Open Access

Phylogenetic analysis of newly isolated grass carp reovirus

  • Xiu-ying Yan1,
  • Ya Wang1,
  • Ling-fang Xiong1,
  • Ji-chang Jian1Email author and
  • Zao-he Wu2
SpringerPlus20143:190

https://doi.org/10.1186/2193-1801-3-190

Received: 13 January 2014

Accepted: 27 March 2014

Published: 15 April 2014

Abstract

Grass carp reovirus (GCRV) is a causative agent of haemorrhagic disease in grass carp that drastically affects grass carp aquaculture. Here we report a novel GCRV isolate isolated from sick grass carp that induces obvious cytopathic effect in CIK cells and name it as GCRV096. A large number of GCRV 096 viral particles were found in the infected CIK cells by electron microscope. The shape, size and the arrangement of this virus were similar to those of grass carp reovirus. With the primers designed according to GCRV 873 genome sequences, specific bands were amplified from sick grass carp and the infected CIK cells. The homology rates among vp4, vp6 and vp7 gene in GCRV 096 and those of some GCRV isolates were over 89%. In this study, the sequences of vp4, vp6 and vp7 were used to analyse sequence variation, phylogenetic relationships and genotypes in twenty five GCRV isolates. The results indicated these twenty five GCRV isolates should be attributed to four genotypes. And there were no obvious characteristics in the geographical distribution of GCRV genotype. The study should provide the exact foundation for developing more effective prevention strategies of grass carp haemorrhagic disease.

Keywords

Grass carp reovirus (GCRV)IdentificationPhylogenetic relationshipGenotype

Introduction

Grass carp reovirus (GCRV), which is known as a member of the Aquareovirus genus and the Reoviridae family, can cause serious haemorrhagic disease in grass carp (Chen and Jiang 1983) and obvious cytopathic effect (CPE) on many cell lines from fish (Zuo et al. 1986; Lu et al. 1990). To date, a number of various GCRV isolates have been isolated from diseased grass carp around the world, including GCRV 873, GCRV 875, GCRV HZ08, GCRV GD108, AGCRV and others (Fang et al. 2002; Chi et al. 2011; Ye et al. 2012; Zeng et al. 2013). These isolates are distinct not only in their levels of virulence and cell culture characteristics, but also in their antigenicity (Fang et al. 2002; Mohd Jaafar et al. 2008; Zhang et al. 2010a).

GCRV is a double-stranded RNA virus that is assigned to the Aquareovirus C species. The genome of GCRV is known to consist of 11 segments of dsRNA contained in a core surrounded with a double-layered icosahedral capsid (Rangel et al. 1999). To our knowledge, there are few published reports about the serotype and genotype of GCRV. Furthermore, there are no uniform criteria for virus genotyping. One of the virus genotyping methods is based on the analysis of the nucleotide sequence.

So far, some gene sequences of GCRV isolates have been reported (Mohd Jaafar et al. 2008; Rangel et al. 1999; Fang et al. 2000; Su et al. 2010; Attoui et al. 2002; Fan et al. 2010). vp4, vp6 and vp7 gene in GCRV encode major outer capsid proteins and are conservative. Moreover, there are many variable sites and informative sites between sequences of vp4, vp6 and vp7 gene in different GCRV isolates. Considering vp4, vp6 and vp7 gene as molecular makers, we investigated sequence variation characteristics, the phylogenetic relationships and genotypes of twenty five GCRV isolates to find the evolutive characteristic of GCRV in the study. In this study, a new GCRV isolate was found and identified from diseased grass carp. This study provides the theoretical basis for the prevention and treatment of haemorrhagic disease in grass carp.

Materials and methods

Virus and cells

GCRV 096 was isolated from the diseased grass carp in Xiaogan, Hubei Province and stored in our laboratory. A widely used GCRV sensitive cell steain, grass carp kidney cells (CIK) were purchased from shenzhen inspection and quarantine bureau in China. CIK is GCRV sensitive cell and are widely used in the related study of GCRV (Ye et al. 2012; Zhang et al. 2010b; Ma et al. 2011).

