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Fisheries Research Services
Virus Diagnostics at FRS Marine Laboratory
Introduction
Fisheries Research Services (FRS) staff carry out diagnostic
identified by an immunofluorescent antibody test (IFAT)
and surveillance tests for five types of virus: infectious
using a specific antibody (Fig. 2). Negative screening
salmon anaemia (ISA), viral haemorrhagic septicaemia
normally takes 28 days.
(VHS), infectious haematopoietic necrosis (IHN), infectious
pancreatic necrosis (IPN) and spring viraemia of carp
(SVC). The group also responds to requests for testing for
other viral pathogens, for example, nodavirus,
alphaviruses and aquareoviruses.
Virus Testing Methods Used
Infectious Salmon Anaemia (ISA)
FRS staff sample heart, kidney, liver and spleen tissues
from all fish for virus testing. ISA virus (ISAV) is isolated
by cell culture using the established fish cell lines salmon
Figure 2
ISAV infected TO cells showing positive staining by IFAT.
head kidney (SHK-1) or a leucocyte line (TO). Cell cultures
Other techniques are also used to provide evidence of
are observed for cytopathic effect (CPE). Since ISAV may
ISAV infection which include staining for viral antigen in
not give rise to CPE in infected cells, an additional
kidney imprints (Fig. 3) and identification of virus specific
detection method for intra-cellular virus is also used. ISAV
nucleic acid sequences.
is a virus which possesses a surface glyco-protein
haemagglutinin to which red blood cells will adhere. This
property can be exploited by overlaying the test cell
cultures with salmon or trout red blood cells. Red cell
clusters are observed which reveal centres of virus
infection (Fig. 1). Virus associated with infected cells is
Figure 3
Highly stained kidney imprint showing severe ISAV infection in
salmon.
Viral Haemorrhagic Septicaemia (VHS) and Infectious
Heamatopoietic Necrosis (IHN)
Brain or heart, spleen and kidney from immature fish,
Figure 1
Red blood cells cluster overlying ISAV infected SHK-1 cells.
and gonadal fluids from mature fish are sampled in the
course of statutory surveillance for VHS virus (VHSV) and
Fisheries Research Services is an agency of the Scottish Executive
FRS Marine Laboratory PO Box 101 375 Victoria Road Aberdeen
tel +44 (0)1224 876544 fax +44 (0)1224 295511
[email protected] http://www.frs-scotland.gov.uk
AB11 9DB
UK
Fisheries Research Services
IHN virus (IHNV). Tests are carried out by cell culture
Spring Viraemia of Carp (SVC)
isolation on bluegill fibroblast (BF-2) or rainbow trout
Brain, spleen and kidney tissues are sampled from all
gonad (RTG-2) cells for VHSV and epithelioma papulosum
sizes of fish for testing. SVC virus is detected by cell
cyprini (EPC) or fathead minnow (FHM) cells for IHNV.
culture isolation using EPC or FHM cells. Cell cultures are
Microscopic examination of cell cultures for CPE is carried
observed microscopically for CPE and virus identification
out.
is made by ELISA. The test normally takes 14 days for
negative screening.
Identification of both viruses is made by an enzyme-linked
immunosorbent assay (ELISA). The test normally takes
14 days for negative screening.
Viral Nervous Necrosis (VNN)
Brain, spinal cord, spleen and kidney tissues are sampled
from all sizes of fish for testing. The causal nodavirus
Infectious Pancreatic Necrosis (IPN) Diagnostics
may affect many different fish species including Atlantic
In diagnostic testing, mid-sections are sampled from sac
salmon, halibut and turbot. Nodavirus can be detected
fry and small fry, for larger fish kidney tissue is sampledand
by cell culture isolation on striped snakehead cells (SSN-
for mature fish gonadal fluids have to be sampled.
1) or a clone of the SSN-1 cell line, named E-11. Cultures
Detection of IPNV is carried out by cell culture isolation
are observed microscopically for CPE over 28 days.
on chinook salmon embryo (CHSE-214) cells. Cell cultures
Nodavirus leads to partial CPE which is recognised by
are observed microscopically for CPE (Fig. 4). Identification
trained readers. Virus identification is carried out by IFAT
of IPNV from the positive cultures is carried out by ELISA.
using a specific antiserum. Negative screening takes
Negative screening normally takes 14 days.
approximately 28 days.
Sleeping Disease Virus (SDV) and Salmon Pancreas
Disease Virus (SPDV)
The two salmonid alphaviruses SDV and SPDV are
genetically very closely related and regarded as types of
the same genus. Kidney, heart, pancreas and serum are
sampled for testing. Both viruses can be detected by cell
culture isolation on CHSE-214 cells; however isolation is
relatively difficult and slow. Careful microscopic
observation of cultures is required to detect CPE which
may only become evident after several passages. Virus
Figure 4
IPNV infected CHSE-214 cells showing CPE.
identification is carried out using PCR. Negative screening
takes a minimum of 42 days.
AAAH10|04|06
Fisheries Research Services is an agency of the Scottish Executive
FRS Marine Laboratory PO Box 101 375 Victoria Road Aberdeen
AB11 9DB
UK
tel +44 (0)1224 876544 fax +44 (0)1224 295511
[email protected] http://www.frs-scotland.gov.uk
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