Drug-resistant Influenza Viruses Research Articles

Platycodin D facilitates antiviral immunity through inhibiting cytokine storm via targeting K63-linked TRAF6 ubiquitination.

Influenza virus infection is a worldwide challenge that causes heavy burdens on public health. The mortality rate of severe influenza patients is often associated with hyperactive immunological abnormalities characterized by hypercytokinemia. Due to the continuous mutations and the occurrence of drug-resistant influenza virus strains, the development of host-directed immunoregulatory drugs is urgently required. Platycodon grandiflorum is among the top 10 herbs of traditional Chinese medicine used to treat pulmonary diseases. As one of the major terpenoid saponins extracted from Platycodon grandiflorum, Platycodin D (PD) has been reported to play several roles, including anti-inflammation, analgesia, anti-cancer, hepatoprotection, and immunoregulation. However, the therapeutic roles of PD to treat influenza virus infection remains unknown. Here, we show that PD can protect the body weight loss in severely infected influenza mice, alleviate lung damage, and thus improve the survival rate. More specifically, PD protects flu mice via decreasing the immune cell infiltration into lungs and downregulating the overactivated inflammatory response. Western blot and immunofluorescence assays exhibited that PD could inhibit the activation of TAK1/IKK/NF-κB and MAPK pathways. Besides that, CETSA, SPR and immunoprecipitation assays indicated that PD binds with TRAF6 to decrease its K63 ubiquitination after R837 stimulation. Additionally, siRNA interference experiments exhibited that PD could inhibit the secretion of IL-1β and TNF-α in TRAF6-dependent manner. Altogether, our results suggested that PD is a promising drug candidate for treating influenza. Our study also offered a scientific explanation for the commonly used Platycodon grandiflorum in many anti-epidemic classic formulas. Due to its host-directed regulatory role, PD may serve as an adjuvant therapeutic drug in conjunction with other antiviral drugs to treat the flu.

Molecular Determinants of Drug Resistance and Mutation Patterns in Influenza Viruses Circulating in Poland Across Multiple Epidemic Seasons: Implications for Vaccination Strategies.

BACKGROUND According to the WHO, up to 650 000 people die each year from seasonal flu-related respiratory illnesses. The most effective method of fighting the virus is seasonal vaccination. However, if an infection does occur, antiviral medications should be used as soon as possible. No studies of drug resistance in influenza viruses circulating in Poland have been systematically conducted. Therefore, the aim of the present study was to investigate the drug resistance and genetic diversity of influenza virus strains circulating in Poland by determining the presence of mutations in the neuraminidase gene. MATERIAL AND METHODS A total of 258 clinical specimens were collected during the 2016-2017, 2017-2018, and 2018-2019 epidemic seasons. The samples containing influenza A and B were analyzed by RT-PCR and Sanger sequencing. RESULTS Differences were found between the influenza virus strains detected in different epidemic seasons, demonstrating the occurrence of mutations. Influenza A virus was found to be more genetically variable than influenza B virus (P<0.001, Kruskal-Wallis test). However, there was no significant difference in the resistance prevalence between the influenza A subtypes A/H1N1/pdm09 (4.8%) and A/H3N2/ (6.1%). In contrast, more mutations of drug-resistance genes were found in the influenza B virus (P<0.001, chi-square test). In addition, resistance mutations appeared en masse in vaccine strains circulating in unvaccinated populations. CONCLUSIONS It seems important to determine whether the influenza virus strains tested for drug resistance as part of global influenza surveillance are equally representative of viruses circulating in populations with high and low vaccination rates, for all countries. Our results suggest that countries with low levels of influenza immunization may constitute reservoirs of drug-resistant influenza viruses.

