Treatment Of Influenza Virus Infection Research Articles

Pharmacoeconomic study of anti-influenza virus drugs in Japan based on a network meta-analysis.

An analysis was conducted in Japan to determine the most cost-effective neuraminidase inhibitor for the treatment of influenza virus infections from the healthcare payer's standpoint. This study reanalysed the findings of a previous study that had some limitations (no probabilistic sensitivity analysis and quality of life scores measured by the EQ-5D-3L instead of the EQ-5D-5L) and used a decision tree model with only three health conditions. This study incorporated new data from a network meta-analysis study into the first examination. The second examination involved constructing a new decision tree model encompassing seven health conditions and identifying costs, which consisted of medical costs and drug prices based on the 2020 version of the Japanese medical fee index. Effectiveness outcomes were measured using EQ-5D-5L questionnaires for adult patients with a history of influenza virus infections within a 14-day time horizon. Deterministic and probabilistic sensitivity analyses were performed to examine the uncertainty. In the first examination, the base-case cost-effectiveness analysis confirmed that oseltamivir outperformed laninamivir, zanamivir and peramivir, making it the most cost-effective neuraminidase inhibitor. The second examination revealed that oseltamivir dominated the other agents. Both deterministic and probabilistic sensitivity analyses showed robust results that validated oseltamivir as the most cost effective among the four neuraminidase inhibitors. This study thus reaffirms oseltamivir's position as the most cost-effective neuraminidase inhibitor for the treatment of influenza virus infections in Japan from the perspective of healthcare payment. These findings can help decision makers and healthcare providers in Japan.

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.

Network pharmacology-based study to investigate the mechanism of compound houttuynia mixture against influenza virus infection by suppressing TLR7/MyD88 signaling pathway

Aim of the studyCompound houttuynia mixture (CHM) has been approved to cure respiratory diseases in China. However, the anti-influenza virus effect and underlying mechanism of CHM remains to be confirmed. Materials and methodsThe in vitro activity of CHM against H3N2 virus was evaluated using MDCK, A549 and THP-1 cells as an in vitro model. The in vivo protective effect on H3N2 virus infection was investigated. Moreover, serum cytokines were measured with high throughput liquid phase protein chip. The anti-influenza mechanism of CHM was predicted by network pharmacology and further validated with immunoblotting. ResultsOur results indicated that CHM has significant inhibitory effect on the replication of influenza A H3N2 virus. Furthermore, CHM could effectively reduce the mortality caused by lethal H3N2 virus infection by prolonging the survival time, reducing lung pathological injury, and suppressing excessive cytokines storm. Network pharmacology revealed that the protective effect of CHM on influenza virus infection was involved in multiple targets and multiple pathways. Mechanistic validation indicated that inhibiting the excessive activation of TLR7/MyD88 signaling pathway may be the critical mechanism of CHM exerting the protective effect against influenza virus infection. ConclusionRegulating of TLR7/MyD88/NF-κB signaling pathway in multiple target cells might be one of key mechanisms of CHM by inhibiting virus replication and excessive inflammatory. Our findings indicated that CHM might be an effective treatment for influenza virus infection.

Arbidol attenuates liver fibrosis and activation of hepatic stellate cells by blocking TGF-β1 signaling

Chronic liver diseases (CLD) impact over 800 million people globally, causing about 2 million deaths annually. Arbidol (ARB), an indole-derivative used to treat influenza virus infection, was extensively used during COVID-19 pandemic in China. In recent years, studies have shown that ARB, compared to other antiviral drugs, exhibits greater liver-protective efficacy, indicating a potential hepatoprotective effect beyond its antiviral activity. However, the mechanism remains unclear. In this study, we investigated the impact of ARB on liver injury/fibrosis in bile duct ligated (BDL) mice and its effect on spontaneous and transforming growth factor β1 (TGF-β1)-induced activation of primary cultured hepatic stellate cells (HSCs). Oral administration of ARB significantly ameliorated BDL-induced liver injury/fibrosis as reflected by decreased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), reduced collagen deposition, and diminished mRNA expression of fibrosis markers. ARB notably inhibited spontaneous and TGF-β1-induced activation of primary cultured HSCs. Moreover, ARB also drastically attenuated mRNA expression levels of platelet-derived growth factor receptor (Pdgfr), transforming growth factor-beta receptor (Tgfbr) 1, Tgfbr2, matrix metalloproteinase (Mmp)-2, and Mmp-9 in activated HSCs. We further demonstrate that ARB mitigated Smad2/3 phosphorylation in both TGF-β1 treated HSCs and BDL mice. These data together demonstrate that the therapeutic efficacy of ARB on liver fibrosis is independent of its antiviral activity and likely is achieved by blocking TGF-β1 signaling-mediated HSC activation.

