Heat shock protein 90 facilitates SARS-CoV-2 structural protein-mediated virion assembly and promotes virus-induced pyroptosis.

Inhibition of heat shock protein 90 (Hsp90), a prominent molecular chaperone, effectively limits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection but little is known about any interaction between Hsp90 and SARS-CoV-2 proteins. Here, we systematically analyzed the effects of the chaperone isoforms Hsp90α and Hsp90ß on individual SARS-CoV-2 viral proteins. Five SARS-CoV-2 proteins, namely nucleocapsid (N), membrane (M), and accessory proteins Orf3, Orf7a, and Orf7b were found to be novel clients of Hsp90ß in particular. Pharmacological inhibition of Hsp90 with 17-DMAG results in N protein proteasome-dependent degradation. Hsp90 depletion-induced N protein degradation is independent of CHIP, a ubiquitin E3 ligase previously identified for Hsp90 client proteins, but alleviated by FBXO10, an E3 ligase identified by subsequent siRNA screening. We also provide evidence that Hsp90 depletion may suppress SARS-CoV-2 assembly partially through induced M or N degradation. Additionally, we found that GSDMD-mediated pyroptotic cell death triggered by SARS-CoV-2 was mitigated by inhibition of Hsp90. These findings collectively highlight a beneficial role for targeting of Hsp90 during SARS-CoV-2 infection, directly inhibiting virion production and reducing inflammatory injury by preventing the pyroptosis that contributes to severe SARS-CoV-2 disease.

Repurposing niclosamide as a novel anti-SARS-Cov-2 drug by restricting entry protein CD147 (preprint)

Background The burst of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the global COVID-19 pandemic. But until today only limited numbers of drugs are discovered to treat COVID-19 patients. Even worse, the rapid mutations of SARS-CoV-2 compromise the effectiveness of existing vaccines and neutralizing antibodies due to the increased viral transmissibility and immune escape. CD147-spike protein, one of the entries of SRAR-CoV-2 into host cells, has been reported as a promising therapeutic target for developing drugs against COVID-19.Methods CRISPR-Cas9 induced gene knockout, western blotting, tet-off protein overexpression, ribonucleoprotein IP and RNA-IP were used to confirm the regulation of HuR on mRNA of CD147. Regulation of niclosamide on HuR nucleo-translocation was assessed by immunofluorescence staining of cell lines, IHC staining of tissue of mouse model and western blotting. Finally, the suppression of niclosamide on SARS-CoV-2 infection induced CD147 was evaluated by ACE2-expressing A549 cells and western blotting.Results We first discovered a novel regulation mechanism of CD147 via the RNA-binding protein HuR. We found that HuR regulates CD147 post-transcription by directly bound to its 3'-UTR. The loss of HuR reduced CD147 in multiple cell lines. Niclosamide inhibited CD147 function by blocking HuR cytoplasmic translocation and diminishing CD147 glycosylation. SARS-CoV-2 infection induced CD147 in ACE2-expressing A549 cells, which could be neutralized by niclosamide in a dose-dependent manner.Conclusion Together, our study reveals a novel regulation mechanism of CD147 and niclosamide can be repurposed as an effective COVID-19 drug by targeting the virus entry, CD147-spike protein.

Heparan sulfate promotes ACE2 super-cluster assembly to enhance SARS-CoV-2-associated syncytium formation (preprint)

The mechanism of syncytium formation, caused by spike-induced cell-cell fusion in severe COVID-19, is largely unclear. Here we combine chemical genetics with 4D confocal imaging to establish the cell surface heparan sulfate (HS) as a critical host factor exploited by SARS-CoV-2 to enhance spike’s fusogenic activity. HS binds spike to facilitate ACE2 clustering, generating synapse-like cell-cell contacts to promote fusion pore formation. ACE2 clustering, and thus, syncytium formation is significantly mitigated by chemical or genetic elimination of cell surface HS, while in a cell-free system consisting of purified HS, spike, and lipid-anchored ACE2, HS directly induces ACE2 clustering. Importantly, the interaction of HS with spike allosterically enables a conserved ACE2 linker in receptor clustering, which concentrates spike at the fusion site to overcome fusion-associated activity loss. This fusion-boosting mechanism can be effectively targeted by an investigational HS-binding drug, which reduces syncytium formation in vitro and viral infection in mice.

