Long-term elevation of complement factors in cerebrospinal fluid of patients with Borna disease virus 1 (BoDV-1) encephalitis.

BACKGROUND: Borna disease virus 1 (BoDV-1) causes rare but severe zoonotic infections in humans, presenting as severe encephalitis. The case-fatality risk is very high and no effective countermeasures have been established so far. An immunopathology is presumed, while data on immune responses in humans are limited. Evidence of a role of the complement system in various neurological disorders and central nervous viral infections is increasing and specific inhibitors are available as therapeutic options. METHODS: In this study, we investigated factors of the complement system in the cerebrospinal fluid (CSF) of patients with BoDV-1 infections (n = 17) in comparison to non-inflammatory control CSF samples (n = 11), using a bead-based multiplex assay. In addition, immunohistochemistry was performed using post-mortem brain tissue samples. RESULTS: We found an intrathecal elevation of complement factors of all complement pathways and an active cascade during human BoDV-1 infections. The increase of certain complement factors such as C1q was persistent and C3 complement deposits were detected in post-mortem brain sections. Intrathecal complement levels were negatively correlated with survival. CONCLUSION: Further investigations are warranted to clarify, whether targeting the complement cascade by specific inhibitors might be beneficial for patients suffering from severe BoDV-1 encephalitis.

One Health in action: Investigation of the first detected local cluster of fatal borna disease virus 1 (BoDV-1) encephalitis, Germany 2022.

BACKGROUND: Zoonotic Borna disease virus 1 (BoDV-1) causes fatal encephalitis in humans and animals. Subsequent to the detection of two paediatric cases in a Bavarian municipality in Germany within three years, we conducted an interdisciplinary One Health investigation. We aimed to explore seroprevalence in a local human population with a risk for BoDV-1 exposure as well as viral presence in environmental samples from local sites and BoDV-1 prevalence within the local small mammal population and its natural reservoir, the bicoloured white-toothed shrew (Crocidura leucodon). METHODS: The municipality's adult residents participated in an anonymised sero-epidemiological study. Potential risk factors and clinical symptoms were assessed by an electronic questionnaire. Small mammals, environmental samples and ticks from the municipality were tested for BoDV-1-RNA. Shrew-derived BoDV-1-sequences together with sequences of the two human cases were phylogenetically analysed. RESULTS: In total, 679 citizens participated (response: 41 %), of whom 38 % reported shrews in their living environment and 19 % direct shrew contact. No anti-BoDV-1 antibodies were detected in human samples. BoDV-1-RNA was also undetectable in 38 environmental samples and 336 ticks. Of 220 collected shrews, twelve of 40 C. leucodon (30%) tested BoDV-1-RNA-positive. BoDV-1-sequences from the previously diagnosed two paediatric patients belonged to two different subclades, that were also present in shrews from the municipality. INTERPRETATION: Our data support the interpretation that human BoDV-1 infections are rare even in endemic areas and primarily manifest as severe encephalitis. Sequence analysis linked both previous paediatric human infections to the local shrew population, but indicated independent infection sources. FUNDING: The project was partly financed by funds of the German Federal Ministry of Education and Research (grant numbers: 01KI2005A, 01KI2005C, 01KI1722A, 01KI1722C, 01KI2002 to MaBe, DR, RGU, DT, BS) as well as by the ReForM-A programme of the University Hospital Regensburg (to MaBa) and by funds of the Bavarian State Ministry of Health, Care and Prevention, project "Zoonotic Bornavirus Focal Point Bavaria - ZooBoFo" (to MaBa, MaBe, BS, MMB, DR, PS, RGU).

Performance of T-Track® SARS-CoV-2, an Innovative Dual Marker RT-qPCR-Based Whole-Blood Assay for the Detection of SARS-CoV-2-Reactive T Cells.

