Recombinant West Caucasian bat virus Glycoprotein G (G)

What is the West Caucasian bat virus glycoprotein G and what is its significance in viral pathogenesis?

The glycoprotein G of West Caucasian bat virus is one of the five main proteins encoded by this negative-sense single-stranded RNA virus. As a structural component, it forms the knobbed spikes that protrude from the viral membrane and plays a crucial role in mediating viral entry into host cells . The glycoprotein is particularly significant because it serves as the primary viral antigen that induces neutralizing antibodies in the host. Unlike other lyssaviruses, WCBV glycoprotein has a longer non-coding region at 697 nucleotides, contributing to its distinct phylogenetic classification . This structural uniqueness likely explains why current rabies vaccines provide no cross-protection against WCBV infection, making the study of this glycoprotein particularly important for public health preparedness .

What expression systems have been successfully used to produce recombinant WCBV glycoprotein G?

The vaccinia virus expression system has been successfully employed to generate recombinant WCBV glycoprotein G. Researchers created recombinant vaccinia viruses expressing the WCBV glycoprotein gene, designated as VV-WG . The methodology involved transfecting Vaccinia Copenhagen (Vacc Cop)-infected cell cultures with recombinant transfer vectors containing the WCBV glycoprotein gene. These constructs were designed with the glycoprotein gene under the regulation of the p7·5 vaccinia virus promoter . Additionally, dual-expression constructs combining WCBV glycoprotein with RABV glycoprotein (VV-RGWG) have been developed using a similar approach, demonstrating the versatility of the vaccinia expression system . The resulting recombinant viruses underwent six rounds of selection under mycophenolic acid resistance conditions and were confirmed for homogeneity using PCR targeting the thymidine kinase region of the Vacc Cop genome .

What are the most effective methods for purifying recombinant WCBV glycoprotein G for immunological studies?

For immunological studies requiring highly purified recombinant WCBV glycoprotein G, a multi-step purification protocol has been established. After propagating the recombinant vaccinia viruses expressing WCBV glycoprotein (VV-WG), semi-purification is achieved through ultracentrifugation at 19,000 g through a 36% sucrose cushion . This initial purification step separates the viral particles from cellular debris. The resulting pellet is then resuspended in minimal essential medium (MEM) containing antibiotics before storage at -80°C .

For further purification of the glycoprotein itself, researchers can employ affinity chromatography techniques using antibodies specific to the WCBV glycoprotein. The expression and purity of the recombinant glycoprotein can be confirmed using indirect immunofluorescence assays (IFA) with mouse anti-WCBV hyperimmune serum or specific monoclonal antibodies . This methodological approach ensures that the purified glycoprotein maintains its structural integrity and immunological properties, which is critical for downstream applications such as vaccine development and antibody production studies.

How can researchers accurately assess the immunogenicity of recombinant WCBV glycoprotein G in experimental models?

To accurately assess the immunogenicity of recombinant WCBV glycoprotein G, researchers should implement a comprehensive approach combining in vivo and in vitro methods. Animal models, particularly Syrian hamsters, have proven valuable as they represent accidental hosts and can be infected via the intramuscular route to mimic natural infection . Following inoculation with recombinant WCBV glycoprotein G, serum samples should be collected at regular intervals to monitor antibody development.

Key methodological steps include:

• Measuring virus-neutralizing antibody (VNAb) responses using standard neutralization assays

• Performing real-time PCR analysis on retrotranscribed cDNAs to evaluate gene expression profiles related to immune response

• Monitoring cellular immune responses by assessing cytokine production and lymphocyte recruitment and activation in the central nervous system

• Comparing immune responses to those elicited by other lyssaviruses such as RABV and DUVV as benchmark standards

Statistical analysis should employ appropriate tests such as the Wilcoxon–Mann–Whitney test for independent groups to evaluate significance of the immunological findings . This multifaceted approach allows for comprehensive characterization of both humoral and cellular immune responses to the recombinant glycoprotein.

What are the key considerations for designing a recombinant vaccine vector expressing WCBV glycoprotein G?

When designing a recombinant vaccine vector expressing WCBV glycoprotein G, several critical factors must be considered to ensure efficacy and safety:

How does the cross-protective potential of recombinant WCBV glycoprotein G compare with other lyssavirus glycoproteins?

Recombinant WCBV glycoprotein G demonstrates distinct cross-protective patterns compared to other lyssavirus glycoproteins, with important implications for vaccine development. Research has shown that while vaccines containing rabies virus (RABV) glycoprotein provide protection against most lyssaviruses, they notably fail to protect against WCBV, demonstrating the immunological divergence of this virus .

