Oseltamivir-resistant Viruses Research Articles

Bifunctional neuraminidase inhibitory and simultaneously anti-Mycoplasma pneumoniae of flavan-3-ols and flavanones: Combined molecular docking, virtual screening, ADMET, and synthesis prediction

After lifting COVID-19 restrictions, pneumonia caused by influenza A virus coinfection with Mycoplasma pneumoniae bacteria emerges as a matter of concern. Only a few reports have recently proposed natural compounds for treating the coinfection of influenza A virus and M. pneumoniae, especially resistant strains. Hence, this study demonstrated in silico investigation of two natural classes (flavan-3-ol and flavanone) that could become future inhibitors for bifunctional neuraminidase and anti-M. pneumoniae. Combining the molecular docking with based-virtual screening strategies for over 900 ligands with neuraminidase protein of oseltamivir-resistant virus strain at H274Y mutation (PDB ID: 3CL0), 03 flavan-3-ols and 46 flavanones interacted with both the active site and the adjacent 430-cavity, and showed lower binding energies than laninamivir (−8.1 kcal/mol). The four most suitable candidates (FL22, FN166, FN316, and ChEMBL1779463) also exhibited potential characteristics for inhibiting M. pneumoniae through some vital targets (30S Ribosome-binding factor A (PDB ID: 1PA4) and immunodominant protein P40/P90 complex (PDB ID: 6TLZ)). Furthermore, the total-synthesis strategy of ChEMBL1779463 was successfully proposed using commercial reagents and modern techniques. Our results will offer potential insights for future methods to identify biflavonoids, especially biflavanone, to simultaneously suppress the resistant strains of influenza A virus and M. pneumoniae.

Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants.

The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 in the neuraminidase (E119V-NA). Early detection of resistant influenza viruses is important for patient management and for the rapid containment of antiviral resistance. The neuraminidase inhibition assay allows the phenotypical identification of resistant strains; however, this test often has limited sensitivity with high variability depending on the virus strain, drugs and assays. Once a mutation such as E119V-NA is known, highly sensitive PCR-based genotypic assays can be used to identify the prevalence of such mutant influenza viruses in clinical samples. In this study, based on an existing reverse transcriptase real-time PCR (RT-qPCR) assay, we developed a reverse transcriptase droplet digital PCR assay (RT-ddPCR) to detect and quantify the frequency of the E119V-NA mutation. Furthermore, reverse genetics viruses carrying this mutation were created to test the performance of the RT-ddPCR assay and compare it to the standard phenotypic NA assay. We also discuss the advantage of using an RT-ddPCR instead of qPCR method in the context of viral diagnostics and surveillance.

Diagnosis and treatment of influenza: a literature review

Alisa Pozniak1 1Vilnius University, Faculty of Medicine, Vilnius, Lithuania Abstract Background. Influenza is an acute viral respiratory infection. Influenza viruses spread through respiratory droplets and are characterized by frequent mutations that lead to antigenicity changes. According to the World Health Organization, seasonal influenza epidemics cause approximately 4 million cases of severe illness and 250,000 to 500,000 deaths each year globally. Aim: to review the diagnosis and treatment of influenza. Methodology: literature sources were selected from PubMed database following dates from 2017 to 2022 using keywords and their combinations: influenza, rapid antigen test, neuraminidase inhibitors, favipiravir, baloxavir. Results. Influenza should be suspected in patients who develop acute respiratory symptoms during the influenza season. Diagnostic tests available for influenza include rapid antigen and molecular tests, polymerase chain reaction tests, virological culture. Antiviral therapy should be initiated in patients who are at increased risk of complications from influenza. The neuraminidase inhibitors zanamivir and oseltamivir are used for specific antiviral therapy. New antivirals such as peramivir, favipiravir, laninamivir and baloxavir have emerged as a promising new treatment approach. Conclusions. Influenza is a serious infectious disease that can lead to life-threatening complications. Virological diagnosis is based on specific epidemiological data, clinical symptoms and laboratory tests. Neuraminidase inhibitors are recommended for early treatment. Several cases of zanamivir and oseltamivir-resistant virus have been reported. That is the reason why different approaches for antiviral treatment are being investigated. Keywords: influenza, rapid antigen test, neuraminidase inhibitors, favipiravir, baloxavir.

