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Contemporary antiviral development, whether by rational drug design or forward pharmacology, primarily strives to produce ‘lock and key’ inhibitors. While the technology to identify druggable targets and create compounds to bind them has improved dramatically over the last century it has always been constrained by the finite availability of suitable binding sites that antiviral compounds can occupy. Here we present a new approach to drug design that utilises compounds devised to alter the microenvironment of the virion surface making it incompatible with virus entry and illustrate this strategy with inhibitors of influenza virus. We show that compounds that produce a proton-rich mantle above the virion surface induce a conformational change in the viral hemagglutinin (HA) rendering the virus unable to interact with cellular receptors and gain entry to the cell. The compounds show exceptional antiviral activity bothin vitroandin vivoand protect against influenza illness in mice and ferrets after a single dosing, either therapeutically or prophylactically. The work presented here lays the foundation for a brand-new category of inhibitors that could be engineered to counter many different viruses and potentially other pathogens.

We have tested the in vitro susceptibility to the neuraminidase (NA) inhibitors of 96 highly pathogenic clade 2.1 A(H5N1) viruses from Indonesia, isolated between 2008 and 2011. HPAI virus samples obtained through the Influenza Virus Monitoring (IVM) surveillance program in Indonesia were tested for susceptibility to oseltamivir and zanamivir. The NAs of four viruses were identified as extreme outliers to oseltamivir, based on statistical analysis by box plots, with IC50 values ranging from 46 to 62 nM. The NAs of two of these viruses from Sumatra and Aceh, had an N294S substitution, while one virus from Sulawesi had an S246N NA substitution. The NAs of all four viruses showed a specific loss of slow binding to oseltamivir in an IC50 kinetics assay. As observed in our previous surveillance, there was only a minimal effect on the sensitivity to zanamivir or peramivir for these mutants or any of the other isolates tested. The continued circulation of subtype H5N1 viruses in avian species poses an on-going zoonotic threat. The fact that we continue to identify avian isolates with naturally occurring mutations conferring reduced oseltamivir susceptibility remains a concern, given oseltamivir will be a key antiviral in the event of a new pandemic emerging.

The International Society for Influenza and other Respiratory Virus Diseases held its 5th Antiviral Group (isirv-AVG) Conference in Shanghai, China, in conjunction with the Shanghai Public Health Center and Fudan University from 14-16 June 2017. The three-day programme encompassed presentations on some of the clinical features, management, immune responses and virology of respiratory infections, including influenza A(H1N1)pdm09 and A(H7N9) viruses, MERS-CoV, SARS-CoV, adenovirus Type 80, enterovirus D68, metapneumovirus and respiratory syncytial virus (RSV). Updates were presented on several therapeutics currently in clinical trials, including influenza polymerase inhibitors pimodivir/JNJ6362387, S033188, favipiravir, monoclonal antibodies MHAA45449A and VIS410, and host directed strategies for influenza including nitazoxanide, and polymerase ALS-008112 and fusion inhibitors AK0529, GS-5806 for RSV. Updates were also given on the use of the currently licensed neuraminidase inhibitors. Given the location in China, there were also presentations on the use of Traditional Chinese Medicines. Following on from the previous conference, there were ongoing discussions on appropriate endpoints for severe influenza in clinical trials from regulators and clinicians, an issue which remains unresolved. The aim of this conference summary is to provide information for not only conference participants, but a detailed referenced review of the current status of clinical trials, and pre-clinical development of therapeutics and vaccines for influenza and other respiratory diseases for a broader audience., Highlights • Details of presentations given at the recent isirv-AVG conference held in Shanghai, June 2017 are summarized. • Topics covered clinical features, management, immune responses and virology of respiratory infections. • Viruses discussed included influenza, RSV, SARS, MERS-CoV, EV-D68, adenovirus Type 80 and metapneumovirus. • Influenza vaccines, polymerase inhibitors, monoclonal antibodies and cell targeted therapies were discussed. • RSV vaccines, polymerase and fusion inhibitors were discussed.

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.

An influenza A(H1N1)pdm09 and an influenza B virus were passaged in 3-fluoro(eq)-4-guanidino difluoro sialic acid (3Feq4Gu DFSA), an inhibitor of the influenza neuraminidase (NA) to determine whether resistant variants could be selected. 3Feq4Gu DFSA is a mechanism-based inhibitor, forming a covalent link to Y406 in the NA active site. Given its similarity to the natural substrate, sialic acid, we predicted resistant variants would be difficult to select. Yields of both viruses decreased with passaging, so that after 12 passages both viruses were only growing to low titers. Drug concentrations were decreased for another three passages. There was no difference in NA sensitivity in the MUNANA fluorescence-based assay, nor in plaque assays for the passaged virus stocks. All influenza B plaques were still wild type in all assays. There were isolated small diffuse plaques in the P15 pdm09 stock, which after purification had barely detectable NA or hemagglutinin (HA) activity. These had a novel non-active site I106M substitution in the NA gene, but unexpectedly no HA changes. The I106M may impact NA function through steric effects on the movement of the 150 and 430-loops. The I106M viruses had similar replication kinetics in MDCK cells as wild type viruses, but their ability to bind to and infect CHO-K1 cells expressing high levels of cell-bound mucin was compromised. The I106M substitution was unstable, with progeny rapidly reverting to wild type by three different mechanisms. Some had reverted to I106, some had V106, both with wild type NA and HA properties. A third group retained the I106M, but had a compensating R363K substitution, which regained almost wild type NA properties. These viruses now agglutinated chicken red blood cells (CRBCs) but unlike the I/V106, they rebound after elution at 37 °C. There were no mutations in the HA, but each phenotype correlated with the NA sequence. We propose that the activity in the I106M mutant is insufficient to remove carbohydrates from the virion HA and NA, sterically limiting HA access to CRBC receptors, thus resulting in poor HA binding.

