were determined for the optimization of ELISA (Anti-Hantavirus Pool ELISA; Euroimmun, Germany) and immunoblot (Euroline Anti-Hanta Profile 1 strips; Euroimmun, Germany) methods. Optimized ELISA method was used for the screening and optimized immunoblot method was used for the confirmation. A total of 84 wild rodent sera that belonged to Apodemus and Microtus species were evaluated with this procedure and the cut-off value, sensitivity and specificity of optimized ELISA method were determined. For the optimization of ELISA 1/50, 1/100 and 1/200 serum dilutions and 1/10.000, 1/20.000 and 1/40.000 conjugate dilutions were tested. For the optimization of immunoblot, 1/50 and 1/100 serum dilutions and 1/5.000 and 1/10.000 conjugate dilutions were tested. The horseradish peroxidase conjugated goat anti-mouse IgG for ELISA and the alkaline phosphatase conjugated goat anti-mouse IgG for immunoblot were used. We followed the manufacturer's recommendations for the incubation parameters, substrate and the number of washes. 1/50 serum dilution and 1/10.000 conjugate dilution for ELISA and 1/100 serum dilution and 1/5.000 conjugate dilution for immunoblot were determined as optimal concentrations. By using the optimized ELISA, 26.2% (22/84) of rodents were found positive for hantavirus antibodies according the determined cut-off value (OD(450/620): 0.325). By using immunoblot as a confirmatory test, 20 out of 22 ELISA positive samples could be studied because of the insufficient amount of sera and 17 of them was found positive in terms of DOBV antibodies. Of these rodents 11 were Apodemus flavicollis, three were Apodemus agrarius, two were Microtus guentheri and one was Apodemus sylvaticus. When the results of ELISA were compared to immunoblot results, the optimized ELISA's sensitivity and specificity were found as 100% and 95%, respectively. In this study, a method that can be used in the screening of rodent sera was constituted which uses commercial antigens that can be provided easily, gives fast and reliable results. Similar serological methods optimized for different types of rodents are of great importance for the realization of active follow-up and monitoring of the studies in the field.

Detection of Dobrava hantavirus RNA in Apodemus mice in Bulgaria.

Several Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in Europe: Dobrava-Belgrade virus (DOBV), Puumala, Saaremaa, Sochi, and Seoul virus. Although HFRS is endemic in Bulgaria, genome sequences of hantaviruses have never been detected in wild rodents. To identify rodent reservoirs, a total of 691 rodents from three endemic regions were trapped in 2011-2012 and screened by TaqMan RT-PCR for detection of hantaviral genomic RNA. Partial small (S) and/or large (L)-segment sequences were recovered from six Apodemus mice: five of the species A. flavicollis and one A. agrarius. Phylogenetic analysis revealed that all recovered sequences belonged to DOBV. On the phylogenetic trees, the novel Bulgarian hantavirus sequences clustered together with sequences of established previously DOBV variants recovered from Bulgarian HFRS patients and also with variants found in wild rodents trapped in Slovenia, Greece, and Slovakia. One of the novel Bulgarian DOBV S-sequences from A. agrarius was related closely to DOBV sequences recovered from A. flavicollis, suggesting a spillover of DOBV from its natural host to A. agrarius mice. The results of this study confirmed the circulation of DOBV in wild rodents in Bulgaria. The complexity of the epidemiological situation in the Balkans requires further studies of hantaviruses in rodent hosts and human HFRS cases.

A short review of hemorrhagic fever with renal syndrome and it's extrarenal complications

Hantaviruses are enveloped tri-segmented negative-stranded RNA viruses. Infection from these viruses is growing in last decades. They are causative agent for two types of diseases: a) Hemorrhagic fever with renal syndrome (HFRS), mostly observed in Eurasia and b) Hantavirus cardiopulmonary syndrome (HPS), mostly observed in American continent. There are several Hantaviruses known to infect humans, and their main hosts are rodents. HFRS is caused from Hantaan, Dobrava, Saaremaa, Seoul and Puumala virus. The main factor, which determines the severity of Hantavirus infection, is the degree of increased permeability of infected endothelium, and the most common clinical findings in those patients are fever, headache, abdominal pains, backache, myalgia and nausea or/and vomiting. There are already numerous publications with regards to the involvement of two major systems such as renal and pulmonary in patients with HFRS. In this paper we would try to make a short review about the involvement of other organs and systems of our body in those patients. Extrarenal manifestations of Hantavirus infection include: acute ocular impairment with visual function, acute myopia, central nervous system (CNS) complications, cardiovascular impairment with myocarditis, gastrointestinal hemorrhages, but liver and pancreas may be also affected.

