Puumala Virus Research Articles

Seroprevalence and incidence of Puumala orthohantavirus in its bank vole (Myodes glareolus) host population in northeastern France: Between-site and seasonal variability

Given the difficulty of measuring pathogen transmission in wildlife, epidemiological studies frequently rely on cross-sectional seroprevalence. However, seropositivity indicates only exposure to a pathogen at an unknown time. By allowing to obtain repeated test results from individuals sampled multiple times over an extended period, longitudinal data help reduce this uncertainty. We used capture-mark-recapture data on bank vole (Myodes glareolus) individuals collected at four sites over ten years in northeastern France to investigate the impact of environmental variables on seroprevalence and incidence of Puumala orthohantavirus (PUUV). PUUV causes a chronic infection without apparent symptoms, that may however impair survival of its rodent host in the wild. Viral transmission between rodents may occur through direct contact or via the environment. Principal component analysis was used to deal with multicollinearity among environmental variables. Incidence and seroprevalence were investigated with either generalized estimating equations or Poisson regression models depending on the number of observations for each season. In spring, only the factor site was found to be significant for seroprevalence, while a principal component including meteorological conditions of the previous winter and the normalized difference vegetation index (NDVI) of both the previous winter and spring had a significant effect on incidence. In autumn, only the factor site was significant for incidence, while two principal components, including either the meteorological conditions of the autumn and previous spring or NDVI of the autumn significantly affected seroprevalence. We discuss these results in light of the particular demography of small mammals. We encourage other researchers to investigate the relationships between demographic parameters of wild host populations and the environment, by using both incidence and seroprevalence.

Analysis of the relationship between replication of the Hokkaido genotype of Puumala orthohantavirus and autophagy

Hantaviruses are potentially fatal zoonotic pathogens of the family Hantaviridae. No human infection by the Hokkaido genotype of Puumala orthohantavirus (PUUV-Hok) has been reported. However, other PUUV genotypes cause hemorrhagic fever with renal syndrome (HFRS) in humans. Autophagy is a highly conserved lysosomal degradation process in eukaryotic cells that affects the replication of various viruses. In this study, we examined the role of autophagy in PUUV-Hok replication. PUUV-Hok infection induced the expression of LC3-II, an autophagosome marker, and the nucleocapsid protein (NP) of PUUV-Hok was colocalized with punctate structures of LC3. Inhibition of autophagy using an siRNA for Atg5, an autophagy-related gene, increased the replication of PUUV-Hok, whereas an autophagy inducer decreased its replication. Inhibition of lysosomal degradation increased the expression of NP and LC3-II. In summary, autophagy was induced by PUUV-Hok infection, which inhibited PUUV-Hok replication in a manner related to the degradation of the NP in lysosomes.

Puumala Orthohantavirus Infection Does Not Affect the Trapping Success of Its Reservoir Host.

Pathogens might affect behavior of infected reservoir hosts and hence their trappability, which could bias population estimates of pathogen prevalence. In this study, we used snap-trapping data on Puumala orthohantavirus (PUUV)-infected (n = 1619) and noninfected (n = 6940) bank voles (Myodes glareolus) from five vole cycles, normally representing increase, peak, and decline phase, to evaluate if infection status affected trapping success. If PUUV infection, as previously suggested, increases activity and/or mobility, we would expect a higher proportion of infected than noninfected specimens in the first trapping night. However, the proportion of PUUV-infected voles did not differ across the three trapping nights. We conclude that PUUV infection did not affect trapping success, confirming snap trapping as an appropriate trapping method for studies on PUUV prevalence and likely other orthohantaviruses.

Neutralizing Antibody Titers in Hospitalized Patients with Acute Puumala Orthohantavirus Infection Do Not Associate with Disease Severity.

Nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome (HFRS), is an acute febrile illness caused by Puumala orthohantavirus (PUUV). NE manifests typically with acute kidney injury (AKI), with a case fatality rate of about 0.1%. The treatment and management of hantavirus infections are mainly supportive, although neutralizing monoclonal antibodies and immune sera therapeutics are under investigation. In order to assess the potential use of antibody therapeutics in NE, we sought to determine the relationship between circulating PUUV neutralizing antibodies, PUUV nucleocapsid protein (N) IgG antibodies, and viral loads with markers of disease severity. The study included serum samples of extensively characterized patient cohorts (n = 116) from Tampere University Hospital, Finland. The results showed that upon hospitalization, most patients already had considerable neutralizing and anti-PUUV-N IgG antibody levels. However, contrary to expectations, neutralizing antibody titers from the first day of hospitalization did not appear to protect from AKI or correlate with more favorable disease outcomes. This indicates that further studies are needed to investigate the applicability of neutralizing antibodies as a therapy for hospitalized NE patients.

Abdominal CT findings in Puumala hantavirus-infected patients.

Previous clinical studies have reported abdominal findings on ultrasonography or MRI in Puumala hantavirus-infected patients. To determine if abdominal computed tomography (CT) can lead to a diagnosis of Puumala virus infection in the presence of a suggestive clinical picture. CT findings were studied retrospectively in 30 patients who presented to the emergency department of two (Belgian) hospitals with serologically confirmed Puumala hantavirus infection. The most frequent finding was perirenal fascial thickening (90%), followed by perirenal fat stranding (87%). Retroperitoneal fat stranding was found in 19 patients (64%) in the perivesical spaces along the fascia of the external iliac vessels with or without involvement of the presacral fat. Half of the patients had pelvic ascites, and pleural fluid was found in 7 of them. The right and left mean pole-to-pole kidney's lengths were respectively 125.7mm and 127.8mm in 28 patients. Retroperitoneal fat stranding, perirenal fascial thickening and/or perirenal fat stranding were found in most patients with acute Puumala virus infection who have undergone CT. Although nonspecific, these findings may help to suggest Puumala hantavirus infection in the right clinical settings.

Expression Profile of Human Renal Mesangial Cells Is Altered by Infection with Pathogenic Puumala Orthohantavirus.

Acute kidney injury (AKI) with proteinuria is a hallmark of infections with Eurasian orthohantaviruses. Different kidney cells are identified as target cells of hantaviruses. Mesangial cells may play a central role in the pathogenesis of AKI by regulation of inflammatory mediators and signaling cascades. Therefore, we examined the characteristics of hantavirus infection on human renal mesangial cells (HRMCs). Receptor expression and infection with pathogenic Puumala virus (PUUV) and low-pathogenic Tula virus (TULV) were explored. To analyze changes in protein expression in infected mesangial cells, we performed a proteome profiler assay analyzing 38 markers of kidney damage. We compared the proteome profile of in vitro-infected HRMCs with the profile detected in urine samples of 11 patients with acute hantavirus infection. We observed effective productive infection of HRMCs with pathogenic PUUV, but only poor abortive infection for low-pathogenic TULV. PUUV infection resulted in the deregulation of proteases, adhesion proteins, and cytokines associated with renal damage. The urinary proteome profile of hantavirus patients demonstrated also massive changes, which in part correspond to the alterations observed in the in vitro infection of HRMCs. The direct infection of mesangial cells may induce a local environment of signal mediators that contributes to AKI in hantavirus infection.

Cluster of human Puumala orthohantavirus infections due to indoor exposure?-An interdisciplinary outbreak investigation.

Puumala orthohantavirus (PUUV) is the most important hantavirus species in Europe, causing the majority of human hantavirus disease cases. In central and western Europe, the occurrence of human infections is mainly driven by bank vole population dynamics influenced by beech mast. In Germany, hantavirus epidemic years are observed in 2‐ to 5‐year intervals. Many of the human infections are recorded in summer and early autumn, coinciding with peaks in bank vole populations. Here, we describe a molecular epidemiological investigation in a small company with eight employees of whom five contracted hantavirus infections in late 2017. Standardized interviews with employees were conducted to assess the circumstances under which the disease cluster occurred, how the employees were exposed and which counteractive measures were taken. Initially, two employees were admitted to hospital and serologically diagnosed with hantavirus infection. Subsequently, further investigations were conducted. By means of a self‐administered questionnaire, three additional symptomatic cases could be identified. The hospital patients' sera were investigated and revealed in one patient a partial PUUV L segment sequence, which was identical to PUUV sequences from several bank voles collected in close proximity to company buildings. This investigation highlights the importance of a One Health approach that combines efforts from human and veterinary medicine, ecology and public health to reveal the origin of hantavirus disease clusters.

