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Tick-borne encephalitis (TBE) virus (TBEV) is transmitted to humans via tick bites. Infection is benign in >90% of the cases but can cause mild (<5%), moderate (<4%), or severe (<1%) encephalitis. We show here that ∼10% of patients hospitalized for severe TBE in cohorts from Austria, Czech Republic, and France carry auto-Abs neutralizing IFN-α2, -β, and/or -ω at the onset of disease, contrasting with only ∼1% of patients with moderate and mild TBE. These auto-Abs were found in two of eight patients who died and none of 13 with silent infection. The odds ratios (OR) for severe TBE in individuals with these auto-Abs relative to those without them in the general population were 4.9 (95% CI: 1.5-15.9, P < 0.0001) for the neutralization of only 100 pg/ml IFN-α2 and/or -ω, and 20.8 (95% CI: 4.5-97.4, P < 0.0001) for the neutralization of 10 ng/ml IFN-α2 and -ω. Auto-Abs neutralizing type I IFNs accounted for ∼10% of severe TBE cases in these three European cohorts., (© 2024 Gervais et al.)

Background: As highlighted by recent pandemic outbreaks, antiviral drugs are crucial resources in the global battle against viral diseases. Unfortunately, most antiviral drugs are characterized by a plethora of side effects and low efficiency/poor bioavailability owing to their insolubility. This also applies to the arylnaphthalide lignin family member, diphyllin (Diph). Diph acts as a vacuolar ATPase inhibitor and has been previously identified as a promising candidate with broad-spectrum antiviral activity. However, its physicochemical properties preclude its efficient administration in vivo, complicating preclinical testing., Methods: We produced human recombinant H - ferritin (HsaFtH) and used it as a delivery vehicle for Diph encapsulation through pH-mediated reversible reassembly of HsaFtH. Diph nanoformulation was subsequently thoroughly characterized and tested for its non-target cytotoxicity and antiviral efficiency using a panel of pathogenic viral strain., Results: We revealed that loading into HsaFtH decreased the undesired cytotoxicity of Diph in mammalian host cells. We also confirmed that encapsulated Diph exhibited slightly lower antiviral activity than free Diph, which may be due to the differential uptake mechanism and kinetics of free Diph and Diph@HsaFtH. Furthermore, we confirmed that the antiviral effect was mediated solely by Diph with no contribution from HsaFtH., Conclusion: It was confirmed that HsaFtH is a suitable vehicle that allows easy loading of Diph and production of highly homogeneous nanoparticles dispersion with promising broad-spectrum antiviral activity., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Vojnikova et al.)

Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) emerged about 30 years ago and continues to cause major economic losses in the pork industry. The lack of effective modified live vaccines (MLV) allows the pandemic to continue., Background and Objective: We have previously shown that wild strains of PRRSV affect the nascent T cell repertoire in the thymus, deplete T cell clones recognizing viral epitopes essential for neutralization, while triggering a chronic, robust, but ineffective antibody response. Therefore, we hypothesized that the current MLV are inappropriate because they cause similar damage and fail to prevent viral-induced dysregulation of adaptive immunity., Methods: We tested three MLV strains to demonstrate that all have a comparable negative effect on thymocytes in vitro . Further in vivo studies compared the development of T cells in the thymus, peripheral lymphocytes, and antibody production in young piglets. These three MLV strains were used in a mixture to determine whether at least some of them behave similarly to the wild virus type 1 or type 2., Results: Both the wild and MLV strains cause the same immune dysregulations. These include depletion of T-cell precursors, alteration of the TCR repertoire, necrobiosis at corticomedullary junctions, low body weight gain, decreased thymic cellularity, lack of virus-neutralizing antibodies, and production of non-neutralizing anti-PRRSV antibodies of different isotypes., Discussion and Conclusion: The results may explain why the use of current MLV in young animals may be ineffective and why their use may be potentially dangerous. Therefore, alternative vaccines, such as subunit or mRNA vaccines or improved MLV, are needed to control the PRRSV pandemic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Stepanova, Toman, Sinkorova, Sinkora, Pfeiferova, Kupcova Skalnikova, Abuhajiar, Moutelikova, Salat, Stepanova, Nechvatalova, Leva, Hermanova, Kratochvilova, Dusankova, Sinkora, Horak, Hudcovic, Butler and Sinkora.)

