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Puumala orthohantavirus (PUUV) is a causative agent of haemorrhagic fever with renal syndrome, mainly transmitted through rodent excretions. Classification of PUUV has traditionally been based on the geographic locations of their discovery, but this system faces challenges due to inconsistencies between the names of lineages and their actual distribution. In this study, we propose a quantitative criterion of "10% nucleotide difference" to distinguish PUUV genotypes, resulting in a subdivision of PUUV into twentyone distinct genotypes. The application of this criterion suggested a further division of the CE, RUS, FIN and N-SCA lineages and was consistent with the current taxonomic position of the S-SCA, LAT, DAN and ALAD lineages. In addition, analysis of reassortment and recombination events showed that PUUV undergoes these processes, but they are relatively rare. Our results emphasise the importance of considering genetic distances in the classification of PUUV and provide a robust criterion for subspecies-level taxonomy. [ABSTRACT FROM AUTHOR]

The Puumala orthohantavirus is present in the body of the bank vole (Myodes glareolus). Humans infected with this virus may develop hemorrhagic fever accompanying renal syndrome. In addition, the infection may further lead to the failure of an immune system completely. The present study aimed to propose a possible vaccine by employing bioinformatics techniques to identify B and T‐cell antigens. The best multi‐epitope of potential immunogenicity was generated by combining epitopes. Additionally, the linkers EAAAK, AAY, and GPGPG were utilized in order to link the epitopes successfully. Further, C‐ImmSim was used to perform in silico immunological simulations upon the vaccine. For the purpose of conducting expression tests in Escherichia coli, the chimeric protein construct was cloned using Snapgene into the pET‐9c vector. The designed vaccine showed adequate results, evidenced by the global population coverage and favorable immune response. The developed vaccine was found to be highly effective and to have excellent population coverage in a number of computer‐based assessments. This work is fully dependent on the development of nucleoprotein‐based vaccines, which would constitute a significant step forward if our findings were used in developing a global vaccination to combat the Puumala virus. [ABSTRACT FROM AUTHOR]

Introduction. Vaccination could be the most effective means of preventing hemorrhagic fever with renal syndrome (HFRS), which is one of the leading zoonoses and a major cause of natural disease in humans. Therefore, research to develop an effective vaccine and the search for new non-toxic and effective adjuvants that can enhance and prolong the immune response, reduce the antigen concentration and reduce the frequency of dose administration is an urgent task. The aim of the study was to determine the dependence of the immunogenic activity of an experimental hantavirus vaccine based on Puumala virus (HV) on the antigen dose both in native form and in combination with carbohydrate adjuvants. Materials and methods. The immunoadjuvant effect of Ac3-LPS S. sonnei and S. flexneri 1B and emulsions of nanoemulsified squalene Sepivac SWE (SWE) as part of HV at the optimal and minimal doses was investigated by induction of neutralising antibodies after 3-fold immunisation (day 0, 14, 182) of guinea pigs (Cavia porcellus). Differences in the immunogenic activity of HV were assessed in the neutralisation reaction by 50% suppression of focal units in Vero E6 cell cultures. Results. A statistically significant increase in neutralising antibodies levels was observed after immunization both with HV at the optimal dose and for its combination with adjuvants. Ac3-LPS S. sonnei and SWE showed the most pronounced immunoadjuvant effect at concentrations of 50 and 100 μg/dose, respectively. The immunoadjuvant effect of the combination of the hantavirus vaccine with Ac3-LPS S. sonnei and SWE contributed to both an enhanced immune response and its duration. The tenfold reduction of the antigen dose in the presence of SWE allows optimization of the immune response to the vaccine. Conclusion. The results of this study show the prospects of using Ac3-LPS S. sonnei and Sepivac SWE in the vaccine for the prevention of HFRS.

Puumala orthohantavirus (PUUV) is a causative agent of haemorrhagic fever with renal syndrome, mainly transmitted through rodent excretions. Classification of PUUV has traditionally been based on the geographic locations of their discovery, but this system faces challenges due to inconsistencies between the names of lineages and their actual distribution. In this study, we propose a quantitative criterion of “10% nucleotide difference” to distinguish PUUV genotypes, resulting in a subdivision of PUUV into twenty-one distinct genotypes. The application of this criterion suggested a further division of the CE, RUS, FIN and N-SCA lineages and was consistent with the current taxonomic position of the S-SCA, LAT, DAN and ALAD lineages. In addition, analysis of reassortment and recombination events showed that PUUV undergoes these processes, but they are relatively rare. Our results emphasise the importance of considering genetic distances in the classification of PUUV and provide a robust criterion for subspecies-level taxonomy.

In 2018, a local case of nephropathia epidemica was reported in Scania, southern Sweden, more than 500 km south of the previously known presence of human hantavirus infections in Sweden. Another case emerged in the same area in 2020. To investigate the zoonotic origin of those cases, we trapped rodents in Ballingslöv, Norra Sandby, and Sörby in southern Sweden during 2020‒2021. We found Puumala virus (PUUV) in lung tissues from 9 of 74 Myodes glareolus bank voles by screening tissues using a hantavirus pan–large segment reverse transcription PCR. Genetic analysis revealed that the PUUV strains were distinct from those found in northern Sweden and Denmark and belonged to the Finnish PUUV lineage. Our findings suggest an introduction of PUUV from Finland or Karelia, causing the human PUUV infections in Scania. This discovery emphasizes the need to understand the evolution, cross-species transmission, and disease outcomes of this newly found PUUV variant.

