Recombinant African swine fever virus Uncharacterized membrane protein KP93L/DP93R (BA71V-002)

What is the basic structural composition of KP93L/DP93R (BA71V-002)?

KP93L/DP93R is a 93-amino acid uncharacterized membrane protein encoded by the African swine fever virus (ASFV). The full amino acid sequence is: MFFFGIFRCNMDHWTTKRQVYIYLCFSLMTIALICYLIHICCHTKKNVVTNALPSNNMALIPYTPSNNMALIPYTPSNNTVPPPYTISGSCPQ . The protein contains a putative transmembrane domain (approximately amino acids 5-25), consistent with its classification as a membrane protein. Recombinant versions of this protein are typically produced with an N-terminal His-tag to facilitate purification and experimental manipulation .

Where are the KP93L and DP93R genes located in the ASFV genome?

The KP93L and DP93R genes are strategically positioned within the terminal inverted repeat (TIR) regions of the ASFV genome. These genes represent an interesting genomic phenomenon wherein they are perfectly repeated, although inverted, at both ends of the genome in some ASFV strains . In the BA71V strain, there is a block of three RTD33 direct tandem repeats that originated from the duplication of one of the two RDT33 repeats found in the virulent BA71 strain . This structural arrangement suggests potential functional significance in viral replication or pathogenesis that remains to be fully elucidated.

How does the recombinant protein differ from the native viral protein?

The recombinant KP93L/DP93R protein available for research (catalog number RFL21122AF) is produced in E. coli expression systems with an N-terminal His-tag . This differs from the native viral protein in several important ways:

• Post-translational modifications: The bacterial expression system lacks the eukaryotic machinery for certain post-translational modifications that may occur in mammalian cells during ASFV infection.

• Protein folding environment: The bacterial cytoplasm provides a different folding environment than the endoplasmic reticulum of mammalian cells where viral membrane proteins are typically processed.

• His-tag addition: The recombinant protein contains an N-terminal His-tag that is not present in the native viral protein, which may subtly alter protein conformation or interaction capability.

• Buffer conditions: The recombinant protein is supplied in a Tris/PBS-based buffer with 6% trehalose at pH 8.0, which differs from the native viral environment .

These differences should be considered when designing experiments to investigate the protein's functional properties.

What are the optimal storage and handling conditions for the recombinant KP93L/DP93R protein?

For optimal preservation of recombinant KP93L/DP93R protein activity, researchers should follow these evidence-based protocols:

• Initial storage: Store the lyophilized powder at -20°C to -80°C upon receipt .

• Reconstitution: Before opening, briefly centrifuge the vial to bring contents to the bottom. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Working aliquots: Add glycerol to a final concentration of 5-50% (standard is 50%) and divide into small working aliquots to prevent repeated freeze-thaw cycles, which significantly reduce protein stability .

• Short-term storage: Working aliquots can be stored at 4°C for up to one week .

• Long-term storage: For extended storage periods, keep aliquots at -20°C to -80°C .

The presence of trehalose (6%) in the storage buffer enhances protein stability during freeze-thaw cycles by preventing protein aggregation and maintaining native conformation.

What experimental approaches are most effective for studying membrane protein localization in ASFV-infected cells?

For investigating KP93L/DP93R localization in infected cells, researchers should employ a multi-technique approach:

• Confocal microscopy with fluorescent antibodies: Using anti-His antibodies for recombinant protein studies or generating specific antibodies against KP93L/DP93R. Co-localization with established subcellular markers (e.g., calnexin for ER, GM130 for Golgi) can reveal trafficking patterns throughout infection.

• Subcellular fractionation and Western blotting: Physical separation of cellular components followed by immunoblotting can quantitatively determine the protein's distribution across different membrane compartments.

• Electron microscopy with immunogold labeling: Provides nanometer-scale resolution of protein localization in relation to viral and cellular structures.

• Live-cell imaging with fluorescent protein fusions: While technically challenging with ASFV, this approach can reveal dynamic trafficking patterns during viral replication.

Recent studies on other ASFV membrane proteins have demonstrated that transmembrane domain-containing proteins often display specific localization patterns during infection that correlate with their function in viral replication . When designing such experiments for KP93L/DP93R, researchers should consider the timing of protein expression during the viral life cycle, as determined through time-course experiments.

How can researchers accurately assess potential post-translational modifications of KP93L/DP93R?