Some GCRV isolates were examined in the present study, which were identified in previous studies (Mohd Jaafar et al. 2008; Rangel et al. 1999; Fang et al. 2000; Su et al. 2010; Attoui et al. 2002; Fan et al. 2010). Table 1 presents information about the specific names of twenty five GCRV isolates, their abbreviations, locations where they were collected, the genes of GCRV and their GenBank accession numbers.
Table 1

Names of GCRV isolates, abbreviations, localities, genes of GCRV used in this study and their GenBank accession numbers

Names

Abbreviations

Localities

Genes

GenBank accession numbers

AGCRV PB01-155

155

America

vp4, vp6, vp7

EF589103, EF589105, EF589107

AGCRV

ARV

America

vp4, vp6, vp7

NC010589, NC010593, NC010594

GCRV 096

096

Hubei, China

vp4, vp6, vp7

JN206664, HQ452490, JN206665

GCRV 104

104

Hubei, China

vp6

HM234682

GCRV 097

097

Shanxi, China

vp4

GQ469997

GCRV 873

873

Hunan, China

vp4, vp6, vp7

AF403392, AF260512, AF260513,

GCRV 875

875

Hubei, China

vp6, vp7

AF403412, AF403409

GCRV 876

876

Jiangxi, China

vp6, vp7

AF403413, AF403410

GCRV 991

991

Hunan, China

vp6, vp7

AF403414, AF403411

GCRV GD108

108

Guangdong, China

vp4, vp6, vp7

HQ231208, HQ231205, HQ231203

GCRV HeNan988

988

Henan, China

vp4, vp6

KC847325, KC847328

GCRV HN12

H12

China

vp6

KC130075

GCRV HS11

H11

China

vp6

KC130076

GCRV HuNan794

794

Hunan, China

vp4, vp6

KC238681, KC238684

GCRV HZ08

H08

Zhejiang, China

vp4, vp6, vp7

GQ896337, GU350746, GU350744

GCRV JS12

J12

China

vp6

KC130077

GCRV NC11

N11

China

vp6

KC130078

GCRV QC11

Q11

China

vp6

KC130079

GCRV QY12

Q12

China

vp6

KC130080

GCRV YX11

Y11

China

vp6

KC130081

GCRV ZS11

Z11

China

vp6

KC130082

GCRV 106

106

China

vp4, vp6

KC201171, KC201174

GCRV 918

918

China

vp4, vp6

KC201182, KC201185

GCRV JX01

J01

Jiangxi, China

vp7

JQ042807

GCRV JX02

J02

Jiangxi, China

vp7

JX263303

Bovine rotavirus B223

Bovine

 

vp4, vp6, vp7

D13394, AF317128, X57852

Virus culture and transmission electron microscopy observation

Cell culture, viral infection and propagation determination were performed as previously described (Fang et al. 1989). GCRV 096 particles were extracted with the differential centrifugation at 250-6000 g, and the supernatant was then ultracentrifuged at 35,000 g at 4°C for 2.5 h. The purified virus pellet was resuspended in phosphate-buffered saline (PBS) with pH 7.4 and then stored at -70°C for the further use. After removing the cell culture medium from the confluent monolayer cell, the monolayer cell was rinsed two times with the PBS buffer and the virus was added with the adsorption for one hour at room temperature. Then, after aspirating off the virus, the maintain solution (M199 containing 2% FBS) was added. The infected CIK cells were incubated at 28°C and observed daily.

Electron microscopic section of the infected CIK cells with CPE was made and observed in transmission electron microscope.

RT-PCR amplification

With viral RNA kit (Takara, Dalian, China), the GCRV 096 genome RNA was extracted from purified GCRV 096 virus. The cDNA of GCRV 096 genome RNA was acquired with RT-PCR kit (Takara, Dalian, China) using the random primers and M-MLV reverse transcriptase.

