PSMD12-Mediated M1 Ubiquitination of Influenza A Virus at K102 Regulates Viral Replication.

The M1 of influenza A virus (IAV) is important for the virus life cycle, especially for the assembly and budding of viruses, which is a multistep process that requires host factors. Identifying novel host proteins that interact with M1 and understanding their functions in IAV replication are of great interest in antiviral drug development. In this study, we identified 19 host proteins in DF1 cells suspected to interact with the M1 protein of an H5N6 virus through immunoprecipitation (IP)/mass spectrometry. Among them, PSMD12, a 26S proteasome regulatory subunit, was shown to interact with influenza M1, acting as a positive host factor in IAV replication in avian and human cells. The data showed that PSMD12 promoted K63-linked ubiquitination of M1 at the K102 site. H5N6 and PR8 with an M1-K102 site mutant displayed a significantly weaker replication ability than the wild-type viruses. Mechanistically, PSMD12 promoted M1-M2 virus-like particle (VLP) release, and an M1-K102 mutation disrupted the formation of supernatant M1-M2 VLPs. An H5N6 M1-K102 site mutation or knockdown PSMD12 disrupted the budding release of the virus in chicken embryo fibroblast (CEF) cells, which was confirmed by transmission electron microscopy. Further study confirmed that M1-K102 site mutation significantly affected the virulence of H5N6 and PR8 viruses in mice. In conclusion, we report the novel host factor PSMD12 which affects the replication of influenza virus by mediating K63-linked ubiquitination of M1 at K102. These findings provide novel insight into the interactions between IAV and host cells, while suggesting an important target for anti-influenza virus drug research. IMPORTANCE M1 is proposed to play multiple biologically important roles in the life cycle of IAV, which relies largely on host factors. This study is the first one to identify that PSMD12 interacts with M1, mediates K63-linked ubiquitination of M1 at the K102 site, and thus positively regulates influenza virus proliferation. PSMD12 promoted M1-M2 VLP egress, and an M1-K102 mutation affected the M1-M2 VLP formation. Furthermore, we demonstrate the importance of this site to the morphology and budding of influenza viruses by obtaining mutant viruses, and the M1 ubiquitination regulator PSMD12 has a similar function to the M1 K102 mutation in regulating virus release and virus morphology. Additionally, we confirm the reduced virulence of H5N6 and PR8 (H1N1) viruses carrying the M1-K102 site mutation in mice. These findings provide novel insights into IAV interactions with host cells and suggest a valid and highly conserved candidate target for antiviral drug development.

Antiviral Activity and Underlying Action Mechanism of Euglena Extract against Influenza Virus.

It is difficult to match annual vaccines against the exact influenza strain that is spreading in any given flu season. Owing to the emergence of drug-resistant viral strains, new approaches for treating influenza are needed. Euglena gracilis (hereinafter Euglena), microalga, used as functional foods and supplements, have been shown to alleviate symptoms of influenza virus infection in mice. However, the mechanism underlying the inhibitory action of microalgae against the influenza virus is unknown. Here, we aimed to study the antiviral activity of Euglena extract against the influenza virus and the underlying action mechanism using Madin-Darby canine kidney (MDCK) cells. Euglena extract strongly inhibited infection by all influenza virus strains examined, including those resistant to the anti-influenza drugs oseltamivir and amantadine. A time-of-addition assay revealed that Euglena extract did not affect the cycle of virus replication, and cell pretreatment or prolonged treatment of infected cells reduced the virus titer. Thus, Euglena extract may activate the host cell defense mechanisms, rather than directly acting on the influenza virus. Moreover, various minerals, mainly zinc, in Euglena extract were found to be involved in the antiviral activity of the extract. In conclusion, Euglena extract could be a potent agent for preventing and treating influenza.

Assessment of the Feasibility of Using Noninvasive Wearable Biometric Monitoring Sensors to Detect Influenza and the Common Cold Before Symptom Onset.

