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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., (Copyright © 2024 Science China Press. Published by Elsevier B.V. All rights reserved.)

Seasonal influenza A virus subtype H3N2 (A/H3N2) circulates globally and has been linked to higher hospitalization rates and summer outbreaks in temperate regions. Here, A/H3N2 circulation in Shanghai, China was systematically studied using data and materials generated by the Shanghai influenza surveillance network from 2005 to 2023. Time-series analysis of incidence and subtyping data showed that A/H3N2 co-circulated with other (sub)types and dominated in multiple seasonal influenza peaks, preferentially in summer. Whole genomes of 528 representative strains were sequenced, and spatiotemporal phylodynamic analysis using these and GISAID-archived sequences demonstrated that in the years before the COVID-19 pandemic, phylogenetically similar strains were circulating locally and elsewhere. However, clade 1a.1 (within 3C.2a.1b.2a), circulated in and only in Shanghai and domestically in 2022, while the sibling clade 2 predominated in other regions. Interestingly, clade 1a.1 was swiftly and completely replaced by clade 2, mostly 2a.3a.1, at the start of 2023. In hemagglutination inhibition and neutralization assays, sera from healthy donors collected in 2022 displayed higher or similar reactivity against 2a.3a.1 compared to 1a.1. By contrast, transcription and replication competence of 2a.3a.1 in MDCK cells was higher than 1a.1. These results indicated that instead of antigenicity differences enabling evasion of pre-existing immunity, higher replicative capability more likely contributed to 2a.3a.1 viruses achieving dominance in China. In addition to summarizing patterns of A/H3N2 local circulation in Shanghai, this work revealed an unusual episode in A/H3N2 global circulation and evolution dynamics in connection to the COVID-19 pandemic and explored possible mechanistic explanations.

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a promising therapeutic approach for the treatment of HBV infection. In this study, the hit compound CDI (IC 50  = 2.46 ± 0.33 μM) was identified by screening of an in-house compound library. And then novel potent benzimidazole derivatives were designed and synthesized as core assembly modulators, and their antiviral effects were evaluated in vitro and in vivo biological experiments. The results indicated that compound 26f displayed the most optimized modulator of HBV capsid assembly (IC 50  = 0.51 ± 0.20 μM, EC 50  = 2.24 ± 0.43 μM, CC 50  = 84.29 μM) and high selectivity index. Moreover, treatment with compound 26f for 14 days significantly decreased serum levels of HBV DNA levels in the Hydrodynamic-Injection (HDI) mouse model. Therefore, compound 26f could be considered as a promising candidate drug for further development of novel HBV CAMs with the desired potency and safety., Competing Interests: Declaration of competing interest The authors declared that there were no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces direct cytopathic effects, complicating the establishment of low-cytotoxicity cell culture models for studying its replication. We initially developed a DNA vector-based replicon system utilizing the CMV promoter to generate a recombinant viral genome bearing reporter genes. However, this system frequently resulted in drug resistance and cytotoxicity, impeding model establishment. Herein, we present a novel cell culture model with SARS-CoV-2 replication induced by Cre/ LoxP -mediated DNA recombination. An engineered SARS-CoV-2 transcription unit was subcloned into a bacterial artificial chromosome (BAC) vector. To enhance biosafety, the viral spike protein gene was deleted, and the nucleocapsid gene was replaced with a reporter gene. An exogenous sequence was inserted within NSP1 as a modulatory cassette that is removable after Cre/ LoxP -mediated DNA recombination and subsequent RNA splicing. Using the PiggyBac transposon strategy, the transcription unit was integrated into host cell chromatin, yielding a stable cell line capable of inducing recombinant SARS-CoV-2 RNA replication. The model exhibited sensitivity to the potential antivirals forsythoside A and verteporfin. An innovative inducible SARS-CoV-2 replicon cell model was introduced to further explore the replication and pathogenesis of the virus and facilitate screening and assessment of anti-SARS-CoV-2 therapeutics., Competing Interests: The authors declare no conflicts of interest.

Vaccines play essential roles in the fight against the COVID-19 pandemic. The development and assessment of COVID-19 vaccines have generally focused on the induction and boosting of neutralizing antibodies targeting the SARS-CoV-2 spike (S) protein. Due to rapid and continuous variation in the S protein, such vaccines need to be regularly updated to match newly emerged dominant variants. T-cell vaccines that target MHC I- or II-restricted epitopes in both structural and non-structural viral proteins have the potential to induce broadly cross-protective and long-lasting responses. In this work, the entire proteome encoded by SARS-CoV-2 (Wuhan-hu-1) is subjected to immunoinformatics-based prediction of HLA-A*02:01-restricted epitopes. The immunogenicity of the predicted epitopes is evaluated using peripheral blood mononuclear cells from convalescent Wuhan-hu-1-infected patients. Furthermore, predicted epitopes that are conserved across major SARS-CoV-2 lineages and variants are used to construct DNA vaccines expressing multi-epitope polypeptides. Most importantly, two DNA vaccine constructs induce epitope-specific CD8 + T-cell responses in a mouse model of HLA-A*02:01 restriction and protect immunized mice from challenge with Wuhan-hu-1 virus after hACE2 transduction. These data provide candidate T-cell epitopes useful for the development of T-cell vaccines against SARS-CoV-2 and demonstrate a strategy for quick T-cell vaccine candidate development applicable to other emerging pathogens.

Chronic hepatitis B virus (HBV) infection (CHB) is a risk factor for the development of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Covalently closed circular DNA serves as the sole transcription template for all viral RNAs and viral transcription is driven and enhanced by viral promoter and enhancer elements, respectively. Interactions between transcription factors and these cis -elements regulate their activities and change the production levels of viral RNAs. Here, we report the identification of homeobox protein MSX-1 (MSX1) as a novel host restriction factor of HBV in liver. In both HBV-transfected and HBV-infected cells, MSX1 suppresses viral gene expression and genome replication. Mechanistically, MSX1 downregulates enhancer II/core promoter (EnII/Cp) activity via direct binding to an MSX1 responsive element within EnII/Cp, and such binding competes with hepatocyte nuclear factor 4α binding to EnII/Cp due to partial overlap between their respective binding sites. Furthermore, CHB patients in immune active phase express higher levels of intrahepatic MSX1 but relatively lower levels of serum and intrahepatic HBV markers compared to those in immune tolerant phase. Finally, MSX1 was demonstrated to induce viral clearance in two mouse models of HBV persistence, suggesting possible therapeutic potential for CHB.IMPORTANCECovalently closed circular DNA plays a key role for the persistence of hepatitis B virus (HBV) since it serves as the template for viral transcription. Identification of transcription factors that regulate HBV transcription not only provides insights into molecular mechanisms of viral life cycle regulation but may also provide potential antiviral targets. In this work, we identified host MSX1 as a novel restriction factor of HBV transcription. Meanwhile, we observed higher intrahepatic MSX1 expression in chronic hepatitis B virus (CHB) patients in immune active phase compared to those in immune tolerant phase, suggesting possible involvement of MSX1 in the regulation of HBV activity by the host. Lastly, intrahepatic overexpression of MSX1 delivered by recombinant adenoviruses into two mouse models of HBV persistence demonstrated MSX1-mediated repression of HBV in vivo , and MSX1-induced clearance of intrahepatic HBV DNA in treated mice suggested its potential as a therapeutic target for the treatment of CHB., Competing Interests: The authors declare no conflict of interest.

