Hantavirus infection-induced B cell activation elevates free light chains levels in circulation.

In humans, orthohantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). An earlier study reported that acute Andes virus HPS caused a massive and transient elevation in the number of circulating plasmablasts with specificity towards both viral and host antigens suggestive of polyclonal B cell activation. Immunoglobulins (Igs), produced by different B cell populations, comprise heavy and light chains; however, a certain amount of free light chains (FLCs) is constantly present in serum. Upregulation of FLCs, especially clonal species, associates with renal pathogenesis by fibril or deposit formations affecting the glomeruli, induction of epithelial cell disorders, or cast formation in the tubular network. We report that acute orthohantavirus infection increases the level of Ig FLCs in serum of both HFRS and HPS patients, and that the increase correlates with the severity of acute kidney injury in HFRS. The fact that the kappa to lambda FLC ratio in the sera of HFRS and HPS patients remained within the normal range suggests polyclonal B cell activation rather than proliferation of a single B cell clone. HFRS patients demonstrated increased urinary excretion of FLCs, and we found plasma cell infiltration in archival patient kidney biopsies that we speculate to contribute to the observed FLC excreta. Analysis of hospitalized HFRS patients' peripheral blood mononuclear cells showed elevated plasmablast levels, a fraction of which stained positive for Puumala virus antigen. Furthermore, B cells isolated from healthy donors were susceptible to Puumala virus in vitro, and the virus infection induced increased production of Igs and FLCs. The findings propose that hantaviruses directly activate B cells, and that the ensuing intense production of polyclonal Igs and FLCs may contribute to acute hantavirus infection-associated pathological findings.

Correction: Monocyte subset redistribution from blood to kidneys in patients with Puumala virus caused hemorrhagic fever with renal syndrome.

[This corrects the article DOI: 10.1371/journal.ppat.1009400.].

Monocyte subset redistribution from blood to kidneys in patients with Puumala virus caused hemorrhagic fever with renal syndrome.

Innate immune cells like monocytes patrol the vasculature and mucosal surfaces, recognize pathogens, rapidly redistribute to affected tissues and cause inflammation by secretion of cytokines. We previously showed that monocytes are reduced in blood but accumulate in the airways of patients with Puumala virus (PUUV) caused hemorrhagic fever with renal syndrome (HFRS). However, the dynamics of monocyte infiltration to the kidneys during HFRS, and its impact on disease severity are currently unknown. Here, we examined longitudinal peripheral blood samples and renal biopsies from HFRS patients and performed in vitro experiments to investigate the fate of monocytes during HFRS. During the early stages of HFRS, circulating CD14-CD16+ nonclassical monocytes (NCMs) that patrol the vasculature were reduced in most patients. Instead, CD14+CD16- classical (CMs) and CD14+CD16+ intermediate monocytes (IMs) were increased in blood, in particular in HFRS patients with more severe disease. Blood monocytes from patients with acute HFRS expressed higher levels of HLA-DR, the endothelial adhesion marker CD62L and the chemokine receptors CCR7 and CCR2, as compared to convalescence, suggesting monocyte activation and migration to peripheral tissues during acute HFRS. Supporting this hypothesis, increased numbers of HLA-DR+, CD14+, CD16+ and CD68+ cells were observed in the renal tissues of acute HFRS patients compared to controls. In vitro, blood CD16+ monocytes upregulated CD62L after direct exposure to PUUV whereas CD16- monocytes upregulated CCR7 after contact with PUUV-infected endothelial cells, suggesting differential mechanisms of activation and response between monocyte subsets. Together, our findings suggest that NCMs are reduced in blood, potentially via CD62L-mediated attachment to endothelial cells and monocytes are recruited to the kidneys during HFRS. Monocyte mobilization, activation and functional impairment together may influence the severity of disease in acute PUUV-HFRS.

Introduction and Rapid Spread of SARS-CoV-2 Omicron Variant and Dynamics of BA.1 and BA.1.1 Sublineages, Finland, December 2021.

Multiple introductions of SARS-COV-2 Omicron variant BA.1 and BA.1.1. lineages to Finland were detected in early December 2021. Within 3 weeks, Omicron overtook Delta as the most common variant in the capital region. Sequence analysis demonstrated the emergence and spread through community transmission of a large cluster of BA.1.1 virus.

