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Host susceptibility to parasites is mediated by intrinsic and external factors such as genetics, ecology, age and season. While waterfowl are considered central to the reservoir community for low pathogenic avian influenza A viruses (LPAIV), the role of host phylogeny has received limited formal attention. Herein, we analysed 12 339 oropharyngeal and cloacal swabs and 10 826 serum samples collected over 11 years from wild birds in Australia. As well as describing age and species-level differences in prevalence and seroprevalence, we reveal that host phylogeny is a key driver in host range. Seasonality effects appear less pronounced than in the Northern Hemisphere, while annual variations are potentially linked to El Niño-Southern Oscillation. Our study provides a uniquely detailed insight into the evolutionary ecology of LPAIV in its avian reservoir community, defining distinctive processes on the continent of Australia and expanding our understanding of LPAIV globally.

Intracellular RIG-I receptors represent key innate sensors of RNA virus infection, and RIG-I activation results in the induction of hundreds of host effector genes, including interferon-stimulated genes (ISGs). Synthetic RNA agonists targeting RIG-I have shown promise as antivirals against a broad spectrum of viruses, including influenza A virus (IAV), in both in vitro and mouse models of infection. Herein, we demonstrate that treatment of a ferret airway epithelial (FRL) cell line with a RIG-I agonist rapidly and potently induced expression of a broad range of ISGs and resulted in potent inhibition of growth of different IAV strains. In ferrets, a single intravenous injection of RIG-I agonist was associated with upregulated ISG expression in peripheral blood mononuclear cells and lung tissue, but not in nasal tissues. In a ferret model of viral contact transmission, a single treatment of recipient animals 24 h prior to cohousing with IAV-infected donors did not reduce virus transmission and shedding but did result in reduced lung virus titers 6 days after treatment. A single treatment of the IAV-infected donor animals also resulted in reduced virus titers in the lungs 2 days later. Thus, a single intravenous treatment with RIG-I agonist prior to infection or to ferrets with an established IAV infection can reduce virus growth in the lungs. These findings support further development of RIG-I agonists as effective antiviral treatments to limit the impact of IAV infections, particularly in reducing virus replication in the lower airways. IMPORTANCE RIG-I agonists have shown potential as broad-spectrum antivirals in vitro and in mouse models of infection. However, their antiviral potential has not been reported in outbred animals such as ferrets, which are widely regarded as the gold standard small animal model for human IAV infections. Herein, we demonstrate that RIG-I agonist treatment of a ferret airway cell line resulted in ISG induction and inhibition of a broad range of human influenza viruses. A single intravenous treatment of ferrets also resulted in systemic induction of ISGs, including in lung tissue, and when delivered to animals prior to IAV exposure or to animals with established IAV infection treatment resulted in reduced virus replication in the lungs. These data demonstrate the effectiveness of single RIG-I treatment against IAV in the ferret model and highlight the importance of future studies to optimize treatment regimens and delivery routes to maximize their ability to ameliorate IAV infections.

Most of our understanding of the ecology and evolution of avian influenza A virus (AIV) in wild birds is derived from studies conducted in the northern hemisphere on waterfowl, with a substantial bias towards dabbling ducks. However, relevant environmental conditions and patterns of avian migration and reproduction are substantially different in the southern hemisphere. Through the sequencing and analysis of 333 unique AIV genomes collected from wild birds collected over 15 years we show that Australia is a global sink for AIV diversity and not integrally linked with the Eurasian gene pool. Rather, AIV are infrequently introduced to Australia, followed by decades of isolated circulation and eventual extinction. The number of co-circulating viral lineages varies per subtype. AIV haemagglutinin (HA) subtypes that are rarely identified at duck-centric study sites (H8-12) had more detected introductions and contemporary co-circulating lineages in Australia. Combined with a lack of duck migration beyond the Australian-Papuan region, these findings suggest introductions by long-distance migratory shorebirds. In addition, on the available data we found no evidence of directional or consistent patterns in virus movement across the Australian continent. This feature corresponds to patterns of bird movement, whereby waterfowl have nomadic and erratic rainfall-dependant distributions rather than consistent intra-continental migratory routes. Finally, we detected high levels of virus gene segment reassortment, with a high diversity of AIV genome constellations across years and locations. These data, in addition to those from other studies in Africa and South America, clearly show that patterns of AIV dynamics in the Southern Hemisphere are distinct from those in the temperate north., Competing Interests: The authors have declared that no competing interests exist.

