Tick-borne encephalitis (TBE) is a severe neuropathological disorder caused by tick-borne encephalitis virus (TBEV). Brain TBEV infection is characterized by extensive pathological neuroinflammation. ...The mechanism by which TBEV causes CNS destruction remains unclear, but growing evidence suggests that it involves both direct neuronal damage by the virus infection and indirect damage caused by the immune response. Here, we aimed to examine the TBEV-infection-induced innate immune response in mice and in human neural cells. We also compared cytokine/chemokine communication between naïve and infected neuronal cells and astrocytes.
We used a multiplexed Luminex system to measure multiple cytokines/chemokines and growth factors in mouse serum samples and brain tissue, and in human neuroblastoma cells (SK-N-SH) and primary cortical astrocytes (HBCA), which were infected with the highly pathogenic TBEV strain Hypr. We also investigated changes in cytokine/chemokine production in naïve HBCA cells treated with virus-free supernatants from TBEV-infected SK-N-SH cells and in naïve SK-N-SH cells treated with virus-free supernatants from TBEV-infected HBCA cells. Additionally, a plaque assay was performed to assess how cytokine/chemokine treatment influenced viral growth following TBEV infection.
TBEV-infected mice exhibited time-dependent increases in serum and brain tissue concentrations of multiple cytokines/chemokines (mainly CXCL10/IP-10, and also CXCL1, G-CSF, IL-6, and others). TBEV-infected SK-N-SH cells exhibited increased production of IL-8 and RANTES and downregulated MCP-1 and HGF. TBEV infection of HBCA cells activated production of a broad spectrum of pro-inflammatory cytokines, chemokines, and growth factors (mainly IL-6, IL-8, CXCL10, RANTES, and G-CSF) and downregulated the expression of VEGF. Treatment of SK-N-SH with supernatants from infected HBCA induced expression of a variety of chemokines and pro-inflammatory cytokines, reduced SK-N-SH mortality after TBEV infection, and decreased virus growth in these cells. Treatment of HBCA with supernatants from infected SK-N-SH had little effect on cytokine/chemokine/growth factor expression but reduced TBEV growth in these cells after infection.
Our results indicated that both neurons and astrocytes are potential sources of pro-inflammatory cytokines in TBEV-infected brain tissue. Infected/activated astrocytes produce cytokines/chemokines that stimulate the innate neuronal immune response, limiting virus replication, and increasing survival of infected neurons.
Tick-borne encephalitis virus (TBEV) targets the central nervous system (CNS), leading to potentially severe neurological complications. The neurovascular unit plays a fundamental role in the CNS and ...in the neuroinvasion of TBEV. However, the role of human brain pericytes, a key component of the neurovascular unit, during TBEV infection has not yet been elucidated. In this study, TBEV infection of the primary human brain perivascular pericytes was investigated with highly virulent Hypr strain and mildly virulent Neudoerfl strain. We used Luminex assay to measure cytokines/chemokines and growth factors. Both viral strains showed comparable replication kinetics, peaking at 3 days post infection (dpi). Intracellular viral RNA copies peaked at 6 dpi for Hypr and 3 dpi for Neudoerfl cultures. According to immunofluorescence staining, only small proportion of pericytes were infected (3% for Hypr and 2% for Neudoerfl), and no cytopathic effect was observed in the infected cells. In cell culture supernatants, IL-6 production was detected at 3 dpi, together with slight increases in IL-15 and IL-4, but IP-10, RANTES and MCP-1 were the main chemokines released after TBEV infection. These chemokines play key roles in both immune defense and immunopathology during TBE. This study suggests that pericytes are an important source of these signaling molecules during TBEV infection in the brain.
The recent Zika virus (ZIKV) outbreak has linked ZIKV with microcephaly and other central nervous system pathologies in humans. Astrocytes are among the first cells to respond to ZIKV infection in ...the brain and are also targets for virus infection. In this study, we investigated the interaction between ZIKV and primary human brain cortical astrocytes (HBCA).
HBCAs were highly sensitive to representatives of both Asian and African ZIKV lineages and produced high viral yields. The infection was associated with limited immune cytokine/chemokine response activation; the highest increase of expression, following infection, was seen in CXCL-10 (IP-10), interleukin-6, 8, 12, and CCL5 (RANTES). Ultrastructural changes in the ZIKV-infected HBCA were characterized by electron tomography (ET). ET reconstructions elucidated high-resolution 3D images of the proliferating and extensively rearranged endoplasmic reticulum (ER) containing viral particles and virus-induced vesicles, tightly juxtaposed to collapsed ER cisternae.
