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The ancestral SARS-CoV-2 strain that initiated the Covid-19 pandemic at the end of 2019 has rapidly mutated into multiple variants of concern with variable pathogenicity and increasing immune escape strategies. However, differences in host cellular antiviral responses upon infection with SARS-CoV-2 variants remain elusive. Leveraging whole-cell proteomics, we determined host signaling pathways that are differentially modulated upon infection with the clinical isolates of the ancestral SARS-CoV-2 B.1 and the variants of concern Delta and Omicron BA.1. Our findings illustrate alterations in the global host proteome landscape upon infection with SARS-CoV-2 variants and the resulting host immune responses. Additionally, viral proteome kinetics reveal declining levels of viral protein expression during Omicron BA.1 infection when compared to ancestral B.1 and Delta variants, consistent with its reduced replication rates. Moreover, molecular assays reveal deferral activation of specific host antiviral signaling upon Omicron BA.1 and BA.2 infections. Our study provides an overview of host proteome profile of multiple SARS-CoV-2 variants and brings forth a better understanding of the instigation of key immune signaling pathways causative for the differential pathogenicity of SARS-CoV-2 variants.
The highly transmissible Omicron (B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in late 2021. Initial Omicron waves were primarily made up of sub-lineages BA.1 and/or BA.2, BA.4, and BA.5 subsequently became dominant in mid-2022, and several descendants of these sub-lineages have since emerged. Omicron infections have generally caused less severe disease on average than those caused by earlier variants of concern in healthy adult populations, at least, in part, due to increased population immunity. Nevertheless, healthcare systems in many countries, particularly those with low population immunity, have been overwhelmed by unprecedented surges in disease prevalence during Omicron waves. Pediatric admissions were also higher during Omicron waves compared with waves of previous variants of concern. All Omicron sub-lineages exhibit partial escape from wild-type (Wuhan-Hu 1) spike-based vaccine-elicited neutralizing antibodies, with sub-lineages with more enhanced immuno-evasive properties emerging over time. Evaluating vaccine effectiveness (VE) against Omicron sub-lineages has become challenging against a complex background of varying vaccine coverage, vaccine platforms, prior infection rates, and hybrid immunity. Original messenger RNA vaccine booster doses substantially improved VE against BA.1 or BA.2 symptomatic disease. However, protection against symptomatic disease waned, with reductions detected from 2 months after booster administration. While original vaccine-elicited CD8+ and CD4+ T-cell responses cross-recognize Omicron sub-lineages, thereby retaining protection against severe outcomes, variant-adapted vaccines are required to expand the breadth of B-cell responses and improve durability of protection. Variant-adapted vaccines were rolled out in late 2022 to increase overall protection against symptomatic and severe infections caused by Omicron sub-lineages and antigenically aligned variants with enhanced immune escape mechanisms.
The antiviral drugs tecovirimat, brincidofovir, and cidofovir are considered for mpox (monkeypox) treatment despite a lack of clinical evidence. Moreover, their use is affected by toxic side-effects (brincidofovir, cidofovir), limited availability (tecovirimat), and potentially by resistance formation. Hence, additional, readily available drugs are needed. Here, therapeutic concentrations of nitroxoline, a hydroxyquinoline antibiotic with a favourable safety profile in humans, inhibited the replication of 12 mpox virus isolates from the current outbreak in primary cultures of human keratinocytes and fibroblasts and a skin explant model by interference with host cell signalling. Tecovirimat, but not nitroxoline, treatment resulted in rapid resistance development. Nitroxoline remained effective against the tecovirimat-resistant strain and increased the anti-mpox virus activity of tecovirimat and brincidofovir. Moreover, nitroxoline inhibited bacterial and viral pathogens that are often co-transmitted with mpox. In conclusion, nitroxoline is a repurposing candidate for the treatment of mpox due to both antiviral and antimicrobial activity.
A Corrigendum on "SARS-CoV-2 Omicron variants: burden of disease, impact on vaccine effectiveness and need for variant-adapted vaccines" by Pather S, Madhi SA, Cowling BJ, Moss P, Kamil JP, Ciesek S, Muik A and Türeci Ö (2023). . 14:1130539. doi: 10.3389/fimmu.2023.1130539
The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication.
Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits.
Omicron is the evolutionarily most distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VOC) to date. We report that Omicron BA.1 breakthrough infection in BNT162b2-vaccinated individuals resulted in strong neutralizing activity against Omicron BA.1, BA.2, and previous SARS-CoV-2 VOCs but not against the Omicron sublineages BA.4 and BA.5. BA.1 breakthrough infection induced a robust recall response, primarily expanding memory B (BMEM) cells against epitopes shared broadly among variants, rather than inducing BA.1-specific B cells. The vaccination-imprinted BMEM cell pool had sufficient plasticity to be remodeled by heterologous SARS-CoV-2 spike glycoprotein exposure. Whereas selective amplification of BMEM cells recognizing shared epitopes allows for effective neutralization of most variants that evade previously established immunity, susceptibility to escape by variants that acquire alterations at hitherto conserved sites may be heightened.
Background: To minimize the risk of disease transmission in cornea transplantation, donor screening for blood-derived viral infections is mandatory. Ideally, pre-mortem blood samples are used, but based on availability, cadaveric blood samples of cornea donors may also be used. However, serological and nucleic acid amplification tests (NATs) need to be validated for the use of cadaveric specimens.
Methods: Hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV) 1/2, and Treponema pallidum (syphilis)-specific serological and/or NAT assays were validated on different platforms (Abbott Alinity i, Alinity m, Roche Cobas 6800, and Roche Cobas AmpliPrep/Cobas TaqMan (CAP/CTM)) using (un)spiked paired pre- and post-mortem cornea donor blood samples from the same individual (up to 23.83 h after death) of 28 individuals in accordance with the specifications of the German Federal Institute for Vaccines and Biomedicines (Paul-Ehrlich-Institut [PEI]). In addition, routinely HBV-, HCV- and HIV-PCR-negative tested post-mortem blood samples of 24 individuals were used to assess NAT specificity.
Results: For the majority of serological parameters on the Abbott Alinity i (HBsAg, anti-HBc, anti-HBs, anti-HCV, anti-HIV, anti-HTLV 1/2, and anti-Treponema pallidum), ratios of generated test results of (un)spiked paired pre- and post-mortem blood samples differed ≤25%, with an agreement of qualitative pre- and post-mortem test results ranging from 91.2 to 100%. For NAT parameters (HBV, HCV, and HIV) on the Cobas 6800, Alinity m, and CAP/CTM, no significant deviation in virus concentrations (factor >5) of spiked pre- and post-mortem blood samples could be observed. Ct-values of corresponding internal controls did also not differ significantly (>1.5 Ct-values). In addition, no false-positive test results were generated when specificity was assessed.
Conclusion: Overall, fluctuations of test results for serological and NAT parameters in pre- and post-mortem blood samples examined in this study, were only limited and within the range of what is also observed when routinely testing fresh patient specimens. We conclude that all examined assays are eligible for the screening of blood samples taken up to about 24 h after the occurrence of death.
Background & Aims: Elimination of chronic HBV/HDV infection remains a major global health challenge. Targeting excessive hepatitis B surface antigen (HBsAg) release may provide an interesting window of opportunity to break immune tolerance and to achieve a functional cure using additional antivirals.
Methods: We evaluated a HBsAg-specific human monoclonal antibody, as part of either a prophylactic or therapeutic strategy, against HBV/HDV infection in cell culture models and in human-liver chimeric mice. To assess prophylactic efficacy, mice were passively immunized prior to infection with HBV or HBV/HDV (coinfection and superinfection setting). Therapeutic efficacy was assessed in HBV and HBV/HDV-coinfected mice receiving 4 weeks of treatment. Viral parameters (HBV DNA, HDV RNA and HBsAg) were assessed in mouse plasma.
Results: The antibody could effectively prevent HBV/HDV infection in a dose-dependent manner with IC50 values of ∼3.5 ng/ml. Passive immunization showed complete protection of mice from both HBV and HBV/HDV coinfection. Moreover, HDV superinfection was either completely prevented or at least attenuated in HBV-infected mice. Finally, antibody treatment in mice with established HBV/HDV infection resulted in a significant decline in viremia and a concomitant drop in on-treatment HBsAg, with a moderate viral rebound following treatment cessation.
Conclusion: We present data on a valuable antibody candidate that could complement other antivirals in strategies aimed at achieving functional cure of chronic HBV and HDV infection.
Impact and implications: Patients chronically infected with HBV may eventually develop liver cancer and are at great risk of being superinfected with HDV, which worsens and accelerates disease progression. Unfortunately, current treatments can rarely eliminate both viruses from chronically infected patients. In this study, we present data on a novel antibody that is able to prevent chronic HBV/HDV infection in a mouse model with a humanized liver. Moreover, antibody treatment of HBV/HDV-infected mice strongly diminishes viral loads during therapy. This antibody is a valuable candidate for further clinical development.
Objectives: Regarding reactogenicity and immunogenicity, heterologous COVID-19 vaccination regimens are considered as an alternative to conventional immunization schemes.
Methods: Individuals receiving either heterologous (ChAdOx1-S [AstraZeneca, Cambridge, UK]/BNT162b2 [Pfizer-BioNTech, Mainz, Germany]; n = 306) or homologous (messenger RNA [mRNA]-1273 [Moderna, Cambridge, Massachusetts, USA]; n = 139) vaccination were asked to participate when receiving their second dose. Reactogenicity was assessed after 1 month, immunogenicity after 1, 3, and/or 6 months, including a third dose, through SARS-CoV-2 antispike immunoglobulin G, surrogate virus neutralization test, and a plaque reduction neutralization test against the Delta (B.1.167.2) and Omicron (B.1.1.529; BA.1) variants of concern.
Results: The overall reactogenicity was lower after heterologous vaccination. In both cohorts, SARS-CoV-2 antispike immunoglobulin G concentrations waned over time with the heterologous vaccination demonstrating higher neutralizing activity than homologous mRNA vaccination after 3 months to low neutralizing levels in the Delta plaque reduction neutralization test after 6 months. At this point, 3.2% of the heterologous and 11.4% of the homologous cohort yielded low neutralizing activity against Omicron. After a third dose of an mRNA vaccine, ≥99% of vaccinees demonstrated positive neutralizing activity against Delta. Depending on the vaccination scheme and against Omicron, 60% to 87.5% of vaccinees demonstrated positive neutralizing activity.
