- Volume 1, Issue 10, 2019

Background: Despite the worldwide re-emergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. Here, we identified a novel class of CHIKV inhibitors i.e. the CHVB series.

Methods: CPE-reduction and virus yield assays were performed in Vero cells. Drug-resistant variants were selected using clonal resistance selection. The enzymatic assays for alphavirus capping were done using the non-structural protein 1 (nsP1) of Semliki Forest virus (SFV) and Venezuelan equine encephalitis (VEEV).

Results: CHVB compounds inhibited the in vitro replication of CHIKV isolates with EC50 values in the low μM range. In virus yield assays, the most potent analogues reduced the viral load with 4-5 log10. CHVB-resistant variants were selected and found to carry (i) two mutations in the gene encoding nsP1 (responsible for viral RNA capping), (ii) one mutation in nsP2 and (iii) one mutation in nsP3. Reverse-engineering suggested that nsP1 is the target of CHVB, since both nsP1 mutations were needed to achieve 10-fold resistance. Interestingly, the CHVBres virus proved cross-resistant to the MADTP series, a class of CHIKV capping inhibitors that we described earlier, suggesting a similar mode of action. In enzymatic assays, CHVB proved a potent inhibitor of the methyltransferase and guanylyltransferase activities of nsP1 of SFV and VEEV.

Conclusion: We identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity and the high barrier to resistance make this chemical scaffold a potential candidate for CHIKV drug development.

Aedes albopictus is an aggressive invasive species and a competent vector for over 20 arboviruses, including Chikungunya, Dengue and Zika viruses. Understanding the molecular and cellular interactions between viruses and vectors is key to implement transmission-blocking strategies to prevent viral outbreaks. However, the mechanisms that shape vector competence are poorly understood. Recent evidence reveals that the genomes of Aedes spp. harbour fragmented viral sequences which produce PIWI-interacting RNAs (piRNAs), suggesting a role in vector competence. Current knowledge of the piRNA pathway in Ae. albopictus is limited, and its possible role in the establishment of persistent infections widely unknown.

We combined cutting-edge bioinformatic analyses based on next-generation sequencing data with molecular biology and virology techniques to characterise the main genes of the piRNA pathway in this mosquito species, assess their polymorphisms and analyse their expression throughout mosquito development and following infection with the Chikungunya and Dengue-1 viruses.

We identified seven piwi genes which displayed high levels of polymorphism across populations and signs of adaptive evolution. Superposition of protein homology models indicate high structure similarity among all Piwi proteins, with high levels of amino acid conservation in the inner regions devoted to RNA binding. On the contrary, solvent-exposed surfaces showed low conservation, with sites under positive selection. Infection experiments indicated specific responses depending on viral species, time of infection and mosquito tissue, highlighting distinct roles for specific Piwi proteins.

In conclusion, this work helps define the role of the piRNA pathway in persistent arboviral infections and understand the evolutionary divergence among piwi proteins.

Aedes albopictus is an aggressive invasive species and a competent vector for over 20 arboviruses, including Chikungunya, Dengue and Zika viruses. Understanding the molecular and cellular interactions between viruses and vectors is key to implement transmission-blocking strategies to prevent viral outbreaks. However, the mechanisms that shape vector competence are poorly understood. Recent evidence reveals that the genomes of Aedes spp. harbour fragmented viral sequences which produce PIWI-interacting RNAs (piRNAs), suggesting a role in vector competence. Current knowledge of the piRNA pathway in Ae. albopictus is limited, and its possible role in the establishment of persistent infections widely unknown.

We combined cutting-edge bioinformatic analyses based on next-generation sequencing data with molecular biology and virology techniques to characterise the main genes of the piRNA pathway in this mosquito species, assess their polymorphisms and analyse their expression throughout mosquito development and following infection with the Chikungunya and Dengue-1 viruses.

We identified seven piwi genes which displayed high levels of polymorphism across populations and signs of adaptive evolution. Superposition of protein homology models indicate high structure similarity among all Piwi proteins, with high levels of amino acid conservation in the inner regions devoted to RNA binding. On the contrary, solvent-exposed surfaces showed low conservation, with sites under positive selection. Infection experiments indicated specific responses depending on viral species, time of infection and mosquito tissue, highlighting distinct roles for specific Piwi proteins.

In conclusion, this work helps define the role of the piRNA pathway in persistent arboviral infections and understand the evolutionary divergence among piwi proteins.

