Accepted Manuscript

First report of spring viremia of carp virus in Percocypris pingi in

China

L.P. Zheng, Y. Geng, Z.H. Yu, K.Y. Wang, Y.P. Ou, D.F. Chen,
X.L. Huang, L.J. Deng, W.X. Gan, J. Fang, Z.J. Zhong, W.M. Lai

PII: S0044-8486(17)32438-9
DOI: doi:10.1016/j.aquaculture.2018.04.056
Reference: AQUA 633220
To appear in: aquaculture
Received date: 8 December 2017
Revised date: 20 April 2018
Accepted date: 29 April 2018

Please cite this article as: L.P. Zheng, Y. Geng, Z.H. Yu, K.Y. Wang, Y.P. Ou, D.F. Chen,
X.L. Huang, L.J. Deng, W.X. Gan, J. Fang, Z.J. Zhong, W.M. Lai , First report of spring
viremia of carp virus in Percocypris pingi in China. The address for the corresponding
author was captured as affiliation for all authors. Please check if appropriate. Aqua(2018),
doi:10.1016/j.aquaculture.2018.04.056

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First report of spring viremia of carp virus in Percocypris pingi in China

Zheng L P1, Geng Y1, Yu Z H1, Wang K Y1, Ou Y P1, Chen D F2, Huang X L2, Deng L J3, Gan W

X3, Fang J1, Zhong Z J1, Lai W M1

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College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Sichuan 611130,

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China

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Department of Aquaculture, Sichuan Agricultural University, Wenjiang Sichuan 611130, China

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Yalong River Hydropower Development Company, LTD, Xichang Sichuan 615000, China

*Corresponding author at:

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Corresponding author: Geng Y
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Address: Huimin Road No. 211, Wenjiang, Sichuan Province, 611130, China

Tel.: 86028-86291162
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Fax: 86028-86291162
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E-mail:gengyisicau@126.com
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Abstract

Spring viraemia of carp (SVC) is a rhabdovirus infection capable of inducing an acute

haemorrhagic and contagious viraemia in cyprinids. In April 2016, an infectious disease of more

than 35% mortality occured on cultured Percocypris pingi in Leshan County, Sichuan Province,

China. Necropsy of diseased fish had signs consistent with spring viremia of carp disease,

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including hemorrhage on the gills, swim bladder and internal organs. Histopathological

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examination revealed that a hemorrhagic and necrotic inflammatory response was observed in all

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major organs, particularly evident in the liver, spleen and kidney. Tissue filtrates of diseased fish

produced cytopathic effects (CPE) in epithelioma papulosum cyprini (EPC) cells and a specific
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606 bp fragment from the glycoprotein gene of SVCV was detected by RT-PCR in tissue filtrates
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of diseased fish and the cell cultures showing CPE. Based on phylogenetic analysis of

glycoprotein genes, the isolated virus was classified into the Ia genogroup and more closely to the
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Chinese isolates. Infection experiment indicated that SVCV was the aetiological agent for this
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natural epizootic event. Our study reported the first SVCV infection in cultured Percocypris pingi
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in China.
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Keywords: spring viraemia of carp virus (SVCV), isolation and identification, pathological
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lesions, phylogenetic analysis, Percocypris pingi

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1. Introduction

Spring viraemia of carp virus (SVCV) is a member of Rhabdoviridae family and belongs to

the genus Sprivivirus(Fijan et al., 1971 ; Ahne W, 2002), which is the causative agent of SVC

disease. The SVC usually occurs in the spring, when the water temperature starts to increase after

a cold winter. The morbidity and mortality are found highest when the water temperature is

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between 10 to 17 °C, however, when the temperature is above 22 °C, it no longer affect(Ahne,

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1986). SVCV has a wide host range and mainly affect cyprinids, include common carp (Cyprinus

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carpio) (Fijan et al., 1971), koi (Cyprinus carpio koi) (Goodwin, 2002), silver carp

(Hypophthalmichthys molitrix) (Jorgensen et al., 1989), bighead carp (Aristichthys nobilis)

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(Shchelkunov & Shchelkunova, 1989), grass carp (Ctenopharyngodon idella) (Ahne, 1975), tench
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(Tinca tinca) (Ahne, 1982), orfe (Leuciscus idus) (Stone et al., 2003) and caspian white fish

(Rutilus frisii kutum) (Ghasemi et al., 2014). Like the other fish Rhabdoviridae, SVCV infection is
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highly lethal which ultimately causes substantial economic losses to the aquaculture industry. It is
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recognized as one of the class-1 disease by animal epidemic prevention law of the people’s
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republic of China, and listed as a notifiable disease by the Office International des Epizooties
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(OIE)(Ashraf et al., 2016).