Conclusion: ChAdOx1-S/BNT162b2 vaccination demonstrated an acceptable reactogenicity and immunogenicity profile. A third dose of an mRNA vaccine is necessary to maintain neutralizing activity against SARS-CoV-2. However, variants of concern-adapted versions of the vaccines would be desirable.
Wastewater-based epidemiology (WBE) has demonstrated its importance to support SARS-CoV-2 epidemiology complementing individual testing strategies. Due to their immune-evasive potential and the resulting significance for public health, close monitoring of SARS-CoV-2 variants of concern (VoC) is required to evaluate the regulation of early local countermeasures. In this study, we demonstrate a rapid workflow for wastewater-based early detection and monitoring of the newly emerging SARS-CoV-2 VoCs Omicron in the end of 2021 at the municipal wastewater treatment plant (WWTP) Emschermuendung (KLEM) in the Federal State of North-Rhine-Westphalia (NRW, Germany).
Initially, available primers detecting Omicron-related mutations were rapidly validated in a central laboratory. Subsequently, RT-qPCR analysis of purified SARS-CoV-2 RNA was performed in a decentral PCR laboratory in close proximity to KLEM. This decentralized approach enabled the early detection of K417N present in Omicron in samples collected on 8th December 2021 and the detection of further mutations (N501Y, Δ69/70) in subsequent biweekly sampling campaigns. The presence of Omicron in wastewater was confirmed by next generation sequencing (NGS) in a central laboratory with samples obtained on 14th December 2021. Moreover, the relative increase of the mutant fraction of Omicron was quantitatively monitored over time by dPCR in a central PCR laboratory starting on 12th December 2021 confirming Omicron as the dominant variant by the end of 2021.
In conclusions, WBE plays a crucial role in surveillance of SARS-CoV-2 variants and is suitable as an early warning system to identify variant emergence. In particular, the successive workflow using RT-qPCR, RT-dPCR and NGS demonstrates the strength of WBE as a versatile tool to monitor variant spreading.
Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. Omicron sub-variants BA.4/5 have spread globally displacing the predeceasing variants. Due to the severe transmissibility and immune escape potential of BA.4/5, early monitoring was required to asses and implement countermeasures in time.
In this study, we monitored the prevalence of SARS-CoV-2 BA.4/5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North-Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability to detect BA.4/5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022 and the presence of BA.4/5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V. Finally, the mutant fractions were quantitatively monitored by digital PCR confirming BA.4/5 as the majority variant by 5th June 2022.
In conclusions, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variant spreading independent of individual test capacities.
Background: In recent months, Omicron variants of SARS-CoV-2 have become dominant in many regions of the world, and case numbers with Omicron subvariants BA.1 and BA.2 continue to increase. Due to numerous mutations in the spike protein, the efficacy of currently available vaccines, which are based on Wuhan-Hu 1 isolate of SARS-CoV-2, is reduced, leading to breakthrough infections. Efficacy of monoclonal antibody therapy is also likely impaired.
Methods: In our in vitro study using A549-AT cells constitutively expressing ACE2 and TMPRSS2, we determined and compared the neutralizing capacity of vaccine-elicited sera, convalescent sera and monoclonal antibodies against authentic SARS-CoV-2 Omicron BA.1 and BA.2 compared with Delta.
Findings: Almost no neutralisation of Omicron BA.1 and BA.2 was observed using sera from individuals vaccinated with two doses 6 months earlier, regardless of the type of vaccine taken. Shortly after the booster dose, most sera from triple BNT162b2-vaccinated individuals were able to neutralise both Omicron variants. In line with waning antibody levels three months after the booster, only weak residual neutralisation was observed for BA.1 (26%, n = 34, 0 median NT50) and BA.2 (44%, n = 34, 0 median NT50). In addition, BA.1 but not BA.2 was resistant to the neutralising monoclonal antibodies casirivimab/imdevimab, while BA.2 exhibited almost a complete evasion from the neutralisation induced by sotrovimab.
Interpretation: Both SARS-CoV-2 Omicron subvariants BA.1 and BA.2 escape antibody-mediated neutralisation elicited by vaccination, previous infection with SARS-CoV-2, and monoclonal antibodies. Waning immunity renders the majority of tested sera obtained three months after booster vaccination negative in BA.1 and BA.2 neutralisation. Omicron subvariant specific resistance to the monoclonal antibodies casirivimab/imdevimab and sotrovimab emphasizes the importance of genotype-surveillance and guided application.
Funding: This study was supported in part by the Goethe-Corona-Fund of the Goethe University Frankfurt (M.W.) and the Federal Ministry of Education and Research (COVIDready; grant 02WRS1621C (M.W.).
The immune response is known to wane after vaccination with BNT162b2, but the role of age, morbidity and body composition is not well understood. We conducted a cross-sectional study in long-term care facilities (LTCFs) for the elderly. All study participants had completed two-dose vaccination with BNT162b2 five to 7 months before sample collection. In 298 residents (median age 86 years, range 75–101), anti-SARS-CoV-2 rector binding IgG antibody (anti-RBD-IgG) concentrations were low and inversely correlated with age (mean 51.60 BAU/ml). We compared the results to Health Care Workers (HCW) aged 18–70 years (n = 114, median age: 53 years), who had a higher mean anti-RBD-IgG concentration of 156.99 BAU/ml. Neutralization against the Delta variant was low in both groups (9.5% in LTCF residents and 31.6% in HCWs). The Charlson Comorbidity Index was inversely correlated with anti-RBD-IgG, but not the body mass index (BMI). A control group of 14 LTCF residents with known breakthrough infection had significant higher antibody concentrations (mean 3,199.65 BAU/ml), and 85.7% had detectable neutralization against the Delta variant. Our results demonstrate low but recoverable markers of immunity in LTCF residents five to 7 months after vaccination.
Background: International travel poses the risk of importing SARS-CoV-2 infections and introducing new viral variants into the country of destination. Established measures include mandatory quarantine with the opportunity to abbreviate it with a negative rapid antigen test (RAT).
Methods: A total of 1,488 returnees were tested for SARS-CoV-2 with both PCR and RAT no earlier than 5 days after arrival. We assessed the sensitivity and specificity of the RAT. Positive samples were evaluated for infectivity in vitro in a cell culture outgrowth assay. We tracked if participants who tested negative were reported positive within 2 weeks of the initial test.
Results: Potential infectiousness was determined based on symptom onset analysis, resulting in a sensitivity of the antigen test of 89% in terms of infectivity. The specificity was 100%. All positive outgrowth assays were preceded by a positive RAT, indicating that all participants with proven in vitro infectivity were correctly identified. None of the negative participants tested positive during the follow-up.
Conclusions: RAT no earlier than the 5th day after arrival was a reliable method for detecting infectious travellers and can be recommended as an appropriate method for managing SARS-CoV-2 travel restrictions. Compliance to the regulations and a high standard of test quality must be ensured.
Recent findings in permanent cell lines suggested that SARS-CoV-2 Omicron BA.1 induces a stronger interferon response than Delta. Here, we show that BA.1 and BA.5 but not Delta induce an antiviral state in air-liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells and primary human monocytes. Both Omicron subvariants caused the production of biologically active type I (α/β) and III (λ) interferons and protected cells from super-infection with influenza A viruses. Notably, abortive Omicron infection of monocytes was sufficient to protect monocytes from influenza A virus infection. Interestingly, while influenza-like illnesses surged during the Delta wave in England, their spread rapidly declined upon the emergence of Omicron. Mechanistically, Omicron-induced interferon signalling was mediated via double-stranded RNA recognition by MDA5, as MDA5 knock-out prevented it. The JAK/ STAT inhibitor baricitinib inhibited the Omicron-mediated antiviral response, suggesting it is caused by MDA5-mediated interferon production, which activates interferon receptors that then trigger JAK/ STAT signalling. In conclusion, our study 1) demonstrates that only Omicron but not Delta induces a substantial interferon response in physiologically relevant models, 2) shows that Omicron infection protects cells from influenza A virus super-infection, and 3) indicates that BA.1 and BA.5 induce comparable antiviral states.
Recently, we have shown that SARS-CoV-2 Omicron virus isolates are less effective at inhibiting the host cell interferon response than Delta viruses. Here, we present further evidence that reduced interferon-antagonising activity explains at least in part why Omicron variant infections are inherently less severe than infections with other SARS-CoV-2 variants. Most importantly, we here also show that Omicron variant viruses display enhanced sensitivity to interferon treatment, which makes interferons promising therapy candidates for Omicron patients, in particular in combination with other antiviral agents.
The SARS-CoV-2 Omicron variant is currently causing a large number of infections in many countries. A number of antiviral agents are approved or in clinical testing for the treatment of COVID-19. Despite the high number of mutations in the Omicron variant, we here show that Omicron isolates display similar sensitivity to eight of the most important anti-SARS-CoV-2 drugs and drug candidates (including remdesivir, molnupiravir, and PF-07321332, the active compound in paxlovid), which is of timely relevance for the treatment of the increasing number of Omicron patients. Most importantly, we also found that the Omicron variant displays a reduced capability of antagonising the host cell interferon response. This provides a potential mechanistic explanation for the clinically observed reduced pathogenicity of Omicron variant viruses compared to Delta variant viruses.
The new variant of concern (VOC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Omicron (B.1.1.529), is genetically very different from other VOCs. We compared Omicron with the preceding VOC Delta (B.1.617.2) and the wildtype strain (B.1) with respect to their interactions with the antiviral type I interferon (IFN-alpha/beta) response in infected cells. Our data indicate that Omicron has gained an elevated capability to suppress IFN-beta induction upon infection and to better withstand the antiviral state imposed by exogenously added IFN-alpha.
Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a read-out for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in a broad range of cell culture models, independently of cytopathogenic effect formation. Compared to other cell culture models, the Caco-2 subline Caco-2-F03 displayed superior performance, as it possesses a stable SARS-CoV-2 susceptible phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of PHGDH, CLK-1, and CSF1R. The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the HK2 inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false positive hits.
Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. Omicron sub-variants BA.4/5 have spread globally displacing the predeceasing variants. Due to the severe transmissibility and immune escape potential of BA.4/5, early monitoring was required to asses and implement countermeasures in time.
In this study, we monitored the prevalence of SARS-CoV-2 BA.4/5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North-Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability to detect BA.4/5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022 and the presence of BA.4/5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V. Finally, the mutant fractions were quantitatively monitored by digital PCR confirming BA.4/5 as the majority variant by 5th June 2022.