Development and Stability Study of Oseltamivir Reconstitutable Dry Suspension from Capsules

Oseltamivir phosphate (OSEL) is recommended for the prevention and treatment of influenza virus infection. In a hospital pharmacy, OSEL liquid extemporaneous preparation is generally compounded on a case-by-case basis from the capsules because it can be stored for a certain period. Therefore, the objective of the current research was to develop an oseltamivir reconstitutable dry suspension (ORDS), and to evaluate the stability of the ORDS. The dry-suspending vehicles were first developed and subjected to stability tests. The stable vehicle was selected to mix with the content of OSEL capsules and dry powder was termed ORDS. The stability of ORDS was then tested in both dried and reconstituted states. It was found that the dry vehicle using xanthan gum as a suspending agent (at 0.25% w/v of the reconstituted suspension) was the most stable formulation at 25°C and 45°C under 75% relative humidity (RH) for 28 days. Upon mixing with OSEL capsule contents, ORDS in the dried state showed no change in either physical or chemical properties after storing at room temperature for 7 days. For the reconstituted state, the ORDS showed no change in odor and pH after storing at 2°C to 8°C in a refrigerator, and 25°C and 45°C under 75%RH for 14 days. However, its color was slightly darker after storing at 45°C. The percentage remaining OSEL at the refrigerator temperatures was 98.13% ± 1.63%, at 25°C was 100.30% ± 0.71% and at 45°C was 97.54% ± 1.38%. In conclusion, ORDS may be a convenient alternative to extemporaneous preparations. Keywords: Oseltamivir capsule, Reconstitutable dry suspension, Extemporaneous, Physico-chemical stability, Influenza

2147. Design of a Ligand-Targeted Immunotherapy for Treatment of Influenza Virus Infections

Abstract Background Nearly 10% of world’s population is affected by influenza virus infections and more than 26 million Americans were infected during the 2022-2023 flu season, with 290,000-640,000 patients requiring hospitalization. While flu vaccines are widely available, they were only 36% effective during the 2021-2022 season. Standard-of-care (SOC) drugs such as Tamiflu and Xofluza are only effective when taken in the early stages of infection requiring novel flu drugs for treating later stages of infection. Schematics of the mechanism of action of EV21 Methods Herein we report a targeted therapy (EV21) with a dual mechanism of action that elicits a host immune response to infections. Because neuraminidase is expressed on both the viral envelope and infected host cell surface, we repurposed the neuraminidase inhibitor zanamivir for use as the targeting ligand. We linked zanamivir to two distinct haptens that bind to two different naturally occurring antibodies in humans. Once recruited, these antibodies recruit the innate immune system to kill both virus and virus-infected cells. Preclinical in vitro and in vivo experiments were performed to evaluate the efficacy, safety, and pharmacokinetics of EV21. Results When tested in BALB/c mice supplemented with physiological levels of human IgG (IVIG) and infected with 10xLD50 of influenza A virus (H1N1, A/Puerto Rico/8/1934), EV21 elicited excellent antiviral activity. A single dose of our EV21 showed much better activity in late-stage infections than daily doses of the SOC drugs. Also, EV21 caused a significantly faster reduction of viral titer in the lungs of infected mice. Although EV21 exerts its antiviral effect by engaging the innate immune system, it did not give rise to any local or systemic cytokine storm while protecting mice from virus-induced cytokine release syndrome. Moreover, EV21 demonstrated excellent safety, pharmacokinetics, and metabolic stability making it a promising clinical candidate. Conclusion Our zanamivir-targeted dual hapten immunotherapy (EV21) has the potential to treat both early and late-stage influenza infections more effectively and rapidly than SOC drugs. Further testing in more clinically relevant ferret models and against multiple influenza strains, however, will be necessary to confirm the above benefits. Disclosures Imrul Shahriar, PhD Candidate, Eradivir Inc.: Ownership Interest|Eradivir Inc.: Stocks/Bonds Charity L. Campbell, n/a, Eradivir Inc.: Salary Ananda K. Kanduluru, PhD, Eradivir Inc.: Salary|Eradivir Inc.: Ownership Interest|Eradivir Inc.: Stocks/Bonds Madduri Srinivasarao, PhD, Eradivir Inc.: Ownership Interest|Eradivir Inc.: Stocks/Bonds Philip S. Low, PhD, Eradivir Inc.: Board Member|Eradivir Inc.: Ownership Interest|Eradivir Inc.: Stocks/Bonds

