bull. eur. ass. fish pathol., 24(4) 2004, 194 isolation of ... 4/24_194.pdf · cyprini cells (epc,...

Bull. Eur. Ass. Fish Pathol., 24(4) 2004, 194

Isolation of spring viraemia of carp virus(SVCV) from cultured koi (Cyprinus carpio koi)

and common carp (C. carpio carpio) inP.R.China

H. Liu12, L. Gao2, X. Shi2, T. Gu, Y. Jiang2, H. Chen1

1Laboratory of Animal Virology of Veterinary Medicine College, Huazhong Agrucultural Universiy,Wuhan Hubei, 430070, P. R. China; 2The Key Lab of Aquatic Animal Diseases, Shenzhen Exit & Entry

Inspection and Quarantine Bureau, 2049 Heping Road, Shenzhen 518001, P. R. China.

AbstractTwo strains (isolates 890 and 992) of spring viraemia of carp virus (SVCV) were isolated sepa-rately from cultured koi (Cyprinus carpio koi), and common carp (C. carpio carpio) in Tianjin re-gion of P. R. China in June 2003. The supernatant of a homogenate of pooled brain, spleen andkidney was inoculated into CO, FHM and EPC cells, and cytopathogenic effects (CPE) appearedin 48 hrs at 20°C. A 714 bp fragment was amplified from the viral RNA with reverse-transcrip-tion polymerase chain reaction (RT-PCR) using primers for the glycoprotein gene of SVCV. Thefragments were cloned and sequenced. A phylogenetic tree was made based on the multiplesequence alignment of the 550 bp nucleotide region of the glycoprotein gene of SVCV isolates890, 992, ATCC VR-1390 and UK isolates 980451 and 980528. The similarity between the 890 and992 strains was 98%. The isolates 890 and 992 shared more than 98% nucleotide identity with980451 and 980528, and less than 90% nucleotide identity with ATCC VR-1390. Phylogeneticanalysis showed that the isolates 890 and 992 share evolutionary direction with UK isolates980451, 980528 and 970469, and are different from 13 other SVCV isolates from European coun-tries. This is the first confirmed identification of SVCV in China.

IntroductionSpring viraemia of carp (SVC) is an impor-tant disease affecting cyprinids, with commoncarp (Cyprinus carpio) being the main host(Fijan, 1999). The disease has caused signifi-cant morbidity and mortality in carp culture(Ahne et al., 2002). The most common exter-nal signs of SVC are hemorrhages of the skin,exophthalmia, abdominal distension, and aninflamed or edematous vent. Internal signs areperitonitis, ascites, catarrhal and hemorrhagicenteritis, edematous viscera and petechialhemorrhages of the internal walls of the

swimbladder and in skeletal muscle (Ahne &Wolf, 1977; Fijan et al., 1971; Negele, 1977).

The causative agent of SVC is spring virae-mia of carp virus (SVCV), which is presentlyclassified as a member of the genusVesiculovirus of the family Rhabdovirudae(Walker et al., 2000). The genomic RNA is11019 bases in length. The virion of SVCVcontains 5 structural proteins: N protein (thenucleoprotein), P protein (the phosphopro-tein), M protein (the membrane protein), Gprotein (the glycoprotein) and L protein (the


RNA-dependent RNA polymerase)(Hoffmann et al., 2002). The G protein ofSVCV acts as the most important viral anti-gen which determines the serological prop-erties of rhabdoviruses (Bishop & Smith, 1977;Hill et al., 1975; Jørgensen et al., 1989). The550 bp partial glycoprotein gene sequencesfrom 15 SVCV isolates have been analysedand divided into 4 subgroups based onphylogenetic trees (Stone et al., 2003).

Until recently, it was believed that SVC oc-curred only in Europe, but in 1998, 2 isolatesof SVCV (980451 and 980528) were obtainedin the UK from koi exported from the Beijingregion, China (Stone et al., 2003). However,despite comprehensive monitoring of regis-tered ornamental fish farms during the periodof 1998-2002 for the virus, there was no evi-dence of SVCV infection in China. In 2003,SVC broke out in farmed koi carp in NorthCarolina and subsequently the virus was de-tected in common carp in Wisconsin, USA.Sequence analysis of partial glycoprotein se-quences revealed that the isolates belong tothe same subgroup of SVCV as the UK iso-lates purported to originate in China (D.Stonepers.comm.).

