Investigation of the innate immune response after infection with Dobrava and Puumala viruses

V Sloveniji hemoragično mrzlico z renalnim sindromom (HMRS) povzročajo hantavirusi Dobrava (DOB), Puumala (PUU) in Dobrava-Kurkino. Potek bolezni je lahko zelo različen, od blage oblike bolezni, do bolezni s težkim potekom, ki se lahko konča tudi s smrtnim izidom. Dosedanje raziskave kažejo na to, da ima pri okužbi s hantavirusi pomembno vlogo odgovor prirojenega imunskega sistema gostitelja. Pokazali so, da patogeni hantavirusi zavirajo zgodnji odziv interferonov tipa I (IFN&#945in IFN&#946), stopnja zaviranja IFN pa je odvisna od stopnje patogenosti hantavirusa. Utišanje prirojenega imunskega odziva posledično privede do hitrega in nenadzorovanega, sistemskega razsoja okužbe. V dostopni literaturi še ni opisanih razlik v prirojenem imunskem odzivu po okužbi z virusom DOB in virusom PUU. V naši raziskavi smo želeli ugotoviti ali obstajajo razlike v prirojenem imunskem odzivu mononuklearnih celic iz periferne krvi (PBMC) pridobljenih iz kliničnih vzorcev bolnikov po okužbi z virusom DOB in virusom PUU ter njihov pomen v patogenezi HMRS. Doktorsko nalogo smo razdelili na tri dele. V prvem delu raziskave smo želeli ugotoviti, ali obstajajo razlike v odzivu IFN tipa I pri PBMC po stimulaciji z virusom DOB in virusom PUU ter ali razlike v odzivu IFN tipa I vplivajo na različen potek bolezni. Z virusom DOB in virusom PUU smo stimulirali PBMC zdravih prostovoljcev in PBMC bolnikov z znanim potekom HMRS. Ugotovili smo, da virus PUU, ne pa virus DOB, stimulira odziv IFN tip I, saj je bilo izražanje genov IFN&#946, STAT-1 in MxA 48 ur po stimulaciji statistično značilno večje kot pri nestimuliranih celicah. Z rezultati raziskave smo potrdili postavljeno hipotezo, da je stopnja odziva IFN tipa I po stimulaciji PBMC z virusom DOB nižja kot po stimulaciji z virusom PUU, kar posledično lahko pripomore k težji klinični sliki HMRS po okužbi z virusom DOB. S stimulacijo PBMC iz vzorcev bolnikov po preboleli HMRS smo ugotovili, da je izražanje gena MxA pri PBMC bolnikov, ki so imeli blažji potek okužbe z virusom PUU statistično značilno višje, kot pri PBMC bolnikov, ki so imeli težji potek okužbe. Iz rezultatov naše raziskave sklepamo, da večje protivirusno delovanje, ki smo ga potrdili z višjim izražanjem gena MxA, pripomore k blažjemu poteku okužbe z virusom PUU. Odziv PBMC pridobljenih iz akutnih vzorcev bolnikov smo primerjali z odzivom PBMC, ki smo jih pridobili iz vzorcev bolnikov po preboleli HMRS, saj nas je zanimala vloga imunskega spomina. Predpostavljali smo, da bo odziv IFN tipa I je bolj zavrt v PBMC med akutno okužbo kot v PBMC po preboleli okužbi. Statistično značilne razlike so se pokazale med PBMC bolnikov, ki so imeli težji potek okužbe z virusom PUU. Ugotovili smo, da je izražanje gena IFN&#946statistično značilno nižje, izražanje gena MxA pa statistično značilno višje pri akutnih PBMC, kot pri PBMC po preboleli okužbi. Iz rezultatov sklepamo, da so PBMC pridobljeni v akutni fazi HMRS bolj odzivni na stimulacijo z virusom PUU, kot PBMC pridobljeni po preboleli bolezni. Menimo, da odziv IFN tipa I pri PBMC ne prispeva k zaščiti pred ponovno okužbo z virusom DOB in virusom PUU, saj je del prirojenega imunskega sistema, ki na patogene deluje z nespecifičnimi odgovori. V drugem delu doktorske naloge smo s pretočno citometrijo primerjali profil PBMC bolnikov s HMRS. Želeli smo ugotoviti, ali okužba z virusom DOB in virusom PUU povzročita različen odziv imunskih celic. Zanimalo nas je tudi, ali so prisotne razlike v limfocitnih populacijah povezane s potekom HMRS. Ugotovili smo, da imajo bolniki okuženi z virusom DOB statistično značilno večji delež celic NK, kot bolniki okuženi z virusom PUU. Prav tako smo potrdili večji delež celic NK pri bolnikih okuženih z virusom PUU, ki so imeli težji potek bolezni, kot pri bolnikih z blažjim potekom okužbe. Dodatno smo ugotovili, da imajo bolniki s težjim potekom okužbe z virusom PUU, višjo koncentracijo citotoksičnih celic NK CD56dim. Iz rezultatov sklepamo, da so celice NK povezane s težjim potekom HMRS. Še posebno so s težjim potekom okužbe z virusom PUU povezane citotoksične celice NK CD56dim. Z analizo limfocitov T smo ugotovili, da imajo bolniki okuženi z virusom PUU višjo koncentracijo limfocitov T kot bolniki okuženi z virusom DOB. Prav tako so rezultati pokazali višjo koncentracijo limfocitov T pri bolnikih okuženih z virusom PUU, ki so imeli blažji potek okužbe, kot pri bolnikih s težjim potekom bolezni. Dodatno smo z našo raziskavo ugotovili, da imajo bolniki okuženih z virusom PUU večjo koncentracijo celic Th ter več aktiviranih limfocitov T, ki izražajo aktivacijski marker CD69, ki se pojavi tik pred proliferacijo in aktivacijski marker HLA-DR, ki se