Recombinant Duvenhage virus Glycoprotein G (G)

Basic Research Questions

• What is Duvenhage virus and how does it relate to other lyssaviruses?

  Duvenhage virus (DUVV) belongs to the genus Lyssavirus within the family Rhabdoviridae. It is classified as genotype 4 in the lyssavirus taxonomy, alongside rabies virus (genotype 1), Lagos bat virus (genotype 2), Mokola virus (genotype 3), European bat lyssaviruses 1 and 2 (genotypes 5 and 6), and Australian bat lyssavirus (genotype 7). Phylogenetic analysis places DUVV in phylogroup I along with rabies virus, European bat lyssaviruses, and Australian bat lyssavirus . Within this phylogroup, the glycoprotein ectodomain shares at least 74% amino acid identity and displays significant cross-neutralization properties. DUVV has caused documented human infections following bat exposures, with two known cases in South Africa separated by 36 years and approximately 80 km geographically .

• What is the structure and function of Duvenhage virus Glycoprotein G?

  Duvenhage virus Glycoprotein G is a membrane protein that forms spikes on the viral surface. It serves three critical functions: (1) binding to host cell receptors to facilitate viral entry, (2) mediating membrane fusion during viral entry, and (3) acting as the primary antigenic determinant recognized by virus-neutralizing antibodies . The glycoprotein undergoes post-translational modifications, particularly glycosylation, which influences its proper folding, trafficking through the endoplasmic reticulum and Golgi apparatus, and antigenic properties . Like other lyssavirus glycoproteins, DUVV G contains important antigenic sites that induce neutralizing antibodies and T-cell responses, making it the major target for vaccine development .

• How do specific amino acid residues in Duvenhage virus Glycoprotein G affect its pathogenicity?

  Specific amino acid residues in DUVV G play crucial roles in determining pathogenicity. Most notably, position 333 is critical for lyssavirus virulence. In phylogroup I viruses including DUVV, an arginine (R333) is essential for neuroinvasiveness and neurovirulence. Research has demonstrated that phylogroup II viruses (Mokola and Lagos bat viruses) naturally have aspartic acid (D333) at this position, which results in attenuated pathogenicity . This difference in amino acid explains why phylogroup I viruses are pathogenic when injected by both intracerebral and intramuscular routes, whereas phylogroup II viruses are only pathogenic via the intracerebral route . Additionally, by analogy with rabies virus, other positions such as amino acid 8 may affect protein conformation and function. Studies with vaccinia-rabies recombinants showed that a proline rather than leucine at position 8 was critical for effective immunization .

• What glycosylation patterns are observed in Duvenhage virus Glycoprotein G?

  Glycosylation is crucial for proper folding, trafficking, and antigenic properties of lyssavirus glycoproteins. DUVV G contains specific N-glycosylation sites that influence its structure and function. Research has identified a potential N-glycosylation site at position 247 that appears to be specific to Duvenhage virus . This differs from other lyssaviruses, which have unique glycosylation patterns - Mokola virus contains a site at position 202, while Lagos bat virus has potential sites at positions 184, 202, and 334 . The minimal glycosylation site sufficient for adequate maturation and routing through the endoplasmic reticulum and Golgi apparatus appears to be located in the G ectodomain of bat lyssaviruses, including DUVV . These differences in glycosylation patterns contribute to the distinct antigenic properties of each lyssavirus and affect their recognition by the immune system.

Methodological Questions

• What techniques are most effective for detecting immune responses to recombinant Duvenhage virus Glycoprotein G?

  Several complementary techniques are used to comprehensively assess immune responses to DUVV G:

  For antibody responses:

  • Virus neutralization assays: The gold standard for measuring functional antibodies that can neutralize virus infectivity. This can be performed with live DUVV or pseudotyped viruses expressing DUVV G .

  • ELISA: Quantifies antibody binding to recombinant G protein or specific epitopes.

  • Immunofluorescence assays: Detect antibodies that bind to G protein expressed on infected cells .

  • Western blotting: Identifies antibodies recognizing linear epitopes on denatured G protein.

  For cellular immune responses:

  • ELISpot assays: Enumerate antigen-specific cytokine-producing T cells.

  • Intracellular cytokine staining: Measure T-cell activation via flow cytometry.

  • Cytotoxic T-lymphocyte (CTL) assays: Assess the ability of CD8+ T cells to kill infected target cells. Studies with recombinant vaccinia-rabies G viruses have shown they effectively prime mice to generate secondary virus-specific CTL responses .

  • Lymphocyte proliferation assays: Measure T-cell proliferation in response to antigen stimulation.

  These methods allow researchers to characterize both humoral (antibody) and cellular immune responses, which are both important for protection against lyssavirus infections.