Tick-borne encephalitis (TBE) is a neuroviral disease that ranges in severity from a mild febrile illness to a severe and life-threatening meningoencephalitis or encephalomyelitis. There is increasing evidence that susceptibility to tick-borne encephalitis virus (TBEV)-induced disease and its severity are largely influenced by host genetic factors, in addition to other virus- and host-related factors. In this study, we investigated the contribution of selected single nucleotide polymorphisms (SNPs) in innate immunity genes to predisposition to TBE in humans. More specifically, we investigated a possible association between SNPs rs304478 and rs303212 in the gene Interferon Induced Protein With Tetratricopeptide Repeats 1 (IFIT1), rs7070001 and rs4934470 in the gene Interferon Induced Protein With Tetratricopeptide Repeats 2 (IFIT2), and RIG-I (Retinoic acid-inducible gene I) encoding gene DDX58 rs311795343, rs10813831, rs17217280 and rs3739674 SNPs with predisposition to TBE in population of the Czech Republic, where TBEV is highly endemic. Genotypic and allelic frequencies for these SNPs were analyzed in 247 nonimmunized TBE patients and compared with 204 control subjects. The analysis showed an association of IFIT1 rs304478 SNP and DDX58 rs3739674 and rs17217280 SNPs with predisposition to TBE in the Czech population indicating novel risk factors for clinical TBE but not for disease severity. These results also highlight the role of innate immunity genes in TBE pathogenesis., (© 2023. The Author(s).)

Tick-borne encephalitis (TBE) is a severe neuroinfection of humans. Dogs are also commonly infected with tick-borne encephalitis virus (TBEV). These infections are usually asymptomatic, but sometimes show clinical signs similar to those seen in humans and can be fatal. To date, there is no TBEV vaccine available for use in dogs. To address this need, a TBEV vaccine candidate for dogs based on inactivated whole virus antigen was developed. The safety, immunogenicity, and efficacy of the vaccine candidate were tested in mice as the preclinical model and in dogs as the target organism. The vaccine was well tolerated in both species and elicited the production of specific anti-TBEV antibodies with virus neutralising activity. Vaccination of mice provided complete protection against the development of fatal TBE. Immunisation of dogs prevented the development of viremia after challenge infection. Therefore, the developed vaccine candidate is promising to protect dogs from severe TBEV infections., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jiri Salat and Milan Hunady report financial support that was provided by Ministry of Industry and Trade of the Czech Republic. Daniel Ruzek reports financial support that was provided by EEA Grants. Daniel Ruzek reports financial support that was provided by European Union. Milan Hunady reports a relationship with Bioveta as that includes: employment. Lucie Strelcova reports a relationship with Bioveta as that includes: employment. Milan Hunady, Daniel Ruzek, Jiri Salat has patent #31355 issued to Utility model issued by the Industrial Property Office, Prague, Czech Republic., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of interests The Rockefeller University filed a patent application in connection with antibodies T025 and T028, on which M.A., D.F.R., and M.C.N. are inventors., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

PRRSV is capable of evading the effective immune response, thus persisting in piglets and throughout the swine herd. We show here that PRRSV invades the thymus and causes depletion of T-cell precursors and alteration of the TCR repertoire. Developing thymocytes are affected during negative selection when they transit from the triple-negative to triple-positive stages at the corticomedullary junction just before entering the medulla. The restriction of repertoire diversification occurs in both helper and cytotoxic αβ-T cells. As a result, critical viral epitopes are tolerated, and infection becomes chronic. However, not all viral epitopes are tolerated. Infected piglets develop antibodies capable of recognizing PRRSV, but these are not virus neutralizing. Further analysis showed that the lack of an effective immune response against the critical viral structures results in the absence of a germinal center response, overactivation of T and B cells in the periphery, robust production of useless antibodies of all isotypes, and the inability to eliminate the virus. Overall, the results show how a respiratory virus that primarily infects and destroys myelomonocytic cells has evolved strategies to disrupt the immune system. These mechanisms may be a prototype for how other viruses can similarly modulate the host immune system., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Magnetic nanorobots offer wireless navigation capability in hard-to-reach areas of the human body for targeted therapy and diagnosis. Though in vivo imaging is required for guidance of the magnetic nanorobots toward the target areas, most of the imaging techniques are inadequate to reveal the potential locomotion routes. This work proposes the use of radiopaque magnetic nanorobots along with microcomputed tomography (microCT) for localized in vivo imaging applications. The nanorobots consist of a contrast agent, barium sulfate (BaSO 4 ), magnetized by the decoration of magnetite (Fe 3 O 4 ) particles. The magnetic features lead to actuation under rotating magnetic fields and enable precise navigation in a microfluidic channel used to simulate confined spaces of the body. In this channel, the intrinsic radiopacity of the nanorobots also provides the possibility to reveal the internal structures by X-ray contrast. Furthermore, in vitro analysis indicates nontoxicity of the nanorobots. In vivo experiments demonstrate localization of the nanorobots in a specific part of the gastrointestinal (GI) tract upon the influence of the magnetic field, indicating the efficient control even in the presence of natural peristaltic movements. The nanorobots reported here highlight that smart nanorobotic contrast agents can improve the current imaging-based diagnosis techniques by providing untethered controllability in vivo., (© 2022 Wiley-VCH GmbH.)