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Percocypris pingi, a member of the Cyprinidae family, living in upper reaches of Yangtze

River. As a result of the deterioration of the ecological environment, the number of the wild

Percocypris pingi declined sharply. It has been rated as one of the first class protection animals in

the Yangtze River (Liu, 2004), and listed as a rare protected fish in Sichuan Province. In May

2015, Percocypris pingi was classified as Endangered (EN) in the Red List of China’s Vertebrates

(Jiang et al., 2016). But realizing the high nutritional, medicinal and economic value, the artificial
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breeding farms of Percocypris pingi are increased. However, the rapid expansion and

intensification of Percocypris pingi farming have triggered higher susceptibility to various

diseases and caused significant losses. In Leshan County, Sichuan Province, China has a serious

infectious disease characterized by systemic hemorrhage outbroke in cultured Percocypris pingi

farms in April 2016, which caused more than 35% mortality rate as well as great economic loss.

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Therefore, the main objective of the present study is to elucidate the aetiology of the infectious

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disease and provide reference for the effective prevention and control of the disease.

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2. Materials and methods

2.1. Examination of diseased Percocypris pingi

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Five Percocypris pingi with typical clinical signs were collected and delivered to the
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laboratory for diagnosis. The gills and body surface were examined microscopically for the

presence of parasites and necropsies in all diseased fish. Samples for histopathological
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examination, including liver, spleen, kidney, intestine and gills, were fixed in 10% neutral buffered
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formalin and processed by standard paraffin wax techniques. Sections (4 μm) were stained with
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hematoxylin and eosin (H&E).

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Samples obtained from the kidney, liver and spleen of infected Percocypris pingi for
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bacteriological examination and streaked directly onto brain heart infusion agar (BHIA) (Solarbio)

then incubated at 28°C for 48 h. Tissues from the kidney and spleen of the diseased fish for

virological examination were homogenized, diluted to 1:10 with M199 medium (Solarbio), and

centrifuged at 2000 g for 20 min. Then, the supernatant was filtered through a 0.22 μm membrane

filter and the filtrate was inoculated onto a monolayer of Epithelioma Papulosum Cyprini (EPC)

cells. After incubation at 16°C for 1 h, inoculum was removed and replaced with fresh M199
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medium supplemented with 2% FBS (Gibco) and 1% Penicillin-Streptomycin (Solarbio). Cells

were incubated at 16°C for 7 days and examined on daily basis for cytopathic effect (CPE). When

CPE was observed, cell cultures were harvested by freezing/thawing for three times and

centrifuged at 2000 g for 10 min at 4°C, and the supernatants were stored at -80°C.

The infected cells were collected for transmission electron microscopy, 3000×g

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centrifugation for 30 min and fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.3, 0.1 M) at

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4 °C for 24h. After post-fixing with 1% osmium tetroxide (OsO4), the specimens were dehydrated

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using a series of graded alcohols and embedded in epoxy resin. The blocks were sectioned at 50

nm, stained with uranyl acetate and lead citrate, using a transmission electron microscope to
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observe the virus particles.
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2.2. RNA extraction and RT-PCR detection for SVCV

Total RNA was extracted from tissue homogenates and infected cell culture supernatants
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using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The extracted RNA
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was amplified using a PrimeScript RT reagent kit (Takara) in accordance with the manufacturer’s
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protocol. The OIE-recommended primers (OIE, 2016) were used to amplify SVCV glycoprotein
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gene. SVCV-F1 and SVCV-R2 primer to amplified 714 bp fragments from the glycoprotein gene
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of SVCV, and SVCV-F1 and SVCV-R4 primer amplified 606bp fragments from this fragment.

The RT-PCR products were visualized by 1% agarose gel electrophoresis.

2.3. G gene amplification, Sequencing and phylogenetic analysis

PCR products were purified by PCR purification kit (Takara), then send to Sangon Biotech

(Shanghai) Co. Ltd. for sequencing. The amplified sequence was further aligned online by BLAST

(Basic Local Alignment Search Tool). Genetic distances analysis were performed using Kimura’s
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two-parameter model and evolutionary trees were constructed using the neighbour-joining method

with MEGA 6.0 program through 1000 replicate bootstrap analysis.