pojavi tik pred klonsko ekspanzijo ter višjo koncentracijo aktiviranih efektorskih celic T (CD25+CD4+) in regulatornih celic T (CD4+CD25+CD127low/-), kot bolniki okuženi z virusom DOB. Iz rezultatov sklepamo, da višja koncentracija limfocitov T in večji specifični T celični odziv pripomoreta k blažjemu poteku HMRS po okužbi z virusom PUU, kot z virusom DOB. Bolniki okuženi z virusom DOB, ki so imeli blažji potek bolezni, so imeli večji delež celic Th, ki so izražale aktivacijski marker HLA-DR, kot bolniki z blažjim potekom okužbe z virusom DOB. Glede na rezultate menimo, da večji delež aktiviranih celic T pomagalk, pripomore k blažji obliki okužbe z virusom DOB. V tretjem delu doktorske naloge smo aktivacijo prirojenega imunskega odziva preverjali na nivoju koncentracije proteina visoko-mobilne skupine B1 (HMGB1). Želeli smo opredeliti vlogo HMGB1 kot napovednega dejavnika pri hantavirusni okužbi. Koncentracijo HMGB1 smo izmerili v supernatantih stimuliranih PBMC zdravih prostovoljcev ter v serumih bolnikov okuženih z virusom DOB in virusom PUU. Ugotovili smo, da je koncentracija HMGB1 višja v supernatantih stimuliranih PBMC z virusom DOB in virusom PUU, kot pri nestimuliranih PBMC, medtem ko razlika v koncentraciji HMGB1 med virusoma ni bila statistično značilna. Rezultate smo potrdili tudi z merjenjem koncentracije HMGB1 v serumih bolnikov s HMRS, pri čemer smo ugotovili, da imajo bolniki okuženi z virusom DOB in virusom PUU povišano koncentracijo HMGB1, med virusoma pa prav tako ni bilo statistično značilnih razlik. Z raziskavo smo prvi pokazali, da ima HMGB1 vlogo tudi v patogenezi HMRS. Pokazali smo, da je HMGB1 uporaben napovedni dejavnik za težji potek okužbe z virusom PUU, saj imajo bolniki s težjim potekom okužbe z virusom PUU višjo koncentracijo HMGB1, kot bolniki z blažjim potekom bolezni. Iz naše raziskave sklepamo, da je odziv prirojenega imunskega sistema bolj aktiviran po okužbi z virusom PUU, kot po okužbi z virusom DOB. Menimo, da zaviranje imunskega odziva gostitelja pripomore k težjemu poteku HMRS po okužbi z virusom DOB, kot po okužbi z virusom PUU. Našo ugotovitev smo v doktorski nalogi potrdili na treh nivojih. Na nivoju proučevanja IFN odziva smo ugotovili, da virus PUU bolj stimulira odziv IFN tipa I, višje izražanje gena MxA pripomore k lažjemu poteku okužbe z virusom PUU. Na nivoju proučevanja limfocitov smo potrdili, da virus PUU bolj aktivira specifični odziv limfocitov T, kot virus DOB. Na biološkem nivoju smo z merjenjem koncentracije beljakovine HMGB1 pokazali, da je HMGB1 vpleten v patogenezo HMRS in da je ustrezen napovedni dejavnik za težji potek bolezni po okužbi z virusom PUU. Zaviranje odziva IFN tipa I z virusom DOB je možen razlog, da HMGB1 ni ustrezen napovedni dejavnik za težji potek okužbe z virusom DOB.Hemorrhagic fever with renal syndrome (HFRS) is an endemic disease in Slovenia and is caused by Dobrava virus (DOBV), Puumala virus (PUUV) and Dobrava-Kurkino virus. The clinical severity of HFRS varies greatly and both mild and severe clinical courses of the disease have been observed, with an overall case fatality rate of 4,5 %. The host innate immune responses play an important role in the infection with hantaviruses. Pathogenic hantaviruses inhibit the early response of type I interferons (IFN&#945and IFN&#946) and the IFN inhibition depends on the hantavirus pathogenicity. Inhibition of innate immune responses consequently leads to a rapid and systemic hantavirus infection. According to available literature, the difference in innate immune response between DOBV and PUUV infection was yet not compared. In our study, differences in activation of the innate immune response in peripheral blood mononuclear cells (PBMCs) from clinical samples of patients infected with DOBV and PUUV were investigated and significance of observed differences in HFRS severity was compared. The doctoral thesis is divided into three sections. In the first part of the study, the difference in the IFN type I-induced antiviral state were investigated in PBMCs in early post-stimulation with DOBV or PUUV. PBMCs from healthy volunteers and HFRS patients were stimulated with DOBV and PUUV. Our results showed that PUUV, but not DOBV, activated the IFN type I-induced antiviral state in stimulated PBMCs, and that IFN&#946, STAT-1, and MxA were highly up-regulated at 48 h post-stimulation. The results of the study confirmed the hypothesis that DOBV activated lower IFN type I response in comparison to PUUV and the delayed IFN type I response could be a contributor to a more severe clinical outcome of the disease. Furthermore, up-regulation of MxA was statistically significant higher in convalescent-phase PBMCs from patients with mild PUUV infection than in patients with severe disease. Higher