Tick-borne Encephalitis Virus Replication Research Articles

Cellular stress is triggered by tick-borne encephalitis virus and limits the virus replication in PMJ2-R mouse macrophage cell line

Viral infection may represent a stress condition to the host cell. Cells react to it by triggering the defence programme to restore homeostasis and these events may in turn impact the viral replication. The knowledge about tick-borne encephalitis virus (TBEV) infection-associated stress is limited. Here we investigated the interplay between TBEV infection and stress pathways in PMJ2-R mouse macrophage cell line, as macrophages are the target cells in early phases of TBEV infection. First, to determine how stress influences TBEV replication, the effect of stress inducers H2O2 and tunicamycin (TM) was tested. Viral multiplication was decreased in the presence of both stress inducers suggesting that the stress and cellular stress responses restrict the virus replication. Second, we investigated the induction of oxidative stress and endoplasmic reticulum (ER) stress upon TBEV infection. The level of oxidative stress was interrogated by measuring the reactive oxygen species (ROS). ROS were intermittently increased in infected cells at 12 hpi and at 72 hpi. As mitochondrial dysfunction may result in increased ROS level, we evaluated the mitochondrial homeostasis by measuring the mitochondrial membrane potential (MMP) and found that TBEV infection induced the hyperpolarization of MMP. Moreover, a transient increase of gene expression of stress-induced antioxidative enzymes, like p62, Gclm and Hmox1, was detected. Next, we evaluated the ER stress upon TBEV infection by analysing unfolded protein responses (UPR). We found that infection induced gene expression of two general sensors BiP and CHOP and activated the IRE1 pathway of UPR. Finally, since the natural transmission route of TBEV from its tick vector to the host is mediated via tick saliva, the impact of tick saliva from Ixodes ricinus on stress pathways in TBEV-infected cells was tested. We observed only marginal potentiation of UPR pathway.In conclusion, we found that TBEV infection of PMJ2-R cells elicits the changes in redox balance and triggers cellular stress defences, including antioxidant responses and the IRE1 pathway of UPR. Importantly, our results revealed the negative effect of stress-evoked events on TBEV replication and only marginal impact of tick saliva on stress cellular pathways.

Inhibition of tick-borne encephalitis virus in cell cultures by ribavirin.

Tick-borne encephalitis virus (TBEV) belonging to arboviruses is a major member of zoonotic pathogens. TBEV infection causes severe human encephalitis without specific antiviral drugs. Due to its use of antiviral drug against a wide range of viruses, we investigated antiviral effect of ribavirin against TBEV in susceptible human cell lines A549 and SH-SY5Y. Ribavirin displayed minor cytotoxicity on multiple cell lines. Ribavirin obviously impaired TBEV replication and protected the infected cells from cytopathic effect. Importantly, ribavirin markedly inhibited TBEV propagation, as evidenced by impairment of TBEV production and viral RNA replication. Treatment with ribavirin (co-treatment and post-treatment) led to a dose-dependent reduction in TBEV titers as well as the viral RNA levels. Antiviral protein myxovirus resistance A mRNA expression was significantly up-regulated and signal transducer and activator of transcription 3 was activated in TBEV-infected A549 cells upon the ribavirin treatment. Induction of inflammatory cytokine tumor necrosis factor alpha by TBEV was decreased in A549 cells with the treatment of ribavirin, whereas interleukin 1 beta release appeared to be unaffected. These results suggest that ribavirin might represent a promising safe and effective antiviral drug against TBEV.

Sterile 20-like kinase 3 promotes tick-borne encephalitis virus assembly by interacting with NS2A and prM and enhancing the NS2A-NS4A association.