Baloxavir is approved in several countries for the treatment of uncomplicated influenza in otherwise-healthy and high-risk patients. Treatment-emergent viruses with reduced susceptibility to baloxavir have been detected in clinical trials, but the likelihood of widespread occurrence depends on replication capacity and onward transmission. We evaluated the fitness of A/H3N2 and A/H1N1pdm09 viruses with the polymerase acidic (PA) I38T-variant conferring reduced susceptibility to baloxavir relative to wild-type (WT) viruses, using a competitive mixture ferret model, recombinant viruses and patient-derived virus isolates. The A/H3N2 PA/I38T virus showed a reduction in within-host fitness but comparable between-host fitness to the WT virus, while the A/H1N1pdm09 PA/I38T virus had broadly similar within-host fitness but substantially lower between-host fitness. Although PA/I38T viruses replicate and transmit between ferrets, our data suggest that viruses with this amino acid substitution have lower fitness relative to WT and this relative fitness cost was greater in A/H1N1pdm09 viruses than in A/H3N2 viruses., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: • Keiko Baba, Takashi Hashimoto, Shinya Omoto, Takao Shishido and Takeki Uehara are employees of Shionogi & Co. • Klaus Kuhlbusch, Steffen Wildum and Aeron C Hurt are employees of F. Hoffmann-La Roche. • Neil Collinson and Barry Clinch are employees of Roche Products Ltd. • Wendy Barclay has received honoraria from Roche, Sanofi Pasteur and Seqirus. • Leo YY Lee has received honoraria from Roche. • Paulina Koszalka, Jie Zhou, Rubaiyea Farrukee, Rebecca Frise, Edin Mifsud, Monica Galiano and Shahjahan Miah have nothing to disclose.

Highly pathogenic avian influenza (HPAI) H5Nx viruses of the goose/Guangdong/96 lineage continue to cause outbreaks in poultry and wild birds globally. Shorebirds, known reservoirs of avian influenza viruses, migrate from Siberia to Australia along the East-Asian-Australasian Flyway. We examined whether migrating shorebirds spending nonbreeding seasons in Australia were exposed to HPAI H5 viruses. We compared those findings with those for a resident duck species. We screened >1,500 blood samples for nucleoprotein antibodies and tested positive samples for specific antibodies against 7 HPAI H5 virus antigens and 2 low pathogenicity avian influenza H5 virus antigens. We demonstrated the presence of hemagglutinin inhibitory antibodies against HPAI H5 virus clade 2.3.4.4 in the red-necked stint (Calidris ruficolis). We did not find hemagglutinin inhibitory antibodies in resident Pacific black ducks (Anas superciliosa). Our study highlights the potential role of long-distance migratory shorebirds in intercontinental spread of HPAI H5 viruses.

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., (Copyright © 2019. Published by Elsevier B.V.)

Background: Influenza virus circulation is monitored through the Cambodian influenza-like illness (ILI) sentinel surveillance system and isolates are characterized by the National Influenza Centre (NIC). Seasonal influenza circulation has previously been characterized by year-round activity and a peak during the rainy season (June-November)., Objectives: We documented the circulation of seasonal influenza in Cambodia for 2012-2015 and investigated genetic, antigenic, and antiviral resistance characteristics of influenza isolates., Patients/methods: Respiratory samples were collected from patients presenting with influenza-like illness (ILI) at 11 hospitals throughout Cambodia. First-line screening was conducted by the National Institute of Public Health and the Armed Forces Research Institute of Medical Sciences. Confirmation of testing and genetic, antigenic and antiviral resistance characterization was conducted by Institute Pasteur in Cambodia, the NIC. Additional virus characterization was conducted by the WHO Collaborating Centre for Reference and Research on Influenza (Melbourne, Australia)., Results: Between 2012 and 2015, 1,238 influenza-positive samples were submitted to the NIC. Influenza A(H3N2) (55.3%) was the dominant subtype, followed by influenza B (30.9%; predominantly B/Yamagata-lineage) and A(H1N1)pdm09 (13.9%). Circulation of influenza viruses began earlier in 2014 and 2015 than previously described, coincident with the emergence of A(H3N2) clades 3C.2a and 3C.3a, respectively. There was high diversity in the antigenicity of A(H3N2) viruses, and to a smaller extent influenza B viruses, during this period, with some mismatches with the northern and southern hemisphere vaccine formulations. All isolates tested were susceptible to the influenza antiviral drugs oseltamivir and zanamivir., Conclusions: Seasonal and year-round co-circulation of multiple influenza types/subtypes were detected in Cambodia during 2012-2015., (© 2019 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8 + T cells confer cross-protection against IAV strains, however the responses of CD8 + T cells to IBV and ICV are understudied. We investigated the breadth of CD8 + T cell cross-recognition and provide evidence of CD8 + T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8 + T cell epitopes from IBVs that were protective in mice and found memory CD8 + T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8 + T cells displayed tissue-resident memory phenotypes. Notably, CD38 + Ki67 + CD8 + effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8 + T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.

Background: Intravenous zanamivir has been used to treat patients with severe influenza. Because the majority of cases (including immunocompromised patients) require the drug for an extended period of treatment, there is a higher risk that the virus will develop resistance. Therefore, knowing the possible amino acid substitutions that may arise in recently circulating influenza strains under prolonged zanamivir exposure and their impact on antiviral susceptibility is important., Methods: Influenza A(H1N1)pdm09, A(H3N2) and B virus were serially passaged under increasing zanamivir pressure in vitro. Neuraminidase (NA) mutations that arose were introduced into recombinant viruses and the susceptibility to oseltamivir, zanamivir, peramivir and laninamivir was determined. The replication fitness of the recombinant variants was assessed in the ferret., Results: NA mutations E119D (N1 numbering) and E117D (B numbering) were detected in A(H1N1)pdm09 and B (Victoria-lineage) viruses respectively and were associated with reduced susceptibility to all four NA inhibitors. No NA mutations were detected in the A(H3N2) or B (Yamagata-lineage) viruses. In ferrets, the A(H1N1)pdm09 E119D variant caused a lower degree of morbidity and the mutation was found to be unstable with E119 reverted virus detected 4 days post-infection of ferrets with the variant E119D virus. In contrast, the influenza B E117D variant was genetically stable in ferrets, caused a noticeable level of morbidity but had a significant reduction in replication fitness compared to wild-type virus., Conclusions: The NA mutations E119D in influenza A(H1N1)pdm09 and E117D in influenza B viruses that arose under zanamivir pressure conferred resistance to multiple NA inhibitors but had compromised viral replication in ferrets compared to wild-type virus without antiviral drug pressure.

