The diagnostic window for detection of viruses infecting salmon in erythrocytes.
Lead Research Organisation:
University of Stirling
Department Name: Institute of Aquaculture
Abstract
Diagnosis of viruses infecting fish is often done by detecting the nucleic acids of the viruses in fish tissues. This approach ignores increasing evidence that these viruses can replicate in blood. If blood could be used as a diagnostic sample it would simplify and possibly also speed up diagnostic testing.
We want to explore the replication of five viruses infecting salmon (ISAV, SAV, PRV,PMCV, IPMV) in salmon erythrocytes. We want to assess which of these viruses show good replication in erythrocytes and if conclusions for a diagnostic window for their detection in live salmon can be drawn. We also want to explore if virus infection of the erythrocytes illicits an immune response, which would show that erythrocytes which are in first contact with the infectious agents are also involved in front line defense against infection. Another feature we would like to monitor are the morphological changes induced in infected erythrocytes.
If replication of these viruses in erythrocytes can be confirmed it would lead the way to simplifying virus diagnostics. To additionally standardize testing we want to develop material for external quality assessments of molecular diagnostics in fish diagnostic laboratories. This is absolutely necessary since accreditation services can not assess how individual laboratories are performing nor are individual laboratories able to compare their performance to others. In short the whole concept of quality assessment, which is well established in the diagnostics of viruses infecting humans, is missing in the laboratory scene testing for fish viruses. To this end we have included QMCD from Glasgow into our consortium which has a long standing expertise in developing EQA panels.
Overall the outcomes of this project will help to simplify and to optimize molecular detection for viruses infecting salmon.
We want to explore the replication of five viruses infecting salmon (ISAV, SAV, PRV,PMCV, IPMV) in salmon erythrocytes. We want to assess which of these viruses show good replication in erythrocytes and if conclusions for a diagnostic window for their detection in live salmon can be drawn. We also want to explore if virus infection of the erythrocytes illicits an immune response, which would show that erythrocytes which are in first contact with the infectious agents are also involved in front line defense against infection. Another feature we would like to monitor are the morphological changes induced in infected erythrocytes.
If replication of these viruses in erythrocytes can be confirmed it would lead the way to simplifying virus diagnostics. To additionally standardize testing we want to develop material for external quality assessments of molecular diagnostics in fish diagnostic laboratories. This is absolutely necessary since accreditation services can not assess how individual laboratories are performing nor are individual laboratories able to compare their performance to others. In short the whole concept of quality assessment, which is well established in the diagnostics of viruses infecting humans, is missing in the laboratory scene testing for fish viruses. To this end we have included QMCD from Glasgow into our consortium which has a long standing expertise in developing EQA panels.
Overall the outcomes of this project will help to simplify and to optimize molecular detection for viruses infecting salmon.
Technical Summary
Objectives
1. To characterise ISAV, SAV, PRV, PMCV and IPNV replication in primary salmon erythrocyte culture
2. To characterise the immune response elicited in salmon erythrocytes during virus infection
3. To develop virus standards which can be used for EQA
Methods used.
ISAV, SAV, and IPNV will be cultured from virus stocsks available at the Institute of Aquaculture, Stirling (IoA) and Marine Scotland, Aberdeen (MS). PRV and PMCV will be isolated by IoA from fish samples provided by MS. Initially pure virus stocks of ISAV, SAV, PRV, PMCV and IPNV will be prepared using ultarcentrifugation. These viruses will be sent for commercial generation of polyclonal sera which will then be used for immunofluorescent staining techniques to detect the viruses inside of the erythrocytes. Primary erythrocyte cultures will be infected with ISAV, SAV, PRV, PMCV and IPNV and the time course of virus replication will be monitored by qPCR and TCID50. The infected erythrocytes will be subjected to several staining techniques to identify morphological changes.
In order to characterize the possible immune response of the nucleated erythrocytes polyribosomal RNA will be purified and screened using selected qPCR panels to detect innate and adaptive immune response markers (Zoology, Aberdeen (ZA)) and RNA.seq for general transcriptome analysis (IoA). All of the mentioned protocols are available at the respective institutions. Purified viruses will be sent to Quality Control for Molecular Diagnostics, Glasgow (QCMD) who will prepare EQA panels using well established techniques developed for human virus EQA panels. These EQA panels will be sent to 5 fish diagnostic laboratories to test their proficiency. Results will be analyzed and published. If successful the EQA panels could be developed into a commercial product by QMCD.
1. To characterise ISAV, SAV, PRV, PMCV and IPNV replication in primary salmon erythrocyte culture
2. To characterise the immune response elicited in salmon erythrocytes during virus infection
3. To develop virus standards which can be used for EQA
Methods used.