Matrix metalloproteinases (MMPs) play an important role in central nervous system infections. We analysed the levels of 8 different MMPs in the cerebrospinal fluid (CSF) of 89 adult patients infected with tick-borne encephalitis (TBE) virus and compared them with the levels in a control group. MMP-9 was the only MMP that showed significantly increased CSF levels in TBE patients. Serum MMP-9 levels were subsequently measured in 101 adult TBE patients at various time points during the neurological phase of TBE and at follow-up. In addition, serum MMP-9 was analysed in 37 paediatric TBE patients. Compared with control levels, both paediatric and adult TBE patients had significantly elevated serum MMP-9 levels. In most adult patients, serum MMP-9 levels peaked at hospital admission, with higher serum MMP-9 levels observed in patients with encephalitis than in patients with meningitis. Elevated serum MMP-9 levels were observed throughout hospitalisation but decreased to normal levels at follow-up. Serum MMP-9 levels correlated with clinical course, especially in patients heterozygous for the single-nucleotide polymorphism rs17576 (A/G; Gln279Arg) in the MMP9 gene. The results highlight the importance of MMP-9 in the pathogenesis of TBE and suggest that serum MMP-9 may serve as a promising bioindicator of TBE in both paediatric and adult TBE patients., Competing Interests: Declaration of Competing Interest We declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Tick-borne encephalitis virus (TBEV) is an arbovirus that causes severe infections in humans, and is endemic to large areas of Europe and Asia. Humans most commonly become infected with TBEV after a tick bite; however, alimentary infection can occur after consumption of unpasteurized dairy products. Milk from sheep and goats can be a source of alimentary TBE infections. In addition, sheep and goats are considered suitable sentinels for surveillance of TBEV-associated risks in endemic areas. Here we conducted a serological survey to determine the prevalence of TBEV infection among sheep and goats in the Czech Republic. In 2019-2020, a total of 310 serum samples were collected from sheep and 418 from goats, in 11 of the 14 administrative districts of the country. Sera were tested for the presence of TBEV-specific IgG by ELISA, and suspected results were validated using a virus neutralization test. Positive samples were identified in 56.7% of goat farms, and 82.4% of sheep farms, and in 9 of the 11 administrative districts examined. The seroprevalence was significantly higher among sheep (32.5%) than goats (19.7%) (p < 0.001). The present results indicate that sheep and goats have a relatively high rate of exposure to TBEV-infected ticks in most of the administrative districts of the Czech Republic. These findings confirm the usefulness of serological testing in small ruminants to determine and monitor the risk of TBEV infection in humans., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

The presence of a non-structural protein 1 (NS1) in tick-borne encephalitis (TBE) vaccines and the possible induction of an NS1-specific immune response in vaccinated individuals remains a somewhat controversial topic. Previously, we detected the presence of NS1 in the Encepur TBE vaccine by mass spectrometry and found the induction of NS1-specific IgG antibodies in mice vaccinated with the FSME-Immun TBE vaccine. Here, in this follow-up study, we examined the dynamics and extent of the NS1-specific IgG response in mice vaccinated with these two vaccines in more detail and compared it with the IgG response to the whole virus (WV). Mice were vaccinated at two-week intervals with a total of six doses of each vaccine, and levels of IgG antibodies to TBE virus WV and NS1 were measured by ELISA after each dose. Both vaccines elicited a robust anti-WV IgG response after two doses. The Encepur vaccine did not elicit NS1-specific IgG even after all six doses. In contrast, the FSME-Immun vaccine triggered the production of NS1-specific IgG after four doses. The results indicate that FSME-Immun is the only vaccine that elicits an NS1-specific antibody response in mice. However, compared to WV-specific IgG, the NS1-specific response is weaker, and a higher number of doses is required to induce detectable levels of NS1-specific IgG antibodies.

The survival of African swine fever virus (ASFV) on different matrices and its infectivity in wild as well as domestic swine is still a matter of interest. ASFV is resistant to environmental effects; this fact is enhanced by the presence of organic material. Therefore, the aim of this work was to determine the ability of laboratory ASFV to survive in soil at different temperatures (4 and 22 °C) and with and without the presence of blood using culture procedures. The suitability of the procedure for determining the viability and titre of the ASFV field strain by the hemadsorption method was also verified, when a higher decrease in virus infectivity in the case of clay compared with peat was demonstrated. The stability of the virus was clearly temperature-dependent, the infectious virus was detected after 112 days, and the viral DNA was still detected in the matrix 210 days after inoculation in a relatively high and stable concentration (between 10 6 and 10 7 genome equivalents/mL). Based on this knowledge, soil and other environmental samples could provide rapid and reliable information on the disease outbreak and serve as indicators of the risk posed by the affected locality.