An ultrasensitive, rapid and portable method for screening oseltamivir-resistant virus based on CRISPR/Cas12a combined with immunochromatographic strips.

Influenza is a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. The neuraminidase inhibitor oseltamivir is used as an antiviral drug in clinical treatment. However, its therapeutic effects can be greatly compromised by the emergence of drug-resistant mutant viruses. Thus, there is an urgent need to distinguish drug-resistant strains with a simple method. To address this, in the present study, we develop a rapid, sensitive and convenient molecular diagnosis method based on CRISPR/Cas12a technology and lateral flow detection (LFD). By targeting mutant sequences amplified by recombinase polymerase amplification (RPA) reaction, crRNA is designed to develop the CRISPR/Cas12a assay, and 2000 copies can be directly observed by the naked eye under blue light-emitting diode (LED) light. Combined with LFD, the limit of detection of RPA-CRISPR/Cas12a-LFD is about 20 copies of target sequence per reaction. Collectively, RPA-CRISPR/Cas12a-LFD method provides a novel alternative for the sensitive, specific and portable detection to diagnose oseltamivir-resistant mutant strains.

Identification and anti-viral activities of butanolide skeleton from Cinnamomum kotoense and Cinnamomum subavenium by inhibiting H5N1 neuraminidase

The human influenza virus and avian influenza virus represents a serious threat to human health. Although zanamivir and oseltamivir against influenza virus neuraminidase and thus the viral replication is not available. The oseltamivir-resistant H5N1 virus has been isolated from patient. Moreover, several cases have been reported where patients developed adverse symptoms due to a long-term use of oseltamivir. Effective antiviral drugs are essential for early control of an influenza pandemic. Natural products have been used as important resources for medicine and healthy food to improve the quality of life. Cinnamon bark is widely used as a spice and herb medicine. In this study, we have isolated and identified three butanolide derivatives, kotomolide A, isolinderanolide B and linderanolide B from Cinnamomum kotoense and Cinnamomum subavenium. Further, enzyme-based screening and anti-viral activity were also examined. Our study provides a starting point for developing new therapeutic or preventive agents from natural products for avian influenza virus.

Frequency and distribution of H1N1 influenza A viruses with oseltamivir-resistant mutations worldwide before and after the 2009 pandemic.

H1N1 influenza has brought serious threats to people's health and a high socioeconomic burden to society. Oseltamivir, a kind of neuraminidase (NA) inhibitor, is the second-generation specific drug that is broadly used currently. However, H1N1 influenza viruses have exhibited oseltamivir resistance in the past decades, which might be a hidden danger. To understand the frequency and distribution laws of oseltamivir-resistant viruses, we conducted a thorough and deep analysis of the available NA protein sequences of H1N1 influenza viruses worldwide from 1918 to 2020. The differences and similarities before and after 2009 were also considered since the dominant viruses changed in this period. Results showed that 3.76% of H1N1 viruses harbored oseltamivir resistance currently. Among various significative mutations, H274Y had the highest frequency of 3.30%, while the frequencies of the other mutations were far below this whether before or after 2009. The oseltamivir resistance was mainly found in three hosts, humans, swine, and avian. Different mutation sites could exhibit different distributions in each host. Our results showed that the resistance level reached a peak during the 2007-2008 influenza season and then quickly decreased in 2009. The resistance also displayed a global distribution. The densely populated countries usually had a high resistance level. However, frequent significative mutations were also found in some small countries. Our findings indicated the necessity of monitoring oseltamivir resistance around the world. The study could provide a unique perspective towardthe cognition of viruses and facilitate the future study of both pandemic and drug development.

