Spring viraemia of carp virus (SVCV) is an infectious viral disease affecting carp and other cyprinid fish. It is caused by SVCV, which is a rhabdovirus. SVCV was initially identified in Yugoslavia in 1971 and has since spread to other parts of Europe, Asia, and North America. Clinical signs in infected fish include lethargy, skin hemorrhaging, and darkening. Mortality is highest between 10-17°C. The virus replicates within host cells and is transmitted horizontally. Multiple genetic variants of SVCV exist worldwide. Effective control relies on good management practices and vaccination.

1. Spring Viraemia of Carp Virus
Presented by-
Avijit Pramanik
AAH-MA-09-02
B.F.Sc, W.B.U.A.F.S.
M.F.Sc, CIFE,Mumbai
A SEMINAR ON-

2. q Spring viraemia of carp (SVC) is an infectious viral disease of carp (Cyprinus carpio)
and other cyprinid fish species.
q The common carp is the principal host, but clinical disease has also been reported
to occur in ornamental koi carp, grass carp, silver carp, crucian carp, goldfish,
tench and sheatfish.
What Is It?

3. Where and When Might it Occur?
Ø SVC was initially diagnosed in Yugoslavia (Fijan et al. 1971).
Ø Since then, it has been identified in other European countries, Russia, Brazil, the Middle
East, China, and North America.
Ø In 2002, Rhabdovirus carpio was first reported in U.S. waters at a North Carolina koi
hatchery.
Ø Outbreaks were reported in cultivated koi in Washington and Missouri in 2004, and in
wild fish in the Upper Mississippi River (Wisconsin, Minnesota) in 2007.
Ø SVC is mainly present in countries which experience low water temperatures.

4. Ø High mortality can occur in all age groups of fish at water temperatures between
10°to 17°C.
Ø Most cases occur in the spring or early summer.
Ø However, fry can be affected at temperatures as high as 23°C.
Aetiological agent
• The aetiological agent of SVC is Spring viraemia of carp virus (SVCV)
• It is 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.
• SVCV exibits the typical bulllet-shaped morphology.
• The virion measures approximately length - 80 to 180 nm
diameter - 60 to 90 nm

5. Ø The genome contains 11,019 nucleotides
Ø Encoding five proteins in the following order:
(a) a nucleoprotein (N) - (38–47 kDa)
(b) a phosphoprotein (P) - (22–26 kDa , formerly designated M1)
(c) a matrix protein (M) - (17–22 kDa, formerly designated M2)
(d) a glycoprotein (G) - (63–80 kDa) and
(e) an RNA-dependent RNA polymerase (L) - (150–225 kDa)
Ø Genomic length - approximately about 11 kb
Ø Among the five structural proteins-
matrix protein (M) and glycoprotein (G) were studied widely for their
significant role in viral pathogenesis.
Ø M protein causes apoptosis and inhibits host cell transcription, whereas G protein was
concerned with viral endocytosis.

7. Schematic of SVCV and its genome. (a) Model of viral structure. (b) Genome organization
showing encoded proteins, gene junctions, and leader and trailer regions. The total genome
length is ∼ 11 kb. The numbers indicate the start and end positions of the respective ORFs.

8. Recently, the availability of sequence data for the entire SVCV genome allows more
accurate predictions of the molecular weights of the structural proteins (ignoring
post-translational modifications) as well as other features of these molecules
(Table )

9. Ø The surface G protein is the most important viral antigenic protein that determines the
infectivity and serological properties of the virus (Hill et al., 1975; Bishop & Smith,
1977; Johnson et al., 1999).
Ø The G protein forms the trimeric spikes or peplomers on the virus outer surface and
helps the virus to induce receptor-mediated endocytosis (Hill et al., 1975; Bishop &
Smith, 1977).
Ø Similar to other vesiculoviruses, SVCV has one type of M protein that provides the
bullet-shaped structure of the virus and binds the nucleocapsid to the cytoplasmic
domains of the G proteins embedded in the viral envelope (Kiuchi & Roy, 1984).
Ø The N protein is a highly abundant viral protein that, in association with viral RNA, gives
a helical symmetry to the nucleocapsid (Sokol & Koprowski, 1975).