Anti-rheumatic colchicine phytochemical exhibits potent antiviral activities against avian and seasonal Influenza A viruses (IAVs) via targeting different stages of IAV replication cycle

BackgroundThe continuous evolution of drug-resistant influenza viruses highlights the necessity for repurposing naturally-derived and safe phytochemicals with anti-influenza activity as novel broad-spectrum anti-influenza medications.MethodsIn this study, nitrogenous alkaloids were tested for their viral inhibitory activity against influenza A/H1N1 and A/H5N1 viruses. The cytotoxicity of tested alkaloids on MDCK showed a high safety range (CC50 > 200 µg/ml), permitting the screening for their anti-influenza potential. ResultsHerein, atropine sulphate, pilocarpine hydrochloride and colchicine displayed anti-H5N1 activities with IC50 values of 2.300, 0.210 and 0.111 µg/ml, respectively. Validation of the IC50 values was further depicted by testing the three highly effective alkaloids, based on their potent IC50 values against seasonal influenza A/H1N1 virus, showing comparable IC50 values of 0.204, 0.637 and 0.326 µg/ml, respectively. Further investigation suggests that colchicine could suppress viral infection by primarily interfering with IAV replication and inhibiting viral adsorption, while atropine sulphate and pilocarpine hydrochloride could directly affect the virus in a cell-free virucidal effect. Interestingly, the in silico molecular docking studies suggest the abilities of atropine, pilocarpine, and colchicine to bind correctly inside the active sites of the neuraminidases of both influenza A/H1N1 and A/H5N1 viruses. The three alkaloids exhibited good binding energies as well as excellent binding modes that were similar to the co-crystallized ligands. On the other hand, consistent with in vitro results, only colchicine could bind correctly against the M2-proton channel of influenza A viruses (IAVs). This might explicate the in vitro antiviral activity of colchicine at the replication stage of the virus replication cycle.ConclusionThis study highlighted the anti-influenza efficacy of biologically active alkaloids including colchicine. Therefore, these alkaloids should be further characterized in vivo (preclinical and clinical studies) to be developed as anti-IAV agents.

Discovery of ligustrazine and chalcone derivatives as novel viral nucleoprotein nuclear export inhibitors against influenza viruses.

Influenza viruses pose a significant threat to human health worldwide due to seasonal epidemics and occasional global pandemics. These viruses can cause severe upper respiratory tract infections that contribute to high morbidity and mortality rates. The emergence of drug-resistant influenza viruses has created the need for the development of novel broad-spectrum antivirals. Here, we present a novel anti-influenza agent with new targets and mechanisms of action to address this problem. Our findings led to the discovery of a novel influenza virus inhibitor, a ligustrazine derivative known as A9. We have found that it exhibits broad-spectrum antiviral properties against influenza A and B viruses (IAV and IBV, respectively), including oseltamivir-resistant strain. Through multiple bioassays such as time-of-addition assay, indirect immunofluorescence assay, and nuclear-cytoplasmic fractionation assay, we demonstrated that A9 inhibits the nuclear export of the viral ribonucleoprotein (vRNP). Furthermore, escape mutant analyses and affinity studies determined by surface plasmon resonanceindicated that A9 specifically targets the nucleoprotein.In addition, four chalcone derivatives developed from A9 (B14, B29, B31, and B32), were found to effectively inhibit the replication of influenza virus through the same mechanism of action. In this manuscript we highlight A9 and its four derivatives as potential leads for the treatment of IAV and IBV infections, and their unique and novel mechanism of action probable benefit the field of anti-influenza drug discovery.

Glycyrrhizic Acid-Based Carbonized Dots Boost Antiviral Activity against Influenza A Virus via Multisite Inhibition Mechanisms

Influenza A virus (IVA) has been continuously causing pandemics in several animal hosts and has become a worldwide public health threat. Currently, antiviral drugs have become associated with a lot of side effects and limited activity against emerging drug-resistant influenza viruses. Therefore, the development of novel antiviral drugs is of great importance. In this study, we synthesized a kind of carbon dots (CDs) with high dispersibility from glycyrrhizic acid (GA) using a simple dry heating method. Compared with glycyrrhizic acid alone, GA-CDs exhibit superior solubility and significantly improve the antiviral property against IVA. Investigation of the mechanism revealed that GA-CDs act against IVA mainly by inhibiting viral internalization, replication of the viral genome, neuraminidase activity, and host inflammatory responses. More importantly, in a mouse model, GA-CDs can significantly alleviate the clinical symptoms and decrease mortality and lung viral titers. In vitro and in vivo experiments demonstrate that GA-CDs possess extraordinary therapeutic effects; therefore, we propose that GA-CDs may be a promising alternative therapy for IVA infection.

Vitisin B inhibits influenza A virus replication by multi-targeting neuraminidase and virus-induced oxidative stress.

