Deepening the Action on Salt Reduction in China-suggestions on strategy and implementation plan / 中华预防医学杂志

Excessive sodium/salt intake is the leading dietary risk factor for the loss of healthy life in the Chinese population. The "Healthy China 2030" Action Plan set the goal of reducing salt intake by 20% by 2030. However, salt intake in China is still at a very high level in the world, with adults reaching 11 g/d, more than twice the recommended limit of 5 g/d. The current policies and action plans of China have targeted catering workers, children, adolescents, and home chefs in salt, oil, and sugar reduction actions. However, there are still obvious deficiencies in the coordinated promotion and implementation. This study, therefore, proposed a set of comprehensive strategies (named CHRPS that is composed of communication and education, salt reduction in home cooking, salt reduction in restaurants, reducing salt content in pre-packaged food, and surveillance and evaluation) and key implementation points for further deepening the salt reduction action in China. These strategies were developed based on the main sources of dietary sodium for Chinese residents, the status of "knowledge, attitude and practice" in salt reduction, evidence of effective intervention measures, existing policies and requirements, and the salt reduction strategies of the World Health Organization and experience from some other countries. As a scientific reference, the CHRPS strategies will help the government and relevant organizations quickly implement salt reduction work and facilitate the earlier realization of China's salt reduction goal.

Burden of hemorrhagic stroke and hypertensive heart disease attributed to alcohol consumption in China / 中华流行病学杂志

Objective: To describe the prevalence of alcohol consumption and the burden of hemorrhagic stroke and hypertensive heart disease attributed to alcohol consumption in adults aged ≥20 years in 31 provinces in China from 2005 to 2018. Methods: Data from several national representative surveys was used to estimate provincial alcohol exposure level of adults aged ≥20 years from 2005 to 2018 by using kriging interpolation and locally weighted regression methods. Global disease burden research method and data, and China's death cause surveillance data were used to calculate the population attributable fraction (PAF) of hemorrhagic stroke and hypertensive heart disease and the deaths due to alcohol consumption in men and women aged ≥20 years in 31 provinces in China. China census data of 2010 were used to calculate the attributable standardized mortality rate. Results: In 2005 and 2018, the prevalence of alcohol consumption was 58.7% (95%CI: 57.8%-59.5%) and 58.4% (95%CI: 57.6%-59.3%), respectively, in men and 17.0% (95%CI: 16.6%-17.4%) and 18.7% (95%CI:18.1%-19.3%), respectively, in women. The daily alcohol intake was 24.6 (95%CI: 23.8-25.3) g and 27.7 (95%CI: 26.8-28.7) g, respectively, in men and 6.3 (95%CI: 6.0-6.5) g and 5.3 (95%CI: 5.0-5.6) g, respectively, in women. Alcohol exposure level was higher in the provinces in central and eastern China than in western provinces. The lowest exposure level was found in northwestern provinces. From 2005 to 2018, the PAF of hemorrhagic stroke death due to alcohol consumption increased from 5.5% to 6.8%, the attributable deaths increased from 50 200 to 59 100, while the PAF of hypertensive heart disease death due to alcohol consumption increased from 7.0% to 7.7%, the attributable deaths increased from 15 200 to 29 300. The PAF of hypertensive heart disease and hemorrhagic stroke was higher in men than in women, and in central and eastern provinces than in western provinces. In 2018, the standardized mortality rates of hemorrhagic stroke and hypertensive heart disease attributed to alcohol consumption were 4.58/100 000 and 2.11/100 000, respectively. Conclusions: The prevalence of alcohol consumption in men and daily alcohol intake of drinkers were relatively high in China, especially in eastern provinces. Alcohol exposure level was lower in women than in men. Regional measures should be taken to reduce the alcohol intakes in men and current drinkers in order to reduce the health problems caused by alcohol consumption.

Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

ACE2 is a major receptor for cell entry of SARS-CoV-2. Despite advances in targeting ACE2 to inhibit SARS-CoV-2s binding, how to efficiently and flexibly control ACE2 levels for prevention of SARS-CoV-2 infection has not been explored. Here, we revealed Vitamin C (VitC) administration as an effective strategy to prevent SARS-CoV-2 infection. VitC reduced ACE2 protein levels in a dose-dependent manner, while partial reduction of ACE2 can greatly restrict SARS-CoV-2 infection. Further studies uncovered that USP50 is a crucial regulator of ACE2 protein levels, and VitC blocks the USP50-ACE2 interaction, thus promoting K48-linked polyubiquitination at Lys788 and degradation of ACE2, without disrupting ACE2 transcriptional expression. Importantly, VitC administration reduced host ACE2 and largely blocked SARS-CoV-2 infection in mice. This study identified an in vivo ACE2 balance controlled by both USP50 and an essential nutrient VitC, and revealed a critical role and application of VitC in daily protection from SARS-CoV-2 infection. HighlightsO_LIVitC reduces ACE2 protein levels in a dose-dependent manner C_LIO_LIVitC and USP50 regulate K48-linked ubiquitination at Lys788 of ACE2 C_LIO_LIVitC blocks the interaction between USP50 and ACE2 C_LIO_LIVitC administration lowers host ACE2 and prevents SARS-CoV-2 infection in vivo C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/499651v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@196682borg.highwire.dtl.DTLVardef@190f14dorg.highwire.dtl.DTLVardef@d22b59org.highwire.dtl.DTLVardef@1c0faa_HPS_FORMAT_FIGEXP M_FIG C_FIG The deubiquitinase USP50 controls ACE2 protein stability and levels, while Vitamin C blocks the USP50-ACE2 interaction and therefore results in ACE2 degradation, offering a flexible and efficient approach to protection of the host from SARS-CoV-2 infection.

Harringtonine has the effects of double blocking SARS-CoV-2 membrane fusion

Fusion with host cell membrane is the main mechanism of infection of SARS-CoV-2. Here, we propose a new strategy to double block SARS-CoV-2 membrane fusion by using Harringtonine (HT), a small-molecule antagonist. By using cell membrane chromatography (CMC), we found that HT specifically targeted the SARS-CoV-2 S protein and host cell TMPRSS2, and then confirmed that HT can inhibit pseudotyped virus membrane fusion. Furthermore, HT successfully blocked SARS-CoV-2 infection, especially in the delta and Omicron mutant. Since HT is a small-molecule antagonist, it is minimally affected by the continuous variation of SARS-CoV-2. Our findings show that HT is a potential small-molecule antagonist with a new mechanism of action against SARS-CoV-2 infection, and thus HT mainly targets the S protein, and thus, greatly reduces the damage of the S proteins autotoxicity to the organ system, has promising advantages in the clinical treatment of COVID-19.

Retrograde intrarenal surgery with holmium laser lithotripsy for the management of calyceal diverticular calculi / 中华泌尿外科杂志

