CHAPTER 2.3.9.

SPRING VIRAEMIA OF CARP

1. Scope1

Spring viraemia of carp (SVC) is a rhabdovirus infection capable of inducing an acute haemorrhagic and contagious
viraemia in several carp species and of some other cyprinid and ictalurid fish species. For the purpose of this chapter,
SVC is considered to be infection with spring viraemia of carp virus (SVCV). Comprehensive references can be found
in reviews by Wolf, 1988, Ahne et al., 2002and Dixon, 2008.

2. Disease information

2.1. Agent factors

2.1.1. Aetiological agent, agent strains

The aetiological agent of SVC is Spring viraemia of carp virus (SVCV), a species in the genus Vesiculovirus
in the virus family Rhabdoviridae (Carsten, 2010). The virus genome is a non-segmented, negative-sense,
single strand of RNA. The genome contains 11,019 nucleotides encoding five proteins in the following order:
a nucleoprotein (N), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G) and an RNA-dependent,
RNA polymerase (L). The genome does not contain a non-virion (NV) gene between the G and L genes as is
found in fish rhabdoviruses of the genus Novirhabdovirus (Ahne et al., 2002). The type strain of SVCV is
available from the American Type Culture Collection (ATCC VR-1390). Two complete genome sequences of
the type strain have been submitted to Genbank (Genbank accession U18101 by Björklund et al., 1996and
Genbank accession AJ318079 by Hoffmann et al., 2002). The complete genome sequence of isolates from
China (People’s Rep. of) has also been deposited in Genbank (Genbank accession DQ097384 by Teng et
al., 2007and Genbank accession EU177782 by Zhang et al., 2009).

Stone et al., 2003used sequence analysis of a 550 nucleotide region of the G-gene to compare 36 isolates
from different fish species and geographical locations previously identified as SVCV or pike fry rhabdovirus
(PFRV) by serology. The analysis showed that the isolates could be separated into four distinct genogroups
and that all of the SVCV isolates could be assigned to genogroup I, sharing <61% nucleotide identity with
viruses in the other three genogroups. Genogroup II comprised a single isolate from grass carp, previously
identified by serology as PFRV, genogroup III comprised the reference PFRV isolate, and genogroup IV
comprised a large number of unassigned isolates and isolates previously identified as PFRV. The latter
genogroup was called the tench rhabdovirus (TenRV) group after the species from which the earliest member
was isolated. Further analysis also showed that SVCV genogroup I could be further subdivided into at least
four subgenogroups. Ahne et al., 1998showed that the two viruses could also be differentiated by a
ribonuclease protection assay using a G-gene probe, suggesting that genetic differences exist between the
two viruses.

Antibodies directed against SVCV cross-react to various degrees with members of the other three
genogroups, indicating that the viruses possess common antigens, whilst being genetically distinct. The
viruses have been shown to share common antigenic determinants on the G, N and M proteins, but can be
differentiated by neutralisation assays (Jørgensen et al., 1989).

2.1.2. Survival outside the host

The virus has been shown to remain viable outside the host for 5 weeks in river water at 10°C, for more than
6 weeks in pond mud at 4°C reducing to 4 days in pond mud at 10°C (Ahne, 1976).

2.1.3. Stability of the agent

The virus is inactivated at 56°C for 30 minutes, at pH 12 for 10 minutes and pH 3 for 2 hours (Ahne, 1986).
Oxidising agents, sodium dodecyl sulphate, non-ionic detergents and lipid solvents are all effective for

1 NB: Version adopted by the World Assembly of Delegates of the OIE in May 2012.

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Chapter 2.3.9. - Spring viraemia of carp

inactivation of SVCV. The following disinfectants are also effective for inactivation: 3% formalin for 5 minutes,
2% sodium hydroxide for 10 minutes, 540 mg litre‒1 chlorine for 20 minutes, 200‒250 ppm (parts per million)
iodine compounds for 30 minutes, 100 ppm benzalkonium chloride for 20 minutes, 350 ppm alkyltoluene for
20 minutes, 100 ppm chlorhexidine gluconate for 20 minutes and 200 ppm cresol for 20 minutes (Ahne, 1982;
Ahne & Held, 1980; Kiryu et al., 2007). The virus can be stored for several months when frozen in medium
containing 2‒5% serum. The virus is most stable at lower temperatures, with little loss of titre for when stored
for 1 month at ‒20°C, or for 6 months at ‒30 or ‒74°C (Ahne, 1976; Kinkelin & Le Berre, 1974). The virus is
stable over four freeze(‒30°C)‒thaw cycles in medium containing 2% serum (Kinkelin & Le Berre, 1974).

2.1.4. Life cycle

The virus appears to enter the host via the gill. A viraemia follows and the virus rapidly spreads to the liver,
kidney, spleen and alimentary tract. The virus can be detected in faeces and is also shed into the water via
faeces and urine.

2.2. Host factors

2.2.1. Susceptible host species

Naturally occurring SVC infections have been recorded from the following cyprinid species: common carp
(Cyprinus carpio carpio) and koi carp (Cyprinus carpio koi), crucian carp (Carassius carassius), silver carp
(Hypophthalmichthys molitrix), bighead carp (Aristichthys nobilis), grass carp (white amur)
(Ctenopharyngodon idella), goldfish (Carassius auratus), orfe (Leuciscus idus), and tench (Tinca tinca) and
bream (Abramis brama) (Basic et al., 2009; Dixon, 2008). Three Indian carp species, merigal (Cirrhinus
merigala [= C.cirrhosus]), rohu (Labeo rohita) and catla (Catla catla [= Gebelion catla]) have been reported to
be hosts of SVCV (Haghighi Khiabanian Asl et al., 2008a), but the nucleotide sequence data from a
confirmatory reverse-transcription polymerase chain reaction (RT-PCR) deposited at Genbank does not align
with known SVCV nucleotide sequence data (D.M Stone, pers. comm.). In addition, the deduced amino acid
sequence shares only limited similarity with SVCV, and therefore further work is required to determine
whether this virus is SVCV in origin. The virus has also been isolated from the non-cyprinid sheatfish (also
known as European catfish or wels) (Silurus glanis) and from pike (Esox lucius); the viral nucleic acid has also
been detected in pike by combined RT-PCR and nested PCR (Koutná et al., 2003).

SVCV has also been reported to have been isolated from Nile tilapia (Sarotherodon niloticus) (Soliman et al.,
2008) and rainbow trout (Oncorhynchus mykiss) (Jeremic et al., 2004; Haghighi Khiabanian Asl et al., 2008b).
Immunohistochemistry constituted the sole basis of identification of SVCV from Nile tilapia; electron
microscopy purported to show virus in the nucleus, which is not a feature of SVCV infection. Haghighi
Khiabanian Asl et al., 2008bused the same RT-PCR to identify the virus in rainbow trout that produced
equivocal results when used to type the virus in Indian carp described above, and so the identity of that virus
in rainbow trout awaits confirmation. The virus isolated from rainbow trout by Jeremic et al., 2004was
subsequently confirmed to be SVCV by nucleotide sequence analysis, but attempts to infect rainbow trout
with the virus by intraperitoneal injection were unsuccessful, although the virus was virulent for common carp
(P.F. Dixon, J. Munro & D.M. Stone, unpublished data). Hence, the status of rainbow trout and tilapia as hosts
for SVCV remains unresolved, and awaits further confirmatory data. Some serological tests do not distinguish
SVCV from members of the other genogroups described by Stone et al., 2003, and it is imperative that
sequence data are used to confirm the identity of putative SVCV isolates from new hosts.