Urokinase plasminogen activator mediates changes in human astrocytes modeling fragile X syndrome.

The function of astrocytes intertwines with the extracellular matrix, whose neuron and glial cell-derived components shape neuronal plasticity. Astrocyte abnormalities have been reported in the brain of the mouse model for fragile X syndrome (FXS), the most common cause of inherited intellectual disability, and a monogenic cause of autism spectrum disorder. We compared human FXS and control astrocytes generated from human induced pluripotent stem cells and we found increased expression of urokinase plasminogen activator (uPA), which modulates degradation of extracellular matrix. Several pathways associated with uPA and its receptor function were activated in FXS astrocytes. Levels of uPA were also increased in conditioned medium collected from FXS hiPSC-derived astrocyte cultures and correlated inversely with intracellular Ca2+ responses to activation of L-type voltage-gated calcium channels in human astrocytes. Increased uPA augmented neuronal phosphorylation of TrkB within the docking site for the phospholipase-Cγ1 (PLCγ1), indicating effects of uPA on neuronal plasticity. Gene expression changes during neuronal differentiation preceding astrogenesis likely contributed to properties of astrocytes with FXS-specific alterations that showed specificity by not affecting differentiation of adenosine triphosphate (ATP)-responsive astrocyte population. To conclude, our studies identified uPA as an important regulator of astrocyte function and demonstrated that increased uPA in human FXS astrocytes modulated astrocytic responses and neuronal plasticity.

Heterozygous TLR3 Mutation in Patients with Hantavirus Encephalitis.

Puumala hantavirus (PUUV) hemorrhagic fever with renal syndrome (HFRS) is common in Northern Europe; this infection is usually self-limited and severe complications are uncommon. PUUV and other hantaviruses, however, can rarely cause encephalitis. The pathogenesis of these rare and severe events is unknown. In this study, we explored the possibility that genetic defects in innate anti-viral immunity, as analogous to Toll-like receptor 3 (TLR3) mutations seen in HSV-1 encephalitis, may explain PUUV encephalitis. We completed exome sequencing of seven adult patients with encephalitis or encephalomyelitis during acute PUUV infection. We found heterozygosity for the TLR3 p.L742F novel variant in two of the seven unrelated patients (29%, p = 0.0195). TLR3-deficient P2.1 fibrosarcoma cell line and SV40-immortalized fibroblasts (SV40-fibroblasts) from patient skin expressing mutant or wild-type TLR3 were tested functionally. The TLR3 p.L742F allele displayed low poly(I:C)-stimulated cytokine induction when expressed in P2.1 cells. SV40-fibroblasts from three healthy controls produced increasing levels of IFN-λ and IL-6 after 24 h of stimulation with increasing concentrations of poly(I:C), whereas the production of the cytokines was impaired in TLR3 L742F/WT patient SV40-fibroblasts. Heterozygous TLR3 mutation may underlie not only HSV-1 encephalitis but also PUUV hantavirus encephalitis. Such possibility should be further explored in encephalitis caused by these and other hantaviruses.

Zoonotic Virus Seroprevalence among Bank Voles, Poland, 2002-2010.

Bank voles in Poland are reservoirs of zoonotic viruses. To determine seroprevalence of hantavirus, arenavirus, and cowpox virus and factors affecting seroprevalence, we screened for antibodies against these viruses over 9 years. Cowpox virus was most prevalent and affected by extrinsic and intrinsic factors. Long-term and multisite surveillance is crucial.

Seroprevalence of lymphocytic choriomeningitis virus and Ljungan virus in Finnish patients with suspected neurological infections.

