Recombinant European bat lyssavirus 1 Glycoprotein G (G)

What is European bat lyssavirus 1 Glycoprotein G and what is its functional significance?

European bat lyssavirus 1 glycoprotein G is the sole surface protein of EBLV-1, a member of the Lyssavirus genus primarily detected in serotine bats (Eptesicus serotinus) and responsible for the majority of bat rabies cases in mainland Europe . As the only membrane protein of lyssaviruses, glycoprotein G is crucial for virus entry both in vitro and in vivo . This protein mediates viral attachment to host cell receptors, facilitates viral entry through membrane fusion, and serves as the primary antigenic determinant that stimulates neutralizing antibody production .

Studies investigating interspecies protein substitution have demonstrated that G protein significantly influences virus fitness, pathogenicity, and neuroinvasiveness . The G protein contains both an ectodomain that interacts with host receptors and a cytoplasmic domain with cellular protein-binding sites that contribute to pathogenicity . Understanding EBLV-1 G protein function is essential for comprehending how this zoonotic pathogen emerges, spreads, and causes disease.

How does EBLV-1 glycoprotein G compare structurally and functionally to other lyssavirus glycoproteins?

EBLV-1 glycoprotein G shares structural and functional similarities with other lyssavirus glycoproteins, particularly rabies virus (RABV) G, but exhibits distinct antigenic properties and pathogenicity profiles. Comparative analysis of phylogroup I lyssaviruses—including EBLV-1, EBLV-2, BBLV, ARAV, KHUV, IRKV, GBLV, DUVV, ABLV, and RABV—has revealed both conserved domains critical for basic viral functions and variable regions that confer virus-specific characteristics .

Antigenic cartography has demonstrated that recombinant viruses expressing EBLV-1 G protein (designated as SN-1) are antigenically indistinguishable from wild-type EBLV-1, regardless of the backbone virus sequence . This confirms that G protein is the primary determinant of antigenic identity in lyssaviruses. The substitution of EBLV-1 G into a RABV backbone affects virus titer, with SN-1 requiring fewer passages to reach 100% infectivity compared to SN-2 (containing EBLV-2 G) .

Molecular evolutionary analysis indicates that EBLV-1 G has been under strong selective constraints, with low dN/dS ratios similar to those observed in other lyssaviruses . The G gene exhibits a lower dN/dS ratio than the P and M genes but higher than the highly conserved N gene, reflecting its balanced evolutionary constraints between functional conservation and adaptation .

What are the primary genetic characteristics of EBLV-1 glycoprotein G?

EBLV-1 glycoprotein G exhibits remarkable genetic stability, with evidence suggesting it has been under purifying selection throughout its evolutionary history . A comprehensive analysis of 53 full-length glycoprotein gene sequences isolated from different hosts across 21 countries over 70 years revealed minimal positive selection pressure on the gene . When examining EBLV-1 sequences from the UK and France, researchers found no evidence of selective pressure driving adaptation to UK serotine bats, despite these representing a naïve host population .

What methodologies are most effective for creating and characterizing recombinant EBLV-1 glycoprotein G constructs?

Creating recombinant viruses containing EBLV-1 glycoprotein G typically involves reverse genetics systems, which have been successfully established for both RABV and EBLV-1 . For the generation of chimeric viruses, researchers have used a recombinant attenuated RABV backbone (SN) with the entire G gene replaced by heterologous proteins from EBLV-1 (SN-1) or EBLV-2 (SN-2) .

The most effective methodology involves:

• Plasmid construction: Creating a plasmid containing the full-length RABV genome with the G gene replaced by EBLV-1 G.

• Rescue system: Using RABV helper plasmids to rescue recombinant viruses through transfection of cell cultures.

• Passage optimization: Determining the optimal number of passages required to reach 100% infectivity (SN-1 required only two passages, similar to the parent SN virus) .

• Sequence verification: Confirming that the appropriate EBLV G sequence is in frame with no errors and no changes due to cell passage .

• Virus amplification: Growing viruses to 100% infectivity and performing titration to determine viral yields.

For characterization, researchers should employ:

• Antigenic cartography: To compare recombinant viruses with wild-type viruses and determine if the recombinant viruses are antigenically indistinguishable from their G protein donors .

• In vitro growth kinetics: To assess the effect of G substitution on virus replication and final titers.

• In vivo pathogenicity studies: Using standardized mouse models to evaluate neuroinvasiveness, virulence, and clinical disease progression .

How does substitution of EBLV-1 glycoprotein G into heterologous viral backbones affect pathogenicity profiles?