Extensive axonal and neuronal loss is the main cause of severe manifestations and poor outcomes in tick-borne encephalitis (TBE). Phosphorylated neurofilament heavy subunit (pNF-H) is an essential component of axons, and its detection in cerebrospinal fluid (CSF) or serum can indicate the degree of neuroaxonal damage. We examined the use of pNF-H as a biomarker of neuroaxonal injury in TBE. In 89 patients with acute TBE, we measured CSF levels of pNF-H and 3 other markers of brain injury (glial fibrillary acidic protein, S100B and ubiquitin C-terminal hydrolase L1) and compared the results to those for patients with meningitis of other aetiology and controls. Serum pNF-H levels were measured in 80 patients and compared with findings for 90 healthy blood donors. TBE patients had significantly ( P <0.001) higher CSF pNF-H levels than controls as early as hospital admission. Serum pNF-H concentrations were significantly higher in samples from TBE patients collected at hospital discharge ( P <0.0001) than in controls. TBE patients with the highest peak values of serum pNF-H, exceeding 10 000 pg ml -1 , had a very severe disease course, with coma or tetraplegia. Patients requiring intensive care had significantly higher serum pNF-H levels than other TBE patients ( P <0.01). Elevated serum pNF-H values were also observed in patients with incomplete recovery ( P <0.05). Peak serum pNF-H levels correlated positively with the duration of hospitalization ( P =0.005). Measurement of pNF-H levels in TBE patients might be useful for assessing disease severity and determining prognosis.

Tick-borne encephalitis (TBE) is one of the most common zoonotic diseases in Europe transmitted by Ixodidae vectors. While small mammals such as bank voles and ticks constitute the main reservoirs for virus transmission, large sylvatic species act as a food source for ticks. Cervids such as roe deer and red deer are considered sentinel species for TBE in natural foci. In addition, an increase of the population size and density of large wild mammals in an area corresponds to an increase in the tick burden and may potentially increase the prevalence of TBE virus (TBEV) in ticks and tick hosts and further exposure risk in humans. Humans are considered accidental hosts. The prevalence of TBE relies on interactions between host, vector and environment. The present study examines the exposure of the largest European herbivore, the European bison (Bison bonasus) to TBEV infection. Assessed using the IMMUNOZYM FSME ELISA (PROGEN), the overall TBEV seroprevalence was 62.7% in the 335 European bison that were studied. ELISA results were confirmed by the gold-standard virus neutralization test (VNT) with 98.7% sensitivity and thus giving a true prevalence of 63.5%. TBEV seroprevalence was significantly correlated to the origin, age group, sex, population type (free living/captive) and sanitary status (healthy/selectively eliminated/found dead/killed in accident) of the European bison in the univariable analysis. The highest seroprevalences were observed in the three largest north-eastern wild populations (Białowieska, Borecka and Knyszyńska forests), which corresponded with the highest incidence of human cases reported in the country. The risk of TBEV seropositivity increased with age and was higher in female and free-ranging European bison. Additionally, to the epidemiological investigation, the continuous detection of TBEV antibodies was studied by repetitive testing of animals over the course of 34 months. Two of six seropositive animals remained seropositive throughout the study. The presence of antibodies was followed throughout the study in seropositive European bison and for at least a year in animals that seroconverted during the observation period., (Copyright © 2021. Published by Elsevier GmbH.)

Dogs are frequently infected with the tick-borne encephalitis virus (TBEV). However, to date, only a few clinically manifest cases of tick-borne encephalitis (TBE) have been reported in dogs. In this study, three-month-old beagle dogs were infected with TBEV through a subcutaneous injection. Body temperature, clinical signs, blood haematology, blood biochemistry, and immune responses were monitored for up to 28 days postinfection (p.i.). No changes in body temperature or clinical signs were observed in the infected dogs. Most haematology and blood biochemistry parameters were unchanged after the infection, except for a slight reduction in blood lymphocyte counts, but they were within the physiological range. Low-titre viraemia was detected in 2/4 infected dogs between days 1 and 3 p.i. All infected dogs developed a robust immune response, in terms of neutralising antibodies. Thus, TBEV infections lead to effective seroconversion in dogs. Next, to assess TBEV exposure in dogs in the TBEV-endemic region of the Czech Republic, we conducted a serosurvey. Virus neutralisation tests revealed TBEV-specific antibodies in 17 of 130 (13.07%) healthy dogs, which confirmed a high, but clinically inappreciable TBEV exposure rate in the endemic area. The seropositivity rate was similar (12.7%; 41 positives out of 323) in a subgroup of dogs with various clinical disorders, and it was 13.4% (23 out of 171) in a subgroup of dogs with signs of acute neurological disease. Two dogs with fatal acute meningoencephalitis showed positive results for TBEV-specific IgM and IgG antibodies. These data extended our understanding of the clinical presentation of TBEV infections.