While the pathology of acute hemorrhagic fever with renal syndrome (HFRS) has been widely researched, details on the chronic HFRS sequelae remain mainly unexplored. In this study, we analyzed the clinical and laboratory characteristics of 30 convalescent HFRS patients 14 years after the disease contraction, mainly emphasizing several endothelial dysfunction parameters. Convalescent HFRS patients exhibited significantly higher serum levels of erythrocyte sedimentation rate, von Willebrand factor, uric acid, C-reactive protein and immunoglobulin A when compared to healthy individuals. Furthermore, 24 h urine analyses revealed significantly lower sodium and potassium urine levels, as well as significantly higher proteinuria, microalbumin levels and β2-microglobulin levels when compared to healthy individuals. First morning urine analysis revealed significantly higher levels of hematuria in convalescent HFRS patients. None of the additional analyzed endothelium dysfunction markers were significantly different in post-HFRS patients and healthy individuals, including serum and urine P-selectin, E-selectin, soluble intercellular adhesion molecule 1, vascular intercellular adhesion molecule 1 (sVCAM-1) and vascular endothelial growth factor (VEGF). However, binary logistic regression revealed a weak association of serum sVCAM-1 and urine VEGF levels with HFRS contraction. Generally, our findings suggest mild chronic inflammation and renal dysfunction levels in convalescent HFRS patients 14 years after the disease contraction. [ABSTRACT FROM AUTHOR]

ABSTRACT Laboratory diagnosis of orthohantavirus infection is primarily based on serology. However, for a confirmed serological diagnosis, evaluation of a follow-up serum sample is essential, which is time consuming and causes delay. Real-time reverse transcription polymerase chain reaction (RT-PCR) tests, if positive, provide an immediate and definitive diagnosis, and accurately identify the causative agent, where the discriminative nature of serology is suboptimal. We re-evaluated sera from orthohantavirus-suspected clinical cases in the Dutch regions of Twente and Achterhoek from July 2014 to April 2016 for the presence of Puumala orthohantavirus (PUUV), Tula orthohantavirus (TULV), and Seoul orthohantavirus (SEOV) RNA. PUUV RNA was detected in 11% of the total number (n = 85) of sera tested, in 50% of sera positive for anti-PUUV/TULV IgM (n = 16), and in 1.4% of sera negative or indeterminate for anti-PUUV/TULV IgM (n = 69). No evidence was found for the presence of TULV or SEOV viral RNA. Based on these findings, we propose two algorithms to implement real-time RT-PCR testing in routine orthohantavirus diagnostics, which optimally provide clinicians with early confirmed diagnoses and could prevent possible further invasive testing and treatment.IMPORTANCEThe addition of a real-time reverse transcription polymerase chain reaction test to routine orthohantavirus diagnostics may better aid clinical decision making than the use of standard serology tests alone. Awareness by clinicians and clinical microbiologists of this advantage may ultimately lead to a reduction in over-hospitalization and unnecessary invasive diagnostic procedures.

We analyzed Puumala virus (PUUV) sequences collected from bank voles from different regions of Russia. Phylogenetic analysis revealed PUUV reassortments in areas with the highest hemorrhagic fever with renal syndrome incidence, indicating reassortment might contribute to pathogenic properties of PUUV. Continued surveillance is needed to assess PUUV pathogenicity in Russia.

We investigated a prospective cohort of 23 patients who had Puumala virus infection in Austria to determine predictors of infection outcomes. We reviewed routinely available clinical and laboratory parameters collected when patients initially sought care. Low absolute lymphocyte count and dyspnea were parameters associated with a severe course of infection.

While the pathology of acute hemorrhagic fever with renal syndrome (HFRS) has been widely researched, details on the chronic HFRS sequelae remain mainly unexplored. In this study, we analyzed the clinical and laboratory characteristics of 30 convalescent HFRS patients 14 years after the disease contraction, mainly emphasizing several endothelial dysfunction parameters. Convalescent HFRS patients exhibited significantly higher serum levels of erythrocyte sedimentation rate, von Willebrand factor, uric acid, C-reactive protein and immunoglobulin A when compared to healthy individuals. Furthermore, 24 h urine analyses revealed significantly lower sodium and potassium urine levels, as well as significantly higher proteinuria, microalbumin levels and β2-microglobulin levels when compared to healthy individuals. First morning urine analysis revealed significantly higher levels of hematuria in convalescent HFRS patients. None of the additional analyzed endothelium dysfunction markers were significantly different in post-HFRS patients and healthy individuals, including serum and urine P-selectin, E-selectin, soluble intercellular adhesion molecule 1, vascular intercellular adhesion molecule 1 (sVCAM-1) and vascular endothelial growth factor (VEGF). However, binary logistic regression revealed a weak association of serum sVCAM-1 and urine VEGF levels with HFRS contraction. Generally, our findings suggest mild chronic inflammation and renal dysfunction levels in convalescent HFRS patients 14 years after the disease contraction.

Hemorrhagic fever with renal syndrome (HFRS) represents a serious zoonotic disease caused by orthohantaviruses in Eurasia. A specific antiviral therapy is not available. HFRS is characterized by acute kidney injury (AKI) with often massive proteinuria. Infection of kidney cells may contribute to the clinical picture. However, orthohantaviral replication in kidney cells is not well characterized. Therefore, we aimed to perform a reliable high-throughput assay that allows the quantification of infection rates and testing of antiviral compounds in different cell types. We quantified relative infection rates of Eurasian pathogenic Puumala virus (PUUV) by staining of nucleocapsid protein (N protein) in an in-cell Western (ICW) assay. Vero E6 cells, derived from the African green monkey and commonly used in viral cell culture studies, and the human podocyte cell line CIHP (conditionally immortalized human podocytes) were used to test the ICW assay for replication kinetics and antiviral drug testing. Quantification of infection by ICW revealed reliable results for both cell types, as shown by their correlation with immunofluorescence quantification results by counting infected cells. Evaluation of antiviral efficacy of ribavirin by ICW assay revealed differences in the toxicity (TC) and inhibitory concentrations (IC) between Vero E6 cells and podocytes. IC5O of ribavirin in podocytes is about 12-fold lower than in Vero E6 cells. In summary, ICW assay together with relevant human target cells represents an important tool for the study of hantaviral replication and drug testing.

In the Russian Federation, the vaccine against hemorrhagic fever with renal syndrome is at the stage of preclinical and clinical trials. The aim of the study was to analyze an effect of vaccine schedule on neutralizing antibodies (nAB) dynamics in guinea pig models applied with experimental Puumala virus based hantavirus vaccine (HV). Quantitative evaluation of neutralizing antibodies was presented as antibody titer geometric mean expressed in binary logarithms (log2) by the 50% reduction of focus-forming units (FRNT50) in Vero cell in the focus reduction neutralization test. The HV dual inoculation to guinea pigs was carried out in 14 day intervals, booster injection was applied on day 182 after the onset, in the thigh muscle tissue by using 0.3 ml undiluted (HV-u/d) and in the 1/10 dilution (HV-1/10). nAB titer on day 14 after the first HV-u/d and HV-1/10 injection was measured to be at titer of 5.50.3 and 4.80.3, respectively. After the second injection, the nAB peak was as high as 90.2 on day 42 after the first HV-u/f injection, and 6.50.2 on day 14 after the HV-1/10 injection. nAB decreased down to 6.20.3 and 50.3, respectively, on day 364 after the first injection. The booster HV-u/d and HV-1/10 injection induced increase in nAB up to 9.50.3 and 6.50.3, respectively. After the booster injection, it induced significantly higher nAB observed on day 238 after the first HV-u/d injection and delayed up to the 294 day for the HV-1/10. The results of the study indicated the early formation of the immune response, long-term nAB persistence and significantly enhanced immune response after the booster injection on day 182, which indicated a potential for the booster injection a year later. The immunological efficacy and protective activity of the vaccine schedule may be finally assessed according to the results of clinical trials.