Experimental data indicates that recombinant vaccinia viruses expressing a single glycoprotein gene (including VV-WG) protect mice against lethal intracranial challenge with homologous virus . Interestingly, dual-expression constructs that combine glycoprotein genes from two different lyssaviruses (such as VV-RGWG combining RABV and WCBV glycoproteins) offer protection against both homologous viruses . This suggests a potential strategy for developing broadly protective vaccines.

Unlike constructs expressing MOKV glycoprotein, which generate virus-neutralizing antibodies (VNAb) that cross-neutralize Lagos bat virus (LBV), antibodies induced by WCBV glycoprotein show more limited cross-reactivity . This pattern aligns with phylogenetic analyses placing WCBV in its own distinct phylogroup, underscoring the importance of including WCBV antigens in next-generation rabies vaccines aimed at providing comprehensive protection against all lyssaviruses .

What structural features of the WCBV glycoprotein G account for its inability to be neutralized by conventional rabies vaccine-induced antibodies?

The structural divergence of WCBV glycoprotein G from other lyssavirus glycoproteins explains why antibodies induced by conventional rabies vaccines fail to neutralize this virus. Several key structural features contribute to this phenomenon:

These structural differences collectively result in limited antigenic overlap between WCBV glycoprotein and those of other lyssaviruses, rendering antibodies induced by standard rabies vaccines ineffective against WCBV. This underscores the importance of developing new vaccine formulations that include WCBV glycoprotein antigens for comprehensive protection against all lyssaviruses .

How do mutations in recombinant WCBV glycoprotein G affect its immunogenicity and pathogenicity in experimental models?

A notable mutation (H496Q) has been identified in the cytoplasmic domain of the WCBV glycoprotein G, though its specific impact on viral pathogenesis or cellular anti-viral response has not been fully characterized . This mutation occurs in a functionally important region that could influence viral assembly, budding, or interaction with host cell components.

When evaluating WCBV pathogenicity in Syrian hamsters as an experimental model for accidental hosts, researchers found that WCBV was highly pathogenic, causing 100% lethality and mild encephalitis . Comparative analyses with other lyssaviruses (RABV and DUVV) revealed that WCBV exhibits an intermediate capacity to:

• Promote cellular antiviral responses

• Produce pro-inflammatory cytokines

• Recruit and activate lymphocytes in the central nervous system

These immunological characteristics likely reflect the structural and functional properties of the glycoprotein, which mediates viral entry and serves as a primary target for immune recognition. Researchers investigating glycoprotein mutations should consider employing reverse genetics approaches to systematically evaluate how specific amino acid changes affect these immunological parameters .

What are the most promising approaches for developing cross-protective vaccines that include WCBV glycoprotein G?

The development of cross-protective vaccines that include WCBV glycoprotein G represents a critical public health need given the ineffectiveness of current rabies vaccines against this virus. Based on available research, several promising approaches emerge:

• Multivalent Recombinant Vectors: Dual-expression constructs containing both RABV and WCBV glycoprotein genes (such as VV-RGWG) have demonstrated protection against both viruses in experimental models . This suggests that a multivalent approach incorporating glycoproteins from phylogenetically distant lyssaviruses could provide broad-spectrum protection.

• Chimeric Glycoprotein Constructs: Engineering chimeric glycoproteins that combine immunodominant epitopes from WCBV with those of other lyssaviruses may induce broader neutralizing antibody responses while maintaining production efficiency.

• Novel Adjuvant Formulations: Exploring advanced adjuvant systems that enhance the immunogenicity of WCBV glycoprotein G could potentially overcome the limited cross-reactivity observed with conventional vaccine formulations.

• Prime-Boost Strategies: Sequential immunization with different lyssavirus glycoproteins, including WCBV glycoprotein G, might broaden the immune response beyond what can be achieved with simultaneous administration.

• Next-Generation Platform Technologies: mRNA or DNA vaccine platforms offer advantages for rapid development and may provide opportunities to deliver optimized WCBV glycoprotein G constructs that induce robust immunity.

Each approach requires systematic evaluation in appropriate animal models, with careful assessment of both humoral and cellular immune responses to determine the most effective strategy for comprehensive protection against the full spectrum of lyssaviruses .

How can recombinant WCBV glycoprotein G be utilized for developing improved diagnostic assays for lyssavirus surveillance?

