Optimizing electrochemically active microorganisms as a key player in the bioelectrochemical system: Identification methods and pathways to large-scale implementation.

The rapid global economic growth driven by industrialization and population expansion has resulted in significant issues, including reliance on fossil fuels, energy scarcity, water crises, and environmental emissions. To address these issues, bioelectrochemical systems (BES) have emerged as a dual-purpose solution, harnessing electrochemical processes and the capabilities of electrochemically active microorganisms (EAM) to simultaneously recover energy and treat wastewater. This review examines critical performance factors in BES, including inoculum selection, pretreatment methods, electrodes, and operational conditions. Further, authors explore innovative approaches to suppress methanogens and simultaneously enhance the EAM in mixed cultures. Additionally, advanced techniques for detecting EAM are discussed. The rapid detection of EAM facilitates the selection of suitable inoculum sources and optimization of enrichment strategies in BESs. This optimization is essential for facilitating the successful scaling up of BES applications, contributing substantially to the realization of clean energy and sustainable wastewater treatment. This analysis introduces a novel viewpoint by amalgamating contemporary research on the selective enrichment of EAM in mixed cultures. It encompasses identification and detection techniques, along with methodologies tailored for the selective enrichment of EAM, geared explicitly toward upscaling applications in BES.

New insights into the neuraminidase-mediated hemagglutination activity of influenza A(H3N2) viruses.

Influenza virus neuraminidase (NA) can act as a receptor-binding protein, a role commonly attributed to hemagglutinin (HA). In influenza A(H3N2) viruses, three NA amino acid residues have previously been associated with NA-mediated hemagglutination: T148, D151, and more recently, H150. These residues are part of the 150-loop of the NA monomer. Substitutions at 148 and 151 arise from virus propagation in laboratory cell cultures, whereas changes at 150 occurred during virus evolution in the human host. In this study, we examined the effect of natural amino acid polymorphism at position 150 on NA-mediated hemagglutination. Using the A/Puerto Rico/8/34 backbone, we generated a comprehensive panel of recombinant A(H3N2) viruses that have different NAs but shared an HA that displays poor binding to red blood cells (RBCs). None of the tested substitutions at 150 (C, H, L, R, and S) promoted NA-binding. However, we identified two new determinants of NA-binding, Q136K and T439R, that emerged during virus culturing. Similar to T148I, both Q136K and T439R reduced NA enzyme activity by 48-86% and inhibition (14- to 173-fold) by the NA inhibitor zanamivir. NA-binding was observed when a virus preparation contained approximately 10% of NA variants with either T148I or T439R, highlighting the benefit of using deep sequencing in virus characterization. Taken together, our findings provide new insights into the molecular mechanisms underlying the ability of NA to function as a binding protein. Information gained may aid in the design of new and improved NA-targeting antivirals.

Antiviral susceptibility of clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses isolated from birds and mammals in the United States, 2022.

Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) A(H5N1) viruses that are responsible for devastating outbreaks in birds and mammals pose a potential threat to public health. Here, we evaluated their susceptibility to influenza antivirals. Of 1,015 sequences of HPAI A(H5N1) viruses collected in the United States during 2022, eight viruses (∼0.8%) had a molecular marker of drug resistance to an FDA-approved antiviral: three adamantane-resistant (M2-V27A), four oseltamivir-resistant (NA-H275Y), and one baloxavir-resistant (PA-I38T). Additionally, 31 viruses contained mutations that may reduce susceptibility to inhibitors of neuraminidase (NA) (n = 20) or cap-dependent endonuclease (CEN) (n = 11). A panel of 22 representative viruses was tested phenotypically. Overall, clade 2.3.4.4b A(H5N1) viruses lacking recognized resistance mutations were susceptible to FDA-approved antivirals. Oseltamivir was least potent at inhibiting NA activity, while the investigational NA inhibitor AV5080 was most potent, including against NA mutants. A novel NA substitution T438N conferred 12-fold reduced inhibition by zanamivir, and in combination with the known marker N295S, synergistically affected susceptibility to all five NA inhibitors. In cell culture-based assays HINT and IRINA, the PA-I38T virus displayed 75- to 108-fold and 37- to 78-fold reduced susceptibility to CEN inhibitors, baloxavir and the investigational AV5116, respectively. Viruses with PA-I38M or PA-A37T showed 5- to 10-fold reduced susceptibilities. As HPAI A(H5N1) viruses continue to circulate and evolve, close monitoring of drug susceptibility is needed for risk assessment and to inform decisions regarding antiviral stockpiling.