Avian influenza virus (AIV) surveillance in Antarctica during 2013 revealed the prevalence of evolutionarily distinct influenza viruses of the H11N2 subtype in Adélie penguins. Here we present results from the continued surveillance of AIV on the Antarctic Peninsula during 2014 and 2015. In addition to the continued detection of H11 subtype viruses in a snowy sheathbill during 2014, we isolated a novel H5N5 subtype virus from a chinstrap penguin during 2015. Gene sequencing and phylogenetic analysis revealed that the H11 virus detected in 2014 had a >99.1% nucleotide similarity to the H11N2 viruses isolated in 2013, suggesting the continued prevalence of this virus in Antarctica over multiple years. However, phylogenetic analysis of the H5N5 virus showed that the genome segments were recently introduced to the continent, except for the NP gene, which was similar to that in the endemic H11N2 viruses. Our analysis indicates geographically diverse origins for the H5N5 virus genes, with the majority of its genome segments derived from North American lineage viruses but the neuraminidase gene derived from a Eurasian lineage virus. In summary, we show the persistence of AIV lineages in Antarctica over multiple years, the recent introduction of gene segments from diverse regions, and reassortment between different AIV lineages in Antarctica, which together significantly increase our understanding of AIV ecology in this fragile and pristine environment., Importance: Analysis of avian influenza viruses (AIVs) detected in Antarctica reveals both the relatively recent introduction of an H5N5 AIV, predominantly of North American-like origin, and the persistence of an evolutionarily divergent H11 AIV. These data demonstrate that the flow of viruses from North America may be more common than initially thought and that, once introduced, these AIVs have the potential to be maintained within Antarctica. The future introduction of AIVs from North America into the Antarctic Peninsula is of particular concern given that highly pathogenic H5Nx viruses have recently been circulating among wild birds in parts of Canada and the Unites States following the movement of these viruses from Eurasia via migratory birds. The introduction of a highly pathogenic influenza virus in penguin colonies within Antarctica might have devastating consequences., (Copyright © 2016 Hurt et al.)

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 (IC50) 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., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Pigs carry receptors for both avian- and human-adapted influenza viruses and have previously been proposed as a mixing and amplification vessel for influenza. Until now, there has been no investigation of influenza A viruses within feral pigs in Australia. We collected samples from feral pigs in Ramsar listed wetlands of South Australia and demonstrated positive antibodies to influenza A viruses. We propose feral pigs, and their control programs, as an available resource for future surveillance for influenza A viruses.

Understanding the spatiotemporal patterns of emergence and circulation of new human seasonal influenza virus variants is a key scientific and public health challenge. The global circulation patterns of influenza A/H3N2 viruses are well characterized, but the patterns of A/H1N1 and B viruses have remained largely unexplored. Here we show that the global circulation patterns of A/H1N1 (up to 2009), B/Victoria, and B/Yamagata viruses differ substantially from those of A/H3N2 viruses, on the basis of analyses of 9,604 haemagglutinin sequences of human seasonal influenza viruses from 2000 to 2012. Whereas genetic variants of A/H3N2 viruses did not persist locally between epidemics and were reseeded from East and Southeast Asia, genetic variants of A/H1N1 and B viruses persisted across several seasons and exhibited complex global dynamics with East and Southeast Asia playing a limited role in disseminating new variants. The less frequent global movement of influenza A/H1N1 and B viruses coincided with slower rates of antigenic evolution, lower ages of infection, and smaller, less frequent epidemics compared to A/H3N2 viruses. Detailed epidemic models support differences in age of infection, combined with the less frequent travel of children, as probable drivers of the differences in the patterns of global circulation, suggesting a complex interaction between virus evolution, epidemiology, and human behaviour.

