Human CD8+ T cell cross-reactivity across influenza A, B and C viruses.

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.

Australia as a global sink for the genetic diversity of avian influenza A virus.

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.

Strong host phylogenetic and ecological effects on host competency for avian influenza in Australian wild birds.

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.

Predicting Permissive Mutations That Improve the Fitness of A(H1N1)pdm09 Viruses Bearing the H275Y Neuraminidase Substitution.

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. IMPORTANCE Oseltamivir (Tamiflu) is the most widely used antiviral for the treatment of influenza infections. Therefore, resistance to oseltamivir is a public health concern. This study is important as it explores the different evolutionary pathways available to current circulating influenza viruses that may lead to widespread oseltamivir resistance. Specifically, this study develops valuable experimental and computational tools to evaluate the fitness landscape of circulating A(H1N1)pmd09 influenza viruses bearing the H275Y mutation. The H275Y substitution is most commonly reported to confer oseltamivir resistance but also leads to loss of virus replication and transmission fitness, which limits its spread. However, it is known from previous influenza seasons that influenza viruses can evolve to overcome this loss of fitness. Therefore, this study aims to prospectively predict how contemporary A(H1N1)pmd09 influenza viruses may evolve to overcome the fitness cost of bearing the H275Y NA substitution, which could result in widespread oseltamivir resistance.

Induction of Interferon-Stimulated Genes Correlates with Reduced Growth of Influenza A Virus in Lungs after RIG-I Agonist Treatment of Ferrets.

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.

Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model.

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.

Understanding the Impact of Resistance to Influenza Antivirals.

Influenza poses a significant burden on society and health care systems. Although antivirals are an integral tool in effective influenza management, the potential for the emergence of antiviral-resistant viruses can lead to uncertainty and hesitation among front-line prescribers and policy makers. Here, we provide an overview of influenza antiviral resistance in context, exploring the key concepts underlying its development and clinical impact. Due to the acute nature of influenza in immunocompetent patients, resistant viruses that develop during antiviral treatment of a single patient ("treatment-emergent resistance") are usually cleared in a relatively short time, with no impact on future antiviral efficacy. In addition, although available data are limited by small numbers of patients, they show that antiviral treatment still provides clinical benefit to the patient within whom resistance emerges. In contrast, the sustained community transmission of resistant variants in the absence of treatment ("acquired resistance") is of greater concern and can potentially render front-line antivirals ineffective. Importantly, however, resistant viruses are usually associated with reduced fitness such that their widespread transmission is relatively rare. Influenza antivirals are an essential part of effective influenza management due to their ability to reduce the risk of complications and death in infected patients. Although antiviral resistance should be taken seriously and requires continuous careful monitoring, it is not comparable to antibiotic resistance in bacteria, which can become permanent and widespread, with far-reaching medical consequences. The benefits of antiviral treatment far outweigh concerns of potential resistance, which in the vast majority of cases does not have a significant clinical impact.

Retinoic Acid-Inducible Gene I Activation Inhibits Human Respiratory Syncytial Virus Replication in Mammalian Cells and in Mouse and Ferret Models of Infection.

Infections caused by human respiratory syncytial virus (RSV) are associated with substantial rates of morbidity and mortality. Treatment options are limited, and there is urgent need for the development of efficient antivirals. Pattern recognition receptors such as the cytoplasmic helicase retinoic acid-inducible gene (RIG) I can be activated by viral nucleic acids, leading to activation of interferon-stimulated genes and generation of an "antiviral state." In the current study, we activated RIG-I with synthetic RNA agonists (3pRNA) to induce resistance to RSV infection in vitro and in vivo. In vitro, pretreatment of human, mouse, and ferret airway cell lines with RIG-I agonist before RSV exposure inhibited virus infection and replication. Moreover, a single intravenous injection of 3pRNA 1 day before RSV infection resulted in potent inhibition of virus replication in the lungs of mice and ferrets, but not in nasal tissues. These studies provide evidence that RIG-I agonists represent a promising antiviral drug for RSV prophylaxis.

Reducing Influenza Virus Transmission: The Potential Value of Antiviral Treatment.

