SARS-CoV-2 entry route impacts a range of downstream viral and cellular processes

SARS-CoV-2 entry is promoted by both cell-surface TMPRSS2 and endolysosomal cathepsins. To investigate the impact of differentially routed virions on host and viral processes, lung epithelial cells expressing distinct combinations of entry factors were infected with authentic viruses. Entry route determined early rates of viral replication and transcription, egress and inhibitor sensitivity, with differences observed between virus strains. Transcriptional profiling revealed that induction of innate immunity was correlated to viral genome and transcript abundance in infected cells. Surface entry triggered early activation of antiviral responses, reducing cumulative virion production, while endolysosomal entry delayed antiviral responses and prolonged virus shedding due to extended cell viability. The likely molecular footprints of escape from antiviral effector targeting were also recorded in viral genomes and correlated with entry route-dependent immune status of cells. TMPRSS2 orthologues from diverse mammals, but not zebra fish, facilitated infection enhancement, which was more pronounced for ancestral strains. Leveraging RNA-seq and scRNA-seq datasets from SARS-CoV-2 infected hamsters, we validate aspects of our model in vivo. In summary, we demonstrate that distinct cellular and viral processes are linked to viral entry route, collectively modulating virus shedding, cell-death rates and viral genome evolution.

Risk of SARS-CoV-2 transmission by fomites: a clinical observational study in highly infectious COVID-19 patients

BackgroundThe contribution of droplet-contaminated surfaces for virus transmission has been discussed controversially in the context of the current Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic. Importantly, the risk of fomite-based transmission has not been systematically addressed. MethodsWe initiated this single-center observational study to evaluate whether hospitalized COVID-19 patients can contaminate stainless steel carriers by coughing or intensive moistening with saliva and to assess the risk of SARS-CoV-2 transmission upon detection of viral loads and infectious virus in cell culture. Fifteen hospitalized patients with a high baseline viral load (CT value [≤] 25) shortly after admission were included. We documented clinical and laboratory parameters and used patient samples to perform virus culture, quantitative PCR and virus sequencing. ResultsNasopharyngeal and oropharyngeal swabs of all patients were positive for viral RNA on the day of the study. Infectious SARS-CoV-2 could be isolated from 6 patient swabs (46.2 %). While after coughing, no infectious virus could be recovered, intensive moistening with saliva resulted in successful viral recovery from steel carriers of 5 patients (38.5 %). ConclusionsTransmission of infectious SARS-CoV-2 via fomites is possible upon extensive moistening, but unlikely to occur in real-life scenarios and from droplet-contaminated fomites.

Breakthrough Infections with SARS-CoV-2 in Vaccinated Students After a Festive Event - Identification of Risk Factors

We report an outbreak with SARS-CoV-2 breakthrough infections related to a festive event in Northern Bavaria, Germany in October 2021, with 24 of 95 participants infected. Correlation analyses among 15 interrogated variables revealed that duration at the event and conversation with the supposed index person were significant risk factors. Article Summary LineThe risk of infection with SARS-CoV-2 in a vaccinated cohort associated with a private festive event was significantly increased upon conversation with the putative index person and positively correlated to the duration of stay at the event.

Risk assessment of banknotes as a fomite of SARS-CoV-2 in cash payment transactions

BackgroundThe COVID 19 pandemic has triggered concerns and assumptions globally about transmission of the SARS-CoV-2 virus via cash transactions. ObjectivesAssess the risk of contracting COVID-19 through exposure to SARS-CoV-2 via cash acting as a fomite in payment transactions. MethodsA quantitative microbial risk assessment was conducted for a worst-case scenario assuming an infectious person at the onset of symptoms, when virion concentrations in coughed droplets are at their highest. This person then contaminates a banknote by coughing on it and immediately hands it over to another person, who might then be infected by transferring the virions with a finger from the contaminated banknote to a facial mucous membrane. The scenario considered transfer efficiency of virions on the banknote to fingertips when droplets were still wet and after having dried up and subsequently being touched by finger printing or rubbing the object. ResultsAccounting for the likelihood of the worst-case scenario to occur by considering 1) a local prevalence of 100 COVID-19 cases/100,000 persons, 2) a maximum of about 1/5th of infected persons transmit high virus loads and 3) the numbers of cash transactions/person/day, the risk of contracting COVID-19 via person-to-person cash transactions was estimated to be much lower than once per 39,000 days (107 years) for a single person. In the general populace, there will be a maximum of 2.6 expected cases/100,000 persons/day. The risk for a cashier at an average point of sale was estimated to be much less than once per 430 working days (21 months). DiscussionThe worst-case scenario is a rare event, therefore, for a single person, the risk of contracting COVID-19 via person-to-person cash transactions is very low. At a point of sale, the risk to the cashier proportionally increases but it is still low.

