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The neuraminidase (NA) inhibitor (NAI) oseltamivir (OST) is the most widely used influenza antiviral drug. Several NA amino acid substitutions are reported to reduce viral susceptibility to OST in in vitro assays. However, whether there is a correlation between the level of reduction in susceptibility in vitro and the efficacy of OST against these viruses in vivo is not well understood. In this study, a ferret model was utilised to evaluate OST efficacy against circulating influenza A and B viruses with a range of in vitro generated 50% inhibitory concentrations (IC50) values for OST. OST efficacy against an A(H1N1)pdm09 and an A(H1N1)pdm09 virus with the H275Y substitution in neuraminidase was also tested in the macaque model. The results from this study showed that OST had a significant impact on virological parameters compared to placebo treatment of ferrets infected with wild-type influenza A viruses with normal IC50 values (~1 nM). However, this efficacy was lower against wild-type influenza B and other viruses with higher IC50 values. Differing pathogenicity of the viruses made evaluation of clinical parameters difficult, although some effect of OST in reducing clinical signs was observed with influenza A(H1N1) and A(H1N1)pdm09 (H275Y) viruses. Viral titres in macaques were too low to draw conclusive results. Analysis of the ferret data revealed a correlation between IC50 and OST efficacy in reducing viral shedding but highlighted that the current WHO guidelines/criteria for defining normal, reduced or highly reduced inhibition in influenza B viruses based on in vitro data are not well aligned with the low in vivo OST efficacy observed for both wild-type influenza B viruses and those with reduced OST susceptibility.

Two lineages of influenza B virus currently co-circulate and have distinct antigenicity, termed Victoria and Yamagata after the B/Victoria/2/87 and B/Yamagata/16/88 strains, respectively. We analyzed antibody titer dynamics following PCR-confirmed influenza B virus infection in a longitudinal community-based cohort study conducted in Hong Kong from 2009-2014 to assess patterns in changes in antibody titers to B/Victoria and B/Yamagata viruses following infections with each lineage. Among 62 PCR-confirmed cases, almost half had undetectable hemagglutination inhibition (HAI) antibody titers to the lineage of infection both pre-infection and post-infection. Among those infected with influenza B/Victoria who showed an HAI titer response after infection, we found strong rises to the lineage of infection, positive but smaller cross-lineage HAI titer boosts, a small dependence of HAI titer boosts on pre-infection titers, and a shorter half-life of HAI titers in adults. Our study is limited by the low HAI sensitivity for non-ether-treated IBV antigen and the incapacity of performing other assays with higher sensitivity, as well as the mismatch between the B/Yamagata lineage circulating strain and the assay strain in one of the study seasons.

Low pathogenic A(H9N2) subtype avian influenza viruses (AIVs) were originally detected in Cambodian poultry in 2013, and now circulate endemically. We sequenced and characterised 64 A(H9N2) AIVs detected in Cambodian poultry (chickens and ducks) from January 2015 to May 2016. All A(H9) viruses collected in 2015 and 2016 belonged to a new BJ/94-like h9-4.2.5 sub-lineage that emerged in the region during or after 2013, and was distinct to previously detected Cambodian viruses. Overall, there was a reduction of genetic diversity of H9N2 since 2013, however two genotypes were detected in circulation, P and V, with extensive reassortment between the viruses. Phylogenetic analysis showed a close relationship between A(H9N2) AIVs detected in Cambodian and Vietnamese poultry, highlighting cross-border trade/movement of live, domestic poultry between the countries. Wild birds may also play a role in A(H9N2) transmission in the region. Some genes of the Cambodian isolates frequently clustered with zoonotic A(H7N9), A(H9N2) and A(H10N8) viruses, suggesting a common ecology. Molecular analysis showed 100% of viruses contained the hemagglutinin (HA) Q226L substitution, which favours mammalian receptor type binding. All viruses were susceptible to the neuraminidase inhibitor antivirals; however, 41% contained the matrix (M2) S31N substitution associated with resistance to adamantanes. Overall, Cambodian A(H9N2) viruses possessed factors known to increase zoonotic potential, and therefore their evolution should be continually monitored.

