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.

Human Infections by Non-Rodent-Associated Hantaviruses in Africa.

Various hantaviruses have been discovered in unconventional hosts (shrews and bats) in Africa. Up to now, it was unknown whether these viruses pose a threat for human health. In this study, using newly established serological assays, we demonstrated evidence of shrew-borne hantavirus infections in humans from Côte d'Ivoire and Gabon.

Novel Arenavirus Isolates from Namaqua Rock Mice, Namibia, Southern Africa.

Arenaviruses are feared as agents that cause viral hemorrhagic fevers. We report the identification, isolation, and genetic characterization of 2 novel arenaviruses from Namaqua rock mice in Namibia. These findings extend knowledge of the distribution and diversity of arenaviruses in Africa.

Sangassou virus, the first hantavirus isolate from Africa, displays genetic and functional properties distinct from those of other murinae-associated hantaviruses.

We have discovered the first indigenous African hantavirus, Sangassou virus (SANGV). The virus was isolated from an African wood mouse (Hylomyscus simus), trapped in a forest habitat in Guinea, West Africa. Here, we report on the characterization of the genetic and functional properties of the virus. The complete genome of SANGV was determined and showed typical hantavirus organization. The small (S), medium (M), and large (L) genome segments containing genes encoding nucleocapsid protein, two envelope glycoproteins, and viral polymerase were found to be 1,746, 3,650, and 6,531 nucleotides long, respectively. The exact 5' and 3' termini for all three segments of the SANGV genome were determined and were predicted to form the panhandle structures typical of bunyaviruses. Phylogenetic analyses of all three segment sequences confirmed SANGV as a Murinae-associated hantavirus most closely related to the European Dobrava-Belgrade virus. We showed, however, that SANGV uses ß(1) integrin rather than ß(3) integrin and decay-accelerating factor (DAF)/CD55 as an entry receptor. In addition, we demonstrated a strong induction of type III lambda interferon (IFN-λ) expression in type I IFN-deficient Vero E6 cells by SANGV. These properties are unique within Murinae-associated hantaviruses and make the virus useful in comparative studies focusing on hantavirus pathogenesis.

Seroepidemiological study reveals regional co-occurrence of Lassa- and Hantavirus antibodies in Upper Guinea, West Africa.

OBJECTIVES: To assess the public health relevance of Lassa arenavirus and hantavirus infections in a subpopulation of recently febrile patients. METHODS: In a human seroprevalence study, we enrolled 253 participants on the basis of reported high fever during the last 3 months. They represented roughly 20% of the population of Bantou and Tanganya villages. Comprehensive serological screening and confirmatory assays (enzyme-linked immunosorbent assay, immunofluorescence assay, Western blot analysis) with several Lassa virus and hantavirus antigens were used to ensure high specificity and broad detection capacity. RESULTS: We found a Lassa IgG prevalence of 40.3% (102/253) and a hantavirus IgG prevalence of 1.2% (3/253). The Lassa IgM prevalence reached 2.8% (7/253). CONCLUSIONS: High Lassa virus seroprevalence in recently febrile patients indicates that Lassa fever is a significant public health problem in the region. Human hantavirus infections also occur in the region but their public health relevance remains to be determined.

Isolation of sochi virus from a fatal case of hantavirus disease with fulminant clinical course.

Sochi virus, a novel genetic variant of Dobrava-Belgrade virus, was isolated in cell culture from a fulminant lethal case of hantavirus disease presenting with shock and combined kidney and lung failure. Sochi virus is transmitted to humans from host reservoir Apodemus ponticus and must be considered a life-threatening emerging agent.

Serological evidence of human hantavirus infections in Guinea, West Africa.

We recently discovered a novel hantavirus, Sangassou virus, in Guinea, West Africa. Using enzyme-linked immunosorbent assays followed by confirmatory and serotyping assays, we retrospectively detected hantavirus antibodies in 3 (4.4%) of 68 patients with fever of unknown origin in Sangassou village, Forest Guinea. A population-based survey in Forest Guinea (n = 649) found the prevalence of hantavirus antibodies to be 1.2%. Specific neutralizing antibodies against Sangassou virus were demonstrated in serum samples from 2 patients and in 2 serum samples of the serosurvey. Our data allow us to conclude that hantavirus infections may be a significant unrecognized medical problem in at least this part of Africa.

A novel cardiotropic murine adenovirus representing a distinct species of mastadenoviruses.