10. Ø The P protein is a component of the nucleocapsid that interacts with N and L proteins to
mediate transcription (Roy, 1981).
Ø The L protein is involved in transcription and viral replication, which are achieved
through interaction of the L protein with the N and P proteins (Roy & Clewley, 1978).
Ø The SVCV genome contains a putative leader region of 59 bases at the 3′ terminal region.
Ø It followed by a consensus start signal sequence (AACAG; anti-genomic orientation) for
initiation of transcription of the N gene (Hoffmann et al., 2002; Warg et al., 2007).
Ø The four SVCV genome junctions are N–P, P–M, M–G and G–L junction
Ø These junctions are evolutionarily conserved, with a polyadenylation or transcription
stop signal sequence [TATG(A)7; anti-genomic orientation] at the end of each
gene.(Hoffmann et al., 2002; Warg et al., 2007)

11. v 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).
v The type strain of SVCV is available from the American Type Culture Collection (ATCC
VR-1390).
v 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).
v 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).

12. Genetic diversity amongst SVCV strains
ØMany SVCV strains have been identified.
ØAs in other RNA viruses that can evolve rapidly, a high level of plasticity has been
reported in the SVCV genome.
ØBroadly, SVCV isolates are divided into two clades: an Asian clade and a European clade
(Stone et al., 2007; Miller et al., 2003).
ØBased on nucleotide sequence analysis of the G gene, SVCV isolates are further classified
into four genogroups: Ia, Ib, Ic and Id (Warg et al., 2007; Zhang et al., 2009).
Ø(a)Genogroup Ia contains isolates from Asia, the UK and the Americas;
(b)Ib and Ic contain isolates from Eastern Europe;
(c) Id contains isolates from the UK and some other European countries
(Stone et al., 2003; Hoffmann et al., 2005; Miller et al., 2007; Zhang et al., 2009; Padhi &
Verghese, 2012).

13. ØThe genetic clustering of SVCV isolates is closely associated with geographical location,
suggesting that the virus has evolved independently in different geographical regions
(Stone et al., 2013).
Ø In addition, different isolates have evolved at varied rates, with the Ia group evolving
the most quickly (Padhi & Verghese, 2012).
ØThe nucleotide substitution rates in the P and G genes of genogroup Ia are ∼ 3.5 times
higher than in the same genes of the Id group (Padhi & Verghese, 2012).
ØCompared with the N and G genes, the P gene exhibits a higher degree of genetic
variation and is considered to be a useful marker for studying SVCV epidemiology (Sokol
& Koprowski, 1975; Miller et al., 2007).

14. Ø The genomes of most identified SVCV strains have been partially sequenced.
Ø To date, only five strains have been completely sequenced: Fijian, Björklund,
SVCV-A1, SVCV-C1 and SVCV-265
( Björklund et al., 1996; Hoffmann et al., 2002; Teng et al., 2007; Zhang et al.,
2009;Xiao et al., 2014).
Ø A comparative analysis of all completely sequenced strains relative to the Fijian
strain as a reference strain is summarized in comming Table . These analyses were
performed by blast analysis (http://www.ncbi.nlm.nih.gov/blast).

15. Nucleotide and amino acid sequence similarity amongst fully sequenced SVCV
strains. All strains compared with Fijian as the reference strain.

16. Replication
Ø Negative sense ssRNA viruses need RNA polymerase to form a positive sense RNA.
Ø The positive-sense RNA acts as a viral mRNA, which is translated into proteins for the
production of new virion materials.
Ø With the newly formed virions, more negative sense RNA molecules are produced.
q Replication of the virion consists of the following steps:
1) A virion enters the host cell and releases its negative RNA into the cytoplasm.
2) The virus uses its own RNA replicase, also known as RNA-dependent RNA polymerase
(RdRp), to form positive RNA template strands through complementary base pairing.
3) The positive RNA acts as mRNA, which is translated into structural capsomere proteins
and viral RdRp by the host's ribosomes.

