Development and validation of a novel enzyme-linked immunosorbent assay for the differentiation of tick-borne encephalitis infections caused by different virus subtypes

Development and validation of a novel enzyme-linked immunosorbent assay for the differentiation of tick-borne encephalitis infections caused by different virus subtypes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development and validation of a novel enzyme-linked immunosorbent assay for the differentiation of tick-borne encephalitis infections caused by different virus subtypes Zane Freimane, Gerhard Dobler, Lidia Chitimia-Dobler, Guntis Karelis, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4546509/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Aug, 2024 Read the published version in Infection → Version 1 posted 9 You are reading this latest preprint version Abstract Objectives Tick-borne encephalitis (TBE) is an infection caused by the tick-borne encephalitis virus (TBEV) that can lead to symptoms of central nervous system inflammation. There are five subtypes of TBEV, three of which – European, Siberian and Far Eastern – occur in Europe. As it is thought that different subtype infections exhibit varying clinical courses and outcomes, serological differentiation of the virus subtypes is clearly important. However, to date, this has proved difficult to achieve. Methods An ELISA format was developed based on TBE virus NS1 antigen against the European, Siberian and Far Eastern subtype. The three NS1 antigens were biotechnologically produced in a human cell line and used for ELISA coating. Sera from German (European subtype) and Russian (Siberian and/or Far Eastern subtypes) TBE patients with positive TBEV IgG were used to test the reactivity against these three NS1 antigens. Results Testing of 14 German and 32 Russian TBEV IgG-positive sera showed that the ELISA was able to differentiate between TBEV European subtype and TBEV Siberian and Far Eastern subtype infections. Conclusions In geographical areas where two or more TBEV subtype infections can occur, the NS1-IgG ELISA developed here constitutes an important diagnostic tool to differentiate between European subtype infections and Siberian/Far Eastern subtype infections and to use the new assay for epidemiological studies to clarify the importance of particular subtype infections in an area. Consequently, it may help to better describe and anticipate the clinical courses and outcomes of particular TBEV subtype infections. NS1 antigen NS1 antibodies ELISA TBEV subtypes flavivirus Figures Figure 1 Figure 2 Figure 3 Introduction Tick-borne encephalitis (TBE) is a viral infectious disease of the central nervous system caused by the tick-borne encephalitis virus (TBEV) [ 1 ]. TBEV is predominantly transmitted to humans via the bite of an Ixodes tick. Less commonly, it can be transmitted by consumption of unpasteurised dairy products [ 1 , 2 ]. TBE is endemic in Northern, Central and Eastern Europe as well as parts of Asia, where it is one of the most common causes of viral meningitis and encephalitis [ 1 – 3 ]. The clinical course of the disease can be unpredictable, with symptoms ranging from febrile illness and mild meningitis through to severe encephalitis and even death [ 1 ]. Currently, there is no specific treatment for TBE. However, highly effective vaccines against TBEV are available in Europe (FSME-Immun®, Pfizer; Encepur®, Bavarian Nordic) and recommended for people living in or travelling to TBE endemic regions [ 4 ]. TBEV is a single-stranded RNA virus belonging to the Flaviviridae family and genus Flavivirus . Based on phylogenetic differences, TBEV can be divided into five different subtypes: European (TBEV-Eu), Siberian (TBEV-Sib), Far Eastern (TBEV-FE), Himalayan (TBEV-Him) and Baikalian (TBEV-Bkl). Three of the five subtypes are clinically relevant to humans, namely TBEV-Eu, TBEV-Sib and TBEV-FE [ 5 ]. At the amino acid level, the genetic difference can be up to 2% within a subtype and 5–6% between subtypes [ 6 ]. These subtypes are associated with different geographical distributions and clinical courses. The geographical distribution of each subtype mostly corresponds to the nominal region; however, there are some exceptions. For instance, the European subtype has been found in South Korea [ 7 ], Altai (Southern Siberia) and Irkutsk (Eastern Siberia). Furthermore, the Siberian subtype has been found to circulate in parts of Scandinavia [ 8 ], the Baltic states [ 9 ], Bosnia and Central Asia [ 10 ]. Additionally, the Far Eastern subtype has been detected in Southern Siberia, the Urals, the Baltic states [ 11 ] and Moldova [ 12 ]. Clinically, the European subtype is reported to be associated with a milder disease and lower case fatality rates (0.5–2%) compared to the Siberian and Far Eastern subtypes (case fatality rates between 5 and 20%) [ 13 , 14 ]. The European Centre for Disease Prevention and Control’s definition of a TBE case is as follows: a person with a laboratory-identified TBEV infection and symptoms of central nervous system inflammation. The laboratory diagnosis of TBE is mainly based on the detection of TBEV-specific IgM antibodies in cerebrospinal fluid (intrathecal production) and/or TBEV-specific IgM and IgG antibodies in serum [ 15 ]. Enzyme-linked immunosorbent assay (ELISA) is considered the standard method for the serological diagnosis of TBE. However, the currently available ELISAs have the fundamental disadvantage of cross-reactivity with other flavivirus infections or vaccination [ 16 ]. Presently, the most specific assay for serological differentiation of flavivirus antibodies is the virus neutralisation test; however, this test is unable to distinguish between different TBEV subtype infections. Therefore, serological differentiation of TBEV subtype infections in geographical regions with co-existence of two or more TBEV subtypes is not possible at this time and consequently data collection on the incidence, prevalence and clinical course of particular TBEV subtype infections is majorly restricted. A new approach has recently been applied to ELISA [ 17 ] and suspension multiplex immunoassay [ 18 , 19 ] to detect antibodies against TBEV non-structural protein 1 (NS1). NS1 is a glycoprotein that is central for viral RNA replication. It also induces an immune response against the virus, which may even play a role in protection [ 20 ]. In contrast to TBEV envelope (E) protein (the antigen commonly used in ELISA), NS1 protein elicits an immune response exclusively after wild-type natural infection, which could possibly be distinguishable between TBEV subtypes. The currently available vaccines against TBEV, namely FSME-Immun® and Encepur®, are highly purified, inactive and do not contain substantial amounts of NS1. Therefore, following vaccination, no TBEV replication occurs and generally there is no formation of NS1 protein nor development of NS1-specific antibodies. These aspects of TBE vaccination are extremely advantageous for the employment of NS1-IgG ELISA in TBE testing [ 17 , 19 ]. The aim of this study was to develop and validate a diagnostic method to detect NS1-IgG antibodies against the three main TBEV subtypes (European, Siberian and Far Eastern). Furthermore, we wanted to establish whether our NS1-IgG ELISA could serologically differentiate infections caused by TBEV-Eu, TBEV-Sib and TBEV-FE, which to date has not been possible. This diagnostic capability of an ELISA is of major importance in order to more fully understand each TBEV subtype’s epidemiology, pathogenicity, disease severity and vaccine effectiveness. Methods Ethics statement The study was approved by the Rīga Stradinš University Ethics Committee (No. 6 − 1/03/19; March 26, 2020). Only anonymous samples or sera for research purposes were used in the present analysis. Development of anti-TBEV NS1 IgG ELISA (NS1 ELISA) Three recombinant TBEV NS1 proteins (European – strain Neudörfl, Siberian – strain MucAr DZIF 22/237 and Far-Eastern – strain Sofjin) were purchased from The Native Antigen Company (United Kingdom). Antigens were produced in HEK-293 human cell lines, highly purified and presented in a hexameric, native folding state. TBEV-Sib-NS1-IgG ELISA and TBEV-FE-NS1-IgG ELISA were prepared as previously described for TBEV-Eu-NS1-IgG ELISA [ 17 ] with some modifications. Polystyrene plates (96-well) (Nunc Immuno MaxiSorp, Thermo Fisher Scientific, Waltham, Massachusetts, USA) were coated overnight at 4°C with 100 µl of the three recombinant TBEV NS1 antigens at a concentration of 1,000 pg/ml in carbonate buffer (0.6 M, pH 9.6) to test the optimal coating concentration. Wells were blocked with gelatine (PanReac AppliChem, Darmstadt, Germany) in phosphate-buffered saline (PBS) for 1 h at room temperature, after which antigen plates were stored at − 80°C until use. Sera were tested in triplicate against all three subtypes in parallel and average optical densities (ODs) of the different antigens were compared. For the screening of sera, TBE-IgM ELISA and TBE-IgG ELISA (both from Euroimmun, Lübeck, Germany) were used according to the instructions of the manufacturer. Study population and serum samples For the validation of NS1 ELISA, we analysed a total of 142 serum samples which were divided into six groups (Table 1 ). All sera, except sera in Group 2, were from the German National Consulting Laboratory for TBE and were anonymised prior to analysis. The samples were from TBE patients and patients with other confirmed diseases (including other flavivirus infections). In addition, samples from TBE vaccine recipients and