BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir.

Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128's primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently-but complementary-with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect-a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

Boosting with Multiple Doses of mRNA Vaccine after Priming with Two Doses of Protein Subunit Vaccine MVC-COV1901 Elicited Robust Humoral and Cellular Immune Responses against Emerging SARS-CoV-2 Variants.

Confronted with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, such as Delta and Omicron, with high infectivity and immune evasion capacity, vaccination remains the most effective tool to prevent infection and severe illness. However, heterologous vaccination of mRNA vaccines primed with protein subunit vaccines had not been evaluated before the current study. Since subunit vaccine MVC-COV1901 (MVC) has been granted emergency use authorization in Taiwan, in this study, we explored the humoral and cellular immune responses to additional third (2× MVC/Mod) and fourth (2× MVC/2× Mod) doses of mRNA-1273 (Mod) after priming with two doses of subunit vaccine MVC against the emerging variants. We found a 12.3- to 16.1-fold increase in antibodies targeting the receptor binding domain (RBD) of the Delta variant with 2× MVC/Mod compared to two doses of MVC (2× MVC) or AZD1222 (2× AZ) regimens and a 26- to 32.2-fold improvement in neutralizing potency against the Omicron variant (BA.1). Besides, the numbers of gamma interferon (IFN-γ)-secreting T cells induced by 2× MVC/Mod were also elevated 3.5-fold and 3.7- to 4.3-fold for the wild type and Delta variant. However, boosting with a fourth dose of Mod (2× MVC/2× Mod) after the 2× MVC/Mod regimen failed to significantly improve the immune responses. Moreover, all vaccination schedules showed reduced neutralizing activity against the Omicron variant. Collectively, our results suggested that the third or fourth dose booster vaccination with mRNA vaccine after priming with two doses of protein subunit vaccine could elicit stronger humoral and cellular immune responses. These findings could provide a future global heterologous boosting strategy against COVID-19. IMPORTANCE Vaccination is the most important strategy to combat the COVID-19 outbreak; however, it remains to be determined whether heterologous prime-boost regimens could induce equal or even stronger immune responses against SARS-CoV-2. Here, we showed that boosting the additional doses of mRNA-1273 (Mod) priming with two doses of MVC-COV1901 (MVC) (2× MVC/Mod) improved humoral and cellular immunity compared to two doses of AZD1222 (2× AZ) or MVC (2× MVC) against SARS-CoV-2 variants. However, the Omicron variant showed strong immune evasion ability for all vaccination schedules. Our findings provided evidence supporting that heterologous vaccination by boosting with mRNA vaccine after priming with two doses of protein subunit vaccine could strongly promote humoral and cellular immune responses against the emerging SARS-CoV-2 variants.

Humoral, Cellular and Cytokine Immune Responses Against SARS-CoV-2 Variants in COVID-19 Convalescent and Confirmed Patients With Different Disease Severities.

Objectives: To assess humoral and cellular immune responses against SARS-CoV-2 variants in COVID-19 convalescent and confirmed patients, to explore the correlation between disease severity, humoral immunity, and cytokines/chemokines in confirmed patients, and to evaluate the ADE risk of SARS-CoV-2. Methods: Anti-RBD IgG were quantified using an ELISA. Neutralization potency was measured using pseudovirus and real virus. Cellular immunity was measured using ELISpot. Cytokine/chemokine levels were detected using multiplex immunoassays. In vitro ADE assays were performed using Raji cells. Results: One-month alpha convalescents exhibited spike-specific antibodies and T cells for alpha and delta variants. Notably, the RBD-specific IgG towards the delta variant decreased by 2.5-fold compared to the alpha variant. Besides, serum from individuals recently experienced COVID-19 showed suboptimal neutralizing activity against the delta and omicron variants. Humoral immune response, IL-6, IP-10 and MCP-1 levels were greater in patients with severe disease. Moreover, neither SARS-CoV-1 nor SARS-CoV-2 convalescent sera significantly enhanced SARS-CoV-2 pseudovirus infection. Conclusions: Significant resistance of the delta and omicron variants to the humoral immune response generated by individuals who recently experienced COVID-19. Furthermore, there was a significant correlation among disease severity, humoral immune response, and specific cytokines/chemokine levels. No evident ADE was observed for SARS-CoV-2.

