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Abstract
Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000–3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3′ untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.
Highlights
Tick-borne encephalitis virus complex represents a group of closely related viruses endemic in Europe and Asia causing serious neuroinfections and hemorrhagic fevers [1]
A comparative analysis of all small interfering RNA (siRNA) target sequences with the genomes of five members of the Tick-borne encephalitis virus complex revealed that siRNA sequences D1, D3, D8, D10 and D13 located within the 39UTR, 59UTR and the core region were strongly conserved with 1 to 2 nucleotide divergence between different viral subtypes
The majority (11/19) of the 19 siRNA sequences screened in this study were capable of reducing Langat virus titer by more than 80% in comparison to nonsense siRNA when transfected into the cells before the infection (Figure 1 B)
Summary
Tick-borne encephalitis virus complex represents a group of closely related viruses endemic in Europe and Asia causing serious neuroinfections and hemorrhagic fevers [1]. Following an incubation period of 3–8 days after a tick bite, the virus replicates locally in epidermal dendritic cells and spreads via lymph vessels to the blood stream where a short but significant viremia occurs and extraneural tissues are infected. It is during this viremic phase that the virus crosses the blood-brain barrier and invades the central nervous system (CNS) by a still unknown mechanism. The most common clinical feature of TBE patients is ataxia followed by paresis or paralysis of one or more extremities [10]
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