In a search for alternative, environmentally friendly and effective disinfecting agents, a commercially available protease—Neutrase®—was tested in this work for inactivation of koi herpesvirus (KHV) and of viral haemorrhagic septicaemia virus (VHSV). For comparison, the stability of these viral pathogens in similar configurations at various pH values and concentrations of peracetic acid or quicklime, typically used for disinfection, was tested. Therefore, virus suspensions were incubated with various concentrations of different agents for 24 hr and the titre of the remaining infectious particles was determined by virus titration. Furthermore, the treatment of both viruses, with the agents at concentrations that were previously appointed as effective, was also examined in the presence of solid material (quartz sand). All procedures investigated in this study, including the protease treatment, were able to reduce the titre of KHV and VHSV below the detection limit of the titration. Although further studies are necessary, this is the first report of the application of a protease for the inactivation of the selected fish pathogens, demonstrating the great potential of the latter for disinfection.

Cholesterol is essential for building and maintaining cell membranes and is critical for several steps in the replication cycle of viruses, especially for enveloped viruses. In mammalian cells virus infections lead to the accumulation of the oxysterol 25-hydroxycholesterol (25HC), an antiviral factor, which is produced from cholesterol by the cholesterol 25 hydroxylase (CH25H). Antiviral responses based on CH25H are not well studied in fish. Therefore, in the present study putative genes encoding for CH25H were identified and amplified in common carp and rainbow trout cells and an HPLC-MS method was applied for determination of oxysterol concentrations in these cells under virus infection. Our results give some evidence that the activation of CH25H could be a part of the antiviral response against a broad spectrum of viruses infecting fish, in both common carp and rainbow trout cells in vitro. Quantification of oxysterols showed that fibroblastic cells are capable of producing 25HC and its metabolite 7α,25diHC. The oxysterol 25HC showed an antiviral activity by blocking the entry of cyprinid herpesvirus 3 (CyHV-3) into KFC cells, but not spring viremia of carp virus (SVCV) or common carp paramyxovirus (Para) in the same cells, or viral haemorrhagic septicaemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) into RTG-2 cells. Despite the fact that the CH25H based antiviral response coincides with type I IFN responses, the stimulation of salmonid cells with recombinant type I IFN proteins from rainbow trout could not induce ch25h_b gene expression. This provided further evidence, that the CH25H-response is not type I IFN dependent. Interestingly, the susceptibility of CyHV-3 to 25HC is counteracted by a downregulation of the expression of the ch25h_b gene in carp fibroblasts during CyHV-3 infection. This shows a unique interplay between oxysterol based immune responses and immunomodulatory abilities of certain viruses.

Cholesterol is a major constituent of cell membranes and exhibits together with its oxidation products (oxysterols) several essential cellular functions. Additionally, it was recently shown to play a role during virus replication in fish. To investigate the involvement of cholesterol and oxysterols, for example, during a viral infection in fish cells, quantitative analysis of these analytes from cell cultures is necessary. A fast method based on solvent extraction followed by reversed‐phase high‐performance liquid chromatography with mass spectrometry for quantitation of cholesterol and seven oxysterols from cells and culture medium is described in this work. Detection limits were in the low ng/mL range, and the intra‐ and inter‐day precisions were above 87% for all analytes. A cholesterol content of approximately 10 μg per 2 × 106 cells, exceeding the concentration of oxysterols by at least 103‐fold, was determined for common carp brain cells. Furthermore, using the method established here, an uptake of externally supplied 25‐hydroxycholesterol by fish cells and its conversion to 7a,25‐dihydroxycholesterol could be shown. In summary, this is the first report on quantification of cholesterol and oxysterols from fish cell cultures, which can help in exploring their function during viral infections.

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