Development of a potential vaccine against Capripox virus implementing reverse vaccinology and pan-genomic immunoinformatics

CPXV is responsible for animal diseases affecting cattle (Lumpy Skin Disease), sheep (Sheeppox), and goats (Goatpox). During outbreaks, these diseases have huge socio-economic effects. Now, no vaccination that is effective against sheeppox, goatpox, and lumpy skin disease is available. This work used an immunoinformatic methodology to discover possible targets for vaccination against CPXV. After the 122 CPXV proteins were obtained from the Vipr database, several investigations into the proteins' virulence, antigenicity, toxicity, solubility, and IFN-g activity were carried out. Three outer membrane and extracellular proteins were selected to predict their B-cell and T-cell epitopes based on certain distinctive features. These epitopes exhibit conservation across three species, namely Sheeppox virus (SPPV), Goatpox virus (GTPV), and Lumpy skin disease virus (LSDV) of CPXV. This will provide more comprehensive immunity against diverse virus strains worldwide. Nine MHC-I, MHC-II, and B-cell epitopes were selected to generate multi-epitope vaccine constructions. These constructs were linked using AAY, GPGPG, and KK linkers and 50S ribosomal protein L7/L12 adjuvants to enhance the immunogenicity of the vaccines. Molecular modeling and structural validation enabled the production of vaccine constructs with high-quality 3D structures. CPXV (Protein A35, Protein Resolve A22, and Scaffold Protein) was selected for further analysis because of its varied immunological and physiochemical properties (Number of Amino Acids, Molecular Weight (Daltons), Theoretical pI Aliphatic index, Grand average of hydropathicity (GRAVY), Instability index GC content, and CAI value) and docking scores. The bacterial expression system showed notable gene expression for the CPXV-V5 vaccine, as shown by computational cloning analysis. Molecular dynamics (MD) simulations revealed structural stability and long-term epitope visibility, implying strong immune responses after delivery. These results suggest that the developed vaccines might be quite safe and effective in practical settings, and they offer a solid foundation for further experimental verification.

The genus Capripoxvirus belongs to the Poxviridae family. The sheeppox, goatpox, and lumpy skin disease viruses are three species of this genus with 96% identity in their genomes. These are financially devastating viral infections among cattle, which cause a reduction in animal products and lead to a loss in livestock industries. In the current study, the phylogenetic analysis was carried out to reveal the evolutionary relationships of Capripoxvirus species (i.e., sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV)) with other viruses from the Poxviridae family with >96% query coverage to find the similarity index among all members. The three viruses (i.e., SPPV, GTPV, and LSDV) joined the clade of Capripoxvirus of the Poxviridae family in the phylogenetic tree and exhibited close evolutionary relationships. The multiple sequence alignment using ClustalOmega revealed significant variations in the protein sequences of the DNA-dependent RNA polymerase of SPPV, GTPV, and LSDV. The three-dimensional structures of five selected bee peptides and DNA-directed RNA polymerase of SPPV, GTPV, and LSDV were predicted using trRosetta and I-TASSER and used for molecular docking and simulation studies. The protein-protein docking was carried out using HADDOCK server to explore the antiviral activity of peptides as honey bee proteins against SPPV, GTPV, and LSDV. In total, five peptides were docked to DNA-directed RNA polymerase of these viruses. The peptides mellitin and secapin-1 displayed the lowest binding scores (-106.9 +/- 7.2 kcal/mol and -101.4 +/- 11.3 kcal/mol, respectively) and the best patterns with stable complexes. The molecular dynamics simulation indicated that the complex of protein DNA-dependent RNA polymerase and the peptide melittin stayed firmly connected and the peptide binding to the receptor protein was stable. The findings of this study provide the evidence of bee peptides as potent antimicrobial agents against sheeppox, goatpox, and lumpy skin disease viruses with no complexity.

Screening and identification of LSDV-specific monoclonal antibodies to establish a double-antibody sandwich ELISA for distinguishing LSDV from SPPV and GTPV.

LSDV126 gene based molecular assays for specific detection and characterization of emerging Lumpy Skin Disease virus

A lumpy skin disease virus deficient of an IL-10 gene homologue provides protective immunity against virulent capripoxvirus challenge in sheep and goats

Sheep pox (SPP) and goat pox (GTP) are viral diseases of sheep and goats caused by sheep pox virus (SPPV) and goat pox virus (GTPV), respectively. SPPV and GTPV belong to the genus Capripoxvirus of the family Poxviridae, together with lumpy skin disease virus (LSDV) of cattle. They have double-stranded DNA genomes of approximately 134–147 kbp. SPPV and GTPV are closely related to LSDV though they possess specific nucleotide differences suggesting distinct phylogeny. SPP and GTP are notifiable diseases to the World Organisation for Animal Health (OIE). They are highly contagious diseases: the viruses spread through direct contact with lesions or contaminated objects, feed, and wool. SPP and GTP are endemic in Africa (except southern Africa), central Asia, the Indian subcontinent, the Middle East, Turkey, Greece, and some eastern European countries. Clinically, the presence of nodular skin lesions, mostly around the mouth and perineum regions, is a typical sign of the disease. Capripoxviruses classification and their nomenclature have been mainly based on the affected host species, creating a challenge for isolates naming. For a more accurate naming, it is better to use molecular methods as support to identify and classify capripoxvirus isolates. Conventional and real-time PCR methods are available that could help with the simultaneous detection and genotyping of the viruses. SPPV and GTPV as well as LSDV cross-react serologically, making it difficult to differentiate them using serological methods. To prevent and control SPP and GTP, illegal animal movement restrictions and vaccination campaigns with adequate vaccines and sufficient vaccination coverage are two very effective measures. The development of a high-throughput serological assay (ELISA) with better sensitivity and specificity and the development of a safe and effective vaccine, which can support the differentiation of infected from vaccinated animals (DIVA), are highly required.

