Inferring Mycobacterium Tuberculosis Drug Resistance and Transmission using Whole-genome Sequencing in a High TB-burden Setting in China

Objective China is among the 30 countries with a high burden of tuberculosis(TB)worldwide,and TB remains a public health concern.Kashgar Prefecture in the southern Xinjiang Autonomous Region is considered as one of the highest TB burden regions in China.However,molecular epidemiological studies of Kashgar are lacking.Methods A population-based retrospective study was conducted using whole-genome sequencing(WGS)to determine the characteristics of drug resistance and the transmission patterns.Results A total of 1,668 isolates collected in 2020 were classified into lineages 2(46.0%),3(27.5%),and 4(26.5%).The drug resistance rates revealed by WGS showed that the top three drugs in terms of the resistance rate were isoniazid(7.4%,124/1,668),streptomycin(6.0%,100/1,668),and rifampicin(3.3%,55/1,668).The rate of rifampicin resistance was 1.8%(23/1,290)in the new cases and 9.4%(32/340)in the previously treated cases.Known resistance mutations were detected more frequently in lineage 2 strains than in lineage 3 or 4 strains,respectively:18.6%vs.8.7 or 9%,P<0.001.The estimated proportion of recent transmissions was 25.9%(432/1,668).Multivariate logistic analyses indicated that sex,age,occupation,lineage,and drug resistance were the risk factors for recent transmission.Despite the low rate of drug resistance,drug-resistant strains had a higher risk of recent transmission than the susceptible strains(adjusted odds ratio,1.414;95%CI,1.023–1.954;P=0.036).Among all patients with drug-resistant tuberculosis(DR-TB),78.4%(171/218)were attributed to the transmission of DR-TB strains.Conclusion Our results suggest that drug-resistant strains are more transmissible than susceptible strains and that transmission is the major driving force of the current DR-TB epidemic in Kashgar.

Origin,virological features,immune evasion and intervention of SARS-CoV-2 Omicron sublineages

Recently,a large number of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)variants continuously emerged and posed a major threat to global public health.Among them,particularly,Omicron variant(B.1.1.529),first identified in November 2021,carried numerous mutations in its spike protein(S),and then quickly spread around the world.Currently,Omicron variant has expanded into more than one hundred sublineages,such as BA.1,BA.2,BA.2.12.1,BA.4 and BA.5,which have already become the globally dominant variants.Different from other variants of concern(VOCs)of SARS-CoV-2,the Omicron variant and its sublineages exhibit increased transmissibility and immune escape from neutralizing antibodies generated through previous infection or vaccination,and have caused numerous re-infections and breakthrough infections.In this prospective,we have focused on the origin,virological features,immune evasion and intervention of Omicron sublineages,which will benefit the development of nextgeneration vaccines and therapeutics,including pan-sarbecovirus and universal anti-CoV therapeutics,to combat currently circulating and future emerging Omicron sublineages as well as other SARS-CoV-2 variants.

Molecular mechanism of interaction between SARS-CoV-2 and host cells and interventional therapy

The pandemic of coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection has resulted in an unprecedented setback for global economy and health.SARS-CoV-2 has an exceptionally high level of transmissibility and extremely broad tissue tropism.However,the underlying molecular mechanism responsible for sustaining this degree of virulence remains largely unexplored.In this article,we review the current knowledge and crucial information about how SARS-CoV-2 attaches on the surface of host cells through a variety of receptors,such as ACE2,neuropilin-1,AXL,and antibody-FcyR complexes.We further explain how its spike(S)protein undergoes conformational transition from prefusion to postfusion with the help of proteases like furin,TMPRSS2,and cathepsins.We then review the ongoing experimental studies and clinical trials of antibodies,peptides,or small-molecule compounds with anti-SARS-CoV-2 activity,and discuss how these antiviral therapies targeting host-pathogen interaction could potentially suppress viral attachment,reduce the exposure of fusion peptide to curtail membrane fusion and block the formation of six-helix bundle(6-HB)fusion core.Finally,the specter of rapidly emerging SARS-CoV-2 variants deserves a serious review of broad-spectrum drugs or vaccines for long-term prevention and control of COVID-19 in the future.