Dobrava Virus Carried by the Yellow-Necked Field Mouse Apodemus flavicollis, Causing Hemorrhagic Fever with Renal Syndrome in Romania

Hemorrhagic fever with renal syndrome (HFRS) has been confirmed by serological methods during recent years in Romania. In the present study, focus-reduction neutralization tests (FRNT) confirmed Dobrava hantavirus (DOBV) as the causative agent in some HFRS cases, but could not distinguish between DOBV and Saaremaa virus (SAAV) infections in other cases. DOBV was detected by a DOBV-specific TaqMan assay in sera of nine patients out of 22 tested. Partial sequences of the M genomic segment of DOBV were obtained from sera of three patients and revealed the circulation of two DOBV lineages in Romania. Investigation of rodents trapped in Romania found three DOBV-positive Apodemus flavicollis out of 83 rodents tested. Two different DOBV lineages were also detected in A. flavicollis as determined from partial sequences of the M and S genomic segments. Sequences of DOBV in A. flavicollis were either identical or closely related to the sequences obtained from the HFRS patients. The DOBV strains circulating in Romania clustered in two monophyletic groups, together with strains from Slovenia and the north of Greece. This is the first evidence for the circulation of DOBV in wild rodents and for a DOBV etiology of HFRS in Romania.

Rodents as reservoirs of human pathogens in Bulgaria

Small mammals are reservoirs of various human pathogens. The aim of this work was to investigate infections with human pathogens in rodents trapped in different regions of Bulgaria. A total of 284 rodents were investigated by PCR for detection of the flagellin gene of borreliae within Borrelia burgdorferi sensu lato complex, ankA gene of Anaplasma phagocytophilum and nucleoprotein gene of hantavirus Dobrava - conventional nested RT-PCR and Real Time PCR with TaqMan probe. B. burgdorferi was detected in 64/284 (22.5%) of the investigated rodents by PCR. Of them, 41 samples originated from Apodemus flavicollis, 20 from A. agrarius, and 3 from A. sylvaticus. Overall, 33 of the investigated 284 rodents were infected with Anaplasma phagocytophilum (11.6%) - 11 Apodemus flavicollis (infectivity rate 8.6% of the 128 investigated), 13 Apodemus agrarius (infectivity rate 13.5% of the 96 tested), and 1 Apodemus sylvaticus (infectivity rate 9% of the 11 tested). Hantavirus RNA was detected in 9 of the rodents. Only Dobrava-Belgrade virus but not Puumala or Saaremaa virus was detected. Almost all infected rodents were A. flavicollis (8/9 PCR-positive rodents). Rodents are important reservoirs of human pathogens. In this study, active infection in rodents was confirmed by detection of microorganism's genome. Remarkably, a high number of rodents from genus Apodemus were infected with borreliae. The high rate of detection of A. phagocytophilum in rodents from A. agrarius species suggested that this species might serve as major reservoir of human anaplasmosis in Bulgaria. Mainly infected with hantaviruses were A. flavicollis mice, known as reservoir of Dobrava hantavirus but hantavirus infections were detected also in A. agrarius mice. Medical authorities should be aware of the risk for humans.

Molecular characterization of Dobrava and Kurkino genotypes of Dobrava-Belgrade hantavirus detected in Hungary and Northern Croatia

Among the Hantavirus genus, Saaremaa virus (SAAV) has been the subject of taxonomical debates. While the International Committee on Taxonomy of Viruses declares SAAV as a distinct species, several European hantavirus experts proposed that SAAV is in fact a genotype of Dobrava-Belgrade virus (DOBV). In the present study we performed S-segment-based phylogenetic analysis of eight DOBV strains identified in rodents in Hungary and Northern Croatia. These new sequences considerably increase the number of complete nucleoprotein gene sequences deposited in the NCBI database. Our phylogenetic analysis clearly support the taxonomical nomenclature recently proposed for DOBV, i.e., genotypes such as Dobrava, Saaremaa, Kurkino, and Sochi should indeed be classified within the DOBV hantavirus species. Moreover, we found that only the Dobrava and Kurkino genotypes of DOBV species are circulating in Hungary while currently there is no evidence for the presence of Saaremaa genotype.