Comparison of clinical efficacy and safety of baloxavir marboxil versus oseltamivir as the treatment for influenza virus infections: A systematic review and meta-analysis

IntroductionBaloxavir marboxil (BXM), a newly developed cap-dependent endonuclease inhibitor, is widely used to treat influenza virus infections in inpatients and outpatients. A previous meta-analysis included only outpatients and patients suspected of having an influenza virus infection based on clinical symptoms. However, whether BXM or oseltamivir is safer and more effective for inpatients remains controversial. Therefore, we conducted a systematic review and meta-analysis validating the effectiveness and safety of BXM versus oseltamivir in inpatients with influenza virus. MethodsThe Scopus, EMBASE, PubMed, Ichushi, and CINAHL databases were systematically searched for articles published until January 2023. The outcomes were mortality, hospitalization period, incidence of BXM- or oseltamivir-related adverse events, illness duration, and changes of virus titers and viral RNA load in patients with influenza virus infections. ResultsTwo randomized controlled trials with 1624 outpatients and two retrospective studies with 874 inpatients were enrolled. No deaths occurred in outpatients treated with BXM or oseltamivir. Among inpatients, BXM reduced mortality (p = 0.06) and significantly shortened hospitalization period (p = 0.01) compared to oseltamivir. In outpatients, BXM had a significantly lower incidence of adverse events (p = 0.03), reductions in influenza virus titers (p < 0.001) and viral RNA loads (p < 0.001), and a tendency to be a shorter illness duration compared with that of oseltamivir (p = 0.27). ConclusionsOur meta-analysis showed that BXM was safer and more effective in patients than oseltamivir; thus, supporting the use of BXM for the initial treatment of patients with proven influenza virus infection.

Schisandra chinensis (Turcz.) Baill. polysaccharide inhibits influenza A virus in vitro and in vivo.

Influenza virus is prone to seasonal spread and widespread outbreaks, which pose important challenges to public health security. Therefore, it is important to effectively prevent and treat influenza virus infection. Schisandra polysaccharide (SPJ) is a polysaccharide derived from the fruit of Schisandra chinensis (Turcz.) Baill. In this study, we evaluated the antiviral activity of SPJ in vitro and in vivo, especially against influenza A virus (IAV) infection. By analyzing SPJ structure and monosaccharide composition, the molecular weight of SPJ was determined to be 115.5 KD, and it is composed of galacturonic acid (89.4%), rhamnose (0.8%), galactose (4.4%), arabinose (3.8%) and glucose (1.7%). Immunofluorescence analysis showed that SPJ treatment reduced the positive rate of viral nucleoproteins in cells, indicating that the compound had an inhibitory effect on influenza virus replication. Furthermore, SPJ therapy improved the survival of infected mice. Lung virus titer assays indicated that SPJ treatment significantly reduced viral-loading in the lung tissue of infected mice, and alleviated the pathological damage caused by influenza virus infection. Moreover, SPJ reduced cytokine expression during influenza virus challenge. In conclusion, SPJ has anti-influenza virus effects, and may have potential as an anti-influenza drug candidate in further clinical studies.