The development of drug-resistant influenza and new pathogenic virus strains underscores the need for antiviral therapeutics. Currently, neuraminidase (NA) inhibitors are commonly used antiviral drugs approved by the US Food and Drug Administration (FDA) for the prevention and treatment of influenza. Here, we show that vitisin B (VB) inhibits NA activity and suppresses H1N1 viral replication in MDCK and A549 cells. Reactive oxygen species (ROS), which frequently occur during viral infection, increase virus replication by activating the NF-κB signaling pathway, downmodulating glucose-6-phosphate dehydrogenase (G6PD) expression, and decreasing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response activity. VB decreased virus-induced ROS generation by increasing G6PD expression and Nrf2 activity, and inhibiting NF-κB translocation to the nucleus through IKK dephosphorylation. In addition, VB reduced body weight loss, increased survival, decreased viral replication and the inflammatory response in the lungs of influenza A virus (IAV)-infected mice. Taken together, our results indicate that VB is a promising therapeutic candidate against IAV infection, complements existing drug limitations targeting viral NA. It modulated the intracellular ROS by G6PD, Nrf2 antioxidant response pathway, and NF-κB signaling pathway. These results demonstrate the feasibility of a multi-targeting drug strategy, providing new approaches for drug discovery against IAV infection.

Pterostilbene effectively inhibits influenza A virus infection by promoting the type I interferon production

With the prevalence of novel strains and drug-resistant influenza viruses, there is an urgent need to develop effective and low-toxicity anti-influenza therapeutics. Regulation of the type I interferon antiviral response is considered an attractive therapeutic strategy for viral infection. Pterostilbene, a 3,5-dimethoxy analog of resveratrol, is known for its remarkable pharmacological activity. Here, we found that pterostilbene effectively inhibited influenza A virus infection and mainly affected the late stages of viral replication. A mechanistic study showed that the antiviral activity of pterostilbene might promote the induction of antiviral type I interferon and expression of its downstream interferon-stimulated genes during viral infection. The same effect of pterostilbene was also observed in the condition of polyinosinic-polycytidylic acid (poly I:C) transfection. Further study showed that pterostilbene interacted with influenza non-structural 1 (NS1) protein, inhibited ubiquitination mediated degradation of RIG-I and activated the downstream antiviral pathway, orchestrating an antiviral state against influenza virus in the cell. Taken together, pterostilbene could be a promising anti-influenza agent for future antiviral drug exploitation and compounds with similar structures may provide new options for the development of novel inhibitors against influenza A virus (IAV).

Assessing the fitness of a dual-antiviral drug resistant human influenza virus in the ferret model

Influenza antivirals are important tools in our fight against annual influenza epidemics and future influenza pandemics. Combinations of antivirals may reduce the likelihood of drug resistance and improve clinical outcomes. Previously, two hospitalised immunocompromised influenza patients, who received a combination of a neuraminidase inhibitor and baloxavir marboxil, shed influenza viruses resistant to both drugs. Here-in, the replicative fitness of one of these A(H1N1)pdm09 virus isolates with dual resistance mutations (NA-H275Y and PA-I38T) was similar to wild type virus (WT) in vitro, but reduced in the upper respiratory tracts of challenged ferrets. The dual-mutant virus transmitted well between ferrets in an airborne transmission model, but was outcompeted by the WT when the two viruses were co-administered. These results indicate the dual-mutant virus had a moderate loss of viral fitness compared to the WT virus, suggesting that while person-to-person transmission of the dual-resistant virus may be possible, widespread community transmission is unlikely.

Anti-influenza virus activity of the REV-ERBα agonist SR9009 and related analogues