17. 5) A replicative complex is formed with RdRp: The positive strands can either function as
mRNA to produce more proteins or as template to make more negative RNA strands.
6) New viral capsids are assembled with the capsomere proteins. The negative RNA
strands combine with capsids and viral RdRp to form new negative RNA virions.
7) After assembly and maturation of nucleocapsid, the new virions exit the cell by
budding or lysing through cell membrane to further infect other cells.

18. CLINICAL SIGNS
Ø Early in the course of SVCV, affected fish may appear
(a) weak and may congregate in areas of slow-flowing water.
(b) clinical signs, including but not limited to lethargy, ascites, exophthalmia, pale gills,
overall darkening of the body surface, persistent fecal casts, skin and branchial hemorrhages,
and distention/protrusion of the vent.
Common carp (Cyprinus carpio) showing external
clinical signs of spring viremia of carp (SVC) after
experimental infection with SVC virus. Note the
exophthalmia, petechial hemorrhage of the skin,
and inflammation of the vent.

19. q On necropsy, affected fish may have (a) generalized edema (the fluid may be sanguineous)
(b) swim bladder (and other organ) hemorrhages,
(c) intestinal inflammation
(d)The gastrointestinal tract may contain mucus and not ingesta
(e)swollen and coarse-textured spleen, hepatic necrosis, enteritis, and
pericarditis (Ghasemi et al., 2014; Misk et al., 2015).

20. Ø Transmission is horizontal, and most cases occur in the spring or early summer,
when the water begins to warm but remains below 15°C.
Ø The virus is often transmitted by the virus remaining in the feces of infected
fish and from blood-sucking parasites.
Transmission
Vectors
Ø Among animate vectors, the parasitic invertebrates
(a) Argulus foliaceus (Crustacea, Branchiura)
(b) 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).

21. Pathogenesis of SVCV
Ø One pathogenic mechanism used by SVCV is the induction of autophagy (Liu et al., 2015).
Ø Epithelioma papulosum cyprini (EPC) cells when infected with SVCV showed the activation
of autophagy pathway.
Ø Autophagy is a cellular pathway that has important roles in viral infection and pathogenesis
(Orvedahl & Levine, 2008).
Ø By using the autophagy pathway SVCV facilitate its own replication by inducing the SVCV G
protein (Liu et al., 2015).
Ø Autophagy enhances the survival of SVCV-infected cultured cells by eliminating damaged
mitochondrial DNA generated during viral infection.

22. Ø The SVCV glycoprotein, rather than viral replication, activates the autophagy pathway.
Ø Autophagy is a self-digesting mechanism responsible for removal of damaged
organelles, malformed proteins during biosynthesis, and nonfunctional long-lived
proteins by lysosome.
Ø SVCV induces autophagy in EPC cells through the ERK/mTOR signalling pathway.
Ø The increase in LC3 lipidation could be due to autophagy activation or defective
degradation.
Ø SVCV utilized the autophagy pathway to facilitate its own genomic RNA replication and
to enhance its titres in the supernatants.
Ø NOTE: For more details plz see - Spring viraemia of carp virus induces autophagy for
necessary viral replication,Liyue Liu,Bibo Zhu,Shusheng Wu,Li Lin,Guangxin Liu,Yang
Zhou,Weimin Wang,Muhammad Asim,Junfa Yuan,
https://doi.org/10.1111/cmi.12387

23. Ø Haem oxygenase 1 (HO-1){ also known as heat-shock protein 32} is a cytoprotective
enzyme protect the body against oxidative stress during inflammatory processes
(Otterbein & Choi, 2000).
Ø HO-1 exhibits antiviral properties against multiple viruses.
Ø Viral infection can suppress expression of HO-1, perhaps contributing to viral
pathogenesis (Marinissen et al., 2006).
Ø In vitro and in vivo studies have demonstrated that expression of HO-1 is downregulated
during SVCV infection (Yuan et al., 2012),
Ø Suggesting that SVCV infection induces host oxidative stress, which may contribute to
tissue injury in affected fish.

