PURPOSE OF REVIEW: This review article aims to explore the GI changes induced by SARS-CoV-2 and how gut microbial homeostasis can influence these changes and affect the lung-gut axis and its relationship with the induction of the cytokine release syndrome in severe COVID-19 patients. RECENT FINDINGS: Coronavirus disease 2019 (COVID-19) affects not only the respiratory system but can produce multi-systemic damage. The expression of angiotensin-converting enzyme 2 (ACE-2) receptors in the gastrointestinal (GI) tract, the high prevalence of GI symptoms in severely ill COVID-19 patients, and the abnormalities described in the gut microbiome in these patients have raised concerns about the influence of GI tract as a risk factor or as a potential modulator to reduce the severity of COVID-19. Understanding the mechanisms by which gut dysbiosis may influence viral transmission and disease progression in COVID-19 may help in shaping how accessible therapies, like diet modulation, can potentially help beat the devastating consequences of COVID-19.

BACKGROUND: Telepresence robots provide real-time audio, video, and mobility features, allowing faculty and students to engage in learning experiences without being physically present. PROBLEM: With multiple students and faculty members needing to quarantine due to the COVID-19 pandemic, a flexible learning environment was essential. APPROACH: The telepresence robots were used as an innovative approach for both faculty and students to engage in learning experiences offered in a variety of settings. OUTCOME: Feedback was obtained from faculty and students about the use of and satisfaction with telepresence robots. The robots were easy to use and posed only a few technological challenges, which were easily overcome. CONCLUSIONS: Telepresence robots were effective tools in overcoming teaching and learning barriers caused by the COVID-19 pandemic. The telepresence robots have many applications, including use in clinical and community settings.

During the past three years, we assisted to the rise of a new pathogen that afflicted the world with a global pandemic. Working in an era of rapid change has posed important challenges and the focus of research has shifted more and more toward topics of greater social utility. However, this period has also brought a new role for wastewater highlighting how it can provide insight into the health of a community. This is the approach of Wastewater-based Epidemiology (WBE). The work presented here aimed to deepen this approach not only at the theoretical level but also contributing with an ongoing monitoring of about 30 months. The main objectives were (i) to collect information on the recently discovered SARS-CoV-2 virus, its biology, transmission mechanism, and role in wastewater treatment plants (WWTP); (ii) set up a surveillance system that would allow to monitor SARS-CoV-2 infections over time, obtaining early information on its spread among the population to support the Health Authority. Starting from a detailed study of the shedding mechanisms of SARS-CoV-2 in the feces of infected patients, we moved on to the evaluation of the viral concentrations in the sewage system and the wastewater entering the WWTP. The possibility of a faecal-oral transmission route of the virus was investigated by evaluating the data about viability and infectivity in wastewater. The natural processes of decay of the virus in wastewater and the reduction of its concentration in the different treatment stages of WWTPs were explored in literature and with experimental data. At the same time, we developed a SARS-CoV-2 surveillance system in wastewater by applying different detection methods. Some practical and scientific aspects of the analysis protocol have been studied in depth such as the choice of the type of sample, the storage temperatures, and the pre-heat treatments aimed at making the analysis safer for the operator. The choice of the concentration method was evaluated to comply with the low concentration of the viral titer and therefore the crucial importance of this phase of the protocol. During the monitoring campaign, we further investigated aspects related to data processing and developed normalization approaches. Samples from WWTPs in the province of Trento were analysed weekly and sampling frequencies and curve smoothing methods deriving from those data were evaluated. The trend curves thus obtained were compared with those deriving from clinical data provided by the local Health Authority and signals of early warnings of virus diffusion trends in the population were highlighted. With the alternation of the different variants of the virus and the evidence of their importance in the development of new waves of infection, a PCR based genotyping method has been devised to rapidly identify the already known variants. In conclusion, this research project addressed a broad spectrum of aspects related to the WBE approach in contrasting the COVID-19 emergency and confirmed that wastewater could be a valuable source of information and management support for this and other emerging pathogens or micropollutants.

No description available.