Staphylococcus aureus can form biofilms on biotic surfaces or implanted materials, leading to biofilm-associated diseases in humans and animals that are refractory to conventional antibiotic treatment. Recent studies indicate that the unique ArlRS regulatory system in S. aureus is a promising target for screening inhibitors that may eradicate formed biofilms, retard virulence and break antimicrobial resistance. In this study, by screening in the library of FDA-approved drugs, tilmicosin was found to inhibit ArlS histidine kinase activity (IC 50 = 1.09 μM). By constructing a promoter-fluorescence reporter system, we found that tilmicosin at a concentration of 0.75 μM or 1.5 μM displayed strong inhibition on the expression of the ArlRS regulon genes spx and mgrA in the S. aureus USA300 strain. Microplate assay and confocal laser scanning microscopy showed that tilmicosin at a sub-minimal inhibitory concentration (MIC) had a potent inhibitory effect on biofilms formed by multiple S. aureus strains and a strong biofilm-forming strain of S. epidermidis . In addition, tilmicosin at three-fold of MIC disrupted USA300 mature biofilms and had a strong bactericidal effect on embedded bacteria. Furthermore, in a BioFlux flow biofilm assay, tilmicosin showed potent anti-biofilm activity and synergized with oxacillin against USA300.

Background: After the eradication of smallpox in China in 1979, vaccination with the vaccinia virus (VACV) Tiantan strain for the general population was stopped in 1980. As the monkeypox virus (MPXV) is rapidly spreading in the world, we would like to investigate whether the individuals with historic VACV Tiantan strain vaccination, even after more than 40 years, could still provide ELISA reactivity and neutralizing protection; and whether the unvaccinated individuals have no antibody reactivity against MPXV at all., Results: We established serologic ELISA to measure the serum anti-MPXV titer by using immunodominant MPXV surface proteins, A35R, B6R, A29L, and M1R. A small proportion of individuals (born before 1980) with historic VACV Tiantan strain vaccination exhibited serum ELISA cross-reactivity against these MPXV surface proteins. Consistently, these donors also showed ELISA seropositivity and serum neutralization against VACV Tiantan strain. However, surprisingly, some unvaccinated young adults (born after 1980) also showed potent serum ELISA activity against MPXV proteins, possibly due to their past infection by some self-limiting Orthopoxvirus (OPXV)., Conclusions: We report the serum ELISA cross-reactivity against MPXV surface protein in a small proportion of individuals both with and without VACV Tiantan strain vaccination history. Combined with our serum neutralization assay against VACV and the recent literature about mice vaccinated with VACV Tiantan strain, our study confirmed the anti-MPXV cross-reactivity and cross-neutralization of smallpox vaccine using VACV Tiantan strain. Therefore, it is necessary to restart the smallpox vaccination program in high risk populations., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The COVID-19 pandemic caused by SARS-CoV-2 continues to pose a great threat to public health while various vaccines are available worldwide. Main protease (M pro ) has been validated as an effective anti-COVID-19 drug target. Using medicinal chemistry and rational drug design strategies, we identified a quinazolin-4-one series of nonpeptidic, noncovalent SARS-CoV-2 M pro inhibitors based on baicalein, 5,6,7-trihydroxy-2-phenyl-4H-chromen-4-one. In particular, compound C7 exhibits superior inhibitory activity against SARS-CoV-2 M pro relative to baicalein (IC 50  = 0.085 ± 0.006 and 0.966 ± 0.065 μM, respectively), as well as improved physicochemical and drug metabolism and pharmacokinetics (DMPK) properties. In addition, C7 inhibits viral replication in SARS-CoV-2-infected Vero E6 cells more effectively than baicalein (EC 50  = 1.10 ± 0.12 and 5.15 ± 1.64 μM, respectively) with low cytotoxicity (CC 50 ＞ 50 μM). An X-ray co-crystal structure reveals a non-covalent mechanism of action, and a noncanonical binding mode not observed by baicalein. These results suggest that C7 represents a promising lead for development of more effective SARS-CoV-2 M pro inhibitors and anti-COVID-19 drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), the transcription template for all viral mRNAs, is highly stable and current treatment options cannot effectively induce its clearance. Previously, we established an HBV persistence mouse model based on a clinical isolate (termed BPS) and identified interleukin-21 (IL-21) as a potent inducer of HBV clearance. Lipid nanoparticle (LNP) mediated delivery of mRNA has proven to be a highly safe and effective delivery platform. This work explored the applicability and effectiveness of the mRNA-LNP platform in IL-21-based HBV therapies. First, LNP-encapsulated murine IL-21 mRNA (LNP-IL-21) was prepared, characterized, and demonstrated to engender IL-21 expression in vitro and in vivo. Next, LNP-IL-21 was shown to induce clearance of both serum and intrahepatic HBV antigen and DNA in two HBV persistence mouse models based on BPS and recombinant cccDNA (rcccDNA), respectively, which was associated with HBV-specific humoral and cellular immune responses. Furthermore, peripheral blood mononuclear cells from BPS persistence mice treated ex vivo with LNP-IL-21 and HBV surface antigen (HBsAg) could induce similar HBV clearance upon infusion into recipient mice. These findings indicated that IL-21 combined with mRNA-LNP platform represents a valid and promising strategy for developing novel therapeutics against chronic HBV infection., (© 2023 Wiley Periodicals LLC.)

BK polyomavirus (BKV) is a small non-enveloped DNA virus. BKV infection or reactivation may cause BKV-associated nephropathy and hemorrhagic cystitis in immunosuppressed transplant recipients. No effective antivirals or prevention strategies are available against BKV infections. The current BKV reverse system employs the transfection of purified full-length linear viral genomes released by enzyme digestion from BKV genomic plasmids. The method is laborious and often results in variable DNA yield and quality, which can affect the efficiency of transfection and subsequent formation of circular viral genomes in cells. In this study, we report the generation of circular viral genomes by Cre-mediated DNA recombination in cells directly transfected with BKV precursor genomic plasmids. The novel system supported efficient viral expression and replication, and produced a higher level of infectious virions compared with the transfection with linear BKV genomes. Furthermore, we successfully constructed recombinant BKV capable of reporter gene expression. In conclusion, the novel BKV reverse genetic system allows for simpler manipulation of BKV genome with better virus yield, providing a tool for the study of BKV life cycle and antiviral screening., (© 2023 Wiley Periodicals LLC.)