individuals vaccinated against other flaviviruses (yellow fever virus, Japanese encephalitis virus) were included for flavivirus diagnostics. Sera were stored at − 80°C until use. Table 1 Study population groups Group Characteristics TBEV infection (SENSITIVITY analysis) Group 1 14 sera from acute TBEV-Eu cases, confirmed by specific IgM and IgG antibodies and TBEV-Eu-NS1-IgG ELISA. Group 2 32 sera from Russian patients with confirmed acute (IgG ELISA positive; neutralisation antibody positive) or past TBEV infection (non-TBEV-Eu; kindly provided by Tatiana Poponnikova in 2007) No TBEV infection (SPECIFICITY analysis) Group 3 9 sera with confirmed dengue fever infection: 6 patients with acute secondary dengue antibody response; 1 with acute primary dengue infection; 2 with past dengue infection. Group 4 7 sera from individuals with yellow fever vaccination and TBE vaccination. Group 5 40 sera from blood donors with confirmed TBEV-Eu vaccination (TBEV-IgG; neutralisation test). Group 6 40 sera from blood donors negative for both infection and vaccination (negative TBEV-IgG; negative neutralisation test). Group 3, consisting of nine sera from confirmed dengue fever cases, was included in the analysis to test the specificity of the assay (Table 2 ). Six sera (TM58, DEN-71, DEN-96, DEN-100, DEN-136, DEN-137) were from patients with an acute dengue fever infection (IgM positive) and a serological pattern of secondary dengue infection (i.e. high level of IgG antibodies against all flaviviruses tested, including anti-TBEV-IgG). One serum (DEN-72) was from a patient with acute primary dengue infection (high level of IgG against dengue virus; no antibodies against other flaviviruses tested). Two sera were from patients with a past dengue infection, one (DEN-131) from a patient with a primary antibody response (high level of specific anti-dengue IgG, no other anti-flavivirus IgG) and one (DEN-84) from a patient with a past secondary dengue infection (IgG cross-reaction against all tested flaviviruses). Table 2 IgG titres of sera in Group 3 against different flaviviruses No. ID Den-IgM Den-IgG YF-IgG WN-IgG TBE-IgG 1 TM58 80 20,480 10,240 2,560 5,120 2 DEN-71 80 1,280 640 640 640 3 DEN-72 40 320 < 10 < 10 < 10 4 DEN-84 20,000 20,000 > 20,000 6 DEN-100 20 2,560 2,560 1,280 640 7 DEN-131 < 10 320 < 10 < 10 < 10 8 DEN-136 40 2,560 2,560 2,560 2,560 9 DEN-137 40 5,120 5,120 10,000 5,120 Dengue fever; YF, yellow fever; WN, West Nile fever; TBE, tick-borne encephalitis. Performance of anti-TBEV NS1 IgG ELISA Each serum sample was diluted 1:100 and applied in triplicate to wells of each of the three TBEV subtypes (TBEV-Eu, TBEV-Sib and TBEV-FE), a total of nine wells. After incubation for 1 h at 37°C, the ELISA plates were washed three times. Then, 100 µl of horseradish peroxidase (HRP)-conjugated detection antibody (polyclonal rabbit anti-human IgG-HRP, Dako, Jena, Germany) was added to each well and incubated for 1 h at 37°C. Following three washes with Phosphate Buffered Saline with Tween (PBS-T), 100 µl of substrate tetramethylbenzidine (TMB; Substrate-Chromogen ready to use, Dako) was added for 6 min at room temperature. The reaction was terminated by adding 50 µl of 0.5 M sulphuric acid. The OD was measured in an ELISA microplate reader (Infinite F50, Tecan, Männedorf, Switzerland) at 450 nm, 620 nm reference. Data analysis Positive and negative controls consisted of pooled serum samples with known anti-TBEV IgG antibody levels, tested by indirect immunofluorescence test (IIFT; Flavivirus Mosaic 1, Euroimmun, AG, Luebeck, Germany). The cut-off was calculated as three times the mean NS1 ELISA OD of the negative serum against all three TBEV-NS1-IgG antigens. The mean cut-off OD + 1 standard deviation (SD; 0.1) was defined as the negative threshold. The mean cut-off OD + 3 SD was defined as the positive threshold. Samples with an OD lower than the negative threshold were considered negative. Samples with an OD higher than the positive threshold were considered positive. Samples with an OD in between the mean negative and positive thresholds were considered borderline. Calculation of sensitivity and specificity Sensitivity was calculated as the proportion of patients acutely ill with TBE (Group 1, Group 2) who were correctly identified as positive by the assay. Specificity was calculated as the proportion of patients without a current or previous TBEV infection (Groups 3–6) that tested negative in the assay. Sensitivity and specificity analyses were also conducted with respect to the NS1 antigen subgroups used, i.e. TBEV-Eu, TBEV-Sib and TBEV-FE. All statistical analyses were conducted with Excel software, IBM SPSS Statistics version 22 and GraphPad Prism V 6 for Windows (GraphPad Software, San Diego, CA, USA). Results Evaluation of NS1 ELISA outcome against the actual subtype of TBEV Group 1: TBEV-Eu-infected sera This group consisted of 14 serum samples from acutely ill patients with a proven TBEV-Eu infection. All sera had previously demonstrated a positive reaction in an anti-TBEV IgM-ELISA and anti-TBEV IgG-ELISA and had also shown reactivity against anti-TBEV NS1-IgG in the assay described by Girl et al [ 17 ]. We found that all sera reacted against TBEV-Eu NS1-IgG; however, all sera also reacted against TBEV-Sib NS1-IgG and TBEV-FE NS1-IgG. However, a comparison showed that each of the sera exhibited a significantly higher OD against TBEV-Eu NS1-IgG than against TBEV-Sib NS1-IgG and TBEV-FE NS1-IgG (Fig. 1 , Fig. 2 ). Thus, using the newly developed subtype-specific NS1-IgG assay, it was possible to clearly differentiate the TBE-Eu cases infected with TBEV-Eu subtype. Group 2: TBEV-Sib/-FE-infected sera Thirty-two sera from Russian patients with a confirmed acute or past TBE infection (positive TBEV-IgG ELISA utilising a TBEV-Eu antigen) were tested for reactivity against the NS1 antigens of TBEV-Eu, TBEV-Sib and TBEV-FE. Of the 32 serum samples, 27 showed a positive result against any of the three NS1 antigens used in the assay. The five remaining sera which had previously shown reactivity in the TBEV-IgG ELISA but were negative in the NS1-IgG assay against all three subtype NS1 antigens, showed positive neutralisation against TBEV strains of the European subtype (K23) and Siberian subtype (Baikal clade) and were subsequently confirmed to be TBEV-IgM positive. These results may indicate early serum sample testing and consequently undetectable levels of TBEV NS1 antibodies in the sera. None of the tested sera showed a higher OD against TBEV-Eu NS1 in comparison to TBEV-Sib NS1 and/or TBEV-FE NS1 (Fig. 1 , Fig. 2 ). Indeed, for 26 of the 27 reactive sera, the OD of TBEV-Eu was significantly lower than the ODs of TBEV-Sib and TBEV-FE; two samples were actually negative against TBEV-Eu NS1. Therefore, these 26 sera could be clearly classified as non-TBEV-Eu. In the case of the remaining reactive serum, we could not differentiate between TBEV-Eu and TBEV-Sib/TBEV-FE as it showed a low, non-discriminating OD against all three subtypes. Regarding the differentiation of TBEV-Sib and TBEV-FE, seven of the 27 reactive sera did not show a significant difference in the ODs against the two NS1 antigens. Ten showed a significantly higher OD against the NS1 of TBEV-Sib than the NS1 of TBEV-FE, while ten showed a significantly higher OD against the NS1 of TBEV-FE compared to the NS1 of TBEV-Sib. Group 3: Flavivirus-positive, TBEV-negative sera Nine sera from patients with acute or past dengue infection were used to test for cross-reactions for NS1-IgG against other flaviviruses. Although all except two sera showed high cross-reacting antibody titers against TBEV in indirect immunofluorescence (up to > 1:20.000), 8/9 sera did not show any positive reactivity against any of the three NS1 antigens (Fig. 3 ). One of these eight sera did show a higher OD than the threshold for a negative result; however, the OD value was still in the upper negative range. This might be due to a former yellow fever vaccination causing somewhat higher ODs against all the TBEV NS1 antigens. The remaining serum (DEN-71) reacted against all three NS1 antigens, with the highest positive OD against TBEV-Eu. We therefore assume that this patient had an unknown prior TBEV infection and during their acute dengue infection they developed a serological secondary-type response due to this former TBEV infection. Group 4: TBEV-Eu-vaccinated, yellow fever-vaccinated sera Seven serum samples from individuals with TBEV-Eu vaccination and yellow fever vaccination were tested. All sera showed a somewhat higher OD than serum samples from individuals with TBEV-Eu vaccination only (Group 5). However, none of the ODs reached the calculated cut-off (Fig. 3 ). Therefore, none of the sera showed a positive or borderline reactivity against any of the three tested TBEV subtype NS1 antigens. Group 5: TBEV-Eu-vaccinated, non-TBEV-infected sera Forty sera from vaccinated and non-infected individuals, as determined by IgG-ELISA, neutralisation test and TBEV-Eu-NS1-IgG, were tested against the NS1-IgG of all three subtypes. All sera showed negative reactivity (Fig. 3 ). Group 6: Non-TBEV-Eu-vaccinated, non-TBEV-infected sera Forty sera from non-vaccinated and non-infected individuals, as determined by a negative IgG-ELISA, were tested. All sera showed negative reactivity against the NS1-IgG of all three subtypes (Fig. 3 ). Overall and subtype-specific sensitivity Sensitivity was evaluated using 46 serum samples from patients acutely ill with TBE (Group 1 and Group 2). Of these serum samples, 14/14 (100%) of TBEV-Eu infection samples tested