A27L Antibody

What is the A27L protein and why is it significant in poxvirus research?

The A27L gene encodes a 14-kDa protein found in the envelope of intracellular mature virus (IMV) of vaccinia virus, which serves multiple critical functions in the viral lifecycle. This protein is essential for virus-cell attachment, virus-cell fusion, and virus release from cells . It mediates vaccinia virus binding to cell surface heparan sulfate during viral infection . Additionally, A27L is crucial for the formation of extracellular enveloped virus (EEV), a vital infectious form that facilitates virus dissemination within cultured cells and infected animal tissues . The protein's significance in poxvirus research stems from its multifunctional nature and its importance as a target for viral neutralization, making it valuable for understanding virus-host interactions and developing anti-viral strategies.

What are the structural characteristics of the A27L protein that determine antibody binding?

The A27L protein has a complex structural organization with distinct functional domains that influence antibody binding. The protein consists of:

• An N-terminal region (residues 21-30) containing the signal sequence

• A heparan sulfate binding domain (residues 21-33)

• A fusion domain (residues 29-43)

• A central coiled-coil region (residues 43-84) responsible for oligomer formation

• A leucine zipper-like third alpha helix (residues 80-101) that interacts with the A17L gene product to anchor the protein to the IMV envelope

Crystal structure studies of an N-terminal fragment of A27 (residues 21-84) revealed that only the central fragment (residues 47-84) is ordered, suggesting flexibility of the N-terminal GAG binding domain . This structural complexity creates multiple epitopes for antibody recognition, with at least four distinct antibody binding groups identified in research studies . The protein forms homotrimers with an antiparallel arrangement, adding further complexity to antibody interactions .

How do different types of A27L antibodies compare in their binding characteristics?

Research has identified at least four distinct epitope groups (I-IV) of anti-A27L antibodies based on cross-blocking experiments and epitope mapping . Their binding characteristics vary considerably:

Group I antibodies are particularly significant as they bind to a region adjacent to the GAG binding site, potentially interfering with the cellular adhesion function of A27L . Crystal structure analysis of antibody-antigen complexes supports the model that antibodies capable of interfering with the functional activity of the antigen (such as Group I) are more likely to confer protection than those binding at the protein's extremities .

What are the most effective applications for A27L antibodies in poxvirus research?

A27L antibodies have proven effective in multiple experimental applications for poxvirus research:

• ELISA (Enzyme-Linked Immunosorbent Assay): All commercial and laboratory-developed anti-A27L antibodies have demonstrated utility in ELISA applications , making this a primary method for detecting the presence of A27L protein or whole vaccinia virus.

• Western Blot (WB): Most A27L antibodies work effectively in Western blot applications , allowing researchers to identify the 14-kDa protein in viral lysates or recombinant preparations.

• Immunofluorescence (IF) and Immunohistochemistry (IHC): A27L antibodies can be used to visualize vaccinia virus particles and infected cells . In immunofluorescence studies, antibodies to the 14-kDa protein have revealed small dots on infected cells corresponding to individual IMVs .

• Virus Neutralization Assays: Specific antibodies (particularly Group I) can neutralize vaccinia virus in a complement-dependent manner , making them useful for functional studies of viral infectivity.

• Fusion-from-Without Assays: These assays with purified virus have confirmed that the fusion process is mediated by the 14-kDa protein, with specific antibodies capable of blocking this activity .

• Competitive Inhibition Studies: A27L antibodies can be used in competitive inhibition experiments with soluble heparin and synthetic peptides to study virus-cell binding mechanisms .

• In vivo Protection Studies: Some A27L antibodies have demonstrated protective effects against vaccinia virus challenge in animal models, making them valuable for vaccine and therapeutic research .

How can researchers optimize the production and purification of A27L antibodies?

