During 2023-2024, highly pathogenic avian influenza A(H5N1) viruses from clade 2.3.2.1c caused human infections in Cambodia and from clade 2.3.4.4b caused human infections in the Americas. We assessed the susceptibility of those viruses to approved and investigational antiviral drugs. Except for 2 viruses isolated from Cambodia, all viruses were susceptible to M2 ion channel-blockers in cell culture-based assays. In the neuraminidase inhibition assay, all viruses displayed susceptibility to neuraminidase inhibitor antiviral drugs oseltamivir, zanamivir, peramivir, laninamivir, and AV5080. Oseltamivir was ≈4-fold less potent at inhibiting the neuraminidase activity of clade 2.3.4.4b than clade 2.3.2.1c viruses. All viruses were susceptible to polymerase inhibitors baloxavir and tivoxavir and to polymerase basic 2 inhibitor pimodivir with 50% effective concentrations in low nanomolar ranges. Because drug-resistant viruses can emerge spontaneously or by reassortment, close monitoring of antiviral susceptibility of H5N1 viruses collected from animals and humans by using sequence-based analysis supplemented with phenotypic testing is essential.

Since 2013, a total of 167 human infections with swine-origin (variant) influenza A viruses of A(H1N1)v, A(H1N2)v, and A(H3N2)v subtypes have been reported in the United States. Analysis of 147 genome sequences revealed that nearly all had S31N substitution, an M2 channel blocker-resistance marker, whereas neuraminidase inhibitor-resistance markers were not found. Two viruses had a polymerase acidic substitution (I38M or E199G) associated with decreased susceptibility to baloxavir, an inhibitor of viral cap-dependent endonuclease (CEN). Using phenotypic assays, we established subtype-specific susceptibility baselines for neuraminidase and CEN inhibitors. When compared with either baseline or CEN-sequence-matched controls, only the I38M substitution decreased baloxavir susceptibility, by 27-fold. Human monoclonal antibodies FI6v3 and CR9114 targeting the hemagglutinin's stem showed variable (0.03 to >10 µg/mL) neutralizing activity toward variant viruses, even within the same clade. Methodology and interpretation of laboratory data described in this study provide information for risk assessment and decision-making on therapeutic control measures.

Since May 2023, a novel combination of neuraminidase mutations, I223V + S247N, has been detected in influenza A(H1N1)pdm09 viruses collected in countries spanning 5 continents, mostly in Europe (67/101). The viruses belong to 2 phylogenetically distinct groups and display ≈13-fold reduced inhibition by oseltamivir while retaining normal susceptibility to other antiviral drugs.

Antiviral susceptibility of influenza viruses was assessed using a high-content imaging-based neutralization test. Cap-dependent endonuclease inhibitors, baloxavir and AV5116, were superior to AV5115 against type A viruses, and AV5116 was most effective against PA mutants tested. However, these three inhibitors displayed comparable activity (EC(50) 8-22 nM) against type C viruses from six lineages. Banana lectin and a monoclonal antibody, YA3, targeting the hemagglutinin-esterase protein effectively neutralized some, but not all, type C viruses.

This report updates previous recommendations by CDC's Advisory Committee on Immunization Practices (ACIP) regarding the use of antiviral agents for the prevention and treatment of influenza (CDC. Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2008;57[No. RR-7]).This report contains information on treatment and chemoprophylaxis of influenza virus infection and provides a summary of the effectiveness and safety of antiviral treatment medications. Highlights include recommendations for use of 1) early antiviral treatment of suspected or confirmed influenza among persons with severe influenza (e.g., those who have severe, complicated, or progressive illness or who require hospitalization); 2) early antiviral treatment of suspected or confirmed influenza among persons at higher risk for influenza complications; and 3) either oseltamivir or zanamivir for persons with influenza caused by 2009 H1N1 virus, influenza A (H3N2) virus, or influenza B virus or when the influenza virus type or influenza A virus subtype is unknown; 4) antiviral medications among children aged <1 year; 5) local influenza testing and influenza surveillance data, when available, to help guide treatment decisions; and 6) consideration of antiviral treatment for outpatients with confirmed or suspected influenza who do not have known risk factors for severe illness, if treatment can be initiated within 48 hours of illness onset. Additional information is available from CDC's influenza website at http://www.cdc.gov/flu, including any updates or supplements to these recommendations that might be required during the 2010-11 influenza season. Health-care providers should be alert to announcements of recommendation updates and should check the CDC influenza website periodically for additional information. Recommendations related to the use of vaccines for the prevention of influenza during the 2010-11 influenza season have been published previously (CDC. Prevention and control of influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices [ACIP], 2010. MMWR 2010;59[No. RR-8]).