In 2021, Slovenia faced an outbreak of hantavirus infection, which causes haemorrhagic fever with renal syndrome. In children, haemorrhagic fever with renal syndrome is rare. In this article, we present a 14-year-old girl with haemorrhagic fever with renal syndrome, which was caused by Puumala virus.

"The Power of We" is a personal tribute to the individuals and organizations that collaborated in the discovery and advancement of knowledge of the hantaviruses following the original isolation of Hantaan virus by Ho Wang Lee. It focuses on the work done primarily at the United States Army Medical Research Institute of Infectious Diseases during the decade of the 1980s under the leadership of Joel Dalrymple, who worked in close partnership with Ho Wang Lee. These early studies helped define the global distribution of Seoul virus and provided seminal information on its maintenance and transmission among urban rats. Other collaborations involved partners in Europe, Asia, and Latin America and resulted in the isolation of novel hantaviruses, a better understanding of their global distribution, and validation of diagnostics and therapeutic interventions for treatment of human diseases. By working in partnership, scientists from around the world made critical discoveries that led to a better understanding of the hantaviruses. "The Power of We" demonstrates that we all benefit when we work together with a shared vision, a common commitment to excellence, and mutual respect. [ABSTRACT FROM AUTHOR]

We aimed to estimate the seroprevalence of Puumala orthohantavirus (PUUV) among forestry workers in northern France, and to explore sociodemographic risk factors. We conducted a random cross-sectional seroprevalence survey among 1777 forestry workers in 2019–2020. The presence of immunoglobulin G against PUUV antigens in serum was assessed using enzyme-linked immunosorbent assay and confirmed using immunofluorescence assay. Poisson regression models were used to explore factors associated with seropositivity. Weighted seroprevalence was 5% (3–6) in northeastern France, 4% (2–6) in north central France, and 1% in two regions located in the center of the country (Auvergne and Limousin). There were no seropositive workers detected in northwestern France. Seropositivity was associated with age, sex, and cumulative seniority in the forestry sector. Seroprevalence was highest in known endemic areas of the northeast and lowest in the northwest. Nevertheless, we found serological evidence of PUUV infection in two regions located in the center of the country, suggesting circulation of the virus in these regions, previously thought to be non-endemic. [ABSTRACT FROM AUTHOR]

Puumala orthohantavirus (PUUV) is endemic in Europe and can cause hemorrhagic fever with renal syndrome (nephropathia epidemica). Disease features include fever, thrombocytopenia, and acute kidney injury (AKI). This retrospective cohort study of forty PUUV patients aims to characterize associations of serum immunological, hemostatic or kidney injury markers to disease severity. While interleukin-18 (IL-18) was significantly increased in severely thrombocytopenic patients (<100 × 109 platelets/L) compared to patients with higher platelet counts, RANTES was significantly decreased in these patients. These data suggest that patients with significant thrombocytopenia might have experienced pronounced Th1 immune responses. When kidney dysfunction was used as the primary disease outcome, recently identified AKI biomarkers (Cystatin C, insulin-like growth factor-binding protein 7, Nephrin, and trefoil factor 3) were significantly upregulated in patients with severe PUUV infection, defined as the estimated glomerular filtration rate (eGFR) below 30 m/min/1.73 m2. The increased expression of these markers specifically indicates pathology in glomeruli and proximal tubuli. Furthermore, E-selectin was significantly higher while interferon gamma-induced protein 10 (IP-10) was significantly lower in PUUV patients with more severe kidney dysfunction compared to patients with higher eGFR-values. Increased E-selectin illustrates the central role of endothelial cell activation, whereas decreased IP-10 could indicate a less important role of this cytokine in the pathogenesis of kidney dysfunction. [ABSTRACT FROM AUTHOR]

Hemorrhagic fever with renal syndrome (HFRS) continues to be a cause of death in Europe. Our aim was to describe the clinical and histopathological features of fatal HFRS in the Udmurt Republic (Udmurtia), located in the European part of Russia. This retrospective observational study included all fatal cases of HFRS that occurred in Udmurtia from January 2010 through December 2019. The most relevant clinical and autopsy data of these cases were recorded through a review of the patients' medical records and autopsy reports. During 2010–2019, Udmurtia had 41 fatal cases of HFRS of a total of 10,312 confirmed cases (case-fatality rate of 0.4%). Twenty-seven patients died in hypotensive and oliguric phases of HFRS due to refractory septic shock and acute respiratory distress syndrome. Fourteen patients died in the polyuric phase of the disease from complications of acute kidney injury or because of hospital-acquired bacterial infections. Multiorgan involvement was noted in all autopsies with variable degrees of generalized venous congestion, interstitial edema, capillary wall thickening, perivascular deposition of plasma proteins, microthrombosis formation, and perivascular hemorrhage. The more prominent histopathological features were seen in kidneys, lungs, and hypophysis. [ABSTRACT FROM AUTHOR]

Background: Previous clinical studies have reported abdominal findings on ultrasonography or MRI in Puumala hantavirus-infected patients. Purpose: To determine if abdominal computed tomography (CT) can lead to a diagnosis of Puumala virus infection in the presence of a suggestive clinical picture. Material and methods: CT findings were studied retrospectively in 30 patients who presented to the emergency department of two (Belgian) hospitals with serologically confirmed Puumala hantavirus infection. Results: 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 125.7 mm and 127.8 mm in 28 patients. Conclusion: 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. [ABSTRACT FROM AUTHOR]

