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In 2018, a 15-year-old female adolescent in Australia was infected with swine influenza A(H3N2) variant virus. The virus contained hemagglutinin and neuraminidase genes derived from 1990s-like human seasonal viruses and internal protein genes from influenza A(H1N1)pdm09 virus, highlighting the potential risk that swine influenza A virus poses to human health in Australia.

As part of its role in the World Health Organization’s (WHO) Global Influenza Surveillance and Response System (GISRS), the WHO Collaborating Centre for Reference and Research on Influenza in Melbourne received a total of 3993 human influenza-positive samples during 2018. Viruses were analysed for their antigenic, genetic and antiviral susceptibility properties. Selected viruses were propagated in qualified cells or hens’ eggs for use as potential seasonal influenza vaccine virus candidates. In 2018, influenza A(H1)pdm09 viruses predominated over influenza A(H3) and B viruses, accounting for a total of 53% of all viruses analysed. The majority of A(H1)pdm09, A(H3) and influenza B viruses analysed at the Centre were found to be antigenically similar to the respective WHO-recommended vaccine strains for the Southern Hemisphere in 2018. However, phylogenetic analysis indicated that a significant proportion of circulating A(H3) viruses had undergone genetic drift relative to the WHO-recommended vaccine strain for 2018. Of 2864 samples tested for susceptibility to the neuraminidase inhibitors oseltamivir and zanamivir, three A(H1)pdm09 viruses showed highly reduced inhibition by oseltamivir, while one B/Victoria virus showed highly reduced inhibition by both oseltamivir and zanamivir.

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.

Background Influenza C is usually considered a minor cause of respiratory illness in humans with many infections being asymptomatic or clinically mild. Large outbreaks can occur periodically resulting in significant morbidity. Objectives This study aimed at analyzing the available influenza C clinical samples from two widely separated states of Australia, collected over a 7-year period and to compare them with influenza C viruses detected in other parts of the world in recent years. Patients/Methods Between 2008 and 2014, 86 respiratory samples that were influenza C positive were collected from subjects with influenza-like illness living in the states of Victoria and Western Australia. A battery of other respiratory viruses were also tested for in these influenza C-positive samples. Virus isolation was attempted on all of these clinical samples, and gene sequencing was performed on all influenza C-positive cultures. Results and conclusions Detections of influenza C in respiratory samples were sporadic in most years studied, but higher rates of infection occurred in 2012 and 2014. Many of the patients with influenza C had coinfections with other respiratory pathogens. Phylogenetic analysis of the full-length hemagglutinin–esterase–fusion (HE) gene found that most of the viruses grouped in the C/Sao Paulo/378/82 clade with the remainder grouping in the C/Kanagawa/1/76 clade.

Influenza A and B viruses are the causative agents of annual influenza epidemics that can be severe, and influenza A viruses intermittently cause pandemics. Sequence information from influenza virus genomes is instrumental in determining mechanisms underpinning antigenic evolution and antiviral resistance. However, due to sequence diversity and the dynamics of influenza virus evolution, rapid and high-throughput sequencing of influenza viruses remains a challenge. We developed a single-reaction influenza A/B virus (FluA/B) multiplex reverse transcription-PCR (RT-PCR) method that amplifies the most critical genomic segments (hemagglutinin [HA], neuraminidase [NA], and matrix [M]) of seasonal influenza A and B viruses for next-generation sequencing, regardless of viral type, subtype, or lineage. Herein, we demonstrate that the strategy is highly sensitive and robust. The strategy was validated on thousands of seasonal influenza A and B virus-positive specimens using multiple next-generation sequencing platforms.

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

Background The isolation and propagation of influenza viruses from clinical specimens are essential tools for comprehensive virologic surveillance. Influenza viruses must be amplified in cell culture for detailed antigenic analysis and for phenotypic assays assessing susceptibility to antiviral drugs or for other assays. Objectives To conduct an external quality assessment (EQA) of proficiency for isolation and identification of influenza viruses using cell culture techniques among National Influenza Centres (NICs) in the World Health Organisation (WHO) South East Asia and Western Pacific Regions. Study design Twenty-one NICs performed routine influenza virus isolation and identification techniques on a proficiency testing panel comprising 16 samples, containing influenza A or B viruses and negative control samples. One sample was used exclusively to determine their capacity to measure hemagglutination titer and the other 15 samples were used for virus isolation and identification. Results All NICs performed influenza virus isolation using Madin Darby canine kidney (MDCK) or MDCK-SIAT-1 cells. If virus growth was detected, the type, subtype and/or lineage of virus present in isolates was determined using immunofluorescence, RT-PCR and/or hemagglutination inhibition (HI) assays. Most participating laboratories could detect influenza virus growth and could identify virus amplified from EQA samples. However, some laboratories failed to isolate and identify viruses from EQA samples that contained lower titres of virus, highlighting issues regarding the sensitivity of influenza virus isolation methods between laboratories. Conclusion This first round of EQA was successfully conducted by NICs in the Asia Pacific Region, revealing good proficiency in influenza virus isolation and identification.