According to the genome sequence of GCRV 873, primers for GCRV 096 vp4, vp6 and vp7 gene amplification were designed based on homologous sequence in GCRV 873: 5″-CACTTCGCACTCTCTCTACAATG-3′ and 5′-AGTACGACACTTCCCGCCGTT-3′, 5′-TGTGATGGCACAGCGTCAG-3′ and 5′-GTTAGA CGAACATCGCCTGC-′3, 5′-TCACCACGATGCCACTTCAC-3′ and 5′-CGGTGCTTAATCGGATGGCT-3′, respectively. Primers were also designed based on homologous sequence from GCRV GD108 and GCRV HZ08 for vp4, vp6 and vp7 gene: 5′-ACTTACGGCCACTATCATGG-3′ and 5′-TCGGTGTACACGACCTAAG-3′, 5′-CTTTGAGTCGACGCACGTAT-3′ and 5′-CCGTCGGGTGGATTAGGTC-3′, 5′-TCTACTGCCAAGATGGCCAC-3′ and 5′-GCACGCACCTTACTTACAGCA-3′. The PCR cycling conditions were an initial denaturation at 95°C for 3 min followed by 30 cycles consisting of 94°C for 30 s, 55°C for 60 s and 72°C for 70 s, and a final extension step of 30 min at 72°C. The composition of the PCR system (50 μl) includes 33 μl sterile water, 3 μl dNTP (each is 2.5 mmol/L), 10 pmol/L primer for 2 μl each, 10 × buffer for 5 μl (containing Mg++), DNA for 100 ng and Taq polymerase for 0.25 μl (5 U/μl). The aimed genes were purified using Gel Extraction Kit (Takara, Dalian, China) from gelose gel and connected with pMD18-T vector at 16°C, then transformed to DH5α E.coli. The recombined plasmid was verified by sequencing.

Gene sequence analysis of GCRV isolates

Sequences of vp4, vp6 and vp7 genes were aligned by using the Clustal V method in DNAstar software. Subsequently, the alignment was manually adjusted. Variable sites, information sites, genetic distances, and homologic rates of segments were calculated with MEGA5.1 (Tamura et al. 2007) and DnaSP5.10 (Rozas and Rozas 1999) software.

Phylogenetic relationships of GCRV isolates

Evolutionary models of vp4, vp6 and vp7 gene in GCRV were separately simulated in ModelTest3.7 (Posada and Crandall 1998). Subsequently, phylogenetic trees were restructured with simulation results. Using bovine rotavirus B223 as the outgroup, maximum parsimony (MP) trees, maximum likelyhood (ML) trees, and UPGMA trees were constructed with MEGA 4.1 (Tamura et al. 2007) software. MP trees were also built in PAUP4.0 (Swofford 1998) by running the heuristic search with TBR branch swapping, 100 random addition sequence replications, and non-parameter bootstrap resampling procedures to get the coincidence of the resultant MP trees. Bayesian analysis were performed with MrBayes3.12 (Huelsenbeck and Ronquist 2001) using the general-time-reversible + gamma + invariants (GTR + G + I) model of sequence evolution and four Markov Chain Monte Carlo (MCMC) sampling to assess phylogenetic relationships. We set the parameters in MrBayes as follows: nst = 6, rate = gamma, basefreq = estimate, generations = 10,000,000, and the posterior probability and branches of the phylogeny were summed by burnin = 500 and contype = allcompat.

Sequence variation analysis of vp4, vp6 and vp7 genes in GCRV isolated to the same genotype

Sequences of vp4, vp6 and vp7 genes in GCRV isolated to the same genotype were aligned by using the Clustal V method in DNAstar software. Alignment was manually adjusted. Variable sites were analysed.

Results

Virus infection in sensitive cellls and particle identification

Three days after the culture of the CIK cells infected by GCRV 096, CPE phenomenon was observed and the shedding and apoptosis occurred in most of the CIK cells five days after the infection. While, the controlled CIK cells without the infection by the virus grew well (Figure 1).
Figure 1

CPE in the CIK cells 3 d after GCRV 096 isolate inoculation (A, A’ 100×) and Crystalline array of viral particles (B 50,000×). Notes: A. The control CIK cells without GCRV096 inoculation. A’. CPE in the CIK cells 3 d after GCRV 096 isolate inoculation. B: Crystalline array of viral particles.

A large number of virus particles without the envelope structure crystalline in CIK cells were detected from transmission electron microscopy ultrathin section of CIK cells infected with GCRV 096 (Figure 1). The shape, size and the arrangement of GCRV 096 were similar to those of grass carp reovirus (Ke et al. 1990).

