Antiviral Research Articles

Comparative study of UV spectroscopy, RP-HPLC and HPTLC methods for quantification of antiviral drug lamivudine in tablet formulation

BackgroundIn the current study, estimation of lamivudine (LMU) by UV spectroscopy, reverse-phase HPLC (RP-HPLC) and HPTLC methods in tablet formulation was developed, and comparative studies between the methods were investigated by analytical results and statistical test analysis of variance (ANOVA) to find out best method. In the UV spectral method, LMU was quantified at 271 nm absorption maxima using methanol as the solvent. In the RP-HPLC method, the Shimadzu C18 column (250 mm × 4.6 mm i.d., 5 µm particle size) was employed for chromatographic separation. The mobile phase used consists of methanol: water (70:30 v/v) in an isocratic mode with a 1.0 mL/min flow rate. In the HPTLC method, the chromatogram was developed on a pre-coated plate of silica gel 60 F254 with a mobile phase composition of chloroform: methanol (8:2 v/v). The quantification was performed at an absorbance mode of 271 nm by densitometry. The methods were validated according to the International Conference on Harmonization (ICH) guideline Q2 (R1). The degradation conditions were employed as per ICH guidelines Q1A(R2) and Q1B which include acid, alkaline, neutral, thermal and photostability to determine the intrinsic stability of the drug in varied environmental conditions.ResultsLMU absorption maxima was found to be 271 nm. The retention time of LMU was 3.125 min, and the total analysis time was 5 min. The Rf value of LMU was 0.49–0.62. The methods were linear within 2–12 μg/mL range. The correlation coefficient (r2) for UV, HPLC and HPTLC was 0.9980, 0.9993 and 0.9988, and percent recoveries were calculated as 98.40–100.52%, 99.27–101.18% and 98.01–100.30%, respectively, with percentage relative standard deviation (RSD) less than 2% showing that methods were precise and accurate.ConclusionDeveloped UV, RP-HPLC and HPTLC methods are free from intervention caused by excipients present in tablets and thus can be used for regular quantitative analysis of LMU in tablet formulation. Based on analytical results and statistical tests, ANOVA, it is inferred that the HPLC method is best for LMU quantification tablet formulation due to its high reproducibility, good retention time and sensitivity; it has a higher percent recovery and has less analysis time, i.e., 5 min. The degradation peaks were well separated from the LMU peak indicating stability of the HPLC method.

Study of key residues in MERS-CoV and SARS-CoV-2 main proteases for resistance against clinically applied inhibitors nirmatrelvir and ensitrelvir

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an epidemic, zoonotically emerging pathogen initially reported in Saudi Arabia in 2012. MERS-CoV has the potential to mutate or recombine with other coronaviruses, thus acquiring the ability to efficiently spread among humans and become pandemic. Its high mortality rate of up to 35% and the absence of effective targeted therapies call for the development of antiviral drugs for this pathogen. Since the beginning of the SARS-CoV-2 pandemic, extensive research has focused on identifying protease inhibitors for the treatment of SARS-CoV-2. Our intention was therefore to assess whether these protease inhibitors are viable options for combating MERS-CoV. To that end, we used previously established protease assays to quantify inhibition of SARS-CoV-2, MERS-CoV and other main proteases. Nirmatrelvir inhibited several of these proteases, whereas ensitrelvir was less broadly active. To simulate nirmatrelvir’s clinical use against MERS-CoV and subsequent resistance development, we applied a safe, surrogate virus-based system. Using the surrogate virus, we previously selected hallmark mutations of SARS-CoV-2-Mpro, such as T21I, M49L, S144A, E166A/K/V and L167F. In the current study, we selected a pool of MERS-CoV-Mpro mutants, characterized the resistance and modelled the steric effect of catalytic site mutants S142G, S142R, S147Y and A171S.

Carbon‐Based Nanomaterials for Antiviral Applications

AbstractIn the antimicrobial resistance era, carbon‐based nanomaterials (CBNs) such as fullerenes, carbon dots, graphene, and their derivatives are promising therapeutic tools in combating viral diseases. This review shows that these materials have broad‐spectrum antiviral activity against 33 viruses belonging to different Baltimore groups. CBNs also exhibit antimicrobial activity against bacteria and fungi and possess a low risk of selecting for resistance, since their primary mode of antimicrobial action involves physically damaging the microbes. CBNs also offer additional promising properties, including enhanced antiviral effectiveness under diverse types of irradiation and facilitating antiviral immune responses. Their potential as antiviral agents is still in its infancy and future research should focus on their toxicity, antiviral mechanisms, pharmacokinetics, and bioavailability. They are also potential antiviral materials for preventing the transmission of viral diseases for use in face masks, shields, hospital and airport surfaces, and elevators, among others. It is anticipated that CBNs will play an increasingly significant role in the fight against viruses and infectious diseases.