In conclusions, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variant spreading independent of individual test capacities.
The antiviral drugs tecovirimat, brincidofovir, and cidofovir are considered for mpox (monkeypox) treatment despite a lack of clinical evidence. Moreover, their use is affected by toxic side-effects (brincidofovir, cidofovir), limited availability (tecovirimat), and potentially by resistance formation. Hence, additional, readily available drugs are needed. Here, therapeutic concentrations of nitroxoline, a hydroxyquinoline antibiotic with a favourable safety profile in humans, inhibited the replication of 12 mpox virus isolates from the current outbreak in primary cultures of human keratinocytes and fibroblasts and a skin explant model by interference with host cell signalling. Tecovirimat, but not nitroxoline, treatment resulted in rapid resistance development. Nitroxoline remained effective against the tecovirimat-resistant strain and increased the anti-mpox virus activity of tecovirimat and brincidofovir. Moreover, nitroxoline inhibited bacterial and viral pathogens that are often co-transmitted with mpox. In conclusion, nitroxoline is a repurposing candidate for the treatment of mpox due to both antiviral and antimicrobial activity.
Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19.
As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging–based SARS-CoV-2 infection assay was developed in VeroE6-eGFP cells and was used to screen a library of 5676 compounds that passed phase 1 clinical trials. Eight candidates (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) with in vitro anti–SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines were identified. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment.
Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19. As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging–based SARS-CoV-2 infection assay was developed in VeroE6 African green monkey kidney epithelial cells expressing a stable enhanced green fluorescent protein (VeroE6-eGFP cells) and was used to screen a library of 5676 compounds that passed Phase 1 clinical trials. Eight drugs (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) were identified as inhibitors of in vitro anti–SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment.
Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities.
The new variant of concern (VOC) of SARS-CoV-2, Omicron (B.1.1.529), is genetically very different from other VOCs. We compared Omicron with the preceding VOC Delta (B.1.617.2) and the wildtype (wt) strain (B.1) with respect to their interactions with the antiviral interferon (IFN-alpha/beta) response in infected cells. Our data indicate that IFN induction by Omicron is low and comparable to the wt, whereas Delta showed an increased IFN induction. However, Omicron exceeded both the wt and the Delta strain with respect to the ability to withstand the antiviral state imposed by IFN-alpha.
Vaccines are central to controlling the coronavirus disease 2019 (COVID-19) pandemic but the durability of protection is limited for currently approved COVID-19 vaccines. Further, the emergence of variants of concern (VoCs) that evade immune recognition has reduced vaccine effectiveness, compounding the problem. Here, we show that a single dose of a murine cytomegalovirus (MCMV)-based vaccine, which expresses the spike (S) protein of the virus circulating early in the pandemic (MCMVS), protects highly susceptible K18-hACE2 mice from clinical symptoms and death upon challenge with a lethal dose of D614G SARS-CoV-2. Moreover, MCMVS vaccination controlled two immune-evading VoCs, the Beta (B.1.135) and the Omicron (BA.1) variants in BALB/c mice, and S-specific immunity was maintained for at least 5 months after immunization, where neutralizing titers against all tested VoCs were higher at 5-months than at 1-month post-vaccination. Thus, cytomegalovirus (CMV)-based vector vaccines might allow for long-term protection against COVID-19.
The long-term effect of protection by two doses of SARS-CoV-2 vaccination in patients receiving chronic intermittent hemodialysis (CIHD) is an urging question. We investigated the humoral and cellular immune response of 42 CIHD patients who had received two doses of SARS-CoV-2 vaccine, and again after a booster vaccine with mRNA-1273 six months later. We measured antibody levels and SARS-CoV-2-specific surrogate neutralizing antibodies (SNA). Functional T cell immune response to vaccination was assessed by quantifying interferon-γ (IFN-γ) and IL-2 secreting T cells specific for SARS-CoV-2 using an ELISpot assay. Our data reveal a moderate immune response after the second dose of vaccination, with significantly decreasing SARS-CoV-2-specific antibody levels and less than half of the study group showed neutralizing antibodies six months afterwards. Booster vaccines increased the humoral response dramatically and led to a response rate of 89.2% for antibody levels and a response rate of 94.6% for SNA. Measurement in a no response/low response (NR/LR) subgroup of our cohort, which differed from the whole group in age and rate of immunosuppressive drugs, indicated failure of a corresponding T cell response after the booster vaccine. We strongly argue in favor of a regular testing of surrogate neutralizing antibodies and consecutive booster vaccinations for CIHD patients to provide a stronger and persistent immunity.
The plaque reduction neutralization test (PRNT) is a preferred method for the detection of functional, SARS-CoV-2 specific neutralizing antibodies from serum samples. Alternatively, surrogate enzyme-linked immunosorbent assays (ELISAs) using ACE2 as the target structure for the detection of neutralization-competent antibodies have been developed. They are capable of high throughput, have a short turnaround time, and can be performed under standard laboratory safety conditions. However, there are very limited data on their clinical performance and how they compare to the PRNT. We evaluated three surrogate immunoassays (GenScript SARS-CoV-2 Surrogate Virus Neutralization Test Kit (GenScript Biotech, Piscataway Township, NJ, USA), the TECO® SARS-CoV-2 Neutralization Antibody Assay (TECOmedical AG, Sissach, Switzerland), and the Leinco COVID-19 ImmunoRank™ Neutralization MICRO-ELISA (Leinco Technologies, Fenton, MO, USA)) and one automated quantitative SARS-CoV-2 Spike protein-based IgG antibody assay (Abbott GmbH, Wiesbaden, Germany) by testing 78 clinical samples, including several follow-up samples of six BNT162b2 (BioNTech/Pfizer, Mainz, Germany/New York, NY, USA) vaccinated individuals. Using the PRNT as a reference method, the overall sensitivity of the examined assays ranged from 93.8 to 100% and specificity ranged from 73.9 to 91.3%. Weighted kappa demonstrated a substantial to almost perfect agreement. The findings of our study allow these assays to be considered when a PRNT is not available. However, the latter still should be the preferred choice. For optimal clinical performance, the cut-off value of the TECO assay should be individually adapted.
Despite the recent availability of vaccines against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there is an urgent need for specific anti-SARS-CoV-2 drugs. Monoclonal neutralizing antibodies are an important drug class in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection and their potential to be used as both prophylactic and therapeutic drugs. Clinically used neutralizing antibodies against respiratory viruses are currently injected intravenously, which can lead to suboptimal pulmonary bioavailability and thus to a lower effectiveness. Here we describe DZIF-10c, a fully human monoclonal neutralizing antibody that binds the receptor-binding domain of the SARS-CoV-2 spike protein. DZIF-10c displays an exceptionally high neutralizing potency against SARS-CoV-2, retains full activity against the variant of concern (VOC) B.1.1.7 and still neutralizes the VOC B.1.351, although with reduced potency. Importantly, not only systemic but also intranasal application of DZIF-10c abolished the presence of infectious particles in the lungs of SARS-CoV-2 infected mice and mitigated lung pathology when administered prophylactically. Along with a favorable pharmacokinetic profile, these results highlight DZIF-10c as a novel human SARS-CoV-2 neutralizing antibody with high in vitro and in vivo antiviral potency. The successful intranasal application of DZIF-10c paves the way for clinical trials investigating topical delivery of anti-SARS-CoV-2 antibodies.
The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Most SARS-CoV-2 infections are mild or even asymptomatic. However, a small fraction of infected individuals develops severe, life-threatening disease, which is caused by an uncontrolled immune response resulting in hyperinflammation. However, the factors predisposing individuals to severe disease remain poorly understood. Here, we show that levels of CD47, which is known to mediate immune escape in cancer and virus-infected cells, are elevated in SARS-CoV-2-infected Caco-2 cells, Calu-3 cells, and air−liquid interface cultures of primary human bronchial epithelial cells. Moreover, SARS-CoV-2 infection increases SIRPalpha levels, the binding partner of CD47, on primary human monocytes. Systematic literature searches further indicated that known risk factors such as older age and diabetes are associated with increased CD47 levels. High CD47 levels contribute to vascular disease, vasoconstriction, and hypertension, conditions that may predispose SARS-CoV-2-infected individuals to COVID-19-related complications such as pulmonary hypertension, lung fibrosis, myocardial injury, stroke, and acute kidney injury. Hence, age-related and virus-induced CD47 expression is a candidate mechanism potentially contributing to severe COVID-19, as well as a therapeutic target, which may be addressed by antibodies and small molecules. Further research will be needed to investigate the potential involvement of CD47 and SIRPalpha in COVID-19 pathology. Our data should encourage other research groups to consider the potential relevance of the CD47/ SIRPalpha axis in their COVID-19 research.
Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings
(2021)
The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In this study, we evaluated lysis buffers that are commonly used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. In addition, viral stability in cell culture media at 4 °C and on display glass and plastic surfaces used in laboratory environment was analyzed. Furthermore, we evaluated chemical and non-chemical inactivation methods including heat inactivation, UV-C light, addition of ethanol, acetone-methanol, and PFA, which might be used as a subsequent inactivation step in the case of insufficient inactivation. We infected susceptible Caco-2 and Vero cells with pre-treated SARS-CoV-2 and determined the tissue culture infection dose 50 (TCID50) using crystal violet staining and microscopy. In addition, lysates of infected cells and virus containing supernatant were subjected to RT-qPCR analysis. We have found that guanidine thiocyanate and most of the tested detergent containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate the virus. In conclusion, careful evaluation of the used inactivation methods is required especially for non-denaturing buffers. Additional inactivation steps might be necessary before removal of lysed viral samples from BSL-3.
Although vaccination campaigns are currently being rolled out to prevent coronavirus disease (COVID-19), antivirals will remain an important adjunct to vaccination. Antivirals against coronaviruses do not exist, hence global drug repurposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID-19. Itraconazole, an antifungal agent, has been reported to have activity against animal coronaviruses. Using cell-based phenotypic assays, the in vitro antiviral activity of itraconazole and 17-OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Itraconazole demonstrated antiviral activity in human Caco-2 cells (EC50 = 2.3 µM; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). Similarly, its primary metabolite, 17-OH itraconazole, showed inhibition of SARS-CoV-2 activity (EC50 = 3.6 µM). Remdesivir inhibited viral replication with an EC50 = 0.4 µM. Itraconazole and 17-OH itraconazole resulted in a viral yield reduction in vitro of approximately 2-log10 and approximately 1-log10, as measured in both Caco-2 cells and VeroE6-eGFP cells, respectively. The viral yield reduction brought about by remdesivir or GS-441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3-log10 drop and >4-log10 drop in Caco-2 cells and VeroE6-eGFP cells, respectively. Itraconazole and 17-OH itraconazole exert in vitro low micromolar activity against SARS-CoV-2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID-19 patients in a clinical study (EudraCT Number: 2020-001243-15).