Based on recent findings regarding other ASFV proteins, potential post-translational modifications (PTMs) of KP93L/DP93R should be systematically investigated using these approaches:

• Palmitoylation analysis: Given that several ASFV proteins have been demonstrated to undergo palmitoylation , researchers should employ metabolic labeling with palmitate analogs (e.g., 17-ODYA) followed by click chemistry and detection. The transmembrane domain and proximity of cysteine residues in KP93L/DP93R are consistent with potential palmitoylation sites.

• Mass spectrometry (MS): High-resolution MS combined with enrichment strategies for specific PTMs can provide comprehensive PTM mapping. For KP93L/DP93R, special attention should be given to:

  • Phosphorylation sites (particularly serine and threonine residues)

  • Glycosylation (N-linked and O-linked sites)

  • Ubiquitination or SUMOylation (regulatory modifications)

• Site-directed mutagenesis: Following identification of putative modification sites, targeted mutagenesis can confirm functional relevance. For instance, if C18 is identified as a potential palmitoylation site (similar to other ASFV proteins ), C18S mutants can determine the functional significance of this modification.

• Inhibitor studies: Employing specific inhibitors of PTM-catalyzing enzymes during viral infection can reveal the importance of modifications for proper protein localization and function.

A recent study with ASFV protein CP123L demonstrated that palmitoylation at C18 was critical for membrane association and function . Similar approaches could reveal whether KP93L/DP93R undergoes comparable modifications.

What is currently known about the function of KP93L/DP93R in the ASFV life cycle?

The function of KP93L/DP93R remains largely uncharacterized despite genomic analysis across multiple ASFV strains. Current evidence suggests several hypotheses regarding its potential roles:

• Genome organization and replication: The presence of KP93L/DP93R in the terminal inverted repeat (TIR) regions of the ASFV genome suggests it may play a role in genome organization, replication, or packaging . The variation in repeat structure between virulent BA71 and attenuated BA71V strains (two versus three RTD33 direct tandem repeats) indicates potential involvement in virulence mechanisms .

• Membrane modification: As a predicted membrane protein, KP93L/DP93R may participate in viral factory formation or modification of host cell membranes during infection. Many ASFV membrane proteins contribute to viral assembly compartments.

• Host-pathogen interactions: The protein may mediate interactions with host cell factors, potentially modulating cellular responses to infection.

Despite these hypotheses, functional studies specifically targeting KP93L/DP93R are notably absent from the literature. This represents a significant knowledge gap that merits investigation, particularly given the conservation of this gene across ASFV isolates and its potential relevance to viral pathogenesis.

How does KP93L/DP93R expression differ between virulent and attenuated ASFV strains?

Comparative genomic analyses between virulent field isolates (such as BA71) and attenuated laboratory strains (such as BA71V) reveal important differences in the KP93L/DP93R genomic region:

• Repeat structure variation: In the virulent BA71 strain, there are two RDT33 repeats in the TIR region, while the attenuated BA71V strain contains a block of three RTD33 direct tandem repeats that originated from duplication of one of the two repeats in BA71 . This structural change affects the KP93L/DP93R genes located in this region.

• Conservation pattern: All European isolates of ASFV whose genome sequences include the TIR region maintain the BA71 structure rather than the BA71V structure , suggesting the BA71V variant represents a laboratory adaptation rather than a common field variant.

• Expression level differences: While specific expression data comparing virulent and attenuated strains is limited, the genomic differences suggest potential alterations in expression levels or production of variant protein forms between strains.

This variation between virulent and attenuated strains suggests that changes in KP93L/DP93R structure or expression may contribute to attenuation, although direct experimental evidence linking these changes to virulence is currently lacking. Studies examining protein expression levels in cells infected with different ASFV strains would help clarify this relationship.

What interactions have been identified between KP93L/DP93R and other viral or host proteins?

• Viral structural proteins: KP93L/DP93R may interact with other ASFV membrane proteins or structural components during viral assembly. Proximity ligation assays or co-immunoprecipitation experiments could identify such interactions.

• Host cell membrane proteins: As a viral membrane protein, KP93L/DP93R may interact with host proteins in the endoplasmic reticulum, Golgi apparatus, or plasma membrane. Mass spectrometry-based interactome analysis following affinity purification could reveal such interaction partners.

• Immune evasion components: Many viral membrane proteins participate in immune evasion strategies. KP93L/DP93R might interact with components of the host immune system, such as MHC class I molecules or pattern recognition receptors.