Abstract Background Transmission of all known pathogenic orthohantaviruses (family Hantaviridae) usually occurs via inhalation of aerosols contaminated with viral particles derived from infected rodents and organ manifestation of infections is characterized by lung and kidney involvement. Orthohantaviruses found in Eurasia cause hemorrhagic fever with renal syndrome (HFRS) and New World orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS). However, cases of infection with Old World orthohantaviruses with severe pulmonary manifestations have also been observed. Therefore, human airway cells may represent initial targets for orthohantavirus infection and may also play a role in the pathogenesis of infections with Eurasian orthohantaviruses. Methods We analyzed the permissiveness of primary endothelial cells of the human pulmonary microvasculature and of primary human epithelial cells derived from bronchi, bronchioles and alveoli for Old World orthohantavirus Puumala virus (PUUV) in vitro. In addition, we examined the expression of orthohantaviral receptors in these cell types. To minimize donor-specific effects, cells from two different donors were tested for each cell type. Results Productive infection with PUUV was observed for endothelial cells of the microvasculature and for the three tested epithelial cell types derived from different sites of the respiratory tract. Interestingly, infection and particle release were also detected in bronchial and bronchiolar epithelial cells although expression of the orthohantaviral receptor integrin β3 was not detectable in these cell types. In addition, replication kinetics and viral release demonstrate enormous donor-specific variations. Conclusions The human respiratory epithelium is among the first targets of orthohantaviral infection and may contribute to virus replication, dissemination and pathogenesis of HFRS-causing orthohantaviruses. Differences in initial pulmonary infection due to donor-specific factors may play a role in the observed broad variance of severity and symptoms of orthohantavirus disease in patients. The absence of detectable levels of integrin αVβ3 surface expression on bronchial and small airway epithelial cells indicates an alternate mode of orthohantaviral entry in these cells that is independent from integrin β3.

There are emerging eosinophil-related considerations concerning viral infections. The role of eosinophils has poorly been evaluated during Hantavirus infection. The aim of this study was to determine the prevalence of eosinophilia (defined as an eosinophil count above 500 cells/mm3) during haemorrhagic fever with renal syndrome (HFRS) in a large cohort of patients, and to identify factors associated with eosinophilia. Among 387 patients hospitalized for HFRS, 98 (25.3%) had eosinophilia. By univariate analysis, eosinophilia was significantly associated with more severe thrombocytopenia, high C-reactive protein level, white blood cell count and neutrophil count and lower nephrotoxic drug intake. As there was a collinearity between white blood cell count and C-reactive protein level, only C-reactive protein level with platelet count and nephrotoxic drug intake were entered in the multivariable analysis. Elevated C-reactive protein concentrations remained independently associated with eosinophilia. Eosinophilia during HFRS affects one quarter of patients, and supports the role of eosinophils in antiviral immunity against hantavirus infection. [ABSTRACT FROM AUTHOR]

We aimed to estimate the seroprevalence of Puumala orthohantavirus (PUUV) among forestry workers in northern France, and to explore sociodemographic risk factors. We conducted a random cross-sectional seroprevalence survey among 1777 forestry workers in 2019–2020. The presence of immunoglobulin G against PUUV antigens in serum was assessed using enzyme-linked immunosorbent assay and confirmed using immunofluorescence assay. Poisson regression models were used to explore factors associated with seropositivity. Weighted seroprevalence was 5% (3–6) in northeastern France, 4% (2–6) in north central France, and 1% in two regions located in the center of the country (Auvergne and Limousin). There were no seropositive workers detected in northwestern France. Seropositivity was associated with age, sex, and cumulative seniority in the forestry sector. Seroprevalence was highest in known endemic areas of the northeast and lowest in the northwest. Nevertheless, we found serological evidence of PUUV infection in two regions located in the center of the country, suggesting circulation of the virus in these regions, previously thought to be non-endemic.

Background: The largest documented outbreak of hemorrhagic fever with renal syndrome occurred in Primorje-Gorski Kotar County, Croatia, in 2021, marking the first-time cases of hantavirus infection recorded outside of the known endemic region in the north of the county. Aim: To identify the factors contributing to the spread of the outbreak and to compare risk factors for acquiring hantavirus infection in the endemic and newly affected regions. Methods and Results: A total of 189 cases were confirmed by positive Puumala IgM/IgG antibodies (93.6%), and 13 probable cases were identified by clinical and epidemiological data (6.4%) using a structured questionnaire. Of the 179 cases with available clinical data, 59 (33.0%) were hospitalized. Three cases received hemodialysis, and no deaths were reported. Among 170 cases with information on exposures, 66 (38.8%) reported occupational risk. Cases in the northern part of county were more likely to have been infected in early spring (OR 27.1, 95% CI 2.93-250.7), to report seeing a rodent (OR 6.5; 95%CI 2.3-18.4), and to know someone with hemorrhagic fever with renal syndrome (HFRS) (OR 3.0; 95%CI 1.2-8.0) than cases from the southern part of the county. Data from Croatian Forests Ltd. suggested that an unusually good production of beech seeds in 2020 may have contributed to an increased rodent population in 2021. However, average temperature, rainfall, and humidity data from 2021 did not illustrate a significant difference from previous years (Kruskal-Wallis p = 0.837, p = 0.999, p = 0.108). Conclusion: The 2021 HFRS outbreak was likely fueled by an abundant rodent population and virus transmission in rodent hosts. Human activity, environmental factors, and the ensuing animal-human interactions have spread hantavirus infection from Croatia's mountainous region to a previously nonendemic coastal area with a Mediterranean climate.

Laboratory diagnosis of orthohantavirus infection is primarily based on serology. However, for a confirmed serological diagnosis, evaluation of a follow-up serum sample is essential, which is time consuming and causes delay. Real-time reverse transcription polymerase chain reaction (RT-PCR) tests, if positive, provide an immediate and definitive diagnosis, and accurately identify the causative agent, where the discriminative nature of serology is suboptimal. We re-evaluated sera from orthohantavirus-suspected clinical cases in the Dutch regions of Twente and Achterhoek from July 2014 to April 2016 for the presence of Puumala orthohantavirus (PUUV), Tula orthohantavirus (TULV), and Seoul orthohantavirus (SEOV) RNA. PUUV RNA was detected in 11% of the total number ( n = 85) of sera tested, in 50% of sera positive for anti-PUUV/TULV IgM ( n = 16), and in 1.4% of sera negative or indeterminate for anti-PUUV/TULV IgM ( n = 69). No evidence was found for the presence of TULV or SEOV viral RNA. Based on these findings, we propose two algorithms to implement real-time RT-PCR testing in routine orthohantavirus diagnostics, which optimally provide clinicians with early confirmed diagnoses and could prevent possible further invasive testing and treatment., Importance: The addition of a real-time reverse transcription polymerase chain reaction test to routine orthohantavirus diagnostics may better aid clinical decision making than the use of standard serology tests alone. Awareness by clinicians and clinical microbiologists of this advantage may ultimately lead to a reduction in over-hospitalization and unnecessary invasive diagnostic procedures., Competing Interests: The authors declare no conflict of interest.