Other cyprinid species have been shown to be susceptible to SVCV by experimental bath infection, including
roach (Rutilus rutilus) (Haenen & Davidse, 1993) whilst zebra fish (Danio rerio) and the golden shiner
(Notemigonus crysoleucas) have been infected with SVCV by intraperitoneal injection (see Dixon, 2008). It is
reasonable to assume that other cyprinid species in temperate waters may be susceptible to infection. Other
species can also be infected experimentally, e.g. guppy (Lebistes reticulatus). The pumpkinseed (Lepomis
gibbosus) has been reported to have been experimentally infected with SVCV, but there are no supporting
data.

The nucleotide sequence of the G gene of a rhabdovirus isolated from the Pacific white shrimp, Litopenaeus
(Penaeus) vannamei, in Hawaii is over 99% identical to that of SVCV (Johnson et al., 1999), and is
serologically related to SVCV. The virus caused mortality in juvenile Pacific blue shrimp, L. stylirostris, fed
food pellets soaked in the virus (Lu & Loh, 1994).

2.2.2. Susceptible stages of the host

Generally, young fish up to 1 year old are most susceptible to clinical disease, but all age groups can be
affected. Moreover, there is a high variability in the degree of susceptibility to SVC among individuals of the

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same fish species. Apart from the physiological state of the fish, the role of which is poorly understood, age
or the age-related status of innate immunity appears to be extremely important: the younger the fish, the
higher the susceptibility to overt disease, although even adult broodfish can be susceptible to infection.

2.2.3. Species or subpopulation predilection (probability of detection)

Common carp varieties are the principal hosts for SVCV and are considered to be most susceptible to SVCV
infection followed, in order of susceptibility, by other carp species (including hybrids), other susceptible
cyprinid species and finally susceptible non-cyprinid fish species. When sampling during surveillance
programmes for SVC, common carp or strains such as koi or ghost (koi × common) carp are preferentially
selected, followed by carp hybrids (e.g. common carp × crucian carp), then other carp species such as crucian
carp, goldfish, grass carp, bighead carp and silver carp. Should these species not be available then other
known susceptible species should be sampled in the following preferential order: tench, orfe, wels catfish and,
finally, any other cyprinid species present. For disease surveillance purposes all cyprinid species should be
considered as potential covert carriers of SVCV. Cyprinid species are commonly mixed together in polyculture
systems and the risk of transmission of SVCV between species during disease outbreaks is high.

2.2.4. Target organs and infected tissue

High titres of virus occur in the liver and kidney of infected fish, but much lower titres occur in the spleen, gills
and brain (Dixon, 2008).

2.2.5. Persistent infection with lifelong carriers

The reservoirs of SVCV are clinically infected fish and covert virus carriers among cultured, feral or wild fish.
Factors affecting persistence and duration of the carrier state have not been studied.

2.2.6. Vectors

Among animate vectors, the parasitic invertebrates Argulus foliaceus (Crustacea, Branchiura) and Piscicola
geometra (Annelida, Hirudinea) transferred SVCV from diseased to healthy fish under experimental
conditions and the virus has been isolated from A. foliaceus removed from infected carp (Ahne et al., 2002;
Dixon, 2008). Herons (Ardea cinerea) were fed SVCV-infected carp and made to regurgitate the fish at
intervals post-feeding. Virus was isolated from fish regurgitated 120 minutes after feeding.

2.2.7. Known or suspected wild aquatic animal carriers

Most reports of SVC have been from cultured fish, but the virus has been isolated from both diseased and
apparently healthy feral carp in lakes.

It has been suggested that a possible mode of transmission of the virus is by the movement of baitfish, but
there are no data to show that this has occurred (Goodwin et al., 2004). The main mode of transmission of
the virus from one area to another is by movements of infected fish. The virus is often found in ornamental
fish such as goldfish and koi carp, which are regularly transported around the world.

2.3. Disease pattern

2.3.1. Transmission mechanisms

The mode of transmission for SVCV is horizontal, but ‘egg-associated’ transmission (usually called 'vertical'
transmission) cannot be ruled out following one report of isolation of SVCV from carp ovarian fluid although
there have been no further such reports. Horizontal transmission may be direct or vectorial, water being the
major abiotic vector (Fijan, 1988). Animate vectors (Section 2.2.6) and fomites may also be involved in
transmission of SVCV (Fijan, 1988). Once SVCV is established in pond stock or pond farm stock, it may be
very difficult to eradicate without destroying all types of life at the fish production site.

2.3.2. Prevalence

There are very few data on the prevalence of SVC, although there have been a small number of surveys of
prevalence of antibody against the virus. In one such survey, carp in 19 of 20 hatcheries surveyed were
positive for antibody against the virus. Data collected over the 10-year period 1992‒2002 from Serbia showed
that the virus had been isolated from carp at 12 of 38 hatcheries. The virus can occur sporadically in different
ponds on one site, and sporadically from year to year at different sites.

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2.3.3. Geographical distribution

For a long time, the geographical range of SVC was limited to countries of the European continent that
experience low water temperatures during winter. Consequently, the disease has been recorded from most
European countries and from certain of the western Independent States of the former Soviet Union (Belarus,
Georgia, Lithuania, Moldova, Russia and the Ukraine) (see Dixon, 2008for references to these and the
following locations). However, in 1998, the disease was recorded in goldfish in a lake in Brazil, in 2002 it was
reported for the first time in two separate sites in the USA, and it was reported from Canada in 2006. Detection
of the virus in carp in China (People’s Rep. of) was confirmed in 2004. Confirmation of the isolation of SVCV
from rainbow trout and Indian carp in Iran, and from Nile tilapia in Egypt awaits more data, and so the addition
of those countries to the geographical range of the virus, also awaits those data.

2.3.4. Mortality and morbidity

Disease patterns are influenced by water temperature, age and condition of the fish, population density and
stress factors. The immune status of the fish is also an important factor with both nonspecific (e.g. interferon)
and specific immunity (serum antibodies, cellular immunity) having important roles. Poor physiological
condition of over-wintered fish may be a contributory factor to disease susceptibility. In European aquaculture,
losses can be up to 70% in young carp (Ahne et al., 2002), but are usually from 1 to 40%. Approximately 20%
of the carp population in a lake in the USA died from SVC during a disease outbreak.

2.3.5. Environmental factors

Disease outbreaks in carp generally occur between 11 and 17°C. They rarely occur below 10°C, and
mortalities, particularly in older fish, decline as the temperature exceeds 22°C (Fijan, 1988). Secondary and
concomitant bacterial and/or parasitic infections can affect the mortality rate and display of signs. In carp, the
disease is often observed in springtime (hence the common name for the disease), particularly in countries
having cold winters. It is believed that the poor condition of the over-wintered fish may be a contributory factor
in disease occurrence. The disease can occur in fish in quarantine following the stress of transportation, even
though there has been no evidence of virus in the fish prior to transportation. The virus was isolated from
apparently healthy fish from a lake in Canada that had been sampled over a 13-day period during which the
water temperature varied between 27.3°C and 24.2°C.