T-cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a central role in the control of the virus. In this study, we evaluated the performance of T-Track® SARS-CoV-2, a novel CE-marked quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay, which relies on the combined evaluation of IFNG and CXCL10 mRNA levels in response to the S1 and NP SARS-CoV-2 antigens, in 335 participants with or without a history of SARS-CoV-2 infection and vaccination, respectively. Of the 62 convalescent donors, 100% responded to S1 and 88.7% to NP antigens. In comparison, of the 68 naïve donors, 4.4% were reactive to S1 and 19.1% to NP. Convalescent donors <50 and ≥50 years of age demonstrated a 100% S1 reactivity and an 89.1% and 87.5% NP reactivity, respectively. T-cell responses by T-Track® SARS-CoV-2 and IgG serology by recomLine SARS-CoV-2 IgG according to the time from the last immunisation (by vaccination or viral infection) were comparable. Both assays showed a persistent cellular and humoral response for at least 36 weeks post immunisation in vaccinated and convalescent donors. Our results demonstrate the very good performance of the T-Track® SARS-CoV-2 molecular assay and suggest that it might be suitable to monitor the SARS-CoV-2-specific T-cell response in COVID-19 vaccinations trials and cross-reactivity studies.

Performance of T-Track® TB, a Novel Dual Marker RT-qPCR-Based Whole-Blood Test for Improved Detection of Active Tuberculosis.

Tuberculosis (TB) is one of the leading causes of death by an infectious disease. It remains a major health burden worldwide, in part due to misdiagnosis. Therefore, improved diagnostic tests allowing the faster and more reliable diagnosis of patients with active TB are urgently needed. This prospective study examined the performance of the new molecular whole-blood test T-Track® TB, which relies on the combined evaluation of IFNG and CXCL10 mRNA levels, and compared it to that of the QuantiFERON®-TB Gold Plus (QFT-Plus) enzyme-linked immunosorbent assay (ELISA). Diagnostic accuracy and agreement analyses were conducted on the whole blood of 181 active TB patients and 163 non-TB controls. T-Track® TB presented sensitivity of 94.9% and specificity of 93.8% for the detection of active TB vs. non-TB controls. In comparison, the QFT-Plus ELISA showed sensitivity of 84.3%. The sensitivity of T-Track® TB was significantly higher (p < 0.001) than that of QFT-Plus. The overall agreement of T-Track® TB with QFT-Plus to diagnose active TB was 87.9%. Out of 21 samples with discordant results, 19 were correctly classified by T-Track® TB while misclassified by QFT-Plus (T-Track® TB-positive/QFT-Plus-negative), and two samples were misclassified by T-Track® TB while correctly classified by QFT-Plus (T-Track® TB-negative/QFT-Plus-positive). Our results demonstrate the excellent performance of the T-Track® TB molecular assay and its suitability to accurately detect TB infection and discriminate active TB patients from non-infected controls.

DNA extraction from clotted blood in genotyping quality.

DNA extraction from frozen blood clots is challenging. Here, the authors applied QIAGEN Clotspin Baskets and the Gentra Puregene Blood Kit for DNA extraction to cellular fraction of 5.5 ml whole blood without anticoagulating additives. The amount and quality of extracted DNA were assessed via spectrophotometer and gel electrophoresis. Results from array-based genotyping were analyzed. All steps were compared with DNA isolated from anticoagulated blood samples from a separate study. The quality and concentration of DNA extracted from clotted blood were comparable to those of DNA extracted from anticoagulated blood. DNA yield was on average 27 µg per ml clotted blood, with an average purity of 1.87 (A260/A280). Genotyping quality was similar for both DNA sources (call rate: 99.56% from clotted vs 99.49% from anticoagulated blood).

Human Infections with Borna Disease Virus 1 (BoDV-1) Primarily Lead to Severe Encephalitis: Further Evidence from the Seroepidemiological BoSOT Study in an Endemic Region in Southern Germany.

More than 40 human cases of severe encephalitis caused by Borna disease virus 1 (BoDV-1) have been reported to German health authorities. In an endemic region in southern Germany, we conducted the seroepidemiological BoSOT study ("BoDV-1 after solid-organ transplantation") to assess whether there are undetected oligo- or asymptomatic courses of infection. A total of 216 healthy blood donors and 280 outpatients after solid organ transplantation were screened by a recombinant BoDV-1 ELISA followed by an indirect immunofluorescence assay (iIFA) as confirmatory test. For comparison, 288 serum and 258 cerebrospinal fluid (CSF) samples with a request for tick-borne encephalitis (TBE) diagnostics were analyzed for BoDV-1 infections. ELISA screening reactivity rates ranged from 3.5% to 18.6% depending on the cohort and the used ELISA antigen, but only one sample of a patient from the cohort with requested TBE diagnostics was confirmed to be positive for anti-BoDV-1-IgG by iIFA. In addition, the corresponding CSF sample of this patient with a three-week history of severe neurological disease tested positive for BoDV-1 RNA. Due to the iIFA results, all other results were interpreted as false-reactive in the ELISA screening. By linear serological epitope mapping, cross-reactions with human and bacterial proteins were identified as possible underlying mechanism for the false-reactive ELISA screening results. In conclusion, no oligo- or asymptomatic infections were detected in the studied cohorts. Serological tests based on a single recombinant BoDV-1 antigen should be interpreted with caution, and an iIFA should always be performed in addition.

