Annual report on influenza viruses received and tested by the Melbourne WHO Collaborating Centre for Reference and Research on Influenza in 2016

As part of its role in the World Health Organization's (WHO) Global Influenza Surveillance and Response System (GISRS), the WHO Collaborating Centre for Reference and Research on Influenza in Melbourne received a total of 4,247 human influenza positive samples during 2016. Viruses were analysed for their antigenic, genetic and antiviral susceptibility properties and also propagated in qualified cells and hens eggs for potential seasonal influenza vaccine virus candidates. In 2016, influenza A(H3) viruses predominated over influenza A(H1)pdm09 and B viruses, accounting for a total of 51% of all viruses analysed. The vast majority of A(H1)pdm09, A(H3) and influenza B viruses analysed at the Centre were found to be antigenically similar to the respective WHO recommended vaccine strains for the Southern Hemisphere in 2016. However, phylogenetic analysis of a selection of viruses indicated that the majority of circulating A(H3) viruses had undergone some genetic drift relative to the WHO recommended strain for 2016. Of more than 3,000 samples tested for resistance to the neuraminidase inhibitors oseltamivir and zanamivir, six A(H1)pdm09 viruses and two B/Victoria lineage viruses showed highly reduced inhibition to oseltamivir.

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors and status of novel antivirals, 2016-2017.

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

Influenza viruses received and tested by the Melbourne WHO Collaborating Centre for Reference and Research on Influenza annual report, 2015.

As part of its role in the World Health Organization's (WHO) Global Influenza Surveillance and Response System, the WHO Collaborating Centre for Reference and Research on Influenza in Melbourne received a total of 5,557 influenza positive samples during 2015. Viruses were analysed for their antigenic, genetic and antiviral susceptibility properties. In 2015, influenza B viruses predominated over influenza A(H1)pdm09 and A(H3) viruses, accounting for a total of 58% of all viruses analysed. The vast majority of A(H1)pdm09, A(H3) and influenza B viruses analysed at the Centre were found to be antigenically similar to the respective WHO recommended vaccine strains for the Southern Hemisphere in 2015. However, phylogenetic analysis of a selection of viruses indicated that the majority of circulating A(H3) viruses were genetically distinct from the WHO recommended strain for 2015, resulting in an update to the recommended vaccine strain for the Southern Hemisphere for 2016. With an increasing predominance of B/Victoria lineage viruses over B/Yamagata lineage viruses through the course of 2015, WHO also updated the recommended influenza B strain in the trivalent influenza vaccine for 2016. Of more than 3,300 samples tested for resistance to the neuraminidase inhibitors oseltamivir and zanamivir, only 1 A(H1)pdm09 virus showed highly reduced inhibition by oseltamivir. The Centre undertook primary isolation of candidate vaccine viruses directly into eggs, and in 2015 a total of 45 viruses were successfully isolated in eggs.

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2015-2016.

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 (IC50) 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.

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2'-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

Affective dysfunction in a mouse model of Rett syndrome: Therapeutic effects of environmental stimulation and physical activity.

Rett syndrome (RTT) is a neurodevelopmental disorder associated with mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2) and consequent dysregulation of brain maturation. Patients suffer from a range of debilitating physical symptoms, however, behavioral and emotional symptoms also severely affect their quality of life. Here, we present previously unreported and clinically relevant affective dysfunction in the female heterozygous Mecp2(tm1Tam) mouse model of RTT (129sv and C57BL6 mixed background). The affective dysfunction and aberrant anxiety-related behavior of the Mecp2(+/-) mice were found to be reversible with environmental enrichment (EE) from 4 weeks of age. The effect of exercise alone (via wheel running) was also explored, providing the first evidence that increased voluntary physical activity in an animal model of RTT is beneficial for some phenotypes. Mecp2(+/-) mutants displayed elevated corticosterone despite decreased Crh expression, demonstrating hypothalamic-pituitary-adrenal axis dysregulation. EE of Mecp2(+/-) mice normalized basal serum corticosterone and hippocampal BDNF protein levels. The enrichment-induced rescue appears independent of the transcriptional regulation of the MeCP2 targets Bdnf exon 4 and Crh. These findings provide new insight into the neurodevelopmental role of MeCP2 and pathogenesis of RTT, in particular the affective dysfunction. The positive outcomes of environmental stimulation and physical exercise have implications for the development of therapies targeting the affective symptoms, as well as behavioral and cognitive dimensions, of this devastating neurodevelopmental disorder.

Zanamivir-resistant influenza viruses with Q136K or Q136R neuraminidase residue mutations can arise during MDCK cell culture creating challenges for antiviral susceptibility monitoring.

