H3N2 Wisconsin

What is the A/Wisconsin/67/2005 (H3N2) strain?

A/Wisconsin/67/2005 (H3N2) is a specific influenza virus strain that has been extensively used in influenza research, particularly in transmission and vaccine studies. This strain represents a significant H3N2 variant that emerged in the mid-2000s and has served as an important reference strain for subsequent H3N2 evolution . The strain has been utilized in human challenge models to assess person-to-person transmission dynamics and has contributed to our understanding of influenza pathogenesis . The virus possesses specific hemagglutinin (HA) and neuraminidase (NA) proteins that determine its binding characteristics, transmissibility, and antigenic properties, making it valuable for comparative studies with more recent H3N2 variants .

How is H3N2 Wisconsin isolated and propagated in laboratory settings?

Isolation and propagation of A/Wisconsin/67/2005 (H3N2) typically employs standard virological techniques with specific considerations. For experimental studies, the virus is often generated using reverse genetics systems. The hemagglutinin (HA) and neuraminidase (NA) genes are cloned into bidirectional plasmid backbones such as pHW2000 to facilitate virus generation . The complete process involves:

• Gene cloning into appropriate expression vectors (e.g., pHW2000)

• Transfection into suitable cell lines

• Harvesting of recombinant virus

• Verification of viral identity through sequencing

• Titration to determine infectious potential

The Wisconsin strain is typically propagated in mammalian cell culture systems rather than eggs to minimize adaptive mutations, particularly in the HA protein that might alter receptor binding properties . When conducting passage experiments, researchers should carefully document any potential mutations that emerge, particularly at key sites like NA position 151, which can significantly impact viral properties .

What are the key genetic characteristics of H3N2 Wisconsin that distinguish it from other H3N2 variants?

The A/Wisconsin/67/2005 (H3N2) strain possesses distinct genetic characteristics that differentiate it from both earlier and later H3N2 variants:

• Hemagglutinin (HA) Properties: The Wisconsin HA exhibits intermediate receptor-binding properties compared to earlier strains like A/Hong Kong/2/1968 and later strains like A/Hanoi/Q118/2007 . These differences in the HA affect how the virus interacts with various sialic acid receptors on host cells.

• Neuraminidase (NA) Characteristics: The Wisconsin strain's NA lacks the polymorphism at position 151 (D151G) that became more common in later H3N2 isolates . This position is significant as it can switch between receptor-cleaving (D151) and receptor-binding (G151) functions.

• Evolutionary Position: The Wisconsin strain represents an important transitional variant in H3N2 evolution, positioned between early strains that had robust receptor binding and later strains that demonstrate reduced affinity for certain sialic acid receptors .

These genetic characteristics make the Wisconsin strain particularly valuable for studying how evolutionary changes in influenza viruses affect transmission dynamics and host adaptation .

What cell culture systems are recommended for studying H3N2 Wisconsin?

For optimal experimental work with A/Wisconsin/67/2005 (H3N2), researchers should consider the following cell culture systems:

• MDCK Cells: Standard Madin-Darby Canine Kidney cells are commonly used for propagation and experimental studies with H3N2 Wisconsin .

• MDCK-SIAT1 Cells: These cells overexpress human α-2,6-sialyltransferase and provide improved growth conditions for human influenza viruses, potentially reducing selective pressure for adaptive mutations .

• Human Airway Epithelial Cells: For more physiologically relevant studies, primary human airway epithelial cells or organoid systems can be employed, though these are more technically demanding.

When analyzing influenza sequence data from databases, it's crucial to consider the passage history, as cell culture adaptations can significantly impact viral genotypes. Analysis of 15,079 sequences from the Global Initiative on Sharing All Influenza Data (GISAID) EpiFlu database showed distinct patterns of polymorphisms (particularly at NA position 151) depending on whether viruses were egg-passaged, cell-culture-passaged, or unpassaged .

How do receptor binding properties of H3N2 Wisconsin compare to contemporary H3N2 strains?

The receptor binding properties of A/Wisconsin/67/2005 (H3N2) show important differences from more contemporary H3N2 strains, which has implications for research applications:

• Binding Affinity: Wisconsin/2005 exhibits stronger receptor binding compared to later strains like A/Hanoi/Q118/2007, which demonstrates the evolutionary trend of reduced sialic acid receptor affinity in more recent human H3N2 viruses .

