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 β3 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 β 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

Molecular characterization and pathogenicity potential of novel hantavirus isolates

Hantaviren, deren Hauptreservoir Nager bilden, sind weltweit verbreitete Pathogene. Nach Übertragung auf den Menschen prägen sie vorwiegend zwei Krankheitsbilder: Das hämorrhagische Fieber mit renalem Syndrom (HFRS) oder das Hantavirus-assoziierte cardiopulmonale Syndrom (HCPS). Je nach Virusstamm variiert die Mortalitätsrate zwischen 0,1% und 50%. Das Dobrava-Belgrad-Virus (DOBV), welches zum HFRS führt, weist eine Mortalitätsrate von bis zu 12% auf und ist somit das lebensbedrohlichste Hantavirus in Europa. Wie ihre natürlichen Wirte, die Mäuse aus dem Genus Apodemus, bilden auch die DOBV unterschiedliche phylogenetische Linien. Das aus A. flavicolis (Af) isolierte DOBV-Af ist im Balkan verbreitet und führt zu schweren Verläufen des HFRS. Das in A. agrarius (Aa) gefundene DOBV-Aa verursacht schwache bis milde HFRS-Erkrankungen in Mitteleuropa und dem zentraleuropäsichen Teil Russlands. Moderate bis schwere HFRS-Verläufe im südeuropäischen Teil Russlands wurden mit Viren der DOBV-Ap-Linie in Verbindung gebracht, welche von A. ponticus (Ap) übertragen werden. In Deutschland ist DOBV endemisch im nördlichen Teil des Landes. Epidemiologische Studien, basierend auf der serologischen Feintypisierung mittels Neutralisationstests und phylogenetischen Analysen Patienten-assoziierter Virussequenzen, zeigen, dass Viren der DOBV-Aa-Linie für die HFRS-Fälle in dieser Region verantwortlich sind. Bislang konnte das für die humane Erkrankung in Deutschland verantwortliche Virus noch nicht in Zellkultur isoliert werden. In der vorliegenden Arbeit wurde das erste Zellkulturisolat eines aus Deutschland stammenden DOBV gewonnen, dessen vollständige Genomsequenz bestimmt und als Greifswald-Virus (GRW/Aa) bezeichnet wurde. In phylogenetischen Analysen bildete GRW/Aa eine gemeinsame Gruppe mit viralen Sequenzen, die aus norddeutschen HFRS-Patienten gewonnen wurden. Folglich kann GRW/Aa als das für HFRS in Deutschland verantwortliche DOBV angesehen werden. Anhand von Wirtszellkulturen konnten wir zeigen, dass GRW/Aa ähnliche Charakteristika aufweist, wie das pathogene Hantaan-Virus; es nutzt b3-Integrine und den decay accelerating factor (DAF) als zelluläre Rezeptoren und induziert die späte Expression von Markern der angeborenen Immunantwort (z. B. IFN-b, IFN-l1, MxA). Obwohl Hantaviren auf nahezu allen Kontinenten als Humanpathogene bekannt sind, wurde das erste afrikanische Hantavirus (Sangassou-Virus, SANGV) erst kürzlich in unserer Arbeitsgruppe isoliert. Aufgrund der engen phylogenetischen Verwandtschaft zwischen SANGV und GRW/Aa haben wir untersucht, ob das potentiell humanpathogene SANGV ähnliche Eigenschaften wie GRW/Aa aufweist. Aufgrund der Tatsache, dass bislang noch kein Tiermodell für die Untersuchung der Hantaviruspathogenese existiert, haben wir die Rezeptorerkennung und Induktion der Marker der angeborenen Immunantwort zur Abschätzung des humanpathogenen Potentials von SANGV, im Vergleich zu GRW/Aa, verwendet. Wir zeigten, dass SANGV ausschließlich b1-Integrine als Rezeptor nutzt und eine starke IFN-l1-Antwort in der infizierten Zelle induziert. In der Zellkultur zeigt SANGV somit andere funktionelle Charakteristika als GRW/Aa. Ob diese in Zusammenhang mit einer durch SANGV vermittelten Pathogenese stehen, muss in zukünftigen Studien untersucht werden.Hantaviruses are worldwide distributed pathogens which are mainly carried by rodents. When transmitted to humans, they can cause two significant diseases: hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS). Depending on the virus strain, the case fatality rates of hantavirus diseases are ranging from 0.1% up to 50%. In Europe Dobrava-Belgrade virus (DOBV) is the most life-threatening hantavirus leading to HFRS with case fatality rates of up to 12%. According to its natural hosts, mice of the genus Apodemus, DOBV forms distinct phylogenetic lineages. DOBV-Af associated with A. flavicollis (Af) causes severe HFRS cases in the Balkan region. DOBV-Aa, found in A. agrarius (Aa), is typical for Central Europe and Central European Russia where it causes mild/moderate disease. Moderate to severe HFRS cases in South European Russia have been associated with virus strains of the DOBV-Ap lineage, transmitted by A. ponticus (Ap). In Germany, DOBV is endemic in the northern part of the country. Seroepidemiological studies involving fine serotyping by neutralization assay as well as phylogenetic analyses of patient-associated virus sequences showed that strains of DOBV-Aa lineage are responsible for HFRS cases in this geographical region. However, the causative agent of human disease from Germany was not isolated in cell culture. Within the current study, we have generated the first cell culture isolate of DOBV from Germany, called Greifswald virus (GRW/Aa), and determined its complete genomic nucleotide sequence. Phylogenetic analyses revealed close clustering of GRW/Aa with sequences derived from Northern German HFRS patients. Consequently, GRW/Aa can be taken as a representative of DOBV strains causing HFRS in Germany. We have demonstrated that in cultivated host cells GRW/Aa exhibits properties similar to pathogenic Hantaan virus; it recognizes b3 integrins and Decay Accelerating Factor (DAF) as cellular entry receptors and induces late expression of innate immunity markers (IFN-b, IFN-l1, MxA). Despite hantaviruses being well recognized human pathogens in almost all continents, the first African hantavirus named Sangassou virus (SANGV) has been only very recently isolated in our group. Important to note that SANGV is most closely related to DOBV in molecular phylogenetic analyses. Therefore, it was interesting to investigate if SANGV, a potential human pathogen, displays properties similar to GRW/Aa. Given that an animal model for studying hantavirus-mediated pathogenesis is not available, we used cellular receptor recognition and induction of innate immunity markers as in vitro determinants to estimate the pathogenic potential of SANGV in comparison to GRW/Aa. We have found that SANGV exclusively recognizes b1 integrins as cellular entry receptors and elicits strong induction of IFN-l1 in infected cells. Therefore, in cultivated host cells SANGV exhibits functional characteristics distinct from GRW/Aa. Whether these properties contribute to SANGV-mediated pathogenesis in humans needs to be elucidated in future studies