A fully human monoclonal antibody possesses antibody-dependent cellular cytotoxicity (ADCC) activity against the H1 subtype of influenza A virus by targeting a conserved epitope at the HA1 protomer interface.

The DiversitabTM system produces target specific high titer fully human polyclonal IgG immunoglobulins from transchromosomic (Tc) bovines shown to be safe and effective against multiple virulent pathogens in animal studies and Phase 1, 2 and 3 human clinical trials. We describe the functional properties of a human monoclonal antibody (mAb), 38C2, identified from this platform, which recognizes recombinant H1 hemagglutinins (HAs) and induces appreciable antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Interestingly, 38C2 monoclonal antibody demonstrated no detectable neutralizing activity against H1N1 virus in both hemagglutination inhibition and virus neutralization assays. Nevertheless, this human monoclonal antibody induced appreciable ADCC against cells infected with multiple H1N1 strains. The HA-binding activity of 38C2 was also demonstrated in flow cytometry using Madin-Darby canine kidney cells infected with multiple influenza A H1N1 viruses. Through further investigation with the enzyme-linked immunosorbent assay involving the HA peptide array and 3-dimensional structural modeling, we demonstrated that 38C2 appears to target a conserved epitope located at the HA1 protomer interface of H1N1 influenza viruses. A novel mode of HA-binding and in vitro ADCCactivity pave the way for further evaluation of 38C2 as a potential therapeutic agent to treat influenza virus infections in humans.

Highly Cross-Reactive and Protective Influenza A Virus H3N2 Hemagglutinin- and Neuraminidase-Specific Human Monoclonal Antibodies.

Due to antigenic drift and shift of influenza A viruses (IAV) and the tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to new strains of seasonal IAV and is at risk from viruses with pandemic potential for which limited or no immunity may exist. The genetic drift of H3N2 IAV is specifically pronounced, resulting in two distinct clades since 2014. Here, we demonstrate that immunization with a seasonal inactivated influenza vaccine (IIV) results in increased levels of H3N2 IAV-specific serum antibodies against hemagglutinin (HA) and neuraminidase (NA). Detailed analysis of the H3N2 B cell response indicated expansion of H3N2-specific peripheral blood plasmablasts 7 days after IIV immunization which expressed monoclonal antibodies (MAbs) with broad and potent antiviral activity against many H3N2 IAV strains as well as prophylactic and therapeutic activity in mice. These H3N2-specific B cell clonal lineages persisted in CD138+ long-lived bone marrow plasma cells. These results demonstrate that IIV-induced H3N2 human MAbs can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IAV H3N2-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development. IMPORTANCE Influenza A virus (IAV) infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets within the influenza virus hemagglutinin and neuraminidase proteins. We have demonstrated that seasonal immunization with inactivated influenza vaccine (IIV) stimulates H3N2-specific monoclonal antibodies in humans that are broad and potent in their neutralization of virus in vitro. These antibodies also provide protection from H3N2 IAV in a mouse model of infection. Furthermore, they persist in the bone marrow, where they are expressed by long-lived antibody-producing plasma cells. This significantly demonstrates that seasonal IIV can induce a subset of H3N2-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine.

Bioassay guided isolation of caffeoylquinic acids from the leaves of Ilex pubescens Hook. et Arn. and investigation of their anti-influenza mechanism.