With the introduction of the influenza specific neuraminidase inhibitors (NAIs) in 1999, there were concerns about the emergence and spread of resistant viruses in the community setting. Surveillance and testing of community isolates for their susceptibility to the NAIs was initially carried out by the Neuraminidase Inhibitor Susceptibility Network (NISN) and has subsequently been taken on by the global WHO influenza network laboratories. During the NISN surveillance, we identified two Yamagata lineage influenza B viruses with amino acid substitutions of H134Y (B/Auckland/2/2001) or W438R (B/Yokohama/12/2005) which had slightly elevated IC50 values for zanamivir and/or oseltamivir, but not sufficiently to be characterized as mild outliers at the time. As it has now been well demonstrated that mixed populations can mask the true magnitude of resistance of a mutant, we re-examined both of these isolates by plaque purification to see if the true susceptibilities were being masked due to mixed populations. Results confirmed that the B/Auckland isolate contained both wild type and H134Y mutant populations, with mutant IC50 values > 250 nM for both oseltamivir and peramivir in the enzyme inhibition assay. The B/Yokohama isolate also contained both wild type and W438R mutant populations, the latter now demonstrating IC50 values > 400 nM for zanamivir, oseltamivir and peramivir. In addition, plaque purification of the B/Yokohama isolate identified viruses with other single neuraminidase substitutions H134Y, H134R, H431R, or T436P. H134R and H431R viruses had IC50 values > 400 nM and >250 nM respectively against all three NAIs. All changes conferred much greater resistance to peramivir than to zanamivir, and less to oseltamivir, and affected the kinetics of binding and dissociation of the NAIs. Most affected affinity (Km) for the MUNANA substrate, but some had decreased while others had increased affinity. Despite resistance in the enzyme assay, no reduced susceptibility was seen in plaque reduction assays in MDCK cells for any of the mutant viruses. None of these substitutions was in the active site. Modelling suggests that these substitutions affect the 150 and 430-loop regions described for influenza A NAs, suggesting they may also be important for substrate and inhibitor binding for influenza B NAs.

The influenza neuraminidase (NA) is a homotetramer with head, stalk, transmembrane and cytoplasmic regions. The structure of the NA head with a stalk has never been determined. The NA head from an N9 subtype influenza A virus, A/tern/Australia/G70C/1975 (H1N9), was expressed with an artificial stalk derived from the tetrabrachion (TB) tetramerization domain fromStaphylothermus marinus. The NA was successfully crystallized both with and without the TB stalk, and the structures were determined to 2.6 and 2.3 Å resolution, respectively. Comparisons of the two NAs with the native N9 NA structure from egg-grown virus showed that the artificial TB stalk maintained the native NA head structure, supporting previous biological observations.

With the constant threat of emergence of a novel influenza virus pandemic, there must be continued evaluation of the molecular mechanisms that contribute to virulence. Although the influenza A virus surface glycoprotein neuraminidase (NA) has been studied mainly in the context of its role in viral release from cells, accumulating evidence suggests it plays an important, multifunctional role in virus infection and fitness. This review investigates the various structural features of NA, linking these with functional outcomes in viral replication. The contribution of evolving NA activity to viral attachment, entry and release of virions from infected cells, and maintenance of functional balance with the viral hemagglutinin are also discussed. Greater insight into the role of this important antiviral drug target is warranted.

The International Society for Influenza and other Respiratory Virus Diseases (isirv) held its 4th Antiviral Group Conference at the University of Texas on 2–4 June, 2015. With emerging resistance to the drugs currently licensed for treatment and prophylaxis of influenza viruses, primarily the neuraminidase inhibitor oseltamivir phosphate (Tamiflu) and the M2 inhibitors amantadine and rimantadine, and the lack of effective interventions against other respiratory viruses, the 3-day programme focused on the discovery and development of inhibitors of several virus targets and key host cell factors involved in virus replication or mediating the inflammatory response. Virus targets included the influenza haemagglutinin, neuraminidase and M2 proteins, and both the respiratory syncytial virus and influenza polymerases and nucleoproteins. Therapies for rhinoviruses and MERS and SARS coronaviruses were also discussed. With the emerging development of monoclonal antibodies as therapeutics, the potential implications of antibody-dependent enhancement of disease were also addressed. Topics covered all aspects from structural and molecular biology to preclinical and clinical studies. The importance of suitable clinical trial endpoints and regulatory issues were also discussed from the perspectives of both industry and government. This meeting summary provides an overview, not only for the conference participants, but also for those interested in the current status of antivirals for respiratory viruses., Highlights • The International Society for Influenza and other Respiratory Viruses held an Antiviral Group conference in June, 2015. • This report covers oral presentations, including therapies against influenza and respiratory syncytial virus infections. • Therapies for rhinovirus, MERS and SARS coronavirus infections were also topics at the conference. • Some speakers focused on monoclonal antibodies as therapeutics and antibody-dependent enhancement of disease. • The importance of suitable clinical trial endpoints and regulatory issues were also discussed.

Competitive inhibitors of the influenza neuraminidase (NA) were discovered almost 20 years ago, with zanamivir and oseltamivir licensed globally. These compounds are based on a transition state analogue of the sialic acid substrate. We recently showed that 5-N-(acetylamino)-2,3,5-trideoxy-2,3-difluoro-d-erythro-β-l-manno-2-nonulopyranosonic acid (DFSA) and its derivatives are also potent inhibitors of the influenza NA. They are mechanism based inhibitors, forming a covalent bond between the C2 of the sugar ring and Y406 in the NA active site, thus inactivating the enzyme. We have now synthesized a series of deoxygenated DFSA derivatives in order to understand the contribution of each hydroxyl in DFSA to binding and inhibition of the influenza NA. We have investigated their relative efficacy in enzyme assays in vitro, in cell culture, and by X-ray crystallography. We found loss of the 8- and 9-OH had the biggest impact on the affinity of binding and antiviral potency.

The neuraminidase (NA) inhibitors oseltamivir and zanamivir are administered twice daily for 5 days for treatment of influenza. Laninamivir is a 7-methoxy derivative of zanamivir, but a single dose is effective when taken as the laninamivir octanoate prodrug. We show here in IC 50 kinetics assays and a solid phase reactivation assay that compared to zanamivir laninamivir also demonstrates slow binding to but slower dissociation from multiple wild type NAs. A D197E mutation in an influenza B and an E119G in an N9 neuraminidase which confer 15- and 150-fold resistance to laninamivir result in faster binding and dissociation. Despite similar IC 50 s our assays demonstrate more rapid dissociation of laninamivir from clade 1 compared to 2 H5N1 NAs.

Objectives: We characterized human H1N1 influenza isolate A/Hokkaido/15/02, which has haemagglutinin and neuraminidase mutations that reduce drug susceptibility to oseltamivir, zanamivir and peramivir. Methods: One wild-type and three mutant viruses were isolated by plaque purification. Viruses were tested in MUNANA-based enzyme assays, cell culture and receptor binding assays. Results: Two viruses had a neuraminidase Y155H mutation that reduced susceptibility in the enzyme inhibition assay to all inhibitors by 30-fold to .100-fold. The Y155H mutation reduced plaque size and affected the stability, Km and pH activity profile of the enzyme. In contrast to previous mutants, this neuraminidase demonstrated a slower rate of inhibitor binding in the IC50 kinetics assay. One virus had both the Y155H mutation and a haemagglutinin D225G mutation that rescued the small-plaque phenotype of the Y155H virus and affected receptor binding and drug susceptibility in cell culture and binding assays. We also isolated a third mutant virus, with both neuraminidase V114I and haemagglutinin D225N mutations, which affected susceptibility in the enzyme inhibition assay and receptor binding, respectively, but to lesser extents than the Y155H and D225G mutations. Conclusions: Neither Y155 nor V114 is conserved across neuraminidase subtypes. Furthermore, Y155 is not conserved even among avian and swine N1 viruses. Structurally, both residues reside far from the neuraminidase active site. D225 forms part of the receptor binding site of the haemagglutinin. We believe this is the first demonstration of a specific haemagglutinin mutation correlating with reduced drug susceptibility in plaque assays in both Madin Darby Canine Kidney and SIAT cells.