Based on established protocols, researchers can optimize A27L antibody production and purification through the following methodological approach:

• Immunization Strategy:

  • Use recombinant vaccinia virus 14 kDa fusion protein (1-110aa) as the immunogen

  • Implement repeated immunization of rabbits or mice until adequate antibody titer is achieved

  • For monoclonal antibody production, infect mice with a sublethal dose of VACV prior to hybridoma generation

• Purification Methodology:

  • Collect serum from immunized animals

  • Employ protein A/G affinity chromatography for IgG purification

  • For monoclonal antibodies, standard hybridoma technology followed by protein A/G purification has proven effective

• Validation Techniques:

  • Confirm antibody specificity by testing against vaccinia virus and A27L-deletion mutants using immunofluorescence

  • Verify reactivity with recombinant A27L protein by ELISA

  • Test functionality in multiple applications (ELISA, WB, immunofluorescence) to ensure versatility

• Storage Recommendations:

  • For short-term storage (less than six months), maintain at 4°C

  • For longer storage, maintain at -20°C

The production process should be tailored to the specific research needs, particularly considering whether a polyclonal or monoclonal approach is more appropriate for the intended applications.

What controls should be included when using A27L antibodies in immunoassays?

To ensure reliability and specificity when using A27L antibodies in immunoassays, researchers should include the following controls:

• Positive Controls:

  • Vaccinia virus (NYCBOH Strain) lysate or purified virions

  • Recombinant A27L protein (either full-length or truncated versions such as A27L 16-100 or A27L 16-110)

  • Cells infected with wild-type vaccinia virus

• Negative Controls:

  • A27L deletion mutant vaccinia virus

  • Uninfected cell lysates

  • Other poxviruses or non-poxviruses to verify specificity

  • Confirmed non-cross-reactive viruses such as parainfluenza 1-3, RSV, adenovirus, influenza A and B, or HSV 1

• Specificity Controls:

  • Competitive inhibition with soluble A27L protein or specific peptides covering identified epitopes

  • Pre-adsorption of antibody with target antigen

  • Secondary antibody-only controls

• Validation Methods:

  • Cross-blocking ELISA to verify epitope specificity for monoclonal antibodies

  • Western blot to confirm recognition of the expected 14-kDa protein

  • Immunofluorescence patterns (characteristic small dots corresponding to individual IMVs)

For quantitative assays, standard curves using known quantities of recombinant A27L protein should be established to enable accurate quantification of target proteins in experimental samples.

How do the binding characteristics of different A27L antibody epitope groups correlate with their functional properties?

Research has revealed significant correlations between A27L antibody binding characteristics and their functional properties:

The crystal structure analysis of antibody-antigen complexes provides molecular insights into these functional differences :

These structure-function relationships explain why Group I antibodies are neutralizing and protective, while antibodies targeting other epitopes lack these functional properties despite strong binding to the A27L protein .

What mechanisms explain the neutralizing activity of specific A27L antibodies?

The neutralizing activity of specific A27L antibodies, particularly Group I antibodies, operates through several mechanisms:

• Interference with GAG Binding: Group I antibodies bind adjacent to the heparin binding domain (residues 21-33) , potentially blocking the interaction between A27L and cell surface heparan sulfate, which is critical for viral attachment .

• Complement-Dependent Neutralization: Group I antibodies neutralize the mature virion in a complement-dependent manner , suggesting they activate the classical complement pathway upon binding to viral particles.

• Inhibition of Fusion Activity: The A27L protein contains a fusion domain (residues 29-43) , and antibodies binding near this region may interfere with the virus-cell fusion process necessary for viral entry.

• Disruption of Protein-Protein Interactions: By binding to A27L, these antibodies may disrupt critical interactions between A27L and other viral proteins, such as the A17L protein that anchors A27L to the viral membrane .

• Steric Hindrance: Even without directly blocking functional domains, large antibody molecules bound to A27L may create steric hindrance that prevents normal virus-cell interactions.

Experimental evidence for these mechanisms comes from:

• Fusion-from-without assays confirming the role of the 14-kDa protein in the fusion process

• Competitive inhibition studies with soluble heparin and synthetic peptides

• Crystal structure analysis showing antibody binding in relation to functional domains

• In vitro neutralization assays demonstrating complement dependence

The epitope-specific nature of neutralization emphasizes the importance of antibody binding location rather than just binding affinity in determining functional outcomes.

How do mutations in the A27L protein affect antibody recognition and viral function?

