Recombinant European bat lyssavirus 2 Glycoprotein G (G)

What is European Bat Lyssavirus 2 Glycoprotein G and what is its significance in viral pathogenesis?

European Bat Lyssavirus 2 Glycoprotein G is a membrane protein that plays a crucial role in the viral life cycle, being responsible for virus entry into host cells and eliciting protective immune responses. EBLV-2 is one of three rabies-virus-like agents of the genus Lyssavirus found predominantly in Daubenton's bats (Myotis daubentonii) in Great Britain and other European countries .

The glycoprotein forms trimeric spikes on the viral surface and mediates attachment to cellular receptors and subsequent fusion of the viral envelope with endosomal membranes. Its significance extends beyond viral attachment as it represents the main target for neutralizing antibodies, making it an important focus for vaccine development and immunological studies .

Evolutionarily, EBLV-2 shows limited diversity compared to classical rabies virus (RABV) and appears well-adapted to its bat host species . This host adaptation may explain the relatively stable genetic composition of the glycoprotein over time, with current diversity estimated to have developed over the last 2000 years .

How do researchers produce recombinant EBLV-2 Glycoprotein G for laboratory studies?

The production of recombinant EBLV-2 Glycoprotein G typically involves the following methodological approach:

• Gene Amplification: The glycoprotein gene is amplified from viral RNA extracted from infected tissue or cultured virus using reverse transcription PCR with specific primers designed based on published sequences.

• Expression System Selection: Common expression systems include:

  • Bacterial systems (E. coli): Useful for producing peptide fragments but often lack proper glycosylation

  • Yeast (Pichia pastoris): Better for folded proteins requiring disulfide bonds

  • Mammalian cells (HEK293, CHO): Preferred for full-length glycoproteins with native conformation

  • Insect cells (Sf9, High Five) with baculovirus vectors: Balance between yield and post-translational modifications

• Vector Construction: The amplified gene is cloned into an appropriate expression vector, typically including:

  • Kozak consensus sequence for efficient translation initiation

  • Signal peptide to direct the protein to the secretory pathway

  • Purification tag (His-tag, GST, etc.)

  • Protease cleavage site to remove the tag if necessary

• Protein Expression and Purification: The recombinant protein is expressed in the chosen system and purified using affinity chromatography, ion-exchange chromatography, and size-exclusion chromatography.

• Validation: The purified protein is validated by Western blotting, ELISA, mass spectrometry, and functional assays to ensure proper folding and antigenicity.

Researchers must optimize conditions specifically for EBLV-2 Glycoprotein G, as lyssavirus glycoproteins can be challenging to express in their native conformation.

What is known about the evolutionary patterns of EBLV-2 Glycoprotein G compared to other lyssavirus glycoproteins?

EBLV-2 Glycoprotein G demonstrates interesting evolutionary patterns that distinguish it from other lyssavirus glycoproteins:

Phylogenetic Relationships:

EBLV-2 forms a monophyletic group separate from other bat-type lyssaviruses, with significant statistical support in phylogenetic analyses . Current evidence indicates that EBLV-2 shares the most recent common ancestry with Bokeloh bat lyssavirus (BBLV) and Khujan virus (KHUV) .

Evolutionary Rate:

EBLV-2 exhibits a remarkably slow tempo of viral evolution compared to other lyssaviruses. Studies estimate that:

• Current EBLV-2 diversity built up during the last 2000 years

• EBLV-2 diverged from KHUV approximately 8000 years ago

Selection Pressure:

The glycoprotein gene of EBLV-2 appears to be under purifying selection rather than positive selection, suggesting that the G gene may not play a significant role in lyssavirus adaptation . Computational analyses found minimal evidence of positive selection on the glycoprotein gene. Only one codon at amino acid residue 483 was found to be under weak positive selection (with marginal probability of 95%) .