Human Puumala and Dobrava Hantavirus Infections in the Black Sea Region of Turkey: A Cross-Sectional Study

This study was carried out to better understand the epidemiology of hantaviruses in a province of Turkey (Giresun) where human hantavirus disease has recently been detected. In this cross-sectional study, a total of 626 blood samples from healthy people aged 15 and 84 years old were collected both in urban and rural areas in 2009. The sera were tested by enzyme-linked immunosorbent assay (ELISA), immunoblotting assay, and the focus reduction neutralization test (FRNT). We screened the samples by an ELISA and found that 65/626 samples reacted positively for the presence of hantavirus-reactive immunoglobulin G (IgG). Twenty of the 65 ELISA-positive samples could be confirmed by an immunobloting assay, and the overall seroprevalence was thereby calculated to 3.2% (20/626). The seroprevalence of the people living in wood areas or adobe houses 9/17 (52.9%) was significantly higher than among people living in concrete houses 10/47 (21.3%) (p=0.014). Finally, 3 of the 20 immunoblot-positive sera were confirmed as specific for the Puumala hantavirus serotype by FRNT, 1 serum was confirmed as Dobrava virus-specific, whereas 1 serum was found to be equally reactive to Dobrava and Saaremaa viruses. We will now focus on further investigations of the ecology and epidemiology of hantaviruses in humans and their carrier animals in Turkey, studies that have already been started and will be further intensified.

Cross-Species Transmission in the Speciation of the Currently Known Murinae-Associated Hantaviruses

To gain more insight into the phylogeny of Dabieshan virus (DBSV), carried by Niviventer confucianus and other Murinae-associated hantaviruses, genome sequences of novel variants of DBSV were recovered from Niviventer rats trapped in the mountainous areas of Wenzhou, China. Genetic analyses show that all known genetic variants of DBSV, including the ones identified in this study, are distinct from other Murinae-associated hantaviruses. DBSV variants show geographic clustering and high intraspecies diversity. The data suggest that DBSV is a distinct species in the genus Hantavirus. Interestingly, DBSV shows the highest sequence identity to Hantaan virus (HTNV), with a >7% difference in the sequences of the N, GPC, and L proteins, while N. confucianus is more closely related to Rattus norvegicus (the host of Seoul virus [SEOV]) than to Apodemus agrarius (the host of HTNV and Saaremaa virus [SAAV]). Further genetic analyses of all known Murinae-associated hantaviruses (both established and tentative species) show that many of them, including DBSV, may have originated from host switching. The estimation of evolutionary rates and divergence time supports the role of cross-species transmission in the evolution of Murinae-associated hantaviruses. The detection of positive selection suggests that genetic drift may contribute to the speciation of Murinae-associated hantaviruses and that adaptation has a role as well.

Hantavirus infections in Europe and their impact on public health

Hantaviruses (genus Hantavirus, family Bunyaviridae) are enveloped tri-segmented negative-stranded RNA viruses each carried by a specific rodent or insectivore host species. Several different hantaviruses known to infect humans circulate in Europe. The most common is Puumala (PUUV) carried by the bank vole; another two important, genetically closely related ones are Dobrava-Belgrade (DOBV) and Saaremaa viruses (SAAV) carried by Apodemus mice (species names follow the International Committee on Taxonomy of Viruses nomenclature). Of the two hantaviral diseases, hemorrhagic fever with renal syndrome (HFRS) and hantaviral cardiopulmonary syndrome, the European viruses cause only HFRS: DOBV with often severe symptoms and a high case fatality rate, and PUUV and SAAV more often mild disease. More than 10,000 HFRS cases are diagnosed annually in Europe and in increasing numbers. Whether this is because of increasing recognition by the medical community or due to environmental factors such as climate change, or both, is not known. Nevertheless, in large areas of Europe, the population has a considerable seroprevalence but only relatively few HFRS cases are reported. Moreover, no epidemiological data are available from many countries. We know now that cardiac, pulmonary, ocular and hormonal disorders are, besides renal changes, common during the acute stage of PUUV and DOBV infection. About 5% of hospitalized PUUV and 16%-48% of DOBV patients require dialysis and some prolonged intensive-care treatment. Although PUUV-HFRS has a low case fatality rate, complications and long-term hormonal, renal, and cardiovascular consequences commonly occur. No vaccine or specific therapy is in general use in Europe. We conclude that hantaviruses have a significant impact on public health in Europe.