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.

Clade 2.3.4.4 b 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 1015 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 2.3.4.4  b 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 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.

The COVID-19 pandemic and subsequent implementation of nonpharmaceutical interventions (e.g., cessation of global travel, mask use, physical distancing, and staying home) reduced the transmission of some viral respiratory pathogens.1 In the United States, influenza activity decreased in March 2020, was historically low through the summer of 2020,2 and remained low during October 2020–May 2021 (<0.4% of respiratory specimens with positive test results for each week of the season). Circulation of other respiratory pathogens, including respiratory syncytial virus (RSV), common human coronaviruses (HCoVs) types OC43, NL63, 229E, and HKU1, and parainfluenza viruses (PIVs) types 1–4 also decreased in early 2020 and did not increase until spring 2021. Human metapneumovirus (HMPV) circulation decreased in March 2020 and remained low through May 2021. Respiratory adenovirus (RAdV) circulated at lower levels throughout 2020 and as of early May 2021. Rhinovirus and enterovirus (RV/EV) circulation decreased in March 2020, remained low until May 2020, and then increased to near prepandemic seasonal levels. Circulation of respiratory viruses could resume at prepandemic levels after COVID-19 mitigation practices become less stringent. Clinicians should be aware of increases in some respiratory virus activity and remain vigilant for off-season increases. In addition to the use of everyday preventive actions, fall influenza vaccination campaigns are an important component of prevention as COVID-19 mitigation measures are relaxed and schools and workplaces resume in-person activities.

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.

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.

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.

It is more than 20 years since the neuraminidase inhibitors, oseltamivir and zanamivir were approved for the treatment and prevention of influenza. Guidelines for global surveillance and methods for evaluating resistance were established initially by the Neuraminidase Inhibitor Susceptibility Network (NISN), which merged 10 years ago with the International Society for influenza and other Respiratory Virus Diseases (isirv) to become the isirv-Antiviral Group (isirv-AVG). With the ongoing development of new influenza polymerase inhibitors and recent approval of baloxavir marboxil, the isirv-AVG held a closed meeting in August 2019 to discuss the impact of resistance to these inhibitors. Following this meeting and review of the current literature, this article is intended to summarize current knowledge regarding the clinical impact of resistance to polymerase inhibitors and approaches for surveillance and methods for laboratory evaluation of resistance, both in vitro and in animal models. We highlight limitations and gaps in current knowledge and suggest some strategies for addressing these gaps, including the need for additional clinical studies of influenza antiviral drug combinations. Lessons learned from influenza resistance monitoring may also be helpful for establishing future drug susceptibility surveillance and testing for SARS-CoV-2.

The COVID-19 pandemic and subsequent implementation of nonpharmaceutical interventions (e.g., cessation of global travel, mask use, physical distancing, and staying home) reduced transmission of some viral respiratory pathogens (1). In the United States, influenza activity decreased in March 2020, was historically low through the summer of 2020 (2), and remained low during October 2020-May 2021 (<0.4% of respiratory specimens with positive test results for each week of the season). Circulation of other respiratory pathogens, including respiratory syncytial virus (RSV), common human coronaviruses (HCoVs) types OC43, NL63, 229E, and HKU1, and parainfluenza viruses (PIVs) types 1-4 also decreased in early 2020 and did not increase until spring 2021. Human metapneumovirus (HMPV) circulation decreased in March 2020 and remained low through May 2021. Respiratory adenovirus (RAdV) circulated at lower levels throughout 2020 and as of early May 2021. Rhinovirus and enterovirus (RV/EV) circulation decreased in March 2020, remained low until May 2020, and then increased to near prepandemic seasonal levels. Circulation of respiratory viruses could resume at prepandemic levels after COVID-19 mitigation practices become less stringent. Clinicians should be aware of increases in some respiratory virus activity and remain vigilant for off-season increases. In addition to the use of everyday preventive actions, fall influenza vaccination campaigns are an important component of prevention as COVID-19 mitigation measures are relaxed and schools and workplaces resume in-person activities.

