Recombinant African swine fever virus Uncharacterized protein F165R (Pret-058)

How does F165R compare to other uncharacterized ASFV proteins, and what experimental approaches have successfully characterized similar proteins?

F165R belongs to a substantial group of uncharacterized ASFV proteins that comprise nearly half of the approximately 150-170 open reading frames in the ASFV genome. Successful characterization of similarly uncharacterized ASFV proteins has employed a multi-faceted approach:

• Comparative genomics: Analysis of presence/absence patterns across attenuated versus virulent strains, such as those performed with the Georgia 2007/1 strain .

• Transcriptomic profiling: Cap Analysis Gene Expression sequencing (CAGE-seq) has successfully mapped transcription start sites and expression patterns of ASFV genes, categorizing them into early and late expression profiles . This approach revealed that MGF genes (to which F165R might belong) are predominantly early-expressed genes.

• Deletion mutant phenotyping: Generation of gene knockout viruses using CRISPR/Cas9-based genome engineering as demonstrated with other ASFV genes (A238L, K145R, and I329L) . For instance, deletion of the dUTPase gene E165R (not to be confused with F165R) demonstrated it was nonessential for viral replication and virulence .

• Protein-protein interaction studies: Identifying binding partners often reveals functional implications, as has been demonstrated with immunomodulatory ASFV proteins.

For F165R specifically, researchers should prioritize determining its temporal expression pattern during infection and generating deletion mutants to assess its contribution to viral fitness.

What synthetic genomics-based approaches can be applied to study F165R function in the context of complete ASFV genome?

Recent advances in ASFV synthetic genomics offer powerful approaches for F165R functional studies. A comprehensive methodology would involve:

• Fragment-based genome assembly: Divide the ASFV genome into manageable fragments (approximately 12-14 pieces as shown in recent studies) , with F165R located within one specific fragment. Using CRISPR-Cas9 editing, researchers can precisely modify the F165R gene within its native genomic context.

• Yeast-E. coli assembly system: The modified fragment containing altered F165R can be assembled with other fragments in yeast, followed by transfer to E. coli for verification and amplification. This approach has successfully generated recombinant ASFVs with multiple modifications across the genome .

• Helper virus reconstitution system: For viral recovery, transfect the assembled genome into permissive cells (such as WSL-gRO61R cells) along with a helper virus system. Specifically, a self-helper virus approach using a Cas9-susceptible helper virus and Cas9-resistant synthetic genome has proven effective .

• Plaque isolation and verification: Select viral plaques and verify genotypes through PCR amplification and sequencing to confirm F165R modifications. This step is critical due to the occurrence of homologous recombination between helper virus and synthetic genomes, requiring careful screening to isolate desired mutants .

• Phenotypic characterization: Compare growth kinetics, plaque morphology, and host range of F165R-modified viruses against wild-type counterparts in both cell culture and primary swine macrophages.

This methodological pipeline mirrors successful approaches used to generate and characterize other ASFV mutants, providing a roadmap for F165R functional studies.

How can transcriptomic and proteomic approaches be integrated to elucidate F165R's role during different stages of viral infection?

A multi-omics approach to characterize F165R function would involve sequential and integrated analyses:

• Temporal transcription profiling:

  • Apply CAGE-seq to map the precise transcription start site of F165R and determine whether it follows early or late expression patterns .

  • Compare F165R transcript levels between virulent isolates (e.g., Georgia 2007/1) and attenuated strains (e.g., ASFV-G/VP110) to identify potential correlations with virulence.

  • Develop a time-course RNA-seq experiment in primary porcine macrophages to establish the exact timing of F165R expression relative to other viral genes.

• Translational dynamics:

  • Employ ribosome profiling to assess translational efficiency of F165R mRNA during infection.

  • Use pulse-chase labeling combined with immunoprecipitation to determine protein half-life and potential post-translational modifications.

• Protein interaction network:

  • Implement proximity-labeling approaches (BioID or APEX2) with F165R as bait to identify proximal proteins during infection.

  • Perform co-immunoprecipitation followed by mass spectrometry to identify stable interaction partners.

  • Validate key interactions using techniques like bimolecular fluorescence complementation or Förster resonance energy transfer.