REV-ERBα is a member of the nuclear receptor superfamily of transcription factors that aids in the regulation of many diseases. However, the prospect of using REV-ERBα for anti-influenza virus treatment remains poorly described, and there is an urgent need to develop effective anti-influenza agents due to the emergence of drug-resistant influenza viruses. In this study, eight SR9009 analogues were designed, synthesized, and evaluated for their biological activities against multiple influenza virus strains (H1N1, H3N2, adamantane- and oseltamivir-resistant H1N1 and influenza B virus), using ribavirin as the positive control. SR9009 and its analogues showed low micromolar or submicromolar EC50 values and exhibited modestly improved antiviral potency compared to that of ribavirin. In particular, compound 5a possessed the most potent inhibitory activity (EC50 = 0.471, 0.644, 1.644, 0.712 and 0.661 μM for A/PR/8/34, A/WSN/33, A/Wisconsin/67/2005, B/Yamagata/16/88 and Hebei/SWL1/2006, respectively). Cotransfection assays showed that all synthesized derivatives efficaciously suppressed transcription driven by the Bmal1 promoter. Mechanistic study results indicated that 5a efficiently inhibited IAV replication and interfered with the ealry stage of influenza virus life cycle. In addition, we found that 5a upregulated the key antiviral interferon-stimulated genes MxA, OAS2 and CH25H. Further in-depth transcriptome analysis revealed a series of upregulated genes that may contribute to the antiviral activities of 5a. These findings may provide an important direction for the development of new host-targeted broad-spectrum antiviral agents.

MicroRNA let-7 Suppresses Influenza A Virus Infection by Targeting RPS16 and Enhancing Type I Interferon Response.

Given the frequent emergence of drug-resistant influenza virus strains and new highly pathogenic influenza virus strains, there is an urgent need to identify new antiviral drugs and targets. We found that influenza A virus (IAV) infection caused a significant decrease of microRNA let-7 expression in host cells; that overexpression of let-7 increased interferon expression and effectively inhibit IAV infection; and that let-7 targets the 3’-untranslated region (UTR) of the ribosomal protein 16 (RPS16) gene, decreasing its expression. Knocking down the expression of RPS16 increased the expression of type I interferon and inhibited viral replication. The present study uncovered the regulatory effect of let-7b and let-7f on influenza A infection, which is a potential biomarker of IAV infection. In addition, let-7 may be a promising therapeutic agent against influenza A.

Role of Glycoconjugates and Mammalian Sialidases Involved in Viral Infection and Neural Function

Glycoconjugates are present in various organisms, ranging from animals to viruses. Glycoconjugates are involved in several biologically significant functions, including viral infection and neurotransmission. However, the role of glycoconjugates in virus replication and neural function remains unknown. We discovered that the influenza A virus (IAV) binds to sulfatide, which lacks a sialic acid residue, and that delivering sulfatide combined with newly synthesized IAV hemagglutinin (HA) to the target plasma membrane induces the translocation of viral ribonucleoprotein complexes from the nucleus to the cytoplasm. Molecular species of sialic acid are largely classified as N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). We discovered that two amino acids in IAV H3 HA play a critical role in the recognition of Neu5Ac and Neu5Gc. Also, we observed that human parainfluenza virus types 1 (hPIV-1) and 3 (hPIV-3) bind to different forms of gangliosides. These viruses preferentially recognized oligosaccharides containing branched N-acetyllactosaminoglycans with terminal Neu5Acα2-3Gal. Neu5Gcα2-3Gal- and NeuAcα2-6Gal-containing receptors were identified by hPIV-3, but not by hPIV-1. We constructed a novel sialidase fluorescent substrate, 2-benzothiazol-2-yl-phenol derivative-based N-acetylneuraminic acid (BTP3-Neu5Ac), which detects sialidase activity in living mammalian tissues and virus-infected cells expressing viral neuraminidase. We discovered that neural activity-dependent desialylation by sialidase contributes to rat hippocampal memory processing. Using BTP3-Neu5Ac, we developed a rapid and sensitive approach for detecting and isolating drug-resistant influenza viruses. This review summarizes the role of sialylglycoconjugates and sulfatide in virus replication as well as mammalian sialidases involved in neural function and insulin secretion.