Hepatitis B virus (HBV) DNA is much higher during HBeAg-positive chronic HBV infection (EP-CBI) than during HBeAg-negative chronic HBV infection (EN-CBI), although the necroinflammation in liver is minimal and the adaptive immune response is similar in both phases. We previously reported that mRNA levels of EVA1A were higher in EN-CBI patients. In this study, we aimed to investigate whether EVA1A inhibits HBV gene expression and examine the underlying mechanisms. The available cell models for HBV replication and model HBV mice were used to investigate how EVA1A regulates HBV replication and the antiviral activity based on gene therapy. The signaling pathway was determined through RNA sequencing analysis. The results demonstrated that EVA1A can inhibit HBV gene expression in vitro and in vivo. In particular, EVA1A overexpression resulted in accelerated HBV RNA degradation and activation of the PI3K-Akt-mTOR pathway, two processes that directly and indirectly inhibiting HBV gene expression. EVA1A is a promising candidate for treating chronic hepatitis B (CHB). In conclusion, EVA1A is a new host restriction factor that regulates the HBV life cycle via a nonimmune process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Neutralizing antibodies exert a potent inhibitory effect on viral entry; however, they are less effective in therapeutic models than in prophylactic models, presumably because of their limited efficacy in eliminating virus-producing cells via Fc-mediated cytotoxicity. Herein, we present a SARS-CoV-2 spike-targeting bispecific T-cell engager (S-BiTE) strategy for controlling SARS-CoV-2 infection. This approach blocks the entry of free virus into permissive cells by competing with membrane receptors and eliminates virus-infected cells via powerful T cell-mediated cytotoxicity. S-BiTE is effective against both the original and Delta variant of SARS-CoV2 with similar efficacy, suggesting its potential application against immune-escaping variants. In addition, in humanized mouse model with live SARS-COV-2 infection, S-BiTE treated mice showed significantly less viral load than neutralization only treated group. The S-BiTE strategy may have broad applications in combating other coronavirus infections., (© 2023. The Author(s).)

Objective: Pre-S1 antigen (pre-S1) is a component of hepatitis B virus large surface antigen (L-HBsAg). This study aimed to investigate the association between clinical pre-S1 antigen (pre-S1) status and adverse prognostic events in chronic hepatitis B (CHB) patients., Methods: This study retrospectively enrolled 840 CHB patients with comprehensive clinical data, including 144 patients with multiple follow-up of pre-S1 status. All patients were tested for serum pre-S1 and divided into pre-S1 positive and negative groups. Single factor and logistic multiple regression analyses were performed to explore the association between pre-S1 and other HBV biomarkers with the risk of hepatocellular carcinoma (HCC) in CHB patients. The pre-S1 region sequences of HBV DNA were obtained from one pre-S1 positive and two pre-S1 negative treatment-naïve patients using polymerase chain reaction (PCR) amplification followed by Sanger sequencing., Results: The quantitative HBsAg level was significantly higher in the pre-S1 positive group than that in the pre-S1 negative group (Z=-15.983, P <0.001). The positive rate of pre-S1 increased significantly with the increase in HBsAg level ( χ 2 =317.963, P<0.001) and HBV DNA load ( χ 2 =15.745, P <0.001). The pre-S1 negative group had a higher HCC risk than the pre-S1 positive group (Z=-2.00, P =0.045, OR=1.61). Moreover, patients in the sustained pre-S1 negative group had a higher HCC risk (Z=-2.56, P =0.011, OR=7.12) than those in the sustained pre-S1 positive group. The sequencing results revealed mutations in the pre-S1 region from samples of pre-S1 negative patients, including frameshift and deletion mutations., Conclusion: Pre-S1 is a biomarker that indicates the presence and replication of HBV. Pre-S1 sustained negativity attributed to pre-S1 mutations in CHB patients may be associated with a higher risk of HCC, which has clinical significance and warrant further investigations., Competing Interests: The authors have no conflict of interest in this work., (© 2023 Zhang et al.)

The emergence of multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of currently approved vaccines and authorized therapeutic monoclonal antibodies (MAbs). It is hence important to continue searching for SARS-CoV-2 broadly neutralizing MAbs and defining their epitopes. Here, we isolate 9 neutralizing mouse MAbs raised against the spike protein of a SARS-CoV-2 prototype strain and evaluate their neutralizing potency towards a panel of variants, including B.1.1.7, B.1.351, B.1.617.1, and B.1.617.2. By using a combination of biochemical, virological, and cryo-EM structural analyses, we identify three types of cross-variant neutralizing MAbs, represented by S5D2, S5G2, and S3H3, respectively, and further define their epitopes. S5D2 binds the top lateral edge of the receptor-binding motif within the receptor-binding domain (RBD) with a binding footprint centred around the loop 477-489 , and efficiently neutralizes all variant pseudoviruses, but the potency against B.1.617.2 was observed to decrease significantly. S5G2 targets the highly conserved RBD core region and exhibits comparable neutralization towards the variant panel. S3H3 binds a previously unreported epitope located within the evolutionarily stable SD1 region and is able to near equally neutralize all of the variants tested. Our work thus defines three distinct cross-variant neutralizing sites on the SARS-CoV-2 spike protein, providing guidance for design and development of broadly effective vaccines and MAb-based therapies.

Tumour cell metabolic plasticity is essential for tumour progression and therapeutic responses, yet the underlying mechanisms remain poorly understood. Here, we identify Prospero-related homeobox 1 (PROX1) as a crucial factor for tumour metabolic plasticity. Notably, PROX1 is reduced by glucose starvation or AMP-activated protein kinase (AMPK) activation and is elevated in liver kinase B1 (LKB1)-deficient tumours. Furthermore, the Ser79 phosphorylation of PROX1 by AMPK enhances the recruitment of CUL4-DDB1 ubiquitin ligase to promote PROX1 degradation. Downregulation of PROX1 activates branched-chain amino acids (BCAA) degradation through mediating epigenetic modifications and inhibits mammalian target-of-rapamycin (mTOR) signalling. Importantly, PROX1 deficiency or Ser79 phosphorylation in liver tumour shows therapeutic resistance to metformin. Clinically, the AMPK-PROX1 axis in human cancers is important for patient clinical outcomes. Collectively, our results demonstrate that deficiency of the LKB1-AMPK axis in cancers reactivates PROX1 to sustain intracellular BCAA pools, resulting in enhanced mTOR signalling, and facilitating tumourigenesis and aggressiveness., (© 2022. The Author(s).)

