H3N2 Wisconsin/67/05

What is the phylogenetic classification of A/Wisconsin/67/05(H3N2) relative to other influenza strains?

A/Wisconsin/67/05(H3N2) belongs to group IV in the evolutionary tree of H3N2 viruses from 2002-2005. Phylogenetic analysis reveals that H3N2 viruses from this period can be divided into four major subgroups: group I (2002 isolates), group II (2003 isolates including A/Fujian/411/02), group III (2003-2005 isolates including A/Wellington/1/04 and A/California/7/04), and group IV (2005 isolates including A/Wisconsin/67/05) . The virus represents a continuation in the evolutionary pathway from previous reference strains like A/Panama/2007/99 and A/Fujian/411/02. The NA gene of A/Wisconsin/67/05 and other 2005 isolates clustered in group II of NA phylogeny (corresponding to groups III and IV for HA) .

What unique receptor binding properties distinguish A/Wisconsin/67/05(H3N2) from earlier H3N2 strains?

A/Wisconsin/67/05(H3N2) and related strains exhibit distinctive receptor binding characteristics resulting from substitutions S193F and D225N in the HA receptor binding site . These mutations reduced binding affinity to avian receptors, manifesting as a loss of ability to agglutinate turkey and chicken red blood cells compared to earlier A/Wellington/1/2004-like viruses . Amino acid positions 193 and 225 are critical determinants of receptor specificity in H3 HAs, with position 225 particularly influencing preferences for different sialic acid linkages.

Interestingly, glycan array studies failed to identify clear differences in receptor binding specificities that correlated with the observed differences in hemagglutination patterns, suggesting complex receptor interactions beyond what could be modeled in artificial glycan arrays .

How do passage conditions affect the receptor binding characteristics of A/Wisconsin/67/05(H3N2)?

Passage conditions dramatically influence the receptor binding properties of A/Wisconsin/67/05(H3N2) through selection of different adaptive mutations:

The egg-selected mutations complement the S193F and D225N substitutions present in A/Wisconsin/67/05-like viruses, enhancing their otherwise poor growth in eggs. MDCK-selected NA mutations compensate for reduced HA binding by providing an alternative attachment mechanism . This differential selection between culture systems is critical for researchers to consider when interpreting receptor binding and antigenic data.

What is the significance of the D151G/N/A mutation in the neuraminidase of A/Wisconsin/67/05(H3N2)?

The D151G/N/A substitution in neuraminidase represents a fascinating adaptation observed primarily in MDCK-passaged A/Wisconsin/67/05(H3N2) isolates. This single amino acid change fundamentally alters NA function by conferring receptor binding capability without significantly compromising enzymatic activity . The mutation causes neuraminidase to acquire affinity for sialic acid receptors that are resistant to catalytic cleavage, resulting in NA-dependent, oseltamivir-sensitive hemagglutination of red blood cells .

This phenomenon explains the poor inhibition of hemagglutination by specific antisera observed with MDCK-grown A/Wisconsin/67/05-like viruses. The conservation of aspartic acid at position 151 in natural isolates suggests an important evolutionary role in maintaining complementarity between HA receptor binding and NA enzymatic activity .

How can researchers differentiate between HA-mediated and NA-mediated hemagglutination in experimental systems?

Researchers can differentiate between HA-mediated and NA-mediated hemagglutination by implementing the following methodological approach:

• Perform parallel hemagglutination inhibition (HI) assays with and without oseltamivir carboxylate (neuraminidase inhibitor)

• In assays containing oseltamivir, NA-dependent agglutination is suppressed, restoring the anti-HA specificity of the HI test

• Compare results between the two conditions - differences indicate NA-mediated binding

This modification allows accurate monitoring of antigenic changes in HA without interference from NA-dependent agglutination . Additionally, virus neutralization assays provide complementary data since anti-HA antibodies effectively neutralize virus infectivity regardless of NA-dependent agglutination, making neutralization a more reliable measure of functional immunity .

