Anti-Influenza Activity of Medicinal Material Extracts from Qinghai-Tibet Plateau.

To discover sources for novel anti-influenza drugs, we evaluated the antiviral potential of nine extracts from eight medicinal plants and one mushroom (Avena sativa L., Hordeum vulgare Linn. var. nudum Hook. f., Hippophae rhamnoides Linn., Lycium ruthenicum Murr., Nitraria tangutorum Bobr., Nitraria tangutorum Bobr. by-products, Potentilla anserina L., Cladina rangiferina (L.) Nyl., and Armillaria luteo-virens) from the Qinghai-Tibetan plateau against the influenza A/H3N2 virus. Concentrations lower than 125 µg/mL of all extracts demonstrated no significant toxicity in MDCK cells. During screening, seven extracts (A. sativa, H. vulgare, H. rhamnoides, L. ruthenicum, N. tangutorum, C. rangiferina, and A. luteo-virens) exhibited antiviral activity, especially the water-soluble polysaccharide from the fruit body of the mushroom A. luteo-virens. These extracts significantly reduced the infectivity of the human influenza A/H3N2 virus in vitro when used at concentrations of 15.6-125 µg/mL. Two extracts (N. tangutorum by-products and P. anserina) had no A/H3N2 virus inhibitory activity. Notably, the extract obtained from the fruits of N. tangutorum and N. tangutorum by-products exhibited different anti-influenza effects. The results suggest that extracts of A. sativa, H. vulgare, H. rhamnoides, L. ruthenicum, N. tangutorum, C. rangiferina, and A. luteo-virens contain substances with antiviral activity, and may be promising sources of new antiviral drugs.

Identification of Nifurtimox and Chrysin as Anti-Influenza Virus Agents by Clinical Transcriptome Signature Reversion.

The rapid development in the field of transcriptomics provides remarkable biomedical insights for drug discovery. In this study, a transcriptome signature reversal approach was conducted to identify the agents against influenza A virus (IAV) infection through dissecting gene expression changes in response to disease or compounds' perturbations. Two compounds, nifurtimox and chrysin, were identified by a modified Kolmogorov-Smirnov test statistic based on the transcriptional signatures from 81 IAV-infected patients and the gene expression profiles of 1309 compounds. Their activities were verified in vitro with half maximal effective concentrations (EC50s) from 9.1 to 19.1 µM against H1N1 or H3N2. It also suggested that the two compounds interfered with multiple sessions in IAV infection by reversing the expression of 28 IAV informative genes. Through network-based analysis of the 28 reversed IAV informative genes, a strong synergistic effect of the two compounds was revealed, which was confirmed in vitro. By using the transcriptome signature reversion (TSR) on clinical datasets, this study provides an efficient scheme for the discovery of drugs targeting multiple host factors regarding clinical signs and symptoms, which may also confer an opportunity for decelerating drug-resistant variant emergence.

Synthesis and structure-activity relationship of L-methionine-coupled 1,3,4-thiadiazole derivatives with activity against influenza virus.

In previous investigations, we identified a class of 1,3,4-thiadiazole derivatives with antiviral activity. N-{3-(Methylsulfanyl)-1-[5-(phenylamino)-1,3,4-thiadiazole-2-yl]propyl}benzamide emerged as a relevant lead compound for designing novel influenza A virus inhibitors. In the present study, we elaborated on this initial lead by performing chemical synthesis and antiviral evaluation of a series of structural analogues. During this research, thirteen novel 1,3,4-thiadiazole derivatives were synthesized by the cyclization of the corresponding thiosemicarbazides as synthetic precursors. The structures and the purities of the synthesized compounds were confirmed through chromatographic and spectral data. Four L-methionine-based 1,3,4-thiadiazole derivatives displayed activity against influenza A virus, the two best compounds being 24 carrying a 5-(4-chlorophenylamino)-1,3,4-thiadiazole moiety and 30 possessing a 5-(benzoylamino)-1,3,4-thiadiazole structure [antiviral EC50 against influenza A/H3N2 virus: 4.8 and 7.4 µM, respectively]. The 1,3,4-thiadiazole derivatives were inactive against influenza B virus and a wide panel of unrelated DNA and RNA viruses. Compound 24 represents a new class of selective influenza A virus inhibitors acting during the virus entry process, as evidenced by our findings in a time-of-addition assay. Molecular descriptors and in silico prediction of ADMET properties of the active compounds were calculated. According to in silico ADMET and drug similarity studies, active compounds have been estimated to be good candidates for oral administration with no apparent toxicity considerations.