• Functional proteomics:

  • Generate F165R deletion mutants and perform comparative proteomics between wild-type and mutant infections to identify downstream effects.

  • Use phosphoproteomics to determine if F165R affects signaling pathways during infection.

• Data integration framework:

  • Develop a computational pipeline to integrate transcriptomic and proteomic datasets.

  • Apply network analysis to position F165R within the temporal cascade of viral gene expression and protein interaction networks.

This integrated approach has been successful in characterizing other viral proteins with unknown functions and would provide comprehensive insights into F165R's role during ASFV infection.

How does F165R sequence conservation compare across ASFV genotypes, and what methods can assess functional conservation?

Analyzing F165R conservation requires a comprehensive approach combining in silico and experimental methods:

• Sequence conservation analysis:

  • Perform multiple sequence alignments of F165R from representative isolates across all 24 known ASFV genotypes.

  • Calculate sequence identity and similarity percentages, with particular focus on comparing isolates with different virulence profiles.

  • Identify conserved motifs that may indicate functional domains using tools like MEME and GLAM2.

• Structural conservation assessment:

  • Use structural prediction algorithms (AlphaFold2, I-TASSER) to generate models of F165R from different genotypes.

  • Calculate structural similarity metrics (TM-score, RMSD) between models to assess three-dimensional conservation beyond primary sequence.

  • Identify spatially conserved surface patches that might represent functional interaction sites.

• Evolutionary analysis:

  • Calculate selective pressure (dN/dS ratios) across the F165R coding sequence to identify regions under purifying or positive selection.

  • Perform phylogenetic analysis to determine if F165R evolution correlates with ASFV genotype divisions based on traditional markers like p72 (B646L).

• Functional conservation testing:

  • Express recombinant F165R from diverse genotypes and assess biochemical activities (if identified).

  • Develop complementation assays where F165R from one genotype is expressed in the context of an F165R-deleted virus from another genotype.

  • Compare host protein interaction profiles of F165R variants to determine if interaction partners are conserved across genotypes.

Leveraging similar approaches used for other ASFV proteins would reveal whether F165R represents a core function conserved across genotypes or contributes to strain-specific properties that might correlate with virulence differences observed between attenuated and virulent strains .

What experimental design would best determine if F165R expression or function differs between attenuated and virulent ASFV strains?

To rigorously investigate potential differences in F165R between attenuated and virulent strains, the following experimental design would be optimal:

• Strain selection and pairing:

  • Identify paired virulent/attenuated strain sets, such as:

    • Original Georgia 2007/1 isolate and its cell culture-adapted attenuated derivative (ASFV-G/VP110)

    • Congo virulent (Congo-v) and attenuated (Congo-a) strains

    • Other naturally occurring strain pairs with documented virulence differences

• Expression analysis:

  • Quantitative RT-PCR targeting F165R mRNA at multiple time points post-infection

  • Western blot analysis using custom antibodies against F165R to compare protein levels

  • Immunofluorescence microscopy to assess subcellular localization differences

  • Polysome profiling to determine translational efficiency of F165R mRNA

• Functional comparative assays:

  • Generate recombinant F165R proteins from both virulent and attenuated strains

  • Conduct comparative biochemical assays based on predicted functions

  • Perform host protein interaction studies using approaches such as:

    • Yeast two-hybrid screening

    • Co-immunoprecipitation followed by mass spectrometry

    • Protein microarrays with host immune components

• Cross-complementation studies:

  • Create F165R deletion mutants in both virulent and attenuated backgrounds

  • Complement each deletion with F165R from the opposite strain

  • Assess whether virulence phenotypes co-segregate with F165R variants

• In vivo validation:

  • Compare virulence of wild-type, deletion mutants, and cross-complemented viruses in swine models

  • Monitor clinical signs, viral replication in tissues, and immune responses

  • Establish correlations between F165R sequence variants and in vivo phenotypes

This comprehensive experimental design would determine whether F165R contributes to the attenuation process during serial passage, similar to what has been observed with specific genomic changes during the development of attenuated ASFV strains .

What methodologies can determine if F165R interacts with components of the porcine immune system, and how might these interactions affect viral pathogenesis?