Directly-transmitted rodent-borne zoonotic viruses, such as lymphocytic choriomeningitis virus (LCMV) can cause nervous system infections. Rodent-borne Ljungan virus (LV) is considered potentially zoonotic possibly causing neurological symptoms. Our objective was to understand the role of these two viruses compared to other pathogens in causing neurological infections in Finnish patients. Routine screening data were available for 400 patients aged 5-50 years, collected from December 2013 to December 2014 with suspected neurological infection. Depending on symptoms, patients were variously tested for herpesviruses, enteroviruses, varicella zoster virus, and Mycoplasma pneumoniae, while those suspected of tick bite were further tested for Borrelia spp. and tick-borne encephalitis virus using antibody and/or nucleic acid tests. For 380 patients, we also screened the RNA and antibody prevalence of LCMV and LV in order to test if either of these viruses were the causative agent. Data collected indicated that the causative microbial agent was confirmed in only 15.5% of all Finnish patients with neurological symptoms, with M. pneumoniae (26 cases) being the most common causative agent found in sera, whereas Borrelia spp. (15), herpes simplex viruses (7), and enteroviruses (5) were the most common agents confirmed in the CSF. The seroprevalences for LV and LCMV were 33.8% and 5.0%, respectively, but no samples were PCR-positive. In this study, M. pneumoniae and Borrelia spp. were the most common causative agents of neurological infections in Finland. No LCMV or LV infections were detected. We conclude there was no association of LV with neurological diseases in this patient cohort.

Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells.

Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-ß-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-ß-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression.

Interferons Induce STAT1-Dependent Expression of Tissue Plasminogen Activator, a Pathogenicity Factor in Puumala Hantavirus Disease.

Hantaviruses are zoonotic viruses that show various degrees of vasculopathy in humans. In this study, we analyzed the regulation of 2 fibrinolytic parameters, tissue plasminogen activator (tPA) and its physiological inhibitor, plasminogen activator inhibitor 1 (PAI-1), in Puumala hantavirus (PUUV)-infected patients and in human microvascular endothelial cells. We detected strong upregulation of tPA in the acute phase of illness and in PUUV-infected macaques and found the tPA level to positively correlate with disease severity. The median levels of PAI-1 during the acute stage did not differ from those during the recovery phase. In concordance, hantaviruses induced tPA but not PAI-1 in microvascular endothelial cells, and the induction was demonstrated to be dependent on type I interferon. Importantly, type I and II interferons directly upregulated tPA through signal transducer and activator of transcription 1 (STAT1), which regulated tPA gene expression via a STAT1-responsive enhancer element. These results suggest that tPA may be a general factor in the immunological response to viruses.

Acute Human Inkoo and Chatanga Virus Infections, Finland.

Inkoo virus (INKV) and Chatanga virus (CHATV), which are circulating in Finland, are mosquitoborne California serogroup orthobunyaviruses that have a high seroprevalence among humans. Worldwide, INKV infection has been poorly described, and CHATV infection has been unknown. Using serum samples collected in Finland from 7,961 patients suspected of having viral neurologic disease or Puumala virus infection during the summers of 2001-2013, we analyzed the samples to detect California serogroup infections. IgM seropositivity revealed 17 acute infections, and cross-neutralization tests confirmed presence of INKV or CHATV infections. All children (<16 years of age) with INKV infection were hospitalized; adults were outpatients with mild disease, except for 1 who was hospitalized with CHATV infection. Symptoms included fever, influenza-like illness, nausea or vomiting, disorientation, nuchal rigidity, headache, drowsiness, and seizures. Although many INKV and CHATV infections appear to be subclinical, these viruses can cause more severe disease, especially in children.

Long-term hormonal follow-up after human Puumala hantavirus infection.

OBJECTIVE: Nephropathia epidemica (NE) is a haemorrhagic fever with renal syndrome (HFRS) caused by Puumala hantavirus (PUUV). Pituitary haemorrhage and hypopituitarism may complicate recovery from acute NE. DESIGN: Forty-seven of our recent cohort of 58 NE patients volunteered to be re-examined in order to estimate the burden of hormonal deficiency 4 to 8 years after the acute illness. Two patients had suffered from pituitary haemorrhage, but many others exhibited pituitary oedema during their acute infection. In this study, we searched for symptoms of hormonal deficiency, performed hormonal laboratory screening, and most patients underwent pituitary MRI examination. RESULTS: The pituitary size had diminished in all patients in whom MRI was performed (P < 0·001). One patient with acute phase haemorrhage had made a complete recovery while the other continued to require hormonal substitution. In addition, hormonal laboratory abnormalities were observed in nine other patients; these being attributable to several reasons, for example independent peripheral hormonal diseases, side effects of medication or other secondary causes such as obesity. None of them had signs of late-onset pituitary insufficiency caused by their previous NE. Health-related quality of life (mean and median 15D score) of patients was comparable to that of age-standardized general population. CONCLUSIONS: None of our patients had developed obvious late-onset hypopituitarism despite of the fact that pituitary gland can be affected during acute NE. We recommend requesting a history of hantavirus infection whenever the possibility of pituitary dysfunction is suspected at least in patients originating from regions with high NE infection rate.

