## Developing Diagnostic Tools for Capripoxvirus and Rift Valley Fever virus Scientists are constantly working on new and improved diagnostic tools for infectious diseases. Two of these diseases, capripoxvirus and Rift Valley fever virus (RVFV), pose important threats to livestock and human health, respectively. ### Targeting specific Proteins for Detection To develop accurate diagnostic tests for these viruses, researchers identified specific proteins as targets. For capripoxvirus, the focus was on a protein homologous to the vaccinia virus C-type lectin-like protein A34 (LSDV ORF 123). This protein had already proven effective in elisas for detecting capripoxvirus antibodies in sheep, goats, and cattle. For RVFV,the target was the nucleocapsid (NP) protein. This protein is crucial for viral replication and is known to trigger a strong immune response. ### Producing and Purifying the Target Proteins The capripoxvirus protein was initially produced using established methods. After successful proof-of-concept testing, the protein production was scaled up commercially by GenScript, Inc. (Piscataway, NJ, USA). For the RVFV NP protein, researchers used a plasmid (pET 32a-RVFV NP) containing the gene for the protein to transform bacterial cells (BL21a cells). This transformation was confirmed through DNA amplification using specific primers. The transformed bacteria were then grown in a nutrient-rich broth and induced to produce the NP protein using specific chemicals (L-arabinose and IPTG).The production process was carefully monitored over a five-hour period to determine optimal expression time. Following cell lysis, the target protein was purified using an affinity purification method. This technique relies on the protein’s His-tag, wich binds specifically to nickel ions immobilized on a column. This allows for the separation and isolation of the NP protein from other cellular components. To ensure the protein remained in its natural, functional conformation, the purification was carried out under native conditions. This involved using specific concentrations of imidazole (10mM for binding,20mM for washing,and 250mM for elution) in the buffers used throughout the purification process. an Amicon concentrator was used for buffer exchange, ensuring the purified protein was in a suitable solution for further use in developing diagnostic tests.
Development and Validation of an ELISA for the Detection of Rift Valley Fever Virus Nucleoprotein Antibodies
Scientists have successfully developed and validated a new Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of antibodies against Rift Valley Fever virus (RVFV) nucleoprotein. This breakthrough will significantly enhance the ability to diagnose RVFV infections in both animals and humans. The ELISA utilizes purified RVFV nucleoprotein as the antigen, ensuring high specificity and sensitivity.RVFV Nucleoprotein Production and Validation
The researchers first produced the RVFV nucleoprotein through a careful purification process.They confirmed its purity and integrity using SDS-PAGE and Western blot analysis, employing an anti-penta His antibody for detection. The presence of antibodies against RVFV nucleoprotein in both goat and cattle sera, previously confirmed to be positive for the virus, was also verified by Western blotting.ELISA development and Optimization
The ELISA protocol was meticulously optimized. A checkerboard titration was performed to determine the optimal concentrations of antigen, serum dilutions, and secondary antibody. The assay was designed to detect the presence of antibodies against RVFV nucleoprotein in serum samples. Once optimized, the ELISA was rigorously validated using a panel of serum samples from animals known to be infected with RVFV and from uninfected animals. The results demonstrated excellent specificity and sensitivity, highlighting the reliability and accuracy of the new assay.Applications and Future Directions
The development of this RVFV nucleoprotein ELISA represents a significant advancement in the field of infectious disease diagnostics. This assay will be invaluable for: * Quickly and accurately diagnosing RVFV infections in both animals and humans. * surveillance and monitoring of RVFV outbreaks. * Tracking the efficacy of RVFV vaccination programs. Further research will focus on evaluating the performance of the ELISA in diverse geographical regions and exploring its potential for point-of-care diagnostics.Evaluating the Accuracy of a Novel Diagnostic Test for Lumpy Skin Disease
This study aimed to evaluate the accuracy of a new diagnostic test for lumpy skin disease (LSD), a significant viral illness affecting cattle. Researchers meticulously gathered a wide range of serum samples to rigorously assess the test’s performance.Serum Samples for Evaluation