Human antibody recognizing a quaternary epitope in the Puumala virus glycoprotein provides broad protection against orthohantaviruses.

The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the "capping loop" of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HFRS-associated hantaviruses, and single doses of this nAb could protect Syrian hamsters and bank voles challenged with the highly virulent HCPS-causing ANDV and HFRS-causing PUUV, respectively. ADI-42898 is a promising candidate for clinical development as a countermeasure for both HCPS and HFRS, and its mode of Gn/Gc recognition informs the development of broadly protective hantavirus vaccines.

Long-Term Consequences of Puumala Hantavirus Infection.

Several viral infections are associated with acute and long-term complications. During the past two years, there have been many reports on post-infectious symptoms of the patients suffering from COVID-19 disease. Serious complications occasionally occur during the acute phase of Puumala orthohantavirus caused nephropathia epidemica. Severe long-term consequences are rare. Fatigue for several weeks is quite common. Hormonal insufficiencies should be excluded if the patient does not recover normally.

Analysis of Puumala orthohantavirus Genome Variants Identified in the Territories of Volga Federal District.

Hemorrhagic fever with renal syndrome (HFRS) is a zoonotic disease commonly diagnosed in the Volga Federal District (VFD). HFRS is caused by Puumala orthohantavirus (PUUV), and this virus is usually detected in bank voles as its natural host (Myodes glareolus). The PUUV genome is composed of the single-stranded, negative-sense RNA containing three segments. The goal of the current study is to identify genome variants of PUUV strains circulating in bank voles captured in the Udmurt Republic (UR) and Ulyanovsk region (ULR). The comparative and phylogenetic analysis of PUUV strains revealed that strains from Varaksino site UR are closely related to strains previously identified in the Pre-Kama area of the Republic of Tatarstan (RT), whilst strains from Kurlan and Mullovka sites ULR are similar to strains from the Trans-Kama area of the RT. It was also found that Barysh ULR strains form a separate distinct group phylogenetically equidistant from Varaksino and Kurlan–Mullovka groups. The identified groups of strains can be considered as separate sub-lineages in the PUUV Russian genetic lineage. In addition, the genomes of the strains from the UR, most likely, were formed as a result of reassortment.

Hantavirus Disease: An Update

In addition to the well-known clinical early symptoms of hantavirus disease (fever, flank and abdominal pain as well as arthralgia), unusual neurological changes in the context of infection come into focus. The spectrum of neurological symptoms ranges from transient myopia to severe pareses in the context of Guillain-Barré syndrome. In endemic areas, rapid IgM tests for initial assessment are of certain value for differential diagnosis. For therapeutic approaches, only supportive measures up to transient dialysis are available.Molecular genetic analysis and comparison of hantavirus strains of patients and mice from the same geographical area allowed molecular characterization of different outbreak regions. In the meantime, the Puumala viruses of the main outbreak regions in Germany are molecularly well characterized; therefore, the nucleotide sequence of the virus strain detected in a patient makes it possible to draw conclusions about the geographic region where the patient's infection took place.The human pathogenic hantaviruses being prevalent in Germany are the Puumala virus (reservoir: bank vole) and the Dobrava-Belgrade virus, genotype Kurkino (reservoir: striped field mouse). Recently, the molecular detection of further hantaviruses in patients with hantavirus disease was achieved. It can be concluded that also the Seoul virus (reservoir: rats) and the Tulavirus (reservoir: field mouse and related species) occasionally cause hantavirus disease in Germany.New results revealed that human infections can occur not only by the generally accepted route of inhalation of virus-containing aerosols, but also by ingestion of virus-containing materials.For patients with hantavirus infection or disease, it can be assumed that they are not infectious for their environment. A new systematic review could not confirm a human-to-human transmission previously postulated for South American hantaviruses.While all known human pathogenic hantaviruses are transmitted by rodents, other hantaviruses have been recently detected in shrews, moles, and bats. The clinical significance of these new viruses is quite unclear as yet.

Cocirculation of Leptospira spp. and multiple orthohantaviruses in rodents, Lithuania, Northern Europe.

