One-step-one-pot hydrothermally derived metal-organic-framework-nanohybrids for integrated point-of-care diagnostics of SARS-CoV-2 viral antigen/pseudovirus utilizing electrochemical biosensor chip.

The COVID-19 pandemic has become a global catastrophe, affecting the health and economy of the human community. It is required to mitigate the impact of pandemics by developing rapid molecular diagnostics for SARS-CoV-2 virus detection. In this context, developing a rapid point-of-care (POC) diagnostic test is a holistic approach to the prevention of COVID-19. In this context, this study aims at presenting a real-time, biosensor chip for improved molecular diagnostics including recombinant SARS-CoV-2 spike glycoprotein and SARS-CoV-2 pseudovirus detection based on one-step-one-pot hydrothermally derived CoFeBDCNH2-CoFe2O4 MOF-nanohybrids. This study was tested on a PalmSens-EmStat Go POC device, showing a limit of detection (LOD) for recombinant SARS-CoV-2 spike glycoprotein of 6.68 fg/mL and 6.20 fg/mL in buffer and 10% serum-containing media, respectively. To validate virus detection in the POC platform, an electrochemical instrument (CHI6116E) was used to perform dose dependent studies under similar experimental conditions to the handheld device. The results obtained from these studies were comparable indicating the capability and high detection electrochemical performance of MOF nanocomposite derived from one-step-one-pot hydrothermal synthesis for SARS-CoV-2 detection for the first time. Further, the performance of the sensor was tested in the presence of Omicron BA.2 and wild-type D614G pseudoviruses.

The 5'UTR of HCoV-OC43 adopts a topologically constrained structure to intrinsically repress translation.

The emergence of SARS-CoV-2, which is responsible for the COVID-19 pandemic, has highlighted the need for rapid characterization of viral mechanisms associated with cellular pathogenesis. Viral UTRs represent conserved genomic elements that contribute to such mechanisms. Structural details of most CoV UTRs are not available, however. Experimental approaches are needed to allow for the facile generation of high-quality viral RNA tertiary structural models, which can facilitate comparative mechanistic efforts. By integrating experimental and computational techniques, we herein report the efficient characterization of conserved RNA structures within the 5'UTR of the HCoV-OC43 genome, a lab-tractable model coronavirus. We provide evidence that the 5'UTR folds into a structure with well-defined stem-loops (SLs) as determined by chemical probing and direct detection of hydrogen bonds by NMR. We combine experimental base-pair restraints with global structural information from SAXS to generate a 3D model that reveals that SL1-4 adopts a topologically constrained structure wherein SLs 3 and 4 coaxially stack. Coaxial stacking is mediated by short linker nucleotides and allows SLs 1 to 2 to sample different cojoint orientations by pivoting about the SL3,4 helical axis. To evaluate the functional relevance of the SL3,4 coaxial helix, we engineered luciferase reporter constructs harboring the HCoV-OC43 5'UTR with mutations designed to abrogate coaxial stacking. Our results reveal that the SL3,4 helix intrinsically represses translation efficiency since the destabilizing mutations correlate with increased luciferase expression relative to wildtype without affecting reporter mRNA levels, thus highlighting how the 5'UTR structure contributes to the viral mechanism.

Factors influencing neutralizing antibody titers elicited by coronavirus disease 2019 vaccines.

The World Health Organization has highlighted the importance of an international standard (IS) for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) neutralizing antibody titer detection to calibrate diagnostic techniques. We applied an IS to calibrate neutralizing antibody titers (NTs) (international units/mL) in response to coronavirus disease 2019 (COVID-19) vaccination. Moreover, the association between different factors and neutralizing antibodies was analyzed. A total of 1667 serum samples were collected from participants receiving different COVID-19 vaccines. Antibody titers were determined by a microneutralization assay using live viruses in a biosafety level 3 (BSL-3) laboratory and a commercial serological MeDiPro kit. The titer determined using the MeDiPro kit was highly correlated with the NT determined using live viruses and calibrated using IS. Fever and antipyretic analgesic treatment were related to neutralizing antibody responses in ChAdOx1-S and BNT162b2 vaccinations. Individuals with diabetes showed a low NT elicited by MVC-COV1901. Individuals with hypertension receiving the BNT162b2 vaccine had lower NTs than those without hypertension. Our study provided the international unit (IU) values of NTs in vaccinated individuals for the development of vaccines and implementation of non-inferiority trials. Correlation of the influencing factors with NTs can provide an indicator for selecting COVID-19 vaccines based on personal attributes.