The results confirm that human astrocytes are sensitive to ZIKV infection and could be a source of proinflammatory cytokines in the ZIKV-infected brain tissue.
Tick-borne encephalitis virus (TBEV) causes serious, potentially fatal neurological infections that affect humans in endemic regions of Europe and Asia. Neurons are the primary target for TBEV ...infection in the central nervous system. However, knowledge about this viral infection and virus-induced neuronal injury is fragmental. Here, we directly examined the pathology that occurs after TBEV infection in human primary neurons. We exploited the advantages of advanced high-pressure freezing and freeze-substitution techniques to achieve optimal preservation of infected cell architecture. Electron tomographic (ET) reconstructions elucidated high-resolution 3D images of the proliferating endoplasmic reticulum, and individual tubule-like structures of different diameters in the endoplasmic reticulum cisternae of single cells. ET revealed direct connections between the tubule-like structures and viral particles in the endoplasmic reticulum. Furthermore, ET showed connections between cellular microtubules and vacuoles that harbored the TBEV virions in neuronal extensions. This study was the first to characterize the 3D topographical organization of membranous whorls and autophagic vacuoles in TBEV-infected human neurons. The functional importance of autophagy during TBEV replication was studied in human neuroblastoma cells; stimulation of autophagy resulted in significantly increased dose-dependent TBEV production, whereas the inhibition of autophagy showed a profound, dose-dependent decrease of the yield of infectious virus.
•A new veterinary tick-borne encephalitis virus (TBEV) vaccine candidate was developed.•Vaccination elicited the production of high levels of virus-neutralizing antibodies.•Vaccination provided full ...protection against lethal TBE in mice.•Vaccination prevented development of TBEV viremia in sheep.•Vaccination prevented presence of TBEV in milk of lactating ewes.
In tick-borne encephalitis (TBE) endemic areas, consumption of unpasteurized milk or milk products from grazing domestic ruminants (goats, cattle, and sheep) represents a risk of TBE virus (TBEV) infection for humans. In addition to vaccination of humans, human alimentary TBEV infections can be avoided by pasteurizing milk or by vaccination of the ruminants. However, there is presently no TBEV vaccine for veterinary use. Here, we developed a new veterinary TBE vaccine candidate based on cell culture-derived, purified, and formaldehyde-inactivated TBEV (strain Hypr). The safety and immunogenicity of the vaccine was evaluated in mice and sheep and was well-tolerated while eliciting the production of high levels of virus-neutralizing antibodies. Vaccination provided full protection against lethal TBE in mice, prevented development of viremia in sheep and presence of TBEV in milk of lactating ewes. This vaccine is a good candidate for immunization of ruminants to prevent alimentary milk-borne TBEV infections in humans.
Tick-borne encephalitis virus (TBEV) is a leading cause of human neuroinfections in Europe and Northeast Asia. There are no antiviral therapies for treating TBEV infection. A series of nucleoside ...analogues was tested for the ability to inhibit the replication of TBEV in porcine kidney cells and human neuroblastoma cells. The interactions of three nucleoside analogues with viral polymerase were simulated using advanced computational methods. The nucleoside analogues 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), 2'-C-methyladenosine (2'-CMA), and 2'-C-methylcytidine (2'-CMC) inhibited TBEV replication. These compounds showed dose-dependent inhibition of TBEV-induced cytopathic effects, TBEV replication (50% effective concentrations EC50of 5.1 ± 0.4 μM for 7-deaza-2'-CMA, 7.1 ± 1.2 μM for 2'-CMA, and 14.2 ± 1.9 μM for 2'-CMC) and viral antigen production. Notably, 2'-CMC was relatively cytotoxic to porcine kidney cells (50% cytotoxic concentration CC50 of ∼50 μM). The anti-TBEV effect of 2'-CMA in cell culture diminished gradually after day 3 posttreatment. 7-Deaza-2'-CMA showed no detectable cellular toxicity (CC50 > 50 μM), and the antiviral effect in culture was stable for >6 days posttreatment. Computational molecular analyses revealed that compared to the other two compounds, 7-deaza-2'-CMA formed a large cluster near the active site of the TBEV polymerase. High antiviral activity and low cytotoxicity suggest that 7-deaza-2'-CMA is a promising candidate for further investigation as a potential therapeutic agent in treating TBEV infection.