Ilex pubescens Hook. et Arn. (Maodongqing, MDQ) is a common herbal tea ingredient in Southern China for heat clearance and anti-inflammation. Our preliminary screening showed that 50% ethanol extract of its leaves has anti-influenza virus activity. In this report, we proceed to identify the active components and clarify the related anti-influenza mechanisms. We aim to isolate and identify the anti-influenza virus phytochemicals from the extract of the MDQ leaves, and study their anti-influenza virus mechanism. Plaque reduction assay was used to test the anti-influenza virus activity of fractions and compounds. Neuraminidase inhibitory assay was used to confirm the target protein. Molecular docking and reverse genetics were used to confirm the acting site of caffeoylquinic acids (CQAs) on viral neuraminidase. Eight CQAs, 3,5-di-O-caffeoylquinic acid methyl ester (Me 3,5-DCQA), 3,4-di-O-caffeoylquinic acid methyl ester (Me 3,4-DCQA), 3,4,5-tri-O-caffeoylquinic acid methyl ester (Me 3,4,5-TCQA), 3,4,5-tri-O-caffeoylquinic acid (3,4,5-TCQA), 4,5-di-O-caffeoylquinic acid (4,5-DCQA), 3,5-di-O-caffeoylquinic acid (3,5-DCQA), 3,4-di-O-caffeoylquinic acid (3,4-DCQA), and 3,5-di-O-caffeoyl-epi-quinic acid (3,5-epi-DCQA) were identified from the MDQ leaves, in which Me 3,5-DCQA, 3,4,5-TCQA and 3,5-epi-DCQA were isolated for the first time. All these eight compounds were found to inhibit neuraminidase (NA) of influenza A virus. The results of molecular docking and reverse genetics indicated that 3,4,5-TCQA interacted with Tyr100, Gln412 and Arg419 of influenza NA, and a novel NA binding groove was found. Eight CQAs isolated from the leaves of MDQ were found to inhibit influenza A virus. 3,4,5-TCQA was found to interact with Tyr100, Gln412 and Arg419 of influenza NA. This study provided scientific evidence on the use of MDQ for treating influenza virus infection, and laid the foundation for the development of CQA derivatives as potential antiviral agents.

A novel E198K substitution in the PA gene of influenza A virus with reduced susceptibility to baloxavir acid.

Baloxavir acid (BXA), the active compound in baloxavir marboxil (BXM), reduces the propagation of influenza A and B viruses by inhibiting the cap-dependent endonuclease activity of the polymerase acidic (PA) subunit. Although BXM has been used to treat influenza virus infections, recently, there has been general concern about the emergence of viruses with low susceptibility to BXA. Here, we identified a novel PA subunit substitution, PA E198K, that reduced susceptibility to BXA. The IC50 of BXA toward influenza A viruses containing PA E198K increased approximately 2- to 6-fold. These findings help to understand the mechanism by which PA substitutions reduce susceptibility to BXA.

Influenza A Virus Production in Mouse Lung Is Inhibited by Inhalation of Aerosol Polyethylenimine/Short Hairpin RNA Plasmid Complexes.

Influenza pandemics are a global threat to human health, with existing vaccines and antiviral drugs providing limited protection. There is an urgent need for new prophylactic and treatment strategies. In this study, 12 short hairpin (sh)RNAs targeting conserved regions of influenza A virus (IAV) matrix protein (M)2, nucleocapsid protein (NP), nonstructural protein (NS), and polymerase acidic (PA) were synthesized, and their effects on IAV replication in cells were investigated using Madin–Darby canine kidney (MDCK) cells transfected with the shRNA plasmids. Additionally, mice were administered a polyethyleneimine PEI/pLKD-NP-391 complex in aerosol form and then infected with AIV, and viral particles in the mouse lung were detected. IAV production was markedly lower in MDCK cells transfected with pLKD-M-121, pLKD-M-935, pLKD-NP-391, pLKD-NP-1291, pLKD-PA-1365, and pLKD-PA-1645 plasmids than in control cells (p < 0.01). The viral load in MDCK cells was decreased by transfection of plasmids pLKD-M-121 (p < 0.05) and pLKD-M-935, pLKD-NP-391, pLKD-NP-1291, pLKD-PA-1365, and pLKD PA-1645 (p < 0.01) compared to an empty plasmid. The viral load was significantly lower in the lungs of mice transfected with pLKD-NP-391 than in the control plasmid and mock transfection groups (p < 0.01 and p < 0.05, respectively). Thus, IAV production was inhibited by shRNAs targeting matrix IAV components; moreover, inhalation of a PEI/pLKD-NP-391 complex in aerosol form suppressed IAV production in infected mice. Thus, these shRNAs can be effective for the prevention and treatment of influenza virus infection.