Interspecies substitution of EBLV-1 glycoprotein G into heterologous viral backbones significantly impacts virus pathogenicity, but in complex ways that depend on multiple factors. In a key study, researchers created a recombinant attenuated RABV (SN) with its entire G gene replaced by EBLV-1 G (SN-1) . When tested in vivo following peripheral infection with a high viral dose (10^4 ffu), animals infected with SN-1 showed reduced survivorship compared to those infected with the parent SN virus, resulting in survival rates more similar to wild-type EBLV-1 infection .

At reduced viral doses (10^3 ffu), all recombinant viruses (SN, SN-1, and SN-2) resulted in 100% survivorship, while clinical disease still developed in mice infected with wild-type EBLVs . This dose-dependent pathogenicity demonstrates that G protein alone cannot fully reconstitute the virulence characteristics of the donor virus.

The data collectively indicate that:

• G protein substitution affects virus titer in vitro, which may influence pathogenicity.

• G protein contributes to neuroinvasiveness and virulence, but does not solely determine the complete pathogenicity profile.

• Other viral proteins and their interactions with G likely modulate the final disease outcome.

What selection pressures have shaped the evolution of EBLV-1 glycoprotein G?

Comprehensive computational analyses suggest that EBLV-1 glycoprotein G has predominantly been under purifying (negative) selection pressure throughout its evolutionary history . When analyzing 53 full-length G sequences isolated from different hosts across 21 countries over a 70-year period, researchers found minimal evidence of positive selection driving adaptive evolution .

Key findings regarding selection pressures include:

• Purifying selection dominance: Most amino acid sites in the G gene show evidence of negative selection, preserving functional constraints and structural integrity . Selection analyses using SLAC, REL, and FEL methods found no strong evidence of positive selection on any site of the G gene .

• Marginal evidence of positive selection: Only one site at position 416 was identified as potentially under weak positive selection by FEL with a marginally significant P-value of 0.0999, but this was not supported by other analytical methods .

• Evolutionary rate constraints: In UK and European EBLV-1 sequences, low dN/dS ratios indicate strong selective constraints, with EBLV-1b subtype exhibiting lower values than EBLV-1a . This pattern is consistent across lyssavirus genes, with the ascending order of dN/dS ratios being N-L-G-P-M .

• Adaptation to new hosts: Despite EBLV-1's introduction into naïve serotine bat populations in the UK, no evidence of positive selection driving adaptation was observed . This contradicts the expectation that host-switching events typically drive molecular adaptation, as previously shown for classical rabies virus when switching from domestic dogs to foxes .

These findings collectively suggest that the G gene has remained functionally conserved despite infecting different hosts over long periods, indicating that G gene evolution may not play a significant role in lyssavirus adaptation to new hosts .

How can researchers accurately measure antigenic differences between wild-type and recombinant EBLV-1 glycoprotein G?

Accurately measuring antigenic differences between wild-type and recombinant EBLV-1 glycoprotein G requires sophisticated serological methods that can quantify subtle variations in antibody recognition. Based on current research, the following methodological approach is recommended:

• Antigenic cartography: This computational technique analyzes serological data to position viruses and antisera in a two-dimensional "map" where distances correspond to antigenic differences . This method has successfully demonstrated that recombinant viruses expressing EBLV-1 G (SN-1) are antigenically indistinguishable from wild-type EBLV-1, confirming that G protein determines antigenic identity .

• Virus neutralization assays: Using panels of monoclonal antibodies against different epitopes of G protein to compare neutralization profiles between wild-type and recombinant viruses. Differences in neutralization titers indicate antigenic variations.

• Cross-reactivity assessments: Testing antisera raised against recombinant G proteins against wild-type viruses, and vice versa, to evaluate reciprocal antigenic relationships.

• Epitope mapping: Using site-directed mutagenesis or peptide arrays to identify specific antigenic sites within G that may differ between wild-type and recombinant proteins.

• Competitive binding assays: Measuring the ability of antibodies to competitively inhibit binding to specific epitopes, revealing structural or conformational differences between recombinant and wild-type G proteins.

What are the optimal protocols for assessing pathogenicity and neuroinvasiveness of recombinant viruses containing EBLV-1 glycoprotein G?