Objective:To investigate the efficacy and safety of retrograde intrarenal surgery(RIRS) with holmium laser lithotripsy in the management of calyceal diverticular calculi.Methods:A retrospective analysis was performed on 56 patients with calyceal diverticular calculi admitted to Huashan Hospital of Fudan University from January 2017 to May 2020. The 56 cases included 25 males and 31 females. The average age was 37.4 (ranging 22-67) years. Calyceal diverticular stones were located in the upper pole of the kidney in 32 cases, middle pole in 16 case and lower pole in 8 cases, with 32 cases on the left side and 24 cases on the right side. Multiple stones occurred in 46 cases, and single stone in 10 cases. The mean diameter of stones was 11.5 (ranging 3.0-17.5)mm. All 56 patients had different degrees of lumbar pain and/or hematuria preoperatively. Among them, 17 patients received extracorporeal shock wave lithotripsy (ESWL) with failure. Moreover, 9 cases suffered with urinary infection. All 56 patients with calyceal diverticular calculi underwent retrograde flexible ureterorenoscopic Ho: YAG laser lithotripsy under general anesthesia. The flexible ureterorenoscope was advanced into the kidney through the ureteral access sheath, looking for the cervical orifice of calyceal diverticulum. After finding renal diverticulum, holmium laser was used to incise and expand the neck or weak part of the diverticulum. The diverticular calculi were fragmented into particles less than 3 mm. Larger fragments were removed through a nitinol stone basket one by one. A F6 D-J stent was indwelled. The intraoperative conditions, postoperative complication rate and stone free rate were statistically analyzed.Results:The calyceal diverticular calculi in all 56 patients were discovered, and the diverticulum orifice were identified in 48 patients(85.7%). 53 of them underwent calyceal diverticular calculi fragmentation successfully. Lithotripsy failed in 3 cases, as the calculi were incarcerated in the lower pole calyceal diverticulum with a long narrow neck and the limitation of flexure at the end of the flexible ureteroscope. Two of them underwent percutaneous nephrolithotomy instead due to the calculi located in the posterior calyx. In another one case, ESWL was performed as the calculi located in the anterior calyx. Of the 17 cases received unsuccessful ESWL, RIRS was successful in 16 cases (94.1%). The mean operative time was 68.1(ranging 37-105)min, and mean hospitalization was 1.8 (ranging 1-3)d. The complication rate was 15.1%(8/53). All of these complications were mild (Clavien Ⅰ-Ⅱ). No serious complications such as perforation of the renal pelvis and ureter or major bleeding were occurred. After mean postoperative follow-up of 6.3(ranging 3-12) months, the stone-free rate was 83.0% (44/53) after the first procedure. 7 cases with residual stones ≥4mm received a second procedure. Among them, 6 cases received flexible ureterorenoscopy and the other one received ESWL and external physical vibration lithecbole therapy. The stone-free rate and symptom remission rate was 92.5% (49/53) and 96.2% (51/53) respectively after the second procedure, and no recurrence of calyceal diverticular calculi was observed during the stage of fllow-up.Conclusions:RIRS with holmium laser lithotripsy in the treatment of calyceal diverticular calculi, using the body's natural cavities, is a minimally invasive, safe and efficient strategy with slight complications. RIRS with holmium laser lithotripsy is an optional treatment for calyceal diverticular calculi.

An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1) and Mu (B.1.621). Furthermore, a heavy chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0x10-12 M) and strong neutralizing activity (IC50 = 0.0017 nM). Together, we developed a tetravalent biparatopic human heavy chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

Inhibition of SAR S-CoV-2 infection and replication by lactoferrin, MUC1 and α-lactalbumin identified in human breastmilk

The global pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection confers great threat to the public health. Human breastmilk is an extremely complex with nutritional composition to nourish infants and protect them from different kinds of infection diseases and also SARS-CoV-2 infection. Previous studies have found that breastmilk exhibited potent antiviral activity against SARS-CoV-2 infection. However, it is still unknown which component(s) in the breastmilk is responsible for its antiviral activity. Here, we identified Lactoferrin (LF), MUC1 and -Lactalbumin (-LA) from human breastmilk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and in vitro confirmation that inhibited SARS-CoV-2 infection and analyzed their antiviral activity using the SARS-CoV-2 pseudovirus system and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP) in the Huh7.5, Vero E6 and Caco-2-N cell lines. Additionally, we found that LF and MUC1 could inhibit viral attachment, entry and post-entry replication, while -LA just inhibit viral attachment and entry. Importantly, LF, MUC1 and -LA possess potent antiviral activities towards not only wild-type but also variants such as B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.1 (kappa). Moreover, LF from other species (e.g., bovine and goat) is still capable of blocking viral attachment to cellular heparan sulfate. Taken together, our study provided the first line of evidence that human breastmilk components (LF, MUC1 and -LA) are promising therapeutic candidates warranting further development or treatingVID-19 given their exceedingly safety levels.

Characterization of SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 on cell entry, host range, and sensitivity to convalescent plasma and ACE2 decoy receptor

Recently, highly transmissible SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 were identified in India with mutations within the spike proteins. The spike protein of Kappa contains four mutations E154K, L452R, E484Q and P681R, and Delta contains L452R, T478K and P681R, while B.1.618 spike harbors mutations {Delta}145-146 and E484K. However, it remains unknown whether these variants have altered in their entry efficiency, host tropism, and sensitivity to neutralizing antibodies as well as entry inhibitors. In this study, we found that Kappa, Delta or B.1.618 spike uses human ACE2 with no or slightly increased efficiency, while gains a significantly increased binding affinity with mouse, marmoset and koala ACE2 orthologs, which exhibits limited binding with WT spike. Furthermore, the P618R mutation leads to enhanced spike cleavage, which could facilitate viral entry. In addition, Kappa, Delta and B.1.618 exhibits a reduced sensitivity to neutralization by convalescent sera owning to the mutation of E484Q, T478K, {Delta}145-146 or E484K, but remains sensitive to entry inhibitors-ACE2-lg decoy receptor. Collectively, our study revealed that enhanced human and mouse ACE2 receptor engagement, increased spike cleavage and reduced sensitivity to neutralization antibodies of Kappa, Delta and B.1.618 may contribute to the rapid spread of these variants and expanded host range. Furthermore, our result also highlighted that ACE2-lg could be developed as broad-spectrum antiviral strategy against SARS-CoV-2 variants.

Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption

COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as "Cluster 5" that contained mutations in the spike protein. However, the functional properties of these "Cluster 5" mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.

Susceptibilities of human ACE2 genetic variants in coronavirus infection

The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in more than 1603 million cases worldwide and 3.4 million deaths (as of May 2021), with varying incidences and death rates among regions/ethnicities. Human genetic variation can affect disease progression and outcome, but little is known about genetic risk factors for SARS-CoV-2 infection. The coronaviruses SARS-CoV, SARS-CoV-2 and HCoV-NL63 all utilize the human protein angiotensin-converting enzyme 2 (ACE2) as the receptor to enter cells. We hypothesized that the genetic variability in ACE2 may contribute to the variable clinical outcomes of COVID-19. To test this hypothesis, we first conducted an in silico investigation of single-nucleotide polymorphisms (SNPs) in the coding region of ACE2 gene. We then applied an integrated approach of genetics, biochemistry and virology to explore the capacity of select ACE2 variants to bind coronavirus spike protein and mediate viral entry. We identified the ACE2 D355N variant that restricts the spike protein-ACE2 interaction and consequently limits infection both in vitro and in vivo. In conclusion, ACE2 polymorphisms could modulate susceptibility to SARS-CoV-2, which may lead to variable disease severity.

Analysis of viral RNA-host protein interactomes enables rapid antiviral drug discovery

RNA viruses including SARS-CoV-2, Ebola virus (EBOV), and Zika virus (ZIKV) constitute a major threat to global public health and society. The interactions between viral genomes and host proteins are essential in the life cycle of RNA viruses and thus provide targets for drug development. However, viral RNA-host protein interactions have remained poorly characterized. Here we applied ChIRP-MS to profile the interactomes of human proteins and the RNA genomes of SARS-CoV-2, EBOV, and ZIKV in infected cells. Integrated interactome analyses revealed interaction patterns that reflect both common and virus-specific host responses, and enabled rapid drug screening to target the vulnerable host factors. We identified Enasidenib as a SARS-CoV-2 specific antiviral agent, and Trifluoperazine and Cepharanthine as broad spectrum antivirals against all three RNA viruses. One Sentence SummaryInteractome analyses of host proteins and the SARS-CoV-2, EBOV, and ZIKV RNA genomes unveil viral biology and drug targets.

Spike mutations in SARS-CoV-2 variants confer resistance to antibody neutralization

New SARS-CoV-2 variants continue to emerge from the current global pandemic, some of which can replicate faster and with greater transmissibility and pathogenicity. In particular, UK501Y.V1 identified in UK, SA501Y.V2 in South Africa, and BR501Y.V3 in Brazil are raising serious concerns as they spread quickly and contain spike protein mutations that may facilitate escape from current antibody therapies and vaccine protection. Here, we constructed a panel of 28 SARS-CoV-2 pseudoviruses bearing single or combined mutations found in the spike protein of these three variants, as well as additional nine mutations that within or close by the major antigenic sites in the spike protein identified in the GISAID database. These pseudoviruses were tested against a panel of monoclonal antibodies (mAbs), including some approved for emergency use to treat SARS-CoV-2 infection, and convalescent patient plasma collected early in the pandemic. SA501Y.V2 pseudovirus was the most resistant, in magnitude and breadth, against mAbs and convalescent plasma, followed by BR501Y.V3, and then UK501Y.V1. This resistance hierarchy corresponds with Y144del and 242-244del mutations in the N-terminal domain as well as K417N/T, E484K and N501Y mutations in the receptor binding domain (RBD). Crystal structural analysis of RBD carrying triple K417N-E484K-N501Y mutations found in SA501Y.V2 bound with mAb P2C-1F11 revealed a molecular basis for antibody neutralization and escape. SA501Y.V2 and BR501Y.V3 also acquired substantial ability to use mouse and mink ACE2 for entry. Taken together, our results clearly demonstrate major antigenic shifts and potentially broadening the host range of SA501Y.V2 and BR501Y.V3, which pose serious challenges to our current antibody therapies and vaccine protection.