Titers and breadth of neutralizing antibodies against SARS-CoV-2 variants after heterologous booster vaccination in health care workers primed with two doses of ChAdOx1 nCov-19: A single-blinded, randomized clinical trial.

OBJECTIVES: We conducted a single-blinded, randomized trial to evaluate the safety, reactogenicity, and immunogenicity of heterologous booster vaccination in health care workers (HCW) who had received two doses of ChAdOx1 nCov-19. METHODS: HCW who had at least 90 days after the second dose were enrolled to receive one of the four vaccines: BNT162b2 (30 µg), half-dose mRNA-1273 (50 µg), mRNA-1273 (100 µg), and MVC-COV1901 (15 µg). The primary outcomes were humoral and cellular immunogenicity and secondary outcomes assessed safety and reactogenicity at 28 days post-booster. RESULTS: MVC-COV1901 Three hundred and forty HCW were enrolled: 83 received BNT162b2 (2 excluded), 85 half-dose mRNA-1273, 85 mRNA-1273, and 85 MVC-COV1901. mRNA vaccines had more reactogenicity than protein vaccine. The fold-rise of anti-spike IgG geometric mean titer was 8.4 (95% CI 6.8-10.4) for MVC-COV1901, 32.2 (27.2-38.1) for BNT162b2, 47.6 (40.8-55.6) for half-dose mRNA-1273 and 63.2 (53.6-74.6) for mRNA-1273. The live virus microneutralization assays (LVMNA) against the wild type, alpha and delta variants were consistent with anti-spike IgG for all booster vaccines. The LVMNA in the four groups against omicron BA.1 variant were 6.4 to 13.5 times lower than those against the wild type. All booster vaccines induced a comparable T cell response. CONCLUSIONS: Third dose booster not only increases neutralizing antibody titer but also enhances antibody breadth against SARS-CoV-2 variants. mRNA vaccines are preferred booster vaccines for those who received primary series of ChAdOx1 nCov-19.

Molecular Virology of SARS-CoV-2 and Related Coronaviruses.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The global COVID-19 pandemic continues to threaten the lives of hundreds of millions of people, with a severe negative impact on the global economy. Although several COVID-19 vaccines are currently being administered, none of them is 100% effective. Moreover, SARS-CoV-2 variants remain an important worldwide public health issue. Hence, the accelerated development of efficacious antiviral agents is urgently needed. Coronavirus depends on various host cell factors for replication. An ongoing research objective is the identification of host factors that could be exploited as targets for drugs and compounds effective against SARS-CoV-2. In the present review, we discuss the molecular mechanisms of SARS-CoV-2 and related coronaviruses, focusing on the host factors or pathways involved in SARS-CoV-2 replication that have been identified by genome-wide CRISPR screening.

Quantifying Neutralizing Antibodies in Patients with COVID-19 by a Two-Variable Generalized Additive Model.

Considering the urgent demand for faster methods to quantify neutralizing antibody titers in patients with coronavirus (CoV) disease 2019 (COVID-19), developing an analytical model or method to replace the conventional virus neutralization test (NT) is essential. Moreover, a "COVID-19 immunity passport" is currently being proposed as a certification for people who travel internationally. Therefore, an enzyme-linked immunosorbent assay (ELISA) was designed to detect severe acute respiratory syndrome CoV 2 (SARS-CoV-2)-neutralizing antibodies in serum, which is based on the binding affinity of SARS-CoV-2 viral spike protein 1 (S1) and the viral spike protein receptor-binding domain (RBD) to antibodies. The RBD is considered the major binding region of neutralizing antibodies. Furthermore, S1 covers the RBD and several other regions, which are also important for neutralizing antibody binding. In this study, we assessed 144 clinical specimens, including those from patients with PCR-confirmed SARS-CoV-2 infections and healthy donors, using both the NT and ELISA. The ELISA results analyzed by spline regression and the two-variable generalized additive model precisely reflected the NT value, and the correlation between predicted and actual NT values was as high as 0.917. Therefore, our method serves as a surrogate to quantify neutralizing antibody titer. The analytic method and platform used in this study present a new perspective for serological testing of SARS-CoV-2 infection and have clinical potential to assess vaccine efficacy. IMPORTANCE Herein, we present a new approach for serological testing for SARS-CoV-2 antibodies using innovative laboratory methods that demonstrate a combination of biology and mathematics. The traditional virus neutralization test is the gold standard method; however, it is time-consuming and poses a risk to medical personnel. Thus, there is a demand for methods that rapidly quantify neutralizing antibody titers in patients with COVID-19 or examine vaccine efficacy at a biosafety level 2 containment facility. Therefore, we used a two-variable generalized additive model to analyze the results of the enzyme-linked immunosorbent assay and found the method to serve as a surrogate to quantify neutralizing antibody titers. This methodology has potential for clinical use in assessing vaccine efficacy.

