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Abstract Tick-borne encephalitis virus (TBEV) vaccine breakthrough (VBT) infections are not uncommon in endemic areas. The clinical and immunological outcomes have been poorly investigated. We assessed the magnitude and specificity of virus-specific antibody and T cell responses after TBE in previously vaccinated subjects and compared the results with those of unvaccinated TBE patients and study subjects that received vaccination without VBT infection. Symptomatic TBEV infection of unvaccinated study subjects induced virus-specific antibody responses to the E protein and non-structural protein 1 (NS1) as well as T cell responses to structural and other non-structural (NS) proteins. After VBT infections, significantly impaired NS1-specific antibody responses were observed, while the virus-specific T cell responses to the NS proteins were relatively strong. VBT infection caused predominantly moderate to severe disease during hospitalization. The level of TBEV EDIII- and NS1-specific antibodies in unvaccinated convalescent patients inversely correlated with TBE severity and neurological symptoms early after infection.

Abstract Background Paediatric patients with autoimmune rheumatic diseases (pARD) have a dysregulated immune system, so infections present a major threat to them. To prevent severe COVID-19 infections we aimed to vaccinate them as soon as possible. Studies have shown that the BNT162b2 vaccine is safe, effective, and immunogenic, however, in a short observation period, only. Methods The main objective was to compare the serological response between three groups of pARD: after SARS-CoV-2 infection, after vaccination against COVID-19 with two doses of the BNT162b2 vaccine, and after experiencing both events. Data on demographics, diagnosis, therapy, and serology (anti-SARS-CoV-2 IgG/IgA) were collected from March 2020 to April 2022. For statistical analysis ANOVA, Mann-Whitney U test, Chi-square test and Fisher’s exact test were applied. To compare adverse events (AE) after vaccination we included a control group of healthy adolescents. Results We collected data from 115 pARD; from 92 after infection and 47 after vaccination. Twenty-four were included in both groups. Serological data were available for 47 pARD after infection, 25 after vaccination, and 21 after both events. Serological response was better after vaccination and after both events compared to after infection only. No effect of medication on the antibody levels was noted. The safety profile of the vaccine was good. Systemic AE after the first dose of the vaccine were more common in healthy adolescents compared to pARD. In the observation period of 41.3 weeks, 60% of vaccinated pARD did not experience a symptomatic COVID-19 infection. Conclusions IgG and IgA anti-SARS-CoV-2 levels were higher after vaccination and after both events compared to after infection only. Six months after vaccination we observed an increase in antibody levels, suggesting that pARD had been exposed to SARS-CoV-2 but remained asymptomatic. Trial registration The study was approved by the Medical Ethics Committee of the Republic of Slovenia (document number: 0120–485/2021/6).

Abstract Background The concurrent circulation of SARS-CoV-2 with other respiratory viruses is unstoppable and represents a new diagnostic reality for clinicians and clinical microbiology laboratories. Multiplexed molecular testing on automated platforms that focus on the simultaneous detection of multiple respiratory viruses in a single tube is a useful approach for current and future diagnosis of respiratory infections in the clinical setting. Methods Two time periods were included in the study: from February to April 2022, an early 2022 period, during the gradual lifting of COVID-19 prevention measures in the country, and from October 2022 to April 2023, the 2022/23 respiratory infections season. We analysed a total of 1,918 samples in the first period and 18,131 respiratory samples in the second period using a multiplex molecular assay for the simultaneous detection of Influenza A (Flu-A), Influenza B (Flu-B), Human Respiratory Syncytial Virus (HRSV) and SARS-CoV-2. Results The results from early 2022 showed a strong dominance of SARS-CoV-2 infections with 1,267/1,918 (66.1%) cases. Flu-A was detected in 30/1,918 (1.6%) samples, HRSV in 14/1,918 (0.7%) samples, and Flu-B in 2/1,918 (0.1%) samples. Flu-A/SARS-CoV-2 co-detections were observed in 11/1,267 (0.9%) samples, and HRSV/SARS-CoV-2 co-detection in 5/1,267 (0.4%) samples. During the 2022/23 winter respiratory season, SARS-CoV-2 was detected in 1,738/18,131 (9.6%), Flu-A in 628/18,131 (3.5%), Flu-B in 106/18,131 (0.6%), and HRSV in 505/18,131 (2.8%) samples. Interestingly, co-detections were present to a similar extent as in early 2022. Conclusion The results show that the multiplex molecular approach is a valuable tool for the simultaneous laboratory diagnosis of SARS-CoV-2, Flu-A/B, and HRSV in hospitalized and outpatients. Infections with Flu-A/B, and HRSV occurred shortly after the COVID-19 control measures were lifted, so a strong reoccurrence of various respiratory infections and co-detections in the post COVID-19 period was to be expected.

ABSTRACTTick-borne encephalitis virus (TBEV) infection may cause acute central nervous system inflammation varying in clinical manifestations and severity. A possible correlation of TBEV-specific antibody and cell-mediated immune responses, shortly after infection, with clinical manifestations, severity and long-term outcome has been poorly investigated. In a cohort of thirty early tick-borne encephalitis (TBE) patients, we assessed the magnitude, specificity and functional properties of TBEV-specific T-cell and antibody responses. These responses early during disease were assessed in view of clinical manifestations, severity and long-term outcome. TBEV-specific T-cell responses to C, E, NS1, and NS5 proteins were significantly lower in patients with severe acute illness than in patients with mild TBE. Lower T-cell responses to E, NS1, and NS5 proteins also correlated with the development of meningoencephalomyelitis. Virus-specific antibody titres early after infection did not correlate with disease severity, clinical manifestations, or long-term outcome in this study, possibly due to the small number of patients of which matching serum and peripheral blood mononuclear cells were available. The findings suggest that virus-specific T cells afford a certain degree of protection against the development of severe TBEV-induced disease.

Patients who have Lyme neuroborreliosis (LNB) might experience lingering symptoms that persist despite antibiotic drug therapy. We tested whether those symptoms are caused by maladaptive immune responses by measuring 20 immune mediators in serum and cerebrospinal fluid (CSF) in 79 LNB patients followed for 1 year. At study entry, most mediators were highly concentrated in CSF, the site of the infection. Those responses resolved with antibiotic therapy, and associations between CSF cytokines and signs and symptoms of LNB were no longer observed. In contrast, subjective symptoms that persisted after use of antibiotics were associated with increased levels of serum interferon-α (IFN-α), which were already observed at study entry, and remained increased at each subsequent timepoint. Highest IFN-α levels corresponded with severe disease. Although the infection serves as the initial trigger, sequelae after antibiotic therapy are associated with unremitting systemic IFN-α levels, consistent with the pathogenic role of this cytokine in interferonopathies in other conditions.