Geographic Distribution and Genetic Clustering:

Finnish EBLV-2 strains cluster with strains from Central Europe, supporting the hypothesis of a Central European origin for EBLV-2 circulating in Finland . Regional strains show patterns of local evolution, with Finnish and Swiss strains estimated to have diverged from other EBLV-2 strains during the last 1000 years .

This slow evolutionary rate and strong purifying selection suggest that EBLV-2 Glycoprotein G is highly specialized and well-adapted to its natural host, Daubenton's bats.

What experimental models are available for studying EBLV-2 Glycoprotein G function and pathogenesis?

Researchers have developed several experimental models to study EBLV-2 Glycoprotein G function and pathogenesis, each with specific advantages and limitations:

In Vivo Models:

• Natural Host (Daubenton's Bats):

  • Most relevant model for understanding natural infection dynamics

  • Challenging due to conservation status and ethical considerations

  • Previous studies have demonstrated that Daubenton's bats show surprising resilience to infection

  • Can be infected through intracranial (i.c.), subdermal (s.d.), or intramuscular (i.m.) routes with varying success rates

• Mouse Models:

  • More accessible than bat models

  • May not fully recapitulate bat-specific aspects of infection

  • Useful for vaccine efficacy and pathogenesis studies

In Vitro Models:

• Primary Bat Cell Cultures:

  • Derived from relevant tissues (brain, salivary glands)

  • Better represent the natural cellular environment

  • Limited availability and standardization challenges

• Established Cell Lines:

  • Neuroblastoma cells (N2a, SH-SY5Y)

  • Bat-derived cell lines (e.g., Tb1-Lu)

  • Human embryonic kidney cells (HEK293T) for expression studies

• Pseudotyped Viruses:

  • VSV or lentiviral vectors pseudotyped with EBLV-2 G

  • Allow study of entry mechanisms under BSL-2 conditions

  • Enable high-throughput screening of entry inhibitors

Biochemical and Structural Models:

• Recombinant Protein Systems:

  • Soluble G ectodomain for structural studies

  • Membrane mimetics (nanodiscs, liposomes) with incorporated G protein

• Protein-Protein Interaction Assays:

  • Surface plasmon resonance (SPR) to study receptor binding

  • ELISA-based systems for antibody interaction studies

Regardless of the model chosen, researchers must consider that EBLV-2 is a biosafety level 3 (BSL-3) pathogen, requiring appropriate containment facilities for work with infectious virus.

What evidence exists for recombination events in EBLV-2 Glycoprotein G genes?

While recombination has been documented in some lyssaviruses, the evidence specifically for recombination in EBLV-2 Glycoprotein G genes is limited:

Methodologies for Detecting Recombination:

Researchers investigating potential recombination in EBLV-2 typically employ:

• Computational Algorithms:

  • RDP4 package (RDP, GENECONV, Bootscan, MaxChi, Chimaera, SiScan)

  • GARD (Genetic Algorithm for Recombination Detection)

  • SimPlot analysis for visual detection of potential breakpoints

• Phylogenetic Incongruence Tests:

  • Analysis of different genome regions to detect topological inconsistencies

  • Statistical evaluation of alternative tree topologies

• Sequence Similarity Plots:

  • Sliding window analysis of sequence identity across the genome

  • Identification of abrupt changes in sequence similarity patterns

The apparent rarity of recombination in EBLV-2 and other lyssaviruses may be due to:

• The nature of the RNA-dependent RNA polymerase

• Limited opportunities for co-infection with different viral strains

• Evolutionary constraints on viable recombinants

• Technical limitations in detecting recombination events in closely related sequences

What are the most effective methods for detecting EBLV-2 Glycoprotein G in clinical or field samples?