Generation, Characterization, and Applications of Influenza A Reporter Viruses.

Secondary experimental procedures such as immunostaining have been utilized to study wild-type influenza A viruses (IAV) but are inadequate to rapidly determine the virus in infected cells or for the high-throughput screening (HTS) of antivirals or neutralizing antibodies. Reverse genetics approaches have allowed the generation of recombinant IAV expressing bioluminescent (BL) reporters or fluorescent proteins (FPs). These approaches can easily track viral infections in cultured cells and in validated animal models of infection using in vivo imaging systems (IVIS). Here, we describe the experimental procedures to generate recombinant monomeric (m)Cherry-expressing influenza A/Puerto Rico/8/34 (PR8-mCherry) H1N1 by altering the non-structural (NS) vRNA segment and its use in mCherry-based microneutralization assays to assess antivirals and neutralizing antibodies. The experimental procedures could be used for the generation of other recombinant influenza virus types (e.g., influenza B) or IAV subtypes (e.g., H3N2) expressing mCherry or other BL reporters or FPs from the NS or other vRNA segment. These recombinant reporter-expressing viruses represent an excellent toolbox for the identification of prophylactics or therapeutics for the treatment of influenza viral infections in HTS settings as well as to study different aspects related with the biology of influenza viruses and/or its interaction with the host.

Potent antiviral activity of the extract of Elaeocarpus sylvestris against influenza A virus in vitro and in vivo

BackgroundElaeocarpus sylvestris (Lour.) Poir. (Elaeocarpaceae) belongs to a genus of tropical and semitropical evergreen trees, which has known biological activities such as antiviral and immunomodulatory activities. However, its antiviral potential against influenza virus infection remains unknown. PurposeIn this study, we investigated the antiviral activity of the 50% aqueous ethanolic extract of E. sylvestris (ESE) against influenza A virus (IAV) infection, which could lead to the development of novel phytomedicine to treat influenza virus infection. MethodsTo investigate the in vitro antiviral activity of ESE and its main ingredients, 1,​2,​3,​4,​6-​penta-​O-​galloyl-β-d-glucose (PGG) and geraniin (GE), the levels of viral RNAs, proteins, and infectious viral particles in IAV-infected MDCK cells were analyzed. Molecular docking analysis was performed to determine the binding energy of PGG and GE for IAV proteins. To investigate in vivo antiviral activity, IAV-infected mice were treated intranasally or intragastrically with ESE, PGG, or GE. ResultsESE and its gallate main ingredients (PGG and GE) strongly inhibited the production of viral RNAs, viral proteins, and infectious viral particles in vitro. Also through the viral attachment on cells, polymerase activity, signaling pathway, we revealed the ESE, PGG, and GE inhibit multiple steps of IAV replication. Molecular docking analysis revealed that PGG and GE could interact with 12 key viral proteins (M1, NP, NS1 effector domain (ED), NS1 RNA-binding domain (RBD), HA pocket A, HA receptor-binding domain (RBD), NA, PA, PB1, PB2 C-terminal domain, PB2 middle domain, and PB2 cap-binding domain) of IAV proteins with stable binding energy. Furthermore, intranasal administration of ESE, PGG, or GE protected mice from IAV-induced mortality and morbidity. Importantly, oral administration of ESE suppressed IAV replication and the expression of inflammatory cytokines such as IFN-γ, TNF-α, and IL-6 in the lungs to a large extent. ConclusionESE and its major components (PGG and PE) exhibited strong antiviral activity in multiple steps against IAV infection in silico, in vivo, and in vitro. Therefore, ESE could be used as a novel natural product derived therapeutic agent to treat influenza virus infection.

Identification of C5-NH2 Modified Oseltamivir Derivatives as Novel Influenza Neuraminidase Inhibitors with Highly Improved Antiviral Activities and Favorable Druggability.