Isolation,identification, and genetic evolution analysis of variant strain CH-HK-2021 of G2a porcine epidemic diarrhea virus

Object: To explore genetic evolution relationship of variant porcine epidemic diarrhea virus(PEDV)and antigenic differential sites among variant strain subtypes,so as to lay a foundation for the development of novel vaccines and diagnostic kits. Method: Three PEDV-positive porcine intestinal samples were inoculated on to confluent Vero cells to isolate PEDV. Virus identification was performed by indirect fluorescence assay(IFA), Western blotting,RT-PCR and whole genome sequencing and electron microscopic observation;virus titer was determined by TCID50and the in vitvo proliferation dynamin curve of the virus was drawn. The genome of the isolated strain was divided into 33 segments for RT-PCR amplification, and the SeqMan of Lasergene was used to splice sequences. Then the genetic evolution analysis was performed with MEGA 7.0, and the antigenicity analysis was performed with Jameson-Wolf algorithm in Protean. Result: Typical cytopathic effect appeared in one PEDV-positive porcine intestinal sample in Vero cells when it was blindly passaged to the 6thgeneration and the sample was designated as CH-HK-2021. IFA and Western blotting results showed that the strain CH-HK-2021 could react with PEDV N monoclonal antibody and expected reads were obtained through RT-PCR amplification, which demonstrated this virus was PEDV. Diameter of strain CH-HK-2021 was 80-120 nm and the surface of the virus particles were in spike-like shape, indicating it was coronavirus. The strain could be stably propagated in Vero cells, and it has been passaged to 100thgeneration. After 24 h of infecting the Vero cells, virus titer of strain CH-HK-2021 reached the highest,105.6TCID50/mL. The size whole genome of strain CH-HK-2021 not including poly(A)tail was 28034 bp, with a similarity of 96.0%-98.9% with nucleotide sequence of the PEDV reference strain and a similarity of 93.1%-99.0% with S-base nucleotide sequence of the reference strain. The strain had the highest similarity with nucleotide sequence of variant strain CH/JX/01(KX058031)and the lowest similarity with nucleotide sequence of classical strain AVCT12(LC053455). Strain CH-HK-2021 was a subtype of G2a and it is spreading in China. Strain G2a and variant strain G2b had 42 nucleotide differential sites in S gene and 6 antigenic differential sites;and main differential sites located in subunit S2.

Chicken or Porcine Aminopeptidase N Mediates Cellular Entry of Pseudoviruses Carrying Spike Glycoprotein from the Avian Deltacoronaviruses HKU11, HKU13, and HKU17.

Members of deltacoronavirus (DCoV) have mostly been identified in diverse avian species as natural reservoirs, though the porcine DCoV (PDCoV) is a major swine enteropathogenic virus with global spread. The important role of aminopeptidase N (APN) orthologues from various mammalian and avian species in PDCoV cellular entry and interspecies transmission has been revealed recently. In this study, comparative analysis indicated that three avian DCoVs, bulbul DCoV HKU11, munia DCoV HKU13, and sparrow DCoV HKU17 (Chinese strain), and PDCoV in the subgenera Buldecovirus are grouped together at whole-genome levels; however, the spike (S) glycoprotein and its S1 subunit of HKU17 are more closely related to night heron DCoV HKU19 in Herdecovirus. Nevertheless, the S1 protein of HKU11, HKU13, or HKU17 bound to or interacted with chicken APN (chAPN) or porcine APN (pAPN) by flow cytometry analysis of cell surface expression of APN and by coimmunoprecipitation in APN-overexpressing cells. Expression of chAPN or pAPN allowed entry of pseudotyped lentiviruses with the S proteins from HKU11, HKU13 and HKU17 into nonsusceptible cells and natural avian and porcine cells, which could be inhibited by the antibody against APN or anti-PDCoV-S1. APN knockdown by siRNA or knockout by CRISPR/Cas9 in chicken or swine cell lines significantly or almost completely blocked infection of these pseudoviruses. Hence, we demonstrate that HKU11, HKU13, and HKU17 with divergent S genes likely engage chAPN or pAPN to enter the cells, suggesting a potential interspecies transmission from wild birds to poultry and from birds to mammals by certain avian DCoVs. IMPORTANCE The receptor usage of avian deltacoronaviruses (DCoVs) has not been investigated thus far, though porcine deltacoronavirus (PDCoV) has been shown to utilize aminopeptidase N (APN) as a cell receptor. We report here that chicken or porcine APN also mediates cellular entry by three avian DCoV (HKU11, HKU13, and HKU17) spike pseudoviruses, and the S1 subunit of three avian DCoVs binds to APN in vitro and in the surface of avian and porcine cells. The results fill the gaps in knowledge about the avian DCoV receptor and elucidate important insights for the monitoring and prevention of potential interspecies transmission of certain avian DCoVs. In view of the diversity of DCoVs, whether this coronavirus genus will cause novel virus to emerge in other mammals from birds, are worthy of further surveillance and investigation.

