Recombinant Lagos bat virus Glycoprotein G (G)

What is Lagos bat virus and what is its taxonomic classification?

Lagos bat virus (LBV) is a lyssavirus of the Rhabdoviridae family that causes rabies-like illness in mammals. It belongs to phylogroup II within the Lyssavirus genus. LBV was first isolated from a fruit bat (Eidolon helvum) from Lagos Island, Nigeria in 1956, representing the first discovery of a rabies-related virus. Until this discovery, rabies was thought to have a single causal agent . The complete taxonomic classification is:

LBV has been isolated from various species including fruit bats, cats, and one dog in southern Africa. Notably, no human cases of LBV infection have been documented to date .

What is the fundamental structure and function of LBV glycoprotein G?

The LBV glycoprotein G is a surface protein that plays essential roles in viral attachment and entry. It spans amino acids 26-522 and functions to attach the virus to host cellular receptors, inducing endocytosis of the virion. Once in the endosome, the acidic pH induces conformational changes in the glycoprotein trimer, which triggers fusion between virus and cell membranes .

The glycoprotein is critical for neuroinvasion and neurovirulence. Research has demonstrated that both the matrix protein (M) and glycoprotein (G) of LBV play significant roles in viral pathogenesis, as recombinant viruses containing these proteins showed increased lethality in mouse models . The antigenic composition of LBV G differs substantially from that of rabies virus (RABV) G, which explains why current rabies vaccines do not provide cross-protection against phylogroup II lyssaviruses like LBV .

How can recombinant LBV glycoprotein G be produced for research purposes?

Recombinant LBV glycoprotein G can be produced using several expression systems, with the E. coli cell-free expression system being a validated approach. The recombinant protein typically includes amino acids 26-522 and can be produced with either a His-tag or in tag-free form. The purity of such products is generally >90%, as determined by SDS-PAGE .

For researchers investigating glycoprotein function, the recombinant protein's biological activity can be measured by its binding ability in functional ELISA assays. When designing expression constructs, it's crucial to consider the following methodological aspects:

• Selection of appropriate expression system (bacterial, mammalian, or insect cells)

• Codon optimization for the expression system

• Signal peptide design for proper protein localization

• Inclusion of purification tags that minimally interfere with protein function

• Validation of proper folding and post-translational modifications

For viral studies requiring functional glycoprotein in its native environment, reverse genetics approaches permit the creation of recombinant viruses containing the LBV G gene .

How do LBV antibodies distribute among bat populations in endemic regions?

Studies investigating the presence of LBV-specific antibodies in megachiroptera from West Africa have revealed variable seroprevalence rates among different bat species. Using fluorescent antibody virus neutralization tests, researchers have detected neutralizing antibodies in:

These findings confirm the presence of LBV in West Africa and suggest that Eidolon helvum may be an important reservoir host for this virus . When designing serological studies for LBV, researchers should consider sampling from multiple bat species across various habitats (urban, savannah, and forest) to obtain comprehensive epidemiological data.

How can reverse genetics be utilized to study LBV glycoprotein function?

Reverse genetics technology has proven invaluable for investigating the role of specific viral proteins in pathogenesis. For LBV glycoprotein studies, researchers have constructed chimeric viruses where genes encoding glycoprotein or matrix protein and glycoprotein of attenuated RABV strains are replaced with those of LBV.

Methodological approach:

• Create plasmid constructs containing the full-length viral genome with targeted gene replacements

• Transfect cells with plasmids encoding viral proteins necessary for initial replication

• Recover recombinant viruses

• Characterize recombinant viruses in vitro and in vivo

For example, researchers created SPBN-LBVG (where only the G gene was replaced) and SPBN-LBVM-LBVG (where both M and G genes were replaced). Pathogenicity studies revealed that all recombinant viruses were lethal to mice after intracranial inoculation, but following intramuscular inoculation, only SPBN-LBVM-LBVG was lethal, indicating that both M and G proteins play critical roles in LBV pathogenesis .

Additionally, a creative approach for immunogenicity studies involved constructing SPBNGAS-LBVG-GAS, where the LBV G was inserted between two mutated RABV G genes. This construction showed potential as a pan-lyssavirus vaccine candidate .