Detection by RT-PCR

vp4, vp6 and vp7 genes were PCR amplified from GCRV 096, subcloned into a pMD18-T vector and sequenced. The length of vp4, vp6 and vp7 genes in GCRV 096 was 1981 bp, 1258 bp, and 855 bp, respectively (GenBank accession numbers: JN206664, HQ452490 , and JN206665).

Sequence analysis

vp4, vp6 and vp7 genes in the these GCRV isolates contain 184, 447 and 375 informative sites, respectively. Table 2 shows the identity and divergence among GCRV isolates based on vp4, vp6 and vp7 genes, respectively. Based on the data shown in Table 2, it is apparent that genetic distances of vp4, vp6 and vp7 genes among GCRV 096, GCRV 873, GCRV 875, GCRV JX01, GCRV 876 and GCRV 991 or AGCRV and AGVRV PB01-155 were small, and their homologous rates were high. Also, genetic distances among GCRV HZ08, GCRV GD108, GCRV 918, GCRV HuNan794, GCRV HeNan988, GCRV 106, GCRV ZS11, GCRV QC11, GCRV HN12, GCRV HS11, GCRV YX11, GCRV JS12, GCRV QY12, GCRV JX02, and GCRV 097 were small, with elevated homologous rates. Other genetic distances were far and the genetic identities were small.
Table 2

Identity (above the diagonal) and divergence (under the diagonal) between GCRV isolates based on the vp4 , vp6 , vp7 gene [×1000]

 

Based on the vp4 gene

Based on the vp7 gene

GCRV

155

ARV

096

097

873

108

988

794

H08

106

918

GCRV

155

AVR

096

873

875

876

991

108

H08

J01

J02

155

 

1000

599

317

603

299

293

292

286

293

296

155

 

1000

203

303

279

287

289

219

214

302

213

ARV

0

 

599

317

603

299

293

292

286

293

296

ARV

0

 

203

303

279

287

289

219

214

302

213

096

453

453

 

295

993

303

299

298

304

298

304

096

1178

1178

 

212

196

197

197

199

208

213

207

097

946

946

827

 

311

981

990

987

994

990

987

873

767

767

920

 

902

999

1000

199

197

994

205

873

445

445

5

827

 

298

297

296

299

296

302

875

856

856

910

100

 

901

902

222

208

899

206

108

967

967

886

20

872

 

970

970

963

971

968

876

776

776

855

1

102

 

999

204

200

993

197

988

971

971

891

10

882

31

 

997

986

998

986

991

782

782

848

0

100

1

 

204

200

995

195

794

977

977

896

13

888

31

3

 

985

998

985

108

1218

1218

920

885

891

865

870

 

986

203

987

H08

941

941

875

6

863

35

14

14

 

986

984

H08

1194

1194

904

863

880

843

848

14

 

202

998

106

973

973

893

10

884

30

2

2

13

 

987

J01

772

772

897

6

103

7

5

890

864

 

193

918

962

962

897

13

888

33

15

15

17

13

 

J02

1300

1300

913

858

876

839

844

14

2

859

 

Based on the vp6 gene

GCRV

155

ARV

096

104

873

875

876

991

108

988

H12

H11

794

H08

J12

N11

Q11

Q12

Y11

Z11

106

918

 

155

 

1000

548

230

548

547

547

548

196

233

241

235

233

204

235

235

235

235

235

233

233

232

 

ARV

0

 

548

230

548

547

547

548

196

233

241

235

233

204

235

235

235

235

235

233

233

232

 

096

500

500

 

220

994

998

998

997

201

242

235

235

241

202

233

233

239

233

233

243

241

241

 

104

936

936

769

 

238

246

246

246

190

232

229

232

232

198

238

238

228

238

232

232

232

223

 

873

503

503

6

751

 

999

999

998

204

243

235

235

242

205

232

232

239

232

232

243

242

242

 

875

516

516

3

751

1

 

1000

999

211

238

231

231

238

203

233

233

236

233

233

238

238

237

 

876

516

516

3

751

1

0

 