Assessment of pathogenicity and neuroinvasiveness of recombinant viruses containing EBLV-1 glycoprotein G requires standardized in vivo models and comprehensive analytical techniques. Based on recent research, the following protocol framework is recommended:

• Animal model selection: Standardized mouse models have been effectively used to compare pathogenicity of different lyssaviruses and recombinant constructs . Both ic (intracranial) and im (intramuscular) inoculation routes should be considered to assess direct neural pathogenicity versus neuroinvasiveness, respectively .

• Dose titration studies: Testing multiple viral doses (e.g., 10^3 ffu and 10^4 ffu) is crucial for revealing dose-dependent pathogenicity differences . At high doses (10^4 ffu), SN-1 showed reduced survivorship compared to SN, while at lower doses (10^3 ffu), all recombinant viruses showed 100% survivorship while wild-type viruses still caused disease .

• Clinical assessment parameters:

  • Survival time and rate

  • Clinical scoring systems for disease progression

  • Weight loss measurements

  • Behavioral changes

• Tissue analysis protocols:

  • Histopathological examination: To assess pathological changes in neural tissues

  • Immunofluorescence imaging: Using antibodies against viral proteins (e.g., P protein) and cell-type markers (e.g., GFAP for astrocytes, NeuN for neurons) to determine cell tropism

  • Antigen distribution mapping: To compare patterns between recombinant and wild-type viruses

  • Viral load quantification: In different tissues to assess spread

• Multifactorial assessment: Combining survival data with histopathological findings and antigen distribution patterns provides a more complete understanding of pathogenicity profiles . For example, SN-1 showed survivorship similar to EBLV-1 but histopathological changes more similar to SN, indicating the complex nature of pathogenicity determinants .

• Serial passage confirmation: For negative results, conducting three consecutive serial passages to confirm the absence of virus .

This comprehensive approach enables researchers to systematically evaluate how EBLV-1 glycoprotein G affects pathogenicity when expressed in recombinant viral contexts, providing insights into both the contribution of G protein to virulence and potential applications in vaccine development.

How should researchers interpret differences in virus titers between recombinant viruses with heterologous glycoproteins?

Interpreting differences in virus titers between recombinant viruses with heterologous glycoproteins requires consideration of multiple factors that influence viral replication efficiency and growth kinetics. Based on experimental data with recombinant lyssaviruses, researchers should employ the following interpretive framework:

This multifaceted approach to data interpretation allows researchers to distinguish between technical limitations and biologically meaningful differences in how heterologous G proteins function within recombinant viral systems.

What does phylogenetic analysis reveal about the evolution and geographic spread of EBLV-1 glycoprotein G variants?

Phylogenetic analysis provides crucial insights into the evolutionary history, geographic spread, and molecular epidemiology of EBLV-1 glycoprotein G variants. Recent comprehensive analyses of EBLV-1 sequences have revealed several key patterns:

• Geographic clustering and transmission: Bayesian phylogenetic analysis of EBLV-1 sequences from multiple European countries demonstrates distinct geographic clustering, with sequences from the same country typically sharing more recent common ancestors . This pattern indicates limited long-distance transmission and suggests that EBLV-1 primarily spreads through local bat populations.

• Single source introductions: The emergence of EBLV-1 in new territories often originates from a single source rather than multiple independent introductions. For example, UK EBLV-1 sequences show extreme similarity (99.9-100%), suggesting a single introduction event followed by local spread .

• Cross-border transmission timelines: Phylogenetic analysis combined with molecular clock approaches can estimate when EBLV-1 crossed geographical boundaries. UK EBLV-1 sequences shared their most recent common ancestor with a sequence from Brittany, France (2001), with an estimated divergence date of 1997 . Within the UK, the earliest sequence divergence was estimated to occur in 2007 .

• Evolutionary rate constraints: Whole-genome analysis reveals that EBLV-1 evolves under strong selective constraints, with mean substitution rates consistent with previous estimates . These low evolutionary rates suggest that EBLV-1 is well-adapted to its primary host and undergoes minimal adaptive evolution even when entering new host populations.

• Subtype differentiation: Phylogenetic analysis distinguishes between EBLV-1a and EBLV-1b subtypes, with EBLV-1b exhibiting lower dN/dS values, indicating stronger purifying selection . This pattern suggests potential differences in host adaptation or functional constraints between subtypes.

• Rare recombination events: Phylogenetic incongruence analysis has identified potential recombination events in lyssavirus G genes, though these appear to be rare . One recombinant (AY987478) was detected with two possible breakpoints, suggesting genetic exchange between viral lineages .

These phylogenetic insights are valuable for understanding EBLV-1 epidemiology, predicting future spread, and developing control strategies for this emerging zoonotic pathogen.