IFN-γ-Based ELISpot as a New Tool to Detect Human Infections with Borna Disease Virus 1 (BoDV-1): A Pilot Study.

More than 40 human infections with the zoonotic Borna disease virus 1 (BoDV-1) have been reported to German health authorities from endemic regions in southern and eastern Germany. Diagnosis of a confirmed case is based on the detection of BoDV-1 RNA or BoDV-1 antigen. In parallel, serological assays such as ELISA, immunoblots, and indirect immunofluorescence are in use to detect the seroconversion of Borna virus-reactive IgG in serum or cerebrospinal fluid (CSF). As immunopathogenesis in BoDV-1 encephalitis appears to be driven by T cells, we addressed the question of whether an IFN-γ-based ELISpot may further corroborate the diagnosis. For three of seven BoDV-1-infected patients, peripheral blood mononuclear cells (PBMC) with sufficient quantity and viability were retrieved. For all three patients, counts in the range from 12 to 20 spot forming units (SFU) per 250,000 cells were detected upon the stimulation of PBMC with a peptide pool covering the nucleocapsid protein of BoDV-1. Additionally, individual patients had elevated SFU upon stimulation with a peptide pool covering X or phosphoprotein. Healthy blood donors (n = 30) and transplant recipients (n = 27) were used as a control and validation cohort, respectively. In this pilot study, the BoDV-1 ELISpot detected cellular immune responses in human patients with BoDV-1 infection. Its role as a helpful diagnostic tool needs further investigation in patients with BoDV-1 encephalitis.

Cerebrospinal fluid in Borna disease virus 1 (BoDV-1) encephalitis.

Borna disease virus 1 (BoDV-1) has been recognized as a rare cause of very severe encephalitis with rapid onset in central Europe. Data on cerebrospinal fluid (CSF) analysis have not yet been analyzed in detail. Here, we present the first study on CSF changes in BoDV-1 encephalitis. We retrospectively analyzed CSFs from 18 BoDV-1 encephalitis cases from Bavaria, Germany, an endemic region, from 1996 to 2021. Data were obtained through review of medical records and institutional databases. We found that white blood cell count (WBC) in CSF is elevated in 13 of our 18 patients at first examination (average 83.2 ± 142.3 leukocytes/µl) and cytology showed predominance of lymphocytes. Patients with typical symptoms of meningoencephalitis had higher WBC in first CSF analyzation (133.5 ± 163.1 vs 4.0 ± 3.2/µl; p = 0.065). BoDV-1 PCR of CSF is not always positive when tested (7 of 9 cases). Four of five patients tested showed a polyvalent reaction against multiple viruses in the CSF suggesting that BoDV-1 may trigger autoimmune mechanisms. CSF changes in BoDV-1 encephalitis seem similar to those of other viral encephalitis and at the beginning WBC can be normal in up to 28%, making the diagnosis even more challenging. All in all, BoDV-1 should be included in the diagnostic workup of patients with rapidly evolving and/or severe encephalitis and patients with severe neuropathy and secondary encephalopathy with and without CSF changes. Repeated CSF examinations as well as BoDV-1 serology and CSF PCR have to be considered in endemic areas.

Higher Infection Risk among Health Care Workers and Lower Risk among Smokers Persistent across SARS-CoV-2 Waves-Longitudinal Results from the Population-Based TiKoCo Seroprevalence Study.