positive for TBEV-Eu NS1-specific IgG antibodies and 27/32 (84%) samples from Russian patients tested positive for TBEV-Sib or TBEV-FE NS1-specific IgG antibodies, resulting in an overall sensitivity of 89%. However, the five samples from Russian patients that tested negative subsequently tested positive when analysed by TBEV-IgM ELISA, indicating an acute infection. It is well established from studies of TBEV-Eu-NS1 IgG in acute TBE patients that TBEV-NS1 is detectable only between 5–7 days after the onset of neurological symptoms. It is therefore plausible to assume that these five serum samples that tested negative in our NS1-IgG assay were taken too early during the symptomatic course of TBE illness to react positive. By omitting these five sera from the analysis, our assay detected 41/41 sera correctly, thus yielding an overall sensitivity of 100%. Regarding subtype-specific sensitivity, TBEV-Eu subtype differentiation sensitivity was 100% (14/14 correctly identified as TBEV-Eu) and TBEV-Sib/-FE differentiation sensitivity was 96% (26/27 correctly identified as non-TBEV-Eu). Overall and subtype-specific specificity Ninety-six serum samples from patients/individuals without TBE infection were included in the study. Forty sera from individuals with a complete TBE vaccination schedule tested negative against each of the three subtype NS1 antigens. Another 40 sera from individuals residing in non-endemic areas and with no history of TBE vaccination also tested negative against each of the three subtype NS1 antigens. Additionally, eight out of nine sera from patients with primary or secondary serological reactivity against dengue infection due to acute or past dengue infection tested negative. However, one serum sample (DEN-71) in this group showed a clearly positive reactivity against all three NS1 antigens, with the highest OD against the TBEV-Eu NS1 antigen. We assume that this patient with an acute dengue infection had an unknown past TBEV-Eu infection and therefore developed a serological secondary-type response during their acute dengue infection. An earlier validation of TBEV-Eu NS1-IgG revealed a weak cross-reactivity with sera from yellow fever-vaccinated individuals [ 17 ]. Therefore, we also included seven sera from individuals with known TBE vaccination and yellow fever vaccination. All seven sera were negative according to the calculated cut-off of the assay. However, they showed a somewhat higher OD than sera from individuals with TBE vaccination only. In summary, the overall specificity of the tested 96 sera was 99% (95/96). By omitting the serum from the patient in Group 3 assumed to have had an unknown past TBEV-Eu infection from the analysis, the overall specificity would be 100% (96/96). This degree of specificity was found against all three subtype antigens. Discussion Since the introduction of sequencing of viral genes and genomes it has been recognized that TBEV can be divided into at least five subtypes (TBEV-Eu, TBEV-Sib, TBEV-FE, TBEV-Baikal, TBEV-Himal).To date, it has not been possible to ascertain TBEV subtype-specific information in a clinical setting from serum samples drawn from patients with TBE infection. However, the ascertainment of this information is important as different virus subtype infections are reported to exhibit different clinical courses and outcomes. This is especially important for many European TBE-endemic countries, where more than one virus subtype is in circulation. Also, a serological subtyping of TBEV infections will facilitate the monitoring of the emergence of TBEV subtypes into new areas and the overall extend of the subtype distribution which has been depending on virus detection and characterization so far, either in patients or in the vectors or mammalian hosts. In the present study, we describe for the first time the development and validation of an anti-TBEV NS1 IgG ELISA that can differentiate subtype-specific anti-TBEV IgG against three clinically relevant TBEV subtypes, namely European, Siberian and Far Eastern. The development of our new immunoassay was based on previous work by two of the authors (Gerhard Dobler and Philipp Girl) which culminated in an anti-TBEV NS1 IgG ELISA based on the European subtype NS1 antigen [ 17 ]. This assay has a high sensitivity (94%) and specificity (93%) for the detection of wild-type TBEV infections in TBEV-Eu subtype-circulating areas. However, the assay may have a more limited use in European countries where TBEV-Sib and TBEV-FE infections are also prevalent as it could prove discriminatory to a certain extent and consequently have a lower sensitivity for the detection of non-TBEV-Eu infections. For the validation of our new anti-TBEV NS1 IgG ELISA coated with all three subtype antigens, serum samples from TBEV-infected patients and other flavivirus-infected patients were tested for broad and cross-reactive antibodies, and the subtyping results were analysed with regard to risk group and geographical origin (samples from Europe and Russia). Our new NS1-IgG ELISA against all three subtypes showed an overall sensitivity of 89%. However, based on IgM ELISA results, five serum samples may have been taken too early during the symptomatic course of TBE illness to react positive. By omitting these five sera from the analysis, the overall sensitivity of the assay would increase to 100%. Furthermore, the expected subtype of TBEV that infected a patient showed the highest OD value in comparison to the other subtypes; TBEV-Eu subtype differentiation sensitivity was 100% and TBEV-Sib/-Fe differentiation sensitivity was 96%. Therefore, the testing of TBEV IgG-positive sera from German and Russian patients showed that the new NS1-IgG ELISA was able to differentiate between TBEV European subtype and TBEV Siberian and Far Eastern subtype infections. The unambiguous discrimination of TBEV-Sib and TBEV-FE was difficult and not possible for many of the sera of Group 2. Among them, one serum showed highest reactivity against the TBEV-Eu NS1 antigen. Unfortunately, any information from the patients nor on their clinical form or subtype of TBEV infection was missing. In our established assay a Baltic TBEV-Sib NS1 antigen was used for coating. There is at least one amino acid exchange from the Baltic NS1 to the Siberian NS1 sequences which might cause some change in binding of Russian sera to this antigen. Testing of TBE patients from the Baltics or from Finland with respective subtype infections would give valuable information further, whether they can be unambiguously differentiated by using homologous NS1 antigen. For the specificity analysis, we tested 96 serum samples from individuals with no prior TBEV infection history. These samples were comprised of nine sera from confirmed dengue fever cases, seven sera from individuals with yellow fever vaccination and TBE vaccination, 40 sera from individuals with a complete TBE vaccination schedule and 40 sera from individuals with no TBE infection nor TBE vaccination history. The overall specificity of the tested 96 sera was 99%. One serum showed a clearly positive reactivity against all three NS1 antigens, with the highest OD against the TBEV-Eu NS1 antigen; however, it is plausible that this patient may have had an unknown past TBEV-Eu infection and thus developed a serological secondary-type response during their acute dengue infection. By omitting this serum from the analysis, the overall specificity of the assay would increase to 100%. This degree of specificity was found against all three subtype antigens. The sensitivity and specificity analyses demonstrate that the new NS1-IgG ELISA against all three subtypes is suitable for a range of practical and scientific purposes. From a practical point of view, the data presented here have proven NS1-IgG ELISA to be an appropriate tool to diagnose TBEV infections in both the acute and convalescence phases, alongside pre-existing common diagnostic methods such as standard ELISA and neutralisation test. However, due to the somewhat delayed antibody kinetics against NS1, commercially available ELISA kits based on the detection of specific antibodies against the whole TBE virus – particularly against the structural E protein – are more suitable for early acute TBE diagnosis. Conceivably, the NS1-IgG ELISA could play more of a supportive role in the early disease stage, particularly in challenging serological situations and for differential diagnosis purposes between other flavivirus infections and replace the complex neutralization test, or for monitoring the emergence of TBEV subtypes for surveillance and prevention by vaccination. The specificity results of our NS1-IgG ELISA are consistent with other published results [ 17 , 19 ], i.e. the assay is exclusively indicative for virus replication in natural infections and differentiates TBEV infection-induced specific antibodies from vaccine-induced antibodies. All 40 sera from TBEV-vaccinated individuals reacted negative against all three subtype NS1-IgG. Therefore, the NS1-IgG ELISA can be used as a valuable tool to accurately detect TBE vaccine failures or interpret serological patterns in people vaccinated in close proximity to the onset of the disease. Due to minimal cross-reaction with other flavivirus infections (e.g. West Nile fever virus, yellow fever virus and dengue fever virus), as well as vaccinated individuals (e.g. yellow fever vaccination), TBEV-Sib NS1-IgG and TBEV-FE NS1-IgG can also be used for TBE differential diagnosis between other flaviviruses, as previously described for TBEV-Eu NS1-IgG [ 17 ]. In conclusion, our newly