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The World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza (WHO CCs) tested 13,312 viruses collected by WHO recognized National Influenza Centres between May 2014 and May 2015 to determine 50% inhibitory concentration (IC 50 ) data for neuraminidase inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. Ninety-four per cent of the viruses tested by the WHO CCs were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 0.5% (n = 68) of viruses showed either highly reduced inhibition (HRI) or reduced inhibition (RI) (n = 56) against at least one of the four NAIs. Of the twelve viruses with HRI, six were A(H1N1)pdm09 viruses, three were A(H3N2) viruses and three were B/Yamagata-lineage viruses. The overall frequency of viruses with RI or HRI by the NAIs was lower than that observed in 2013–14 (1.9%), but similar to the 2012–13 period (0.6%). Based on the current analysis, the NAIs remain an appropriate choice for the treatment and prophylaxis of influenza virus infections. [ABSTRACT FROM AUTHOR]

Significant changes in the circulation of antiviral-resistant influenza viruses have occurred over the last decade. The emergence and continued circulation of adamantane-resistant A(H3N2) and A(H1N1)pdm09 viruses mean that the adamantanes are no longer recommended for use. Resistance to the newer class of drugs, the neuraminidase inhibitors, is typically associated with poorer viral replication and transmission. But ‘permissive’ mutations, that compensated for impairment of viral function in A(H1N1) viruses during 2007/2008, enabled them to acquire the H275Y NA resistance mutation without fitness loss, resulting in their rapid global spread. Permissive mutations now appear to be present in A(H1N1)pdm09 viruses thereby increasing the risk that oseltamivir-resistant A(H1N1)pdm09 viruses may also spread globally, a concerning scenario given that oseltamivir is the most widely used influenza antiviral. [ABSTRACT FROM AUTHOR]

Summary: Influenza A H1N1 2009 virus caused the first pandemic in an era when neuraminidase inhibitor antiviral drugs were available in many countries. The experiences of detecting and responding to resistance during the pandemic provided important lessons for public health, laboratory testing, and clinical management. We propose recommendations for antiviral susceptibility testing, reporting results, and management of patients infected with 2009 pandemic influenza A H1N1. Sustained global monitoring for antiviral resistance among circulating influenza viruses is crucial to inform public health and clinical recommendations for antiviral use, especially since community spread of oseltamivir-resistant A H1N1 2009 virus remains a concern. Further studies are needed to better understand influenza management in specific patient groups, such as severely immunocompromised hosts, including optimisation of antiviral treatment, rapid sample testing, and timely reporting of susceptibility results. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

With the recent emergence and spread of influenza A(H1N2) viruses which appear to have arisen by reassortment of circulating A(H1N1) and A(H3N2) strains, there is a need in epidemiological studies to determine the neuraminidase type in order to differentiate between influenza A(H1N2) and A(H1N1) strains. A fluorescence-based neuraminidase enzyme inhibition assay that has been developed to screen influenza viruses for potential resistance to the neuraminidase inhibitor drugs appears to be suitable for this purpose. When used with the neuraminidase inhibitor zanamivir the assay was able to provide a positive predictive value of 93.5% for the identification of neuraminidase type N1 or N2. This assay enables a large number of influenza A viruses to be screened at low cost to determine relative levels of A(H1N2) or A(H1N1) viruses circulating in the population. [Copyright &y& Elsevier]

Human influenza viruses isolated from Australasia (Australia and New Zealand) and South East Asia were analysed to determine their sensitivity to the NA inhibitor drugs, zanamivir and oseltamivir. A total of 532 strains isolated between 1998 and 2002 were tested using a fluorescence-based assay to measure the relative inhibition of NA activity over a range of drug concentrations. Based on median IC50 values, influenza A viruses (with neuraminidase subtypes N1 and N2) were more sensitive to both the NA inhibitors than were influenza B strains. Influenza A viruses with a N1 subtype and influenza B strains both demonstrated a greater sensitivity to zanamivir than to oseltamivir carboxylate, whereas influenza A strains with a N2 subtype were more susceptible to oseltamivir carboxylate. For each of the neuraminidase types, IC50 values for viruses from Australasia and South East Asia were found to be comparable. Based on the data prior to and following the licensing of the drugs into the respective regions, the use of the NA inhibitors did not appear to have a significant impact on the susceptibility of the viruses tested to zanamivir or oseltamivir carboxylate. [Copyright &y& Elsevier]