The study utilized four distinct groups of bovine serum samples: * **LSD-Positive Sera:** 77 samples were collected from cattle naturally infected with LSD in Bulgaria and the Republic of North Macedonia.These samples were confirmed as positive through a virus neutralization test (VNT). * **LSD-Negative Sera:** 59 samples were gathered from countries where LSD is not typically found – France, Austria, and the Republic of North Macedonia (collected before 2010). These samples served as negative controls and were confirmed negative through VNT. * **Capripoxvirus-Positive sera:** 27 samples from experimentally infected sheep and goats, originating from research institutions in the UK, mali, and Ethiopia, were included. These samples were also confirmed positive by VNT. * **Capripoxvirus-Negative Sera:** 181 samples from Austria, a country without a history of capripoxvirus, were incorporated as negative controls. For further analysis of cross-reactivity, sera from animals infected with other parapoxviruses were used.This included 12 samples from goats naturally infected with Orf virus, 4 samples from cattle infected with pseudocowpox (PCP) from Zambia, and one sample from a cow with bovine papular stomatitis (BPS) from Austria.Longitudinal Studies
The researchers also included samples from longitudinal studies, tracking antibody development over time in cattle experimentally infected with LSD virus. ## Capripoxvirus and RVF Serum samples Used in a Diagnostic Study This study utilized three sets of capripoxvirus samples for analysis. The first set consisted of samples from cattle infected with the highly virulent South African LSDV Neethling strain. These samples were collected at 0, 7, 14, 21, and 28 days post-infection (DPI). The second set of samples came from goats experimentally infected with GTPV Oman 84. Collections took place at 0, 7, 14, 21, 28, 35, 42, 49, and 56 DPI, as part of a larger study investigating GTPV in Ethiopian goats. The third set consisted of samples from a single sheep infected with LCV (mali) SPPV algeria/93 Djelfa. Samples were collected at 0, 7, 14, 21, and 28 DPI. ## RVF Serum Samples Four distinct types of reference populations provided RVF serum samples: (a) Bovine RVF-positive sera (n=6) originated from Zambia,field-infected cattle from Côte d’Ivoire,and commercially available bovine RVF-positive serum samples from IDVet,Montpellier,France. Confirmation of positivity was achieved through cELISA testing conducted by IDvet, Montpellier, France. (b) A total of 56 bovine negative serum samples were sourced from countries where RVF is not historically present – France, Austria, and the United Kingdom.IDVet, Montpellier, France, provided the French samples, while AGES, Austria, supplied the Austrian samples, and the Pirbright Institute provided the UK samples. (c) Samples of RVF-positive sheep and goat sera (n=13) came from a goat experimental infection in Teramo, Italy, courtesy of Chiara pinoni from Diagnostica e Sorveglianza Malattie Esotiche, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G.Caporale.” Additional positive sheep and goat samples were collected during routine screening at the Laboratoire Vétérinaire de Kinshasa, DRC.Development of a Luminex multiplex Assay for Detecting Rift valley Fever Virus and Capripoxvirus Antibodies
researchers have developed a novel Luminex multiplex serological assay for the simultaneous detection of antibodies against Rift Valley fever virus (RVFV) and capripoxvirus (CaPV).This innovative diagnostic tool offers significant advantages over customary single-antigen assays, allowing for the efficient and accurate screening of animal populations for exposure to these significant livestock pathogens. The assay relies on coupling specific viral target antigens to polystyrene carboxyl magnetic beads.coupling of Antigens to Beads
The process began by washing and activating carboxyl magnetic beads. Sulfo-NHS and EDC, chemicals used for covalent coupling, were added to the beads and incubated in the dark.The beads were then washed and resuspended in coupling buffer. Purified RVFV nucleoprotein (NP) or A34-CaPV antigens were added to corresponding beads designated for each virus. The bead-antigen mixtures were incubated, washed, and resuspended in a storage buffer. The number of microspheres recovered after the coupling process was carefully counted.This assay involved a multi-step process to accurately measure antigen levels. Each well of a microplate was prepared by rinsing with wash buffer and then pre-filled with a specific volume of BB1 buffer. Coupled beads, designed to target specific antigens, were then added to the wells along with diluted serum samples.Bovine serum samples were diluted 1:500 in BB1, while sheep and goat samples were diluted 1:2000. The plate was sealed, covered with foil, and incubated overnight at 4°C on a shaker.
Following incubation, the liquid from each well was removed, and the wells were washed repeatedly with wash buffer before being treated with a diluted, species-specific biotin-conjugated secondary antibody. This antibody was designed to bind to the target antigen. After incubation and further washing, a diluted streptavidin–PE solution was added to each well. This solution binds to the biotin on the secondary antibody, allowing for detection.
Following another round of washing, reading buffer was added to each well, and the plate was shaken before being analyzed in a BioPlex 200 Luminex machine. this machine measures the fluorescence intensity emitted by each bead, providing a quantitative measure of the target antigen present in the sample.
All samples were analyzed in duplicate, and the average reading was calculated. To ensure accuracy, background fluorescence levels were accounted for by subtracting the signal from wells containing no serum from the signal obtained from wells containing serum.