developed NS1-IgG ELISA provides a very important tool for use in TBEV diagnostics. It has the capability to differentiate three TBEV subtypes (TBEV-Eu, TBEV-Sib, TBEV-FE) and to detect wild-type TBE infection. Furthermore, it has the potential to be used in surveillance studies, epidemiological studies and vaccine safety studies, especially in TBE endemic regions with high vaccination rates, where TBEV subtypes overlap. Future studies need to evaluate the sensitivity and specificity of NS1-IgG ELISA when used in populations/countries in which all three TBEV subtypes co-circulate. Additionally, a better understanding of NS1 protein and its role in immune protection in TBEV-infected individuals and vaccinees is urgently required. Declarations Author Contribution Z.F., D.Z., G.K., G.D., P.G., L. C.D., W.E., S.K. and O.S. wrote the main manuscript text. Z.F. and G.D. prepared tables 1 and 2. P.G. prepared figures 1-3. All authors reviewed the manuscript. Acknowledgements None Transparency declaration: Rīga Stradinš University acted as the sponsor of the study. Study received funding (Investigator Sponsored Research grant) from a commercial source, Pfizer; the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. GD is a lecturer for Pfizer Vaccine, Inc. and Bavarian Nordic – both producers of TBE vaccine – and holds investigator-sponsored research grants from both companies. All authors remain committed to the transparency and integrity of their research. References Lindquist L, Vapalahti O. Tick-borne encephalitis. Lancet. 2008;371(9627):1861–71. 10.1016/S0140-6736(08)60800-4 . Süss J. Tick-borne encephalitis 2010: epidemiology, risk areas, and virus strains in Europe and Asia-an overview. Ticks Tick Borne Dis. 2011;2(1):2–15. 10.1016/j.ttbdis.2010.10.007 . Lyons JL. Viral Meningitis and Encephalitis. Continuum (Minneap Minn) . 2018;24(5, Neuroinfectious Disease):1284–1297. 10.1212/CON.0000000000000650 . Who Publication. 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Genetic diversity and geographical distribution of the Siberian subtype of the tick-borne encephalitis virus. Ticks Tick Borne Dis. 2020;11(2):101327. 10.1016/j.ttbdis.2019.101327 . Epub 2019 Nov 7. PMID: 31767494. Dörrbecker B, Dobler G, Spiegel M, Hufert FT. Tick-borne encephalitis virus and the immune response of the mammalian host. Travel Med Infect Dis. 2010;8(4):213–22. 10.1016/j.tmaid.2010.05.010 . Epub 2010 Jun 25. PMID: 20970724. Mansfield KL, Johnson N, Phipps LP, Stephenson JR, Fooks AR, Solomon T. Tick-borne encephalitis virus - a review of an emerging zoonosis. J Gen Virol. 2009;90(Pt 8):1781–94. 10.1099/vir.0.011437-0 . Epub 2009 May 6. PMID: 19420159. Commission Implementing Decision (EU). 2018/945 of 22 June 2018 on the communicable diseases and related special health issues to be covered by epidemiological surveillance as well as relevant case definitions. C/2018/3868. Chan KR, Ismail AA, Thergarajan G, Raju CS, Yam HC, Rishya M, Sekaran SD. Serological cross-reactivity among common flaviviruses. Front Cell Infect Microbiol. 2022;12:975398. 10.3389/fcimb.2022.975398 . PMID: 36189346; PMCID: PMC9519894. Girl P, Bestehorn-Willmann M, Zange S, Borde JP, Dobler G, von Buttlar H. Tick-Borne Encephalitis Virus Nonstructural Protein 1 IgG Enzyme-Linked Immunosorbent Assay for Differentiating Infection versus Vaccination Antibody Responses. J Clin Microbiol. 2020;58(4):e01783–19. 10.1128/JCM.01783-19 . PMID: 31969423; PMCID: PMC7098735. Albinsson B, Rönnberg B, Vene S, Lundkvist Å. Antibody responses to tick-borne encephalitis virus non-structural protein 1 and whole virus antigen-a new tool in the assessment of suspected vaccine failure patients. Infect Ecol Epidemiol. 2019;9(1):1696132. PMID: 31839903; PMCID: PMC6896504. Albinsson B, Vene S, Rombo L, Blomberg J, Lundkvist Å, Rönnberg B. Distinction between serological responses following tick-borne encephalitis virus (TBEV) infection vs vaccination, Sweden 2017. Euro Surveill. 2018;23(3):17–00838. PMID: 29386094; PMCID: PMC5792698. Beicht J, Kubinski M, Zdora I, Puff C, Biermann J, Gerlach T, Baumgärtner W, Sutter G, Osterhaus ADME, Prajeeth CK, Rimmelzwaan GF. Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice. Front Immunol. 2023;14:1177324. 10.3389/fimmu.2023.1177324 . PMID: 37483628; PMCID: PMC10360051. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4546509","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":316517769,"identity":"94da8203-5441-426a-9852-16efd0f3f177","order_by":0,"name":"Zane Freimane","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYBAC+wYgwfiPgYENzK0AEcwNeLUwNkAJiJYzcDEitEB4bURoYWY/e/ABY8M9OT6xw88efJx3z97gAGObBD4tbDx5yQaMP4qN2aTTzA1nbitO3EBICw9DjpkEY0NCYpt0gpk077aEBKAtzQb4tEjwvzH/AdRS3yad/k3675wEe4JaDCRyzID+TUhgk84xkwYyGIEOa3yAX8sbY4nEhgTDNumcMsmeYwmJMw8T0GLfn2P44WNDgrz87PRtEj9qEuz5jjcfOIBPCxgkoPCYCaofBaNgFIyCUUAIAADzukWKEo0fHgAAAABJRU5ErkJggg==","orcid":"","institution":"Children’s Clinical University Hospital, Rīga Stradinš University","correspondingAuthor":true,"prefix":"","firstName":"Zane","middleName":"","lastName":"Freimane","suffix":""},{"id":316517770,"identity":"71124638-5fdc-41b3-bbff-d19b06b0e2ed","order_by":1,"name":"Gerhard Dobler","email":"","orcid":"","institution":"Bundeswehr Institute of Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Gerhard","middleName":"","lastName":"Dobler","suffix":""},{"id":316517771,"identity":"ec63f38b-d010-4149-92b0-f837352e6413","order_by":2,"name":"Lidia Chitimia-Dobler","email":"","orcid":"","institution":"Bundeswehr Institute of Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Lidia","middleName":"","lastName":"Chitimia-Dobler","suffix":""},{"id":316517772,"identity":"e62cdcab-3efd-4920-8e50-c3ee6500b3eb","order_by":3,"name":"Guntis Karelis","email":"","orcid":"","institution":"Riga East University Hospital, Rīga Stradinš University","correspondingAuthor":false,"prefix":"","firstName":"Guntis","middleName":"","lastName":"Karelis","suffix":""},{"id":316517773,"identity":"608e7c15-9b8d-4e27-b14d-c43d9327f53c","order_by":4,"name":"Philipp Girl","email":"","orcid":"","institution":"Bundeswehr Institute of Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Philipp","middleName":"","lastName":"Girl","suffix":""},{"id":316517774,"identity":"ced26646-c824-4fcd-8818-2ac3411f5b1a","order_by":5,"name":"Sanita Kuzmane","email":"","orcid":"","institution":"Riga East University Hospital, Rīga Stradinš University","correspondingAuthor":false,"prefix":"","firstName":"Sanita","middleName":"","lastName":"Kuzmane","suffix":""},{"id":316517775,"identity":"92a2cb19-330b-46c7-9a02-965ea7ffc6d4","order_by":6,"name":"Oksana Savicka","email":"","orcid":"","institution":"Riga East University Hospital, Rīga Stradinš University","correspondingAuthor":false,"prefix":"","firstName":"Oksana","middleName":"","lastName":"Savicka","suffix":""},{"id":316517776,"identity":"c59355c9-c952-448a-9906-0a797abe4fca","order_by":7,"name":"Wilhelm Erber","email":"","orcid":"","institution":"Independent consultant vaccines","correspondingAuthor":false,"prefix":"","firstName":"Wilhelm","middleName":"","lastName":"Erber","suffix":""},{"id":316517777,"identity":"990984ce-dede-4e70-9f81-b29df838ee3b","order_by":8,"name":"Dace Zavadska","email":"","orcid":"","institution":"Children’s Clinical University Hospital, Rīga Stradinš University","correspondingAuthor":false,"prefix":"","firstName":"Dace","middleName":"","lastName":"Zavadska","suffix":""}],"badges":[],"createdAt":"2024-06-07 13:46:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4546509/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4546509/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s15010-024-02370-2","type":"published","date":"2024-08-23T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59216721,"identity":"32c7193e-b303-40b9-b350-8a2e4563e926","added_by":"auto","created_at":"2024-06-27 19:09:34","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":65308,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e: Mean ODs of TBEV-Eu sera tested against TBEV-Eu NS1, TBEV-Sib NS1, and TBEV-FE NS1. \u003cstrong\u003eB\u003c/strong\u003e: ODs of TBEV-Sib/TBEV-FE sera tested against TBEV-Eu NS1, TBEV-Sib NS1, and TBEV-FE NS1 (**** highly significant; p\u0026lt;0.0001, paired t-test).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4546509/v1/1b026b39c4529673e2f32a12.jpeg"},{"id":59216103,"identity":"31d824f5-a8d8-4d20-9187-337f493b91fa","added_by":"auto","created_at":"2024-06-27 19:01:34","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":168004,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA: C\u003c/strong\u003eomparison of individual serum ODs of TBEV-Eu sera against NS1 antigen of TBEV-Eu, TBEV-Sib and TBEV-FE. \u003cstrong\u003eB: \u003c/strong\u003eComparison of individual serum ODs of TBEV-Sib/TBEV-FE sera against NS1 antigen of TBEV-Eu, TBEV-Sib and TBEV-FE. Green lines: OD drop between TBEV-Eu and Sib and between Sib and FE; Red lines: OD drop between Eu and Sib, with increase between Sib and FE; Blue lines: Increase in OD between Eu and Sib and drop between Sib and FE; Yellow lines: Increase in OD between Eu and Sib and increase between Sib and FE.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4546509/v1/25fb58fe618faee8fbb10349.jpeg"},{"id":59216102,"identity":"e7989fb4-26cd-4ac3-b490-53f37f78903b","added_by":"auto","created_at":"2024-06-27 19:01:34","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":99473,"visible":true,"origin":"","legend":"\u003cp\u003eMean ODs of sera of all groups tested against NS1 antigen of TBEV-Eu, TBEV-Sib, and TBEV-FE.