A broad influenza virus inhibitor acting via IMP dehydrogenase and in synergism with ribavirin.

To suppress serious influenza infections in persons showing insufficient protection from the vaccines, antiviral drugs are of vital importance. There is a need for novel agents with broad activity against influenza A (IAV) and B (IBV) viruses and with targets that differ from those of the current antivirals. We here report a new small molecule influenza virus inhibitor referred to as CPD A (chemical name: N-(pyridin-3-yl)thiophene-2-carboxamide). In an influenza virus minigenome assay, this non-nucleoside compound inhibited RNA synthesis of IAV and IBV with EC50 values of 2.3 µM and 2.6 µM, respectively. Robust in vitro activity was noted against a broad panel of IAV (H1N1 and H3N2) and IBV strains, with a median EC50 value of 0.20 µM, which is 185-fold below the 50% cytotoxic concentration. The action point in the viral replication cycle was located between 1 and 5 h p.i., showing a similar profile as ribavirin. Like this nucleoside analogue, CPD A was shown to cause strong depletion of the cellular GTP pool and, accordingly, its antiviral activity was antagonized when this pool was restored with exogenous guanosine. This aligns with the observed inhibition in a cell-based IMP dehydrogenase (IMPDH) assay, which seems to require metabolic activation of CPD A since no direct inhibition was seen in an enzymatic IMPDH assay. The combination of CPD A with ribavirin, another IMPDH inhibitor, proved strongly synergistic. To conclude, we established CPD A as a new inhibitor of influenza A and B virus replication and RNA synthesis, and support the potential of IMPDH inhibitors for influenza therapy with acceptable safety profile.

Identification of novel influenza polymerase PB2 inhibitors using virtual screening approach and molecular dynamics simulation analysis of active compounds.

Hierarchical virtual screening combined with ADME prediction and cluster analysis methods were used to identify influenza virus PB2 inhibitors with high activity, good druggability properties, and diverse structures. The 200,000 molecules in the ChemDiv core library were narrowed down to a final set of 97 molecules, of which six compounds were found to rescue cells from both H1N1 and H3N2 virus-induced CPE with EC50 values ranging from 5.81 µM to 42.77 µM, and could bind to the PB2 CBD of H1N1, with Kd values of 0.11 µM to 6.4 µM. The six compounds have novel structures and low molecular weight and are, thus, suitable serve as lead compounds for development as PB2 inhibitors. A receptor-based pharmacophore model was successfully constructed using key amino acid residues for the binding of inhibitors to PB2, provided by the MD simulations. This pharmacophore model suggested that to improve the activity of our active compounds, we should mainly focus on optimizing their existing structures with the aim of increasing their adaptability to the binding site, rather than adding chemical fragments to increase their binding to adjacent sites. This pharmacophore construction method facilitates the creation of a reasonable pharmacophore model without the need to fully understand the structure-activity relationships, and our descriptions provide a useful reference for similar research.

Effect of Tilorone on the Dynamics of Viral Load and the Levels of Interferons and Interleukin-1ß in the Lung Tissue and Blood Serum of Mice with Experimental Influenza.

We studied the effect of tilorone on the dynamics of IFNα, IFNγ, and IL-1ß levels in the lung tissue and blood serum in relation to viral load in the lungs of BALB/c mice with pneumonia caused by influenza virus A/Aichi/2/68 (H3N2). Tilorone was administered per os in doses of 40, 150, and 540 µg per mouse 6, 30, and 78 h postinfection, which simulated the drug regimen used in the clinic for the treatment of influenza and acute respiratory viral infections in Russia and post-Soviet countries. Tilorone reduced viral load with the maximum amplitude (2-3 lg) after 1-2 administrations. The results of studying the dynamics of the cytokine levels in the infected animals in general support the previous hypothesis that, in repeated dosing, tilorone enhances the IFN response (compensates for its deficiency) at the early stages of acute respiratory viral infections and suppresses (damps) excessive production of IFN and proinflammatory cytokines at the later stages.