SARS-CoV-2 seroprevalence was reported as substantially increased in medical personnel and decreased in smokers after the first wave in spring 2020, including in our population-based Tirschenreuth Study (TiKoCo). However, it is unclear whether these associations were limited to the early pandemic and whether the decrease in smokers was due to reduced infection or antibody response. We evaluated the association of occupation and smoking with period-specific seropositivity: for the first wave until July 2020 (baseline, BL), the low infection period in summer (follow-up 1, FU1, November 2020), and the second/third wave (FU2, April 2021). We measured binding antibodies directed to SARS-CoV-2 nucleoprotein (N), viral spike protein (S), and neutralizing antibodies at BL, FU1, and FU2. Previous infection, vaccination, smoking, and occupation were assessed by questionnaires. The 4181 participants (3513/3374 at FU1/FU2) included 6.5% medical personnel and 20.4% current smokers. At all three timepoints, new seropositivity was higher in medical personnel with ORs = 1.99 (95%-CI = 1.36-2.93), 1.41 (0.29-6.80), and 3.17 (1.92-5.24) at BL, FU1, and FU2, respectively, and nearly halved among current smokers with ORs = 0.47 (95%-CI = 0.33-0.66), 0.40 (0.09-1.81), and 0.56 (0.33-0.94). Current smokers compared to never-smokers had similar antibody levels after infection or vaccination and reduced odds of a positive SARS-CoV-2 result among tested. Our data suggest that decreased seroprevalence among smokers results from fewer infections rather than reduced antibody response. The persistently higher infection risk of medical staff across infection waves, despite improved means of protection over time, underscores the burden for health care personnel.

Human Borna disease virus 1 encephalitis shows marked pro-inflammatory biomarker and tissue immunoactivation during the course of disease.

Human Borna disease virus 1 (BoDV-1) encephalitis is a severe emerging disease with a very high case-fatality rate. While the clinical disease, case definitions, diagnostic algorithms and neuropathology have been described, very little is known about the immunological processes of human BoDV-1 encephalitis. Here, we analyzed serum and cerebrospinal fluid (CSF) samples from 10 patients with fatal BoDV-1 encephalitis for changes of different cytokines, chemokines, growth factors and other biomarkers over time. From one of these individuals, also autoptic formalin-fixed brain tissue was analyzed for the expression of inflammatory biomarkers by mRNA levels and immunostaining; in a further patient, only formalin-fixed brain tissue was available and examined in addition. A marked and increasing immune activation from the initial phase to the last phase of acute BoDV-1 encephalitis is shown in serum and CSF, characterized by cytokine concentration changes (IFNγ, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-13, IL-18, TGF-ß1) with a predominantly pro-inflammatory pattern over time. IFNγ production was demonstrated in endothelial cells, astrocytes and microglia, IL-6 in activated microglia, and TGF-ß1 in endothelial cells, activated astrocytes and microglia. This was paralleled by an increase of chemokines (CCL-2, CCL-5, CXCL-10, IL-8) to attract immune cells to the site of infection, contributing to inflammation and tissue damage. Pathologically low growth factor levels (BDNF, ß-NGF, PDGF) were seen. Changed levels of arginase and sTREM further fostered the pro-inflammatory state. This dysbalanced, pro-inflammatory state likely contributes importantly to the fatal outcome of human BoDV-1 encephalitis, and might be a key target for possible treatment attempts.

Time Trend in SARS-CoV-2 Seropositivity, Surveillance Detection- and Infection Fatality Ratio until Spring 2021 in the Tirschenreuth County-Results from a Population-Based Longitudinal Study in Germany.

Herein, we provide results from a prospective population-based longitudinal follow-up (FU) SARS-CoV-2 serosurveillance study in Tirschenreuth, the county which was hit hardest in Germany in spring 2020 and early 2021. Of 4203 individuals aged 14 years or older enrolled at baseline (BL, June 2020), 3546 participated at FU1 (November 2020) and 3391 at FU2 (April 2021). Key metrics comprising standardized seroprevalence, surveillance detection ratio (SDR), infection fatality ratio (IFR) and success of the vaccination campaign were derived using the Roche N- and S-Elecsys anti-SARS-CoV-2 test together with a self-administered questionnaire. N-seropositivity at BL was 9.2% (1st wave). While we observed a low new seropositivity between BL and FU1 (0.9%), the combined 2nd and 3rd wave accounted for 6.1% new N-seropositives between FU1 and FU2 (ever seropositives at FU2: 15.4%). The SDR decreased from 5.4 (BL) to 1.1 (FU2) highlighting the success of massively increased testing in the population. The IFR based on a combination of serology and registration data resulted in 3.3% between November 2020 and April 2021 compared to 2.3% until June 2020. Although IFRs were consistently higher at FU2 compared to BL across age-groups, highest among individuals aged 70+ (18.3% versus 10.7%, respectively), observed differences were within statistical uncertainty bounds. While municipalities with senior care homes showed a higher IFR at BL (3.0% with senior care home vs. 0.7% w/o), this effect diminished at FU2 (3.4% vs. 2.9%). In April 2021 (FU2), vaccination rate in the elderly was high (>77.4%, age-group 80+).

