Perth 16/09

What is A/Perth/16/2009 and why is it significant in influenza research?

A/Perth/16/2009 is an influenza A virus of the H3N2 subtype that was isolated in Perth, Australia in 2009. It gained prominence when it was selected as the H3N2 component of the 2010-2011 seasonal influenza vaccine. This strain serves as an important reference point for monitoring antigenic drift and evolution of H3N2 viruses. Researchers use A/Perth/16/2009 as a benchmark when characterizing newer circulating strains to assess genetic and antigenic changes that may impact vaccine effectiveness. The strain belongs to a specific evolutionary lineage within H3N2 viruses that has since diversified into multiple clades with varying degrees of antigenic similarity to the original isolate .

How is the A/Perth/16/2009 strain classified within influenza taxonomy?

A/Perth/16/2009 follows the standard influenza virus naming convention: A indicates the type (influenza A virus), Perth indicates the geographic location of first isolation, 16 is the isolate number, and 2009 is the year of isolation. It belongs to the H3N2 subtype, where H3 refers to the hemagglutinin surface protein and N2 to the neuraminidase surface protein. Within the evolutionary context of H3N2 viruses, A/Perth/16/2009 represents a specific clade that was predominant during the 2009-2010 period. Phylogenetic analyses show that A/Perth/16/2009 subsequently gave rise to the Victoria/208/2009 clade, which became more prevalent in later seasons. Genetic classification of H3N2 viruses is continuously updated as new variants emerge through antigenic drift .

What reagents are available for studying A/Perth/16/2009?

Several standardized reagents are available for studying A/Perth/16/2009, including:

• Influenza Anti-A/Perth/16/09-like HA serum (such as NIBSC code: 10/182), prepared specifically for single radial diffusion assay of A/Perth/16/09-like antigens. This antiserum reagent was prepared in sheep (designated as 526, 527, and 528) to the purified HA of A/Victoria/210/09 (NYMCX-187), which is antigenically similar to A/Perth/16/09 .

• Recombinant A/Perth/16/09 proteins synthesized from cDNA sequences encoding the viral proteins. These can be used for various applications including serological assays and structural studies .

• Reference viruses grown in embryonated chicken eggs or cell culture systems that maintain the antigenic characteristics of the original isolate.

These reagents enable consistent analysis across different laboratories and are essential for standardized characterization of influenza viruses in research and surveillance settings.

How does one perform antigenic characterization of influenza isolates compared to A/Perth/16/2009?

Antigenic characterization of influenza isolates compared to A/Perth/16/2009 is commonly performed using the hemagglutination inhibition (HI) assay. The methodology involves:

• Preparation of standardized amounts of virus (typically 4 hemagglutination units)

• Incubation with serial dilutions of post-infection ferret antisera against A/Perth/16/2009

• Addition of 0.5% v/v turkey red blood cells

• Reading of HI titers, defined as the reciprocal of the highest dilution of serum that completely inhibits hemagglutination

An eightfold or greater reduction in HI titer compared to the reference A/Perth/16/2009 strain is generally considered significant and indicative of antigenic drift. This methodology allows researchers to quantify how antigenically distant a new isolate is from the reference strain. The HI assay is the gold standard for antigenic characterization in influenza surveillance networks globally and guides decisions on vaccine strain updates. The specific procedure used by reference laboratories includes careful standardization of reagents and turkey red blood cells to ensure reproducible results across different testing sites .

What techniques are used to analyze the genetic relationship between circulating H3N2 strains and A/Perth/16/2009?

The genetic relationship between circulating H3N2 strains and A/Perth/16/2009 is primarily analyzed through several complementary molecular techniques:

These analyses help track the evolution of influenza viruses over time and identify emerging variants that may necessitate updates to vaccine strains.

How can structural modeling enhance our understanding of A/Perth/16/2009 hemagglutinin antigenic sites?

Structural modeling of the A/Perth/16/2009 hemagglutinin provides valuable insights into the three-dimensional configuration of antigenic sites and how mutations might affect antibody recognition. The methodology involves:

• Template selection: Identifying appropriate crystal structures of related H3 hemagglutinins, such as A/Aichi/2/1968 (Protein Data Bank accession number 3HMG).

• Homology modeling: Using computational tools to generate a structural model of A/Perth/16/2009 HA based on the template structure.

• Mutation mapping: Visualizing the locations of amino acid substitutions in circulating variants relative to A/Perth/16/2009, particularly focusing on the five recognized antigenic sites (A-E) of H3 hemagglutinin.

• Epitope analysis: Assessing how specific mutations might alter antibody binding sites through changes in surface topology, charge distribution, or glycosylation patterns.

• Visualization: Using molecular visualization software such as PyMOL to generate interactive three-dimensional models that can be manipulated to examine specific regions of interest.

This approach allows researchers to predict how genetic changes might translate to altered antigenicity and helps explain serological results from hemagglutination inhibition assays. Studies have shown that mutations across all five H3 antigenic sites can be observed when comparing evolved strains to A/Perth/16/2009, with significant implications for immune recognition .