Reduced sialidase activity of influenza A(H3N2) neuraminidase associated with positively charged amino acid substitutions.

Neuraminidase (NA) inhibitors (NAI), oseltamivir and zanamivir, are the main antiviral medications for influenza and monitoring of susceptibility to these antivirals is routinely done by determining 50 % inhibitory concentrations (IC50) with MUNANA substrate. During 2010-2019, levels of A(H3N2) viruses presenting reduced NAI inhibition (RI) were low (~0.75 %) but varied year-on-year. The highest proportions of viruses showing RI were observed during the 2013-2014, 2016-2017 and 2017-2018 Northern Hemisphere seasons. The majority of RI viruses were found to contain positively charged NA amino acid substitutions of N329K, K/S329R, S331R or S334R, being notably higher during the 2016-2017 season. Sialidase activity kinetics were determined for viruses of RI phenotype and contemporary wild-type (WT) viruses showing close genetic relatedness and displaying normal inhibition (NI). RI phenotypes resulted from reduced sialidase activity compared to relevant WT viruses. Those containing S329R or N329K or S331R showed markedly higher Km for the substrate and Ki values for NAIs, while those with S334R showed smaller effects. Substitutions at N329 and S331 disrupt a glycosylation sequon (NDS), confirmed to be utilised by mass spectrometry. However, gain of positive charge at all three positions was the major factor influencing the kinetic effects, not loss of glycosylation. Because of the altered enzyme characteristics NAs carrying these substitutions cannot be assessed reliably for susceptibility to NAIs using standard MUNANA-based assays due to reductions in the affinity of the enzyme for its substrate and the concentration of the substrate usually used.

Replication and transmission of an influenza A(H3N2) virus harboring the polymerase acidic I38T substitution, in guinea pigs.

The polymerase acidic (PA) I38T substitution is a dominant marker of resistance to baloxavir. We evaluated the impact of I38T on the fitness of a contemporary influenza A(H3N2) virus. Influenza A/Switzerland/9715293/2013 (H3N2) wild-type (WT) virus and its I38T mutant were rescued by reverse genetics. Replication kinetics were compared using ST6GalI-MDCK and A549 cells and infectivity/contact transmissibility were evaluated in guinea pigs. Nasal wash (NW) viral titres were determined by TCID50 ml-1 in ST6GalI-MDCK cells. Competition experiments were performed and the evolution of viral population was assessed by droplet digital RT-PCR. I38T did not alter in vitro replication. I38T induced comparable titres vs the WT in guinea pigs NWs and the two viruses transmitted equally by direct contact. However, a 50 %:50 % mixture inoculum evolved to mean WT/I38T ratios of 71 %:29 % and 66.4 %:33.6 % on days 4 and 6 p.i., respectively. Contemporary influenza A(H3N2)-I38T PA variants may conserve a significant level of viral fitness.

Inactivated influenza vaccine effectiveness among department of defense beneficiaries aged 6 months-17 years, 2016-2017 through 2019-2020 influenza seasons.

A test-negative case-control study was conducted to assess inactivated influenza vaccine effectiveness (VE) in children aged 6 months-17 years. The database was developed from the US Department of Defense Global Respiratory Pathogen Surveillance Program over four consecutive influenza seasons from 2016 to 2020. A total of 9,385 children including 4,063 medically attended, laboratory-confirmed influenza-positive cases were identified for VE analysis. A generalized linear mixed model with logit link and binomial distribution was used to estimate the VE. The adjusted VE for children was 42% [95% confidence interval (CI): 37-47%] overall, including 55% (95% CI: 47-61%) for influenza A(H1N1)pdm09, 37% (95% CI: 28-45%) for influenza A(H3N2), and 49% (95% CI: 41-55%) for influenza B. The analysis by age groups indicated that the adjusted VE in children aged 6 months-4 years was higher against influenza A(H1N1)pdm09 and influenza B, and comparable against influenza A(H3N2), compared to those in children aged 5-17 years. Further age-stratified analysis showed that the VE against any types of influenza was low and non-significant for children aged 6-11 months (33%; 95% CI:-2-56%), but it was high (54%; 95% CI: 34-67%) in children aged 12-23 months, and then declined linearly with increasing age. In conclusion, the inactivated influenza vaccination was moderately effective against influenza infection, based on the analysis from a large number of children aged 6 months-17 years over multiple influenza seasons.