The genomes of Aedes spp. mosquitoes contain integrated sequences from nonretroviral RNA viruses that are enriched in piRNA clusters, are embedded next to transposable elements (TEs) and produce piRNAs. The parallelism between TEs and viral integrations led to the hypothesis that viral integrations may constitute an archive of past viral infections and potentially have an immunity impact on novel infection with cognate viruses, similarly to how the piRNA pathway interacts with TEs. A corollary of this hypothesis is that the landscape of viral integrations should be variable across populations depending on their viral exposure. The highly repetitive nature of Aedes spp. genomes make the discovery of viral integrations from whole genome sequencing data of wild mosquitoes a daunting task.

Here we describe a novel bioinformatic pipeline to rigorously identify viral integrations using Next Generation Sequencing (NGS) data. Libraries from single or pools of mosquitoes, reference genome statistics, the landscape of TEs and the geographic origin of the analyzed samples are the actors of the analysis.

This pipeline has been tested in Ae. aegypti and Ae. albopictus mosquitoes, allowing to compare the performance of the analyses on genome assemblies of different completeness. We identified novel viral integrations in both genomes. Additionally, we show that the landscape of viral integrations is dynamic, with a population-specific behavior that we can leverage to formulate hypothesis on mechanisms of integration and the biological role of viral integrations.

Powassan virus (POWV) is the only tick-borne flavivirus (TBFV) known to circulate in North America. Although there are relatively few documented cases of POWV disease, investigations into POWV are justified due to the increasing incidence of infection and significant case fatality rate associated with this virus. To better describe the molecular biology of the POWV replication cycle in mammalian cells, we performed genome-scale screens to uncover host factors required for viral replication. Putative proviral host factors were identified by infecting pools of cells containing knockout mutations in non-essential genes with POWV, followed by analysis of cells resistant to virus-induced cell death. Many endoplasmic reticulum membrane complex proteins were revealed in these screens, suggesting that TBFVs share some common host cell hijacking strategies with mosquito-borne flaviviruses. Candidate proteins that function in cell-matrix adhesion, glycosylation, or RNA binding in uninfected cells were the focus of validation studies. We used single-gene knockout cell lines to investigate possible proviral roles for specific proteins in the replication cycle of either POWV or Langat virus (LGTV), a non-pathogenic and model TBFV. Proteins identified in the POWV screens were not necessarily critical for LGTV replication. These results suggest that our screens were able to identify both pan-flaviviral, as well as POWV-specific, host gene products exploited during virus replication. Ongoing work is focused on characterizing distinct host-cell requirements of diverse flaviviruses. This genetic assessment of POWV replication factors, in combination with ensuing mechanistic studies, will provide possible avenues for the development of host-targeting countermeasures.

Powassan virus (POWV) is the only tick-borne flavivirus (TBFV) known to circulate in North America. Although there are relatively few documented cases of POWV disease, investigations into POWV are justified due to the increasing incidence of infection and significant case fatality rate associated with this virus. To better describe the molecular biology of the POWV replication cycle in mammalian cells, we performed genome-scale screens to uncover host factors required for viral replication. Putative proviral host factors were identified by infecting pools of cells containing knockout mutations in non-essential genes with POWV, followed by analysis of cells resistant to virus-induced cell death. Many endoplasmic reticulum membrane complex proteins were revealed in these screens, suggesting that TBFVs share some common host cell hijacking strategies with mosquito-borne flaviviruses. Candidate proteins that function in cell-matrix adhesion, glycosylation, or RNA binding in uninfected cells were the focus of validation studies. We used single-gene knockout cell lines to investigate possible proviral roles for specific proteins in the replication cycle of either POWV or Langat virus (LGTV), a non-pathogenic and model TBFV. Proteins identified in the POWV screens were not necessarily critical for LGTV replication. These results suggest that our screens were able to identify both pan-flaviviral, as well as POWV-specific, host gene products exploited during virus replication. Ongoing work is focused on characterizing distinct host-cell requirements of diverse flaviviruses. This genetic assessment of POWV replication factors, in combination with ensuing mechanistic studies, will provide possible avenues for the development of host-targeting countermeasures.