Serological survey of Seewis virus antibodies in patients suspected for hantavirus infection in Finland; a cross-reaction between Puumala virus antiserum with Seewis virus N protein?

Puumala virus (PUUV, carried by Myodes glareolus) co-circulates with Seewis virus (SWSV, carried by Sorex araneus) in Finland. While PUUV causes 1000-3000 nephropathia epidemica (NE) cases annually, the pathogenicity of SWSV to man is unknown. To study the prevalence of SWSV antibodies in hantavirus fever-like patients' sera, we used recombinant SWSV nucleocapsid (N) protein as the antigen in ELISA, immunofluorescence assay (IFA) and immunoblotting. While characterizing the recombinant SWSV N protein, we observed that a polyclonal rabbit antiserum against PUUV N protein cross-reacted with SWSV N protein and vice versa. We initially screened 486 (450 PUUV-seronegative and 36 PUUV-seropositive) samples sent to Helsinki University Hospital Laboratory for PUUV serodiagnosis during 2002 and 2007 in an SWSV N protein IgG ELISA. In total, 4.2 % (19/450) of the PUUV-seronegative samples were reactive in the SWSV N protein IgG ELISA and none of the tested samples [43 PUUV-seronegative (weakly reactive in the SWSV IgG ELISA) and 15 random] were reactive in the SWSV N protein IgM ELISA. None of the IgG reactions could be confirmed by IFA or immunoblotting. Furthermore, among the 36 PUUV-seropositive samples three were reactive in SWSV N protein IgG and ten in SWSV N protein IgM ELISA. One PUUV-seropositive sample reacted with SWSV N protein in IFA and four in immunoblotting. Finally, we applied competitive ELISA to confirm that the observed reactivity was due to cross-reactivity rather than a true SWSV response. In conclusion, no evidence of SWSV infection was found among the 486 samples studied; however, we did demonstrate that PUUV antiserum cross-reacted with shrew-borne hantavirus N protein.

Rapid homogeneous immunoassay based on time-resolved Förster resonance energy transfer for serodiagnosis of acute hantavirus infection.

We recently introduced a homogeneous immunoassay based on time-resolved Förster resonance energy transfer (TR-FRET) elicited by fluorophore-labeled antigen and fluorophore-labeled protein L, bound by an immunoglobulin. As the first clinical application, we employ this approach (LFRET) in serodiagnosis of Puumala hantavirus (PUUV) infection. A reference panel containing serum from individuals with acute (n = 21) or past (n = 17) PUUV infection and from PUUV-seronegative individuals (n = 20) was used to define the parameters. The clinical assay performance was evaluated with a prospectively collected serum panel (panel 2; n = 153). Based on the results for panel 1, the threshold for positivity was set at a signal level that was 3-fold over background, while those with a signal <3-fold over the background level were considered PUUV seronegative. With panel 1, 20/21 acute- and 7/10 past-infection samples induced positive signals, compared to 0/20 seronegatives. With panel 2, a positive signal was obtained in 39/40 acute- and 4/10 past-infection samples, as opposed to 7/103 seronegatives. However, after IgG depletion, 58/61 acute-infection samples were LFRET positive, while all past-infection and seronegative samples were negative, corresponding to 100% specificity and 95% sensitivity in detection of acute PUUV infection. We demonstrate that the novel immunoassay is a promising tool for rapid serodiagnosis of acute Puumala virus infection.

Competitive Homogeneous Immunoassay for Rapid Serodiagnosis of Hantavirus Disease.