Importance: Currently, there are no presymptomatic screening methods to identify individuals infected with a respiratory virus to prevent disease spread and to predict their trajectory for resource allocation. Objective: To evaluate the feasibility of using noninvasive, wrist-worn wearable biometric monitoring sensors to detect presymptomatic viral infection after exposure and predict infection severity in patients exposed to H1N1 influenza or human rhinovirus. Design, Setting, and Participants: The cohort H1N1 viral challenge study was conducted during 2018; data were collected from September 11, 2017, to May 4, 2018. The cohort rhinovirus challenge study was conducted during 2015; data were collected from September 14 to 21, 2015. A total of 39 adult participants were recruited for the H1N1 challenge study, and 24 adult participants were recruited for the rhinovirus challenge study. Exclusion criteria for both challenges included chronic respiratory illness and high levels of serum antibodies. Participants in the H1N1 challenge study were isolated in a clinic for a minimum of 8 days after inoculation. The rhinovirus challenge took place on a college campus, and participants were not isolated. Exposures: Participants in the H1N1 challenge study were inoculated via intranasal drops of diluted influenza A/California/03/09 (H1N1) virus with a mean count of 106 using the median tissue culture infectious dose (TCID50) assay. Participants in the rhinovirus challenge study were inoculated via intranasal drops of diluted human rhinovirus strain type 16 with a count of 100 using the TCID50 assay. Main Outcomes and Measures: The primary outcome measures included cross-validated performance metrics of random forest models to screen for presymptomatic infection and predict infection severity, including accuracy, precision, sensitivity, specificity, F1 score, and area under the receiver operating characteristic curve (AUC). Results: A total of 31 participants with H1N1 (24 men [77.4%]; mean [SD] age, 34.7 [12.3] years) and 18 participants with rhinovirus (11 men [61.1%]; mean [SD] age, 21.7 [3.1] years) were included in the analysis after data preprocessing. Separate H1N1 and rhinovirus detection models, using only data on wearble devices as input, were able to distinguish between infection and noninfection with accuracies of up to 92% for H1N1 (90% precision, 90% sensitivity, 93% specificity, and 90% F1 score, 0.85 [95% CI, 0.70-1.00] AUC) and 88% for rhinovirus (100% precision, 78% sensitivity, 100% specificity, 88% F1 score, and 0.96 [95% CI, 0.85-1.00] AUC). The infection severity prediction model was able to distinguish between mild and moderate infection 24 hours prior to symptom onset with an accuracy of 90% for H1N1 (88% precision, 88% sensitivity, 92% specificity, 88% F1 score, and 0.88 [95% CI, 0.72-1.00] AUC) and 89% for rhinovirus (100% precision, 75% sensitivity, 100% specificity, 86% F1 score, and 0.95 [95% CI, 0.79-1.00] AUC). Conclusions and Relevance: This cohort study suggests that the use of a noninvasive, wrist-worn wearable device to predict an individual's response to viral exposure prior to symptoms is feasible. Harnessing this technology would support early interventions to limit presymptomatic spread of viral respiratory infections, which is timely in the era of COVID-19.

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells.

Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log10 (HAU/100 µl) were achieved using fast-growing MDCK cells at concentrations up to 9.5 × 106 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based steric-exclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.

Sulfated glycans containing NeuAcα2-3Gal facilitate the propagation of human H1N1 influenza A viruses in eggs.

When human influenza viruses are isolated and passaged in chicken embryos, variants with amino acid substitutions around the receptor binding site of hemagglutinin (HA) are selected; however, the mechanisms that underlie this phenomenon have yet to be elucidated. Here, we analyzed the receptor structures that contributed to propagation of egg-passaged human H1N1 viruses. The analysis included seasonal and 2009 pandemic strains, both of which have amino acid substitutions of HA found in strains isolated or passaged in eggs. These viruses exhibited high binding to sulfated glycans containing NeuAcα2-3Gal. In MDCK cells overexpressing the sulfotransferase that synthesize Galß1-4(SO3--6)GlcNAc, production of human H1N1 viruses was increased up to 90-fold. Furthermore, these sulfated glycans were expressed on the allantoic and amniotic membranes of chicken embryos. These results suggest that 6-sulfo sialyl Lewis X and/or NeuAcα2-3Galß1-4(SO3--6)GlcNAc are involved in efficient propagation of human H1N1 viruses in chicken embryos.

Utility of Three Nebulizers in Investigating the Infectivity of Airborne Viruses.