The current emergence of monkeypox:The recurrence of another smallpox?

Since its first confirmation in London on 12 May 2022,many monkeypox cases have been reported worldwide.Noticeably,the epidemiology,pathology,and clinical features of the current emergence have been compared to those of smallpox,a severe contagious disease historically epidemic worldwide for nearly 3,000 years.However,some characteristics of the present outbreak differed from those of previous monkeypox outbreaks.Herein,we ask if this emergence of monkeypox could cause another global pandemic similar to smallpox or influenza or if it is only the re-emergence of a new strain.To address these questions,we reviewed its virology,transmission,clinical characteristics,experimental diagnosis,and prevention and intervention,giving our commentary along the way.

Structure-based design of pan-coronavirus inhibitors targeting host cathepsin L and calpain-1

Respiratory disease caused by coronavirus infection remains a global health crisis.Although several SARS-CoV-2-specific vaccines and direct-acting antivirals are available,their effcacy on emerging coronaviruses in the future,including SARS-CoV-2 variants,might be compromised.Host-targeting antivirals provide preventive and therapeutic strategies to overcome resistance and manage future outbreak of emerging coronaviruses.Cathepsin L(CTSL)and calpain-1(CAPN1)are host cysteine proteases which play crucial roles in coronaviral entrance into cells and infection-related immune response.Here,two peptidomimetic a-ketoamide compounds,14a and 14b,were identified as potent dual target inhibitors against CTSL and CAPN1.The X-ray crystal structures of human CTSL and CAPN1 in complex with 14a and 14b revealed the covalent binding of a-ketoamide groups of 14a and 14b to C25 of CTSL and C115 of CAPN1.Both showed potent and broad-spectrum anticoronaviral activities in vitro,and it is worth noting that they exhibited low nanomolar potency against SARS-CoV-2 and its variants of concern(VOCs)with ECso values ranging from 0.80 to 161.7 nM in various cells.Preliminary mechanistic exploration indicated that they exhibited anticoronaviral activity through blocking viral entrance.Moreover,14a and 14b exhibited good oral pharmacokinetic properties in mice,rats and dogs,and favorable safety in mice.In addition,both 14a and 14b treatments demonstrated potent antiviral potency against SARS-CoV-2 XBB 1.16 variant infection in a K18-hACE2 transgenic mouse model.And 14b also showed effective antiviral activity against HCoV-OC43 infection in a mouse model with a final survival rate of 60%.Further evaluation showed that 14a and 14b exhibited excellent antiinflammatory effects in Raw 264.7 mouse macrophages and in mice with acute pneumonia.Taken together,these results suggested that 14a and 14b are promising drug candidates,providing novel insight into developing pan-coronavirus inhibitors with antiviral and anti-inflammatory properties.

Optimized RNA interference therapeutics combined with interleukin-2 mRNA for treating hepatitis B virus infection

This study aimed to develop a pan-genotypic and multifunctional small interfering RNA(siRNA)against hepatitis B virus(HBV)with an efficient delivery system for treating chronic hepatitis B(CHB),and explore combined RNA interference(RNAi)and immune modulatory modalities for better viral control.Twenty synthetic siRNAs targeting consensus motifs distributed across the whole HBV genome were designed and evaluated.The lipid nanoparticle(LNP)formulation was optimized by adopting HO-PEG_(2000)-DMG lipid and modifying the molar ratio of traditional polyethylene glycol(PEG)lipid in LNP prescriptions.The efficacy and safety of this formulation in delivering siHBV(tLNP/siHBV)along with the mouse IL-2(mIL-2)mRNA(tLNP/siHBVIL2)were evaluated in the rAAVHBV1.3 mouse model.A siRNA combination(terms“siHBV”)with a genotypic coverage of 98.55%was selected,chemically modified,and encapsulated within an optimized LNP(tLNP)of high efficacy and security to fabricate a therapeutic formulation for CHB.The results revealed that tLNP/siHBV significantly reduced the expression of viral antigens and DNA(up to 3log_(10)reduction;vs PBS)in dose-and time-dependent manners at single-dose or multi-dose frequencies,with satisfactory safety profiles.Further studies showed that tLNP/siHBVIL2 enables additive antigenic and immune control of the virus,via introducing potent HBsAg clearance through RNAi and triggering strong HBV-specific CD4^(+)and CD8^(+)T cell responses by expressed mIL-2 protein.By adopting tLNP as nucleic acid nanocarriers,the co-delivery of siHBV and mIL-2 mRNA enables synergistic antigenic and immune control of HBV,thus offering a promising translational therapeutic strategy for treating CHB.