Studies with mutant forms of the A27L protein have provided valuable insights into the relationship between protein structure, antibody recognition, and viral function:

• N-terminal Deletion Mutants:

  • A27L protein lacking the first 29 amino acids (14K-A-Δ29) fails to form extracellular enveloped virus (EEV)

  • This mutation eliminates the heparin binding domain (residues 21-33) and fusion domain (residues 29-43)

  • Antibodies targeting these regions cannot bind to the mutant protein

• Cysteine-to-Alanine Mutations:

  • Replacement of cysteine residues at positions 71 and 72 with alanines (14K-A protein) affects disulfide bond formation

  • These mutations do not prevent oligomer formation, as the coiled-coil structure is maintained

  • Antibodies recognizing conformational epitopes may show altered binding to these mutants

• Leucine Zipper Mutations:

  • Point mutation Leu89Ala (14K-A-L89A) affects the leucine zipper domain involved in interaction with the A17L protein

  • This may alter membrane anchoring of the A27L protein

  • Antibodies recognizing the C-terminal region may display different binding patterns

How should researchers design experiments to evaluate the protective efficacy of A27L antibodies?

When evaluating the protective efficacy of A27L antibodies, researchers should implement a comprehensive experimental design that includes:

• In Vitro Neutralization Assays:

  • Test antibody neutralization of vaccinia virus with and without complement to distinguish complement-dependent mechanisms

  • Use plaque reduction assays to quantify neutralization potency

  • Include both mature virion (MV) and extracellular enveloped virus (EEV) forms to assess form-specific neutralization

• Cell Binding Inhibition Studies:

  • Evaluate the ability of antibodies to block viral attachment to target cells

  • Use biotinylated A27L protein in cell binding assays with competitive inhibition by antibodies

  • Compare with heparin inhibition as a positive control for attachment blocking

• Animal Protection Models:

  • SCID mouse model with intravenous infection (as used for testing anti-A27 MAbs)

  • Intranasal challenge model for respiratory infection assessment

  • Measure both morbidity (weight loss, clinical signs) and mortality endpoints

  • Include appropriate dose-ranging studies to determine minimum protective dose

• Comparative Analysis:

  • Compare A27L antibodies with antibodies targeting other viral proteins (e.g., H3 protein antibodies)

  • Test combinations of antibodies targeting different epitopes to assess synergistic effects

  • Include positive control antibodies with established protective efficacy

• Mechanism Investigation:

  • Perform fusion-from-without assays to assess antibody effects on viral fusion

  • Use immunofluorescence to track virus location in the presence of antibodies

  • Evaluate effects on EEV formation using appropriate viral mutants as controls

The experimental design should account for variability in virus strains, cell types, and animal models to ensure robust and translatable findings regarding protective efficacy.

What methodological approaches can distinguish between antibodies targeting different epitopes of A27L?

To differentiate antibodies targeting distinct epitopes of the A27L protein, researchers should employ multiple complementary approaches:

• Cross-Blocking ELISA:

  • Immobilize recombinant A27L protein on plates

  • Pre-incubate with unlabeled candidate antibodies

  • Probe with biotinylated or differently labeled reference antibodies

  • Cluster antibodies based on blocking patterns into distinct epitope groups

• Peptide Mapping:

  • Synthesize overlapping peptides spanning the entire A27L sequence

  • Test antibody binding to each peptide via ELISA

  • Identify the minimal peptide sequence required for binding

  • Distinguish linear epitope-binding antibodies from those requiring conformational epitopes

• Alanine Scanning Mutagenesis:

  • Create a panel of A27L mutants with systematic alanine substitutions

  • Express and purify these mutant proteins

  • Assess antibody binding to each mutant

  • Identify critical residues for antibody recognition

• Structural Analysis:

  • Determine crystal structures of antibody-antigen complexes

  • Identify specific contact residues between antibody and antigen

  • Compare contact patterns between different antibodies

  • Map binding sites onto the three-dimensional structure of A27L

• Functional Assays:

  • Test each antibody in neutralization assays

  • Assess ability to block heparin binding

  • Evaluate inhibition of cell attachment and fusion

  • Correlate functional properties with epitope binding

By integrating data from these approaches, researchers can create a comprehensive epitope map of the A27L protein and classify antibodies according to their binding sites and functional properties, as demonstrated in previous studies identifying four distinct epitope groups .

How can researchers effectively use A27L antibodies to study the mechanisms of poxvirus entry and spread?