In contrast to TH2-biased approaches, TH1 COVID-19 vaccines protect Syrian hamsters from severe disease in the absence of dexamethasone-treatable vaccine-associated enhanced respiratory pathology

Since December 2019, the novel human coronavirus SARS-CoV-2 has spread globally, causing millions of deaths. Unprecedented efforts have enabled development and authorization of a range of vaccines, which reduce transmission rates and confer protection against the associated disease COVID-19. These vaccines are conceptually diverse, including e.g. classical adjuvanted whole-inactivated virus, viral vectors, and mRNA vaccines. We have analysed two prototypic model vaccines, the strongly TH1-biased measles vaccine-derived candidate MeVvac2-SARS2-S(H) and a TH2-biased Alum-adjuvanted, non-stabilized Spike (S) protein side-by-side, for their ability to protect Syrian hamsters upon challenge with a low-passage SARS-CoV-2 patient isolate. As expected, the MeVvac2-SARS2-S(H) vaccine protected the hamsters safely from severe disease. In contrast, the protein vaccine induced vaccine-associated enhanced respiratory disease (VAERD) with massive infiltration of eosinophils into the lungs. Global RNA-Seq analysis of hamster lungs revealed reduced viral RNA and less host dysregulation in MeVvac2-SARS2-S(H) vaccinated animals, while S protein vaccination triggered enhanced host gene dysregulation compared to unvaccinated control animals. Of note, mRNAs encoding the major eosinophil attractant CCL-11, the TH2 response-driving cytokine IL-19, as well as TH2-cytokines IL-4, IL-5, and IL-13 were exclusively up-regulated in the lungs of S protein vaccinated animals, consistent with previously described VAERD induced by RSV vaccine candidates. IL-4, IL-5, and IL-13 were also up-regulated in S-specific splenocytes after protein vaccination. Using scRNA-Seq, T cells and innate lymphoid cells were identified as the source of these cytokines, while Ccl11 and Il19 mRNAs were expressed in lung macrophages displaying an activated phenotype. Interestingly, the amount of viral reads in this macrophage population correlated with the abundance of Fc-receptor reads. These findings suggest that VAERD is triggered by induction of TH2-type helper cells secreting IL-4, IL-5, and IL-13, together with stimulation of macrophage subsets dependent on non-neutralizing antibodies. Via this mechanism, uncontrolled eosinophil recruitment to the infected tissue occurs, a hallmark of VAERD immunopathogenesis. These effects could effectively be treated using dexamethasone and were not observed in animals vaccinated with MeVvac2-SARS2-S(H). Taken together, our data validate the potential of TH2-biased COVID-19 vaccines and identify the transcriptional mediators that underlie VAERD, but confirm safety of TH1-biased vaccine concepts such as vector-based or mRNA vaccines. Dexamethasone, which is already in use for treatment of severe COVID-19, may alleviate such VAERD, but in-depth scrutiny of any next-generation protein-based vaccine candidates is required, prior and after their regulatory approval.

Differential interferon-α subtype immune signatures suppress SARS-CoV-2 infection

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies and have been successfully employed for the treatment of viral diseases. Humans express twelve IFN-alpha () subtypes, which activate downstream signalling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in type I IFN immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19, therefore, early administration of type I IFNs may be protective against life-threatening disease. Here we comprehensively analysed the antiviral activity of all IFN subtypes against SARS-CoV-2 to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFN subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate and low antiviral IFNs. In particular IFN5 showed superior antiviral activity against SARS-CoV-2 infection. Dose-dependency studies further displayed additive effects upon co-administered with the broad antiviral drug remdesivir in cell culture. Transcriptomics of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting and prototypical genes of individual IFN subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in type I IFN signalling pathways, negative regulation of viral processes and immune effector processes for the potent antiviral IFN5. Taken together, our data provide a systemic, multi-modular definition of antiviral host responses mediated by defined type I IFNs. This knowledge shall support the development of novel therapeutic approaches against SARS-CoV-2.