BackgroundDespite decreasing COVID-19 disease severity during the Omicron waves, a proportion of patients still require hospitalization and intensive care.ObjectiveTo compare demographic characteristics, comorbidities, vaccination status, and previous infections in patients hospitalized for community-associated COVID-19 (CAC) in predominantly Delta, Omicron BA.1 and BA.4/5 SARS-CoV-2 waves.MethodsData were extracted from three national databases—the National COVID-19 Database, National Vaccination Registry and National Registry of Hospitalizations.ResultsAmong the hospitalized CAC patients analyzed in this study, 5,512 were infected with Delta, 1,120 with Omicron BA.1, and 1,143 with the Omicron BA.4/5 variant. The age and sex structure changed from Delta to BA.4/5, with the proportion of women (9.5% increase), children and adolescents (10.4% increase), and octa- and nonagenarians increasing significantly (24.5% increase). Significantly more patients had comorbidities (measured by the Charlson Comorbidity Index), 30.3% in Delta and 43% in BA.4/5 period. The need for non-invasive ventilatory support (NiVS), ICU admission, mechanical ventilation (MV), and in-hospital mortality (IHM) decreased from Delta to Omicron BA.4/5 period for 12.6, 13.5, 11.5, and 6.3%, respectively. Multivariate analysis revealed significantly lower odds for ICU admission (OR 0.68, CI 0.54–0.84, p < 0.001) and IHM (OR 0.74, CI 0.58–0.93, p = 0.011) during the Delta period in patients who had been fully vaccinated or boosted with a COVID-19 vaccine within the previous 6 months. In the BA.1 variant period, patients who had less than 6 months elapsed between the last vaccine dose and SARS-CoV-2 positivity had lower odds for MV (OR 0.38, CI 0.18-0.72, p = 0.005) and IHM (OR 0.56, CI 0.37- 0.83, p = 0.005), but not for NIVS or ICU admission.ConclusionThe likelihood of developing severe CAC in hospitalized patients was higher in those with the Delta and Omicron BA.1 variant compared to BA.4/5.

IntroductionTick-borne encephalitis (TBE) is an emerging vector-borne and food-borne disease caused by the tick-borne encephalitis virus (TBEV; Orthoflavivirus encephalitidis), with a distribution spanning the Eurasian continent. Despite its significant public health impact in various European regions, TBE remains largely underdiagnosed in Serbia due to limited awareness and diagnostic challenges. In response to this, our study aimed to comprehensively assess TBEV exposure in individuals infested with ticks and to identify potential TBEV foci within Serbia.Materials and methodsFrom 2019 to 2021, we conducted an observational study involving 450 patients who reported tick infestations.ResultsOur demographic analysis revealed a median age of 38 years, with a slight male predominance among the participants. We documented tick infestations in 38 municipalities across 14 districts of Serbia, with a notable concentration in proximity to Fruška Gora Mountain. The ticks most frequently removed were Ixodes ricinus, with nymphs and adult females being the predominant stages. On average, nymphs were removed after about 27.1 hours of feeding, while adult females remained attached for approximately 44.4 hours. Notably, we found age as a significant predictor of infestation time for both nymphs and adult females. Furthermore, we detected TBEV-neutralizing antibodies in 0.66% of the serum samples, shedding light on potential TBEV foci, particularly in Fruška Gora Mountain and other regions of Serbia.ConclusionOur study emphasizes the urgent need for active TBE surveillance programs, especially in areas suspected of hosting TBEV foci, in order to assess the true TBE burden, identify at-risk populations, and implement effective preventive measures.

Abstract Background Paediatric patients with autoimmune rheumatic diseases (pARD) are often immunocompromised because of the disease and/or the therapy they receive. At the beginning of COVID-19 pandemic there was a great concern about the possibility of severe SARS-CoV-2 infection in these patients. The best method of protection is vaccination, so as soon as vaccine was licenced, we aimed to vaccinate them. Data on disease relapse rate after COVID-19 infection and vaccination are scarce, but they play important role in everyday clinical decisions. Methods The aim of this study was to determine the relapse rate of autoimmune rheumatic disease (ARD) after COVID-19 infection and vaccination. Data on demographic, diagnosis, disease activity, therapy, clinical presentation of the infection and serology were collected from pARD who had COVID-19 and from pARD who were vaccinated against COVID-19, from March 2020 to April 2022. All vaccinated patients received two doses of the BNT162b2 BioNTech vaccine, on average, 3.7 (S.D.=1.4) weeks apart. Activity of the ARD was followed prospectively. Relapse was defined as a worsening of the ARD in a time frame of 8 weeks after infection or vaccination. For statistical analysis, Fisher’s exact test and Mann-Whitney U test were used. Results We collected data from 115 pARD, which we divided into two groups. We included 92 pARD after infection and 47 after vaccination, with 24 in both groups (they were infected before/after vaccination). In 92 pARD we registered 103 SARS-CoV-2 infections. Infection was asymptomatic in 14%, mild in 67% and moderate in 18%, 1% required hospitalization; 10% had a relapse of ARD after infection and 6% after vaccination. There was a trend towards higher disease relapse rate after infection compared to vaccination, but the difference was not statistically significant (p = 0.76). No statistically significant difference was detected in the relapse rate depending on the clinical presentation of the infection (p = 0.25) or the severity of the clinical presentation of COVID-19 between vaccinated and unvaccinated pARD (p = 0.31). Conclusions There is a trend towards a higher relapse rate in pARD after infection compared to vaccination and connection between the severity of COVID-19 and vaccination status is plausible. Our results were, however, not statistically significant.

Monkeypox virus was imported into Finland during late May–early June 2022. Intrahost viral genome variation in a sample from 1 patient comprised a major variant with 3 lineage B.1.3–specific mutations and a minor variant with ancestral B.1 nucleotides. Results suggest either ongoing APOBEC3 enzyme–mediated evolution or co-infection.