Rosmarinic acid interferes with influenza virus A entry and replication by decreasing GSK3ß and phosphorylated AKT expression levels.

BACKGROUND: The purpose of this study was to examine the in vivo activity of rosmarinic acid (RA) - a phytochemical with antioxidant, anti-inflammatory, and antiviral properties - against influenza virus (IAV). An antibody-based kinase array and different in vitro functional assays were also applied to identify the mechanistic underpinnings by which RA may exert its anti-IAV activity. METHODS: We initially examined the potential efficacy of RA using an in vivo mouse model. A time-of-addition assay and an antibody-based kinase array were subsequently applied to investigate mechanism-of-action targets for RA. The hemagglutination inhibition assay, neuraminidase inhibition assay, and cellular entry assay were also performed. RESULTS: RA increased survival and prevented body weight loss in IAV-infected mice. In vitro experiments revealed that RA inhibited different IAV viruses - including oseltamivir-resistant strains. From a mechanistic point of view, RA downregulated the GSK3ß and Akt signaling pathways - which are known to facilitate IAV entry and replication into host cells. CONCLUSIONS: RA has promising preclinical efficacy against IAV, primarily by interfering with the GSK3ß and Akt signaling pathways.

Methotrexate inhibition of SARS-CoV-2 entry, infection and inflammation revealed by bioinformatics approach and a hamster model.

Background: Drug repurposing is a fast and effective way to develop drugs for an emerging disease such as COVID-19. The main challenges of effective drug repurposing are the discoveries of the right therapeutic targets and the right drugs for combating the disease. Methods: Here, we present a systematic repurposing approach, combining Homopharma and hierarchal systems biology networks (HiSBiN), to predict 327 therapeutic targets and 21,233 drug-target interactions of 1,592 FDA drugs for COVID-19. Among these multi-target drugs, eight candidates (along with pimozide and valsartan) were tested and methotrexate was identified to affect 14 therapeutic targets suppressing SARS-CoV-2 entry, viral replication, and COVID-19 pathologies. Through the use of in vitro (EC50 = 0.4 µM) and in vivo models, we show that methotrexate is able to inhibit COVID-19 via multiple mechanisms. Results: Our in vitro studies illustrate that methotrexate can suppress SARS-CoV-2 entry and replication by targeting furin and DHFR of the host, respectively. Additionally, methotrexate inhibits all four SARS-CoV-2 variants of concern. In a Syrian hamster model for COVID-19, methotrexate reduced virus replication, inflammation in the infected lungs. By analysis of transcriptomic analysis of collected samples from hamster lung, we uncovered that neutrophil infiltration and the pathways of innate immune response, adaptive immune response and thrombosis are modulated in the treated animals. Conclusions: We demonstrate that this systematic repurposing approach is potentially useful to identify pharmaceutical targets, multi-target drugs and regulated pathways for a complex disease. Our findings indicate that methotrexate is established as a promising drug against SARS-CoV-2 variants and can be used to treat lung damage and inflammation in COVID-19, warranting future evaluation in clinical trials.

Perilla (Perilla frutescens) leaf extract inhibits SARS-CoV-2 via direct virus inactivation.

BACKGROUND: While severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presents with mild or no symptoms in most cases, a significant number of patients become critically ill. Remdesivir has been approved for the treatment of coronavirus disease 2019 (COVID-19) in several countries, but its use as monotherapy has not substantially lowered mortality rates. Because agents from traditional Chinese medicine (TCM) have been successfully utilized to treat pandemic and endemic diseases, we designed the current study to identify novel anti-SARS-CoV-2 agents from TCM. METHODS: We initially used an antivirus-induced cell death assay to screen a panel of herbal extracts. The inhibition of the viral infection step was investigated through a time-of-drug-addition assay, whereas a plaque reduction assay was carried out to validate the antiviral activity. Direct interaction of the candidate TCM compound with viral particles was assessed using a viral inactivation assay. Finally, the potential synergistic efficacy of remdesivir and the TCM compound was examined with a combination assay. RESULTS: The herbal medicine Perilla leaf extract (PLE, approval number 022427 issued by the Ministry of Health and Welfare, Taiwan) had EC50 of 0.12 ± 0.06 mg/mL against SARS-CoV-2 in Vero E6 cells - with a selectivity index of 40.65. Non-cytotoxic PLE concentrations were capable of blocking viral RNA and protein synthesis. In addition, they significantly decreased virus-induced cytokine release and viral protein/RNA levels in the human lung epithelial cell line Calu-3. PLE inhibited viral replication by inactivating the virion and showed additive-to-synergistic efficacy against SARS-CoV-2 when used in combination with remdesivir. CONCLUSION: Our results demonstrate for the first time that PLE is capable of inhibiting SARS-CoV-2 replication by inactivating the virion. Our data may prompt additional investigation on the clinical usefulness of PLE for preventing or treating COVID-19.

