A novel immunoinformatics approach for developing a poly-epitope vaccine targeting foot and mouth disease virus, exploiting structural VP proteins.

Foot and mouth Disease virus (FMDV) belongs to Picornaviridae family and Aphthovirus genus causing Foot and mouth disease (FMD) in cloven-hoofed animals. FMDV, a prevalent virus induces both acute and chronic infections with high mutation rates resulting in seven primary serotypes, making vaccine development indispensable. Due to time and cost effectiveness of the immunoinformatic approach, we designed in-silico polyepitope vaccine (PEV) for the curtailment of FMDV. Structural and immunogenic parts of FMDV (Viral Protein 1 (VP1), Viral Protein 2 (VP2), Viral Protein 3 (VP3), and Viral Protein 4 (VP4)) were used to design the cytotoxic T Lymphocyte (CTL), Helper T Lymphocyte (HTL), and B-cell epitopes, followed by screening for antigenic, non-allergenic, Interferon (IFN) simulator, and non-toxicity, which narrowed down to 7 CTL, 3 HTL, and 12 B-cell epitopes. These selected epitopes were linked using appropriate linkers and Cholera Toxin B (CTB) adjuvant for immunological modulation. The physiochemical analyses followed by the structure prediction demonstrated the stability, hydrophilicity and solubility of the PEV. The interactions and stability between the vaccine, Toll like Receptor 3 (TLR3) and Toll like receptor 7 (TLR7) were revealed by molecular docking and Molecular Mechanics/Poisson Boltzmann Surface Area (MMPBSA) with high stability and compactness verified by MD simulation. In-silico immune simulation demonstrated a strong immunological response. FMDV-PEV (Poly epitope vaccine) will be effectively produced in an E. coli system, as codon optimization and cloning in an expression vector was performed. The effectiveness, safety, and immunogenicity profile of FMDV-PEV may be confirmed by further experimental validations.Communicated by Ramaswamy H. Sarma.

The structural and immunogenic parts of FMDV were targeted for developing VaccineCTB-adjuvant and appropriate linkers, enhancing the immunogenicity of the PEVMinimal deformability and high stability of Vaccine using immunoinformaticsStrong antigen-specific humoral and cellular immune response of potential vaccineResults indicating the effectiveness, safety, and immunogenicity of the PEV.

Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression.

Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the ßC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vß, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T1 plants harbored a single copy of the Vß transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vß-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vß-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.

Sugarcane Mosaic Virus-Based Gene Silencing in Nicotiana benthamiana.

Background: Potyvirus-based virus-induced gene silencing (VIGS) is used for knocking down the expression of a target gene in numerous plant species. Sugarcane mosaic virus (SCMV) is a monopartite, positive single strand RNA virus. Objectives: pBINTRA6 vector was modifi ed by inserting a gene segment of SCMV in place of Tobacco rattle virus (TRV) genome part 1 (TRV1 or RNA1) and the two nonstructural proteins of TRV2(RNA2). Materials and Methods: SCMV construct was inoculated into 3-4 weeks Nicotiana benthamiana plant leaves either by using a needleless syringe or applying pricking with a toothpick. Results: The construct (SCMV-RNA2) successfully induced post-transcriptional gene silencing (PTGS) of the target genes GFP and ChlI through agroinoculation proving that SCMV is a substitute of the RNA1, which plays a pivotal role in the systemic gene silencing. 2-3-weeks of post inoculation, target genes' silencing was observed in the newly developed noninoculated leaves. Conclusions: The newly developed construct expresses the knocked down of the endogenous as well as exogenous genes and only four weeks are required for the transient expression of the gene silencing based on SCMV-VIGS system.