Our previous efforts have proved that modifications targeting the 150-cavity of influenza neuraminidase can achieve more potent and more selective inhibitors. In this work, four subseries of C5-NH2 modified oseltamivir derivatives were designed and synthesized to explore every region inside the 150-cavity. Among them, compound 23d was exceptionally potent against the whole panel of Group-1 NAs with IC50 values ranging from 0.26 to 0.73 nM, being 15-53 times better than oseltamivir carboxylate (OSC) and 7-11 times better than zanamivir. In cellular assays, 23d showed more potent or equipotent antiviral activities against corresponding virus strains compared to OSC with no cytotoxicity. Furthermore, 23d exhibited high metabolic stability in human liver microsomes (HLM) and low inhibitory effect on main cytochrome P450 enzymes. Notably, 23d displayed favorable druggability in vivo and potent antiviral efficacy in the embryonated egg model and mice model. Overall, 23d appears to be a promising candidate for the treatment of influenza virus infection.

Antiviral activity of diallyl trisulfide against H9N2 avian influenza virus infection in vitro and in vivo

BackgroundDiallyl trisulfide (DATS) is a garlic-derived organosulfur compound. As it has been shown to have anti-viral activity, we hypothesized that it may alleviate infections caused by H9N2 avian influenza virus (AIV), which is prevalent in poultry with pandemic potential.MethodsHuman lung A549 epithelial cells were treated with three different concentrations of DATS 24 h before (pre-treatment) or one hour after (post-treatment) H9N2 AIV infection. Culture supernatants were collected 24 h and 48 h post-infection and analyzed for viral titers and levels of inflammatory and anti-viral immune responses. For in vivo experiments, BABL/c mice were administered daily by intraperitoneal injection with DATS (30 mg/kg) for 2 weeks starting 1 day after H9N2 AIV infection. Clinical signs, lung pathology, and inflammatory and anti-viral immune responses were assessed 2, 4, and 6 days after infection.ResultsBoth pre-treatment and post-treatment of A549 cells with DATS resulted in reduced viral loads, increased expression of anti-viral genes (RIG-I, IRF-3, and interferon-β), and decreased expression of inflammatory cytokines (TNF-α and IL-6). These effects were also observed in H9N2 AIV-infected mice treated with DATS. Such treatment also reduced lung edema and inflammation in mice.ConclusionsResults suggest that DATS has anti-viral activity against H9N2 AIV and may be used as an alternative treatment for influenza virus infection.

Diagnostic Stewardship of Respiratory Pathogen Panel Utilization

Background: Diagnostic stewardship modifies the ordering, performing, and reporting of diagnostic tests to optimize clinical care and infection prevention while conserving healthcare resources. Timely and accurate diagnosis of respiratory virus infections can optimize the use of antibiotics, antivirals, ancillary tests, and inpatient beds. Influenza-like illnesses (ILIs) are frequently caused by viruses. However, before COVID-19, specific antiviral medication was commonly used only for the treatment of influenza virus infections. Methods: Eskenazi Health (EH) had 2 respiratory PCR assays: influenza/RSV ($58.18 per assay) and a 20-pathogen respiratory pathogens panel (RPP) ($129 per assay). An inpatient ILI algorithm was developed and implemented in the electronic health record (EHR) in October 2018 to guide the selection of the appropriate assay (Figure 1). Ambulatory testing defaulted to the influenza/RSV assay. Prescribers retained the ability to override recommendations. We performed a retrospective chart review of all orders for RPP and influenza/RSV assays before implementation of the ILI algorithm (October 1, 2017, to September 30, 2018) and after implementation (October 1, 2018, to September 30, 2019). The primary end point was the number of RPP assays ordered. The secondary end point was the appropriateness of RPP assays ordered (ie, met ≥1 criteria) and number of influenza/RSVs assays ordered with virus detected. Results: Before the implementation of the intervention, 1,882 orders were reviewed. After implementation 1,621 orders were reviewed. All influenza/RSV and RPP assays were included if they were ordered between October 1, 2017, and September 30, 2019, at EH. There were no exclusion criteria. After implementation, RPP assays decreased ~40% (Table 1), with associated cost savings of $35,368.68 (22.6% of total assay costs; $163,742.88 before implementation and $128,374.20 after implementation). Although some of this reduction could be attributed to the lower number of overall assays ordered, the 40% reduction in RPP assays exceeded the 14% decrease in overall orders, demonstrating improvement in utilization of RPP assays. A corresponding increase in influenza/RSV assay orders was not observed; both groups had similar total influenza/RSV orders. Both groups also had similar percentages of viruses detected with influenza/RSV and RPP (33% before vs 31% after). After implementation, 1,522 (94%) of 1,621 orders followed the recommendations of the ILI algorithm (Table 2). Several prescribers ordered influenza/RSV despite the patient meeting criteria for RPP assay; of these 26 assays, 4 (15%) resulted in virus detection. Of the 73 instances in which prescribers bypassed recommendations for the influenza/RSV assay and ordered an RPP assay, 14 (19%) of the assays resulted in virus detection; only 1 of 14 was a virus that would have been detected by the influenza/RSV. We were unable to identify any trends that would assist in developing additional order questions to capture these patients. Conclusions: Implementation of the ILI algorithm was associated with high adherence, improvement in the appropriateness of ordering, and significant cost savings.Funding: NoDisclosures: None