Tick-borne encephalitis virus (TBEV) is the causative agent of a potentially fatal neurological infection in humans. Investigating virus-host interaction is important for understanding the pathogenesis of TBEV and developing effective antiviral drugs against this virus. Here, we report that mammalian ste20-like kinase 3 (MST3) is involved in the regulation of TBEV infection. The knockdown or knockout of MST3, but not other mammalian ste20-like kinase family members, inhibited TBEV replication. The knockdown of MST3 also significantly reduced TBEV replication in mouse primary astrocytes. Life cycle analysis indicated that MST3 remarkably impaired virion assembly efficiency and specific infectivity by respectively 59% and 95% in MST3-knockout cells. We further found that MST3 interacts with the viral proteins NS2A and prM; and MST3 enhances the interaction of NS2A-NS4A. Thus, MST3-NS2A complex plays a major role in recruiting prM-E heterodimers and NS4A and mediates the virion assembly. Additionally, we found that MST3 was biotinylated and combined with other proteins (e.g., ATG5, Sec24A, and SNX4) that are associated with the cellular membrane required for TBEV infection. Overall, our study revealed a novel function for MST3 in TBEV infection and identified as a novel host factor supporting TBEV assembly.

Functional characterization of 5' untranslated region (UTR) secondary RNA structures in the replication of tick-borne encephalitis virus in mammalian cells.

Tick-borne Encephalitis Virus (TBEV) is an emerging flavivirus that causes neurological disorders including viral encephalitis of varying severity. Whilst secondary RNA structures within the 5' untranslated regions (UTRs) of many flaviviruses determine both virus replication and pathogenic outcomes in humans, these elements have not been systematically investigated for TBEV. In this study, we investigated the role of predicted RNA secondary elements of the first 107 nucleotides (nts) of the viral genome forming the stem-loop A (SLA). Experiments were performed in replicons and infectious TBEV system. This region comprises three distinct structures: 5' stem 0 (S0), stem-loop 1 (SL1) and stem-loop 2 (SL2). S0 was found to be essential for virus infection as mutations in the lower stem of this region significantly reduced virus replication. Point mutations in SL1 that preserved the Y-shape confirmation delayed viral RNA replication but did not abolish virus infectivity. Deletion of SL2 did not abolish infectivity but had a negligible effect on virus propagation. No correlation was observed between in vitro translation efficiency and virus infectivity, suggesting that the 5'UTR functions independently to virus translation. Together, these findings reveal distinct RNA elements within the 5'UTR that are essential for the stability and replication of viral RNA. We further identify changes in RNA folding that lead to altered TBEV infectivity and pathogenesis.

Flavivirus prM interacts with MDA5 and MAVS to inhibit RLR antiviral signaling

BackgroundVector-borne flaviviruses, including tick-borne encephalitis virus (TBEV), Zika virus (ZIKV), West Nile virus (WNV), yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV), pose a growing threat to public health worldwide, and have evolved complex mechanisms to overcome host antiviral innate immunity. However, the underlying mechanisms of flavivirus structural proteins to evade host immune response remain elusive.ResultsWe showed that TBEV structural protein, pre-membrane (prM) protein, could inhibit type I interferon (IFN-I) production. Mechanically, TBEV prM interacted with both MDA5 and MAVS and interfered with the formation of MDA5-MAVS complex, thereby impeding the nuclear translocation and dimerization of IRF3 to inhibit RLR antiviral signaling. ZIKV and WNV prM was also demonstrated to interact with both MDA5 and MAVS, while dengue virus serotype 2 (DENV2) and YFV prM associated only with MDA5 or MAVS to suppress IFN-I production. In contrast, JEV prM could not suppress IFN-I production. Overexpression of TBEV and ZIKV prM significantly promoted the replication of TBEV and Sendai virus.ConclusionOur findings reveal the immune evasion mechanisms of flavivirus prM, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention and vaccine development.