Genetic characterization of seoul hantavirus originated from norway rats (Rattus norvegicus) captured in Belgium

Hantaviruses (genus Hantavirus, family Bunyaviridae) cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus (cardio)pulmonary syndrome (HCPS) in the Americas. So far, in Europe, four pathogenic hantaviruses have been found, often in co-circulation: Puumala virus (PUUV), Dobrava virus (DOBV), Saaremaa virus (SAAV), and Seoul virus (SEOV). Of those, only PUUV was found in Belgium. Recently, in our search for hantaviruses in the Flanders region of Belgium we collected genetic and serological evidence for the presence of SEOV virus in local brown rats. In this article, the results of (phylo)genetic analysis of wild-type SEOV strain from the Flanders are presented. The analysis based on the complete S segment sequence and also partial M- and L-segment sequences revealed that the Belgian SEOV strain was related most closely to strains from France, Indonesia, Japan, Singapore, Cambodia (those associated with the species Rattus norvegicus) and Vietnam. Such a clustering was in perfect agreement with the results of direct sequence comparison and suggested the same evolutionary history for all three genome segments of the Belgian SEOV strain (i.e., no reassortment of genome segments). So far, SEOV has been found in two European countries, France and Belgium, and there is every reason to believe that the area of the virus distribution in Europe is not restricted to those countries.

Second External Quality Assurance Study for the Serological Diagnosis of Hantaviruses in Europe

Hantaviruses are endemic throughout the world and hosted by rodents and insectivores. Two human zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), are caused by hantaviruses and case fatality rates have reached 12% for HFRS and 50% for HPS in some outbreaks. Symptomatic hantavirus infections in Europe are summarised as HFRS mainly due to Puumala, Dobrava-Belgrade and Saaremaa virus. While HFRS has an overall low incidence in Europe, the number of cases varies from 100 per year in all Eastern and Southern Europe up to 1,000 per year only in Finland. To assess the quality of hantavirus diagnostics, the European Network for the Diagnostics of “Imported” Viral Diseases (ENIVD) organised a first external quality assurance (EQA) in 2002. The purpose of this second EQA study is to collect updated information on the efficiency and accurateness of hantavirus serological methods applied by expert laboratories. A serum panel of 14 samples was sent to 28 participants in Europe of which 27 sent results. Performance in hantavirus diagnosis varied not only on the method used but also on the laboratories and the subclass of antibodies tested. Commercial and in-house assays performed almost equally. Enzyme immunoassays were mainly used but did not show the best performances while immunoblot assays were the less employed and showed overall better performances. IgM antibodies were not detected in 61% of the positive IgM samples and IgM detection was not performed by 7% of the laboratories indicating a risk of overlooking acute infections in patients. Uneven performances using the same method is indicating that there is still a need for improving testing conditions and standardizing protocols.

Genetic evidence for the presence of two distinct hantaviruses associated with Apodemus mice in Croatia and analysis of local strains

In Europe, Dobrava-Belgrade (DOBV), Saaremaa (SAAV), and Puumala (PUUV) viruses are known to cause hemorrhagic fever with renal syndrome (HFRS). All three hantaviruses are now found in Croatia. Lung tissue samples of 315 Apodemus mice trapped in 2003-2004 were screened for the presence of hantaviral N-Ag and 20 mice (6.3%) were found either strongly positive or weak/suspected-positive. Partial sequences of hantavirus M and S segments were recovered by RT-PCR from six mice and subjected to (phylo)genetic analysis that revealed the presence of four novel strains of DOBV and one of SAAV. Curiously, one of the newly described DOBV strains was found in Apodemus agrarius mouse, that is, not in the traditional host, A. flavicollis mice, suggesting a spillover event. S segment sequences recovered previously from HFRS cases [Markotić et al., 2002] were confirmed as DOBV sequences; one of which appeared particularly close to the prototype Slovenian DOBV isolate. Taken together with earlier data on PUUV in Croatia, these results show a co-circulation of three European hantavirus pathogens in this country. So far, not a single SAAV sequence has been recovered from HFRS patients either in Croatia or neighboring Slovenia and Hungary nor in Slovakia suggesting a somewhat lower fequency of acute SAAV infection in humans in this part of Europe than for example in the Baltics.