Breakthrough infection with Omicron subvariant BF.7 induces broadly neutralizing antibodies to SARS-CoV-2 variants including XBB lineage (preprint)

Since China eased its COVID-19 response strategies in late 2022, we have been witnessing a rapid and wide spread of SARS-CoV-2 infection across the major cities, including capital Beijing, where Omicron subvariant BF.7 has been dominating the infection. Here, we show that such expansion is unlikely due to a higher binding affinity of BF.7 to human receptor angiotensin-converting enzyme 2 (ACE2) as the similar binding activities were found for other Omicron subvariants tested such as BA.1, BA.5.2, BQ.1, BQ.1.1, XBB, and XBB.1. Additionally, through study of antibody response among six different clinical cohorts, we found that primary infection with BF.7 among the unvaccinated individuals only elicited type-specific neutralizing antibodies to the infecting virus and its close related strains. By a distinct contrast, breakthrough infection with BF.7 among the vaccinated individuals, particularly those severe cases, induced strong and broadly neutralizing antibodies to a diverse panel of SARS-CoV-2 variants and Omicron subvariants including the XBB lineage. A deeper understanding of how these broadly neutralizing antibodies were generated or boosted by BF.7 breakthrough infection will hold the key for augmenting antibody immunity against diverse SARS-CoV-2 variants.

A Generalized Multinomial Probabilistic Model for SARS-CoV-2 Infection Prediction and Public Health Intervention Assessment in an Indoor Environment (preprint)

SARS-CoV-2 Omicron has become the predominant variant globally. Current infection models are limited by the need for large datasets or calibration to specific contexts, making them difficult to cater for different settings. To ensure public health decision-makers can easily consider different public health interventions (PHIs) over a wide range of scenarios, we propose a generalized multinomial probabilistic model of airborne infection to systematically capture group characteristics, epidemiology, viral loads, social activities, environmental conditions, and PHIs, with assumptions made on social distancing and contact duration, and estimate infectivity over short time-span group gatherings. This study is related to our 2021 work published in Nature Scientific Reports that modelled airborne SARS-CoV-2 infection (Han, Lam, Li, et al., 2021). It is differentiated from former works on probabilistic infection modelling in terms of the following: (1) predicting new cases arising from more than one infectious in a gathering, (2) incorporating additional key infection factors, and (3) evaluating the effectiveness of multiple PHIs on SARS-CoV-2 infection simultaneously. Although our results reveal that limiting group size has an impact on infection, improving ventilation has a much greater positive health impact. Our model is versatile and can flexibly accommodate other scenarios by allowing new factors to be added, to support public health decision-making.

