Seroprevalence of Old World Hantaviruses and Crimean Congo Hemorrhagic Fever Viruses in Human Populations in Northwestern Ukraine.

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.

Molecular Epidemiology of Hantaviruses in the Czech Republic.

During 2008-2018, we collected samples from rodents and patients throughout the Czech Republic and characterized hantavirus isolates. We detected Dobrava-Belgrade and Puumala orthohantaviruses in patients and Dobrava-Belgrade, Tula, and Seewis orthohantaviruses in rodents. Increased knowledge of eco-epidemiology of hantaviruses will improve awareness among physicians and better outcomes of patients.

Molecular and epidemiological characteristics of human Puumala and Dobrava-Belgrade hantavirus infections, Germany, 2001 to 2017.

IntroductionTwo hantavirus species, Puumala (PUUV) and Dobrava-Belgrade (DOBV) virus (genotype Kurkino), are endemic in Germany. Recent PUUV outbreaks raised questions concerning increasing frequency of outbreaks and expansion of PUUV endemic areas.AimsTo describe the epidemiology of human PUUV and DOBV infections in Germany.MethodsWe conducted an observational retrospective study analysing national hantavirus surveillance data notified to the national public health institute and hantavirus nucleotide sequences from patients collected at the national consultation laboratory between 2001 and 2017. Matching molecular sequences with surveillance data, we conducted epidemiological, phylogenetic and phylogeographic analyses.ResultsIn total, 12,148 cases of symptomatic hantavirus infection were notified 2001-17 (mean annual incidence: 0.87/100,000; range: 0.09-3.51). PUUV infections showed a highly variable space-time disease incidence pattern, causing large outbreaks every 2-3 years with peaks in early summer and up to 3,000 annually reported cases. Sex-specific differences in disease presentation were observed. Of 202 PUUV nucleotide sequences obtained from cases, 189 (93.6%) fall into well-supported phylogenetic clusters corresponding to different endemic areas in Germany. DOBV infections caused few, mostly sporadic cases in autumn and winter in the north and east of Germany.ConclusionsThe frequency of PUUV outbreaks increased between 2001 and 2017 but our data does not support the suggested expansion of endemic areas. The epidemiology of PUUV and DOBV-Kurkino infections differs in several aspects. Moreover, the latter are relatively rare and combining efforts and data of several countries to identify risk factors and develop specific recommendations for prevention could be worthwhile.

Two Point Mutations in Old World Hantavirus Glycoproteins Afford the Generation of Highly Infectious Recombinant Vesicular Stomatitis Virus Vectors.

Rodent-to-human transmission of hantaviruses is associated with severe disease. Currently, no FDA-approved, specific antivirals or vaccines are available, and the requirement for high biocontainment (biosafety level 3 [BSL-3]) laboratories limits hantavirus research. To study hantavirus entry in a BSL-2 laboratory, we set out to generate replication-competent, recombinant vesicular stomatitis viruses (rVSVs) bearing the Gn and Gc (Gn/Gc) entry glycoproteins. As previously reported, rVSVs bearing New World hantavirus Gn/Gc were readily rescued from cDNAs, but their counterparts bearing Gn/Gc from the Old World hantaviruses, Hantaan virus (HTNV) or Dobrava-Belgrade virus (DOBV), were refractory to rescue. However, serial passage of the rescued rVSV-HTNV Gn/Gc virus markedly increased its infectivity and capacity for cell-to-cell spread. This gain in viral fitness was associated with the acquisition of two point mutations: I532K in the cytoplasmic tail of Gn and S1094L in the membrane-proximal stem of Gc. Follow-up experiments with rVSVs and single-cycle VSV pseudotypes confirmed these results. Mechanistic studies revealed that both mutations were determinative and contributed to viral infectivity in a synergistic manner. Our findings indicate that the primary mode of action of these mutations is to relocalize HTNV Gn/Gc from the Golgi complex to the cell surface, thereby affording significantly enhanced Gn/Gc incorporation into budding VSV particles. Finally, I532K/S1094L mutations in DOBV Gn/Gc permitted the rescue of rVSV-DOBV Gn/Gc, demonstrating that incorporation of cognate mutations into other hantaviral Gn/Gc proteins could afford the generation of rVSVs that are otherwise challenging to rescue. The robust replication-competent rVSVs, bearing HTNV and DOBV Gn/Gc, reported herein may also have utility as vaccines.IMPORTANCE Human hantavirus infections cause hantavirus pulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome (HFRS) in Eurasia. No FDA-approved vaccines and therapeutics exist for these deadly viruses, and their development is limited by the requirement for high biocontainment. In this study, we identified and characterized key amino acid changes in the surface glycoproteins of HFRS-causing Hantaan virus that enhance their incorporation into recombinant vesicular stomatitis virus (rVSV) particles. The replication-competent rVSVs encoding Hantaan virus and Dobrava-Belgrade virus glycoproteins described in this work provide a powerful and facile system to study hantavirus entry under lower biocontainment and may have utility as hantavirus vaccines.