Hepatitis B virus core protein as a Rab-GAP suppressor driving liver disease progression

Chronic hepatitis B virus(HBV)infection can lead to advanced liver pathology.Here,we establish a transgenic murine model expressing a basic core promoter(BCP)-mutated HBV genome.Unlike previous studies on the wild-type virus,the BCP-mutated HBV transgenic mice manifest chronic liver injury that culminates in cirrhosis and tumor development with age.Notably,agonistic anti-Fas treatment induces fulminant hepatitis in these mice even at a negligible dose.As the BCP mutant exhibits a striking increase in HBV core protein(HBc)expression,we posit that HBc is actively involved in hepatocellular injury.Accordingly,HBc interferes with Fis1-stimulated mitochondrial recruitment of Tre-2/Bub2/Cdc16 domain family member 15(TBC1D15).HBc may also inhibit multiple Rab GTPase-activating proteins,including Rab7-specific TBC1D15 and TBC1D5,by binding to their conserved catalytic domain.In cells under mitochondrial stress,HBc thus perturbs mitochondrial dynamics and prevents the recycling of damaged mitochondria.Moreover,sustained HBc expression causes lysosomal consumption via Rab7 hyperactivation,which further hampers late-stage autophagy and substantially increases apoptotic cell death.Finally,we show that adenovirally expressed HBc in a mouse model is directly cytopathic and causes profound liver injury,independent of antigen-specific immune clearance.These findings reveal an unexpected cytopathic role of HBc,making it a pivotal target for HBV-associated liver disease treatment.The BCP-mutated HBV transgenic mice also provide a valuable model for understanding chronic hepatitis B progression and for the assessment of therapeutic strategies.

Mutations in the SARS-CoV-2 spike receptor binding domain and their delicate balance between ACE2 affinity and antibody evasion

Intensive selection pressure constrains the evolutionary trajectory of SARS-CoV-2 genomes and results in various novel variants with distinct mutation profiles.Point mutations,particularly those within the receptor binding domain(RBD)of SARS-CoV-2 spike(S)protein,lead to the functional alteration in both receptor engagement and monoclonal antibody(mAb)recognition.Here,we review the data of the RBD point mutations possessed by major SARS-CoV-2 variants and discuss their individual effects on ACE2 affinity and immune evasion.Many single amino acid substitutions within RBD epitopes crucial for the antibody evasion capacity may conversely weaken ACE2 binding affinity.However,this weakened effect could be largely compensated by specific epistatic mutations,such as N501Y,thus maintaining the overall ACE2 affinity for the spike protein of all major variants.The predominant direction of SARS-CoV-2 evolution lies neither in promoting ACE2 affinity nor evading mAb neutralization but in maintaining a delicate balance between these two dimensions.Together,this review interprets how RBD mutations efficiently resist antibody neutralization and meanwhile how the affinity between ACE2 and spike protein is maintained,emphasizing the significance of comprehensive assessment of spike mutations.

gp120-derived amyloidogenic peptides form amyloid fibrils that increase HIV-1 infectivity