Depression and Anxiety among nursing students in the post COVID-19 pandemic in Inner Mongolia: An online cross-sectional survey (preprint)

Background: COVID-19 pandemic had considerable impacts on each aspect of worldwide, especially psychological disorders that would remain influenced in the post-pandemic era. Nursing students were also influenced by some special factors as facing unprecedented challenges. Objectives: The aim of this study was to evaluate the psychological status and explore the independent influencing factors of pandemic-related experiences, feelings, finance and protective behaviors among nursing students in Inner Mongolia Minzu University in post-COVID-19 era. Design: The online cross-sectional survey. Settings: Population-based study in China. Participants: Nursing undergraduate students (I-Ⅲyear). Methodology: It was conducted by WeChat platform from December 2021 to January 2022. The questionnaire included General demographic characteristics, Self-Rating Anxiety Scale (SAS) and Self-Rating Depression Scale (SDS). Cronbach's alpha, Bartlett's sphericity tests and KMO were tested the reliability and validity of scales. Descriptive analyses were completed by Mean and Standard Deviation. T-tests and ANOVA were conducted to test influencing factors. And general linear regression analyses were performed to identify the significant independent influencing factors of psychological disorders based on statistically significant results of univariate analysis. Results: 495 effective questionnaires were received. The prevalence of depression and anxiety disorders among participants was 14.7% and 9.1%, respectively. In our study, “parents with chronic illnesses”, “feeling very stressful due to the specialty of major”, “unstable family incomes” and “paying less attention to protective behaviors” had higher anxiety and depressive levels. Besides, “feeling fearful and unknown about the pandemic development” was significantly impacted on depression only. Conclusion: The findings obtained that depression was more prevalent among Chinese nursing students than anxiety in post-COVID-19 pandemic. It is essential to take appropriate measurements to alleviate psychological disorders by financial and family-related supports for medical-related students, in order to better respond to public health emergencies in further.

SARS-CoV-2 Omicron Variants Reduce Antibody Neutralization and Acquire Usage of Mouse ACE2

Striking number of mutations found in the spike protein of recently emerged SARS-CoV-2 Omicron subvariants BA.1, BA.2, BA.3 and BA.4/5 has raised serious concerns regarding the escape from current antibody therapies and vaccine protection. Here, we conducted comprehensive analysis on the extent of two major Omicron lineages BA.1/BA.1.1 and BA.2 to escape neutralization from the therapeutic antibodies approved by the regulatory authorities and convalescent plasma from SARS-CoV-2 patients infected during initial wave of pandemic in early 2020. We showed that Omicron BA.1/BA.1.1 were the most resistant in both magnitude and breadth against antibodies and convalescent plasma, followed by Beta, BA.2, Gamma, Delta and Alpha. While the majority of therapeutic antibodies lost binding and neutralization to Omicron variants, BRII combo (BRII-196 + BRII-198), S309, and AZ combo (COV2-2196 + COV2-2130) maintained neutralization despite of reduction due to either conserved epitope or combinational effect between the two designated antibodies. A single intraperitoneal injection of BRII combo as a prophylactic treatment protected animals from Omicron infection. Treated animals manifested normal body weight, survived infection up to 14 days, undetectable levels of infectious viruses in the lungs, and reduced lung pathology compared to the controls. Analyzing ACE2 from diverse host species showed that Omicron variants acquired ability to use mouse ACE2 for entry. These results demonstrate major antigenic shifts and potentially broadening the host range of two major Omicron lineages BA.1/BA.1.1 and BA.2, posing serious challenges to current antibody therapies and vaccine protection as well as increasing danger of spillover into the wildlife.

Broadly neutralizing and protective nanobodies against diverse sarbecoviruses (preprint)

As SARS-CoV-2 Omicron and other variants of concern continue spreading around the world, development of antibodies and vaccines to confer broad and protective activity is a global priority. Here, we report on the identification of a special group of nanobodies from immunized alpaca with exceptional breadth and potency against diverse sarbecoviruses including SARS-CoV-1, Omicron BA.1, and BA.2. Crystal structure analysis of one representative nanobody, 3-2A2-4, revealed a highly conserved epitope between the cryptic and the outer face of the receptor binding domain (RBD). The epitope is readily accessible regardless of RBD in up or down conformation and distinctive from the receptor ACE2 binding site. Passive delivery of 3-2A2-4 protected K18-hACE2 mice from infection of authentic SARS-CoV-2 Delta and Omicron. This group of nanobodies and the epitope identified should provide invaluable reference for the development of next generation antibody therapies and vaccines against wide varieties of SARS-CoV-2 infection and beyond.