Shortly after the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cases of viral, bacterial, and fungal coinfections in hospitalized patients became evident. This retrospective study investigates the prevalence of multiple pathogen co-detections in 1472 lower respiratory tract (LRT) samples from 229 SARS-CoV-2-positive patients treated in the largest intensive care unit (ICU) in Slovenia. In addition to SARS-CoV-2, (rt)RT-PCR tests were used to detect cytomegalovirus (CMV), Epstein–Barr virus (EBV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), and atypical bacteria: Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila/spp. At least one co-detection was observed in 89.1% of patients. EBV, HSV-1, and CMV were the most common, with 74.7%, 58.1%, and 38.0% of positive patients, respectively. The median detection time of EBV, HSV-1, and CMV after initial SARS-CoV-2 confirmation was 11 to 20 days. Bronchoalveolar lavage (BAL) and tracheal aspirate (TA) samples showed equivalent performance for the detection of EBV, CMV, and HSV-1 in patients with both available samples. Our results indicate that SARS-CoV-2 infection could be a risk factor for latent herpesvirus reactivation, especially HSV-1, EBV, and CMV. However, additional studies are needed to elucidate the clinical importance of these findings.

Tick-borne encephalitis (TBE) usually has a biphasic course which begins with unspecific febrile illness, followed by central nervous system involvement. Because TBE is not yet suspected during the initial phase, knowledge of early TBE pathogenesis is incomplete. Herein we evaluated laboratory and immune findings in the initial and second (meningoencephalitic) phase of TBE in 88 well-defined adult patients. Comparison of nine laboratory blood parameters in both phases of TBE revealed that laboratory abnormalities, consisting of low leukocyte and platelet counts and increased liver enzymes levels, were predominately associated with the initial phase of TBE and resolved thereafter. Assessment of 29 immune mediators in serum during the initial phase, and in serum and cerebrospinal fluid (CSF) during the second phase of TBE revealed highly distinct clustering patterns among the three groups. In the initial phase of TBE, the primary finding in serum was a rather heterogeneous immune response involving innate (CXCL11), B cell (CXCL13, BAFF), and T cell mediators (IL-27 and IL-4). During the second phase of TBE, growth factors associated with angiogenesis (GRO-α and VEGF-A) were the predominant characteristic in serum, whereas innate and Th1 mediators were the defining feature of immune responses in CSF. These findings imply that distinct immune processes play a role in the pathophysiology of different phases of TBE and in different compartments.

In this article, we report on a rare case of acute respiratory distress syndrome (ARDS) caused by the Puumala orthohantavirus (PUUV), which is typically associated with hemorrhagic fever with renal syndrome (HFRS). This is the first documented case of PUUV-associated ARDS in Southeast Europe. The diagnosis was confirmed by serum RT-PCR and serology and corroborated by phylogenetic analysis and chemokine profiling. The patient was a 23-year-old male from Zagreb, Croatia, who had recently traveled throughout Europe. He presented with fever, headache, abdominal pain, and sudden onset of ARDS. Treatment involved high-flow nasal cannula oxygen therapy and glucocorticoids, which resulted in a full recovery. A systematic literature review identified 10 cases of hantavirus pulmonary syndrome (HPS) caused by PUUV in various European countries and Turkey between 2002 and 2023. The median age of patients was 53 years (range 24–73), and six of the patients were male. Most patients were treated in intensive care units, but none received antiviral therapy targeting PUUV. Eight patients survived hospitalization. The presented case highlights the importance of considering HPS in the differential diagnosis of ARDS, even in areas where HFRS is the dominant form of hantavirus infection.

Tick-borne encephalitis (TBE) is a viral infection of the human central nervous system caused by the TBE virus (TBEV). The most effective protective measure against TBE is vaccination. Despite the highly immunogenic vaccine, cases of vaccine breakthroughs (VBTs) occur. One of the first targets of infection is dendritic cells (DC), which represent a fundamental bridge between innate and adaptive immunity through antigen presentation, costimulation, and cytokine production. Therefore, we investigated the activation and maturation of DCs and cytokine production after in vitro TBEV stimulation of peripheral blood mononuclear cells (PBMCs) obtained from VBT and unvaccinated TBE patients. Our results showed that the expression of HLA-DR and CD86 on DCs, was upregulated to a similar extent in both vaccinated and unvaccinated TBE patients but differed in cytokine production after stimulation with TBEV. PBMCs from patients with VBT TBE responded with lower levels of IFN-α and the proinflammatory cytokines IL-12 (p70) and IL-15 after 24- and 48-hour in vitro stimulation with TBEV, possibly facilitating viral replication and influencing the development of cell-mediated immunity. On the other hand, significantly higher levels of IL-6 in addition to an observed trend of higher expression of TNF-α measured after 6 days of in vitro stimulation of PBMC could support disruption of the blood–brain barrier and promote viral and immune cell influx into the CNS, leading to more severe disease in VBT TBE patients.

Lyme neuroborreliosis (LNB) in Europe may manifest with painful meningoradiculoneuritis (also known as Bannwarth syndrome) or lymphocytic meningitis with or without cranial neuritis (peripheral facial palsy). We assessed host immune responses and the prevalence of TLR1 (toll-like receptor 1)–1805GG polymorphism to gain insights into the pathophysiology of these conditions. Regardless of LNB manifestation, most mediators associated with innate and adaptive immune responses were concentrated in cerebrospinal fluid; serum levels were unremarkable. When stratified by specific clinical manifestation, patients with meningoradiculoneuritis had higher levels of B-cell chemoattractants CXC motif chemokine ligand (CXCL) 12 and CXCL13 and T-cell–associated mediators CXCL9, CXCL10, and interleukin 17, compared with those without radicular pain. Moreover, these patients had a higher frequency of TLR1–1805GG polymorphism and more constitutional symptoms. These findings demonstrate that meningoradiculoneuritis is a distinct clinical entity with unique immune and genetic pathophysiology, providing new considerations for the study of LNB and borrelial meningoradiculitis.

Information on febrile illness caused by tick-borne encephalitis virus (TBEV) without central nervous system involvement is limited. We characterized 98 patients who had TBEV RNA in their blood but no central nervous system involvement at the time of evaluation. Median duration of illness was 7 days; 37 (38%) patients were hospitalized. The most frequent findings were malaise or fatigue (98%), fever (97%), headache (86%), and myalgias (54%); common laboratory findings were leukopenia (88%), thrombocytopenia (59%), and abnormal liver test results (63%). During the illness, blood leukocyte counts tended to improve, whereas thrombocytopenia and liver enzymes tended to deteriorate. At the time of positive PCR findings, 0/98 patients had serum IgG TBEV and 7 serum IgM TBEV; all patients later seroconverted. Viral RNA load was higher in patients with more severe illness but did not differ substantially in relation to several other factors. Illness progressed to tick-borne encephalitis in 84% of patients within 18 days after defervescence.