In Europe, zoonotic Leptospira spp. and orthohantaviruses are mainly associated with specific rodent hosts. These pathogens cause febrile human diseases with similar symptoms and disease progression. In Lithuania, the presence of Dobrava-Belgrade orthohantavirus (DOBV), Tula orthohantavirus (TULV) and Leptospira spp. in rodent reservoirs is still unknown, and Puumala orthohantavirus (PUUV) was detected in bank voles (Clethrionomys glareolus) at only one site. Therefore, we collected and screened 1617 rodents and insectivores from Lithuania for zoonotic (re-)emerging Leptospira and orthohantaviruses. We detected Leptospira DNA in six rodent species, namely striped field mouse (Apodemus agrarius), yellow-necked mouse (Apodemus flavicollis), bank vole, common vole (Microtus arvalis), field vole (Microtus agrestis) and root vole (Microtus oeconomus). Leptospira DNA was detected with an overall mean prevalence of 4.4% (range 3.7%-7.9% per rodent species). We detected DOBV RNA in 5.6% of the striped field mice, PUUV RNA in 1% of bank voles and TULV RNA in 4.6% of common voles, but no Leptospira DNA in shrews and no hantavirus-Leptospira coinfections in rodents. Based on the complete coding sequences of the three genome segments, two distant DOBV phylogenetic lineages in striped field mice, one PUUV strain in bank voles and two TULV strains in common voles were identified. The Leptospira prevalence for striped field mice and yellow-necked mice indicated a significant negative effect of the distance to water points. The detection of (re-)emerging human pathogenic Leptospira and three orthohantaviruses in rodent reservoirs in Lithuania calls for increased awareness of public health institutions and allows the improvement of molecular diagnostics for pathogen identification.

Characterization of Hantavirus N Protein Intracellular Dynamics and Localization.

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation, and several other crucial processes during hantavirus infection. In this study, we generated fluorescently tagged N protein constructs derived from Puumalavirus (PUUV), the dominant hantavirus species in Central, Northern, and Eastern Europe. We comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We observed formation of large, fibrillar PUUV N protein aggregates, rapidly coalescing from early punctate and spike-like assemblies. Moreover, we found significant spatial correlation of N with vimentin, actin, and P-bodies but not with microtubules. N constructs also co-localized with Gn and Gc albeit not as strongly as the glycoproteins associated with each other. Finally, we assessed oligomerization of N constructs, observing efficient and concentration-dependent multimerization, with complexes comprising more than 10 individual proteins.

Increased Heparanase Levels in Urine during Acute Puumala Orthohantavirus Infection Are Associated with Disease Severity.

Old–world orthohantaviruses cause hemorrhagic fever with renal syndrome (HFRS), characterized by acute kidney injury (AKI) with transient proteinuria. It seems plausible that proteinuria during acute HFRS is mediated by the disruption of the glomerular filtration barrier (GFB) due to vascular leakage, a hallmark of orthohantavirus–caused diseases. However, direct infection of endothelial cells by orthohantaviruses does not result in increased endothelial permeability, and alternative explanations for vascular leakage and diminished GFB function are necessary. Vascular integrity is partly dependent on an intact endothelial glycocalyx, which is susceptible to cleavage by heparanase (HPSE). To understand the role of glycocalyx degradation in HFRS–associated proteinuria, we investigated the levels of HPSE in urine and plasma during acute, convalescent and recovery stages of HFRS caused by Puumala orthohantavirus. HPSE levels in urine during acute HFRS were significantly increased and strongly associated with the severity of AKI and other markers of disease severity. Furthermore, increased expression of HPSE was detected in vitro in orthohantavirus–infected podocytes, which line the outer surfaces of glomerular capillaries. Taken together, these findings suggest the local activation of HPSE in the kidneys of orthohantavirus–infected patients with the potential to disrupt the endothelial glycocalyx, leading to increased protein leakage through the GFB, resulting in high amounts of proteinuria.