Three members of Capripoxvirus (CaPV) genus, including lumpy skin disease virus (LSDV), goatpox virus (GTPV), and sheeppox virus (SPPV), are mentioned as notifiable forms by World Organization for Animal Health. These viruses have negatively impacted ruminant farming industry worldwide, causing great economic losses. Although SPPV and GTPV cause more severe clinical disease in only one animal species, they can transfer between sheep and goats. Both homologous and heterologous immunization strategies are used to protect animals against CaPVs. However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of CaPV infection. Therefore, we developed a novel triplex real-time PCR (qPCR) for the differentiation of LSDV, GTPV, and SPPV. Universal primers were designed to detect pan-CaPV sequences. Species-specific minor groove binder (MGB)-based probes were designed, which were labeled with FAM for LSDV, HEX for GTPV, and ROX for SPPV. The sensitivity, specificity, reproducibility, and ability of detecting mixed infections were evaluated for the triplex qPCR. Further, 226 clinical samples of the infection and negative controls were subjected to the triplex qPCR, and the results were verified using PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing methods for PRO30 gene. The triplex qPCR could successfully distinguish LSDV, GTPV, and SPPV in one reaction, and the assay sensitivity was 5.41, 27.70, and 17.28 copies/μL, respectively. No cross-reactivity was observed with other viruses causing common ruminant diseases, including des petits ruminants virus, foot-and-mouth disease virus, bluetongue virus, ovine contagious pustular dermatitis virus, infectious bovine rhinotracheitis virus, and bovine viral diarrhea-mucosal disease virus. Inter-and intra-assay variabilities were < 2.5%. The results indicated that the triplex qPCR was highly specific, sensitive, and reproducible. Simulation experiments revealed that this assay could successfully distinguish two or three viruses in case of mixed infections without any cross-reaction. For clinical samples, the results were completely consistent with the results of PCR-RFLP and sequencing. This demonstrated that the assay was reliable for clinical application. The triplex qPCR is a robust, rapid, and simple tool for identifying various types of CaPV as it can successfully distinguish LSDV, GTPV, and SPPV in one reaction. Furthermore, the assay can facilitate more accurate disease diagnosis and surveillance for better control of CaPV infection.

Sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) affect small ruminants and cattle causing sheeppox (SPP), goatpox (GTP) and lumpy skin disease (LSD) respectively. In endemic areas, vaccination with live attenuated vaccines derived from SPPV, GTPV or LSDV provides protection from SPP and GTP. As live poxviruses may cause adverse reactions in vaccinated animals, it is imperative to develop new diagnostic tools for the differentiation of SPPV field strains from attenuated vaccine strains. Within the capripoxvirus (CaPV) homolog of the variola virus B22R gene, we identified a unique region in SPPV vaccines with two deletions of 21 and 27 nucleotides and developed a High-Resolution Melting (HRM)-based assay. The HRM assay produces four distinct melting peaks, enabling the differentiation between SPPV vaccines, SPPV field isolates, GTPV and LSDV. This HRM assay is sensitive, specific, and provides a cost-effective means for the detection and classification of CaPVs and the differentiation of SPPV vaccines from SPPV field isolates.

Comparative sensitivity study of primary cells, vero, OA3.Ts and ESH-L cell lines to lumpy skin disease, sheeppox, and goatpox viruses detection and growth

Lumpy skin disease (LSD) is an important infectious disease caused by lumpy skin disease virus (LSDV) in bovine. LSDV, sheep pox virus (SPPV), and goat pox virus (GTPV) from the same genus Capripoxvirus (CaPV) of the Poxviridae family exhibit a nucleotide sequence similarity of up to 97%. Therefore, attenuated vaccines of GTPV and SPPV are often used to vaccinate cattle against LSD. However, available serological testing methods cannot accurately differentiate cattle vaccinated with GTPV from those infected with LSDV, posing a significant risk for disease spread. In this study, we developed a synthesized gene unique to LSDV as a differential antigen to detect serum antibodies specific to LSDV and differentiate naturally infected from vaccinated animals (DIVA). We used it for an in-house indirect enzyme-linked immunosorbent assay (iELISA), and no cross-reaction with positive sera for bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), Mycobacterium bovis (M. Tb), Pasteurella multocida (P. multocida), and Mycoplasma bovis (M. bovis). The cut-off value (S/P%) was 30% for in-house iELISA. The corresponding diagnostic specificity was 100% (95% CI: 88.43-100), and the diagnostic sensitivity was 93.3% (95% CI: 77.93-99.18). The intra-assay coefficient of variation (CV) ranged from 1.08% to 4.11%, and the interassay CV was 0.00%-8.90%. Furthermore, 200 clinical serum samples were examined, in the vaccinated herd, there were no positive samples (0/141) indicating the strong differentiation ability of this method. On the other hand, in the infected herds, the overall positivity was 33.90% (20/59) (95% CI: 22.08-47.39). In summary, a valuable synthesized protein unique to LSDV was developed and showed a promising application in an iELISA with high specificity and sensitivity in differentiating cattle infected with LSDV from those vaccinated with GTPV.