We report a case of natural infection with severe acute respiratory syndrome coronavirus 2 transmitted from an owner to a pet ferret in the same household in Slovenia. The ferret had onset of gastroenteritis with severe dehydration. Whole-genome sequencing of the viruses isolated from the owner and ferret revealed a 2-nt difference.

BackgroundSARS-CoV-2 infection does not confer long immunity. However, studies suggest that prior infection is associated with lower risk of reinfection and milder outcomes of recurrent infections. The aims of this retrospective observational case-control study were to describe the clinical and molecular characteristics of genetically confirmed Delta reinfection cases and to assess the potential protective role of preceding infection on the severity of reinfection.MethodsWe used next generation sequencing (NGS) to explore if cases with two positive real time RT-PCR tests > 90 days apart were infected with a different SARS-CoV-2 variant. Cases with confirmed reinfection between August 1st and October 31st, 2021 (the Delta wave) in Slovenia were matched 1:4 by age, sex and timeframe (week of positive test) with individuals with primary infection. Sociodemographic and epidemiologic data, vaccination status, and data on hospitalization and outcome of infection were retrieved from several centralized and standardized national databases. Additional epidemiologic surveys were performed on a limited number of cases and controls.ResultsWe identified 628 cases of genetically confirmed reinfection during the study period and matched them with 2,512 control subjects with Delta primary infection. Primary infections in individuals with reinfection were mainly caused by B.1.258.17 (51.1%), followed by B.1.1.7 (15.1%) and reinfection was detected on average 271 days after primary infection (range 101–477 days). Our results show a substantially lower probability of hospitalization in cases with reinfection compared with controls (OR: 0.21, p = 0.017), but no significant difference was observed in intensive care unit admission and deaths. We observed a significantly lower proportion of vaccinated individuals among cases compared to controls (4.5% vs. 28.2%), suggesting that hybrid immunity leads to lower probability of reinfection. Detailed analysis of the temporal distribution of variants, responsible for reinfections, showed no significant differences in reinfection potential.ConclusionReinfection with the SARS-CoV-2 Delta variant resulted in fewer hospitalizations compared to the primary Delta infection, suggesting that primary infection may, to some extent, produce at least short lasting protective immunity. This study provides additional insight into the reinfection dynamics that may allow appropriate public health measures to be taken in subsequent waves of the COVID-19 pandemic.

Tularemia is a zoonosis caused by the highly invasive bacterium Francisella tularensis. It is transmitted to humans by direct contact with infected animals or by vectors, such as ticks, mosquitos, and flies. Even though it is well-known as a tick-borne disease, it is usually not immediately recognised after a tick bite. In Slovenia, tularemia is rare, with 1–3 cases reported annually; however, the incidence seems to be increasing. Ulceroglandular tularemia is one of its most common forms, with cervical colliquative lymphadenopathy as a frequent manifestation. The diagnosis of tularemia largely relies on epidemiological information, clinical examination, imaging, and molecular studies. Physicians should consider this disease a differential diagnosis for a neck mass, especially after a tick bite, as its management significantly differs from that of other causes. Tularemia-associated lymphadenitis is treated with antibiotics and surgical drainage of the colliquated lymph nodes. Additionally, tularemia should be noted for its potential use in bioterrorism on behalf of the causative agents’ low infectious dose, possible aerosol formation, no effective vaccine at disposal, and the ability to produce severe disease. This article reviews the recent literature on tularemia and presents a case of an adult male with tick-borne cervical ulceroglandular tularemia.

Escherichia coli is known to be an important uropathogenic agent. Several models were developed for investigating the uropathogensis of E. coli, including the recent biomimetic porcine urothelial in vitro model. The aim of this study was to assess the cytokine response of the cells of the biomimetic porcine urothelial model to different E. coli strains. The production of nine different cytokines in response to E. coli infection was evaluated using the commercial pre-configured immunoassay multiplex Cytokine & Chemokine 9-Plex Porcine ProcartaPlex™ Panel 1 kit. Our results showed that cells of the biomimetic porcine urothelial model reacted to the presence of all the employed different E. coli strains, albeit with some differences in levels and types of cytokines produced. Increased production of IL-10, IL-8, TNF-α, IL-1β, IL-4 and IL-12p40 was observed. Statistical analysis (Fisher’s exact test) revealed a correlation between the high fold change in the immune response and the presence of the cnf1 gene that encodes the cytotoxic necrotizing factor. Our results shed light on the cytokine response of normal urothelial cells to different E. coli strains and have the potential to fuel the search for understanding the mechanisms behind the different cytokine responses to different E. coli strains.

The clinical symptoms caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are nonspecific and can be associated with most other respiratory viruses that cause acute respiratory tract infections (ARI). Because the clinical differentiation of COVID-19 patients from those with other respiratory viruses is difficult, the evaluation of automated methods to detect important respiratory viruses together with SARS-CoV-2 seems necessary. Therefore, this study compares two molecular assays for the detection of respiratory viruses, including SARS-CoV-2: the Respiratory Viruses 16-Well Assay (AusDiagnostics, Pty Ltd., Mascot, Australia) and the Allplex™ RV Essential Assay coupled with the Allplex™-nCoV Assay (Seegene Inc., Seoul, Korea). The two methods (AusDiagnostics and AlplexTM-nCoV Assay SARS-CoV-2) had 98.6% agreement with the reference method, cobas 6800, for the detection of SARS-CoV-2. Agreement between the AusDiagnostics assay and the AlplexTM RV Essential Assay for the detection of seven respiratory viruses was 99%. In our experience, the Respiratory Viruses 16-Well Assay proved to be the most valuable and useful medium-throughput method for simultaneous detection of important respiratory viruses and SARS-CoV-2. The main advantages of the method are high specificity for all targets included and their simultaneous detection and medium throughput with the option of having multiple instruments provide a constant run.