What approaches can be used to optimize single radial diffusion assays for accurate quantification of A/Perth/16/09-like antigens?

Optimization of single radial diffusion (SRD) assays for A/Perth/16/09-like antigens requires careful attention to multiple parameters:

• Antibody selection and standardization: Use of high-quality antisera specifically developed for A/Perth/16/09-like antigens, such as the NIBSC reagent 10/182. This antiserum is prepared from sheep immunized with purified HA of A/Victoria/210/09 (NYMCX-187), which is antigenically similar to A/Perth/16/09 .

• Agarose gel preparation:

  • Optimal agarose concentration (typically 1%)

  • Uniform antibody distribution within the gel

  • Consistent gel thickness (typically 1.5mm)

  • Controlled temperature during gel preparation

• Antigen preparation:

  • Standardized detergent treatment (typically Zwittergent 3-14) to release and expose HA proteins

  • Proper controls including reference antigens

  • Preparation of a dilution series for standard curves

• Assay conditions:

  • Controlled temperature during incubation (typically 20-22°C)

  • Optimal incubation time (18-24 hours)

  • Humidity control to prevent gel drying

• Analysis:

  • Precise measurement of precipitation ring diameters

  • Standard curve construction

  • Statistical analysis of technical replicates

The SRD assay is particularly valuable for quantitative analysis of influenza antigens in vaccine preparations and can complement other methods such as ELISA and hemagglutination assays when properly optimized with strain-specific reagents.

How does one map and analyze mutations in the antigenic sites of H3N2 viruses that evolved from the A/Perth/16/2009 lineage?

Mapping and analyzing mutations in the antigenic sites of H3N2 viruses evolved from the A/Perth/16/2009 lineage involves a comprehensive approach:

• Sequence alignment: Multiple sequence alignment of HA genes from circulating isolates with the A/Perth/16/2009 reference sequence to identify amino acid substitutions.

• Antigenic site mapping: Classification of mutations according to their location within the five recognized antigenic sites of H3 hemagglutinin (sites A-E), based on their positions in the protein sequence.

• Frequency analysis: Determination of mutation prevalence across geographical regions and time periods to identify emerging patterns.

• Phenotypic impact assessment: Correlation of specific mutations with changes in antigenic properties as measured by hemagglutination inhibition assays.

• Structural context evaluation: Interpretation of mutations in the context of the three-dimensional structure of HA to understand their potential impact on antibody binding.

Research has documented mutations at 28 different amino acid positions across all five H3 antigenic sites when comparing evolved strains to A/Perth/16/2009. Individual isolates typically exhibited 5-11 mutations across these antigenic sites, with several mutational patterns defining specific genetic groups within the Victoria/208/2009 clade that emerged from the A/Perth/16/2009 lineage .

What methodologies are employed to predict whether emerging H3N2 variants will necessitate an update from the A/Perth/16/2009 vaccine strain?

Prediction of whether emerging H3N2 variants necessitate a vaccine strain update involves multiple integrated approaches:

• Antigenic cartography: Mathematical modeling of hemagglutination inhibition data to visualize antigenic distances between strains in a two-dimensional map. Strains clustering far from A/Perth/16/2009 may indicate a need for vaccine updates.

• Serological cross-reactivity studies: Evaluating whether antisera raised against A/Perth/16/2009 effectively neutralize emerging variants, with an 8-fold or greater reduction in HI titer generally suggesting significant antigenic drift.

• Genetic cluster analysis: Identifying emerging clades with characteristic mutations and increased prevalence across geographic regions. For example, the emergence of the Victoria/208/2009 clade, and subsequently the Hong Kong/2121/2010 subclade (defined by mutations D53N, Y94H, I230V, and E280A), signaled significant evolution away from the A/Perth/16/2009 strain .

• Vaccine effectiveness monitoring: Analysis of clinical and epidemiological data to assess how well A/Perth/16/2009-based vaccines protect against circulating strains.

• Epitope-specific antibody escape analysis: Evaluation of mutations in key antibody binding sites to predict changes in immune recognition.

These complementary approaches inform the biannual WHO vaccine strain selection process, which aims to ensure optimal match between vaccine strains and circulating viruses.

What are the key genetic changes observed in H3N2 viruses that evolved from the A/Perth/16/2009 lineage?

The key genetic changes observed in H3N2 viruses that evolved from the A/Perth/16/2009 lineage reveal distinct evolutionary patterns:

Analysis of Ontario isolates from 2010-2011 revealed that 93% fell within the Victoria/208/2009 clade, with only 7% remaining in the Perth/16/2009 clade. Within the Victoria/208/2009 clade, four recognizable genetic groups emerged based on specific non-synonymous substitutions. Most notably, three of these genetic groups (II, III, and IV) representing 86% of isolates clustered with the Hong Kong/2121/2010 subclade, indicating rapid evolution away from the vaccine strain .

How do amino acid substitutions in the hemagglutinin of evolved H3N2 variants impact their antigenicity compared to A/Perth/16/2009?