There are two major classes of antivirals available for the treatment and prevention of influenza, the M2 inhibitors and the neuraminidase inhibitors (NAIs). The M2 inhibitors are cheap, but they are only effective against influenza A viruses, and resistance arises rapidly. The current influenza A H3N2 and pandemic A(H1N1)pdm09 viruses are already resistant to the M2 inhibitors as are many H5N1 viruses. There are four NAIs licensed in some parts of the world, zanamivir, oseltamivir, peramivir, and a long-acting NAI, laninamivir. This review focuses on resistance to the NAIs. Because of differences in their chemistry and subtle differences in NA structures, resistance can be both NAI- and subtype specific. This results in different drug resistance profiles, for example, the H274Y mutation confers resistance to oseltamivir and peramivir, but not to zanamivir, and only in N1 NAs. Mutations at E119, D198, I222, R292, and N294 can also reduce NAI sensitivity. In the winter of 2007-2008, an oseltamivir-resistant seasonal influenza A(H1N1) strain with an H274Y mutation emerged in the northern hemisphere and spread rapidly around the world. In contrast to earlier evidence of such resistant viruses being unfit, this mutant virus remained fully transmissible and pathogenic and became the major seasonal A(H1N1) virus globally within a year. This resistant A(H1N1) virus was displaced by the sensitive A(H1N1)pdm09 virus. Approximately 0.5-1.0% of community A(H1N1)pdm09 isolates are currently resistant to oseltamivir. It is now apparent that variation in non-active site amino acids can affect the fitness of the enzyme and compensate for mutations that confer high-level oseltamivir resistance resulting in minimal impact on enzyme function.

Objectives: Pandemic H1N1/09 viruses with the neuraminidase H274Y mutation have emerged in untreated patients or following oseltamivir therapy or prophylaxis. There have been no reports yet of zanamivir-resistant H1N1/09 viruses in previously healthy patients. We wanted to determine whether we could select for neuraminidase mutations conferring high-level resistance to zanamivir by in vitro passage of the virus. We also wanted to investigate if passaging in a combination of zanamivir and oseltamivir could prevent the emergence of the H274Y mutation. Methods: An H1N1/09 virus was passaged in cell culture in increasing concentrations of either zanamivir or a combination of zanamivir and oseltamivir. Results: Passage in zanamivir selected a virus with N146S neuraminidase and G158E haemagglutinin mutations. The neuraminidase mutation only reduced drug susceptibility by 2-fold in enzyme assays. The haemagglutinin mutation conferred drug dependence and drug resistance in cells to oseltamivir and zanamivir and reduced binding to red blood cells. After four passages in zanamivir and oseltamivir, virus with a D198G neuraminidase mutation was selected with around 10-fold reduced susceptibility to oseltamivir, zanamivir and peramivir in the enzyme assay. Further passaging selected a virus with both D198G and H274Y mutations that was highly resistant to oseltamivir and peramivir, but not zanamivir. All mutations affected growth in cell culture and decreased affinities of the neuraminidases for substrate. Conclusions: We did not select a virus with a neuraminidase mutation conferring high-level resistance to zanamivir. Dual exposure to zanamivir and oseltamivir was not sufficient to prevent selection of the H274Y mutation.

Human parainfluenza virus type 1 (hPIV1) generally does not show visible plaques in common cell lines, including Lewis lung carcinoma-monkey kidney (LLC-MK(2)) cells, by plaque formation assays for human parainfluenza virus type 3 (hPIV3) and Sendai virus. In several conditions of the plaque formation assay, complete elimination of serum proteins in the overlay medium was necessary for visualization of hPIV1-induced plaque formation in LLC-MK(2) cells. We developed a plaque formation assay for hPIV1 isolation and titration in LLC-MK(2) cells using an initial overlay medium of bovine serum albumin-free Eagle's minimum essential medium containing agarose and acetylated trypsin for 4-6 d followed by a second overlay staining medium containing agarose and neutral red. The assay allowed both laboratory and clinical hPIV1 strains to form large plaques. The plaque reduction assay was also performed with rabbit anti-hPIV1 antibody as a general evaluation model of viral inhibitors to decrease both the plaque number and size. The results indicate that the plaque formation assay is useful for hPIV1 isolation, titration, evaluation of antiviral reagents and epidemiologic research.

We have identified a virus, B/Perth/211/2001, with a spontaneous mutation, D197E in the neuraminidase (NA), which confers cross-resistance to all NA inhibitors. We analyzed enzyme properties of the D197 and E197 NAs and compared these to a D197N NA, known to arise after oseltamivir treatment. Zanamivir and peramivir bound slowly to the wild type NA, but binding of oseltamivir was more rapid. The D197E/N mutations resulted in faster binding of all three inhibitors. Analysis of the crystal structures of D197 and E197 NAs with and without inhibitors showed that the D197E mutation compromised the interaction of neighboring R150 with the N-acetyl group, common to the substrate sialic acid and all NA inhibitors. Although rotation of the E275 in the NA active site occurs upon binding peramivir in both the D197 and E197 NAs, this does not occur upon binding oseltamivir in the E197 NA. Lack of the E275 rotation would also account for the loss of slow binding and the partial resistance of influenza B wild type NAs to oseltamivir.

With the global spread of the pandemic H1N1 and the ongoing pandemic potential of the H5N1 subtype, the influenza virus represents one of the most alarming viruses spreading worldwide. The influenza virus sialidase is an effective drug target, and a number of inhibitors are clinically effective against the virus (zanamivir, oseltamivir, peramivir). Here we report structural and biochemical studies of the human cytosolic sialidase Neu2 with influenza virus sialidase-targeting drugs and related compounds.