Prompt antiviral treatment has the potential to reduce influenza virus transmission to close contacts, but rigorous data on the magnitude of treatment effects on transmission are limited. Animal model data indicate that rapid reductions in viral replication after antiviral treatment reduce the risk of transmission. Observational and clinical trial data with oseltamivir and other neuraminidase inhibitors indicate that prompt treatment of household index patients seems to reduce the risk of illness in contacts, although the magnitude of the reported effects has varied widely across studies. In addition, the potential risk of transmitting drug-resistant variants exists with all approved classes of influenza antivirals. A controlled trial examining baloxavir treatment efficacy to reduce transmission, including the risk of transmitting virus with reduced baloxavir susceptibility, is currently in progress. If reduced transmission risk is confirmed, modeling studies indicate that early treatment could have major epidemiologic benefits in seasonal and pandemic influenza.

Reassortment and Persistence of Influenza A Viruses from Diverse Geographic Origins within Australian Wild Birds: Evidence from a Small, Isolated Population of Ruddy Turnstones.

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.IMPORTANCE High prevalence of avian influenza viruses (AIVs) was detected in a small, low-density, isolated population of ruddy turnstones in Australia. Analysis of these viruses revealed relatively recent introductions of viral gene segments from both Eurasia and North America, as well as long-term persistence of introduced gene segments in Australian wild birds. These data demonstrate that the flow of viruses into Australia may be more common than initially thought and that, once introduced, these AIVs have the potential to be maintained within the continent. These findings add to a growing body of evidence suggesting that Australian wild birds are unlikely to be ecologically isolated from the highly pathogenic H5Nx viruses circulating among wild birds throughout the Northern Hemisphere.

Utilising animal models to evaluate oseltamivir efficacy against influenza A and B viruses with reduced in vitro susceptibility.

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.

Baloxavir treatment of ferrets infected with influenza A(H1N1)pdm09 virus reduces onward transmission.

Influenza viruses cause seasonal outbreaks and pose a continuous pandemic threat. Although vaccines are available for influenza control, their efficacy varies each season and a vaccine for a novel pandemic virus manufactured using current technology will not be available fast enough to mitigate the effect of the first pandemic wave. Antivirals can be effective against many different influenza viruses but have not thus far been used extensively for outbreak control. Baloxavir, a recently licensed antiviral drug that targets the influenza virus endonuclease, has been shown to reduce virus shedding more effectively than oseltamivir, a widely used neuraminidase inhibitor drug. Thus it is possible that treatment with baloxavir might also interrupt onward virus transmission. To test this, we utilized the ferret model, which is the most commonly used animal model to study influenza virus transmission. We established a subcutaneous baloxavir administration method in ferrets which achieved similar pharmacokinetics to the approved human oral dose. Transmission studies were then conducted in two different locations with different experimental setups to compare the onward transmission of A(H1N1)pdm09 virus from infected ferrets treated with baloxavir, oseltamivir or placebo to naïve sentinel ferrets exposed either indirectly in adjacent cages or directly by co-housing. We found that baloxavir treatment reduced infectious viral shedding in the upper respiratory tract of ferrets compared to placebo, and reduced the frequency of transmission amongst sentinels in both experimental setups, even when treatment was delayed until 2 days post-infection. In contrast, oseltamivir treatment did not substantially affect viral shedding or transmission compared to placebo. We did not detect the emergence of baloxavir-resistant variants in treated animals or in untreated sentinels. Our results support the concept that antivirals which decrease viral shedding could also reduce influenza transmission in the community.

Baloxavir Marboxil for Uncomplicated Influenza in Adults and Adolescents.

