Evaluation of the Antiviral Efficacy of Subcutaneous Nafamostat Formulated with Glycyrrhizic Acid against SARS-CoV-2 in a Murine Model.

The ongoing COVID-19 pandemic highlights the urgent need for effective antiviral agents and vaccines. Drug repositioning, which involves modifying existing drugs, offers a promising approach for expediting the development of novel therapeutics. In this study, we developed a new drug, MDB-MDB-601a-NM, by modifying the existing drug nafamostat (NM) with the incorporation of glycyrrhizic acid (GA). We assessed the pharmacokinetic profiles of MDB-601a-NM and nafamostat in Sprague-Dawley rats, revealing rapid clearance of nafamostat and sustained drug concentration of MDB-601a-NM after subcutaneous administration. Single-dose toxicity studies showed potential toxicity and persistent swelling at the injection site with high-dose administration of MDB-601a-NM. Furthermore, we evaluated the efficacy of MDB-601a-NM in protecting against SARS-CoV-2 infection using the K18 hACE-2 transgenic mouse model. Mice treated with 60 mg/kg and 100 mg/kg of MDB-601a-NM exhibited improved protectivity in terms of weight loss and survival rates compared to the nafamostat-treated group. Histopathological analysis revealed dose-dependent improvements in histopathological changes and enhanced inhibitory efficacy in MDB-601a-NM-treated groups. Notably, no viral replication was detected in the brain tissue when mice were treated with 60 mg/kg and 100 mg/kg of MDB-601a-NM. Our developed MDB-601a-NM, a modified Nafamostat with glycyrrhizic acid, shows improved protectivity against SARS-CoV-2 infection. Its sustained drug concentration after subcutaneous administration and dose-dependent improvements makes it a promising therapeutic option.

A Rapid Method for Generating Infectious SARS-CoV-2 and Variants Using Mutagenesis and Circular Polymerase Extension Cloning.

The appearance of SARS-CoV-2 variants in late 2020 raised alarming global public health concerns. Despite continued scientific progress, the genetic profiles of these variants bring changes in viral properties that threaten vaccine efficacy. Thus, it is critically important to investigate the biologic profiles and significance of these evolving variants. In this study, we demonstrate the application of circular polymerase extension cloning (CPEC) to the generation of full-length clones of SARS-CoV-2. We report that, combined with a specific primer design scheme, this yields a simpler, uncomplicated, and versatile approach for engineering SARS-CoV-2 variants with high viral recovery efficiency. This new strategy for genomic engineering of SARS-CoV-2 variants was implemented and evaluated for its efficiency in generating point mutations (K417N, L452R, E484K, N501Y, D614G, P681H, P681R, Δ69-70, Δ157-158, E484K+N501Y, and Ins-38F) and multiple mutations (N501Y/D614G and E484K/N501Y/D614G), as well as a large truncation (ΔORF7A) and insertion (GFP). The application of CPEC to mutagenesis also allows the inclusion of a confirmatory step prior to assembly and transfection. This method could be of value in the molecular characterization of emerging SARS-CoV-2 variants as well as the development and testing of vaccines, therapeutic antibodies, and antivirals. IMPORTANCE Since the first emergence of the SARS-CoV-2 variant in late 2020, novel variants have been continuously introduced to the human population, causing severe public health threats. In general, because these variants acquire new genetic mutation/s, it is critical to analyze the biological function of viruses that such mutations can confer. Therefore, we devised a method that can construct SARS-CoV-2 infectious clones and their variants rapidly and efficiently. The method was developed based on a PCR-based circular polymerase extension cloning (CPEC) combined with a specific primer design scheme. The efficiency of the newly designed method was evaluated by generating SARS-CoV-2 variants with single point mutations, multiple point mutations, and a large truncation and insertion. This method could be of value for the molecular characterization of emerging SARS-CoV-2 variants and the development and testing of vaccines and antiviral agents.

Antiviral Activities of High Energy E-Beam Induced Copper Nanoparticles against H1N1 Influenza Virus.