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4546509/v1/c4e68be1e90d0b7c8f773859.jpeg"},{"id":63300667,"identity":"eef85c8f-1d37-44fb-b2a6-bbc7d127327d","added_by":"auto","created_at":"2024-08-26 16:16:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":920083,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4546509/v1/d3db45f0-141b-4050-a61a-72eb43639abe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and validation of a novel enzyme-linked immunosorbent assay for the differentiation of tick-borne encephalitis infections caused by different virus subtypes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTick-borne encephalitis (TBE) is a viral infectious disease of the central nervous system caused by the tick-borne encephalitis virus (TBEV) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. TBEV is predominantly transmitted to humans via the bite of an \u003cem\u003eIxodes\u003c/em\u003e tick. Less commonly, it can be transmitted by consumption of unpasteurised dairy products [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. TBE is endemic in Northern, Central and Eastern Europe as well as parts of Asia, where it is one of the most common causes of viral meningitis and encephalitis [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The clinical course of the disease can be unpredictable, with symptoms ranging from febrile illness and mild meningitis through to severe encephalitis and even death [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Currently, there is no specific treatment for TBE. However, highly effective vaccines against TBEV are available in Europe (FSME-Immun\u0026reg;, Pfizer; Encepur\u0026reg;, Bavarian Nordic) and recommended for people living in or travelling to TBE endemic regions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTBEV is a single-stranded RNA virus belonging to the Flaviviridae family and genus \u003cem\u003eFlavivirus\u003c/em\u003e. Based on phylogenetic differences, TBEV can be divided into five different subtypes: European (TBEV-Eu), Siberian (TBEV-Sib), Far Eastern (TBEV-FE), Himalayan (TBEV-Him) and Baikalian (TBEV-Bkl). Three of the five subtypes are clinically relevant to humans, namely TBEV-Eu, TBEV-Sib and TBEV-FE [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. At the amino acid level, the genetic difference can be up to 2% within a subtype and 5\u0026ndash;6% between subtypes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These subtypes are associated with different geographical distributions and clinical courses. The geographical distribution of each subtype mostly corresponds to the nominal region; however, there are some exceptions. For instance, the European subtype has been found in South Korea [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], Altai (Southern Siberia) and Irkutsk (Eastern Siberia). Furthermore, the Siberian subtype has been found to circulate in parts of Scandinavia [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the Baltic states [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], Bosnia and Central Asia [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Additionally, the Far Eastern subtype has been detected in Southern Siberia, the Urals, the Baltic states [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and Moldova [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Clinically, the European subtype is reported to be associated with a milder disease and lower case fatality rates (0.5\u0026ndash;2%) compared to the Siberian and Far Eastern subtypes (case fatality rates between 5 and 20%) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe European Centre for Disease Prevention and Control\u0026rsquo;s definition of a TBE case is as follows: a person with a laboratory-identified TBEV infection and symptoms of central nervous system inflammation. The laboratory diagnosis of TBE is mainly based on the detection of TBEV-specific IgM antibodies in cerebrospinal fluid (intrathecal production) and/or TBEV-specific IgM and IgG antibodies in serum [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Enzyme-linked immunosorbent assay (ELISA) is considered the standard method for the serological diagnosis of TBE. However, the currently available ELISAs have the fundamental disadvantage of cross-reactivity with other flavivirus infections or vaccination [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Presently, the most specific assay for serological differentiation of flavivirus antibodies is the virus neutralisation test; however, this test is unable to distinguish between different TBEV subtype infections. Therefore, serological differentiation of TBEV subtype infections in geographical regions with co-existence of two or more TBEV subtypes is not possible at this time and consequently data collection on the incidence, prevalence and clinical course of particular TBEV subtype infections is majorly restricted.\u003c/p\u003e \u003cp\u003eA new approach has recently been applied to ELISA [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and suspension multiplex immunoassay [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] to detect antibodies against TBEV non-structural protein 1 (NS1). NS1 is a glycoprotein that is central for viral RNA replication. It also induces an immune response against the virus, which may even play a role in protection [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In contrast to TBEV envelope (E) protein (the antigen commonly used in ELISA), NS1 protein elicits an immune response exclusively after wild-type natural infection, which could possibly be distinguishable between TBEV subtypes. The currently available vaccines against TBEV, namely FSME-Immun\u0026reg; and Encepur\u0026reg;, are highly purified, inactive and do not contain substantial amounts of NS1. Therefore, following vaccination, no TBEV replication occurs and generally there is no formation of NS1 protein nor development of NS1-specific antibodies. These aspects of TBE vaccination are extremely advantageous for the employment of NS1-IgG ELISA in TBE testing [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe aim of this study was to develop and validate a diagnostic method to detect NS1-IgG antibodies against the three main TBEV subtypes (European, Siberian and Far Eastern). Furthermore, we wanted to establish whether our NS1-IgG ELISA could serologically differentiate infections caused by TBEV-Eu, TBEV-Sib and TBEV-FE, which to date has not been possible. This diagnostic capability of an ELISA is of major importance in order to more fully understand each TBEV subtype\u0026rsquo;s epidemiology, pathogenicity, disease severity and vaccine effectiveness.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e The study was approved by the Rīga Stradinš University Ethics Committee (No. 6\u0026thinsp;\u0026minus;\u0026thinsp;1/03/19; March 26, 2020). Only anonymous samples or sera for research purposes were used in the present analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment of anti-TBEV NS1 IgG ELISA (NS1 ELISA)\u003c/h2\u003e \u003cp\u003eThree recombinant TBEV NS1 proteins (European \u0026ndash; strain Neud\u0026ouml;rfl, Siberian \u0026ndash; strain MucAr DZIF 22/237 and Far-Eastern \u0026ndash; strain Sofjin) were purchased from The Native Antigen Company (United Kingdom). Antigens were produced in HEK-293 human cell lines, highly purified and presented in a hexameric, native folding state.\u003c/p\u003e \u003cp\u003eTBEV-Sib-NS1-IgG ELISA and TBEV-FE-NS1-IgG ELISA were prepared as previously described for TBEV-Eu-NS1-IgG ELISA [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] with some modifications. Polystyrene plates (96-well) (Nunc Immuno MaxiSorp, Thermo Fisher Scientific, Waltham, Massachusetts, USA) were coated overnight at 4\u0026deg;C with 100 \u0026micro;l of the three recombinant TBEV NS1 antigens at a concentration of 1,000 pg/ml in carbonate buffer (0.6 M, pH 9.6) to test the optimal coating concentration. Wells were blocked with gelatine (PanReac AppliChem, Darmstadt, Germany) in phosphate-buffered saline (PBS) for 1 h at room temperature, after which antigen plates were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use. Sera were tested in triplicate against all three subtypes in parallel and average optical densities (ODs) of the different antigens were compared.\u003c/p\u003e \u003cp\u003eFor the screening of sera, TBE-IgM ELISA and TBE-IgG ELISA (both from Euroimmun, L\u0026uuml;beck, Germany) were used according to the instructions of the manufacturer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStudy population and serum samples\u003c/h2\u003e \u003cp\u003eFor the validation of NS1 ELISA, we analysed a total of 142 serum samples which were divided into six groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All sera, except sera in Group 2, were from the German National Consulting Laboratory for TBE and were anonymised prior to analysis. The samples were from TBE patients and patients with other confirmed diseases (including other flavivirus infections). In addition, samples from TBE vaccine recipients and individuals vaccinated against other flaviviruses (yellow fever virus, Japanese encephalitis virus) were included for flavivirus diagnostics. Sera were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStudy population groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTBEV infection (SENSITIVITY analysis)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 sera from acute TBEV-Eu cases, confirmed by specific IgM and IgG antibodies and TBEV-Eu-NS1-IgG ELISA.