Bovine nodular dermatitis (Bovine lumpy skin disease) is a transmissible disease of cattle (Bos indicus and B. taurus) and Asian water buffaloes (Bubalus bubalis), that has been classified as a notifiable disease by the World Organization for Animal Health. Bovine lumpy skin disease virus belongs to the Poxviridae family. The poxviridae family can be divided into 2 subfamilies: Chordopoxviridae, which infects vertebrates, and Entopoxviridae, which infects insects. The causative agent of lumpy skin disease belongs to the subfamily Chordopoxviridae, the genus capripoxvirus, that also includes the causative agent of sheep and goat pox virus [1,2]. The genomes of goat and sheep pox viruses are very similar to the genome of bovine lumpy dermatitis virus. They have about 97% nucleotide identity. All genes for sheep pox and goat pox viruses are evident in bovine lumpy disease. The work was carried out using bioinformative and molecular genetic research methods. We have developed a technique for detecting capripoxvirus DNA by the method of isothermal loop amplification of the p32 / ld121 gene region under conditions of 30-40 min. amplification at 60 ° C. The developed technique is sensitive (analytical sensitivity corresponds to the activity of the virus 2 lg TCID50 / ml), specific and reproducible, and the developed primers do not hybridize with heterologous DNA templates.

A real time high-resolution melting PCR assay for detection and differentiation among sheep pox virus, goat pox virus, field and vaccine strains of lumpy skin disease virus

BackgroundCapripox viruses are economically important pathogens in goat and sheep producing areas of the world, with specific focus on goat pox virus (GTPV), sheep pox virus (SPPV) and the Lumpy Skin Disease virus (LSDV). Clinically, sheep pox and goat pox have the same symptoms and cannot be distinguished serologically. This presents a real need for a rapid, inexpensive, and easy to operate and maintain genotyping tool to facilitate accurate disease diagnosis and surveillance for better management of Capripox outbreaks.ResultsA LAMP method was developed for the specific differential detection of GTPV and SPPV using three sets of LAMP primers designed on the basis of ITR sequences. Reactions were performed at 62°C for either 45 or 60 min, and specificity confirmed by successful differential detection of several GTPV and SPPV isolates. No cross reactivity with Orf virus, foot-and-mouth disease virus (FMDV), A. marginale Lushi isolate, Mycoplasma mycoides subsp. capri, Chlamydophila psittaci, Theileria ovis, T. luwenshuni, T. uilenbergi or Babesia sp was noted. RFLP-PCR analysis of 135 preserved epidemic materials revealed 48 samples infected with goat pox and 87 infected with sheep pox, with LAMP test results showing a positive detection for all samples. When utilizing GTPV and SPPV genomic DNA, the universal LAMP primers (GSPV) and GTPV LAMP primers displayed a 100% detection rate; while the SPPV LAMP detection rate was 98.8%, consistent with the laboratory tested results.ConclusionsIn summary, the three sets of LAMP primers when combined provide an analytically robust method able to fully distinguish between GTPV and SPPV. The presented LAMP method provides a specific, sensitive and rapid diagnostic tool for the distinction of GTPV and SPPV infections, with the potential to be standardized as a detection method for Capripox viruses in endemic areas.

Capripoxvirus-vectored vaccines against livestock diseases in Africa

Lumpy Skin Disease (LSD) is a notifiable viral disease caused by Lumpy Skin Disease virus (LSDV). It is usually associated with high economic losses, including a loss of productivity, infertility, and death. LSDV shares genetic and antigenic similarities with Sheep pox virus (SPV) and Goat pox (GPV) virus. Hence, the LSDV traditional diagnostic tools faced many limitations regarding sensitivity, specificity, and cross-reactivity. Herein, we fabricated a paper-based turn-on fluorescent Molecularly Imprinted Polymer (MIP) sensor for the rapid detection of LSDV. The LSDV-MIPs sensor showed strong fluorescent intensity signal enhancement in response to the presence of the virus within minutes. Our sensor showed a limit of detection of 101 log10 TCID50/mL. Moreover, it showed significantly higher specificity to LSDV relative to other viruses, especially SPV. To our knowledge, this is the first record of a paper-based rapid detection test for LSDV depending on fluorescent turn-on behavior.