2.4. Infection experiment

Thirty healthy Percocypris pingi (body weight 151.4 ± 5.2 g) were collected from a farm in

Sichuan province and the specimen found negative for SVCV by RT-PCR. All the fish were

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allowed to culture in the water temperature at 16 °C. After 7 days of acclimatization, half of the

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fish were injected intraperitoneally (IP) with 100 μL virus culture solution (107 TCID50 ml 1),

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while the remaining fish served as the control group which were injected with equivalent amount

of M199 medium. All fish were checked on daily basis for clinical signs and mortalities for 2
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weeks. After the death of the Percocypris pingi, necropsy examinations were performed and
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tissues samples (liver, kidney and spleen) were collected for RT-PCR detection.

3. Results
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3.1. Examination of diseased Percocypris pingi

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Clinical signs of diseased fish exhibited loss of appetite, listless swimming and sluggish near
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the water surface. External lesions included exophthalmia, abdominal extension, redness and
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swelling of the vent, gills pale and petechial-hemorrhage (Fig.1 A). After dissection, infected fish
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showed ascites, swelling and diffuse hemorrhage in liver, splenomegaly (Fig.1 B); edematous and

enlarged kidney (Fig.1 C); petechial hemorrhages in the wall of the swim bladder (Fig.1 D) and

enteritis.

Histopathological examination showed severe lesions in all major organs of the diseased fish.

Hepatocyte swelling with vacuolar degeneration and steatosis (Fig.2 A), multifocal necrosis (Fig.2

B), spleen showed hemorrhage, lymphocyte necrosis, as well as a considerable hyperplasia of

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macrophages and the reticuloendothelium (Fig.2 C), renal tubular epithelial cells degeneration and

necrosis with interstitial nephritis (Fig.2 D), recrotizing enteritis (Fig.2 E), necrosis and

hemorrhage in the gill (Fig.2 F), which were consistent with reported in the literature.

No parasite was exposed from the gills or body surface of diseased fish, and neither

bacterium was isolated from the the kidney, spleen or liver. Nevertheless, tissue filtrates from

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kidney and spleen produced CPE in EPC cells (Fig.3 B). The viral isolates from the filtered spleen

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and kidney tissue homogenates could grow on EPC cells at 16°C, and after cultured of 3 days, the

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cells begin to condense, granular cell grape-like clusters were appeared in EPC cells, showed

obvious CPE. There were no obvious changes in the control cells (Fig.3 A). The isolated virus was
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provisionally designated as ZLP160415.
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Electron microscopy revealed the typical bullet-shaped rhabdovirus in infected cells, 80-200

nm long and 50-80 nm wide. The transverse and longitudinal sections of the isolate are shown in
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Fig.4.
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Fig.1. Gross lesions in infected Percocypris pingi. A. Gills pale and hemorrhage; B. Hepatomegaly and
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splenomegaly, with hemorrhage; C. Kidney tumefaction and hemorrhage; D. Petechial hemorrhages in the wall of
the swim bladder.

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Fig.2. Pathologic lesions in infected Percocypris pingi. A. Hyperaemia and vacuolar degeneration in the liver
(bar=50μm). B. Focal necrosis with karyopyknosis and karyolysis (arrows) of liver cells (bar=10μm). C.The
spleen was hyperaemia and hemorrhage, with hyperplasia of macrophages and the reticuloendothelium
(bar=50μm). D. Degeneration and necrosis of the renal tubular epithelium, and necrosis of the haematopoietic
tissue (bar=50μm). E. Catarrhal enteritis with necrosis and sloughing of the epithelium (bar=100μm). F. Necrosis
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and hemorrhage in the gill (bar=50μm).

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Fig.3. CPE appeared in EPC infected with isolate ZLP160415. A.control EPC cell; B.cytopathic effect (CPE) of

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SVCV in EPC cell line, 72h post-inoculation.

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Fig.4. Electron micrograph of SVCV isolate ZLP160415. Bar = 100 nm.

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3.2. RT-PCR, sequencing and phylogenetic analysis

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A semi-nested RT-PCR (OIE, 2016) was performed to amplify the glycoprotein gene of

SVCV and the target band was amplified for both tissue filtrates of diseased fish and infected cell

culture samples. In the first round of PCR amplified approximately 714 bp fragments were noted,

while in the second round of PCR amplification approximately 606 bp fragments were identified.

No product was generated with the negative control (Fig.5).

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Fig.5. The electrophoresis of SVCV glycoprotein gene RT-PCR amplification. M: DL2000 DNA marker; 1,4:

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negative control (No fragment); 2,3: the first round PCR product (714bp); 5,6: the second round PCR product
(606bp).