The clinical presentation of tick-borne encephalitis virus (TBEV) infection varies from asymptomatic to severe meningoencephalitis or meningoencephalomyelitis. The TBEV subtype has been suggested as one of the most important risk factors for disease severity, but TBEV genetic characterization is difficult. Infection is usually diagnosed in the post-viremic phase, and so relevant clinical samples of TBEV are extremely rare and, when present, are associated with low viral loads. To date, only two complete TBEV genomes sequenced directly from patient clinical samples are publicly available. The aim of this study was to develop novel protocols for the direct sequencing of the TBEV genome, enabling studies of viral genetic determinants that influence disease severity. We developed a novel oligonucleotide primer scheme for amplification of the complete TBEV genome. The primer set was tested on 21 clinical samples with various viral loads and collected over a 15-year period using the two most common sequencing platforms. The amplicon-based strategy was compared to direct shotgun sequencing. Using the novel primer set, we successfully obtained nearly complete TBEV genomes (>90% of genome) from all clinical samples, including those with extremely low viral loads. Comparison of consensus sequences of the TBEV genome generated using the novel amplicon-based strategy and shotgun sequencing showed no difference. We conclude that the novel primer set is a powerful tool for future studies on genetic determinants of TBEV that influence disease severity and will lead to a better understanding of TBE pathogenesis.

At the end of 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was discovered in China, causing a new coronavirus disease, termed COVID-19 by the WHO on February 11, 2020. At the time of this paper (January 31, 2021), more than 100 million cases have been recorded, which have claimed over 2 million lives worldwide. The most important clinical presentation of COVID-19 is severe pneumonia; however, many patients present various neurological symptoms, ranging from loss of olfaction, nausea, dizziness, and headache to encephalopathy and stroke, with a high prevalence of inflammatory central nervous system (CNS) syndromes. SARS-CoV-2 may also target the respiratory center in the brainstem and cause silent hypoxemia. However, the neurotropic mechanism(s) by which SARS-CoV-2 affects the CNS remain(s) unclear. In this paper, we first address the involvement of astrocytes in COVID-19 and then elucidate the present knowledge on SARS-CoV-2 as a neurotropic virus as well as several other neurotropic flaviviruses (with a particular emphasis on the West Nile virus, tick-borne encephalitis virus, and Zika virus) to highlight the neurotropic mechanisms that target astroglial cells in the CNS. These key homeostasis-providing cells in the CNS exhibit many functions that act as a favorable milieu for virus replication and possibly a favorable environment for SARS-CoV-2 as well. The role of astrocytes in COVID-19 pathology, related to aging and neurodegenerative disorders, and environmental factors, is discussed. Understanding these mechanisms is key to better understanding the pathophysiology of COVID-19 and for developing new strategies to mitigate the neurotropic manifestations of COVID-19.

Orthohantaviruses (family Hantaviridae, order Bunyavirales) can cause two serious syndromes in humans: hemorrhagic fever with renal syndrome (HFRS), associated with the Old World orthohantaviruses, and hantavirus cardiopulmonary syndrome (HCPS), associated with orthohantaviruses in the Americas. In Europe, four different orthohantaviruses (DOBV, PUUV, SEOV, and TULV) are associated with human disease. As disease severity and zoonotic source differ between orthohantavirus species, conclusive determination of the infecting species by either RT-PCR or comparative virus neutralization test (VNT) is of importance. Currently, the focus reduction neutralization test (FRNT) is considered the ‘Gold Standard’ for orthohantavirus VNTs, however this test is laborious and time-consuming. Consequently, more high-throughput alternatives are needed. In this study, we developed a comparative orthohantavirus microneutralization test (MNT) including all four human pathogenic orthohantavirus species circulating in Europe. The assay was validated using RT-PCR-confirmed rodent (n=17) and human sera (n=17), DOBV-suspected human sera (n=3) and cohorts of orthohantavirus-negative rodent (n=3) and human sera (n=85). 16/17 RT-PCR-confirmed rodent sera and 18/20 of the RT-PCR-confirmed and DOBV-suspected human sera were serotyped successfully, while for the remaining rodent (n=1) and human sera (n=2) no neutralizing titers could be detected. All negative control sera tested negative in the MNT. The assay was subsequently evaluated using a clinical cohort of 50 orthohantavirus patients. Orthohantavirus infection was confirmed in all 50 patients, and 47/50 (94%) sera were serotyped successfully, confirming PUUV as the major cause of orthohantavirus infections in Netherlands. Notably, two previously unrecognized SEOV cases from 2013 were diagnosed using the MNT, underlining the added value of the MNT in a diagnostic setting. In conclusion, we demonstrate the successful development and clinical implementation of a comparative European orthohantavirus MNT to determine the infecting virus species in European HFRS patients. Identification of the causative species is needed for an adequate Public Health response and can support individual patient care. For many labs, the implementation of orthohantavirus neutralization tests has not been a straightforward procedure. This issue will be addressed by the rollout of the comparative MNT to multiple European laboratories to support patient diagnostics, surveillance and Public Health responses.

Hemorrhagic fever with renal syndrome (HFRS), caused by Dobrava (DOBV) and Puumala (PUUV) orthohantaviruses, is an endemic disease in Slovenia. DOBV is mainly responsible for a more severe disease, whereas PUUV usually causes a milder form. Therefore, the aim of our study was to determine whether any differences in lymphocyte population in patients infected with these two viruses exist. Mononuclear cells from peripheral blood (PBMCs) were isolated from DOBV or PUUV infected patients and different lymphocyte subpopulations were analyzed with flow cytometry. Decreased concentrations of lymphocyte subpopulation were observed in DOBV and in PUUV infected patients compared with a healthy control, which was especially evident in DOBV infected patients. The lower values of T cells are likely due to the extravasation of the activated cells from the circulation to the infected tissue. Higher percentage of NK cells were detected in DOBV infected patients in comparison to PUUV infected patients, which could be associated with a more severe HFRS caused by DOBV. PUUV infected patients had a significantly higher concentration of activated T cell subsets, expressing markers CD25, CD69, and HLA-DR in comparison to DOBV infected patients. Higher activation of T cell subsets in PUUV infected patients could be a contributor to a milder HFRS. Further studies are necessary to elucidate the relation between the protective and the harmful role of activated lymphocytes subsets in HFRS pathogenesis.