PROPOZYCJA MONITORINGU NORNICY RUDEJ (MYODES GLAREOLU, SCHREBER 1780)

There are many reasons why it might be necessary to monitor the bank voles. It causes damage to trees and eats seeds in orchards and tree plantations, and the determination of its presence as well as the effectiveness of the measures taken to minimize losses is very important in the activity of the above-mentioned. The rodent is also the main for central Europe reservoir of the Puumala virus, which is dangerous to humans, so observing the trends in the number of voles may be crucial. The small number of the species may indicate environmental poisoning, especially with copper. And finally, it is a very important species for the environment, both transformed by man and natural, so it is very important to preserve it despite the climate changes. The bank vole is a small mammal from the Cricetidae family. It occurs all over Europe. It lives mainly in deciduous forests of medium density and rich undergrowth, although it is also found in other areas, including anthropogenic ones. The proposed monitoring is based on catching with the use of wooden box traps and the analysis of pellets. The indicators used would be: the number of individuals caught per 100 trap-nights, the percentage of the species in total number of small mammals and the age structure of the population. In the first method one should remember about practices that will reduce or eliminate the mortality of captured animals – the appropriate setting of traps and selection of weather conditions. Also collecting pellets, one should bear in mind the limitations of this method and the differences between the spring-summer and autumn-winter material. The determination of the condition of the habitat may be based on the percentage of deciduous trees, density of trees and the appearance of the undergrowth.

Hantavirus Associated with Hemorrhagic Fever with Renal Syndrome Outbreak in the Saratov Region in 2019

Pathogenic hantaviruses, belonging to the family Hantaviridae, genus Orthohantavirus, are widely spread in many regions of the world and cause hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia. In the European Russia, the most active HFRS foci are located in the optimum habitat area of natural reservoir of the Puumala virus (PUUV), bank voles (Myodes glareolus), – in the Middle Volga and Cis-Urals. The largest number of cases of HFRS was registered in the Volga Federal District. In 2019, an outbreak of HFRS was registered among the residents of Saratov and the Saratov Region, the number of cases was 2702. Objective of the study was genetic identification of hantaviruses from HFRS patients and rodent carriers and phylogenetic analysis of full-size genomes from natural hosts during HFRS outbreak in the Saratov Region, 2019. Materials and methods. Blood samples of 8 HFRS patients from Saratov and 3 lung samples of bank voles captured in the suburb of Saratov were analyzed using reverse transcription polymerase chain reaction, followed by sequencing and phylogenetic analysis. Results and discussion. A total of 6 viral RNA isolates from HFRS patients were genetically typed, full-length RNA-isolate genomes were obtained for 3 natural carriers. Our data indicate that PUUV virus was associated with HFRS outbreak in Saratov. Genetic analysis revealed that the virus belonged to RUS lineage of PUUV, related most closely to strains from Udmurtia and Tatarstan and differed from strains circulating in the territory of Bashkortostan and Samara Region.

Immunogenic Properties of MVs Containing Structural Hantaviral Proteins: An Original Study.

Hemorrhagic fever with renal syndrome (HFRS) is an emerging infectious disease that remains a global public health threat. The highest incidence rate is among zoonotic disease cases in Russia. Most cases of HFRS are reported in the Volga region of Russia, which commonly identifies the Puumala virus (PUUV) as a pathogen. HFRS management is especially challenging due to the lack of specific treatments and vaccines. This study aims to develop new approaches for HFRS prevention. Our goal is to test the efficacy of microvesicles (MVs) as PUUV nucleocapsid (N) and glycoproteins (Gn/Gc) delivery vehicles. Our findings show that MVs could deliver the PUUV N and Gn/Gc proteins in vitro. We have also demonstrated that MVs loaded with PUUV proteins could elicit a specific humoral and cellular immune response in vivo. These data suggest that an MV-based vaccine could control HFRS.

Changes in the Epidemiology of Zoonotic Infections in Children: A Nationwide Register Study in Finland.

Zoonotic infections are difficult to recognize in children. The age distributions and seasonal occurrences of these infections vary substantially, even among those transmitted by the same vectors, and their epidemiology may change over time. The aim was to report the incidences and trends of Borrelia burgdorferi, Puumala virus, Francisella tularensis and tick-borne encephalitis (TBE) virus infections in the pediatric population (age 0-19) of Finland. A nationwide survey based on the National Infectious Disease Register was conducted from 1996 to 2019 and all laboratory-confirmed cases were included. Age-stratified incidences per 100,000 person-years were calculated. Cumulative incidences were B. burgdorferi 11.2, TBE 0.4, Puumala virus 6.4 and F. tularensis 2.5 per 100,000 person-years. An increasing trend in the incidences of B. burgdorferi and TBE was observed. Borrelia expanded geographically northward and inland. Tularemia follows a 2-4-year epidemic cycle and rates are similar across age groups. Puumala incidences are highest in the older children. Borrelia infections increased most rapidly in children 5-9 years of age and overall expanded geographically in Finland. Tularemia epidemic cycles were shorter than those previously reported. These results will help clinicians to identify these infections in different geographic areas and age groups in Finland.