Identification of sensitive indicators in immune response for leprosy affected patients: An observational clinical study of safety and immunogenicity of influenza vaccine.

ABSTRACT: Cured leprosy patients have special physical conditions, which could pose challenges for safety and immunogenicity after immunization. We performed an observational clinical study aimed to identify the safety and immunogenicity of influenza vaccine in cured leprosy patients. A total of 65 participants from a leprosarium were recruited into leprosy cured group or control group, and received a 0.5 ml dose of the inactivated split-virion trivalent influenza vaccine and a follow-up 28 days proactive observation of any adverse events. Hemagglutination and hemagglutination inhibition test was performed to evaluate serum antibody titer, flow cytometry was conducted to screen of cytokines level. The total rate of reactogenicity was 0.0% [0/41] in leprosy cured group and 37.5% [9/24] in control group. The seroconversion rate for H1N1 was difference between leprosy cured group and control group (41.83% vs 79.17%, P = .0082), but not for H3N2 (34.25% vs 50.00%, P = .4468). At day 0, leprosy cured group have relatively high concentration of interleukin-6, interleukin-10, tumor necrosis factor, interferon-γ, and interleukin-17 compared to control group. The interleukin-2 concentration increased 2 weeks after vaccination compared to pre-vaccination in leprosy cured group, but declined in control group (0.92 pg/ml vs -0.02 pg/ml, P = .0147). Leprosy cured group showed a more rapid down-regulation of interleukin-6 when influenza virus was challenged compared to control group (-144.38 pg/ml vs -11.52 pg/ml, P < .0001). Subgroup analysis revealed that the immunization administration declined interleukin-17 concentration in Tuberculoid type subgroup, but not in Lepromatous type subgroup or control group. Clinically cured leprosy patients are relatively safe for influenza vaccine. Leprosy cured patient have immune deficit in producing antibody. Interleukin-6 and interleukin-17 were 2 sensitive indicators in immune response for leprosy affected patients. The identification of indicators might be help management of leprosy and used as predictive markers in leprosy early symptom monitoring.

Intranasal vaccination with influenza HA/GO-PEI nanoparticles provides immune protection against homo- and heterologous strains.

Intranasal (i.n.) immunization is a promising vaccination route for infectious respiratory diseases such as influenza. Recombinant protein vaccines can overcome the safety concerns and long production phase of virus-based influenza vaccines. However, soluble protein vaccines are poorly immunogenic if administered by an i.n. route. Here, we report that polyethyleneimine-functionalized graphene oxide nanoparticles (GP nanoparticles) showed high antigen-loading capacities and superior immunoenhancing properties. Via a facile electrostatic adsorption approach, influenza hemagglutinin (HA) was incorporated into GP nanoparticles and maintained structural integrity and antigenicity. The resulting GP nanoparticles enhanced antigen internalization and promoted inflammatory cytokine production and JAWS II dendritic cell maturation. Compared with soluble HA, GP nanoparticle formulations induced significantly enhanced and cross-reactive immune responses at both systemic sites and mucosal surfaces in mice after i.n. immunization. In the absence of any additional adjuvant, the GP nanoparticle significantly boosted antigen-specific humoral and cellular immune responses, comparable to the acknowledged potent mucosal immunomodulator CpG. The robust immune responses conferred immune protection against challenges by homologous and heterologous viruses. Additionally, the solid self-adjuvant effect of GP nanoparticles may mask the role of CpG when coincorporated. In the absence of currently approved mucosal adjuvants, GP nanoparticles can be developed into potent i.n. influenza vaccines, providing broad protection. With versatility and flexibility, the GP nanoplatform can be easily adapted for constructing mucosal vaccines for different respiratory pathogens.