999

211

238

231

231

238

203

233

233

236

233

233

238

238

237

 

991

514

514

4

755

2

1

1

 

211

238

231

236

238

200

233

233

236

233

233

238

238

237

 

108

1427

1427

1340

1527

1261

1236

1236

1236

 

207

197

203

207

965

204

204

203

204

197

200

200

199

 

988

834

834

682

939

693

665

665

665

1157

 

975

972

998

208

971

971

990

971

973

998

998

995

 

H12

844

844

680

959

692

659

659

659

1214

26

 

991

975

210

990

990

979

990

992

975

976

973

 

H11

850

850

683

962

694

662

662

662

1211

29

9

 

973

205

998

998

981

998

999

973

974

971

 

794

838

838

685

931

697

670

670

670

1149

2

25

28

 

206

972

972

990

972

974

998

999

996

 

H08

1400

1400

1288

1510

1210

1196

1196

1196

32

1113

1165

1162

1106

 

204

204

200

204

205

206

206

204

 

J12

850

850

683

958

694

662

662

662

1228

29

10

2

29

1178

 

1000

980

1000

998

972

973

970

 

N11

850

850

683

958

694

662

662

662

1228

29

10

2

29

1178

0

 

980

1000

998

972

973

970

 

Q11

834

834

689

923

701

670

670

670

1142

10

22

19

10

1099

20

20

 

980

982

992

991

988

 

Q12

850

850

683

958

694

662

662

662

1228

29

10

2

29

1178

0

20

20

 

988

972

973

970

 

Y11

854

854

686

958

698

667

667

667

1219

28

8

1

27

1170

2

2

19

2

 

974

975

971

 

Z11

834

834

682

939

693

665

665

665

1142

2

25

28

2

1099

29

29

8

29

27

 

999

996

 

106

838

838

685

935

697

670

670

670

1149

2

24

27

1

1106

28

28

9

28

26

1

 

997

 

918

851

851

692

946

704

677

677

677

1154

5

28

30

4

1124

31

31

12

31

29

4

3

  

Simulation results of evolutionary model and phylogenetic relationships of GCRV isolates

Simulation results of the GCRV evolutionary model based on vp4, vp6 and vp7 gene from ModelTest3.7 (Posada and Crandall 1998) are shown in Table 3. The simulation results are used to construct phylogenetic trees.
Table 3

Simulation results of the evolutionary model

   

vp4 gene

vp6 gene

vp7 gene

Model selected:

HKY + G

GTR + G

HKY + G

TVMef + G

K80 + G

  -lnL =

10960.6680

10947.8525

10486.8574

10480.6113

6139.4121

  K =

5

9

5

8

2

  AIC =

 

21913.7051

  

20977.2227

Base frequencies:

     

  freqA =

0.2726

0.2724

0.2561

0.2530

 

  freqC =

0.2541

0.2476

0.2612

0.2586

 

  freqG =

0.2361

0.2401

0.2285

0.2313

 

  freqT =

0.2371

0.2400

0.2541

0.2571

 

Substitution model:

     

  R(a) [A-C] =

 

1.9847

 

1.8471

 

  R(b) [A-G] =

 

3.8446

 

7.0961

 

  R(c) [A-T] =

 

1.3223

 

1.3157

 

  R(d) [C-G] =

 

1.7384

 

1.3664

 

  R(e) [C-T] =

 

4.4150

 

7.0961

 

  R(f) [G-T] =

 

1.0000

 

1.0000

 

  i/tv ratio =

1.3563

 

2.5439

  

Proportion of invariable

     

 sites =

0

0

0

0

0

Gamma distribution

     

shape parameter =

4.2112

4.3849

3.8529

3.7611

3.8868

Topological structures of constructed phylogenetic trees, based on vp4, vp6 and vp7 genes of GCRV in this article are basically coincident. According to evolutionary simulation results, there is the UPGMA tree constructed based on vp4 gene in Figure 2. The results showed that the cluster on the top of the UPGMA tree consisted of GCRV 106, GCRV HeNan988, GCRV HuNan794, GCRV 097, GCRV 918, GCRV GD108 and GCRV HZ08. The second cluster was AGCRV PB01-155 and AGCRV. The third cluster contained GCRV 096 and GCRV 873.
Figure 2

The constructed UPGMA tree based on the vp4 gene (Numbers indicate degree of confidence) was created first in MEGA software and completed with Microsoft Paint program.