Laboratory-generated bioaerosols are widely used in aerobiology studies of viruses; however, few comparisons of alternative nebulizers exist. We compared aerosol production and virus survival for a Collison nebulizer, vibrating mesh nebulizer (VMN), and hydraulic spray atomizer (HSA). We also measured the dry size distribution of the aerosols produced and calculated the droplet sizes before evaporation and the dry size distribution from normal saline solution. Dry count median diameters of 0.11, 0.22, and 0.30 µm were found for normal saline from the Collison nebulizer, VMN, and HSA, respectively. The volume median diameters were 0.323, 1.70, and 1.30 µm, respectively. The effect of nebulization on the viability of two influenza A viruses (IAVs) (H1N1 and H3N2) and human rhinovirus 16 (HRV-16) was assessed by nebulization into an SKC BioSampler. The HSA had the least impact on surviving fractions (SFs) of H1N1 and H3N2 (89% ± 3% and 94% ± 2%, respectively), followed by the Collison nebulizer (83% ± 1% and 82% ± 2%, respectively). The VMN yielded SFs of 78% ± 2% and 76% ± 2%, respectively. Conversely, for HRV-16, the VMN produced higher SFs (87% ± 8%). Our findings indicate that there were no statistical differences between SFs of the viruses nebulized by these nebulizers. However, VMN produced higher aerosol concentrations within the airborne size range, making it more suitable where high aerosol mass production is required. IMPORTANCE Viral respiratory tract infections cause millions of lost days of work and physician visits globally, accounting for significant morbidity and mortality. Respiratory droplets and droplet nuclei from infected hosts are the potential carriers of such viruses within indoor environments. Laboratory-generated bioaerosols are applied in understanding the transmission and infection of viruses, modeling the physiological aspects of bioaerosol generation in a controlled environment. However, little comparative characterization exists for nebulizers used in infectious disease aerobiology, including Collison nebulizer, vibrating mesh nebulizer, and hydraulic spray atomizer. This study characterized the physical features of aerosols generated by laboratory nebulizers and their performance in producing aerosols at a size relevant to airborne transmission used in infectious disease aerobiology. We also determined the impact of nebulization mechanisms of these nebulizers on the viability of human respiratory viruses, including IAV H1N1, IAV H3N2, and HRV-16.

Antimicrobial Terpenoids from South China Sea Soft Coral Lemnalia sp.

Chemical investigation of the South China Sea soft coral Lemnalia sp. afforded 13 structurally diverse terpenoids, including three new neolemnane sesquiterpene lineolemnene, E-G (1-3); a new aristolane-type sesquiterpenoid, 2-acetoxy-aristolane (4); four new decalin-type bicyclic diterpenes, named biofloranate A-D (5-8); a new serrulatane, named euplexaurene D (9); and a new aromadendrane-type diterpenoid cneorubin K (10), together with three known related compounds (11-13). The structures of the new compounds were elucidated by NMR spectroscopy, the Mosher's method, and ECD analysis. Compounds 1-13 were tested in a wide panel of biological assays. Lineolemnene J (3) showed weak cytotoxicity against the CCRF-CEM cancer cell line. The isolated new diterpenes, except euplexaurene D (9), demonstrated moderate antimicrobial activity against Bacillus subtilis and Staphylococcus aureus with a MIC of 4-64 µg/mL. Compound 2 exhibited a mild inhibitory effect against influenza A H1N1 virus (IC50 = 5.9 µM).

CircRNA expression profiling and bioinformatics analysis indicate the potential biological role and clinical significance of circRNA in influenza A virus-induced lung injury.

Circular RNA (circRNA) plays an important role in the regulation of multiple biological processes. However, circRNA profiling and the potential biological role of circRNA in influenza A virus (IAV)-induced lung injury have not been investigated. In the present study, circRNA expression profiles in lung tissues from mice with and without IAV-induced lung injury were analyzed using high-throughput sequencing, and differentially expressed circRNAs were verified by quantitative PCR. The gene homology of candidate circRNAs was investigated and the expression of plasma circRNAs from patients with IAV-induced acute respiratory distress syndrome (ARDS) was detected. The target microRNAs (miRNAs) of circRNAs were predicted. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. In total, 781 circRNAs were differentially expressed between ARDS mice and control (467 were up-regulated and 314 were down-regulated). Moreover, the candidate circRNAs (Slco3a1, Nfatc2, Wdr33, and Dmd) expression showed the same trend with the sequencing results. The isoforms of circRNA Slco3a1 and Wdr33 were highly conserved between humans and mice. Plasma circRNA Slco3a1 and Wdr33 presented differential expression in patients with IAV-induced ARDS compared to control. The circRNAmiRNA interaction network and GO and KEGG analyses indicated the potential biological role of circRNAs in the development of IAV-induced lung injury. Taken together, a large number of differentially expressed circRNAs were identified in our study. CircRNA Slco3a1 and Wdr33 had significantly different expression in specimens from mice and humans, and showed a potential biological role in IAV-induced lung injury by bioinformatics analysis.