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.

Dimeric clerodane diterpenoids and antiviral constituents of Dodonaea viscosa.

Three unprecedented dimeric clerodane diterpenoids, dodovisdimers A-C (1-3), along with six known clerodane monomers (4-9), were isolated from Dodonaea viscosa. Compounds 1-3 may be biosynthetically formed via an intermolecular Diels-Alder [4+2] cycloaddition between the coexisting monomers 4-7. The structures of these clerodanes were characterized by spectroscopic techniques, X-ray crystallographic study, and ECD calculations. Some isolates exerted antiviral effects on human influenza A virus (H3N2) in vitro.

The cleavage of spike protein НА0→НА1/HA2 by trypsin permits activation of the M2 channel without its proteolytic cleavage in the influenza A virus.

M2 plays numerous regulatory roles in influenza A virus infection confirming the old adage: "a little body often harbors a great sense". The comment here demonstrates that a small viral protein M2, having 14 kD m.w. and situating in the virion at a minor amount of only about 40 molecules per virus particle is resistant to trypsin at concentrations initiating the HA0 cleavage and virus infectivity activation. A mechanism involving a programmed disassembly by cascade-type transmembrane signaling of the HA-M2-M1-RNP cooperation during virus entry into the infected cell is proposed.

Antiviral Activity of 3D, a Butene Lactone Derivative Against Influenza A Virus In Vitro and In Vivo.

Influenza A virus is a highly variable and contagious respiratory pathogen that can cause annual epidemics and it poses an enormous threat to public health. Therefore, there is an urgent need for a new generation of antiviral drugs to combat the emergence of drug-resistant strains of the influenza virus. A novel series of butene lactone derivatives were screened and the compound 3D was selected, as it exhibited in vitro potential antiviral activity against A/Weiss/43 H1N1 virus with low toxicity. In addition, 3D dose-dependently inhibited the viral replication, expression of viral mRNA and viral proteins. 3D exerted a suppressive effect on A/Virginia/ATCC2/2009 H1N1 and A/California/2/2014 H3N2 in vitro. The time-of-addition analysis indicated that 3D suppressed H1N1 in the early stage of its life cycle. A/Weiss/43 H1N1-induced apoptosis in A549 cells was reduced by 3D via the mitochondrial apoptosis pathway. 3D could decrease the production of H1N1-induced pro-inflammatory cytokines that are induced by H1N1 in vitro and in vivo. The administration of 3D reduced lung lesions and virus load in vivo. These results suggest that 3D, which is a butene lactone derivative, is a promising agent for the treatment of influenza A virus infection.

Bioactive prenylated C6-C3 derivatives from the stems and leaves of Illicium fargesii.

Seventeen new prenylated C6-C3 derivatives, namely, illifargeins A-M (1-13), including three pairs of enantiomers (1, 5, and 12) and one norillifargeal A (14), together with eight known analogues (15-22), were isolated from the stems and leaves of Illicium fargesii. The structures of the new compounds were elucidated using spectroscopic data (UV, IR, 1D and 2D NMR, and HRESIMS). Their absolute configurations were determined by using experimental and calculated ECD data analysis, as well as a modified Mosher's method. Compounds 1a, 1b, 2, 3, 5a, 7, 10, 11, 15, 16, 19, and 20 showed potential activity against Coxsackie virus B3, with IC50 values ranging from 6.23 to 33.33 µM. Compounds 9 and 15 exhibited potential activity against influenza virus A, with IC50 values of 11.11 and 19.24 µM, respectively. Compounds 2, 3, and 18 exhibited potential anti-oxidant activity, with IC50 values ranging from 1.43 to 6.71 µM.

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection.

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in naïve human populations.

Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses.

