The Environmental Stability of SARS-CoV-2 Variants Omicron BA.1 and BA.5 on the Surfaces of Widely Used Transport Packaging Materials.

The increased transmissibility of SARS-CoV-2 variants of concern (VOCs) has raised questions regarding the environmental stability of these viruses. Although a prolonged survival time has been reported for SARS-CoV-2, how long new variants can persist on contaminated surfaces and how environmental factors affect the persistence time are not fully characterized. The present study provides a comprehensive assessment of the stability of Omicron variants BA.1 and BA.5, which are currently circulating strains, on the surfaces of widely used transport packaging materials. By monitoring viable virus detection over a 7-day period under different environmental conditions, it was found that the environmental stability of SARS-CoV-2 Omicron variants depended heavily on the surface type, temperature, and virus concentration. In addition, virus nucleic acid exhibited high stability on the material surface independent of whether viable virus was detected. These findings provide useful information for logistics practitioners and the general public to appropriately deal with transport items under different conditions to minimize the risk of epidemic transmission. IMPORTANCE This study shows the environmental stability of SARS-CoV-2 Variants Omicron BA.1 and BA.5 on surfaces of widely used transport packaging materials. The findings demonstrate that the environmental stability of the SARS-CoV-2 Omicron variants varies based on material type. The viability of SARS-CoV-2 on material surfaces depends heavily on temperature and viral titer. Low temperatures and high viral titers promote virus survival. Moreover, in contrast to virus viability, virus nucleic acid exhibits high stability on the surfaces of widely used materials, making the detection of virus nucleic acid unsuitable for evaluating the risk of epidemic transmission.

Omicron BA.1 breakthrough infections in inactivated COVID-19 vaccine recipients induced distinct pattern of antibody and T cell responses to different Omicron sublineages.

The adaptive immunity against SARS-CoV-2 prototype strain and Omicron sublineages induced by BA.1 breakthrough infection in vaccinees of inactivated COVID-19 vaccines have not been well characterized. Here, we report that BA.1 breakthrough infection induced mucosal sIgA and resulted in higher IgG titers against prototype strain and Omicron sublineages in vaccinees than in vaccine naïve-infected individuals. BA.1 breakthrough infection boosted antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis to prototype strain and BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.2.75 but not BA.4/5 and induced neutralization against prototype strain and BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, and BA.4/5 but not BF.7, BQ.1, and XBB. In total, BA.1 breakthrough infection individuals produced less extensive sIgA, plasma IgG and NAb responses against Omicron sublineages compared with those against prototype strain. Further, BA.1 breakthrough infection induced recall B cell response to prototype strain and Omicron variant, primarily targeting memory B cells producing conserved epitopes. Memory T cell responses against Omicron is largely preserved. Individuals with vaccine booster did not induce more beneficial immune responses to Omicron sublineages upon BA.1 breakthrough infection than those with primary vaccine dose only. The breakthrough infection individuals produced stronger adaptive immunity than those of inactivated vaccine-healthy individuals. These data have important implications for understanding the vaccine effectiveness and adaptive immunity to breakthrough infection in individuals fully immunized with inactivated vaccines. Omicron sublineages, especially for those emerged after BA.4/5 strain, evade NAb responses induced by BA.1 breakthrough infection. It is urgent to optimize the vaccine immunogen design and formulations to SARS-CoV-2 variants.

SARS-CoV-2 hijacks cellular kinase CDK2 to promote viral RNA synthesis.

The coronavirus disease 2019 (COVID-19) pandemic has devastated global health. Identifying key host factors essential for SARS-CoV-2 RNA replication is expected to unravel cellular targets for the development of broad-spectrum antiviral drugs which have been quested for the preparedness of future viral outbreaks. Here, we have identified host proteins that associate with nonstructural protein 12 (nsp12), the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 using a mass spectrometry (MS)-based proteomic approach. Among the candidate factors, CDK2 (Cyclin-dependent kinase 2), a member of cyclin-dependent kinases, interacts with nsp12 and causes its phosphorylation at T20, thus facilitating the assembly of the RdRp complex consisting of nsp12, nsp7 and nsp8 and promoting efficient synthesis of viral RNA. The crucial role of CDK2 in viral RdRp function is further supported by our observation that CDK2 inhibitors potently impair viral RNA synthesis and SARS-CoV-2 infection. Taken together, we have discovered CDK2 as a key host factor of SARS-CoV-2 RdRp complex, thus serving a promising target for the development of SARS-CoV-2 RdRp inhibitors.

