Human coronavirus 229E infection inactivates pyroptosis executioner gasdermin D but ultimately leads to lytic cell death partly mediated by gasdermin E (preprint)

Human coronavirus 229E (HCoV-229E) is associated with upper respiratory tract infections and causes local respiratory symptoms. It has been reported that HCoV-229E can cause cell death in a variety of cells in vitro. However, the molecular pathways that lead to virus-induced cell death remain poorly characterized. Here, we show that the main protease (Mpro) of HCoV-229E can cleave the pyroptosis executioner gasdermin D (GSDMD) within its active N-terminal domain at two different sites (Q29 and Q193) to generate fragments unable to cause pyroptosis. Despite GSDMD cleavage by HCoV-229E Mpro, we show that HCoV-229E infection leads to lytic cell death. We further demonstrate that virus-induced lytic cell death is partially dependent on the activation of caspases-3 and -8. Interestingly, inhibition of caspases does not only reduce lytic cell death upon infection, but also sustains the release of virus particles over time, which suggests that caspase-mediated cell death is a mechanism to limit virus replication and spread. Finally, we show that pyroptosis is partially dependent on another gasdermin family member, gasdermin E (GSDME). During HCoV-229E infection, GSDME is cleaved to yield its N-terminal pore-forming domain (p30). Accordingly, GSDME knockout cells show a significant decrease in lytic cell death upon virus, whereas this is not the case for GSDMD knockout cells, which aligns with the observation that GSDMD is also inactivated by caspase-3 during infection. These results suggest that GSDMD is inactivated during HCoV-229E infection, and point to GSDME as an important player in the execution of virus-induced cell death.

Induction of antiviral gene expression by cyclosporine A, but not inhibition of cyclophilin A or B, contributes to its restriction of human coronavirus 229E infection in a lung epithelial cell line (preprint)

The development of antivirals with an extended spectrum of activity is an attractive possibility to protect against future emerging coronaviruses (CoVs). Cyclosporine A (CsA), a clinically approved immunosuppressive drug, has established antiviral activity against diverse unrelated viruses, including several CoVs. However, its antiviral mechanisms of action against CoV infection have remained elusive, precluding the rational design of non-immunosuppressive derivatives with improved antiviral activities. In this study, we evaluated the mechanisms of CsA against HCoV-229E infection in a human lung epithelial cell line. We demonstrate that the antiviral activity of CsA against HCoV-229E is independent of classical CsA target proteins, cyclophilin A or B, which are not required host factors for HCoV-229E in A549 cells. Instead, CsA treatment induces expression of antiviral genes in a manner dependent on interferon regulatory factor 1, but independent of classical interferon responses, which contributes to its inhibitory effect against HCoV-229E infection. Our results also point to a role for the HCoV-229E nucleoprotein in antagonizing activation of type I interferon, but we show that CsA treatment does not affect evasion of innate immune signaling pathways by HCoV-229E. Overall, our findings further the understanding of the antiviral mechanisms of CsA against CoV infection and highlight a novel immunomodulatory strategy to inhibit CoV infection that may inform future drug development efforts.

Meta-analysis of diagnostic performance of serological tests for SARS-CoV-2 antibodies up to 25 April 2020 and public health implications.

We reviewed the diagnostic accuracy of SARS-CoV-2 serological tests. Random-effects models yielded a summary sensitivity of 82% for IgM, and 85% for IgG and total antibodies. For specificity, the pooled estimate were 98% for IgM and 99% for IgG and total antibodies. In populations with ≤ 5% of seroconverted individuals, unless the assays have perfect (i.e. 100%) specificity, the positive predictive value would be ≤ 88%. Serological tests should be used for prevalence surveys only in hard-hit areas.

