Recombinant Ovine respiratory syncytial virus Major surface glycoprotein G (G)

What is the genomic structure of the ovine RSV G glycoprotein gene?

The ovine RSV G gene contains 838 nucleotides with a major open reading frame encoding a protein of 263 amino acid residues. The predicted molecular weight of this polypeptide is approximately 29 kDa. The gene shares 73% nucleotide sequence identity with that of bovine RSV strain A51908, but only 60% amino acid identity at the protein level, indicating significant divergence between these related viruses . The ovine RSV G protein has the same length as bovine RSV strain A51908 but contains an additional six amino acids at the carboxyl terminus compared to bovine RSV strain 391-2 .

How does ovine RSV G glycoprotein differ from bovine and human RSV G proteins?

The amino acid sequence of ovine RSV G protein shows only 60% identity with the bovine RSV G protein, with even lower identity (53%) between their extracellular domains . Despite this low level of identity, there are similarities in the predicted hydropathy profiles of the G proteins from ovine and bovine RSV . The intergenic sequences for the SH-G and G-F gene junctions of ovine RSV show 64% and 57% identity respectively with the corresponding regions of bovine RSV . These differences suggest that ovine and bovine RSV might be classified as two subgroups of an ungulate RSV rather than as two distinct viruses .

What are the key functional domains of the RSV G glycoprotein?

Based on available research, the RSV G protein is proposed to have three main functional domains (though the information from the search results is incomplete on this specific point) . In human RSV, the central region of the G protein (amino acids 149-200) has been identified as immunologically significant, as it can prime mice for polarized type 2 T-cell responses, and peripheral blood T cells from most human donors recognize epitopes within this region .

What are the most effective expression systems for producing recombinant ovine RSV G protein?

While the search results don't specifically address ovine RSV G protein expression systems, related research on RSV fusion (F) glycoprotein demonstrates successful use of baculovirus vectors for expression in Sf9 insect cells . For recombinant RSV G protein, researchers would likely need to:

• Clone the G gene from ovine RSV isolates using RT-PCR

• Insert the gene into an appropriate expression vector

• Express the protein in a cellular system such as insect cells or mammalian cells

• Purify the recombinant protein using affinity chromatography

The choice between bacterial, yeast, insect, or mammalian expression systems would depend on the research goals, particularly whether proper glycosylation is required for the intended studies.

How can researchers optimize the yield and purity of recombinant ovine RSV G protein?

Based on principles applied to similar viral glycoproteins:

• Codon optimization for the chosen expression system

• Inclusion of appropriate secretion signals if extracellular expression is desired

• Selection of optimal cell culture conditions (temperature, pH, media composition)

• Development of a multi-step purification protocol that may include:

  • Affinity chromatography (using specific antibodies or tags)

  • Ion exchange chromatography

  • Size exclusion chromatography

• Implementation of quality control measures to ensure protein integrity and activity

What methods are most effective for analyzing the glycosylation patterns of recombinant ovine RSV G protein?

For comprehensive glycosylation analysis, researchers should consider:

• Mass spectrometry (MS) techniques:

  • MALDI-TOF MS for molecular weight determination

  • LC-MS/MS for detailed glycan structural analysis

• Enzymatic digestion with specific glycosidases followed by gel mobility shift analysis

• Lectin binding assays to identify specific glycan structures

• Nuclear magnetic resonance (NMR) spectroscopy for detailed structural characterization

• Comparison with native ovine RSV G protein to assess glycosylation fidelity

How can researchers effectively characterize the antigenic epitopes of recombinant ovine RSV G protein?

Epitope mapping techniques would include:

• Generation of monoclonal antibodies against different regions of the G protein

• Creation of a panel of deletion mutants or overlapping peptides

• Competitive binding assays to determine epitope relationships

• X-ray crystallography of antibody-antigen complexes when possible

• Phage display technology to identify peptide mimics of conformational epitopes

• Bioinformatic prediction tools combined with experimental validation

What strategies can be used to generate recombinant ovine RSV with modifications in the G protein gene?