Analysis on Mechanism of Astragali Radix in Treating IgA Nephropathy Based on Network Pharmacology and in Vitro Cell Experiment / 中国实验方剂学杂志

Objective:To explore the multi-component， multi-target and multi-pathway mechanism of Astragali Radix against immunoglobulin A nephropathy （IgAN） by network pharmacology， aiming to provide evidence for its basic research and clinical application. Method:The active chemical components and targets of Astragali Radix and targets associated with IgAN were obtained by literature mining and GeneCards， Traditinal Chinese Medicine Integrated Database （TCMID）， Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） databases. Cytoscape 3.7.1 software was used to draw network interaction diagrams. The key targets of Astragali Radix against IgAN were searched by network topology. Gene ontology （GO） analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway analysis involved in the targets were analyzed by different packages in R programming language. On this basis， cell experiments <italic>in vitro</italic> were carried out to verify the activation effect of astragaloside Ⅳ on phosphatidylinositol 3-kinase/protein kinase B/tumor suppressor gene protein 53 （PI3K/Akt/p53） signaling pathway of human mesangial cells. Result:A total of 25 active components and 49 ingredient-disease targets of Astragali Radix were screened. The GO enrichment analysis included 84 items， which were related to nuclear hormone receptor binding， nuclear receptor activity， deoxyribonucleic acid binding transcriptional activation activity and other aspects. The KEGG pathway enrichment analysis included 88 KEGG pathways， which were closely related to PI3K/Akt signaling pathway， hypoxia inducible factor-1 （HIF-1） signaling pathway， advanced glycation end product/receptor of advanced glycation end product （AGE/RAGE） signaling pathway and others. Cell experiments <italic>in vitro </italic>confirmed that astragaloside Ⅳ could effectively inhibit the platelet derived growth factor-BB （PDGF-BB）-induced proliferation of human mesangial cells by regulating PI3K/Akt/p53 signaling pathway. Conclusion:The active ingredients of Astragali Radix may play a role in the treatment of IgAN by acting on targets and pathways related to apoptosis， oxidative stress， inflammation response and others， providing ideas and directions for the new drug development and mechanism study of IgAN.

Malnutrition in Relation with Dietary, Geographical, and Socioeconomic Factors among Older Chinese / 生物医学与环境科学（英文）

Objective@#Nutrition is closely related to the health of the elderly population. This study aimed to provide a comprehensive picture of the nutrition status of elderly Chinese and its related dietary, geographical, and socioeconomic factors.@*Methods@#A total of 13,987 ≥ 60-year-old persons from the 2010-2013 Chinese National Nutrition and Health Survey were included to evaluate various aspects of malnutrition, including underweight, overweight or obesity, and micronutrient inadequacy.@*Results@#Overall, the prevalence of obesity, overweight, and underweight was 12.4%, 34.8%, and 5.7%, respectively, with disparities both geographically and socioeconomically. The prevalence of underweight was higher among the older old (≥ 75 years), rural residents and those with low income, with low education status, and residing in undeveloped West areas. More than 75% of the elderly do not meet the Dietary Reference Intakes for vitamins A, B @*Conclusions@#Obesity epidemic, inadequacy of micronutrient intake, and high prevalence of underweight and anemia in susceptible older people are the major nutrition challenges for the rapidly aging population in China.

A novel cell culture system modeling the SARS-CoV-2 life cycle

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19, and no effective antiviral agents and vaccines are available. SARS-CoV-2 is classified as a biosafety level-3 (BLS-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2-GFP/{Delta}N trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. The single mutation that allowed for mouse ACE2 to serve as a viral receptor provides a potential avenue for the development of SARS-CoV-2 mouse model.