In human astrocytes neurotropic flaviviruses increase autophagy, yet their replication is autophagy-independent

Astrocytes, an abundant type of glial cells, are the key cells providing homeostasis in the central nervous system. Due to their susceptibility to infection, combined with high resilience to virus-induced cell death, astrocytes are now considered one of the principal types of cells, responsible for virus retention and dissemination within the brain. Autophagy plays an important role in elimination of intracellular components and in maintaining cellular homeostasis and is also intertwined with the life cycle of viruses. The physiological significance of autophagy in astrocytes, in connection with the life cycle and transmission of viruses, remains poorly investigated. In the present study, we investigated flavivirus-induced modulation of autophagy in human astrocytes by monitoring a tandem fluorescent-tagged LC3 probe (mRFP-EGFP-LC3) with confocal and super-resolution fluorescence microscopy. Astrocytes were infected with tick-borne encephalitis virus (TBEV) or West Nile virus (WNV), both pathogenic flaviviruses, and with mosquito-only flavivirus (MOF), which is considered non-pathogenic. The results revealed that human astrocytes are susceptible to infection with TBEV, WNV and to a much lower extent also to MOF. Infection and replication rates of TBEV and WNV are paralleled by increased rate of autophagy, whereas autophagosome maturation and the size of autophagic compartments are not affected. Modulation of autophagy by rapamycin and wortmannin does not influence TBEV and WNV replication rate, whereas bafilomycin A1 attenuates their replication and infectivity. In human astrocytes infected with MOF, the low infectivity and the lack of efficient replication of this flavivirus are mirrored by the absence of an autophagic response.

Guanine quadruplexes in the RNA genome of the tick-borne encephalitis virus: their role as a new antiviral target andin virus biology.

We have identified seven putative guanine quadruplexes (G4) in the RNA genome of tick-borne encephalitis virus (TBEV), a flavivirus causing thousands of human infections and numerous deaths every year. The formation of G4s was confirmed by biophysical methods on synthetic oligonucleotides derived from the predicted TBEV sequences. TBEV-5, located at the NS4b/NS5 boundary and conserved among all known flaviviruses, was tested along with its mutated variants for interactions with a panel of known G4 ligands, for the ability to affect RNA synthesis by the flaviviral RNA-dependent RNA polymerase (RdRp) and for effects on TBEV replication fitness in cells. G4-stabilizing TBEV-5 mutations strongly inhibited RdRp RNA synthesis and exhibited substantially reduced replication fitness, different plaque morphology and increased sensitivity to G4-binding ligands in cell-based systems. In contrast, strongly destabilizing TBEV-5 G4 mutations caused rapid reversion to the wild-type genotype. Our results suggest that there is a threshold of stability for G4 sequences in the TBEV genome, with any deviation resulting in either dramatic changes in viral phenotype or a rapid return to this optimal level of G4 stability. The data indicate that G4s are critical elements for efficient TBEV replication and are suitable targets to tackle TBEV infection.

Dual control of tick-borne encephalitis virus replication by autophagy in mouse macrophages

Autophagy is a lysosomal degradative pathway responsible for recycling cytosolic proteins and organelles and also functions as an innate defense mechanism that host cells use against viral infection. While many viruses have evolved mechanisms to antagonize the antiviral effects of the autophagy pathway, others subvert autophagy to facilitate replication. For flaviviruses, both the positive and negative role of autophagy in virus replication has been reported. The interplay between autophagy and tick-borne encephalitis virus (TBEV) in innate immune cells is largely unknown. Here we report the relationship between an autophagy and TBEV replication in mouse macrophage cell line PMJ2-R using Hypr strain of TBEV. First, we examined the effect of Hypr infection on the autophagy pathway. We detected a mild and a temporary increase of autophagy marker LC3-II in Hypr-infected cells. The role of autophagy in TBEV replication was evaluated in autophagy related gene 5 (Atg5) knockdown cells (shAtg5). Our results showed that during an early stage of Hypr infection the viral titers were increased, while later on, at 72 hpi, the titers have declined in shAtg5 cells compared to control. Moreover, the higher number of virus-positive cells was observed in shAtg5 cells in early stage of infection and correlated with enhanced virus entry. Finally, we found an increased production of IFN-β in Hypr-infected shAtg5 cells in comparison to control at 48 and 72 hpi implicating that autophagy restricts the amount of IFN produced by TBEV-infected macrophages. To conclude, in mouse macrophages TBEV replication is controlled by autophagy in time dependent manner, having temporally an antiviral and then a pro-viral role during infection. Our study points out to a delicate and complex involvement of autophagy machinery at level of virus entry and IFN-β production when controlling TBEV infection.

