Skip to main content
PMC home page
SpringerPlus logoLink to SpringerPlus
. 2014 Apr 15;3:190. doi: 10.1186/2193-1801-3-190

Phylogenetic analysis of newly isolated grass carp reovirus

Xiu-ying Yan 1, Ya Wang 1, Ling-fang Xiong 1, Ji-chang Jian 1,✉, Zao-he Wu 2
PMCID: PMC4021032  PMID: 24834372

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), Identification, Phylogenetic relationship, Genotype

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
Open in a new tab

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.

Figure 1

Open in a new tab

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
Open in a new tab

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
Open in a new tab

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.

Figure 2

Open in a new tab

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.

Figure 3

Open in a new tab

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.

Figure 4

Open in a new tab

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.

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.

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.

Footnotes

Competing interests

The authors declare that they have no competing interests. The authors alone are responsible for the content and writing of the paper.

Authors’ contributions

YXY, JJC and WZH conceived and designed the experiments described in this work and wrote the manuscript. YXY, WY and XLF performed and analyzed the data. JJC and WZH supervised the work and analyzed the results. All the authors read and approved the final manuscript.

Contributor Information

Xiu-ying Yan, Email: yanxiuying1201@126.com.

Ya Wang, Email: yawang_1688@126.com.

Ling-fang Xiong, Email: xlf530@126.com.

Ji-chang Jian, Email: jianjc@gmail.com.

Zao-he Wu, Email: wuzaohe@163.com.