Enveloped RNA viruses like human immunodeficiency virus type-1 (HIV-1) and SARS-CoV-2 enter host cells through fusion with the plasma membrane, a process facilitated by specific viral envelope proteins that recognize and bind to receptors expressed on the host cell surface. These receptors can diverge based on the type of cell and virus. For HIV-1, the primary receptors on myeloid cells are CD4 and CCR5 or CXCR4. For SARS-CoV-2, although the primary receptor is ACE2, other myeloid-cell specific sialic acid binding lectins can also facilitate entry. Following cellular invasion, different viral RNA species can be detected by distinct host nucleic acid sensors, resulting in type I interferons and pro-inflammatory cytokine induction. While these innate immune responses are essential for controlling viral infections, overactivation can lead to chronic inflammation, tissue damage, and disease pathogenesis. Herein, I examine the contribution of HIV-1 and SARS-CoV-2 de-novo RNA expression and the molecular mechanisms that contribute to innate immune activation in myeloid cells. Despite advancements in combination antiretroviral therapy (ART) in suppressing systemic viral replication in individuals infected with HIV, residual viral RNA expression in tissue reservoirs remains a significant hindrance to curative efforts. I hypothesized that persistent expression of viral RNAs in myeloid cells triggers dysregulated innate immune activation, and inflammasomes activation. This study centers on the long-lived tissue-resident innate immune cells - macrophages and microglia, which, owing to their self-renewing nature, operate as reservoirs of viral RNA production, and are thought to lead to chronic immune activation even in the absence of productive replication. Our previous studies suggest that de novo expression of unspliced intron-containing HIV-1 RNA (herein referred to as icRNA) triggers activation of pro-inflammatory cytokines in myeloid cells. Here, I demonstrate that cytosolic expression of HIV-1 icRNA, but not multiply-spliced viral RNAs induces inflammasome activation, LDH release and IL-1β secretion in productively infected monocyte-derived macrophages (MDM) and induced pluripotent stem cell (iPSC)-derived microglia. Interestingly, knockdown of RLRs, RIG-I and MDA5 or endosomal TLRs failed to abrogate HIV-1 icRNA-induced IL-1β secretion. Rather, knockdown of NLRP1, but not NLRP3, inflammasome resulted in a significant reduction in IL-1β secretion, underscoring NLRP1's pivotal role in the HIV-1 icRNA-induced IL-1β secretion. Furthermore, Rev-Crm1-dependent nucleocytoplasmic export of HIV-1 icRNA was required for NLRP1-mediated Caspase-1 activation, IL-1β secretion, LDH release and cell death. Similarly, SARS-CoV-2, while not establishing productive infection in macrophages, can activate these cells, contributing to a hyper-inflammatory response marked by the heightened expression of pro-inflammatory cytokines, which is understood to be a principal driver of COVID-19 pathology. SARS-CoV-2 established an abortive infection in macrophages. CD169, a macrophage-specific sialic-acid binding lectin, mediated ACE2-independent SARS-CoV-2 entry in human macrophages and establishment of restricted infection. Interestingly, CD169-mediated SARS-CoV-2 entry in macrophages led to the expression of viral genomic and subgenomic RNAs, with negligible viral protein expression and no release of infectious virus particles, implying a post-entry restriction to SARS-CoV-2 replication in macrophages that was curbed by exogenous ACE2 expression. Despite restricted viral RNA expression, cytoplasmic RLRs, RIG-I and MDA5, sensed abortive viral transcripts, and induced pro-inflammatory responses in a MAVS dependent manner. This dissertation reveals striking parallels between the role of viral RNAs in driving pro-inflammatory responses in HIV-1 and SARS-CoV-2 infections. These findings collectively underscore the central role of cytoplasmic sensing of viral RNAs and their contribution to chronic inflammation in virus-infected myeloid cells. Elucidating these molecular mechanisms further may pave the way for novel therapeutic interventions to mitigate the persistent innate immune activation and immunopathology detected in HIV-1 and SARS-CoV-2 infected individuals.