Sequential MAVS and MyD88/TRIF signaling triggers anti-viral responses of tick-borne encephalitis virus-infected murine astrocytes.

Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is typically transmitted upon tick bite and can cause meningitis and encephalitis in humans. In TBEV-infected mice, mitochondrial antiviral-signaling protein (MAVS), the downstream adaptor of retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling, is needed to induce early type I interferon (IFN) responses and to confer protection. To characterize the brain-resident cell subset that produces protective IFN-β in TBEV-infected mice, we isolated neurons, astrocytes, and microglia from mice and exposed these cell types to TBEV in vitro. Under such conditions, neurons showed the highest percentage of infected cells, whereas astrocytes and microglia were infected to a lesser extent. In the supernatant (SN) of infected neurons, IFN-β was not detectable, while infected astrocytes showed high and microglia low IFN-β expression. Transcriptome analyses of astrocytes implied that MAVS signaling was needed early after TBEV infection. Accordingly, MAVS-deficient astrocytes showed enhanced TBEV infection and significantly reduced early IFN-β responses. Nevertheless, at later time points, moderate amounts of IFN-β were detected in the SN of infected MAVS-deficient astrocytes. Transcriptome analyses indicated that MAVS deficiency negatively affected the induction of early anti-viral responses, which resulted in significantly increased TBEV replication. Treatment with MyD88 and TRIF inhibiting peptides reduced only late IFN-β responses of TBEV-infected WT astrocytes and blocked entirely IFN-β responses of infected MAVS-deficient astrocytes. Thus, upon TBEV exposure of brain-resident cells, astrocytes are important IFN-β producers showing biphasic IFN-β induction that initially depends on MAVS and later on MyD88/TRIF signaling.

ADAM15 Participates in Tick-Borne Encephalitis Virus Replication.

Tick-borne encephalitis virus (TBEV), a major tick-borne viral pathogen of humans, is known to cause neurological diseases such as meningitis, encephalitis, and meningoencephalitis. However, the life cycle and pathogenesis of TBEV are not well understood. Here, we show that the knockdown or knockout of ADAM15 (a disintegrin and metalloproteinase 15), a host protein involved in neuroblastoma diseases, leads to TBEV replication and assembly defects. We characterized the disintegrin domain in ADAM15 and found that the ADAM15 subcellular localization was changed following TBEV infection. RNA interference (RNAi) screen analysis confirmed ADAM's nonredundant functions and identified a specific role for ADAM15 in TBEV infection. An RNA-sequencing analysis was also conducted to understand the causal link between TBEV infection and the cellular endomembrane network, namely, the generation of replication organelles promoting viral genome replication and virus production. Our data demonstrated that TBEV infection changes ADAM15 cellular localization, which contributes to membrane reorganization and viral replication.IMPORTANCE Tick populations are increasing, and their geographic ranges are expanding. Increases in tick-borne disease prevalence and transmission are important public health issues. Tick-borne encephalitis virus (TBEV) often results in meningitis, encephalitis, and meningoencephalitis. TBEV causes clinical disease in more than 20,000 humans in Europe and Asia per year. An increased incidence of TBE has been noted in Europe and Asia, as a consequence of climate and socioeconomic changes. The need to investigate the mechanism(s) of interaction between the virus and the host factors is apparent, as it will help us to understand the roles of host factors in the life cycle of TBEV. The significance of our research is in identifying the ADAM15 for TBEV replication, which will greatly enhance our understanding of TBEV life cycle and highlight a target for pharmaceutical consideration.