Tick-borne encephalitis virus (TBEV) is a medically important representative of the Flaviviridae family. The TBEV genome encodes a single polyprotein, which is co/post-translationally cleaved into three structural and seven non-structural proteins. Of the non-structural proteins, NS5, contains an RNA-dependent RNA polymerase (RdRp) domain that is highly conserved and is responsible for the genome replication. Screening for potential antivirals was done using a hybrid receptor and ligand-based pharmacophore search likely targeting the RdRp domain. For the identification of pharmacophores, a mixture of small probe molecules and nucleotide triphosphates were used. The ligand/receptor interaction screenings of structures from the ZINC database resulted in five compounds. Zinc 3677 and 7151 exhibited lower cytotoxicity and were tested for their antiviral effect against TBEV in vitro. Zinc 3677 inhibited TBEV at micromolar concentrations. The results indicate that Zinc 3677 represents a good target for structure-activity optimizations leading potentially to a discovery of effective TBEV antivirals., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Vector-borne flaviviruses (VBFs) affect human health worldwide, but no approved drugs are available specifically to treat VBF-associated infections. Here, we performed in silico screening of a library of U.S. Food and Drug Administration-approved antiviral drugs for their interaction with Zika virus proteins. Twelve hit drugs were identified by the docking experiments and tested in cell-based antiviral assay systems. Efavirenz, tipranavir, and dasabuvir at micromolar concentrations were identified to inhibit all VBFs tested; i.e., two representatives of mosquito-borne flaviviruses (Zika and West Nile viruses) and one representative of flaviviruses transmitted by ticks (tick-borne encephalitis virus). The results warrant further research into these drugs, either individually or in combination, as possible pan-flavivirus inhibitors., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

This dataset, gathered during the RETRO-BMC cruise, reports multiple-scale measurements at the Confluence of the Brazil and Malvinas Currents. The cruise was carried out between 8 and 28 April 2017 onboard R/V Hespérides, departing from Ushuaia and arriving to Santos. Along its track, the vessel recorded near-surface temperature and salinity, as well as the horizontal flow from 20 m down to about 800 m. A total of 33 hydrographic stations were completed in a region off the Patagonian Shelf, within 41.2°S-35.9°S and out to 53.0°W. At each station, a multiparametric probe and velocity sensors were deployed inside the frame of a rosette used to collect water samples at selected depths; these samples were later used for several water analyses, including inorganic nutrient concentrations. Microstructure measurements were carried out in 11 of these hydrographic stations. In addition, two high-resolution three-dimensional surveys were conducted with an instrumented undulating vehicle between 40.6°S-39.0°S and 55.6°W-53.8°W. Lastly, eight high-frequency vertical profilers were deployed in the region and five position-transmitting drifters were launched. These data allow the description of the Confluence from the regional scale to the microscale, and provide a view of the variability of the frontal region on time scales from days to weeks., (© 2020 The Author(s).)

Vaccination against tick-borne encephalitis (TBE) is based on the use of formalin-inactivated, culture-derived whole-virus vaccines. Immune response following vaccination is primarily directed to the viral envelope (E) protein, the major viral surface antigen. In Europe, two TBE vaccines are available in adult and pediatric formulations, namely FSME-IMMUN ® (Pfizer) and Encepur ® (GlaxoSmithKline). Herein, we analyzed the content of these vaccines using mass spectrometry (MS). The MS analysis revealed that the Encepur vaccine contains not only proteins of the whole virus particle, but also viral non-structural protein 1 (NS1). MS analysis of the FSME-IMMUN vaccine failed due to the high content of human serum albumin used as a stabilizer in the vaccine. However, the presence of NS1 in FSME-IMMUN was confirmed by immunization of mice with six doses of this vaccine, which led to a robust anti-NS1 antibody response. NS1-specific Western blot analysis also detected anti-NS1 antibodies in sera of humans who received multiple doses of either of these two vaccines; however, most vaccinees who received ≤3 doses were negative for NS1-specific antibodies. The contribution of NS1-specific antibodies to protection against TBE was demonstrated by immunization of mice with purified NS1 antigen, which led to a significant ( p < 0.01) prolongation of the mean survival time after lethal virus challenge. This indicates that stimulation of anti-NS1 immunity by the TBE vaccines may increase their protective effect., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Tick-borne encephalitis (TBE), a disease caused by tick-borne encephalitis virus (TBEV), represents a serious neural infection of humans in Europe and Asia. The main reservoir hosts of TBEV are ticks, rodents and insectivores, but domestic animals may also be infected with the virus. This review summarizes what is known about TBE in domestic species (e.g., dogs, horses), in which infection may manifest with clinical signs similar to those seen in severe human cases. We also focus on TBE in ruminants where TBE infections are typically asymptomatic and do not cause health problems in the infected hosts. However, the risk to human health is the main problem of asymptomatic infection, because the presence of TBEV in the milk of infected ruminants can serve as a source of TBE infection via the alimentary route. An experimental veterinary vaccine was developed recently, and future vaccination of selected domestic animals is proposed to avoid the development of severe TBE symptoms in sensitive animals (e.g., dogs, horses) or to decrease the risk of alimentary infection in humans (e.g., goats and sheep). Keywords: tick-borne encephalitis; dog; goat; sheep; cow; horse.