The human parainfluenza virus type 3 (hPIV3) hemagglutinin-neuraminidase (HN) has opposing functions of binding sialic acid receptors and cleaving them, facilitating virus release. The crystal structure of hPIV3 HN complexed with the substrate analogue difluorosialic acid (DFSA) revealed that catalysis by HN involves the formation of a covalently linked sialosyl-enzyme intermediate which was trapped along with a transition-state analogue resembling an oxocarbenium ion. This mechanism of enzyme catalysis was also confirmed in the crystal structure of the influenza N9 neuraminidase complexed with DFSA. Additionally, novel secondary receptor binding sites were identified in the hPIV3 HN-DFSA complex including one near the catalytic cavity which upon binding DFSA imposes subtle changes and may help the HN balance the opposing functions. Multiple receptor binding sites may increase avidity to facilitate cell binding and fusion promotion. The secondary receptor binding sites in the paramyxoviruses are so far unique to each virus type.

The X-ray structure of influenza virus neuraminidase (NA) isolated from whale, subtype N9, has been determined at 2.2 A resolution and contains a tetrameric protein in the asymmetric unit. In structures of NA determined previously, a calcium ion is observed to coordinate amino acids near the substrate-binding site. In three of the NA monomers determined here this calcium is absent, resulting in structural alterations near the substrate-binding site. These changes affect the conformation of residues that participate in several key interactions between the enzyme and substrate and provide at a molecular level the basis of the structural and functional role of calcium in substrate and inhibitor binding. Several sulfate ions were identified in complex with the protein. These are located in the active site, occupying the space reserved for the substrate (sialic acid) carboxylate, and in positions leading away from the substrate-binding site. These sites offer a new opportunity for the design of inhibitors of influenza virus NA.

Background: Viruses resistant to zanamivir have been generated in vitro, but no resistant virus has yet been isolated from a zanamivir-treated immunocompetent patient. In contrast most resistant viruses isolated from oseltamivir-treated patients correspond to those selected in vitro. However, despite mutations being in conserved residues in the neuraminidase (NA) they do not confer resistance in all NA subtypes. Objectives and methods: We have used reverse genetics and the recombinant baculovirus expression system for investigating reasons for the lack of isolation of zanamivir-resistant H3N2 viruses and for further exploring subtype-specific oseltamivir resistance. Results: H3N2 viruses generated by reverse genetics with H274Y, R292K E119V and E119D mutations were rescued. Those with E119G, E119A or R152K mutations could only be rescued in the presence of exogenous NA and after passage in the absence of exogenous NA only isolates that had reverted to the wild-type NA or, surprisingly, E119G/A to E119V NA were isolated. Mutations conferring zanamivir resistance significantly affected enzyme activity, virus replication or NA thermal stability. E119V viruses were stable and grew to similar titres as wild-type virus, consistent with their isolation from oseltamivirtreated patients. Mutations conferring oseltamivir resistance in N1 (H274Y) and B (R152K) NAs also conferred resistance in recombinant G70C N9 NA expressed in insect cells. Conclusions: These data suggest that zanamivir-resistant H3N2 viruses may not readily arise in vivo due to their poor viability. The G70C N9 NA may also provide a useful model for understanding the structural basis of subtype-specific drug resistance.

Due to their generally small size, detection of respiratory syncytial virus (RSV) plaques commonly relies on immunostaining using either polyclonal antisera or a pool of monoclonal antibodies against the surface fusion glycoprotein. Disadvantages include the costs of the antibodies and substrate, and microscopic examination is often needed for counting of plaques. By optimizing many parameters, greatly improved plaque assays for RSV A2, Long and RSV B 18537 strains using Vero or HEp-2 cells have been developed. Although many groups use Vero cells for plaquing, after optimization plaques were much larger in HEp-2 cells. Both cell types needed to be confluent at infection and agarose was more suitable than carboxymethyl cellulose for the overlay. Overlay medium for HEp-2 was DMEM/F12, 0.3% agarose, and for Veros it was Liebovitz L15, 5% fetal calf serum, 0.3-0.5% agarose. After 5-7 days, monolayers were fixed with 1% formal saline overnight, agarose was removed and monolayers were stained with 0.05% neutral red in water. Plaques were readily visible by eye and plates could be dried and stored permanently. Parainfluenza virus type 3 also formed large plaques in HEp-2 cells under the conditions optimal for RSV.

A total of 126 influenza B isolates isolated between 1998 and 2002 from Australasia and the Asia-Pacific region were tested for their sensitivity to the neuraminidase (NA) inhibitor drugs zanamivir and oseltamivir carboxylate using a fluorescence-based enzyme assay. The mean (+/-1 S.D.) 50% inhibitory concentration (IC50) of the influenza B viruses tested was 1.41+/-0.53 nM against zanamivir and 14.91+/-14.31 nM with oseltamivir carboxylate. However, a single type B isolate (B/Perth/211/2001) from an infant who had not been treated with either of the NA inhibitor drugs, showed a nine-fold lower sensitivity to zanamivir and a 14-fold lower sensitivity to oseltamivir carboxylate compared with the mean IC50 of influenza B strains. A decrease in sensitivity to oseltamivir carboxylate and RWJ-270201 was also seen in both: a chemiluminescent assay and a second different fluorescent assay. Sequence analysis of the haemagglutinin HA1 region and the neuraminidase gene of B/Perth/211/2001 revealed no amino acid changes in sites that have previously been reported to confer resistance to either of the NAI drugs. Further investigations are in progress to identify the basis for this reduced sensitivity.

The three-dimensional structure of the haemagglutinin-neuraminidase (HN) from a human parainfluenza virus is described at ca 2.0 A resolution, both in native form and in complex with three substrate analogues. In support of earlier work on the structure of the homologous protein from the avian pathogen Newcastle disease virus (NDV), we observe a dimer of β-propellers and find no evidence for spatially separated sites performing the receptor-binding and neuraminidase functions of the protein. As with the NDV HN, the active site of the HN of parainfluenza viruses is structurally flexible, suggesting that it may be able to switch between a receptor-binding state and a catalytic state. However, in contrast to the NDV structures, we observe no ligand-induced structural changes that extend beyond the active site and modify the dimer interface.