Co‐circulation of three pathogenic hantaviruses: Puumala, Dobrava, and Saaremaa in Hungary

Hantaviruses (Bunyaviridae) cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus (cardio)pulmonary syndrome (HCPS) in the Americas. HFRS is caused by Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV), Saaremaa virus (SAAV), and Puumala virus (PUUV). Of those, only HTNV is not present in Europe. In recent years, hantaviruses, described in other parts of Europe, were also detected at various locations in Hungary. To study the genetic properties of Hungarian hantaviruses in detail, sequences of the viral S and M segments were recovered from bank voles (Myodes glareolus), yellow-necked mice (Apodemus flavicollis), and striped field mice (Apodemus agrarius) trapped in the Transdanubian region. As expected, the sequences recovered belonged, respectively, to PUUV (two strains), DOBV (one strain), and SAAV (one strain). On phylogenetic trees two new Hungarian PUUV strains located within the well- supported Alpe-Adrian (ALAD) genetic lineage that included also Austrian, Slovenian, and Croatian strains. Analysis of the Hungarian SAAV and DOBV genetic variants showed host-specific clustering and also geographical clustering within each of these hantavirus species. Hungarian SAAV and DOBV strains were related most closely to strains from Slovenia (Prekmurje region). This study confirms that multiple hantaviruses can co-circulate in the same locality and can be maintained side-by-side in different rodent species.

The hantaviral load in tissues of naturally infected rodents

Hantaviruses cause a lifelong and asymptomatic infection in naturally infected hosts as well as in experimentally infected rodents. Understanding the ecology and pathogenesis of hantaviruses requires an interdisciplinary research approach, which links laboratory experiments with results gained from field studies. Although several studies report hantavirus persistence and tissue infection patterns for experimentally infected rodents, field data is very limited. For this reason, the aim of our study was to investigate Puumala, Dobrava and Saaremaa virus RNA loads and tissue infection patterns in their natural reservoirs. Hantavirus RNA was demonstrated in all tested internal organs and blood samples of 14 naturally infected rodent hosts. However, the concentration of a specific virus differs depending on the virus, the host and the organ tested. Above all, the Dobrava virus showed a considerably higher viral load in all internal organs and blood samples of infected Apodemus flavicollis hosts. Results obtained in the study support the thesis that virus RNA load reaches its peak in the first month after infection, presumably after the virus has spread throughout all internal organs. This also implies that recently infected rodents are more important for transmission of the virus in the community.

Characterization of Hemorrhagic Fever with Renal Syndrome Caused by Hantaviruses, Estonia

Thirty cases of hemorrhagic fever with renal syndrome (HFRS) due to Puumala virus (PUUV), Saaremaa virus (SAAV), and Dobrava virus infection were confirmed in Estonia. Except for the levels of serum creatinine, no remarkable differences were found in the clinical course of HFRS caused by PUUV and SAAV.

Serological Survey for Viral Pathogens in Turkish Rodents

Wild rodents (n = 330) were trapped around the villages of Altindere and Coşandere (Maçka, Trabzon Province), Ayder, Ortan, and Yolkiyi (Camlihemşin, Rize Province), and Bozdag (Odemiş, Izmir Province) in northeastern and western Turkey during April 2004. Samples were tested for arenavirus, hantavirus, and cowpox virus (family Poxviridae, genus Orthopoxvirus, CPXV) antibodies by using immunofluorescence assays (IFAs). Antibodies against arenaviruses were found in eight of 330 (2.4%) rodents. Arenavirus sero-positive animals were found from all study sites. Antibodies to Puumala virus (family Bunyaviridae, genus Hantavirus, PUUV) were detected in four of 65 Microtus voles tested. Of the PUUV-IFA-positive voles, one Microtus guentheri lydius was caught from Izmir, and one Microtus roberti and two Microtus rossiaemeridionalis were captured near Trabzon. All 264 Apodemus spp. mice tested negative for antibodies to Saaremaa virus (family Bunyaviridae, genus Hantavirus, SAAV); the single Dryomys nitedula tested negative for both PUUV and SAAV antibodies. Only one (0.3%) of the rodents, an Apodemus sylvaticus from Trabzon area, tested seropositive to CPXV. This is the first serologic survey for rodent-borne viruses in their natural hosts in Turkey. Although these preliminary results support presence of several virus groups with zoonotic potential, additional studies are needed to identify the specific viruses that are present in these populations.

