Inhibitors of dihydroorotate dehydrogenase cooperate with molnupiravir and N4-hydroxycytidine to suppress SARS-CoV-2 replication

The nucleoside analog N4-hydroxycytidine (NHC) is the active metabolite of the prodrug molnupiravir, which has been approved for the treatment of COVID-19. SARS-CoV-2 incorporates NHC into its RNA, resulting in defective virus genomes. Likewise, inhibitors of dihydroorotate dehydrogenase (DHODH) reduce virus yield upon infection, by suppressing the cellular synthesis of pyrimidines. Here, we show that NHC and DHODH inhibitors strongly synergize in the inhibition of SARS-CoV-2 replication in vitro. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC into nascent viral RNA. This concept is supported by the rescue of virus replication upon addition of pyrimidine nucleosides to the media. DHODH inhibitors increased the antiviral efficiency of molnupiravir not only in organoids of human lung, but also in Syrian Gold hamsters and in K18-hACE2 mice. Combining molnupiravir with DHODH inhibitors may thus improve available therapy options for COVID-19

Migraine and the development of additional psychiatric and pain disorders in the transition from adolescence to adulthood

Introduction: The transition from childhood to adolescence and from adolescence to adulthood are vulnerable phases in life. In these phases, late or insufficient treatment of diseases may lead to chronification and favor development of additional disorders. In adolescents, migraine often has a highly negative impact on school performance and everyday life. The hypothesis of the present study was that adolescents with migraine have a higher risk for developing additional disorders such as psychiatric disorders or other pain syndromes in the course of the disease. Materials and methods: In this study, we analyzed health insurance data of 56,597 German adolescents at the age of 15 years in the year 2006. By using the International Classification of Diseases (ICD 10), we determined a group with migraine diagnosis in the year 2006 and a control group without any headache diagnosis in 2006. We then compared both groups regarding the development of additional disorders (based on the ICD 10) during the following 10 years (2007 to 2016). Results: Adolescents with migraine had a 2.1 fold higher risk than persons without migraine diagnosis to develop an additional affective or mood disorder, a 1.8 fold higher risk to obtain neurotic, stress-related and somatoform disorders, a 1.8 fold higher risk to subsequently suffer from behavioral syndromes, a 1.6 higher risk to get back pain and a 1.5 fold higher risk for irritable bowel syndrome during the next 10 years. Conclusion: Adolescents with migraine are at risk for developing additional disorders later. Considering and addressing the patient’s risks and potential medical and psychosocial problems might improve the long-term outcome significantly

MicroRNAs modulate SARS-CoV-2 infection of primary human hepatocytes by regulating the entry factors ACE2 and TMPRSS2.

BACKGROUND AND AIMS Severe acute respiratory syndrome coronavirus (SARS-CoV-2) preferentially infects the respiratory tract; however, several studies have implicated a multi-organ involvement. Hepatic dysfunctions caused by SARS-CoV-2 infection have been increasingly recognized and described to correlate with disease severity. To elucidate molecular factors that could contribute towards hepatic infection, we concentrated on microRNAs (miRNAs), a class of small non-coding RNAs that modulate various cellular processes and which are reported to be differentially regulated during liver injury. We aimed to study the infection of primary human hepatocytes (PHH) with SARS-CoV-2 and to evaluate the potential of miRNAs for modulating viral infection. METHODS We analysed liver autopsies from a coronavirus disease 19 (COVID-19)-positive cohort for the presence of viral RNA using Nanopore sequencing. PHH were used for the infection with SARS-CoV-2. The candidate miRNAs targeting angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) were identified using in silico approaches. To discover the potential regulatory mechanism, transfection experiments, qRT-PCRs, western blots and luciferase reporter assays were performed. RESULTS We could detect SARS-CoV-2 RNA in COVID-19-positive liver autopsies. We show that PHH express ACE2 and TMPRSS2 and can be readily infected with SARS-CoV-2, resulting in robust replication. Transfection of selected miRNA mimics reduced SARS-CoV-2 receptor expression and SARS-CoV-2 burden in PHH. In silico and biochemical analyses supported a potential direct binding of miR-141-3p to the SARS-CoV-2 genome. CONCLUSION We confirm that PHH are susceptible to SARS-CoV-2 infection and demonstrate selected miRNAs targeting SARS-CoV-2 entry factors and/or the viral genome reduce viral loads. These data provide novel insights into hepatic susceptibility to SARS-CoV-2 and associated dysfunctions in COVID-19

Inhibitors of dihydroorotate dehydrogenase cooperate with molnupiravir and N4-hydroxycytidine to suppress SARS-CoV-2 replication

Funding Information: We thank Thorsten Wolff, Daniel Bourquain, Jessica Schulz, and Christian Mache from the Robert-Koch Institute and Martin Beer from the Friedrich Loeffler Institute (FLI) for providing isolates of SARS-CoV-2 variants. We thank Anna Kraft and Gabriele Czerwinski (both FLI) for support in the preparation of samples for pathology, and Catherine Hambly (University of Aberdeen) for help with daily energy expenditure measurements. We would like to thank Cathrin Bierwirth (University Medical Center Göttingen), Isabell Schulz, Anne-Kathrin Donner, and Frank-Thorben Peters for excellent technician assistance and Jasmin Fertey and Alexandra Rockstroh for providing the virus stocks for the mice experiment (Fraunhofer Institute IZI Leipzig). We acknowledge support by the Open Access Publication Funds of the Göttingen University. KMS was a member of the Göttingen Graduate School GGNB during this work. This work was funded by the COVID-19 Forschungsnetzwerk Niedersachsen (COFONI) to MD, by the Federal Ministry of Education and Research Germany ( Bundesministerium für Bildung und Forschung; BMBF ; OrganSARS , 01KI2058 ) to SP and TM, and by a grant of the Max Planck Foundation to DG. Declaration of interests AS, HK, EP, and DV are employees of Immunic AG and own shares and/or stock-options of the parent company of Immunic AG, Immunic Inc. Some of the Immunic AG employees also hold patents for the Immunic compounds described in this manuscript (WO2012/001,148, WO03006425). KMS, AD, and MD are employees of University Medical Center Göttingen, which has signed a License Agreement with Immunic AG covering the combination of DHODH inhibitors and nucleoside analogs to treat viral infections, including COVID-19 (inventors: MD, KMS, and AD). The other authors declare no conflict of interest.Peer reviewedPublisher PD