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32 sera from Russian patients with confirmed acute (IgG ELISA positive; neutralisation antibody positive) or past TBEV infection (non-TBEV-Eu; kindly provided by Tatiana Poponnikova in 2007)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNo TBEV infection (SPECIFICITY analysis)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 sera with confirmed dengue fever infection: 6 patients with acute secondary dengue antibody response; 1 with acute primary dengue infection; 2 with past dengue infection.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 sera from individuals with yellow fever vaccination and TBE vaccination.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40 sera from blood donors with confirmed TBEV-Eu vaccination (TBEV-IgG; neutralisation test).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40 sera from blood donors negative for both infection and vaccination (negative TBEV-IgG; negative neutralisation test).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGroup 3, consisting of nine sera from confirmed dengue fever cases, was included in the analysis to test the specificity of the assay (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Six sera (TM58, DEN-71, DEN-96, DEN-100, DEN-136, DEN-137) were from patients with an acute dengue fever infection (IgM positive) and a serological pattern of secondary dengue infection (i.e. high level of IgG antibodies against all flaviviruses tested, including anti-TBEV-IgG). One serum (DEN-72) was from a patient with acute primary dengue infection (high level of IgG against dengue virus; no antibodies against other flaviviruses tested). Two sera were from patients with a past dengue infection, one (DEN-131) from a patient with a primary antibody response (high level of specific anti-dengue IgG, no other anti-flavivirus IgG) and one (DEN-84) from a patient with a past secondary dengue infection (IgG cross-reaction against all tested flaviviruses).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIgG titres of sera in Group 3 against different flaviviruses\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDen-IgM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDen-IgG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYF-IgG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWN-IgG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTBE-IgG\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTM58\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20,480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10,240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2,560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5,120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDEN-71\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1,280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e640\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDEN-72\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDEN-84\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e640\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDEN-96\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20,000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDEN-100\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2,560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2,560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1,280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e640\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDEN-131\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDEN-136\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2,560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2,560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2,560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2,560\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDEN-137\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5,120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5,120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5,120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDengue fever; YF, yellow fever; WN, West Nile fever; TBE, tick-borne encephalitis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePerformance of anti-TBEV NS1 IgG ELISA\u003c/h2\u003e \u003cp\u003eEach serum sample was diluted 1:100 and applied in triplicate to wells of each of the three TBEV subtypes (TBEV-Eu, TBEV-Sib and TBEV-FE), a total of nine wells. After incubation for 1 h at 37\u0026deg;C, the ELISA plates were washed three times. Then, 100 \u0026micro;l of horseradish peroxidase (HRP)-conjugated detection antibody (polyclonal rabbit anti-human IgG-HRP, Dako, Jena, Germany) was added to each well and incubated for 1 h at 37\u0026deg;C. Following three washes with Phosphate Buffered Saline with Tween (PBS-T), 100 \u0026micro;l of substrate tetramethylbenzidine (TMB; Substrate-Chromogen ready to use, Dako) was added for 6 min at room temperature. The reaction was terminated by adding 50 \u0026micro;l of 0.5 M sulphuric acid. The OD was measured in an ELISA microplate reader (Infinite F50, Tecan, M\u0026auml;nnedorf, Switzerland) at 450 nm, 620 nm reference.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003ePositive and negative controls consisted of pooled serum samples with known anti-TBEV IgG antibody levels, tested by indirect immunofluorescence test (IIFT; Flavivirus Mosaic 1, Euroimmun, AG, Luebeck, Germany). The cut-off was calculated as three times the mean NS1 ELISA OD of the negative serum against all three TBEV-NS1-IgG antigens. The mean cut-off OD\u0026thinsp;+\u0026thinsp;1 standard deviation (SD; 0.1) was defined as the negative threshold. The mean cut-off OD\u0026thinsp;+\u0026thinsp;3 SD was defined as the positive threshold. Samples with an OD lower than the negative threshold were considered negative. Samples with an OD higher than the positive threshold were considered positive. Samples with an OD in between the mean negative and positive thresholds were considered borderline.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCalculation of sensitivity and specificity\u003c/h2\u003e \u003cp\u003eSensitivity was calculated as the proportion of patients acutely ill with TBE (Group 1, Group 2) who were correctly identified as positive by the assay. Specificity was calculated as the proportion of patients without a current or previous TBEV infection (Groups 3\u0026ndash;6) that tested negative in the assay. Sensitivity and specificity analyses were also conducted with respect to the NS1 antigen subgroups used, i.e. TBEV-Eu, TBEV-Sib and TBEV-FE. All statistical analyses were conducted with Excel software, IBM SPSS Statistics version 22 and GraphPad Prism V 6 for Windows (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of NS1 ELISA outcome against the actual subtype of TBEV\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003eGroup 1: TBEV-Eu-infected sera\u003c/h2\u003e \u003cp\u003eThis group consisted of 14 serum samples from acutely ill patients with a proven TBEV-Eu infection. All sera had previously demonstrated a positive reaction in an anti-TBEV IgM-ELISA and anti-TBEV IgG-ELISA and had also shown reactivity against anti-TBEV NS1-IgG in the assay described by Girl et al [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We found that all sera reacted against TBEV-Eu NS1-IgG; however, all sera also reacted against TBEV-Sib NS1-IgG and TBEV-FE NS1-IgG. However, a comparison showed that each of the sera exhibited a significantly higher OD against TBEV-Eu NS1-IgG than against TBEV-Sib NS1-IgG and TBEV-FE NS1-IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thus, using the newly developed subtype-specific NS1-IgG assay, it was possible to clearly differentiate the TBE-Eu cases infected with TBEV-Eu subtype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGroup 2: TBEV-Sib/-FE-infected sera\u003c/h2\u003e \u003cp\u003eThirty-two sera from Russian patients with a confirmed acute or past TBE infection (positive TBEV-IgG ELISA utilising a TBEV-Eu antigen) were tested for reactivity against the NS1 antigens of TBEV-Eu, TBEV-Sib and TBEV-FE. Of the 32 serum samples, 27 showed a positive result against any of the three NS1 antigens used in the assay. The five remaining sera which had previously shown reactivity in the TBEV-IgG ELISA but were negative in the NS1-IgG assay against all three subtype NS1 antigens, showed positive neutralisation against TBEV strains of the European subtype (K23) and Siberian subtype (Baikal clade) and were subsequently confirmed to be TBEV-IgM positive. These results may indicate early serum sample testing and consequently undetectable levels of TBEV NS1 antibodies in the sera.\u003c/p\u003e \u003cp\u003eNone of the tested sera showed a higher OD against TBEV-Eu NS1 in comparison to TBEV-Sib NS1 and/or TBEV-FE NS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Indeed, for 26 of the 27 reactive sera, the OD of TBEV-Eu was significantly lower than the ODs of TBEV-Sib and TBEV-FE; two samples were actually negative against TBEV-Eu NS1. Therefore, these 26 sera could be clearly classified as non-TBEV-Eu. In the case of the remaining reactive serum, we could not differentiate between TBEV-Eu and TBEV-Sib/TBEV-FE as it showed a low, non-discriminating OD against all three subtypes.\u003c/p\u003e \u003cp\u003eRegarding the differentiation of TBEV-Sib and TBEV-FE, seven of the 27 reactive sera did not show a significant difference in the ODs against the two NS1 antigens. Ten showed a significantly higher OD against the NS1 of TBEV-Sib than the NS1 of TBEV-FE, while ten showed a significantly higher OD against the NS1 of TBEV-FE compared to the NS1 of TBEV-Sib.