Design of Neuraminidase-Targeted Imaging and Therapeutic Agents for the Diagnosis and Treatment of Influenza Virus Infections.

The last step in influenza virus replication involves the assembly of viral components on the infected cell's plasma membrane followed by budding of intact virus from the host cell surface. Because viral neuraminidase and hemagglutinin are both inserted into the host cell's membrane during this process, influenza virus-infected cells are distinguished from uninfected cells by the presence of viral neuraminidase and hemagglutinin on their cell surfaces. In an effort to exploit this difference in cell surface markers for development of diagnostic and therapeutic agents, we have modified an influenza neuraminidase inhibitor, zanamivir, for targeting of attached imaging and therapeutic agents selectively to influenza viruses and virus-infected cells. We have designed here a zanamivir-conjugated rhodamine dye that allows visual monitoring of binding, internalization, and intracellular trafficking of the fluorescence-labeled neuraminidase in virus-infected cells. We also synthesize a zanamivir-99mTc radioimaging conjugate that permits whole body imaging of the virus's biodistribution and abundance in infected mice. Finally, we create both a zanamivir-targeted cytotoxic drug (i.e., zanamivir-tubulysin B) and a viral neuraminidase-targeted CAR T cell and demonstrate that they are both able to kill viral neuraminidase-expressing cells without damaging healthy cells. Taken together, these data suggest that the influenza virus neuraminidase inhibitor, zanamivir, can be exploited to improve the diagnosis, imaging, and treatment of influenza virus infections.

Design of Ligand-Targeted Imaging and Therapeutic Agents for the Diagnosis and Treatment of Influenza Virus Infections

The last step in influenza virus replication involves the assembly of viral components on the infected cell’s plasma membrane followed by budding of intact virus from the host cell surface. Because viral neuraminidase and hemagglutinin are both inserted into the host cell’s membrane during this process, influenza virus-infected cells are distinguished from uninfected cells by the presence of viral neuraminidase and hemagglutinin on their cell surfaces. In an effort to exploit this difference in cell surface markers for development of diagnostic and therapeutic agents, we have modified an influenza neuraminidase inhibitor, zanamivir, for targeting of attached imaging and therapeutic agents selectively to influenza viruses and virus-infected cells. We design here a zanamivir-conjugated rhodamine dye that allows visual monitoring of binding, internalization and intracellular trafficking of the fluorescence-labeled neuraminidase in virus-infected cells. We also synthesize a zanamivir-99mTc radioimaging conjugate that permits whole body imaging of the virus’ biodistribution and abundance in infected mice. Finally, we create both a zanamivir-targeted cytotoxic drug (i.e. zanamivir-tubulysin B) and a viral neuraminidase-targeted CAR T cell and demonstrate that they are both able to kill viral neuraminidase-expressing cells without damaging healthy cells. Taken together, these data suggest that the influenza virus neuraminidase inhibitor, zanamivir, can be exploited to improve the diagnosis, imaging and treatment of influenza virus infections.