Acyl-Coenzyme A Synthetase Long-Chain Family Member 4 Is Involved in Viral Replication Organelle Formation and Facilitates Virus Replication via Ferroptosis.

Enterovirus infections can cause severe complications, such as poliomyelitis, encephalitis, myocarditis, meningitis, neurological pulmonary edema, and even death. Here, we used genome-wide CRISPR screens to gain new insight into the mechanism by which enteroviruses co-opt host pathways to potentiate replication and propagation. We found that acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) is involved in viral replication organelle formation. ACSL4 is a key component of ferroptosis, an iron-dependent, nonapoptotic programmed cell death. Our results indicated that enteroviruses and coronaviruses can induce ferroptosis via ACSL4. Most importantly, ferroptosis inhibitors, including two FDA-approved drugs, rosiglitazone (ROSI; ACSL4 inhibitor) and pioglitazone (PIO; ACSL4 inhibitor), decreased the viral load of human enteroviruses and coronaviruses, suggesting that ACSL4 is a target for counteracting viral infection. IMPORTANCE We provide the first evidence for the role of ACSL4 in enterovirus replication organelle formation. Moreover, both enteroviruses and coronaviruses induce ferroptosis via ACSL4. These findings establish a novel regulatory mechanism for viral replication. The inhibition of ACSL4 by ferroptosis inhibitors can reduce viral yields of enteroviruses and coronaviruses, including SARS-CoV-2, implying that ACSL4-mediated ferroptosis is a promising therapeutic target for viral diseases.

Stability of SARS-CoV-2 Spike G614 Variant Surpasses That of the D614 Variant after Cold Storage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carrying the D614G mutation on the spike protein is the predominant circulating variant and is associated with enhanced infectivity. However, whether this dominant variant can potentially spread through the cold chain and whether the spike protein affects virus stability after cold storage remain unclear. To compare the infectivity of two SARS-CoV-2 variants, namely, SARS-CoV-2 variants with spike protein with the D614 mutation (S-D614) and G614 mutation (S-G614), after different periods of refrigeration (4°C) and freezing (-20°C). We also determined the integrity of the viral RNA and the ability of the spike protein to bind angiotensin-converting enzyme 2 (ACE2) after storage at these conditions. The results showed that SARS-CoV-2 was more stable and infectious after storage at -20°C than at 4°C. Particularly, the S-G614 variant was found to be more stable than the S-D614 variant. The spike protein of the S-G614 variant had better binding ability with the ACE2 receptor than that of the S-D614 variant after storage at -20°C for up to 30 days. Our findings revealed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, and their stability and infectivity up to 30 days depends on the spike variant. Stability and infectivity are related to each other, and the higher stability of S-G614 compared to that of S-D614 may contribute to rapid viral spread of the S-G614 variant.IMPORTANCE It has been observed that variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more stable and infectious after storage at -20°C than at 4°C. A SARS-CoV-2 S-D614G variant is currently the most dominant variant in circulation and is associated with enhanced infectivity. We compared the stability of two SARS-CoV-2 variants: the early S-D614 variant carrying the D614 spike protein and the new S-G614 variant carrying the G614 spike protein, stored at both 4°C and -20°C for different periods. We observed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, which further depends on the spike variant, that is, the ability of the spike protein to bind with the ACE2 receptor with higher efficiency. The high stability of the S-G614 variant also explains its rapid spread and infectivity. Therefore, precautions should be taken during and after handling food preserved under cold conditions.