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

Objective: It is unclear whether and to what extent COVID-19 infection poses health risks and a chronic impairment of performance in athletes. Identification of individual health risk is an important decision-making basis for managing the pandemic risk of infection with SARS-CoV-2 in sports and return to play (RTP). Methods: This study aims 1) to analyze the longitudinal rate of seroprevalence of SARSCoV- 2 in German athletes, 2) to assess health-related consequences in athletes infected with SARS-CoV-2, and 3) to reveal effects of the COVID-19 pandemic in general and of a cleared SARS-CoV-2 infection on exercise performance. CoSmo-S is a prospective observational multicenter study establishing two cohorts: 1) athletes diagnosed positive for COVID-19 (cohort 1) and 2) federal squad athletes who perform their annual sports medical preparticipation screening (cohort 2). Comprehensive diagnostics including physical examination, laboratory blood analyses and blood biobanking, resting and exercise electrocardiogram (ECG), echocardiography, spirometry and exercise testing added by questionnaires are conducted at baseline and follow-up. Results and Conclusion: We expect that the results obtained, will allow us to formulate recommendations regarding RTP on a more evidence-based level.

No description available.

In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by the cellular angisin-converting enzyme 2 (ACE2). The virus abundantly replicates in the epithelia of the upper respiratory tract. Beyond vaccines for immunization, there is an imminent need for novel treatment options in COVID-19 patients. So far, only a few drugs have found their way into the clinics, often withmodest success. Specific gene silencing based on small interfering RNA (siRNA) has emerged as a promising strategy for therapeutic intervention, preventing/limiting SARS-CoV-2 entry into host cells or interfering with viral replication. Here, we pursued both strategies. We designed and screened nine siRNAs (siA1-9) targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90% knockdown of the ACE2 mRNA and protein for at least six days. In vitro, siA1 application was found to protect Vero E6 and Huh-7 cells from infectionwith SARS-CoV-2 with an up to ~92% reduction of the viral burden indicating that the treatment targets both the endosomal and the viral entry at the cytoplasmic membrane. Since the RNAencoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analysed eight siRNAs (siV1-8) directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). As a significant hallmark of this study, we identified siV1 (siRNA against leader protein of SARS-CoV-2), which targets the nsp1- encoding sequence (a.k.a. ‘host shutoff factor’) as particularly efficient. SiV1 inhibited SARSCoV- 2 replication in Vero E6 or Huh-7 cells by more than 99% or 97%, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention.