First molecular characterization of Dobrava-Belgrade virus found in Apodemus flavicollis in Poland.

INTRODUCTION: Dobrava-Belgrade virus (DOBV) is one of the emerging pathogens which have been reported during the last decades in Europe and have attracted the attention of researchers. The course of infection among humans may have a varied course - from the completely asymptomatic to the more severe forms, such as haemorrhagic fever with renal syndrome (HFRS). DOBV is hosted and carried by rodents like Apodemus flavicollis or A. agrarius, which occur commonly in Europe. OBJECTIVE: To-date, orthohantaviruses have been reported in Poland, both in humans and animals, but detailed country-scale studies have not yet been carried out. The aim of the study was molecular characterization of a strain which was found in A. flavicollis in south-eastern Poland. MATERIAL AND METHODS: The phylogenetic analysis of the first Dobrava-Belgrade virus found in A. flavicollis in the subcarpathian region of south-eastern Poland, presented in this study, was performed after virus proliferation in cell culture and sequencing of specific PCR products. RESULTS: Based on genetic sequences of fragments of three segments (S, M and L), the isolated virus was assigned to the Dobrava genotype, taking into consideration the most current classification of the DOBV species. CONCLUSIONS: The Dobrava-Belgrade virus strain isolated from A. flavicollis in the subcarpathian region of south-eastern Poland, has been molecularly characterized and assigned to Dobrava genotype, thereby the occurrence of that genotype in Poland has been confirmed by molecular techniques.

Molecular epidemiology of Dobrava-Belgrade virus in Greece.

In order to gain an insight into the genetic relatedness of the Dobrava-Belgrade virus (DOBV) in Greece, a phylogenetic analysis was performed based on all currently available DOBV sequences obtained from hospitalized cases with hemorrhagic fever with renal syndrome (HFRS). Most cases occurred in northwestern and north central part of the country. Two sequence datasets consisted of 41 S and 12 M partial DOBV RNA segment sequences were analyzed. All DOBV strains belong to Dobrava genotype which is associated with the rodent Apodemus flavicollis. In both phylogenetic trees (S and M segments), two main clusters of Greek strains could be distinguished. Phylogenetic analysis showed a spatial rather than temporal relation of the strains, since their genetic clustering was highly associated with the geographic distribution of the cases. Besides previous characterized endemic foci, novel ones have been identified, expanding our knowledge on the epidemiology of HFRS in Greece.

Proteinuria and the Clinical Course of Dobrava-Belgrade Hantavirus Infection.

PURPOSE: Human infection with Dobrava-Belgrade virus (DOBV) in Northern Germany causes a mild form of hantavirus disease predominantly characterized by acute kidney injury due to interstitial nephritis. We evaluated the largest number of DOBV-infected patients so far regarding clinical course, proteinuria, and prognostic markers. PATIENTS AND METHODS: Patients with DOBV-associated hantavirus disease admitted to the Renal Division of the University of Lübeck (Germany) between 1997 and 2012 were included in this study. Symptoms, clinical course, laboratory parameters, and urinary protein analysis were investigated at admission (baseline, t0), 3-5 days (t3-5), 10-17 days (t10-17), and after 1 year of follow-up (t365). RESULTS: Of the 34 patients (male/female ratio: 23/11; age: 41 ± 14 years) included in the study, 4 underwent hemodialysis (HD). Glomerular filtration rate was 17 ± 14 mL/min at t0 and increased to 27 ± 26 mL/min (t3-5), 57 ± 20 mL/min (t10-17), and 84 ± 16 mL/min (t365). Albuminuria and tubular proteinuria (α1- and ß2-microglobulin) decreased during follow-up; the urinary α1-microglobulin concentration in patients who required HD was significantly higher than that in patients not requiring HD (t0: 186 ± 51 vs. 45 ± 26 mg/g creatinine; t3-5: 87 ± 14 vs. 32 ± 16 mg/g creatinine; t10-17: 63 ± 18 vs. 28 ± 12 mg/g creatinine; p < 0.001). CONCLUSIONS: DOBV infection of inpatients in Northern Germany is associated with severe kidney injury that recovers within a few weeks and normalizes within 1 year. Tubular proteinuria is associated with the severity of kidney injury and the necessity of renal replacement therapy in these DOBV-infected patients.