To effectively leverage A27L antibodies for studying poxvirus entry and spread mechanisms, researchers should implement the following methodological approaches:

• Viral Attachment Studies:

  • Utilize biotinylated A27L protein in cell binding assays with different cell types

  • Perform competitive inhibition with antibodies and soluble heparin to assess GAG-dependent binding

  • Use antibodies targeting different epitopes to identify critical binding regions

  • Employ cells treated with sodium chlorate to produce undersulfated GAGs as a control system

• Fusion Mechanism Analysis:

  • Conduct fusion-from-without assays using purified virus with and without A27L antibodies

  • Use antibodies binding to different regions to map the fusion domain precisely

  • Combine with synthetic peptides corresponding to the fusion domain (residues 29-43) for competitive studies

  • Visualize fusion events using fluorescent membrane dyes and confocal microscopy

• Viral Trafficking Investigations:

  • Implement live-cell imaging with fluorescently labeled antibodies to track viral particles

  • Use pulse-chase experiments with antibodies to distinguish surface-bound from internalized virus

  • Combine with markers for different cellular compartments to track the viral entry pathway

  • Compare intracellular mature virus (IMV) and extracellular enveloped virus (EEV) trafficking

• Viral Spread Analysis:

  • Utilize antibodies in plaque size reduction assays to assess effects on cell-to-cell spread

  • Employ recombinant viruses expressing fluorescent proteins to visualize spread in real-time

  • Compare antibodies against A27L with those targeting other envelope proteins

  • Test spread in different cell types to assess tissue-specific mechanisms

• Structural-Functional Mapping:

  • Use the crystal structure of A27L in complex with antibodies to guide mutagenesis studies

  • Generate viral mutants with alterations in key A27L functional domains

  • Test these mutants for antibody binding, cell attachment, fusion, and spread

  • Correlate structural features with functional outcomes in the presence of different antibodies

By systematically applying these approaches, researchers can dissect the complex roles of A27L in poxvirus entry and spread, as demonstrated by studies showing its involvement in heparan sulfate binding , membrane fusion , and EEV formation .

How should researchers interpret contradictory results between different antibody-based assays targeting A27L?

When faced with contradictory results between different antibody-based assays targeting A27L, researchers should systematically analyze potential sources of discrepancy:

• Epitope Accessibility Variations:

  • Different assay formats may expose or mask specific epitopes

  • In Western blots (denaturing conditions), conformational epitopes are lost while linear epitopes remain accessible

  • Native conditions in ELISA or immunofluorescence preserve conformational epitopes

  • Solution: Use multiple antibodies targeting different epitopes (Groups I-IV) to obtain comprehensive results

• Oligomerization State Effects:

  • A27L forms homotrimers with antiparallel arrangement

  • Some epitopes may be obscured in the oligomeric state

  • Chemical cross-linking experiments can verify oligomerization state

  • Solution: Compare results using conditions that maintain or disrupt oligomers

• Post-translational Modifications:

  • Different expression systems may yield variations in protein glycosylation or other modifications

  • These modifications can affect antibody recognition

  • Solution: Characterize the A27L protein source using mass spectrometry to identify modifications

• Virus Form Specificity:

  • A27L presentation differs between intracellular mature virus (IMV) and extracellular enveloped virus (EEV)

  • A27L may interact differently with other viral proteins in different virus forms

  • Solution: Specify and compare results between different viral forms

• Methodological Considerations:

When reporting results, researchers should clearly describe the experimental conditions, antibody epitope group, virus form, and assay format to facilitate accurate interpretation and reproducibility of findings.

What are the common pitfalls in using A27L antibodies for virus detection, and how can they be overcome?

Researchers using A27L antibodies for virus detection should be aware of several common pitfalls and implement appropriate solutions:

• Cross-Reactivity Issues:

  • Pitfall: A27L proteins are conserved across orthopoxviruses, leading to potential cross-reactivity

  • Solution: Verify antibody specificity against multiple poxviruses; some A27L antibodies have documented cross-reactivity with monkeypox virus

  • Validation: Test against a panel of related viruses, including vaccinia, variola, monkeypox, and cowpox

• Sensitivity Limitations:

  • Pitfall: A27L is not the most abundant viral protein, potentially limiting detection sensitivity

  • Solution: Implement signal amplification methods such as tyramide signal amplification for immunohistochemistry

  • Alternative: Consider detecting multiple viral antigens simultaneously to increase sensitivity

• Epitope Masking in Clinical Samples:

  • Pitfall: Immune complexes or host factors may mask A27L epitopes in clinical specimens