To be successfully transmitted to another susceptible human host by Aedes aegypti, dengue virus (DENV) must first successfully infect the mosquito midgut. The virus-host interactions that enable successful midgut infection, however, is not well understood. To understand the important interactions for successful midgut infection, we took advantage of the wild-type DENV2 16681 and its attenuated derivative PDK53, which has been shown to be refractory in mosquito infection. Using oral infectious-blood feeding, we observed that PDK53 failed to produce infectious progenies in the midgut, despite detectable viral genome replication. Furthermore, we found that the foci of PDK53 infection in the midgut, detected by immunofluorescence staining, was limited in both size and number, compared to its parent, 16681. Transcriptional analysis of the mosquito midgut revealed increased expression of genes in multiple innate immune pathways upon PDK53 infection but not 16681. To pinpoint the mutation responsible for this phenotype, we constructed an infectious clone of 16681 and used site-directed mutagenesis to substitute each of the known mutations in PDK53 into the 16681 genomic backbone. This approach pinpointed the NS1 G53D mutation as the single most important attenuating mutation in PDK53 in engendering refractoriness to mosquito midgut infection. Mechanistically, our data also suggests that this mutation affected the virus-ER-resident protein interactions that impacted the efficiency of DENV replication and hence induction of the innate immune response. Our findings reveal insights into the pathogenesis of dengue and adds to the body of knowledge on critical virushost interactions that govern epidemiological fitness of DENV.

To be successfully transmitted to another susceptible human host by Aedes aegypti, dengue virus (DENV) must first successfully infect the mosquito midgut. The virus-host interactions that enable successful midgut infection, however, is not well understood. To understand the important interactions for successful midgut infection, we took advantage of the wild-type DENV2 16681 and its attenuated derivative PDK53, which has been shown to be refractory in mosquito infection. Using oral infectious-blood feeding, we observed that PDK53 failed to produce infectious progenies in the midgut, despite detectable viral genome replication. Furthermore, we found that the foci of PDK53 infection in the midgut, detected by immunofluorescence staining, was limited in both size and number, compared to its parent, 16681. Transcriptional analysis of the mosquito midgut revealed increased expression of genes in multiple innate immune pathways upon PDK53 infection but not 16681. To pinpoint the mutation responsible for this phenotype, we constructed an infectious clone of 16681 and used site-directed mutagenesis to substitute each of the known mutations in PDK53 into the 16681 genomic backbone. This approach pinpointed the NS1 G53D mutation as the single most important attenuating mutation in PDK53 in engendering refractoriness to mosquito midgut infection. Mechanistically, our data also suggests that this mutation affected the virus-ER-resident protein interactions that impacted the efficiency of DENV replication and hence induction of the innate immune response. Our findings reveal insights into the pathogenesis of dengue and adds to the body of knowledge on critical virushost interactions that govern epidemiological fitness of DENV.

To combat DENV transmission, we aim to screen for molecules that target mosquito host cell factors playing a role in the viral replication, consequently inhibiting the replication of virus in the mosquito. Use case scenarios for such compounds include human mass drug administration in endemic populations, application in sugar baits or bed-nets.

In order to accomplish this, we have set up high throughput screening (HTS) using viral replication inhibition assays. We describe here the generation of a novel stable Nanoluciferase-reporter based dengue replicon system in U4.4 mosquito cells. The U4.4-DENV1 replicon cell line has been stably and successfully maintained for over 30 passages without significant loss of reporter signal. In order to characterize the cell line further, the cells were subjected to treatment with antiviral compound and viral inhibition was observed with ribavirin (IC50 =1.69 × 10-6M) and siRNA against NS3. For the purpose of HTS, viral inhibition, cytotoxicity and luciferase interference assays were established on a 384-well plate using the cryopreserved U4.4_DENV1 replicon cells. The latter two were used as deselection assays to differentiate between false- and true- positives. To conclude, we have developed a robust screening cascade to identify small molecules that may act as transmission blocking compounds. Screening of a chemical diversity library is ongoing and the results will be presented at the meeting.

To combat DENV transmission, we aim to screen for molecules that target mosquito host cell factors playing a role in the viral replication, consequently inhibiting the replication of virus in the mosquito. Use case scenarios for such compounds include human mass drug administration in endemic populations, application in sugar baits or bed-nets.