In vitro and in vivo antiviral activity of Maqian (Zanthoxylum myriacanthum var. pubescens) essential oil and its major constituents against strains of influenza virus

Zanthoxylum myriacanthum var. pubescens (Maqian) is an economically important plant that is widely consumed as a flavoring agent or indigenous medicine by local ethnic groups in Southwest China. The pharmacological activities, such as the anti-inflammatory activity of Maqian essential oil, have been highlighted in recent years. In this study, the in vitro and in vivo anti-influenza activities of Maqian essential oil and its major ingredient, d/L-limonene, were assessed. The Maqian essential oil and d/L-limonene were effective against the influenza A and influenza B viruses in vitro. Notably, the Maqian essential oil was highly inhibitive against the drug-resistant influenza virus strain with an EC50 of 1.87 ± 1.04 μg/ml and a selective index of 85.13. Mode-of-action analysis revealed that Maqian essential oil and d/L-limonene inhibited the influenza virus at the stage of intracellular replication. For in vivo therapeutic efficacy, both intragastric and intranasal administration of d/L-limonene protected over 50% of mice from lethal influenza virus challenge. Both infectious viral particles and viral genome RNA were decreased approximately 2-fold in the d/L-limonene-treated mice compared with the virus group. Treatment with d/L-limonene potently improved the pathological features of the influenza virus-infected mice. Analysis of the gene expression level revealed that the major proinflammatory cytokines, including IL-1β, IL-6 and TNF-α, were significantly decreased in the d/L-limonene treated mouse lung tissues. Interestingly, we found that D-limonene was more potent than L-limonene in inhibiting proinflammatory cytokines. Transcriptome analysis revealed 32 host genes that participated in the antiviral process, most of which were related to the immune system and signal transduction. Combined with the antiviral and immunoregulatory effects of limonene, we believe that this multi-functional monoterpene has potential to become a novel candidate against influenza virus infections.

Zanamivir-Cholesterol Conjugate: A Long-Acting Neuraminidase Inhibitor with Potent Efficacy against Drug-Resistant Influenza Viruses.

Antiviral therapy of influenza virus infections depends heavily on two viral neuraminidase (NA) inhibitors, oseltamivir (OSV) and zanamivir (ZNV). The efficacy of OSV is challenged by the development of viral resistance, while the clinical use of ZNV is limited by its poor pharmacokinetic profile and requirement for twice-daily intranasal administration. We have developed a novel NA inhibitor by conjugating ZNV to cholesterol. The ZNV-cholesterol conjugate showed markedly improved antiviral efficacy and plasma half-life compared with ZNV. Single-dose administration of the conjugate protected the mice from lethal challenges with wild-type or mutant H1N1 influenza viruses bearing an OSV-resistant H275Y-substitution. Mechanistic studies showed that the conjugate targeted the cell membrane and entered the host cells, thereby inhibiting the NA function and the assembly of progeny virions. The ZNV-cholesterol conjugate represents a potential new treatment for influenza infections with sustained effect. Cholesterol conjugation may be an effective strategy for improving the pharmacokinetics and efficacy of other small-molecule therapeutics.

Bleomycin-Fe(II) agent with potentiality for treating drug-resistant H1N1 influenza virus: A study using electrochemical RNA beacons

Rapid emergence of new strains of drug-resistant H1N1 influenza viruses calls for effective drugs for the controls prior to their outbreaks. In the present work, electrochemical H1N1 RNA beacons have been newly designed for exploring the potentiality of an anticancer agent of Bleomycin (BLM) with Fe (ΙΙ) ions (BLM-Fe(ΙΙ)) alternatively the treatment of drug-resistant H1N1 strains with H274Y gene mutation. Herein, biotinylated (−) ssRNA of H1N1 virus and its complementary (+) ssRNA were labeled with electrochemical signal probes of ferrocene and anthraquinone, respectively. The resultants were hybridized and conjugated with avidin-modified magnetic beads to create electrochemical RNA beacons. The electrochemical signal variation of the H1N1 RNA beacon treated with the RNA degradation agent of BLM-Fe(ΙΙ) were monitored. Results indicate that the BLM-Fe(ΙΙ) agent could effectively cleave both H1N1 dsRNAs and ssRNAs at selective cutting sites, as evidenced by the mass spectrometry analysis. This indicates that the BLM-Fe(II) agent could be utilized to block the viral-host infection process by curbing the host-cell viral RNA-mRNA transcription or inactivate the viruses through the cleavage of viral genomes. The efficiency of the BLM-Fe(ΙΙ) agent was verified with clinical seasonal H1N1 samples using real-time polymerase chain reaction. The therapeutic gene drug of BLM-Fe(ΙΙ) holds great potential for controlling new strains of H1N1 virus resistant to clinical antiviral drugs. More importantly, the so designed RNA beacons may provide a rapid, sensitive and cost-effective platform of drug screening by monitoring the drug-DNA/RNA interactions.