Die neuartige Infektionskrankheit Covid-19 breitet sich seit Dezember 2019 von Wuhan, China, in zahlreiche Länder aller Kontinente aus. Durch die rasche Verbreitung der Viruserkrankung und die schnell steigende Zahl an infizierten Personen, rief die WHO im Januar 2020 die „Gesundheitliche Notlage internationaler Tragweite“ aus. Im März desselben Jahres wurde die Situation als Pandemie deklariert. Die Infektionskrankheit wird durch das SARS-CoV-2 hervorgerufen, ein Vertreter aus der Gattung der Betacoronaviren, dessen Genom durch einzelsträngige RNA positiver Polarität (ssRNA) gekennzeichnet ist. Ein zoonotischer Ursprung des Virus aus Fledermäusen wird derzeit aufgrund hoher genetischer Übereinstimmungen als wahrscheinlichste initiale Quelle betrachtet. Die Transmission des SARS-CoV-2 erfolgt über die respiratorische Aufnahme von virushaltigem Aerosol und ruft Symptome wie Husten, Fieber, Geschmacks- und Geruchsstörungen sowie bei schwerem Verlauf auch Pneumonien mit beatmungspflichtigem ARDS (Acute Respiratory Distress Syndrome) hervor. Eine spezifische Therapie für Covid-19 ist bisher nicht etabliert, jedoch sind supportive Maßnahmen sinnvoll, der Einsatz von Remdesivir von der Europäischen Arzneimittel-Agentur zugelassen und die Verwendung von systemischen Kortikosteroiden von der WHO empfohlen. Um die Basisreproduktionszahl für das SARS-CoV-2 gering zu halten, sind infektionspräventive Maßnahmen wie Mund-Nase-Bedeckung, Mindestabstand, regelmäßiger Luftaustausch und das strenge Einhalten von Hygieneregeln wirksam. Die ersten Impfstoffe für die Viruserkrankung Covid-19 stehen seit Dezember 2020 bereit. Da die Vakzine bislang begrenzt zur Verfügung stehen, ist eine frühzeitige Diagnostik und die Nachverfolgung des Infektionsgeschehens weiterhin von großer Bedeutung. Der direkte Erregernachweis des SARS-CoV-2 erfolgt durch einen Nasen-Rachen-Abstrich, der mittels RT-PCR auf replizierendes Virusgenom untersucht wird. Für die Erfassung der asymptomatisch bzw. subklinisch infizierten Personen sind neben dem Antigen-Schnelltest auch serologische Nachweisverfahren notwendig, um die reale Ausbreitung des SARS-CoV-2 nachvollziehen zu können. Ziel dieser Arbeit war es, durch zahlreiche Optimierungen ein valides Protokoll für einen SARS-CoV-2-N IgG Antikörper ELISA zu etablieren. Der Test sollte einen qualitativen Nachweis zur Detektion der SARS-CoV-2-Immunantwort gegen das Nukleokapsidprotein des Virus möglich machen. Die in das Protokoll aufgenommenen Optimierungen umfassen u. a. die Konzentration des verwendeten Antigens N-MBP, die Zusammensetzung der Beschichtungs- und Blocklösung, die Probenvolumina, die Verdünnung des Sekundärantikörpers und die Zeit der Substratinkubation für die Farbentwicklung. Für das Protokoll des SARS-CoV-2-N IgG Antikörper ELISA wurde ein Kalibrator entwickelt, um einen normierten Grenzwert für Seropositivität festlegen zu können und ein Antikörper-Konzentrationsstandard etabliert, um die Ergebnisse des Immunassays auch quantitativ interpretieren zu können. Zur Validierung des SARS-CoV-2-N IgG Antikörper ELISA wurden 73 Covid-19-Seren von Probanden mit zuvor positivem RT-PCR Testergebnis und 180 Negativkontrollseren bzw. -plasmen verwendet. Die Covid-19-Patienten zeigten eine milde bis moderate Erkrankung oder einen asymptomatischen Infektionsverlauf. Die Covid-19-Seren wurden 2 - 3 Wochen (n = 25) oder über 4 Wochen (n = 48) nach Symptombeginn bzw. positivem RNA-Test gewonnen. Die Spezifität des SARS-CoV-2-N IgG Antikörpertests betrug 99,44 % und die Sensitivität wurde mit 80,82 % ermittelt. Nach 2 - 3 Wochen entnommene Covid-19-Seren wurden dabei mit einer Sensitivität