BACKGROUND: Many serologic studies were done during and after the 2009 influenza pandemic, to estimate the cumulative incidence of influenza A(H1N1)pdm09 virus infections, but there are few comparative estimates of the incidence of influenza A(H3N2) virus infections during epidemics. METHODS: We conducted a longitudinal serologic study in Hong Kong. We collected sera annually and tested samples from 2009-13 by HAI against the A/Perth/16/2009(H3N2) virus, and samples from 2013-15 against the A/Victoria/361/2011(H3N2) virus using the hemagglutination inhibition (HAI) assay. We estimated the cumulative incidence of infections based on 4-fold or greater rises in HAI titers in consecutive sera. RESULTS: There were four major H3N2 epidemics: (1) Aug-Oct 2010; (2) Mar-Jun 2012; (3) Jul-Oct 2013; and (4) Jun-Jul 2014. Between 516 and 619 relevant pairs of sera were available for each epidemic. We estimated that 9%, 19%, 7% and 7% of the population were infected in each epidemic, respectively, with higher incidence in children in epidemics 1 and 4. CONCLUSIONS: We found that re-infections in each of the four H3N2 epidemics that occurred from 2010 through 2014 were rare. The largest H3N2 epidemic occurred with the lowest level of pre-epidemic immunity.

Influenza is an infectious disease that primarily attacks the respiratory system. Innate immunity provides both a very early defense to influenza virus invasion and an effective control of viral growth. Previous modelling studies of virus-innate immune response interactions have focused on infection with a single virus and, while improving our understanding of viral and immune dynamics, have been unable to effectively evaluate the relative feasibility of different hypothesised mechanisms of antiviral immunity. In recent experiments, we have applied consecutive exposures to different virus strains in a ferret model, and demonstrated that viruses differed in their ability to induce a state of temporary immunity or viral interference capable of modifying the infection kinetics of the subsequent exposure. These results imply that virus-induced early immune responses may be responsible for the observed viral hierarchy. Here we introduce and analyse a family of within-host models of re-infection viral kinetics which allow for different viruses to stimulate the innate immune response to different degrees. The proposed models differ in their hypothesised mechanisms of action of the non-specific innate immune response. We compare these alternative models in terms of their abilities to reproduce the re-exposure data. Our results show that 1) a model with viral control mediated solely by a virus-resistant state, as commonly considered in the literature, is not able to reproduce the observed viral hierarchy; 2) the synchronised and desynchronised behaviour of consecutive virus infections is highly dependent upon the interval between primary virus and challenge virus exposures and is consistent with virus-dependent stimulation of the innate immune response. Our study provides the first mechanistic explanation for the recently observed influenza viral hierarchies and demonstrates the importance of understanding the host response to multi-strain viral infections. Re-exposure exper

Ferrets are the preferred animal model to assess influenza virus infection, virulence and transmission as they display similar clinical symptoms and pathogenesis to those of humans. Measures of disease severity in the ferret include weight loss, temperature rise, sneezing, viral shedding and reduced activity. To date, the only available method for activity measurement has been the assignment of an arbitrary score by a 'blind' observer based on pre-defined responsiveness scale. This manual scoring method is subjective and can be prone to bias. In this study, we described a novel video-tracking methodology for determining activity changes in a ferret model of influenza infection. This method eliminates the various limitations of manual scoring, which include the need for a sole 'blind' observer and the requirement to recognise the 'normal' activity of ferrets in order to assign relative activity scores. In ferrets infected with an A(H1N1)pdm09 virus, video-tracking was more sensitive than manual scoring in detecting ferret activity changes. Using this video-tracking method, oseltamivir treatment was found to ameliorate the effect of influenza infection on activity in ferret. Oseltamivir treatment of animals was associated with an improvement in clinical symptoms, including reduced inflammatory responses in the upper respiratory tract, lower body weight loss and a smaller rise in body temperature, despite there being no significant reduction in viral shedding. In summary, this novel video-tracking is an easy-to-use, objective and sensitive methodology for measuring ferret activity.

The factors that determine the characteristic seasonality of influenza remain enigmatic. Current models predict that occurrences of influenza outside the normal surveillance season within a temperate region largely reflect the importation of viruses from the alternate hemisphere or from equatorial regions in Asia. To help reveal the drivers of seasonality we investigated the origins and evolution of influenza viruses sampled during inter-seasonal periods in Australia. To this end we conducted an expansive phylogenetic analysis of 9912, 3804, and 3941 hemagglutinnin (HA) sequences from influenza A/H1N1pdm, A/H3N2, and B, respectively, collected globally during the period 2009-2014. Of the 1475 viruses sampled from Australia, 396 (26.8% of Australian, or 2.2% of global set) were sampled outside the monitored temperate influenza surveillance season (1 May - 31 October). Notably, rather than simply reflecting short-lived importations of virus from global localities with higher influenza prevalence, we documented a variety of more complex inter-seasonal transmission patterns including "stragglers" from the preceding season and "heralds" of the forthcoming season, and which included viruses sampled from clearly temperate regions within Australia. We also provide evidence for the persistence of influenza B virus between epidemic seasons, in which transmission of a viral lineage begins in one season and continues throughout the inter-seasonal period into the following season. Strikingly, a disproportionately high number of inter-seasonal influenza transmission events occurred in tropical and subtropical regions of Australia, providing further evidence that climate plays an important role in shaping patterns of influenza seasonality.