In this study, we describe a competitive homogeneous immunoassay that makes use of Förster resonance energy transfer (FRET) in rapid detection of pathogen-specific antibodies. The assay principle is based on competition between a monoclonal antibody (MAb) and serum antibodies to a given antigen. In the assay, named competitive FRET immunoassay (CFRET-IA), the FRET signal is induced if MAb carrying a donor label binds to an acceptor-labeled antigen. Specific antibodies in serum compete for antigen binding, resulting in reduced FRET signal. The proof-of-principle for the assay was obtained using donor-labeled Puumala virus nucleocapsid protein (PUUV-N) and acceptor-labeled anti-PUUV-N MAb. The assay was evaluated by analyzing 329 clinical samples comprising 101 from individuals with acute PUUV infection, 42 from individuals with past infection, and 186 from individuals with PUUV-seronegative sera, and the results were compared to those of reference tests. The rapid serodiagnostic test we introduced herein performed with 100% sensitivity and 99% specificity for diagnosing acute hantavirus disease.

Ezrin is down-regulated in diabetic kidney glomeruli and regulates actin reorganization and glucose uptake via GLUT1 in cultured podocytes.

Diabetic nephropathy is a complication of diabetes and a major cause of end-stage renal disease. To characterize the early pathophysiological mechanisms leading to glomerular podocyte injury in diabetic nephropathy, we performed quantitative proteomic profiling of glomeruli isolated from rats with streptozotocin-induced diabetes and controls. Fluorescence-based two-dimensional difference gel electrophoresis, coupled with mass spectrometry, identified 29 differentially expressed spots, including actin-binding protein ezrin and its interaction partner, NHERF2, which were down-regulated in the streptozotocin group. Knockdown of ezrin by siRNA in cultured podocytes increased glucose uptake compared with control siRNA-transfected cells, apparently by increasing translocation of glucose transporter GLUT1 to the plasma membrane. Knockdown of ezrin also induced actin remodeling under basal conditions, but reduced insulin-stimulated actin reorganization. Ezrin-dependent actin remodeling involved cofilin-1 that is essential for the turnover and reorganization of actin filaments. Phosphorylated, inactive cofilin-1 was up-regulated in diabetic glomeruli, suggesting altered actin dynamics. Furthermore, IHC analysis revealed reduced expression of ezrin in the podocytes of patients with diabetes. Our findings suggest that ezrin may play a role in the development of the renal complication in diabetes by regulating transport of glucose and organization of the actin cytoskeleton in podocytes.

Smoking is associated with aggravated kidney injury in Puumala hantavirus-induced haemorrhagic fever with renal syndrome.

BACKGROUND: Previous studies indicate that smoking affects the outcome of some infections and is a risk factor for Puumala virus (PUUV) infection. The aim of this study was to assess the effect of smoking on the clinical severity of PUUV infection and the prevalence of smoking in patients with PUUV infection. METHODS: A questionnaire on smoking habits was sent to 494 patients in 2012, who had been treated in Tampere University Hospital, Finland, for serologically confirmed PUUV infection during years 1982-2012. RESULTS: Of all patients, 357 (72%) participated. Maximum plasma creatinine level measured during acute illness was significantly higher in current smokers than in non-smokers (median: 273 versus 184 µmol/L, P < 0.001). Current smokers had a higher maximum blood leucocyte count than non-smokers (median: 10.8 versus 8.9 × 10(9)/L, P < 0.001) and they were younger than non-smokers (38 versus 45 years, P < 0.001). There were no differences between current smokers and non-smokers in the other variables reflecting the severity of PUUV infection. Altogether 51% were current smokers at the time of onset of the illness, 57% of males and 36% of females. During these years in Finland, smoking among males in the same aged population has decreased from 33 to 22% and among females, smoking has varied between 14 and 20%. CONCLUSIONS: Smoking is common in patients with PUUV infection. Current smokers suffer from more severe acute kidney injury (AKI) and they have higher leucocyte count than non-smokers in PUUV infection. Smoking cessation decreases the risk of severe AKI to the same level as observed in never-smokers.

Acute hantavirus infection induces galectin-3-binding protein.