Chronic infection by hepatitis B virus (HBV) is associated with high risks of liver fibrosis, cirrhosis and hepatocellular carcinoma. HBV covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocyte serves as transcription template. Neither natural resolution of acute infection nor current treatment options for chronic infection are believed to cause cccDNA clearance. We previously showed that injection of IL-33-expressing plasmid facilitated clearance of intrahepatic HBV DNA in a mouse model of HBV persistence. In this work, HBV-targeting therapeutic effects of IL-33 were further explored. Murine IL-33 delivered by recombinant adeno-associated virus (AAV-mIL-33) induced clearance of both serum HBV markers and intrahepatic HBV DNA in two mouse models of HBV persistence based on replicon plasmid and recombinant cccDNA (rcccDNA) respectively. Clearance was associated with serum ALT elevations and liver infiltrations by CD4 + and CD8 + T cells, indicating IL-33-induced cellular immune responses against HBV-harboring cells. Adoptive transfer of splenocytes from AAV-mIL-33-cured mice was indeed sufficient to engender similar clearance in recipient mice. In vitro, intracellular, instead of extracellular, IL-33 was mainly responsible for repressing viral transcription, protein production and genome replication in Huh7 cells transfected with HBV replicon or rcccDNA. IL-33 was shown to be recruited onto rcccDNA minichromosome accompanied by loss of transcriptional activation epigenetic marks. Finally, transfection of IL-33 into HBV-infected HepG2/NTCP cells resulted in reduced transcription, antigen expression and genome replication, suggesting repression of canonical cccDNA. These data demonstrated diverse inhibitory effects on HBV and HBV-infected cells mediated by IL-33, and suggest IL-33 as an interesting therapeutic candidate., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

New threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design. Here, we identified a receptor-binding domain (RBD)-binding antibody, XG014, which potently neutralizes β-coronavirus lineage B (β-CoV-B), including SARS-CoV-2, its circulating variants, SARS-CoV and bat SARSr-CoV WIV1. Interestingly, antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibody-dependent SARS-CoV-2 spike (S) protein-mediated cell-cell fusion, suggesting a unique mode of recognition by XG014. Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional "down" conformation, while its family member XG005 directly competes with ACE2 binding and position the RBD "up". Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo. Our findings suggest the potential to develop XG014 as pan-β-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines against β-CoV-B and newly emerging SARS-CoV-2 variants of concern., (© 2021. The Author(s).)

Hepatitis B virus (HBV) is a primary cause of chronic liver diseases in humans. HBV infection exhibits strict host and tissue tropism. HBV core promoter (Cp) drives transcription of pregenomic RNA (pgRNA) and plays a key role in the viral life cycle. Hepatocyte nuclear factor 4α (HNF4α) acts as a major transcriptional factor that stimulates Cp. In this work, we reported that BEL7404 ​cell line displayed a high efficiency of DNA transfection and high levels of HBV antigen expression after transfection of HBV replicons without prominent viral replication. The introduction of exogenous HNF4α and human sodium taurocholate cotransporting polypeptide (hNTCP) expression into BEL7404 made it permissive for HBV replication and susceptible to HBV infection. BEL7404-derived cell lines with induced HBV permissiveness and susceptibility were constructed by stable co-transfection of hNTCP and Tet-inducible HNF4α followed by limiting dilution cloning. HBV replication in such cells was sensitive to inhibition by nucleotide analog tenofovir, while the infection was inhibited by HBV entry inhibitors. This cell culture system provides a new and additional tool for the study of HBV replication and infection as well as the characterization of antiviral agents., (Copyright © 2022 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Background: Pancreatic cancer (PC) is a highly metastatic and lethal cancer with a very low overall 5-year survival rate. There is an urgent need for identifying new therapeutic agents for this deadly disease. Cardiac glycosides (CGs) have been traditionally used for their potent cardiovascular activities and have also recently been reported to exhibit anti-tumor effects. Proscillaridin A (Pro A), a natural CG, has been shown to display anti-tumor effects on multiple cancer types., Methods: The cytotoxic effect of Pro A on PC cells was determined using cell viability assay, colony formation assay and transwell assay in vitro . Cell apoptosis, cell cycle, reactive oxygen species (ROS) generation, intracellular Ca 2+ levels and mitochondrial membrane potential (MMP) were assayed by flow cytometry. Panc-1-xenografted mice model was used to evaluate Pro A's effect in tumor growth. Mitochondria morphology was observed by transmission electron microscopy. LC3 aggregation was assessed by GFP-LC3 fluorescence microscopy. Gene expression was assayed by western blot or real-time quantitative polymerase chain reaction (qPCR)., Results: Pro A inhibits the proliferation, migration and invasion of Panc-1, BxPC-3 and AsPC-1 PC cells in vitro , and Panc-1 cells display the highest sensitivity with an IC50 at the nano-molar level. In vivo , Pro A treatment inhibits tumor progression in Panc-1 xenograft nude mice. Pro A treatment promotes both cell apoptosis and autophagy, and Pro A-treated PC cells display characteristics of mitochondrial damage including increased ROS generation, intracellular Ca 2+ levels and disruption of MMP. In addition, high sensitivity towards Pro A of Panc-1 cells compared to BxPC-3 and AsPC-1 cells could be partially attributed to the loss of endogenous SMAD4 expression in the latter., Conclusions: Our findings suggest that Pro A constitutes a promising therapeutic candidate for certain types of PC., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-1085/coif). The authors have no conflicts of interest to declare., (2022 Annals of Translational Medicine. All rights reserved.)

Frequent outbreaks of coronaviruses underscore the need for antivirals and vaccines that can counter a broad range of coronavirus types. We isolated a human antibody named 76E1 from a COVID-19 convalescent patient, and report that it has broad-range neutralizing activity against multiple α- and β-coronaviruses, including the SARS-CoV-2 variants. 76E1 also binds its epitope in peptides from γ- and δ-coronaviruses. 76E1 cross-protects against SARS-CoV-2 and HCoV-OC43 infection in both prophylactic and therapeutic murine animal models. Structural and functional studies revealed that 76E1 targets a unique epitope within the spike protein that comprises the highly conserved S2' site and the fusion peptide. The epitope that 76E1 binds is partially buried in the structure of the SARS-CoV-2 spike trimer in the prefusion state, but is exposed when the spike protein binds to ACE2. This observation suggests that 76E1 binds to the epitope at an intermediate state of the spike trimer during the transition from the prefusion to the postfusion state, thereby blocking membrane fusion and viral entry. We hope that the identification of this crucial epitope, which can be recognized by 76E1, will guide epitope-based design of next-generation pan-coronavirus vaccines and antivirals., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies have been limited by the continuous emergence of viral variants and by the restricted diffusion of antibodies from circulation into the sites of respiratory virus infection. Here, we report the identification of two highly conserved regions on the Omicron variant receptor-binding domain recognized by broadly neutralizing antibodies. Furthermore, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant receptor-binding domain as revealed by cryo-EM structures. We demonstrated that this bispecific antibody can be effectively delivered to lung via inhalation administration and exhibits exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. Importantly, this study also deciphered an uncommon and highly conserved cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics., Competing Interests: Declaration of interests C.L., Y.W., and T.Y. are listed as inventors on two patent applications related to this work., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Hepatitis B virus exposure in children usually develops into chronic hepatitis B (CHB). Although hepatitis B surface antigen (HBsAg)-specific CD8+ T cells contribute to resolve HBV infection, they are preferentially undetected in CHB patients. Moreover, the mechanism for this rarely detected HBsAg-specific CD8+ T cells remains unexplored. We herein found that the frequency of HBsAg-specific CD8+ T cells was inversely correlated with expansion of monocytic myeloid-derived suppressor cells (mMDSCs) in young rather than in adult CHB patients, and CCR9 was upregulated by HBsAg on mMDSCs via activation of ERK1/2 and IL-6. Sequentially, the interaction between CCL25 and CCR9 mediated thymic homing of mMDSCs, which caused the cross-presentation, transferring of peripheral HBsAg into the thymic medulla, and then promoted death of HBsAg-specific CD8+ thymocytes. In mice, adoptive transfer of mMDSCs selectively obliterated HBsAg-specific CD8+ T cells and facilitated persistence of HBV in a CCR9-dependent manner. Taken together, our results uncovered a novel mechanism for establishing specific CD8+ tolerance to HBsAg in chronic HBV infection., (© 2022 Fang et al.)