Apart from mediating viral entry,the function of the free HIV-1 envelope protein(gp120)has yet to be elucidated.Our group previously showed that EP2 derived from oneβ-strand in gp120 can form amyloid fibrils that increase HIV-1 infectivity.Importantly,gp120 contains~30β-strands.We examined whether gp120 might serve as a precursor protein for the proteolytic release of amyloidogenic fragments that form amyloid fibrils,thereby promoting viral infection.Peptide array scanning,enzyme degradation assays,and viral infection experiments in vitro confirmed that manyβ-stranded peptides derived from gp120 can indeed form amyloid fibrils that increase HIV-1 infectivity.These gp120-derived amyloidogenic peptides,or GAPs,which were confirmed to form amyloid fibrils,were termed gp120-derived enhancers of viral infection(GEVIs).GEVIs specifically capture HIV-1 virions and promote their attachment to target cells,thereby increasing HIV-1 infectivity.Different GAPs can cross-interact to form heterogeneous fibrils that retain the ability to increase HIV-1 infectivity.GEVIs even suppressed the antiviral activity of a panel of antiretroviral agents.Notably,endogenous GAPs and GEVIs were found in the lymphatic fluid,lymph nodes,and cerebrospinal fluid(CSF)of AIDS patients in vivo.Overall,gp120-derived amyloid fibrils might play a crucial role in the process of HIV-1 infectivity and thus represent novel targets for anti-HIV therapeutics.

Asialoglycoprotein receptor 1 promotes SARS-CoV-2 infection of human normal hepatocytes

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)causes multi-organ damage,which includes hepatic dysfunction,as observed in over 50%of COVID-19 patients.Angiotensin I converting enzyme(peptidyl-dipeptidase A)2(ACE2)is the primary receptor for SARS-CoV-2 entry into host cells,and studies have shown the presence of intracellular virus particles in human hepatocytes that express ACE2,but at extremely low levels.Consequently,we asked if hepatocytes might express receptors other than ACE2 capable of promoting the entry of SARS-CoV-2 into cells.To address this question,we performed a genome-wide CRISPR-Cas9 activation library screening and found that Asialoglycoprotein receptor 1(ASGR1)promoted SARS-CoV-2 pseudovirus infection of HeLa cells.In Huh-7 cells,simultaneous knockout of ACE2 and ASGR1 prevented SARS-CoV-2 pseudovirus infection.In the immortalized THLE-2 hepatocyte cell line and primary hepatic parenchymal cells,both of which barely expressed ACE2,SARSCoV-2 pseudovirus could successfully establish an infection.However,after treatment with ASGR1 antibody or siRNA targeting ASGR1,the infection rate significantly dropped,suggesting that SARS-CoV-2 pseudovirus infects hepatic parenchymal cells mainly through an ASGR1-dependent mechanism.We confirmed that ASGR1 could interact with Spike protein,which depends on receptor binding domain(RBD)and N-terminal domain(NTD).Finally,we also used Immunohistochemistry and electron microscopy to verify that SARS-CoV-2 could infect primary hepatic parenchymal cells.After inhibiting ASGR1 in primary hepatic parenchymal cells by siRNA,the infection efficiency of the live virus decreased significantly.Collectively,these findings indicate that ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of hepatic parenchymal cells.

Characterization of the receptor-binding domain(RBD)of 2019 novel coronavirus:implication for development of RBD protein as a viral attachment inhibitor and vaccine

The outbreak of Coronavirus Disease 2019(COVID-19)has posed a serious threat to global public health,calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent,severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),also known as 2019 novel coronavirus(2019-nCoV).The CoV spike(S)protein plays the most important roles in viral attachment,fusion and entry,and serves as a target for development of antibodies,entry inhibitors and vaccines.Here,we identified the receptor-binding domain(RBD)in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2(ACE2)receptors.SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and,hence,attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells,thus inhibiting their infection to host cells.SARS-CoV RBD-specific antibodies could crossreact with SARS-CoV-2 RBD protein,and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2,suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.