Dynamic residue interaction network analysis of the oseltamivir binding site of N1 neuraminidase and its H274Y mutation site conferring drug resistance in influenza A virus.

BackgroundOseltamivir (OTV)-resistant influenza virus exhibits His-to-Tyr mutation at residue 274 (H274Y) in N1 neuraminidase (NA). However, the molecular mechanisms by which the H274Y mutation in NA reduces its binding affinity to OTV have not been fully elucidated.MethodsIn this study, we used dynamic residue interaction network (dRIN) analysis based on molecular dynamics simulation to investigate the correlation between the OTV binding site of NA and its H274Y mutation site.ResultsdRIN analysis revealed that the OTV binding site and H274Y mutation site of NA interact via the three interface residues connecting them. H274Y mutation significantly enhanced the interaction between residue 274 and the three interface residues in NA, thereby significantly decreasing the interaction between OTV and its surrounding loop 150 residues. Thus, we concluded that such changes in residue interactions could reduce the binding affinity of OTV to NA, resulting in drug resistant influenza viruses. Using dRIN analysis, we succeeded in understanding the characteristic changes in residue interactions due to H274Y mutation, which can elucidate the molecular mechanism of reduction in OTV binding affinity to influenza NA. Finally, the dRIN analysis used in this study can be widely applied to various systems such as individual proteins, protein-ligand complexes, and protein-protein complexes, to characterize the dynamic aspects of the interactions.

Interferon Lambda Delays the Emergence of Influenza Virus Resistance to Oseltamivir.

Influenza viruses are a leading cause of morbidity and mortality worldwide. These air-borne pathogens are able to cross the species barrier, leading to regular seasonal epidemics and sporadic pandemics. Influenza viruses also possess a high genetic variability, which allows for the acquisition of resistance mutations to antivirals. Combination therapies with two or more drugs targeting different mechanisms of viral replication have been considered an advantageous option to not only enhance the effectiveness of the individual treatments, but also reduce the likelihood of resistance emergence. Using an in vitro infection model, we assessed the barrier to viral resistance of a combination therapy with the neuraminidase inhibitor oseltamivir and human interferon lambda against the pandemic H1N1 A/Netherlands/602/2009 (H1N1pdm09) virus. We serially passaged the virus in a cell line derived from human bronchial epithelial cells in the presence or absence of increasing concentrations of oseltamivir alone or oseltamivir plus interferon lambda. While the treatment with oseltamivir alone quickly induced the emergence of antiviral resistance through a single mutation in the neuraminidase gene, the co-administration of interferon lambda delayed the emergence of drug-resistant influenza virus variants. Our results suggest a possible clinical application of interferon lambda in combination with oseltamivir to treat influenza.

Development of 6E3 antibody-mediated SERS immunoassay for drug-resistant influenza virus

Influenza viruses are responsible for several pandemics and seasonal epidemics and pose a major public health threat. Even after a major outbreak, the emergence of drug-resistant influenza viruses can pose disease control problems. Here we report a novel 6E3 monoclonal antibody capable of recognizing and binding to the H275Y neuraminidase (NA) mutation, which has been associated with reduced susceptibility of influenza viruses to NA inhibitors. The 6E3 antibody had a KD of 72.74 μM for wild-type NA and 32.76 pM for H275Y NA, suggesting that it can identify drug-resistant pandemic H1N1 (pH1N1) influenza virus. Molecular modeling studies also suggest the high-affinity binding of this antibody to pH1N1 H275Y NA. This antibody was also subject to dot-blot, enzyme-linked immunosorbent assay, bare-eye detection, and lateral flow assay to demonstrate its specificity to drug-resistant pH1N1. Furthermore, it was immobilized on Au nanoplate and nanoparticles, enabling surface-enhanced Raman scattering (SERS)-based detection of the H275Y mutant pH1N1. Using 6E3 antibody-mediated SERS immunoassay, the drug-resistant influenza virus can be detected at a low concentration of 102 plaque-forming units/mL. We also detected pH1N1 in human nasopharyngeal aspirate samples, suggesting that the 6E3-mediated SERS assay has the potential for diagnostic application. We anticipate that this newly developed antibody and SERS-based immunoassay will contribute to the diagnosis of drug-resistant influenza viruses and improve treatment strategies for influenza patients.