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., Competing Interests: The authors have no conflicts of interest to declare., (© 2022 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

The emergence of SARS-CoV-2 variants and potentially other highly pathogenic sarbecoviruses in the future highlights the need for pan-sarbecovirus vaccines. Here, we discovered a new STING agonist, CF501, and found that CF501-adjuvanted RBD-Fc vaccine (CF501/RBD-Fc) elicited significantly stronger neutralizing antibody (nAb) and T cell responses than Alum- and cGAMP-adjuvanted RBD-Fc in mice. Vaccination of rabbits and rhesus macaques (nonhuman primates, NHPs) with CF501/RBD-Fc elicited exceptionally potent nAb responses against SARS-CoV-2 and its nine variants and 41 S-mutants, SARS-CoV and bat SARSr-CoVs. CF501/RBD-Fc-immunized hACE2-transgenic mice were almost completely protected against SARS-CoV-2 challenge, even 6 months after the initial immunization. NHPs immunized with a single dose of CF501/RBD-Fc produced high titers of nAbs. The immunized macaques also exhibited durable humoral and cellular immune responses and showed remarkably reduced viral load in the upper and lower airways upon SARS-CoV-2 challenge even at 108 days post the final immunization. Thus, CF501/RBD-Fc can be further developed as a novel pan-sarbecovirus vaccine to combat current and future outbreaks of sarbecovirus diseases., (© 2022. The Author(s).)

The detection of samples at ultralow concentrations (one to ten copies in 100 μl) in biofluids is hampered by the orders-of-magnitude higher amounts of 'background' biomolecules. Here we report a molecular system, immobilized on a liquid-gated graphene field-effect transistor and consisting of an aptamer probe bound to a flexible single-stranded DNA cantilever linked to a self-assembled stiff tetrahedral double-stranded DNA structure, for the rapid and ultrasensitive electromechanical detection (down to one to two copies in 100 μl) of unamplified nucleic acids in biofluids, and also of ions, small molecules and proteins, as we show for Hg 2+ , adenosine 5'-triphosphate and thrombin. We implemented an electromechanical biosensor for the detection of SARS-CoV-2 into an integrated and portable prototype device, and show that it detected SARS-CoV-2 RNA in less than four minutes in all nasopharyngeal samples from 33 patients with COVID-19 (with cycle threshold values of 24.9-41.3) and in none of the 54 COVID-19-negative controls, without the need for RNA extraction or nucleic acid amplification., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Chronic infection with hepatitis B virus (HBV) is associated with liver cirrhosis and hepatocellular carcinoma. Upon infection of hepatocytes, HBV covalently closed circular DNA (cccDNA) exists as histone-bound mini-chromosome, subjected to transcriptional regulation similar to chromosomal DNA. Here we identify high mobility group AT-hook 1 (HMGA1) protein as a positive regulator of HBV transcription that binds to a conserved ATTGG site within enhancer II/core promoter (EII/Cp) and recruits transcription factors FOXO3α and PGC1α. HMGA1-mediated upregulation of EII/Cp results in enhanced viral gene expression and genome replication. Notably, expression of endogenous HMGA1 was also demonstrated to be upregulated by HBV, which involves HBV X protein (HBx) interacting with SP1 transcription factor to activate HMGA1 promoter. Consistent with these in vitro results, chronic hepatitis B patients in immune tolerant phase display both higher intrahepatic HMGA1 protein levels and higher serum HBV markers compared to patients in inactive carrier phase. Finally, using a mouse model of HBV persistence, we show that targeting endogenous HMGA1 through RNA interference facilitated HBV clearance. These data establish HMGA1 as an important positive regulator of HBV that is reciprocally upregulated by HBV via HBx and also suggest the HMGA1-HBV positive feedback loop as a potential therapeutic target., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Since late 2019, SARS-CoV-2 infection has resulted in COVID-19 accompanied by diverse clinical manifestations. However, the underlying mechanism of how SARS-CoV-2 interacts with host and develops multiple symptoms is largely unexplored., Methods: Bioinformatics analysis determined the sequence similarity between SARS-CoV-2 and human genomes. Diverse fragments of SARS-CoV-2 genome containing Human Identical Sequences (HIS) were cloned into the lentiviral vector. HEK293T, MRC5 and HUVEC were infected with laboratory-packaged lentivirus or transfected with plasmids or antagomirs for HIS. Quantitative RT-PCR and chromatin immunoprecipitation assay detected gene expression and H3K27ac enrichment, respectively. UV-Vis spectroscopy assessed the interaction between HIS and their target locus. Enzyme-linked immunosorbent assay evaluated the hyaluronan (HA) levels of culture supernatant and plasma of COVID-19 patients., Findings: Five short sequences (24-27 nt length) sharing identity between SARS-CoV-2 and human genome were identified. These RNA elements were highly conserved in primates. The genomic fragments containing HIS were predicted to form hairpin structures in silico similar to miRNA precursors. HIS may function through direct genomic interaction leading to activation of host enhancers, and upregulation of adjacent and distant genes, including cytokine genes and hyaluronan synthase 2 (HAS2). HIS antagomirs and Cas13d-mediated HIS degradation reduced HAS2 expression. Severe COVID-19 patients displayed decreased lymphocytes and elevated D-dimer, and C-reactive proteins, as well as increased plasma hyaluronan. Hymecromone inhibited hyaluronan production in vitro, and thus could be further investigated as a therapeutic option for preventing severe outcome in COVID-19 patients., Interpretation: HIS of SARS-CoV-2 could promote COVID-19 progression by upregulating hyaluronan, providing novel targets for treatment., Funding: The National Key R&D Program of China (2018YFC1005004), Major Special Projects of Basic Research of Shanghai Science and Technology Commission (18JC1411101), and the National Natural Science Foundation of China (31872814, 32000505)., Competing Interests: Declaration of interests Wenqiang Yu, Wei Li, Jianqing Xu, Hailin Wang, Cheng Lian, Peng Xu, Shuai Yang, and Daoping Ru are listed as inventors on patents' application related to this work; no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Host cellular receptors play key roles in the determination of virus tropism and pathogenesis. However, little is known about SARS-CoV-2 host receptors with the exception of ACE2. Furthermore, ACE2 alone cannot explain the multi-organ tropism of SARS-CoV-2 nor the clinical differences between SARS-CoV-2 and SARS-CoV, suggesting the involvement of other receptor(s). Here, we performed genomic receptor profiling to screen 5054 human membrane proteins individually for interaction with the SARS-CoV-2 capsid spike (S) protein. Twelve proteins, including ACE2, ASGR1, and KREMEN1, were identified with diverse S-binding affinities and patterns. ASGR1 or KREMEN1 is sufficient for the entry of SARS-CoV-2 but not SARS-CoV in vitro and in vivo. SARS-CoV-2 utilizes distinct ACE2/ASGR1/KREMEN1 (ASK) receptor combinations to enter different cell types, and the expression of ASK together displays a markedly stronger correlation with virus susceptibility than that of any individual receptor at both the cell and tissue levels. The cocktail of ASK-related neutralizing antibodies provides the most substantial blockage of SARS-CoV-2 infection in human lung organoids when compared to individual antibodies. Our study revealed an interacting host receptome of SARS-CoV-2, and identified ASGR1 and KREMEN1 as alternative functional receptors that play essential roles in ACE2-independent virus entry, providing insight into SARS-CoV-2 tropism and pathogenesis, as well as a community resource and potential therapeutic strategies for further COVID-19 investigations., (© 2021. The Author(s), under exclusive licence to Center for Excellence in Molecular Cell Science, CAS.)