Novel transcription and replication-competent virus-like particles system modelling the Nipah virus life cycle

ABSTRACT Nipah virus (NiV), a highly pathogenic Henipavirus in humans, has been responsible for annual outbreaks in recent years. Experiments involving live NiV are highly restricted to biosafety level 4 (BSL-4) laboratories, which impedes NiV research. In this study, we developed transcription and replication-competent NiV-like particles (trVLP-NiV) lacking N, P, and L genes. This trVLP-NiV exhibited the ability to infect and continuously passage in cells ectopically expressing N, P, and L proteins while maintaining stable genetic characteristics. Moreover, the trVLP-NiV displayed a favourable safety profile in hamsters. Using the system, we found the NiV nucleoprotein residues interacting with viral RNA backbone affected viral replication in opposite patterns. This engineered system was sensitive to well-established antiviral drugs, innate host antiviral factors, and neutralizing antibodies. We then established a high-throughput screening platform utilizing the trVLP-NiV, leading to the identification of tunicamycin as a potential anti-NiV compound. Evidence showed that tunicamycin inhibited NiV replication by decreasing the infectivity of progeny virions. In conclusion, this trVLP-NiV system provided a convenient and versatile molecular tool for investigating NiV molecular biology and conducting antiviral drug screening under BSL-2 conditions. Its application will contribute to the development of medical countermeasures against NiV infections.

High temperature exposure causes sexual dimorphism in immune responses against largemouth bass ranavirus (LMBV) in Micropterus salmoides

Largemouth bass (Micropterus salmoides) is an important freshwater fish species in aquaculture. Largemouth bass ranavirus (LMBV) infection at high temperature results in a considerably higher mortality rate in females than that in males. This study compared the spleen transcriptomes and analyzed the impact of sexual dimorphism on immunity and metabolism of largemouth bass infected with LMBV at high temperatures to gain a comprehensive understanding of the sex-based differences in immunity and metabolism of this species. (1) Compared with the ambient temperature group (W20), the high temperature group (W30) exhibited multiple significant differences in metabolic and immune terms, with several immunity-related pathways, including phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt) signaling pathway. Most identified pathways were associated with the estrogen signaling pathway, which also differed significantly. (2) Differences in glycolipid metabolism, immunity, and the endocrine and complex adaptive systems were distinctly different between male groups (W20M vs. W30M), whereas the female groups (W20F vs. W30F) were not significantly different. The male groups had a significantly higher number of enriched immunity-related KEGG pathways and differentially expressed genes (DEGs) and pathways related to the estrogen signaling pathway. (3) When comparing male and female samples infected with LMBV at the same temperature, no significant difference was observed between the ambient temperature groups (W20F vs. W20M). The high-temperature groups (W30F vs. W30M) had 18 immunity-related pathways among the top 25 enriched KEGG pathways, and gene expression correlation analysis of the DEGs showed positive correlations. This study demonstrates that the combined effects of high temperature and LMBV affect multiple metabolic and immune pathways in largemouth bass, thereby modulating immune responses by regulating the estrogen signaling pathway and influencing the expression of estrogen and its receptors. Under high-temperature conditions, male fish may have stronger estrogen signaling pathway-associated immune responses, which regulate immune pathways, including antigen processing and presentation, complement system, and phagocytosis. Consequently, males may have higher disease resistance than that of females. Our result contribute to mono-sex breeding and antiviral drug therapies available in largemouth bass, thereby markedly improving breeding efficiency.

Macrocyclic Azapeptide Nitriles: Structure-Based Discovery of Potent SARS-CoV-2 Main Protease Inhibitors as Antiviral Drugs.

Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M-1s-1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents.

Novel insights into the host cell glycan binding profile of human metapneumovirus.

Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galβ1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates.IMPORTANCEAll cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease.

Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.