Inhibition of Tick-Borne Encephalitis Virus Replication with Eprosartan and Ribavirin In Vitro and In Vivo

Active search for new antiviral substances is currently underway. The purpose of this work is to identify the inhibitory activity of eprosartan medication in comparison with ribavirin in vitro and in vivo in relation to tick-borne encephalitis virus. The value of the half the maximum cytotoxic concentration (CC50) for eprosartan (8.8±1.2 mg/ml) and ribavirin (1.074±0.16 mg/ml) was established. To obtain a medium effective virus-inhibiting concentration (IC50) of the medications, EIA data were used. Using nonlinear regression analysis of the percentage of antigen positive samples, IC50 values of the studied substances were obtained, which for eprosartan was 0.64±0.23 mg/ml in the treatment regimen. The selective index (SI) or chemotherapeutic index (CTI) was 13.7. The IC50 of ribavirin was 0.0067±0.0015 mg/ml, SI or CTI was 160. The suppression of viral reproduction 2.0 log TCID50 occurred in PEK cell culture under the influence of eprosartan at concentrations of 1.2–3.0 mg/ml (treatment regimen), under the influence of ribavirin — 0.2 mg/ml (prophylactic regimen) and 0.2–0.0125 mg/ml (treatment regimen). Samples with eprosartan (1.5 and 0.6 mg/ml) showed an increase in survival of mice by 50% and 20% compared with the virus control group in the in vivo model and, accordingly, an increase in average life expectancy of 5.2 and 2.1 days. Samples with ribavirin (0.05 and 0.025 mg/ml) increased the survival of mice by 60% and 40% and, accordingly, increased the life expectancy by 7.3 and 4.8 days. The data obtained allow recommending eprosartan as an active agent against tick-borne encephalitis virus along with ribavirin.

Molecular and Genetic Bases of Inhibition of Tick-Borne Encephalitis Virus Replication by Eprosartan and Ribavirin.

The antiviral activity of eprosartan (compound selected in silico) towards highly and low-virulent strains of tick-borne encephalitis virus was compared in vitro with activity of ribavirin. Study of the cytopathogenic activity of the virus on SPEV cells by ELISA, IFAT, and PCR showed similar results: both substances (eprosartan and ribavirin) promoted elimination of tick-borne encephalitis virus. Ribavirin exhibited intracellular inhibition towards both strains: the selectivity index for highly virulent Dal'negorsk strain was 160, for low-virulent Primorye-437 strain - 113. Eprosartan inhibited intracellular replication of Dal'negorsk strain (13.7) and less so that of Primorye-437 strain (2.9). The efficiency of virtual screening of the ligand (eprosartan) was demonstrated for highly virulent, but not low virulent tick-borne encephalitis strain.

Characterization and invitro assembly of tick-borne encephalitis virus C protein.

Tick-borne encephalitis virus (TBEV), a member of flaviviruses, represents a serious health threat by causing human encephalitis mainly in central and eastern Europe, Russia, and northeastern Asia. As no specific therapy is available, there is an urgent need to understand all steps of the TBEV replication cycle at the molecular level. One of the critical events is the packaging of flaviviral genomic RNA by TBEV C protein to form a nucleocapsid. We purified recombinant TBEV C protein and used a combination of physical-chemical approaches, such as size-exclusion chromatography, circular dichroism, NMR spectroscopies, and transmission electron microscopy, to analyze its structural stability and its ability to dimerize/oligomerize. We compared the ability of TBEV C protein to assemble invitro into a nucleocapsid-like structure with that of dengue C protein.

Inhibitory Activity of Scutellaria baicalensis Flavonoids against Tick-Borne Encephalitis Virus.