Orthohantaviruses, transmitted primarily by rodents, cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome in the Americas. These viruses, with documented human-to-human transmission, exhibit a wide case-fatality rate, 0.5-40 %, depending on the virus species, and no vaccine or effective treatment for severe Orthohantavirus infections exists. In Europe, the Puumala virus (PUUV), carried by the bank vole Myodes glareolus, causes a milder form of HFRS. Despite the reliance on serology and PCR for diagnosis, the three genomic segments of Swedish wild-type PUUV have yet to be completely sequenced. We have developed a targeted hybrid-capture method aimed at comprehensive genomic sequencing of wild-type PUUV isolates and the identification of other Orthohantaviruses. Our custom-designed panel includes >11,200 probes covering the entire Orthohantavirus genus. Using this panel, we sequenced complete viral genomes from bank vole lung tissue, human plasma samples, and cell-cultured reference strains. Analysis revealed that Swedish PUUV isolates belong to the Northern Scandinavian lineage, with nucleotide diversity ranging from 2.8 % to 3.7 % among them. Notably, no significant genotypic differences were observed between the viral sequences from reservoirs and human cases except in the nonstructural protein. Despite the high endemicity of PUUV in Northern Sweden, these are the first complete Swedish wild-type PUUV genomes and substantially increase our understanding of PUUV evolution and epidemiology. The panel's sensitivity enables genomic sequencing of human samples with viral RNA levels reflecting the natural progression of infection and underscores our panel's diagnostic value, and could help to uncover novel Orthohantavirus transmission routes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Pathogenic Eurasian hantaviruses cause hemorrhagic fever with renal syndrome (HFRS), which is characterized by acute kidney injury. The clinical course shows a broad range of severity and is influenced by direct and immune-mediated effects. The neutrophil-to-lymphocyte ratio (NLR) is a marker of systemic inflammation and predicts severity and outcome in various diseases. Therefore, we examined the role of NLR in HFRS caused by hantavirus Puumala (PUUV) and its association with disease severity and kidney injury. We detected elevated NLR levels on admission (NLR adm : median 3.82, range 1.75-7.59), which increased during acute HFRS. Maximum NLR levels (NLR max : median 4.19, range 1.75-13.16) were 2.38-fold higher compared to the reference NLR level of 1.76 in the general population. NLR levels on admission correlate with markers of severity (length of hospital stay, serum creatinine) but not with other markers of severity (leukocytes, platelets, C-reactive protein, lactate dehydrogenase, serum albumin, proteinuria). Interestingly, levels of nephrin, which is a specific marker of podocyte damage in kidney injury, are highest on admission and correlate with NLR max , but not with NLR adm . Together, we observed a correlation between systemic inflammation and the severity of HFRS, but our results also revealed that podocyte damage precedes these inflammatory processes., (© 2024 Wiley Periodicals LLC.)

We report 3 cases of Puumala virus infection in a family in Switzerland in January 2019. Clinical manifestations of the infection ranged from mild influenza-like illness to fatal disease. This cluster illustrates the wide range of clinical manifestations of Old World hantavirus infections and the challenge of diagnosing travel-related hemorrhagic fevers.

Background: Transmission of all known pathogenic orthohantaviruses (family Hantaviridae) usually occurs via inhalation of aerosols contaminated with viral particles derived from infected rodents and organ manifestation of infections is characterized by lung and kidney involvement. Orthohantaviruses found in Eurasia cause hemorrhagic fever with renal syndrome (HFRS) and New World orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS). However, cases of infection with Old World orthohantaviruses with severe pulmonary manifestations have also been observed. Therefore, human airway cells may represent initial targets for orthohantavirus infection and may also play a role in the pathogenesis of infections with Eurasian orthohantaviruses. Methods: We analyzed the permissiveness of primary endothelial cells of the human pulmonary microvasculature and of primary human epithelial cells derived from bronchi, bronchioles and alveoli for Old World orthohantavirus Puumala virus (PUUV) in vitro. In addition, we examined the expression of orthohantaviral receptors in these cell types. To minimize donor-specific effects, cells from two different donors were tested for each cell type. Results: Productive infection with PUUV was observed for endothelial cells of the microvasculature and for the three tested epithelial cell types derived from different sites of the respiratory tract. Interestingly, infection and particle release were also detected in bronchial and bronchiolar epithelial cells although expression of the orthohantaviral receptor integrin β3 was not detectable in these cell types. In addition, replication kinetics and viral release demonstrate enormous donor-specific variations. Conclusions: The human respiratory epithelium is among the first targets of orthohantaviral infection and may contribute to virus replication, dissemination and pathogenesis of HFRS-causing orthohantaviruses. Differences in initial pulmonary infection due to donor-specific factors may play a role in the observed broad variance of severity and symptoms of orthohantavirus disease in patients. The absence of detectable levels of integrin αVβ3 surface expression on bronchial and small airway epithelial cells indicates an alternate mode of orthohantaviral entry in these cells that is independent from integrin β3. [ABSTRACT FROM AUTHOR]

Relevance. Hemorrhagic fever with renal syndrome (HFRS) is a non-transmissible viral zoonosis widespread in Eurasia, and in Russia it occupies a leading position among zoonotic viral infections and one of the first places among all natural focal human diseases. Aim. Obtaining evidence of the safety, quality and efficacy of a polyvalent vaccine against HFRS as a result of its preclinical studies using scientific assessment methods that meet the requirements and rules of good laboratory practice. Materials and methods. For preclinical studies of the polyvalent vaccine against HFRS, the materials and methods were used in strict accordance with the requirements of the regulatory documents, as well as previously described methods used to control the vaccine at the technological stages of its manufacture. Results. The data obtained as a result of preclinical studies of the polyvalent vaccine agains HFRS indicate a high immunogenicity and stability of the vaccine, the absence of: acute and chronic toxicity, allergenic, immunotoxic and mutagenic, as well as toxic effects on the reproductive organs of animals, embryo development and offspring, born to females who received the vaccine within 20 days of gestation. Conclusion. The obtained results of preclinical studies comply with the requirements for immunobiological medical preparations designed for humans, and are the basis for conducting the 1st phase of clinical trials the polyvalent vaccine against HFRS.

