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

ABSTRACT: 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 ?3 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 ? and ? 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.

SUBMITTER: Popugaeva E

PROVIDER: S-EPMC3335829 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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

Popugaeva Elena E Witkowski Peter T PT Schlegel Mathias M Ulrich Rainer G RG Auste Brita B Rang Andreas A Krüger Detlev H DH Klempa Boris B

PloS one 20120424 4

<h4>Background</h4>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.<h4>Methodology/principal findings</h4>Here we report on characterization of a ...[more]

PMID: 22545121

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Project description:We used an in vitro model to study the impact of hantavirus infection on the cellular gene expression profile. To do so, A549 cells were infected with pathogenic DOBV genotypes Dobrava, Kurkino, and Sochi and less-pathogenic TULV . A549 cells were chosen because of their high susceptibility towards hantavirus infection and because many fundamental studies on hantavirus biology and on host gene expression changes following infection have been performed with this cell line. Cells were infected at a high multiplicity of infection of 5 focus forming units (FFU) per cell to guarantee uniform infection of all cells. Total RNA from infected and mock-infected control cells was isolated at 12 h post infection. This point of time was chosen to allow enough time for establishment of infection and progression to early viral gene expression but to avoid the risk of missing gene expression changes associated with the onset of the cellular innate immune response towards infection. The distinct modulation of cellular transcription of A549 cells was analyzed by means of a whole genome cRNA microarray. All experiments were performed in duplicate using RNA samples from two independently infected cell cultures for each analysis. To compare the gene expression profiles, ratios were calculated by dividing the merged normalized signal intensities of infected samples by mock-control signal intensities. Genes that exhibited a ≥2-fold change (FC) in gene expression and signal intensities that were significantly above the background with p-values ≤ 0.01 were chosen for further analysis. The successful infection of A549 cells with all viruses and the uniform progression of infection were confirmed by immunofluorescence microscopy of cells infected in parallel. At 12 h post infection with each virus nearly all cells were infected without showing cytopathic effects. Since different clinical courses are caused by infection with distinct DOBV genotypes, genotype-specific regulation of cellular transcripts were investigated that may be crucial for pathogenesis in vitro. Selected infection regulated candidates were verified by qPCR at different time points after infection.

Project description:We used an in vitro model to study the impact of hantavirus infection on the cellular gene expression profile. To do so, A549 cells were infected with pathogenic DOBV genotypes Dobrava, Kurkino, and Sochi and less-pathogenic TULV . A549 cells were chosen because of their high susceptibility towards hantavirus infection and because many fundamental studies on hantavirus biology and on host gene expression changes following infection have been performed with this cell line. Cells were infected at a high multiplicity of infection of 5 focus forming units (FFU) per cell to guarantee uniform infection of all cells. Total RNA from infected and mock-infected control cells was isolated at 12 h post infection. This point of time was chosen to allow enough time for establishment of infection and progression to early viral gene expression but to avoid the risk of missing gene expression changes associated with the onset of the cellular innate immune response towards infection. The distinct modulation of cellular transcription of A549 cells was analyzed by means of a whole genome cRNA microarray. All experiments were performed in duplicate using RNA samples from two independently infected cell cultures for each analysis. To compare the gene expression profiles, ratios were calculated by dividing the merged normalized signal intensities of infected samples by mock-control signal intensities. Genes that exhibited a â¥2-fold change (FC) in gene expression and signal intensities that were significantly above the background with p-values â¤ 0.01 were chosen for further analysis. The successful infection of A549 cells with all viruses and the uniform progression of infection were confirmed by immunofluorescence microscopy of cells infected in parallel. At 12 h post infection with each virus nearly all cells were infected without showing cytopathic effects. Since different clinical courses are caused by infection with distinct DOBV genotypes, genotype-specific regulation of cellular transcripts were investigated that may be crucial for pathogenesis in vitro. Selected infection regulated candidates were verified by qPCR at different time points after infection. We analyzed the gene expression profile of A549 cells wich were either mock-infected or infected with Dobrava Virus (DOBV) Dobrava, Kurkino or Sochi, or with Tula Virus (TULV) at a multiplicity of infection of 5. Experiments were performed in duplicate. At 12 h post infection total RNA was isolated from infected cells and used for microarray analysis.

Dataset Information

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

Publications

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

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