Flaviviruses are relevant animal and human pathogens of increasing importance worldwide. The similarities of the initial clinical symptoms and the serological cross-reactivity of viral structural antigens make a laboratory diagnosis of flavivirus infection problematic. The main aim of the present study was the comparative specificity and sensitivity analysis of the non-structural protein NS1 as an antigen to detect flavivirus antibodies in sera from exposed individuals. A strategy for the purification of native recombinant non-structural protein 1 of representative flaviviruses including tick-borne encephalitis, West Nile, Zika and dengue virus was developed. The immunological properties of the purified antigens were analyzed using sera of immunized mice and of infected individuals in comparison with standard commercial assays. Recombinant NS1 protein was confirmed as a valuable option for the detection of flavivirus antibodies with reduced cross-reactivity and high sensitivity offering additional advantages for the detection of vaccine breakthrough cases.

Abstract Background In the past decade, many new paramyxoviruses that do not belong to any of the seven established genera in the family Paramyxoviridae have been discovered. Amongst them are J-virus (JPV), Beilong virus (BeiPV) and Tailam virus (TlmPV), three paramyxovirus species found in rodents. Based on their similarities, it has been suggested that these viruses should compose a new genus, tentatively called ‘Jeilongvirus’. Results Here we present the complete genomes of three newly discovered paramyxoviruses, one found in a bank vole (Myodes glareolus) from Slovenia and two in a single, co-infected Rungwe brush-furred rat (Lophuromys machangui) from Mozambique, that represent three new, separate species within the putative genus ‘Jeilongvirus’. The genome organization of these viruses is similar to other paramyxoviruses, but like JPV, BeiPV and TlmPV, they possess an additional open reading frame, encoding a transmembrane protein, that is located between the F and G genes. As is the case for all Jeilongviruses, the G genes of the viruses described here are unusually large, and their encoded proteins are characterized by a remarkable amino acid composition pattern that is not seen in other paramyxoviruses, but resembles certain motifs found in Orthopneumovirus G proteins. Conclusions The phylogenetic clustering of JPV, BeiPV and TlmPV with the viruses described here, as well as their shared features that set them apart from other paramyxoviruses, provide additional support for the recognition of the genus ‘Jeilongvirus’.

Tick-borne encephalitis: One vaccine protects against multiple flaviviruses Prior exposure to tick-borne encephalitis virus confers immunity to distinct, but genetically related, significant human pathogens. The tick-borne encephalitis complex comprises multiple ‘flaviviruses’; however, a licensed vaccine only exists for one: tick-borne encephalitis virus (TBEV). Alexander McAuley, of the University of Texas Medical Branch, USA, and collaborators tested human serum samples from donors who had either received the TBEV vaccine, been infected with the virus naturally, or had contracted the virus despite prior vaccination. The team tested the sera against six genetically related flaviviruses, looking for antibodies that indicate viral immunity. The results showed that TBEV exposure induce production of antibodies that inhibit other flaviviruses, the efficacy of which decreases as the genetic variation from TBEV increases. Additionally, sera from infected individuals generated more antibodies than sera from vaccinated donors. This study provides an insight into the relationship between genetic similarity and vaccine cross-protection.

Monitoring infectious diseases is a crucial part of preventive veterinary medicine in zoological collections. This zoo environment contains a great variety of animal species that are in contact with wildlife species as a potential source of infectious diseases. Wild birds may be a source of West Nile virus (WNV) and Usutu (USUV) virus, which are both emerging pathogens of rising concern. The aim of this study was to use zoo animals as sentinels for the early detection of WNV and USUV in Slovenia. In total, 501 sera from 261 animals of 84 animal species (including birds, rodents, lagomorphs, carnivores, ungulates, reptiles, equids, and primates) collected for 17 years (2002–2018) were tested for antibodies to WNV and USUV. Antibodies to WNV were detected by indirect immunofluorescence tests in 16 (6.1%) of 261 animals representing 10 species, which were sampled prior to the first active cases of WNV described in 2018 in Slovenia in humans, a horse, and a hooded crow (Corvus cornix). Antibodies to USUV were detected in 14 out of 261 animals tested (5.4%) that were positive prior to the first positive cases of USUV infection in common blackbirds (Turdus merula) in Slovenia. The study illustrates the value of zoological collections as a predictor of future emerging diseases.

The biphasic course of tick-borne encephalitis (TBE) is well described, but information on the monophasic course is limited. We assessed and compared the clinical presentation, laboratory findings, and immune responses in 705 adult TBE patients: 283 with monophasic and 422 with biphasic course. Patients with the monophasic course were significantly (p ≤ 0.002) older (57 vs. 50 years), more often vaccinated against TBE (7.4% vs. 0.9%), more often had comorbidities (52% vs. 37%), and were more often treated in the intensive care unit (12.4% vs. 5.2%). Multivariate logistic regression found strong association between the monophasic TBE course and previous TBE vaccination (OR = 18.45), presence of underlying illness (OR = 1.85), duration of neurologic involvement before cerebrospinal fluid (CSF) examination (OR = 1.39), and patients’ age (OR = 1.02). Furthermore, patients with monophasic TBE had higher CSF levels of immune mediators associated with innate and adaptive (Th1 and B-cell) immune responses, and they had more pronounced disruption of the blood–brain barrier. However, the long-term outcome 2–7 years after TBE was comparable. In summary, the monophasic course is a frequent and distinct presentation of TBE that is associated with more difficult disease course and higher levels of inflammatory mediators in CSF than the biphasic course; however, the long-term outcome is similar.

Tick-borne encephalitis (TBE) is associated with a range of disease severity. The reasons for this heterogeneity are not clear. Levels of serum IgG antibodies to TBE virus (TBEV) were determined in 691 adult patients during the meningoencephalitic phase of TBE and correlated with detailed clinical and laboratory parameters during acute illness and with the presence of post-encephalitic syndrome (PES) 2–7 years after TBE. Specific IgG antibody levels ranged from below cut-off value (in 32/691 patients, 4.6%), to 896 U/mL (median = 37.3 U/mL). Patients with meningoencephalomyelitis were more often seronegative (24.3%; 9/37) than those with meningoencephalitis (4.7%; 20/428) or meningitis (1.3%; 3/226). Moreover, patients with antibody levels below cut-off had longer hospitalization (13 versus 8 days); more often required intensive care unit treatment (22% versus 8%) and artificial ventilation (71% versus 21%); and had a higher fatality rate (3/32; 9.4% versus 1/659; 0.2%) than seropositive patients. These results were confirmed when antibody levels, rather than cut-off values, were correlated with clinical parameters including the likelihood to develop PES. Low serum IgG antibody responses against TBEV at the onset of neurologic involvement are associated with a more difficult clinical course and unfavorable long-term outcome of TBE, providing a diagnostic and clinical challenge for physicians.