The Administration for exit-entry inspectionand quarantine of the Peoples Republic ofChina adopted a stricter SVC surveillanceprogramme throughout China in 2003. Twostrains of SVCV were isolated separately fromthe koi and common carp cultured in Tianjinregion, Northern China. In this paper, the 714bp fragments of the glycoprotein gene of thesetwo isolates were amplified, sequenced andcompared with the published sequences fora number of SVCV strains. The evolution ofthese two strains was also analysed. The find-

ings will provide useful epidemiology infor-mation in order to have a better understand-ing of this economically important disease.

Materials and MethodsFishKoi and common carp samples were collectedfrom over 65 ornamental fish farms in differ-ent regions in P. R. China. Among them, koi(sample no. 890) and common carp (sampleno. 992) were collected separately from twoindependent fish farms in Tianjin in June. Theaverage body size was 25-40 cm in koi and10-25 cm in common carp. No external or in-ternal clinical signs of SVC disease were ob-served.

Cell linesOvary of grass carp (CO), kidney of grass carp(CK), embryo cells of chinook salmon (CHSE,Lannan et al. 1984), caudal trunk cells of fat-head minnow (FHM, Gravell andMarlsberger, 1965), epithelioma papillosumcyprini cells (EPC, Fijan et al., 1983), wereused. The cells were grown at 20 and 25°C inTC199 medium containing Earle‘s salts, 10%fetal bovine serum, 100 IU penicillin ml-1, and100 mg streptomycin ml-1.

Virus isolationKidney, brain and spleen were excised fromthe fish, pooled, homogenized with a mortarand pestle and then diluted 1:10 in TC199medium containing 10% FCS and antibiotics.After centrifugation at 3,000 rpm for 20 min,10-fold diluted supernatant fluid was inocu-lated onto monolayers of several fish cell lines.Inoculated cell cultures in 96-well plate wereincubated at 20 and 25°C and observed dailyfor two weeks.


Preparation of viral nucleic acidA 450 ml aliquot of virus suspension was fro-zen and thawed and mixed with 450 ml ofCTAB buffer (2% w/v hexadecyl trimethyl-ammonium bromide, 1.4 M NaCl, 20 mMEDTA, 100 mM Tris-HCl pH 7.5 and 0.25% v/v 2-mercaptoethanol added just before use)in a 1.5 ml microfuge tube and incubated at25°C. After 2.5 h, 600 ml of Phenol/chloro-form/iso-amyl alcohol (25:24:1) was added tothe tube and mixed vigorously for 30 s. Thephases were separated by centrifugation at12,000 g for 5 min. 750 ml of the upper aque-ous phase was then removed to a clean tubeand 750 ml of Chloroform/iso-amyl alcohol(24:1) added and mixed vigorously for 30 s.600 ml of the upper aqueous phase was thenremoved to another clean tube and 900 ml ofcold ethanol added. This was then mixed gen-tly and the solution left at –20°C overnight.Next day the tube was centrifuged at 12,000 gfor 15 min., the supernatant discarded and thevirus RNA pellet air-dried. The RNA pelletwas re-suspended with 12ì l of water for useas the template in the RT-PCR.

Reverse-transcription and PCR protocolThe master mix for one 25 ml reverse-tran-scription PCR was prepared as follows: 3 mlof upstream primer SVCV-R2 (5’-AGA TGGTAT GGA CCC CAA TAC ATH ACN CAY-3’,40mmoL), 10 ml of RNA template and 2 ml ofwater was heated to 90°C for 5 min to dena-ture viral RNA, cooled on ice for 5 min, and 5ml of AMV buffer, 1ml of AMV reverse tran-scriptase (TAKARA, 5 U/ml), 1 ml of RNasin(40 U/ml) , 2 ml of 10 mM dNTP and 1 ml ofwater added. The tubes were then centrifugedbriefly to ensure the mastermix was at thebottom of the tube and placed in a thermal

cycler and heated at 70°C. After 1 h, 2.5 ml ofthe primer SVCV-R2, 2.5 ml of the down-stream primer SVCV-F1 (5’- TCT TGG AGCCAA ATA GCT CAR RTC-3’, 40mmoL), 2 mlof 10 mM dNTP, 8 ml of 10x DNA Taqpolymerase buffer, 8 ml of 25 mmoL/L MgCl2,1ml of 5U/ml DNA Taq polymerase and 58ml of water was added to each tube. The 100ml of reaction mixture was then overlaid with50 ml of mineral oil and subjected to 35 cy-cles of 1 min at 94°C, 1 min at 55°C and 1 minat 72°C followed by a final extension step of10 min at 72°C.