IFN type I-induced antiviral state could be a contributor to mild HFRS, particularly in PUUV infection. The IFN type I response were compared between acute-phase PBMCs and convalescent-phase cells from the same HFRS patients, and the role of immunological memory was investigated. Higher inhibition of IFN type I response was hypothesized in acute-phase PBMCs. Statistically significant differences were found between acute- and convalescent-phase PBMCs from patients with severe PUUV infection. Our result showed significant lower expression of IFN&#946and significant higher expression of MxA in acute-phase PBMCs compared to convalescent-phase PBMCs, which indicated higher responsiveness of acute-phase PBMCs to PUUV stimulation. Results indicated no contribution of IFN type I response to the protection against DOBV or PUUV re-infection, as it is part of the innate immune system, with non-specific defense against pathogens. In the second part of the doctoral thesis, the PBMCs profile of patients with HFRS was analyzed by the flow cytometry. The different response of lymphocyte populations was investigated between DOBV and PUUV patients, and connection to HFRS severity was examined. Results showed significantly higher count of NK cells in patients with DOBV infection than in patients with PUUV infection. Higher count of NK cells was detected in patients with severe PUUV infection compared to patients with mild course of infection. Additionally, higher concentration of cytotoxic NK cells CD56dim was detected in patients with severe PUUV infection. Results indicated that NK cells could be associated with severe course of HFRS, especially higher amount of cytotoxic NK cells CD56dim could contribute to the more severe PUUV infection. Analysis showed higher concentration of T lymphocyte, especially higher concentration of Th cells, in PUUV infected patients in comparison to DOBV infected patients. Moreover, results indicated higher concentration of T lymphocyte in PUUV infected patients with mild HFRS compared to those with severe clinical course of the disease. Additionally, PUUV infected patients indicated higher concentration of effector T cells (CD25+CD4+), regulatory T cells (CD4+CD25+CD127low/-) and higher T lymphocyte activation, expressing activation markers CD69 and HLA-DR, in comparison to DOBV infected patients. Higher T lymphocyte concentration and higher specific T cell response contribute to milder HFRS after PUUV infection in comparison to DOBV infection. Results indicated higher amount of Th cell, expressing activation marker HLA-DR in patients with mild DOBV infection in comparison to patients with severe infection. According to results of our study, higher amount of activated Th cell could contribute to milder HFRS after DOBV infection. In the third part of the study, the concentration of protein High mobility group box 1 (HMGB1) was measured and the role of HMGB1 as a prognostic marker for HFRS severity was investigated. The HMGB1 concentration was measured in supernatants of stimulated PBMCs from healthy donors and in serum samples of HFRS patients. Result showed significantly higher concentration of HMGB1 in supernatants of PBMCs stimulated with DOBV and PUUV in comparison to unstimulated PBMCs, but the difference between viruses was not statistically significant. Results of our in vitro experiment was confirmed by measurement of HMGB1 concentration in patients’ serum samples. HMGB1 concentration was significantly elevated in HFRS patients compared to healthy donors, and no statistically significant difference was observed between the viruses. Our study is the first indicating the role of HMGB1 in HFRS pathogenesis. We have demonstrated potential usefulness of HMGB1 as a biomarker for severity in PUUV infection, as patients with a severe PUUV infection had significantly higher concentration of HMGB1 than patients with mild disease. In conclusion, results of our study indicated higher activation of innate immune responses after PUUV infection in comparison to DOBV infection. The inhibition of innate immune response could be one of the contributors to a more severe clinical outcome after DOBV infection. In the doctoral thesis, the differences in innate immunity between DOBV and PUUV infection were confirmed on three different biological levels. Results of PBMCs stimulation indicated high activation of the IFN type I-induced antiviral state after PUUV infection. Flow cytometry analyses showed high specific T lymphocyte activation in patients infected with PUUV. Our study indicated potential usefulness of HMGB1 as a prognostic marker for PUUV severity. Inhibition of IFN&#946by DOBV could be a possible reason that HMGB1 is not the appropriate biomarker for severity in DOBV infection