Emergence of influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAIs) is sporadic, often follows exposure to NAIs, but occasionally occurs in the absence of NAI pressure. The emergence and global spread in 2007/2008 of A(H1N1) influenza viruses showing clinical resistance to oseltamivir due to neuraminidase (NA) H275Y substitution, in the absence of drug pressure, warrants continued vigilance and monitoring for similar viruses. 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 11,387 viruses collected by WHO-recognized National Influenza Centres (NIC) between May 2012 and May 2013 to determine 50% inhibitory concentration (IC50) data for oseltamivir, zanamivir, peramivir and laninamivir. The data were evaluated using normalized IC50 fold-changes rather than raw IC50 data. Nearly 90% of the 11,387 viruses were from three WHO regions: Western Pacific, the Americas and Europe. Only 0.2% (n=27) showed highly reduced inhibition (HRI) against at least one of the four NAIs, usually oseltamivir, while 0.3% (n=39) showed reduced inhibition (RI). NA sequence data, available from the WHO CCs and from sequence databases (n=3661), were screened for amino acid substitutions associated with reduced NAI susceptibility. Those showing HRI were A(H1N1)pdm09 with NA H275Y (n=18), A(H3N2) with NA E119V (n=3) or NA R292K (n=1) and B/Victoria-lineage with NA H273Y (n=2); amino acid position numbering is A subtype and B type specific. Overall, approximately 99% of circulating viruses tested during the 2012-2013 period were sensitive to all four NAIs. Consequently, these drugs remain an appropriate choice for the treatment and prophylaxis of influenza virus infections., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

ABSTRACT Distinct lineages of avian influenza viruses (AIVs) are harbored by spatially segregated birds, yet significant surveillance gaps exist around the globe. Virtually nothing is known from the Antarctic. Using virus culture, molecular analysis, full genome sequencing, and serology of samples from Adélie penguins in Antarctica, we confirmed infection by H11N2 subtype AIVs. Their genetic segments were distinct from all known contemporary influenza viruses, including South American AIVs, suggesting spatial separation from other lineages. Only in the matrix and polymerase acidic gene phylogenies did the Antarctic sequences form a sister relationship to South American AIVs, whereas distant phylogenetic relationships were evident in all other gene segments. Interestingly, their neuraminidase genes formed a distant relationship to all avian and human influenza lineages, and the polymerase basic 1 and polymerase acidic formed a sister relationship to the equine H3N8 influenza virus lineage that emerged during 1963 and whose avian origins were previously unknown. We also estimated that each gene segment had diverged for 49 to 80 years from its most closely related sequences, highlighting a significant gap in our AIV knowledge in the region. We also show that the receptor binding properties of the H11N2 viruses are predominantly avian and that they were unable to replicate efficiently in experimentally inoculated ferrets, suggesting their continuous evolution in avian hosts. These findings add substantially to our understanding of both the ecology and the intra- and intercontinental movement of Antarctic AIVs and highlight the potential risk of an incursion of highly pathogenic AIVs into this fragile environment. IMPORTANCE Avian influenza viruses (AIVs) are typically maintained and spread by migratory birds, resulting in the existence of distinctly different viruses around the world. However, AIVs have not previously been detected in Antarctica. In this study, we characterized H11N2 viruses sampled from Adélie penguins from two geographically different sites in Antarctica and show that the segmented AIV genome diverged between 49 and 80 years ago from other AIVs, with several genes showing similarity and shared ancestry with H3N8 equine influenza viruses. This study provides the first insight into the ecology of AIVs in Antarctica and highlights the potential risk of an introduction of highly pathogenic AIVs into the continent.

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.

Influenza A H10N7 virus with a hemagglutinin gene of North American origin was detected in Australian chickens and poultry abattoir workers in New South Wales, Australia, in 2010 and in chickens in Queensland, Australia, on a mixed chicken and domestic duck farm in 2012. We investigated their genomic origins by sequencing full and partial genomes of H10 viruses isolated from wild aquatic birds and poultry in Australia and analyzed them with all available avian influenza virus sequences from Oceania and representative viruses from North America and Eurasia. Our analysis showed that the H10N7 viruses isolated from poultry were similar to those that have been circulating since 2009 in Australian aquatic birds and that their initial transmission into Australia occurred during 2007 and 2008. The H10 viruses that appear to have developed endemicity in Australian wild aquatic birds were derived from several viruses circulating in waterfowl along various flyways. Their hemagglutinin gene was derived from aquatic birds in the western states of the United States, whereas the neuraminidase was closely related to that from viruses previously detected in waterfowl in Japan. The remaining genes were derived from Eurasian avian influenza virus lineages. Our analysis of virological data spanning 40 years in Oceania indicates that the long-term evolutionary dynamics of avian influenza viruses in Australia may be determined by climatic changes. The introduction and long-term persistence of avian influenza virus lineages were observed during periods with increased rainfall, whereas bottlenecks and extinction were observed during phases of widespread decreases in rainfall. These results extend our understanding of factors affecting the dynamics of avian influenza and provide important considerations for surveillance and disease control strategies.

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., (Copyright © 2012. Published by Elsevier B.V.)

We investigated carriage of avian influenza viruses by wild birds in Australia, 2005-2008, to assess the risks to poultry industries and human health. We collected 21,858 (7,357 cloacal, 14,501 fecal) samples and detected 300 viruses, representing a detection rate of ≈1.4%. Rates were highest in autumn (March-May) and differed substantially between bird types, areas, and years. We typed 107 avian influenza viruses and identified 19 H5, 8 H7, and 16 H9 (40% of typed viruses). All were of low pathogenicity. These viruses formed clearly different phylogenetic clades to lineages from Eurasia or North America, suggesting the potential existence of Australian lineages. H7 viruses were similar to highly pathogenic H7 strains that caused outbreaks in poultry in Australia. Several periods of increased detection rates (numbers or subtypes of viruses) were identified. This study demonstrates the need for ongoing surveillance to detect emerging pathogenic strains and facilitate prevention of outbreaks.