Omicron's binding to sotrovimab, casirivimab, imdevimab, CR3022, and sera from previously infected or vaccinated individuals.

SARS-CoV-2 Omicron is the first pandemic variant of concern exhibiting an abrupt accumulation of mutations particularly in the receptor-binding domain that is a critical target of vaccination induced and therapeutic antibodies. Omicron's mutations did only marginally affect the binding of ACE2, and the two antibodies Sotrovimab and CR3022 but strongly impaired the binding of Casirivimab and Imdevimab. Moreover, as compared with Wuhan, there is reduced serum reactivity and a pronounced loss of competitive surrogate virus neutralization (sVN) against Omicron in naïve vaccinees and in COVID-19 convalescents after infection and subsequent vaccination. Finally, although the booster vaccination response conferred higher titers and better sVN, the effect was nonetheless significantly lower compared with responses against Wuhan. Overall, our data suggest that the antigenicity of Omicrons receptor binding motive has largely changed but antibodies such as Sotrovimab targeting other conserved sites maintain binding and therefore hold potential in prophylaxis and treatment of Omicron-induced COVID-19.

Comparative Immunogenicity of COVID-19 Vaccines in a Population-Based Cohort Study with SARS-CoV-2-Infected and Uninfected Participants.

To assess vaccine immunogenicity in non-infected and previously infected individuals in a real-world scenario, SARS-CoV-2 antibody responses were determined during follow-up 2 (April 2021) of the population-based Tirschenreuth COVID-19 cohort study comprising 3378 inhabitants of the Tirschenreuth county aged 14 years or older. Seronegative participants vaccinated once with Vaxzevria, Comirnaty, or Spikevax had median neutralizing antibody titers ranging from ID50 = 25 to 75. Individuals with two immunizations with Comirnaty or Spikevax had higher median ID50s (of 253 and 554, respectively). Regression analysis indicated that both increased age and increased time since vaccination independently decreased RBD binding and neutralizing antibody levels. Unvaccinated participants with detectable N-antibodies at baseline (June 2020) revealed a median ID50 of 72 at the April 2021 follow-up. Previously infected participants that received one dose of Vaxzevria or Comirnaty had median ID50 to 929 and 2502, respectively. Individuals with a second dose of Comirnaty given in a three-week interval after the first dose did not have higher median antibody levels than individuals with one dose. Prior infection also primed for high systemic IgA levels in response to one dose of Comirnaty that exceeded IgA levels observed after two doses of Comirnaty in previously uninfected participants. Neutralizing antibody levels targeting the spike protein of Beta and Delta variants were diminished compared to the wild type in vaccinated and infected participants.

Antibodies against viral nucleo-, phospho-, and X protein contribute to serological diagnosis of fatal Borna disease virus 1 infections.

Borna disease virus 1 (BoDV-1) causes rare but often fatal encephalitis in humans. Late diagnosis prohibits an experimental therapeutic approach. Here, we report a recent case of fatal BoDV-1 infection diagnosed on day 12 after hospitalization by detection of BoDV-1 RNA in the cerebrospinal fluid. In a retrospective analysis, we detect BoDV-1 RNA 1 day after hospital admission when the cell count in the cerebrospinal fluid is still normal. We develop a new ELISA using recombinant BoDV-1 nucleoprotein, phosphoprotein, and accessory protein X to detect seroconversion on day 12. Antibody responses are also shown in seven previously confirmed cases. The individual BoDV-1 antibody profiles show variability, but the usage of three different BoDV-1 antigens results in a more sensitive diagnostic tool. Our findings demonstrate that early detection of BoDV-1 RNA in cerebrospinal fluid and the presence of antibodies against at least two different viral antigens contribute to BoDV-1 diagnosis. Physicians in endemic regions should consider BoDV-1 infection in cases of unclear encephalopathy and initiate appropriate diagnostics at an early stage.

Spectrum Bias and Individual Strengths of SARS-CoV-2 Serological Tests-A Population-Based Evaluation.