The increasing use of influenza virus neuraminidase (NA) inhibitors (NIs) necessitates the development of reliable methods for assessing the NI susceptibility of clinical isolates. We evaluated three NA inhibition assays against a panel of five clinical isolates each of influenza virus A/H1N1, A/H3N2, and B strains and four viruses with a defined resistance genotype (R292K, H274Y, R152K, and E119V). For fluorometric enzyme assay (FA) 1 (FA-1), 2′-(4-methylumbelliferyl)-α- d - N -acetylneuraminic acid (MUNANA) at 100 μM was used as the substrate, with pretitration of the virus input. For FA-2, MUNANA at 200 μM was used as the substrate, with a fixed 1:10 dilution of input virus. For the chemiluminescence (CL) assay, the 1,2-dioxetane derivative of sialic acid at 100 μM was used as the substrate, with pretitration of the virus. Four different operators repeated the assays several times in a blinded fashion with both zanamivir and oseltamivir carboxylate (GS4071) to determine intra- and interassay variations. Mean 50% inhibitory concentration (IC 50 ) values were lower and generally less variable with the CL assay. FA-1 displayed greater variation than the CL assay or FA-2 and the highest IC 50 values with zanamivir; FA-2 showed the highest values with oseltamivir, particularly for influenza virus B, and was more variable with zanamivir than was the CL assay. All three assays detected 40-fold or greater changes in IC 50 values for the resistant viruses with at least one drug. Mixing experiments, whereby increasing fractions (0, 20, 40, 60, 80, and 100%) of NA from a known NI-resistant virus were mixed with the corresponding NI-sensitive parental NA, indicated that the resolution of IC 50 values was clearer with the CL assay than with FA-2 for two of the resistant variants (R152K and E119V). The FA and CL methods were reliable for the detection of NI resistance, but all assays have certain limitations. Based on reproducibility, ease of automation, time required for the assay, and greater sensitivity, the CL assay was selected for future susceptibility testing of influenza virus isolates circulating globally.

The impact of influenza virus infection is estimated to run into billions of dollars worldwide. Vaccination plays a key role in prevention; however, vaccines do not provide complete protection against influenza due to the constant mutation of the virus responsible. Unlike amantadine and rimantadine, which are only effective against influenza A, the new neuraminidase inhibitors zanamivir (Relenza), GlaxoSmithKline) and oseltamivir (Tamiflu), Gilead/Roche) are potent and specific inhibitors of influenza types A and B and have minimal side effects. The greatest benefit is derived if treatment commences as soon as possible after symptoms develop. In order for these inhibitors to have a significant impact on the disease, clinicians and the general public need to be made more aware of the symptoms of influenza and the availability of these new drugs.

Previously, three influenza A(H3N2) isolates with a reduced susceptibility to the neuraminidase inhibitors (NAIs) zanamivir and oseltamivir were identified during screening by the Neuraminidase Inhibitor Susceptibility Network (NISN). The isolates were from untreated patients from the first three years post licensure of the NAIs. We plaque-purified progeny from each of these isolates and determined the NAI sensitivity of each plaqued population. Sequencing and serology for each population revealed that the isolates contained a mix of wild type influenza A(H3N2) and influenza B. The NAI susceptibility reductions that had originally been reported were a consequence of influenza B neuraminidases that have lower relative NAI sensitivities, rather than being due to resistant influenza A(H3N2) viruses. Our study highlights the need to check for mixed influenza infections when isolates with potentially lower sensitivities to NAIs are identified.

Structure-based inhibitor design has resulted in the development of highly selective inhibitors of the influenza virus neuraminidase (NA). So far, two compounds have been approved for therapeutic use, zanamivir and oseltamivir, but others are under development. However, it is important to establish whether resistance arises readily to these new inhibitors. Unlike amantadine, it takes several passages in culture before resistant variants are isolated. Mutants have also been isolated from oseltamivir-treated patients and from an immunocompromised zanamivir-treated child. Mutations are found in both the NA and the hemagglutinin (HA). In vitro two HA or an HA and NA mutation can act synergistically to increase resistance. The NA mutations are in previously conserved catalytic and structural residues, Glu 119, Arg 152, Arg 292, and His 274. The NA mutations have an adverse effect on NA activity or stability. There are significant quantitative differences observed between the inhibitors upon binding to these mutant NAs. The HA mutations tend to map to regions associated with receptor binding of the HA. The effect of the HA mutations appears to be to reduce the affinity of the HA for the cellular receptor. Their role in vivo is not yet known.

Influenza virus is a negative stranded RNA virus. It contains two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA exists as a trimer and is responsible for binding to the terminal sialic acid bound to receptors on the surface of the target cell, leading to attachment and subsequent penetration by the virus into the cell. Influenza virus isolates from different animals appear to have a preference for specific receptor linkages. Equine and avian isolates bind preferentially to the a2,3 galactose structure, while human isolates bind preferentially to the a2,6 galactose structure (Leigh et al., 1995). A single amino acid mutation is sufficient to change receptor specificity (Rogers and Paulson, 1983; Nobusawa and Nakajima, 1988; Martin et al., 1998). Sequence analysis and alignment has identified key residues conserved across all HA subtypes, which are involved in receptor binding (Nobusawa et al., 1991). The sialic acid binding site forms a groove across the top of the HA surrounded by antibody binding sites (Weis et al., 1988). Residues 134–138 form the right side of the ligand binding site, and residues 224–228 form the left side. Other conserved residues appear to play a role in orienting several of the surface atoms for binding to the sialic acid, these include Tyr 98, Trp 153, His 183, Glu 190, Leu 194 and Tyr at 195 (Weis et al., 1988; Nobusawa et al., 1991). After replication of the virus, progeny virions bud from the cell surface. NA is thought to be responsible for cleavage of terminal sialic acid moieties from receptors, to facilitate elution of progeny virions from the infected cell. Since they are also glycosylated, newly synthesized HA and NA on virions may also contain sialic acid residues on their oligosaccharide chains. Removal of these terminal sugars is therefore also necessary to prevent self-aggregation, due to the HA of one virion binding to the sialic acids on an adjacent * Fax: +61-3-96627101. E-mail address: jennyb@bioresi.com.au (J.L. McKimmBreschkin).

We have previously reported the isolation and characterization of an influenza virus variant with decreased sensitivity to the neuraminidase-specific inhibitor zanamivir. This variant, which has a mutation in the active site, Glu 119 Gly (E119G), has the same specific activity as the wild-type neuraminidase (NA), but is inherently unstable, as measured by loss of both enzyme activity and NC10 monoclonal antibody reactivity. However, despite the instability of the NA, replication of the virus in liquid culture is not adversely affected. We demonstrate here that in addition to enhanced temperature sensitivity the mutant NA was significantly more sensitive to formaldehyde and to specimen preparation for electron microscopy. Substrate, inhibitor, or monoclonal antibodies stabilized the NA against all methods of denaturation. These results suggest that the instability of the variant is primarily at the level of polypeptide chain folding rather than at the level of association of monomers into tetramers. Furthermore the presence of high levels of substrate, either cell or virus associated, may be sufficient to stabilize the NA during virus replication.