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

The goals of our study were to compare the immune response to different killed and modified live vaccines against PRRS virus and to monitor the antibody production and the cell mediated immunity both at the systemic and local level. In the experiment, we immunized four groups of piglets with two commercial inactivated (A1-Progressis, A2-Suivac) and two modified live vaccines (B3-Amervac, B4-Porcilis). Twenty-one days after the final vaccination, all piglets, including the control non-immunized group (C5), were i.n., infected with the Lelystad strain of PRRS virus. The serum antibody response (IgM and IgG) was the strongest in group A1 followed by two MLV (B3 and B4) groups. Locally, we demonstrated the highest level of IgG antibodies in bronchoalveolar lavages (BALF), and saliva in group A1, whereas low IgA antibody responses in BALF and feces were detected in all groups. We have found virus neutralization antibody at DPV 21 (days post vaccination) and higher levels in all groups including the control at DPI 21 (days post infection). Positive antigen specific cell-mediated response in lymphocyte transformation test (LTT) was observed in groups B3 and B4 at DPV 7 and in group B4 at DPV 21 and in all intervals after infection. The IFN-γ producing lymphocytes after antigen stimulation were found in CD4 - CD8 + and CD4 + CD8 + subsets of all immunized groups 7 days after infection. After infection, there were obvious differences in virus excretion. The virus was detected in all groups of piglets in serum, saliva, and occasionally in feces at DPI 3. Significantly lower virus load was found in groups A1 and B3 at DPI 21. Negative samples appeared at DPI 21 in B3 group in saliva. It can be concluded that antibodies after immunization and infection, and the virus after infection can be detected in all the compartments monitored. Immunization with inactivated vaccine A1-Progressis induces high levels of antibodies produced both systemically and locally. Immunization with MLV-vaccines (Amervac and Porcilis) produces sufficient antibody levels and also cell-mediated immunity. After infection virus secretion gradually decreases in group B3, indicating tendency to induce sterile immunity.

Rabies is a deadly viral disease with an extremely high fatality rate in humans. Previously, it was suggested that an enhancement of the blood-brain barrier (BBB) permeability, which allows immune cells and/or antibodies to enter the central nervous system (CNS) tissue, is critical to clear the infection. In this study, we utilised mannitol to increase BBB permeability in mice infected with highly pathogenic silver-haired bat rabies virus (SHBRV). We found that intraperitoneal injection of mannitol causes a slight, transient increase of BBB permeability in the treated mice. SHBRV-infected mice were treated with intraperitoneally administered mannitol daily from day 3 or day 4 post-infection, but no effect of this treatment on the time of disease onset, clinical signs or survival was observed. This data indicates that the increase of BBB permeability by mannitol is not efficient in promoting CNS virus clearance in SHBRV-infected mice., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Kyasanur Forest disease virus (KFDV) is a highly pathogenic tick-borne flavivirus enzootic to India. In humans, KFDV causes a severe febrile disease. In some infected individuals, hemorrhagic manifestations, such as bleeding from the nose and gums and gastrointestinal bleeding with hematemesis and/or blood in the stool, have been reported. However, the mechanisms underlying these hemorrhagic complications remain unknown, and there is no information about the specific target cells for KFDV. We investigated the interaction of KFDV with vascular endothelial cells (ECs) and monocyte-derived dendritic cells (moDCs), which are key targets for several other hemorrhagic viruses. Here, we report that ECs are permissive to KFDV infection, which leads to their activation, as demonstrated by the upregulation of E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 at the mRNA and protein levels. Increased expression of these adhesive molecules correlated with increased leukocyte adhesion. Infected ECs upregulated the expression of interleukin (IL)-6 but not IL-8. Additionally, moDCs were permissive to KFDV infection, leading to increased release of IL-6 and tumor necrosis factor-α. Supernatants from KFDV-infected moDCs caused EC activation, as measured by leukocyte adhesion. The results indicate that ECs and moDCs can be targets for KFDV and that both direct and indirect mechanisms can contribute to EC activation.