Saaremaa Hantavirus Should Not Be Confused with Its Dangerous Relative, Dobrava Virus

In a recent paper, Klempa and coauthors described an isolate from a striped field mouse (Apodemus agrarius) captured in Slovakia (4). They concluded that this hantavirus, which they call Dobrava virus (DOBV)-Aa, is responsible for most of the DOBV-caused cases of hemorrhagic fever with renal syndrome (HFRS) in central Europe. While we appreciate this contribution, we also feel that in that paper some opinions on the controversial issue of DOBV and Saaremaa virus (SAAV) have not been represented. We wish to correct these in the following paragraphs. First, we will discuss nomenclature. The name SAAV, first suggested in 2000 (1), is in line with the old tradition in arbovirus and robovirus (for rodent borne) taxonomy; discoverers of a new virus name it after the geographic area where it was first found. Had we named the newly discovered virus properly at the beginning, i.e., in 1997 (13), there would not be any problem. But the name SAAV was not coined until 1999, when, in a collaborative study, we detected the cocirculation of DOBV and SAAV in Slovenia in association with their respective rodent hosts, A. flavicollis and A. agrarius. Reproductive isolation of these two hantaviruses—one is allowed to use such a term after the virus species concept has been introduced (see, e.g., reference 16)—was a crucial piece of information to draw a demarcation line. These results were published in 2000 (1). By that time, the first SAAV isolate had been reported from Estonia (9) and the virus was also found in Russia (12). In a paper published in the next year (14), SAAV was reported in Slovakia and it was suggested to call it “DOBV-Aa” (from A. agrarius). We feel that this name is not correct, and not for priority reasons only; it is also misleading. DOBV is known to be one of the most dangerous European viral pathogens, with a fatality rate of associated HFRS of around 10% (Table ​(Table1).1). In contrast, no fatalities have so far been associated with SAAV, not even in Estonia and Latvia, where human anti-SAAV seroprevalence is about 3% (6, 17). In addition to the paper by Klempa et al. and other studies from the same laboratory (e.g., reference 14), our unpublished results on HFRS patients in Germany, Slovakia, and Estonia confirm the low pathogenicity of SAAV for humans. Clinical data indicate that SAAV infections are much less severe than DOBV infections, perhaps even milder than Puumala virus infections. In line with this, our studies with animal models clearly distinguished SAAV from DOBV; i.e., SAAV infections did not harm mice, while DOBV killed 100% of the animals infected (5). It obviously makes a major difference for patients to be diagnosed with SAAV and not DOBV infection. TABLE 1. Differences between SAAV and DOBV In their paper, Klempa et al. stated that the SAAV isolate from Estonia is “apparently a reassortant.” This conclusion is based on the conflicting phylogenies inferred for the S/L and M segment sequences of the SAAV (DOBV-Aa) and DOBV (DOBV-Af) strains. We have discussed this specific issue in detail elsewhere (15). Here we wish to emphasize that there is an alternative point of view (7, 11, 15) which is based on the results of in-depth phylogenetic analyses using not only the TreePuzzle method (which Klempa et al. used) but also the classic maximum-likelihood, maximum-parsimony, and distance matrix methods. The important conclusion from these studies is that all SAAV (DOBV-Aa) S sequences are monophyletic; i.e., they share a most recent common ancestor which is different from the most recent common ancestor shared by all DOBV strains (DOBV-Af). The main reason for difficulties in inferring correct S segment-based phylogeny might be a host switch which probably occurred in the evolution of DOBV and SAAV (8, 11). A likely scenario is that pre-DOBV colonized another host, A. agrarius, establishing pre-SAAV. In the course of evolution, the housekeeping N and L proteins (and the encoding S and L genome RNA segments) of the two viruses have been diverging more slowly than surface glycoproteins G1 and G2 (and the encoding M segment), which are involved in the recognition of a host cell receptor(s). Consequently, M/G1G2 sequences have accumulated more mutations than S/N and Lsegm/Lprot sequences, making phylogenetic reconstructions easier. By the way, the L segment-based phylogeny inferred by Klempa et al. is currently based on a short part of the sequence (541 out of 6,500 nucleotides) and therefore awaits further investigation. In spite of this controversy, it seems that we may agree that SAAV (DOBV-Aa) and DOBV (DOBV-Af) are distinct entities. We have summarized their differences in Table ​Table1.1. It is important to make the distinction since the two viruses differ drastically in pathogenicity and circulate in the same geographic regions in central and southeastern Europe.