BACKGROUND: Baloxavir marboxil is a selective inhibitor of influenza cap-dependent endonuclease. It has shown therapeutic activity in preclinical models of influenza A and B virus infections, including strains resistant to current antiviral agents. METHODS: We conducted two randomized, double-blind, controlled trials involving otherwise healthy outpatients with acute uncomplicated influenza. After a dose-ranging (10 to 40 mg) placebo-controlled trial, we undertook a placebo- and oseltamivir-controlled trial of single, weight-based doses of baloxavir (40 or 80 mg) in patients 12 to 64 years of age during the 2016-2017 season. The dose of oseltamivir was 75 mg twice daily for 5 days. The primary efficacy end point was the time to alleviation of influenza symptoms in the intention-to-treat infected population. RESULTS: In the phase 2 trial, the median time to alleviation of influenza symptoms was 23.4 to 28.2 hours shorter in the baloxavir groups than in the placebo group (P<0.05). In the phase 3 trial, the intention-to-treat infected population included 1064 patients; 84.8 to 88.1% of patients in each group had influenza A(H3N2) infection. The median time to alleviation of symptoms was 53.7 hours (95% confidence interval [CI], 49.5 to 58.5) with baloxavir, as compared with 80.2 hours (95% CI, 72.6 to 87.1) with placebo (P<0.001). The time to alleviation of symptoms was similar with baloxavir and oseltamivir. Baloxavir was associated with greater reductions in viral load 1 day after initiation of the regimen than placebo or oseltamivir. Adverse events were reported in 20.7% of baloxavir recipients, 24.6% of placebo recipients, and 24.8% of oseltamivir recipients. The emergence of polymerase acidic protein variants with I38T/M/F substitutions conferring reduced susceptibility to baloxavir occurred in 2.2% and 9.7% of baloxavir recipients in the phase 2 trial and phase 3 trial, respectively. CONCLUSIONS: Single-dose baloxavir was without evident safety concerns, was superior to placebo in alleviating influenza symptoms, and was superior to both oseltamivir and placebo in reducing the viral load 1 day after initiation of the trial regimen in patients with uncomplicated influenza. Evidence for the development of decreased susceptibility to baloxavir after treatment was also observed. (Funded by Shionogi; JapicCTI number, 153090, and CAPSTONE-1 ClinicalTrials.gov number, NCT02954354 .).

Global circulation patterns of seasonal influenza viruses vary with antigenic drift.

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.

Treatment-Emergent Influenza Variant Viruses With Reduced Baloxavir Susceptibility: Impact on Clinical and Virologic Outcomes in Uncomplicated Influenza.

BACKGROUND: Single-dose baloxavir rapidly reduces influenza virus titers and symptoms in patients with uncomplicated influenza, but viruses with reduced in vitro susceptibility due to amino acid substitutions at position 38 of polymerase acidic protein (PA/I38X) sometimes emerge. METHODS: We evaluated the kinetics, risk factors, and effects on clinical and virologic outcomes of emergence of PA/I38X-substituted viruses. RESULTS: Viruses containing PA/I38X substitutions were identified 3-9 days after baloxavir treatment in 9.7% (36/370) of patients, of whom 85.3% had transient virus titer rises. Median time to sustained cessation of infectious virus detection was 192, 48, and 96 hours in the baloxavir recipients with PA/I38X-substituted viruses, without PA/I38X-substituted viruses, and placebo recipients, respectively. The corresponding median times to alleviation of symptoms were 63.1, 51.0, and 80.2 hours, respectively. After day 5, symptom increases occurred in 11.5%, 8.0%, and 13.0%, respectively, and in 8.9% of oseltamivir recipients. Variant virus emergence was associated with lower baseline neutralizing antibody titers. CONCLUSIONS: The emergence of viruses with PA/I38X substitutions following baloxavir treatment was associated with transient rises in infectious virus titers, prolongation of virus detectability, initial delay in symptom alleviation, and uncommonly with symptom rebound. The potential transmissibility of PA/I38X-substituted viruses requires careful study. CLINICAL TRIAL REGISTRATION: NCT02954354.

Antarctic Penguins as Reservoirs of Diversity for Avian Avulaviruses.

Wild birds harbor a huge diversity of avian avulaviruses (formerly avian paramyxoviruses). Antarctic penguin species have been screened for avian avulaviruses since the 1980s and, as such, are known hosts of these viruses. In this study, we screened three penguin species from the South Shetland Islands and the Antarctic Peninsula for avian avulaviruses. We show that Adelie penguins (Pygoscelis adeliae) are hosts for four different avian avulavirus species, the recently described avian avulaviruses 17 to 19 and avian avulavirus 10-like, never before isolated in Antarctica. A total of 24 viruses were isolated and sequenced; avian avulavirus 17 was the most common, and phylogenetic analysis demonstrated patterns of occurrence, with different genetic clusters corresponding to penguin age and location. Following infection in specific-pathogen-free (SPF) chickens, all four avian avulavirus species were shed from the oral cavity for up to 7 days postinfection. There was limited shedding from the cloaca in a proportion of infected chickens, and all but one bird seroconverted by day 21. No clinical signs were observed. Taken together, we propose that penguin species, including Antarctic penguins, may be the central reservoir for a diversity of avian avulavirus species and that these viruses have the potential to infect other avian hosts.IMPORTANCE Approximately 99% of all viruses are still to be described, and in our changing world, any one of these unknown viruses could potentially expand their host range and cause epidemic disease in wildlife, agricultural animals, or humans. Avian avulavirus 1 causes outbreaks in wild birds and poultry and is thus well described. However, for many avulavirus species, only a single specimen has been described, and their viral ecology and epidemiology are unknown. Through the detection of avian avulaviruses in penguins from Antarctica, we have been able to expand upon our understanding of three avian avulavirus species (avian avulaviruses 17 to 19) and report a potentially novel avulavirus species. Importantly, we show that penguins appear to play a key role in the epidemiology of avian avulaviruses, and we encourage additional sampling of this avian group.

