Improved assessment of fish virus survival in seawater using a standardized in vivo-based test system
1. Purpose.
Water is the primary medium for pathogen transmission in aquatic organisms. As obligate parasites, fish viruses cannot replicate outside a suitable fish and the transfer between hosts is basically a matter of staying infective, i.e. ‘survive’ to ensure persistence and spread. Virus can survive in water for a few minutes to several weeks, depending on environmental factors. Many of previous fish virus survival studies focused on directly evaluating inactivation by performing cell culture assays or qPCR analyses, or challenging fish with virus-spiked water. The studies have included manipulation/handling of the virus that likely have influenced the reliability of obtained measures. Certain fish viruses, such as PMCV, PRV and SGPV, cannot be cultured in vitro and infectivity can hence only be evaluated by in vivo methodology.
The objective of this study is to determine the survival of ISAV and SAV3 in seawater. To this end, a highly standardized in vivo benchmarking system using salmon fry will be applied. Salmon fry are very susceptible to the viruses and hence constitute a highly sensitive monitoring of virus survival. Importantly, this in vivo methodology will allow the avoidance of any manipulation of viruses that could interfere with the produced virus survival estimates. As a necessary preliminary experiment, minimal infectious doses (MID) for ISAV and SAV3 in our benchmark system will be determined. Such standardized MID data will be very important by their own right, allowing improved epidemiological understanding of the viruses.
2. Distress
The experiment will cause distress and morbidity in fish, but since it will terminated at an early time point, mortality is not expected.
3. Expected benefit.
Improved and standardized estimates of biophysical virus parameters that are crucial for understanding the epidemiology of two viruses that have severe effects in aquaculture, from where they also pose a risk to wild salmon populations.
4. Number of animals, and what kind
6320 Atlantic salmon (Salmo salar) fry
5. How to adhere to 3R
The salinity and high content of bacteria/fungi make seawater highly incompatible with direct use in in vitro cell culture asays 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. Appliance with 3R is hence mainly restricted to performing short experiments that are terminated before the fish develop severe morbidity and any mortality. Further, to use strict humane end points and to ensure an experimental design that maximizes the likelihood of producing important and statistical significant scientific results.
Extension of FOTS validity to June 15, 2025 is requested because of an unavoidable delay in conducting some of the experiments (SAV3 stability study in seawater postponed last October 2023 due to low titer virus production).
Water is the primary medium for pathogen transmission in aquatic organisms. As obligate parasites, fish viruses cannot replicate outside a suitable fish and the transfer between hosts is basically a matter of staying infective, i.e. ‘survive’ to ensure persistence and spread. Virus can survive in water for a few minutes to several weeks, depending on environmental factors. Many of previous fish virus survival studies focused on directly evaluating inactivation by performing cell culture assays or qPCR analyses, or challenging fish with virus-spiked water. The studies have included manipulation/handling of the virus that likely have influenced the reliability of obtained measures. Certain fish viruses, such as PMCV, PRV and SGPV, cannot be cultured in vitro and infectivity can hence only be evaluated by in vivo methodology.
The objective of this study is to determine the survival of ISAV and SAV3 in seawater. To this end, a highly standardized in vivo benchmarking system using salmon fry will be applied. Salmon fry are very susceptible to the viruses and hence constitute a highly sensitive monitoring of virus survival. Importantly, this in vivo methodology will allow the avoidance of any manipulation of viruses that could interfere with the produced virus survival estimates. As a necessary preliminary experiment, minimal infectious doses (MID) for ISAV and SAV3 in our benchmark system will be determined. Such standardized MID data will be very important by their own right, allowing improved epidemiological understanding of the viruses.
2. Distress
The experiment will cause distress and morbidity in fish, but since it will terminated at an early time point, mortality is not expected.
3. Expected benefit.
Improved and standardized estimates of biophysical virus parameters that are crucial for understanding the epidemiology of two viruses that have severe effects in aquaculture, from where they also pose a risk to wild salmon populations.
4. Number of animals, and what kind
6320 Atlantic salmon (Salmo salar) fry
5. How to adhere to 3R
The salinity and high content of bacteria/fungi make seawater highly incompatible with direct use in in vitro cell culture asays 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. Appliance with 3R is hence mainly restricted to performing short experiments that are terminated before the fish develop severe morbidity and any mortality. Further, to use strict humane end points and to ensure an experimental design that maximizes the likelihood of producing important and statistical significant scientific results.
Extension of FOTS validity to June 15, 2025 is requested because of an unavoidable delay in conducting some of the experiments (SAV3 stability study in seawater postponed last October 2023 due to low titer virus production).