• Role in Viral Cooperation: When Wisconsin/2005 HA was used in experiments examining cooperation between NA variants (D151 and G151), the cooperative effect observed with later strain HAs was eliminated . This indicates that the Wisconsin HA possesses sufficient binding activity that it doesn't require additional binding support from NA variants.

• Experimental Implications: For researchers, these properties make Wisconsin/2005 valuable for comparative studies examining how receptor binding changes affect transmission, pathogenesis, and evolutionary dynamics .

The binding properties also influence experimental design decisions, as contemporary H3N2 strains may require different approaches for successful propagation and challenge models compared to the Wisconsin strain .

What methodologies have been employed to study H3N2 Wisconsin transmission in humans?

Human challenge models have provided valuable insights into A/Wisconsin/67/2005 (H3N2) transmission. A notable proof-of-concept study demonstrated the following methodological approach:

• Subject Selection: Healthy adults were screened for serological susceptibility to A/H3N2/Wisconsin/67/2005.

• Challenge Design:

  • "Donor" subjects were intranasally inoculated with the virus

  • When symptoms developed, "Recipient" subjects were exposed to Donors

  • Exposure occurred through close contact (playing games, eating meals together) for 28 hours over a 2-day period

• Outcome Assessment:

  • Infection status was confirmed through multiple sampling methods

  • 78% (7/9) of Donors became infected following inoculation

  • 20% (3/15) of Recipients became infected following exposure to Donors

This methodology established that experimentally induced A/Wisconsin/67/2005 infection is transmissible between humans in controlled settings, providing a valuable model for studying transmission dynamics .

Additional methodologies have examined environmental factors in transmission:

• Surface swabs around infected individuals revealed 38% of subject locations were contaminated

• Room air sampling detected viral RNA in 42% of air samples, including in particles ≤4μm that could reach the distal lung

How do viral shedding patterns affect H3N2 Wisconsin transmission dynamics?

Detailed studies of A/Wisconsin/67/2005 (H3N2) viral shedding have revealed important patterns relevant to transmission dynamics:

• Duration of Shedding:

  • Mean shedding duration: 6.2 days by PCR detection

  • Mean shedding duration: 4.6 days by viral culture

  • Over 25% of cases remained potentially infectious for at least 5 days

• Correlation with Symptoms:

  • Symptom scores and viral shedding were poorly correlated

  • This suggests asymptomatic or mildly symptomatic individuals may still transmit efficiently

• Environmental Contamination:

  • Subjects with higher nasal viral loads on day 3 of illness produced significantly more environmental contamination

  • Subjects with more prominent respiratory symptoms also showed increased environmental contamination

• Aerosol Generation:

  • Viral RNA was detected in room air samples in 42% of subjects

  • Particles small enough to reach distal lung (≤4μm) contained viral RNA

  • This supports both contact and aerosol transmission routes

These findings have important implications for infection control strategies and experimental design when working with H3N2 Wisconsin and related strains.

How do cooperative interactions between viral variants affect H3N2 studies?

Research with A/Wisconsin/67/2005 (H3N2) has revealed complex cooperative interactions between viral variants that significantly impact experimental outcomes:

• NA Polymorphism Effects: Studies have identified a critical polymorphism at position 151 in neuraminidase (NA) that produces distinct variants:

  • D151 variant: Maintains normal receptor-cleaving activity

  • G151 variant: Gains receptor-binding activity but loses cleaving function

• Strain-Specific Cooperation:

  • In viruses with later H3N2 HAs (like A/Hanoi/Q118/2007), mixed populations of D151 and G151 NA variants show enhanced fitness compared to either variant alone

  • When the same NA variants were tested with Wisconsin/2005 HA, this cooperative effect was eliminated

• Experimental Implications:

  • The absence of cooperation with Wisconsin/2005 HA suggests its stronger receptor binding eliminates the advantage of G151 NA binding

  • This strain-specific interaction demonstrates how HA evolution influences viral population dynamics and adaptation

• Methodological Considerations:

  • Researchers should genotype NA position 151 when working with H3N2 strains

  • Mixed populations may emerge during passage and affect experimental results

  • Single-virus isolates may behave differently than the mixed viral populations that often exist in nature

This research illustrates the importance of considering viral quasispecies dynamics when designing and interpreting experiments with H3N2 Wisconsin and other influenza strains.

What immunological responses are elicited by H3N2 Wisconsin compared to newer vaccine approaches?