Insights into the synthesis strategies of plant-derived cyclotides.

Cyclotides are plant peptides characterized with a head-to-tail cyclized backbone and three interlocking disulfide bonds, known as a cyclic cysteine knot. Despite the variations in cyclotides peptide sequences, this core structure is conserved, underlying their most useful feature: stability against thermal and chemical breakdown. Cyclotides are the only natural peptides known to date that are orally bioavailable and able to cross cell membranes. Cyclotides also display bioactivities that have been exploited and expanded to develop as potential therapeutic reagents for a wide range of conditions (e.g., HIV, inflammatory conditions, multiple sclerosis, etc.). As such, in vitro production of cyclotides is of the utmost importance since it could assist further research on this peptide class, specifically the structure-activity relationship and its mechanism of action. The information obtained could be utilized to assist drug development and optimization. Here, we discuss several strategies for the synthesis of cyclotides using both chemical and biological routes.

Cysteine-rich peptides: From bioactivity to bioinsecticide applications.

Greater levels of insect resistance and constraints on the use of current pesticides have recently led to increased crop losses in agricultural production. Further, the health and environmental impacts of pesticides now restrict their application. Biologics based on peptides are gaining popularity as efficient crop protection agents with low environmental toxicity. Cysteine-rich peptides (whether originated from venoms or plant defense substances) are chemically stable and effective as insecticides in agricultural applications. Cysteine-rich peptides fulfill the stability and efficacy requirements for commercial uses and provide an environmentally benign alternative to small-molecule insecticides. In this article, cysteine-rich insecticidal peptide classes identified from plants and venoms will be highlighted, focusing on their structural stability, bioactivity and production.

Pretreatments of lignocellulosic and algal biomasses for sustainable biohydrogen production: Recent progress, carbon neutrality, and circular economy.

Lignocellulosic and algal biomasses are known to be vital feedstocks to establish a green hydrogen supply chain toward achieving a carbon-neutral society. However, one of the most pressing issues to be addressed is the low digestibility of these biomasses in biorefinery processes, such as dark fermentation, to produce green hydrogen. To date, various pretreatment approaches, such as physical, chemical, and biological methods, have been examined to enhance feedstock digestibility. However, neither systematic reviews of pretreatment to promote biohydrogen production in dark fermentation nor economic feasibility analyses have been conducted. Thus, this study offers a comprehensive review of current biomass pretreatment methods to promote biohydrogen production in dark fermentation. In addition, this review has provided comparative analyses of the technological and economic feasibility of existing pretreatment techniques and discussed the prospects of the pretreatments from the standpoint of carbon neutrality and circular economy.

The transcription factors Junb and Fosl2 cooperate to regulate Cdh3 expression in 15P-1 Sertoli cells.

In Sertoli cells of the testis, cadherins (Cdh) are important cell-to-cell interaction proteins and contribute to the formation of the blood-testis barrier being essential for germ cells' protection. P-cadherin or Cdh3 is only expressed in Sertoli cells from embryonic to prepubertal development. Interestingly, the expression profile of Cdh3 correlates with that of activating protein-1 (AP-1) transcription factors during Sertoli cells development. To assess their potential implications in the regulation of Cdh3, different AP-1 transcription factors were overexpressed in 15P-1 Sertoli cells. We found that the overexpressions of Junb and Fosl2 activated Cdh3 promoter. ChIP-qPCR assay and luciferase reporter assay with 5' promoter deletions and site-directed mutagenesis confirmed the recruitment of Junb and Fosl2 to an AP-1 regulatory element at -47 bp in the proximal region of Cdh3 promoter in 15P-1 cells. These findings were further supported by histone modification markers and chromatin accessibility surrounding Cdh3 promoter in mouse testis. Moreover, the knockdowns of Junb and/or Fosl2 by siRNA decreased Cdh3 protein levels. Taken together, these data suggest that in 15P-1 Sertoli cells, the AP-1 family members Junb and Fosl2 are responsible for the regulation of Cdh3 expression, which requires the recruitment of both factors to the proximal region of the Cdh3 promoter.