Innate Antiviral Cytokine Response to Swine Influenza Virus by Swine Respiratory Epithelial Cells.

Swine influenza virus (SIV) can cause respiratory illness in swine. Swine contribute to influenza virus reassortment, as avian, human, and/or swine influenza viruses can infect swine and reassort, and new viruses can emerge. Thus, it is important to determine the host antiviral responses that affect SIV replication. In this study, we examined the innate antiviral cytokine response to SIV by swine respiratory epithelial cells, focusing on the expression of interferon (IFN) and interferon-stimulated genes (ISGs). Both primary and transformed swine nasal and tracheal respiratory epithelial cells were examined following infection with field isolates. The results show that IFN and ISG expression is maximal at 12 h postinfection (hpi) and is dependent on cell type and virus genotype. IMPORTANCE Swine are considered intermediate hosts that have facilitated influenza virus reassortment events that have given rise pandemics or genetically related viruses have become established in swine. In this study, we examine the innate antiviral response to swine influenza virus in primary and immortalized swine nasal and tracheal epithelial cells, and show virus strain- and host cell type-dependent differential expression of key interferons and interferon-stimulated genes.

Inhibitory effects of aprotinin on influenza A and B viruses in vitro and in vivo.

Influenza viruses cause significant morbidity and mortality worldwide. Long-term or frequent use of approved anti-influenza agents has resulted in drug-resistant strains, thereby necessitating the discovery of new drugs. In this study, we found aprotinin, a serine protease inhibitor, as an anti-influenza candidate through screening of compound libraries. Aprotinin has been previously reported to show inhibitory effects on a few influenza A virus (IAV) subtypes (e.g., seasonal H1N1 and H3N2). However, because there were no reports of its inhibitory effects on the other types of influenza viruses, we investigated the inhibitory effects of aprotinin in vitro on a wide range of influenza viruses, including avian and oseltamivir-resistant influenza virus strains. Our cell-based assay showed that aprotinin had inhibitory effects on seasonal human IAVs (H1N1 and H3N2 subtypes), avian IAVs (H5N2, H6N5, and H9N2 subtypes), an oseltamivir-resistant IAV, and a currently circulating influenza B virus. We have also confirmed its activity in mice infected with a lethal dose of influenza virus, showing a significant increase in survival rate. Our findings suggest that aprotinin has the capacity to inhibit a wide range of influenza virus subtypes and should be considered for development as a therapeutic agent against influenza.

Airway basal stem cells generate distinct subpopulations of PNECs.

Pulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.

Glucosylceramide synthase inhibitors prevent replication of SARS-CoV-2 and influenza virus.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health. Vaccines are ideal solutions to prevent infection, but treatments are also needed for those who have contracted the virus to limit negative outcomes, when vaccines are not applicable. Viruses must cross host cell membranes during their life cycle, creating a dependency on processes involving membrane dynamics. Thus, in this study, we examined whether the synthetic machinery for glycosphingolipids, biologically active components of cell membranes, can serve as a therapeutic target to combat SARS-CoV-2. We examined the antiviral effect of two specific inhibitors of glucosylceramide synthase (GCS): (i) Genz-123346, an analogue of the United States Food and Drug Administration-approved drug Cerdelga and (ii) GENZ-667161, an analogue of venglustat, which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit replication of SARS-CoV-2. Moreover, these inhibitors also disrupt replication of influenza virus A/PR/8/34 (H1N1). Our data imply that synthesis of glycosphingolipids is necessary to support viral life cycles and suggest that GCS inhibitors should be further explored as antiviral therapies.

Progenitor identification and SARS-CoV-2 infection in human distal lung organoids.

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.

In Vitro and In Vivo Antiviral Activity of Gingerenone A on Influenza A Virus Is Mediated by Targeting Janus Kinase 2.