2.4. Control and prevention

Methods to control SVC disease mainly rely on avoiding exposure to the virus coupled with good hygiene practices.
This is feasible on small farms supplied by spring or borehole water and a secure system to prevent fish entering
the farm via the discharge water. Hygiene measures should include disinfection of eggs by iodophor treatment
(Ahne & Held, 1980), until it has been confirmed unequivocally that vertical transmission does not occur, regular
disinfection of ponds, chemical disinfection of farm equipment, careful handling of fish to avoid stress and safe
disposal of dead fish. Reducing fish stocking density during winter and early spring will reduce the spread of the
virus. In rearing facilities with a controlled environment, elevation of water temperature above 19‒20°C may stop
or prevent SVC outbreaks. A safe and effective vaccine is not currently available. However, a number of
experimental inactivated preparations, live attenuated vaccines and DNA vaccines have given encouraging results
(Dixon, 2008).

2.4.1. Vaccination

A number of studies have reported the efficacy of vaccination, and vaccination trials in the field have been
reported from the former Yugoslavia, Austria and the former Czechoslovakia (Fijan, 1988); a vaccine was
once marketed in the latter country, but is no longer available. Laboratory trials have shown that DNA
vaccination can protect fish (Dixon, 2008; Emmenegger & Kurath, 2008), but further developmental work is
required.

2.4.2. Chemotherapy

Methisoprinol inhibits the replication of SVCV in vitro, but has not been tested under carp culture conditions.

2.4.3. Immunostimulation

Injection into carp of single-stranded and double-stranded RNA (which is an interferon inducer) protected carp
for longer than 3 weeks, but the treatment is not effective by bath administration.

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2.4.4. Resistance breeding

The “Krasnodar” strain of common carp has been bred for increased resistance to SVCV.

2.4.5. Restocking with resistant species

Not reported. The wide host range of the virus means that rigorous selection procedures would have to be
applied to prospective alternative species.

2.4.6. Blocking agents

Not identified.

2.4.7. Disinfection of eggs and larvae

The virus is not considered to be transmitted via the egg, but if thought necessary, eggs could be disinfected
by iodophor treatment (Ahne & Held, 1980).

2.4.8. General husbandry practices

Ponds should be disinfected regularly and effective disease biosecurity practices should be used. Equipment,
particularly nets, should not be used in different ponds unless first disinfected. Practices that might cause
stress should be minimised, and high stocking densities should be avoided.

3. Sampling

3.1. Selection of individual specimens

3.1.1. Diseased fish

Moribund fish or fish exhibiting clinical signs of the disease should be collected; fish should be alive when
collected. However, there are no pathognomonic gross lesions and there may be no clinical signs in cases of
sudden mortality (see Section 4.1.1). An identification label that includes information on the place, time, date,
species, number of samples collected, dead/moribund state on collection, and the name and contact
information of the individual collecting the sample(s) should be attached to the sample(s). A general approach
to surveillance and sampling is provided in Chapter 1.4. of the Aquatic Animal Health Code. See also the OIE
Guide for Aquatic Animal Health Surveillance (2009).

3.1.2. Fish appear to be clinically normal

Fish collection should encompass a statistically significant number of specimens, but it is obvious that failure
to detect certain pathogens from the sample does not guarantee the absence of these agents in the specimen
examined or in the stock. This is particularly true of free-ranging or feral stocks from which it is difficult to
collect a representative and random sample. However, the risk of a pathogen escaping the surveillance
system is reduced in fish farms whose fish stocks have been inspected and checked for pathogens for several
years (at least two), insofar as they are not exposed to possible recontamination by feral fish.

Samples should comprise all susceptible species on the site with each lot of a species being represented in
the sample group. A lot is defined as a group of the same fish species that shares a common water supply
and that originates from the same broodfish or spawning population.

Any moribund fish present in the fish population to be sampled should be selected first for sample collection
and the remainder of the sample should comprise randomly selected live fish from all rearing units that
represent the lot being examined.

A general approach to surveillance and sampling is provided in Chapter 1.4. of the Aquatic Animal Health
Code. See also the OIE Guide for Aquatic Animal Health Surveillance (2009).

3.2. Preservation of samples for submission

Samples for virus isolation should be transported to the laboratory at 4°C using refrigerated containers or on ice,
preferably in virus transport medium (Chapter 2.3.0, Section A.2.2.1.), and tested within 24 hours or, in exceptional
circumstances, 48 hours. The shipment of organ samples is preferred, but live or whole dead fish can be submitted

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to the testing laboratory if necessary. If this is not possible, samples can be frozen, but there may be loss of virus
viability on thawing the samples. Repeated freeze‒thawing of the sample must be avoided. Samples for RT-PCR
can be preserved in commercially available RNA preservation solutions according to the manufacturers’
recommendations, or, alternatively, samples can be fixed in ethanol.

3.3. Pooling of samples

Samples from up to five fish can be pooled.

3.4. Best organs or tissues

Subclinically infected fish (apparently healthy fish): kidney, spleen, gill and encephalon (any size fish).

Clinically affected fish: whole alevin (body length ≤ 4 cm), entire viscera including kidney and encephalon (> 4 cm
body length ≤ 6 cm) or, for larger sized fish, liver, kidney, spleen and encephalon.

3.5. Samples/tissues that are not suitable

It can be difficult to isolate virus from subclinically infected carrier fish and, in particular, from fish surviving a
disease outbreak with increasing time after the disease occurrence. Likewise, isolation of virus from such fish at
temperatures outside the clinical range for the disease is problematic. It may be possible to detect antibody against
the virus in such fish (Dixon, 2008), but see the caveat in Section 4below. Virus isolation may not be possible from
decomposed clinical samples, so the presence of signs of SVC disease and a positive indirect immunofluorescent
antibody test (IFAT) or enzyme-linked immunosorbent assay (ELISA) may be considered sufficient to initiate
control measures. A number of studies in which attempts were made to isolate virus from reproductive fluids were
unsuccessful, although the virus has been isolated at low frequency from ovarian, but not seminal, fluids.

4. Diagnostic methods

Diagnosis of SVC in clinically affected fish may be achieved by virus isolation or, more rapidly, by IFAT or ELISA on
infected tissues. Ideally, direct diagnosis by IFAT or ELISA should be confirmed by virus isolation followed by a virus
neutralisation (VN) test or RT-PCR and sequence analysis.

The detection of fish antibodies to viruses has not thus far been accepted as a routine screening method for assessing
the viral status of fish populations because of insufficient knowledge of the serological responses of fish to virus
infections. However, the validation of some serological techniques for certain fish virus infections could arise in the near
future, rendering the use of fish serology more widely acceptable for health screening purposes. As SVCV cannot be
detected at all times of the year, or with confidence from all carrier fish, there are occasions when detection of fish
antibody may provide useful information for epidemiological studies or risk assessments. However, it must be borne in
mind that the presence of specific antibody only indicates previous exposure to the virus, and is not an indicator of the
current presence of virus in a fish. Antibody surveys are best used at the population level, rather than the individual level
as an indicator of previous exposure to the virus.

