Infectious salmon anaemia virus (ISAV) is an enveloped, segmented, negative sense ssRNA virus. ISAV is a WOAH listed virus and is a major threat to salmonid farming. Horizontal transmission is the major route for transmission of ISAV in fish. Hence, determination of virus survival under different environmental conditions is important for risk assessment, disease modelling to develop preventive and control measures for disease management. The primary challenge in virus survival studies lies in the inconsistency regarding the types and sources of water used, virus quantification methods, fish strains, and evaluation techniques (Oidtmann et al., 2018). Furthermore, the reliance on cell culture-based systems presents a limitation, particularly in direct seawater applications. Seawater contains salts and inorganic substances, live and dead organic matter, including bacteria and fungi and their toxic products, that would have a very bad influence on cells. Removing salt, organic matter, toxins, bacteria, fungi et al. will inherently be done by methods that will influence the integrity/infectiousness of virus – and hence these can not be an option aiming at determining exactly that. To overcome this limitation, sterile seawater is employed in cell culture-based virus survival assessments. Moreover, the sterile seawater is adjusted for pH and supplemented with Hanks balanced salt solution, HEPES, antibiotics, antimycotics, and 5% FBS (Tapia et al., 2013). While this approach mimics more of the cell culture media, allowing for prolonged virus survival, and it deviates significantly from real-life conditions in seawater. Thus, estimating virus survival under such controlled conditions does not accurately reflect the challenges posed by natural seawater environments. At IMR, a standardized in vivo system was developed to test virus survival wherein virus manipulation was kept to a minimum. Moreover, the same fish breed and fish size was used. We can also use this standardized method to estimate virus survival for nonculturable viruses. With this system, in a previous experiment, the minimum infectious dose (MID) and survival of ISAV at 10°C in seawater was determined (not published FOTS27817). With this background, the main goal of this study is to determine the virus survival at different temperatures (4-17°C) and salinities (0-35 ppt) using this in vivo system. The hypothesis is that the increase in temperature and salinity will reduce the ISAV survival.

All the virus-spiked seawater incubation containers will be taken out of incubator on the day just before the virus challenge. 40 Atlantic salmon fry will be netted, counted and moved to 3.5 L tanks with 200 ml of freshwater. After that 200 ml seawater with virus will be added to the respective tanks and incubated for 6 hrs. The tanks will be aerated and monitored continuously during the bath challenge period. The fish has shown tolerance 50% seawater without any abnormality and mortality in previous experiments (FOTS27817). In addition, the naïve cohabitant fish will be sedated with tricaine methanesulfonate (Finquel MS-222) (50 mg/L) buffered with sodium bicarbonate (50 mg/L) added to the tanks with bath challenged fish after 2 days of primary infection. During the sampling, fish will be netted from the tanks and euthanized with overdose of tricaine methanesulfonate (Finquel MS-222). From the tanks for water sampling. The water flow will be stopped from day 4, morning for one hour and tanks will be aerated, and 1 liter of water will be taken from the tanks for water filtration and virus estimation from day four to 10. Survival experiment is for 7/8 days, however for shedding experiment To get more data about shedding, we are extending two days more for this filtration experiment. The fish will be sampled until day 10 and fish will be sampled at day 11. The water filtration experiment will be terminated if we see any morality in the tanks.

The salinity, presence of bacteria/fungi and other organic and inorganic materials make seawater highly incompatible with direct use in in vitro cell culture assays aimed at quantifying viable viruses. Methodological procedures employed to circumvent these problems will inevitably interfere with virus numbers and virus viability and hence compromise the essence of the study aim, leading to inferior data output.