Recombinant WCBV glycoprotein G offers significant potential for enhancing lyssavirus surveillance through the development of more comprehensive diagnostic assays:

• Expanded Serological Testing: Current rabies serology tests may miss antibodies specific to divergent lyssaviruses like WCBV. Incorporating recombinant WCBV glycoprotein G into multiplex serological assays would enable detection of antibodies against this phylogenetically distant virus, providing a more complete picture of lyssavirus exposure in surveillance populations .

• Differential Diagnosis Platforms: The development of protein microarrays or multiplex bead-based assays incorporating recombinant WCBV glycoprotein G alongside other lyssavirus glycoproteins would allow simultaneous testing for antibodies against multiple lyssavirus species, facilitating rapid differential diagnosis.

• Improved Virus Neutralization Tests: Pseudotyped viruses expressing WCBV glycoprotein G can be utilized in biosafe neutralization assays to evaluate protective antibody levels against this virus without requiring BSL-3 containment.

• Monoclonal Antibody Development: Recombinant WCBV glycoprotein G can serve as an immunogen for producing monoclonal antibodies useful in developing antigen capture assays specific for WCBV detection in field samples.

• Ecological Surveillance Tools: Given WCBV's association with specific bat species (Miniopterus schreibersii, Rousettus aegyptiacus, and Eidolon helvum) and recent spillover to a domestic cat, non-invasive sampling combined with WCBV-specific diagnostics could enhance ecological surveillance efforts .

Implementation of these advanced diagnostic approaches would significantly improve our ability to monitor the circulation of WCBV and other divergent lyssaviruses, providing early warning of potential spillover events that threaten public health .

What are the critical knowledge gaps in understanding the role of WCBV glycoprotein G in cross-species transmission?

Despite growing research on WCBV, several critical knowledge gaps remain regarding the role of its glycoprotein G in cross-species transmission:

• Receptor Binding Specificity: The precise cellular receptors that WCBV glycoprotein G interacts with across different mammalian species remain poorly characterized. Understanding these interactions is crucial for predicting host range and potential spillover risks .

• Adaptive Mutations: While a mutation (H496Q) in the cytoplasmic domain of WCBV glycoprotein G has been identified, we lack comprehensive data on which specific mutations facilitate adaptation to new host species . Comparative genomic analyses of multiple WCBV isolates from different hosts would help identify key adaptive changes.

• Immunological Evasion Mechanisms: How WCBV glycoprotein G interacts with host immune defenses, particularly the innate immune response in different mammalian species, requires further investigation to understand its capacity for successful cross-species transmission .

• Tissue Tropism Determinants: The specific regions of WCBV glycoprotein G that determine tissue tropism across different host species need clarification. Although studies with related lyssaviruses suggest conservation of entry receptors across mammals, WCBV-specific data is limited .

• Transmission Dynamics: Given the recent spillover event to a domestic cat, understanding how glycoprotein properties influence viral shedding, transmission routes, and infectious potential in accidental hosts is crucial for risk assessment .

• Evolutionary Constraints: The evolutionary pressures acting on WCBV glycoprotein G during host adaptation remain unclear. Research comparing glycoprotein sequences from natural hosts versus accidental hosts would provide insights into selection pressures during cross-species transmission events .

Addressing these knowledge gaps through focused research would significantly enhance our understanding of WCBV's zoonotic potential and inform public health preparedness efforts .

What are the optimal conditions for expressing and analyzing WCBV glycoprotein G in various experimental systems?

When working with WCBV glycoprotein G in research settings, optimizing expression and analysis conditions is crucial for generating reliable data. Based on existing methodologies, the following approaches are recommended:

For vaccinia virus-based expression systems:

• Use the p7.5 vaccinia virus promoter, which has been successfully employed to drive expression of WCBV glycoprotein G

• Perform plaque purification in the presence of selection medium containing 25 μg/ml mycophenolic acid, 250 μg/ml xanthine and 15 μg/ml hypoxanthine

• Conduct six rounds of selection to ensure homogeneity of the recombinant isolates

• Verify recombinant constructs by PCR targeting the thymidine kinase region of the vaccinia genome

For protein expression confirmation:

• Employ indirect immunofluorescence assays (IFA) using mouse anti-WCBV hyperimmune serum or specific monoclonal antibodies

• Perform comprehensive sequencing analysis to confirm the integrity of all constructs and cloned genes

For purification and storage:

• Propagate recombinant viruses and semi-purify through ultracentrifugation at 19,000 g through a 36% sucrose cushion

• Resuspend in MEM with 1× antibiotics and store in aliquots at −80°C for long-term viability

For functional analysis:

• Evaluate immunogenicity through animal models, particularly Syrian hamsters, using intramuscular inoculation to mimic natural infection routes

• Employ real-time PCR on retrotranscribed cDNAs using a two-step PCR approach for gene expression analysis

• Normalize target gene Ct values to β-actin housekeeping gene and evaluate relative mRNA expression levels as fold increase compared to control animals

What experimental designs best elucidate the role of WCBV glycoprotein G in pathogenesis and host immune responses?