An AIE and ESIPT based neuraminidase fluorescent probe for influenza virus detection and imaging

Neuraminidase (NA), an important enzyme for influenza virus replication, is a main target for influenza virus inhibition and detection. In this work, the aggregation-induced emission (AIE) and excited state intramolecular proton transfer (ESIPT)-active neuraminidase fluorescent probe SABP was firstly developed for influenza virus detection. The probe SABP showed high sensitivity towards NA with strong green fluorescence, which could be used for influenza virus detection and living cell imaging. In the presence of NA, SABP exhibited increased fluorescence signal by up to 30-fold at 524 nm. SABP detected NA activity in the range 0–5.0 U/mL, with a limit of detection (LOD) of 0.024 U/mL and detect influenza virus in the range of 2−4–26 HAU with a LOD of 2−1 HAU. Moreover, SABP was successfully applied to distinguish oseltamivir-resistant influenza virus from wild type.

Inhibition of influenza virus replication by Apiaceae plants, with special reference to Peucedanum japonicum (Sacna) constituents

Ethnopharmacological relevanceApiaceae plants possess various pharmacological properties, such as antimicrobial, antioxidant, hypoglycemic, hypolipidemic, anxiolytic, analgesic, anti-inflammatory, anti-convulsant, and anti-cancer activities; however, data on their antiviral activity are limited. Peucedanum japonicum, also known as Sacna, is a plant used as food and as a traditional folk medicine for treating coughs. However, the active components in the leaves of this plant are yet unexplored. Aim of the studyTo assess Apiaceae plants, especially Peucedanum japonicum, with anti-viral activity, and the function and antiviral potential of Sacna constituents, considering the emergence of influenza virus strains resistant to the currently available drugs. Materials and methodsWe prepared grinds of the freeze-dried leaves and roots of the Apiaceae family and the hot water extracts. The antiviral activities of the extracts were determined by focus formation reduction assay. In the time-of-addition assay, the test medium containing Sacna extract at 2 mg/mL was added at −1 to 0 h (adsorption) or from 0 to 4, 4 to 8, or 0 to 8 h (replication). The Sacna extract was separated by reversed-phase flash column chromatography using an Isolera Spektra system. The antiviral activity of each fraction was then determined using the focus formation reduction assay. The active fraction was analyzed using an LC20ADXR high performance liquid chromatography system equipped with a microTOF-QII quadrupole time-of-flight tandem mass spectrometer. ResultsAll examined extracts of Apiaceae plants showed anti-influenza activity. Sacna extract most strongly inhibited the replication of influenza viruses. Individual components of Sacna possess antiviral activities against the influenza A/PR/8/34 virus. Sacna was found to inhibit the multiplication of A (H1N1 and H3N2) types and B types of influenza viruses, including amantadine-resistant and oseltamivir-resistant viruses. Sacna also inhibited influenza infection during viral replication. However, Sacna did not inhibit influenza infection during cell adsorption and did not suppress hemagglutination inhibition or cell fusion. Further, our findings suggest that the antiviral compounds in Sacna include flavonoids (quercetin and luteolin) and other polyphenols (caffeic acid, hymecromone, and umbelliferone). Although several effective compounds in Sacna inhibit multiple steps of viral replication, caffeic acid, which was increased by heat treatment at the time of extraction, significantly inhibited only the late period of viral growth, similar to the Sacna extract, indicating that it is the major component responsible for the antiviral activity of Sacna. ConclusionsApiaceae plants possess antiviral activity. Caffeic acid is the major component responsible for the antiviral activity of Sacna. To our knowledge, this is the first report regarding the anti-influenza virus activity of Sacna. Overall, these results indicate that Sacna has potential as a novel treatment against influenza A and B viruses.

Detection of H275Y Mutation Conferring Oseltamivir Drug Resistance in Influenza A (H1N1) pdm09 Virus