Antibody testing for determining the SARS-CoV-2 serostatus was rapidly introduced in early 2020 and since then has been gaining special emphasis regarding correlates of protection. With limited access to representative samples with known SARS-CoV-2 infection status during the initial period of test development and validation, spectrum bias has to be considered when moving from a "test establishment setting" to population-based settings, in which antibody testing is currently implemented. To provide insights into the presence and magnitude of spectrum bias and to estimate performance measures of antibody testing in a population-based environment, we compared SARS-CoV-2 neutralization to a battery of serological tests and latent class analyses (LCA) in a subgroup (n = 856) of the larger population based TiKoCo-19 cohort (n = 4185). Regarding spectrum bias, we could proof notable differences in test sensitivities and specificities when moving to a population-based setting, with larger effects visible in earlier registered tests. While in the population-based setting the two Roche ELECSYS anti-SARS-CoV-2 tests outperformed every other test and even LCA regarding sensitivity and specificity in dichotomous testing, they didn't provide satisfying quantitative correlation with neutralization capacity. In contrast, our in-house anti SARS-CoV-2-Spike receptor binding domain (RBD) IgG-ELISA (enzyme-linked-immunosorbant assay) though inferior in dichotomous testing, provided satisfactory quantitative correlation and may thus represent a better correlate of protection. In summary, all tests, led by the two Roche tests, provided sufficient accuracy for dichotomous identification of neutralizing sera, with increasing spectrum bias visible in earlier registered tests, while the majority of tests, except the RBD-ELISA, didn't provide satisfactory quantitative correlations.

Estimates and Determinants of SARS-Cov-2 Seroprevalence and Infection Fatality Ratio Using Latent Class Analysis: The Population-Based Tirschenreuth Study in the Hardest-Hit German County in Spring 2020.

SARS-CoV-2 infection fatality ratios (IFR) remain controversially discussed with implications for political measures. The German county of Tirschenreuth suffered a severe SARS-CoV-2 outbreak in spring 2020, with particularly high case fatality ratio (CFR). To estimate seroprevalence, underreported infections, and IFR for the Tirschenreuth population aged ≥14 years in June/July 2020, we conducted a population-based study including home visits for the elderly, and analyzed 4203 participants for SARS-CoV-2 antibodies via three antibody tests. Latent class analysis yielded 8.6% standardized county-wide seroprevalence, a factor of underreported infections of 5.0, and 2.5% overall IFR. Seroprevalence was two-fold higher among medical workers and one third among current smokers with similar proportions of registered infections. While seroprevalence did not show an age-trend, the factor of underreported infections was 12.2 in the young versus 1.7 for ≥85-year-old. Age-specific IFRs were <0.5% below 60 years of age, 1.0% for age 60-69, and 13.2% for age 70+. Senior care homes accounted for 45% of COVID-19-related deaths, reflected by an IFR of 7.5% among individuals aged 70+ and an overall IFR of 1.4% when excluding senior care home residents from our computation. Our data underscore senior care home infections as key determinant of IFR additionally to age, insufficient targeted testing in the young, and the need for further investigations on behavioral or molecular causes of the fewer infections among current smokers.

Lactonization of the Oncometabolite D-2-Hydroxyglutarate Produces a Novel Endogenous Metabolite.

In recent years, onco-metabolites like D-2-hydroxyglutarate, which is produced in isocitrate dehydrogenase-mutated tumors, have gained increasing interest. Here, we report a metabolite in human specimens that is closely related to 2-hydroxyglutarate: the intramolecular ester of 2-hydroxyglutarate, 2-hydroxyglutarate-γ-lactone. Using 13C5-L-glutamine tracer analysis, we showed that 2-hydroxyglutarate is the endogenous precursor of 2-hydroxyglutarate-lactone and that there is a high exchange between these two metabolites. Lactone formation does not depend on mutated isocitrate dehydrogenase, but its formation is most probably linked to transport processes across the cell membrane and favored at low environmental pH. Furthermore, human macrophages showed not only striking differences in uptake of 2-hydroxyglutarate and its lactone but also in the enantiospecific hydrolysis of the latter. Consequently, 2-hydroxyglutarate-lactone may play a critical role in the modulation of the tumor microenvironment.

Active Case Finding of Current Bornavirus Infections in Human Encephalitis Cases of Unknown Etiology, Germany, 2018-2020.