During cell culture isolation experiments to recover Dobrava hantavirus from a suspension of liver from a striped field mouse (Apodemus agrarius), an unknown virus was coisolated. Atypically for hantaviruses, it had extensive cytopathic effects. Using a random PCR approach, it was identified as a novel murine adenovirus, MAdV-3 (for MAdV type 3). A plaque-purified virus clone was prepared and further characterized. The complete genome sequence of MAdV-3 was determined to be 30,570 bp in length. Sequence comparisons to other adenovirus species revealed highest similarity to MAdV-1, the representative of the murine adenovirus A species. However, substantial differences were found in the E1, E3, and E4 genomic regions. The phylogenetic distance of MAdV-3 amino acid sequences for pVIII, protease, polymerase, and hexon from MAdV-1 is markedly higher than 0.1 exchange per position, and, based on our cross-neutralization experiments, MAdV-3 and MAdV-1 can be regarded as different serotypes. Therefore, we propose to classify MAdV-3 as the first isolate of a novel adenovirus species, designated murine adenovirus C (MAdV-C). The novel MAdV-3 virus is not only genetically and serologically distinct from MAdV-1 but also shows a unique organ tropism in infected mice. In contrast to MAdV-1, the virus was not detectable in brain but predominantly infected heart tissue. Thus, infection of mice with cardiotropic MAdV-3 might be an interesting animal model of adenovirus-induced myocarditis.

Dobrava-belgrade virus spillover infections, Germany.

We present the molecular identification of Apodemus agrarius (striped field mouse) as reservoir host of the Dobrava-Belgrade virus (DOBV) lineage DOBV-Aa in 3 federal states of Germany. Phylogenetic analyses provided evidence for multiple spillover of DOBV-Aa to A. flavicollis, a crucial prerequisite for host switch and genetic reassortment.

Molecular diagnostics of hemorrhagic fever with renal syndrome during a Dobrava virus infection outbreak in the European part of Russia.

A large outbreak of hemorrhagic fever with renal syndrome (HFRS) occurred in the winter of 2006-2007 in a region southeast of Moscow in Central European Russia. Of the 422 patients with HFRS investigated in this study, 58 patients were found to be infected by Puumala virus, whereas as many as 364 were infected by Dobrava-Belgrade virus (DOBV). Early serum samples from 10 DOBV-infected patients were used for nucleic acid amplification, which was successful for 5 patients. Molecular analyses demonstrated that the causative hantavirus belongs to the DOBV-Aa genetic lineage, which is carried by the striped field mouse (Apodemus agrarius) as the natural reservoir host. Neutralization assays with convalescent-phase sera from these patients confirmed infection by DOBV-Aa; related viruses, such as the Dobrava-Slovenia virus (DOBV-Af) and the Dobrava-Sochi virus (DOBV-Ap), were neutralized at lower efficiencies. The clinical courses of the 205 patients enrolled in the study were found to be mostly mild to moderate; however, an unexpectedly high fraction (27%) of patients exhibited severe illness. One patient died from kidney failure and showed symptoms of generalized subcutaneous hemorrhage. The results provide molecular, serodiagnostic, and clinical evidence that DOBV-Aa is a common pathogen in East Europe that causes large outbreaks of HFRS.

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.

Human seroprevalence indicating hantavirus infections in tropical rainforests of Côte d'Ivoire and Democratic Republic of Congo.

Hantaviruses are members of the Bunyaviridae family carried by small mammals and causing human hemorrhagic fevers worldwide. In Western Africa, where a variety of hemorrhagic fever viruses occurs, indigenous hantaviruses have been molecularly found in animal reservoirs such as rodents, shrews, and bats since 2006. To investigate the human contact to hantaviruses carried by these hosts and to assess the public health relevance of hantaviruses for humans living in the tropical rainforest regions of Western and Central Africa, we performed a cross-sectional seroprevalence study in the region of Taï National Park in Côte d'Ivoire and the Bandundu region near the Salonga National Park in the Democratic Republic (DR) of Congo. Serum samples were initially screened with enzyme-linked immunosorbent assays using nucleoproteins of several hantaviruses as diagnostic antigens. Positive results were confirmed by Western blotting and immunofluorescence testing. Seroprevalence rates of 3.9% (27/687) and 2.4% (7/295), respectively, were found in the investigated regions in Côte d'Ivoire and the DR Congo. In Côte d'Ivoire, this value was significantly higher than the seroprevalence rates previously reported from the neighboring country Guinea as well as from South Africa. Our study indicates an exposure of humans to hantaviruses in West and Central African tropical rainforest areas. In order to pinpoint the possible existence and frequency of clinical disease caused by hantaviruses in this region of the world, systematic investigations of patients with fever and renal or respiratory symptoms are required.