Cell Surface Nucleocapsid Protein Expression: A Betacoronavirus Immunomodulatory Strategy (preprint)

We recently reported that SARS-CoV-2 Nucleocapsid (N) protein is abundantly expressed on the surface of both infected and neighboring uninfected cells, where it enables activation of Fc receptor-bearing immune cells with anti-N antibodies (Abs) and inhibits leukocyte chemotaxis by binding chemokines (CHKs). Here, we extend these findings to N from the seasonal human coronavirus (HCoV)-OC43, which is also robustly expressed on the surface of infected and non-infected cells by binding heparan-sulfate/heparin (HS/H). HCoV-OC43 N binds with high affinity to the same set of 11 human CHKs as SARS-CoV-2 N, but also to a non-overlapping set of 6 cytokines (CKs). As with SARS-CoV-2 N, HCoV-OC43 N inhibits CXCL12{beta}-mediated leukocyte migration in chemotaxis assays, as do all highly pathogenic and endemic HCoV N proteins. Together, our findings indicate that cell surface HCoV N plays important evolutionary conserved roles in manipulating host innate immunity and as a target for adaptive immunity.

CompCorona: A Web Portal for Comparative Analysis of the Host Transcriptome of PBMC and Lung SARS-CoV-2, SARS-CoV, and MERS-CoV (preprint)

Motivation: Understanding the host response to SARS-CoV-2 infection is crucial for deciding on the correct treatment of this epidemic disease. Although several recent studies reported the comparative transcriptome analyses of the three coronaviridae (CoV) members; namely SARS-CoV, MERS-CoV, and SARS-CoV-2, there is yet to exist a web-tool to compare increasing number of host transcriptome response datasets against the pre-processed CoV member datasets. Therefore, we developed a web application called CompCorona, which allows users to compare their own transcriptome data of infected host cells with our pre-built datasets of the three epidemic CoVs, as well as perform functional enrichment and principal component analyses (PCA). Results: Comparative analyses of the transcriptome profiles of the three CoVs revealed that numerous differentially regulated genes directly or indirectly related to several diseases (e.g., hypertension, male fertility, ALS, and epithelial dysfunction) are altered in response to CoV infections. Transcriptome similarities and differences between the host PBMC and lung tissue infected by SARS-CoV-2 are presented. Most of our findings are congruent with the clinical cases recorded in the literature. Hence, we anticipate that our results will significantly contribute to ongoing studies investigating the pre-and/or post-implications of SARS-CoV-2 infection. In addition, we implemented a user-friendly public website, CompCorona for biomedical researchers to compare users own CoV-infected host transcriptome data against the built-in CoV datasets and visualize their results via interactive PCA, UpSet and Pathway plots. Availability: CompCorona is freely available on the web at http://compcorona.mu.edu.tr Contact: tugbasuzek@mu.edu.tr

Human coronaviruses activate and hijack the proteostasis guardian HSF1 to enhance viral replication (preprint)

Organisms respond to proteotoxic stress by activating a cellular defense mechanism, known as the heat shock response (HSR), that triggers the expression of cytoprotective heat shock proteins (HSP) to counteract the damaging effects of proteostasis disruption. The HSR is regulated by a family of transcription factors (heat shock factors, HSFs); among six human HSFs, HSF1 acts as a proteostasis guardian regulating acute and severe stress-driven transcriptional responses. Seasonal coronaviruses HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1 (sHCoV) are globally circulating in the human population. Although sHCoV generally cause only mild upper respiratory diseases in immunocompetent hosts, severe complications may occur in specific populations. There is no effective treatment for sHCoV infections, also due to the limited knowledge on sHCoV biology. We now show that both Alpha- and Beta- sHCoV are potent inducers of HSF1, selectively promoting HSF1 phosphorylation at serine-326 residue and nuclear translocation, and triggering a powerful HSF1-driven transcriptional response in infected cells at late stages of infection. Despite the coronavirus-mediated shut-down of the host cell translational machinery, high levels of selected canonical and non-canonical HSF1-target genes products, including HSP70, HSPA6 and the zinc-finger AN1-type domain-2a gene/AIRAP, were found in HCoV-infected cells. Interestingly, silencing experiments demonstrate that HSR activation does not merely reflect a cellular defense response to viral infection, but that sHCoV activate and hijack the HSF1-pathway for their own gain. Notably, nuclear HSF1 pools depletion via Direct-Targeted HSF1 inhibitor (DTHIB) treatment was highly effective in hindering sHCoV replication in lung cells. Altogether the results open new scenarios for the search of innovative antiviral strategies in the treatment of coronavirus infections.