Human bornavirus encephalitis is a severe and often fatal infection caused by variegated squirrel bornavirus 1 (VSBV-1) and Borna disease virus 1 (BoDV-1). We conducted a prospective study of bornavirus etiology of encephalitis cases in Germany during 2018-2020 by using a serologic testing scheme applied along proposed graded case definitions for VSBV-1, BoDV-1, and unspecified bornavirus encephalitis. Of 103 encephalitis cases of unknown etiology, 4 bornavirus infections were detected serologically. One chronic case was caused by VSBV-1 after occupational-related contact of a person with exotic squirrels, and 3 acute cases were caused by BoDV-1 in virus-endemic areas. All 4 case-patients died. Bornavirus etiology could be confirmed by molecular methods. Serologic testing for these cases was virus specific, discriminatory, and a practical diagnostic option for living patients if no brain tissue samples are available. This testing should be guided by clinical and epidemiologic suspicions, such as residence in virus-endemic areas and animal exposure.

A highly specific and sensitive serological assay detects SARS-CoV-2 antibody levels in COVID-19 patients that correlate with neutralization.

OBJECTIVE: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic challenges national health systems and the global economy. Monitoring of infection rates and seroprevalence can guide public health measures to combat the pandemic. This depends on reliable tests on active and former infections. Here, we set out to develop and validate a specific and sensitive enzyme linked immunosorbent assay (ELISA) for detection of anti-SARS-CoV-2 antibody levels. METHODS: In our ELISA, we used SARS-CoV-2 receptor-binding domain (RBD) and a stabilized version of the spike (S) ectodomain as antigens. We assessed sera from patients infected with seasonal coronaviruses, SARS-CoV-2 and controls. We determined and monitored IgM-, IgA- and IgG-antibody responses towards these antigens. In addition, for a panel of 22 sera, virus neutralization and ELISA parameters were measured and correlated. RESULTS: The RBD-based ELISA detected SARS-CoV-2-directed antibodies, did not cross-react with seasonal coronavirus antibodies and correlated with virus neutralization (R2 = 0.89). Seroconversion started at 5 days after symptom onset and led to robust antibody levels at 10 days after symptom onset. We demonstrate high specificity (99.3%; N = 1000) and sensitivity (92% for IgA, 96% for IgG and 98% for IgM; > 10 days after PCR-proven infection; N = 53) in serum. CONCLUSIONS: With the described RBD-based ELISA protocol, we provide a reliable test for seroepidemiological surveys. Due to high specificity and strong correlation with virus neutralization, the RBD ELISA holds great potential to become a preferred tool to assess thresholds of protective immunity after infection and vaccination.

Manufacturing of convalescent plasma of COVID-19 patients: Aspects of quality.

The ongoing coronavirus disease 2019 (COVID-19) pandemic emerged in December 2019. Convalescent plasma represents a promising COVID-19 treatment. Here, we report on the manufacturing of a plasma-based product containing antibodies specific to SARS-CoV-2 obtained from recently recovered COVID-19 patients. Convalescent plasma donors were screened as follows: 1) previously confirmed SARS-CoV-2 infection (by real-time PCR (RT-PCR)); 2) a subsequent negative PCR test followed by a 2-week waiting period; 3) an additional negative PCR test prior to plasmapheresis; and 4) confirmation of the presence of SARS-CoV-2 specific antibodies. Convalescent plasma was stored fresh (2-6°C) for up to 5 days or frozen (-30°C) for long-term storage. Donor peripheral blood and final plasma product were assayed for binding antibodies targeting the SARS-CoV-2 S-protein receptor-binding domain (RBD) and their titers measured by an enzyme-linked immunosorbent assay (ELISA). We performed 72 plasmaphereses resulting in 248 final products. Convalescent plasma contained an RBD-specific antibody titer (IgG) ranging from 1:100 to 1:3200 (median 1:800). The titer was congruent to the titer of the blood (n = 34) before collection (1:100-1:6400, median 1:800). Levels of IL-8 and LBP of donors were slightly increased. Therapeutic products derived from a human origin must undergo rigorous testing to ensure uniform quality and patient safety. Whilst previous publications recommended RBD-specific binding antibody titers of ≥ 1:320, we selected a minimum titer of 1:800 in order to maximize antibody delivery. Production of highly standardized convalescent plasma was safe, feasible and was readily implemented in the treatment of severely ill COVID-19 patients.