Amino acid substitutions in hemagglutinin significantly impact the antigenicity of evolved H3N2 variants compared to A/Perth/16/2009:

Research has documented mutations at 28 different amino acid positions across all five H3 antigenic sites when comparing evolved strains to A/Perth/16/2009. The cumulative effect of multiple mutations across different antigenic sites contributes to reduced recognition by antibodies generated against A/Perth/16/2009. Strains with 8-fold or greater reductions in hemagglutination inhibition titers are considered antigenically distinct enough to potentially compromise vaccine effectiveness .

What is the relationship between genetic groups within the Victoria/208/2009 clade and their antigenic properties?

The relationship between genetic groups within the Victoria/208/2009 clade and their antigenic properties reveals important patterns in H3N2 evolution:

Phylogenetic analysis shows that the Victoria/208/2009 clade rapidly became dominant, with 93% of isolates in the 2010-2011 season belonging to this clade rather than the vaccine strain clade. Within this clade, the Hong Kong/2121/2010 subclade (defined by mutations D53N, Y94H, I230V, and E280A) emerged as the predominant evolutionary branch, containing 86% of the analyzed isolates. This rapid genetic diversification was accompanied by antigenic changes that reduced cross-reactivity with A/Perth/16/2009 antisera, highlighting the continuous challenge of maintaining vaccine effectiveness against evolving influenza viruses .

How can researchers overcome challenges in the antigenic characterization of A/Perth/16/2009-like viruses?

Researchers face several challenges in antigenic characterization of A/Perth/16/2009-like viruses and can employ these methodological solutions:

The use of standardized reagents, such as the NIBSC Influenza Anti-A/Perth/16/09-like HA serum (code: 10/182), specifically developed for assaying A/Perth/16/09-like antigens, is crucial for reliable antigenic characterization. This antiserum is prepared under controlled conditions in sheep to the purified HA of A/Victoria/210/09, which is antigenically similar to A/Perth/16/09 .

What considerations are important when using recombinant A/Perth/16/2009 proteins in research applications?

When using recombinant A/Perth/16/2009 proteins in research applications, several important considerations must be addressed:

Recombinant A/Perth/16/2009 proteins provide valuable tools for various applications, from antigenic characterization to vaccine development. A typical approach involves constructing a cDNA sequence encoding the Perth 16/09 protein of interest and using it to recombinantly synthesize the protein in an appropriate expression system . Careful attention to these considerations ensures that the recombinant proteins accurately represent the properties of the native viral proteins.

What lessons from the A/Perth/16/2009 strain have informed current influenza surveillance and vaccine development strategies?

The evolution of H3N2 viruses from the A/Perth/16/2009 strain has provided valuable insights that continue to influence influenza surveillance and vaccine development:

• Enhanced surveillance sensitivity: The rapid emergence of the Victoria/208/2009 clade and subsequent Hong Kong/2121/2010 subclade demonstrated the need for more frequent and geographically diverse sampling to detect novel variants earlier.

• Integration of genetic and antigenic data: The experience with A/Perth/16/2009 highlighted the importance of combining sequence analysis with antigenic characterization to fully understand virus evolution. The observation that 93% of isolates had shifted to the Victoria/208/2009 clade within a single season, with specific genetic groups defined by mutations like I192T, S199A, and I140V, underscores how quickly influenza viruses can evolve .

• Improved structural modeling: The analysis of mutations across the five antigenic sites of H3 hemagglutinin has led to better understanding of structure-function relationships and more accurate prediction of which mutations will impact antigenicity.

• Reagent standardization: Development of reference reagents like the NIBSC Influenza Anti-A/Perth/16/09-like HA serum (code: 10/182) has facilitated more consistent antigenic characterization across different laboratories .

• Accelerated vaccine strain selection: The rapid evolution observed with A/Perth/16/2009 has informed more agile approaches to vaccine strain updates, with increased attention to emerging genetic clades that show evidence of antigenic drift.

These lessons continue to shape our approach to influenza surveillance and vaccine development, with the ultimate goal of improving the match between vaccine strains and circulating viruses to maximize public health impact.

What are the priorities for future research building on our understanding of A/Perth/16/2009 and its evolved variants?

Future research priorities building on our understanding of A/Perth/16/2009 and its evolved variants should focus on several key areas:

• Comprehensive epitope mapping: Detailed characterization of antibody binding sites on the A/Perth/16/2009 hemagglutinin and how they are affected by specific mutations observed in evolved variants.

• Improved predictive models: Development of computational approaches that can more accurately predict which emerging genetic variants will cause significant antigenic drift.

• Cross-protective immunity: Investigation of immune responses that might provide protection against both A/Perth/16/2009 and its drift variants, informing universal vaccine approaches.

• Long-term evolutionary patterns: Analysis of how H3N2 evolution from A/Perth/16/2009 to subsequent strains fits into broader patterns of influenza virus evolution and antigenic drift.

• Enhanced surveillance methods: Development of more sensitive approaches to detect and characterize emerging variants before they become widespread.

• Improved vaccine platforms: Exploration of new vaccine technologies that might provide broader protection against diverse H3N2 variants or allow faster updates when new strains emerge.