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.

Surveillance data indicate that most circulating A(H1N1)pdm09 influenza viruses have remained antigenically similar since they emerged in humans in 2009. However, antigenic drift is likely to occur in the future in response to increasing population immunity induced by infection or vaccination. In this study, sequential passaging of A(H1N1)pdm09 virus by contact transmission through two independent series of suboptimally vaccinated ferrets resulted in selection of variant viruses with an amino acid substitution (N156K, H1 numbering without signal peptide; N159K, H3 numbering without signal peptide; N173K, H1 numbering from first methionine) in a known antigenic site of the viral HA. The N156K HA variant replicated and transmitted efficiently between naïve ferrets and outgrew wildtype virus in vivo in ferrets in the presence and absence of immune pressure. In vitro, in a range of cell culture systems, the N156K variant rapidly adapted, acquiring additional mutations in the viral HA that also potentially affected antigenic properties. The N156K escape mutant was antigenically distinct from wildtype virus as shown by binding of HA-specific antibodies. Glycan binding assays demonstrated the N156K escape mutant had altered receptor binding preferences compared to wildtype virus, which was supported by computational modeling predictions. The N156K substitution, and culture adaptations, have been detected in human A(H1N1)pdm09 viruses with N156K preferentially reported in sequences from original clinical samples rather than cultured isolates. This study demonstrates the ability of the A(H1N1)pdm09 virus to undergo rapid antigenic change to evade a low level vaccine response, while remaining fit in a ferret transmission model of immunization and infection. Furthermore, the potential changes in receptor binding properties that accompany antigenic changes highlight the importance of routine characterization of clinical samples in human A(H1N1)pdm09 influenza surveillance.

INTRODUCTION: We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses. FINDINGS: A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of non-specific primer-dimer was evident and affected the overall melting temperature (T(m)) of the amplified products. Due to primer-dimer appearance at >30 cycles we found that if the cycle threshold (C(T)) for a dilution was >30, the HRM assay did not consistently discriminate mutant from wildtype. Where the C(T) was <30 we noted an inverse relationship between C(T) and T(m) and fitted quadratic curves allowed the discrimination of wildtype, mutant and 30∶70 mutant∶wildtype virus mixtures. We compared the C(T) values for a TaqMan H1N1 09 detection assay with those for the HRM assay using 59 clinical samples and demonstrated that samples with a TaqMan detection assay C(T)>32.98 would have an H275Y assay C(T)>30. Analysis of the TaqMan C(T) values for 609 consecutive clinical samples predicted that 207 (34%) of the samples would result in an HRM assay C(T)>30 and therefore not be amenable to the HRM assay. CONCLUSIONS: The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting T(m) against C(T). Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations.

BACKGROUND: Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance. METHODOLOGY/PRINCIPAL FINDINGS: A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses. CONCLUSIONS/SIGNIFICANCE: In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches.

BACKGROUND: We describe the serological response following H1N1-2009 influenza A infections confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). METHODOLOGY AND PRINCIPAL FINDINGS: The study included patients admitted to hospital, subjects of a seroepidemiologic cohort study, and participants identified from outbreak studies in Singapore. Baseline (first available blood sample) and follow-up blood samples were analyzed for antibody titers to H1N1-2009 and recently circulating seasonal influenza A virus strains by hemagglutination inhibition (HI) and virus micro-neutralization (VM) assays. 267 samples from 118 cases of H1N1-2009 were analyzed. Geometric mean titers by HI peaked at 123 (95% confidence interval, CI 43-356) between days 30 to 39. The chance of observing seroconversion (four-fold or greater increase of antibodies) was maximized when restricting analysis to 45 participants with baseline sera collected within 5 days of onset and follow-up sera collected 15 or more days after onset; for these participants, 82% and 89% seroconverted to A/California/7/2009 H1N1 by HI and VM respectively. A four-fold or greater increase in cross-reactive antibody titers to seasonal A/Brisbane/59/2007 H1N1, A/Brisbane/10/2007 H3N2 and A/Wisconsin/15/2009 H3N2 occurred in 20%, 18% and 16% of participants respectively. CONCLUSIONS AND SIGNIFICANCE: Appropriately timed paired serology detects 80-90% RT-PCR confirmed H1N1-2009; Antibodies from infection with H1N1-2009 cross-reacted with seasonal influenza viruses.