Novel Acyl Thiourea-Based Hydrophobic Tagging Degraders Exert Potent Anti-Influenza Activity through Two Distinct Endonuclease Polymerase Acidic-Targeted Degradation Pathways.

The spread of the influenza virus has caused devastating pandemics and huge economic losses worldwide. Antiviral drugs with diverse action modes are urgently required to overcome the challenges of viral mutation and drug resistance, and targeted protein degradation strategies constitute excellent candidates for this purpose. Herein, the first degradation of the influenza virus polymerase acidic (PA) protein using small-molecule degraders developed by hydrophobic tagging (HyT) technology to effectively combat the influenza virus was reported. The SAR results revealed that compound 19b with Boc2-(L)-Lys demonstrated excellent inhibitory activity against A/WSN/33/H1N1 (EC50 = 0.015 μM) and amantadine-resistant strain (A/PR/8/H1N1), low cytotoxicity, high selectivity, substantial degradation ability, and good drug-like properties. Mechanistic studies demonstrated that the proteasome system and autophagic lysosome pathway were the potential drivers of these HyT degraders. Thus, this study provides a powerful tool for investigating the targeted degradation of influenza virus proteins and for antiviral drug development.

Pharmacological activation of TLR7 exerts inhibition on the replication of EV-D68 in respiratory cells.

The globally reemerging respiratory pathogen enterovirus D68 (EV-D68) is implicated in outbreaks of severe respiratory illness and associated with acute flaccid myelitis. However, there remains a lack of effective treatments for EV-D68 infection. In this work, we found that the host Toll-like receptor 7 (TLR7) proteins, which function as powerful innate immune sensors, were selectively elevated in expression in response to EV-D68 infection. Subsequently, we investigated the impact of Vesatolimod (GS-9620), a Toll-like receptor 7 agonist, on EV-D68 replication. Our findings revealed that EV-D68 infection resulted in increased mRNA levels of TLR7. Treatment with Vesatolimod significantly inhibited EV-D68 replication [half maximal effective concentration (EC50) = 0.1427 µM] without inducing significant cytotoxicity at virucidal concentrations. Although Vesatolimod exhibited limited impact on EV-D68 attachment, it suppressed RNA replication and viral protein synthesis after virus entry. Vesatolimod broadly inhibited the replication of circulating isolated strains of EV-D68. Furthermore, our findings demonstrated that treatment with Vesatolimod conferred resistance to both respiratory and neural cells against EV-D68 infection. Overall, these results present a promising strategy for drug development by pharmacologically activating TLR7 to initiate an antiviral state in EV-D68-infected cells selectively.IMPORTANCEConventional strategies for antiviral drug development primarily focus on directly targeting viral proteases or key components, as well as host proteins involved in viral replication. In this study, based on our intriguing discovery that enterovirus D68 (EV-D68) infection specifically upregulates the expression of immune sensor Toll-like receptor 7 (TLR7) protein, which is either absent or expressed at low levels in respiratory cells, we propose a potential antiviral approach utilizing TLR7 agonists to activate EV-D68-infected cells into an anti-viral defense state. Notably, our findings demonstrate that pharmacological activation of TLR7 effectively suppresses EV-D68 replication in respiratory tract cells through a TLR7/MyD88-dependent mechanism. This study not only presents a promising drug candidate and target against EV-D68 dissemination but also highlights the potential to exploit unique alterations in cellular innate immune responses induced by viral infections, selectively inducing a defensive state in infected cells while safeguarding uninfected normal cells from potential adverse effects associated with therapeutic interventions.

Adherence in Hepatitis C Virus Treatment: What We Know.

Although therapy with direct-acting antiviral (DAA) agents achieves high hepatitis C virus (HCV) cure rates and is forgiving of missed doses, certain patient populations, such as people who inject drugs (PWID), are often denied therapy because of a perceived high risk of nonadherence. However, a relationship between adherence to DAAs for various patient populations and efficacy has not been well defined. The lack of a standardized method for evaluating adherence complicates making comparisons between studies, making it difficult to develop and implement novel measures that may improve adherent behavior. Traditional methods for assessing adherence may overestimate medication adherence, while newer, technology-based methods may assist with accurately assessing and maintaining patient adherence to therapy. Data demonstrate that special populations of patients with HCV, such as PWID, can be successfully treated, with relatively high rates of sustained virologic response (SVR) despite less-than-optimal adherence. While rates of adherence, and subsequently SVR, can be improved, antiviral therapy should not be withheld because of fear of nonadherence. This article addresses medication adherence and forgiveness of DAA regimens, such as sofosbuvir/velpatasvir and glecaprevir/pibrentasvir, in different patient populations with HCV. Considerations in evaluating adherence in HCV therapy and available methods for assessing adherence are detailed.