Since the end of 2019, the outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has evolved into a global pandemic. There is an urgent need for effective and low-toxic antiviral drugs to remedy Remdesivir's limitation. Hydroxychloroquine, a broad spectrum anti-viral drug, showed inhibitory activity against SARS-CoV-2 in some studies. Thus, we adopted a drug repurposing strategy, and further investigated hydroxychloroquine. We obtained different configurations of hydroxychloroquine side chains by using chiral resolution technique, and successfully furnished R-/S-hydroxychloroquine sulfate through chemical synthesis. The R configuration of hydroxychloroquine was found to exhibit higher antiviral activity (EC50 = 3.05 μM) and lower toxicity in vivo. Therefore, R-HCQ is a promising lead compound against SARS-CoV-2. Our research provides new strategy for the subsequent research on small molecule inhibitors against SARS-CoV-2., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Human hepatitis B virus (HBV) and hepatitis D virus (HDV) cause acute and chronic infections. The latter poses a serious public health threat as it is the major cause of chronic hepatitis, liver failure, cirrhosis, and hepatocellular carcinoma (HCC). In nature, HBV and HDV have a narrow host range and highly hepatotropic, only infecting the hepatocytes of humans and a few primates. The elucidation of cell entry mechanism by HBV has made great progress in recent years, which strongly promotes the establishment of new HBV infection models and the development of viral entry inhibitors. The study of HBV entry inhibitors has culminated in the first direct antiviral treatment for HDV. This review provides a concise introduction on the progress of HBV/HDV entry inhibitors in the recent years., (© 2022. Springer Nature Singapore Pte Ltd.)

To curb the pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), multiple platforms have been employed toward a safe and highly effective vaccine. Here, we develop a novel cell-based vaccine candidate, namely K562-S, by utilizing human cell K562 as a cellular carrier to display Spike (S) protein of SARS-CoV-2 on the membrane. Analogous to the traditional inactivated vaccine, K562-S cells can be propagated to a large scale by culturing and completely lose their viability after exposure to X-ray irradiation or formalin. We in turn demonstrated high immunogenicity of formalin-inactivated K562-S vaccine in both mouse and non-human primates and its protective efficacy in mice. In mice, immunization with inactivated K562-S vaccines can elicit potent neutralizing antibody (nAb) responses persisting longer than 5 months. We consequently showed in a hACE2 mouse model of SARS-CoV-2 infection that a two-shot vaccination with adjuvanted K562-S rendered greater than 3 log reduction in viral lung load and concomitant ameliorated lung pathology. Of importance, the administration of the same regimen in non-human primates was able to induce a neutralizing antibody titer averaging three-fold higher relative to human convalescent serum. These results together support the promise of K562-based, S-protein-expressing vaccines as a novel vaccination approach against SARS-CoV-2. Importantly, with a powerful capacity to carry external genes for cell-based vectors, this platform could rapidly generate two- and multiple-valent vaccines by incorporating SARS-CoV-2 mutants, SARS-CoV, or MERS-CoV.

COVID-19 pandemic has severely impacted the public health and social economy worldwide. A safe, effective, and affordable vaccine against SARS-CoV-2 infections/diseases is urgently needed. We have been developing a recombinant vaccine based on a prefusion-stabilized spike trimer of SARS-CoV-2 and formulated with aluminium hydroxide and CpG 7909. The spike protein was expressed in Chinese hamster ovary (CHO) cells, purified, and prepared as a stable formulation with the dual adjuvant. Immunogenicity studies showed that candidate vaccines elicited robust neutralizing antibody responses and substantial CD4 + T cell responses in both mice and non-human primates. And vaccine-induced neutralizing antibodies persisted at high level for at least 6 months. Challenge studies demonstrated that candidate vaccine reduced the viral loads and inflammation in the lungs of SARS-CoV-2 infected golden Syrian hamsters significantly. In addition, the vaccine-induced antibodies showed cross-neutralization activity against B.1.1.7 and B.1.351 variants. These data suggest candidate vaccine is efficacious in preventing SARS-CoV-2 infections and associated pneumonia, thereby justifying ongoing phase I/II clinical studies in China (NCT04982068 and NCT04990544)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

Direct SARS-CoV-2 nucleic acid testing with fast speed and high frequency is crucial for controlling the COVID-19 pandemic. Here, direct testing of SARS-CoV-2 nucleic acid is realized by field-effect transistors (FETs) with an electro-enrichable liquid gate (LG) anchored by tetrahedral DNA nanostructures (TDNs). The applied gate bias electrostatically preconcentrates nucleic acids, while the liquid gate with TDNs provides efficient analyte recognition and signal transduction. The average diagnosis time is ∼80 s, and the limit of detection approaches 1-2 copies in 100 μL of clinical samples without nucleic acid extraction and amplification. As such, TDN-LG FETs solve the dilemma of COVID-19 testing on mass scale that diagnosis accuracy and speed undergo trade-off. In addition, TDN-LG FETs achieve unamplified 10-in-1 pooled nucleic acid testing for the first time, and the results are consistent with PCR. Thus, this technology promises on-site and wide population COVID-19 screening and ensures safe world-reopening.