Population dynamics of wild rodents induce stochastic fadeouts of a zoonotic pathogen.

Stochastic processes play an important role in the infectious disease dynamics of wildlife, especially in species subject to large population oscillations. Here, we study the case of a free ranging population of yellow-necked mice (Apodemus flavicollis) in northern Italy, where circulation of Dobrava-Belgrade hantavirus (DOBV) has been detected intermittently since 2001, until an outbreak emerged in 2010. We analysed the transmission dynamics of the recent outbreak using a computational model that accounts for seasonal changes of the host population and territorial behaviour. Model parameters were informed by capture-mark-recapture data collected over 14 years and longitudinal seroprevalence data from 2010 to 2013. The intermittent observation of DOBV before 2010 can be interpreted as repeated stochastic fadeouts after multiple introductions of infectious rodents migrating from neighbouring areas. We estimated that only 20% of introductions in a naïve host population results in sustained transmission after 2 years, despite an effective reproduction number well above the epidemic threshold (mean 4·5, 95% credible intervals, CI: 0·65-15·8). Following the 2010 outbreak, DOBV has become endemic in the study area, but we predict a constant probability of about 4·7% per year that infection dies out, following large population drops in winter. In the absence of stochastic fadeout, viral prevalence is predicted to continue its growth to an oscillating equilibrium around a value of 24% (95% CI: 3-57). We presented an example of invasion dynamics of a zoonotic virus where stochastic fadeout have played a major role and may induce future extinction of the endemic infection.

Clinical aspects of hantavirus infections in Bulgaria.

BACKGROUND: Hantaviruses cause two distinct human diseases: hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe and hantavirus pulmonary syndrome (HPS) in America. In Europe, mainly Puumala, Dobrava and Seoul viruses cause HFRS. A total of 23 cases of HFRS were detected in Bulgaria over a 2­year period 2013-2014. The aim of the study was to present epidemiology, clinical manifestations and laboratory findings of these patients. METHODS: Patients with HFRS were diagnosed using PCR, ELISA and immunoblotting tests. RESULTS: Dobrava-Belgrade virus (DOBV) was revealed as etiological agent in 16 (69.6%) patients and Puumala virus (PUUV) in 7 (30.4%) patients. All 23 patients were men aged 22-66 years of which 6 (26.1%) patients originated from regions in northern and western Bulgaria previously thought to be non-endemic. Patients with HFRS, despite the infecting hantavirus, manifested acute renal failure, asthenia and less pronounced hemorrhagic syndrome. Patients with DOBV infection were much more likely to present with arthromyalgia, severe headache, severe to moderately severe asthenoadynamia, abdominal pain, vomiting, hypotension, nervous system disorders as well as kidney enlargement, leucopenia and higher levels of blood creatinine, requiring hemodialysis procedures more often and for a longer period of time than patients with PUUV infection. CONCLUSIONS: The present report describes for the first time comparative analysis of epidemiological features, clinical manifestations and laboratory findings of DOBV and PUUV infections in Bulgaria.

Clinical characterization of two severe cases of hemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses Puumala and Dobrava-Belgrade genotype Sochi.

BACKGROUND: Hantavirus disease belongs to the emerging infections. The clinical picture and severity of infections differ between hantavirus species and may even vary between hantavirus genotypes. The mechanisms that lead to the broad variance of severity in infected patients are not completely understood. Host- and virus-specific factors are considered. CASE PRESENTATION: We analyzed severe cases of hantavirus disease in two young women. The first case was caused by Puumala virus (PUUV) infection in Germany; the second case describes the infection with Dobrava-Belgrade virus (DOBV) in Russia. Symptoms, laboratory parameters and cytokine levels were analyzed and compared between the two patients. Serological and sequence analysis revealed that PUUV was the infecting agent for the German patient and the infection of the Russian patient was caused by Dobrava-Belgrade virus genotype Sochi (DOBV-Sochi). The symptoms in the initial phase of the diseases did not differ noticeably between both patients. However, deterioration of laboratory parameter values was prolonged and stronger in DOBV-Sochi than in PUUV infection. Circulating endothelial progenitor cells (cEPCs), known to be responsible for endothelial repair, were mobilized in both infections. Striking differences were observed in the temporal course and level of cytokine upregulation. Levels of angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), and stromal derived factor-1 (SDF-1α) were increased in both infections; but, sustained and more pronounced elevation was observed in DOBV-Sochi infection. CONCLUSIONS: Severe hantavirus disease caused by different hantavirus species did not differ in the general symptoms and clinical characteristics. However, we observed a prolonged clinical course and a late and enhanced mobilization of cytokines in DOBV-Sochi infection. The differences in cytokine deregulation may contribute to the observed variation in the clinical course.