Uncovering Flexible Active Site Conformations of SARS-CoV-2 3CL Proteases through Protease Pharmacophore Clusters and COVID-19 Drug Repurposing.

The infectious SARS-CoV-2 causes COVID-19, which is now a global pandemic. Aiming for effective treatments, we focused on the key drug target, the viral 3C-like (3CL) protease. We modeled a big dataset with 42 SARS-CoV-2 3CL protease-ligand complex structures from ∼98.7% similar SARS-CoV 3CL protease with abundant complex structures. The diverse flexible active site conformations identified in the dataset were clustered into six protease pharmacophore clusters (PPCs). For the PPCs with distinct flexible protease active sites and diverse interaction environments, we identified pharmacophore anchor hotspots. A total of 11 "PPC consensus anchors" (a distinct set observed in each PPC) were observed, of which three "PPC core anchors" EHV2, HV1, and V3 are strongly conserved across PPCs. The six PPC cavities were then applied in virtual screening of 2122 FDA drugs for repurposing, using core anchor-derived "PPC scoring S" to yield seven drug candidates. Experimental testing by SARS-CoV-2 3CL protease inhibition assay and antiviral cytopathic effect assays discovered active hits, Boceprevir and Telaprevir (HCV drugs) and Nelfinavir (HIV drug). Specifically, Boceprevir showed strong protease inhibition with micromolar IC50 of 1.42 µM and an antiviral activity with EC50 of 49.89 µM, whereas Telaprevir showed moderate protease inhibition only with an IC50 of 11.47 µM. Nelfinavir solely showed antiviral activity with a micromolar EC50 value of 3.28 µM. Analysis of binding mechanisms of protease inhibitors revealed the role of PPC core anchors. Our PPCs revealed the flexible protease active site conformations, which successfully enabled drug repurposing.

Interplay of COVID-19 and physiological dysfunctions.

The outbreak of the global coronavirus disease 2019 (COVID-19) pandemic continues to impact the socioeconomic fabric and the general well-being of numerous populations and communities around the world. As cases continue to rise exponentially, gaining a better understanding of the pathophysiology and the associated clinical implications of SARS-CoV-2, the causative agent of COVID-19, becomes increasingly necessary. In this article, we delineate the role of COVID-19 in physiological and immunological dysfunction. Specifically, we highlight the various possible mechanisms and effects of SARS-CoV-2 infections on major organ systems as well as their contribution toward multiorgan system failure. By analyzing studies and statistics regarding various comorbidities in COVID-19 patients, we make inferences on the linkage between COVID-19, immune injury, multiorgan system damage, and disease progression.

Mosquito Cell-Derived Japanese Encephalitis Virus-Like Particles Induce Specific Humoral and Cellular Immune Responses in Mice.

The Japanese encephalitis virus (JEV) is the major cause of an acute encephalitis syndrome in many Asian countries, despite the fact that an effective vaccine has been developed. Virus-like particles (VLPs) are self-assembled multi-subunit protein structures which possess specific epitope antigenicities related to corresponding native viruses. These properties mean that VLPs are considered safe antigens that can be used in clinical applications. In this study, we developed a novel baculovirus/mosquito (BacMos) expression system which potentially enables the scalable production of JEV genotype III (GIII) VLPs (which are secreted from mosquito cells). The mosquito-cell-derived JEV VLPs comprised 30-nm spherical particles as well as precursor membrane protein (prM) and envelope (E) proteins with densities that ranged from 30% to 55% across a sucrose gradient. We used IgM antibody-capture enzyme-linked immunosorbent assays to assess the resemblance between VLPs and authentic virions and thereby characterized the epitope specific antigenicity of VLPs. VLP immunization was found to elicit a specific immune response toward a balanced IgG2a/IgG1 ratio. This response effectively neutralized both JEV GI and GIII and elicited a mixed Th1/Th2 response in mice. This study supports the development of mosquito cell-derived JEV VLPs to serve as candidate vaccines against JEV.

dsRNA Binding Domain of PKR Is Proteolytically Released by Enterovirus A71 to Facilitate Viral Replication.