Background: 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)., Results: 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., Conclusions: 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.

Marine biogeochemistry dynamics in coastal marine areas is strongly influenced by episodic events such as rain, intense winds, river discharges and anthropogenic activities. We evaluated in this study the importance of these forcing events on modulating seasonal changes in the marine biogeochemistry of the northwestern coast of the Mediterranean Sea, based on data gathered from a fixed coastal sampling station in the area. A 4-year (2011-2014) monthly sampling at four depths (0.5m, 20m, 50m and 80m) was performed to examine the time variability of several oceanographic variables: seawater temperature, salinity, inorganic nutrient concentrations (NO 3 - , PO 4 3- and SiO 2 ), chlorophyll a (Chl a), dissolved organic carbon (DOC) and fluorescent dissolved organic matter (FDOM). FDOM dynamics was predominantly influenced by upwelling events and mixing processes, driven by strong and characteristic wind episodes. SW wind episodes favored the upwelling of deeper and denser waters into the shallower shelf, providing a surplus of autochthonous humic-like material and inorganic nutrients, whereas northerlies favored the homogenization of the whole shelf water column by cooling and evaporation. These different wind-induced processes (deep water intrusion or mixing), reported along the four sampled years, determined a high interannual environmental variability in comparison with other Mediterranean sampling sites., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Little is known about the occurrence of tick-borne encephalitis in Romania. Sheep are an infection source for humans and are useful sentinels for risk analysis. We demonstrate high antibody prevalence (15.02%) among sheep used as sentinels for this disease in 80% of the tested localities in 5 counties of northwestern Romania.

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'- C -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'- C -methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'- C -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'- C -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'- C -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. IMPORTANCE This study found that the nucleoside analog 7-deaza-2'- C -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'- C -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., (Copyright © 2017 American Society for Microbiology.)

MicroRNAs are subject to precise regulation and have key roles in tumorigenesis. In contrast to the oncogenic role of miR-22 reported in myelodysplastic syndrome (MDS) and breast cancer, here we show that miR-22 is an essential anti-tumour gatekeeper in de novo acute myeloid leukaemia (AML) where it is significantly downregulated. Forced expression of miR-22 significantly suppresses leukaemic cell viability and growth in vitro, and substantially inhibits leukaemia development and maintenance in vivo. Mechanistically, miR-22 targets multiple oncogenes, including CRTC1, FLT3 and MYCBP, and thus represses the CREB and MYC pathways. The downregulation of miR-22 in AML is caused by TET1/GFI1/EZH2/SIN3A-mediated epigenetic repression and/or DNA copy-number loss. Furthermore, nanoparticles carrying miR-22 oligos significantly inhibit leukaemia progression in vivo. Together, our study uncovers a TET1/GFI1/EZH2/SIN3A/miR-22/CREB-MYC signalling circuit and thereby provides insights into epigenetic/genetic mechanisms underlying the pathogenesis of AML, and also highlights the clinical potential of miR-22-based AML therapy.

Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic malignancies with variable response to treatment. AMLs bearing MLL (mixed lineage leukemia) rearrangements are associated with intermediate or poor survival. MicroRNAs (miRNAs), a class of small noncoding RNAs, have been postulated to be important gene expression regulators virtually in all biological processes, including leukemogenesis. Through a large-scale, genome-wide miRNA expression profiling assay of 85 human AML and 15 normal control samples, we show that among 48 miRNAs that are significantly differentially expressed between MLL- and non-MLL-rearranged AML samples, only one (miR-495) is expressed at a lower level in MLL-rearranged AML than in non-MLL-rearranged AML; meanwhile, miR-495 is also significantly down-regulated in MLL-rearranged AML samples compared with normal control samples. Through in vitro colony-forming/replating assays and in vivo bone marrow transplantation studies, we show that forced expression of miR-495 significantly inhibits MLL-fusion-mediated cell transformation in vitro and leukemogenesis in vivo. In human leukemic cells carrying MLL rearrangements, ectopic expression of miR-495 greatly inhibits cell viability and increases cell apoptosis. Furthermore, our studies demonstrate that PBX3 and MEIS1 are two direct target genes of miR-495, and forced expression of either of them can reverse the effects of miR-495 overexpression on inhibiting cell viability and promoting apoptosis of human MLL-rearranged leukemic cells. Thus, our data indicate that miR-495 likely functions as a tumor suppressor in AML with MLL rearrangements by targeting essential leukemia-related genes.

