Molecular characterization and genogrouping of VP1 of aquatic birnavirus GC1 isolated from rockfish Sebastes schlegeli in Korea.

The cDNA nucleotide sequence of genome segment B encoding the VP1 protein was determined for the aquatic birnavirus GC1 isolated from the rockfish Sebastes schlegeli in Korea. The VP1 protein of GC1 contains a 2,538 bp open reading frame, which encodes a protein comprising 846 amino acid residues that has a predicted MW of 94 kDa. The sequence contains 6 potential Asn-X-Ser/Thr motifs. Eight potential Ser phosphorylation sites and 1 potential Tyr phophorylation site were also identified. GC1 contains the Leu-Lys-Asn (LKN) motif instead of the typical Gly-Asp-Asp (GDD) motif found in other aquatic birnaviruses. We also identified the GLPYIGKT motif, the putative GTPbinding site at amino acid position 248. In total, the VP1 regions of 22 birnavirus strains were compared for analyzing the genetic relationship among the family Birnaviridae. Based on the deduced amino acid sequences, GC1 was observed to be more closely related to the infectious pancreatic necrosis virus (IPNV) from the USA, Japan, and Korea than the IPNV from Europe. Further, aquatic birnaviruses containing GC1 and IPNV have genogroups that are distinct from those in the genus Avibirnaviruses and Entomo-birnaviruses. The birnavirusstrains were clustered into 5 genogroups based on their amino acid sequences. The marine aquatic birnaviruses (MABVs) containing GC1 were included in the MABV genogroup; the IPNV strains isolated from Korea, Japan, and the USA were included in genogroup 1 and the IPNV strains isolated primarily from Europe were included in genogroup 2. Avibirnaviruses and entomobirnaviruses were included in genogroup 3 and 4, respectively.

Introduction

Members of the family Birnaviridae have 2-segmented genomes - A and B. This family comprises 3 main genera, including the <span class="Disease">genus Aquabirnavirus, Avibirnavirus, and Entomobirnavirus [4,19]. The type species of the <span class="Disease">genus Aquabirnavirus is the infectious pancreatic necrosis virus (IPNV); the genus comprises marine aquatic birnaviruses (MABV) of fish and shellfish [3]. Other members of the family Birnaviridae include infectious bursal disease virus (IBDV) belongs to the genus Avibirnavirus, and Drosophila X virus (DVX) that belongs to the genus Entomobirnavirus. Aquatic birnaviruses are the largest and most diverse group of viruses within the family Birnaviridae. The first reported MABV was isolated from the yellowtail Seriola quinqueradiatain Japan [22], other MABVs have been subsequently isolated from various marine fishes in Korea and Japan, and their characteristics have been investigated [7,8,14,18,23,24]. The genome segment B of birnaviruses encodes the VP1 protein, which is the presumptive virion-associated RNA-dependent RNA polymerase (RdRp) [13,15]. Some researchers reported the characteristics of VP1 and compared the VP1 region among birnaviruses [4,25]. They identified several conserved domains associated with RdRps and GTP-binding proteinsin the IPNV strains; these domains were the same as those in other RNA viruses. However, they also discovered that the typical Gly-Asp-Asp (GDD) motif that is found in all RNA viruses was absent in the VP1 region of some IPNV [4] IBDV, and DXV [2] strains.

The physical, antigenic, and genetic features of the VP2/NS junction region of the aquatic birnavirus <span class="Gene">GC1 isolated from the rockfish <span class="Species">Sebastes (S.) schlegeli, which is the second most important in the aquaculture industry in Korea, has been studied [8,9,20].

In the present study, we investigated the genetic characteristics of the VP1 protein and compared the genetic relationship between aquatic birnaviruses and other genuses within family Birnaviridae.

Materials and Methods

Virus and cell

<span class="Gene">GC1 was isolated from the rockfish <span class="Species">S. schlegeli, and it was grown in the Chinook Salmon Embryo-214 cell line supplemented with Eagle's minimum essential medium. The sources of VP1 sequence cited in this study are listed in Table 1.