¿Cuál es el impacto de las campañas comunicacionales para prevenir el Virus Hanta?: síntesis rápida de evidencia / What is the impact of communication campaigns to prevent Hantavirus?: rapid evidence synthesis

ANTECEDENTES Y OBJETIVO El Virus Hanta es una infección viral que se transmite principalmente por el contacto con roedores que se encuentran fundamentalmente en zonas rurales. Para prevenir su transmisión, una alternativa es realizar campañas comunicacionales con información relevante. En este contexto el Subsecretario de Salud Pública solicita esta síntesis de evidencia con el objetivo de estudiar el impacto de estas campañas comunicacionales en la prevención de este virus, y de esta manera informar la toma de decisiones respecto del impacto de las campañas comunicacionales para prevenir el virus Hanta en la población. METODOLOGÍA Se formuló una estrategia de búsqueda para ser utilizada en las bases de datos Epistemonikos, Health Systems Evidence, Health Evidence, la Biblioteca Cochrane y PubMed con el objetivo de identificar revisiones sistemáticas del tema. que abordaran la pregunta formulada. Al no encontrarse, se buscaron estudios primarios en PubMed y CENTRAL. Se utiliza la metodología de la certeza de la evidencia GRADE. Consultando con expertos en el área, se decidió excluir todos los artículos que contemplaran campañas comunicacionales para otro tipo de enfermedades. RESULTADOS Se utiliza 1 estudio primario, del cual se obtiene los siguientes resultados. -No se puede concluir sobre el impacto de las campañas comunicacionales para promover conductas de prevención del virus Hanta, porque la certeza en la evidencia es muy baja. -Se podría considerar evaluar formalmente el impacto que las campañas comunicacionales ya realizadas han tenido sobre la conducta de la población, y la incidencia del virus en nuestro país.

Infection of human airway epithelial cells by different subtypes of Dobrava-Belgrade virus reveals gene expression patterns corresponding to their virulence potential.

Dobrava-Belgrade virus (DOBV) is a pathogen causing hemorrhagic fever with renal syndrome in Europe. Virulence and case fatality rate are associated with virus genotype; however the reasons for these differences are not well understood. In this work we present virus-specific effects on the gene expression profiles of human lung epithelial cells (A549) infected with different genotypes of DOBV (Dobrava, Kurkino, and Sochi), as well as the low-virulent Tula virus (TULV). The data was collected by whole-genome gene expression microarrays and confirmed by quantitative real-time PCR. Despite their close genetic relationship, the expression profiles induced by infection with different hantaviruses are significantly varying. Major differences were observed in regulation of immune response genes, which were especially induced by highly virulent DOBV genotypes Dobrava and Sochi in contrast to less virulent DOBV-Kurkino and TULV. This work gives first insights into the differences of virus - host interactions of DOBV on genotype level.

Does proficiency testing improve the quality of hantavirus serodiagnostics? Experiences with INSTAND EQA schemes.

Hantavirus infections in Germany appear periodically with peak numbers every 2-3 years. The reported cases in the years 2007, 2010 and 2012 exceeded many times over those in the years in-between. In order to reveal faults of certain in vitro diagnostic assays (IVDs), to harmonize the performances of the individual assays and to improve the users' competence in interpreting the results, the National Consiliary Laboratory for Hantaviruses and INSTAND e.V. (Society for Promoting Quality Assurance in Medical Laboratories e.V.) established an external quality assessment (EQA) scheme for proficiency testing of hantavirus serodiagnostics. The first EQA scheme (pilot study) started in March 2009 with 58 participating laboratories from Germany and neighboring countries. Twice a year four serum samples were sent out to the participants to investigate whether the sample reflects an acute or past infection and to distinguish between infections with the hantavirus types Puumala virus (PUUV) and Dobrava-Belgrade virus (DOBV), both endemic in Central Europe. In addition, samples negative for anti-hantavirus antibodies were tested in order to examine the specificity of the IVDs applied in the participating laboratories. An increasing number of laboratories participated, with a maximum of 92 in March 2014. When summarizing in total 2592 test results, the laboratories reached an overall specificity of 96.7% and a sensitivity of 95% in their detection of a hantavirus infection. A correct distinction between acute and past infections was forwarded in 90-96% of replies of laboratories. Exact serotyping (PUUV vs. DOBV) of the infection was reported in 81-96% of replies with the lowest accuracy for past DOBV infections; cross-reactivities between diagnostic antigens of the two viruses as well as persistent IgM titers in humans may interfere with exact testing. The EQAs revealed acceptable results for the serodiagnostic of hantavirus infection including serotyping but further improvement is still needed.