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGroup 3: Flavivirus-positive, TBEV-negative sera\u003c/h2\u003e \u003cp\u003eNine sera from patients with acute or past dengue infection were used to test for cross-reactions for NS1-IgG against other flaviviruses. Although all except two sera showed high cross-reacting antibody titers against TBEV in indirect immunofluorescence (up to \u0026gt;\u0026thinsp;1:20.000), 8/9 sera did not show any positive reactivity against any of the three NS1 antigens (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). One of these eight sera did show a higher OD than the threshold for a negative result; however, the OD value was still in the upper negative range. This might be due to a former yellow fever vaccination causing somewhat higher ODs against all the TBEV NS1 antigens. The remaining serum (DEN-71) reacted against all three NS1 antigens, with the highest positive OD against TBEV-Eu. We therefore assume that this patient had an unknown prior TBEV infection and during their acute dengue infection they developed a serological secondary-type response due to this former TBEV infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGroup 4: TBEV-Eu-vaccinated, yellow fever-vaccinated sera\u003c/h2\u003e \u003cp\u003eSeven serum samples from individuals with TBEV-Eu vaccination and yellow fever vaccination were tested. All sera showed a somewhat higher OD than serum samples from individuals with TBEV-Eu vaccination only (Group 5). However, none of the ODs reached the calculated cut-off (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, none of the sera showed a positive or borderline reactivity against any of the three tested TBEV subtype NS1 antigens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGroup 5: TBEV-Eu-vaccinated, non-TBEV-infected sera\u003c/h2\u003e \u003cp\u003eForty sera from vaccinated and non-infected individuals, as determined by IgG-ELISA, neutralisation test and TBEV-Eu-NS1-IgG, were tested against the NS1-IgG of all three subtypes. All sera showed negative reactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGroup 6: Non-TBEV-Eu-vaccinated, non-TBEV-infected sera\u003c/h2\u003e \u003cp\u003eForty sera from non-vaccinated and non-infected individuals, as determined by a negative IgG-ELISA, were tested. All sera showed negative reactivity against the NS1-IgG of all three subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOverall and subtype-specific sensitivity\u003c/h2\u003e \u003cp\u003eSensitivity was evaluated using 46 serum samples from patients acutely ill with TBE (Group 1 and Group 2). Of these serum samples, 14/14 (100%) of TBEV-Eu infection samples tested positive for TBEV-Eu NS1-specific IgG antibodies and 27/32 (84%) samples from Russian patients tested positive for TBEV-Sib or TBEV-FE NS1-specific IgG antibodies, resulting in an overall sensitivity of 89%. However, the five samples from Russian patients that tested negative subsequently tested positive when analysed by TBEV-IgM ELISA, indicating an acute infection. It is well established from studies of TBEV-Eu-NS1 IgG in acute TBE patients that TBEV-NS1 is detectable only between 5\u0026ndash;7 days after the onset of neurological symptoms. It is therefore plausible to assume that these five serum samples that tested negative in our NS1-IgG assay were taken too early during the symptomatic course of TBE illness to react positive. By omitting these five sera from the analysis, our assay detected 41/41 sera correctly, thus yielding an overall sensitivity of 100%. Regarding subtype-specific sensitivity, TBEV-Eu subtype differentiation sensitivity was 100% (14/14 correctly identified as TBEV-Eu) and TBEV-Sib/-FE differentiation sensitivity was 96% (26/27 correctly identified as non-TBEV-Eu).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eOverall and subtype-specific specificity\u003c/h2\u003e \u003cp\u003eNinety-six serum samples from patients/individuals without TBE infection were included in the study. Forty sera from individuals with a complete TBE vaccination schedule tested negative against each of the three subtype NS1 antigens. Another 40 sera from individuals residing in non-endemic areas and with no history of TBE vaccination also tested negative against each of the three subtype NS1 antigens. Additionally, eight out of nine sera from patients with primary or secondary serological reactivity against dengue infection due to acute or past dengue infection tested negative. However, one serum sample (DEN-71) in this group showed a clearly positive reactivity against all three NS1 antigens, with the highest OD against the TBEV-Eu NS1 antigen. We assume that this patient with an acute dengue infection had an unknown past TBEV-Eu infection and therefore developed a serological secondary-type response during their acute dengue infection.\u003c/p\u003e \u003cp\u003eAn earlier validation of TBEV-Eu NS1-IgG revealed a weak cross-reactivity with sera from yellow fever-vaccinated individuals [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, we also included seven sera from individuals with known TBE vaccination and yellow fever vaccination. All seven sera were negative according to the calculated cut-off of the assay. However, they showed a somewhat higher OD than sera from individuals with TBE vaccination only.\u003c/p\u003e \u003cp\u003eIn summary, the overall specificity of the tested 96 sera was 99% (95/96). By omitting the serum from the patient in Group 3 assumed to have had an unknown past TBEV-Eu infection from the analysis, the overall specificity would be 100% (96/96). This degree of specificity was found against all three subtype antigens.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSince the introduction of sequencing of viral genes and genomes it has been recognized that TBEV can be divided into at least five subtypes (TBEV-Eu, TBEV-Sib, TBEV-FE, TBEV-Baikal, TBEV-Himal).To date, it has not been possible to ascertain TBEV subtype-specific information in a clinical setting from serum samples drawn from patients with TBE infection. However, the ascertainment of this information is important as different virus subtype infections are reported to exhibit different clinical courses and outcomes. This is especially important for many European TBE-endemic countries, where more than one virus subtype is in circulation. Also, a serological subtyping of TBEV infections will facilitate the monitoring of the emergence of TBEV subtypes into new areas and the overall extend of the subtype distribution which has been depending on virus detection and characterization so far, either in patients or in the vectors or mammalian hosts. In the present study, we describe for the first time the development and validation of an anti-TBEV NS1 IgG ELISA that can differentiate subtype-specific anti-TBEV IgG against three clinically relevant TBEV subtypes, namely European, Siberian and Far Eastern.\u003c/p\u003e \u003cp\u003eThe development of our new immunoassay was based on previous work by two of the authors (Gerhard Dobler and Philipp Girl) which culminated in an anti-TBEV NS1 IgG ELISA based on the European subtype NS1 antigen [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This assay has a high sensitivity (94%) and specificity (93%) for the detection of wild-type TBEV infections in TBEV-Eu subtype-circulating areas. However, the assay may have a more limited use in European countries where TBEV-Sib and TBEV-FE infections are also prevalent as it could prove discriminatory to a certain extent and consequently have a lower sensitivity for the detection of non-TBEV-Eu infections.\u003c/p\u003e \u003cp\u003eFor the validation of our new anti-TBEV NS1 IgG ELISA coated with all three subtype antigens, serum samples from TBEV-infected patients and other flavivirus-infected patients were tested for broad and cross-reactive antibodies, and the subtyping results were analysed with regard to risk group and geographical origin (samples from Europe and Russia). Our new NS1-IgG ELISA against all three subtypes showed an overall sensitivity of 89%. However, based on IgM ELISA results, five serum samples may have been taken too early during the symptomatic course of TBE illness to react positive. By omitting these five sera from the analysis, the overall sensitivity of the assay would increase to 100%. Furthermore, the expected subtype of TBEV that infected a patient showed the highest OD value in comparison to the other subtypes; TBEV-Eu subtype differentiation sensitivity was 100% and TBEV-Sib/-Fe differentiation sensitivity was 96%. Therefore, the testing of TBEV IgG-positive sera from German and Russian patients showed that the new NS1-IgG ELISA was able to differentiate between TBEV European subtype and TBEV Siberian and Far Eastern subtype infections.\u003c/p\u003e \u003cp\u003eThe unambiguous discrimination of TBEV-Sib and TBEV-FE was difficult and not possible for many of the sera of Group 2. Among them, one serum showed highest reactivity against the TBEV-Eu NS1 antigen. Unfortunately, any information from the patients nor on their clinical form or subtype of TBEV infection was missing. In our established assay a Baltic TBEV-Sib NS1 antigen was used for coating. There is at least one amino acid exchange from the Baltic NS1 to the Siberian NS1 sequences which might cause some change in binding of Russian sera to this antigen. Testing of TBE patients from the Baltics or from Finland with respective subtype infections would give valuable information further, whether they can be unambiguously differentiated by using homologous NS1 antigen.