In Figure 3, the MP tree was constructed based on vp6 gene. On the MP tree, the cluster on the top consisted of GCRV 106, GCRV HeNan988, GCRV HuNan794, GCRV 918, GCRV ZS11, GCRV QC11, GCRV HN12, GCRV HS11, GCRV YX11, GCRV JS12, GCRV QY12, GCRV GD108 and GCRV HZ08. The second cluster was GCRV 104. The next cluster was AGCRV PB01-155 and AGCRV. The last cluster contained GCRV 096, GCRV 875, GCRV 876, GCRV 991 and GCRV 873.
Figure 3

The constructed MP tree based on the vp6 gene (Numbers indicate the degree of confidence) was created first in PAUP software and completed with Microsoft Paint program.

In Figure 4, the UPGMA tree was constructed based on vp7 gene. The cluster on the top of this tree consisted of GCRV 096, GCRV 875, GCRV 876, GCRV JX01, GCRV 991 and GCRV 873. The second cluster was GCRV GD108, GCRV HZ08 and GCRV JX02. The last cluster contained AGCRV PB01-155 and AGCRV. The phylogenetic relationships of GCRV096, GCRV 991, GCRV 876, GCRV 873, GCRV JX01, and GCRV 875 or GCRV HZ08, GCRV JX02, and GCRV GD108 are relatively close.
Figure 4

The constructed UPGMA tree based on the vp7 gene (Numbers indicate the degree of confidence) was created first in MEGA software and completed with Microsoft Paint program.

Sequence analysis of vp4, vp6 and vp7 genes in GCRV isolated to the same genotype

By analysing variable sites, we found the ratios of variation sites located on the third condon and transitions were respectively 71.6% and 82.1% in vp4 gene; 57.1% and 80.0% in vp6 gene; 77.3% and 89.3% in vp7 gene.

Discussion

Amongst all aquareovirus isolates, GCRV is one of the most pathogenic agents (Fang et al. 2002). GCRV can cause fatal epidemics of haemorrhagic disease in grass carp, and affects approximately 85% of fingerling and yearling populations (Jiang and Ahne 1989). Many GCRV isolates have been isolated in recent years, and various of them have been reported to exhibit distinctive differences in virulence (Fang et al. 2002). Moreover, new GCRV isolates were found constantly. In this study, GCRV 096 is a new GCRV isolate similar to GCRV 873, GCRV 875, GCRV 876, GCRV 991 and GCRV JX01.

In order to analyse the difference among GCRV isolates as well as their evolutionary relatiohship, it is necessary to genotyping. Currently, uniform criteria in place for virus genotyping are still unavailable. In hepatitis C virus, a more than 30% nucleotide sequence divergence between genotypes is generally considered standard (Simmonds 2004). The genetic heterogeneity among genotypes of hepatitis E virus has been shown to be more than 20% (Schlauder and Mushahwar 2001). In GCRV, relatively conservative vp4, vp6 and vp7 gene encode major outer capsid proteins and consist of many variable sites (Rangel et al. 1999). So, vp4, vp6 and vp7 gene could be used for GCRV genotyping.