24. ANTIGENIC PROPERTIES OF SVCV
Ø Several studies showed that carp develop a humoral immune response to SVCV (Fijan &
Mata$in 1980, Fijan 1988).
Ø Rhabdovirus-neutralizing antibodies are directed against the surface glycoprotein of the
virus (Kelly et al. 1972).
Ø Induction of humoral antibodies against SVCV in carp is influenced by the age,condition of
carp, by the route of infection and most importantly, by the temperature of water (Fig.
4).
Ø In carp which were infected by waterborne exposure to low doses of SVCV and kept at
20°C, SVCV neutralizing antibodies appeared 7 d after infection.
Ø In contrast, carp infected in the same way, but kept at 13°C, showed first detectable
antibodies 7 wk after infection.

25. Ø At 13°C, carp developed a subclinical
infection with presence of virus in the blood
of carp for about 10 wk.
Ø Neutralizing antibodies which appeared 8
to 10 wk after infection lead to a rapid
decline of the amount of virus in the blood
(Ahne 1979, 1980, 1986).
Ø The biological properties of the SVCV
neutralizing antibodies have not been
studied in detail, but probably resemble the
tetrameric antibody typically found in
teleost fishes (Fijan et al. 1977a, Kaattari &
Piganelli 1996).

26. Virus isolation
q The virus replicates in cell cultures originating from fish, birds and mammals
cell systems.
q These optimal cell systems are derived from cyprinid fish such as
(a) epithelioma papulosum cyprini (EPC) (Fijan, Sulimanovic, Bearzotti, Muzinic,
Zwillenberg, Chilmonczyk, Vautherot & de Kinkelin 1983) and
(b) fathead minnow (FHM) (Gravell & Malsberger 1965) cell lines that are grown at
20–22°C.
q The cytopathic effect (CPE) is characterized by rounding, detachment and lysis of cells.
q The isolated virus is best identified utilizing a SVCV-specific reverse transcription
polymerase chain reaction (RT-PCR) assay that differentiates SVCV from the closely related
pike fry rhabdovirus (PFRV) (Stone, Ahne, Denham, Dixon, Liu, Sheppard, Taylor & Way
2003).

27. q Conventional serological techniques used to detect SVCV include the virus
neutralization test, immunoperoxidase assay, indirect immunofluorescence assay
and ELISA.
q Moreover, the indirect immunofluorescence assay and ELISA appear to cross-react
with other rhabdoviruses (Way, 1991), leading to possible false-positive diagnoses.
q Monoclonal antibodies were generated against M and G proteins that are useful in
the development of SVCV diagnostic methods. (Chen et al., 2008; Luo et al., 2014; Li
et al., 2015).
q Recently, a single-chain fragment variable antibody against SVCV has been
developed using phage display technology and employed for rapid detection of SVCV
(Liu et al., 2013). This antibody reacted specifically with SVCV, but did not cross-react
with other viruses.
Diagnoses

28. Ø A single-chain variable fragment (scFv) is not actually a fragment of an antibody, but
instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL)
of immunoglobulins, connected with a short linker peptide of ten to about 25 amino
acids.
Ø Antibody-displaying phage library was selected after three rounds of panning against
spring viraemia of carp virus (SVCV) by phage display technology.
Ø Eight positive clones which could produce soluble single-chain fragment variable (scFv)
antibody induced by isopropyl-beta-d-thiogalactopyranoside (IPTG) were obtained.
Ø Dot blot results showed that the eight scFv antibodies could recognize SVCV.
Ø The soluble scFv antibodies showed a molecular weight 29 kD by Western blot.