Background: Inhibitors of the influenza virus neuraminidase have been shown to be effective antiviral agents in humans. Several studies have reported the selection of novel influenza strains when the virus is cultured with neuraminidase inhibitors in vitro . These resistant viruses have mutations either in the neuraminidase or in the viral haemagglutinin. Inhibitors in which the glycerol sidechain at position 6 of 2-deoxy-2,3-dehydro- N -acetylneuraminic acid (Neu5Ac2en) has been replaced by carboxamide-linked hydrophobic substituents have recently been reported and shown to select neuraminidase variants. This study seeks to clarify the structural and functional consequences of replacing the glycerol sidechain of the inhibitor with other chemical constituents. Results: The neuraminidase variant Arg292→Lys is modified in one of three arginine residues that encircle the carboxylate group of the substrate. The structure of this variant in complex with the carboxamide inhibitor used for its selection, and with other Neu5Ac2en analogues, is reported here at high resolution. The structural consequences of the mutation correlate with altered inhibitory activity of the compounds compared with wild-type neuraminidase. Conclusions: The Arg292→Lys variant of influenza neuraminidase affects the binding of substrate by modification of the interaction with the substrate carboxylate. This may be one of the structural correlates of the reduced enzyme activity of the variant. Inhibitors that have replacements for the glycerol at position 6 are further affected in the Arg292→Lys variant because of structural changes in the binding site that apparently raise the energy barrier for the conformational change in the enzyme required to accommodate such inhibitors. These results provide evidence that a general strategy for drug design when the target has a high mutation frequency is to design the inhibitor to be as closely related as possible to the natural ligands of the target.

An influenza B virus from an infant with no history of treatment or contact with neuraminidase inhibitors demonstrated a significant reduction in sensitivity to these drugs. Here, we describe the analysis of a mixed viral population that contained a novel D197E amino acid substitution that was responsible for this reduction.

Adding to the Antiviral Arsenal The envelope of influenza virus contains two immunodominant glycoproteins: hemagglutinin and neuraminidase (NA). Existing antivirals like zanamivir (Relenza) and oseltamivir (Tamiflu) target NA; however, the development of drug resistance is a problem. Kim et al. (p. 71 , published online 21 February) now report a different class of NA inhibitors. NA catalyzes the removal of sialic acids from the surface of host cells to initiate entry. Discovery of a NA–sialic acid intermediate led to the production of sialic acid analogs that bound covalently to NA and inhibited its enzymatic activity. These compounds showed activity against a wide variety of influenza strains, inhibited viral replication in cell culture, and were able to protect mice against influenza infection. Protection of mice was equivalent to protection seen from zanamivir. Moreover, the compounds showed activity against drug-resistant strains in vitro. These compounds represent a potentially useful addition to the arsenal of antivirals used to treat influenza infection.

In 1988 the preceding journal of Nature Biotechnology, Bio/Technology, reported a work by Hopp and co-workers about a new tag system for the identification and purification of recombinant proteins: the FLAG-tag. Beside the extensively used hexa-his tag system the FLAG-tag has gained broad popularity due to its small size, its high solubility, the presence of an internal Enterokinase cleavage site, and the commercial availability of high-affinity anti-FLAG antibodies. Surprisingly, considering the heavy use of FLAG in numerous laboratories world-wide, we identified in insect cells a post-translational modification (PTM) that abolishes the FLAG-anti-FLAG interaction rendering this tag system ineffectual for secreted proteins. The present publication shows that the tyrosine that is part of the crucial FLAG epitope DYK is highly susceptible to sulfation, a PTM catalysed by the enzyme family of Tyrosylprotein-Sulfo-transferases (TPSTs). We showed that this modification can result in less than 20% of secreted FLAG-tagged protein being accessible for purification questioning the universal applicability of this established tag system.

Background While it is well known that different antibodies can be produced against a particular antigen, and even against a particular site on an antigen, up until now there have been no structural studies of cross-reacting antibodies of this type. One antibody– antigen complex whose structure is known is that of the influenza virus antigen, neuraminidase, in complex with the NC41 antibody. Another anti-neuraminidase antibody, NC10, binds to an overlapping site on the antigen. The structure of the complex formed by this antibody with neuraminidase is described here and compared with the NC41-containing complex. Results The crystal structure of the NC10 Fab– neuraminidase complex has been refined to a nominal resolution of 2.5a. Approximately 80% of the binding site of the NC10 antibody on neuraminidase overlaps with that of the NC41 antibody. The epitope residues of neuraminidase are often engaged in quite different interactions with the two antibodies. Although the NC10 and NC41 antibodies have identical amino acid sequences within the first complementarity determining region of their heavy chains, this is not the basis of the cross-reaction. Conclusions The capacity of two different proteins to bind to the same target structure on a third protein need not be based on the existence of identical or homologous amino acid sequences within those proteins. As we have demonstrated, amino acid residues on the common target structure may be in quite different chemical environments, and may also adopt different conformations within two protein– protein complexes.

The influenza neuraminidase (NA) inhibitors zanamivir, oseltamivir and peramivir were all designed based on the knowledge that the transition state analogue of the cleaved sialic acid, 2-deoxy,2,3-dehydro N-acetyl neuraminic acid (DANA) was a weak inhibitor of NA. While DANA bound rapidly to the NA, modifications leading to the improved potency of these new inhibitors also conferred a time dependent or slow binding phenotype. Many mutations in the NA leading to decreased susceptibility result in loss of slow binding, hence this is a phenotypic marker of many but not all resistant NAs. We present here a simplified approach to determine whether an inhibitor is fast or slow binding by extending the endpoint fluorescent enzyme inhibition assay to a real time assay and monitoring the changes in IC(50)s with time. We carried out two reactions, one with a 30 min preincubation with inhibitor and the second without. The enzymatic reaction was started via addition of substrate and IC(50)s were calculated after each 10 min interval up to 60 min. Results showed that without preincubation IC(50)s for the wild type viruses started high and although they decreased continuously over the 60 min reaction time the final IC(50)s remained higher than for pre-incubated samples. These results indicate a slow equilibrium of association and dissociation and are consistent with slow binding of the inhibitors. In contrast, for viruses with decreased susceptibility, preincubation had minimal effect on the IC(50)s, consistent with fast binding. Therefore this modified assay provides additional phenotypic information about the rate of inhibitor binding in addition to the IC(50), and critically demonstrates the differential effect of incubation times on the IC(50) and K(i) values of wild type and mutant viruses for each of the inhibitors.

Crystallographic studies of neuraminidase-sialic acid complexes indicate that sialic acid is distorted on binding the enzyme. Three arginine residues on the enzyme interact with the carboxylate group of the sugar which is observed to be equatorial to the saccharide ring as a consequence of its distorted geometry. The glycosidic oxygen is positioned within hydrogen-bonding distance of Asp-151, implicating this residue in catalysis.