We studied virus-inhibiting activity of Baikal skullcap (Scutellaria baicalensis) flavonoids against tick-borne encephalitis virus using various model schemes. The half-maximum cytotoxic concentration (CC50) for the plant extract was found (363.9±58.6 μg/ml). Based on the CC50 and IC50, selective index (SI) was calculated for viricidal (53.4), preventive (50.5), and direct antiviral actions (39.1) and for-intracellular replication of the virus (40.4). Suppression of virus reproduction ≥2.0 lg TCID50 was observed at extract concentration ≥5 μg/ml (viricidal effect), ≥11.2 μg/ml (preventive and direct antiviral effects), and ≥9 μg/ml (intracellular replication). Flavonoids of Baikal skullcap extract produced an in vitro inhibitory effect on tick-borne encephalitis virus due to their direct viricidal activity and direct inhibition of adsorption and intracellular replication of tick-borne encephalitis virus, which determines their value as highly effective antiviral drugs.

Comprehensive assessment of specific antibodies on infectious activity of tick-borne encephalitis virus

Vaccines for prophylactic immunization provide the most reliable and effective protection against the vast majority of infectious diseases. Tick-borne encephalitis (TBE) represents a high-priority medical issue at the territory of the Eurasian continent. Of great importance is assessing a role of distinct antibody titers especially low titers, observed quite often in vaccinated individuals, sometimes posing obstacles in determining a threshold of seropositivity as well as the level of specific protection against TBE virus. We aimed at obtaining data to assess antiviral activity of virus-specific antibodies with distinct titer levels based on the in vitro, ex vivo and in vivo experimental studies with a highly virulent Far-Eastern strain of tick-borne encephalitis virus. The in vitro, ex vivo and in vivo comprehensive experimental studies with a highly virulent Far-Eastern strain of tick-borne encephalitis virus (TBEV) were conducted and the dynamics of antiviral activity of virus-specific antibodies at variable titers (1:100–1:3200) was measured (timeframe ranged within 1–96 hours p.i.) to provide a rationale for evaluating the antiviral immune response. It was found that the in vitro experiments demonstrated that the IgG at 1:100 titer exerted a weak anti-TBEV neutralizing effect at all time-points examined. The IgG 1:400 titer caused a 2 log PFU/mL decline in TBEV Dal strain yield at 72 h post-infection, whereas at 1:3200 titer it completely suppressed TBEV replication throughout the observation period. The ex vivo experiments with blood serum obtained from vaccinated subjects demonstrating a range of TBEV antibody titers (sera from vaccinated individuals with varying anti-TBEV antibody titers) and in vivo (outinbred white mice) experiments revealed a delayed virus elimination for antibody titers at 1:100 and 1:200 as well as rapid virus elimination (1–2 days p.i.) for antibody titers greater than 1:400. Thus, antibody titer at 1:400 may be considered as the universal anti-TBEV protection threshold. In order to properly conclude regarding the revaccination schedule it is advised to start with testing blood serum for durability of anti-TBEV immune response. Subjects with TBEV antibody titers at 1:100 and 1:200 should be strongly recommended to undergo a mandatory revaccination. Such an approach is believed to be the most effective way toward enhancing efficacy of vaccine-mediated protection against TBE.

Model System for the Formation of Tick-Borne Encephalitis Virus Replication Compartments without Viral RNA Replication.

