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Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: definition of genotypes and their characteristics.

ABSTRACT: Dobrava-Belgrade virus (DOBV) is a human pathogen that has evolved in, and is hosted by, mice of several species of the genus Apodemus. We propose a subdivision of the species Dobrava-Belgrade virus into four related genotypes - Dobrava, Kurkino, Saaremaa, and Sochi - that show characteristic differences in their phylogeny, specific host reservoirs, geographical distribution, and pathogenicity for humans.

SUBMITTER: Klempa B

PROVIDER: S-EPMC3586401 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: definition of genotypes and their characteristics.

Klempa Boris B Avsic-Zupanc Tatjana T Clement Jan J Dzagurova Tamara K TK Henttonen Heikki H Heyman Paul P Jakab Ferenc F Kruger Detlev H DH Maes Piet P Papa Anna A Tkachenko Evgeniy A EA Ulrich Rainer G RG Vapalahti Olli O Vaheri Antti A

Archives of virology 20121023 3

Dobrava-Belgrade virus (DOBV) is a human pathogen that has evolved in, and is hosted by, mice of several species of the genus Apodemus. We propose a subdivision of the species Dobrava-Belgrade virus into four related genotypes - Dobrava, Kurkino, Saaremaa, and Sochi - that show characteristic differences in their phylogeny, specific host reservoirs, geographical distribution, and pathogenicity for humans. ...[more]

PMID: 23090188

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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

Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: definition of genotypes and their characteristics.

Publications

Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: definition of genotypes and their characteristics.

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