The factors that determine the characteristic seasonality of influenza remain enigmatic. Current models predict that occurrences of influenza outside the normal surveillance season within a temperate region largely reflect the importation of viruses from the alternate hemisphere or from equatorial regions in Asia. To help reveal the drivers of seasonality we investigated the origins and evolution of influenza viruses sampled during inter-seasonal periods in Australia. To this end we conducted an expansive phylogenetic analysis of 9912, 3804, and 3941 hemagglutinnin (HA) sequences from influenza A/H1N1pdm, A/H3N2, and B, respectively, collected globally during the period 2009-2014. Of the 1475 viruses sampled from Australia, 396 (26.8% of Australian, or 2.2% of global set) were sampled outside the monitored temperate influenza surveillance season (1 May – 31 October). Notably, rather than simply reflecting short-lived importations of virus from global localities with higher influenza prevalence, we documented a variety of more complex inter-seasonal transmission patterns including “stragglers” from the preceding season and “heralds” of the forthcoming season, and which included viruses sampled from clearly temperate regions within Australia. We also provide evidence for the persistence of influenza B virus between epidemic seasons, in which transmission of a viral lineage begins in one season and continues throughout the inter-seasonal period into the following season. Strikingly, a disproportionately high number of inter-seasonal influenza transmission events occurred in tropical and subtropical regions of Australia, providing further evidence that climate plays an important role in shaping patterns of influenza seasonality., Author Summary Human influenza virus commonly causes disease in the winter months of temperate countries, but exhibits more complex patterns in tropical localities. Most studies of this complex seasonality have only considered viruses sampled within the “normal” influenza season. To help reveal the drivers of influenza seasonality we utilized viruses sampled outside of the normal influenza season, focusing on Australia which is characterized by a wide range of climates. Using a phylogenetic approach we revealed more complex patterns of influenza transmission than previously anticipated, particularly that the virus is able to transmit for extended periods and even persist locally within Australia throughout the virus “off-season”. In addition, we found that inter-seasonal influenza was more frequent in tropical and sub-tropical than temperate regions, adding weight to theories that climate likely plays an important role in influenza seasonality.

Background Full genome sequencing of influenza A viruses (IAV), including those that arise from annual influenza epidemics, is undertaken to determine if reassorting has occurred or if other pathogenic traits are present. Traditionally IAV sequencing has been biased toward the major surface glycoproteins haemagglutinin and neuraminidase, while the internal genes are often ignored. Despite the development of next generation sequencing (NGS), many laboratories are still reliant on conventional Sanger sequencing to sequence IAV. Objectives To develop a minimal and robust set of primers for Sanger sequencing of the full genome of IAV currently circulating in humans. Study design A set of 13 primer pairs was designed that enabled amplification of the six internal genes of multiple human IAV subtypes including the recent avian influenza A(H7N9) virus from China. Specific primers were designed to amplify the HA and NA genes of each IAV subtype of interest. Each of the primers also incorporated a binding site at its 5′-end for either a forward or reverse M13 primer, such that only two M13 primers were required for all subsequent sequencing reactions. Results This minimal set of primers was suitable for sequencing the six internal genes of all currently circulating human seasonal influenza A subtypes as well as the avian A(H7N9) viruses that have infected humans in China. Conclusions This streamlined Sanger sequencing protocol could be used to generate full genome sequence data more rapidly and easily than existing influenza genome sequencing protocols.

A complex interplay of viral, host, and ecological factors shapes the spatio-temporal incidence and evolution of human influenza viruses. Although considerable attention has been paid to influenza A viruses, a lack of equivalent data means that an integrated evolutionary and epidemiological framework has until now not been available for influenza B viruses, despite their significant disease burden. Through the analysis of over 900 full genomes from an epidemiological collection of more than 26,000 strains from Australia and New Zealand, we reveal fundamental differences in the phylodynamics of the two co-circulating lineages of influenza B virus (Victoria and Yamagata), showing that their individual dynamics are determined by a complex relationship between virus transmission, age of infection, and receptor binding preference. In sum, this work identifies new factors that are important determinants of influenza B evolution and epidemiology., eLife, 4, ISSN:2050-084X