Enterovirus 71 (EV-A71) causes hand, foot and mouth disease in young children and infants, but can also cause severe neurological complications or even death. The double-stranded RNA (dsRNA)-dependent protein kinase R (PKR), an interferon-induced antiviral protein, phosphorylates the regulatory α-subunit of the eukaryotic translation initiation factor 2 in response to viral infection, thereby blocking the translation of cellular and viral mRNA and promoting apoptosis. The cleavage of PKR after infection with poliovirus, a prototype enterovirus, has been reported by others, but the underlying mechanism of this cleavage and its role in viral replication remain unclear. In the present study, we show that viral 3C protease cleaves PKR at a site, Q188, which differs from the site cleaved during apoptosis, D251. In contrast to the conventional phosphorylation of PKR by dsRNA, EV-A71 3C physically interacts with PKR to mediate the phosphorylation of PKR; this effect is dependent on 3C protease activity. Overexpression of a catalytically inactive PKR mutant (K296H) accelerates viral protein accumulation and increases virus titer, whereas a K64E substitution in the dsRNA binding site abolishes this advantage. We also demonstrate that PKR cleavage mediated by EV-A71 3C protease produces a short N-terminal PKR fragment that can enhance EV-A71 replication, in terms of viral RNA, viral protein, and viral titers. We conclude that PKR is co-opted by EV-A71 via viral protease 3C-mediated proteolytic activation to facilitate viral replication.

Microbial contaminations of laboratory mice and rats in Taiwan from 2004 to 2007.

Limited data are available on the pathogen status of contemporary rodent colonies in Taiwan. Here we summarized the rodent pathogen diagnostic records of the Taiwan National Laboratory Animal Center during a 4-y period that representing approximately 10% of the rodent colonies in Taiwan. Demand for pathogen diagnostic service increased continuously from 2004 to 2007, with a 20% increase each year. In 2007, more than 20% of the mouse colonies were positive for mouse parvovirus, mouse hepatitis virus, Theiler murine encephalomyelitis virus, and Mycoplasma pulmonis, with fewer colonies diagnosed as having infections of pneumonia virus of mice, mouse adenovirus, lymphocytic choriomeningitis virus, and reovirus. Almost 40% of tested rat colonies were positive for Mycoplasma pulmonis and rat parvovirus, with fewer colonies containing Kilham rat virus, sialodacryoadenitis virus, pneumonia virus of mice, Sendai virus, and Syphacia spp. These data provide a sound overall picture of the health status of mouse and rat colonies in Taiwan.

PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells.

Strains of species in the Bacillus cereus group are potentially enterotoxic. Thus, the detection of all B. cereus group strains is important. As 16S ribosomal DNA sequence analysis cannot adequately differentiate species of the B. cereus group, we explored the potential of the groEL gene as a phylogenetic marker. A phylogenetic analysis of the groEL sequences of 78 B. cereus group strains revealed that the B. cereus group strains were split into two major clusters, one including six B. mycoides and one B. pseudomycoides (cluster II) and the other including two B. mycoides and the rest of the B. cereus group strains (cluster I). Cluster I was further differentiated into two subclusters, Ia and Ib. The sodA gene sequences of representative strains from different clusters were also compared. The phylogenetic tree constructed from the sodA sequences showed substantial similarity to the tree constructed from the groEL sequences. Based on the groEL sequences, a PCR assay for detection and identification of B. cereus group strains was developed. Subsequent restriction fragment length polymorphism (RFLP) analysis verified the PCR amplicons and the differentiation of the B. cereus group strains. RFLP with MboI was identical for all the B. cereus group strains analyzed, while RFLP with MfeI or PstI classified all B. cereus and B. thuringiensis strains into two groups. All cluster II B. mycoides and B. pseudomycoides strains could be discriminated from other B. cereus group bacteria by restriction analysis with TspRI.