In-depth analysis of T cell immunity and antibody responses in heterologous prime-boost-boost vaccine regimens against SARS-CoV-2 and Omicron variant.

With the emergence of novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Variants of Concern (VOCs), vaccination studies that elucidate the efficiency and effectiveness of a vaccination campaign are critical to assess the durability and the protective immunity provided by vaccines. SARS-CoV-2 vaccines have been found to induce robust humoral and cell-mediated immunity in individuals vaccinated with homologous vaccination regimens. Recent studies also suggest improved immune response against SARS-CoV-2 when heterologous vaccination strategies are employed. Yet, few data exist on the extent to which heterologous prime-boost-boost vaccinations with two different vaccine platforms have an impact on the T cell-mediated immune responses with a special emphasis on the currently dominantly circulating Omicron strain. In this study, we collected serum and peripheral blood mononuclear cells (PBMCs) from 57 study participants of median 35-year old's working in the health care field, who have received different vaccination regimens. Neutralization assays revealed robust but decreased neutralization of Omicron VOC, including BA.1 and BA.4/5, compared to WT SARS-CoV-2 in all vaccine groups and increased WT SARS-CoV-2 binding and neutralizing antibodies titers in homologous mRNA prime-boost-boost study participants. By investigating cytokine production, we found that homologous and heterologous prime-boost-boost-vaccination induces a robust cytokine response of CD4+ and CD8+ T cells. Collectively, our results indicate robust humoral and T cell mediated immunity against Omicron in homologous and heterologous prime-boost-boost vaccinated study participants, which might serve as a guide for policy decisions

TMPRSS2-mediated SARS-CoV-2 uptake boosts innate immune activation, enhances cytopathology, and drives convergent virus evolution.

The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models

In Vitro Lung Models and Their Application to Study SARS-CoV-2 Pathogenesis and Disease

SARS-CoV-2 has spread across the globe with an astonishing velocity and lethality that has put scientist and pharmaceutical companies worldwide on the spot to develop novel treatment options and reliable vaccination for billions of people. To combat its associated disease COVID-19 and potentially newly emerging coronaviruses, numerous pre-clinical cell culture techniques have progressively been used, which allow the study of SARS-CoV-2 pathogenesis, basic replication mechanisms, and drug efficiency in the most authentic context. Hence, this review was designed to summarize and discuss currently used in vitro and ex vivo cell culture systems and will illustrate how these systems will help us to face the challenges imposed by the current SARS-CoV-2 pandemic

Short Communication: The Role of Different Body-Related Visual Cues in Synchronized Trampolining

Visual information is thought to be a dominant information source when performing whole-body movements in gymnastics. Visual information can furthermore facilitate performance when being engaged in situations of interpersonal coordination that may occur in sports such as synchronized trampolining. The question arises, which body-related visual cues are most important in the emergence of behavioral synchronization between two gymnasts in synchronized trampolining? To address this question, advanced gymnasts were asked to synchronize their performance to video-sequences of a model gymnast, while body-related visual cues of the model gymnast were systematically manipulated. Results revealed, first that biological motion cues are more important than nonbiological motion cues, and second that the perceived motion of the arms seems to drive synchronicity in synchronized trampolining. It is stated, that biological visual information of the arms is a dominant visual information source when performing leaps in synchronized trampolining

Antiviral Effect of Budesonide against SARS-CoV-2

Treatment options for COVID-19, a disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, are currently severely limited. Therefore, antiviral drugs that efficiently reduce SARS-CoV-2 replication or alleviate COVID-19 symptoms are urgently needed. Inhaled glucocorticoids are currently being discussed in the context of treatment for COVID-19, partly based on a previous study that reported reduced recovery times in cases of mild COVID-19 after inhalative administration of the glucocorticoid budesonide. Given various reports that describe the potential antiviral activity of glucocorticoids against respiratory viruses, we aimed to analyze a potential antiviral activity of budesonide against SARS-CoV-2 and circulating variants of concern (VOC) B.1.1.7 (alpha) and B.1.351 (beta). We demonstrate a dose-dependent inhibition of SARS-CoV-2 that was comparable between all viral variants tested while cell viability remains unaffected. Our results are encouraging as they could indicate a multimodal mode of action of budesonide against SARS-CoV-2 and COVID-19, which could contribute to an improved clinical performance

In vitro lung models and their application to study SARS-CoV-2 pathogenesis and disease

SARS-CoV-2 has spread across the globe with an astonishing velocity and lethality that has put scientist and pharmaceutical companies worldwide on the spot to develop novel treatment options and reliable vaccination for billions of people. To combat its associated disease COVID-19 and potentially newly emerging coronaviruses, numerous pre-clinical cell culture techniques have progressively been used, which allow the study of SARS-CoV-2 pathogenesis, basic replication mechanisms, and drug efficiency in the most authentic context. Hence, this review was designed to summarize and discuss currently used in vitro and ex vivo cell culture systems and will illustrate how these systems will help us to face the challenges imposed by the current SARS-CoV-2 pandemic