Development of flexible electrochemical impedance spectroscopy-based biosensing platform for rapid screening of SARS-CoV-2 inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the cells through the binding of its spike protein (S-protein) to the cell surface-expressing angiotensin-converting enzyme 2 (ACE2). Thus, inhibition of S-protein-ACE2 binding may impede SARS-CoV-2 cell entry and attenuate the progression of Coronavirus disease 2019 (COVID-19). In this study, an electrochemical impedance spectroscopy-based biosensing platform consisting of a recombinant ACE2-coated palladium nano-thin-film electrode as the core sensing element was fabricated for the screening of potential inhibitors against S-protein-ACE2 binding. The platform could detect interference of small analytes against S-protein-ACE2 binding at low analyte concentration and small volume (0.1 µg/mL and ~1 µL, estimated total analyte consumption < 4 pg) within 21 min. Thus, a few potential inhibitors of S-protein-ACE2 binding were identified. This includes (2S,3aS,6aS)-1-((S)-N-((S)-1-Carboxy-3-phenylpropyl)alanyl)tetrahydrocyclopenta[b] pyrrole-2-carboxylic acid (ramiprilat) and (2S,3aS,7aS)-1-[(2S)-2-[[(2S)-1-Carboxybutyl]amino]propanoyl]-2,3,3a,4,5,6,7,7a-octahydroindole-2-carboxylic acid (perindoprilat) that reduced the binding affinity of S-protein to ACE2 by 72% and 67%; and SARS-CoV-2 in vitro infectivity to the ACE2-expressing human oral cavity squamous carcinoma cells (OEC-M1) by 36.4 and 20.1%, respectively, compared to the PBS control. These findings demonstrated the usefulness of the developed biosensing platform for the rapid screening of modulators for S-protein-ACE2 binding.

Discovery of M Protease Inhibitors Encoded by SARS-CoV-2.

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.

Engineering a novel IgG-like bispecific antibody against enterovirus A71.

Frequent outbreaks of enterovirus A71 (EVA71) occur in the Asia-Pacific area, and these are closely associated with severe neurological symptoms in young children. No effective antiviral therapy is currently available for the treatment of EVA71 infection. The development of monoclonal antibodies (mAbs) has demonstrated promise as a novel therapy for the prevention and treatment of infectious diseases. Several medical conditions have been treated using bispecific or multi-specific antibodies that recognize two or more distinct epitopes simultaneously. However, bispecific or multi-specific antibodies often encounter protein expression and product stability problems. In this study, we developed an IgG-like bispecific antibody (E18-F1) comprising two anti-EVA71 antibodies: E18 mAb and llama-derived F1 single-domain antibody. E18-F1 was demonstrated to exhibit superior binding affinity and antiviral activity compared with E18 or F1. Additionally, E18-F1 not only improved survival rate, but also reduced clinical signs in human SCARB2 receptor (hSCARB2) transgenic mice challenged with a lethal dose of EVA71. Altogether, our results reveal that E18-F1 is a simple format bispecific antibody with promising antiviral activity for EVA71.

Antivirals and vaccines for Enterovirus A71.

Enterovirus A71 (EV-A71) is an important emerging virus posing a threat to children under five years old. EV-A71 infection in infants or young children can cause hand-foot-and-mouth disease, herpangina, or severe neurological complications. However, there are still no effective antivirals for treatment of these infections. In this review, we summarize the antiviral compounds developed to date based on various targets of the EV-A71 life cycle. Moreover, development of a vaccine would be the most effective approach to prevent EV-A71 infection. Therefore, we also summarize the development and clinical progress of various candidate EV-A71 vaccines, including inactivated whole virus, recombinant VP1 protein, synthetic peptides, viral-like particles, and live attenuated vaccines.

Control of the negative IRES trans-acting factor KHSRP by ubiquitination.

Cells and viruses can utilize internal ribosome entry sites (IRES) to drive translation when cap-dependent translation is inhibited by stress or viral factors. IRES trans-acting factors (ITAFs) are known to participate in such cap-independent translation, but there are gaps in the understanding as to how ITAFs, particularly negative ITAFs, regulate IRES-driven translation. This study found that Lys109, Lys121 and Lys122 represent critical ubiquitination sites for far upstream element-binding protein 2 (KHSRP, also known as KH-type splicing regulatory protein or FBP2), a negative ITAF. Mutations at these sites subsequently reduced KHSRP ubiquitination and abolished its inhibitory effect on IRES-driven translation. We further found that interaction between the Kelch domain of Kelch-like protein 12 (KLHL12) and the C-terminal domain of KHSRP contributed to KHSRP ubiquitination, leading to downregulation of enterovirus IRES-mediated translation in infected cells and increased competition against other positive ITAFs. Together, these results show that ubiquitination can exert control over IRES-driven translation via modification of ITAFs, and to the best of our knowledge, this is the first description of such a regulatory mechanism for IRES-dependent translation.