The experimental verification of these potential interactions represents an important area for future research that could significantly advance our understanding of KP93L/DP93R function in ASFV biology.

How can CRISPR-Cas9 genome editing be applied to study KP93L/DP93R function in the context of ASFV infection?

CRISPR-Cas9 technology offers powerful approaches to investigate KP93L/DP93R function, though its application to ASFV presents unique challenges that require specialized methodologies:

• Generation of KP93L/DP93R knockout viruses:

  • Design guide RNAs targeting conserved regions of the KP93L/DP93R genes

  • Introduce CRISPR-Cas9 components into cells along with infectious ASFV

  • Isolate and verify viral mutants through plaque purification and sequencing

  • Challenge: The repetitive nature of the TIR regions where KP93L/DP93R resides requires careful guide RNA design to ensure specificity

• Complementation studies:

  • Establish cell lines stably expressing KP93L/DP93R

  • Determine if these cells can complement growth defects of knockout viruses

  • Introduce tagged or mutant versions to identify functional domains

• Conditional knockdown approaches:

  • Develop inducible CRISPR systems that allow temporal control of gene disruption

  • Monitor viral replication kinetics following induced knockout at different stages of infection

• Challenges and considerations:

  • ASFV's complex genome structure requires careful validation of CRISPR edits

  • The presence of KP93L/DP93R at both genomic termini may necessitate simultaneous targeting of both copies

  • The essential or non-essential nature of these genes remains undetermined, potentially requiring careful titration of knockout efficiency

Recent successful applications of CRISPR-Cas9 to other large DNA viruses provide methodological frameworks that could be adapted for ASFV KP93L/DP93R studies.

What are the primary challenges in developing neutralizing antibodies against KP93L/DP93R for diagnostic or therapeutic purposes?

Developing effective neutralizing antibodies against KP93L/DP93R presents several significant challenges that researchers must address:

• Transmembrane topology constraints:

  • The predicted transmembrane domain (amino acids 5-25) suggests that much of the protein may be embedded in membranes

  • Accessible epitopes may be limited to small extracellular or intracellular domains

  • Solution: Focus antibody development on predicted exposed regions using epitope mapping and accessibility prediction algorithms

• Protein conformation in native environments:

  • Recombinant KP93L/DP93R produced in E. coli may not adopt the same conformation as in infected cells

  • Solution: Consider mammalian expression systems for immunogen production that preserve native folding and post-translational modifications

• Cross-reactivity concerns:

  • Sequence similarity with other viral or host proteins may lead to cross-reactivity

  • Solution: Careful epitope selection and extensive validation for specificity

• Functional relevance:

  • Without established functional data, it remains unclear whether antibodies against KP93L/DP93R would have neutralizing potential

  • Solution: Paired functional studies to determine protein role before investing in therapeutic antibody development

• Strain variability:

  • Differences in KP93L/DP93R between ASFV strains may limit broad applicability of antibodies

  • Solution: Target conserved regions identified through multiple sequence alignment of diverse ASFV strains

These challenges necessitate a systematic approach beginning with comprehensive structural and functional characterization before pursuing antibody development for diagnostic or therapeutic applications.

How might structural biology approaches advance our understanding of KP93L/DP93R function?

Structural biology techniques offer powerful insights into protein function, particularly for uncharacterized proteins like KP93L/DP93R. A systematic approach should include:

• X-ray crystallography challenges and solutions:

  • Challenge: Membrane proteins are notoriously difficult to crystallize

  • Solution: Consider crystallizing soluble domains separately or using lipidic cubic phase crystallization methods specifically designed for membrane proteins

  • Expected outcome: High-resolution structures revealing functional motifs and potential interaction sites

• Cryo-electron microscopy (cryo-EM) applications:

  • Advantages: Can visualize proteins in near-native environments without crystallization

  • Methodology: Reconstitution into nanodiscs or liposomes to maintain membrane environment

  • Integration: Combine with subtomogram averaging to visualize KP93L/DP93R in the context of viral particles

• NMR spectroscopy for dynamic analysis:

  • Focus on: Solution NMR of isolated domains or solid-state NMR of membrane-embedded protein

  • Target information: Binding interfaces, conformational changes, and dynamic properties

• Computational structural biology integration:

  • AlphaFold2 or RoseTTAFold predictions can provide initial structural models

  • Molecular dynamics simulations can reveal membrane interactions and conformational flexibility

  • Virtual screening could identify potential binding partners or inhibitors

• Correlative approaches:

  • Combine structural data with functional assays and site-directed mutagenesis

  • Map conservation patterns onto structural models to identify functionally important residues

Recent advances in membrane protein structural biology, particularly in cryo-EM, make this a promising approach for unraveling KP93L/DP93R function despite the technical challenges involved.