• How can animal models be used to evaluate the efficacy of vaccines based on recombinant Duvenhage virus Glycoprotein G?

  Animal models are crucial for evaluating vaccine efficacy before human trials. For DUVV G-based vaccines, several approaches can be used:

  Mouse models:

  • Challenge studies: Immunized mice are challenged with virulent DUVV via intracerebral or peripheral routes to assess protection. Survival rates and clinical scores are primary endpoints .

  • Dose-response studies: Determine minimum protective doses - studies with vaccinia-rabies glycoprotein recombinants found that 10^4 plaque-forming units were required for effective immunization .

  • Cross-protection studies: Test protection against other lyssaviruses to assess breadth of immunity. Vaccinia-rabies glycoprotein recombinants have shown protection against not only rabies virus but also Duvenhage virus, though not against Mokola virus .

  Critical parameters to measure:

  • Neutralizing antibody titers: Strong correlate of protection against lyssavirus infection.

  • T-cell responses: Both CD4+ and CD8+ T-cell responses contribute to protection.

  • Duration of immunity: Long-term protection is essential for effective vaccines.

  • Safety parameters: Weight loss, local reactions, systemic adverse events.

  Special considerations:

  • Both live and inactivated preparations should be evaluated - β-propiolactone-inactivated preparations of vaccinia-rabies recombinants have successfully induced neutralizing antibodies and protection .

  • Multiple challenge strains should be tested to ensure broad protection, as demonstrated in studies showing protection against several strains of rabies virus .

• What molecular techniques can be used to study the interactions between Duvenhage virus Glycoprotein G and host cell receptors?

  Understanding G protein-receptor interactions requires sophisticated molecular approaches:

  Receptor identification methods:

  • Co-immunoprecipitation with crosslinking to capture transient interactions

  • CRISPR-Cas9 screening to identify host factors essential for viral entry

  • Proximity labeling techniques (BioID, APEX) to identify proteins in close proximity to G during entry

  • RNA interference screening to identify host factors required for viral entry

  Binding affinity measurements:

  • Surface plasmon resonance (SPR) to measure real-time binding kinetics

  • Bio-layer interferometry (BLI) for label-free detection of biomolecular interactions

  • Isothermal titration calorimetry (ITC) to measure thermodynamic parameters of binding

  Structural characterization:

  • X-ray crystallography of G protein-receptor complexes to determine atomic structure

  • Cryo-electron microscopy for structural analysis of larger complexes

  • Computational modeling and molecular dynamics simulations to predict interactions

  Functional assays:

  • Cell-cell fusion assays to assess fusion activity mediated by G protein

  • Pseudotyped virus entry assays using viruses bearing DUVV G

  • Site-directed mutagenesis to identify critical binding residues

  • Competition assays with peptides or antibodies to block specific interactions

  These approaches would help identify whether DUVV G interacts with known lyssavirus receptors (nicotinic acetylcholine receptor, neural cell adhesion molecule, or p75 neurotrophin receptor) or utilizes unique receptors for cell entry.

• How can immunoinformatics approaches be used to design effective vaccines against Duvenhage virus?

  Immunoinformatics combines computational tools and immunological knowledge to accelerate vaccine design:

  Epitope prediction:

  • B-cell epitope prediction: Algorithms analyze protein sequences for properties associated with antibody recognition (hydrophilicity, accessibility, flexibility)

  • T-cell epitope prediction: Tools like those in the Immune Epitope Database (IEDB) predict peptide binding to MHC molecules

  • Conserved epitope identification: Comparing sequences across lyssavirus strains to identify invariant regions

  Population coverage analysis:

  • HLA allele frequency data is used to predict what percentage of a population might respond to specific epitopes

  • Research on DUVV G identified the epitope "YFLIGVSAV" with binding to 18 alleles and 99.36% worldwide population coverage

  Vaccine design strategies:

  • Multi-epitope vaccines combining B-cell and T-cell epitopes

  • Consensus sequence approaches to address viral variation

  • Structure-based design focusing on exposing neutralizing epitopes

  In silico validation:

  • Molecular dynamics simulations to assess epitope stability

  • Docking studies to predict epitope-antibody or epitope-MHC interactions

  • Homology modeling of epitope-MHC complexes

  Studies have successfully applied these approaches to DUVV G, identifying promising epitopes for peptide vaccine development with predicted high immunogenicity and low allergenicity . These computational predictions require experimental validation but can significantly accelerate the vaccine development process.

Research Data and Tables

Table 1: Comparison of Key Properties of Lyssavirus Phylogroups

Table 2: Glycosylation Sites in Lyssavirus Glycoproteins