Janus kinase (JAK) inhibitors have been developed as novel immunomodulatory drugs and primarily used for treating rheumatoid arthritis and other inflammatory diseases. Recent studies have suggested that this category of anti-inflammatory drugs could be potentially useful for the control of inflammation "storms" in respiratory virus infections. In addition to their role in regulating immune cell functions, JAK1 and JAK2 have been recently identified as crucial cellular factors involved in influenza A virus (IAV) replication and could be potentially targeted for antiviral therapy. Gingerenone A (Gin A) is a compound derived from ginger roots and a dual inhibitor of JAK2 and p70 S6 kinase (S6K1). Our present study aimed to determine the antiviral activity of Gin A on influenza A virus (IAV) and to understand its mechanisms of action. Here, we reported that Gin A suppressed the replication of three IAV subtypes (H1N1, H5N1, H9N2) in four cell lines. IAV replication was also inhibited by Ruxolitinib (Rux), a JAK inhibitor, but not by PF-4708671, an S6K1 inhibitor. JAK2 overexpression enhanced H5N1 virus replication and attenuated Gin A-mediated antiviral activity. In vivo experiments revealed that Gin A treatment suppressed IAV replication in the lungs of H5N1 virus-infected mice, alleviated their body weight loss, and prolonged their survival. Our study suggests that Gin A restricts IAV replication by inhibiting JAK2 activity; Gin A could be potentially useful for the control of influenza virus infections.

Anti-Influenza Virus Activity and Chemical Components from the Parasitic Plant Cuscuta japonica Choisy on Dimocarpus longans Lour.

Dodder (Cuscuta spp.) is a parasitic weed damaging many plants and agricultural production. The native obligate parasite Cuscuta japonica Choisy (Japanese dodder) parasitizes Dimocarpus longans Lour., Ficus septica Burm. F., Ficus microcarpa L.f., Mikania micrantha H.B.K. and Melia azedarach Linn, respectively. Five Japanese dodders growing on different plants exhibit slightly different metabolites and amounts which present different pharmacological effects. Among these plants, a significant antiviral activity against influenza A virus (IAV) was found in Japanese dodder parasitizing on D. longans Lour. (CL). To further explore methanol extract components in Japanese dodder (CL), four undescribed aromatic glycosides, cuscutasides A-D (compounds 1-4) were isolated, together with twenty-six known compounds 5-30. The chemical structures of 1-4 were elucidated using a combination of spectroscopic techniques. The eighteen isolated compounds were evaluated for antiviral activity against IAV activity. Among them, 1-monopalmitin (29) displayed potent activity against influenza A virus (A/WSN/1933(H1N1)) with EC50 2.28 ± 0.04 µM and without noteworthy cytotoxicity in MDCK cells. The interrupt step of 29 on the IAV life cycle was determined. These data provide invaluable information for new applications for this otherwise harmful weed.

Thapsigargin at Non-Cytotoxic Levels Induces a Potent Host Antiviral Response that Blocks Influenza A Virus Replication.

Influenza A virus is a major global pathogen of humans, and there is an unmet need for effective antivirals. Current antivirals against influenza A virus directly target the virus and are vulnerable to mutational resistance. Harnessing an effective host antiviral response is an attractive alternative. We show that brief exposure to low, non-toxic doses of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, promptly elicits an extended antiviral state that dramatically blocks influenza A virus production. Crucially, oral administration of TG protected mice against lethal virus infection and reduced virus titres in the lungs of treated mice. TG-induced ER stress unfolded protein response appears as a key driver responsible for activating a spectrum of host antiviral defences that include an enhanced type I/III interferon response. Our findings suggest that TG is potentially a viable host-centric antiviral for the treatment of influenza A virus infection without the inherent problem of drug resistance.