Surveillance of circulating influenza strains for antiviral susceptibility is important to ensure patient treatment guidelines remain appropriate. Influenza A(H3N2) and A(H1N1)pdm09 virus isolates containing mutations at the Q136 residue of the neuraminidase (NA) that conferred reduced susceptibility to the NA inhibitor (NAI) zanamivir were detected during antiviral susceptibility monitoring. Interestingly, the mutations were not detectable in the viruses from respective clinical specimens, only in the cultured isolates. We showed that variant viruses containing the Q136K and Q136R NA mutations were preferentially selected in Madin-Darby canine kidney epithelial (MDCK) cells, but were less well supported in MDCK-SIAT1 cells and embryonated eggs. The effect of Q136K, Q136R, Q136H and Q136L substitutions in NA subtypes N1 and N2 on NAI susceptibility and in vitro viral fitness was assessed. This study highlights the challenges that cell culture derived mutations can pose to the NAI susceptibility analysis and interpretation and reaffirms the need to sequence viruses from respective clinical specimens to avoid misdiagnosis. However, we also demonstrate that NA mutations at residue Q136 can confer reduced zanamivir, peramivir or laninamivir susceptibility, and therefore close monitoring of viruses for mutations at this site from patients being treated with these antivirals is important.

Influenza viruses with B/Yamagata- and B/Victoria-like neuraminidases are differentially affected by mutations that alter antiviral susceptibility.

OBJECTIVES: The burden of disease due to influenza B is often underestimated. Clinical studies have shown that oseltamivir, a widely used neuraminidase inhibitor (NAI) antiviral drug, may have reduced effectiveness against influenza B viruses. Therefore, it is important to study the effect of neuraminidase mutations in influenza B viruses that may further reduce NAI susceptibility, and to determine whether these mutations have the same effect in the two lineages of influenza B viruses that are currently circulating (B/Yamagata-like and B/Victoria-like). METHODS: We characterized the effect of 16 amino acid substitutions across five framework residues and four monomeric interface residues on the susceptibility to four different NAIs (oseltamivir, zanamivir, peramivir and laninamivir). RESULTS: Framework residue mutations E117A and E117G conferred highly reduced inhibition to three of the four NAIs, but substantially reduced neuraminidase activity, whereas other framework mutations retained a greater level of NA activity. Mutations E105K, P139S and G140R of the monomeric interface were also found to cause highly reduced inhibition, but, interestingly, their effect was substantially greater in a B/Victoria-like neuraminidase than in a B/Yamagata-like neuraminidase, with some susceptibility values being up to 1000-fold different between lineages. CONCLUSIONS: The frequency and the effect of key neuraminidase mutations on neuraminidase activity and NAI susceptibility can differ substantially between the two influenza B lineages. Therefore, future surveillance, analysis and interpretation of influenza B virus NAI susceptibility should consider the B lineage of the neuraminidase in the same manner as already occurs for different influenza A neuraminidase subtypes.

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2013-2014.

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 (IC50) 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.

Evaluation of oseltamivir prophylaxis regimens for reducing influenza virus infection, transmission and disease severity in a ferret model of household contact.

OBJECTIVES: The emergence of the pandemic influenza A(H1N1)pdm09 virus in 2009 saw a significant increase in the therapeutic and prophylactic use of neuraminidase inhibitors (NAIs) to mitigate the impact of this highly transmissible virus. Prior to the pandemic, many countries stockpiled NAIs and developed pandemic plans for the use of antiviral drugs, based on either treatment of high-risk individuals and/or prophylaxis of contacts. However, to date there has been a lack of in vivo models to test the efficacy of treatment or prophylaxis with NAIs, for influenza-infected individuals or exposed contacts, in a household setting. METHODS: A ferret model of household contact was developed to study the efficacy of different prophylaxis regimens in preventing infection in contact ferrets exposed to influenza A(H1N1)pdm09-infected index ferrets. RESULTS: Among the different prophylactic regimens, contact ferrets receiving oseltamivir prophylaxis twice daily showed better outcomes than those receiving oseltamivir once daily. Benefits included a significant delay in the time to secondary infection, lower weight loss and higher activity levels. The treatment of index ferrets at 36 h post-infection did not influence either secondary infection rates or clinical symptoms in exposed contact ferrets. Neither prophylaxis nor treatment prevented infection or reduced the duration of viral shedding, although clinical symptoms did improve in infected animals receiving prophylaxis. CONCLUSIONS: Different oseltamivir prophylaxis regimens did not prevent infections, but consistently resulted in a reduction in symptoms in infected ferrets. However, oseltamivir prophylaxis failed to reduce viral titres, which warrants further investigation in humans.