Hantaviruses are zoonotic viruses that cause life-threatening diseases when transmitted to humans. Severe hantavirus infection is manifested by impairment of renal function, pulmonary oedema and capillary leakage. Both innate and adaptive immune responses contribute to the pathogenesis, but the underlying mechanisms are not fully understood. Here, we showed that galectin-3-binding protein (Gal-3BP) was upregulated as a result of hantavirus infection both in vitro and in vivo. Gal-3BP is a secreted glycoprotein found in human serum, and increased Gal-3BP levels have been reported in chronic viral infections and in several types of cancer. Our in vitro experiments showed that, whilst Vero E6 cells (an African green monkey kidney cell line) constitutively expressed and secreted Gal-3BP, this protein was detected in primary human cells only as a result of hantavirus infection. Analysis of Gal-3BP levels in serum samples of cynomolgus macaques infected experimentally with hantavirus indicated that hantavirus infection induced Gal-3BP also in vivo. Finally, analysis of plasma samples collected from patients hospitalized because of acute hantavirus infection showed higher Gal-3BP levels during the acute than the convalescent phase. Furthermore, the Gal-3BP levels in patients with haemorrhagic fever with renal syndrome correlated with increased complement activation and with clinical variables reflecting the severity of acute hantavirus infection.

Isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body disease.

Boid inclusion body disease (BIBD) is a progressive, usually fatal disease of constrictor snakes, characterized by cytoplasmic inclusion bodies (IB) in a wide range of cell types. To identify the causative agent of the disease, we established cell cultures from BIBD-positive and -negative boa constrictors. The IB phenotype was maintained in cultured cells of affected animals, and supernatants from these cultures caused the phenotype in cultures originating from BIBD-negative snakes. Viruses were purified from the supernatants by ultracentrifugation and subsequently identified as arenaviruses. Purified virus also induced the IB phenotype in naive cells, which fulfilled Koch's postulates in vitro. One isolate, tentatively designated University of Helsinki virus (UHV), was studied in depth. Sequencing confirmed that UHV is a novel arenavirus species that is distinct from other known arenaviruses including those recently identified in snakes with BIBD. The morphology of UHV was established by cryoelectron tomography and subtomographic averaging, revealing the trimeric arenavirus spike structure at 3.2-nm resolution. Immunofluorescence, immunohistochemistry, and immunoblotting with a polyclonal rabbit antiserum against UHV and reverse transcription-PCR (RT-PCR) revealed the presence of genetically diverse arenaviruses in a large cohort of snakes with BIBD, confirming the causative role of arenaviruses. Some snakes were also found to carry arenavirus antibodies. Furthermore, mammalian cells (Vero E6) were productively infected with UHV, demonstrating the potential of arenaviruses to cross species barriers. In conclusion, we propose the newly identified lineage of arenaviruses associated with BIBD as a novel taxonomic entity, boid inclusion body disease-associated arenaviruses (BIBDAV), in the family Arenaviridae.

Identification of linear human B-cell epitopes of tick-borne encephalitis virus.

BACKGROUND: Tick-borne encephalitis (TBE) is a central nervous system infection transmitted to humans by ticks. The causative agent, tick-borne encephalitis virus (TBEV), belongs to the genus Flavivirus (family Flaviviridae), which includes globally important arthropod-borne viruses, such as dengue, Yellow fever, Japanese encephalitis and West Nile viruses. Flaviviruses are highly cross-reactive in serological tests that are currently based on viral envelope proteins. The envelope (E) protein is the major antigenic determinant and it is known to induce neutralizing antibody responses. METHODS: We synthesized the full-length TBEV proteome as overlapping synthetic 18-mer peptides to find dominant linear IgG epitopes. To distinguish natural TBEV infections from responses to TBE immunization or other flavivirus infections, the peptides were probed with sera of patients infected with TBEV, West Nile virus (WNV) or dengue virus (DENV), sera from TBE vaccinees and negative control sera by SPOT array technique. RESULTS: We identified novel linear TBEV IgG epitopes in the E protein and in the nonstructural protein 5 (NS5). CONCLUSIONS: In this study, we screened TBEV structural and nonstructural proteins to find linear epitopes specific for TBEV. We found 11 such epitopes and characterized specifically two of them to be potential for differential diagnostics. This is the first report of identifying dominant linear human B-cell epitopes of the whole TBEV genome. The identified peptide epitopes have potential as antigens for diagnosing TBEV and to serologically distinguish flavivirus infections from each other.