First evidence that an emerging mammalian alphacoronavirus is able to infect an avian species.

A novel swine enteric alphacoronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), related to Rhinolophus bat CoV HKU2 in the subgenus Rhinacovirus emerged in southern China in 2017, causing diarrhoea in newborn piglets, and critical questions remain about the pathogenicity, cross-species transmission and potential animal reservoirs. Our laboratory's previous research has shown that SADS-CoV can replicate in various cell types from different species, including chickens. Here, we systematically explore the susceptibility of chickens to a cell-adapted SADS-CoV strain both in vitro and in vivo. First, evidence of SADS-CoV replication in primary chicken cells, including cytopathic effects, immunofluorescence staining, growth curves and structural protein expression, was proven. Furthermore, we observed that SADS-CoV replicated in chicken embryos without causing gross lesions and that experimental infection of chicks resulted in mild respiratory symptoms. More importantly, SADS-CoV shedding and viral distribution in the lungs, spleens, small intestines and large intestines of infected chickens were confirmed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The genomic sequence of the original SADS-CoV from the pig source sample in 2017 was determined to have nine nucleotide differences compared to the cell-adapted strain used; among these were three nonsynonymous mutations in the spike gene. These results collectively demonstrate that chickens are susceptible to SADS-CoV infection, suggesting that they are a potential animal reservoir. To our knowledge, this study provides the first experimental evidence of cross-species infection in which a mammalian alphacoronavirus is able to infect an avian species.

A cross-sectional study of the psychological status of 33,706 hospital workers at the late stage of the COVID-19 outbreak.

BACKGROUND: Hospital workers have been under intense psychological pressure since the COVID-19 outbreak. We analyzed the psychological status of hospital staff in the late period of the COVID-19 to provide a basis for the construction of global health care after the COVID-19 outbreak. METHODS: We used online surveys to assess participants' self-reported symptoms at the late stage of the outbreak. This study collected data on sociodemographic characteristics, epidemic-related factors, psychological status (PHQ-9, GAD-7, and PHQ-15), psychological assistance needs, perceived stress and support, PTSD symptoms (PCL-C) and suicidal and self-injurious ideation (SSI). Participants were hospital workers in all positions from 46 hospitals. Chi-square tests to compare the scales and logistic regression analysis were used to identify risk factors for PTSD and SSI. RESULTS: Among the 33,706 participants, the prevalences of depression, anxiety, somatic symptoms, PTSD symptoms, and SSI were 35.8%, 24.4%, 49.7%, 5.0%, and 1.3%, respectively. Logistic regression analysis showed that work in a general ward, attention to the epidemic, high education, work in non-first-line departments, insufficient social support, and anxiety and somatization symptoms were influencing factors of PTSD (P<0.05). The independent risk factors for SSI were female gender; psychological assistance needs; contact with severe COVID-19 patients; high stress at work; single or divorced marital status; insufficient social support; and depression, anxiety or PTSD symptoms (P<0.05). LIMITATIONS: This cross-sectional study could not reveal causality, and voluntary participation may have led to selection bias. The longer longitudinal studies are needed to determine the long-term psychological impact. CONCLUSION: This COVID-19 pandemic had a sustained, strong psychological impact on hospital workers, and hospital workers with PTSD symptoms were a high-risk group for SSI in the later period of the epidemic. Continuous attention and positive psychological intervention are of great significance for specific populations.