Antiviral activity of furanocoumarins isolated from Angelica dahurica against influenza a viruses H1N1 and H9N2.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. (Umbelliferae family) is an herbaceous, perennial plant native to northern and eastern Asia. The root of A. dahurica has traditionally been used under the name "Bai Zhi" as a medicinal plant for colds, dizziness, ulcers, and rheumatism. Moreover, it is also an important ingredient of various prescriptions, such as Gumiganghwal-Tang, for the common cold and influenza. AIM OF THE STUDY: Even though various biological activities of the root of A. dahurica have been reported along with its chemical components, the detailed mechanism of how it exerts anti-influenza activity at the compound level has not been studied. Therefore, we investigated the anti-influenza properties of furanocoumarins purified by bioactivity-guided isolation. MATERIALS AND METHODS: Bioactivity-guided isolation from a 70% EtOH extract of the root of A. dahurica was performed to produce four active furanocoumarins. The inhibition of cytopathic effects (CPEs) was evaluated to ascertain the antiviral activity of these compounds against influenza A (H1N1 and H9N2) viruses. The most potent compound was subjected to detailed mechanistic studies such as the inhibition of viral protein synthesis, CPE inhibition in different phases of the viral replication cycle, neuraminidase (NA) inhibition, antiapoptotic activity using flow cytometry, and immunofluorescence. RESULTS: The bioactivity-guided isolation produced four active furanocoumarins, isoimperatorin (1), oxypeucedanin (2), oxypeucedanin hydrate (3) and imperatorin (4) from the n-BuOH fraction. Among them, compound 2 (followed by compounds 1, 4 and 3) showed a significant CPE inhibition effect, which was stronger than that of the positive control ribavirin, against both H1N1 and H9N2 with an EC50 (µM) of 5.98 ± 0.71 and 4.52 ± 0.39, respectively. Compound 2 inhibited the synthesis of NA and nucleoprotein (NP) in a dose-dependent manner. In the time course assays, the cytopathic effects of influenza A-infected MDCK cells were reduced by 80-90% when treated with compound 2 for 1 and 2 h after infection and declined drastically 3 h after infection. The level of viral NA and NP production was markedly reduced to less than 20% for both proteins in compound 2 (20 µM)-treated cells compared to untreated cells at 2 h after infection. In the molecular docking analysis, compound 2 showed a stronger binding affinity for the C-terminus of polymerase acidic protein (PAC; -36.28 kcal/mol) than the other two polymerase subunits. Compound 2 also exerted an antiapoptotic effect on virus infected cells and significantly inhibited the mRNA expression of caspase-3 and Bax. CONCLUSION: Our results suggest that compound 2 might exert anti-influenza A activity via the inhibition of the early phase of the viral replication cycle, not direct neutralization of surface proteins, such as hemagglutinin and NA, and abnormal apoptosis induced by virus infection. Taken together, these findings suggest that furanocoumarins predominant in A. dahurica play a pivotal role in its antiviral activity. These findings can also explain the reasons for the ethnopharmacological uses of this plant as an important ingredient in many antiviral prescriptions in traditional Chinese medicine (TCM).

Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.

The seafood Musculus senhousei shows anti-influenza A virus activity by targeting virion envelope lipids.

Marine environments are known to be a new source of structurally diverse bioactive molecules. In this paper, we identified a porphyrin derivative of Pyropheophorbide a (PPa) from the mussel Musculus senhousei (M. senhousei) that showed broad anti-influenza A virus activity in vitro against a panel of influenza A viral strains. The analysis of the mechanism of action indicated that PPa functions in the early stage of virus infection by interacting with the lipid bilayer of the virion, resulting in an alteration of membrane-associated functions, thereby blocking the entry of enveloped viruses into host cells. In addition, the anti-influenza A virus activity of PPa was further assessed in mice infected with the influenza A virus. The survival rate and mean survival time of mice were apparently prolonged compared with the control group which was not treated with the drug. Therefore, PPa and its derivatives may represent lead compounds for controlling influenza A virus infection.

Performance of an acoustic settler versus a hollow fiber-based ATF technology for influenza virus production in perfusion.

Process intensification and integration is crucial regarding an ever increasing pressure on manufacturing costs and capacities in biologics manufacturing. For virus production in perfusion mode, membrane-based alternating tangential flow filtration (ATF) and acoustic settler are the commonly described cell retention technologies. While acoustic settlers allow for continuous influenza virus harvesting, the use of commercially available membranes for ATF systems typically results in the accumulation of virus particles in the bioreactor vessel. Accordingly, with one single harvest at the end of a cultivation, this increases the risk of lowering the product quality. To assess which cell retention device would be most suitable for influenza A virus production, we compared various key performance figures using AGE1.CR.pIX cells at concentrations between 25 and 50 × 106 cells/mL at similar infection conditions using either an ATF system or an acoustic settler. Production yields, process-related impurities, and aggregation of viruses and other large molecules were evaluated. Taking into account the total number of virions from both the bioreactor and the harvest vessel, a 1.5-3.0-fold higher volumetric virus yield was obtained for the acoustic settler. In addition, fewer large-sized aggregates (virus particles and other molecules) were observed in the harvest taken directly from the bioreactor. In contrast, similar levels of process-related impurities (host cell dsDNA, total protein) were obtained in the harvest for both retention systems. Overall, a clear advantage was observed for continuous virus harvesting after the acoustic settler operation mode was optimized. This development may also allow direct integration of subsequent downstream processing steps. KEY POINTS: • High suspension cell density, immortalized avian cell line, influenza vaccine.