ATG10S promotes IFNL1 expression and autophagic degradation of multiple viral proteins mediated by IFNL1

ABSTRACT ATG10S is a newly discovered subtype of the autophagy protein ATG10. It promotes complete macroautophagy/autophagy, degrades multiple viral proteins, and increases the expression of type III interferons. Here, we aimed to investigate the mechanism of ATG10S cooperation with IFNL1 to degrade viral proteins from different viruses. Using western blot, immunoprecipitation (IP), tandem sensor RFP-GFP-LC3B and in situ proximity ligation assays, we showed that exogenous recombinant ATG10S protein (rHsATG10S) could enter into cells through clathrin, and ATG10S combined with ATG7 with IFNL1 assistance to facilitate ATG12–ATG5 conjugation, thereby contributing to the autophagosome formation in multiple cell lines containing different virions or viral proteins. The results of DNA IP and luciferase assays also showed that ATG10S was able to directly bind to a core motif (CAAGGG) within a binding site of transcription factor ZNF460 on the IFNL1 promoter, by which IFNL1 transcription was activated. These results clarified that ATG10S promoted autophagosome formation with the assistance of IFNL1 to ensure autophagy flux and autophagic degradation of multiple viral proteins and that ATG10S could also act as a novel transcription factor to promote IFNL1 gene expression. Importantly, this study further explored the antiviral mechanism of ATG10S interaction with type III interferon and provided a theoretical basis for the development of ATG10S into a new broad-spectrum antiviral protein drug. Abbreviation: ATG: autophagy related; ATG10S: the shorter isoform of autophagy-related 10; CC50: half cytotoxicity concentration; CCV: clathrin-coated transport vesicle; CLTC: clathrin heavy chain; CM: core motif; co-IP: co-immunoprecipitation; CPZ: chlorpromazine; ER: endoplasmic reticulum; HCV: hepatitis C virus; HBV: hepatitis B virus; HsCoV-OC43: Human coronavirus OC43; IFN: interferon; PLA: proximity ligation assay; rHsATG10S: recombinant human ATG10S protein; RLU: relative light unit; SQSTM1: sequestosome 1; ZNF: zinc finger protein

Colloidal Aggregation Confounds Cell-Based Covid-19 Antiviral Screens.

Colloidal aggregation is one of the largest contributors to false positives in early drug discovery. Here, we consider aggregation's role in cell-based infectivity assays in Covid-19 drug repurposing. We investigated the potential aggregation of 41 drug candidates reported as SARs-CoV-2 entry inhibitors. Of these, 17 formed colloidal particles by dynamic light scattering and exhibited detergent-dependent enzyme inhibition. To evaluate the impact of aggregation on antiviral efficacy in cells, we presaturated the colloidal drug suspensions with BSA or spun them down by centrifugation and measured the effects on spike pseudovirus infectivity. Antiviral potencies diminished by at least 10-fold following both BSA and centrifugation treatments, supporting a colloid-based mechanism. Aggregates induced puncta of the labeled spike protein in fluorescence microscopy, consistent with sequestration of the protein on the colloidal particles. These observations suggest that colloidal aggregation is common among cell-based antiviral drug repurposing and offers rapid counter-screens to detect and eliminate these artifacts.

Medicinal Chemistry of Antisense Oligonucleotides for Therapeutic Use in SARS-CoV-2: Design Strategies and Challenges for Targeted Delivery