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile cellular infection model that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The model is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression (A549-AT). Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV-2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected A549-AT cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel model cell line allows rapid and sensitive monitoring of SARS-CoV-2 infection and the screening for host factors essential for viral replication. HIGHLIGHTS: Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection; The versatile model cell line A549-AT is suitable for rapid and sensitive high-throughput assays; Additional gene specific expression cassettes allow the screening for compounds and cellular factors limiting SARS-CoV-2 replication.
Due to globally rising numbers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, resources for real-time reverse-transcription polymerase chain reaction (rRT-PCR)-based testing have been exhausted. In order to meet the demands of testing and reduce transmission, SARS-CoV-2 antigen-detecting rapid diagnostic tests (Ag-RDTs) are being considered. These tests are fast, inexpensive, and simple to use, but whether they detect potentially infectious cases has not been well studied. We evaluated three lateral flow assays (RIDA®QUICK SARS-CoV-2 Antigen (R-Biopharm), SARS-CoV-2 Rapid Antigen Test (Roche)), and NADAL® COVID-19 Ag Test (Nal von Minden GmbH, Regensburg, Germany) and one microfluidic immunofluorescence assay (SARS-CoV-2 Ag Test (LumiraDx GmbH, Cologne, Germany)) using 100 clinical samples. Diagnostic rRT-PCR and cell culture testing as a marker for infectivity were performed in parallel. The overall Ag-RDT sensitivity for rRT-PCR-positive samples ranged from 24.3% to 50%. However, for samples with a viral load of more than 6 log10 RNA copies/mL (22/100), typically seen in infectious individuals, Ag-RDT positivity was between 81.8% and 100%. Only 51.6% (33/64) of the rRT-PCR-positive samples were infectious in cell culture. In contrast, three Ag-RDTs demonstrated a more significant correlation with cell culture infectivity (61.8–82.4%). Our findings suggest that large-scale SARS-CoV-2 Ag-RDT-based testing can be considered for detecting potentially infective individuals and reducing the virus spread.
Background: Due to the coronavirus disease 2019 (COVID-19) pandemic, interventions in the upper airways are considered high-risk procedures for otolaryngologists and their colleagues. The purpose of this study was to evaluate limitations in hearing and communication when using a powered air-purifying respirator (PAPR) system to protect against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) transmission and to assess the benefit of a headset. Methods: Acoustic properties of the PAPR system were measured using a head and torso simulator. Audiological tests (tone audiometry, Freiburg speech test, Oldenburg sentence test (OLSA)) were performed in normal-hearing subjects (n = 10) to assess hearing with PAPR. The audiological test setup also included simulation of conditions in which the target speaker used either a PAPR, a filtering face piece (FFP) 3 respirator, or a surgical face mask. Results: Audiological measurements revealed that sound insulation by the PAPR headtop and noise, generated by the blower-assisted respiratory protection system, resulted in significantly deteriorated hearing thresholds (4.0 ± 7.2 dB hearing level (HL) vs. 49.2 ± 11.0
The duration of infectivity of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in living patients has been demarcated. In contrast, a possible SARS-CoV-2 infectivity of corpses and subsequently its duration under post mortem circumstances remain to be elucidated. The aim of this study was to investigate the infectivity and its duration of deceased COVID-19 (coronavirus disease) patients. Four SARS-CoV-2 infected deceased patients were subjected to medicolegal autopsy. Post mortem intervals (PMI) of 1, 4, 9 and 17 days, respectively, were documented. During autopsy, swabs and organ samples were taken and examined by RT-qPCR (real-time reverse transcription-polymerase chain reaction) for the detection of SARS-CoV-2 ribonucleic acid (RNA). Determination of infectivity was performed by means of virus isolation in cell culture. In two cases, virus isolation was successful for swabs and tissue samples of the respiratory tract (PMI 4 and 17 days). The two infectious cases showed a shorter duration of COVID-19 until death than the two non-infectious cases (2 and 11 days, respectively, compared to > 19 days), which correlates with studies of living patients, in which infectivity could be narrowed to about 6 days before to 12 days after symptom onset. Most notably, infectivity was still present in one of the COVID-19 corpses after a post-mortem interval of 17 days and despite already visible signs of decomposition. To prevent SARS-CoV-2 infections in all professional groups involved in the handling and examination of COVID-19 corpses, adequate personal safety standards (reducing or avoiding aerosol formation and wearing FFP3 [filtering face piece class 3] masks) have to be enforced for routine procedures.
Aims: Patients with cardiovascular comorbidities have a significantly increased risk for a critical course of COVID-19. As the SARS-CoV2 virus enters cells via the angiotensin-converting enzyme receptor II (ACE2), drugs which interact with the renin angiotensin aldosterone system (RAAS) were suspected to influence disease severity.
Methods and results: We analyzed 1946 consecutive patients with cardiovascular comorbidities or hypertension enrolled in one of the largest European COVID-19 registries, the Lean European Open Survey on SARS-CoV-2 (LEOSS) registry. Here, we show that angiotensin II receptor blocker intake is associated with decreased mortality in patients with COVID-19 [OR 0.75 (95% CI 0,59–0.96; p = 0.013)]. This effect was mainly driven by patients, who presented in an early phase of COVID-19 at baseline [OR 0,64 (95% CI 0,43–0,96; p = 0.029)]. Kaplan-Meier analysis revealed a significantly lower incidence of death in patients on an angiotensin receptor blocker (ARB) (n = 33/318;10,4%) compared to patients using an angiotensin-converting enzyme inhibitor (ACEi) (n = 60/348;17,2%) or patients who received neither an ACE-inhibitor nor an ARB at baseline in the uncomplicated phase (n = 90/466; 19,3%; p<0.034). Patients taking an ARB were significantly less frequently reaching the mortality predicting threshold for leukocytes (p<0.001), neutrophils (p = 0.002) and the inflammatory markers CRP (p = 0.021), procalcitonin (p = 0.001) and IL-6 (p = 0.049). ACE2 expression levels in human lung samples were not altered in patients taking RAAS modulators.
Conclusion: These data suggest a beneficial effect of ARBs on disease severity in patients with cardiovascular comorbidities and COVID-19, which is linked to dampened systemic inflammatory activity.
Aim: It can be challenging to distinguish COVID-19 in children from other common infections. We set out to determine the rate at which children consulting a primary care paediatrician with an acute infection are infected with SARS-CoV-2 and to compare distinct findings. Method: In seven out-patient clinics, children aged 0–13 years with any new respiratory or gastrointestinal symptoms and presumed infection were invited to be tested for SARS-CoV-2. Factors that were correlated with testing positive were determined. Samples were collected from 25 January 2021 to 01 April 2021. Results: Seven hundred and eighty-three children participated in the study (median age 3 years and 0 months, range 1 month to 12 years and 11 months). Three hundred and fifty-eight were female (45.7%). SARS-CoV-2 RNA was detected in 19 (2.4%). The most common symptoms in children with as well as without detectable SARS-CoV-2 RNA were rhinitis, fever and cough. Known recent exposure to a case of COVID-19 was significantly correlated with testing positive, but symptoms or clinical findings were not. Conclusion: COVID-19 among the children with symptoms of an acute infection was uncommon, and the clinical presentation did not differ significantly between children with and without evidence of an infection with SARS-CoV-2.
Aim: It can be challenging to distinguish COVID-19 in children from other common infections. We set out to determine the rate at which children consulting a primary care paediatrician with an acute infection are infected with SARS-CoV-2 and to compare distinct findings. Method: In seven out-patient clinics, children aged 0–13 years with any new respiratory or gastrointestinal symptoms and presumed infection were invited to be tested for SARS-CoV-2. Factors that were correlated with testing positive were determined. Samples were collected from 25 January 2021 to 01 April 2021. Results: Seven hundred and eighty-three children participated in the study (median age 3 years and 0 months, range 1 month to 12 years and 11 months). Three hundred and fifty-eight were female (45.7%). SARS-CoV-2 RNA was detected in 19 (2.4%). The most common symptoms in children with as well as without detectable SARS-CoV-2 RNA were rhinitis, fever and cough. Known recent exposure to a case of COVID-19 was significantly correlated with testing positive, but symptoms or clinical findings were not. Conclusion: COVID-19 among the children with symptoms of an acute infection was uncommon, and the clinical presentation did not differ significantly between children with and without evidence of an infection with SARS-CoV-2.
Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection
(2021)
Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.
Blood-pressure-lowering drugs are proposed to foster SARS-CoV-2 infection by pharmacological upregulation of angiotensin-converting enzyme 2 (ACE2), the binding partner of the virus spike (S) protein, located on the surface of the host cells. Conversely, it is postulated that angiotensin–renin system antagonists may prevent lung damage caused by SARS-CoV-2 infection, by reducing angiotensin II levels, which can induce permeability of lung endothelial barrier via its interaction with the AT1 receptor (AT1R). Methods: We have investigated the influence of the ACE inhibitors (lisinopril, captopril) and the AT1 antagonists (telmisartan, olmesartan) on the level of ACE2 mRNA and protein expression as well as their influence on the cytopathic effect of SARS-CoV-2 and on the cell barrier integrity in a Caco-2 cell model. Results: The drugs revealed no effect on ACE2 mRNA and protein expression. ACE inhibitors and AT1R antagonist olmesartan did not influence the infection rate of SARS-CoV-2 and were unable to prevent the SARS-CoV-2-induced cell barrier disturbance. A concentration of 25 µg/mL telmisartan significantly reduced the virus replication rate. Conclusion: ACE inhibitors and AT1R antagonist showed neither beneficial nor detrimental effects on SARS-CoV-2-infection and cell barrier integrity in vitro at pharmacologically relevant concentrations.
Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant.