HMGB1 Is a Potential Biomarker for Severe Viral Hemorrhagic Fevers.

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

Performance of the rapid high-throughput automated electrochemiluminescence immunoassay targeting total antibodies to the SARS-CoV-2 spike protein receptor binding domain in comparison to the neutralization assay

Background: Neutralization tests (NT) are the gold standard for detecting and quantifying anti-SARS-CoV-2 neutralizing antibodies (NAb), but their complexity restricts them to research settings or reference laboratories. Antibodies against S protein receptor binding domain (RBD) have been shown to confer a neutralizing activity against SARS-CoV-2. Assays quantitatively measuring anti-S1-RBD-SARS-CoV-2 antibodies could be of great value for NAb screening of potential donors for convalescent-phase plasma therapy, assessing natural or vaccine-induced immunity, stratifying individuals for vaccine receipt, and documenting vaccine response. Methods: Elecsys Anti-SARS-CoV-2 S (Elecsys-S), a high-throughput automated electrochemiluminescence double-antigen sandwich immunoassay for quantitative measurement of pan-anti-S1-RBD-SARS-CoV-2 antibodies, was evaluated against NT on 357 patients with PCR-confirmed SARS-CoV-2 infection. NT was performed in a BSL-3 laboratory using a Slovenian SARS-CoV-2 isolatethe NT titer ≥1:20 was considered positive. Results: Elecsys-S detected pan-anti-S1-RBD-SARS-CoV-2 antibodies in 352/357 (98.6 %) samples. NAb were identified by NT in 257/357 (72 %) samples. The Elecsys-S/NT agreement was moderate (Cohen’s kappa 0.56). High NT titer antibodies (≥1:160) were detected in 106/357 (30 %) samples. Elecsys-S’s pan-anti-S1-RBD-SARS-CoV-2 antibody concentrations correlated with individual NT titer categories (the lowest concentrations were identified in NT-negative samples and the highest in samples with NT titer 1:1,280), and the Elecsys-S cutoff value for reasonable prediction of NAb generated after natural infection was established (133 BAU/mL). Conclusion: Although NT should remain the gold standard for assessing candidates for convalescent-phase plasma donors, selected commercial anti-SARS-CoV-2 assays with optimized cutoff, like Elecsys-S, could be used for rapid, automated, and large-scale screening of individuals with clinically relevant NAb levels as suitable donors

Comparison of serum HMGB1 concentrations in patients with CCHFV infection according to the clinical course of the disease.