In the treatment of influenza, Neuraminidase inhibitors (NAIs) (Oseltamivir and Zanamivir) play a major role. The emergence of variants of influenza A (H1N1) pdm09 virus resistant to Oseltamivir is a matter of great concern as it limits its usage. Therefore, vigilant monitoring for Oseltamivir-resistant viruses has been recommended by the World Health Organization (WHO). Our study aimed to screen the influenza A (H1N1) pdm09 virus for NAI drug resistance during the outbreak of 2015-16 in North-Western India. A total of 640 H1N1pdm09 virus-positive samples were screened for drug resistance to Oseltamivir by WHO allelic discrimination real-time RT-PCR protocol. The allelic discrimination PCR protocol can detect the presence of single nucleotide polymorphisms (SNPs), the H275Y mutation is detected by this method which causes resistance to Oseltamivir. Sanger sequencing of partial fragment of NA gene (fragment IV), of 90 samples were performed to confirm the presence of NA-H275Y mutation. Neuraminidase susceptibility of 20 randomly selected isolates to Oseltamivir was tested using NA inhibition chemiluminiscence based assay. Among 640 H1N1pdm09 positive samples tested, H275Y mutation was detected in one sample (0.15%) by PCR and confirmed by Sanger sequencing also. All the 20 isolates tested for NAI susceptibility by NA star assay were found to be sensitive to Oseltamivir. WHO allelic discrimination PCR is an easy, rapid and sensitive method for high-throughput detection of resistance to Oseltamivir. Systematic regular drug resistance surveillance of Influenza A is essential to monitor the emergence and spread of drug-resistant strains.

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.

Nanoparticle composite TPNT1 is effective against SARS-CoV-2 and influenza viruses

A metal nanoparticle composite, namely TPNT1, which contains Au-NP (1 ppm), Ag-NP (5 ppm), ZnO-NP (60 ppm) and ClO2 (42.5 ppm) in aqueous solution was prepared and characterized by spectroscopy, transmission electron microscopy, dynamic light scattering analysis and potentiometric titration. Based on the in vitro cell-based assay, TPNT1 inhibited six major clades of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with effective concentration within the range to be used as food additives. TPNT1 was shown to block viral entry by inhibiting the binding of SARS-CoV-2 spike proteins to the angiotensin-converting enzyme 2 (ACE2) receptor and to interfere with the syncytium formation. In addition, TPNT1 also effectively reduced the cytopathic effects induced by human (H1N1) and avian (H5N1) influenza viruses, including the wild-type and oseltamivir-resistant virus isolates. Together with previously demonstrated efficacy as antimicrobials, TPNT1 can block viral entry and inhibit or prevent viral infection to provide prophylactic effects against both SARS-CoV-2 and opportunistic infections.

Inhibitory Activity of Honeysuckle Extracts against Influenza A Virus In Vitro and In Vivo.

Honeysuckle has been used in the treatment of influenza virus infection for thousands of years in China. However, its main active components and the functional mechanisms remain to be elucidated. Here, four honeysuckle extracts, including acids extract, flavonoids extract, total extract and acids-flavonoids mixture, were prepared to clarify the main active antiviral components. The cytopathic effect reduction assay showed that all the four extracts inhibited the replication of influenza viruses H1N1, H3N2 and the oseltamivir-resistant mutant strain H1N1-H275Y. The acids-flavonoids mixture had the strongest inhibitory effects in vitro with EC50 values of 3.8, 4.1, and > 20μg/mL against H1N1, H3N2 and H1N1-H275Y, respectively, showing competitive antiviral activity with oseltamivir and ribavirin. Honeysuckle acids extract alsoshowed the most significant antiviral activity in vivo. Oral administration of the acids extract at a dosage of 600mg/kg/d effectively alleviated viral pneumonia, maintained body weight and improved the survival rate to 30% of the mice infected with a lethal dose of H1N1. The results of time-of-drug addition experiment and neuraminidase (NA) inhibition assay showed that honeysuckle extracts had a broad-spectrum inhibitory effect against influenza virus NAs. The flavonoid extract showed the strongest inhibitory effect on the NA of influenza virus H7N9 with an IC50 of 24.7μg/mL. These results suggested that these extracts might exert their antiviral activity by suppressing the release of influenza viruses. Briefly, our findings demonstrate that acids and flavonoids extracts of honeysuckle are the major antiviral active components, and the acids extract has the potential to be developed into an antiviral agent against influenza virus, especially for oseltamivir-resistant viruses.

Punicalagin is a neuraminidase inhibitor of influenza viruses.