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.

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 (<0.01%; 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 IC(50) values in a nanomolar range. Median IC(50) 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. 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.

Baloxavir, a new antiviral drug targeting cap-dependent endonuclease activity of polymerase acidic (PA) protein of influenza viruses, is now approved in multiple countries. Several substitutions at isoleucine 38 in PA protein (e.g., PA-I38T) have been associated with decreased baloxavir susceptibility in vitro and in vivo. In recent years, next generation sequencing (NGS) analysis and pyrosequencing have been used by CDC and U.S. Public Health Laboratories to monitor drug susceptibility of influenza viruses. Here we described an improved pyrosequencing assay for detecting influenza A viruses carrying substitutions at PA-38. Cyclic and customized orders of nucleotide dispensation were evaluated, and pyrosequencing results were compared to those generated using NGS. Our data showed that the customized nucleotide dispensation has improved the pyrosequencing assay performance in identification of double mixtures (e.g., PA-38I/T); however, identification of PA-38 variants in triple mixtures remains a challenge. While NGS analysis indicated the presence of PA-I38K in one clinical specimen and isolate, our attempts to detect this mutation by pyrosequencing or recover the virus carrying PA-I38K in cell culture were unsuccessful, raising a possibility of a rarely occurring sequencing error. Overall, pyrosequencing provides a convenient means to detect baloxavir resistant influenza viruses when NGS is unavailable or a faster turnaround time is required.

The neuraminidase (NA) of influenza A and B viruses plays a distinct role in viral replication and has a highly conserved catalytic site. Numerous sialic (neuraminic) acid analogs that competitively bind to the NA active site and potently inhibit enzyme activity have been synthesized and tested. Four NA inhibitors are now licensed in various parts of the world (zanamivir, oseltamivir, peramivir, and laninamivir) to treat influenza A and B infections. NA changes, naturally occurring or acquired under selective pressure, have been shown to reduce drug binding, thereby affecting the effectiveness of NA inhibitors. Drug resistance and other drawbacks have prompted the search for the next-generation NA-targeting therapeutics. One of the promising approaches is the identification of monoclonal antibodies (mAbs) targeting the conserved NA epitopes. Anti-NA mAbs demonstrate Fab-based antiviral activity supplemented with Fc-mediated immune effector functions. Antiviral Fc-conjugates offer another cutting-edge strategy that is based on a multimodal mechanism of action. These novel antiviral agents are composed of a small-molecule NA inhibitor and an Fc-region that simultaneously engages the immune system. The significant advancements made in recent years further support the value of NA as an attractive target for the antiviral development.

The global analysis of neuraminidase inhibitor (NAI) susceptibility of influenza viruses has been conducted since the 2012-13 period. In 2018 a novel cap-dependent endonuclease inhibitor, baloxavir, that targets polymerase acidic subunit (PA) was approved for the treatment of influenza virus infection in Japan and the United States. For this annual report, the susceptibilities of influenza viruses to NAIs and baloxavir were analyzed. A total of 15409 viruses, collected by World Health Organization (WHO) recognized National Influenza Centers and other laboratories between May 2017 and May 2018, were assessed for phenotypic NAI susceptibility by five WHO Collaborating Centers (CCs). The 50% inhibitory concentration (IC(50)) was determined for oseltamivir, zanamivir, peramivir and laninamivir. Reduced inhibition (RI) or highly reduced inhibition (HRI) by one or more NAIs was exhibited by 0.8% of viruses tested (n = 122). The frequency of viruses with RI or HRI has remained low since this global analysis began (2012-13: 0.6%; 2013-14: 1.9%; 2014-15: 0.5%; 2015-16: 0.8%; 2016-17: 0.2%). PA gene sequence data, available from public databases (n = 13523), were screened for amino acid substitutions associated with reduced susceptibility to baloxavir (PA E23G/K/R, PA A36V, PA A37T, PA I38F/M/T/L, PA E119D, PA E199G): 11 (0.08%) viruses possessed such substitutions. Five of them were included in phenotypic baloxavir susceptibility analysis by two WHO CCs and IC(50) values were determined. The PA variant viruses showed 6-17-fold reduced susceptibility to baloxavir. Overall, in the 2017-18 period the frequency of circulating influenza viruses with reduced susceptibility to NAIs or baloxavir was low, but continued monitoring is important.