  • Solution: Include sample pretreatment steps (mild denaturation, pH adjustment) to expose epitopes

  • Validation: Include known positive samples with potentially interfering substances

• Viral Variant Considerations:

  • Pitfall: Mutations in the A27L gene may affect antibody recognition

  • Solution: Use antibodies targeting conserved epitopes or implement multiple antibodies targeting different regions

  • Verification: Sequence the A27L gene from clinical isolates to identify potential variations

• Technical Optimization Requirements:

• Reference Standard Issues:

  • Pitfall: Lack of standardized positive controls across laboratories

  • Solution: Develop and validate quantitative standards using recombinant A27L protein

  • Implementation: Express, purify, and characterize recombinant A27L protein using established protocols

By addressing these potential pitfalls through careful experimental design and validation, researchers can enhance the reliability and reproducibility of virus detection using A27L antibodies.

How can researchers differentiate between vaccinia virus and other orthopoxviruses using A27L antibodies?

Differentiating between vaccinia virus and other orthopoxviruses using A27L antibodies requires strategic approaches to overcome the high conservation of A27L across the orthopoxvirus genus:

• Epitope-Specific Antibody Selection:

  • Identify and target variable regions within the A27L protein that differ between orthopoxviruses

  • Develop monoclonal antibodies against these differential epitopes

  • Some commercial antibodies have documented specificity; for example, PA1-7258 detects vaccinia virus without cross-reacting with other viruses like parainfluenza, RSV, adenovirus, influenza, or HSV

• Competitive Binding Assays:

  • Implement differential competitive ELISA using species-specific peptides

  • Measure antibody binding in the presence of competing peptides from different orthopoxviruses

  • Quantify displacement patterns to identify virus-specific signatures

• High-Resolution Methods:

  • Employ techniques with higher resolution for epitope recognition:

  • Surface plasmon resonance (SPR) to measure binding kinetics and affinities

  • Peptide arrays with single amino acid resolution

  • These methods can detect subtle differences in antibody binding patterns

• Antibody Panel Approach:

  • Use combinations of antibodies targeting multiple viral proteins

  • Create a "fingerprint" pattern specific to each orthopoxvirus

  • Include antibodies against A27L along with other differential markers like B5R and A33R

• Complementary Molecular Methods:

  • Combine antibody detection with PCR or sequencing techniques

  • Target A27L gene regions with known sequence differences between orthopoxviruses

  • Implement multiplex detection systems

• Quantitative Differential Analysis:

By implementing these strategic approaches, researchers can enhance their ability to differentiate between closely related orthopoxviruses despite the high conservation of the A27L protein, ultimately improving diagnostic accuracy and research specificity in orthopoxvirus studies.

What emerging technologies might enhance the specificity and utility of A27L antibodies for poxvirus research?

Several emerging technologies hold promise for enhancing the specificity and utility of A27L antibodies in poxvirus research:

• Single-Domain Antibodies (Nanobodies):

  • Derived from camelid heavy-chain-only antibodies

  • Smaller size (~15 kDa) allows access to cryptic epitopes on A27L

  • Potential for higher specificity and tissue penetration

  • May recognize epitopes inaccessible to conventional antibodies

• Antibody Engineering Approaches:

  • Guided by crystal structures of A27L-antibody complexes

  • Structure-based design of antibodies with enhanced affinity to specific epitopes

  • Development of bispecific antibodies targeting A27L and other viral proteins simultaneously

  • Engineering of antibody fragments (Fab, scFv) for specialized applications

• Advanced Imaging Technologies:

  • Super-resolution microscopy using fluorescently labeled A27L antibodies

  • Single-molecule tracking to monitor real-time virus-cell interactions

  • Correlative light and electron microscopy (CLEM) to visualize virus-antibody interactions at nanometer resolution

  • Live-cell imaging to track viral entry and intracellular trafficking

• High-Throughput Epitope Mapping:

  • Next-generation phage display libraries for comprehensive epitope mapping

  • Microfluidic systems for rapid screening of antibody-antigen interactions

  • Machine learning algorithms to predict novel functional epitopes

  • Massively parallel antibody generation and characterization

• Therapeutic Applications:

  • Development of A27L antibodies as potential therapeutics against orthopoxvirus infections

  • Antibody-drug conjugates targeting A27L for selective delivery of antivirals

  • Combination therapy approaches with antibodies targeting multiple viral proteins

  • Prophylactic applications in high-risk populations

These emerging technologies could significantly advance our understanding of A27L biology and expand the toolkit available for poxvirus research and potential therapeutic interventions.