A Graph Convolutional Network-based screening strategy for rapid identification of SARS-CoV-2 cell-entry inhibitors (preprint)

The cell entry of SARS-CoV-2 has emerged as an attractive drug development target. We previously reported that the entry of SARS-CoV-2 depends on the cell surface heparan sulfate proteoglycan (HSPG) and the cortex actin, which can be targeted by therapeutic agents identified by conventional drug repurposing screens. However, this drug identification strategy requires laborious library screening, which is time-consuming and often limited number of compounds can be screened. As an alternative approach, we developed and trained a graph convolutional network (GCN)-based classification model using information extracted from experimentally identified HSPG and actin inhibitors. This method allowed us to virtually screen 170,000 compounds, resulting in ~2000 potential hits. A hit confirmation assay with the uptake of a fluorescently labeled HSPG cargo further shortlisted 256 active compounds. Among them, 16 compounds had modest to strong inhibitory activities against the entry of SARS-CoV-2 pseudotyped particles into Vero E6 cells. These results establish a GCN-based virtual screen workflow for rapid identification of new small molecule inhibitors against validated drug targets.

Identification and Characterization of MRNAs, lncRNAs, and MiRNAs Expression Profiles and ceRNA Networks in PEDV Infection (preprint)

Background: Long non-coding RNAs (LncRNAs) are transcripts longer than 200 nucleotides with no protein-coding ability and exert crucial effects on viral infection and host immune responses. Porcine Epidemic Diarrhea Virus (PEDV) is a coronavirus that seriously affects the swine industry. However, our understanding of the function of lncRNA involved in host-PEDV interaction is limited. Results: : A total of 1197 mRNA transcripts, 539 lncRNA transcripts, and 208 miRNA transcripts were differentially regulated at 24 h and 48 h post-infection. Moreover, gene ontology (GO) and KEGG pathway enrichment analysis showed that DE mRNAs and DE lncRNAs were mainly involved in biosynthesis, innate immunity, and lipid metabolism. Ten differentially expressed genes were randomly selected and validated by reverse-transcription qRT-PCR. In addition, we constructed a miRNA-mRNA-pathway network followed by a lncRNA-miRNA-mRNA ceRNA network. Conclusions: : The present study is the first to reveal the global expression profiles of mRNAs, lncRNAs, and miRNAs during PEDV infection. We comprehensively characterize the ceRNA networks which can provide new insights into the pathogenesis of PEDV.

Differentiation and treatment of COVID-19 with traditional Chinese medicine

Corona Virus Disease 2019(COVID-19) is highly infectious and has high mortality. There is no specific drug for it at present. Chinese medicine has achieved certain results in the treatment of this disease. COVID-19 can be classified as 'plague' in traditional Chinese medicine. It originates from the pestilential plagues and abnormal climates in the four seasons, and invade the body through mouth, nose and eyes. The disease position is mainly in the lung, may involve the membrane and spread throughout San Jiao(the Three Warmer). The pathogenesis can be characterized by 'warm-heat-humidity-toxin'. According to the clinical manifestations and the regular pattern of disease transmission, NCP can be divided into four stages, namely, the initial stage, the progressive stage, the critical stage and the recovery stage. The principles of syndrome differentiation, disease differentiation and constitution differentiation should be followed in the treatment. At the same time, we should also pay attention to the details of drug decocting, medication methods and medication duration.

Mitoxantrone modulates a glycosaminoglycan-spike complex to inhibit SARS-CoV-2 infection (preprint)

Spike-mediated entry of SARS-CoV-2 into human airway epithelial cells is an attractive therapeutic target for COVID-19. In addition to protein receptors, the SARS-CoV-2 spike (S) protein also interacts with heparan sulfate, a negatively charged glycosaminoglycan (GAG) attached to certain membrane proteins on the cell surface. This interaction facilitates the engagement of spike with a downstream receptor to promote viral entry. Here, we show that Mitoxantrone, an FDA-approved topoisomerase inhibitor, targets a spike-GAG complex to compromise the fusogenic function of spike in viral entry. As a single agent, Mitoxantrone inhibits the infection of an authentic SARS-CoV-2 strain in a cell-based model and in human lung EpiAirway 3D tissues. Gene expression profiling supports the plasma membrane as a major target of Mitoxantrone but also underscores an undesired activity targeting nucleosome dynamics. We propose that Mitoxantrone analogs bearing similar GAG-binding activities but with reduced affinity for DNA topoisomerase may offer an alternative therapy to overcome breakthrough infections in the post-vaccine era.