An optimized cell-based assay to assess influenza virus replication by measuring neuraminidase activity and its applications for virological surveillance.

Year-round virological characterization of circulating epidemic influenza viruses is conducted worldwide to detect the emergence of viruses that may escape pre-existing immunity or acquire resistance to antivirals. High throughput phenotypic assays are needed to complement the sequence-based analysis of circulating viruses and improve pandemic preparedness. The recent entry of a polymerase inhibitor, baloxavir, into the global market further highlighted this need. Here, we optimized a cell-based assay that considerably streamlines antiviral and antigenic testing by replacing lengthy immunostaining and imaging procedures used in current assay with measuring the enzymatic activity of nascent neuraminidase (NA) molecules expressed on the surface of virus-infected cells. For convenience, this new assay was named IRINA (Influenza Replication Inhibition Neuraminidase-based Assay). IRINA was successfully validated to assess inhibitory activity of baloxavir on virus replication by testing a large set (>150) of influenza A and B viruses, including drug resistant strains and viruses collected during 2017-2022. To test its versatility, IRINA was utilized to evaluate neutralization activity of a broadly reactive human anti-HA monoclonal antibody, FI6, and post-infection ferret antisera, as well as the inhibition of NA enzyme activity by NA inhibitors. Performance of IRINA was tested in parallel using respective conventional assays. IRINA offers an attractive alternative to current phenotypic assays, while maintaining reproducibility and high throughput capacity. Additionally, the improved turnaround time may prove to be advantageous when conducting time sensitive studies, such as investigating a new virus outbreak. This assay can meet the needs of surveillance laboratories by providing a streamlined and cost-effective approach for virus characterization.

Factors associated with depression, anxiety, and stress symptoms among men in a rural area in Vietnam during COVID-19.

Background: The COVID-19 pandemic has affected health and well-being worldwide, and its psychological effects are receiving substantial attention in the scientific literature. Research to date shows that the pandemic has increased prevalences of depression, anxiety, and stress. This study aimed to estimate the prevalence of mental health symptoms and identify the associated factors among men in a rural area of Vietnam during the COVID-19 pandemic. Methods and findings: During July 15-31, 2020, we conducted a cross-sectional survey of 1,085 men from 18 years old in 11 rural districts in Thanh Hoa province, Vietnam, and assessed their mental health using the Depression, Anxiety and Stress Scale - 21 Items (DASS-21). Outcomes assessed were have a symptom of depression, anxiety, and stress; risk factors measured included age, religion, marital status, education, occupation, and financial status. Multiple linear regression was performed to determine the statistical significance of associations between risk factors and mental health symptoms. Findings showed that the prevalences of having a symptom of depression, anxiety and stress among participants were 6.39, 9.72, and 5.65%, respectively. Regression model showed being younger (95% CI: -0.030; -0.004, p = 0.001), men had high school degree (95% CI: -0.671; -0.074, p = 0.014), men living in nearly poor houshoulds (95% CI: 0.067, 1.905, p < 0.05) and poor housholds (95% CI: 0.608; 2.721, p < 0.05) had significantly lower depression scores than others. Conclusion: Prevalences of having symptoms of depression, anxiety and stress were much higher than in similar previous research in rural Vietnam, suggesting that mental health problems among men in this setting became more common during the COVID-19 pandemic. Age, religion, level of education and family income status were statistically significant predictors of mental health problems. These findings provide useful insights into the impact of pandemics on mental health.

Synthesis, Biological Evaluation, and Molecular Modeling Studies of 1-Aryl-1H-pyrazole-Fused Curcumin Analogues as Anticancer Agents.