Background: The evolution of novel Severe Acute Respiratory Syndrome-related Coronavirus 2 (SARS-CoV-2) strains with greater degrees of infectivity, resistance to vaccine-induced acquired immunity, and more severe morbidity have contributed to the recent spread of COVID-19. In light of this, novel therapeutic alternatives with improved effectiveness and fewer side effects have become a necessity. Despite many new or repurposed antiviral agents recommended for Coronavirus disease (COVID-19) therapy, this objective remains unfulfilled. Under these circumstances, the scientific community holds the significant responsibility to develop classes of novel therapeutic modalities to combat SARS-CoV-2 with the least harmful side effects. Objective: Antisense Oligonucleotides (ASOs) are short single-stranded oligonucleotides that allow the specific targeting of RNA, leading to its degradation. They may also prevent cellular factors or machinery from binding to the target RNA. It is possible to improve the pharmacokinetics and pharmacodynamics of ASOs by chemical modification or bioconjugation, which may provide conditions for customization of a particular clinical target. This study aimed to outline the potential use of ASOs in the treatment of COVID-19 disease, along with the use of antisense stabilization and transfer methods, as well as future challenges and limitations. Methods: We have reviewed the structure and properties of ASOs containing nucleobase, sugar, or backbone modifications, and provided an overview of the therapeutic potential, delivery challenges, and strategies of ASOs in the treatment of COVID-19. Results: The first-line therapy for COVID-19-infected individuals, as well as the development of oligonucleotide-based drugs, warrants further investigation. Chemical changes in the oligonucleotide structure can affect the biological processes. These chemical alterations may lead to enhanced potency, while changing the pharmacokinetics and pharmacodynamics. Conclusion: ASOs can be designed to target both coding and non-coding regions of the viral genome to disrupt or completely degrade the genomic RNA and thereby eliminate SARS-CoV-2. They may be very effective in areas, where vaccine distribution is challenging, and they may be helpful for future coronavirus pandemics.

Antiviral activity of pimecrolimus against dengue virus type 2 infection in vitro and in vivo

Dengue virus (DENV) infection is a public health concern in several countries and is associated with severe diseases, such as dengue hemorrhagic fever and dengue shock syndrome. DENVs are transmitted to humans via the bites of infected Aedes mosquitoes, and no antiviral therapeutics are currently available. In this work, we aimed to identify antiviral drugs against DENV type 2 (DENV2) infections and selected pimecrolimus as a potential antiviral drug candidate. Pimecrolimus significantly inhibited DENV2-mediated cell death and replication in vitro. We also confirmed a decrease in the number of plaques formed as well as in the envelope protein levels of DENV2. The time-of-addition and course experiments revealed that pimecrolimus inhibited DENV2 infection during the early stages of the virus replication cycle. In an experimental mouse model, orally administered pimecrolimus alleviated body weight loss and lethality caused by DENV2 infection, which we used as readouts of the drug’s antiviral potency. Furthermore, pimecrolimus significantly inhibited the DENV2 load and ameliorated focal necrosis in the liver and spleen. Taken together, our in vitro and in vivo findings suggest that pimecrolimus is a promising antiviral drug candidate for the treatment of DENV2 infection.

Effect of treatment of chronic hepatitis c virus patients with direct-acting anti-retroviral drugs on semen and hormonal parameters

Objective: Hepatitis C virus (HCV) infection is known to influence the seminal and hormonal parameters of infected men. This study was performed to assess the effects of HCV clearance using direct-acting antiviral (DAA) agents on semen and hormonal parameters. Methods: A total of 50 patients with chronic HCV were enrolled, and conventional semen analysis was performed according to World Health Organization guidelines. Basal levels of total testosterone, free testosterone (FT), follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), prolactin, and sex hormone-binding globulin (SHBG) were assessed before and 3 months after treatment with DAAs.Results: Following DAA treatment, statistically significant increases were observed in sperm motility and the proportion of grade A sperm. Additionally, the percentage of abnormal forms was significantly decreased after treatment (p=0.000). However, no significant differences were observed in semen volume, concentration, or total sperm count. Sex hormone analysis of patients after DAA treatment revealed significant increases in FT, LH, and FSH levels, along with significant decreases in SHBG, prolactin, and E2 levels.Conclusion: Following HCV clearance, we noted an improvement in sperm motility and an increase in the percentage of sperm with normal morphology. Treatment with DAAs was also associated with increased levels of FT and LH, along with decreased levels of SHBG, prolactin, and E2.