Background: The rapid diagnosis of influenza can alter the management of a patient's illness, resulting in reduced antibiotic usage, correct use of influenza antivirals and reduced length of stay in hospital emergency departments. The rapid tests have also been used to detect outbreaks in institutions and may play a role in pandemic influenza control., Objectives: To test six different rapid influenza tests, in a head-to-head comparison for the detection of seasonal influenza types A and B, compared to laboratory-based tests., Study Design: One hundred and seventy-seven clinical specimens taken from mostly paediatric patients between June and October 2006 were tested using six influenza diagnostic tests and three laboratory-based techniques (immunofluorescence, cell culture and real-time RT-PCR)., Results and Conclusion: Compared with cell culture, five of the rapid tests (Binax Now Influenza A&B, Directigen EZ Flu A+B, Denka Seiken Quick Ex-Flu, Fujirebio Espline Influenza A&B-N, and Quidel QuickVue Influenza A+B Test) demonstrated a similar influenza A sensitivity of between 67-71% and a specificity of 99-100%, however one rapid test (Rockeby Influenza A Antigen Test) had a significantly lower influenza A sensitivity of only 10% (specificity was 100%). For the five kits that detected influenza B antigen, sensitivity was considerably lower than that seen for influenza A (sensitivity for all the kits was 30%), although the number of specimens containing influenza B viruses was low.

The rapid and accurate detection of influenza A and B in a hospital setting is useful to confirm infection, exclude other diseases and assist in the management of patient illness including the possible use of specific antiviral therapy. We evaluated the use of the Directigen Flu A+B in a paediatric hospital laboratory in comparison with the established diagnostic tests direct immunofluorescence, viral culture and reverse transcriptase-polymerase chain reaction. A total of 193 respiratory specimens were examined and the Directigen test detected positive samples with an 80.8 per cent sensitivity and a specificity of 100 per cent. This study confirms other paediatric studies which have found the Directigen Flu A+B to be less sensitive than traditional laboratory tests but nevertheless to have a potential role in patient management especially when a positive result is obtained.

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.

Two hundred and forty-five human influenza A and B viruses isolated in Australia between 1996 and 2003 were tested for their sensitivity to the NA inhibitor drugs, zanamivir and oseltamivir using a fluorescence-based neuraminidase inhibition assay. Based on mean 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. A comparison of IC50 values for viruses isolated before and after the release of the NA inhibitors in Australia, found there was no significant difference in the sensitivity of strains to either neuraminidase inhibitor and none of the isolates tested showed clinically significant resistance.

Host susceptibility to parasites is mediated by intrinsic and external factors such as genetics, ecology, age and season. While waterfowl are considered central to the reservoir community for low pathogenic avian influenza A viruses (LPAIV), the role of host phylogeny has received limited formal attention. Herein, we analysed 12 339 oropharyngeal and cloacal swabs and 10 826 serum samples collected over 11 years from wild birds in Australia. As well as describing age and species-level differences in prevalence and seroprevalence, we reveal that host phylogeny is a key driver in host range. Seasonality effects appear less pronounced than in the Northern Hemisphere, while annual variations are potentially linked to El Niño–Southern Oscillation. Our study provides a uniquely detailed insight into the evolutionary ecology of LPAIV in its avian reservoir community, defining distinctive processes on the continent of Australia and expanding our understanding of LPAIV globally. [ABSTRACT FROM AUTHOR]

In a post‐hoc analysis of the phase 3 BLOCKSTONE study (JapicCTI‐184180), we investigated household transmission of baloxavir‐resistant (PA/I38X) influenza viruses. Using baloxavir resistance rates from prior clinical trials and the rate of influenza transmission observed in the study, the predicted number of PA/I38X transmission events was 4.8, assuming wild type and PA/I38X viruses were equally transmissible. However, no PA/I38X viruses were observed. These results suggest a low potential for baloxavir‐resistant influenza virus transmission from treated to untreated individuals, potentially due to reduced viral/transmission fitness for PA/I38X viruses and/or low viral titres at the time when resistant viruses arise. [ABSTRACT FROM AUTHOR]

Influenza antivirals are important tools in our fight against annual influenza epidemics and future influenza pandemics. Combinations of antivirals may reduce the likelihood of drug resistance and improve clinical outcomes. Previously, two hospitalised immunocompromised influenza patients, who received a combination of a neuraminidase inhibitor and baloxavir marboxil, shed influenza viruses resistant to both drugs. Here-in, the replicative fitness of one of these A(H1N1)pdm09 virus isolates with dual resistance mutations (NA-H275Y and PA-I38T) was similar to wild type virus (WT) in vitro, but reduced in the upper respiratory tracts of challenged ferrets. The dual-mutant virus transmitted well between ferrets in an airborne transmission model, but was outcompeted by the WT when the two viruses were co-administered. These results indicate the dual-mutant virus had a moderate loss of viral fitness compared to the WT virus, suggesting that while person-to-person transmission of the dual-resistant virus may be possible, widespread community transmission is unlikely. A clinical influenza isolate with dual antiviral resistant mutations displays a moderate loss of viral fitness in ferrets compared to wild-type virus, suggesting widespread community transmission of this dual mutant is unlikely. [ABSTRACT FROM AUTHOR]