Comparable environmental stability and disinfection profiles of the currently circulating SARS-CoV-2 variants of concern B.1.1.7 and B.1.351

The emergence of novel SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern with increased transmission dynamics has raised questions regarding stability and disinfection of these viruses. In this study, we analyzed surface stability and disinfection of the currently circulating SARS-CoV-2 variants B.1.1.7 and B.1.351 compared to the wildtype. Treatment with heat, soap and ethanol revealed similar inactivation profiles indicative of a comparable susceptibility towards disinfection. Furthermore, we observed comparable surface stability on steel, silver, copper and face masks. Overall, our data support the application of currently recommended hygiene concepts to minimize the risk of B.1.1.7 and B.1.351 transmission.

A realistic touch-transfer method reveals low risk of transmission for SARS-CoV-2 by contaminated coins and bank notes

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a significant threat to global health. While respiratory aerosols or droplets are considered as the main route of human-to-human transmission, secretions expelled by infected individuals can also contaminate surfaces and objects, potentially creating the risk of fomite-based transmission. Consequently, frequently touched objects such as paper currency and coins have been suspected as a potential transmission vehicle. To assess the risk of SARS-CoV-2 transmission by banknotes and coins, we examined the stability of SARS-CoV-2 and bovine coronavirus (BCoV), as surrogate with lower biosafety restrictions, on these different means of payment and developed a touch transfer method to examine transfer efficiency from contaminated surfaces to skin. Although we observed prolonged virus stability, our results, including a novel touch transfer method, indicate that the transmission of SARS-CoV-2 via contaminated coins and banknotes is unlikely and requires high viral loads and a timely order of specific events.

SARS-CoV-2 N gene dropout and N gene Ct value shift as indicator for the presence of B.1.1.7 lineage in a widely used commercial multiplex PCR assay

ObjectivesIncreased importance in detection and surveillance of SARS-CoV-2 has been demonstrated due to the emergence of variants of concern (VOCs). In this study we evaluated if a commercially available real-time SARS-CoV-2 PCR assay can identify B.1.1.7 lineage samples by a specific N gene dropout or Ct value shift compared to the S or RdRP gene. MethodsPatients samples with confirmed B.1.1.7 variant by whole-genome sequencing and variant-specific PCR (n=48) and non-B.1.1.7 samples (n=53) were tested by the Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay for presence of S, RdRP and N gene of SARS CoV-2. The N gene coding sequence of SARS-CoV-2 with and without D3L mutation (specific for B.1.1.7) were cloned into pCR(R)-TOPO vectors and Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay was performed. ResultsAll studied B.1.1.7 patient samples showed significantly higher Ct values ({Delta} 6-10, N-gene dropout on Ct values >29) in the N gene compared to the respective values of S and RdRP gene. Receiver operating characteristic (ROC) curve analysis resulted in 100% sensitivity and specificity for {Delta}Ct N/RdRP and {Delta}Ct N/S. As a result of the reversed genetic experiments we found also the shift in Ct values for the 3L variant N-gene. ConclusionsN gene dropout or Ct value shift is specific for B.1.1.7 positive samples using the Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay. This approach can be used as a rapid tool for B.1.1.7 detection in single assay high throughput diagnostics.