Influenza B viruses cause significant morbidity and mortality, particularly in children, but the awareness of their impact is often less than influenza A viruses partly due to their lack of pandemic potential. Here, we summarise the biology, epidemiology and disease burden of influenza B, and review existing data on available antivirals for its management. There has long been uncertainty surrounding the clinical efficacy of neuraminidase inhibitors (NAIs) for influenza B treatment. In this article, we bring together the existing data on NAIs and discuss these alongside recent large randomised controlled trial data for the new polymerase inhibitor baloxavir in high-risk influenza B patients. Finally, we offer considerations for the clinical management of influenza B, with a focus on children and high-risk patients where disease burden is highest. • Influenza B viruses are associated with significant morbidity and mortality, especially in children. • Available evidence suggests neuraminidase inhibitors are less effective at treating influenza B versus A infections. • Phase 3 trial data show the polymerase inhibitor baloxavir is effective in influenza B patients and resistance is rare. • Single oral dose baloxavir offers a more effective and convenient treatment of influenza B than neuraminidase inhibitors. [ABSTRACT FROM AUTHOR]

In late 2009 transplant organizations recommended that kidney recipients be vaccinated for pandemic H1N1 influenza (pH1N1); however, the vaccine efficacy was unknown. We had offered a monovalent non-adjuvanted pH1N1 vaccine to transplant recipients. Here we compared the pre- and post-vaccination seroresponses of 151 transplant recipients to that of 71 hemodialysis patients and 30 healthy controls. Baseline seroprotection was similar between groups but was significantly different at 1 month (44, 56, and 87%, respectively). Seroconversion was significantly less common for transplant recipients (32%) than dialysis patients (45%) and healthy controls (77%). After adjusting for age and gender, dialysis patients were significantly more likely (2.7-fold) to achieve new seroprotection than transplant recipients. The likelihood of seroprotection in transplant recipients was significantly reduced by mycophenolate use (adjusted odds ratio 0.24), in a dose-dependent manner, and by reduced eGFR (adjusted odds ratio 0.16 for worst to best). Seroprotection and geometric mean antibody titers increased substantially in 49 transplant recipients who subsequently received the 2010 seasonal influenza vaccine. Thus, patients requiring renal replacement therapy had reduced seroresponses to vaccination with the monovalent vaccine compared with healthy controls. Transplant recipient responses were further reduced if they were receiving mycophenolate or had significantly lower graft function. [ABSTRACT FROM AUTHOR]

Current influenza antivirals have limitations with regard to their effectiveness and the potential emergence of resistance. Encouragingly, several new compounds which inhibit the polymerase of influenza viruses have recently been shown to have enhanced pre-clinical and clinical effectiveness compared to the neuraminidase inhibitors, the mainstay of influenza antiviral therapy over the last two decades. In this review we focus on four compounds which inhibit polymerase function, baloxavir marboxil, favipiravir, pimodivir and AL-794 and discuss their clinical and virological effectiveness, their propensity to select for resistance and their potential for future combination therapy with the most commonly used neuraminidase inhibitor, oseltamivir. • Antiviral compounds targeting components of the polymerase complex PB1, PB2, and PA, are undergoing clinical investigation. • Baloxavir targets endonuclease activity of the PA polymerase subunit and was recently liscensed in Japan and USA for the treatment of influenza A and B viruses. • Favipiravir, a broad spectrum antiviral compound inhibits viral RNA polymerases and has limited licensure in Japan. • Pimodivir targets PB2 subunit of influenza A viruses preventing binding to 7-methyl GTP cap structures. • AL-794 binds to the endonuclease domain of PA but is no longer being evaluated for clinical use. [ABSTRACT FROM AUTHOR]