Influenza A virus (IAV) causes great morbidity and mortality worldwide every year. However, there are only a limited number of drugs clinically available against IAV infection. Further, emergence of drug-resistant strains can render those drugs ineffective. Thus there is an unmet medical need to develop new anti-influenza agents. In this study, we show that punicalagin from plants possesses strong anti-influenza activity with a low micromolar IC50 value in tissue culture. Using a battery of bioassays such as single-cycle replication assay, neuraminidase (NA) inhibition assay, and virus yield reduction assay, we demonstrate that the primary mechanism of action (MOA) of punicalagin is the NA-mediated viral release. Moreover, punicalagin can inhibit replication of different strains of influenza A and B viruses, including oseltamivir-resistant virus (NA/H274Y), indicating that punicalagin is a broad spectrum antiviral against both IAV and IBV. Further, although punicalagin targets NA like oseltamivir, it has a different MOA. These results suggest that punicalagin is an influenza NA inhibitor that may be further developed as a novel antiviral against influenza viruses.

A retrospective observational study of the treatment of a nosocomial infection caused by oseltamivir-resistant influenza virus A with baloxavir marboxil.

Nosocomial (hospital-acquired) influenza A virus infection is a very important clinical issue. The objective of this study is to describe the effect of baloxavir marboxil in controlling an outbreak of this infection. A retrospective observational study was performed to assess the effectiveness of baloxavir marboxil in the treatment of nosocomial infections caused by oseltamivir-resistant influenza virus A. In September 2019, there was an outbreak of nosocomial influenza A(H1N1)pdm09 viral infection in one out of three facility wards for inpatients at the Okinawa Nanbu Regional Center for Children with Special Needs. Symptomatic staff members were kept off duty until they remained afebrile for 2 days. Prophylactic oseltamivir was administered to inpatients (n=37) and to staff members (n=16) who voluntarily requested the drug. However, both inpatients and staff members showed influenza A infection during prophylactic use of oseltamivir. The A(H1N1)pdm09 virus sample obtained from one patient was shown to be oseltamivir-resistant. After administration of baloxavir marboxil, the nosocomial outbreak gradually ceased. Moreover, the time (hours) to alleviation of fever in the oseltamivir group (n=11) and baloxavir marboxil group (n=13) was significantly different (p=0.0034). Our report provides evidence for the usefulness of baloxavir marboxil in treating influenza A patients who have received prophylactic doses of oseltamivir. This is the first report describing the successful use of baloxavir marboxil for of a nosocomial outbreak caused by oseltamivir-resistant influenza A virus.

Host-targeted nitazoxanide has a high barrier to resistance but does not reduce the emergence or proliferation of oseltamivir-resistant influenza viruses in vitro or in vivo when used in combination with oseltamivir

A major limitation of the currently available influenza antivirals is the potential development of drug resistance. The adamantanes, neuraminidase inhibitors, and more recently polymerase inhibitors, have all been associated with the emergence of viral resistance in preclinical, clinical studies or in clinical use. As a result, host-targeted drugs that act on cellular proteins or functions have become an attractive option for influenza treatment as they are less likely to select for resistance. Nitazoxanide (NTZ) is a host-targeted antiviral that is currently in Phase III clinical trials for the treatment of influenza. In this study, we investigated the propensity for circulating influenza viruses to develop resistance to nitazoxanide in vitro by serially passaging viruses under selective pressure. Phenotypic and genotypic analysis of viruses passaged ten times in the presence of up to 20 μM tizoxanide (TIZ; the active metabolite of nitazoxanide) showed that none had a significant change in TIZ susceptibility, and amino acid substitutions arising that were unique to TIZ passaged viruses, did not alter TIZ susceptibility.Combination therapy, particularly utilising drugs with different mechanisms of action, is another option for combatting antiviral resistance, and while combination therapy has been shown to improve antiviral effects, the effect of reducing the emergence and selection of drug-resistant virus has been less widely investigated. Here we examined the use of TIZ in combination with oseltamivir, both in vitro and using the ferret model for influenza infection and found that the combination of the two drugs did not provide significant benefit in reducing the emergence or selection of oseltamivir-resistant virus.These in vitro findings suggest that clinical use of NTZ may be significantly less likely to select for resistance in circulating influenza viruses compared to virus-targeted antivirals, and although the combination of NTZ with oseltamivir did not reduce the emergence of oseltamivir-resistant virus in vitro or in vivo, combination therapy with NTZ and other newer classes of influenza antiviral drugs should be considered due to NTZ's higher host-based barrier to resistance.