Detection of EBLV-2 Glycoprotein G in clinical or field samples requires sensitive and specific methods. The following approaches are currently employed in research and surveillance settings:

Molecular Detection Methods:

• RT-PCR and qRT-PCR:

  • Target conserved regions of the G gene

  • Can detect viral RNA in saliva, brain tissue, and other samples

  • Quantitative PCR allows estimation of viral load

  • Successfully applied to detect EBLV-2 RNA in bat saliva during experimental infections

• Digital Droplet PCR (ddPCR):

  • Higher sensitivity than conventional qPCR

  • Particularly useful for samples with low viral load

  • Absolute quantification without standard curves

• Next-Generation Sequencing (NGS):

  • Allows detection and full genome characterization simultaneously

  • Metagenomic approaches can detect novel variants

  • Useful for evolutionary studies and surveillance

Protein Detection Methods:

• Immunohistochemistry (IHC):

  • Detection of viral antigens in fixed tissues

  • Successfully used to detect lyssavirus nucleoprotein in brain and spinal cord of infected bats

  • Specific monoclonal antibodies against EBLV-2 G improve specificity

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Direct antigen-capture ELISA using anti-G antibodies

  • Useful for high-throughput screening of samples

• Western Blotting:

  • Confirmation of protein size and specificity

  • Less sensitive than ELISA but more specific

Virus Isolation:

• Cell Culture:

  • Neuroblastoma cells (N2a) or other susceptible cell lines

  • Detection of cytopathic effect and confirmation by immunofluorescence

  • Has been successfully used to isolate EBLV-2 from bat saliva samples

Serological Methods:

• Virus Neutralization Assay (VNA):

  • Gold standard for detecting protective antibodies

  • Requires live virus or pseudotyped alternatives

  • Has been utilized with heterologous WHO international standard immunoglobulin as control

• Indirect ELISA:

  • Detection of anti-G antibodies in serum

  • Useful for surveillance studies in bat populations

Method Selection Guide:

For field surveillance, portable devices employing isothermal amplification (LAMP) or microfluidic technologies are being developed to enable rapid detection without sophisticated laboratory infrastructure.

How has the understanding of EBLV-2 Glycoprotein G selection pressure informed vaccine development strategies?

The understanding of selection pressure on EBLV-2 Glycoprotein G has significantly influenced vaccine development strategies in several ways:

Implications for Vaccine Development:

• Conservation of Antigenic Sites:
  The predominance of purifying selection suggests that critical epitopes are likely conserved across EBLV-2 strains, indicating that vaccines targeting these regions would have broad efficacy against diverse isolates.

• Cross-Protection Potential:
  Given the evolutionary relationship between EBLV-2 and other lyssaviruses like Bokeloh bat lyssavirus (BBLV) and Khujan virus (KHUV) , vaccines developed against conserved epitopes might offer cross-protection against related viruses.

• Rational Antigen Design:
  Knowledge of the few sites under positive selection allows researchers to:

  • Focus on highly conserved regions for vaccine design

  • Potentially include multiple variants of variable regions

  • Design chimeric proteins that present multiple critical epitopes

• Stability of Vaccine Antigens:
  The evolutionary stability of EBLV-2 G suggests that vaccines developed now are likely to remain effective against future strains for an extended period, reducing the need for frequent reformulation.

Practical Vaccine Development Approaches:

The limited evidence for recombination in EBLV-2 further supports the feasibility of developing vaccines with long-term efficacy, as genetic stability reduces the likelihood of vaccine escape through recombination events.

What challenges exist in expressing functional recombinant EBLV-2 Glycoprotein G for structural studies?

Expressing functional recombinant EBLV-2 Glycoprotein G for structural studies presents numerous technical challenges that researchers must overcome:

Expression System Challenges:

• Post-Translational Modifications:

  • EBLV-2 G is heavily glycosylated, requiring eukaryotic expression systems

  • Different expression systems produce varying glycosylation patterns

  • Proper folding depends on correct disulfide bond formation

  • Mammalian or insect cell systems typically yield more native-like protein than bacterial systems

• Membrane Protein Solubility:

  • As an integral membrane protein, G protein contains hydrophobic transmembrane domains

  • These domains cause aggregation when expressed outside a membrane environment

  • Strategies include expressing soluble ectodomain only or using membrane mimetics (detergents, nanodiscs)