Testing for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by RT-PCR is a vital public health tool in the pandemic. Self-collected samples are increasingly used as an alternative to nasopharyngeal swabs. Several studies suggested that they are sufficiently sensitive to be a useful alternative. However, there are limited data directly comparing several different types of self-collected materials to determine which material is preferable. A total of 102 predominantly symptomatic adults with a confirmed SARS-CoV-2 infection self-collected native saliva, a tongue swab, a mid-turbinate nasal swab, saliva obtained by chewing a cotton pad and gargle lavage, within 48 h of initial diagnosis. Sample collection was unsupervised. Both native saliva and gargling with tap water had high diagnostic sensitivity of 92.8% and 89.1%, respectively. Nasal swabs had a sensitivity of 85.1%, which was not significantly inferior to saliva (p = 0.092), but 16.6% of participants reported they had difficult in self-collection of this sample. A tongue swab and saliva obtained by chewing a cotton pad had a significantly lower sensitivity of 74.2% and 70.2%, respectively. Diagnostic sensitivity was not related to the presence of clinical symptoms or to age. When comparing self-collected specimens from different material, saliva, gargle lavage or mid-turbinate nasal swabs may be considered for most symptomatic patients. However, complementary experiments are required to verify that differences in performance observed among the five sampling modes were not attributed to collection impairment.
SARS-CoV-2 is causing the coronavirus disease 2019 (COVID-19) pandemic, for which effective pharmacological therapies are needed. SARS-CoV-2 induces a shift of the host cell metabolism towards glycolysis, and the glycolysis inhibitor 2-deoxy-d-glucose (2DG), which interferes with SARS-CoV-2 infection, is under development for the treatment of COVID-19 patients. The glycolytic pathway generates intermediates that supply the non-oxidative branch of the pentose phosphate pathway (PPP). In this study, the analysis of proteomics data indicated increased transketolase (TKT) levels in SARS-CoV-2-infected cells, suggesting that a role is played by the non-oxidative PPP. In agreement, the TKT inhibitor benfooxythiamine (BOT) inhibited SARS-CoV-2 replication and increased the anti-SARS-CoV-2 activity of 2DG. In conclusion, SARS-CoV-2 infection is associated with changes in the regulation of the PPP. The TKT inhibitor BOT inhibited SARS-CoV-2 replication and increased the activity of the glycolysis inhibitor 2DG. Notably, metabolic drugs like BOT and 2DG may also interfere with COVID-19-associated immunopathology by modifying the metabolism of immune cells in addition to inhibiting SARS-CoV-2 replication. Hence, they may improve COVID-19 therapy outcomes by exerting antiviral and immunomodulatory effects.
The capacity of convalescent and vaccine-elicited sera and monoclonal antibodies (mAb) to neutralize SARS-CoV-2 variants is currently of high relevance to assess the protection against infections.
We performed a cell culture-based neutralization assay focusing on authentic SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.427/B.1.429 (Epsilon), all harboring the spike substitution L452R.
We found that authentic SARS-CoV-2 variants harboring L452R had reduced susceptibility to convalescent and vaccine-elicited sera and mAbs. Compared to B.1, Kappa and Delta showed a reduced neutralization by convalescent sera by a factor of 5.71 and 3.64, respectively, which constitutes a 2-fold greater reduction when compared to Epsilon. BNT2b2 and mRNA1273 vaccine-elicited sera were less effective against Kappa, Delta, and Epsilon compared to B.1. No difference was observed between Kappa and Delta towards vaccine-elicited sera, whereas convalescent sera were 1.6-fold less effective against Delta, respectively. Both B.1.617 variants Kappa (+E484Q) and Delta (+T478K) were less susceptible to either casirivimab or imdevimab.
In conclusion, in contrast to the parallel circulating Kappa variant, the neutralization efficiency of convalescent and vaccine-elicited sera against Delta was moderately reduced. Delta was resistant to imdevimab, which however, might be circumvented by a combination therapy with casirivimab together.
The capacity of convalescent and vaccine-elicited sera and monoclonal antibodies (mAb) to neutralize SARS-CoV-2 variants is currently of high relevance to assess the protection against infections. We performed a cell culture-based neutralization assay focusing on authentic SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.427/B.1.429 (Epsilon), all harboring the spike substitution L452R. We found that authentic SARS-CoV-2 variants harboring L452R had reduced susceptibility to convalescent and vaccine-elicited sera and mAbs. Compared to B.1, Kappa and Delta showed a reduced neutralization by convalescent sera by a factor of 8.00 and 5.33, respectively, which constitutes a 2-fold greater reduction when compared to Epsilon. BNT2b2 and mRNA1273 vaccine-elicited sera were less effective against Kappa, Delta, and Epsilon compared to B.1. No difference was observed between Kappa and Delta towards vaccine-elicited sera, whereas convalescent sera were 1.51-fold less effective against Delta, respectively. Both B.1.617 variants Kappa (+E484Q) and Delta (+T478K) were less susceptible to either casirivimab or imdevimab. In conclusion, in contrast to the parallel circulating Kappa variant, the neutralization efficiency of convalescent and vaccine-elicited sera against Delta was moderately reduced. Delta was resistant to imdevimab, which, however, might be circumvented by combination therapy with casirivimab together.
Background: International travel is a major driver of the introduction and spread of SARS- CoV-2. Aim: To investigate SARS-CoV-2 genetic diversity in the region of a major transport hub in Germany, we characterized the viral sequence diversity of the SARS-CoV-2 variants circulating in Frankfurt am Main, the city with the largest airport in Germany, from the end of October to the end of December 2020. Methods: In total, we recovered 136 SARS-CoV-2 genomes from nasopharyngeal swab samples. We isolated 104 isolates that were grown in cell culture and RNA from the recovered viruses and subjected them to full-genome sequence analysis. In addition, 32 nasopharyngeal swab samples were directly sequenced. Results and conclusion: We found 28 different lineages of SARS- CoV-2 circulating during the study period, including the variant of concern B.1.1.7 (∆69/70, N501Y). Six of the lineages had not previously been observed in Germany. We detected the spike protein (S) deletion ∆69/∆70 in 15% of all sequences, a four base pair (bp) deletion (in 2.9% of sequences) and a single bp deletion (in 0.7% of sequences) in ORF3a, leading to ORF3a truncations. In four sequences (2.9%), an amino acid deletion at position 210 in S was identified. In a single sample (0.7%), both a 9 bp deletion in ORF1ab and a 7 bp deletion in ORF7a were identified. One sequence in lineage B.1.1.70 had an N501Y substitution while lacking the ∆69/70 in S. The high diversity of sequences observed over two months in Frankfurt am Main highlights the persisting need for continuous SARS-CoV-2 surveillance using full-genome sequencing, particularly in cities with international airport connections.
Postmortem detection of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) after the exhumation of a corpse can become important, e.g. in the case of subsequent medical malpractice allegations. To date, data on possible detection periods [e.g. by reverse transcription polymerase chain reaction (RT-PCR)] or on the potential infectivity of the virus after an exhumation are rare. In the present study, these parameters were examined in two cases with a time span of approximately 4 months between day of death and exhumation. Using SARS-CoV-2 RT-PCR on swabs of both lungs and the oropharynx detection was possible with cycle threshold (Ct) values of about 30 despite signs of beginning decay. RT-PCR testing of perioral and perinasal swabs and swabs collected from the inside of the body bag, taken to estimate the risk of infection of those involved in the exhumation, was negative. Cell culture-based infectivity testing was negative for both, lung and oropharyngeal swabs. In one case, RT-PCR testing at the day of death of an oropharyngeal swab showed almost identical Ct values as postmortem testing of an oropharyngeal swab, impressively demonstrating the stability of viral RNA in the intact corpse. However, favorable climatic conditions in the grave have to be taken into account, as it was wintertime with constant low temperatures. Nevertheless, it was possible to demonstrate successful postmortem detection of SARS-CoV-2 infection following exhumation even after months in an earth grave.
The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Most SARS-CoV-2 infections are mild or even asymptomatic. However, a small fraction of infected individuals develops severe, life-threatening disease, which is caused by an uncontrolled immune response resulting in hyperinflammation. Antiviral interventions are only effective prior to the onset of hyperinflammation. Hence, biomarkers are needed for the early identification and treatment of high-risk patients. Here, we show in a range of model systems and data from post mortem samples that SARS-CoV-2 infection results in increased levels of CD47, which is known to mediate immune escape in cancer and virus-infected cells. Systematic literature searches also indicated that known risk factors such as older age and diabetes are associated with increased CD47 levels. High CD47 levels contribute to vascular disease, vasoconstriction, and hypertension, conditions which may predispose SARS-CoV-2-infected individuals to COVID-19-related complications such as pulmonary hypertension, lung fibrosis, myocardial injury, stroke, and acute kidney injury. Hence, CD47 is a candidate biomarker for severe COVID-19. Further research will have to show whether CD47 is a reliable diagnostic marker for the early identification of COVID-19 patients requiring antiviral therapy.
The capacity of convalescent and vaccine-elicited sera and monoclonal antibodies (mAb) to neutralize SARS-CoV-2 variants is currently of high relevance to assess the protection against infections.
We performed a cell culture-based neutralization assay focusing on authentic SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.427/B.1.429 (Epsilon), all harboring the spike substitution L452R.
We found that authentic SARS-CoV-2 variants harboring L452R had reduced susceptibility to convalescent and vaccine-elicited sera and mAbs. Compared to B.1, Kappa and Delta showed a reduced neutralization by convalescent sera by a factor of 8.00 and 5.33, respectively, which constitutes a 2-fold greater reduction when compared to Epsilon. BNT2b2 and mRNA1273 vaccine-elicited sera were less effective against Kappa, Delta, and Epsilon compared to B.1. No difference was observed between Kappa and Delta towards vaccine-elicited sera, whereas convalescent sera were 1.5-fold less effective against Delta, respectively. Both B.1.617 variants Kappa (+E484Q) and Delta (+T478K) were less susceptible to either casirivimab or imdevimab.
In conclusion, in contrast to the parallel circulating Kappa variant, the neutralization efficiency of convalescent and vaccine-elicited sera against Delta was moderately reduced. Delta was resistant to imdevimab, which however, might be circumvented by a combination therapy with casirivimab together.
Purpose: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) replicates predominantly in the upper respiratory tract and is primarily transmitted by droplets and aerosols. Taking the medical history for typical COVID-19 symptoms and PCR-based SARS-CoV-2 testing have become established as screening procedures. The aim of this work was to describe the clinical appearance of SARS-CoV-2-PCR positive patients and to determine the SARS-CoV-2 contact risk for health care workers (HCW).