• NA gene sequencing of 67 HPAI H5N1 viruses revealed NA mutations in nine viruses. • A novel NA I117T mutation of HPAI H5N1 virus conferred resistance to NA inhibitors. • NAI susceptibility studied using fluorometric MUNANA assays and in ovo assays. • The I117T and I117V variants showed different levels of NAI susceptibility. • Natural occurrence of NAI-resistant I117T-mutant H5N1 virus is a cause of concern. Occurrence of avian influenza (AI) with Neuraminidase (NA) mutations which confer reduced neuraminidase inhibitor (NAI) susceptibility has remained a cause of concern. The susceptibility to NAIs of 67 highly pathogenic avian influenza H5N1 viruses isolated during 2006–2012 in India was tested in phenotypic fluorescence-based NA inhibition assay, sequence analysis and in ovo. One isolate showed a novel NA I117T amino acid substitution (N2 numbering) and eight isolates showed previously known NAI-resistance marker mutations (I117V, E119D, N294S, total 9/67). The overall incidence of resistant variants was 13.4%. The novel I117T substitution reduced oseltamivir susceptibility by 18.6-fold and zanamivir susceptibility by 11.8-fold, compared to the wild type AI H5N1virus, thus showed cross-resistance to both oseltamivir and zanamivir in NA inhibition assays. However, the other two isolates with I117V substitution were sensitive to both the NAIs. In addition, the comparison of growth of the I117T and I117V variants in presence of NAI's in the in ovo assays exhibited difference in growth levels. The present study reports the natural occurrence of a novel I117T mutation in AI H5N1 virus conferring cross-resistance to oseltamivir and zanamivir highlighting the urgent need of antiviral surveillance of AI viruses. [ABSTRACT FROM AUTHOR]

Abstract Baloxavir Marboxil (BXM) is an influenza polymerase inhibitor antiviral that binds to the endonuclease region in the PA subunit of influenza A and B viruses. To establish the baseline susceptibility of viruses circulating prior to licensure of BXM and to monitor for susceptibility post-BXM use, a cell culture-based focus reduction assay was developed to determine the susceptibility of 286 circulating seasonal influenza viruses, A(H1N1)pdm09, A(H3N2), B (Yamagata/Victoria) lineage viruses, including neuraminidase inhibitor (NAI) resistant viruses, to Baloxavir Acid (BXA), the active metabolic form of BXM. BXA was effective against all influenza subtypes tested with mean EC 50 values (minimum-maximum) of 0.7 ± 0.5 nM (0.1–2.1 nM), 1.2 ± 0.6 nM (0.1–2.4), 7.2 ± 3.5 nM (0.7–14.8), and 5.8 ± 4.5 nM (1.8–15.5) obtained for A(H1N1)pdm09, A(H3N2), B(Victoria lineage), and B(Yamagata lineage) influenza viruses, respectively. Using reverse genetics, amino acid substitutions known to alter BXA susceptibility were introduced into the PA protein resulting in EC 50 fold change increases that ranged from 2 to 65. Our study demonstrates that currently circulating viruses are susceptible to BXA and that the newly developed focus reduction assay is well suited to susceptibility monitoring in reference laboratories. Highlights • A focus reduction assay was developed to determine baloxavir susceptibility in seasonal influenza viruses. • 286 viruses of all seasonal influenza subtypes and lineages tested were susceptible to baloxavir. • The focus reduction assay was amenable to detecting viruses with reduced susceptibility to baloxavir. • PA N protein substitutions at position 38 derived by reverse genetics resulted in up to a 65 fold reduction in baloxavir EC 50. [ABSTRACT FROM AUTHOR]

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 (IC 50 ) 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. [ABSTRACT FROM AUTHOR]

Neuraminidase inhibitors (NAIs) are the main class of antivirals currently used for the treatment of influenza infections. As influenza viruses are constantly evolving, drug-resistance can emerge resulting in reduced effectiveness of treatment. This study evaluated the presence of molecular markers associated with NAI susceptibility in 724 influenza A(H1N1)pdm09 positive samples from Brazilian surveillance system from the 2014–2016 seasons, including 76 isolates tested for oseltamivir (OST) susceptibility and 23 isolates also tested for zanamivir, peramivir and laninamivir susceptibility. We identified the H275Y (n = 3) and I223K (n = 1) NA substitutions, associated with reduced inhibition (RI) by the NAIs. Noteworthy, no epidemiological links were identified among the patients infected with the mutant viruses. Phylogenetic analysis from NA and hemagglutinin genes showed that mutant viruses were not clustered. All mutant virus strains carried the permissive substitutions V241I and N369K, in addition to the N386K, which has been shown to destabilize the NA structure. Functional NA analysis of one virus containing the H275Y mutation confirmed its highly RI profile to OST and peramivir and demonstrated that it had decreased viral replication and NA thermostability compared to the wild type virus. The remaining tested isolates presented normal inhibition profile to the NAIs tested. In conclusion, the overall frequency of influenza A(H1N1)pdm09 viruses bearing mutations associated with NAI RI was 0.6%, similar to what has been observed in recent global studies. [ABSTRACT FROM AUTHOR]