References

  1. Chen YS, Jiang YL. Morphology and physical and chemical properties of grass carp hemorrhage virus. KeXue Tong-Bao. 1983;18:1138–1140. [Google Scholar]
  2. Zuo WG, Qian HX, Xu YF, Du SY, Yang XL. A cell line derived from the kidney of grass carp (ctenopharyngodon idellus) J Fish China. 1986;10(1):11–17. [Google Scholar]
  3. Lu YA, Lannan CN, Rohovec JS, Fryer JL. Fish cell lines: establishment and characterization of three new cell lines from grass carp (Ctenopharyngodon idella) Vitro Cell Dev Biol. 1990;26(3 Pt 1):275–279. doi: 10.1007/BF02624457. [DOI] [PubMed] [Google Scholar]
  4. Fang Q, Xiao TY, Li L, Zou GP, Zhang HY, Wang YP. Infection Characterizations of four Grass Carp Reovirus (GCRV) strains. Virol Sin. 2002;17:182–184. [Google Scholar]
  5. Chi YY, Tian YY, Ye X, Deng GC, Li J, Wang HJ. Molecular properties of grass carp reovirus in southern China and establishment of duplex PCR detection method. Bing Du Xue Bao. 2011;27(4):358–365. [PubMed] [Google Scholar]
  6. Ye X, Tian YY, Deng GC, Chi YY, Jiang XY. Complete genomic sequence of a reovirus isolated from grass carp in China. Virus Res. 2012;163(1):275–283. doi: 10.1016/j.virusres.2011.10.014. [DOI] [PubMed] [Google Scholar]
  7. Zeng WW, Wang Q, Wang YY, Xu DH, Wu SQ. A one-step molecular biology method for simple and rapid detection of grass carpCtenopharyngodon idella reovirus (GCRV) HZ08 strain. J Fish Biol. 2013;82(5):1545–1555. doi: 10.1111/jfb.12088. [DOI] [PubMed] [Google Scholar]
  8. Mohd Jaafar F, Goodwin AE, Belhouchet M, Merry G, Fang Q, Cantaloube JF, Biagini P, de Micco P, Mertens PPC, Attoui H. Complete characterisation of the American grass carp reovirus genome (genus Aquareovirus: family Reoviridae) reveals an evolutionary link between aquareoviruses and coltiviruses. Virology. 2008;373(2):310–321. doi: 10.1016/j.virol.2007.12.006. [DOI] [PubMed] [Google Scholar]
  9. Zhang LL, Luo Q, Fang Q, Wang YP. An improved RT-PCR assay for rapid and sensitive detection of grass carp reovirus. J Virol Methods. 2010a;169(1):28–33. doi: 10.1016/j.jviromet.2010.06.009. [DOI] [PubMed] [Google Scholar]
  10. Rangel AA, Rockemann DD, Hetrick FM, Samal SK. Identification of grass carp haemorrhage virus as a new genogroup of aquareovirus. J Gen Virol. 1999;80(9):2399–2402. doi: 10.1099/0022-1317-80-9-2399. [DOI] [PubMed] [Google Scholar]
  11. Fang Q, Attoui H, Cantaloube JF, Biagini P, Zhu ZY, De Micco P, De Lamballerie X. Sequence of genome segments 1, 2, and 3 of the grass carp reovirus (Genus Aquareovirus, Family Reoviridae) Biochem Biophys Res Commun. 2000;274(3):762–766. doi: 10.1006/bbrc.2000.3215. [DOI] [PubMed] [Google Scholar]
  12. Su JG, Huang T, Dong J, Heng JF, Zhang RF, Peng LM. Molecular cloning and immune responsive expression of MDA5 gene, a pivotal member of the RLR gene family from grass carp Ctenopharyngodon idella. Fish Shellfish Immun. 2010;28(4):712–718. doi: 10.1016/j.fsi.2010.01.009. [DOI] [PubMed] [Google Scholar]
  13. Attoui H, Fang Q, Mohd Jaafar F, Cantaloube JF, Biagini P, de Micco P, de Lamballerie X. Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus (genus Aquareovirus, family Reoviridae) J Gen Virol. 2002;83(8):1941–1951. doi: 10.1099/0022-1317-83-8-1941. [DOI] [PubMed] [Google Scholar]
  14. Fan C, Shao L, Fang Q. Characterization of the nonstructural protein NS80 of grass carp reovirus. Arch Virol. 2010;155(11):1755–1763. doi: 10.1007/s00705-010-0753-6. [DOI] [PubMed] [Google Scholar]
  15. Zhang C, Wang Q, Shi CB, Zeng WW, Liu Y, Wu SQ. Molecular analysis of grass carp reovirus HZ08 genome segments 1-3 and 5-6. Virus Genes. 2010b;41(1):102–104. doi: 10.1007/s11262-010-0489-0. [DOI] [PubMed] [Google Scholar]
  16. Ma J, Wang W, Zeng L, Fan Y, Xu J, Zhou Y. Inhibition of the replication of grass carp reovirus in CIK cells with plasmid-transcribed shRNAs. J Virol Methods. 2011;175(2):182–187. doi: 10.1016/j.jviromet.2011.05.008. [DOI] [PubMed] [Google Scholar]
  17. Fang Q, Ke LH, Cai YQ. Growth characteristics and high titer culture of grass carp hemorrhage virus (GCHV)-873 in vitro. Virol Sinion. 1989;3:314–319. [Google Scholar]
  18. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24(8):1596–1599. doi: 10.1093/molbev/msm092. [DOI] [PubMed] [Google Scholar]
  19. Rozas J, Rozas R. DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics. 1999;15(2):174–175. doi: 10.1093/bioinformatics/15.2.174. [DOI] [PubMed] [Google Scholar]
  20. Posada D, Crandall K. Modeltest: testing the model of DNA substitution. Bioinformatics. 1998;14(9):817–818. doi: 10.1093/bioinformatics/14.9.817. [DOI] [PubMed] [Google Scholar]
  21. Swofford DL. PAUP: Phylogenetic analysis using parsimony and other methods Associates. 1998. [Google Scholar]
  22. Huelsenbeck JP, Ronquist F. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics. 2001;17(8):754–755. doi: 10.1093/bioinformatics/17.8.754. [DOI] [PubMed] [Google Scholar]
  23. Ke LH, Fang Q, Cai YQ. Characteristics of a novel isolate of grass carp hemorrhagic virus. ACTA Hydrobiol Sin. 1990;14(2):153–159. [Google Scholar]
  24. Jiang Y, Ahne W. Some properties of the etiological agent of the hemorrhagic disease of grass carp and black carp. In: Ahne W, Kurstak E, editors. Viruses of Lower Vertebrates. Berlin: Springer-Verlag; 1989. pp. 227–239. [Google Scholar]
  25. Simmonds P. Genetic diversity and evolution of hepatitis C virus–15 years on. J Gen Virol. 2004;85(11):3173–3188. doi: 10.1099/vir.0.80401-0. [DOI] [PubMed] [Google Scholar]
  26. Schlauder GG, Mushahwar IK. Genetic heterogeneity of hepatitis E virus. J Med Virol. 2001;65(2):282–292. doi: 10.1002/jmv.2031. [DOI] [PubMed] [Google Scholar]
  27. Wang Q, Zeng WW, Liu C, Zhang C, Wang YY, Shi CB, Wu SQ. Complete genome sequence of a reovirus isolated from grass carp, indicating different genotype of GCRV in China. J Virol. 2012a;86(22):12466. doi: 10.1128/JVI.02333-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Goodwin AE, Merry GE, Attoui H. Detection and prevalence of the nonsyncytial American grass carp reovirus Aquareovirus G by quantitative reverse transcriptase polymerase chain reaction. J Aquat Anim Health. 2010;22(1):8–13. doi: 10.1577/H09-025.1. [DOI] [PubMed] [Google Scholar]
  29. Wang T, Xu D, Lv LQ. Detection of the co-infection of different grass carp reovirus strains using dsRNA sequencing technology. J Shanghai Ocean Univ. 2012;21(5):756–762. [Google Scholar]

Articles from SpringerPlus are provided here courtesy of Springer-Verlag

RESOURCES