The SARS-CoV-2 subgenome landscape and its novel regulatory features.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global pandemic. CoVs are known to generate negative subgenomes (subgenomic RNAs [sgRNAs]) through transcription-regulating sequence (TRS)-dependent template switching, but the global dynamic landscapes of coronaviral subgenomes and regulatory rules remain unclear. Here, using next-generation sequencing (NGS) short-read and Nanopore long-read poly(A) RNA sequencing in two cell types at multiple time points after infection with SARS-CoV-2, we identified hundreds of template switches and constructed the dynamic landscapes of SARS-CoV-2 subgenomes. Interestingly, template switching could occur in a bidirectional manner, with diverse SARS-CoV-2 subgenomes generated from successive template-switching events. The majority of template switches result from RNA-RNA interactions, including seed and compensatory modes, with terminal pairing status as a key determinant. Two TRS-independent template switch modes are also responsible for subgenome biogenesis. Our findings reveal the subgenome landscape of SARS-CoV-2 and its regulatory features, providing a molecular basis for understanding subgenome biogenesis and developing novel anti-viral strategies.

Recombinant expression of the precursor of rat lung surfactant protein B in Escherichia coli and its antibacterial mechanism.

While the discovery of antibiotics has made a huge contribution to medicine, bacteria that are resistant to many antibiotics pose new challenges to medicine. Antimicrobial peptides (AMPs), a new kind of antibiotics, have attracted people's attention because they are not prone to drug resistance. In this study, glutathione transferase (GST) was used as a fusion partner to recombinantly expressed rat lung surfactant protein B precursor (proSP-B) in E. coli pLySs. Cck-8 evaluated the cytotoxicity of the fusion protein and calculated its 50% inhibitory concentration (IC50). The purified peptides showed broad-spectrum antibacterial activity using filter paper method and MIC, and propidium iodide (PI) was used to explore the antibacterial mechanism against Staphylococcus aureus. In addition, the pEGFP-N2-proSP-B vector was constructed to explore the localization of proSP-B in CCL-149 cells. We found that proSP-B has obvious antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and fungi, and has broad-spectrum antibacterial activity. Besides, proSP-B fusion protein has low toxicity and can change the permeability of Staphylococcus aureus cell membrane to realize its antibacterial. For these reasons, proSP-B can be used as a potential natural antibacterial drug.

AIMP2 restricts EV71 replication by recruiting SMURF2 to promote the degradation of 3D polymerase.

Hand, foot and mouth disease (HFMD), mainly caused by enterovirus 71 (EV71), have frequently occurred in the Asia-Pacific region, posing a significant threat to the health of infants and young children. Therefore, research on the infection mechanism and pathogenicity of enteroviruses is increasingly becoming important. The 3D polymerase, as the most critical RNA-dependent RNA polymerase (RdRp) for EV71 replication, is widely targeted to inhibit EV71 infection. In this study, we identified a novel host protein, AIMP2, capable of binding to 3D polymerase and inhibiting EV71 infection. Subsequent investigations revealed that AIMP2 recruits the E3 ligase SMURF2, which mediates the polyubiquitination and degradation of 3D polymerase. Furthermore, the antiviral effect of AIMP2 extended to the CVA16 and CVB1 serotypes. Our research uncovered the dynamic regulatory function of AIMP2 during EV71 infection, revealing a novel antiviral mechanism and providing new insights for the development of antienteroviral therapeutic strategies.

Vaccination with Span, an antigen guided by SARS-CoV-2 S protein evolution, protects against challenge with viral variants in mice.

SARS-CoV-2 continues to accumulate mutations to evade immunity, leading to breakthrough infections after vaccination. How researchers can anticipate the evolutionary trajectory of the virus in advance in the design of next-generation vaccines requires investigation. Here, we performed a comprehensive study of 11,650,487 SARS-CoV-2 sequences, which revealed that the SARS-CoV-2 spike (S) protein evolved not randomly but into directional paths of either high infectivity plus low immune resistance or low infectivity plus high immune resistance. The viral infectivity and immune resistance of variants are generally incompatible, except for limited variants such as Beta and Kappa. The Omicron variant has the highest immune resistance but showed high infectivity in only one of the tested cell lines. To provide cross-clade immunity against variants that undergo diverse evolutionary pathways, we designed a new pan-vaccine antigen (Span). Span was designed by analyzing the homology of 2675 SARS-CoV-2 S protein sequences from the NCBI database before the Delta variant emerged. The refined Span protein harbors high-frequency residues at given positions that reflect cross-clade generality in sequence evolution. Compared with a prototype wild-type (Swt) vaccine, which, when administered to mice, induced serum with decreased neutralization activity against emerging variants, Span vaccination of mice elicited broad immunity to a wide range of variants, including those that emerged after our design. Moreover, vaccinating mice with a heterologous Span booster conferred complete protection against lethal infection with the Omicron variant. Our results highlight the importance and feasibility of a universal vaccine to fight against SARS-CoV-2 antigenic drift.

Disaster propagation in interdependent networks with different link patterns.

Disaster propagation in complex, interdependent, and multilayered networks has attracted considerable research interest in recent years. In this paper, we propose a model that combines two dynamic mechanisms, i.e., the spreading of failure in layer-dependent networks, where each node in a layer depends on one in another layer. We first investigate the robustness of the Erdos-Rényi (ER)-ER, scale-free (sf)-ER, and sf-sf pattern of interdependent networks against cascading failure with different probabilities of triggering, and then use the random link, assortative link, and disassortative link patterns between the networks to analyze the scope of propagation of failure. The numerical results show that with increasing probability of triggering, the number of damaged nodes in both layers increased and the robustness of the scale-free network to random failures decreased due to the interdependence. Regardless of the topological structure, the two layers eventually tended to have similar failure characteristics due to their interdependence. In addition, the different link patterns had a significant effect on enhancing disaster propagation in interdependent networks.