29. Ø All scFv antibodies could recognize SVCV proteins specifically without cross-reaction
with other virus proteins by ELISA.
Ø Indirect immunofluorescence results showed that all of these scFv antibodies reacted
positively with virus in the SVCV-infected cells.
Ø These scFv antibodies will be useful tools to establish immunological detection methods
for SVCV.
NOTE: For more details see: Selection and characterization of single-chain recombinant
antibodies against spring viraemia of carp virus from mouse phage display library,
panelHong,LiuacXiaocong,ZhengaFeng,ZhangbLi,YuaXiaohua,ZhangbHeping,DaibQunyi,Hua
aXiujie,
https://doi.org/10.1016/j.jviromet.2013.08.017

30. q Various PCR-based assays have also been used to detect SVCV owing to their high
sensitivity.
q These include- reverse transcription (RT)-PCR combined with nested PCR (Koutná et
al., 2003)
-multiplex real-time quantitative RT-PCR (Liu et al., 2008a) and
- one-step TaqMan real-time quantitative RT-PCR (Yue et al., 2008).
Ø These assays have clearly improved the specificity and sensitivity of detection (Kim,
2012).
q Two studies have used RT-LAMP to detect SVCV based on nucleotide sequences of the
G and M genes (Shivappa et al., 2008; Liu et al., 2008b).
q RT-LAMP can provide higher specificity and sensitivity than nested RT-PCR.

31. The development of diverse and susceptible fish cell lines is also necessary for detection,
isolation, and characterization of SVCV.
q SVCV has been reported to infect and multiply in various fish cell lines, including
1. epithelioma papulosum cyprinid cells from carp (Gotesman et al., 2015);
2. skin cells from Ussuri catfish (Pseudobargus ussuriensis) and
3. red-spotted grouper (Epinephelus akaara) (Lei et al., 2014; Ou et al., 2012);
4. heart cells from giant grouper (Epinephelus lanceolatus) (Guo et al., 2015);
5. haploid embryonic cells from medaka (Oryzias latipes) (Yuan et al., 2013);
6. muscle and fin cells from bluefin trevally (Caranx melampygus) (Zhao & Lu, 2006);
7. swim bladder, fin and snout cells from grass carp (Lu et al., 1990);
q SVCV propagation in these cells has been analysed by RT-PCR, electron microscopy,
immunofluorescence assay.
q The data obtained suggest that established cell lines can potentially serve as a useful
tool for the isolation and detection of SVCV.
Cell culture

32. (A) Common carp after 2 weeks of experimental SVCV infection showing ascitis (arrowhead) with
haemorrhagic prolapsed anal opening (arrow).
(B) Pancreas of common carp after 2 weeks of experimental SVCV infection showing green fluorescence
(arrows) concentrated at peripancreatic acinar area and surrounding vacuolated hepatic tissue. Immuno-
Fluorescence Technique.
HISTOPATHOLOGY

33. (E) Spleen of common carp after 3 weeks of experimental SVCV infection showing multifocal depletion of
white pulp and hemorrhages (arrows). H&E.
(F) Spleen of common carp after 3 weeks of experimental SVCV infection showing brown staining (arrows)
within splenocyes.

34. Control :
q SVC has been designated a notifiable disease by the Office International des Epizootics
(OIE).
q Good biosecurity and sanitation measure
q Iodophore treatment of eggs
q SVCV is susceptible to oxidizing agents, sodium dodecyl sulphate, lipid solvents
q It can be inactivated with formalin- 3% for 5 mint
chlorine- 500 ppm
iodine- 0.01%
UV irradiation- 254 nm
gamma irradiation -103 krads

35. REFERENCE
1. W. Ahne1, H. V. Bjorklund2, S. Essbauer3,*, N. Fijan4, G. Kurath5, J. R. Winton5 REVIEW of Spring viremia
of carp (SVC), DISEASES OF AQUATIC ORGANISMS Dis Aquat Org, Published December 10, Vol. 52:
261–272, 2002
2. 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.
3. 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.
4. Ahne W (1978) Uptake and multiplication of spring viremia of carp virus in carp, Cyprinus carpio L. J Fish
Dis 1:265–268
5. ZHANG N.Z., ZHANG L.F., JIANG Y.N., ZHANG T. & XIA C. (2009). Molecular analysis of spring viraemia of
carp virus in China: A fatal aquatic viral disease that might spread in East Asian. PLoS ONE, 4, 1‒9.
6. Spring viraemia of carp - OIE
7. Spring viraemia of carp - Wikipedia
8. Spring viraemia of carp - NCBI

36. THANK YOU