In order to accomplish this, we have set up high throughput screening (HTS) using viral replication inhibition assays. We describe here the generation of a novel stable Nanoluciferase-reporter based dengue replicon system in U4.4 mosquito cells. The U4.4-DENV1 replicon cell line has been stably and successfully maintained for over 30 passages without significant loss of reporter signal. In order to characterize the cell line further, the cells were subjected to treatment with antiviral compound and viral inhibition was observed with ribavirin (IC50 =1.69 × 10-6M) and siRNA against NS3. For the purpose of HTS, viral inhibition, cytotoxicity and luciferase interference assays were established on a 384-well plate using the cryopreserved U4.4_DENV1 replicon cells. The latter two were used as deselection assays to differentiate between false- and true- positives. To conclude, we have developed a robust screening cascade to identify small molecules that may act as transmission blocking compounds. Screening of a chemical diversity library is ongoing and the results will be presented at the meeting.

The National Collection of Pathogenic Viruses (NCPV) is one of four repositories making up Public Health England Culture Collections. It was established two decades ago to offer safe and secure facilities for storage of patents and deposits as well as to supply standardised virus products to the wider scientific community. Within the collection are strains of both medical and veterinary importance, with representation from a wide variety of virus families, classified as Hazard Groups 2, 3 and 4 by the Advisory Committee for Dangerous Pathogens (ACDP). These include arboviruses such as Dengue, Yellow-fever, Oropouche and Sandfly fever Naples.

Throughout the year, the collection receives virus deposits. The depositing process is free and simple. The quality of every virus material received in the collection is assessed through a series of tests to confirm viability and absence of microbial contamination (including mycoplasmas). Virus identity is confirmed by nucleic acid profiling. Once standards are met, the virus products are made available to the wider scientific community. In addition to facilitating access to viruses and their associated products as well as secure storage for patents, NCPV also undertakes collaborative projects and contract research/developmental work with academic and commercial partners, by providing expertise in the handling of viral pathogens.

A repository for well-characterised, authenticated viruses is a useful resource for the scientific community, aiding in the understanding of circulating viral pathogens and providing access to quality-assured reference reagents for the identification and development of vaccines and therapeutics in the event of global viral outbreaks.

The National Collection of Pathogenic Viruses (NCPV) is one of four repositories making up Public Health England Culture Collections. It was established two decades ago to offer safe and secure facilities for storage of patents and deposits as well as to supply standardised virus products to the wider scientific community. Within the collection are strains of both medical and veterinary importance, with representation from a wide variety of virus families, classified as Hazard Groups 2, 3 and 4 by the Advisory Committee for Dangerous Pathogens (ACDP). These include arboviruses such as Dengue, Yellow-fever, Oropouche and Sandfly fever Naples.

Throughout the year, the collection receives virus deposits. The depositing process is free and simple. The quality of every virus material received in the collection is assessed through a series of tests to confirm viability and absence of microbial contamination (including mycoplasmas). Virus identity is confirmed by nucleic acid profiling. Once standards are met, the virus products are made available to the wider scientific community. In addition to facilitating access to viruses and their associated products as well as secure storage for patents, NCPV also undertakes collaborative projects and contract research/developmental work with academic and commercial partners, by providing expertise in the handling of viral pathogens.

A repository for well-characterised, authenticated viruses is a useful resource for the scientific community, aiding in the understanding of circulating viral pathogens and providing access to quality-assured reference reagents for the identification and development of vaccines and therapeutics in the event of global viral outbreaks.

Deciphering how dengue pathogenesis influences vector transmission will improve our understanding on virulence evolution, epidemiology and design of transmission-blocking tools. Here, we demonstrate that addition to blood meal of plasmin, the human fibrinolytic factor, increased permeability and DENV infectivity in mosquito midgut, resulting in higher infection rate and dissemination in whole mosquitoes. Further, we show that a plasmin-selective mosquito Kazal-type protease inhibitor, AaTI reverted this enhanced infection by inhibiting proteolysis. We also determined that DENV or E-protein, plasmin (not plasminogen) and AaTI can interact to form a tripartite complex using biolayer interferometry, suggesting physical interaction between DENV E-protein and kringle domain of plasmin. Our study suggests that (a) DENV recruit plasmin in solution to increase local proteolytic activity in midgut, thus enhancing DENV infection and (b) AaTI can act as a transmission-blocking agent, which could also alleviate hemorrhagic patients. By discovering that dengue pathogenesis can enhance DENV fitness by increasing mosquito infectivity, our results provide the first evidence of mosquito-based evolutionary pressures on dengue virulence in human. We are currently determining the effect of plasmin and AaTI on DENV infection in cellular and mouse models. H/D exchange mass spectrometry is being employed to identify which kringle domain of plasmin is interacting with DENV E-protein. The structure of the complex formed between the kringle domain of plasmin and DENV E-protein and between the catalytic domain of plasmin and AaTI are in progress.