• Protein Yield Optimization:

  • Low expression levels are common with viral membrane proteins

  • Codon optimization for the expression host can improve yields

  • Signal sequence optimization affects translocation efficiency

  • Temperature, induction timing, and media composition require optimization

Purification Challenges:

• Maintaining Native Conformation:

  • Harsh purification conditions can denature the protein

  • Detergent selection is critical for maintaining structure

  • pH and ionic strength affect stability and oligomeric state

  • Low-temperature purification processes help preserve structure

• Oligomeric State Preservation:

  • Native G protein forms trimers on the viral surface

  • Purification processes can disrupt these higher-order structures

  • Crosslinking or specialized detergents may help maintain oligomeric states

• Homogeneity Requirements:

  • Structural studies require highly homogeneous protein preparations

  • Glycoprotein heterogeneity due to variable glycosylation complicates crystallization

  • Enzymatic deglycosylation or expression in glycosylation-deficient cells may be necessary

Structural Analysis Methods and Challenges:

Functional Validation Approaches:

• Receptor Binding Assays:

  • Surface plasmon resonance (SPR) with purified protein

  • Cell-based binding assays with fluorescently labeled protein

  • Competition assays with known ligands

• Fusion Activity Assessment:

  • Syncytia formation assays in transfected cells

  • Pseudotype virus entry assays

  • Liposome fusion assays with reconstituted protein

• Antibody Recognition:

  • ELISA with conformation-dependent antibodies

  • Western blotting under non-reducing conditions

  • Flow cytometry of surface-expressed protein

Researchers have had success with modular approaches, such as expressing and characterizing domains separately or using stabilizing mutations to improve protein behavior in vitro. Recent advances in membrane protein structural biology, particularly in cryo-EM, offer promising alternatives to traditional crystallography for challenging proteins like EBLV-2 Glycoprotein G.

How does EBLV-2 Glycoprotein G contribute to viral host range and tissue tropism?

EBLV-2 Glycoprotein G plays a central role in determining viral host range and tissue tropism through multiple mechanisms:

Receptor Recognition and Binding:

• Primary Receptor Interactions:

  • EBLV-2 G, like other lyssavirus glycoproteins, recognizes neuronal receptors

  • The nicotinic acetylcholine receptor (nAChR) serves as a receptor for several lyssaviruses

  • Neural cell adhesion molecule (NCAM) and p75 neurotrophin receptor (p75NTR) may also be involved

  • Specific amino acid residues in the receptor-binding domains determine receptor specificity

• Host-Specific Receptor Variations:

  • EBLV-2's adaptation to Daubenton's bats may reflect specialized binding to bat-specific receptor variants

  • The limited host range (primarily Daubenton's bats with occasional spillover to humans) suggests specialized receptor interactions

  • The high degree of conservation in EBLV-2 G sequences indicates adaptation to a specific ecological niche

Post-Entry Contributions to Tropism:

• Fusion Mechanism:

  • G protein mediates pH-dependent fusion in endosomes

  • Cell-specific endosomal properties may influence fusion efficiency

  • Differences in fusion thresholds can contribute to tissue tropism

• Intracellular Trafficking:

  • Following endocytosis, the path of the virion is influenced by G protein interactions

  • Retrograde transport to the central nervous system is facilitated by G protein

Experimental Evidence of Tissue Tropism:

• In Vivo Distribution:

  • Experimental studies in Daubenton's bats show EBLV-2 antigen predominantly in brain and spinal cord tissues

  • Immunohistochemistry reveals specific immunolabelling in the perikarion of infected bats

  • EBLV-2 RNA has been detected in saliva, bladder, and kidney samples of infected bats, though at low levels

• Viral Shedding Patterns:

  • EBLV-2 RNA and viable virus have been isolated from bat saliva in experimental infections

  • The relatively low levels of virus shed in saliva (below 100 genome copies per μg extracted RNA) suggest tissue-specific replication efficiency