To effectively investigate the role of WCBV glycoprotein G in pathogenesis and host immune responses, researchers should consider the following experimental design approaches:

• Comparative Infection Models:

  • Use intramuscular inoculation in Syrian hamsters to mimic natural infection routes

  • Include controls with parental virus strains alongside recombinant constructs expressing WCBV glycoprotein G

  • Compare WCBV with other lyssaviruses (RABV and DUVV) as standards to contextualize pathogenicity findings

• Immunological Profiling:

  • Monitor both humoral and cellular immune responses over time

  • Evaluate virus-neutralizing antibody development in serum samples

  • Assess cytokine production and lymphocyte recruitment/activation in the central nervous system

  • Use real-time PCR to measure expression of immune response genes, normalizing to housekeeping genes like β-actin

• Pathogenesis Assessment:

  • Monitor clinical progression, particularly focusing on neurological symptoms

  • Collect tissue samples (brain, salivary glands, brown fat, lung, kidney, and bladder) post-mortem for comprehensive analysis

  • Test for presence of viral RNA and infectious virus in different tissues to track viral spread

• Route-Dependent Studies:

  • Compare different inoculation routes (intramuscular in different locations, oral) to understand how infection route affects pathogenesis

  • This approach has revealed that WCBV infection progression depends significantly on the location of inoculation

• Statistical Analysis:

  • Apply appropriate statistical tests such as the Mantel-Cox test for survival curves and the Wilcoxon–Mann–Whitney test for independent groups

  • Consider p-values <0.05 as statistically significant

These experimental approaches will provide comprehensive insights into how WCBV glycoprotein G contributes to viral pathogenesis and shapes host immune responses, which is crucial for vaccine development and public health preparedness .

How can researchers effectively address technical challenges in working with recombinant WCBV glycoprotein G?

Researchers working with recombinant WCBV glycoprotein G face several technical challenges that require specific methodological solutions:

• Limited Availability of Reference Strains:

  • Challenge: The reference strain (EF614258) isolated from the reservoir host (M. schreibersii) may not be readily available as original brain material or as low-passage batches suitable for experiments .

  • Solution: Establish collaborations with reference laboratories to access authentic material or develop reverse genetics systems to reconstruct the virus based on published sequences.

• Antigenic Characterization Difficulties:

  • Challenge: The divergence of WCBV from other lyssaviruses complicates serological testing and antigenic characterization.

  • Solution: Develop custom monoclonal antibodies specific to WCBV glycoprotein G and establish standardized immunoassays for consistent detection and characterization.

• Purification Complexities:

  • Challenge: Viral batches may contain non-viral antigens that could confound immunological studies .

  • Solution: Implement more stringent purification protocols beyond ultracentrifugation, such as affinity chromatography or size-exclusion methods to obtain highly purified recombinant glycoprotein preparations.

• Expression System Optimization:

  • Challenge: Achieving optimal expression levels of functional WCBV glycoprotein G.

  • Solution: Evaluate multiple expression systems beyond vaccinia virus, including baculovirus, mammalian cell-based systems, or cell-free protein synthesis approaches, comparing yield and functionality of the expressed protein.

• Safety Considerations:

  • Challenge: Working with a highly pathogenic virus that causes 100% lethality in experimental models .

  • Solution: Develop pseudotyped virus systems expressing only WCBV glycoprotein G on a non-pathogenic viral backbone for safer handling in BSL-2 facilities.

• Strain-Specific vs. Species-General Findings:

  • Challenge: Determining whether observed findings relate to the specific strain under study or represent species-level characteristics .

  • Solution: When possible, compare multiple WCBV isolates, including those from natural hosts versus accidental hosts, and both cell-adapted versus animal-passaged variants.

By systematically addressing these technical challenges, researchers can generate more reliable and comprehensive data on recombinant WCBV glycoprotein G, advancing our understanding of this divergent lyssavirus and its potential public health implications .