Purification, cloning and sequencingAliquots (6 ml ) of the amplified productswere electrophoresed in a 1.5 % (w/v) agarose/TBE (40 mM Tris - boric acid, pH 8.0, 1 mMEDTA) gel containing 1.0 mg/mL ethidiumbromide and visualized by UV irradiation.PCR products (714 bp) were purified usingPCR product purifying kit (Takara, Dalian,China) and ligated into the PT7 blue T vectorusing the standard protocol. The vector weretransformed into the E.coli and cultured onL-agar medium containing X-Gal, IPTG andAmp. The white clone was selected and am-plified using M13 universal sequencing prim-ers and sequenced in TAKARA (Dalian,China).

Multiple alignmentMultiple alignment was analysed by thesoftwares of DNAsis and Sequencher. Thesequences of partial G-pro gene of 890 and992 were compared with G-pro gene of otherSVCV strains in Gene-bank (AJ318079, thecomplete genomic RNA of reference strain,VR-1390; AJ538066, partial G gene for glyco-protein of isolate 980528; AJ538065, partial Ggene for glycoprotein of isolate 980451).


Phylogenetic analysisPhylogenetic trees were generated byNeighbor-Joining of 18 X 550 bp partial glyco-protein gene sequences of SVCV strains iso-lated in this study and of other SVCV strains.The detailed information of these isolates arelisted in the article of Stone et al. (2003).

ResultsIsolationCO cell monolayers inoculated with samples890 and 992 developed cytopathic effects(CPE) at 20°C in 48 hrs. No CPE appeared inthe samples of koi and common carp from theother 63 farms, which are distributed in dif-ferent regions in China. The CPE developedvery quickly with cell monolayer destructionand cell lysis reaching completion in 3 days.Infectivity titres of about 106 and 108 TCID50 /ml were obtained separately with the 890 and992 isolates respectively. The same type ofCPE was observed in FHM and EPC cellmonolayers (Figure 1).

RT-PCR amplication and sequencing ofSVCV G-protein genesAmplifed products of 714 bp were observedunder UV light (Figure 2) after RT-PCR am-plification, with the primers F1 andR2, of RNAextracted from cell cultures inoculated withisolates 890 and 992. The products were thenpurified and sequenced (Figure 3).

Multiple alignment of sequencesIsolates 890 and 992 shared 98% nucleotideidentity with only 11 nucleotide differencesover the 550 bp region studied. Isolate 890shared 99% (5 nucleotide differences) and99.2% (3 nucleotide differences) nucleotide

Figure 1. CPE appeared in CO (left), EPC (middle) and FHM (right) infected with isolate 992.

714bp

Figure 2. DNA fragments of 714 bp was amplifiedfrom viral suspension of 890 and 992 isolates afterRT-PCR. From left to right: Lane 1: DNA marker(DL2000, from up to down, 2000 bp, 1000 bp, 750bp, 500 bp and 250 bp); Lane 2: Amplified productsof isolate 890; Lane 3: Amplified products of isolate992; Lane 4: Amplified products of SVCV isolate10/3.


identity with isolate 980451 and 980528 re-spectively and 992 shared 98.1% (10 nucle-otide differences) nucleotide identity with980451 and 98.3% (9 nucleotide differences)identity with 980528. Isolates 890, 992, 980451and 980528 shared <90.0 % identity with anSVCV reference strain (880062) with >55 nu-

Figure 3. Multiple alignment of the sequences of 550 bp fragment of 890 and 992 strain with an equivalentregion from the reference strain (AJ318079) and two isolates thought to have their origins in China, 980451(AF538065) and isolate 980528 (AJ538066).

cleotide differences (Figure 3). The resultsshowed that 890 and 992 strain has a closerrelationship with 980451 and 980528, but amore distant relationship with the EuropeanSVCV strain, 880062.


DiscussionSVC has caused great losses in carp culturein some European countries (Ahne et al.,2002), while the occurrence of SVC was pre-viously unknown in Asian countries. In 1998,CEFAS in the UK isolated SVCV from Koi carpand goldfish exported from Beijing region,China (Stone et al., 2003). The inspectors ofthe Chinese quarantine department carriedout an investigation on ornamental fish farmsin the suspected production region, but SVCVwas not isolated. From then on, all of the ex-porting ornamental fish farms were registeredand monitored under strict inspection. The

Figure 3. Continued.