Nitazoxanide is a thiazolide compound that was originally developed as an anti-parasitic agent, but has recently been repurposed for the treatment of influenza virus infections. Thought to exert its anti-influenza activity via the inhibition of hemagglutinin maturation and intracellular trafficking in infected cells, the effectiveness of nitazoxanide in treating patients with non-complicated influenza is currently being assessed in phase III clinical trials. Here, we describe the susceptibility of 210 seasonal influenza viruses to tizoxanide, the active circulating metabolite of nitazoxanide. An optimised cell culture-based focus reduction assay was used to determine the susceptibility of A(H1N1)pdm09, A(H3N2), and influenza B viruses circulating in the southern hemisphere from the period March 2014 to August 2016. Tizoxanide showed potent in vitro antiviral activity against all influenza viruses tested, including neuraminidase inhibitor-resistant viruses, allowing the establishment of a baseline level of susceptibility for each subtype. Median EC 50 values (±IQR) of 0.48 μM (0.33–0.71), 0.62 μM (0.56–0.75), 0.66 μM (0.62–0.69), and 0.60 μM (0.51–0.67) were obtained for A(H1N1)pdm09, A(H3N2), B(Victoria lineage), and B(Yamagata lineage) influenza viruses respectively. There was no significant difference in the median baseline tizoxanide susceptibility for each influenza subtype tested. This is the first report on the susceptibility of circulating viruses to tizoxanide. The focus reduction assay format described is sensitive, robust, and less laborious than traditional cell based antiviral assays, making it highly suitable for the surveillance of tizoxanide susceptibility in circulating seasonal influenza viruses. [ABSTRACT FROM AUTHOR]

Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) assessed antiviral susceptibility of 14,330 influenza A and B viruses collected by WHO-recognized National Influenza Centres (NICs) between May 2015 and May 2016. Neuraminidase (NA) inhibition assay was used to determine 50% inhibitory concentration (IC 50 ) data for NA inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. Furthermore, NA sequences from 13,484 influenza viruses were retrieved from public sequence databases and screened for amino acid substitutions (AAS) associated with reduced inhibition (RI) or highly reduced inhibition (HRI) by NAIs. Of the viruses tested by WHO CCs 93% were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 0.8% (n = 113) exhibited either RI or HRI by at least one of four NAIs. As in previous seasons, the most common NA AAS was H275Y in A(H1N1)pdm09 viruses, which confers HRI by oseltamivir and peramivir. Two A(H1N1)pdm09 viruses carried a rare NA AAS, S247R, shown in this study to confer RI/HRI by the four NAIs. The overall frequency of A(H1N1)pdm09 viruses containing NA AAS associated with RI/HRI was approximately 1.8% (125/6915), which is slightly higher than in the previous 2014-15 season (0.5%). Three B/Victoria-lineage viruses contained a new AAS, NA H134N, which conferred HRI by zanamivir and laninamivir, and borderline HRI by peramivir. A single B/Victoria-lineage virus harboured NA G104E, which was associated with HRI by all four NAIs. The overall frequency of RI/HRI phenotype among type B viruses was approximately 0.6% (43/7677), which is lower than that in the previous season. Overall, the vast majority (>99%) of the viruses tested by WHO CCs were susceptible to all four NAIs, showing normal inhibition (NI). Hence, NAIs remain the recommended antivirals for treatment of influenza virus infections. Nevertheless, our data indicate that it is prudent to continue drug susceptibility monitoring using both NAI assay and sequence analysis. [ABSTRACT FROM AUTHOR]