Addressing the growing burden of cancer and the shortcomings of chemotherapy in cancer treatment are the current research goals. Research to overcome the limitations of curcumin and to improve its anticancer activity via its heterocycle-fused monocarbonyl analogues (MACs) has immense potential. In this study, 32 asymmetric MACs fused with 1-aryl-1H-pyrazole (7a-10h) were synthesized and characterized to develop new curcumin analogues. Subsequently, via initial screening for cytotoxic activity, nine compounds exhibited potential growth inhibition against MDA-MB-231 (IC50 2.43-7.84 µM) and HepG2 (IC50 4.98-14.65 µM), in which seven compounds showing higher selectivities on two cancer cell lines than the noncancerous LLC-PK1 were selected for cell-free in vitro screening for effects on microtubule assembly activity. Among those, compounds 7d, 7h, and 10c showed effective inhibitions of microtubule assembly at 20.0 µM (40.76-52.03%), indicating that they could act as microtubule-destabilizing agents. From the screening results, three most potential compounds, 7d, 7h, and 10c, were selected for further evaluation of cellular effects on breast cancer MDA-MB-231 cells. The apoptosis-inducing study indicated that these three compounds could cause morphological changes at 1.0 µM and could enhance caspase-3 activity (1.33-1.57 times) at 10.0 µM in MDA-MB-231 cells, confirming their apoptosis-inducing activities. Additionally, in cell cycle analysis, compounds 7d and 7h at 2.5 µM and 10c at 5.0 µM also arrested MDA-MB-231 cells in the G2/M phase. Finally, the results from in silico studies revealed that the predicted absorption, distribution, metabolism, excretion, and the toxicity (ADMET) profile of the most potent MACs might have several advantages in addition to potential disadvantages, and compound 7h could bind into (ΔG -10.08 kcal·mol-1) and access wider space at the colchicine-binding site (CBS) than that of colchicine or nocodazole via molecular docking studies. In conclusion, our study serves as a basis for the design of promising synthetic compounds as anticancer agents in the future.

Thalassemia in Viet Nam.

The population of Viet Nam, is 96.2 million, of which 13.8% are carriers of thalassemia genes. Thalassemia/hemoglobinopathies carriers exist at different frequencies in all 54 ethnic groups of the country. Gene carrier rate and globin gene mutation rate varies ethnically and topographically. The ethnic groups in the Northern Highland region have high rates of α0- and ß0-thalassemia (α0- and ß0-thal), while those in the Southern Middle region have high rates of α+-thalassemia (α+-thal) and Hb E (or codon 26) (HBB: c.79G>A). The lowest is found in La Hu (0.23%), while the highest is found in Raglai (88.6%). Thalassemia prevention and control programs were introduced using prenatal and neonatal diagnosis for the prevention of new thalassemic births. Most existing thalassemia patients are undergoing supportive treatment with regular blood transfusions and iron chelation. Curative treatment by hematopoietic stem cell transplantation is available but is limited to a minority of the patients.

Influenza Activity and Composition of the 2022-23 Influenza Vaccine - United States, 2021-22 Season.

Before the emergence of SARS-CoV-2, the virus that causes COVID-19, influenza activity in the United States typically began to increase in the fall and peaked in February. During the 2021-22 season, influenza activity began to increase in November and remained elevated until mid-June, featuring two distinct waves, with A(H3N2) viruses predominating for the entire season. This report summarizes influenza activity during October 3, 2021-June 11, 2022, in the United States and describes the composition of the Northern Hemisphere 2022-23 influenza vaccine. Although influenza activity is decreasing and circulation during summer is typically low, remaining vigilant for influenza infections, performing testing for seasonal influenza viruses, and monitoring for novel influenza A virus infections are important. An outbreak of highly pathogenic avian influenza A(H5N1) is ongoing; health care providers and persons with exposure to sick or infected birds should remain vigilant for onset of symptoms consistent with influenza. Receiving a seasonal influenza vaccine each year remains the best way to protect against seasonal influenza and its potentially severe consequences.

Case Report: Successful Treatment of a Child With COVID-19 Reinfection-Induced Fulminant Myocarditis by Cytokine-Adsorbing oXiris® Hemofilter Continuous Veno-Venous Hemofiltration and Extracorporeal Membrane Oxygenation.

