1 French Food Safety Agency, Fish Infectious Diseases and Parasitology Unit, BP 70, F-29 280 Plouzané, France
2 French Food Safety Agency, Viral Genetics and Biosafety Unit, BP 53, F-22 440 Ploufragan, France
Correspondence
Richard Thiéry
r.thiery{at}brest.afssa.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ698093AJ698113.
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INTRODUCTION |
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Betanodaviruses are small, spherical, non-enveloped viruses with a genome that is composed of two single-stranded, positive-sense RNA molecules. The larger genomic segment, RNA1 (3·1 kbp), encodes the RNA-dependent RNA polymerase (Chi et al., 2001; Nagai & Nishizawa, 1999
; Tan et al., 2001
), whilst the coat protein is encoded by RNA 2 (1·4 kbp) (Delsert et al., 1997
; Nishizawa et al., 1994
).
Comparison of the coat protein gene of five fish nodaviruses identified a highly conserved region (aa 83216) and a variable region (aa 235316) with amino acid sequence identities of 93 and 62 %, respectively (Nishizawa et al., 1995). A classification of betanodaviruses based on comparison of the variable region of the coat protein gene among 25 isolates from farmed fish from Japan, Thailand, Italy and Australia was proposed by the same group (Nishizawa et al., 1997
). According to this, betanodaviruses could be classified into four types, designated striped jack nervous necrosis virus (SJNNV), barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV) and red-spotted grouper nervous necrosis virus (RGNNV). The same classification was subsequently used by several groups. New fish nodavirus isolates from Europe and Asia were characterized and the majority of the isolates were classified as the RGNNV type (Skliris et al., 2001
). Another study of nodaviruses collected from fish farms in the Mediterranean basin has classified all isolates as the RGNNV type, whatever fish species they originated from (European sea bass, Dicentrarchus labrax, and shi drum, Umbrina cirrosa; Dalla Valle et al., 2001
). Recently, all betanodaviruses isolated from aquatic organisms in Taiwan have also been classified as the RGNNV type (Chi et al., 2003
). On the other hand, betanodaviruses isolated from cold-water fish species [e.g. Atlantic halibut (Hippoglossus hippoglossus) and Atlantic cod (Gadus morhua) from Canada and the UK, and Dover sole (Solea solea)] were classified as the BFNNV type (Aspehaug et al., 1999
; Johnson et al., 2002
; Starkey et al., 2000
).
Previously, we reported that two isolates from European sea bass had distinct genomes (Thiéry et al., 1999). The phylogenetic classification of one of these isolates, obtained from fish farmed on the Atlantic coast of France, was uncertain, as it segregated as the earliest branch in the RGNNV group in several studies (Aspehaug et al., 1999
; Dalla Valle et al., 2001
, R. Thiéry, unpublished observations).
In the present study, we have classified 21 new isolates obtained from various fish species farmed in France or the Mediterranean basin or from wild fish caught in Tahiti. Phylogenetic analysis of the variable region of the coat protein indicated that the new isolates could be classified into four main types or subtypes. For the first time, one isolate obtained from sea bass was classified as the BFNNV type, whereas an isolate from Senegalese sole (Solea senagalensis) farmed in Spain was classified as the SJNNV type, which was previously found only in Japan. Grouping of the isolates mainly correlated with their geographical origin rather than the fish species from which they were obtained, and a new classification nomenclature is suggested.
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METHODS |
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RT-PCR.
Aliquots (3 µl) of total RNA samples were subjected to RT-PCR as described previously (Thiéry et al., 1999), using primer sets that allowed amplification of the variable part of the coat protein gene, also referred to as the T4 region (Nishizawa et al., 1994
). Some RT-PCRs were also performed by using Ready-to-go RT-PCR beads (Amersham Biosciences) with an uninterrupted one-step protocol, following the manufacturer's instructions. After amplification, PCR products (420 bp) were analysed by electrophoresis on a 2 % agarose gel and stained with ethidium bromide. The entire coat protein gene sequences of two isolates (V26 and BB09) were also determined after cloning by using specific primers. For isolate V26, primers Vac1 and Vac2, which contain restriction sites, were used (Table 2
). The amplified product was then cloned into pcDNAI (Invitrogen). For isolate BB09, several primers (Table 2
) were designed according to the sequence of Atlantic halibut nodavirus (strain AH95NorA) (AH95NorA; GenBank accession no. AJ245641) and used to amplify different portions of the gene, which were cloned into pCRII-TOPO (Invitrogen).
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After exclusion of the primer sequences, nucleotide sequences were aligned by using CLUSTAL_X (Thompson et al., 1997) or PILEUP as implemented in the GCG Wisconsin Package version 10.3 (Accelrys). The multiple nucleotide sequence alignment was inspected and finally edited manually by using GENEDOC (www.psc.edu/biomed/genedoc). Phylogenetic trees were inferred by several methods using PHYLOWIN (Galtier et al., 1996
) or the evolutionary programs implemented in GCG. Distance-based trees were constructed by using the neighbour-joining algorithm (Saitou & Nei, 1987
) and 1000 bootstrap resamplings. Maximum-parsimony analysis was also performed by using the heuristic tree search option and 100 bootstrap resamplings with a PAUP search (GCG). Maximum likelihood analysis was performed by using a PAUP search with the nucleotide frequencies set to: fA=0·22, fC=0·303 and fG=0·237; the transition/transversion ratio set to 1; and the following exchange rates: fAC=1, fAG=2·96, fAT=1, fCG=1 and fCT=4·92. The trees were printed by using the TREEVIEW program (Page, 1996
).