A screen for modulation of nucleocapsid protein condensation identifies small molecules with anti-coronavirus activity (preprint)

Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase separated condensates that are hypothesized to play critical roles in viral replication, transcription and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43 and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.

Prevalence of neutralizing antibody to human coronavirus 229E in Taiwan (preprint)

Background: Four members in the Coronaviruses family including 229E circulating in the community were known to cause mild respiratory tract infections in humans. The epidemiologic information of the seasonal human coronavirus (HCoV) may help gain insight into the development of the ongoing pandemic of coronavirus disease since 2019 (COVID-19). Methods Plasma collection containing 1558 samples was obtained in 2010 for an estimate of the prevalence and severity of 2009 pandemic influenza A H1N1 in Taiwan. Of 1558 samples, 200 were randomly selected from those aged < 1 year to > 60 years. The neutralizing antibody titers to HCoV-229E were determined in the serums using live virus ATCC VR_740TM cultivating in the Huh_ 7 cell line. Results Seroconversion of HCoV-229E (titer ≥ 1:2) was identified as early as less than 5 years of age. Among 140 subjects aged younger than or equal to 40 years, all of them had uniformly low titers (< 1:10) and the geometric mean titers (GMTs) were not significantly different for those aged 0-5, 6-12, 13-18 and 19-40 years (P > 0.1). For 60 subjects greater than 40 years old, a majority (39, 65%) of them had high titers ≥ 1:10 and the GMTs were significantly increased with advanced age (P < 0.0001). Age was the most significant factor predicting seropositivity in the multivariate analysis, with an adjusted odds ratio of 1.107 and a 95% adjusted confidence interval of 1.061-1.155 (P < 0.0001). Conclusion HCoV-229E infection occurred as early as younger than 5 years old in Taiwanese and the magnitudes of neutralizing titers against HCoV-229E increased with advanced age beyond 40 years.

Presence of Recombinant Bat Coronavirus GCCDC1 in Cambodian Bats.

Bats have been recognized as an exceptional viral reservoir, especially for coronaviruses. At least three bat zoonotic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) have been shown to cause severe diseases in humans and it is expected more will emerge. One of the major features of CoVs is that they are all highly prone to recombination. An extreme example is the insertion of the P10 gene from reoviruses in the bat CoV GCCDC1, first discovered in Rousettus leschenaultii bats in China. Here, we report the detection of GCCDC1 in four different bat species (Eonycteris spelaea, Cynopterus sphinx, Rhinolophus shameli and Rousettus sp.) in Cambodia. This finding demonstrates a much broader geographic and bat species range for this virus and indicates common cross-species transmission. Interestingly, one of the bat samples showed a co-infection with an Alpha CoV most closely related to RsYN14, a virus recently discovered in the same genus (Rhinolophus) of bat in Yunnan, China, 2020. Taken together, our latest findings highlight the need to conduct active surveillance in bats to assess the risk of emerging CoVs, especially in Southeast Asia.

Live biotherapeutic throat spray for respiratory virus inhibition and interferon pathway induction (preprint)

Respiratory viruses such as influenza viruses, respiratory syncytial virus (RSV), and coronaviruses initiate infection at the mucosal surfaces of the upper respiratory tract (URT), where the resident respiratory microbiome has an important gatekeeper function. In contrast to gut-targeting administration of beneficial bacteria against respiratory viral disease, topical URT administration of probiotics is currently underexplored, especially for the prevention and/or treatment of viral infections. Here, we report the selection and formulation of a broad-acting throat spray with live lactobacilli which induce interferon regulatory pathways and are able to inhibit respiratory viruses. Rational selection of Lactobacillaceae strains was based on safety, applicability, and potential antiviral and immunostimulatory efficacy in the URT. Three strains, Lacticaseibacillus casei AMBR2, Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum WCFS1 significantly reduced the cytopathogenic effects of RSV, influenza A/H1N1 and B viruses, and HCoV-229E coronavirus in co-culture models with bacteria, virus and host cells. Subsequently, these strains were formulated in a throat spray and human monocytes were employed to confirm the formulation process did not reduce the interferon regulatory pathway-inducing capacity. Administration of the throat spray in healthy volunteers revealed that the lactobacilli were capable of temporary colonization of the throat in a metabolically active form.