While vaccines remain the best tool for preventing influenza virus infections, they have demonstrated low to moderate effectiveness in recent years. Seasonal influenza vaccines typically consist of wild-type influenza A and B viruses that are limited in their ability to elicit protective immune responses against co-circulating influenza virus variant strains. Improved influenza virus vaccines need to elicit protective immune responses against multiple influenza virus drift variants within each season. Broadly reactive vaccine candidates potentially provide a solution to this problem, but their efficacy may begin to wane as influenza viruses naturally mutate through processes that mediates drift. Thus, it is necessary to develop a method that commercial vaccine manufacturers can use to update broadly reactive vaccine antigens to better protect against future and currently circulating viral variants. Building upon the COBRA technology, nine next-generation H3N2 influenza hemagglutinin (HA) vaccines were designed using a next generation algorithm and design methodology. These next-generation broadly reactive COBRA H3 HA vaccines were superior to wild-type HA vaccines at eliciting antibodies with high HAI activity against a panel of historical and co-circulating H3N2 influenza viruses isolated over the last 15 years, as well as the ability to neutralize future emerging H3N2 isolates.

Secondary substitutions in the hemagglutinin and neuraminidase genes associated with neuraminidase inhibitor resistance are rare in the Influenza Resistance Information Study (IRIS).

Amino acid substitutions in influenza virus neuraminidase (NA) that cause resistance to neuraminidase inhibitors (NAI) generally result in virus attenuation. However, influenza viruses may acquire secondary substitutions in the NA and hemagglutinin (HA) proteins that can restore viral fitness. To assess to which extent this happens, the emergence of NAI resistance substitutions and secondary - potentially compensatory - substitutions was quantified in influenza viruses of immunocompetent individuals included in the Influenza Resistance Information Study (IRIS; NCT00884117). Known resistance substitutions were detected by mutation specific RT-PCR in viruses of 57 of 1803 (3.2%) oseltamivir-treated individuals, including 39 individuals infected with A/H1N1pdm09 [H275Y] virus and 18 with A/H3N2 [R292K] virus. A total of fifteen and ten other amino acid substitutions were acquired in HA and NA respectively, of A/H1N1pdm09, A/H3N2 and influenza B viruses upon treatment with oseltamivir but none of these was associated with resistance to oseltamivir. All cultured viruses with the known resistance substitutions H275Y or R292K showed reduced susceptibility to oseltamivir in the NA-star assay. Upon next-generation sequencing, the vast majority of NAI resistant A/H1N1pdm09 and A/H3N2 viruses had no resistance-associated secondary substitutions at high frequency. Only in two A/H1N1pdm09 [H275Y] viruses, the potentially compensatory secondary substitutions HA-D52N and NA-R152K were detected. We conclude that the emergence of secondary substitutions that may restore viral fitness upon the emergence of known influenza virus NAI resistance substitutions was a rare event in this immunocompetent population.

Susceptibility of widely diverse influenza a viruses to PB2 polymerase inhibitor pimodivir.

Pimodivir exerts an antiviral effect on the early stages of influenza A virus replication by inhibiting the cap-binding function of polymerase basic protein 2 (PB2). In this study, we used a combination of sequence analysis and phenotypic methods to evaluate pimodivir susceptibility of influenza A viruses collected from humans and other hosts. Screening PB2 sequences for substitutions previously associated with reduced pimodivir susceptibility revealed a very low frequency among seasonal viruses circulating in the U.S. during 2015-2020 (<0.03%; 3/11,934) and among non-seasonal viruses collected in various countries during the same period (0.2%; 18/8971). Pimodivir potently inhibited virus replication in two assays, a single-cycle HINT and a multi-cycle FRA, with IC50 values in a nanomolar range. Median IC50 values determined by HINT were similar for both subtypes of seasonal viruses, A(H1N1)pdm09 and A(H3N2), across three seasons. Human seasonal viruses with PB2 substitutions S324C, S324R, or N510K displayed a 27-317-fold reduced pimodivir susceptibility by HINT. In addition, pimodivir was effective at inhibiting replication of a diverse group of animal-origin viruses that have pandemic potential, including avian viruses of A(H5N6) and A(H7N9) subtypes. A rare PB2 substitution H357N was identified in an A(H4N2) subtype poultry virus that displayed >100-fold reduced pimodivir susceptibility. Our findings demonstrate a broad inhibitory activity of pimodivir and expand the existing knowledge of amino acid substitutions that can reduce susceptibility to this investigational antiviral.