Laninamivir is a long-acting antiviral requiring only a single dose for the treatment of influenza infection, making it an attractive alternative to existing neuraminidase inhibitors that require multiple doses over many days. Like zanamivir, laninamivir is administered to patients by inhalation of dry powder. To date, studies investigating the effectiveness of laninamivir or zanamivir in a ferret model of influenza infection have administered the drug in a solubilised form. To better mimic the delivery action of laninamivir in humans, we assessed the applicability of a Dry Powder Insufflator™ (DPI) as a delivery method for laninamivir octanoate (LO) in ferrets to determine the effectiveness of this drug in reducing influenza A and B virus infections. In vitro characterisation of the DPI showed that both the small particle sized LO (0.7–6.0 μm diameter) and the large particle sized lactose carrier (20–100 μm diameter) were effectively discharged. However, LO delivered to ferrets via the DPI prior to infection with either A(H1N1)pdm09 or B viruses had a limited effect on nasal inflammation, clinical symptoms and viral shedding compared to placebo. Our preliminary findings indicate the feasibility of administering powder drugs into ferrets, but a better understanding of the pharmacokinetics and pharmacodynamics of LO in ferrets following delivery by the DPI is warranted prior to further studies. [ABSTRACT FROM AUTHOR]

Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) tested 10,641 viruses collected by WHO-recognized National Influenza Centres between May 2013 and May 2014 to determine 50% inhibitory concentration (IC 50 ) data for neuraminidase inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. In addition, neuraminidase (NA) sequence data, available from the WHO CCs and from sequence databases ( n = 3206), were screened for amino acid substitutions associated with reduced NAI susceptibility. Ninety-five per cent of the viruses tested by the WHO CCs were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 2% ( n = 172) showed highly reduced inhibition (HRI) against at least one of the four NAIs, commonly oseltamivir, while 0.3% ( n = 32) showed reduced inhibition (RI). Those showing HRI were A(H1N1)pdm09 with NA H275Y ( n = 169), A(H3N2) with NA E119V ( n = 1), B/Victoria-lineage with NA E117G ( n = 1) and B/Yamagata-lineage with NA H273Y ( n = 1); amino acid position numbering is A subtype and B type specific. Although approximately 98% of circulating viruses tested during the 2013–2014 period were sensitive to all four NAIs, a large community cluster of A(H1N1)pdm09 viruses with the NA H275Y substitution from patients with no previous exposure to antivirals was detected in Hokkaido, Japan. Significant numbers of A(H1N1)pdm09 NA H275Y viruses were also detected in China and the United States: phylogenetic analyses showed that the Chinese viruses were similar to those from Japan, while the United States viruses clustered separately from those of the Hokkaido outbreak, indicative of multiple resistance-emergence events. Consequently, global surveillance of influenza antiviral susceptibility should be continued from a public health perspective. [ABSTRACT FROM AUTHOR]

A contemporary influenza type B virus was passaged in vitro in the presence of increasing concentrations of the neuraminidase inhibitors, zanamivir and oseltamivir carboxylate (0.1–1000 μM over nine passages). After the fifth passage in the presence of zanamivir (10 μM), the virus acquired a Glu 119 Asp neuraminidase mutation (influenza A N2 subtype numbering) in the enzyme active site. After a further three passages, in which growth occurred in 100 μM of zanamivir, a Gln 218 Lys mutation (A (H3) numbering) in the HA1 domain of the haemagglutinin was found. In a fluorescence-based neuraminidase inhibition assay, viruses with the Glu 119 Asp NA mutation had a 32,000-fold reduction in sensitivity to the NA inhibitor zanamivir compared to the wild-type virus, while the mutation resulted in a 105-fold reduction in sensitivity to oseltamivir carboxylate. Viruses grown in the presence of 1000 μM oseltamivir carboxylate did not acquire any neuraminidase mutations but did have a His 103 Gln substitution (A (H3) numbering) in the HA1 region of the haemagglutinin which was demonstrated to significantly reduce receptor binding strength in vitro. Tissue culture assays demonstrated that the HA mutation caused a seven-fold reduction in sensitivity to oseltamivir carboxylate, and a 90-fold reduction in sensitivity to zanamivir. [Copyright &y& Elsevier]