Hantaviruses in Africa.

This paper summarizes the progress in the search for hantaviruses and hantavirus infections in Africa. After having collected molecular evidence of an indigenous African hantavirus in 2006, an intensive investigation for new hantaviruses has been started in small mammals. Various novel hantaviruses have been molecularly identified not only in rodents but also in shrews and bats. In addition, the first African hantavirus, Sangassou virus, has been isolated and functionally characterized in cell culture. Less is known about the ability of these hantaviruses to infect humans and to cause diseases. To date, no hantavirus genetic material could be amplified from patients' specimens collected in Africa. Serological studies in West Africa, based on a battery of screening and confirmatory assays, led to the detection of hantavirus antibodies in the human population and in patients with putative hantavirus disease. In addition to this overview, we present original data from seroepidemiological and field studies conducted in the Southern part of Africa. A human seroprevalence rate of 1.0% (n=1442) was detected in the South African Cape Region whereas no molecular evidence for the presence of hantavirus was found in 2500 small animals trapped in South Africa and Namibia.

Dobrava-Belgrade hantavirus from Germany shows receptor usage and innate immunity induction consistent with the pathogenicity of the virus in humans.

BACKGROUND: Dobrava-Belgrade virus (DOBV) is a European hantavirus causing hemorrhagic fever with renal syndrome (HFRS) in humans with fatality rates of up to 12%. DOBV-associated clinical cases typically occur also in the northern part of Germany where the virus is carried by the striped field mouse (Apodemus agrarius). However, the causative agent responsible for human illness has not been previously isolated. METHODOLOGY/PRINCIPAL FINDINGS: Here we report on characterization of a novel cell culture isolate from Germany obtained from a lung tissue of "spillover" infected yellow necked mouse (A. flavicollis) trapped near the city of Greifswald. Phylogenetic analyses demonstrated close clustering of the new strain, designated Greifswald/Aa (GRW/Aa) with the nucleotide sequence obtained from a northern German HFRS patient. The virus was effectively blocked by specific antibodies directed against ß3 integrins and Decay Accelerating Factor (DAF) indicating that the virus uses same receptors as the highly pathogenic Hantaan virus (HTNV). In addition, activation of selected innate immunity markers as interferon ß and λ and antiviral protein MxA after viral infection of A549 cells was investigated and showed that the virus modulates the first-line antiviral response in a similar way as HTNV. CONCLUSIONS/SIGNIFICANCE: In summary, our study reveals novel data on DOBV receptor usage and innate immunity induction in relationship to virus pathogenicity and underlines the potency of German DOBV strains to act as human pathogen.

Hantavirus disease outbreak in Germany: limitations of routine serological diagnostics and clustering of virus sequences of human and rodent origin.

In Europe, hemorrhagic fever with renal syndrome results mainly from infection with Puumala virus (PUUV) or Dobrava virus. For 31 patients from a hantavirus disease outbreak in Lower Bavaria, a district in southeast Germany, serodiagnosis was undertaken by enzyme-linked immunosorbent assay, immunofluorescence assay, and immunoblot analysis. In a few of these cases, however, PUUV-specific typing of antibodies by these standard assays failed and a virus neutralization assay under biosafety level 3 conditions was required to verify the infection by this virus type. PUUV RNA was amplified by reverse transcription-PCR from acute-phase sera of three patients and was found to be very closely related to virus sequences obtained from bank voles (Clethrionomys glareolus) trapped in the same area. These findings link the outbreak with a novel PUUV lineage, "Bavaria," circulating in the local rodent population. The Bavaria lineage associated with the outbreak is only distantly related to other PUUV lineages from Germany.

Hantavirus in African wood mouse, Guinea.

Hantaviruses are rodent-borne, emerging viruses that cause life-threatening human diseases in Eurasia and the Americas. We detected hantavirus genome sequences in an African wood mouse (Hylomyscus simus) captured in Sangassou, Guinea. Sequence and phylogenetic analyses of the genetic material demonstrate a novel hantavirus species, which we propose to name "Sangassou virus."

First molecular identification of human Dobrava virus infection in central Europe.

Viral RNA was amplified by reverse transcription-PCR from a patient suffering from hemorrhagic fever with renal syndrome (HFRS) in Germany. The virus strain could be assigned to the Dobrava hantavirus (DOBV). This is the first molecular identification of human infection by DOBV in central Europe and the first proof that a virus strain related to the DOBV-Aa lineage, carried by Apodemus agrarius rodents, is able to cause HFRS.