Genetic variations on 31 and 450 residues of influenza A nucleoprotein affect viral replication and translation.

BACKGROUND: Influenza A viruses cause epidemics/severe pandemics that pose a great global health threat. Among eight viral RNA segments, the multiple functions of nucleoprotein (NP) play important roles in viral replication and transcription. METHODS: To understand how NP contributes to the virus evolution, we analyzed the NP gene of H3N2 viruses in Taiwan and 14,220 NP sequences collected from Influenza Research Database. The identified genetic variations were further analyzed by mini-genome assay, virus growth assay, viral RNA and protein expression as well as ferret model to analyze their impacts on viral replication properties. RESULTS: The NP genetic analysis by Taiwan and global sequences showed similar evolution pattern that the NP backbones changed through time accompanied with specific residue substitutions from 1999 to 2018. Other than the conserved residues, fifteen sporadic substitutions were observed in which the 31R, 377G and 450S showed higher frequency. We found 31R and 450S decreased polymerase activity while the dominant residues (31 K and 450G) had higher activity. The 31 K and 450G showed better viral translation and replication in vitro and in vivo. CONCLUSIONS: These findings indicated variations identified in evolution have roles in modulating viral replication in vitro and in vivo. This study demonstrates that the interaction between variations of NP during virus evolution deserves future attention.

Meta-transcriptomics reveals a diverse antibiotic resistance gene pool in avian microbiomes.

BACKGROUND: Antibiotic resistance is rendering common bacterial infections untreatable. Wildlife can incorporate and disperse antibiotic-resistant bacteria in the environment, such as water systems, which in turn serve as reservoirs of resistance genes for human pathogens. Anthropogenic activity may contribute to the spread of bacterial resistance cycling through natural environments, including through the release of human waste, as sewage treatment only partially removes antibiotic-resistant bacteria. However, empirical data supporting these effects are currently limited. Here we used bulk RNA-sequencing (meta-transcriptomics) to assess the diversity and expression levels of functionally viable resistance genes in the gut microbiome of birds with aquatic habits in diverse locations. RESULTS: We found antibiotic resistance genes in birds from all localities, from penguins in Antarctica to ducks in a wastewater treatment plant in Australia. Comparative analysis revealed that birds feeding at the wastewater treatment plant carried the greatest resistance gene burden, including genes typically associated with multidrug resistance plasmids as the aac(6)-Ib-cr gene. Differences in resistance gene burden also reflected aspects of bird ecology, taxonomy, and microbial function. Notably, ducks, which feed by dabbling, carried a higher abundance and diversity of resistance genes than turnstones, avocets, and penguins, which usually prey on more pristine waters. CONCLUSIONS: These transcriptome data suggest that human waste, even if it undergoes treatment, might contribute to the spread of antibiotic resistance genes to the wild. Differences in microbiome functioning across different bird lineages may also play a role in the antibiotic resistance burden carried by wild birds. In summary, we reveal the complex factors explaining the distribution of resistance genes and their exchange routes between humans and wildlife, and show that meta-transcriptomics is a valuable tool to access functional resistance genes in whole microbial communities.

Serologic Evidence of Exposure to Highly Pathogenic Avian Influenza H5 Viruses in Migratory Shorebirds, Australia.

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.

Untargeted metabolomics analysis of the upper respiratory tract of ferrets following influenza A virus infection and oseltamivir treatment.

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.