Recombinant African swine fever virus Protein MGF 110-1L (Ken-007)

What is the Recombinant African Swine Fever Virus Protein MGF 110-1L (Ken-007)?

Recombinant African Swine Fever Virus Protein MGF 110-1L (Ken-007) is a protein derived from African swine fever virus (ASFV), specifically from the isolate Pig/Kenya/KEN-50/1950. It belongs to the multigene family 110 (MGF110) group of proteins encoded by the ASFV genome. The protein is identified in the UniProt database with ID P0C9G2 . ASFV is a member of the Asfarviridae family and is endemic to sub-Saharan Africa, persisting in wildlife through a cycle of infection between ticks and wild pigs, bushpigs, and warthogs. The virus causes a disease with clinical symptoms similar to classical swine fever, requiring laboratory diagnosis for differentiation . The recombinant form of this protein can be used for research applications, particularly in vaccine development studies.

What is the genomic conservation of MGF110-1L across ASFV isolates?

MGF110-1L is uniquely conserved across all known ASFV genomes, suggesting it may play an essential role in viral survival and function. This conservation pattern is distinctive as other MGF110 family genes show significant variation between isolates. Multiple sequence alignments across published ASFV isolates reveal high conservation of the MGF110-1L protein sequence, particularly within genotype groups .

How is MGF110-1L expressed during ASFV infection?

Transcriptional analysis of the MGF110 family of genes, including MGF110-1L, has revealed that these genes are expressed throughout the ASFV infection cycle. Microarray data analysis has shown detection of MGF110 family RNA transcripts at all time points during infection . The expression pattern shows a characteristic fluctuation, with levels decreasing from 3 to 9 hours post-infection (hpi), followed by an increase at 12 to 18 hpi.

This transcriptional pattern is similar to that observed for the early protein p30 (CP204L) . The findings suggest that MGF110-1L is abundantly expressed early in the virus replication cycle, which may indicate its potential role in early stages of infection or in establishing conditions favorable for viral replication.

What experimental approaches have been used to study MGF110-1L function?

The primary experimental approach to study MGF110-1L function has been gene deletion and phenotypic characterization of the resultant mutant virus. Researchers have developed a deletion mutant (ASFV-G-ΔMGF110-1L) through homologous recombination between the parental ASFV genome and a recombination transfer vector . This genetic modification technique involved:

• Construction of a recombination transfer vector (p72mCherryΔMGF110-1L) containing:

  • Flanking genomic regions: left arm (genomic positions 6003-7003) and right arm (genomic positions 7649-8649)

  • A reporter gene cassette with mCherry fluorescent protein under the control of the ASFV p72 late gene promoter

• Infection of macrophage cell cultures with ASFV-G and transfection with the recombination transfer vector

• Purification of the deletion mutant through successive rounds of limiting dilution, using mCherry expression as a marker

• Verification of the genetic modifications by next-generation sequencing (NGS) to confirm:

  • Complete deletion of the MGF110-1L ORF

  • Insertion of the p72-mCherry cassette

  • Absence of additional mutations or parental virus contamination

This methodological approach enabled the generation of a clean deletion mutant for functional studies both in vitro and in vivo, allowing researchers to assess the role of MGF110-1L in viral replication and virulence.

How does deletion of MGF110-1L affect viral replication in vitro?

Despite the conservation of MGF110-1L across all ASFV isolates, experimental evidence indicates that deletion of this gene does not significantly affect viral replication in vitro. Growth kinetics analysis of ASFV-G-ΔMGF110-1L in primary swine macrophage cultures showed remarkably similar replication patterns compared to the parental ASFV-G strain .

In multistep growth curve experiments, macrophage cultures were infected at a multiplicity of infection (MOI) of 0.01, and samples were collected at 2, 24, 48, 72, and 96 hours post-infection (hpi). The results demonstrated that ASFV-G-ΔMGF110-1L displayed replication kinetics comparable to the parental ASFV-G . This finding was surprising considering the universal conservation of MGF110-1L across ASFV genomes, which typically suggests an essential function.

Researchers have proposed that the function of MGF110-1L might be duplicated by other MGF110 family genes or compensated by other viral genes, explaining the lack of replication defects in the deletion mutant . This redundancy in function could be an evolutionary strategy to ensure viral fitness even if mutations occur in individual genes.

What is the impact of MGF110-1L deletion on ASFV virulence in swine?

The impact of MGF110-1L deletion on ASFV virulence has been assessed through experimental infection of domestic pigs. In a controlled study, 36-41 kilogram commercial breed swine were intramuscularly inoculated with 10² HAD₅₀ of either ASFV-G-ΔMGF110-1L or the parental ASFV-G virus . The clinical outcomes and disease progression were then monitored and compared between the groups.