To avoid extensive manipulation for the purification of RNA from cells, several methods were evaluated for the direct release of RNA from influenza virus infected cells and supernatants using slot blot hybridization and non-radioactive probes. Treatment with an equal volume of 10 M aqueous guanidine hydrochloride produced the best hybridization signal. Less, but significant amounts of RNA were also released using the following treatments: dilute alkali (final concentration of 0.16 M NaOH) or 100 degrees C/5 min or RNA sample buffer containing formamide/formaldehyde, then heating at 65 degrees C/10 min. Despite the presence of large amounts of cell debris, RNA from guanidine hydrochloride treated whole cell extracts bound quantitatively to the positively charged nylon membranes. The sensitivity of RNA detection when whole cell extracts treated with guanidine hydrochloride were probed with a digoxigenin labelled cDNA probe was similar to the detection of RNA in highly purified, protein free samples. Three positively charged membranes were tested (from Amersham, ICN and Boehringer Mannheim) using two alkaline phosphatase substrates, NBT-X phos, and a chemiluminescent substrate, 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoyloxy)-phenyl- 1-1,2-dioextane (AMPPD) and a peroxidase substrate, tetramethylbenzidine (TMB). The Boehringer Mannheim membrane had the highest sensitivity for the alkaline phosphatase substrates, but the peroxidase reaction with the TMB substrate was the most consistently sensitive, irrespective of which membrane was used. The ability to quantitatively detect RNA from whole cells without any purification will allow the rapid screening of large numbers of samples for specific RNA species in research or diagnostic laboratories.

Background High usage of the neuraminidase inhibitor (NAI) oseltamivir in Japan since 2003 led the Neuraminidase Inhibitor Susceptibility Network to assess the susceptibility of community isolates of influenza viruses to oseltamivir and zanamivir. Methods Isolates were tested by the enzyme inhibition assay and by neuraminidase (NA) sequence analysis. Results Among 1,141 A(H3N2) viruses and 171 type B viruses collected in Japan during the 2003–2004 season, 3 (0.3%) A(H3N2) isolates showed high 50% inhibitory concentrations (IC50) to oseltamivir. Each possessed a known resistance NA mutation at R292K or E119V. During the 2004–2005 season, no resistance was found among 567 influenza A(H3N2) or 60 A(H1N1) isolates, but 1 of 58 influenza B isolates had an NAI resistance mutation (D197N). Sequence analysis found that 4 (3%) of 132 A(H1N1) viruses from 2005–2006 had known NA resistance mutations (all H274Y), but no additional resistant isolates were detected from that or the subsequent 2006–2007 season. Concurrent testing of a selection of 500 influenza B viruses from 2000 to 2006 showed significant variations between seasons in both oseltamivir and zanamivir IC50 values, but no persistent increases over this period. Conclusions Our findings suggested possible low-level transmission of resistant variants from oseltamivir-treated patients in several seasons in Japan but no sustained reductions in NAI susceptibility or consistently increased frequency of detecting resistant variants for any strain or subtype, despite high levels of drug use. In particular, although oseltamivir-resistant A(H1N1) viruses with the H274Y mutation spread globally in 2007–2008, we found little evidence for increasing levels of resistant A(H1N1) variants in Japan in preceding years.

A rapid new method for the purification of neuraminidase (NA) heads from influenza A virus is described. Virus was pelleted directly from allantoic fluid and was digested with pronase. The cores were removed by centrifugation, redigested and the released NA heads were pooled and concentrated. The NA was separated from all contaminating proteins in a single step on a Superose 12 column. The purified material was suitable for both crystallography and for the production of monospecific antisera.

We tested the neuraminidase drug sensitivity of clade 1 and clade 2 influenza A virus (H5N1). All viruses demonstrated similar sensitivity to zanamivir, but compared to the 2004 clade 1 viruses, the Cambodian 2005 viruses were 6-fold less sensitive and the Indonesian clade 2 viruses were up to 30-fold less sensitive to oseltamivir.

A rapid, sensitive, solid-phase immunoassay, using horseradish peroxidase and 3,3',5,5'-tetramethylbenzidine, was found to be more sensitive and the colour developed was more stable compared with HRPO using 4-chloro-1-naphthol or diaminobenzidine. Assay sensitivity was equal to or greater than that obtained with the alkaline phosphatase reaction. The essential step was to incubate filters in 1% dextran sulphate for 10 min in a pH 5 (10 mM citrate-EDTA) buffer before reacting with TMB (0.05 mg/ml in the same buffer).

The nucleoprotein (NP) gene from influenza virus A/Shearwater/Australia/72 has been expressed intracellularly in both E. coli and insect cells. E. coli-derived NP was identified by Western blot analysis as a 56 kDa protein which co-migrates with virion-derived NP. This protein was purified by immunoaffinity chromatography and a nitrocellulose binding assay showed that NP formed complexes with positive- and negative-sense influenza neuraminidase RNA transcribed in vitro. ELISA and Western blot analysis revealed that recombinant NP of 56 kDa was produced in high yields in insect cells using a baculovirus vector. Immunofluorescence microscopy revealed that NP was localised to the nucleus of infected insect cells.