The analysis of the nucleoprotein gene of 77 Puumala hantavirus strains detected in human samples in France during 2012–2016 showed that all belonged to the Central European lineage. We observed 2 main clusters, geographically structured; one included strains with the Q64 signature and the other strains with the R64 signature.

Puumala orthohantavirus (PUUV) is endemic in Europe and can cause hemorrhagic fever with renal syndrome (nephropathia epidemica). Disease features include fever, thrombocytopenia, and acute kidney injury (AKI). This retrospective cohort study of forty PUUV patients aims to characterize associations of serum immunological, hemostatic or kidney injury markers to disease severity. While interleukin-18 (IL-18) was significantly increased in severely thrombocytopenic patients (9 platelets/L) compared to patients with higher platelet counts, RANTES was significantly decreased in these patients. These data suggest that patients with significant thrombocytopenia might have experienced pronounced Th1 immune responses. When kidney dysfunction was used as the primary disease outcome, recently identified AKI biomarkers (Cystatin C, insulin-like growth factor-binding protein 7, Nephrin, and trefoil factor 3) were significantly upregulated in patients with severe PUUV infection, defined as the estimated glomerular filtration rate (eGFR) below 30 m/min/1.73 m2. The increased expression of these markers specifically indicates pathology in glomeruli and proximal tubuli. Furthermore, E-selectin was significantly higher while interferon gamma-induced protein 10 (IP-10) was significantly lower in PUUV patients with more severe kidney dysfunction compared to patients with higher eGFR-values. Increased E-selectin illustrates the central role of endothelial cell activation, whereas decreased IP-10 could indicate a less important role of this cytokine in the pathogenesis of kidney dysfunction.

Persistence of immune memory in humans is a crucial yet poorly understood aspect of immunology. Here we have studied the effect of Puumala hantavirus infection on unrelated, pre-existing immune memory by studying T cell- and antibody responses against toxoid vaccine antigens of diphtheria, tetanus and pertussis in a cohort of 45 patients. We found that tetanus- and pertussis -specific IgG concentrations elevate during acute Puumala virus infection. Increase in vaccine IgG was associated with proliferation of heterologous T cells. Interestingly, increases in tetanus-specific IgG persisted a year after the infection while pertussis-specific IgG declined rapidly; a difference in IgG kinetics resembling the difference seen after vaccination against tetanus and pertussis. These results suggest that persistence of immune memory is facilitated by heterologous boosting of old memory during memory formation against newly encountered antigens. They also show that different toxoid antigens may be treated differently. Our study gives new insight into how immune memory formation may alter pre-existing immune memory, and also shows that heterologous immunity may have an impact on vaccination outcomes. [ABSTRACT FROM AUTHOR]

Orthohantaviruses (family Hantaviridae, order Bunyavirales) can cause two serious syndromes in humans: hemorrhagic fever with renal syndrome (HFRS), associated with the Old World orthohantaviruses, and hantavirus cardiopulmonary syndrome (HCPS), associated with orthohantaviruses in the Americas. In Europe, four different orthohantaviruses (DOBV, PUUV, SEOV, and TULV) are associated with human disease. As disease severity and zoonotic source differ between orthohantavirus species, conclusive determination of the infecting species by either RT-PCR or comparative virus neutralization test (VNT) is of importance. Currently, the focus reduction neutralization test (FRNT) is considered the ‘Gold Standard’ for orthohantavirus VNTs, however this test is laborious and time-consuming. Consequently, more high-throughput alternatives are needed. In this study, we developed a comparative orthohantavirus microneutralization test (MNT) including all four human pathogenic orthohantavirus species circulating in Europe. The assay was validated using RT-PCR-confirmed rodent (n=17) and human sera (n=17), DOBV-suspected human sera (n=3) and cohorts of orthohantavirus-negative rodent (n=3) and human sera (n=85). 16/17 RT-PCR-confirmed rodent sera and 18/20 of the RT-PCR-confirmed and DOBV-suspected human sera were serotyped successfully, while for the remaining rodent (n=1) and human sera (n=2) no neutralizing titers could be detected. All negative control sera tested negative in the MNT. The assay was subsequently evaluated using a clinical cohort of 50 orthohantavirus patients. Orthohantavirus infection was confirmed in all 50 patients, and 47/50 (94%) sera were serotyped successfully, confirming PUUV as the major cause of orthohantavirus infections in Netherlands. Notably, two previously unrecognized SEOV cases from 2013 were diagnosed using the MNT, underlining the added value of the MNT in a diagnostic setting. In conclusion, we demonstrate the successful development and clinical implementation of a comparative European orthohantavirus MNT to determine the infecting virus species in European HFRS patients. Identification of the causative species is needed for an adequate Public Health response and can support individual patient care. For many labs, the implementation of orthohantavirus neutralization tests has not been a straightforward procedure. This issue will be addressed by the rollout of the comparative MNT to multiple European laboratories to support patient diagnostics, surveillance and Public Health responses.