Potential Drug Candidates to Treat TRPC6 Channel Deficiencies in the Pathophysiology of Alzheimer’s Disease and Brain Ischemia

Alzheimer’s disease and cerebral ischemia are among the many causative neurodegenerative diseases that lead to disabilities in the middle-aged and elderly population. There are no effective disease-preventing therapies for these pathologies. Recent in vitro and in vivo studies have revealed the TRPC6 channel to be a promising molecular target for the development of neuroprotective agents. TRPC6 channel is a non-selective cation plasma membrane channel that is permeable to Ca2+. Its Ca2+-dependent pharmacological effect is associated with the stabilization and protection of excitatory synapses. Downregulation as well as upregulation of TRPC6 channel functions have been observed in Alzheimer’s disease and brain ischemia models. Thus, in order to protect neurons from Alzheimer’s disease and cerebral ischemia, proper TRPC6 channels modulators have to be used. TRPC6 channels modulators are an emerging research field. New chemical structures modulating the activity of TRPC6 channels are being currently discovered. The recent publication of the cryo-EM structure of TRPC6 channels should speed up the discovery process even more. This review summarizes the currently available information about potential drug candidates that may be used as basic structures to develop selective, highly potent TRPC6 channel modulators to treat neurodegenerative disorders, such as Alzheimer’s disease and cerebral ischemia

Piperazine Derivative Stabilizes Actin Filaments in Primary Fibroblasts and Binds G-Actin In Silico

Alzheimer’s disease (AD) is characterized by synaptic dysfunction, which is expressed through the loss of dendritic spines and changes in their morphology. Pharmacological compounds that are able to protect spines in the AD brain are suggested to be novel drugs that would be able to slow down the disease progression. We have recently shown that a positive modulator of transient receptor potential cation channel subfamily C member 6 (TRPC6), the compound N-(2-chlorophenyl)-2-(4-phenylpiperazine-1-yl) acetamide (51164), causes the upregulation of postsynaptic neuronal store-operated calcium entry, maintains mushroom spine percentage, and recovers synaptic plasticity in amyloidogenic mouse models of Alzheimer’s disease. Here, using confocal microscopy and calcium imaging methods, we present the experimental data indicating that 51164 possesses an alternative mechanism of action. We demonstrated that 51164 can increase the mushroom spine percentage in neurons with the downregulated activity of TRPC6-dependent neuronal store-operated calcium entry. Moreover, we report the binding of 51164 to G-actin in silico. We observed that 51164 interacts with Lys 336, Asp157, and Ser14 of G-actin, amino acids involved in the stabilization/polymerization of the G-actin structure. We showed that interactions of 51164 with G-actin are much stronger in comparison to the well-characterized F-actin stabilizing and polymerizing drug, jasplakinolide. The obtained results suggest an alternative protective mechanism of 51164 that is related to the preservation of actin filaments in vitro