Methods: The retrospective study included n = 2283 SARS-CoV-2 PCR tests from n = 1725 patients with otorhinolaryngological (ORL) diseases performed from March to November 2020 prior to inpatient treatment. In addition, demographic data and medical history were assessed.
Results: n = 13 PCR tests (0.6%) were positive for SARS-CoV-2 RNA. The positive rate showed a significant increase during the observation period (p < 0.01). None of the patients had clinical symptoms that led to a suspected diagnosis of COVID-19 before PCR testing. The patients were either asymptomatic (n = 4) or had symptoms that were interpreted as symptoms typical of the ORL disease or secondary diagnoses (n = 9).
Conclusion: The identification of SARS-CoV-2-positive patients is a considerable challenge in clinical practice. Our findings illustrate that taking a medical history alone is of limited value and cannot replace molecular SARS-CoV-2 testing, especially for patients with ORL diseases. Our data also demonstrate that there is a high probability of contact with SARS-CoV-2-positive patients in everyday clinical practice, so that the use of personal protective equipment, even in apparently “routine cases”, is highly recommended.
TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.
The SARS-CoV-2 pandemic has challenged researchers at a global scale. The scientific community’s massive response has resulted in a flood of experiments, analyses, hypotheses, and publications, especially in the field of drug repurposing. However, many of the proposed therapeutic compounds obtained from SARS-CoV-2 specific assays are not in agreement and thus demonstrate the need for a singular source of COVID-19 related information from which a rational selection of drug repurposing candidates can be made. In this paper, we present the COVID-19 PHARMACOME, a comprehensive drug-target-mechanism graph generated from a compilation of 10 separate disease maps and sources of experimental data focused on SARS-CoV-2 / COVID-19 pathophysiology. By applying our systematic approach, we were able to predict the synergistic effect of specific drug pairs, such as Remdesivir and Thioguanosine or Nelfinavir and Raloxifene, on SARS-CoV-2 infection. Experimental validation of our results demonstrate that our graph can be used to not only explore the involved mechanistic pathways, but also to identify novel combinations of drug repurposing candidates.
Reduced neutralization of SARS-CoV-2 Omicron variant by vaccine sera and monoclonal antibodies
(2021)
Due to numerous mutations in the spike protein, the SARS-CoV-2 variant of concern Omicron (B.1.1.529) raises serious concerns since it may significantly limit the antibody-mediated neutralization and increase the risk of reinfections. While a rapid increase in the number of cases is being reported worldwide, until now there has been uncertainty about the efficacy of vaccinations and monoclonal antibodies. Our in vitro findings using authentic SARS-CoV-2 variants indicate that in contrast to the currently circulating Delta variant, the neutralization efficacy of vaccine-elicited sera against Omicron was severely reduced highlighting T-cell mediated immunity as essential barrier to prevent severe COVID-19. Since SARS-CoV-2 Omicron was resistant to casirivimab and imdevimab, genotyping of SARS-CoV-2 may be needed before initiating mAb treatment. Variant-specific vaccines and mAb agents may be required to treat COVID-19 due to Omicron and other emerging variants of concern.
Reduced neutralization of SARS-CoV-2 Omicron variant by vaccine sera and monoclonal antibodies
(2021)
Due to numerous mutations in the spike protein, the SARS-CoV-2 variant of concern Omicron (B.1.1.529) raises serious concerns since it may significantly limit the antibody-mediated neutralization and increase the risk of reinfections. While a rapid increase in the number of cases is being reported worldwide, until now there has been uncertainty about the efficacy of vaccinations and monoclonal antibodies. Our in vitro findings using authentic SARS-CoV-2 variants indicate that in contrast to the currently circulating Delta variant, the neutralization efficacy of vaccine-elicited sera against Omicron was severely reduced highlighting T-cell mediated immunity as essential barrier to prevent severe COVID-19. Since SARS-CoV-2 Omicron was resistant to casirivimab and imdevimab, genotyping of SARS-CoV-2 may be needed before initiating mAb treatment. Variant-specific vaccines and mAb agents may be required to treat COVID-19 due to Omicron and other emerging variants of concern.
Introduction: Reliable and cost-effective diagnostics for hepatitis E virus (HEV) infection are necessary. The aim of our study was to investigate which diagnostic test is most accurate to detect HEV infection in immunocompetent and immunosuppressed patients in a real world setting. Patients and Methods: We performed a retrospective analysis of 1165 patients tested for HEV antibodies and HEV PCR at the same time point. Clinical, laboratory and virological data were taken from patient charts. HEV IgA was measured in a subgroup of 185 patients. Results: HEV RNA was detectable in 61 patients (5.2%); most of them (n = 49, 80.3%/n = 43, 70.5%) were HEV IgM+ and IgG+; however, 12 patients (19.6%) were HEV RNA positive/HEV IgM negative and 17 patients (27.8%) were HEV RNA positive/HEV IgG negative. Ten HEV RNA positive patients (16.4%) had neither HEV IgG nor IgM antibodies. Importantly, all of them were immunosuppressed. HEV IgA testing was less sensitive than HEV IgM for HEV diagnosis. Conclusions: HEV infection can be overlooked in patients without HEV specific antibodies. Performing PCR is necessary to diagnose or exclude HEV infection in immunocompromised hosts. In immunocompetent patients, a screening based on HEV antibodies (IgG/IgM) is sufficient.
Human cytomegalovirus (CMV) is a significant cause of morbidity and mortality in patient groups at risk. We have previously shown that the anti-CMV IgG seroprevalence in an urban region of Germany has changed over the last decades. Overall, a decline from 63.7 to 57.25% had been observed between 1988–1997 and 1998–2008 (p < 0,001). Here, we continuously follow the trends to the most recent decade 2009 to 2018. In a retrospective analysis, we determined the seroprevalence of CMV IgG antibodies in our patient cohort, stratified by gender and selected groups at risk (e.g., patients with HIV infection; women of childbearing age). The overall prevalence of anti-CMV IgG non-significantly declined further from 57.25% in 1998–2008 to 56.48% in 2009–2018 (p = 0.881). Looking at gender differences, overall CMV seroprevalence in males declined to 52.82% (from 55.54% in 1998–2008; p = 0.0254), while it non-significantly increased in females to 59.80%. The high seroprevalence in patients with a known HIV infection further increased from 87.46% in 1998–2008 to 92.93% in the current period (p = 0.9999). In women of childbearing age, no significant changes over the last three decades could be observed. The CMV seroprevalence in oncological patients was determined to be 60.64%. Overall, the former significant decline of CMV seroprevalence between the decades 1988–1997 and 1998–2008 in this urban region of Germany slowed down to a non-significant decrease of 0.77% (1998–2008 vs. 2009–2018). This might be an indicator that CMV seroprevalence has reached a plateau.
Objectives: Given the high need and the absence of specific antivirals for treatment of COVID-19 (the disease caused by severe acute respiratory syndrome-associated coronavirus-2 [SARS-CoV-2]), human immunodeficiency virus (HIV) protease inhibitors are being considered as therapeutic alternatives. Methods: Prezcobix/Rezolsta is a fixed-dose combination of 800 mg of the HIV protease inhibitor darunavir (DRV) and 150 mg cobicistat, a CYP3A4 inhibitor, which is indicated in combination with other antiretroviral agents for the treatment of HIV infection. There are currently no definitive data on the safety and efficacy of DRV/cobicistat for the treatment of COVID-19. The in vitro antiviral activity of darunavir against a clinical isolate from a patient infected with SARS-CoV-2 was assessed. Results: DRV showed no antiviral activity against SARS-CoV-2 at clinically relevant concentrations (EC50 > 100 μM). Remdesivir, used as a positive control, demonstrated potent antiviral activity (EC50 = 0.38 μM). Conclusions: Overall, the data do not support the use of DRV for the treatment of COVID-19.
The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.
Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air–liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease.
SARS-CoV-2 is the causative agent of COVID-19. Severe COVID-19 disease has been associated with disseminated intravascular coagulation and thrombosis, but the mechanisms underlying COVID-19-related coagulopathy remain unknown. The risk of severe COVID-19 disease is higher in males than in females and increases with age. To identify gene products that may contribute to COVID-19-related coagulopathy, we analyzed the expression of genes associated with the Gene Ontology (GO) term “blood coagulation” in the Genotype-Tissue Expression (GTEx) database and identified four procoagulants, whose expression is higher in males and increases with age (ADAMTS13, F11, HGFAC, KLKB1), and two anticoagulants, whose expression is higher in females and decreases with age (C1QTNF1, SERPINA5). However, the expression of none of these genes was regulated in a proteomics dataset of SARS-CoV-2-infected cells and none of the proteins have been identified as a binding partner of SARS-CoV-2 proteins. Hence, they may rather generally predispose individuals to thrombosis without directly contributing to COVID-19-related coagulopathy. In contrast, the expression of the procoagulant transferrin (not associated to the GO term “blood coagulation”) was higher in males, increased with age, and was upregulated upon SARS-CoV-2 infection. Hence, transferrin warrants further examination in ongoing clinic-pathological investigations.
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) serological assays are urgently needed for rapid diagnosis, contact tracing, and for epidemiological studies. So far, there is limited data on how commercially available tests perform with real patient samples, and if positive tested samples show neutralizing abilities. Focusing on IgG antibodies, we demonstrate the performance of two enzyme‐linked immunosorbent assay (ELISA) assays (Euroimmun SARS‐CoV‐2 IgG and Vircell COVID‐19 ELISA IgG) in comparison to one lateral flow assay (FaStep COVID‐19 IgG/IgM Rapid Test Device) and two in‐house developed assays (immunofluorescence assay [IFA] and plaque reduction neutralization test [PRNT]). We tested follow up serum/plasma samples of individuals polymerase chain reaction‐diagnosed with COVID‐19. Most of the SARS‐CoV‐2 samples were from individuals with moderate to the severe clinical course, who required an in‐patient hospital stay. For all examined assays, the sensitivity ranged from 58.8 to 76.5% for the early phase of infection (days 5‐9) and from 93.8% to 100% for the later period (days 10‐18).