The First Disease X is Caused by a Highly Transmissible Acute Respiratory Syndrome Coronavirus

Based on the announcement of the World Health Organization(WHO) in 2018, the Wuhan pneumonia caused by an unknown etiology should be recognized as the first Disease X. Later, the pathogen was identified to be a novel coronavirus denoted 2019-nCoV, which has 79.5% and 96% whole genome sequence identify to SARS-CoV and bat SARS-related coronavirus(SARSr-CoV-RaTG13), respectively, suggesting its potential bat origin. With high human-to-human transmission rate(R0), 2019-nCoV has quickly spread in China and other countries, resulting in 34,953 confirmed cases and 725 deaths as of 8 February 2020, thus calling for urgent development of therapeutics and prophylactics. Here we suggest renaming 2019-nCoV as "transmissible acute respiratory syndrome coronavirus(TARS-CoV)" and briefly review the advancement of research and development of neutralizing antibodies and vaccines targeting the receptor-binding domain(RBD) and viral fusion inhibitors targeting the heptad repeat 1(HR1) domain in spike protein of 2019-nCoV.

Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein

Very recently,a novel coronavirus,2019-nCoV,emerged in Wuhan,China and then quickly spread worldwide,resulting in>17,388 confirmed cases and 361 deaths as of 3 February 2020,thus calling for the development of safe and effective therapeutics and prophylatics.1,2 Similar to severe acute respiratory syndrome(SARS)-CoV,2019-nCoV belongs to lineage B betacoronavirus,and it has the ability to utilize human angiotensin-converting enzyme 2(ACE2)as a receptor to infect human cells.

RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response

The pandemic of coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has posed serious threats to global health and economy,thus calling for the development of safe and effective vaccines.The receptorbinding domain(RBD)in the spike protein of SARS-CoV-2 is responsible for its binding to angiotensin-converting enzyme 2(ACE2)receptor.It contains multiple dominant neutralizing epitopes and serves as an important antigen for the development of COVID-19 vaccines.Here,we showed that immunization of mice with a candidate subunit vaccine consisting of SARS-CoV-2 RBD and Fc fragment of human IgG,as an immunopotentiator,elicited high titer of RBD-specific antibodies with robust neutralizing activity against both pseudotyped and live SARS-CoV-2 infections.The mouse antisera could also effectively neutralize infection by pseudotyped SARS-CoV-2 with several natural mutations in RBD and the IgG extracted from the mouse antisera could also show neutralization against pseudotyped SARS-CoV and SARS-related coronavirus(SARSr-CoV).Vaccination of human ACE2 transgenic mice with RBD-Fc could effectively protect mice from the SARS-CoV-2 challenge.These results suggest that SARS-CoV-2 RBD-Fc has good potential to be further developed as an effective and broad-spectrum vaccine to prevent infection of the current SARS-CoV-2 and its mutants,as well as future emerging SARSr-CoVs and re-emerging SARS-CoV.

A highly potent and stable pan-coronavirus fusion inhibitor as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases

The development of broad-spectrum antivirals against human coronaviruses(HCoVs)is critical to combat the current coronavirus disease 2019(COVID-19)pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and its variants,as well as future outbreaks of emerging CoVs.We have previously identified a polyethylene glycol-conjugated(PEGylated)lipopeptide,EK1C4,with potent pan-CoV fusion inhibitory activity.However,PEG linkers in peptide or protein drugs may reduce stability or induce anti-PEG antibodies in vivo.Therefore,we herein report the design and synthesis of a series of dePEGylated lipopeptide-based pan-CoV fusion inhibitors featuring the replacement of the PEG linker with amino acids in the heptad repeat 2 C-terminal fragment(HR2-CF)of HCoV-OC43.Among these lipopeptides,EKL1C showed the most potent inhibitory activity against infection by SARS-CoV-2 and its spike(S)mutants,as well as other HCoVs and some bat SARS-related coronaviruses(SARSr-CoVs)tested.The dePEGylated lipopeptide EKL1C exhibited significantly stronger resistance to proteolytic enzymes,better metabolic stability in mouse serum,higher thermostability than the PEGylated lipopeptide EK1C4,suggesting that EKL1C could be further developed as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases.