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

Combination therapy is an alternative approach to reduce viral shedding and improve clinical outcomes following influenza virus infections. In this study we used oseltamivir (OST), a neuraminidase inhibitor and nitazoxanide (NTZ), a host directed drug, and found in vitro that the combination of these two antivirals have a synergistic relationship. Using the ferret model of (A/Perth/265/2009, (H1N1)pdm09), virus infections, we found that the combination of NTZ and OST was more effective than either NTZ or OST independently in preventing infection and reducing duration of viral shedding. However, these benefits were only seen if treatment was administered prophylactically, as opposed to therapeutically. We also found that if prophylactically treated ferrets that had detectable virus in the upper respiratory tract, no virus was detected in the lower respiratory tract. This benefit was not observed with NTZ or OST alone. The combination of NTZ and OST enhances the antiviral effect of OST, which is the standard of care in most settings. • Nitazoxanide is a host-directed compound currently licensed to treat parasitic infections. • Prophylactic treatment with nitazoxanide and oseltamivir reduces the likelihood of influenza infection in ferrets. • Nitazoxanide and oseltamivir prophylaxis restricted influenza A(H1N1)pdm09 virus spread to the lungs of ferrets. • Combination of nitazoxanide and oseltamivir was not effective when administered therapeutically to ferrets. [ABSTRACT FROM AUTHOR]

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. • Influenza viruses collected worldwide were analyzed for susceptibilities to NA and PA inhibitors, May 2017–May 2018. • A total of 15,409 viruses were assessed for NA inhibitor phenotypic susceptibility. • A total of 13,523 PA sequences were screened for mutations associated with reduced PA inhibitor baloxavir susceptibility. • The frequency of viruses with reduced susceptibility to NA (0.8%) or PA (0.08%) inhibitors was low. • Global surveillance of influenza antiviral susceptibility should be continued. [ABSTRACT FROM AUTHOR]

The persistent circulation of avian influenza viruses (AIVs) is an ongoing problem for many countries in South East Asia, causing large economic losses to both the agricultural and health sectors. This review analyses AIV diversity, evolution and the risk of AIV emergence in humans in countries of the Greater Mekong Subregion (GMS): Cambodia, Laos, Myanmar, Thailand and Vietnam (excluding China). The analysis was based on AIV sequencing data, serological studies, published journal articles and AIV outbreak reports available from January 2003 to December 2018. All countries of the GMS have suffered losses due repeated outbreaks of highly pathogenic (HP) H5N1 that has also caused human cases in all GMS countries. In Laos, Myanmar and Vietnam AIV outbreaks in domestic poultry have also been caused by clade 2.3.4.4 H5N6. A diverse range of low pathogenic AIVs (H1-H12) have been detected in poultry and wild bird species, though surveillance for and characterization of these subtypes is limited. Subtype H3, H4, H6 and H11 viruses have been detected over prolonged periods; whilst H1, H2, H7, H8, H10 and H12 viruses have only been detected transiently. H9 AIVs circulate endemically in Cambodia and Vietnam with seroprevalence data indicating human exposure to H9 AIVs in Cambodia, Thailand and Vietnam. As surveillance studies focus heavily on the detection of H5 AIVs in domestic poultry further research is needed to understand the true level of AIV diversity and the risk AIVs pose to humans in the GMS. • Risk for avian influenza virus (AIV) emergence in Greater Mekong Subregion (GMS). • Evidence of high diversity of AIVs circulating in the GMS • Reports mostly focus on the circulation of H5N1 in domestic poultry. • A need for improved surveillance of AIVs in the GMS. [ABSTRACT FROM AUTHOR]

Abstract: Annual influenza epidemics in humans affect 5–15% of the population, causing an estimated half million deaths worldwide per year [Stohr K. Influenza—WHO cares. Lancet Infectious Diseases 2002;2(9):517]. The virus can infect this proportion of people year after year because the virus has an extensive capacity to evolve and thus evade the immune response. For example, since the influenza A(H3N2) subtype entered the human population in 1968 the A(H3N2) component of the influenza vaccine has had to be updated almost 30 times to track the evolution of the viruses and remain effective. The World Health Organization Global Influenza Surveillance Network (WHO GISN) tracks and analyzes the evolution and epidemiology of influenza viruses for the primary purpose of vaccine strain selection and to improve the strain selection process through studies aimed at better understanding virus evolution and epidemiology. Here we give an overview of the strain selection process and outline recent investigations into the global migration of seasonal influenza viruses. [Copyright &y& Elsevier]