West Nile virus (WNV) is a flavivirus transmitted by mosquitoes. Birds are the reservoir for the virus; humans, horses and other mammals are dead-end hosts. Infections caused by WNV in humans can vary from asymptomatic infections to West Nile fever (WNF) or West Nile neuroinvasive disease (WNND). In 1995, a serosurvey was performed in Slovenia on forest workers, and WNV specific IgG antibodies were confirmed in 6.8% of the screened samples, indicating that WNV is circulating in Slovenia. No human disease cases were detected in Slovenia until 2013, when the first case of WNV infection was confirmed in a retrospective study in a 79-year old man with meningitis. In 2018, three patients with WNND were confirmed by laboratory tests, with detection of IgM antibodies in the cerebrospinal fluid of the patients. In one of the patients, WNV RNA was detected in the urine sample. In 2017, 2018 and 2019, a mosquito study was performed in Slovenia. Mosquitoes were sampled on 14 control locations and 35 additional locations in 2019. No WNV was detected in mosquitoes in 2017 and 2019, but we confirmed the virus in a pool of Culex sp. mosquitoes in 2018. The virus was successfully isolated, and complete genome sequence was acquired. The whole genome of the WNV was also sequenced from the patient’s urine sample. The whole genome sequences of the WNV virus detected in Slovenian patient and mosquito indicate the virus most likely spread from the north, because of the geographic proximity and because the sequences cluster with the Austrian and Hungarian sequences. A sentinel study was performed on dog sera samples, and we were able to confirm IgG antibodies in 1.8% and 4.3% of the samples in 2017 and 2018, respectively. Though Slovenia is not a highly endemic country for WNV, we have established that the virus circulates in Slovenia.

BACKGROUND:Information on the sequential appearance, duration, and magnitude of clinical and laboratory parameters in hemorrhagic fever with renal syndrome (HFRS) is limited. METHODS:Analysis of clinical and laboratory parameters obtained serially in 81 patients with HFRS, of whom 15 were infected with Dobrava virus and 66 with Puumala virus. RESULTS:The initial signs/symptoms, appearing on median day 1 of illness, were fever, headache, and myalgia. These were present in 86%, 65%, and 40% of patients and had a median duration of 4, 4, and 5.5 days, respectively. The signs/symptoms were followed by myopia (appearance on day 5), insomnia (day 6), oliguria/anuria (day 6), polyuria (day 9), and sinus bradycardia (day 9.5). These were present in 35%, 30%, 28%, 91%, and 35% of patients; their median duration was 2, 2, 2, 7, and 1 day, respectively. Laboratory abnormalities, including thrombocytopenia, elevated alanine aminotransferase, CRP, procalcitonin, creatinine, diminished glomerular filtration rate, and leukocytosis, were ascertained on admission to hospital or on the following day (day 5 or 6 of illness) and were established in 95%, 87%, 99%, 91%, 94%, 87%, and 55% of patients, and had a median duration of 4, 3, 7, 3, 9, 8, and 2 days, respectively. Comparison of patients infected with Dobrava and Puumala viruses found several differences in the frequency, magnitude, and duration of abnormalities, indicating that Dobrava virus causes the more severe HFRS. CONCLUSIONS:In the majority of patients, the classic clinical distinction into febrile, hypotonic, oliguric, polyuric, and convalescent phases of illness is unclear.

OBJECTIVES:Although statins have anti-inflammatory and potentially also antimicrobial (including antiviral) activity, their therapeutic impact on infectious diseases is controversial. In this study, we evaluated whether pre-existing statin use influenced the course and outcome of tick-borne encephalitis. METHODS:To assess the influence of statin usage on the severity of acute illness and the outcome of tick-borne encephalitis, univariate and multivariable analyses were performed for 700 adult patients with tick-borne encephalitis of whom 77 (11%) were being treated with statins, and for 410 patients of whom 53 (13%) were receiving statins, respectively. RESULTS:Multivariable analyses found no statistically significant association between statin usage and having a milder acute illness. There was also no statistically significant benefit with respect to a favorable outcome defined by the absence of post-encephalitic syndrome (ORs for a favorable outcome at 6 months was 0.96, 95% CI: 0.46-2.04, P = 0.926; at 12 months 0.29, 95% CI: 0.06-1.33, P = 0.111; at 2-7 years after acute illness 0.44, 95% CI: 0.09-2.22, P = 0.321), by a reduction in the frequency of six nonspecific symptoms (fatigue, myalgia/arthralgia memory disturbances, headache, concentration disturbances, irritability) occurring during the 4 week period before the last examination, or by higher SF-36 scores in any of the eight separate domains of health as well as in the physical and mental global overall component. Furthermore, there were no significant differences between patients receiving statins and those who were not in the cerebrospinal fluid or serum levels for any of the 24 cytokines/chemokines measured. CONCLUSIONS:In this observational study, we could not prove that pre-existing use of statins affected either the severity of the acute illness or the long-term outcome of tick-borne encephalitis.

Information on the association of inflammatory immune responses and disease outcome after tick-borne encephalitis (TBE) is limited. In the present study, we assessed the levels of 24 cytokines/chemokines associated with innate and adaptive immune responses in matched serum and cerebrospinal fluid (CSF) samples of 81 patients at first visit, and in serum at follow-up time points. Serum levels of several cytokines/chemokines obtained during the meningoencephalitic phase of TBE differed compared to the levels at a follow-up visit 2 months later; several significant differences were also found in cytokine/chemokine levels in serum at 2 months compared to the last time point, 2−7 years after acute illness. Cytokines/chemokines levels in CSF or serum obtained at the time of acute illness or serum levels obtained 2 months after the onset of TBE did not have predictive value for an unfavorable outcome 2−7 years later. In contrast, serum levels of mediators associated with Th17 responses were lower in patients with unfavorable outcome whereas those associated with other adaptive or innate immune responses were higher at the last visit in those with an unfavorable outcome. These findings provide new insights into the immunopathogenesis of TBE and implicate inflammatory immune responses with post-encephalitic syndrome years after the initial infection.