A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets

Over the past 20 years, the emergence of three highly pathogenic coronaviruses (CoV) - SARS-CoV, MERS-CoV, and most recently SARS-CoV-2 - has shown that CoVs pose a serious risk to human health and highlighted the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycle. Here, we conducted two independent genome-wide CRISPR/Cas9 knockout screens to identify pan-CoV host factors required for the replication of both endemic and emerging CoVs, including the novel CoV SARS-CoV-2. Strikingly, we found that several autophagy-related genes, including the immunophilin FKBP8, TMEM41B, and MINAR1, were common host factors required for CoV replication. Importantly, inhibition of the immunophilin family with the compounds Tacrolimus, Cyclosporin A, and the non-immunosuppressive derivative Alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures that resemble the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrate that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

Glycyrrhizin effectively neutralizes SARS-CoV-2 in vitro by inhibiting the viral main protease

The newly emerged coronavirus, which was designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 disease. High effective and well-tolerated medication for hospitalized and non-hospitalized patients is urgently needed. Traditional herbal medicine substances were discussed as promising candidates for the complementary treatment of viral diseases and recently suggested for the treatment of COVID-19. In the present study, we investigated aqueous licorice root extract for its neutralizing activity against SARS-CoV-2 in vitro, identified the active compound glycyrrhizin and uncovered the respective mechanism of viral neutralization. We demonstrated that glycyrrhizin, the primary active ingredient of the licorice root, potently neutralizes SARS-CoV-2 by inhibiting the viral main protease. Our experiments highlight glycyrrhizin as a potential antiviral compound that should be further investigated for the treatment of COVID-19.

Virucidal efficacy of different formulations for hand and surface disinfection targeting SARS CoV-2

In the ongoing SARS CoV-2 pandemic effective measures are needed, and guidance based on the methodological framework of the European committee for standardization (CEN) can help to choose effective disinfectants on an immediate basis. This study demonstrates that two commercially available formulations for surface disinfection and one formulation for hand disinfection claiming "virucidal activity against enveloped viruses" are effectively inactivating SARS-CoV-2. This study emphasizes that chemical disinfectants claiming "virucidal activity against enveloped viruses" are an effective choice to target enveloped SARS-CoV-2 as a preventive measure.

Comparison of the in vitro-efficacy of different mouthwash solutions targeting SARS-CoV-2 based on the European Standard EN 14476

The SARS-Cov-2 pandemic is triggering a global health emergency alert, and recent research is indicating the relevance of aerosols in the spread of SARS-CoV-2. Thus, in this study antiseptic mouthwashes based on the actives chlorhexidine (CHX) and octenidine (OCT) were investigated regarding their efficacy against SARS-CoV-2 using EN 14476. Based on the requirement of EN 14476 (i.e. reduction of viral titer by [≥] 4 log 10), the OCT-based formulation was effective within only 15 sec against SARS-CoV-2, and thus constitutes an interesting candidate for future clinical studies to prove its effectiveness in a potential prevention of SARS-CoV-2 transmission by aerosols.

Holder Pasteurization Inactivates SARS-CoV-2 in Human Breast Milk

SARS-CoV-2 RNA has been detected in the human breast milk of infected mothers, raising concerns regarding the safety of breastfeeding upon infection. We here show that holder pasteurization inactivates SARS-CoV-2 and provides an alternative and safe option for infected mothers to continue feeding breast milk to their infants.

Efficient inactivation of SARS-CoV-2 by WHO-recommended hand rub formulations and alcohols

The recent emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 is a major burden for health care systems worldwide. It is important to address if the current infection control instructions based on active ingredients are sufficient. We therefore determined the virucidal activity of two alcohol-based hand rub solutions for hand disinfection recommended by the World Health Organization (WHO), as well as commercially available alcohols. Efficient SARS-CoV-2 inactivation was demonstrated for all tested alcohol-based disinfectants. These findings show the successful inactivation of SARS-CoV-2 for the first time and provide confidence in its use for the control of COVID-19. ImportanceThe current COVID-19 outbreak puts a huge burden on the worlds health care systems. Without effective therapeutics or vaccines being available, effective hygiene measure are of utmost importance to prevent viral spreading. It is therefore crucial to evaluate current infection control strategies against SARS-CoV-2. We show the inactivation of the novel coronavirus for the first time and endorse the importance of disinfectant-based hand hygiene to reduce SARS-CoV-2 transmission.

LY6E impairs coronavirus fusion and confers immune control of viral disease

Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. Host immune responses to CoV are complex and regulated in part through antiviral interferons. However, the interferon-stimulated gene products that inhibit CoV are not well characterized2. Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo, knowledge that could help inform strategies to combat infection by emerging CoV.