The current COVID-19 pandemic has heavily burdened the global public health system and may keep simmering for years. The frequent emergence of immune escape variants have spurred the search for prophylactic vaccines and therapeutic antibodies that confer broad protection against SARS-CoV-2 variants. Here we show that the bivalency of an affinity maturated fully human single-domain antibody (n3113.1-Fc) exhibits exquisite neutralizing potency against SARS-CoV-2 pseudovirus, and confers effective prophylactic and therapeutic protection against authentic SARS-CoV-2 in the host cell receptor angiotensin-converting enzyme 2 (ACE2) humanized mice. The crystal structure of n3113 in complex with the receptor-binding domain (RBD) of SARS-CoV-2, combined with the cryo-EM structures of n3113 and spike ecto-domain, reveals that n3113 binds to the side surface of up-state RBD with no competition with ACE2. The binding of n3113 to this novel epitope stabilizes spike in up-state conformations but inhibits SARS-CoV-2 S mediated membrane fusion, expanding our recognition of neutralization by antibodies against SARS-CoV-2. Binding assay and pseudovirus neutralization assay show no evasion of recently prevalent SARS-CoV-2 lineages, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) for n3113.1-Fc with Y58L mutation, demonstrating the potential of n3113.1-Fc (Y58L) as a promising candidate for clinical development to treat COVID-19., (© 2021. The Author(s).)

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus display remarkable efficacy against authentic B.1.351 virus. Surprisingly, structural analysis has revealed that 58G6 and 13G9 both recognize the steric region S 470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly binds to another region S 450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrate prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. Together, we have evidenced 2 potent neutralizing Abs with unique mechanism targeting authentic SARS-CoV-2 mutants, which can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic., (© 2021. The Author(s).)

YycFG, one of the two-component systems involved in the regulation of biofilm formation, has attracted increasing interest as a potential target of antibacterial and antibiofilm agents. YycG inhibitors for Staphylococcus aureus and Staphylococcus epidermidis have been developed, but Enterococcus faecalis remains underexplored. Herein, we selected and identified novel candidate molecules against E. faecalis targeting histidine kinase YycG using high-throughput virtual screening; six molecules (compound-16, -30, -42, -46, -59, and -62) with low cytotoxicity toward mammalian cells were verified as potential YycG inhibitors through an autophosphorylation test and binding kinetics. Compound-16 inhibited planktonic cells of E. faecalis , including the vancomycin- or linezolid-resistant strains. In contrast, compound-62 did not affect planktonic growth but significantly inhibited biofilm formation in static and dynamic conditions. Compound-62 combined with ampicillin could synergistically eradicate the biofilm-embedded viable bacteria. The study demonstrates that YycG inhibitors may be valuable approaches for the development of novel antimicrobial agents for difficult-to-treat bacterial infections.

Rapid screening of infected individuals from a large population is an effective means in epidemiology, especially to contain outbreaks such as COVID-19. The gold standard assays for COVID-19 diagnostics are mainly based on the reverse transcription polymerase chain reaction, which mismatches the requirements for wide-population screening due to time-consuming nucleic acid extraction and amplification procedures. Here, we report a direct nucleic acid assay by using a graphene field-effect transistor (g-FET) with Y-shaped DNA dual probes (Y-dual probes). The assay relies on Y-dual probes modified on g-FET simultaneously targeting ORF1ab and N genes of SARS-CoV-2 nucleic acid, enabling high a recognition ratio and a limit of detection (0.03 copy μL -1 ) 1-2 orders of magnitude lower than existing nucleic acid assays. The assay realizes the fastest nucleic acid testing (∼1 min) and achieves direct 5-in-1 pooled testing for the first time. Owing to its rapid, ultrasensitive, easily operated features as well as capability in pooled testing, it holds great promise as a comprehensive tool for population-wide screening of COVID-19 and other epidemics.

Background: The receptor-binding domain (RBD) variants of SARS-CoV-2 could impair antibody-mediated neutralization of the virus by host immunity; thus, prospective surveillance of antibody escape mutants and understanding the evolution of RBD are urgently needed., Methods: Using the single B cell cloning technology, we isolated and characterized 93 RBD-specific antibodies from the memory B cells of four COVID-19 convalescent individuals in the early stage of the pandemic. Then, global RBD alanine scanning with a panel of 19 selected neutralizing antibodies (NAbs), including several broadly reactive NAbs, was performed. Furthermore, we assessed the impact of single natural mutation or co-mutations of concern at key positions of RBD on the neutralization escape and ACE2 binding function by recombinant proteins and pseudoviruses., Results: Thirty-three amino acid positions within four independent antigenic sites (1 to 4) of RBD were identified as valuable indicators of antigenic changes in the RBD. The comprehensive escape mutation map not only confirms the widely circulating strains carrying important immune escape RBD mutations such as K417N, E484K, and L452R, but also facilitates the discovery of new immune escape-enabling mutations such as F486L, N450K, F490S, and R346S. Of note, these escape mutations could not affect the ACE2 binding affinity of RBD, among which L452R even enhanced binding. Furthermore, we showed that RBD co-mutations K417N, E484K, and N501Y present in B.1.351 appear more resistant to NAbs and human convalescent plasma from the early stage of the pandemic, possibly due to an additive effect. Conversely, double mutations E484Q and L452R present in B.1.617.1 variant show partial antibody evasion with no evidence for an additive effect., Conclusions: Our study provides a global view of the determinants for neutralizing antibody recognition, antigenic conservation, and RBD conformation. The in-depth escape maps may have value for prospective surveillance of SARS-CoV-2 immune escape variants. Special attention should be paid to the accumulation of co-mutations at distinct major antigenic sites. Finally, the new broadly reactive NAbs described here represent new potential opportunities for the prevention and treatment of COVID-19., (© 2021. The Author(s).)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus causing acute respiratory tract infection in humans. The virus has the characteristics of rapid transmission, long incubation period and strong pathogenicity, and has spread all over the world. Therefore, it is of great significance to select appropriate animal models for antiviral drug development and therapeutic effect evaluation. Here, we review and compare the current animal models of SARS-CoV-2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shou, Liu, Yang, Kang, Song, Tan, Liu, Wang, Liu and Xie.)