Hemorrhagic fever with renal syndrome (HFRS) and Crimean-Congo hemorrhagic fever (CCHF) are important viral hemorrhagic fevers (VHF), especially in the Balkan region. Infections with Dobrava or Puumala orthohantavirus and Crimean-Congo hemorrhagic fever orthonairovirus can vary from a mild, nonspecific febrile illness, to a severe disease with a fatal outcome. The pathogenesis of both diseases is poorly understood, but it has been suggested that a host’s immune mechanism might influence the pathogenesis of the diseases and survival. The aim of our study is to characterize cytokine response in patients with VHF in association with the disease progression and viral load. Forty soluble mediators of the immune response, coagulation, and endothelial dysfunction were measured in acute serum samples in 100 HFRS patients and 70 CCHF patients. HFRS and CCHF patients had significantly increased levels of IL-6, IL-12p70, IP-10, INF-γ, TNF-α, GM-CSF, MCP-3, and MIP-1b in comparison to the control group. Interestingly, HFRS patients had higher concentrations of serum MIP-1α, MIP-1β, which promote activation of macrophages and NK cells. HFRS patients had increased concentrations of IFN-γ and TNF-α, while CCHF patients had significantly higher concentrations of IFN-α and IL-8. In both, CCHF and HFRS patients’ viral load significantly correlated with IP-10. Patients with fatal outcome had significantly elevated concentrations of IL-6, IFN-α2 and MIP-1α, while GRO-α, chemokine related to activation of neutrophils and basophils, was downregulated. Our study provided a comprehensive characterization of biomarkers released in the acute stages of CCHF and HFRS.

Slovenia is a very diverse country from a natural geography point of view, with many different habitats within a relatively small area, in addition to major geological and climatic differences. It is therefore not surprising that several small mammal species have been confirmed to harbour hantaviruses: A. flavicollis (Dobrava virus), A. agrarius (Dobrava virus–Kurkino), M. glareolus (Puumala virus), S. areanus (Seewis virus),M. agrestis, M. arvalis and M. subterraneus (Tula virus). Three of the viruses, namely the Dobrava, Dobrava–Kurkino and Puumala viruses, cause disease in humans, with significant differences in the severity of symptoms. Due to changes in haemorrhagic fever with renal syndrome cases (HFRS) epidemiology, a detailed study on phylogenetic diversity and molecular epidemiology of pathogenic and non-pathogenic hantaviruses circulating in ecologically diverse endemic regions was performed. The study presents one of the largest collections of hantavirus L, M and S sequences obtained from hosts and patients within a single country. Several genetic lineages were determined for each hantavirus species, with higher diversity among non-pathogenic compared to pathogenic viruses. For pathogenic hantaviruses, a significant geographic clustering of human- and rodent-derived sequences was confirmed. Several geographic and ecological factors were recognized as influencing and limiting the formation of endemic areas.

Crimean-Congo hemorrhagic fever (CCHF) is a highly contagious viral tick-borne disease with case-fatality rates as high as 50%. We describe a collaborative evaluation of the characteristics, performance, and on-site applicability of serologic and molecular assays for diagnosis of CCHF. We evaluated ELISA, immunofluorescence, quantitative reverse transcription PCR, and low-density macroarray assays for detection of CCHF virus using precharacterized archived patient serum samples. Compared with results of local, in-house methods, test sensitivities were 87.8%–93.9% for IgM serology, 80.4%–86.1% for IgG serology, and 79.6%–83.3% for genome detection. Specificity was excellent for all assays; molecular test results were influenced by patient country of origin. Our findings demonstrate that well-characterized, reliable tools are available for CCHF diagnosis and surveillance. The on-site use of such assays by health laboratories would greatly diminish the time, costs, and risks posed by the handling, packaging, and shipping of highly infectious biologic material.

AbstractBackground: Tick-borne encephalitis is endemic in Slovenia, but still less than 10 % of people are regularly vaccinated. The proportion of vaccinated individuals was significantly influenced by obligatory vaccination for all Slovenian military conscripts between 1993 and 2003.Methods: Our study includes 73 men from the Celje region, who were vaccinated with three doses of vaccine against tick-borne meningoencephalitis FSME-Immun® (Baxter), but afterwards they stopped the vaccination for a period of 8 to 16 years. Participating men were serologically tested before and after the first booster dose. We used the enzyme immunoassay Enzygnost®.Results: The result of the analysis was, that with most of the participants the value of titer of antibodies before receiving revaccination was protective (n=67; 91,8 %), while after receiving a booster dose, the protective value was exceeded for all participants who have submitted a second blood sample (n=69; 94,5 %). Geometric mean concentration before the booster dose was 56 U/ml and 314 U/ml after the booster dose.Conclusions: Our study confirmed the long-term protection after primary vaccination with three doses against tick-borne encephalitis with men younger than 50 years, by using enzyme immunoassay. The results support the careful consideration of currently recommended revaccination interval.

Hemorrhagic fever with renal syndrome (HFRS) and Crimean-Congo hemorrhagic fever (CCHF) are common representatives of viral hemorrhagic fevers still often neglected in some parts of the world. Infection with Dobrava or Puumala virus (HFRS) and Crimean-Congo hemorrhagic fever virus (CCHFV) can result in a mild, nonspecific febrile illness or as a severe disease with hemorrhaging and high fatality rate. An important factor in optimizing survival rate in patients with VHF is instant recognition of the severe form of the disease for which significant biomarkers need to be elucidated. To determine the prognostic value of High Mobility Group Box 1 (HMGB1) as a biomarker for disease severity, we tested acute serum samples of patients with HFRS or CCHF. Our results showed that HMGB1 levels are increased in patients with CCHFV, DOBV or PUUV infection. Above that, concentration of HMGB1 is higher in patients with severe disease progression when compared to the mild clinical course of the disease. Our results indicate that HMGB1 could be a useful prognostic biomarker for disease severity in PUUV and CCHFV infection, where the difference between the mild and severe patients group was highly significant. Even in patients with severe DOBV infection concentrations of HMGB1 were 2.8-times higher than in the mild group, but the difference was not statistically significant. Our results indicated HMGB1 as a potential biomarker for severe hemorrhagic fevers.

Tick-borne encephalitis (TBE) developed in 3 persons in Slovenia who drank raw milk; a fourth person, who had been vaccinated against TBE, remained healthy. TBE virus RNA was detected in serum and milk of the source goat. Persons in TBE-endemic areas should be encouraged to drink only boiled/pasteurized milk and to be vaccinated.