Mean similarities between the sequences of the betanodaviruses were determined by using the PLOTSIMILARITY program of the GCG Wisconsin Package version 10.3.
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RESULTS |
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Cluster Ib contained French isolates only. Strain Y235 is the same strain that was reported previously as DlEV (Delsert et al., 1997), SBNNV-Atl (Thiéry et al., 1999
) and SB1 (Breuil et al., 2001
) and was obtained in 1991 from sea bass that was cultured on the Atlantic coast of France. Another isolate obtained 3 years later in the same farm was highly related (V67). Strains X199 and Y55 were obtained from meagre and sea bass, respectively, grown on a different farm. Several isolates had the same nucleotide sequence as Y55 (X82, X137, X149, V68, W62, Y154 and Y193) and were either obtained from the same farm or from different farms that exchanged live fish.
Cluster II contained several isolates that were obtained from cold-water species: Atlantic halibut (Norway and Scotland), Atlantic cod (Canada) and barfin flounder (Japan). Surprisingly, strain BB09 obtained from sea bass in France during a VER outbreak that occurred at a temperature of about 15 °C and was associated with high mortality also clustered into this genotype. To our knowledge, this is the first description of such a nodavirus in European sea bass. Several subtypes were clearly defined within cluster II and correlated with the geographical origin of the isolates. Subtype IIa contained the Atlantic cod strain from Canada (AF445800). Subtype IIb contained a barfin flounder isolate from Japan. Subtype IIc contained European isolates that were obtained from different fish species. These subtypes were supported by high bootstrap values. Cluster II corresponded to the BFNNV type (Nishizawa et al., 1997).
Cluster III was represented here by a single isolate, TPNNV, obtained from tiger puffer in Japan and is the type species of the TPNNV type (Nishizawa et al., 1997).
Cluster IV (SJNNV type) contained strains that were isolated from Japanese striped jack (Nishizawa et al., 1997; Skliris et al., 2001
). Surprisingly, strain 03-160, obtained from Senegalese sole (Spain), also clustered within this genotype and is the first strain belonging to this genotype to be found in Europe. This strain was associated with mortality at 30 °C.
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DISCUSSION |
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The phylogeny among 21 new betanodavirus isolates was subsequently studied by using a smaller region of the coat protein gene, which was shown to be highly variable, in agreement with Nishizawa et al. (1995). Comparison of the sequences of these isolates with other sequences available in GenBank and phylogenetic analysis indicated that they clustered into four types or subtypes. Previously, we reported that farmed sea bass could be infected by two genetically distinct betanodavirus strains (Thiéry et al., 1999
). One of these strains was also isolated from asymptomatic sea bream, whereas it was associated with mortality in sea bass (Castric et al., 2001
). In the present work, a third genetically distinct strain (BB09) causing mortality in sea bass at low temperature was characterized. It clustered into a distinct group (II), which contained isolates that were shown to induce the disease in cold-water fish species in several geographical areas (Japan, Norway, the UK and Canada). It was related closely to the other European isolates from this cluster described so far, here designated IIc. Although all strains from cluster II have a common ancestor, the distinct subtypes correlated strongly with their geographical origins, indicating that they subsequently evolved independently. Fish infected by strain BB09 were the progeny of brood stock that was caught in the wild with no history of the disease at higher temperatures. They suffered from encephalopathy and retinopathy when a sudden drop in temperature occurred accidentally in the premises. As they were not in contact with other farmed cold-water species, the infection presumably occurred in the wild in the brood stock and was subsequently transmitted vertically. This would concur with the observation that vertical transmission of the disease can occur in sea bass at low temperature (15 °C; Breuil et al., 2002
).