Up to 170 tick-borne viruses (TBVs) have been identified to date. However, there is a paucity of information regarding TBVs and their interaction with respective vectors, limiting the development of ...new effective and urgently needed control methods. To overcome this gap of knowledge, it is essential to reproduce transmission cycles under controlled laboratory conditions. In this study we assessed an artificial feeding system (AFS) and an immersion technique (IT) to infect Ixodes ricinus ticks with tick-borne encephalitis (TBE) and Kemerovo (KEM) virus, both known to be transmitted predominantly by ixodid ticks. Both methods permitted TBEV acquisition by ticks and we further confirmed virus trans-stadial transmission and onward transmission to a vertebrate host. However, only artificial feeding system allowed to demonstrate both acquisition by ticks and trans-stadial transmission for KEMV. Yet we did not observe transmission of KEMV to mice (IFNAR
or BALB/c). Artificial infection methods of ticks are important tools to study tick-virus interactions. When optimally used under laboratory settings, they provide important insights into tick-borne virus transmission cycles.
Tick-borne encephalitis virus (TBEV) is a flavivirus that causes human neuroinfections and represents a growing health problem. The human monoclonal antibody T025 targets envelope protein domain III ...(EDIII) of TBEV and related tick-borne flaviviruses, potently neutralizing TBEV in vitro and in preclinical models, representing a promising candidate for clinical development. We demonstrate that TBEV escape in the presence of T025 or T028 (another EDIII-targeting human monoclonal antibody) results in virus variants of reduced pathogenicity, characterized by distinct sets of amino acid changes in EDII and EDIII that are jointly needed to confer resistance. EDIII substitution K311N impairs formation of a salt bridge critical for T025-epitope interaction. EDII substitution E230K is not on the T025 epitope but likely induces quaternary rearrangements of the virus surface because of repulsion of positively charged residues on the adjacent EDI. A combination of T025 and T028 prevents virus escape and improves neutralization.
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•TBEV escape from T025 or T028 results in virus variants with reduced pathogenicity•Escape mutants display a combination of amino acid changes essential for resistance•Mutations within and distal to the antibody epitope can synergize for the resistance•A combination of T025 and T028 prevents virus escape and improves neutralization
Svoboda et al. investigate tick-borne encephalitis virus escape from two potent human monoclonal antibodies. The mutants, resistant to individual antibodies, have distinct sets of amino acid changes that are jointly needed to confer full resistance. A combination of the two monoclonal antibodies effectively prevents virus escape.
The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying ...TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the mCherry reporter protein. The resulting TBEV reporter virus (named mCherry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the mCherry-TBEV virus produced smaller plaques. The magnitude of mCherry expression correlated well with progeny virus production but remained stable over <4 passages in cell culture. Using well-characterized antiviral compounds known to inhibit TBEV, 2′-C-methyladenosine and 2′-deoxy-2′-β-hydroxy-4′-azidocytidine (RO-9187), we demonstrated that mCherry-TBEV is suitable for high-throughput screening of antiviral drugs. Serum samples from a TBEV-vaccinated human and a TBEV-infected dog were used to evaluate the mCherry-based neutralization test. Collectively, recombinant mCherry-TBEV reporter virus described here provides a powerful tool to facilitate the identification of potential antiviral agents, and to measure levels of neutralizing antibodies in human and animal sera.
•The mCherry-TBEV reporter virus was established.•mCherry-TBEV and wild-type TBEV exhibit similar growth kinetics in cultured cells.•mCherry-TBEV is suitable for high-throughput screening of antiviral drugs.•mCherry-TBEV can be used to measure levels of neutralizing antibodies in human and animal sera.
The tick-borne encephalitis virus (TBEV) causes a most important viral life-threatening illness transmitted by ticks. The interactions between the virus and ticks are largely unexplored, indicating a ...lack of experimental tools and systematic studies. One such tool is recombinant reporter TBEV, offering antibody-free visualization to facilitate studies of transmission and interactions between a tick vector and a virus. In this paper, we utilized a recently developed recombinant TBEV expressing the reporter gene mCherry to study its fitness in various tick-derived
cell cultures and live unfed nymphal
ticks. The reporter virus was successfully replicated in tick cell lines and live ticks as confirmed by the plaque assay and the mCherry-specific polymerase chain reaction (PCR). Although a strong mCherry signal determined by fluorescence microscopy was detected in several tick cell lines, the fluorescence of the reporter was not observed in the live ticks, corroborated also by immunoblotting. Our data indicate that the mCherry reporter TBEV might be an excellent tool for studying TBEV-tick interactions using a tick
model. However, physiological attributes of a live tick, likely contributing to the inactivity of the reporter, warrant further development of reporter-tagged viruses to study TBEV in ticks
.
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