The results can be summarized in the following table:

Animals infected with the parental ASFV-G developed high fever (>40°C) by day 4-5 post-infection, followed by the rapid appearance of ASF-associated clinical signs including anorexia, depression, purple skin discoloration, staggering gait, and diarrhea. Disease progression was severe, requiring euthanasia in extremis by day 5-6 post-infection .

Animals receiving ASFV-G-ΔMGF110-1L presented with a very similar clinical disease pattern in terms of both the timing and severity of clinical signs. Consequently, the study concluded that deletion of the MGF110-1L gene does not significantly alter the virulence phenotype of the highly virulent ASFV-G isolate . This result, combined with the in vitro findings, suggests that despite its conservation across ASFV isolates, MGF110-1L is not a major determinant of viral replication efficiency or virulence in the Georgia 2007 strain.

What are the methodological considerations for constructing MGF110-1L deletion mutants?

Constructing MGF110-1L deletion mutants requires careful consideration of several methodological aspects to ensure successful generation of a clean mutant virus. Based on the experimental approach described in the literature, the following considerations are critical:

• Design of recombination transfer vector: The vector should contain:

  • Homologous flanking regions of sufficient length (approximately 1000 bp) on each side of the target gene to facilitate efficient recombination

  • A reporter gene cassette (e.g., fluorescent protein such as mCherry) under a strong viral promoter (e.g., p72 late promoter) for easy identification of recombinant viruses

  • Precise deletion boundaries to avoid affecting adjacent genes

• Infection and transfection protocol:

  • Primary swine macrophages should be infected with parental virus at appropriate MOI

  • Optimal timing for transfection of the recombination vector after infection

  • Appropriate transfection reagent selection for primary macrophages

• Purification strategy:

  • Limiting dilution approach using the highest dilution with detectable amounts of the reporter gene

  • Multiple rounds of purification to ensure homogeneity

  • Verification of purity through methods such as PCR and sequencing

• Genetic verification:

  • Full-genome sequencing using next-generation sequencing technology

  • Confirmation of the expected deletion and insertion

  • Verification of the absence of additional mutations or parental virus contamination

  • Analysis using appropriate software (e.g., CLC Genomics Workbench)

• Phenotypic characterization:

  • In vitro growth kinetics in relevant cell types (primary swine macrophages)

  • In vivo virulence assessment in the natural host (domestic pigs)

  • Appropriate controls (parental virus) and statistical analysis

These methodological considerations ensure the generation of a clean deletion mutant that can be reliably used to study the specific role of MGF110-1L in ASFV biology, without confounding factors from unwanted mutations or mixed virus populations.

How can recombinant MGF110-1L be used in vaccine development strategies?

Recombinant ASFV MGF110-1L protein has potential applications in vaccine development strategies against African swine fever. While the search results indicate that deletion of MGF110-1L alone does not attenuate the highly virulent Georgia strain, the protein's universal conservation across ASFV isolates makes it a potential target for vaccine development approaches . Several strategies could be explored:

• Subunit vaccine component: Recombinant MGF110-1L protein could be used as one component in a subunit vaccine formulation. The high conservation of this protein across ASFV isolates suggests it might induce broadly reactive immune responses. Recombinant versions of the protein, such as the ASFV Ken-007 Protein (aa 1-270), are already being produced for research purposes .

• Combination with other mutations: While MGF110-1L deletion alone does not attenuate ASFV, combining this deletion with modifications to other viral genes might produce suitably attenuated strains for live-attenuated vaccine candidates. The MGF110-1L deletion could potentially contribute to genetic stability of vaccine strains due to its non-essential nature for viral replication.

• Marker vaccine development: The deletion of MGF110-1L combined with the insertion of reporter genes (as demonstrated with the mCherry cassette) could facilitate the development of marked vaccines that allow differentiation between infected and vaccinated animals (DIVA strategy) .

• Antigen for diagnostic assays: The recombinant protein could be used to develop serological assays that detect antibodies against MGF110-1L, potentially contributing to diagnostic tools that might complement vaccination strategies .

• Structure-based vaccine design: Further structural and functional characterization of MGF110-1L could reveal epitopes that might be targeted in rational vaccine design approaches, potentially leading to peptide-based or epitope-focused vaccines.

It's important to note that while recombinant MGF110-1L offers these potential applications, comprehensive evaluation of immunogenicity, protective efficacy, and safety would be necessary before advancing any MGF110-1L-based vaccine strategy to field application.