The potential therapeutic value of terpenes

Terpenes form part of a huge and diverse class of naturally occurring and volatile secondary metabolites produced by many plants, fruits, animals, insects, and other organisms. They are the largest group of naturally occurring metabolites, with over 55,000 types of terpenes produced by plants alone, primarily as essential oils. In humans, they contain significant biological properties such as antifungal, antiviral, antimicrobial, anti-inflammatory, antiparasitic, antihyperglycemic, anti-cancer, and analgesic agents. In plants, terpenes also play significant roles in defensive mechanisms against herbivores and invasive plants, disease resistance, chemical signaling and communication between plants, protection against photo-oxidation, plant-environment mediation, thermo-protection, and the attraction of pollinators. In addition, terpenes are responsible for a plant’s scent, taste, flavor, and pigmentation, leading to their commercial use as fragrances and food dyes. Terpenes are also used in the production of synthetic polymers, natural rubbers (polyisoprene), organic solvents, varnishes, inks, adhesives, cleaning products, biofuels, pesticides, and food and drink products. For these reasons, terpenes have significant value in modern medicine, pharmacy, nutraceuticals, cosmetics, and other industries.

Canthin-6-one analogs block Newcastle disease virus proliferation via suppressing the Akt and ERK pathways

Newcastle disease virus, a member of the Paramyxoviridae family, causes significant economic losses in poultry worldwide. To identify novel antiviral agents against NDV, 36 canthin-6-one analogs were evaluated in this study. Our data showed that 8 compounds exhibited excellent inhibitory effects on NDV replication with IC50 values in the range of 5.26 to 11.76 μM. Besides, these analogs inhibited multiple NDV strains with IC50 values within 12 μM and exerted antiviral activity against peste des petits ruminants virus (PPRV) and canine distemper virus (CDV). Among these analogs, 16 presented the strongest anti-NDV activity (IC50 = 5.26 μM) and minimum cytotoxicity (CC50 > 200 μM) in DF-1 cells. Furthermore, 16 displayed antiviral activity in different cell lines. Our results showed that 16 did not affect the viral adsorption while it can inhibit the entry of NDV by suppressing the Akt pathway. Further study found that 16-treatment inhibited the NDV-activated ERK pathway, thereby promoting the expression of interferon-related genes. Our findings reveal an antiviral mechanism of canthin-6-one analogs through inhibition of the Akt and ERK signaling pathways. These results point to the potential value of canthin-6-one analogs to serve as candidate antiviral agents for NDV.

Role of inflammatory cytokine burst in neuro-invasion of Japanese Encephalitis virus infection: an immunotherapeutic approaches.

Japanese Encephalitis remains a significant global health concern, contributing to millions of deaths annually worldwide. Microglial cells, as key innate immune cells within the central nervous system (CNS), exhibit intricate cellular structures and possess molecular phenotypic plasticity, playing pivotal roles in immune responses during CNS viral infections. Particularly under viral inflammatory conditions, microglial cells orchestrate innate and adaptive immune responses to mitigate viral invasion and dampen inflammatory reactions. This review article comprehensively summarizes the pathophysiology of viral invasion into the CNS and the cellular interactions involved, elucidating the roles of various immune mediators, including pro-inflammatory cytokines, in neuroinflammation. Leveraging this knowledge, strategies for modulating inflammatory responses and attenuating hyperactivation of glial cells to mitigate viral replication within the brain are discussed. Furthermore, current chemotherapeutic and antiviral drugs are examined, elucidating their mechanisms of action against viral replication. This review aims to provide insights into therapeutic interventions for Japanese Encephalitis and related viral infections, ultimately contributing to improved outcomes for affected individuals.

Structural basis of rosmarinic acid inhibitory mechanism on SARS-CoV-2 main protease

The SARS-CoV-2 coronavirus is characterized by high mutation rates and significant infectivity, posing ongoing challenges for therapeutic intervention. To address potential challenges in the future, the continued development of effective drugs targeting SARS-CoV-2 remains an important task for the scientific as well as the pharmaceutical community. The main protease (Mpro) of SARS-CoV-2 is an ideal therapeutic target for COVID-19 drug development, leading to the introduction of various inhibitors, both covalent and non-covalent, each characterized by unique mechanisms of action and possessing inherent strengths and limitations. Natural products, being compounds naturally present in the environment, offer advantages such as low toxicity and diverse activities, presenting a viable source for antiviral drug development. Here, we identified a natural compound, rosmarinic acid, which exhibits significant inhibitory effects on the Mpro of the SARS-CoV-2. Through detailed structural biology analysis, we elucidated the precise crystal structure of the complex formed between rosmarinic acid and SARS-CoV-2 Mpro, revealing the molecular basis of its inhibitory mechanism. These findings not only enhance our understanding of the antiviral action of rosmarinic acid, but also provide valuable structural information and mechanistic insights for the further development of therapeutic strategies against SARS-CoV-2.