The pandemic outbreak of COVID-19 in the year of 2020 that drastically changed everyone's life has raised the urgent and intense need for the development of more efficacious antiviral material. This study was designed to develop copper nanoparticles (Cu NPs) as an antiviral agent and to validate the antiviral activities of developed copper NP. The Cu NPs were synthesized using a high energy electron beam, and the characteristic morphologies and antiviral activities of Cu NPs were evaluated. We found that Cu NPs are of spherical shape and uniformly distributed, with a diameter of around 100 nm, as opposed to the irregular shape of commercially available copper microparticles (Cu MPs). An X-ray diffraction analysis showed the presence of Cu and no copper oxide II and I in the Cu NPs. A virus inactivation assay revealed no visible viral DNA after 10- and 30-min treatment of H1N1 virus with the Cu NPs. The infectivity of the Cu NPs-treated H1N1 virus significantly decreased compared with that of the Cu MPs-treated H1N1 virus. The viability of A549 bronchial and Madin-Darby Canine Kidney (MDCK) cells infected with Cu NPs-treated H1N1 was significantly higher than those infected with Cu MPs-treated H1N1 virus. We also found cells infected with Cu NPs-treated H1N1 virus exhibited a markedly decreased presence of virus nucleoprotein (NuP), an influenza virus-specific structural protein, compared with cells infected with Cu MPs-treated H1N1 virus. Taken together, our study shows that Cu NPs are a more effective and efficacious antiviral agent compared with Cu MPs and offer promising opportunities for the prevention of devastatingly infectious diseases.

Self-limited myocarditis presenting with chest pain and ST segment elevation in adolescents after vaccination with the BNT162b2 mRNA vaccine.

Two adolescent males presented within 3 days after the first and second dose of the BNT162b2 vaccine with chest pain. Elevated troponin levels, ST segment elevation, and enhancement of the myocardium in cardiac MRI suggested myocarditis. Left ventricular function remained normal, symptoms resolved, and patients were discharged in 4 days. BNT162b2 vaccine may be associated with self-limited myocarditis in youth.

Peptide Nucleic Acid (PNA)-Enhanced Specificity of a Dual-Target Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Assay for the Detection and Differentiation of SARS-CoV-2 from Related Viruses.

The threat posed by coronaviruses to human health has necessitated the development of a highly specific and sensitive viral detection method that could differentiate between the currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related coronaviruses (SARSr-CoVs). In this study, we developed a peptide nucleic acid (PNA)-based real-time quantitative polymerase chain reaction (RT-qPCR) assay targeting the N gene to efficiently discriminate SARS-CoV-2 from other SARSr-CoVs in human clinical samples. Without compromising the sensitivity, this method significantly enhanced the specificity of SARS-CoV-2 detection by 100-fold as compared to conventional RT-qPCR. In addition, we designed an RT-qPCR method for the sensitive and universal detection of ORF3ab-E genes of SARSr-CoV with a limit of detection (LOD) of 3.3 RNA copies per microliter. Thus, the developed assay serves as a confirmative dual-target detection method. Our PNA-mediated dual-target RT-qPCR assay can detect clinical SARS-CoV-2 samples in the range of 18.10-35.19 Ct values with an 82.6-100% detection rate. Furthermore, our assay showed no cross-reactions with other coronaviruses such as human coronaviruses (229E, NL63, and OC43) and Middle East respiratory syndrome coronavirus, influenza viruses (Type B, H1N1, H3N2, HPAI H5Nx, and H7N9), and other respiratory disease-causing viruses (MPV, RSV A, RSV B, PIV, AdV, and HRV). We, thus, developed a PNA-based RT-qPCR assay that differentiates emerging pathogens such as SARS-CoV-2 from closely related viruses such as SARSr-CoV and allows diagnosis of infections related to already identified or new coronavirus strains.

Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2.

The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 102 RNA copies close to that of qRT-PCR. Notably, the assay has exhibited a rapid detection span of 30 min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9), and other respiratory disease-causing viruses (RSVA, RSVB, ADV, PIV, MPV, and HRV). Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.