Dissection of the NKG2C NK cell response against Puumala Orthohantavirus.

BackgroundInfections with the Puumala orthohantavirus (PUUV) in humans may cause hemorrhagic fever with renal syndrome (HFRS), known as nephropathia epidemica (NE), which is associated with acute renal failure in severe cases. In response to PUUV-infections, a subset of potent antiviral NKG2C+ NK cells expand, whose role in virus defence and pathogenesis of NE is unclear. NKG2C+ NK cell proliferation is mediated by binding of NKG2C/CD94 to HLA-E on infected cells. The proliferation and activation of NKG2C+ NK cells via the NKG2C/HLA-E axis is affected by different NKG2C (NKG2Cwt/del) and HLA-E (HLA-E*0101/0103) alleles, which naturally occur in the human host. Homozygous (NKG2Cdel/del) and heterozygous (NKG2Cwt/del) deletions of the NKG2C receptor results in an impaired NKG2C/CD94 mediated proliferation and activation of NKG2C+ cells. We therefore analyzed the PUUV-mediated NKG2C+ NK cell responses and the impact of different NKG2C and HLA-E alleles in NE patients.Methodology/Principal findingsNKG2C+ NK cell expansion and effector functions in PUUV-infected cells were investigated using flow cytometry and it was shown that PUUV-infected endothelial cells led to a NKG2C/CD94 mediated NKG2C+ NK cell activation and expansion, dependent on the HLA-G-mediated upregulation of HLA-E. Furthermore, the NKG2Cdel and HLA-E*0101/0103 alleles were determined in 130 NE patients and 130 matched controls, and it was shown that in NE patients the NKG2Cwt/del allele was significantly overrepresented, compared to the NKG2Cwt/wt variant (p = 0.01). In addition, in vitro analysis revealed that NKG2Cwt/del NK cells exhibited on overall a lower proliferation (p = 0.002) and lower IFNγ expression (p = 0.004) than NKG2Cwt/wt NK cells.Conclusions/SignificanceOur results corroborate the substantial impact of the NKG2C/HLA-E axis on PUUV-specific NK cell responses. A weak NKG2C+ NK cell response, as reflected by NKG2Cwt/del variant, may be associated with a higher risk for a severe hantavirus infections.

Comparison of indirect immunofluorescence and western blot method in the diagnosis of hantavirus infections.

BACKGROUNDSerologic cross-reactivity between hantaviruses often complicates the interpretation of the results.AIMTo analyze the diagnostic value of indirect immunofluorescence assay (IFA) and western blot (WB) in the diagnosis of hantavirus infections.METHODSOne hundred eighty-eight serum samples from Puumala (PUUV) and Dobrava (DOBV) orthohantavirus infected patients were analyzed. Serology was performed using commercial tests (Euroimmun, Lübeck, Germany).RESULTSUsing IFA, 49.5% of acute-phase samples showed a monotypic response to PUUV, while 50.5% cross-reacted with other hantaviruses. The overall cross-reactivity was higher for immunoglobulin G (IgG) (50.0%) than for immunoglobulin M (IgM) (25.5%). PUUV IgM/IgG antibodies showed low/moderate reactivity with orthohantaviruses Hantaan (12.3%/31.5%), Seoul (7.5%/17.8%), DOBV (5.4%/ 28.1%), and Saaremaa (4.8%/15.7%). Both DOBV IgM and IgG antibodies were broadly reactive with Hantaan (76.2%/95.2%), Saaremaa (80.9%/83.3%), and Seoul (78.6%/85.7%) and moderate with PUUV (28.5%/38.1%). Using a WB, serotyping was successful in most cross-reactive samples (89.5%). CONCLUSIONThe presented results indicate that WB is more specific than IFA in the diagnosis of hantavirus infections, confirming serotype in most IFA cross-reactive samples.