It iswell-established that influenza virus infections predispose individuals to secondary bacterial infections (SBIs), which may result in a range of clinical outcomes from relatively mild (e.g. sinusitis and otitis media) to severe (e.g. pneumonia and septicaemia). The most common bacterial pathogen associated with SBI following influenza virus infections is Streptococcus pneumoniae (SPN). Of circulating human seasonal influenza viruses, influenza A viruses (IAV) of both the A(H1N1)pdm09 and A(H3N2) subtypes are associated with severe disease but have differing hospitalisation and complication rates. To study the interplay of these two IAV subtypes with SBI, we used a ferret model of influenza infection followed by secondary challenge with a clinical strain of SPN to determine the severity and the period of susceptibility for SBI. Ferrets challenged with SPN 5 days after infectionwith A(H3N2) or A(H1N1)pdm09 viruses developed severe disease that required euthanasia. When the time between viral infection and bacterial challengewas extended, A/H1N1pdm09-infected animals remained susceptible to SBI- for up to 10 days after the viral infection. For A(H3N2)- but not A(H1N1)pdm09-infected ferrets, susceptibility to SBI-associated disease could be extended out to 16 days postviral infection. While caution should be taken when extrapolating animal models to human infections, the differences between A(H3N2) and A(H1N1)pdm09 strains in duration of susceptibility to SBI observed in the ferret model, may provide some insight regarding the higher rates of SBI-associated disease associated with some strains of A(H3N2) viruses in humans. [ABSTRACT FROM AUTHOR]

Background: Influenza viruses cause significant morbidity and mortality, especially in young children, elderly, pregnant women and individuals with co‐morbidities. Patients with severe influenza disease are typically treated with one neuraminidase inhibitor, oseltamivir or zanamivir. These antivirals need to be taken early to be most effective and often lead to the emergence of drug resistance and/or decreased drug susceptibility. Combining oseltamivir with another antiviral with an alternative mode of action has the potential to improve clinical effectiveness and reduce drug resistance. Methods: In this study, we utilized a host‐targeting molecule RM‐5061, a second‐generation thiazolide, in combination with oseltamivir to determine whether these compounds could reduce viral burden and understand their effects on the immune response to influenza virus infection in mice, compared with either monotherapy or placebo. Results: The combination of RM‐5061 and OST administered for 5 days after influenza infection reduced viral burden at day 5 post‐infection, when compared to placebo and RM‐5061 monotherapy, but was not significantly different from oseltamivir monotherapy. The inflammatory cytokine milieu was also reduced in animals which received a combination therapy when compared to RM‐5061 and placebo‐treated animals. Antiviral treatment in all groups led to a reduction in CD8+ T‐cell responses in the BAL when compared to placebo. Conclusions: To our knowledge, this is the first time a combination of a host‐targeting compound, RM‐5061, and neuraminidase inhibitor, OST, has been tested in vivo. This antiviral combination was safe in mice and led to reduced inflammatory responses following viral infection when compared to untreated animals. [ABSTRACT FROM AUTHOR]

Although seasonal influenza viruses circulate globally, prevention and treatment occur at the level of regions, cities, and communities. At these scales, the timing, duration and magnitude of epidemics vary substantially, but the underlying causes of this variation are poorly understood. Here, based on analyses of a 15-year city-level dataset of 18,250 laboratory-confirmed and antigenically-characterised influenza virus infections from Australia, we investigate the effects of previously hypothesised environmental and virological drivers of influenza epidemics. We find that anomalous fluctuations in temperature and humidity do not predict local epidemic onset timings. We also find that virus antigenic change has no consistent effect on epidemic size. In contrast, epidemic onset time and heterosubtypic competition have substantial effects on epidemic size and composition. Our findings suggest that the relationship between influenza population immunity and epidemiology is more complex than previously supposed and that the strong influence of short-term processes may hinder long-term epidemiological forecasts. Seasonal influenza epidemics vary in timing and size, but the causes of the variation remain unclear. Here, the authors analyse a 15-year city-level data set, and find that fluctuations in climatic factors do not predict onset timing, and that while antigenic change does not have a consistent effect on epidemic size, the timing of onset and heterosubtypic competition do. [ABSTRACT FROM AUTHOR]