4.1. Field diagnostic methods

4.1.1. Clinical signs

During an outbreak of SVC there will be a noticeable increase in mortality in the population. Diseased fish
usually appear darker in colour. Typical clinical signs include exophthalmia, pale gills, haemorrhages on the
skin, base of the fins and the vent, abdominal distension or dropsy and a protruding vent (anus), often with
trailing mucoid faecal casts. All these clinical signs may not be present in individual fish, and they may not all
be present in the affected population. Some of these signs may be present in diseases caused by other
pathogens. There may be no clinical signs in cases with a sudden onset of mortality.

4.1.2. Behavioural changes

Generally, young fish up to 1 year are most susceptible to clinical disease, but all age groups can be affected.
Fish become lethargic, separate from the shoal and gather at the water inlet or sides of a pond and some may
experience loss of equilibrium.

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4.2. Clinical methods

4.2.1. Gross pathology

There are no pathognomonic gross lesions. Final diagnosis must await direct detection of viral antigen
or nucleic acid in tissues or virus isolation and identification. Lesions may be absent in cases of sudden
mortality. Gross pathologies are mainly documented for common carp and may include excess ascitic fluid in
the abdominal cavity, usually containing blood, degeneration of the gill lamellae and inflammation of the
intestine, which contains mucus instead of food. Oedema and haemorrhage of the visceral organs is
commonly observed. Focal haemorrhages may be seen in the muscle and fat tissue, as well as in the swim
bladder.

4.2.2. Clinical chemistry

In the absence of large-scale studies, clinical chemistry is an unreliable means of indicating SVC disease.
The data presented below are only indicative of nonspecific disease processes.

Some groups of sheatfish experimentally infected with the virus exhibited lowered haematocrit values, but in
other groups the values remained unchanged. Transaminase activity increased in all groups.

During 3 months following an outbreak of SVC in carp in ponds there was an increase in neutrophils,
monocytes, eosinophils, and basophils. The numbers of lymphocytes declined then rose back to the starting
levels. Over the same period, fish with signs of SVC had an increase in plasma levels of Ca2+, inorganic
phosphate levels, total bilirubin, alanine aminotransferase activity, lactic acid dehydrogenase activity and
α-hydroxybutyryl dehydrogenase activity. Levels of total protein, cholesterol and alkaline phosphatase activity
decreased.

4.2.3. Microscopic pathology

Histopathological changes can be observed in all major organs. In the liver, blood vessels show oedematous
perivasculitis progressing to necrosis. Liver parenchyma shows hyperaemia with multiple focal necroses
and degeneration. The heart shows pericarditis and infiltration of the myocardium progressing to
focal degeneration and necrosis. The spleen shows hyperaemia with hyperplasia of the
reticuloendothelium and enlarged melanomacrophage centres, and the pancreas is inflamed with
multifocal necrosis. In the kidney, damage is seen to excretory and haematopoietic tissue. Renal tubules are
clogged with casts and the cells undergo hyaline degeneration and vacuolation. The intestine shows
perivascular inflammation, desquamation of the epithelium and atrophy of the villi. The peritoneum is inflamed
and lymph vessels are filled with detritus and macrophages. In the swim bladder, the epithelial lamina
changes from a monolayer to a discontinuous multi-layer and vessels in the submucosa are dilated with
nearby lymphocyte infiltration.

4.2.4. Wet mounts

Not relevant.

4.2.5. Smears

Only of value if an immunohistological procedure such as the IFAT (see Section 4.3.1.2.2.1) or
immunoperoxidase procedure is used but see caveats in Section 4.3.1.2

4.2.6. Fixed sections

See Section 4.2.3Fixed sections can also be used for immunohistochemical procedures as in 4.2.5, but see
caveats in Section 4.3.1.2

4.2.7. Electron microscopy/cytopathology

The virus has the typical bullet shape of a rhabdovirus and is approximately 60‒90 nm wide by 90‒180 nm
long, following negative staining. The virus comprises a nucleocapsid surrounded by an envelope.

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4.3. Agent detection and identification methods

See the following sections in Chapter 2.3.0.:

• Section A.2.2.1. for further details of transportation.

• Section A.2.2.2. for virus extraction and obtaining organ homogenates.

4.3.1. Direct detection methods

The virus can be observed directly by electron microscopy, but this will only indicate the presence of a
rhabdovirus and further identification will be needed. Virus antigen and nucleic acid can potentially be
identified in extracts of tissues from clinically infected fish, and virus can usually be isolated from those fish.
However, it is much less likely that virus antigen or nucleic acid will be detected directly from tissues from
subclinically infected carrier fish. Virus isolation is the preferred method for detecting such fish, but is not
100% effective.

4.3.1.1. Microscopic methods

Microscopic methods by themselves are not recommended for diagnosis of SVC as the histopathological
picture is not specific for the disease. They may, however, provide supporting evidence, particularly, when
immunohistological methods are used, but see caveats in Section 4.3.1.2

4.3.1.1.1. Wet mounts

Not relevant.

4.3.1.1.2. Smears/tissue imprints

Only of value if an immunohistological procedure such as the IFAT (see Section 4.3.1.2.2.1) or
immunoperoxidase procedure is used but see caveats in Section 4.3.1.2

4.3.1.1.3. Fixed sections

Only of value if an immunohistological procedure such as the IFAT (see Section 4.3.1.2.1.2) or
immunoperoxidase procedure is used but see caveats in Section 4.3.1.2See Chapter 2.3.0., Section
B.3.3.1. for details of fixation of specimens.

4.3.1.2. Agent isolation and identification

Following isolation, the virus must be identified and this can be achieved by antigen detection methods,
virus neutralisation or nucleic acid identification methods. The former two methods must be regarded as
presumptive unless fully validated monoclonal or polyclonal antibodies are used, as cross reactions with
other viruses occur (Section 2.1.1and Section 5). Commercially available kits using polyclonal antibodies
may lack specificity, and those using monoclonal antibodies may not detect all subgenogroups of SVCV
(Dixon & Longshaw, 2005). Nucleic acid detection methods must always be followed up by sequencing or
use of a method such as reverse hybridisation (Sheppard et al., 2007) to confirm the identity of the virus.

4.3.1.2.1. Cell culture/artificial media

4.3.1.2.1.1. Cell line to be used

EPC or FHM (Chapter 2.3.0., Section B.1.1.).

4.3.1.2.1.2. Virus extraction

Use the procedure described in Chapter 2.3.0, Section A.2.2.2.

4.3.1.2.1.3. Inoculation of cell monolayers

Make two serial tenfold dilutions of the 1/10 organ homogenate supernatants in cell culture medium (i.e.
the homogenate supernatants will be 1/100 and 1/1000 dilutions of the original organ material) and
transfer an appropriate volume of each of these two dilutions on to 24-hour-old cell monolayers drained
of their culture medium. Alternatively, make a single tenfold dilution of the 1/10 organ homogenate (i.e.
a 1/100 dilution of the original organ material) and add an appropriate volume of both the 1/10 and 1/100

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dilutions directly to undrained 24-hour-old cell monolayers, to effect 1/100 and 1/1000 final dilutions of
the organ homogenate. Should toxicity of the sample be a problem, make two serial tenfold dilutions of
the 1/10 organ homogenate supernatants in cell culture medium as described above and inoculate at
least 2 cm2 of drained cell monolayer with 100 µl of each dilution. Allow to adsorb for 0.5‒1 hour at 10‒
15°C, withdraw the inoculum and add cell culture medium buffered at pH 7.6 and supplemented with 2%
fetal calf serum (FCS) (1 ml/well for 24-well cell culture plates). Incubate at 20°C.