Table 1

Descriptions of VP1 sequences of aquabirnaviruses cited in this study

Viral RNA extraction and primers

The viral genomic RNA was extracted using the methods described by Joh et al. [8]. Briefly, GC1-infected cells were frozen and thawed 3 times and clarified by centrifugation. Viral dsRNA was then extracted with phenol and chloroform, followed by digestion with proteinase K. Seven primer pairs were used for reverse transcription-polymerase chain reaction (RT-PCR). The oligonucleotide sequences were deduced according to the published dsRNA sequences of the Western Buxton strains (AF078669) (Table 2).

Table 2

RT-PCR primer sets and amplified cDNA fragments used for sequencing

*Map position of the primers based on the published sequence of Western buxton (AF078669).

cDNA synthesis by RT-PCR

The RT-PCR procedure used in this study was a modification of the method previously described by Joh et al. [10]. The RT-PCR solution was heated to 95℃ for 3 min and passed <span class="Chemical">through 35 cycles under the following conditions: 1 min at 95℃ for denaturation; 1 min at 54℃ to -58℃ (depending on the primer) to allow annealing; 1 min at 72℃ for extension and final amplification at 72℃ for 3 min. The <span class="Chemical">ethidium bromide-stained PCR products were electrophoresed on a 1.5% agarose gel and were visualized by UV fluorescence.

Construction of recombinant plasmids

Each resulting product was gel purified and then cloned into pCR2.1 TA cloning vectors (Invitrogen, USA) according to the manufacturer's instructions. All the clones were amplified by transformation into competent DH5 α cells. Clones with correct in<span class="Chemical">serts were confirmed by restriction enzyme digestion of the recombinant vectors.

Nucleotide sequencing and analysis of the VP1

Nucleotide sequencing was carried out on an ABI 377 sequencer (Applied Biosystems, USA) by the <span class="Chemical">dideoxynucleotide chain termination method by using T7 DNA and <span class="Chemical">SP6 DNA polymerase. The nucleotide and deduced amino acid sequences were analyzedby Vector NTI ver 10.0 (Hitachi, Japan) and were compared with the corresponding sequences of previously reported cite accession numbers of aquabirnaviruses in Table 1.

Results

Nucleotide and amino acid sequences of the VP1 protein

The nucleotide sequence of <span class="Gene">GC1 was found to be 2,776 bp long. The VP1 open reading frame (ORF) gene starts at nucleotide 101 and ends with a single TAA termination codon at nucleotide 2,638. The predicted molecular weight of this virus is 94,263 Daltons, and it contains a single large ORF encoding the 846-amino acid VP1 protein. The VP1 sequence starts with the nucleotide sequence 'GGAAA' and contains the inverted terminal repeats 'GGGTCAAGTTGGTGG' and 'GTGCCACCAAC-TGACCC' near the 5' and 3' terminal sequences, respn>ectively.

Characterization of the VP1 protein

The amino acid composition of VP1 was determined. The VP1 amino acid sequence was scanned for several functional motifs, and the results are summarized in Table 3. We ob<span class="Chemical">served that the VP1 sequence contained 6 potential <span class="Chemical">Asn-X-Ser/Thr motifs. These motifs were presumed to contain an N-linked glycosylation site. There were 8 potential Ser phosphorylation sitesand 1 Tyr phophorylation site. The amino acid sequence of VP1 did not contain the GDD motif, which exists commonly in the RdRps of RNA viruses; however, we could identifythe Leu-Lys-Asn (LKN) motif at position 521 (Table 3). Further, we confirmed the 'GLPYIGKT'motif at amino acid position 248; this motif is the putative GTP-binding site that is commonly found in other aquatic birnaviruses.