What experimental approaches can determine cross-neutralization between LBV and RABV antibodies?

The substantial antigenic differences between LBV G and RABV G result in limited cross-protection by conventional rabies vaccines against LBV infection. Researchers investigating this phenomenon should consider the following methodological approaches:

• Fluorescent antibody virus neutralization tests: This technique can detect neutralizing antibodies against different lyssavirus species, allowing for comparison of cross-neutralization.

• Recombinant virus construction: Creating chimeric viruses containing LBV G in a RABV backbone (like SPBNGAS-LBVG-GAS) and evaluating neutralization by different antisera.

• Challenge studies: Immunizing animals with various vaccine candidates and challenging with virulent virus to assess cross-protection.

Research has demonstrated that serum collected from mice inoculated intramuscularly with SPBNGAS-LBVG-GAS neutralized both phylogroup I and II lyssaviruses, including RABV, Duvenhage virus (DUVV), LBV, and Mokola virus (MOKV) . This suggests potential for developing pan-lyssavirus vaccines through strategic incorporation of LBV G.

How does LBV glycoprotein contribute to differential viral pathogenicity in various hosts?

Studies of LBV pathogenicity reveal complex host-virus interactions that differ between species. While LBV shows high peripheral pathogenicity in murine models, similar to RABV , different patterns emerge in natural bat hosts.

The experimental approach to investigating this should include:

• Comparing virus replication in bat versus non-bat cell lines

• Analyzing differences in receptor binding and cell entry

• Evaluating host immune responses to infection

• Conducting comparative pathology studies in different animal models

Research findings indicate enhanced LBV propagation in Eidolon helvum lung cell lines compared to human A549 lung cells at later time points, suggesting effective viral countermeasures against cellular defense mechanisms in bat cells . Furthermore, comparison of amino acid substitutions among viral glycoproteins has demonstrated significant differences within two antigenic sites between different phylogenetic lineages of LBV. Such molecular variability potentially contributes to differences in peripheral pathogenicity of lyssaviruses .

What are the mechanisms of LBV phosphoprotein's reduced IFN-β inhibitory activity in bats?

Recent research indicates that bats exhibit reduced inflammatory responses to viral infections while maintaining robust interferon (IFN)-related antiviral mechanisms. This unique immune balance may explain why bats like Eidolon helvum can serve as reservoir hosts for highly pathogenic lyssaviruses with limited disease manifestation.

Lyssavirus phosphoproteins typically inhibit the IFN response with virus strain-specific efficiency. Studies comparing the IFN-β inhibitory activity of LBV phosphoprotein with other lyssavirus phosphoproteins reveal reduced inhibitory capability in bat cells. This suggests that the virus-host adaptation in bats may involve distinct interactions with innate immune pathways .

Methodological approaches for investigating this phenomenon include:

• Utilizing bat-specific IFN-promoter activation assays

• Comparing viral protein function between bat and human cell cultures

• Analyzing molecular interactions between viral proteins and host immune factors

• Gene expression profiling to identify differential immune response patterns

What diagnostic and research challenges exist in differentiating LBV from other lyssaviruses?

Differentiating LBV from other lyssaviruses presents several challenges for both diagnostics and research. While the fluorescent antibody test (FAT) reliably detects lyssavirus antigens in brain material, specific identification of LBV requires specialized approaches.

The following methodological approaches are recommended:

• Monoclonal antibody panels: Using specific antibodies like N-MAb M612, which reacts highly specifically with LBV and does not react with other lyssaviruses .

• Mouse inoculation testing: Inoculating suckling mice intracranially with suspected samples and observing pathogenicity patterns (mice typically die around 9 days post-inoculation with LBV) .

• Molecular characterization: DNA sequence analysis of viral isolates to confirm identity and determine phylogenetic relationships.

• Antigenic profiling: Evaluating cross-reactivity patterns with reference antisera to distinguish LBV from other lyssaviruses.

It's worth noting that brain samples from LBV-infected animals show poor cross-reactivity to rabies antibodies despite being closely related to rabies virus , highlighting the need for LBV-specific diagnostic approaches.