4.3.1.2.1.4. Monitoring incubation

Follow the course of infection in positive controls and other inoculated cell cultures by microscopic
examination at ×40‒100 magnification for 7 days. The use of a phase-contrast microscope is
recommended.

Maintain the pH of the cell culture medium at between 7.3 and 7.6 during incubation. This can be
achieved by the addition to the inoculated medium of sterile bicarbonate buffer (for tightly closed cell
culture flasks) or HEPES-buffered medium (HEPES = N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic
acid) or 2 M Tris (Tris [hydroxymethy]) aminomethane)/HCl buffer solution (for cell culture plates).

If a cytopathic effect (CPE) appears in those cell cultures inoculated with the dilutions of the tested
homogenate supernatants, identification procedures must be undertaken immediately (see Sections
4.3.1.2.1.1, 4.3.1.2.1.2, 4.3.1.2.1.3and 4.3.1.2.3.1below).

If no CPE develops in the inoculated cultures (despite normal progression of CPE in the virus controls),
the inoculated cultures should be subcultured for a further 7 days. Should the virus control fail to develop
CPE, the process should be repeated with fresh susceptible cells and new batches of samples.

4.3.1.2.1.5. Subcultivation procedures

Using a pipette, try to dislodge cells from the cell culture vessels and collect aliquots of cell culture
medium plus cells from all inoculated monolayers, keeping different groups separate. The aliquots of the
1/100 and 1/000 dilutions are pooled and inoculated on to fresh 24-hour-old cell cultures to effect 1/10
and 1/100 final dilutions of the pooled aliquots. Incubate and monitor as described above. If no CPE
occurs, the test may be declared negative.

4.3.1.2.1.6. Confirmation of virus identity by neutralisation

i) Collect the culture medium of the cell monolayers exhibiting CPE and centrifuge at 2000 g for
15 minutes at 4°C, or filter through a 0.45 µm pore membrane to remove cell debris.
ii) Dilute the virus-containing medium from 10‒2 to 10‒4.
iii) Mix aliquots of each dilution with equal volumes of an antibody solution against SVCV, and
similarly treat aliquots of each virus dilution with cell culture medium. The neutralising antibody
(NAb) solution must have a 50% plaque reduction titre of at least 2000 based on neutralisation of
50‒100 plaque-forming units (PFU) of SVCV.
iv) In parallel, other neutralisation tests must be performed against:

• a homologous virus strain (positive neutralisation test),

• a heterologous virus strain (negative neutralisation test).

v) Incubate all the mixtures at 20°C for 1 hour.
vi) Transfer aliquots of each of the above mixtures on to cell monolayers (inoculate two cell cultures
per dilution) and allow adsorption to occur for 0.5‒1 hour at 15‒20°C; 24- or 12-well cell culture
plates are suitable for this purpose, using a 50 µl inoculum.
vii) When adsorption is completed, add cell culture medium, supplemented with 2% FCS and buffered
at pH 7.4‒7.6, to each well and incubate at 20°C.
viii) Check the cell cultures for the onset of CPE and read the results as soon as it occurs in
non-neutralised controls (cell monolayers being protected in positive neutralisation controls).
Results are recorded either after a simple microscopic examination (phase-contrast preferable) or
after discarding the cell culture medium and staining the cell monolayers with a solution of 1%
crystal violet in 20% ethanol.

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ix) The tested virus is identified as SVCV when CPE is prevented or noticeably delayed in the cell
cultures that received the virus suspension treated with the SVCV-specific antibody, whereas CPE
is evident in all other cell cultures.

NOTE: Presumptive SVCV isolates identified by the ELISA or the IFAT may not be neutralised by NAb
to SVCV. Also, some SVCV subgenogroups may not be completely neutralised by NAb prepared
against an isolate from a different subgenogroup. Where neutralisation by NAb to SVCV is absent or
incomplete, confirmation by the RT-PCR and nucleotide sequence analysis of RT-PCR products is
recommended to confirm the presence of SVCV.

4.3.1.2.1.7. Confirmation of virus identity by the indirect fluorescent antibody test (IFAT)
i) Prepare monolayers of cells in 2 cm2 wells of plastic cell culture plates, flasks or on cover-slips or
glass slides in order to reach approximately 80% confluency within 24 hours of incubation at 25°C
(seed six cell monolayers per virus isolate to be identified, plus two for positive and two for negative
controls). The FCS content of the cell culture medium can be reduced to 2‒4%. If numerous virus
isolates have to be identified, the use of Terasaki plates is strongly recommended.
ii) When the cell monolayers are ready for infection, i.e. on the same day or on the day after seeding,
inoculate the virus suspensions to be identified by making tenfold dilution steps directly in the cell
culture wells or flasks. For tests using cells cultured on glass cover-slips or slides, the dilutions are
made in sterile containers and then used to inoculate the cells.
iii) Dilute the control virus suspension of SVCV in a similar way, in order to obtain a virus titre of about
5000‒10,000 PFU ml‒1 in the cell culture medium.
iv) Incubate at 20°C for 24 hours.
v) Remove the cell culture medium, rinse once with 0.01 M phosphate-buffered saline (PBS), pH 7.2,
then three times briefly with cold acetone (stored at ‒20°C) for slides or cover-slips or 80% acetone
in water or 30% acetone in ethanol, also at ‒20°C, for cells on plastic substrates. Let the fixative
act for 15 minutes. A volume of 0.5 ml is adequate for 2 cm2 of cell monolayer.
vi) Allow the cell monolayers to air-dry for at least 30 minutes and process immediately or freeze at ‒
20°C.
vii) Rehydrate the dried cell monolayers, if they have been stored frozen, by four rinsing steps with
PBS containing 0.05% Tween 20 (PBST) and remove this buffer completely after the last rinse.
Block with 5% skim milk or 1% bovine serum albumin, in PBST for 30 minutes at 37°C.
viii) Rinse four times with PBST, 5 minutes for each rinse. The slides or plastic culture plates can be
gently agitated during the rinses.
ix) Prepare a solution of purified antibody or serum to SVCV in PBST, at the appropriate dilution
(which has been established previously or as given by the reagent supplier).
x) Incubate the cell monolayers with the antibody solution for 1 hour at 37°C in a humid chamber and
do not allow evaporation to occur.
xi) Rinse four times with PBST.
xii) Incubate the cell monolayers with a solution of fluorescein isothiocyanate (FITC)-conjugated
antibody to the immunoglobulin used in the first layer and prepared according to the instructions of
the supplier. These FITC antibodies are most often rabbit or goat antibodies.
xiii) Rinse four times with PBST.
xiv) View the treated cell monolayers on plastic substrates immediately, or mount the slides or
cover-slips using glycerol saline at pH 8.5, or a commercially available mountant.
xv) Examine under incident ultraviolet (UV) light using a microscope with ×10 eye pieces and ×20 or
×40 objective lenses having numerical apertures of >0.65 and >1.3, respectively. Positive and
negative controls must be found to give the expected results prior to any other observation.