<p>The Mann-Whitney U test was used for statistically analyses. A <i>p</i>-values below 0.05 were considered statistically significant.</p

Relationship between circulating vascular endothelial growth factor and its soluble receptor in patients with hemorrhagic fever with renal syndrome

Hemorrhagic fever with renal syndrome (HFRS) is characterized by endothelial dysfunction with capillary leakage without obvious cytopathology in the capillary endothelium. The aim of the study was to analyze the kinetics of vascular endothelial growth factor (VEGF) and its soluble receptor (sVEGFR-2) in HFRS patients infected with Dobrava (DOBV) or Puumala virus (PUUV). VEGF and sVEGFR-2 levels were measured in daily plasma and urine samples of 73 patients with HFRS (58 with PUUV, 15 with DOBV) and evaluated in relation to clinical and laboratory variables. In comparison with the healthy controls, initial samples (obtained in the first week of illness) from patients with HFRS had higher plasma and urine VEGF levels, whereas sVEGFR-2 levels were lower in plasma but higher in urine. VEGF levels did not differ in relation to hantavirus species, viral load, or the severity of HFRS. The comparison of VEGF dynamics in plasma and urine showed the pronounced secretion of VEGF in urine. Significant correlations were found between daily VEGF/sVEGFR-2 levels and platelet counts, as well as with diuresis: the correlations were positive for plasma VEGF/sVEGFR-2 levels and negative for urine levels. In addition, patients with hemorrhagic manifestations had very high plasma and urine VEGF, together with high urine sVEGFR-2. Measuring the local secretion of sVEGFR-2 in urine might be a useful biomarker for identifying HFRS patients who will progress to severe disease

HMGB1 concentration in patients with hemorrhagic fevers.

<p>HMGB1 concentration in patients with hemorrhagic fevers.</p

Comparative Evaluation of Six SARS-CoV-2 Real-Time RT-PCR Diagnostic Approaches Shows Substantial Genomic Variant&ndash;Dependent Intra- and Inter-Test Variability, Poor Interchangeability of Cycle Threshold and Complementary Turn-Around Times

Several professional societies advise against using real-time Reverse-Transcription PCR (rtRT-PCR) cycle threshold (Ct) values to guide clinical decisions. We comparatively assessed the variability of Ct values generated by six diagnostic approaches by testing serial dilutions of well-characterized isolates of 10 clinically most relevant SARS-CoV-2 genomic variants: Alpha, Beta, Gamma, Delta, Eta, Iota, Omicron, A.27, B.1.258.17, and B.1 with D614G mutation. Comparison of three fully automated rtRT-PCR analyzers and a reference manual rtRT-PCR assay using RNA isolated with three different nucleic acid isolation instruments showed substantial inter-variant intra-test and intra-variant inter-test variability. Ct value differences were dependent on both the rtRT-PCR platform and SARS-CoV-2 genomic variant. Differences ranging from 2.0 to 8.4 Ct values were observed when testing equal concentrations of different SARS-CoV-2 variants. Results confirm that Ct values are an unreliable surrogate for viral load and should not be used as a proxy of infectivity and transmissibility, especially when different rtRT-PCR assays are used in parallel and multiple SARS-CoV-2 variants are circulating. A detailed turn-around time (TAT) comparative assessment showed substantially different TATs, but parallel use of different diagnostic approaches was beneficial and complementary, allowing release of results for more than 81% of non-priority samples within 8 h after admission

Fetal death from SARS-CoV-2 mediated acute placental failure

Introduction: We demonstrate the nonlinear severity of symptoms of SARS-CoV-2 infection in the mother leading to fetal death after acute placental failure. Methods: Careful clinical evaluation, real-time RT-PCR molecular microbiologic testing, isolation of a viable virus, and autopsy with histologic results were used to investigate the possible vertical transmission of SARS-CoV-2 infection from mother to fetus. Results: Histologic changes in the placenta correlate with SARS-CoV-2 infection. Total nucleic acid isolated from vaginal swabs, fresh placental tissue, and deparaffinized tissue showed a high viral load of SARS-CoV-2. Complete genome sequencing confirmed the presence of the SARS-CoV-2 Delta variant. Discussion: Several methods have been used to confirm SARS-CoV-2-mediated acute placental failure, all of which were conclusive. It should be noted that careful periodic fetal well-being checks are required in women infected with SARS-CoV-2, regardless of the severity of symptoms. Most of the cases described with fetal death occurred in the third trimester