Investigating potential interactions between F165R and porcine immune components requires a systematic approach:

• Identification of potential interaction partners:

  • Affinity purification-mass spectrometry (AP-MS) using F165R as bait in porcine macrophage lysates

  • Yeast two-hybrid screening against a library of porcine immune system proteins

  • Protein microarrays containing recombinant porcine immune molecules probed with F165R

  • In silico prediction of protein-protein interactions based on structural modeling

• Validation of identified interactions:

  • Co-immunoprecipitation of F165R with candidate partners from infected cells

  • ELISA-based binding assays with purified recombinant proteins

  • Bimolecular fluorescence complementation (BiFC) or FRET-based approaches in live cells

  • Surface plasmon resonance to determine binding kinetics and affinity

• Functional consequences assessment:

  • Reporter assays for immune signaling pathways (NF-κB, IRF3/7, JAK-STAT) in the presence of F165R

  • Analysis of cytokine production in macrophages expressing F165R

  • Flow cytometry to assess effects on immune cell activation markers

  • RNA-seq of macrophages infected with wild-type versus F165R-deleted viruses

• In vivo relevance determination:

  • Compare immune responses in pigs infected with wild-type versus F165R-deleted viruses

  • Focus on:

    • Cytokine profiles

    • Lymphocyte counts and activation status

    • Histopathological changes in lymphoid tissues

    • Viral distribution in immune system organs

This approach would build on existing knowledge about ASFV immune evasion strategies. For context, several ASFV proteins have been characterized as immune modulators, including A238L (inhibits NF-κB signaling) and CD2v (involved in immune escape and lymphocyte inhibition) . Determining whether F165R plays a similar role would contribute to understanding ASFV's complex manipulation of host immune responses, which typically involve early lymphopenia followed by neutrophilia during infection .

How might the uncharacterized protein F165R contribute to ASFV virulence, and what experimental models would best test this hypothesis?

Investigating F165R's potential contribution to ASFV virulence requires a comprehensive experimental approach:

• Generation of recombinant viruses:

  • Create F165R deletion mutants using CRISPR/Cas9-based reverse genetics

  • Develop revertant viruses to control for unintended mutations

  • Generate point mutants targeting predicted functional domains

  • Construct GFP-tagged versions for tracking during infection

• In vitro virulence indicators:

  • Growth kinetics comparison in primary porcine macrophages

  • Cytopathic effect analysis (timing and morphology)

  • Cell death mechanism assessment (apoptosis, necrosis, pyroptosis)

  • Cytokine and chemokine production profiles

• Ex vivo models:

  • Precision-cut lung slices from porcine tissue to assess tissue tropism

  • Porcine peripheral blood mononuclear cell infection models

  • 3D organoid cultures of relevant porcine tissues

• In vivo experimental design:

  • Swine infection model comparing:

    • Wild-type ASFV

    • F165R deletion mutant

    • Revertant control virus

  • Experimental parameters to monitor:

    • Clinical score (fever, anorexia, depression, skin discoloration)

    • Viremia levels and tissue distribution

    • Survival rates and time to death

    • Immunopathological changes in tissues

    • Transmission potential to contact animals

• Comparative analysis with known virulence factors:

  • Side-by-side comparison with deletion mutants of established virulence factors

  • Double-knockout studies to assess potential redundancy or synergy

  • Transcriptomic and proteomic comparison of host responses

This experimental framework follows established approaches used to identify other ASFV virulence determinants, such as MGF genes and immune modulators . The progressive adaptation model of the Georgia isolate (ASFV-G) through cell culture passages provides a valuable comparative system, as virulence attenuation correlated with specific genomic changes . Analysis of whether F165R is affected during attenuation would provide important context for interpreting virulence studies.

How can F165R be evaluated as a potential component in ASFV subunit or vector vaccine strategies?