Flavivirus is a positive-sense, single-stranded RNA viral genus, with members causing severe diseases in humans such as tick-borne encephalitis, yellow fever, and dengue fever. Flaviviruses are known to cause remodeling of intracellular membranes into small cavities, where replication of the viral RNA takes place. Nonstructural (NS) proteins are not part of the virus coat and are thought to participate in the formation of these viral replication compartments (RCs). Here, we used tick-borne encephalitis virus (TBEV) as a model for the flaviviruses and developed a stable human cell line in which the expression of NS proteins can be induced without viral RNA replication. The model system described provides a novel and benign tool for studies of the viral components under controlled expression levels. We show that the expression of six NS proteins is sufficient to induce infection-like dilation of the endoplasmic reticulum (ER) and the formation of RC-like membrane invaginations. The NS proteins form a membrane-associated complex in the ER, and electron tomography reveals that the dilated areas of the ER are closely associated with lipid droplets and mitochondria. We propose that the NS proteins drive the remodeling of ER membranes and that viral RNA, RNA replication, viral polymerase, and TBEV structural proteins are not required.IMPORTANCE TBEV infection causes a broad spectrum of symptoms, ranging from mild fever to severe encephalitis. Similar to other flaviviruses, TBEV exploits intracellular membranes to build RCs for viral replication. The viral NS proteins have been suggested to be involved in this process; however, the mechanism of RC formation and the roles of individual NS proteins remain unclear. To study how TBEV induces membrane remodeling, we developed an inducible stable cell system expressing the TBEV NS polyprotein in the absence of viral RNA replication. Using this system, we were able to reproduce RC-like vesicles that resembled the RCs formed in flavivirus-infected cells, in terms of morphology and size. This cell system is a robust tool to facilitate studies of flavivirus RC formation and is an ideal model for the screening of antiviral agents at a lower biosafety level.

Evaluation of antivirals against tick-borne encephalitis virus in organotypic brain slices of rat cerebellum.

Neurotropic tick borne encephalitis virus (TBEV) causes life-threatening disease, and accounts for most cases of tick-transmitted viral infections in Central and Eastern Europe and Russia. No specific treatment for TBEV infections exists, and vaccination is recommended for people at risk. So far, various nucleoside analogues have been investigated in vitro as potential candidates for treatment of TBEV infections. However, in vitro experiments with more complex cell culture systems, such as organotypic culture slices which model the sophisticated architecture of the target tissue are lacking. Using TBEV as a model, we investigated the suitability of rat organotypic cerebellum slices (OCS) to study the effectiveness of nucleoside analogues with a well-known anti-TBEV activity. In these OCS, 50 μM of the nucleoside analogues 2’-C-methyladenosine (2’-CMA) and especially 7-deaza-2’-C-methyladenosine (7-deaza-2’-CMA) exhibited strong inhibitory effects on TBEV replication, reducing viral titers to an average of 103-fold and TBEV RNA content 60-90-fold. In contrast, the influence of 2’-C-methylcytidine (2’-CMC) on TBEV replication was very weak, reducing virus titers by 10-fold and TBEV RNA content by 3-fold. In agreement with other studies, there was no noticeable difference in TBEV titers between OCS treated with 50 μM of Ribavirin and the DMSO treated controls. All tested nucleoside analogues exhibited excellent cytotoxicity profiles at concentrations of 50 μM. Our findings in OCS were highly comparable to data obtained in cell line culture systems. Therefore, OCS represent an ideal in vitro approach to study antivirals against TBEV and possibly other neurotropic viruses.

Erratum for Panayiotou et al., "Viperin Restricts Zika Virus and Tick-Borne Encephalitis Virus Replication by Targeting NS3 for Proteasomal Degradation".

Volume 92, no. 7, e02054-17, 2018, [https://doi.org/10.1128/JVI.02054-17][1]. Page 7: In Fig. 4A, “Input” panels, “NS3” should read “FLAG,” and “FLAG” should read “NS3”. [1]: /lookup/doi/10.1128/JVI.02054-17

Cell-type- and region-specific restriction of neurotropic flavivirus infection by viperin