Based on the approaches used for similar recombinant RSV strains:

• Reverse genetics systems that allow manipulation of the full viral genome

• Creation of specific deletions in the G protein central domain, similar to the rA2cpΔG150-222 or rA2cpΔG177-220 recombinant viruses described for human RSV

• Site-directed mutagenesis to modify specific amino acids

• Introduction of reporter genes or epitope tags for tracking purposes

• Validation of modified viruses through sequencing and functional assays

How does deletion of specific regions in the G protein affect viral replication and immunogenicity?

Research on human RSV with G protein modifications shows that:

• Recombinant RSV strains with deletions in the central ectodomain of the G protein (amino acids 151-221 or 178-219) can still replicate effectively in human lung epithelial cell lines

• These modified viruses show normal replication at 37°C but develop pinpoint morphology at 39°C

• They can replicate in the respiratory tracts of mouse models and elicit serum neutralization and anti-F-protein IgG titers comparable to those of parent viruses

• Such modifications might alter the T-cell response profile, potentially reducing unwanted type 2 T-cell polarization that has been associated with respiratory disease enhancement

How can recombinant ovine RSV G protein be used to develop improved diagnostic assays?

Researchers can develop:

• Subgroup-specific peptide-based enzyme-linked immunosorbent assays (ELISAs) using recombinant G protein or specific peptides

• Multiplex assays that can differentiate between ovine and bovine RSV infections

• Lateral flow immunoassays for rapid field testing

• PCR-based assays targeting the G gene to distinguish viral lineages

• Serological assays for epidemiological studies

Such diagnostics would need validation using serum panels from animals with confirmed infections.

What is the potential of recombinant ovine RSV G protein as a vaccine component?

Based on RSV vaccine research principles:

• Recombinant G protein could serve as a subunit vaccine component, potentially combined with other viral proteins like the F protein

• Modified G proteins with deletions in immunomodulatory domains might provide protection without priming for enhanced disease

• Nanoparticle formulations containing G protein, similar to the RSV F nanoparticles described in the literature, might enhance immunogenicity

• The differential immune responses to various domains of the G protein would need careful evaluation to avoid vaccine-enhanced disease

• Both humoral and cell-mediated immune responses should be assessed in appropriate animal models

How do post-translational modifications of recombinant ovine RSV G protein affect its immunological properties?

Researchers investigating this question should examine:

• The impact of different glycosylation patterns on antibody recognition

• Whether O-linked and N-linked glycans differentially affect immune responses

• The role of glycosylation in masking or revealing critical epitopes

• How expression systems affect post-translational modifications

• Methods to produce recombinant G protein with native-like modifications

What molecular mechanisms underlie the species-specific differences in RSV G protein and their impact on host range?

This advanced research question requires:

• Comparative sequence analysis of G proteins from different RSV strains (human, bovine, ovine)

• Identification of regions associated with host cell receptor binding

• Cross-species infection studies to determine barriers to transmission

• Structural biology approaches to understand receptor-ligand interactions

• Creation of chimeric viruses with G proteins from different species to map determinants of host specificity

What are the optimal conditions for analyzing recombinant ovine RSV G protein-induced immune responses in animal models?

Researchers should consider:

• Selection of appropriate animal models:

  Animal Model	Advantages	Limitations
  Sheep	Natural host, relevant immune responses	Expensive, limited reagents
  Cotton rats	Semi-permissive for RSV, established model	Not natural host for ovine RSV
  BALB/c mice	Well-characterized, many reagents available	Limited replication of RSV
  Lambs	Age-relevant natural host	Logistical challenges, cost

• Immunization protocols with appropriate controls

• Comprehensive immune response evaluation:

  • Antibody responses (neutralizing and non-neutralizing)

  • T-cell responses (Th1/Th2 balance, CD8+ responses)

  • Mucosal immunity in respiratory tract

  • Protection against challenge

• Assessment of potential enhanced disease upon challenge

How can researchers effectively compare results from different studies on recombinant ovine RSV G protein?

To ensure comparability across studies:

• Standardize protein characterization methods:

  • SDS-PAGE to confirm size and purity

  • Western blotting with reference antibodies

  • Glycosylation analysis

  • Activity/functionality assays

• Use common reference materials when possible

• Implement consistent reporting of:

  • Expression systems and purification methods

  • Protein concentration determination techniques

  • Storage conditions and stability data

  • Endotoxin levels for proteins used in immunological studies

• Conduct side-by-side comparisons with previous preparations when introducing modifications