We have previously demonstrated that two salivary cysteine protease inhibitors from the Borrelia burgdorferi (Lyme disease) vector Ixodes scapularis- namely sialostatins L and L2 - play an important role in tick biology, as demonstrated by the fact that silencing of both sialostatins in tandem results in severe feeding defects. Here we show that sialostatin L2 - but not sialostatin L - facilitates the growth of B. burgdorferi in murine skin. To examine the structural basis underlying these differential effects of the two sialostatins, we have determined the crystal structures of both sialostatin L and L2. This is the first structural analysis of cystatins from an invertebrate source. Sialostatin L2 crystallizes as a monomer with an 'unusual' conformation of the N-terminus, while sialostatin L crystallizes as a domain-swapped dimer with an N-terminal conformation similar to other cystatins. Deletion of the 'unusual' N-terminal five residues of sialostatin L2 results in marked changes in its selectivity, suggesting that this region is a particularly important determinant of the biochemical activity of sialostatin L2. Collectively, our results reveal the structure of two tick salivary components that facilitate vector blood feeding and that one of them also supports pathogen transmission to the vertebrate host.

Summer conditions in the Mediterranean Sea are characterized by high temperatures and low food availability. This leads to "summer dormancy" in many benthic suspension feeders due to energetic constraints. Analysis of the most recent 33-year temperature time series demonstrated enhanced stratification due to global warming, which produced a approximately 40% lengthening of summer conditions. Many biological processes are expected to be affected by this trend, culminating in such events as mass mortality of invertebrates. Climatic anomalies concomitant with the occurrence of these events represent prolonged exposure to warmer summer conditions coupled with reduced food resources. Simulation of the effects of these conditions on a model organism demonstrated a biomass loss of >35%. Losses of this magnitude result in mortality similar to that noted in field observations during mass mortality events. These results indicate that temperature anomalies are the underlying cause of the events, with energetic constraints serving as the main triggering mechanism.

In the present paper, a normalization technique to scale data that exhibit an allometric growth is presented and the way it has to be used is described. It is shown how the method has been derived from the theoretical equations of allometric growth. Consequently, the method completely removes all the information related to size, not only scaling all individuals to the same size, but also adjusting their shape to that they would have in the new size according to allometry. In the particular case of isometry when the measures are of identical dimension, this normalization coincides with ratios (one of the most popular methods but only valid in this particular case). This procedure is a theoretical generalization of the technique used by Thorpe (1975, Biol. J. Linn. Soc.7, 27-43; 1976, Biol. Rev.51, 407-452) which was recorded as one of the most efficient methods in the empirical evaluation done by Reist (1985, Can. J. Zool.63, 1429-1439)., (Copyright 2000 Academic Press.)

After they had analysed 50 cases of varicocoele the authors decided to put in writing some of their ideas. They conclude that varicocoele is often sub-clinical, localised to the right side, bilateral only in a number of cases. Although neither the sperm count nor the histology of the testes show specific pathological features, treatment of the condition brings worthwhile rewards. About 40% of patients father pregnancies.

The levels of oestradiol and of testosterone were estimated in the spermatic and in the perpheral veins in two series of 13 subjects. The one was a control group and the other stimulated with 10,000 International Units of chorionic gonadontrophir (II.C.G). The follwing conclusions can be drawn. --the first is that the testis secretes simultaneously testosterone and oestradiol 17 beta --the second is that the level of oestradiol is significantly raised after the administration of II.C.G. whereas the level of testosterone hardly alters. This leads on to the supposition that oestradiol does not arise from the same source in the testis as testosterone.

When a pregnant woman presented with a syndrome suggesting obstruction and fever together with jaundice and oliguria the authors remembered the difficulty of clinical diagnosis and the biology of pancreatitis. They studied the connection of this condition with pregnancy, its pathogenesis, its prognosis and its treatment, as well as the various factors that were favourable (treatment with diuretics, the right ovarian vein syndrome).

Giant haemangiomas combined with a haemostasis disorder of the Kasabach-Meritt type are rare and even more rare are the problems posed during labour when vulvo-vagino-uterine sites are involved. Two cases are reported. After summary of the clinical and biochemical studies, the authors report attempts to improve the haemostasis disorder, first with heparin and then with antifibrinolytic preparations, and to improve the angiomas with antifibrinolytic preparations in the hope of stimulating intrasacular thrombosis. In particular, the obstetric and surgical problems are discussed in the context of labour. In one case the outcome of labour was successful only moderate transfusion being required, and in the other the outcome was fatal despite the use of about 100 bottles of blood in the first 24 hours. In both cases the infants were normal.