The immunological responses to A/Wisconsin/67/2005 (H3N2) have been compared with newer vaccine approaches, particularly the M2SR (M2-deficient single replication) vaccine platform:

• Antibody Responses:

  • Traditional responses to Wisconsin H3N2 typically focus on strain-specific hemagglutination inhibition (HAI) antibodies

  • Newer M2SR approaches induce broader responses, including:

    • Microneutralization (MN) antibodies against drifted strains

    • Mucosal antibody responses

    • Cellular immune responses

• Cross-Reactivity:

  • Studies with M2SR H3N2 vaccines showed significant cross-reactivity:

    • Single high-dose (10^9 TCID50) intranasal M2SR vaccine induced ≥2-fold increases in MN antibodies against drifted strain (Belgium2015) in 80.6% of subjects

    • This cross-reactive response is significantly higher than typically seen with traditional vaccines

• Response Profile Comparison:

• Comparative Advantage:

  • The high proportion of serosusceptible adults responding to intranasal M2SR (71% HAI seroconversion) contrasts sharply with the reported response rate for FluMist in similar populations (10%; 95% CI: 8-11%)

These findings illustrate how newer vaccine approaches targeting H3N2 can generate broader and more robust immune responses compared to traditional approaches, with potential implications for protection against drifted strains.

What biosafety considerations are specific to H3N2 Wisconsin research?

Research with A/Wisconsin/67/2005 (H3N2) requires careful attention to biosafety, particularly when conducting studies that may involve:

• Human Challenge Studies:

  • Require clinical protocols approved by institutional review boards

  • Must follow Good Clinical Practice principles based on the Declaration of Helsinki

  • Necessitate informed consent from all participants

  • Need clear inclusion/exclusion criteria (e.g., screening for serological susceptibility)

• Gain-of-Function Research:

  • Modification of H3N2 Wisconsin may trigger additional oversight

  • Institutional Biosafety Committees (IBCs) play a crucial role in risk assessment

  • Disagreements about risk levels may occur between institutions and funding agencies

  • Case example: The University of Wisconsin faced controversy regarding influenza research safety assessments, with the National Institute of Allergy and Infectious Diseases (NIAID) overruling the university's initial risk assessment in one case

• Environmental Contamination Control:

  • Studies have demonstrated that H3N2 Wisconsin can contaminate surfaces (38% of subject locations)

  • Viable virus can be recovered from surfaces, though at low rates (0.3% of samples)

  • Air sampling has detected viral RNA in 42% of room air samples

  • These findings support the need for comprehensive decontamination protocols

• Laboratory Passage Considerations:

  • Cell culture passage can select for mixed viral populations, particularly at NA position 151

  • Careful genotyping is recommended to monitor potential adaptive mutations

  • Passage history should be meticulously documented

Researchers should consult current institutional and national guidelines, as requirements may evolve based on emerging understanding of risks and benefits.

How can H3N2 Wisconsin be used in experimental vaccine evaluation?

A/Wisconsin/67/2005 (H3N2) serves as a valuable platform for experimental vaccine evaluation through several methodological approaches:

• Human Challenge Models:

  • Wisconsin H3N2 has been successfully used in human challenge studies

  • This allows direct assessment of vaccine efficacy against controlled viral challenge

  • Protocol example: screen for serological susceptibility, vaccinate, then challenge with controlled intranasal inoculation

• Immunological Correlate Identification:

  • Studies with newer vaccine technologies like M2SR have identified that:

    • ≥2-fold increases in microneutralization (MN) antibodies against drifted strains correlate with protection

    • This marker likely represents broader immune activation beyond serum antibodies

• Cross-Protection Assessment:

  • Wisconsin H3N2 serves as a reference point for evaluating protection against drifted strains

  • Example methodology from M2SR studies:

    • Vaccinate with H3N2 antigen

    • Measure responses against both homologous (vaccine) and heterologous (drifted) strains

    • Assess multiple immune parameters (serum antibodies, mucosal responses, cellular immunity)

• Immune Response Measurement Protocol:

  • Serum antibody assessment:

    • Hemagglutination inhibition (HAI) assays (seroconversion defined as ≥4-fold increase)

    • Microneutralization (MN) assays (response defined as ≥2-fold increase)

  • Mucosal immunity: measure IgA in nasal secretions

  • Cellular immunity: assess T-cell responses via appropriate assays

This multifaceted approach enables comprehensive evaluation of vaccine candidates, particularly those designed to provide broader protection against drifted influenza strains.