Inhibition of glycogen synthase kinase-3-beta (GSK3β) blocks nucleocapsid phosphorylation and SARS-CoV-2 replication (preprint)

GSK3β has been proposed to have an essential role in Coronaviridae infection. Screening of a targeted library of GSK3β inhibitors against SARS-CoV-2 and HCoV-229E resulted in identification of high proportion of active compounds with low toxicity to host cells. A select lead compound, T-1686568, showed dose-dependent activity against SARS-CoV-2 transcription, translation and viral particle release in multiple cell lines and primary organoids. A protein kinase substrate profiling assay combined with western blot analysis showed that SARS-CoV-2 nucleocapsid is phosphorylated by GSK3β on S180/S184, S190/S194 and T198 which have already been primed in the adjacent phospho-sites S188, T198 and S206 respectively. Inhibition by T-1686568 resulted in reduction of the S1 Spike protein levels, an accumulation of the Nucleocapsid (N) protein and maintenance of the non-structural (NSP2) level in infected Huh-7.5.1 cells, indicating that N phosphorylation might serve as a critical precursor for processing and release of mature viruses.

Modeling SARS-CoV-2 propagation using rat coronavirus-associated shedding and transmission.

At present, global immunity to SARS-CoV-2 resides within a heterogeneous combination of susceptible, naturally infected and vaccinated individuals. The extent to which viral shedding and transmission occurs on re-exposure to SARS-CoV-2 is an important determinant of the rate at which COVID-19 achieves endemic stability. We used Sialodacryoadenitis Virus (SDAV) in rats to model the extent to which immune protection afforded by prior natural infection via high risk (inoculation; direct contact) or low risk (fomite) exposure, or by vaccination, influenced viral shedding and transmission on re-exposure. On initial infection, we confirmed that amount, duration and consistency of viral shedding, and seroconversion rates were correlated with exposure risk. Animals were reinfected after 3.7-5.5 months using the same exposure paradigm. 59% of seropositive animals shed virus, although at lower amounts. Previously exposed seropositive reinfected animals were able to transmit virus to 25% of naive recipient rats after 24-hour exposure by direct contact. Rats vaccinated intranasally with a related virus (Parker's Rat Coronavirus) were able to transmit SDAV to only 4.7% of naive animals after a 7-day direct contact exposure, despite comparable viral shedding. Cycle threshold values associated with transmission in both groups ranged from 29-36 cycles. Observed shedding was not a prerequisite for transmission. Results indicate that low-level shedding in both naturally infected and vaccinated seropositive animals can propagate infection in susceptible individuals. Extrapolated to COVID-19, our results suggest that continued propagation of SARS-CoV-2 by seropositive previously infected or vaccinated individuals is possible.

Antiviral activity of Pacific oyster (Crassostrea gigas) hemolymph against a human coronavirus. (preprint)

Coronaviruses can can cause severe respiratory infections in humans. This study aimed to assess the antiviral activity of Pacific oyster (Crassostrea gigas) hemolymph against a human coronavirus, HCoV-229E. An eight-fold reduction in infectivity of HCoV-229E on Huh-7 cells was observed in the presence of 10% C. gigas hemolymph. Antiviral activity of C. gigas hemolymph positively correlated with its concentration and appears to be active during an intracellular stage of HCoV-229E infection.