4.3.1.2.1.8. Confirmation of virus identity by enzyme-linked immunosorbent assay (ELISA)

i) Coat the wells of microplates designed for ELISAs with appropriate dilutions of purified
immunoglobulins (Ig) specific for SVCV, in 0.02 M carbonate buffer, pH 9.5 (200 µl/well). Ig may
be polyclonal or monoclonal Ig originating most often from rabbit or mouse, respectively. For the
identification of SVCV, monoclonal antibodies (MAbs) specific for certain domains of the
nucleocapsid (N) protein are suitable.

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ii) Incubate overnight at 4°C.

iii) Rinse four times with PBST.
iv) Block with skim milk (5% in carbonate buffer) or other blocking solution for 1 hour at 37°C
(300 µl/well).
v) Rinse four times with PBST.
vi) Add 2% non-ionic detergent (Triton X-100 or Nonidet P-40) to the virus suspension to be identified.
vii) Dispense 100 µl/well of two- or four-step dilutions of the virus to be identified, and of the
non-infected cell culture harvest (negative control). Also include SVCV positive control virus.
Incubate for 1 hour at 37°C.
viii) Rinse four times with PBST.
ix) Add to the wells, 200 µl of horseradish peroxidase (HRPO)-conjugated MAb or polyclonal antibody
to SVCV; or polyclonal IgG to SVCV. A MAb to N protein specific for a domain different from the
one of the coating MAb and previously conjugated with biotin can also be used. Incubate for 1 hour
at 37°C.
x) Rinse four times with PBST.
xi) If HRPO-conjugated antibody has been used, go to step xiii. Otherwise, add 200 µl of
HRPO-conjugated streptavidin or ExtrAvidin (Sigma) to those wells that have received the
biotin-conjugated antibody and incubate for 1 hour at 37°C.
xii) Rinse four times with PBST.
xiii) Add 200 µl of a suitable substrate and chromogen, such as tetramethylbenzidine dihydrochloride.
Stop the course of the test when positive controls react, and read the results.

4.3.1.2.2. Antibody-based antigen detection methods directly on fish tissues

4.3.1.2.2.1. Indirect fluorescent antibody test

i) Bleed the fish thoroughly.
ii) Make kidney imprints on cleaned glass slides or at the bottom of the wells of a plastic cell culture
plate.
iii) Store and transport the kidney pieces as indicated in Chapter 2.3.0., Section A. 2.2.1.) together
with the other organs required for virus isolation.
iv) Allow the imprint to air-dry for 20 minutes.
v) Fix with cold acetone (stored at ‒20°C) for glass slides or 80% acetone in water or 30% acetone
in ethanol, also at ‒20°C, for plastic wells. Let the fixative act for 15 minutes. Allow the imprints to
air-dry for at least 30 minutes and process immediately or freeze at ‒20°C.
vi) Rehydrate the imprints if they have been stored frozen by four rinsing steps with PBST, and
remove this buffer completely after the last rinse. Block with 5% skim milk or 1% bovine serum
albumin, in PBST for 30 minutes at 37°C.
vii) Rinse four times with PBST, 5 minutes for each rinse. The slides or plastic culture plates can be
gently agitated during the rinses.
viii) Prepare a solution of purified antibody or serum to SVCV in PBST, at the appropriate dilution
(which has been established previously or as given by the reagent supplier).
ix) Incubate the imprints with the antibody solution for 1 hour at 37°C in a humid chamber and do not
allow evaporation to occur.
x) Rinse four times with PBST.
xi) Incubate the imprints with a solution of FITC-conjugated antibody to the immunoglobulin used in
the first layer and prepared according to the instructions of the supplier. These FITC antibodies are
most often rabbit or goat antibodies.
xii) Rinse four times with PBST.
xiii) View the treated imprints on plastic plates immediately, or mount the slides with cover-slips using
glycerol saline at pH 8.5, or a commercially-available mountant.

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xiv) Examine under incident ultraviolet (UV) light using a microscope with ×10 eye pieces and ×20 or
×40 objective lenses having numerical aperture of >0.65 and >1.3, respectively. Positive and
negative controls must be found to give the expected results prior to any other observation.

4.3.1.2.2.2. Enzyme-linked immunosorbent assay (ELISA)

See Chapter 2.3.0., Section A.2.2.2. for obtaining organ homogenates.

i) Coat the wells of microplates designed for ELISAs with appropriate dilutions of purified
immunoglobulins (Ig) specific for SVCV, in 0.02 M carbonate buffer, pH 9.5 (200 µl/well). Ig may
be polyclonal or monoclonal Ig originating most often from rabbit or mouse, respectively. For the
identification of SVCV, monoclonal antibodies (MAbs) specific for certain domains of the
nucleocapsid (N) protein are suitable.
ii) Incubate overnight at 4°C.
iii) Rinse four times with PBST.
iv) Block with skim milk (5% in carbonate buffer) or other blocking solution for 1 hour at 37°C
(300 µl/well).
v) Rinse four times with PBST.
vi) Store a 1/4 aliquot of each homogenate at 4°C, in case the test is negative and virus isolation in
cell culture is required.
vii) Treat the remaining part of the homogenate with 2% Triton X-100 or Nonidet P-40 and 2 mM of
phenyl methyl sulphonide fluoride; mix gently.
viii) Dispense 100 µl/well of two- or four-step dilutions of the sample to be identified, and of negative
control tissues. Also include an SVCV positive control virus. Incubate for 1 hour at 37°C.
ix) Rinse four times with PBST.
x) Add to the wells, 200 µl of horseradish peroxidase (HRPO)-conjugated MAb or polyclonal antibody
to SVCV; or polyclonal IgG to SVCV. A MAb to N protein specific for a domain different from the
one of the coating MAb and previously conjugated with biotin can also be used. Incubate for 1 hour
at 37°C.
xi) Rinse four times with PBST.
xii) If HRPO-conjugated antibody has been used, go to step xiv. Otherwise, add 200 µl of
HRPO-conjugated streptavidin or ExtrAvidin (Sigma) to those wells that have received the
biotin-conjugated antibody and incubate for 1 hour at 37°C.
xiii) Rinse four times with PBST.
xiv) Add 200 µl of a suitable substrate and chromogen, such as tetramethylbenzidine dihydrochloride.
Stop the course of the test when positive controls react, and read the results.
xv) If the test is negative, process the organ samples stored at 4°C, for virus isolation in cell culture as
described in Section 4.3.1.2.1

4.3.1.2.3. Molecular techniques

4.3.1.2.3.1. Reverse-transcription polymerase chain reaction (RT-PCR) (confirmation of virus identity or

directly from fish tissue extracts)

The genome of SVCV consists of a single strand of RNA of approximately 11 kb, with negative polarity.
Amplification of a 714 bp fragment of SVCV cDNA is performed using primers derived from sequences
of the region coding for the glycoprotein gene: 5’-TCT-TGG-AGC-CAA-ATA-GCT-CAR*-R*TC-3’ (SVCV
F1) and 5’-AGA-TGG-TAT-GGA-CCC-CAA-TAC-ATH*-ACN*-CAY*-3’ SVCV R2), using a modification
of the method of Stone et al., 2003.
i) Total RNA is extracted from 100 µl of supernatant from cell cultures exhibiting CPE or 100 µl of fish
tissue extract and dissolved in 40 µl molecular biology grade DNase- and RNase-free water.
A number of total RNA extraction kits are available commercially that will produce high quality RNA
suitable for RT-PCR. Examples are Trizol ReagentT (RL, Life Technologies, Paisley, UK), SV Total
RNA isolation system (Promega) and Nucleospin® RNA AB gene).