Host Adaptation and Evolutionary Considerations:

• Host Resilience to Infection:

  • Daubenton's bats show surprising resilience to EBLV-2 infection

  • Only one of seven bats inoculated by the subdermal route developed disease in experimental studies

  • This suggests co-evolutionary adaptation between virus and natural host

• Route of Infection Effects:

  • Experimental studies show differences in infection success based on inoculation route:

    • Intracranial route is most effective at inducing disease

    • Subdermal route has limited success

    • Intramuscular route shows minimal efficacy

  • These differences reflect the importance of G protein interactions with specific tissues

• Evolutionary Stability:

  • The slow evolutionary rate of EBLV-2 G (current diversity estimated to have developed over 2000 years)

  • Limited positive selection on G gene

  • These factors suggest a stable virus-host relationship with optimized tissue tropism

Understanding the molecular basis of EBLV-2 G's contribution to host range and tissue tropism is essential for assessing zoonotic potential and developing preventive measures for human exposure, particularly for those regularly handling bats .

What are the latest findings on neutralizing epitopes in EBLV-2 Glycoprotein G for therapeutic antibody development?

Research on neutralizing epitopes in EBLV-2 Glycoprotein G has yielded important insights for therapeutic antibody development:

Key Neutralizing Epitope Regions:

• Antigenic Site II:

  • Located in the ectodomain of the glycoprotein

  • Contains highly conserved residues across lyssaviruses

  • Target of several cross-neutralizing antibodies

• Conformational Epitopes:

  • Many neutralizing antibodies recognize three-dimensional structures rather than linear sequences

  • Pre- and post-fusion conformations expose different epitopes

  • Antibodies stabilizing pre-fusion form can prevent the conformational changes required for fusion

• Receptor-Binding Domain (RBD):

  • Antibodies targeting the RBD can directly block virus attachment

  • This region shows some conservation among phylogroup I lyssaviruses

Antibody Cross-Reactivity Patterns:

Studies have demonstrated that antibodies raised against EBLV-2 G show varying patterns of cross-reactivity with other lyssaviruses. This has important implications for therapeutic development:

The virus neutralization assay (VNA) has been employed with heterologous WHO international standard immunoglobulin as a control to assess neutralizing antibody responses , providing a standardized approach for comparative studies.

Therapeutic Antibody Development Approaches:

• Monoclonal Antibody Isolation:

  • Phage display libraries from immunized animals or humans

  • Single B-cell sorting from convalescent or vaccinated subjects

  • Humanization of mouse-derived antibodies with neutralizing activity

• Structure-Guided Design:

  • Computational modeling of antibody-epitope interactions

  • Optimization of binding affinity and pharmacokinetic properties

  • Design of bispecific antibodies targeting multiple epitopes

• Cocktail Approaches:

  • Combinations of antibodies targeting different epitopes

  • Reduced risk of escape mutant emergence

  • Enhanced breadth of protection across lyssavirus phylogroups

Experimental Validation Methods:

• In Vitro Neutralization:

  • Pseudotype neutralization assays (safer, BSL-2)

  • Classical virus neutralization with live EBLV-2 (requires BSL-3)

  • Cell-cell fusion inhibition assays

• Animal Model Testing:

  • Mouse models for initial efficacy

  • Hamster models for pathogenesis studies

  • Therapeutic window assessment (time post-exposure)

• Mechanism of Action Studies:

  • Pre- vs. post-attachment neutralization

  • Inhibition of fusion vs. receptor binding

  • Fc-dependent effector functions (ADCC, CDC)

The limited diversity of EBLV-2 G compared to RABV suggests that antibody therapies targeting conserved epitopes could have broad efficacy against circulating EBLV-2 strains. Additionally, the understanding that EBLV-2 G has been under purifying selection indicates that therapeutic antibodies targeting conserved epitopes would likely remain effective over time, as these regions are less prone to mutational escape.