Evaluating F165R as a vaccine component requires a systematic progression through immunogenicity assessment, protective efficacy testing, and delivery platform optimization:

• Immunogenicity evaluation:

  • Express and purify recombinant F165R protein from E. coli or eukaryotic systems

  • Formulate with different adjuvants (oil-in-water emulsions, TLR agonists, saponin-based)

  • Immunize pigs and assess:

    • Antibody responses (titer, isotype, avidity, neutralization capacity)

    • Cell-mediated immunity (IFN-γ ELISPOT, proliferation assays, intracellular cytokine staining)

    • Duration of immune response

  • Compare responses to F165R with established immunogenic ASFV proteins like p72 and CD2v

• Vector platform assessment:

  • Develop recombinant viral vectors expressing F165R, using established systems such as:

    • Modified Vaccinia Ankara (MVA)

    • Adenovirus

    • Alphavirus replicons

    • Pseudorabies virus (similar to CD2v expression systems)

  • Evaluate vector-induced immune responses, focusing on quality and persistence

• Combination strategy development:

  • Test F165R in combination with other ASFV antigens

  • Design multi-epitope constructs incorporating conserved F165R regions

  • Employ prime-boost strategies combining protein and vector delivery

• Challenge studies:

  • Immunize pigs with optimized F165R-based candidates

  • Challenge with virulent ASFV strains of homologous and heterologous genotypes

  • Evaluate protection parameters:

    • Clinical protection (symptom reduction)

    • Virological protection (reduced viremia and tissue viral loads)

    • Sterile immunity (prevention of shedding and transmission)

• Correlates of protection identification:

  • Perform comprehensive immune profiling of protected versus unprotected animals

  • Identify specific immune responses associated with protection

  • Develop in vitro assays that predict in vivo protection

This approach builds on methodologies that have been applied to evaluate other ASFV proteins as vaccine candidates. For example, the CD2v protein delivered by a recombinant pseudorabies virus vector (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) successfully induced both humoral and cellular immune responses . Similar strategies could determine whether F165R contributes meaningful protection alone or as part of a multi-antigen formulation.

What safety and efficacy parameters must be evaluated when testing F165R-deleted ASFV as a potential live-attenuated vaccine candidate?

Development of an F165R-deleted ASFV as a live-attenuated vaccine candidate would require comprehensive safety and efficacy evaluation:

Safety Assessment:

• Attenuation stability:

  • Serial passage in cell culture (minimum 20 passages) with whole genome sequencing at regular intervals to detect reversion or compensatory mutations

  • Assessment of genetic stability in porcine macrophages, the natural target cells

  • Monitoring homologous recombination potential, which has been identified as a concern for ASFV vaccines

• In vivo safety parameters:

  • Dose escalation studies in pigs to identify maximum safe dose

  • Extended observation period (minimum 28 days) monitoring:

    • Clinical signs (fever patterns, appetite, depression, skin discoloration)

    • Viremia levels and duration

    • Virus shedding in secretions and excretions

    • Tissue distribution through sequential necropsy

  • Specific monitoring of immune dysregulation (lymphopenia, neutrophilia)

• Transmission studies:

  • Contact transmission assessment between vaccinated and naive animals

  • Environmental sampling to detect virus shedding

  • Monitoring for potential carrier states or persistent infection

• Safety in pregnant animals:

  • Reproductive safety assessment including potential for vertical transmission

  • Monitoring for embryonic/fetal effects

Efficacy Evaluation:

• Immune response characterization:

  • Antibody response profile (kinetics, magnitude, isotype distribution)

  • Cell-mediated immunity assessment (T-cell proliferation, IFN-γ production)

  • Innate immune response measurement (cytokine profiles, NK cell activation)

• Challenge studies design:

  • Challenge timing optimization (14, 28, and 180 days post-vaccination)

  • Homologous challenge with parental strain

  • Heterologous challenge with distinct genotypes

  • Dose-ranging challenge studies to determine protection threshold

• Protection parameters:

  • Clinical protection scoring system based on temperature, clinical signs, and weight change

  • Virological protection measured by quantitative PCR in blood and tissues

  • Complete protection assessment (prevention of disease, infection, and transmission)

• Comparative studies:

  • Side-by-side comparison with other attenuated ASFV candidates

  • Comparison with previously studied deletion mutants (e.g., ASFV-G-ΔI177L)

This comprehensive evaluation framework follows established approaches used for other ASFV live-attenuated candidates, such as those with deletions in the DP148R and I177L genes, which demonstrated attenuated phenotypes while maintaining immunogenicity . The successful attenuation of ASFV Georgia strain through sequential passage (ASFV-G/VP110) provides a methodological template for evaluating attenuated phenotypes.