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ii) For cDNA synthesis, a reverse transcription reaction is performed at 37°C for 1 hour in a 20 µl
volume consisting of 1 × M-MLV RT reaction buffer (50 mM Tris, pH 8.3, 75 mM KCl, 10 mM DTT,
3 mM MgCl2) containing 1 mM dNTP, 100 pmol SVCV R2 primer, 20 units M-MLV reverse
transcriptase (Promega, Southampton, UK) or equivalent reverse transcriptase and 1/10 of the
total RNA extracted above.
iii) PCR is performed in a 50 µl reaction volume 1 × PCR buffer (50 mM KCl, 10 mM Tris/HCl, pH 9.0,
and 0.1% Triton X-100) containing 2.5 mM MgCl2, 200 µM dNTPs, 50 pmol each of the SVCV R2
and SVCV F1 primers, 1.25 units of Taq DNA polymerase, and 2.5 µl reverse transcription reaction
mix. The reaction mix is overlaid with mineral oil and subjected to 35 temperature cycles of:
1 minute at 95°C, 1 minute at 55°C and 1 minute at 72°C followed by a final extension step of
10 minutes at 72°C. Amplified DNA (714 bp) is analysed by agarose gel electrophoresis.
iv) If the CPE in culture is not extensive it is possible that a product will not be generated using a single
round of amplification. To avoid such problems, use the semi-nested assay using primers:
5’-TCT-TGG-AGC-CAA-ATA-GCT-CAR*-R*TC-3’ (SVCV F1) and
5’-CTG-GGG-TTT-CCN*-CCT-CAA-AGY*-TGY*-3’ (SVC R4) according to Stone et al., 2003.
v) The second round of PCR is performed in a 50 µl reaction volume 1 × PCR buffer (50 mM KCl,
10 mM Tris/HCl, pH 9.0, and 0.1% Triton X-100) containing 2.5 mM MgCl2, 200 µM dNTPs,
50 pmol each of the SVCV R4 and SVCV F1 primers, 1.25 units Taq DNA polymerase, and 2.5 µl
of the first round product. The reaction mix is overlaid with mineral oil and subjected to
35 temperature cycles of: 1 minute at 95°C, 1 minute at 55°C and 1 minute at 72°C followed by a
final extension step of 10 minutes at 72°C. Amplified DNA (606 bp) is analysed by agarose gel
electrophoresis.
vi) All amplified products are confirmed as SVCV in origin by sequencing, and the SVCV subtype
(Ia-Id) is identified using a BLAST search (http://www.ebi.ac.uk/Tools/sss/ncbiblast/) or by
phylogenetic analysis using the SVCV sequences available in public sequence databases.
Phylogenetic analysis is undertaken using a 426 bp region corresponding to nucleotides 429‒855
of the glycoprotein gene.
vii) In some cases where the CPE is extensive and the virus replicates to a high titre, sufficient PCR
amplicon will be available for direct sequencing. Where the amplified product is weak it is
recommended that the product be inserted into an appropriate sequencing vector (e.g. pGEM-T,
pCR® 4- TOPO®) prior to undertaking the sequencing. At least two independent amplification and
sequencing events should be undertaken to eliminate potential sequence errors introduced by the
Taq polymerase.

NOTE: SVCV primer-annealing sites were identified by the alignment of the published amino acid
sequences for the glycoprotein of SVCV (Björklund et al., 1996; Genbank accession U18101), and the
vesicular stomatitis virus (VSV) New Jersey (Gallione & Rose, 1983; Genbank accession V01214), and
Piry strains (Genbank accession D26175). Primers were then designed to anneal to the regions
encoding the conserved amino acids using the published sequence for SVCV (Björklund et al., 1996) as
a skeleton, and introducing degenerate bases at the 3' termini to allow for potential differences in codon
usage. The appropriate IUB codes have been used where appropriate and are indicated by an asterisk
(*).

4.3.1.2.4. Agent purification

The virus can be purified as described by Hill et al., 1975.

i) Harvest medium from infected cell cultures.
ii) Clarify by centrifugation at 2000 g for 15 minutes at 4°C. Remove the supernatant.
iii) Sediment the virus by centrifuging the supernatant at 40,000 g for 1 hour.
iv) Remove and discard the supernatant. Resuspend the pellet in a small volume of PBS or TNE
(0.01 M Tris, 0.1 M NaCl, 1 mM ethylenediaminetetra-acetic acid, pH 7.2). The volume will depend
on the original amount of cell culture medium and the size of tube used to make the gradient. If the
supernatant fluids have been centrifuged in several tubes, combine the resuspended pellets.
v) Prepare a 15‒45% sucrose gradient made up in the buffer used in step iv.
vi) Gently overlay the resuspended pellets and centrifuge at 40,000 g for 2 hours at 4°C.
vii) An opalescent band of virus should be visible in the gradient. Harvest the band and dilute at least
tenfold with the buffer in use.

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viii) Centrifuge at 40,000 g for 2 hours at 4°C.

ix) Resuspend the pellet in the buffer in use.

4.3.2. Serological methods

Fish produce an immune response following infection with SVCV, and this has been studied mainly by
following antibody development. The antibody response is influenced by water temperature. Following SVCV
infection at low temperatures such as 10°C, antibody may not be detected, or may be present at low titre and
may take several weeks to develop, whereas at 20°C, antibody develops sooner (after 1 week), and high titres
can be present (Dixon, 2008). The duration of the antibody response is not known, but antibody has been
detected 1 year after a natural infection in a fishery, and after over 2 years following experimental infection
(unpublished observations). Detection of neutralising antibody has been used in many surveys for the virus,
but ELISA methods are more sensitive. Dixon et al., 1994developed a competitive ELISA, which is applicable
to detection of antibody in a wide range of hosts.

The suitability of antibody detection has been discussed in Sections 3.5and 4

5. Rating of tests against purpose of use

The methods currently available for targeted surveillance and diagnosis of SVC are listed in Table 5.1.The designations
used in the Table indicate: a = the method is the recommended method for reasons of availability, utility, and diagnostic
specificity and sensitivity; b = the method is a standard method with good diagnostic sensitivity and specificity; c = the
method has application in some situations, but cost, accuracy, or other factors severely limits its application; and d = the
method is presently not recommended for this purpose. These are somewhat subjective as suitability involves issues of
reliability, sensitivity, specificity and utility. Although not all of the tests listed as category ‛a’ or ‛b’ have undergone formal
standardisation and validation, their routine nature and the fact that they have been used widely without dubious results,
makes them acceptable.

Table 5.1. Methods for targeted surveillance and diagnosis

Targeted surveillance Presumptive Confirmatory

Method
diagnosis diagnosis
Juveniles Adults

Gross signs d d b d

Histopathology d d b c

Transmission EM d d d d

Isolation in cell culture a a a a

Test for virus antigen d d a c

Test for fish antibody against the virus c c c d

RT-PCR c c a a

Sequence na na a a

EM = electron microscopy; RT-PCR = reverse-transcription polymerase chain reaction; na = not applicable.