How might research on A27L antibodies inform the development of next-generation poxvirus vaccines?

Research on A27L antibodies provides crucial insights for the development of next-generation poxvirus vaccines through several mechanisms:

• Epitope-Focused Vaccine Design:

  • Crystal structures of neutralizing antibodies (e.g., Group I) bound to A27L reveal critical protective epitopes

  • These structures can guide the design of vaccines that present these specific epitopes

  • Structural vaccinology approaches could create immunogens that elicit antibodies similar to the protective Group I antibodies

  • Multi-epitope vaccines could include the protective A27L epitopes alongside epitopes from other viral proteins

• Correlates of Protection Studies:

  • Understanding which A27L antibody responses correlate with protection

  • Identification of Group I antibodies as neutralizing and protective provides a measurable correlate

  • Vaccine trials could monitor the development of these specific antibody responses

  • Standardized assays to measure epitope-specific antibody responses could be developed

• Adjuvant Selection and Formulation:

  • Different adjuvants may enhance the production of antibodies targeting specific A27L epitopes

  • Formulations that promote neutralizing antibody responses (Group I-like) would be prioritized

  • Analysis of isotype distribution and functionality of elicited antibodies

  • Optimization for complement-fixing antibodies, as Group I antibodies neutralize in a complement-dependent manner

• Rational Attenuation Strategies:

  • A27L mutants lacking specific functional domains could serve as attenuated vaccine candidates

  • Understanding of A27L's role in viral spread and pathogenesis informs attenuation strategies

  • Mutations that maintain protective epitopes while reducing virulence could be engineered

  • The A27L-A-Δ29 mutant, which fails to form EEV but maintains immunogenicity, represents one potential approach

• Novel Vaccine Platforms:

  • mRNA vaccines encoding optimized A27L sequences

  • Viral vector vaccines expressing A27L alongside other immunogenic proteins

  • Virus-like particles displaying key A27L epitopes

  • DNA vaccines encoding A27L with optimized epitope presentation

By leveraging these insights from A27L antibody research, next-generation poxvirus vaccines could achieve higher efficacy, better safety profiles, and more precise immune responses than traditional vaccines.

What are the key unresolved questions about A27L protein function that could be addressed using antibody-based approaches?

Despite significant advances in understanding A27L protein, several crucial questions remain unresolved that could be addressed using sophisticated antibody-based approaches:

• Dynamic Conformational Changes During Virus Entry:

  • Question: How does A27L protein change conformation during virus attachment and fusion?

  • Antibody Approach: Develop conformation-specific antibodies that recognize distinct states

  • Methodology: Single-molecule FRET combined with antibody labeling to track conformational transitions

  • Significance: Could reveal triggering mechanisms for fusion activation

• Regulation of A27L-Mediated Functions:

  • Question: How are the multiple functions of A27L (attachment, fusion, EEV formation) regulated?

  • Antibody Approach: Domain-specific antibodies that selectively block individual functions

  • Methodology: Generate antibodies against distinct functional domains and assess specific inhibition patterns

  • Significance: Could identify regulatory switches that control distinct A27L activities

• Interactome Mapping:

  • Question: What is the complete set of viral and cellular proteins that interact with A27L?

  • Antibody Approach: Antibody-based pull-down assays followed by mass spectrometry

  • Methodology: Use antibodies against different A27L domains as affinity reagents to isolate protein complexes

  • Significance: Would reveal the broader functional network of A27L in viral replication

• Temporal Dynamics of A27L During Infection:

  • Question: How does A27L localization and function change throughout the viral life cycle?

  • Antibody Approach: Time-course studies with domain-specific antibodies

  • Methodology: Live-cell imaging with fluorescently labeled antibody fragments

  • Significance: Could identify stage-specific functions and regulatory mechanisms

• Host Range Determinants:

  • Question: How does A27L contribute to orthopoxvirus host range and tissue tropism?

  • Antibody Approach: Species-specific binding studies with A27L antibodies

  • Methodology: Compare antibody inhibition patterns across cells from different species and tissues

  • Significance: Could identify determinants of host range restriction