Additive Promotion of Viral Internal Ribosome Entry Site-Mediated Translation by Far Upstream Element-Binding Protein 1 and an Enterovirus 71-Induced Cleavage Product.

The 5' untranslated region (5' UTR) of the enterovirus 71 (EV71) RNA genome contains an internal ribosome entry site (IRES) that is indispensable for viral protein translation. Due to the limited coding capacity of their RNA genomes, EV71 and other picornaviruses typically recruit host factors, known as IRES trans-acting factors (ITAFs), to mediate IRES-dependent translation. Here, we show that EV71 viral proteinase 2A is capable of cleaving far upstream element-binding protein 1 (FBP1), a positive ITAF that directly binds to the EV71 5' UTR linker region to promote viral IRES-driven translation. The cleavage occurs at the Gly-371 residue of FBP1 during the EV71 infection process, and this generates a functional cleavage product, FBP11-371. Interestingly, the cleavage product acts to promote viral IRES activity. Footprinting analysis and gel mobility shift assay results showed that FBP11-371 similarly binds to the EV71 5' UTR linker region, but at a different site from full-length FBP1; moreover, FBP1 and FBP11-371 were found to act additively to promote IRES-mediated translation and virus yield. Our findings expand the current understanding of virus-host interactions with regard to viral recruitment and modulation of ITAFs, and provide new insights into translational control during viral infection.

A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells.

The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (>10(5) copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5' untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES.

Update on the development of enterovirus 71 vaccines.

INTRODUCTION: Enterovirus 71 (EV71) is an etiological agent that causes severe neurological complications in children. EV71 outbreaks have occurred throughout the Asia-Pacific region, posing a severe global public health threat; however, no specific therapeutic strategy exists for treating EV71-infected children. AREAS COVERED: Five manufacturers have produced inactivated EV71 whole virus vaccines in mainland China, Taiwan, and Singapore, which have completed Phase III (mainland China) and Phase I (Taiwan and Singapore) clinical trials. Various EV71 vaccine candidates are being researched in animal models, including live-attenuated virus vaccine, recombinant VP1 vaccine, VP1-based DNA vaccine, synthetic peptide vaccine and virus-like particle vaccine. In this review, the present situation is summarized, and feasible improvements to the EV71 vaccine are explored. EXPERT OPINION: Although inactivated EV71 vaccines are safe, efficient and elicit strong immune responses to protect adults, children and infants against infection, the quality control of production is critical.

Enterovirus 71 infection cleaves a negative regulator for viral internal ribosomal entry site-driven translation.

Far-upstream element-binding protein 2 (FBP2) is an internal ribosomal entry site (IRES) trans-acting factor (ITAF) that negatively regulates enterovirus 71 (EV71) translation. This study shows that EV71 infection cleaved FBP2. Live EV71 and the EV71 replicon (but not UV-inactivated virus particles) induced FBP2 cleavage, suggesting that viral replication results in FBP2 cleavage. The results also showed that virus-induced proteasome, autophagy, and caspase activity co-contribute to EV71-induced FBP2 cleavage. Using FLAG-fused FBP2, we mapped the potential cleavage fragments of FBP2 in infected cells. We also found that FBP2 altered its function when its carboxyl terminus was cleaved. This study presents a mechanism for virus-induced cellular events to cleave a negative regulator for viral IRES-driven translation.

Far upstream element binding protein 1 binds the internal ribosomal entry site of enterovirus 71 and enhances viral translation and viral growth.

Enterovirus 71 (EV71) is associated with severe neurological disorders in children, and has been implicated as the infectious agent in several large-scale outbreaks with mortalities. Upon infection, the viral RNA is translated in a cap-independent manner to yield a large polyprotein precursor. This mechanism relies on the presence of an internal ribosome entry site (IRES) element within the 5'-untranslated region. Virus-host interactions in EV71-infected cells are crucial in assisting this process. We identified a novel positive IRES trans-acting factor, far upstream element binding protein 1 (FBP1). Using binding assays, we mapped the RNA determinants within the EV71 IRES responsible for FBP1 binding and mapped the protein domains involved in this interaction. We also demonstrated that during EV71 infection, the nuclear protein FBP1 is enriched in cytoplasm where viral replication occurs. Moreover, we showed that FBP1 acts as a positive regulator of EV71 replication by competing with negative ITAF for EV71 IRES binding. These new findings may provide a route to new anti-viral therapy.