What are the optimal cell culture systems for A/Wisconsin/67/05(H3N2) propagation and what methodological considerations are critical?

The two primary systems for propagating A/Wisconsin/67/05(H3N2) are MDCK cells and embryonated eggs, each with specific methodological considerations:

For MDCK cell culture:

• Maintain cells in Eagles minimal essential medium supplemented with L-glutamine (2 mM), gentamycin (1%), and trypsin (without fetal calf serum) at 37°C, 5% CO₂

• Be aware that MDCK passage frequently selects for D151G/N/A NA mutations

• For virus titration, use 96-well plates with 90-95% confluent MDCK monolayers

• Calculate TCID₅₀ after 48 hours using the Reed and Muench method

For egg propagation:

• Expect selection of HA mutations (commonly L194P) that increase binding to avian receptors

• These egg-adaptive mutations complement the S193F and D225N substitutions in A/Wisconsin/67/05-like viruses

• Document passage history meticulously as it significantly impacts virus characteristics

The choice of propagation system should be guided by the specific research question, with clear documentation of passage history essential for proper interpretation of results.

What quantitative methods should researchers use to detect and measure A/Wisconsin/67/05(H3N2) in experimental samples?

Several complementary methodologies are available for quantifying A/Wisconsin/67/05(H3N2) in experimental samples:

• Real-time RT-PCR (highest sensitivity and specificity):

  • RNA extraction followed by amplification using specific primers and TaqMan probes

  • Typical thermal cycling: 20 min at 50°C, 15 min at 95°C, then 40 cycles (15 sec at 95°C, 60 sec at 55°C)

  • Quantification via standard curve with known virus copy numbers

• ELISA for viral antigen detection:

  • Coat plates with purified virus or recombinant proteins (typically 1 μg HA/ml)

  • Detect using species-specific antibodies followed by enzyme conjugates

  • Calculate endpoint titers as reciprocal sera dilution of last positive signal

• Virus titration in cell culture:

  • Serial dilutions in MDCK cells to determine TCID₅₀

  • Observe cytopathic effect or detect viral antigen after 48 hours

For A/Wisconsin/67/05(H3N2) specifically, researchers should consider the potential impact of NA D151G/N/A mutations on hemagglutination-based assays and include appropriate controls (such as parallel tests with oseltamivir) when relevant.

What experimental approaches have been used to develop and evaluate vaccines based on A/Wisconsin/67/05(H3N2)?

Multiple experimental approaches have been employed to develop and evaluate A/Wisconsin/67/05(H3N2) vaccines, including innovative DNA vaccine platforms:

• DNA vaccine development methodology:

  • Gene synthesis based on MDCK-cultivated virus sequences

  • Codon optimization for expression in target species (ferrets/humans)

  • Cloning into expression vectors (pWRG7079 or pKCMV) containing:

    • Kozak ribosomal signal sequence

    • Strong constitutive CMV-IE promoter

    • Polyadenylation signals

    • Intron A sequence

• Validation of expression and functionality:

  • In vitro expression verified by radio immunoprecipitation assay

  • Functionality of expressed HA confirmed via hemagglutination assay

• Immune response evaluation:

  • ELISA to measure antibody responses

  • Hemagglutination inhibition assays

  • Viral challenge studies in animal models

These approaches have demonstrated that vaccines based on A/Wisconsin/67/05(H3N2) can induce protective immune responses that may provide cross-protection against other influenza strains when properly designed and administered.

How effective is A/Wisconsin/67/05(H3N2) as a reference strain for studying cross-protective immunity?

A/Wisconsin/67/05(H3N2) serves as a valuable reference strain for investigating cross-protective immunity, particularly in research examining immune responses across influenza subtypes. Studies have utilized this strain alongside pandemic influenza viruses to assess cross-reactivity potential. For example, research has shown that DNA vaccines encoding proteins from the 1918 H1N1 pandemic virus induced protective cross-reactive immune responses in ferrets, with A/Wisconsin/67/05(H3N2) serving as a contemporary comparison strain .