Direct acting antivirals (DAAs) revolutionized the therapy of chronic hepatitis C infection. However, unexpected high recurrence rates of hepatocellular carcinoma (HCC) after DAA treatment became an issue in patients with advanced cirrhosis and fibrosis. In this study, we aimed to investigate an impact of DAA treatment on the molecular changes related to HCC development and progression in hepatoma cell lines and primary human hepatocytes. We found that treatment with sofosbuvir (SOF), a backbone of DAA therapy, caused an increase in EGFR expression and phosphorylation. As a result, enhanced translocation of EGFR into the nucleus and transactivation of factors associated with cell cycle progression, B-MYB and Cyclin D1, was detected. Serine/threonine kinase profiling identified additional pathways, especially the MAPK pathway, also activated during SOF treatment. Importantly, the blocking of EGFR kinase activity by erlotinib during SOF treatment prevented all downstream events. Altogether, our findings suggest that SOF may have an impact on pathological processes in the liver via the induction of EGFR signaling. Notably, zidovudine, another nucleoside analogue, exerted a similar cell phenotype, suggesting that the observed effects may be induced by additional members of this drug class.
This case series assessed a commercial airline flight from Tel Aviv, Israel, to Frankfurt, Germany, that occurred on March 9th, 2020. Among 102 passengers on a Boeing 737-900 aircraft were 24 members of a tourist group. Starting 7 days earlier, the group had contact with a hotel manager who later received a diagnosis of coronavirus disease 2019 (COVID-19). No member of the group had received a diagnosis of COVID-19 before the flight, and no measures to prevent transmission (eg, wearing of masks) had been applied. The flight duration was 4 hours 40 minutes.
Previous studies reported on the safety and applicability of mesenchymal stem/stromal cells (MSCs) to ameliorate pulmonary inflammation in acute respiratory distress syndrome (ARDS). Thus, multiple clinical trials assessing the potential of MSCs for COVID-19 treatment are underway. Yet, as SARS-inducing coronaviruses infect stem/progenitor cells, it is unclear whether MSCs could be infected by SARS-CoV-2 upon transplantation to COVID-19 patients. We found that MSCs from bone marrow, amniotic fluid, and adipose tissue carry angiotensin-converting enzyme 2 and transmembrane protease serine subtype 2 at low levels on the cell surface under steady-state and inflammatory conditions. We did not observe SARS-CoV-2 infection or replication in MSCs at steady state under inflammatory conditions, or in direct contact with SARS-CoV-2-infected Caco-2 cells. Further, indoleamine 2,3-dioxygenase 1 production in MSCs was not impaired in the presence of SARS-CoV-2. We show that MSCs are resistant to SARS-CoV-2 infection and retain their immunomodulation potential, supporting their potential applicability for COVID-19 treatment.
Background In the pandemic, testing for SARS-CoV-2 by RT-PCR in one of the pillars on which countermeasures are based. Factors limiting the output of laboratories interfere with the effectiveness of public health measures. Conserving reagents by pooling samples in low-probability settings is proposed, but may cause dilution and loss of sensitivity.
Methods We tested an alternate approach (FACT) by simultaneously incubating multiple respiratory swabs in a single tube. This protocol was evaluated by serial incubation of a respiratory swab in up to 10 tubes. The analytics validity of this concept was demonstrated in a five-sample mini pool set-up. It was consequently applied in the testing of 50 symptomatic patients (five-sample pools) as well as 100 asymptomatic residents of a nursing home (ten-sample pools).
Results Serial incubation of a respiratory swab in up to 10 tubes did not lead to a significant decline in viral concentration. The novel FACT-protocol did not cause a false negative result in a five-sample mini-pool setup, with non-significantly differing Ct values between single sample and mini-pool NAT. In two routine applications, all mini pools containing positive patient samples were correctly identified.
Conclusions Our proposed FACT-protocol did not cause a significant loss in analytic or diagnostic sensitivity compared to single sample testing in multiple setups. It reduced the amount of reagents needed by up to 40%, and also reduced hands-on time. This method could enhance testing efficiency, especially in groups with a low pretest-probability, such as systemically relevant professional groups.
It becomes more and more obvious that deregulation of host metabolism play an important role in SARS-CoV-2 pathogenesis with implication for increased risk of severe course of COVID-19. Furthermore, it is expected that COVID-19 patients recovered from severe disease may experience long-term metabolic disorders. Thereby understanding the consequences of SARS-CoV-2 infection on host metabolism can facilitate efforts for effective treatment option. We have previously shown that SARS-CoV-2-infected cells undergo a shift towards glycolysis and that 2-deoxy-D-glucose (2DG) inhibits SARS-CoV-2 replication. Here, we show that also pentose phosphate pathway (PPP) is remarkably deregulated. Since PPP supplies ribonucleotides for SARS-CoV-2 replication, this could represent an attractive target for an intervention. On that account, we employed the transketolase inhibitor benfooxythiamine and showed dose-dependent inhibition of SARS-CoV-2 in non-toxic concentrations. Importantly, the antiviral efficacy of benfooxythiamine was further increased in combination with 2DG.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can spread from symptomatic patients with COVID-19, but also from asymptomatic individuals. Therefore, robust surveillance and timely interventions are essential for the control of virus spread within the community. In this regard the frequency of testing and speed of reporting, but not the test sensitivity alone, play a crucial role. In order to reduce the costs and meet the expanding demands in real-time RT-PCR (rRT-PCR) testing for SARS-CoV-2, complementary assays, such as rapid antigen tests, have been developed. Rigorous analysis under varying conditions is required to assess the clinical performance of these tests and to ensure reproducible results. We evaluated the sensitivity and specificity of a recently licensed rapid antigen test using 137 clinical samples in two institutions. Test sensitivity was between 88.2-89.6% when applied to samples with viral loads typically seen in infectious patients. Of 32 rRT-PCR positive samples, 19 demonstrated infectivity in cell culture, and 84% of these samples were reactive with the antigen test. Seven full-genome sequenced SARS-CoV-2 isolates and SARS-CoV-1 were detected with this antigen test, with no cross-reactivity against other common respiratory viruses. Numerous antigen tests are available for SARS-CoV-2 testing and their performance to detect infectious individuals may vary. Head-to-head comparison along with cell culture testing for infectivity may prove useful to identify better performing antigen tests. The antigen test analyzed in this study is easy-to-use, inexpensive, and scalable. It can be helpful in monitoring infection trends and thus has potential to reduce transmission.
SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinder therapy development. We employed a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phospho-proteomics. We identified viral protein phosphorylation and defined phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways were activated. Drug-protein network analyses revealed GFR signaling as key pathway targetable by approved drugs. Inhibition of GFR downstream signaling by five compounds prevented SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as central pathway essential for SARS-CoV-2 replication. It provides with novel strategies for COVID-19 treatment.
Background: In the pandemic, testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time polymerase chain reaction is one of the pillars on which countermeasures are based. Factors limiting the output of laboratories interfere with the effectiveness of public health measures. Conserving reagents by pooling samples in low-probability settings is proposed but may cause dilution and loss of sensitivity. Blood transfusion services had experience in performance of high throughput nucleic acid testing (NAT) analysis and can support the national health system by screening of the inhabitants for SARS-COV-2.
Methods: We evaluated a new approach of a multiple-swab method by simultaneously incubating multiple respiratory swabs in a single tube. Analytical sensitivity was constant up to a total number of 50 swabs. It was consequently applied in the testing of 50 symptomatic patients (5-sample pools) as well as 100 asymptomatic residents of a nursing home (10-sample pools).
Results: The novel method did not cause false-negative results with nonsignificantly differing cycle threshold values between single-swab and multiple-swab NAT. In two routine applications, all minipools containing positive patient samples were correctly identified.
Conclusions: The new method enables countries to increase the total number of testing significantly. The multiple-swab method is able to screen system relevant groups of employees frequently. The example in Germany shows that blood transfusion services can support general health systems with their experience in NAT and their high-throughput instruments. Screening of a huge number of inhabitants is currently the only option to prevent a second infection wave and enable exit strategies in many countries.
As the current SARS-CoV-2 pandemic continues, serological assays are urgently needed for rapid diagnosis, contact tracing and for epidemiological studies. So far, there is little data on how commercially available tests perform with real patient samples and if detected IgG antibodies provide protective immunity. Focusing on IgG antibodies, we demonstrate the performance of two ELISA assays (Euroimmun SARS-CoV-2 IgG & Vircell COVID-19 ELISA IgG) in comparison to one lateral flow assay ((LFA) FaStep COVID-19 IgG/IgM Rapid Test Device) and two in-house developed assays (immunofluorescence assay (IFA) and plaque reduction neutralization test (PRNT)). We tested follow up serum/plasma samples of individuals PCR-diagnosed with COVID-19. Most of the SARS-CoV-2 samples were from individuals with moderate to severe clinical course, who required an in-patient hospital stay.
For all examined assays, the sensitivity ranged from 58.8 to 76.5% for the early phase of infection (days 5-9) and from 93.8 to 100% for the later period (days 10-18) after PCR-diagnosed with COVID-19. With exception of one sample, all positive tested samples in the analysed cohort, using the commercially available assays examined (including the in-house developed IFA), demonstrated neutralizing (protective) properties in the PRNT, indicating a potential protective immunity to SARS-CoV-2. Regarding specificity, there was evidence that samples of endemic coronavirus (HCoV-OC43, HCoV-229E) and Epstein Barr virus (EBV) infected individuals cross-reacted in the ELISA assays and IFA, in one case generating a false positive result (may giving a false sense of security). This need to be further investigated.
Oral swabs, sputum and blood samples from 18 patients with SARS-CoV-2 infection were examined using real-time reverse transcription polymerase chain reaction (RT-PCR) testing. Whereas oral swabs or sputum from the lower respiratory tract were tested RT-PCR positive in all patients, RNAemia was neither detected in 3 patients without symptoms nor in 14 patients with flu-like symptoms, fever or pneumonia. The only patient with RNAemia suffered from acute respiratory distress syndrome (ARDS) and was artificially ventilated in an intensive care unit. Risk for SARS-CoV-2 transmission through blood components in asymptomatic SARS-CoV-2 infected individuals therefore seems negligible but further studies are needed.