Background: Indirect cardiomyocyte damage-related hyperinflammatory response is one of the key mechanisms in COVID-19-induced fulminant myocarditis. In addition to the clinical benefit of using cytokines absorption hemofiltration, the effectiveness of instituting veno-arterial extracorporeal membrane oxygenation (VA-ECMO) support for cardiac compromise has been reported. However, current literature enunciates a paucity of available data on the effectiveness of these novel modalities. Case Presentation: We reported a 9-year-old boy with recurrent COVID-19 infection-causing fulminant myocarditis, who was treated successfully by using novel modalities of oXiris ® hemofilter continuous venovenous hemofiltration (CVVH) and VA-ECMO. The patient made a full recovery without any sequelae. Conclusion: We conclude that the novel highly-absorptive hemofilter CVVH and VA-ECMO may be effective treatment modalities in managing SARS-CoV-2-induced fulminant myocarditis. Our report highlights the need for further well-designed investigations to confirm this extrapolation.

Global update on the susceptibilities of human influenza viruses to neuraminidase inhibitors and the cap-dependent endonuclease inhibitor baloxavir, 2018-2020.

Global analysis of the susceptibility of influenza viruses to neuraminidase (NA) inhibitors (NAIs) and the polymerase acidic (PA) inhibitor (PAI) baloxavir was conducted by five World Health Organization Collaborating Centres for Reference and Research on Influenza during two periods (May 2018-May 2019 and May 2019-May 2020). Combined phenotypic and NA sequence-based analysis revealed that the global frequency of viruses displaying reduced or highly reduced inhibition (RI or HRI) or potential to show RI/HRI by NAIs remained low, 0.5% (165/35045) and 0.6% (159/26010) for the 2018-2019 and 2019-2020 periods, respectively. The most common amino acid substitution was NA-H275Y (N1 numbering) conferring HRI by oseltamivir and peramivir in A(H1N1)pdm09 viruses. Combined phenotypic and PA sequence-based analysis showed that the global frequency of viruses showing reduced susceptibility to baloxavir or carrying substitutions associated with reduced susceptibility was low, 0.5% (72/15906) and 0.1% (18/15692) for the 2018-2019 and 2019-2020 periods, respectively. Most (n = 61) of these viruses had I38→T/F/M/S/L/V PA amino acid substitutions. In Japan, where baloxavir use was highest, the rate was 4.5% (41/919) in the 2018-2019 period and most of the viruses (n = 32) had PA-I38T. Zoonotic viruses isolated from humans (n = 32) in different countries did not contain substitutions in NA associated with NAI RI/HRI phenotypes. One A(H5N6) virus had a dual substitution PA-I38V + PA-E199G, which may reduce susceptibility to baloxavir. Therefore, NAIs and baloxavir remain appropriate choices for the treatment of influenza virus infections, but close monitoring of antiviral susceptibility is warranted.

Comparative efficacy and safety of pharmacologic interventions to prevent mother-to-child transmission of hepatitis B virus: a systematic review and network meta-analysis.