Genetic analysis of Dobrava-Belgrade virus from western Serbia--a newly detected focus in the Balkan Peninsula.

Dobrava-Belgrade virus (DOBV) is a hantavirus species that causes the most severe form of haemorrhagic fever with renal syndrome (HFRS) in Europe. DOBV has been detected in three Apodemus rodents: A. flavicollis, A. agrarius and A. ponticus. These emerging viruses appear throughout the Balkan Peninsula including Serbia as its central part. In this study, we examined the seroprevalence, molecular epidemiology and phylogenetics of DOBV from A. flavicollis captured at six Serbian localities. Furthermore, we applied microsatellite typing of host animal genome to analyse the role of host kinship in DOBV animal transmission. The overall IgG seropositivity rate over 3 years (2008-2010) was 11.9% (22/185). All seropositive samples were subjected to RT-PCR and DNA sequencing for S and L genome segments (pos. 291-1079 nt and 2999-3316 nt, respectively). DOBV was genetically detected in three samples from mountain Tara in western Serbia, a newly detected DOBV focus in the Balkans. No sequence data from human cases from Serbia are available for the studied period. However, collected DOBV isolates in this work phylogenetically clustered together with isolates from Serbian human cases dating from 2002, with 1.9% nucleotide divergence. We determined the level of kinship between seropositive and seronegative animal groups and found no significant difference, suggesting that horizontal virus transmission in the studied population was the same within and among the hatches. Our findings are the first genetic detection of DOBV in rodents in Serbia. We confirm wide and continuous hantavirus presence in the examined parts of the Balkans, underlying the necessity of continual monitoring of hantavirus circulation in A. flavicollis.

Human Dobrava-Belgrade hantavirus infection, Kosovo.

Here we describe an acute Dobrava-Belgrade virus (DOBV) infection that presented as severe hemorrhagic fever with renal syndrome (HFRS) in an active-duty U.S. soldier. The infection was acquired in northern Kosovo in spring 2013. Amplification of DOBV genome segments directly from the patient's serum sample was successfully performed. Phylogenetic analysis demonstrated that the strain belong to DOBV genotype Dobrava and is closely related to strains circulating in Southeast Europe and Slovakia. Thus, our case confirms that DOBV genotype Dobrava is able to cause a severe form of HFRS, especially when compared to the other less pathogenic DOBV genotypes.

Dobrava-Belgrade virus in Apodemus flavicollis and A. uralensis mice, Turkey.

In 2009, human Dobrava-Belgrade virus (DOBV) infections were reported on the Black Sea coast of Turkey. Serologic and molecular studies of potential rodent reservoirs demonstrated DOBV infections in Apodemus flavicollis and A. uralensis mice. Phylogenetic analysis of DOBV strains showed their similarity to A. flavicollis mice-borne DOBV in Greece, Slovenia, and Slovakia.

Hantavirus disease in Germany due to infection with Dobrava-Belgrade virus genotype Kurkino.

Members of the Dobrava-Belgrade virus (DOBV) species are hantaviruses carried by different Apodemus mice as reservoir hosts and causing haemorrhagic fever with renal syndrome (HFRS) in humans. In Central Europe, the Kurkino genotype of DOBV, associated with the striped field mouse, Apodemus agrarius, is prevalent. This paper presents the first extensive study of the serological and molecular diagnostics, epidemiology and clinics of DOBV-Kurkino infections in Central Europe. Serum samples from 570 German patients living in the habitat of A. agrarius (north and northeast Germany) and exhibiting febrile disease, were analysed. All samples were tested by ELISA, subsets of samples were also analysed by immunoblot, neutralization assay, and RT-PCR. A group of 86 individuals was confirmed as DOBV-infected. The virus neutralization assay allowed a reliable identification of DOBV antibodies during both acute and convalescent phases of infection. However, differentiation of relevant DOBV genotypes was not possible by neutralization test but required molecular analysis. Whereas DOBV IgM antibodies tend to persist in the infected organism, RNAaemia seems to be short. Nucleotide sequences were amplified from four patients, and their analysis demonstrated infection by DOBV-Kurkino. With respect to the initial results, the high degree of identity of local patient-derived and A. agrarius-derived virus sequences may allow a closer allocation of the geographical place where the human infection occurred. In contrast to moderate/severe HFRS caused by the DOBV genotypes Dobrava or Sochi, all available data showed a mild clinical course of HFRS caused by DOBV-Kurkino infection without lethal outcomes.