Serosurvey for SARS-CoV-2 among blood donors in Wuhan, China from September to December 2019.

The emerging of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused COVID-19 pandemic. The first case of COVID-19 was reported at early December in 2019 in Wuhan City, China. To examine specific antibodies against SARS-CoV-2 in biological samples before December 2019 would give clues when the epidemic of SARS-CoV-2 might start to circulate in populations. We obtained all 88,517 plasmas from 76,844 blood donors in Wuhan between 1 September and 31 December 2019. We first evaluated the pan-immunoglobin (pan-Ig) against SARS-CoV-2 in 43,850 samples from 32,484 blood donors with suitable sample quality and enough volume. Two hundred and sixty-four samples from 213 donors were pan-Ig reactive, then further tested IgG and IgM, and validated by neutralizing antibodies against SARS-CoV-2. Two hundred and thirteen samples (from 175 donors) were only pan-Ig reactive, 8 (from 4 donors) were pan-Ig and IgG reactive, and 43 (from 34 donors) were pan-Ig and IgM reactive. Microneutralization assay showed all negative results. In addition, 213 screened reactive donors were analyzed and did not show obviously temporal or regional tendency, but the distribution of age showed a difference compared with all tested donors. Then we reviewed SARS-CoV-2 antibody results from these donors who donated several times from September 2019 to June 2020, partly tested in a previous published study, no one was found a significant increase in S/CO of antibodies against SARS-CoV-2. Our findings showed no SARS-CoV-2-specific antibodies existing among blood donors in Wuhan, China before 2020, indicating no evidence of transmission of COVID-19 before December 2019 in Wuhan, China.

SARS-CoV-2 fusion-inhibitory lipopeptides maintain high potency against divergent variants of concern including Omicron.

The emergence of SARS-CoV-2 Omicron and other variants of concern (VOCs) has brought huge challenges to control the COVID-19 pandemic, calling for urgent development of effective vaccines and therapeutic drugs. In this study, we focused on characterizing the impacts of divergent VOCs on the antiviral activity of lipopeptide-based fusion inhibitors that we previously developed. First, we found that pseudoviruses bearing the S proteins of five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) and one variant of interest (Lambda) exhibited greatly decreased infectivity relative to the wild-type (WT) strain or single D614G mutant, especially the Omicron pseudovirus. Differently, the most of variants exhibited an S protein with significantly enhanced cell fusion activity, whereas the S protein of Omicron still mediated decreased cell-cell fusion. Next, we verified that two lipopeptide-based fusion inhibitors, IPB02V3 and IPB24, maintained the highly potent activities in inhibiting various S proteins-driven cell fusion and pseudovirus infection. Surprisingly, both IPB02V3 and IPB24 lipopeptides displayed greatly increased potencies against the infection of authentic Omicron strain relative to the WT virus. The results suggest that Omicron variant evolves with a reduced cell fusion capacity and is more sensitive to the inhibition of fusion-inhibitory lipopeptides; thus, IPB02V3 and IPB24 can be further developed as potent, broad-spectrum antivirals for combating Omicron and the potential future outbreak of other emerging variants.

Screening for inhibitors against SARS-CoV-2 and its variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, generating new variants that pose a threat to global health; therefore, it is imperative to obtain safe and broad-spectrum antivirals against SARS-CoV-2 and its variants. To this end, we screened compounds for their ability to inhibit viral entry, which is a critical step in virus infection. Twenty compounds that have been previously reported to inhibit SARS-CoV-2 replication were tested by using pseudoviruses containing the spike protein from the original strain (SARS-CoV-2-WH01). The cytotoxicity of these compounds was determined. Furthermore, we identified six compounds with strong antagonistic activity against the WH01 pseudovirus, and low cytotoxicity was identified. These compounds were then evaluated for their efficacy against pseudoviruses expressing the spike protein from B.1.617.2 (Delta) and B.1.1.529 (Omicron), the two most prevalent circulating strains. These assays demonstrated that two phenothiazine compounds, trifluoperazine 2HCl and thioridazine HCl, inhibit the infection of Delta and Omicron pseudoviruses. Finally, we discovered that these two compounds were highly effective against authentic SARS-CoV-2 viruses, including the WH01, Delta, and Omicron strains. Our study identified potential broad-spectrum SARS-CoV-2 inhibitors and provided insights into the development of novel therapeutics.