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The second round of PCR product were purified and recovered, then referred to Sangon

Biotech (Shanghai) Co., Ltd. for sequencing (GenBank Accession number: MG020142). A
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GenBank BLAST search showed 99% sequence identity with SVCV. Based on the phylogenetic
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analysis of partial glycoprotein genes sequence of SVCV, the ZLP160415 isolate was classified

into the Ia genogroup , closely related to the Chinese isolates (Fig.6).

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Fig.6. The phylogenetic tree of SVCV based on the partial glycoprotein gene sequence, generated by using the
neighbour-joining method; Maximum Composite Likelihood; 1000 bootstrap replicates.

3.3. Infection experiment

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To confirm the pathogenicity, EPC-grown SVCV was used to infect fifteen Percocypris pingi
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by IP inoculation. The cumulative mortality of the challenged Percocypris pingi reached to 80%
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within 2 weeks. In contrast, there were no signs of disease or mortalities observed in the control
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group of fish during the experiment (Fig.7). The clinical signs and postmortem lesions in infected
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fish were found similar to those observed in naturally infected along with symptoms of bleeding

on the body surface and internal organs. Beside this the SVCV were detected in all dead

Percocypris pingi by RT-PCR.

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100

Cumulation mortality rate (%)

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0

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1 2 3 4 5 6 7 8 9 10 11 12 13 14
Day after infection
Virus culture solution injection group

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M199 medium injection group
Fig.7. Cumulative mortality of Percocypris pingi after IP injection with virus culture solution and M199 medium.

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4. Discussion

SVCV was first detected in Yugoslavia in 1971 and subsequently it has been reported in
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many other European countries (Fijan et al., 1971), including Netherlands, England, France,
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Austria, Italy, Spain, Germany, and Poland, etc (Ashraf et al., 2016). In China, SVCV was first

isolated in Tianjin region, Northern part of China, in 2003 (Miller et al., 2007). The prevalence
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and occurrence of the diseas cause a great threat to the aquaculture industry, especially the carp
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breed aquatics. Recently, a serious infectious disease characterized by systemic hemorrhage

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outbroke in cultured Percocypris pingi farms in Leshan County, Sichuan Province, China.
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According to its onset water temperature (15-18°C) and symptoms, preliminary diagnosed as a
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SVCV infection, subsequently used the histopathological examination, TEM examination, cell

culture and molecular techniques confirmed as SVCV infection. As far as we know, this is the first

report of SVCV infection in cultured Percocypris pingi in China.

The SVCV genome has been reported to have high plasticity and all SVCV strains can be

divided into two clades: the Asian and the European clade (Miller et al., 2007 ; Stone et al.,

2003). Based on nucleotide sequences of the G gene, SVCV strains can be further classified into
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four genogroups: Ia, Ib, Ic, and Id. Genogroup Ia contains isolates from Asia, USA, UK and

Cannada; genogroups Ib and Ic contain isolates from Eastern Europe; and genogroup Id contains

isolates from the UK and some other European countries (Zhang et al., 2009). Genetic clustering

of SVCV strains is closely linked to their geographic origins, while genogroup Ia own the widest

circulation. In this study, based on phylogenetic analysis of partial glycoprotein genes, the isolate

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belonged to Ia genogroup, which are closely related to the Chinese isolates.

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At present, effective treatment of fish viral diseases has remained an unreached goal in

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aquaculture. Current strategies to prevent and control fish viral diseases have consisted of

preventing the spread of viral pathogens from infected areas, destroying all the fish at affected
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farms, and in some cases vaccination (Ashraf et al., 2016). Recently, some new technique and
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vaccines, such as RNA interference (Gotesman et al., 2015), virus like particles (VLPs) (CAO et

al., 2014) and oral vaccine (Cui et al., 2015), were used to prevent SVCV infections, which are
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getting significant application results. In the future, the genetic selection of carp, the varieties and
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susceptible species for resistance to SVC and other diseases using genetic markers can be
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investigated. Bside this the field of research can also be extended to SVCV gene regulation,
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immune evasion mechanism and so on, which will provide new strategies for prevention and
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control of SVCV.

Acknowledgements

This work was supported by Sichuan Innovation Team Project of Agricultural

Industry Techology System (No.2017SICAD002), and Fish Diseases Prevention and

Control System Project of JPDC (No.D2017050).

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Highlights

 First report on spring viremia of carp virus(SVCV) infection associated with mass

mortality in Percocypris pingi in China.

 The Clinical signs and pathologic lesions were described of SVCV infected

Percocypris pingi.

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 A pathogenic SVCV (MG020142) was isolated from the affected Percocypris pingi,

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and identified by the RT-PCR and G gene sequence analysis.

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