\u003c/p\u003e \u003cp\u003eFor the specificity analysis, we tested 96 serum samples from individuals with no prior TBEV infection history. These samples were comprised of nine sera from confirmed dengue fever cases, seven sera from individuals with yellow fever vaccination and TBE vaccination, 40 sera from individuals with a complete TBE vaccination schedule and 40 sera from individuals with no TBE infection nor TBE vaccination history. The overall specificity of the tested 96 sera was 99%. One serum showed a clearly positive reactivity against all three NS1 antigens, with the highest OD against the TBEV-Eu NS1 antigen; however, it is plausible that this patient may have had an unknown past TBEV-Eu infection and thus developed a serological secondary-type response during their acute dengue infection. By omitting this serum from the analysis, the overall specificity of the assay would increase to 100%. This degree of specificity was found against all three subtype antigens.\u003c/p\u003e \u003cp\u003eThe sensitivity and specificity analyses demonstrate that the new NS1-IgG ELISA against all three subtypes is suitable for a range of practical and scientific purposes. From a practical point of view, the data presented here have proven NS1-IgG ELISA to be an appropriate tool to diagnose TBEV infections in both the acute and convalescence phases, alongside pre-existing common diagnostic methods such as standard ELISA and neutralisation test. However, due to the somewhat delayed antibody kinetics against NS1, commercially available ELISA kits based on the detection of specific antibodies against the whole TBE virus \u0026ndash; particularly against the structural E protein \u0026ndash; are more suitable for early acute TBE diagnosis. Conceivably, the NS1-IgG ELISA could play more of a supportive role in the early disease stage, particularly in challenging serological situations and for differential diagnosis purposes between other flavivirus infections and replace the complex neutralization test, or for monitoring the emergence of TBEV subtypes for surveillance and prevention by vaccination.\u003c/p\u003e \u003cp\u003eThe specificity results of our NS1-IgG ELISA are consistent with other published results [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], i.e. the assay is exclusively indicative for virus replication in natural infections and differentiates TBEV infection-induced specific antibodies from vaccine-induced antibodies. All 40 sera from TBEV-vaccinated individuals reacted negative against all three subtype NS1-IgG. Therefore, the NS1-IgG ELISA can be used as a valuable tool to accurately detect TBE vaccine failures or interpret serological patterns in people vaccinated in close proximity to the onset of the disease. Due to minimal cross-reaction with other flavivirus infections (e.g. West Nile fever virus, yellow fever virus and dengue fever virus), as well as vaccinated individuals (e.g. yellow fever vaccination), TBEV-Sib NS1-IgG and TBEV-FE NS1-IgG can also be used for TBE differential diagnosis between other flaviviruses, as previously described for TBEV-Eu NS1-IgG [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, our newly developed NS1-IgG ELISA provides a very important tool for use in TBEV diagnostics. It has the capability to differentiate three TBEV subtypes (TBEV-Eu, TBEV-Sib, TBEV-FE) and to detect wild-type TBE infection. Furthermore, it has the potential to be used in surveillance studies, epidemiological studies and vaccine safety studies, especially in TBE endemic regions with high vaccination rates, where TBEV subtypes overlap.\u003c/p\u003e \u003cp\u003eFuture studies need to evaluate the sensitivity and specificity of NS1-IgG ELISA when used in populations/countries in which all three TBEV subtypes co-circulate. Additionally, a better understanding of NS1 protein and its role in immune protection in TBEV-infected individuals and vaccinees is urgently required.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZ.F., D.Z., G.K., G.D., P.G., L. C.D., W.E., S.K. and O.S. wrote the main manuscript text. Z.F. and G.D. prepared tables 1 and 2. P.G. prepared figures 1-3. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNone\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTransparency declaration:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRīga Stradin\u0026scaron; University acted as the sponsor of the study. Study received funding (Investigator Sponsored Research grant) from a commercial source, Pfizer; the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. GD is a lecturer for Pfizer Vaccine, Inc. and Bavarian Nordic \u0026ndash; both producers of TBE vaccine \u0026ndash; and holds investigator-sponsored research grants from both companies. All authors remain committed to the transparency and integrity of their research.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLindquist L, Vapalahti O. Tick-borne encephalitis. 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PMID: 37483628; PMCID: PMC10360051.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"infection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infe","sideBox":"Learn more about [Infection](http://link.springer.com/journal/15010)","snPcode":"15010","submissionUrl":"https://submission.nature.com/new-submission/15010/3","title":"Infection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"NS1 antigen, NS1 antibodies, ELISA, TBEV subtypes, flavivirus","lastPublishedDoi":"10.21203/rs.3.rs-4546509/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4546509/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eTick-borne encephalitis (TBE) is an infection caused by the tick-borne encephalitis virus (TBEV) that can lead to symptoms of central nervous system inflammation. There are five subtypes of TBEV, three of which \u0026ndash; European, Siberian and Far Eastern \u0026ndash; occur in Europe. As it is thought that different subtype infections exhibit varying clinical courses and outcomes, serological differentiation of the virus subtypes is clearly important. However, to date, this has proved difficult to achieve.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAn ELISA format was developed based on TBE virus NS1 antigen against the European, Siberian and Far Eastern subtype. The three NS1 antigens were biotechnologically produced in a human cell line and used for ELISA coating. Sera from German (European subtype) and Russian (Siberian and/or Far Eastern subtypes) TBE patients with positive TBEV IgG were used to test the reactivity against these three NS1 antigens.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTesting of 14 German and 32 Russian TBEV IgG-positive sera showed that the ELISA was able to differentiate between TBEV European subtype and TBEV Siberian and Far Eastern subtype infections.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn geographical areas where two or more TBEV subtype infections can occur, the NS1-IgG ELISA developed here constitutes an important diagnostic tool to differentiate between European subtype infections and Siberian/Far Eastern subtype infections and to use the new assay for epidemiological studies to clarify the importance of particular subtype infections in an area. Consequently, it may help to better describe and anticipate the clinical courses and outcomes of particular TBEV subtype infections.\u003c/p\u003e","manuscriptTitle":"Development and validation of a novel enzyme-linked immunosorbent assay for the differentiation of tick-borne encephalitis infections caused by different virus subtypes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-27 19:01:29","doi":"10.21203/rs.3.rs-4546509/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-29T06:45:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-28T14:23:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"281052518511146072126554756532185168508","date":"2024-06-19T16:39:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-19T11:31:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32376813302021126443736667777746561405","date":"2024-06-11T15:13:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-08T16:40:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-08T16:09:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-08T06:46:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Infection","date":"2024-06-07T13:45:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"infection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infe","sideBox":"Learn more about [Infection](http://link.springer.com/journal/15010)","snPcode":"15010","submissionUrl":"https://submission.nature.com/new-submission/15010/3","title":"Infection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7418ff66-91d8-4a50-affb-266b99c54d5b","owner":[],"postedDate":"June 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:09:08+00:00","versionOfRecord":{"articleIdentity":"rs-4546509","link":"https://doi.org/10.1007/s15010-024-02370-2","journal":{"identity":"infection","isVorOnly":false,"title":"Infection"},"publishedOn":"2024-08-23 15:57:36","publishedOnDateReadable":"August 23rd, 2024"},"versionCreatedAt":"2024-06-27 19:01:29","video":"","vorDoi":"10.1007/s15010-024-02370-2","vorDoiUrl":"https://doi.org/10.1007/s15010-024-02370-2","workflowStages":[]},"version":"v1","identity":"rs-4546509","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4546509","identity":"rs-4546509","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}