The methodological approach typically involves:

• Immunizing animal models with vaccines based on different influenza strains

• Challenging with heterologous viruses to assess cross-protection

• Evaluating immune responses through multiple assays:

  • Antibody titers (ELISA, hemagglutination inhibition)

  • Viral load quantification post-challenge

  • Clinical sign monitoring

These studies help identify conserved epitopes that might confer broad protection across influenza subtypes, informing the development of universal influenza vaccines with A/Wisconsin/67/05(H3N2) serving as a well-characterized benchmark.

Does A/Wisconsin/67/05(H3N2) demonstrate tropism for human pancreatic cells, and what experimental systems have been used to investigate this?

A/Wisconsin/67/05(H3N2) has been studied for its potential to infect human pancreatic cells, expanding our understanding of influenza's extrapulmonary tropism. Research has employed multiple experimental systems to investigate this property:

• Human pancreatic cell culture models:

  • Established human pancreatic cell lines (insulinoma and pancreatic duct cell lines)

  • Primary cultures of human pancreatic islets

• Infection protocol:

  • Virus stocks serially expanded in MDCK cells prior to use

  • Monitoring for viral replication and cellular effects

• Complementary animal models:

  • Avian models with monitoring of pancreatic function

  • Assessment of hyperglycemia and increased lipase levels as markers of pancreatic involvement

  • Histopathological and immunohistochemical examination of pancreatic tissue on days 8 and 17 post-infection

These studies provide insights into the potential role of seasonal influenza viruses like A/Wisconsin/67/05(H3N2) in pancreatic pathology, expanding our understanding beyond traditional respiratory manifestations of influenza infection.

How can researchers resolve contradictory data when studying A/Wisconsin/67/05(H3N2) in different experimental systems?

Resolving contradictory data when studying A/Wisconsin/67/05(H3N2) requires systematic methodological approaches:

• Genetic verification of virus stocks:

  • Sequence to confirm presence/absence of D151G/N/A mutations in NA

  • Document passage history meticulously

  • Check for adaptive mutations in HA that affect binding properties

• Modified hemagglutination assays:

  • Perform parallel experiments with/without neuraminidase inhibitors

  • Use different red blood cell species to assess binding preferences

  • Include appropriate controls for NA-mediated agglutination

• Complementary functional assays:

  • Combine HI, neutralization, and ELISA data for comprehensive analysis

  • Neutralization assays are particularly valuable as they measure functional inhibition of virus infectivity

• Advanced molecular characterization:

  • Use pyrosequencing or deep sequencing to identify virus subpopulations

  • Quantify proportions of variants with different NA residues at position 151

  • Clinical isolates often contain mixtures of viruses with different NA compositions

By implementing these approaches, researchers can identify the sources of experimental variability and develop a more complete understanding of A/Wisconsin/67/05(H3N2) biology across different experimental systems.

What structure-function relationships in hemagglutinin and neuraminidase determine the unique properties of A/Wisconsin/67/05(H3N2)?

Critical structure-function relationships in A/Wisconsin/67/05(H3N2) surface glycoproteins include:

In hemagglutinin (HA):

• S193F and D225N substitutions in the receptor binding site alter specificity and affinity

• Position 225 particularly influences preferences for different sialic acid linkages

• These mutations reduce binding to avian red blood cells

In neuraminidase (NA):

• D151G/N/A substitution creates dual functionality:

  • Maintains enzymatic activity for receptor destruction

  • Acquires binding capacity for sialic acid receptors resistant to catalytic cleavage

• Position 151 appears crucial for maintaining the complementary balance between HA binding and NA enzymatic activities

These molecular features explain the distinctive phenotypic characteristics of A/Wisconsin/67/05(H3N2), including its altered hemagglutination patterns and the emergence of NA-dependent binding in MDCK-passaged viruses. The high conservation of aspartic acid at position 151 in natural isolates suggests strong evolutionary pressure to maintain the specialized role of NA in receptor destruction rather than binding.