How do researchers reconcile contradictory findings regarding the presence of KP93L/DP93R across different ASFV isolates?

Researchers face several sources of apparent contradictions in KP93L/DP93R data across ASFV isolates that require systematic resolution approaches:

• Genome annotation inconsistencies:

  • Problem: KP93L/DP93R homologs may be annotated differently or remain unannotated in some genome sequences

  • Resolution approach: Conduct comprehensive sequence similarity searches (BLAST, HMM profiles) across all available ASFV genomes to identify potential homologs regardless of annotation

  • Evidence: In some ASFV isolates, homologous genes have been annotated as "KP86R/DP86L, KP93L/DP93R" indicating potential naming variations

• Terminal region sequencing challenges:

  • Problem: "The TIR region is not present in all the ASFV genome sequences present in the databases"

  • Resolution approach: Distinguish between actual absence versus technical limitations in sequencing terminal regions

  • Methodological solution: Employ specialized sequencing approaches specifically designed for capturing terminal sequences of viral genomes

• Strain-specific structural variations:

  • Problem: Substantial variations in the number and arrangement of repeat elements affect KP93L/DP93R organization

  • Resolution approach: Group ASFV isolates based on their terminal repeat structure rather than treating contradictions as binary presence/absence

  • Example: The three types of repeat structures observed in BA71 versus BA71V represent evolutionary alternatives rather than contradictory data

• Functional versus vestigial presence:

  • Problem: Some strains may contain non-functional variants or pseudogenes

  • Resolution approach: Assess coding potential, conservation of critical residues, and expression evidence when comparing across strains

A comprehensive meta-analysis of available genomic and proteomic data using standardized annotation criteria would significantly help resolve these apparent contradictions.

What experimental design considerations are crucial when comparing KP93L/DP93R function between different ASFV strains?

When designing comparative studies of KP93L/DP93R across different ASFV strains, researchers should implement these critical experimental design elements:

• Strain selection strategy:

  • Include representatives from different genotypic groups with known sequence variations in KP93L/DP93R

  • Balance between virulent field isolates (e.g., BA71) and attenuated laboratory strains (e.g., BA71V)

  • Consider strains with natural variations in the terminal repeat regions

• Standardized expression systems:

  • Use identical expression vectors and cellular backgrounds for comparative studies

  • Control for codon optimization when expressing genes from different strains

  • Employ inducible systems to normalize expression levels

• Controlled infection conditions:

  • Standardize multiplicity of infection (MOI), cell types, and time points for infection studies

  • Include appropriate controls for strain-specific differences in replication kinetics

  • Measure viral replication efficiency alongside KP93L/DP93R expression

• Comprehensive functional readouts:

  • Assess multiple aspects of KP93L/DP93R function (localization, interactions, effect on replication)

  • Employ both gain-of-function and loss-of-function approaches

  • Utilize chimeric constructs to map strain-specific functional differences to specific protein domains

• Statistical robustness:

  • Design experiments with sufficient biological and technical replicates

  • Employ appropriate statistical tests for comparative analyses

  • Pre-register experimental protocols and analysis plans to avoid bias

By implementing these design considerations, researchers can generate more reliable comparative data on KP93L/DP93R function across ASFV strains, minimizing the impact of confounding variables that could lead to contradictory findings.

How should researchers interpret the potential role of KP93L/DP93R in ASFV attenuation based on current genomic data?

The interpretation of KP93L/DP93R's potential role in ASFV attenuation requires careful analysis of available genomic evidence and consideration of multiple hypotheses:

The most defensible interpretation based on current evidence is that KP93L/DP93R variations represent one of multiple genomic changes associated with attenuation, but their specific contribution requires further experimental validation through targeted genetic manipulation.

What emerging technologies hold the most promise for elucidating KP93L/DP93R function?