OBJECTIVE: This study investigated the efficacy and safety of pharmacologic interventions to prevent vertical transmission of the hepatitis B virus. DATA SOURCES: Medline, Cochrane, and Scopus databases were searched up to October 28, 2020. STUDY ELIGIBILITY CRITERIA: All randomized controlled trials reporting vertical hepatitis B virus transmission with pharmacologic intervention were included. METHODS: Risk of bias was assessed using the Cochrane Risk-of-Bias tool, version 2. Treatment efficacy was estimated using stratified network meta-analysis on the basis of maternal hepatitis B envelope antigen status. RESULTS: Nineteen studies were included for mothers positive for hepatitis B surface and envelope antigens. Pooling indicated that a combination of hepatitis B vaccination and hepatitis B immunoglobulin in infants significantly reduced transmission risk compared with vaccination alone, with a risk ratio of 0.52 (95% confidence interval; 0.30-0.91). Only the addition of maternal tenofovir disoproxil fumarate, but not telbivudine, lamivudine, or maternal hepatitis B immunoglobulin further reduced transmission risk compared with a combination of hepatitis B vaccination and hepatitis B immunoglobulin in infants, with a pooled risk ratio of 0.10 (0.03-0.35). Twelve studies conducted in mothers with hepatitis B surface antigen positivity and mixed, unknown, or negative hepatitis B envelope antigen status provided limited evidence to suggest that maternal hepatitis B immunoglobulin combined with hepatitis B vaccination and immunoglobulin in infants was the likely best treatment, but this failed to reach statistical significance compared with a combination of hepatitis B vaccination and immunoglobulin in infants. Similarly, infant hepatitis B immunoglobulin, added to vaccination, likely provides additional benefit but failed to reach statistical significance. CONCLUSION: A combination of hepatitis B vaccination and immunoglobulin in infants is the cornerstone for prevention of vertical transmission for mothers positive for both hepatitis B surface and envelope antigens. The addition of maternal tenofovir to this infant combination regimen was considered the likely most effective treatment. For infants of mothers with hepatitis B surface antigen positivity and mixed, unknown, or negative hepatitis B envelop antigen status, no additional agents provided further benefit beyond hepatitis B vaccination alone.

Cluster of Oseltamivir-Resistant and Hemagglutinin Antigenically Drifted Influenza A(H1N1)pdm09 Viruses, Texas, USA, January 2020.

Four cases of oseltamivir-resistant influenza A(H1N1)pdm09 virus infection were detected among inhabitants of a border detention center in Texas, USA. Hemagglutinin of these viruses belongs to 6B.1A5A-156K subclade, which may enable viral escape from preexisting immunity. Our finding highlights the necessity to monitor both drug resistance and antigenic drift of circulating viruses.

Reference-free transcriptome signatures for prostate cancer prognosis.

BACKGROUND: RNA-seq data are increasingly used to derive prognostic signatures for cancer outcome prediction. A limitation of current predictors is their reliance on reference gene annotations, which amounts to ignoring large numbers of non-canonical RNAs produced in disease tissues. A recently introduced kind of transcriptome classifier operates entirely in a reference-free manner, relying on k-mers extracted from patient RNA-seq data. METHODS: In this paper, we set out to compare conventional and reference-free signatures in risk and relapse prediction of prostate cancer. To compare the two approaches as fairly as possible, we set up a common procedure that takes as input either a k-mer count matrix or a gene expression matrix, extracts a signature and evaluates this signature in an independent dataset. RESULTS: We find that both gene-based and k-mer based classifiers had similarly high performances for risk prediction and a markedly lower performance for relapse prediction. Interestingly, the reference-free signatures included a set of sequences mapping to novel lncRNAs or variable regions of cancer driver genes that were not part of gene-based signatures. CONCLUSIONS: Reference-free classifiers are thus a promising strategy for the identification of novel prognostic RNA biomarkers.

Sociodemographic Factors Associated with Request for Labor Epidural Analgesia in a Tertiary Obstetric Hospital in Vietnam.

This study is aimed at examining the sociodemographic factors associated with the utilization of labor epidural analgesia at a large obstetric and gynecology hospital in Vietnam. This was a cross-sectional study of women who underwent vaginal delivery in September 2018 at the Hanoi Obstetrics and Gynecology Hospital. The utilization of epidural analgesia during labor was determined. Univariate and multivariate regression models were applied to evaluate the association between patient demographic and socioeconomic factors and request for labor epidural analgesia. A total of 417 women had vaginal deliveries during the study period. 207 women utilized epidural analgesia for pain relief during labor, and 210 did not. Parturients older than 35 years of age (OR 2.84, 95% CI 1.11-8.17), multiparous women (OR 2.8 95% CI 1.85-4.25), women living from an urban area, women with higher income (OR 6.47, 95% CI 2.59-19.23), and women with higher level of education were more likely to utilize labor epidurals. Factors related to a parturient request for epidural analgesia during labor at our tertiary obstetric hospital included age greater than 35 years, multiparity, and high income and education levels. Educational outreach to women about the benefits of epidural analgesia can target women who do not share these demographic characteristics.

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.