SARS-CoV-2 is a novel coronavirus currently causing a pandemic. We show that the majority of amino acid positions, which differ between SARS-CoV-2 and the closely related SARS-CoV, are differentially conserved suggesting differences in biological behaviour. In agreement, novel cell culture models revealed differences between the tropism of SARS-CoV-2 and SARS-CoV. Moreover, cellular ACE2 (SARS-CoV-2 receptor) and TMPRSS2 (enables virus entry via S protein cleavage) levels did not reliably indicate cell susceptibility to SARS-CoV-2. SARS-CoV-2 and SARS-CoV further differed in their drug sensitivity profiles. Thus, only drug testing using SARS-CoV-2 reliably identifies therapy candidates. Therapeutic concentrations of the approved protease inhibitor aprotinin displayed anti-SARS-CoV-2 activity. The efficacy of aprotinin and of remdesivir (currently under clinical investigation against SARS-CoV-2) were further enhanced by therapeutic concentrations of the proton pump inhibitor omeprazole (aprotinin 2.7-fold, remdesivir 10-fold). Hence, our study has also identified anti-SARS-CoV-2 therapy candidates that can be readily tested in patients.
Multicentre comparison of quantitative PCR-based assays to detect SARS-CoV-2, Germany, March 2020
(2020)
Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we compare 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic.
Yellow fever virus (YFV) represents a re-emerging zoonotic pathogen, transmitted by mosquito vectors to humans from primate reservoirs. Sporadic outbreaks of YFV occur in endemic tropical regions, causing a viral hemorrhagic fever (VHF) associated with high mortality rates. Despite a highly effective vaccine, no antiviral treatments currently exist. Therefore, YFV represents a neglected tropical disease and is chronically understudied, with many aspects of YFV biology incompletely defined including host range, host–virus interactions and correlates of host immunity and pathogenicity. In this article, we review the current state of YFV research, focusing on the viral lifecycle, host responses to infection, species tropism and the success and associated limitations of the YFV-17D vaccine. In addition, we highlight the current lack of available treatments and use publicly available sequence and structural data to assess global patterns of YFV sequence diversity and identify potential drug targets. Finally, we discuss how technological advances, including real-time epidemiological monitoring of outbreaks using next-generation sequencing and CRISPR/Cas9 modification of vector species, could be utilized in future battles against this re-emerging pathogen which continues to cause devastating disease.
Hepatitis B virus (HBV) reactivation in immunosuppressed patients can cause considerable morbidity and mortality. The aim of our study was to evaluate factors associated with acute liver failure (ALF) in HBV reactivation. Clinical, laboratory, and virological data of 87 patients with HBV reactivation were analyzed retrospectively. Teno torque virus (TTV) plasma loads were measured as a measure of immune competence. HBV genomes isolated from 47 patients were analyzed by next-generation sequencing. A functional analysis of identified HBsAg mutants was performed. In patients with ALF the diagnosis was significantly later confirmed than in the non-ALF group. Patients diagnosed during immunosuppression had a milder clinical course compared to later diagnosed patients (p = 0.018, OR = 4.17). TTV viral loads did not differ significantly between the two groups. The HBV genomes isolated from ALF patients had higher viral complexity. A mutation in C-region of HBsAg (L216*), was associated with reduced HBsAg production and secretion. Patients diagnosed with HBV reactivation during immunosuppression had a milder clinical course compared to patients diagnosed during immune reconstitution. ALF was associated with higher viral complexity. An HBsAg mutation (L216*) was found to be more frequent in ALF patients and was associated with reduced HBsAg production and secretion.
Filoviruses infect a wide range of cell types with the exception of lymphocytes. The intracellular proteins cathepsin B and L, two-pore channel 1 and 2, and bona fide receptor Niemann–Pick Disease C1 (NPC1) are essential for the endosomal phase of cell entry. However, earlier steps of filoviral infection remain poorly characterized. Numerous plasma membrane proteins have been implicated in attachment but it is still unclear which ones are sufficient for productive entry. To define a minimal set of host factors required for filoviral glycoprotein-driven cell entry, we screened twelve cell lines and identified the nonlymphocytic cell line SH-SY5Y to be specifically resistant to filovirus infection. Heterokaryons of SH-SY5Y cells fused to susceptible cells were susceptible to filoviruses, indicating that SH-SY5Y cells do not express a restriction factor but lack an enabling factor critical for filovirus entry. However, all tested cell lines expressed functional intracellular factors. Global gene expression profiling of known cell surface entry factors and protein expression levels of analyzed attachment factors did not reveal any correlation between susceptibility and expression of a specific host factor. Using binding assays with recombinant filovirus glycoprotein, we identified cell attachment as the step impaired in filovirus entry in SH-SY5Y cells. Individual overexpression of attachment factors T-cell immunoglobulin and mucin domain 1 (TIM-1), Axl, Mer, or dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) rendered SH-SY5Y cells susceptible to filovirus glycoprotein-driven transduction. Our study reveals that a lack of attachment factors limits filovirus entry and provides direct experimental support for a model of filoviral cell attachment where host factor usage at the cell surface is highly promiscuous.
Environmental stability and infectivity of hepatitis C virus (HCV) in different human body fluids
(2018)
Background: Hepatitis C virus (HCV) is a hepatotropic, blood-borne virus, but in up to one-third of infections of the transmission route remained unidentified. Viral genome copies of HCV have been identified in several body fluids, however, non-parental transmission upon exposure to contaminated body fluids seems to be rare. Several body fluids, e.g., tears and saliva, are renowned for their antimicrobial and antiviral properties, nevertheless, HCV stability has never been systematically analyzed in those fluids.
Methods: We used state of the art infectious HCV cell culture techniques to investigate the stability of HCV in different body fluids to estimate the potential risk of transmission via patient body fluid material. In addition, we mimicked a potential contamination of HCV in tear fluid and analyzed which impact commercially available contact lens solutions might have in such a scenario.
Results: We could demonstrate that HCV remains infectious over several days in body fluids like tears, saliva, semen, and cerebrospinal fluid. Only hydrogen-peroxide contact lens solutions were able to efficiently inactivate HCV in a suspension test.
Conclusion: These results indicate that HCV, once it is present in various body fluids of infected patients, remains infective and could potentially contribute to transmission upon direct contact.
Objective: Chronically HCV-infected orthotopic liver transplantation (OLT) recipients appear to have improved outcomes when their immunosuppressive regimen includes a mammalian target of rapamycin (mTOR) inhibitor. The mechanism underlying this observation is unknown.
Design: We used virological assays to investigate mTOR signalling on the HCV replication cycle. Furthermore, we analysed HCV RNA levels of 42 HCV-positive transplanted patients treated with an mTOR inhibitor as part of their immunosuppressive regimen.
Results: The mTOR inhibitor rapamycin was found to be a potent inhibitor for HCV RNA replication in Huh-7.5 cells as well as primary human hepatocytes. Half-maximal inhibition was observed at 0.01 µg/mL, a concentration that is in the range of serum levels seen in transplant recipients and does not affect cell proliferation. Early replication cycle steps such as cell entry and RNA translation were not affected. Knockdown of raptor, an essential component of mTORC1, but not rictor, an essential component of mTORC2, inhibited viral RNA replication. In addition, overexpression of raptor led to higher viral RNA replication, demonstrating that mTORC1, but not mTORC2, is required for HCV RNA replication. In 42 HCV-infected liver-transplanted or kidney-transplanted patients who were switched to an mTOR inhibitor, we could verify that mTOR inhibition decreased HCV RNA levels in vivo.
Conclusions: Our data identify mTORC1 as a novel HCV replication factor. These findings suggest an underlying mechanism for the observed benefits of mTOR inhibition in HCV-positive OLT recipients and potentiate further investigation of mTOR-containing regimens in HCV-positive recipients of solid organ transplants.
Background & Aims: Hepatitis C virus (HCV) cell entry is mediated by several cell surface receptors, including scavenger receptor class B type I (SR-BI). Oxidized low density lipoprotein (oxLDL) inhibits the interaction between HCV and SR-BI in a noncompetitive manner. We tested whether serum oxLDL levels correlate with sustained virologic response (SVR) rates after interferon-based treatment of chronic hepatitis C.
Methods: Baseline oxLDL was determined in 379 participants with chronic HCV genotype 1 infection from the INDIV-2 study using a commercial enzyme-linked immunosorbent assay. The mechanistic in vitro studies used full-length and subgenomic HCV genomes replicating in hepatoma cells.
Results: In the multivariate analysis, oxLDL was found to be an independent predictor of SVR. Oxidized LDL did not correlate with markers of inflammation (alanine transaminase, ferritin), nor was serum oxLDL affected by exogenous interferon administration. Also, oxLDL did not alter the sensitivity of HCV replication to interferon. However, oxLDL was found to be a potent inhibitor of cell-to-cell spread of HCV between adjacent cells in vitro. It could thus reduce the rate at which new cells are infected by HCV through either the cell-free or cell-to-cell route. Finally, serum oxLDL was significantly associated with the estimated infected cell loss rate under treatment.
Conclusions: Oxidized LDL is a novel predictor of SVR after interferon-based therapy and may explain the previously observed association of LDL with SVR. Rather than being a marker of activated antiviral defenses it may improve chances of SVR by limiting spread of infection to naive cells through the cell-to-cell route.
Background: The sodium-taurocholate cotransporting polypeptide (NTCP) is both a key bile acid (BA) transporter mediating uptake of BA into hepatocytes and an essential receptor for hepatitis B virus (HBV) and hepatitis D virus (HDV). In this study we aimed to characterize to what extent and through what mechanism BA affect HDV cell entry.
Methods: HuH-7 cells stably expressing NTCP (HuH-7/NTCP) and primary human hepatocytes (PHH) were infected with in vitro generated HDV particles. Infectivity in the absence or presence of compounds was assessed using immunofluorescence staining for HDV antigen, standard 50% tissue culture infectious dose (TCID50) assays and quantitative PCR.
Results: Addition of primary conjugated and unconjugated BA resulted in a dose dependent reduction in the number of infected cells while secondary, tertiary and synthetic BA had a lesser effect. This effect was observed both in HuH-7/NTCP and in PHH. Other replication cycle steps such as replication and particle assembly and release were unaffected. Moreover, inhibitory BA competed with a fragment from the large HBV envelope protein for binding to NTCP-expressing cells. Conversely, the sodium/BA-cotransporter function of NTCP seemed not to be required for HDV infection since infection was similar in the presence or absence of a sodium gradient across the plasma membrane. When chenodeoxycolic acid (15 mg per kg body weight) was administered to three chronically HDV infected individuals over a period of up to 16 days there was no change in serum HDV RNA.
Conclusions: Primary BA inhibit NTCP-mediated HDV entry into hepatocytes suggesting that modulation of the BA pool may affect HDV infection of hepatocytes.