Several cutting-edge technologies show exceptional promise for uncovering the function of KP93L/DP93R:

• Single-cell proteomics and transcriptomics:

  • Application: Reveal cell-to-cell variation in KP93L/DP93R expression and associated host responses during infection

  • Advantage: Can identify subtle phenotypes masked in bulk population studies

  • Implementation: Combine with trajectory analysis to map temporal dynamics of protein function

• Proximity labeling proteomics:

  • Application: Identify proteins in close proximity to KP93L/DP93R in living cells

  • Methodology: Express KP93L/DP93R fused to enzymes like BioID or APEX2 that biotinylate nearby proteins

  • Outcome: Comprehensive interactome revealing functional associations within viral factories or host membrane complexes

• Advanced imaging techniques:

  • Super-resolution microscopy: Visualize KP93L/DP93R distribution at nanoscale resolution

  • Correlative light and electron microscopy (CLEM): Connect protein localization with ultrastructural features

  • Lattice light-sheet microscopy: Capture dynamic localization during live infection with minimal phototoxicity

• Systems biology integration platforms:

  • Multi-omics integration: Combine proteomics, transcriptomics, and metabolomics data

  • Network analysis: Position KP93L/DP93R within viral-host interaction networks

  • Machine learning approaches: Predict functional associations based on integrated datasets

• Organoid and physiologically relevant models:

  • Porcine intestinal organoids: Study KP93L/DP93R function in more physiologically relevant systems

  • Microfluidic organ-on-chip technology: Examine function in dynamic tissue environments

These emerging technologies, particularly when applied in combination, offer unprecedented opportunities to move beyond correlative observations and establish causal relationships between KP93L/DP93R structure, localization, and function in ASFV biology.

What strategies could enhance the yield and stability of recombinant KP93L/DP93R for structural studies?

Optimizing recombinant KP93L/DP93R production for structural studies requires addressing the specific challenges of membrane protein expression:

• Expression system optimization:

  • Eukaryotic alternatives: Replace E. coli with insect cell (Sf9, Hi5) or mammalian expression systems that provide superior membrane protein folding environments

  • Cell-free expression: Consider membrane-mimetic cell-free systems optimized for hydrophobic proteins

  • Strain engineering: Use specialized E. coli strains (C41/C43, Lemo21) specifically developed for membrane protein expression

• Construct design refinements:

  • Fusion partners: Incorporate stability-enhancing fusion partners (MBP, SUMO, GFP) with cleavable linkers

  • Domain isolation: Express stable subdomains separately if full-length protein proves recalcitrant

  • Codon optimization: Adjust codon usage to match expression host while maintaining critical secondary structure elements

• Solubilization strategy development:

  • Detergent screening: Systematically test multiple detergent classes (maltoside, glucoside, phosphocholine-based)

  • Nanodisc incorporation: Reconstitute into membrane scaffold protein (MSP) nanodiscs with defined lipid composition

  • Amphipol stabilization: Transfer from detergent to amphipathic polymers for enhanced stability

• Purification process optimization:

  • Temperature modulation: Perform extraction and purification steps at reduced temperatures (4-16°C)

  • Buffer optimization: Screen additives (glycerol, specific lipids, cholesterol) that maintain native conformation

  • Chromatography sequence: Develop multi-step purification protocols specific to membrane proteins

• Stability assessment and enhancement:

  • Thermal shift assays: Monitor protein stability under various conditions

  • Site-directed mutagenesis: Introduce stabilizing mutations based on computational predictions

  • Nanobody co-crystallization: Develop conformational-specific nanobodies to stabilize flexible regions

Implementation of these strategies, guided by systematic optimization rather than trial-and-error approaches, would significantly enhance the prospects for obtaining sufficient quantities of stable KP93L/DP93R protein suitable for high-resolution structural studies.

How might artificial intelligence approaches accelerate research on KP93L/DP93R and other uncharacterized ASFV proteins?

Artificial intelligence (AI) offers transformative potential for accelerating research on uncharacterized proteins like KP93L/DP93R through multiple complementary approaches:

• Structure prediction and analysis:

  • Implementation: Apply AlphaFold2, RoseTTAFold, or other AI-driven structure prediction tools to generate high-confidence structural models

  • Enhancement: Use predicted structures to identify functional motifs, potential binding sites, and structural homology to proteins of known function

  • Integration: Compare predictions across multiple ASFV strains to identify structurally conserved features despite sequence variation

• Functional annotation through deep learning:

  • Methodology: Develop specialized neural networks trained on known viral protein functions and sequence/structural features

  • Application: Predict potential functions based on subtle patterns not evident through conventional sequence analysis