What experimental approaches can elucidate the molecular basis for A/Wisconsin/67/05(H3N2)'s receptor binding characteristics?

Multiple complementary experimental approaches can reveal the molecular basis of A/Wisconsin/67/05(H3N2)'s receptor binding characteristics:

• Reverse genetics:

  • Generate recombinant viruses with specific mutations

  • Evaluate how individual amino acid changes affect receptor binding

  • Create chimeric viruses to map binding determinants

• Hemagglutination assays with modified conditions:

  • Use red blood cells from different species (turkey, guinea pig, chicken)

  • Apply enzymatic treatments to modify sialic acid content

  • Include neuraminidase inhibitors to distinguish HA-mediated from NA-mediated binding

• Glycan array analysis:

  • Screen binding to panels of sialylated glycans

  • Identify specific receptor structures recognized by the virus

  • Note: Previous studies with A/Wisconsin/67/05-like viruses showed limited correlation between glycan binding profiles and hemagglutination patterns

• Structural biology approaches:

  • X-ray crystallography of HA and NA with receptor analogs

  • Cryo-electron microscopy to visualize protein-receptor interactions

  • Molecular dynamics simulations to model binding energetics

These approaches, when used in combination, provide comprehensive insights into the molecular mechanisms underlying the distinct receptor binding properties of A/Wisconsin/67/05(H3N2) and how they influence viral tropism, transmission, and pathogenesis.

How has A/Wisconsin/67/05(H3N2) contributed to our understanding of influenza virus evolution and adaptation?

A/Wisconsin/67/05(H3N2) has made significant contributions to our understanding of influenza virus evolution and adaptation through several key insights:

• Receptor binding adaptations:

  • Demonstrated how S193F and D225N mutations in HA can alter receptor specificity

  • Revealed the balance between immune evasion and receptor binding functionality

  • Illustrated evolutionary trade-offs between antigenic drift and receptor affinity

• NA functional plasticity:

  • Revealed the capacity of NA to acquire receptor binding function through D151G/N/A mutations

  • Demonstrated how a single amino acid change can fundamentally alter protein function

  • Highlighted the importance of D151 in restricting NA specificity to complement HA function

• Host adaptation mechanisms:

  • Showed different adaptive pathways in different culture systems (eggs vs. MDCK cells)

  • Illustrated how passage conditions can select for distinct viral subpopulations

  • Provided insights into constraints on influenza evolution in different host environments

These findings have enhanced our understanding of influenza virus adaptation mechanisms and the molecular determinants of host range, with implications for surveillance, vaccine development, and pandemic preparedness.

What lessons from A/Wisconsin/67/05(H3N2) research can be applied to current challenges in influenza surveillance and vaccine development?

Research on A/Wisconsin/67/05(H3N2) has yielded valuable lessons applicable to current challenges in influenza surveillance and vaccine development:

• Antigen characterization methodology:

  • Include neuraminidase inhibitors in hemagglutination inhibition assays when characterizing H3N2 viruses

  • Use multiple assay systems (HI, neutralization, ELISA) for comprehensive antigenic analysis

  • Consider both HA and NA contributions to viral antigenicity

• Vaccine production considerations:

  • Be aware of passage-dependent mutations that may alter viral properties

  • Understand how growth substrate (eggs vs. cell culture) affects vaccine strain characteristics

  • Consider alternative platforms like DNA vaccines that avoid adaptive mutations during production

• Surveillance implications:

  • Monitor both HA and NA sequences for mutations affecting receptor binding

  • Recognize that minor viral subpopulations may have significant functional impacts

  • Use deep sequencing to detect mixed viral populations in clinical samples

• Cross-protection strategies:

  • Design vaccines targeting conserved epitopes identified through comparative studies

  • Consider multivalent approaches that induce broader immunity

  • Evaluate cross-protective potential against diverse influenza strains

These lessons highlight the importance of comprehensive characterization of influenza viruses and adapting methodologies to account for the unique properties of each strain, ultimately improving our ability to monitor antigenic drift and develop effective vaccines.