Design, synthesis and invitro anti-influenza A virus evaluation of novel quinazoline derivatives containing S-acetamide and NH-acetamide moieties at C-4.

It is an urgent need to develop more effective anti-influenza agents due to the emergence of highly pathogenic and drug-resistant influenza viruses. Herein, a series of 2,4-disubstituted quinazoline derivatives were designed, synthesized and their antiviral activities against influenza A virus were evaluated. Nine compounds (10a2, 16a, 16e, 16i, 16j, 16n, 16o, 16p and 16r) showed potent activity against influenza A virus (IAV) with IC50 at the low-micromole level (1.29-9.04μM). Particularly, 16e and 16r possess good anti-IAV activity (IC50: 1.29μM and 3.43μM, respectively) and acceptable cytotoxicity, and inhibit the transcription and replication of viral RNA. Together with reasonable PK profiles of 16e, these results suggest their promising potential as candidates for further investigation.

Characterization of neuraminidase inhibitor-resistant influenza virus isolates from immunocompromised patients in the Republic of Korea

BackgroundThe emergence of influenza viruses resistant to anti-influenza drugs is a threat to global public health. The Korea Centers for Disease Control and Prevention operates the Korea Influenza and Respiratory Viruses Surveillance System (KINRESS) to monitor epidemics of influenza and Severe Acute Respiratory Infection (SARI) to identify mutated influenza viruses affecting drug resistance, pathogenesis, and transmission.MethodsOropharyngeal swab samples were collected from KINRESS and SARI during the 2018–2019 season. The specimens confirmed influenza virus using real-time RT-PCR on inoculated MDCK cells. HA and NA sequences of the influenza viruses were analyzed for phylogeny and mutations. Neuraminidase inhibition and hemagglutination inhibition assays were utilized to characterize the isolates.ResultsTwo A(H1N1)pdm09 isolates harboring an H275Y substitution in the neuraminidase sequence were detected in patients with acute hematologic cancer. They had prolonged respiratory symptoms, with the virus present in the respiratory tract despite oseltamivir and peramivir treatment. Through the neuraminidase inhibition assay, both viruses were found to be resistant to oseltamivir and peramivir, but not to zanamivir. Although hemagglutinin and neuraminidase phylogenetic analyses suggested that the 2 A(H1N1)pdm09 isolates were not identical, their antigenicity was similar to that of the 2018–19 influenza vaccine virus.ConclusionsOur data indicate the utility of monitoring influenza-infected immunocompromised patients in general hospitals for the early detection of emerging neuraminidase inhibitor-resistant viruses and maintaining continuous laboratory surveillance of patients with influenza-like illness in sentinel clinics to monitor the spread of such new variants. Finally, characterization of the virus can inform the risk assessment for future epidemics and pandemics caused by drug-resistant influenza viruses.

Virtual Screening Identifies Chebulagic Acid as an Inhibitor of the M2(S31N) Viral Ion Channel and Influenza A Virus.

The increasing prevalence of drug-resistant influenza viruses emphasizes the need for new antiviral countermeasures. The M2 protein of influenza A is a proton-gated, proton-selective ion channel, which is essential for influenza replication and an established antiviral target. However, all currently circulating influenza A virus strains are now resistant to licensed M2-targeting adamantane drugs, primarily due to the widespread prevalence of an M2 variant encoding a serine to asparagine 31 mutation (S31N). To identify new chemical leads that may target M2(S31N), we performed a virtual screen of molecules from two natural product libraries and identified chebulagic acid as a candidate M2(S31N) inhibitor and influenza antiviral. Chebulagic acid selectively restores growth of M2(S31N)-expressing yeast. Molecular modeling also suggests that chebulagic acid hydrolysis fragments preferentially interact with the highly-conserved histidine residue within the pore of M2(S31N) but not adamantane-sensitive M2(S31). In contrast, chebulagic acid inhibits in vitro influenza A replication regardless of M2 sequence, suggesting that it also acts on other influenza targets. Taken together, results implicate chebulagic acid and/or its hydrolysis fragments as new chemical leads for M2(S31N) and influenza-directed antiviral development.