The genetic distances among GCRV 096, GCRV JX01, GCRV 873, GCRV 875, GCRV 876 and GCRV 991 were small with high homologous rates. Furthermore, these isolates clustered together into one cluster on constructed phylogenetic trees. These results present that GCRV 096, GCRV JX01, GCRV 873, GCRV 875, GCRV 876 and GCRV 991 are attributed to the same genotype, i.e. genotype І. Genetic distances between AGCRV PB01-155 and AGVRV were small and their homologous rates were also high. On phylogenetic trees, AGCRV and AGCRV PB01-155 separately clustered into one cluster. These results indicate that AGCRV and AGCRV PB01-155 are attributed to a new genotype, i.e. genotype II. Genetic distances among GCRV HZ08, GCRV GD108, GCRV 918, GCRV HuNan794, GCRV HeNan988, GCRV 106, GCRV ZS11, GCRV QC11, GCRV HN12, GCRV HS11, GCRV YX11, GCRV JS12, GCRV QY12, GCRV JX02, and GCRV 097 were extremely small with especially high homologous rates. Furthermore, these isolates clustered together into one cluster on phylogenetic trees. GCRV HZ08, GCRV GD108, GCRV 918, GCRV HuNan794, GCRV HeNan988, GCRV 106, GCRV ZS11, GCRV QC11, GCRV HN12, GCRV HS11, GCRV YX11, GCRV JS12, GCRV QY12, GCRV JX02, and GCRV 097 were attributed to another new genotype, i.e. genotype III. In contrast, genetic distances between GCRV 104 and other GCRV isolates were large, and their homologous rates were small. On the phylogenetic tree (Figure 4), GCRV 104 separately clustered into one cluster. GCRV 104 is attributed to a new genotype, i.e. genotype IV.

The genotyping results obtained are consistent with previous research conclusions. The study of Wang indicated there were different genotypes of GCRV in China (Wang et al. 2012a). The biological characteristics of GCRV isolates belonging to the same genotype indicated they were analogous. For example, in an artificial infection test, GCRV HZ08 and GCRV GD108 can cause mortality of 60–80% of the yearly grass carp (approx. 10 cm in length), without obvious CPE in CIK cells (Ye et al. 2012; Zhang et al. 2010b). However, American grass carp reovirus (AGCRV) is not strongly connected with infectious disease in fish, although it is commonly detected by cell culture during routine inspections of healthy fish (Goodwin et al. 2010). GCRV 873, GCRV 096, GCRV 875, GCRV 876, GCRV 991 and GCRV JX01 can arouse significant CPE in CIK cells (Zhang et al. 2010a; Wang et al. 2012b). Furthermore, other characteristics of these two isolates were also similar. The genomic segments pattern of GCRV 875 was found to be similar to that of GCRV 873 (Fang et al. 2002). Polyacrylamide gel electrophoresis atlases of GCRV 873, GCRV 875, GCRV 876 and GCRV 991 were also the same (Fang et al. 2002).

The comparative analysis of the geographic location (Table 1) of collected GCRV isolates together with the difference between GCRV isolates and GCRV genotyping indicated there was no obvious relationship between the evolution of GCRV and geographical distribution of GCRV. In the same genotype, the ratios of variation sites on the third condon and the transitions in vp4, vp6 and vp7 gene were high.

Hemorragic disease of grass carp outbreaks seriously in China. Many isolates of grass carp reovirus have been discovered while new isolates are being isolated constantly. The systematic difference comparison of the different GCRV isolates has not been reported. In this study, we have verified the diference among various GCRV genotypes. GCRV genotyping has important significance to diagnosis and treatment in hemorrhagic disease of grass carp, especially to vaccine development. Comparison of different GCRV isolates and genotyping are helpful to further our understanding in GCRV genetic variation and evolution and the development of more effective preventative strategies against GCRV.

This study provides a foundation for revealing differences among GCRV isolates. Simultaneously, it is significant for the further research on genetically engineered vaccines against grass carp haemorrhagic disease and grass carp breeding for disease resistance.

Abbreviations

bp: 

Base pairs

CIK: 

Grass carp kidney cell line

CPE: 

Cytopathic effect

dNTPs: 

Deoxynucleotide triphosphates

FBS: 

Fetal bovine serum

GCRV: 

Grass carp reovirus

MgCl2: 

Magnesim chloride

min: 

Minute

ML: 

Maximum likelihood

MP: 

Maximum parsimony

PBS: 

Phosphate-buffered saline

PCR: 

Polymerase chain reaction

RT-PCR: 

Reverse transcription-PCR

TBR: 

Tree-bisection-recorrnection.

Declarations

Acknowledgements

The study was supported by the National 973 Plan Project in China (No. 2009CB118704). The authors would like to thank Dr. JY Shen for her help.

Authors’ Affiliations

(1)
Guangdong Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Guangdong Ocean University
(2)
Zhongkai University of Agriculture and Engineering

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© Yan et al.; licensee Springer. 2014

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