von 80,0 % und mehr als 4 Wochen nach Diagnosestellung gewonnene Seren mit einer Positivrate von 81,3 % erkannt. Weiterhin wurde die Empfindlichkeit des In-house ELISA in einer prospektiven diagnostischen Studie mit der Sensitivität von sieben kommerziellen Nukleokapsid- oder S-Glykoprotein-basierten Antikörpertests verglichen, um den Nutzen der Tests für den klinischen Einsatz bewerten zu können. Die Sensitivitäten der Antikörperassays lagen bei 64,4 - 93,2 %. Die empfindlichsten Tests erkannten 95,8 - 100 % der über 4 Wochen nach Symptombeginn gewonnenen Covid-19-Seren als positiv. Seren, die 2 - 3 Wochen nach positivem RNA-Test entnommen wurden, wurden mit einer geringeren Sensitivität erkannt, was darauf hindeutet, dass der optimale Zeitpunkt für serologische Testungen später als 3 Wochen nach Ausbruch der Infektionskrankheit liegen sollte. Antikörpertests, die das Nukleokapsid- bzw. das S-Glykoprotein als Antigen verwendeten, zeigten vergleichbare Sensitivitätswerte auf. Dies bedeutet, dass sowohl N- als auch S-basierte Antiköpertests für die serologische Diagnostik geeignet sind. Nukleokapsidprotein und S-Glykoprotein-basierte Antikörperassays zeigten außerdem Unterschiede in der Detektion einer positiven oder negativen Immunreaktion bei den untersuchten Covid-19-Seren. Eine kombinierte Auswertung von seriellen Tests unter separater Verwendung beider Antigene könnte somit die Positivrate bei der Untersuchung von Covid-19-Seren steigern.:INHALTSVERZEICHNIS ABKÜRZUNGSVERZEICHNIS III 1 EINFÜHRUNG 1 1.1 SARS-CoV-2: Virale Struktur und Replikation 1 1.2 Covid-19: Pathogenese und Krankheitsbild 3 1.3 Epidemiologie und Herkunft 6 1.4 Diagnostik 8 1.5 Therapie 11 1.6 Prävention 12 1.7 Impfung 12 2 AUFGABENSTELLUNG 15 3 MATERIALIEN UND METHODEN 16 3.1 Materialien 16 3.1.1 Geräte 16 3.1.2 Chemikalien 16 3.1.3 Proteine 17 3.1.4 Seren 17 3.1.5 Immunoreagenzien 18 3.1.6 Sonstige Materialien 18 3.2 Methoden 18 3.2.1 Allgemein verwendete Lösungen 18 3.2.2 Ethikantrag 19 3.2.3 Probanden 19 3.2.4 Probenentnahme 20 3.2.5 Probenaufarbeitung 20 3.2.6 Nukleokapsidprotein (N) und Maltose-bindendes Protein (MBP) 20 3.2.7 Enzyme-Linked Immunosorbent Assay (ELISA) 22 3.2.8 Kommerzielle Antikörpertests 23 3.2.9 Auswertung und Statistik 24 4 ERGEBNISSE 28 4.1 Optimierung des SARS-CoV-2-N IgG Antikörper ELISA 28 4.1.1 Beschichtung mit Nukleokapsid-Fusionsprotein 28 4.1.2 Einfluss der Antigenkonzentration in der Beschichtungslösung 29 4.1.3 Einfluss der Auftauhäufigkeit des N-MBP-Antigens 31 4.1.4 Variation der Beschichtungslösung 33 4.1.5 Beschichtungs- und Probenvolumina 34 4.1.6 Blocklösung 35 4.1.7 Substratinkubationszeit 36 4.1.8 Sekundärantikörperverdünnung 38 4.1.9 Stabilisierung des Protokolls mit CANDOR®-Reagenzien 40 4.2 Validierung des SARS-CoV-2-N IgG Antikörper ELISA 42 4.2.1 Herstellung eines Kalibrators 42 4.2.2 Bestimmung der Sensitivität 44 4.2.3 Bestimmung der Spezifität 46 4.2.4 Herstellung eines Antikörper-Konzentrationsstandards 47 4.2.5 Intra-Assay und Inter-Assay-Variabilität 49 4.3 Protokoll des SARS-CoV-2-N IgG Antikörper ELISA 50 4.4 Vergleich der diagnostischen Sensitivität von SARS-CoV-2-Nukleoprotein- und Glykoprotein-basierten Antikörpertests 53 5 DISKUSSION 58 5.1 Ergebnisse der diagnostischen Validierungsstudie 58 5.2 Eignung und Verwendung des Nukleokapsidproteins als Antigen für den SARS-CoV-2 IgG Antikörper ELISA 61 5.3 Validität und Limitationen 62 5.4 Ausblick und Bedeutung des SARS-CoV-2-N IgG Antikörpertests 64 6 ZUSAMMENFASSUNG 67 LITERATURVERZEICHNIS 70 ANLAGEN 89 ERKLÄRUNG ÜBER DIE EIGENSTÄNDIGE ABFASSUNG DER ARBEIT 92 LEBENSLAUF 93 PUBLIKATIONSVERZEICHNIS 95 DANKSAGUNG 96