A strain infecting farmed Senegalese sole in Spain (in the Mediterranean Sea) was detected and assigned to cluster IV (SJNNV). This is the first description of this genotype in Europe, as it has only been described previously in farmed fish from Japan: striped jack and red sea bream (Nishizawa et al., 1997). According to the Fish Base (www.fishbase.com), the natural area of distribution of Senegalese sole ranges from the Bay of Biscay to the coastal waters of Senegal and the Canary Islands; thus, there is a risk of dissemination of this strain to other farmed fish species. However, the sequence of this virus was distinct from the virus sequences from Japan, suggesting that a different strain belonging to this genotype circulates in eastern Atlantic waters. It should be noticed that the Senegalese sole sample was considered to be nodavirus-free by using a cell-culture assay (Frerichs et al., 1996
) followed by an indirect fluorescent-antibody test (IFAT) using a polyclonal antibody raised against sea bass isolates (J. Castric, personal communication). Thus, the evidence of a nodavirus infection came from the sequence of PCR products obtained after amplification of RNA samples extracted from fish tissues. The reason why no virus could be detected using cell culture and IFAT is not completely understood. It is likely that the virus was present in high quantities, as mortality was severe. It was reported that IFAT using a rabbit polyclonal serum raised against SJNNV was able to detect nodavirus in sections of virus-infected barramundi larvae (Munday et al., 1994
), demonstrating some antigenic similarity between the two viruses. However, the same serum used in ELISA is only specific for SJNNV (OIE, 1995
) and several nodavirus isolates were neutralized by a polyclonal antibody raised against a nodavirus isolated from sea bass with the exception of one isolate obtained from striped jack (Skliris et al., 2001
). Taken together, these data suggest that antigenic differences among nodavirus strains may influence antibody recognition in diagnostic procedures.
Most of the isolates clustered into group I, previously designated RGNNV. However, this cluster could be divided into two subtypes with good support values. It was particularly interesting that subtype Ib only contained isolates that were obtained from fish species raised in France. These strains were obtained from three different fish species (sea bass, shi drum and meagre), which suggested that this subtype may also spread to several fish species. Before this study, only one strain belonging to this subtype had been described (Delsert et al., 1997; Thiéry et al., 1999
), originating from a sea bass farm located on the French Atlantic coast and corresponding to strain Y235 described here. Thus, we have now provided evidence that this subtype is distributed among several French farms. Previous phylogenetic studies including this strain have classified it into the RGNNV type (Aspehaug et al., 1999
; Dalla Valle et al., 2001
), as have we. Interestingly, strains V26 and Y235, belonging to clusters Ia and Ib, respectively, showed distinct pathogenicity to sea bass larvae (Breuil et al., 2001
). Moreover, strain Y235 could be transmitted from experimentally infected sea bass females to the eggs and larvae, whereas strain V26 could not (Breuil et al., 2002
). The different biological properties probably rely on different temperature optima needed for replication. Thus, it seems that there are sufficient lines of evidence to classify these strains into different subtypes. Whether subtype Ib should be considered as a new genotype depends on the level of identity that is required to define a genotype, which varies considerably from one virus family to another.
The rest of the isolates clustered into group Ia and tended to segregate on different sub-branches according to their geographical origin, although bootstrap values were low. A noticeable exception was that strains from Tunisia grouped with strains obtained from wild fish species caught in Tahiti. Four isolates from France (V26, W80, V113 and X130) were in the same subgroup as those obtained from sea bass in Spain, Italy and Greece. All of these strains were highly related to strain RGNNV (98 % nucleotide sequence identities), as reported previously (Nishizawa et al., 1997; Sideris, 1997
; Thiéry et al., 1999
). The sequence of three isolates obtained from two different fish species from the same site were identical (V26, W80 and V113), but different from that of strain X130, which was obtained from another species (meagre) and another site. Interestingly, strain X130 belonged to a different subtype from the other isolates found in the same site, which all gathered in cluster Ib. These data suggested that at least two different viral introductions occurred at the farm. All other isolates from cluster Ia were obtained in fish species raised in countries from south-east Asia: grouper species from Taiwan (Lin et al., 2001
) and Singapore (Hegde et al., 2002
), barramundi (Lates calcarifer) from Thailand (Skliris et al., 2001
) and the recently reported guppy nervous necrosis virus (GNNV) strain from guppy (Poecilia reticulata) in Singapore (Hegde et al., 2003
).
Overall, the topology of the phylogenetic trees obtained in this study was similar to those obtained in previous studies on betanodaviruses using varying lengths of nucleotide sequences from the same gene (Dalla Valle et al., 2001; Nishizawa et al., 1997
; Skliris et al., 2001
). Thus, the T4 coat protein gene region (Nishizawa et al., 1997
) is sufficiently informative to assess phylogenetic relationships among betanodaviruses. Nevertheless, it should be noted that several isolates that were obtained at an interval of several years from fish grown at the same site had the same sequence. This suggests that the coat protein gene has a low evolutionary rate. On the other hand, some isolates obtained from different farm sites shared the same T4 nucleotide sequence: Y55 (site c), X137 and Y193 (site d), and X149 (site e). Commercial exchange between these sites could explain this observation. Thus, comparison of the T4 region of the coat protein gene between isolates could also provide an interesting epidemiological tool to investigate the origin of an infection.
At present, all reported phylogenetic studies on betanodaviruses have used sequences derived from the coat protein gene. The genome of nodaviruses is composed of two single-stranded RNAs that encode the RNA-dependent RNA polymerase and the coat protein. Available sequences derived from the RNA polymerase gene are still scarce (Nagai & Nishizawa, 1999; Tan et al., 2001
), but further characterization of this gene for an increasing number of isolates will provide complementary data to assess the evolutionary relationships among betanodaviruses in greater detail.
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ACKNOWLEDGEMENTS |
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Received 7 May 2004;
accepted 5 July 2004.
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