Massive production of efficacious SARS-CoV-2 vaccines is essential for controlling the ongoing COVID-19 pandemic. We report here the preclinical development of yeast-produced receptor-binding domain (RBD)-based recombinant protein SARS-CoV-2 vaccines. We found that monomeric RBD of SARS-CoV-2 could be efficiently produced as a secreted protein from transformed Pichia pastoris (P. pastoris) yeast. Yeast-derived RBD-monomer possessed functional conformation and was able to elicit protective level of neutralizing antibodies in mice. We further designed and expressed a genetically linked dimeric RBD protein in yeast. The engineered dimeric RBD was more potent than the monomeric RBD in inducing long-lasting neutralizing antibodies. Mice immunized with either monomeric RBD or dimeric RBD were effectively protected from live SARS-CoV-2 virus challenge even at 18 weeks after the last vaccine dose. Importantly, we found that the antisera raised against the RBD of a single SARS-CoV-2 prototype strain could effectively neutralize the two predominant circulating variants B.1.1.7 and B.1.351, implying broad-spectrum protective potential of the RBD-based vaccines. Our data demonstrate that yeast-derived RBD-based recombinant SARS-CoV-2 vaccines are feasible and efficacious, opening up a new avenue for rapid and cost-effective production of SARS-CoV-2 vaccines to achieve global immunization., (© 2021. The Author(s).)

The COVID-19 pandemic poses a global threat to public health and economy. The continuously emerging SARS-CoV-2 variants present a major challenge to the development of antiviral agents and vaccines. In this study, we identified that EK1 and cholesterol-coupled derivative of EK1, EK1C4, as pan-CoV fusion inhibitors, exhibit potent antiviral activity against SARS-CoV-2 infection in both lung- and intestine-derived cell lines (Calu-3 and Caco2, respectively). They are also effective against infection of pseudotyped SARS-CoV-2 variants B.1.1.7 (Alpha) and B.1.1.248 (Gamma) as well as those with mutations in S protein, including N417T, E484K, N501Y, and D614G, which are common in South African and Brazilian variants. Crystal structure revealed that EK1 targets the HR1 domain in the SARS-CoV-2 S protein to block virus-cell fusion and provide mechanistic insights into its broad and effective antiviral activity. Nasal administration of EK1 peptides to hACE2 transgenic mice significantly reduced viral titers in lung and intestinal tissues. EK1 showed good safety profiles in various animal models, supporting further clinical development of EK1-based pan-CoV fusion inhibitors against SARS-CoV-2 and its variants., (© 2021. The Author(s).)

Chronic infection of hepatitis B virus (HBV) is a high risk factor for hepatic diseases, such as liver fibrosis, cirrhosis and hepatocellular carcinoma. Non-responders and hyporesponders to HBV vaccine are not protected from HBV infection. Patients that achieve autonomous or treatment-induced recovery are at risk of reactivation due to persistence of HBV covalently closed circular DNA (cccDNA) in hepatocytes. Interleukin 21 (IL-21) is a key regulator of HBV clearance in mouse models of HBV persistence: IL-21-based therapies effectively induces HBV clearance and protects mice from subsequent re-challenge. In this study, we explore the possibility of using IL-21 as prophylaxis against HBV by using mouse models of HBV persistence. HBV-naïve mice were transiently exposed to exogenous IL-21 through injection with recombinant adeno-associated virus expressing mouse IL-21 (AAV-IL-21). After extraneous IL-21 protein and DNA had become undetectable, mice were challenged with persistence-inducing HBV replicon plasmid through hydrodynamic injection. Viral persistence was analyzed by measuring viral antigens and DNA markers in serum and intrahepatic HBV DNA. For mechanistic studies, CD8 + T cell functions were blocked by repeated intraperitoneal injections of CD8 monoclonal antibodies in HBV-challenged mice. AAV-IL-21-injected mice quickly cleared HBV after HBV replicon challenge. In contrast, untreated mice and mice injected with control virus (AAV-Ctrl) allowed establishment of HBV persistence. Mechanistically, mice with prior IL-21 exposure displayed marked intrahepatic CD8 + T cell infiltrations, and CD8 blocking experiments demonstrated that CD8 + T cell responses functionally contributed toward clearance., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The SARS-CoV-2 infection is spreading rapidly worldwide. Efficacious antiviral therapeutics against SARS-CoV-2 is urgently needed. Here, we discovered that protoporphyrin IX (PpIX) and verteporfin, two Food and Drug Administration (FDA)-approved drugs, completely inhibited the cytopathic effect produced by SARS-CoV-2 infection at 1.25 μmol/L and 0.31 μmol/L, respectively, and their EC50 values of reduction of viral RNA were at nanomolar concentrations. The selectivity indices of PpIX and verteporfin were 952.74 and 368.93, respectively, suggesting a broad margin of safety. Importantly, PpIX and verteporfin prevented SARS-CoV-2 infection in mice adenovirally transduced with human angiotensin-converting enzyme 2 (ACE2). The compounds, sharing a porphyrin ring structure, were shown to bind viral receptor ACE2 and interfere with the interaction between ACE2 and the receptor-binding domain of viral S protein. Our study suggests that PpIX and verteporfin are potent antiviral agents against SARS-CoV-2 infection and sheds new light on developing novel chemoprophylaxis and chemotherapy against SARS-CoV-2., Competing Interests: The authors declare that they have no conflict of interest., (© 2020 Science China Press. Published by Elsevier B.V. and Science China Press. All rights reserved.)

DNA transfection is routinely used for delivering expression of gene of interest to target cells. Transfected DNA has been known to activate cellular DNA sensor(s) and innate immune responses, but the effects of such responses on transfected DNA are not fully understood. STING (stimulator of interferon genes) is an important adaptor protein in cellular innate immune response to various DNA and RNA stimuli and upon activation induces significant type I interferon responses. In this work, we characterized the effects of STING on gene expression driven by transfected double-stranded DNA. We observed that gene expression from transfected DNA was repressed in the presence of overexpressed STING, but increased if endogenous STING was knocked down through RNA interference. Endogenous chromosomal genes and chromosome-integrated exogenous genes were not affected by such STING-mediated restriction, which did not depend on DNA circularity or linearity, promoter used, or bacterial sequences in transfected DNA. Mechanistically, STING-mediated repression of transfected DNA correlates with reduced mRNA levels, and partially involves the induction of interferon β production by STING. Collectively, these data indicate that episomal double-stranded DNA is targeted by STING-mediated cell defense., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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. Our study shed more lights into the genetic determinants of ACE2 as the functional receptor of SARS-CoV-2, which facilitates our understanding of viral entry., Competing Interests: The authors have declared that no competing interests exist.

The pandemic of COVID-19, caused by SARS-CoV-2, is a major global health threat. Epidemiological studies suggest that bats ( Rhinolophus affinis ) 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. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. By analyzing the conservation of five residues in two virus-binding hotspots of ACE2 (hotspot 31Lys and hotspot 353Lys), we predicted 80 ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 ACE2 orthologs among them for functional analysis, and showed that 44 of these orthologs-including domestic animals, pets, livestock, and animals commonly found in zoos and aquaria-could bind the SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind the 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. 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., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)