SULM – Schweizerische Union für Labormedizin | Union Suisse de Médecine de Laboratoire | Swiss Union of Laboratory Medicine

Abstracts SGM 2016


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E. AJUH1, T. BETHGE1, H.H. HIRSCH1,2

1Transplantation & Clinical Virology, Department of Biomedicine-Haus Petersplatz, University of Basel, CH-4009 Basel, Switzerland, 2Infectious Diseases & Hospital Epidemiology, University Hospital Basel, Basel Switzerland

Introduction: The regulation of the early viral gene region (EVGR) and late viral gene region (LVGR) of human polyomavirus (HPyV) infections has been best studied for BKPyV causing nephropathy (PyVAN) and hemorrhagic cystitis in transplant patients, and JCPyV causing progressive multitfocal leukoencephalopathy (PML). For BKPyV and JCPyV, EVGR and LVGR expression is coordinated by the non-coding control region (NCCR). In patients with PyVAN and PML, however, the NCCR of BKPyV and JCPyV have been found to be rearranged, and constitutively increase EVGR expression and accelerate viral replication in cell culture. Little information is available for any of the 11 novel HPyVs. Moreover, none of the 11 HPyV has been effectively propagated in cell culture. Too close this gap, we compared NCCR-driven EVGR and LVGR expression of the novel HPyVs in different cell culture systems.
Method: NCCRs of all 13 HPyVs were cloned into a bidirectional reporter vector (pRG13D12) bearing the RFP and GFP as markers of EVGR and LVGR, respectively. Expression was assayed in HEK293, HEK293T, and HEK293TT and CV-1 and, Cos-7 to estimate the potential effect of SV40 large T-antigen (LTag). Expression was also monitored in selected cell lines reflecting different human organs using fluorescence microscopy and flow cytometry analysis.
Results: Our results show that a hierarchy of EVGR expression, whereby by MCPyV and HPyV12 were the strongest in 6 of the 7 cell lines. KIPyV EVGR was strong in 3 of the 7 cell lines transfected. HPyV 6, 7 and 9 displayed lowest EVGR expression in most of the cell lines. LVGR expression was generally stronger than EVGR for all archetypes HPyV NCCRs. BKPyV, JCPyV and TSPyV showed strongest LVGR expression in most cell lines. Examining natural variants with „rearranged“ or point mutant HPyV NCCRs from patients, we found that HPyV7-PITT1, HPyV9-UF1 and MCPyV-MCVw156 displayed slightly higher EVGR and LVGR activity than the corresponding “archetype“. HEK293T, HEK293TT, and COS-7 constitutively expressing SV40 LTag increased the NCCR activity of most HPyVs suggesting that the reporter results were modulated as expected in the viral life cycle.
Conclusion: A hierarchy of HPyV NCCR activity could be established. Natural HPyV NCCR variants from patients with HPyV disease showed higher activity suggesting that NCCR are important pathogenicity determinants. LTag expression modulates NCCR activity as expected in the viral life cycle. Together, the results suggest that factors of cell differentiation and activation including small molecules or cytokine should permit identifying suitable cell culture conditions for the novel HPyV, and identify pathogenetic cofactors, and potential inhibitors.

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