Dobrava, but not Saaremaa, hantavirus is lethal and induces nitric oxide production in suckling mice

Hantaviruses are the causative agents of HFRS and HCPS (hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome), two severe, and often fatal human diseases. Mortality from HFRS varies between hantaviruses; Hantaan and Dobrava show the highest, Seoul intermediate, and Puumala low mortality. Saaremaa, genetically closely related to Dobrava, is also known to induce HFRS, with low or no mortality. In this study, mice were inoculated with Dobrava and Saaremaa viruses to test for infectibility, lethality, viremia, nitric oxide production and antibody responses. Out of suckling mice intracerebrally inoculated with 50, 500 and 5000 focus-forming units of Dobrava virus, respectively, 1/8, 2/8 and 7/8 died within 18–26 days. In all but one of the lethally infected mice high levels of replicating virus were detected, and most were positive for neutralizing antibodies and showed elevated levels of nitric oxide production. All suckling mice intracerebrally inoculated with 50, 500, or 5000 focus-forming units of Saaremaa virus survived and all seroconverted. Clearly lower viral titers were observed for the Saaremaa virus-inoculated mice, also when sacrificed at day 18 after infection, compared to those in mice that died following Dobrava virus infection. Dobrava, Saaremaa, Puumala and Hantaan virus infections of adult mice were asymptomatic, and the anti-nucleocapsid protein IgG2a/IgG1-titer ratio was higher in mice inoculated with Dobrava virus than in those inoculated with Saaremaa virus. Elevated nitric oxide production was not detected in asymptomatically infected mice, and iNOS−/− mice, like normal mice, cleared viremia. In conclusion, we show that Dobrava virus and Saaremaa virus induce distinct differences in terms of survival, viremia, nitric oxide production and antibody responses in mice.

Phylogenetic evidence for the distinction of Saaremaa and Dobrava hantaviruses

Dobrava virus (DOBV) and Saaremaa virus (SAAV) are two closely related hantaviruses carried by different rodent species. The distinction of these two viruses has been a matter of debate. While the phylogenies based on the viral M segment sequences were repeatedly showing monophyly of SAAV strains, some trees based on the S segment sequences were not, thus causing questions on the demarcation between these two viruses. In order to clarify this issue, the current collection of the virus S segment sequences was subjected to extensive phylogenetic analysis using maximum likelihood, maximum parsimony and distant matrix methods. In all inferred phylogenies, the SAAV sequences were monophyletic and separated from DOBV sequences, thus supporting the view that SAAV and DOBV are distinct hantavirus species. Since collection of the S segment sequences used in this study "obeyed" the molecular clock, calculations of the split of DOBV and SAAV were now repeated resulting in an estimation of 3.0–3.7 MYA that is very close to the values obtained earlier.

Is there an association ofPneumocystisinfection with the presence of arena-, hanta-, and poxvirus antibodies in wild mice and shrews in Finland?

As part of studies on the nature of the endemic virus infections in natural rodent hosts, the possible association of cyst forms of Pneumocystis spp. with the presence of hanta-, cowpox-, and arenavirus antibodies in wild mice (Apodemus flavicollis, N=105; Apodemus agrarius, N=63; Micromys minutus, N=50) and the common shrew (Sorex araneus, N=101) was studied in south-central Finland. One hantavirus (Saaremaa virus, SAAV) seropositive A. agrarius, and 2 cowpoxvirus (CPXV) seropositive S. araneus were detected, and antibodies against an arenavirus (Lymphocytic choriomeningitis virus, LCMV) were found in all 3 mouse species but not in shrews. Cyst forms of Pneumocystis spp. were detected in all species except A. agrarius. There was no significant association between virus antibodies (LCMV in mice, and CPXV in shrews) and cyst forms of Pneumocystis in any of the species. Concurrent presence of virus antibodies (LCMV) and cyst forms of Pneumocystis were detected only in 1 M. minutus. In conclusion, we found no evidence of any association between Pneumocystis and antibodies to any of the viruses tested.