Baloxavir showed broad-spectrum in vitro replication inhibition of 4 types of influenza viruses (90% effective concentration range 1.2-98.3 nmol/L); susceptibility pattern was influenza A > B > C > D. This drug also inhibited influenza A viruses of avian and swine origin, including viruses that have pandemic potential and those resistant to neuraminidase inhibitors.

During May 19-September 28, 2019,* low levels of influenza activity were reported in the United States, with cocirculation of influenza A and influenza B viruses. In the Southern Hemisphere seasonal influenza viruses circulated widely, with influenza A(H3) predominating in many regions; however, influenza A(H1N1)pdm09 and influenza B viruses were predominant in some countries. In late September, the World Health Organization (WHO) recommended components for the 2020 Southern Hemisphere influenza vaccine and included an update to the A(H3N2) and B/Victoria-lineage components. Annual influenza vaccination is the best means for preventing influenza illness and its complications, and vaccination before influenza activity increases is optimal. Health care providers should recommend vaccination for all persons aged >/=6 months who do not have contraindications to vaccination (1).

Susceptibility of influenza A viruses to baloxavir can be affected by changes at amino acid residue 38 in polymerase acidic (PA) protein. Information on replicative fitness of PA-I38-substituted viruses remains sparse. We demonstrated that substitutions I38L/M/S/T not only had a differential effect on baloxavir susceptibility (9- to 116-fold), but also on in vitro replicative fitness. While I38L conferred undiminished growth, other substitutions led to mild attenuation. In a ferret model, control viruses outcompeted those carrying I38M or I38T substitutions, although their advantage was limited. These findings offer insights into the attributes of baloxavir resistant viruses needed for informed risk assessment.

Drug resistance is a topic of significant concern in the treatment of infectious diseases caused by rapidly evolving RNA viruses that can persist (eg, human immunodeficiency virus and hepatitis C virus) or reinfect (eg, influenza virus). Combination drug therapy is standard of care for the treatment of infections by rapidly mutating RNA viruses [1, 2]. However, it is not a common approach for treating influenza virus infections, partly because of the limited number of anti-influenza drugs and drug targets. We now know that all of the classes of anti-influenza drugs—M2 blockers, neuraminidase inhibitors (NAIs), and the newly licensed cap-dependent endonuclease inhibitor (baloxavir marboxil)—have low genetic barriers to resistance: 1 or 2 amino acid substitutions are sufficient to gain resistance [3, 4].

Influenza activity* in the United States during the 2018-19 season (September 30, 2018-May 18, 2019) was of moderate severity (1). Nationally, influenza-like illness (ILI)(dagger) activity began increasing in November, peaked during mid-February, and returned to below baseline in mid-April; the season lasted 21 weeks,( section sign) making it the longest season in 10 years. Illness attributed to influenza A viruses predominated, with very little influenza B activity. Two waves of influenza A were notable during this extended season: influenza A(H1N1)pdm09 viruses from October 2018 to mid-February 2019 and influenza A(H3N2) viruses from February through May 2019. Compared with the 2017-18 influenza season, rates of hospitalization this season were lower for adults, but were similar for children. Although influenza activity is currently below surveillance baselines, testing for seasonal influenza viruses and monitoring for novel influenza A virus infections should continue year-round. Receiving a seasonal influenza vaccine each year remains the best way to protect against seasonal influenza and its potentially severe consequences.