Phylogenetic analysisThe 18 isolates used in the phylogenetic analy-sis were divided into two big clusters (Figure4). 890, 992, 980451, 980528 and 980469 be-long to one cluster, which have obvious evo-lutionary divergence from another clustercomposed of another 13 SVCV isolates. Theisolates RHV, P4, N1-5 and 2/90 formed a sub-cluster, different in its evolutionary directionwhen compared with the sub-cluster com-posed of the isolates 880062, N3-14, 860115,M2-78, S30, 880124, 770346, 970395, and880062.


discovery of SVC in the USA attracted ourattention and an epidemiological investiga-tion on SVC in the whole of China was initi-ated. The cultured cyprinid fish, both diseasedfish and fish without clinical disease signs,from registered and non-registered farms,were collected in the spring and autumn. Twostrains of SVCV (890 and 992) were isolatedin this epidemiological investigation from twofish farms in the Tianjin region in NorthernChina.

Because there are not enough water resourcesin northern China, fish farms have extractedwater from deep wells and no farm effluentwas released without being disinfected. Infact, the fish farms in this region are totally

Figure 4. Phylogenetic tree generated by Neighbour-Joining analysis based on 550 bp of G-protein genesequences of 890, 992 and other SVCV isolates in Gene-bank. The corresponding sequence of Piry virusGenBank accession no. D26175) was used as an outgroup. The accession number of partial glycoproteingene sequences of isolates 860115, 880062, 880124, 770346, 970395, RHV, P4, 970469, N1-5, M2-78, N3-14,S30, 2/90 in Gene-bank are AJ538080, AJ538079, AJ538078, AJ538077, AJ538076, AJ538075, AJ538074,AJ538067,.AJ538064, AJ538063, AJ538062, AJ538061 andAJ538080 respectively.

isolated from each other. These farms areregistered officially and have been monitoredfor more than 2 years. In earlier investigations,many fish farms in the Tianjin region weretested, but no SVCV was found. The sourceof the SVC virus isolates and how the virusesentered the farms are important questions thatneed to be answered.

In general, the cell lines of cyprinid fish wereused for isolation of SVCV, such as EPC (Fijanet al. 1983) and FHM (Gravell & Malsberger1965), these cell lines are recommended in theOIE Diagnostic Manual of Aquatic AnimalDiseases (OIE, 2000). The CO cell line used inthis investigation is derived from grass carpovary and produces CPE after infection with


SVCV. In this study, obvious CPE was ob-served in CO cell monolayers, which indicatesthat the CO cell line is highly susceptible toSVCV.

The glycoprotein (G-pro) of SVCV formstrimeric peplomers or spikes on the virus sur-face that bind to cellular receptors and induceviral endocytosis. The surface glycoproteinacts as the most important viral antigen thatdetermines the serological properties ofrhabdovirus (Hill et al., 1975; Bishop & Smith,1977; Jørgensen et al., 1989). Part of G-pro gene(550 bp) has been used in the study on geno-group analysis and phylogenetic tree genera-tion (Stone et al., 2003). The SVCV isolates980451 and 980528 belong to geno-group Iaand are deduced to have originated in P. R.China and 970469 in the same geno-group iso-lated from the fish held on a site that had re-ceived several consignment of fish from P. R.China (Stone et al. 2003). The multiple se-quence alignment and phylogentic analysisbased on the the 550 partial glycoprotein genesequences, revealed high nucleotide identityand the same evolutionary direction amongthe isolates 890, 992, 980451, 980528 and970469, which show that these isolates have aclose relationship. Another evolutionary di-rection has been found among the 13 isolatesfrom the fish in the European countries. Sinceno SVCV has been isolated in P. R. China -until the isolation of 890 and 992, the originof SVCV isolates should be explored basedon more information of nucleotide and aminoacid sequences.

This is the first isolation of SVCV from“healthy” koi and common carp in China. Ithas been suspected since 1998 that ornamen-tal fish cultured in China may carry SVCV.

But this assumption has not been confirmeduntil now. Koi culture is an important part ofthe ornamental fish industry in China, and theculture of common carp in China has takenplace for thousands of years. The presence ofSVCV could have significant impact on fishculture in China and disease control measuressuch as quarantine program will be imposed.

Since no outbreak of clinical SVC has beenobserved in China, the virulence of strains 890and 992 needs to be tested in challenge ex-periments. In this study, we just isolated theSVCV strains and amplified partial nucleotideof SVCV G-pro gene. More research, such asthe pathogenicity, infectivity andimmunogenicity of SVCV strains isolated inChina, should be done in the near future andthe surveillance throughout the countryshould be continued. In this study, RT-PCRwas used as an identification test in this studyand showed high sensitivity and specificity.It is suggested that RT-PCR be used as thescreening method in further SVCV epidemio-logical investigations.

AcknowledgmentThe authors would like to thank B. J. Hill,CEFAS Weymouth Laboratory, UK for provid-ing the SVCV isolate-10/3 used as a referencein this investigation.

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