Structure, receptor recognition and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein (preprint)

The recent isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. Here, we determined cryo-electron microscopy structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and identified that it binds canine, feline and porcine aminopeptidase N (APN encoded by ANPEP) orthologs which serve as entry receptors. Introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single nucleotide polymorphisms could account for the detection of this virus in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 S-mediated entry, indicating elicitation of cross-neutralizing activity among -coronaviruses. These data provide a blueprint of the CCoV-HuPn-2018 infection machinery, unveil the viral entry receptor and pave the way for vaccine and therapeutic development targeting this zoonotic pathogen.

Estimating the age of the subfamily Orthocoronavirinae using host divergence times as calibration ages at two internal nodes.

Viruses of the subfamily Orthocoronavirinae can cause mild to severe disease in people, including COVID-19, MERS and SARS. Their most common natural hosts are bat and bird species, which are mostly split across four virus genera. Molecular clock analyses of orthocoronaviruses suggested the most recent common ancestor of these viruses might have emerged either around 10,000 years ago or, using models accounting for selection, many millions of years. Here, we reassess the evolutionary history of these viruses. We present time-aware phylogenetic analyses of a RNA-dependent RNA polymerase locus from 123 orthocoronaviruses isolated from birds and bats, including those in New Zealand, which were geographically isolated from other bats around 35 million years ago. We used this age, as well as the age of the avian-mammals split, to calibrate the molecular clocks, under the assumption that these ages are applicable to the analyzed viruses. We found that the time to the most recent ancestor common for all orthocoronaviruses is likely 150 or more million years, supporting clock analyses that account for selection.

Role of Multifunctional Cytoskeletal Filaments in Coronaviridae Infections: Therapeutic Opportunities for COVID-19 in a Nutshell.

A novel coronavirus discovered in 2019 is a new strain of the Coronaviridae family (CoVs) that had not been previously identified in humans. It is known as SARS-CoV-2 for Severe Acute Respiratory Syndrome Coronavirus-2, whilst COVID-19 is the name of the disease associated with the virus. SARS-CoV-2 emerged over one year ago and still haunts the human community throughout the world, causing both healthcare and socioeconomic problems. SARS-CoV-2 is spreading with many uncertainties about treatment and prevention: the data available are limited and there are few randomized controlled trial data on the efficacy of antiviral or immunomodulatory agents. SARS-CoV-2 and its mutants are considered as unique within the Coronaviridae family insofar as they spread rapidly and can have severe effects on health. Although the scientific world has been succeeding in developing vaccines and medicines to combat COVID-19, the appearance and the spread of new, more aggressive mutants are posing extra problems for treatment. Nevertheless, our understanding of pandemics is increasing significantly due to this outbreak and is leading to the development of many different pharmacological, immunological and other treatments. This Review focuses on a subset of COVID-19 research, primarily the cytoskeleton-related physiological and pathological processes in which coronaviruses such as SARS-CoV-2 are intimately involved. The discovery of the exact mechanisms of the subversion of host cells by SARS-CoV-2 is critical to the validation of specific drug targets and effective treatments.

Improving comparability of studies using HCoV-OC43 as a surrogate for SARS-CoV-2 (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-COV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus, targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose50(TCID50)/ml or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of ~107 plaque forming units/ml at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of ~106.5 TCID50/ml. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts.

In Vitro Models for Studying Entry, Tissue Tropism, and Therapeutic Approaches of Highly Pathogenic Coronaviruses.

Coronaviruses (CoVs) are enveloped nonsegmented positive-sense RNA viruses belonging to the family Coronaviridae that contain the largest genome among RNA viruses. Their genome encodes 4 major structural proteins, and among them, the Spike (S) protein plays a crucial role in determining the viral tropism. It mediates viral attachment to the host cell, fusion to the membranes, and cell entry using cellular proteases as activators. Several in vitro models have been developed to study the CoVs entry, pathogenesis, and possible therapeutic approaches. This article is aimed at summarizing the current knowledge about the use of relevant methodologies and cell lines permissive for CoV life cycle studies. The synthesis of this information can be useful for setting up specific experimental procedures. We also discuss different strategies for inhibiting the binding of the S protein to the cell receptors and the fusion process which may offer opportunities for therapeutic intervention.