NOTE: Isolation in cell culture can only be regarded as presumptive until the identity of the isolated virus is confirmed by
a suitable method.

Four genogroups of piscine rhabdoviruses have been described (Stone et al., 2003): genogroup I (SVCV), genogroup II
(grass carp rhabdovirus), genogroup III (pike fry rhabdovirus) and genogroup IV (tench rhabdovirus). Further analysis
also showed that the SVCV genogroup could be further subdivided into at least four subgenogroups. Antibodies directed
against SVCV cross-react to various degrees with all of the rhabdoviruses in the other three genogroups. The ability to
confirm SVCV based on results from serological tests, such as ELISA, IFAT and serum neutralisation, relies on the
specificity of the antibodies used in the tests. Results from those serological tests can only be accepted as confirming
the presence of SVCV if the antisera used have been validated as detecting viruses in all four subgenogroups of
genogroup I and do not cross-react with isolates from the other three genogroups.

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Many diagnostic laboratories have encountered difficulties in obtaining antibodies against SVCV that are suitable for use
in serological tests and have turned to commercially available test kits. Two commercial test kits are available for
identification of SVCV, the TestLine ELISA kit (TestLine, Brno, Czech Republic) and the Bio-X IFAT kit (Bio-X
Diagnostics, Jemelle, Belgium). Recently the tests have been assessed for their specificity against virus isolates from
genogroups I, II, III and IV by Dixon & Longshaw, 2005who found that the TestLine ELISA, which uses a polyclonal rabbit
antibody, was nonspecific and could not distinguish SVCV from viruses in the other three genogroups. Conversely, the
Bio-X IFAT, which uses a monoclonal mouse antibody, was too specific and could only detect SVCV isolates from one
of the four SVCV subgenogroups. These commercial test kits can be applied for presumptive diagnosis of SVC, but the
problems of specificity severely limit their application for confirmatory diagnosis.

It is recommended that RT-PCR and nucleotide sequence analysis of the PCR products are used for confirmatory
identification of SVCV.

6. Test(s) recommended for targeted surveillance to declare freedom from spring viraemia of
carp

The method for surveillance of susceptible fish populations for declaration of freedom from SVC is inoculation of cell
culture with tissue extracts (as described in Section Cell culture/artificial mediaabove) to demonstrate absence of the
virus.

7. Corroborative diagnostic criteria

7.1. Definition of suspect case

SVC should be considered as a cause of disease when rapid mortalities and significant numbers of mortalities
occur in a population of susceptible fish species, particularly if accompanied by clinical signs of SVC.

A suspect case of SVC disease is defined as the presence of typical clinical signs of the disease in a population of
susceptible fish OR presentation of typical histopathology in tissue sections OR typical CPE in cell cultures without
identification of the causative agent OR a single positive result from one of the diagnostic assays described above.

7.2. Definition of confirmed case

The first case of the disease in a new area, or in an area where SVC has occurred before but has not been identified
over a 2-year surveillance period is described as the index case. A confirmed index case is defined as a suspect
case that has produced a typical CPE in cell culture with subsequent identification of the causative agent by one
of the serological tests using validated antisera or RT-PCR plus sequencing described above OR a second positive
result from a separate and different diagnostic assay described above. If a serological test is used, the antisera
must be “fit for purpose” as indicated in Section 5. If RT-PCR is used, the product obtained must be sequenced in
order to confirm SVCV; if not the case is suspect SVCV.

During follow-up investigations after a confirmed index case, a case can be confirmed on the basis of RT-PCR plus
sequencing alone.

8. References

AHNE W. (1976). Untersuchungen über die Stabilität des karpfenpathogenen Virusstammes 10/3. Fisch und Umwelt,
2, 121‒127.

AHNE W. (1982). Vergleichende Untersuchungen über die Stabilität von vier fischpathogenen Viren (VHSV, PFR,
SVCV, IPNV). Zbl. Vet. Med. B, 29, 457‒476.

AHNE W. (1986). Unterschiedliche biologische Eigenschaften 4 cyprinidenpathogener Rhabdovirusisolate. J. Vet.

Med. B, 33, 253‒259.

AHNE W., BJÖRKLUND H.V., ESSBAUER S., FIJAN N., KURATH G. & WINTON J.R. (2002). Spring viremia of carp
(SVC). Dis. aquat. Org., 52, 261‒272.

AHNE W. & HELD C. (1980). Untersuchungen über die viruzide Wirkung von Actomar® K30 auf fischpathogene Viren.
Tierärztl. Umsch., 35, 308‒318.

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Chapter 2.3.9. - Spring viraemia of carp

AHNE W., KURATH G. & WINTON J. (1998). A ribonuclease protection assay can distinguish spring viremia of carp
virus from pike fry rhabdovirus. Bull. Eur. Ass. Fish Pathol., 18, 220‒224.

BASIC A., SCHACHNER O., BILIC I. & HESS M. (2009). Phylogenetic analysis of spring viraemia of carp virus isolates
from Austria indicates the existence of at least two subgroups within genogroup Id. Dis. aquat. Org., 85, 31‒40.

BJÖRKLUND H.V., HIGMAN K.H. & KURATH G. (1996). The glycoprotein genes and gene junctions of the fish
rhabdoviruses spring viremia of carp virus and hirame rhabdovirus - analysis of relationships with other rhabdoviruses.
Virus Res., 42, 65‒80.

CARSTEN E.B. (2010). Ratification vote on taxonomic proposals to the International Committee on Taxonomy of
Viruses (2009). Arch. Virol., 155, 133‒146.

DIXON P.F. (2008). Virus diseases of cyprinids.In: Fish Diseases, Vol. 1. Eiras J.C., Segner H., Wahli T. & Kapoor B.G.
eds. Science Publishers, Enfield, New Hampshire, USA, 87‒184.

DIXON P.F., HATTENBERGER-BAUDOY A.-M. & WAY K. (1994). Detection of carp antibodies to spring viraemia of
carp virus by a competitive immunoassay. Dis. Aquat. Org., 19, 181‒186.

DIXON P.F. & LONGSHAW C.B. (2005). Assessment of commercial test kits for identification of spring viraemia of carp
virus. Dis. Aquat. Org., 67, 25‒29.

EMMENEGGER E.J. & KURATH G. (2008). DNA vaccine protects ornamental koi (Cyprinus carpio koi) against North
American spring viremia of carp virus. Vaccine, 26, 6415‒6421.

FIJAN N. (1988). Vaccination against spring viraemia of carp.In: Fish Vaccination. Ellis A.E., ed. Academic Press,
London, UK, 204‒215.

GALLIONE C.J. & ROSE J.K. (1983). Nucleotide sequence of a clone encoding the entire glycoprotein from the New
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*
* *
NB: There are OIE Reference Laboratories for Spring viraemia of carp
(see Table at the end of this Aquatic Manual or consult the OIE web site for the most up-to-date list:
http://www.oie.int/en/scientific-expertise/reference-laboratories/list-of-laboratories/).
Please contact the OIE Reference Laboratories for any further information on Spring viraemia of carp

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Chapter 2.3.9. - Spring viraemia of carp

18 2019 © OIE - Manual of Diagnostic Tests for Aquatic Animals - 19/08/2019