Novel oseltamivir-resistant mutations distant from the active site of influenza B neuraminidase

We performed a neuraminidase sequence analysis of thirty-two pediatric patients with influenza B who visited Teikyo University Hospital from January 2016 to March 2017, and found oseltamivir-resistant samples belonging to the Yamagata and Victoria lineages. Comparison with the neuraminidase sequence of oseltamivir-susceptible B/Brisbane/60/2008 revealed 5 common amino acid substitutions in many of these samples. According to the binding free energy calculation, the N340D and E358K substitutions reduced the affinity of oseltamivir to neuraminidase. Unexpectedly, these substitutions were located distant from the oseltamivir-binding site in neuraminidase. According to the molecular dynamics simulations, the N340D substitution rearranged complicated hydrogen bond networks in an extensive surface region of neuraminidase. The E358K substitution extensively altered the electrostatic potential map of the overall neuraminidase structure. Through these novel mechanisms, the N340D and E358K substitutions indirectly influenced the affinity reduction. These results may be useful for designing drugs for the treatment of oseltamivir-resistant virus infections. Communicated by Ramaswamy H. Sarma

Rapid identification and isolation of neuraminidase inhibitors from mockstrawberry (Duchesnea indica Andr.) based on ligand fishing combined with HR-ESI-Q-TOF-MS

Neuraminidase inhibitors (NAIs) are the mainstay antiviral drugs against influenza infection. In this study, a ligand fishing protocol was developed to screen NAIs using neuraminidase immobilized magnetic beads (NA-MB). After verifying the feasibility of NA-MB with an artificial mixture including NA inhibitors and non-inhibitors, the developed ligand fishing protocol was applied to screen NAIs from the crude extracts of Duchesnea indica Andr. Twenty-four NA binding compounds were identified from the normal butanol (n-BuOH) extract of D. indica as potential NAIs by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC–Q-TOF-MS) assisted with Compound Structure Identification (CSI):FingerID, including 12 ellagitannins, 4 brevifolin derivatives, 3 ellagic acid derivatives, and 4 flavonoids. Among them, 9 compounds were isolated and tested for in vitro NA inhibitory activities against NA from Clostridium perfringens, and from oseltamivir sensitive and resistant influenza A virus strains. The results indicate that compound B23 has the NA inhibitory activities in both the oseltamivir sensitive and resistant viral NA, with half maximal inhibitory concentration (IC50) values of 197.9 and 125.4 μmol/L, respectively. Moreover, B23 can obviously reduce the replication of oseltamivir sensitive and resistant viruses in Madin–Darby canine kidney (MDCK) cells at the concentrations of 40 and 200 μmol/L.

Epidemic of influenza A(H1N1)pdm09 analyzed by full genome sequences and the first case of oseltamivir-resistant strain in Myanmar 2017.

A community outbreak of human influenza A(H1N1)pdm09 virus strains was observed in Myanmar in 2017. We investigated the circulation patterns, antigenicity, and drug resistance of 2017 influenza A(H1N1)pdm09 viruses from Myanmar and characterized the full genome of influenza virus strains in Myanmar from in-patients and out-patients to assess the pathogenicity of the viruses. Nasopharyngeal swabs were collected from out-patients and in-patients with acute respiratory tract infections in Yangon and Pyinmana City in Myanmar during January-December 2017. A total of 215 out-patients and 18 in-patients infected with A(H1N1)pdm09 were detected by virus isolation and real-time RT-PCR. Among the positive patients, 90.6% were less than 14 years old. Hemagglutination inhibition (HI) antibody titers against A(H1N1)pdm09 viruses in Myanmar were similar to the recommended Japanese influenza vaccine strain for 2017-2018 seasons (A/Singapore/GP1908/2015) and WHO recommended 2017 southern hemisphere vaccine component (A/Michigan/45/2015). Phylogenetic analysis of the hemagglutinin sequence showed that the Myanmar strains belonged to the genetic subclade 6B.1, possessing mutations of S162N and S164T at potential antigenic sites. However, the amino acid mutation at position 222, which may enhance the severity of disease and mortality, was not found. One case with no prior history of oseltamivir treatment possessed H275Y mutated virus in neuraminidase (NA), which confers resistance to oseltamivir and peramivir with elevated IC50 values. The full genome sequence of Myanmar strains showed no difference between samples from in-patients and out-patients, suggesting no additional viral mutations associated with patient severity. Several amino acid changes were observed in PB2, PB1, and M2 of Myanmar strains when compared to the vaccine strain and other Asian strains. However, no mutations associated with pathogenicity were found in the Myanmar strains, suggesting that viral factors cannot explain the underlying reasons of the massive outbreak in Myanmar. This study reported the first detection of an oseltamivir-resistant influenza virus in Myanmar, highlighting the importance of continuous antiviral monitoring and genetic characterization of the influenza virus in Myanmar.

