Report on influenza viruses received and tested by the Melbourne WHO Collaborating Centre for Reference and Research on Influenza in 2017

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 record total of 5866 human influenza positive samples during 2017. Viruses were analysed for their antigenic, genetic and antiviral susceptibility properties and were propagated in qualified cells and hens' eggs for use as potential seasonal influenza vaccine virus candidates. In 2017, influenza A(H3) viruses predominated over influenza A(H1)pdm09 and B viruses, accounting for a total of 54% 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 2017. However, phylogenetic analysis indicated that the majority of circulating A(H3) viruses had undergone genetic drift relative to the WHO recommended vaccine strain for 2017. Of 3733 samples tested for susceptibility to the neuraminidase inhibitors oseltamivir and zanamivir, only two A(H1)pdm09 viruses and one A(H3) virus showed highly reduced inhibition by oseltamivir, while just one A(H1)pdm09 virus showed highly reduced inhibition by zanamivir.

A simplified Sanger sequencing method for full genome sequencing of multiple subtypes of human influenza A viruses.

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.

Acute emergence and reversion of influenza A virus quasispecies within CD8+ T cell antigenic peptides.

Influenza A virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) provide a degree of cross-strain protection that is potentially subverted by mutation. Here we describe the sequential emergence of such variants within CTL epitopes for a persistently infected, immunocompromised infant. Further analysis in immunodeficient and wild-type mice supports the view that CTL escape variants arise frequently in influenza, accumulate with time and revert in the absence of immune pressure under MHCI-mismatched conditions. Viral fitness, the abundance of endogenous CD8(+) T cell responses and T cell receptor repertoire diversity influence the nature of these de novo mutants. Structural characterization of dominant escape variants shows how the peptide-MHCI interaction is modified to affect variant-MHCI stability. The mechanism of influenza virus escape thus looks comparable to that recognized for chronic RNA viruses like HIV and HCV, suggesting that immunocompromised patients with prolonged viral infection could have an important part in the emergence of influenza quasispecies.

The recent establishment of North American H10 lineage influenza viruses in Australian wild waterfowl and the evolution of Australian avian influenza viruses.

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.

The use of pyrosequencer-generated sequence-signatures to identify the influenza B-lineage and the subclade of the B/Yamataga-lineage viruses from currently circulating human influenza B viruses.

BACKGROUND: Influenza B viruses belong to two antigenically and genetically distinct lineages which co-circulate in varying proportions in many countries. OBJECTIVE: To develop simple, rapid, accurate and robust methods to detect and differentiate currently circulating B-lineage viruses in respiratory samples and virus isolates. STUDY DESIGN: Haemagglutinin (HA) gene sequences from more than 6300 influenza B strains were analysed to identify signature sequences that could be used to distinguish between B-lineages and sublineages. RESULTS: Pyrosequencing and a real time PCR assays were developed to detect the major B-lineages (B/Victoria/2/87 or B/Yamagata/16/88) and pyrosequencing for a unique mutation was used to further differentiate the B/Yamagata viruses into two currently co-circulating subgroups. More than 300 influenza virus-containing samples, including original specimens, cell and egg grown viruses, were tested with a 100% accuracy. Furthermore, when the same PCR primers were used in an rRT-PCR assay, the two lineages could be differentiated by their distinct ranges of melting temperature with an overall accuracy of 99% for 158 samples tested. CONCLUSIONS: These new pyrosequencing and rRT-PCR methods have the potential to aid the rapid identification of influenza B-lineages for surveillance purposes and to increase the available data for bi-annual selection of viruses for updating influenza vaccines.

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

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

Influenza virus A (H10N7) in chickens and poultry abattoir workers, Australia.

In March 2010, an outbreak of low pathogenicity avian influenza A (H10N7) occurred on a chicken farm in Australia. After processing clinically normal birds from the farm, 7 abattoir workers reported conjunctivitis and minor upper respiratory tract symptoms. Influenza virus A subtype H10 infection was detected in 2 workers.

Transmission of influenza A(H1N1) 2009 pandemic viruses in Australian swine.

BACKGROUND: Swine have receptors for both human and avian influenza viruses and are a natural host for influenza A viruses. The 2009 influenza A(H1N1) pandemic (H1N1pdm) virus that was derived from avian, human and swine influenza viruses has infected pigs in various countries. OBJECTIVES: To investigate the relationship between the H1N1pdm viruses isolated from piggery outbreaks in Australia and human samples associated with one of the outbreaks by phylogenetic analysis, and to determine whether there was any reassortment event occurring during the human-pig interspecies transmission. METHODS: Real-time RT-PCR and full genome sequencing were carried out on RNA isolated from nasal swabs and/or virus cultures. Phylogenetic analysis was performed using the Geneious package. RESULTS: The influenza H1N1pdm outbreaks were detected in three pig farms located in three different states in Australia. Further analysis of the Queensland outbreak led to the identification of two distinct virus strains in the pigs. Two staff working in the same piggery were also infected with the same two strains found in the pigs. Full genome sequence analysis on the viruses isolated from pigs and humans did not identify any reassortment of these H1N1pdm viruses with seasonal or avian influenza A viruses. CONCLUSIONS: This is the first report of swine infected with influenza in Australia and marked the end of the influenza-free era for the Australian swine industry. Although no reassortment was detected in these cases, the ability of these viruses to cross between pigs and humans highlights the importance of monitoring swine for novel influenza infections.

Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in Oceania, South East Asia and South Africa.

The neuraminidase inhibitors (NAIs) are an effective class of antiviral drugs for the treatment of influenza A and B infections. Until recently, only a low prevalence of NAI resistance (<1%) had been detected in circulating viruses. However, surveillance in Europe in late 2007 revealed significant numbers of A(H1N1) influenza strains with a H274Y neuraminidase mutation that were highly resistant to the NAI oseltamivir. We examined 264 A(H1N1) viruses collected in 2008 from South Africa, Oceania and SE Asia for their susceptibility to NAIs oseltamivir, zanamivir and peramivir in a fluorescence-based neuraminidase inhibition assay. Viruses with reduced oseltamivir susceptibility were further analysed by pyrosequencing assay. The frequency of the oseltamivir-resistant H274Y mutant increased significantly after May 2008, resulting in an overall proportion of 64% (168/264) resistance among A(H1N1) strains, although this subtype represented only 11.6% of all isolates received during 2008. H274Y mutant viruses demonstrated on average a 1466-fold reduction in oseltamivir susceptibility and 527-fold reduction in peramivir sensitivity compared to wild-type A(H1N1) viruses. The mutation had no impact on zanamivir susceptibility. Ongoing surveillance is essential to monitor how these strains may spread or persist in the future and to evaluate the effectiveness of treatments against them.

Neuraminidase inhibitor-resistant and -sensitive influenza B viruses isolated from an untreated human patient.

An influenza B virus from an infant with no history of treatment or contact with neuraminidase inhibitors demonstrated a significant reduction in sensitivity to these drugs. Here, we describe the analysis of a mixed viral population that contained a novel D197E amino acid substitution that was responsible for this reduction.