Flaviviruses are a family of viruses that cause many diseases in humans. Their similarity in the antigenic structure causes a cross‐reaction, which complicates the precise diagnostic of disease ...causing agents. Tick‐borne encephalitis virus (TBEV), a member of the flavivirus family, is the cause of tick‐borne encephalitis (TBE). Worldwide the awareness of this disease is raising, however, in many countries such as the Republic of Kazakhstan (KZ) there is a lack of serological investigation of flaviviruses in humans. In our study, we focused on two TBE endemic regions of KZ (East Kazakhstan Oblast (EKO) and Almaty (AO)) and a region where TBE cases were registered only since 2010 (Akmola Oblast (AkO)). In KZ, up to 400 cases of serous meningitis of unknown origin were registered annually in the period from 2017 to 2019. Our goals were to calculate the prevalence of antibodies against TBEV in patients with suspected meningitis. We collected 179 sera and 130 cerebrospinal fluid (CSF) samples from patients and included a questionnaire with focus on socio‐demographical factors and observed tick bites. The human samples were tested with TBEV and West‐Nile fever virus (WNFV) IgM and IgG ELISA, by immunofluorescence assay using a flavivirus biochip, and TBEV‐specific real‐time RT‐PCR. We found TBEV and WNFV antibodies in 31 samples by serological and molecular techniques. Seven serum samples out of 31 showed TBEV‐specific antibodies, and three serum pairs had WNFV antibodies. Correlating the serological results with the information gained from the questionnaires it becomes apparent that the number of tick bites is a significant factor for a TBEV infection. This result has an impact on diagnostic in KZ and physicians should be aware that both flaviviruses play a role for serous meningitis of unknown origin in KZ.
Tick-borne encephalitis virus (TBEV) is primarily transmitted to humans through tick bites or oral consumption of accordingly contaminated unpasteurized milk or milk products. The detection of TBEV ...RNA in various body fluids in immunosuppressed human patients is documented. However, the risk of direct contact exposure remains unclear. Interferon-alpha receptor-1 deficient (
) mice, which are lacking the interferon-α/β responses, develop neurologic manifestations after infection with TBEV and Langat virus (LGTV). We showed that subcutaneous, intranasal, and peroral infection of LGTV lead to disease, whereas mice with intragastric application of LGTV showed no disease signs. With LGTV infected mice exhibit seroconversion and significant viral RNA levels was detected in saliva, eye smear, feces and urine. As a result, TBEV and LGTV are transmitted between mice from infected to naïve co-caged sentinel animals. Although intranasal inoculation of LGTV is entirely sufficient to establish the disease in mice, the virus is not transmitted by aerosols. These pooled results from animal models highlight the risks of exposure to TBEV contaminants and the possibility for close contact transmission of TBEV in interferon-alpha receptor-1 deficient laboratory mice.
Tick-borne encephalitis is a severe disease of the central nervous system caused by the tick-borne encephalitis virus (TBEV). Every year between 10,000-12,000 people become infected with this flavivirus. The TBEV is usually transmitted to humans via the bite of a tick, but infections due to consumption of infectious milk products are increasingly being reported. Since there is no therapy for an TBEV infection and mechanisms of virus persistence in reservoir animals are unclear, it is important to highlight the risk of exposure to TBEV contaminants and know possible routes of transmission of this virus. The significance of our research is in identifying other infection routes of TBEV and LGTV, and the possibility of close contact transmission.
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.
Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we ...demonstrate that treatment with a nucleoside analog, 7-deaza-2'-
-methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'-
-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-
-azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'-
-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'-
-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus.
This study found that the nucleoside analog 7-deaza-2'-
-methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'-
-methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.
Tick-borne encephalitis virus (TBEV) is a growing health concern. It causes a severe disease that can lead to permanent neurological complications or death and the incidence of TBEV infections is ...constantly rising. Our understanding of TBEV's structure lags behind that of other flaviviruses, but has advanced recently with the publication of a high-resolution structure of the TBEV virion. The gaps in our knowledge include: aspects of receptor binding, replication and virus assembly. Furthermore, TBEV has mostly been studied in mammalian systems, even though the virus' interaction with its tick hosts is a central part of its life cycle. Elucidating these aspects of TBEV biology are crucial for the development of TBEV antivirals, as well as the improvement of diagnostics. In this review, we summarise the current structural knowledge on TBEV, bringing attention to the current gaps in our understanding, and propose further research that is needed to truly understand the structural-functional relationship of the virus and its hosts.