Recent advances in developing small-molecule inhibitors against SARS-CoV-2

The COVID-19 pandemic caused by the novel SARS-CoV-2 virus has caused havoc across the entire world.Even though several COVID-19 vaccines are currently in distribution worldwide,with others in the pipeline,treatment modalities lag behind.Accordingly,researchers have been working hard to understand the nature of the virus,its mutant strains,and the pathogenesis of the disease in order to uncover possible drug targets and effective therapeutic agents.As the research continues,we now know the genome structure,epidemiological and clinical features,and pathogenic mechanism of SARS-CoV-2.Here,we summarized the potential therapeutic targets involved in the life cycle of the virus.On the basis of these targets,small-molecule prophylactic and therapeutic agents have been or are being developed for prevention and treatment of SARS-CoV-2 infection.

The role of furin cleavage site in SARS-CoV-2 spike protein-mediated membrane fusion in the presence or absence of trypsin

Dear Editor,The rapid spread of SARS-CoV-2(also known as 2019-nCoV and HCoV-191),a novel lineage B betacoronavirus(βCoV),has caused a global pandemic of coronavirus disease(COVID-19).It has been speculated that RRAR,a unique furin-like cleavage site(FCS)in the spike protein(S),which is absent in other lineage BβCoVs,such as SARS-CoV,is responsible for its high infectivity and transmissibility.

Salvianolic acid C potently inhibits SARS-CoV-2 infection by blocking the formation of six-helix bundle core of spike protein

Dear Editor,The pandemic of COVID-19 caused by SARS-CoV-2 infection has posed a serious threat to global public health and the economy.Up to now,although several potentially effective antiviral drugs are under evaluating in clinical trials around the world,1 there are still no specific antiviral countermeasures beyond supportive therapies have been established.We herein report that the hydrophilic compound Salvianolic acid C(Sal-C)from Danshen,a traditional Chinese medicine(TCM),potently inhibit SARS-CoV-2 infection by blocking the formation of six-helix bundle(6-HB)core of spike(S)protein.

Development of oncolytic virotherapy:from genetic modification to combination therapy

Oncolytic virotherapy(OVT)is a novel form of immunotherapy using natural or genetically modified viruses to selectively replicate in and kill malignant cells.Many genetically modified oncolytic viruses(OVs)with enhanced tumor targeting,antitumor efficacy,and safety have been generated,and some of which have been assessed in clinical trials.Combining OVT with other immunotherapies can remarkably enhance the antitumor efficacy.In this work,we review the use of wild-type viruses in OVT and the strategies for OV genetic modification.We also review and discuss the combinations of OVT with other immunotherapies.

Antibody Cocktail Exhibits Broad Neutralization Activity Against SARS-CoV-2 and SARS-CoV-2 Variants

Severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)has precipitated multiple variants resistant to therapeutic antibodies.In this study,12 high-affinity antibodies were generated from convalescent donors in early outbreaks using immune antibody phage display libraries.Of them,two RBD-binding antibodies(F61 and H121)showed high-affinity neutralization against SARS-Co V-2,whereas three S2-target antibodies failed to neutralize SARS-Co V-2.Following structure analysis,F61 identified a linear epitope located in residues G446–S494,which overlapped with angiotensinconverting enzyme 2(ACE2)binding sites,while H121 recognized a conformational epitope located on the side face of RBD,outside from ACE2 binding domain.Hence the cocktail of the two antibodies achieved better performance of neutralization to SARS-Co V-2.Importantly,these two antibodies also showed efficient neutralizing activities to the variants including B.1.1.7 and B.1.351,and reacted with mutations of N501 Y,E484 K,and L452 R,indicated that it may also neutralize the recent India endemic strain B.1.617.The unchanged binding activity of F61 and H121 to RBD with multiple mutations revealed a broad neutralizing activity against variants,which mitigated the risk of viral escape.Our findings revealed the therapeutic basis of cocktail antibodies against constantly emerging SARS-Co V-2 variants and provided promising candidate antibodies to clinical treatment of COVID-19 patients infected with broad SARS-Co V-2 variants.