Mutations in the influenza virus neuraminidase (NA) that cause reduced susceptibility to the NA inhibitor (NAI) oseltamivir may occur naturally or following antiviral treatment. Currently, detection uses either a traditional NA inhibition assay or gene sequencing to identify known markers associated with reduced inhibition by oseltamivir. Both methods are laborious and require trained personnel. The influenza antiviral resistance test (iART), a prototype system developed by Becton, Dickinson and Company for research use only, offers a rapid and simple method to identify such viruses. This study investigated application of iART to influenza A viruses isolated from non-human hosts with a variety of NA subtypes (N1-N9).

Influenza A(H3N2) viruses evade human immunity primarily by acquiring antigenic changes in the haemagglutinin (HA). HA receptor-binding features of contemporary A(H3N2) viruses hinder traditional antigenic characterization using haemagglutination inhibition and promote selection of HA mutants. Thus, alternative approaches are needed to reliably assess antigenic relatedness between circulating viruses and vaccines. We developed a high content imaging-based neutralization test (HINT) to reduce antigenic mischaracterization resulting from virus adaptation to cell culture. Ferret reference antisera were raised using clinical specimens containing viruses representing recent vaccine strains. Analysis of viruses circulating during 2011-2018 showed that gain of an N158-linked glycosylation in HA was a molecular determinant of antigenic distancing between A/Hong Kong/4801/2014-like (clade 3C.2a) and A/Texas/50/2012-like viruses (clade 3C.1), while multiple evolutionary HA F193S substitution were linked to antigenic distancing from A/Switzerland/97152963/2013-like (clade 3C.3a) and further antigenic distancing from A/Texas/50/2012-like viruses. Additionally, a few viruses carrying HA T135K and/or I192T showed reduced neutralization by A/Hong Kong/4801/2014-like antiserum. Notably, this technique elucidated the antigenic characteristics of clinical specimens, enabling direct characterization of viruses produced in vivo, and eliminating in vitro culture, which rapidly alters the genotype/phenotype. HINT is a valuable new antigenic analysis tool for vaccine strain selection.

CDC collects, compiles, and analyzes data on influenza activity and viruses in the United States. During September 30, 2018-February 2, 2019,* influenza activity(dagger) in the United States was low during October and November, increased in late December, and remained elevated through early February. As of February 2, 2019, this has been a low-severity influenza season (1), with a lower percentage of outpatient visits for influenza-like illness (ILI), lower rates of hospitalization, and fewer deaths attributed to pneumonia and influenza, compared with recent seasons. Influenza-associated hospitalization rates among children are similar to those observed in influenza A(H1N1)pdm09 predominant seasons; 28 influenza-associated pediatric deaths occurring during the 2018-19 season have been reported to CDC. Whereas influenza A(H1N1)pdm09 viruses predominated in most areas of the country, influenza A(H3N2) viruses have predominated in the southeastern United States, and in recent weeks accounted for a growing proportion of influenza viruses detected in several other regions. Small numbers of influenza B viruses (<3% of all influenza-positive tests performed by public health laboratories) also were reported. The majority of the influenza viruses characterized antigenically are similar to the cell culture-propagated reference viruses representing the 2018-19 Northern Hemisphere influenza vaccine viruses. Health care providers should continue to offer and encourage vaccination to all unvaccinated persons aged >/=6 months as long as influenza viruses are circulating. Finally, regardless of vaccination status, it is important that persons with confirmed or suspected influenza who have severe, complicated, or progressive illness; who require hospitalization; or who are at high risk for influenza complications be treated with antiviral medications.

The anti-influenza therapeutic baloxavir targets cap-dependent endonuclease activity of polymerase acidic (PA) protein. We monitored baloxavir susceptibility in the United States with next generation sequencing analysis supplemented by phenotypic one-cycle infection assay. Analysis of PA sequences of 6,891 influenza A and B viruses collected during 2016/17 and 2017/18 seasons showed amino acid substitutions: I38L (two A(H1N1)pdm09 viruses), E23G (two A(H1N1)pdm09 viruses) and I38M (one A(H3N2) virus); conferring 4-10-fold reduced susceptibility to baloxavir.