Background: Puumala hantavirus (PUUV) causes nephropathia epidemica (NE), an endemic form of transient acute renal injury (AKI). Serological testing is the mainstay of diagnosis. It was the aim of the present study to assist decision-making for serological testing by constructing a simple tool that predicts the likelihood of PUUV positivity., Methods: We conducted a comparative cohort study of all PUUV-tested cases at Aachen University tertiary care center in Germany between mid-2013 and mid-2021. N = 293 qualified for inclusion; N = 30 had a positive test result and clinical NE; N = 263 were negative. Two predictive point scores, the Aachen PUUV Score (APS) 1 and 2, respectively, were derived with the aid of logistic regression and receiver operating characteristic (ROC) analysis by determining the presence of four admission parameters. For internal validation, the internal Monte Carlo method was applied. In addition, partial external validation was performed using an independent historic cohort of N = 41 positive cases of NE., Results: APS1 is recommended for clinical use as it estimated the probability of PUUV positivity in the entire medical population tested. With a range from 0 to 6 points, it yielded an area under the curve of 0.94 by allotting 2 points each for fever or headache and 1 point each for AKI or LDH>300 U/L. A point sum of 0-2 safely predicted negativity for PUUV, as was confirmed in the NE validation cohort., Conclusion: Here, we present a novel, easy-to-use tool to guide the diagnostic management of suspected PUUV infection/NE and to safely avoid unnecessary serological testing, as indicated by point sum class 0-2. Since 67% of the cohort fell into this stratum, half of the testing should be avoidable in the future., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Brockmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The fluctuation of human infections by the Puumala orthohantavirus (PUUV) in Germany has been linked to weather and phenology parameters that drive the population growth of its host species. We quantified the annual PUUV-outbreaks at the district level by binarizing the reported infections in the period 2006-2021. With these labels we trained a model based on a support vector machine classifier for predicting local outbreaks and incidence well in advance. The feature selection for the optimal model was performed by a heuristic method and identified five monthly weather variables from the previous two years plus the beech flowering intensity of the previous year. The predictive power of the optimal model was assessed by a leave-one-out cross-validation in 16 years that led to an 82.8% accuracy for the outbreak and a 0.457 coefficient of determination for the incidence. Prediction risk maps for the entire endemic area in Germany will be annually available on a freely-accessible permanent online platform of the German Environment Agency. The model correctly identified 2022 as a year with low outbreak risk, whereas its prediction for large-scale high outbreak risk in 2023 was not confirmed., (© 2024. The Author(s).)

In 2018, a local case of nephropathia epidemica was reported in Scania, southern Sweden, more than 500 km south of the previously known presence of human hantavirus infections in Sweden. Another case emerged in the same area in 2020. To investigate the zoonotic origin of those cases, we trapped rodents in Ballingslöv, Norra Sandby, and Sörby in southern Sweden during 2020‒2021. We found Puumala virus (PUUV) in lung tissues from 9 of 74 Myodes glareolus bank voles by screening tissues using a hantavirus pan-large segment reverse transcription PCR. Genetic analysis revealed that the PUUV strains were distinct from those found in northern Sweden and Denmark and belonged to the Finnish PUUV lineage. Our findings suggest an introduction of PUUV from Finland or Karelia, causing the human PUUV infections in Scania. This discovery emphasizes the need to understand the evolution, cross-species transmission, and disease outcomes of this newly found PUUV variant.

Orthohantaviruses (family Hantaviridae , order Bunyavirales) can cause two serious syndromes in humans: hemorrhagic fever with renal syndrome (HFRS), associated with the Old World orthohantaviruses, and hantavirus cardiopulmonary syndrome (HCPS), associated with orthohantaviruses in the Americas. In Europe, four different orthohantaviruses (DOBV, PUUV, SEOV, and TULV) are associated with human disease. As disease severity and zoonotic source differ between orthohantavirus species, conclusive determination of the infecting species by either RT-PCR or comparative virus neutralization test (VNT) is of importance. Currently, the focus reduction neutralization test (FRNT) is considered the 'Gold Standard' for orthohantavirus VNTs, however this test is laborious and time-consuming. Consequently, more high-throughput alternatives are needed. In this study, we developed a comparative orthohantavirus microneutralization test (MNT) including all four human pathogenic orthohantavirus species circulating in Europe. The assay was validated using RT-PCR-confirmed rodent (n=17) and human sera (n=17), DOBV-suspected human sera (n=3) and cohorts of orthohantavirus-negative rodent (n=3) and human sera (n=85). 16/17 RT-PCR-confirmed rodent sera and 18/20 of the RT-PCR-confirmed and DOBV-suspected human sera were serotyped successfully, while for the remaining rodent (n=1) and human sera (n=2) no neutralizing titers could be detected. All negative control sera tested negative in the MNT. The assay was subsequently evaluated using a clinical cohort of 50 orthohantavirus patients. Orthohantavirus infection was confirmed in all 50 patients, and 47/50 (94%) sera were serotyped successfully, confirming PUUV as the major cause of orthohantavirus infections in Netherlands. Notably, two previously unrecognized SEOV cases from 2013 were diagnosed using the MNT, underlining the added value of the MNT in a diagnostic setting. In conclusion, we demonstrate the successful development and clinical implementation of a comparative European orthohantavirus MNT to determine the infecting virus species in European HFRS patients. Identification of the causative species is needed for an adequate Public Health response and can support individual patient care. For many labs, the implementation of orthohantavirus neutralization tests has not been a straightforward procedure. This issue will be addressed by the rollout of the comparative MNT to multiple European laboratories to support patient diagnostics, surveillance and Public Health responses. [ABSTRACT FROM AUTHOR]

Hemorrhagic fever with renal syndrome (HFRS), caused by Dobrava (DOBV) and Puumala (PUUV) orthohantaviruses, is an endemic disease in Slovenia. DOBV is mainly responsible for a more severe disease, whereas PUUV usually causes a milder form. Therefore, the aim of our study was to determine whether any differences in lymphocyte population in patients infected with these two viruses exist. Mononuclear cells from peripheral blood (PBMCs) were isolated from DOBV or PUUV infected patients and different lymphocyte subpopulations were analyzed with flow cytometry. Decreased concentrations of lymphocyte subpopulation were observed in DOBV and in PUUV infected patients compared with a healthy control, which was especially evident in DOBV infected patients. The lower values of T cells are likely due to the extravasation of the activated cells from the circulation to the infected tissue. Higher percentage of NK cells were detected in DOBV infected patients in comparison to PUUV infected patients, which could be associated with a more severe HFRS caused by DOBV. PUUV infected patients had a significantly higher concentration of activated T cell subsets, expressing markers CD25, CD69, and HLA-DR in comparison to DOBV infected patients. Higher activation of T cell subsets in PUUV infected patients could be a contributor to a milder HFRS. Further studies are necessary to elucidate the relation between the protective and the harmful role of activated lymphocytes subsets in HFRS pathogenesis.