BackgroundFlaviviruses are a group of diverse and emerging arboviruses and an immense global health problem. A number of flaviviruses are neurotropic, causing severe encephalitis and even death. Type I interferons (IFNs) are the first line of defense of the innate immune system against flavivirus infection. IFNs elicit the concerted action of numerous interferon-stimulated genes (ISGs) to restrict both virus infection and replication. Viperin (virus-inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an ISG with broad-spectrum antiviral activity against multiple flaviviruses in vitro. Its activity in vivo restricts neurotropic infections to specific regions of the central nervous system (CNS). However, the cell types in which viperin activity is required are unknown. Here we have examined both the regional and cell-type specificity of viperin in the defense against infection by several model neurotropic flaviviruses.MethodsViral burden and IFN induction were analyzed in vivo in wild-type and viperin−/− mice infected with Langat virus (LGTV). The effects of IFN pretreatment were tested in vitro in primary neural cultures from different brain regions in response to infection with tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Zika virus (ZIKV).ResultsViperin activity restricted nonlethal LGTV infection in the spleen and the olfactory bulb following infection via a peripheral route. Viperin activity was also necessary to restrict LGTV replication in the olfactory bulb and the cerebrum following CNS infection, but not in the cerebellum. In vitro, viperin could restrict TBEV replication in primary cortical neurons, but not in the cerebellar granule cell neurons. Interferon-induced viperin was also very important in primary cortical neurons to control TBEV, WNV, and ZIKV.ConclusionsOur findings show that viperin restricts replication of neurotropic flaviviruses in the CNS in a region- and cell-type-specific manner. The most important sites of activity are the olfactory bulb and cerebrum. Activity within the cerebrum is required in the cortical neurons in order to restrict spread. This study exemplifies cell type and regional diversity of the IFN response within the CNS and shows the importance of a potent broad-spectrum antiviral ISG.

Viperin Restricts Zika Virus and Tick-Borne Encephalitis Virus Replication by Targeting NS3 for Proteasomal Degradation.

Flaviviruses are arthropod-borne viruses that constitute a major global health problem, with millions of human infections annually. Their pathogenesis ranges from mild illness to severe manifestations such as hemorrhagic fever and fatal encephalitis. Type I interferons (IFNs) are induced in response to viral infection and stimulate the expression of interferon-stimulated genes (ISGs), including that encoding viperin (virus-inhibitory protein, endoplasmic reticulum associated, IFN inducible), which shows antiviral activity against a broad spectrum of viruses, including several flaviviruses. Here we describe a novel antiviral mechanism employed by viperin against two prominent flaviviruses, tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin was found to interact and colocalize with the structural proteins premembrane (prM) and envelope (E) of TBEV, as well as with nonstructural (NS) proteins NS2A, NS2B, and NS3. Interestingly, viperin expression reduced the NS3 protein level, and the stability of the other interacting viral proteins, but only in the presence of NS3. We also found that although viperin interacted with NS3 of mosquito-borne flaviviruses (ZIKV, Japanese encephalitis virus, and yellow fever virus), only ZIKV was sensitive to the antiviral effect of viperin. This sensitivity correlated with viperin's ability to induce proteasome-dependent degradation of NS3. ZIKV and TBEV replication was rescued completely when NS3 was overexpressed, suggesting that the viral NS3 is the specific target of viperin. In summary, we present here a novel antiviral mechanism of viperin that is selective for specific viruses in the genus Flavivirus, affording the possible availability of new drug targets that can be used for therapeutic intervention.IMPORTANCE Flaviviruses are a group of enveloped RNA viruses that cause severe diseases in humans and animals worldwide, but no antiviral treatment is yet available. Viperin, a host protein produced in response to infection, effectively restricts the replication of several flaviviruses, but the exact molecular mechanisms have not been elucidated. Here we have identified a novel mechanism employed by viperin to inhibit the replication of two flaviviruses: tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin induced selective degradation via the proteasome of TBEV and ZIKV nonstructural 3 (NS3) protein, which is involved in several steps of the viral life cycle. Furthermore, viperin also reduced the stability of several other viral proteins in a NS3-dependent manner, suggesting a central role of NS3 in viperin's antiflavivirus activity. Taking the results together, our work shows important similarities and differences among the members of the genus Flavivirus and could lead to the possibility of therapeutic intervention.