DURING THE LAST 2 YEARS, THE MEDICAL COMMUnity has been tensely following the emergence of resistance to oseltamivir in strains of influenza A. The clinical relevance and transmissibility of these resistant variants has been unknown, and planning for epidemic and pandemic influenza thus far has ignored the issue due to lack of evidence that it will be of medical consequence. Neuraminidase inhibitors are presently the only reliable antiviral option for treatment of influenza infection: the usefulness of the adamantanes (amantadine and rimantidine) has been virtually eliminated by the development of resistance. Until now, little spontaneous resistance to neuraminidase inhibitors has been documented, and no spontaneously resistant influenza viruses were identified in the world prior to the introduction of the drugs. This may now be changing. In this issue of JAMA, the report from Japan by Hatakeyama and colleagues documenting influenza B viruses with decreased sensitivity to oseltamivir and zanamivir increases concerns about the possibility and clinical significance of resistance. The article describes influenza B viruses partially resistant to neuraminidase inhibitors in individuals who had not been treated with antiviral medications and strongly suggests personto-person transmission of these resistant viruses, either within families or in the community. Japan has the highest use of neuraminidase inhibitors of any country in the world, with more than 90% of the prescriptions being for oseltamivir. While the mutations reported in the resistant isolates in the report are mainly those previously associated with selective pressure from oseltamivir, it is not certain whether the mutated viruses came from other treated individuals in the community or if they emerged spontaneously. The emergence of the variants having low-level resistance discussed in the study is also of concern because influenza B has lower baseline sensitivity to neuraminidase inhibitors compared with influenza A, and the presence of low-level resistance sets the stage for selective pressure for development of high-level resistance. In Australia, 2 influenza viruses with mutations conferring high-level resistance to neuraminidase inhibitors have been isolated from untreated patients. One of these viruses had a mutation in the neuraminidase that is known to confer high-level oseltamivir-specific resistance. Since there is little use of neuraminidase inhibitors in Australia, these viruses likely both arose due to spontaneous mutations. Part of the complacency about neuraminidase inhibitor– resistant influenza has been fueled by experiments in vitro and in animal models that have generally found neuraminidase inhibitor–resistant influenza viruses to be compromised in infectivity and transmissibility. This has led to the belief that significant transmission is unlikely to occur among humans. A 2003 model of the spread of resistant viruses in a pandemic concluded that community spread of such variants would be negligible because of decreases in biological fitness and transmissibility of drug-resistant viruses. However, a recent report predicted that, while use of antiviral drugs could significantly slow or stop transmission in the absence of drug resistance, the emergence of resistance could seriously diminish this benefit, depending on the cost of biological fitness: if there were a modest biological fitness cost and transmissibility were high, the effectiveness of antiviral use would plummet. Even if resistant strains emerge de novo at extremely low frequencies in individuals receiving antiviral drugs for treatment or prophylaxis, these strains may well make a significant contribution in an epidemic or pandemic setting. These concepts, together with the findings reported by Hatakeyama et al suggesting that partiallyresistant influenza B viruses are circulating, mean it is no longer possible to be confident that resistant strains will have little effect on epidemic or pandemic influenza. Resistance to oseltamivir has been predicted by in vitro and structural studies to be more likely to arise than resistance to zanamivir under the selective pressure of drug treatment. Zanamivir is more structurally similar to the natural substrate of neuraminidase and fits directly into the active site. For oseltamivir, a rearrangement of amino acids in the active site is necessary to accommodate the drug’s hydrophobic side chain; mutations that prevent this rearrangement might lead to resistance to oseltamivir but not to zanamivir. It has also

SummaryEndoplasmic reticulum (ER) stress caused by misfolded proteins or cytotoxic drugs can kill cells and although activation of this pathway has been implicated in the etiology of certain degenerative disorders its mechanism remains unresolved. Bim, a proapoptotic BH3-only member of the Bcl-2 family is required for initiation of apoptosis induced by cytokine deprivation or certain stress stimuli. Its proapoptotic activity can be regulated by several transcriptional or posttranslational mechanisms, such as ERK-mediated phosphorylation, promoting its ubiquitination and proteasomal degradation. We found that Bim is essential for ER stress-induced apoptosis in a diverse range of cell types both in culture and within the whole animal. ER stress activates Bim through two novel pathways, involving protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and proteasomal degradation and CHOP-C/EBPα-mediated direct transcriptional induction. These results define the molecular mechanisms of ER stress-induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases.

Emergence of influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAIs) develops at a low level following drug treatment, and person-to-person transmission of resistant virus has not been recognized to date. The Neuraminidase Inhibitor Susceptibility Network (NISN) was established to follow susceptibility of isolates and occurrence of NAI resistance at a population level in various parts of the world. Isolates from the WHO influenza collaborating centers were screened for susceptibilities to oseltamivir and zanamivir by a chemiluminescent enzyme inhibition assay, and those considered potentially resistant were analyzed by sequence analysis of the neuraminidase genes. During the first 3 years of NAI use (1999 to 2002), 2,287 isolates were tested. Among them, eight (0.33%) viruses had a >10-fold decrease in susceptibility to oseltamivir, one (0.22%) in 1999 to 2000, three (0.36%) in 2000 to 2001, and four (0.41%) in 2001 to 2002. Six had unique changes in the neuraminidase gene compared to neuraminidases of the same subtype in the influenza sequence database. Although only one of the mutations had previously been recognized in persons receiving NAIs, none were from patients who were known to have received the drugs. During the 3 years preceding NAI use, no resistant variants were detected among 1,054 viruses. Drug use was relatively stable during the period, except for an approximate 10-fold increase in oseltamivir use in Japan during the third year. The frequency of variants with decreased sensitivity to the NAIs did not increase significantly during this period, but continued surveillance is required, especially in regions with higher NAI use.

Although influenza vaccination remains the primary method for the prevention of influenza, efficacy may be limited by a poor match between the vaccine and circulating strains and the poor response of elderly patients. Hence, there is an important role for antiviral therapy in the management of influenza. While amantadine and rimantadine have been available for the treatment of influenza in some countries for several years, they are only effective against influenza A viruses, they can have neurological and gastrointestinal adverse effects, and resistant virus is rapidly generated. Neuraminidase inhibitors, a new class of drug, are potent and specific inhibitors of all strains of influenza virus, and they have minimal adverse effects. The greatest benefit is seen in those patients presenting

Zanamivir (4-guanidino-Neu5Ac2en [4-GU-DANA]) inhibits not only the neuraminidase activity but also the receptor interaction of the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN), blocking receptor binding and subsequent fusion promotion. All activities of the HPIV3 variant ZM1 HN (T193I/I567V) are less sensitive to 4-GU-DANA's effects. The T193I mutation in HN confers both increased receptor binding and increased neuraminidase activity, as well as reduced sensitivities of both activities to 4-GU-DANA inhibition, consistent with a single site on the HN molecule carrying out both catalysis and binding. We now provide evidence that the HPIV3 variant's resistance to receptor-binding inhibition by 4-GU-DANA is related to a reduced affinity of the HN receptor-binding site for this compound as well as to an increase in the avidity of HN for the receptor. Newcastle disease virus (NDV) HN and HPIV3 HN respond differently to inhibition in ways that suggest a fundamental distinction between them. NDV HN-receptor binding is less sensitive than HPIV3 HN-receptor binding to 4-GU-DANA, while its neuraminidase activity is highly sensitive. Both HPIV3 and NDV HNs are sensitive to receptor-binding inhibition by the smaller molecule DANA. However, for NDV HN, some receptor binding cannot be inhibited. These data are consistent with the presence in NDV HN of a second receptor-binding site that is devoid of enzyme activity and has a negligible, if any, affinity for 4-GU-DANA. Avidity for the receptor contributes to resistance by allowing the receptor to compete effectively with inhibitors for interaction with HN, while the further determinant of resistance is the reduced binding of the inhibitor molecule to the binding pocket on HN. Based upon our data and recent three-dimensional structural information on the HPIV3 and NDV HNs, we propose mechanisms for the observed sensitivity and resistance of HN to receptor-binding inhibition and discuss the implications of these mechanisms for the distribution of HN functions.