In Ukraine, a retrospective review of clinical case reports by public health officials suggest that human cases of febrile illnesses associated with hemorrhage may be due to infections of Crimean-Congo hemorrhagic fever virus (CCHFV) and Old World hantaviruses. In a serosurvey of 966 healthy individuals in the Lviv Oblast, Ukraine, bordering Poland, we found that 1.6% showed cross-reactivity to hantaviral antigens by an immunofluorescence assay (IFA) and 1.7% of the study participants had antibodies cross-reactive to CCHFV by enzyme-linked immunosorbent assay (ELISA). Demographic variables and history of exposures obtained through questionnaires were assessed by logistic regression models for association with seroprevalence for both viruses with no significant risk factors found. Analysis of spatial distribution identified two clusters of samples positive for antibodies to both hantaviruses and CCHFV, which, however, were not statistically significant (p > 0.05). In general, the study results suggest that the population of the study area is exposed to hantaviruses and CCHFV. Further surveillance for respective pathogens in Ukraine is warranted and prospective surveillance of febrile patients with unidentified febrile illness.

For wildlife diseases, one often relies on host density to predict host infection prevalence and the subsequent force of infection to humans in the case of zoonoses. Indeed, if transmission is mainly indirect, i.e., by way of the environment, the force of infection is expected to increase with host density, yet the laborious field data supporting this theoretical claim are often absent. Hantaviruses are among those zoonoses that have been studied extensively over the past decades, as they pose a significant threat to humans. In Europe, the most widespread hantavirus is the Puumala virus (PUUV), which is carried by the bank vole and causes nephropathia epidemica (NE) in humans. Extensive field campaigns have been carried out in Central Finland to shed light on this supposed relationship between bank vole density and PUUV prevalence and to identify other drivers for the infection dynamics. This resulted in the surprising observation that the relationship between bank vole density and PUUV prevalence is not purely monotonic on an annual basis, contrary to what previous models predicted: a higher vole density does not necessary result in a higher infection prevalence, nor in an increased number of humans reported having NE. Here, we advance a novel individual-based spatially-explicit model which takes into account the immunity provided by maternal antibodies and which simulates the spatial behavior of the host, both possible causes for this discrepancy that were not accounted for in previous models. We show that the reduced prevalence in peak years can be attributed to transient immunity, and that the density-dependent spatial vole behavior, i.e., the fact that home ranges are smaller in high density years, plays only a minor role. The applicability of the model is not limited to the study and prediction of PUUV (and NE) occurrence in Europe, as it could be easily adapted to model other rodent-borne diseases, either with indirect or direct transmission.

Purpose: To describe early chest imaging abnormalities in patients with acute Puumala virus infection. Materials and Methods: This retrospective study (2005–2017) comprised 64 patients who were admitted to the emergency department of a Belgian hospital. These patients were diagnosed with serologically confirmed acute Puumala virus infection and had at least one chest X-ray (CRX). Imaging studies were evaluated by two experienced chest radiologists reaching agreement by consensus, and abnormalities were reported according to the Fleischner Society glossary of terms for thoracic imaging. When a patient underwent multiple CRX, only the findings of the first were recorded. Six patients underwent chest high-resolution computed tomography (HRCT). Results: CRX showed abnormal findings in 33 patients (51.5%). Most common findings were linear atelectasis (29.7%) and small pleural effusion (20.3%). HRCT showed interlobular septal thickening in four patients and crazy-paving pattern with consolidations in one patient with adult respiratory distress syndrome. Conclusions: Early CRX commonly showed linear atelectasis and small pleural effusion in Puumala virus infected patients above 30 years of age. Chest HRCT most frequently showed atelectasis and smooth interlobular septal thickening. While uncommon, early and severe pulmonary involvement can be associated with Puumala virus infection, albeit these findings are not specific.

We conducted a multicenter, retrospective cohort study of hospitalized patients with serologically proven nephropathia epidemica (NE) living in Ardennes Department, France, during 2000–2014 to develop a bioclinical test predictive of severe disease. Among 205 patients, 45 (22.0%) had severe NE. We found the following factors predictive of severe NE: nephrotoxic drug exposure (p = 0.005, point value 10); visual disorders (p = 0.02, point value 8); microscopic or macroscopic hematuria (p = 0.04, point value 7); leukocyte count >10 × 109 cells/L (p = 0.01, point value 9); and thrombocytopenia 20 identified high-risk patients (45.3% had severe disease). If validated in future studies, this test could be used to stratify patients by severity in research studies and in clinical practice.

Annually, over 10,000 cases of hemorrhagic fever with renal syndrome (HFRS) are diagnosed in Europe. Puumala hantavirus (PUUV) causes most of the European HFRS cases. PUUV causes usually a relatively mild disease, which is rarely fatal. However, the severity of the infection varies greatly, and factors affecting the severity are mostly unrevealed. Host genes are known to have an effect. The typical clinical features in PUUV infection include acute kidney injury, thrombocytopenia, and increased vascular permeability. The primary target of hantavirus is the endothelium of the vessels of different organs. Although PUUV does not cause direct cytopathology of the endothelial cells, remarkable changes in both the barrier function of the endothelium and the function of the infected endothelial cells occur. Host immune or inflammatory mechanisms are probably important in the development of the capillary leakage. Several immunoinflammatory biomarkers have been studied in the context of assessing the severity of HFRS caused by PUUV. Most of them are not used in clinical practice, but the increasing knowledge about the biomarkers has elucidated the pathogenesis of PUUV infection.

In Ukraine, a retrospective review of clinical case reports by public health officials suggest that human cases of febrile illnesses associated with hemorrhage may be due to infections of Crimean-Congo hemorrhagic fever virus (CCHFV) and Old World hantaviruses. In a serosurvey of 966 healthy individuals in the Lviv Oblast, Ukraine, bordering Poland, we found that 1.6% showed cross-reactivity to hantaviral antigens by an immunofluorescence assay (IFA) and 1.7% of the study participants had antibodies cross-reactive to CCHFV by enzyme-linked immunosorbent assay (ELISA). Demographic variables and history of exposures obtained through questionnaires were assessed by logistic regression models for association with seroprevalence for both viruses with no significant risk factors found. Analysis of spatial distribution identified two clusters of samples positive for antibodies to both hantaviruses and CCHFV, which, however, were not statistically significant (p > 0.05). In general, the study results suggest that the population of the study area is exposed to hantaviruses and CCHFV. Further surveillance for respective pathogens in Ukraine is warranted and prospective surveillance of febrile patients with unidentified febrile illness. [ABSTRACT FROM AUTHOR]