Fusion Expression and Fibrinolytic Activity of rPA/SP-B.

BACKGROUND: Pulmonary surfactant dysfunction is an important pathological factor in acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF). OBJECTIVE: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity in acute lung injury cell model were studied. METHODS: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion protein was determined by fibrin-agarose plate method. RESULTS: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149 cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and the fusion protein still had good enzyme activity. CONCLUSION: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model.

ANXA2 Facilitates Enterovirus 71 Infection by Interacting with 3D Polymerase and PI4KB to Assist the Assembly of Replication Organelles.

Similar to that of other enteroviruses, the replication of enterovirus 71 (EV71) occurs on rearranged membranous structures called replication organelles (ROs). Phosphatidylinositol 4-kinase III (PI4KB), which is required by enteroviruses for RO formation, yields phosphatidylinositol-4-phosphate (PI4P) on ROs. PI4P then binds and induces conformational changes in the RNA-dependent RNA polymerase (RdRp) to modulate RdRp activity. Here, we targeted 3D polymerase, the core enzyme of EV71 ROs, and found that the host factor Annexin A2 (ANXA2) can interact with 3D polymerase and promote the replication of EV71. Then, an experiment showed that the annexin domain of ANXA2, which possesses membrane-binding capacity, mediates the interaction of ANXA2 with EV71 3D polymerase. Further research showed that ANXA2 is localized on ROs and interacts with PI4KB. Overexpression of ANXA2 stimulated the formation of PI4P, and the level of PI4P was decreased in ANXA2-knockout cells. Furthermore, ANXA2, PI4KB, and 3D were shown to be localized to the viral RNA replication site, where they form a higher-order protein complex, and the presence of ANXA2 promoted the PI4KB-3D interaction. Altogether, our data provide new insight into the role of ANXA2 in facilitating formation of the EV71 RNA replication complex.

Identification of dibucaine derivatives as novel potent enterovirus 2C helicase inhibitors: In vitro, in vivo, and combination therapy study.

Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot and mouth disease (HFMD) which seriously threatened the safety and lives of infants and young children. However, there are no licensed direct antiviral agents to cure the HFMD. In this study, a series of quinoline formamide analogues as effective enterovirus inhibitors were developed, subsequent systematic structure-activity relationship (SAR) studies demonstrated that these quinoline formamide analogues exhibited good potency to treat EV-A71 infection. As described, the most efficient EV-A71 inhibitor 6i showed good anti-EV-A71 activity (EC50 = 1.238 µM) in RD cells. Furthermore, compound 6i could effectively prevent death of virus infected mice at dose of 6 mg/kg. When combined with emetine (0.1 mg/kg), this treatment could completely prevent the clinical symptoms and death of virus infected mice. Mechanism study indicated that compound 6i inhibited EV-A71 via targeting 2C helicase, thus impeding RNA remodeling and metabolism. Taken together, these data indicated that 6i is a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.

Analytical comparisons of SARS-COV-2 detection by qRT-PCR and ddPCR with multiple primer/probe sets.

Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10-4 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.

ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens.

Quantitative real time PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load specimens and the limitations of RT-PCR, significant numbers of false negative reports are inevitable, which results in failure to timely diagnose, cut off transmission, and assess discharge criteria. To improve this situation, an optimized droplet digital PCR (ddPCR) was used for detection of SARS-CoV-2, which showed that the limit of detection of ddPCR is significantly lower than that of RT-PCR. We further explored the feasibility of ddPCR to detect SARS-CoV-2 RNA from 77 patients, and compared with RT-PCR in terms of the diagnostic accuracy based on the results of follow-up survey. 26 patients of COVID-19 with negative RT-PCR reports were reported as positive by ddPCR. The sensitivity, specificity, PPV, NPV, negative likelihood ratio (NLR) and accuracy were improved from 40% (95% CI: 27-55%), 100% (95% CI: 54-100%), 100%, 16% (95% CI: 13-19%), 0.6 (95% CI: 0.48-0.75) and 47% (95% CI: 33-60%) for RT-PCR to 94% (95% CI: 83-99%), 100% (95% CI: 48-100%), 100%, 63% (95% CI: 36-83%), 0.06 (95% CI: 0.02-0.18), and 95% (95% CI: 84-99%) for ddPCR, respectively. Moreover, 6/14 (42.9%) convalescents were detected as positive by ddPCR at 5-12 days post discharge. Overall, ddPCR shows superiority for clinical diagnosis of SARS-CoV-2 to reduce the false negative reports, which could be a powerful complement to the RT-PCR.

Luminescence behaviour of Mn(4+) ions in seven coordination environments of K3ZrF7.

A single-phased Mn(4+) doped fluorozirconate red phosphor, K3ZrF7:Mn(4+), has been successfully synthesized. Its structure, morphology, composition and optical properties were investigated by X-ray diffraction, scanning electron microscopy, energy-dispersive X-ray spectroscopy, atomic absorption spectroscopy, diffuse reflectance spectroscopy, photoluminescence spectroscopy and by using luminescence decay curves. It was found that Mn(4+) ions only coordinating with seven F(-) anions in a K3ZrF7 crystal field can possess intense red emission under blue light illumination. Mixing the obtained K3ZrF7:Mn(4+) red phosphor with commercial Y3Al5O12:Ce(3+) and coating the mixture on a blue-GaN chip, obvious warm white light with a low correlated color temperature (2970 K) and a high color rendering index (Ra = 91.4 and R9 = 72) were achieved from white light-emitting diode devices.