The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission

The global spread of SARS-CoV-2 is posing major public health challenges. One unique feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site, the function of which remains uncertain. We found that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site instead utilizes a less efficient endosomal entry pathway. This idea was supported by the identification of a suite of endosomal entry factors specific to Sdel virus by a genome-wide CRISPR-Cas9 screen. A panel of host factors regulating the surface expression of ACE2 was identified for both viruses. Using a hamster model, animal-to-animal transmission with the Sdel virus was almost completely abrogated, unlike with Sfull. These findings highlight the critical role of the S1/S2 boundary of the SARS-CoV-2 spike protein in modulating virus entry and transmission.

In vivo structural characterization of the whole SARS-CoV-2 RNA genome identifies host cell target proteins vulnerable to re-purposed drugs

SUMMARYSARS-CoV-2 is an RNA virus of the Coronaviridae family that is the causal pathogen of the ongoing Coronavirus Disease 2019 pandemic. There are currently no antiviral drugs or vaccines to treat COVID-19, and the failure to identify effective interventions can be blamed on our incomplete understanding of the nature of this virus and its host cell infection process. Here, we experimentally determined structural maps of the SARS-CoV-2 RNA genome in infected human cells and also characterized in vitro refolded RNA structures for SARS-CoV-2 and 6 other coronaviruses. Our in vivo data confirms several structural elements predicted from theoretical analysis and goes much further in revealing many previously unknown structural features that functionally impact viral translation and discontinuous transcription in cells. Importantly, we harnessed our in vivo structure data alongside a deep-learning tool and accurately predicted several dozen functionally related host cell proteins that bind to the SARS-CoV-2 RNA genome, none of which were known previously. Thus, our in vivo structural study lays a foundation for coronavirus RNA biology and indicates promising directions for the rapid development of therapeutics to treat COVID-19.HIGHLIGHTSWe mapped the in vivo structure and built secondary structural models of the SARS-CoV-2 RNA genomeWe discovered functionally impactful structural features in the RNA genomes of multiple coronavirusesWe predicted and validated host cell proteins that bind to the SARS-CoV-2 RNA genome based on our in vivo RNA structural data using a deep-learning toolCompeting Interest StatementThe authors have declared no competing interest.View Full Text

Broad-spectrum virucidal activity of bacterial secreted lipases against flaviviruses, SARS-CoV-2 and other enveloped viruses

Viruses are the major aetiological agents of acute and chronic severe human diseases that place a tremendous burden on global public health and economy; however, for most viruses, effective prophylactics and therapeutics are lacking, in particular, broad-spectrum antiviral agents. Herein, we identified 2 secreted bacterial lipases from a Chromobacterium bacterium, named Chromobacterium antiviral effector-1 (CbAE-1) and CbAE-2, with a broad-spectrum virucidal activity against dengue virus (DENV), Zika virus (ZIKV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV) and herpes simplex virus (HSV). The CbAEs potently blocked viral infection in the extracellular milieu through their lipase activity. Mechanistic studies showed that this lipase activity directly disrupted the viral envelope structure, thus inactivating infectivity. A mutation of CbAE-1 in its lipase motif fully abrogated the virucidal ability. Furthermore, CbAE-2 presented low toxicity in vivo and in vitro, highlighting its potential as a broad-spectrum antiviral drug.

Functional and Genetic Analysis of Viral Receptor ACE2 Orthologs Reveals Broad Potential Host Range of SARS-CoV-2

The pandemic of Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major global health threat. Epidemiological studies suggest that bats are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. It has been shown that human, palm civet, pig and bat ACE2 can support virus entry, while the murine ortholog cannot. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. We found that ACE2 is expressed in a wide range of species, with especially high conservation in mammals. By analyzing amino acid residues of ACE2 critical for virus entry, based on structure of SARS-CoV spike protein interaction with human, bat, palm civet, pig and ferret ACE2, we identified approximately eighty ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 representative ACE2 orthologs among eighty orthologs for functional analysis and it showed that 44 of these mammalian ACE2 orthologs, including those of domestic animals, pets, livestock, and animals commonly found in zoos and aquaria, could bind SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. In summary, our study demonstrates that ACE2 from a remarkably broad range of species can facilitate SARS-CoV-2 entry. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.