ISAV, SAV, and IPNV will be cultured from virus stocsks available at the Institute of Aquaculture, Stirling (IoA) and Marine Scotland, Aberdeen (MS). PRV and PMCV will be isolated by IoA from fish samples provided by MS. Initially pure virus stocks of ISAV, SAV, PRV, PMCV and IPNV will be prepared using ultarcentrifugation. These viruses will be sent for commercial generation of polyclonal sera which will then be used for immunofluorescent staining techniques to detect the viruses inside of the erythrocytes. Primary erythrocyte cultures will be infected with ISAV, SAV, PRV, PMCV and IPNV and the time course of virus replication will be monitored by qPCR and TCID50. The infected erythrocytes will be subjected to several staining techniques to identify morphological changes.
In order to characterize the possible immune response of the nucleated erythrocytes polyribosomal RNA will be purified and screened using selected qPCR panels to detect innate and adaptive immune response markers (Zoology, Aberdeen (ZA)) and RNA.seq for general transcriptome analysis (IoA). All of the mentioned protocols are available at the respective institutions. Purified viruses will be sent to Quality Control for Molecular Diagnostics, Glasgow (QCMD) who will prepare EQA panels using well established techniques developed for human virus EQA panels. These EQA panels will be sent to 5 fish diagnostic laboratories to test their proficiency. Results will be analyzed and published. If successful the EQA panels could be developed into a commercial product by QMCD.
Planned Impact
The project will provide data on the diagnostic window for ISAV, SAV, PRV, PMCV and IPNV during their viraemic phase. It will also provide an external quality assessment panel (EQA panel) through the cooperation of IoA, MSS and QMCD consisting of purified inactivated viruses.
Currently the Scottish government (MSS) and the aquaculture industry, in particular Marine Harvest, Scottish Salmon Company, Scottish Sea F,arms operate company owned diagnostic laboratories that use real time qPCR for the detection of various viruses infecting salmon. It is fair to assume that they all try their best but there is no way to verify the performance of their assays. If the new EQA panel is used by QMCD for an EQA proficiency assessment with these laboratories they will, for the first time, be able to assess their performance and if need be to improve their assays.
Since the virus detection results produced by these laboratories are used for major health management decisions, which have commercial consequences for the aquaculture industry, the results of these project may have an important impact on these decisions with all the economic and societal impact attached to these decisions.
Currently the Scottish government (MSS) and the aquaculture industry, in particular Marine Harvest, Scottish Salmon Company, Scottish Sea F,arms operate company owned diagnostic laboratories that use real time qPCR for the detection of various viruses infecting salmon. It is fair to assume that they all try their best but there is no way to verify the performance of their assays. If the new EQA panel is used by QMCD for an EQA proficiency assessment with these laboratories they will, for the first time, be able to assess their performance and if need be to improve their assays.
Since the virus detection results produced by these laboratories are used for major health management decisions, which have commercial consequences for the aquaculture industry, the results of these project may have an important impact on these decisions with all the economic and societal impact attached to these decisions.
Organisations
Publications
McGowan M
(2021)
Testing of NKA expression by mobile real time PCR is an efficient indicator of smoltification status of farmed Atlantic salmon.
in Aquaculture (Amsterdam, Netherlands)
| Description | We have developed a panel of virus materials which can be sent out to test the quality of molecular diagnostic labs . We secondly have shown that nucleated salmon erythrocytes can be infected by viruses. The viruses replicate and induce an immune response in the erythrocytes. This so called innate immune response has a surprising general high level of activity in the salmon erythrocytes even if not infected. Nevertheless viruses manage to infect these erythrocytes and kick selected innate immune response factors into activity. This allows to speculate that erythrocytes in fish are a first line of defence. |
| Exploitation Route | We will develop a new proposal to look at the erythrocyte response ein more detail and to address the question of why viruses still replicate in erythrocytes inspire of the high background activity of the innate immune response. |
| Sectors | Agriculture, Food and Drink,Other |
| Title | EQA panel for salmon viruses |
| Description | An external quality assessment panel for viruses affecting salmon was developed in collaboration with partner QMCD. Final validation is pending. It may be considered for commercial rise by QMCD |
| Type Of Material | Biological samples |
| Year Produced | 2017 |
| Provided To Others? | No |
| Impact | External quality assessments for molecular diagnostics in virology have made all the difference ein human virology. In Aquaculture molecular diagnostics there are no commercially available EQA panels. If successful this tool should help to improve the quality of molecular diagnostic services. |