Circular RNA vaccines against SARS-CoV-2 and emerging variants.

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNARBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2.

Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection.

The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. How the host immune system senses and responds to SARS-CoV-2 infection remain largely unresolved. Here, we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway. SARS-CoV-2 infection induces the cellular level of 2'3'-cGAMP associated with STING activation. cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection. We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion. Furthermore, cytoplasmic chromatin-cGAS-STING pathway, but not MAVS-mediated viral RNA sensing pathway, contributes to interferon and pro-inflammatory gene expression upon cell fusion. Finally, we show that cGAS is required for host antiviral responses against SARS-CoV-2, and a STING-activating compound potently inhibits viral replication. Together, our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection, mediated by cytoplasmic chromatin from the infected cells. Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19. In addition, these findings extend our knowledge in host defense against viral infection by showing that host cells' self-nucleic acids can be employed as a "danger signal" to alarm the immune system.

Genome-wide CRISPR activation screen identifies candidate receptors for SARS-CoV-2 entry.

The outbreak of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has created a global health crisis. SARS-CoV-2 infects varieties of tissues where the known receptor ACE2 is low or almost absent, suggesting the existence of alternative viral entry pathways. Here, we performed a genome-wide barcoded-CRISPRa screen to identify novel host factors that enable SARS-CoV-2 infection. Beyond known host proteins, i.e., ACE2, TMPRSS2, and NRP1, we identified multiple host components, among which LDLRAD3, TMEM30A, and CLEC4G were confirmed as functional receptors for SARS-CoV-2. All these membrane proteins bind directly to spike's N-terminal domain (NTD). Their essential and physiological roles have been confirmed in either neuron or liver cells. In particular, LDLRAD3 and CLEC4G mediate SARS-CoV-2 entry and infection in an ACE2-independent fashion. The identification of the novel receptors and entry mechanisms could advance our understanding of the multiorgan tropism of SARS-CoV-2, and may shed light on the development of COVID-19 countermeasures.

Cross-reactive antibody against human coronavirus OC43 spike protein correlates with disease severity in COVID-19 patients: a retrospective study.

Seasonal human coronaviruses (HCoVs) including HCoV-229E, -OC43, -NL63, and -HKU1 widely spread in global human populations. However, the relevance of humoral response against seasonal HCoVs to COVID-19 pathogenesis is elusive. In this study, we profiled the temporal changes of IgG antibody against spike proteins (S-IgG) of SARS-CoV-2 and seasonal HCoVs in 838 plasma samples collected from 344 COVID-19 patients. We tested the antigenic cross-reactivities of S protein between SARS-CoV-2 and seasonal HCoVs and evaluated the correlations between the levels of HCoV-OC43 S-IgG and the disease severity in COVID-19 patients. We found that SARS-CoV-2 S-IgG titres mounted until days 22-28, whereas HCoV-OC43 antibody titres increased until days 15-21 and then plateaued until day 46. However, IgG titres against HCoV-NL63, -229E, and -HKU1 showed no significant increase. A two-way cross-reactivity was identified between SARS-CoV-2 and HCoV-OC43. Neutralizing antibodies against SARS-CoV-2 were not detectable in healthy controls who were positive for HCoV-OC43 S-IgG. HCoV-OC43 S-IgG titres were significantly higher in patients with severe disease than those in mild patients at days 1-21 post symptom onset (PSO). Higher levels of HCoV-OC43 S-IgG were also observed in patients requiring mechanical ventilation. At days 1-10 PSO, HCoV-OC43 S-IgG titres correlated to disease severity in the age group over 60. Our data indicate that there is a correlation between cross-reactive antibody against HCoV-OC43 spike protein and disease severity in COVID-19 patients.

SARS-CoV-2 nsp12 attenuates type I interferon production by inhibiting IRF3 nuclear translocation.