Little is known about the factors that shape the ecology of RNA viruses in nature. Wild birds are an important case in point, as other than influenza A virus, avian samples are rarely tested for viruses, especially in the absence of overt disease. Using bulk RNA‐sequencing ("meta‐transcriptomics"), we revealed the viral diversity present in Australian wild birds through the lens of the ecological factors that may determine virome structure and abundance. A meta‐transcriptomic analysis of four Anseriformes (waterfowl) and Charadriiformes (shorebird) species sampled in temperate and arid Australia revealed the presence of 27 RNA virus genomes, 18 of which represent newly described species. The viruses identified included a previously described gammacoronavirus and influenza A viruses. Additionally, we identified novel virus species from the families Astroviridae, Caliciviridae, Reoviridae, Rhabdoviridae, Picobirnaviridae and Picornaviridae. We noted differences in virome structure that reflected underlying differences in location and influenza A infection status. Red‐necked Avocets (Recurvirostra novaehollandiae) from Australia's arid interior possessed the greatest viral diversity and abundance, markedly higher than individuals sampled in temperate Australia. In Ruddy Turnstones (Arenaria interpres) and dabbling ducks (Anas spp.), viral abundance and diversity were higher and more similar in hosts that were positive for influenza A infection compared to those that were negative for this virus, despite samples being collected on the same day and from the same location. This study highlights the extent and diversity of RNA viruses in wild birds and lays the foundation for understanding the factors that determine virome structure in wild populations. [ABSTRACT FROM AUTHOR]

Shipping influenza virus specimens, isolates or purified RNA is normally conducted at ultra-low temperatures using dry ice to ensure minimal degradation of the samples but this is expensive and requires special packaging and shipping conditions. Therefore, alternative methods for shipping influenza viruses or RNA at ambient temperatures would be desirable. The RNASound RNA Sampling Card (FortiusBio LLC, San Diego, CA, USA) is a device that enables specimens or isolates to be applied to a card, whereby viruses are inactivated, while RNA is preserved and purified RNA can also easily be eluted. To evaluate this card, we applied influenza virus cell culture isolate supernatants to either the RNASound card or Whatman Grade No. 1 filter paper (GE Healthcare, Rydalmere, NSW, Australia) and compared the preservation to that of material stored in liquid form. Preservation was tested using influenza A and B viruses at two different storage temperatures [cool (2–8∘C) or room temperature (18–22∘C)] and these were compared with control material stored at -80∘C, for 7, 14, or 28 days. The quality of the RNA recovered was assessed using real time RT-PCR and Sanger sequencing. The RNASound card was effective in preserving influenza RNA at room temperature for up to 28 days, with only a minor change in real-time RT-PCR cycle threshold values for selected gene targets when comparing between viruses applied to the card or stored at -80∘C. Similar results were obtained with filter paper, whilst virus in liquid form performed the worst. Nevertheless, as the RNASound card also has the capability to inactivate viruses in addition to preserving RNA at room temperature for many weeks, this makes it feasible to send samples to laboratories using regular mail, and thus avoid the need for expensive shipping conditions requiring biohazard containers and dry ice. Moreover, the quick and simple RNA recovery from the RNASound card allows recipient labs to obtain RNA without the need for special reagents or equipment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

During the 2007 equine infl uenza outbreak in Australia, respiratory disease in dogs in close contact with infected horses was noted; influenza (H3N8) virus infection was confirmed. Nucleotide sequence of the virus from dogs was identical to that from horses. No evidence of dog-to-dog transmission or virus persistence in dogs was found. [ABSTRACT FROM AUTHOR]

Introduction: Influenza is a highly contagious respiratory disease that causes high global morbidity and mortality each year. The dynamics of an influenza infection on the host metabolism, and how metabolism is altered in response to neuraminidase inhibitor drug therapy, is still in its infancy but of great importance. Objectives: We aim to investigate the suitability of ferret nasal wash samples for metabolomics-based analysis and characterization of influenza infections and oseltamivir treatment. Methods: Virological and metabolic analyses were performed on nasal wash samples collected from ferrets treated with oseltamivir or a placebo. Untargeted metabolomics was performed using a gas chromatography coupled with mass spectrometery (GC-MS) based protocol that comprised a retention time (RT) locked method and the use of a commercial metabolomics library. Results: Ferret activity was reduced at 2–3 days post infection, which coincided with the highest influenza viral titre. The metabolomics data indicated a shift in metabolism during various stages of infection. The neuraminidase inhibitor oseltamivir created considerable downregulation of energy center metabolites (glucose, sucrose, glycine and glutamine), which generated high levels of branched amino acids. This further increased branched amino acid degradation and deregulation via glycerate-type intermediates and biosynthesis of fatty acids in oseltamivir-treated animals where abrogated weight loss was observed. Conclusion: Metabolomics was used to profile influenza infection and antiviral drug treatment in ferrets. This has the potential to provide indicators for the early diagnosis of influenza infection and assess the effectiveness of drug therapies. [ABSTRACT FROM AUTHOR]

