National Science Centre, OPUS 25, “Are virucidal plant extracts able to prevent common carp (Cyprinus carpio L) infection by CyHV-3 virus (Cyprinid herpesvirus 3)?” 2024-09-02 – 2028-09-01

Project Manager: Agnieszka Dorota Troszok, PhD

Funding: PLN 2,154,160.00

Project Description: Cyprinid herpesvirus 3 (CyHV-3) is a virus that infects common carp and its ornamental variety, koi carp. CyHV-3, also known as KHV, causes KHVD disease, leading to mass mortality in carp, reaching up to 100% of the population. This virus emerged in the late 1990s and continues to cause enormous losses in aquaculture. For many years, scientists have been trying to find a solution to this problem. Numerous studies are being conducted to strengthen the immunity of farmed carp through immunostimulants, vaccinations, and the selection of individuals with the highest resistance for breeding. All solutions proposed so far remain in the research phase, and none have yet become widely available. These solutions should be safe, economical, and easy to apply in aquaculture. The results of preliminary pilot studies showed that ethanolic extracts of St. John’s wort and plantain inactivated the CyHV-3 virus in an extracellular environment, meaning they had virucidal activity. St. John’s wort and plantain are plants commonly used in folk medicine for centuries due to their health-promoting properties. Administering extracts from these plants in feed has had a positive effect on the health of Atlantic salmon and rainbow trout, so it can be expected that these extracts will also be safe for carp. In the pilot experiment, under in vitro conditions, ethanolic extracts of St. John’s wort and plantain showed very strong virucidal activity, therefore their activity will be investigated under in vivo conditions in this project. The composition of plant extracts (PEs) is highly dependent on the extraction method and conditions. Differences in the composition of extracts may result in them having different biological properties. The plant material will be subjected to extraction under various conditions using the classical method with ethanol, as well as the supercritical carbon dioxide (scCO2) method. The scCO2 extraction method is highly efficient and environmentally friendly, but it is unknown whether PEs obtained by this method will also show the activity observed in the pilot study, as ethanolic extracts were used in the pilot study. The virucidal activity of all PEs will be compared under in vitro conditions. Based on the results obtained, a method will be selected that yields PEs with the strongest virucidal properties. The composition of all PEs will also be examined to identify components that may play a key role in the virucidal action of PEs. The activity of these components will be confirmed in in vitro studies, both individually and in combinations, due to the possible synergistic effect of compounds contained in PEs. As a result, information will be obtained to enable the standardization of virucidal PEs. The most effective PEs will be used in in vivo experiments. The main entry point for CyHV-3 virus infection is the carp’s skin, which is covered with a layer of mucus. This mucus delays the moment of contact between the virus and the fish’s epidermal cells – thus delaying the moment of infection. It seems probable that the virus disseminated by infected individuals into the environment can be inactivated by PEs before the virus manages to infect the fish. This assumption will be tested in in vivo experiments. Extracts will be added to the water where uninfected carp will be kept. Subsequently, CyHV-3 infected carp will be placed in the same tank. The planned studies will clarify whether PEs applied to the water at a concentration safe for carp will prevent the transmission of CyHV-3 virus from infected to uninfected fish. The activity of PEs at different concentrations will be tested to determine the lowest PE concentration that inhibits virus transmission in the carp population. This will allow for the determination of the maximum possible scale of PE application in preventing virus transmission from infected to uninfected fish (large ponds, or smaller tanks used for keeping koi carp). Studies conducted with Atlantic salmon and rainbow trout have shown that PEs from St. John’s wort and plantain, administered with feed, had an immunomodulatory effect on these fish. The skin and gills belong to the mucous surfaces of fish, similar to the intestines, so it can be expected that PEs added to the water where carp live may modulate their immune response. Furthermore, contact of mucous surfaces (skin, gills, intestines) with the antigen of a given pathogen can also modulate the fish’s immune response against that pathogen. Plantain has been found to contain saponins, substances that acted as adjuvants, enhancing the specific immune response in carp. Considering these findings, it can be suspected that the CyHV-3 virus, which will be released by infected fish and then inactivated by virucidal PEs, may act as a mucosal vaccine. During the implementation of this project, the immune response in carp infected with CyHV-3 virus in the presence of PEs in the aquatic environment will be investigated. The studies planned in this project will provide fundamental knowledge that may prove useful for developing a method to prevent the spread of the virus in the carp population, as well as a new method for vaccinating carp. There is a high probability that both methods will be safe, convenient to use, environmentally friendly, and economical.