Tick‐borne encephalitis virus (TBEV) and West Nile virus (WNV) are important arthropod‐borne zoonotic flaviviruses. Due to the emergence of WNV in TBEV‐endemic regions co‐circulation of both viruses ...is increasing. Flaviviruses are structurally highly similar, which leads to cross‐reacting antibodies upon infection. Currently available serological assays for TBEV and WNV infections are therefore compromised by false‐positive results, especially in IgG measurements. In order to discriminate both infections novel diagnostic methods are needed. We describe an ELISA to measure IgG antibodies specific for TBEV and WNV, applicable to human and horse sera. Mutant envelope proteins were generated, that lack conserved parts of the fusion loop domain, a predominant target for cross‐reacting antibodies. These were incubated with equine and human sera with known TBEV, WNV or other flavivirus infections. For WNV IgG, specificities and sensitivities were 100% and 87.9%, respectively, for horse sera, and 94.4% and 92.5%, respectively, for human sera. TBEV IgG was detected with specificities and sensitivities of 95% and 96.7%, respectively, in horses, and 98.9% and 100%, respectively, in humans. Specificities increased to 100% by comparing individual samples on both antigens. The antigens could form the basis for serological TBEV‐ and WNV‐assays with improved specificities.
Background.
Aptamers are small single-stranded DNA or RNA molecules that have an affinity for a specific target molecule. The main method of aptamers construction is the technology of systematic ...evolution of ligands with exponential enrichment (SELEX). However, the exact approach depends on the nature of target molecules, and is selected and optimized by each researcher independently. The article describes the technique of production of aptamers to the tick-borne encephalitis virus (TBEV) using membrane ultrafiltration with a molecular weight cut-off of 100 kDa. As a result, the pool of aptamers with observable affinity for TBEV is successfully selected and enriched.
The aim.
To develop the technique suitable for selection of specific DNA aptamers to a live, crude TBEV suspension directly in cell culture supernatant.
Materials and methods.
The selection of aptamers was carried out using a modified SELEX DNA aptamer technology in combination with semipermeable membrane ultrafiltration using Vivaspin 6 (Sartorius, Germany) concentrators of molecular weight cut-off of 100 kDa. Enrichment of a specific pool of aptamers was performed using real time polymerase chain reaction. Aptamers were sequenced with automated Sanger sequencing method. The direct virucidal effect of the aptamers was determined by the decrease in the titer of the infectious virus after incubation with the aptamer.
Results.
The pool of aptamers to TBEV was selected and enriched. This aptamer pool expressed affinity both to the infectious TBEV and to the TBEV antigen. Sixteen aptamers were sequenced from this pool and four of them were synthesized and tested for antiviral activity against TBEV. No antiviral activity was observed.
Conclusions.
The technique developed that can be successfully used to select aptamers to a live virus culture for the viruses comparable in size to TBEV or larger.
The genetic diversities of mammalian tick-borne flaviviruses are poorly understood. We used next-generation sequencing (NGS) to deep sequence different viruses and strains belonging to this group of ...flaviviruses, including Central European tick-borne encephalitis virus (TBEV-Eur), Far Eastern TBEV (TBEV-FE), Langat (LGTV), Powassan (POWV), Deer Tick (DTV), Kyasanur Forest Disease (KFDV), Alkhurma hemorrhagic fever (AHFV), and Omsk hemorrhagic fever (OHFV) viruses. DTV, AHFV, and KFDV had the lowest genetic diversity, while POWV strains LEIV-5530 and LB, OHFV, TBEV-Eur, and TBEV-FE had higher genetic diversities. These findings are compatible with the phylogenetic relationships between the viruses. For DTV and POWV, the amount of genetic diversity could be explained by the number of tick vector species and amplification hosts each virus can occupy, with low diversity DTV having a more limited vector and host pool, while POWV with higher genetic diversities has been isolated from different tick species and mammals. It is speculated that high genetic diversity may contribute to the survival of the virus as it encounters these different environments.
•Diagnosing tick-borne encephalitis relies on antibody testing.•TBEV specific IgM testing by ELISA is highly specific in serum and CSF.•A CSF-serum antibody index can confirm the diagnosis TBE.•TBEV ...IgG ELISAs lack specificity and require confirmation by virus neutralization.
Tick-borne encephalitis (TBE) is an infectious disease endemic to large parts of Europe and Asia. Diagnosing TBE often relies on the detection of TBEV-specific antibodies in serum and cerebrospinal fluid (CSF) as viral genome is mostly not detectable once neurological symptoms occur.
We evaluated the performance of TBEV IgM and IgG ELISAs in both serum and CSF of confirmed TBEV patients and discuss the role of (CSF) serology in TBEV diagnostics.
For the assay evaluation we collected specimen from confirmed TBEV patients. Assay specificity was assessed using sera from patients with a related flavivirus infection or other acute infection. A selected ELISA assay was used to analyze TBEV-specific antibodies in CSF and to evaluate the use in confirming TBE diagnosis.
In this study the overall sensitivity of the IgM TBEV ELISAs was acceptable (94 -100 %). Four out of five IgM ELISA’s demonstrated an excellent overall specificity from 94 -100% whereas a low overall specificity was observed for the IgG TBEV ELISAs (30-71%). Intrathecal antibody production against TBEV was demonstrated in a subset of TBE patients.
In four out of five ELISAs, IgM testing in serum and CSF of TBE patients is specific and confirmative. The lack of IgG specificity in all ELISAs emphasizes the need of confirmatory testing by virus neutralisation, depending on the patient’s background and the geographic location of exposure to TBEV. A CSF-serum IgG antibody index can support the diagnosis specifically in chronic disease or once IgM has disappeared.
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