During May 20-October 13, 2018,* low levels of influenza activity were reported in the United States, with a mix of influenza A and B viruses circulating. Seasonal influenza activity in the Southern Hemisphere was low overall, with influenza A(H1N1)pdm09 predominating in many regions. Antigenic testing of available influenza A and B viruses indicated that no significant antigenic drift in circulating viruses had emerged. In late September, the components for the 2019 Southern Hemisphere influenza vaccine were selected and included an incremental update to the A(H3N2) vaccine virus used in egg-based vaccine manufacturing; no change was recommended for the A(H3N2) component of cell-manufactured or recombinant influenza vaccines. Annual influenza vaccination is the best method for preventing influenza illness and its complications, and all persons aged >/=6 months who do not have contraindications should receive influenza vaccine, preferably before the onset of influenza circulation in their community, which often begins in October and peaks during December-February. Health care providers should offer vaccination by the end of October and should continue to recommend and administer influenza vaccine to previously unvaccinated patients throughout the 2018-19 influenza season (1). In addition, during May 20-October 13, a small number of nonhuman influenza "variant" virus infections(dagger) were reported in the United States; most were associated with exposure to swine. Although limited human-to-human transmission might have occurred in one instance, no ongoing community transmission was identified. Vulnerable populations, especially young children and other persons at high risk for serious influenza complications, should avoid swine barns at agricultural fairs, or close contact with swine.

Influenza virologic surveillance is critical each season for tracking influenza circulation, following trends in antiviral drug resistance, detecting novel influenza infections in humans, and selecting viruses for use in annual seasonal vaccine production. We developed a framework and process map for characterizing the landscape of US influenza virologic surveillance into 5 tiers of influenza testing: outpatient settings (tier 1), inpatient settings and commercial laboratories (tier 2), state public health laboratories (tier 3), National Influenza Reference Center laboratories (tier 4), and Centers for Disease Control and Prevention laboratories (tier 5). During the 2015-16 season, the numbers of influenza tests directly contributing to virologic surveillance were 804,000 in tiers 1 and 2; 78,000 in tier 3; 2,800 in tier 4; and 3,400 in tier 5. With the release of the 2017 US Pandemic Influenza Plan, the proposed framework will support public health officials in modeling, surveillance, and pandemic planning and response.

A total of 13672 viruses, collected by World Health Organization recognised National Influenza Centres between May 2016 and May 2017, were assessed for neuraminidase inhibitor susceptibility by four WHO Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance Epidemiology and Control of Influenza. The 50% inhibitory concentration (IC50) was determined for oseltamivir and zanamivir for all viruses, and for peramivir and laninamivir in a subset (n=8457). Of the viruses tested, 94% were obtained from the Western Pacific, Americas and European WHO regions, while limited viruses were available from the Eastern Mediterranean, African and South East Asian regions. Reduced inhibition (RI) by one or more neuraminidase inhibitor was exhibited by 0.2% of viruses tested (n=32). The frequency of viruses with RI has remained low since this global analysis began (2015/16: 0.8%, 2014/15: 0.5%; 2013/14: 1.9%; 2012/13: 0.6%) but 2016/17 has the lowest frequency observed to date. Analysis of 13581 neuraminidase sequences retrieved from public databases, of which 5243 sequences were from viruses not included in the phenotypic analyses, identified 58 further viruses (29 without phenotypic analyses) with amino acid substitutions associated with RI by at least one neuraminidase inhibitor. Bringing the total proportion to 0.5% (90/18915). This 2016/17 analysis demonstrates that neuraminidase inhibitors remain suitable for treatment and prophylaxis of influenza virus infections, but continued monitoring is important. An expansion of surveillance testing is paramount since several novel influenza antivirals are in late stage clinical trials with some resistance already having been identified.