engleski

Pathogenic and non-pathogenic hantaviruses use different apoptotic pathways

Hantaviruses are maintained in nature in persistently infected rodents and can also persistently infect cultured mammalian cells, causing little or no cytopathology. An unexpected outcome was our observation of cytophatic effect (CPE) and apoptosis in hantavirus-infected human embryonic kidney cells (HEK293). Many viruses cause apoptosis, or programmed cell death, which is an important mechanism for eliminating virus-infected cells from the host. One possible mechanism for induction of apoptosis could be mediated by TNF and the members of TNF superfamily. However, our initial results indicated that members of the TNF receptor superfamily did not contribute to the apoptosis that we saw in the HEK293 cells infected with pathogenic hantaviruses. Further research with pathogenic hantaviruses and HEK293 cells at the gene expression level indicated that mitochondrial apoptosis pathway could be important. The aim of this study was to find out whether non-pathogenic hantaviruses cause CPE in HEK293 cells and to detect possible apoptotic mechanisms at the gene expression level. Further we would like to confirm our hypothesis that mitochondrial pathway is important in apoptosis caused by pathogenic hantaviruses. HEK293 cells were infected with non-pathogenic Tula virus or pathogenic viruses Hantaan or Andes and samples were collected in different time points. For the gene expression analysis we used Focused Gene Expression cDNA Array Analysis (GEArrays, SuperArray Bioscience, Frederick, MD, USA). GEArray Q series Human Apoptosis Gene Array was used. Selected genes identified by GEArray analysis will be also analyzed by quantitative real-time PCR to verify transcriptional responses. Additionally, ELISA tests for the quantitative detection of caspase-3 and caspase-9 (Bender MedSystems, Vienna, Austria) were used. In cells infected with Tula virus we found significant gene expression for several members of TNF superfamily (FAS, TNFR, caspase-8) suggesting that non-pathogenic hantaviruses cause apoptosis induced by activation of cell surface death receptors (`extrinsic pathway'). In contrary, our experiments showed increase in caspase-3 and caspase-9 release in cell infected with Hantaan and Andes viruses, telling us that mitochondria play a central role in an `intrinsic' pathway of apoptosis caused by pathogenic hantaviruses. We also showed that hantavirus replication is important in proces of apoptosis in HEK293 cells. Further experiments are in process to confrim our findings.

pathogenic hantaviruses; non-pathogenic hantaviruses; apoptotic pathways; superarray

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