Table 3

Kinds of potential motif exist in VP1 of GC1

Comparative studies of nucleotide and amino acid sequences of the VP1 protein

On comparing the nucleotide sequences of VP1 in 22 birnavirus strains, it was found that <span class="Gene">GC1 shares 97-98% homology with MABVs; 86% homology with the <span class="Species">IPNV strains of aquabirnaviruses isolated mainly from the USA, Japan, and Korea; 80-82% homology with the IPNV strains of aquabirnaviruses from Spain; 54-56% homology with the avibirnaviruses; and 46% homology with entomobirnaviruses (Table 4). On comparing the amino acid sequence of VP1, it was found that GC1 shares 97-98% homology with MABVs; 94% homology with the IPNV strains of aquabirnaviruses found mainly in the USA, Japan, and Korea; 87-89% homology with the IPNV strains of aquabirnaviruses from Spain; 46-47% homology with the avibirnaviruses; and 29% homology with the entomobirnaviruses (Table 5).

Table 4

Pairwise similarity and distances among the VP1 nucleotide sequences of 22 birnavirus strains

Table 5

Pairwise similarity and distances among the VP1 amino acid sequences of 22 birnavirus strains

Phylogenetic relationships

In the phylogenetic cladograms that were based on both nucleotide and amino acid sequences, the genetic relationships among the 22 birnaviruses were established and the viruses, including <span class="Gene">GC1, were clustered into 5 genogroups that generally correlated with the geographic origin of the viruses and the water environment of the host. The MABV genogroup consisted of strains such as <span class="Gene">GC1 and NC1 from Korea and YT01A, H1, AY98, and Y6 from Japan. Genogroup 1 mostly consisted of strains from the Pacific coastal nations; DRT is from Korea, WB from the USA, Jasper from Canada, and AM98 from Japan. The isolates of 1146, 88R, 20G1, 2290, and 6B1A from Spain and Sp from Denmark comprised genogroup 2. The 2 avibirnaviruses UPM976/61 from Malaysia and CLV from Vietnamformed genogroup 3, and 1 entomobirnavirus, DVX, formed genogroup 4 (Fig. 1).

Fig. 1

Cladogram representing phylogenetic relationships between birnaviruses based on deduced amino acid sequences of VP1. The length of each pair of branches represents the distance between the sequence pairs, and the numbers in parentheses indicate the bootstrap values.

Discussion

The viral B segment encodes VP1, which is approximately 90 kDa in weight [2,11-13]. The estimated molecular weight of VP1 ranges from 95 kDa for the J<span class="Chemical">asper isolate [4] to 89 kDa for the Sp and Ab isolates of <span class="Species">IPNV [6]. The molecular weight of GC1 has been estimated as 94 kDa and has been shown to be similar to that of the Jasper strain.

Some researchers have reported that the sequence GXXXXGKS/T is a constant motif in <span class="Chemical">GTP-binding proteins [1,16] and is observed in several viral proteins that have a tentative role in RNA replication [15]. The same motif was present in the IPNV strains [4] and in GC1 between residues 248 and 255 (GLPYIGKT). We believe that this motif represents a potential GTP-binding site in the VP1 protein, and has been conserved in GC1, including aquatic birnaviruses.

As reported previously [1,5,17], the GDD sequence is a highly conserved motif that is present in almost all putative RdRps. Researchers have found that the Asp-Asp (DD) sequence lacking Gly, is conserved in IBDV, and also that IPNV does not contain the typical GDD motif in the corresponding region of its VP1 [4,21]. Some IPNV strains containedthe Leu-Lys-Asp (LKD) or LKN motifs instead of the typical GDD motif [4]. GC1 contains the LKN motif instead of the typical GDD motif, which is present in other aquatic birnaviruses.

The study of genetic relationships using a phylogenetic cladogram revealed that <span class="Gene">GC1 is more closely related to genogroup 1 than genogroup 2. This result indicatesthat genetic relationships may be influenced by the geographical distributions of the isolates. Aquatic birnaviruses, including <span class="Gene">GC1 and IPNV, also belong to genogroups that are distinct from those of the avibirnaviruses and Entomo-birnaviruses. This result may thus indicate that the genus Birnavirus has evolved in different ways resulting in the formation of distinct genogroups.