Deciphering how dengue pathogenesis influences vector transmission will improve our understanding on virulence evolution, epidemiology and design of transmission-blocking tools. Here, we demonstrate that addition to blood meal of plasmin, the human fibrinolytic factor, increased permeability and DENV infectivity in mosquito midgut, resulting in higher infection rate and dissemination in whole mosquitoes. Further, we show that a plasmin-selective mosquito Kazal-type protease inhibitor, AaTI reverted this enhanced infection by inhibiting proteolysis. We also determined that DENV or E-protein, plasmin (not plasminogen) and AaTI can interact to form a tripartite complex using biolayer interferometry, suggesting physical interaction between DENV E-protein and kringle domain of plasmin. Our study suggests that (a) DENV recruit plasmin in solution to increase local proteolytic activity in midgut, thus enhancing DENV infection and (b) AaTI can act as a transmission-blocking agent, which could also alleviate hemorrhagic patients. By discovering that dengue pathogenesis can enhance DENV fitness by increasing mosquito infectivity, our results provide the first evidence of mosquito-based evolutionary pressures on dengue virulence in human. We are currently determining the effect of plasmin and AaTI on DENV infection in cellular and mouse models. H/D exchange mass spectrometry is being employed to identify which kringle domain of plasmin is interacting with DENV E-protein. The structure of the complex formed between the kringle domain of plasmin and DENV E-protein and between the catalytic domain of plasmin and AaTI are in progress.

Zika virus (ZIKV) is a member of the Flaviviridae family and was until recently a relatively obscure tropical disease. Subsequently, ZIKV has been shown to be the causative agent of fetal abnormalities and Guillain-Barré syndrome in outbreaks across the Americas and so efforts towards delineating important factors in the viral lifecycle have increased. Combining protein pull-down with mass spectrometry, it was found that ZIKV envelope (Env) interacts with the endoplasmic reticulum (ER) resident chaperone, glucose regulated protein 78 (GRP78) in A549 cells. Flaviviruses such as Japanese encephalitis virus and dengue virus are known to co-opt ER resident proteins and members of the unfolded protein response, including GRP78, to enhance viral infectivity and propagation. The role these proteins play during the ZIKV lifecycle has yet to be elucidated.

To determine the importance of this interaction during ZIKV infection, A549 cells were treated with GRP78-specific siRNAs prior to infection with a NanoLuc expressing reporter virus or a wild-type virus. Depletion of GRP78 significantly reduced both virus luciferase readings and viral titres, indicating that GRP78 is necessary for efficient infection of mammalian cell culture. In contrast, inhibition of GRP78 with small molecule inhibitors did not reduce ZIKV infection. Interestingly, immunofluorescence of ZIKV infected cells reveal that GRP78 re-localises following infection and co-localises with Env. Depletion of GRP78 abrogated localisation of viral replication factories. Further experiments have shown that GRP78 is important for infection post entry and replication, and that putative GRP78 interactions partners are also required during infection.

Zika virus (ZIKV) is a member of the Flaviviridae family and was until recently a relatively obscure tropical disease. Subsequently, ZIKV has been shown to be the causative agent of fetal abnormalities and Guillain-Barré syndrome in outbreaks across the Americas and so efforts towards delineating important factors in the viral lifecycle have increased. Combining protein pull-down with mass spectrometry, it was found that ZIKV envelope (Env) interacts with the endoplasmic reticulum (ER) resident chaperone, glucose regulated protein 78 (GRP78) in A549 cells. Flaviviruses such as Japanese encephalitis virus and dengue virus are known to co-opt ER resident proteins and members of the unfolded protein response, including GRP78, to enhance viral infectivity and propagation. The role these proteins play during the ZIKV lifecycle has yet to be elucidated.

To determine the importance of this interaction during ZIKV infection, A549 cells were treated with GRP78-specific siRNAs prior to infection with a NanoLuc expressing reporter virus or a wild-type virus. Depletion of GRP78 significantly reduced both virus luciferase readings and viral titres, indicating that GRP78 is necessary for efficient infection of mammalian cell culture. In contrast, inhibition of GRP78 with small molecule inhibitors did not reduce ZIKV infection. Interestingly, immunofluorescence of ZIKV infected cells reveal that GRP78 re-localises following infection and co-localises with Env. Depletion of GRP78 abrogated localisation of viral replication factories. Further experiments have shown that GRP78 is important for infection post entry and replication, and that putative GRP78 interactions partners are also required during infection.