Peramivir and laninamivir susceptibility of circulating influenza A and B viruses.

Influenza viruses collected from regions of Asia, Africa and Oceania between 2009 and 2012 were tested for their susceptibility to two new neuraminidase inhibitors, peramivir and laninamivir. All viruses tested had normal laninamivir inhibition. However, 3·2% (19/599) of A(H1N1)pdm09 viruses had highly reduced peramivir inhibition (due to H275Y NA mutation) and <1% (6/1238) of influenza B viruses had reduced or highly reduced peramivir inhibition, with single occurrence of variants containing I221T, A245T, K360E, A395E, D432G and a combined G145R+Y142H mutation. These data demonstrate that despite an increase in H275Y variants in 2011, there was no marked change in the frequency of peramivir- or laninamivir-resistant variants following the market release of the drugs in Japan in 2010.

Estimating the fitness advantage conferred by permissive neuraminidase mutations in recent oseltamivir-resistant A(H1N1)pdm09 influenza viruses.

Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.

Progressive emergence of an oseltamivir-resistant A(H3N2) virus over two courses of oseltamivir treatment in an immunocompromised paediatric patient.

A minor viral population of oseltamivir-resistant A(H3N2) viruses (E119V neuraminidase mutation) was selected and maintained in a continually infected immunocompromised child following initial oseltamivir treatment. A subsequent course of oseltamivir given 7 weeks later rapidly selected for the E119V variant resulting in a near-pure population of the resistant virus. The study highlights the challenges of oseltamivir treatment of immunocompromised patients that are continually shedding virus and demonstrates the ability of the E119V oseltamivir-resistant virus to be maintained for prolonged periods even in the absence of drug-selective pressure.

Influenza antiviral resistance in the Asia-Pacific region during 2011.

Despite greater than 99% of influenza A viruses circulating in the Asia-Pacific region being resistant to the adamantane antiviral drugs in 2011, the large majority of influenza A (>97%) and B strains (∼99%) remained susceptible to the neuraminidase inhibitors oseltamivir and zanamivir. However, compared to the first year of the 2009 pandemic, cases of oseltamivir-resistant A(H1N1)pdm09 viruses with the H275Y neuraminidase mutation increased in 2011, primarily due to an outbreak of oseltamivir-resistant viruses that occurred in Newcastle, as reported in Hurt et al. (2011c, 2012a), where the majority of the resistant viruses were from community patients not being treated with oseltamivir. A small number of influenza B viruses with reduced oseltamivir or zanamivir susceptibility were also detected. The increased detection of neuraminidase inhibitor resistant strains circulating in the community and the detection of novel variants with reduced susceptibility are reminders that monitoring of influenza viruses is important to ensure that antiviral treatment guidelines remain appropriate.

Mutations I117V and I117M and oseltamivir sensitivity of pandemic (H1N1) 2009 viruses.

Analysis of mutations I117V and I117M in the neuraminidase of influenza A pandemic (H1N1) 2009 viruses showed that I117V confers a mild reduction in oseltamivir sensitivity and has a synergistic effect of further increasing resistance when combined with H275Y. Contrary to recent reports, the I117M mutation does not alter oseltamivir sensitivity.

Both E protein glycans adversely affect dengue virus infectivity but are beneficial for virion release.

The E protein of most flaviviruses is modified by Asn-linked glycosylation at residue 153/154 and in the case of the four dengue virus (DENV) serotypes by a second glycan at residue 67. However, the absence of E protein glycosylation among numerous natural isolates of different flaviviruses suggests that the glycan, per se, is not critically important in the virus life cycle. Consistent with this notion, we show that ablation of both glycans from the DENV-2 E protein reduces but does not prevent growth of the variant in mammalian and mosquito cells. We found a pronounced and opposing effect of glycan ablation on two stages of the virus growth cycle: infectivity and release. Loss of either of the two DENV E protein glycans markedly enhanced infectivity of variants for mosquito cells at the expense of efficient virion release. The variants also displayed reduced release in mammalian cells, which was more prominent for viruses lacking the Asn 67-linked glycan than for those lacking the Asn 153-linked glycan, without a marked change in infectivity. Mutations, which compensated for the defect in virus morphogenesis associated with ablation of the Asn 67-linked glycan in mammalian cells but interestingly not in mosquito cells, were identified at the glycosylation acceptor motif and a second site in E protein domain II. The dueling influences of infectivity and release on virus growth affected by the glycans may explain the plasticity in E protein glycosylation among the flaviviruses.