  • Validation approach: Prioritize predictions for experimental validation based on confidence scores

• Interaction network prediction:

  • Tools: Employ protein-protein interaction prediction algorithms to map potential viral-viral and viral-host interactions

  • Integration: Combine with existing interactome data to position KP93L/DP93R within functional networks

  • Experimental design: Generate testable hypotheses about protein function based on predicted interaction partners

• Literature mining and knowledge extraction:

  • Implementation: Use natural language processing to extract relevant information from scientific literature

  • Integration: Build knowledge graphs connecting KP93L/DP93R to related proteins, pathways, and functions

  • Gap analysis: Identify knowledge gaps to prioritize research questions

• Experimental design optimization:

  • Application: Design optimal mutagenesis strategies to maximize information gain with minimal experimental effort

  • Implementation: Use active learning approaches to iteratively improve predictions based on experimental feedback

  • Resource allocation: Prioritize experiments with highest expected information value

These AI approaches would be most effective when implemented as part of an integrated research program that combines computational prediction with targeted experimental validation, creating a virtuous cycle of hypothesis generation and testing that could rapidly advance our understanding of KP93L/DP93R function.

What are the most critical unanswered questions regarding KP93L/DP93R that should be prioritized by the research community?

Based on the current state of knowledge, these research questions represent the highest priorities for advancing understanding of KP93L/DP93R:

• Essential nature determination: Is KP93L/DP93R essential for ASFV replication in primary macrophages and/or in vivo? This fundamental question would establish the protein's basic importance in viral biology and potential as a therapeutic target.

• Subcellular localization mapping: What is the precise subcellular localization of KP93L/DP93R throughout the ASFV replication cycle? Understanding localization patterns would provide critical clues to function.

• Host and viral interaction partners: What proteins (both viral and host) interact with KP93L/DP93R during infection? A comprehensive interactome would position the protein within functional networks.

• Post-translational modification characterization: Does KP93L/DP93R undergo palmitoylation or other post-translational modifications similar to other ASFV membrane proteins , and if so, how do these modifications affect function?

• Contribution to virulence: Does variation in KP93L/DP93R structure between virulent strains (BA71) and attenuated strains (BA71V) directly contribute to differences in pathogenicity ?

• Structural biology: What is the three-dimensional structure of KP93L/DP93R, and how does it relate to function?

• Evolutionary conservation: How conserved is KP93L/DP93R across the diversity of ASFV isolates, and what does this reveal about its importance in viral biology?

Addressing these questions through coordinated research efforts would substantially advance our understanding of this uncharacterized protein and potentially reveal new insights into ASFV biology and pathogenesis.

How can research on KP93L/DP93R contribute to broader ASFV vaccine and antiviral development efforts?

Research on KP93L/DP93R has significant potential to contribute to ASFV countermeasure development through several distinct pathways:

• Attenuated vaccine development:

  • The observation that KP93L/DP93R structure differs between virulent (BA71) and attenuated (BA71V) strains suggests that targeted modification of this region could contribute to rational attenuation strategies

  • Understanding its potential role in attenuation mechanisms could lead to more precisely engineered vaccine candidates with optimal safety and efficacy profiles

  • Potential approach: Combine KP93L/DP93R modifications with other established attenuation strategies for synergistic effects

• Antiviral target assessment:

  • If determined to be essential, KP93L/DP93R could represent a novel target for antiviral development

  • Structure-based drug design approaches could identify small molecules that disrupt essential functions

  • Interaction interface targeting: If critical viral-viral or viral-host protein interactions are identified, these interfaces could become therapeutic targets

• Diagnostic development:

  • Recombinant KP93L/DP93R protein could serve as an antigen in serological assays

  • Antibodies against conserved epitopes could enhance detection of diverse ASFV strains

  • Differential detection of variant forms might distinguish between virulent and attenuated strains

• Fundamental understanding of attenuation:

  • Comprehensive studies of KP93L/DP93R could reveal previously unrecognized mechanisms of attenuation

  • The finding that "OURT88/3 and NH/P68 possess a larger deletion in the same region [as BA71V], which produces a considerably smaller attenuation" suggests complex relationships between genomic changes and attenuation that warrant further investigation

• Vector development platform:

  • If non-essential, the KP93L/DP93R locus could potentially serve as an insertion site for vaccine antigens

  • This approach could leverage the established safety profile of attenuated ASFV while delivering heterologous antigens