The neuraminidase inhibitors zanamivir and oseltamivir are marketed for the treatment and prophylaxis of influenza and have been stockpiled by many countries for use in a pandemic. Although recent surveillance has identified a striking increase in the frequency of oseltamivir-resistant seasonal influenza A (H1N1) viruses in Europe, the United States, Oceania, and South Africa, to date there have been no reports of significant zanamivir resistance among influenza A (H1N1) viruses or any other human influenza viruses. We investigated the frequency of oseltamivir and zanamivir resistance in circulating seasonal influenza A (H1N1) viruses in Australasia and Southeast Asia. Analysis of 391 influenza A (H1N1) viruses isolated between 2006 and early 2008 from Australasia and Southeast Asia revealed nine viruses (2.3%) that demonstrated markedly reduced zanamivir susceptibility and contained a previously undescribed Gln136Lys (Q136K) neuraminidase mutation. The mutation had no effect on oseltamivir susceptibility but caused approximately a 300-fold and a 70-fold reduction in zanamivir and peramivir susceptibility, respectively. The role of the Q136K mutation in conferring zanamivir resistance was confirmed using reverse genetics. Interestingly, the mutation was not detected in the primary clinical specimens from which these mutant isolates were grown, suggesting that the resistant viruses either occurred in very low proportions in the primary clinical specimens or arose during MDCK cell culture passage. Compared to susceptible influenza A (H1N1) viruses, the Q136K mutant strains displayed greater viral fitness than the wild-type virus in MDCK cells but equivalent infectivity and transmissibility in a ferret model. [ABSTRACT FROM AUTHOR]

Oseltamivir-resistant influenza viruses arise due to amino acid mutations in key residues of the viral neuraminidase (NA). These changes often come at a fitness cost; however, it is known that permissive mutations in the viral NA can overcome this cost. This result was observed in former seasonal A(H1N1) viruses in 2007 which expressed the H275Y substitution (N1 numbering) with no apparent fitness cost and lead to widespread oseltamivir resistance. Therefore, this study aims to predict permissive mutations that may similarly enable fit H275Y variants to arise in currently circulating A(H1N1)pdm09 viruses. The first approach in this study utilized in silico analyses to predict potentially permissive mutations. The second approach involved the generation of a virus library which encompassed all possible NA mutations while keeping H275Y fixed. Fit variants were then selected by serially passaging the virus library either through ferrets by transmission or passaging once in vitro. The fitness impact of selected substitutions was further evaluated experimentally. The computational approach predicted three candidate permissive NA mutations which, in combination with each other, restored the replicative fitness of an H275Y variant. The second approach identified a stringent bottleneck during transmission between ferrets; however, three further substitutions were identified which may improve transmissibility. A comparison of fit H275Y variants in vitro and in experimentally infected animals showed a statistically significant correlation in the variants that were positively selected. Overall, this study provides valuable tools and insights into potential permissive mutations that may facilitate the emergence of a fit H275Y A(H1N1)pdm09 variant. [ABSTRACT FROM AUTHOR]

Australian lineages of avian influenza A viruses (AIVs) are thought to be phylogenetically distinct from those circulating in Eurasia and the Americas, suggesting the circulation of endemic viruses seeded by occasional introductions from other regions. However, processes underlying the introduction, evolution and maintenance of AIVs in Australia remain poorly understood. Waders (order Charadriiformes, family Scolopacidae) may play a unique role in the ecology and evolution of AIVs, particularly in Australia, where ducks, geese, and swans (order Anseriformes, family Anatidae) rarely undertake intercontinental migrations. Across a 5-year surveillance period (2011 to 2015), ruddy turnstones (Arenaria interpres) that "overwinter" during the Austral summer in southeastern Australia showed generally low levels of AIV prevalence (0 to 2%). However, in March 2014, we detected AIVs in 32% (95% confidence interval [CI], 25 to 39%) of individuals in a small, low-density, island population 90 km from the Australian mainland. This epizootic comprised three distinct AIV genotypes, each of which represent a unique reassortment of Australian-, recently introduced Eurasian-, and recently introduced American-lineage gene segments. Strikingly, the Australian-lineage gene segments showed high similarity to those of H10N7 viruses isolated in 2010 and 2012 from poultry outbreaks 900 to 1,500 km to the north. Together with the diverse geographic origins of the American and Eurasian gene segments, these findings suggest extensive circulation and reassortment of AIVs within Australian wild birds over vast geographic distances. Our findings indicate that long-term surveillance in waders may yield unique insights into AIV gene flow, especially in geographic regions like Oceania, where Anatidae species do not display regular inter- or intracontinental migration. [ABSTRACT FROM AUTHOR]

Highlights: [•] The influenza virus neuraminidase evolves in an unpredictable fashion. [•] The influenza viral neuraminidases have only one universally conserved epitope which plays crucial roles in virus replication. [•] Monoclonal antibody targeting this epitope inhibits all 9 NA subtypes of virus. [Copyright &y& Elsevier]

Abstract: The post-exposure therapeutic efficacy of injectable peramivir against highly pathogenic avian influenza type A H5N1 was evaluated in mice and in ferrets. Seventy to eighty percent of the H5N1-infected peramivir-treated mice, and 70% in the oseltamivir treated mice survived the 15-day study period, as compared to 36% in control (vehicle) group. Ferrets were infected intranasally with H5N1 followed by treatment with multiple doses of peramivir. In two of three trials, a statistically significant increase in survival over a 16–18 day period resulted from peramivir treatment, with improved survival of 40–64% in comparison to mock-treated or untreated animals. Injected peramivir mitigates virus-induced disease, reduces infectious virus titers in the lungs and brains and promotes survival in ferrets infected intranasally with this highly neurovirulent isolate. A single intramuscular peramivir injection protected mice against severe disease outcomes following infection with highly pathogenic avian influenza and multi-dose treatment was efficacious in ferrets. [Copyright &y& Elsevier]

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]