Morin Hydrate Inhibits Influenza Virus entry into Host Cells and Has Anti-inflammatory Effect in Influenza-infected Mice

Influenza virus is the major cause of seasonal and pandemic flu. Currently, oseltamivir, a potent and selective inhibitor of neuraminidase of influenza A and B viruses, is the drug of choice for treating patients with influenza virus infection. However, recent emergence of oseltamivir-resistant influenza viruses has limited its efficacy. Morin hydrate (3,5,7,2′,4′-pentahydroxyflavone) is a flavonoid isolated from Morus alba L. It has antioxidant, anti-inflammatory, neuroprotective, and anticancer effects partly by the inhibition of the NF-кB signaling pathway. However, its effects on influenza virus have not been studied. We evaluated the antiviral activity of morin hydrate against influenza A/Puerto Rico/8/1934 (A/PR/8; H1N1) and oseltamivir-resistant A/PR/8 influenza viruses in vitro. To determine its mode of action, we carried out time course experiments, and time of addition, hemolysis inhibition, and hemagglutination assays. The effects of the co-administration of morin hydrate and oseltamivir were assessed using the murine model of A/PR/8 infection. We found that morin hydrate reduced hemagglutination by A/PR/8 in vitro. It alleviated the symptoms of A/PR/8-infection, and reduced the levels of pro-inflammatory cytokines and chemokines, such as TNF-α and CCL2, in infected mice. Co-administration of morin hydrate and oseltamivir phosphate reduced the virus titers and attenuated pulmonary inflammation. Our results suggest that morin hydrate exhibits antiviral activity by inhibiting the entry of the virus.

Antiviral Activity of Benzoic Acid Derivative NC-5 Against Influenza A Virus and Its Neuraminidase Inhibition.

The currently available drugs against influenza A virus primarily target neuraminidase (NA) or the matrix protein 2 (M2) ion channel. The emergence of drug-resistant viruses requires the development of new antiviral chemicals. Our study applied a cell-based approach to evaluate the antiviral activity of a series of newly synthesized benzoic acid derivatives, and 4-(2,2-Bis(hydroxymethyl)-5-oxopyrrolidin-l-yl)-3-(5-cyclohexyl-4H-1,2,4-triazol-3-yl)amino). benzoic acid, termed NC-5, was found to possess antiviral activity. NC-5 inhibited influenza A viruses A/FM/1/47 (H1N1), A/Beijing/32/92 (H3N2) and oseltamivir-resistant mutant A/FM/1/47-H275Y (H1N1-H275Y) in a dose-dependent manner. The 50% effective concentrations (EC50) for H1N1 and H1N1-H275Y were 33.6 μM and 32.8 μM, respectively, which showed that NC-5 had a great advantage over oseltamivir in drug-resistant virus infections. The 50% cytotoxic concentration (CC50) of NC-5 was greater than 640 μM. Orally administered NC-5 protected mice infected with H1N1 and H1N1-H275Y, conferring 80% and 60% survival at 100 mg/kg/d, reducing body weight loss, and alleviating virus-induced lung injury. NC-5 could suppress NP and M1 protein expression levels during the late stages of viral biosynthesis and inhibit NA activity, which may influence virus release. Our study proved that NC-5 has potent anti-influenza activity in vivo and in vitro, meaning that it could be regarded as a promising drug candidate to treat infection with influenza viruses, including oseltamivir-resistant viruses.