SARS-CoV-2 is the pathogenic agent of COVID-19, which has evolved into a global pandemic. Compared with some other respiratory RNA viruses, SARS-CoV-2 is a poor inducer of type I interferon (IFN). Here, we report that SARS-CoV-2 nsp12, the viral RNA-dependent RNA polymerase (RdRp), suppresses host antiviral responses. SARS-CoV-2 nsp12 attenuated Sendai virus (SeV)- or poly(I:C)-induced IFN-ß promoter activation in a dose-dependent manner. It also inhibited IFN promoter activation triggered by RIG-I, MDA5, MAVS, and IRF3 overexpression. Nsp12 did not impair IRF3 phosphorylation but suppressed the nuclear translocation of IRF3. Mutational analyses suggested that this suppression was not dependent on the polymerase activity of nsp12. Given these findings, our study reveals that SARS-CoV-2 RdRp can antagonize host antiviral innate immunity and thus provides insights into viral pathogenesis.

Corilagin inhibits SARS-CoV-2 replication by targeting viral RNA-dependent RNA polymerase.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has become one major threat to human population health. The RNA-dependent RNA polymerase (RdRp) presents an ideal target of antivirals, whereas nucleoside analogs inhibitor is hindered by the proofreading activity of coronavirus. Herein, we report that corilagin (RAI-S-37) as a non-nucleoside inhibitor of SARS-CoV-2 RdRp, binds directly to RdRp, effectively inhibits the polymerase activity in both cell-free and cell-based assays, fully resists the proofreading activity and potently inhibits SARS-CoV-2 infection with a low 50% effective concentration (EC50) value of 0.13 µmol/L. Computation modeling predicts that RAI-S-37 lands at the palm domain of RdRp and prevents conformational changes required for nucleotide incorporation by RdRp. In addition, combination of RAI-S-37 with remdesivir exhibits additive activity against anti-SARS-CoV-2 RdRp. Together with the current data available on the safety and pharmacokinetics of corilagin as a medicinal herbal agent, these results demonstrate the potential of being developed into one of the much-needed SARS-CoV-2 therapeutics.

Identification of Potent and Safe Antiviral Therapeutic Candidates Against SARS-CoV-2.

COVID-19 pandemic has infected millions of people with mortality exceeding >1 million. There is an urgent need to find therapeutic agents that can help clear the virus to prevent severe disease and death. Identifying effective and safer drugs can provide more options to treat COVID-19 infections either alone or in combination. Here, we performed a high throughput screening of approximately 1,700 US FDA-approved compounds to identify novel therapeutic agents that can effectively inhibit replication of coronaviruses including SARS-CoV-2. Our two-step screen first used a human coronavirus strain OC43 to identify compounds with anti-coronaviral activities. The effective compounds were then screened for their effectiveness in inhibiting SARS-CoV-2. These screens have identified 20 anti-SARS-CoV-2 drugs including previously reported compounds such as hydroxychloroquine, amlodipine besylate, arbidol hydrochloride, tilorone 2HCl, dronedarone hydrochloride, mefloquine, and thioridazine hydrochloride. Five of the newly identified drugs had a safety index (cytotoxic/effective concentration) of >600, indicating a wide therapeutic window compared to hydroxychloroquine which had a safety index of 22 in similar experiments. Mechanistically, five of the effective compounds (fendiline HCl, monensin sodium salt, vortioxetine, sertraline HCl, and salifungin) were found to block SARS-CoV-2 S protein-mediated cell fusion. These FDA-approved compounds can provide much needed therapeutic options that we urgently need during the midst of the pandemic.

Activation and evasion of type I interferon responses by SARS-CoV-2.

The pandemic of COVID-19 has posed an unprecedented threat to global public health. However, the interplay between the viral pathogen of COVID-19, SARS-CoV-2, and host innate immunity is poorly understood. Here we show that SARS-CoV-2 induces overt but delayed type-I interferon (IFN) responses. By screening 23 viral proteins, we find that SARS-CoV-2 NSP1, NSP3, NSP12, NSP13, NSP14, ORF3, ORF6 and M protein inhibit Sendai virus-induced IFN-ß promoter activation, whereas NSP2 and S protein exert opposite effects. Further analyses suggest that ORF6 inhibits both type I IFN production and downstream signaling, and that the C-terminus region of ORF6 is critical for its antagonistic effect. Finally, we find that IFN-ß treatment effectively blocks SARS-CoV-2 replication. In summary, our study shows that SARS-CoV-2 perturbs host innate immune response via both its structural and nonstructural proteins, and thus provides insights into the pathogenesis of SARS-CoV-2.