Mosquito-transmitted alphaviruses are distributed globally and include human pathogens that can cause severe long-term arthritogenic or neurological complications. There are currently no market-approved antivirals or vaccines to treat or prevent these infections. Outbreaks are difficult to predict as they can emerge spontaneously in susceptible human populations. Furthermore, it is challenging to determine the causative pathogen due to great similarities in clinical features of alphavirus-associated diseases. Therefore, drugs with broad-spectrum anti-alphavirus activity could serve as a fast first-line therapy in case of an outbreak. We have performed high-throughput screenings of broad-chemical space libraries and identified a series of small molecules with antiviral activity against different chikungunya virus lineages. Interestingly, we demonstrated that this series exerts broad-spectrum anti-alphavirus activity against a range of arthritogenic alphaviruses. Time-of-addition studies showed that this series has in vitro antiviral activity early in the viral RNA replication stage. Furthermore, these molecules were not cross-resistant with favipiravir-resistant chikungunya virus, a compound that inhibits the viral RNA polymerase. To investigate the mechanism of action in more detail, in vitro resistance selection is currently ongoing. Elucidation of the specific antiviral target of this newly identified series could reveal a new target in the alphavirus proteome, which could be valuable for the development of broad-spectrum anti-alphavirus drugs.

Mosquito-transmitted alphaviruses are distributed globally and include human pathogens that can cause severe long-term arthritogenic or neurological complications. There are currently no market-approved antivirals or vaccines to treat or prevent these infections. Outbreaks are difficult to predict as they can emerge spontaneously in susceptible human populations. Furthermore, it is challenging to determine the causative pathogen due to great similarities in clinical features of alphavirus-associated diseases. Therefore, drugs with broad-spectrum anti-alphavirus activity could serve as a fast first-line therapy in case of an outbreak. We have performed high-throughput screenings of broad-chemical space libraries and identified a series of small molecules with antiviral activity against different chikungunya virus lineages. Interestingly, we demonstrated that this series exerts broad-spectrum anti-alphavirus activity against a range of arthritogenic alphaviruses. Time-of-addition studies showed that this series has in vitro antiviral activity early in the viral RNA replication stage. Furthermore, these molecules were not cross-resistant with favipiravir-resistant chikungunya virus, a compound that inhibits the viral RNA polymerase. To investigate the mechanism of action in more detail, in vitro resistance selection is currently ongoing. Elucidation of the specific antiviral target of this newly identified series could reveal a new target in the alphavirus proteome, which could be valuable for the development of broad-spectrum anti-alphavirus drugs.

Background. Ticks have developed defense mechanisms and pathways against transmitted infections, including tick-borne encephalitis virus (TBEV). An important issue is to reveal mechanisms allowing them to control the virus at a level which does not hinder ticks’ fitness and development.

Methods. Biotyping was performed on an Autoflex Speed MALDI-TOF/TOF (Bruker Daltonik). Protein digests were analyzed using Synapt G2-Si High Definition mass spectrometer (Waters).

Results. MS profiles of TBEV-infected and non-infected IRE/CTVM19 cells were analyzed using principal component analysis. Obtained spectra were clustered based on the cultivation time, but not the infection status. Nevertheless, analysis of loading plots revealed different factors to be important for clustering of infected and non-infected cells. Out of them, nine were assigned with proteins: five and four for non-infected and infected cells, respectively. Peak with m/z 8565 was found to be of interest from viewpoint of tick-virus interaction and assigned to proteasome subunit alpha type (B7QE67).

Conclusion. MALDI-TOF MS was shown to be useful for characterization of tick cell lines and studying tick-virus interactions. Signals in MS profiles discriminating cell aging and those affected by TBEV were revealed, and matched with proteins.

We thank Dr Lesley Bell-Sakyi and the Tick Cell Biobank for provision of IRE/CTVM19 cells.

This study was supported by the MŠMT ČR INTER-ACTION project (LTARF 18021); GAČR (18-27204S), European Regional Development Fund Project (CZ.02.1.01/0.0/0.0/15_003/0000441), and MSHE RF (#14.616.21.0094, RFMEFI61618X0094). Access to instruments and other facilities was supported by the Czech research infrastructure for systems biology C4SYS (LM2015055).