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Phylogenetic and antigenic characterization of new fish nodavirus isolates from Europe and Asia
Virus Research 75 (2001) 59 – 67 www.elsevier.com/locate/virusres
Phylogenetic and antigenic characterization of new fish nodavirus isolates from Europe and Asia George P. Skliris a, John V. Krondiris b, Diamantis C. Sideris b, Andrew P. Shinn a, William G. Starkey a, Randolph H. Richards a,* b
a Virology Department, The Institute of Aquaculture, Uni6ersity of Stirling, Scotland FK9 4LA, UK Uni6ersity of Athens, School of Biology, Department of Biochemistry and Molecular Biology, Panepistimioupolis, 15701 Athens, Greece
Received 19 September 2000; received in revised form 29 December 2000; accepted 5 January 2001
Abstract Nodaviruses are widespread causative agents of viral nervous necrosis in fish. Based on the coat protein sequence, fish nodaviruses are categorized into four different genotypes. In this study, we present data on the phylogenetic and antigenic characterization of 12 new isolates, eight European and four of Asian origin, from farmed and wild species of fish. Phylogenetic analysis based on the nucleotide sequence (688 bases) of the coat protein classified the majority of these new isolates to the RGNNV genotype. Geographic or host-species specificities were not revealed by this study. Neutralizing assay experiments, further confirmed the genotypic classification, supporting the possibility that the different nodavirus genotypes can also be serologically distinguishable. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Fish nodavirus; Nervous necrosis virus; Viral encephalopathy
1. Introduction The nodaviridae is a family of small, non-enveloped, icosahedral RNA viruses, which infect a wide range of insects and fish (Kaesbergh, 1987; Mori et al., 1992). Fish nodaviruses belonging to the genus betanodavirus are the causative agents of viral nervous necrosis (VNN), also referred to
* Corresponding author. Tel.: + 44-1786-467873; fax: +441786-472133. E-mail address: [email protected] (R.H. Richards).
as viral encephalopathy and retinopathy (VER), and fish encephalitis. This disease is characterised by abnormal swimming behaviour, and vacuolating lesions within the brain and retinal tissue of affected fish. VNN has been associated with high mortalities in several marine fish species of economic importance to the aquaculture industry in Europe, Asia, Japan, and Australia (Nakai et al., 1994; Munday and Nakai 1997; Skliris and Richards 1999). European sea bass production has been seriously affected by VNN, with significant losses occurring in stocks of juvenile and ongrowing fish (Le Breton et al., 1996).
0168-1702/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 0 1 ) 0 0 2 2 5 - 8
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The genome of fish nodaviruses is bipartite, comprising two molecules of single stranded, positive polarity RNA (Mori et al., 1992; Comps et al., 1994). RNA 1 (1.01 ×106 Da) encodes a non-structural protein of approximately 110 kDa (Nagai and Nishizawa 1999), while RNA 2 ( 0.49 ×106 Da) encodes the 42 kDa coat protein precursor. Both RNA molecules are nonpolyadenylated (Mori et al., 1992). The coat protein gene of a striped jack nervous necrosis virus (SJNNV) is 1410 nucleotides in length, containing an open reading frame of 1023 nucleotides, encoding a protein of 340 amino acids, and 5% and 3%-non-coding regions of 16 and 371 nucleotides respectively (Nishizawa et al., 1995). A potential neutralizing determinant has been mapped to amino acid residues 254 – 256 of the coat protein of SJNNV (Nishizawa et al., 1999). Analysis of the genetic variation exhibited by fish nodaviruses is required for the rational development of effective vaccines and diagnostic reagents. The nucleotide sequence homology between the coat protein genes of insect and fish nodaviruses have previously been investigated (Nishizawa et al., 1995). Based on partial sequences of the coat protein gene fish nodaviruses have been classified into four clusters: striped jack nervous necrosis virus (SJNNV), tiger puffer jack nervous necrosis virus (TPNNV), barfin flounder nervous necrosis virus (BFNNV) and red-spotted
grouper nervous necrosis virus (RGNNV) (Nishizawa et al., 1997). However, these studies have predominantly focused on Japanese nodavirus isolates. In this report, we have studied nucleotide sequence and serological relationships of a collection of nodaviruses isolated from Europe, Asia, and Japan. This collection included eight isolates from Sea Bass obtained from European fish farms. Phylogenetic and serological analysis was performed to characterise the relationships between the nodavirus isolates studied in this report, and other previously characterised betanodaviruses.
2. Materials and methods
2.1. Virus isolation and propagation The source and date of isolation of the nodaviruses studied in this report are listed in Table 1. All viruses were isolated from farmed species, with the exception of It/24/Sdr, isolated from Shi Drum (Umbrina cirrosa), which is a wild species. Viruses were propagated in SSN-1 cells, (derived from Striped Snakehead, Channa striatus), grown in Leibovitz L-15 medium (Gibco BRL, UK) containing 5% foetal bovine serum (Gibco BRL) and incubated at 25°C. Culture fluids were harvested from monolayers exhibiting cytopathic ef-
Table 1 Sources of fish nodavirus isolates used in the present study and GenBank accession number for the coat protein sequences Virus isolate
Host species
Country of origin
Year of isolation
Accession number
Mt/01/Sba Gr/02/Sba Gr/12/Sba Pt/08/Sba It/23/Sba It/24/Sdr It/19/Sba Jp/06/SJ Jp/15/Rp Th/07/Bgr Sg/14/Bar Sp/20/Sba Gr/16/Sba
Sea Bass (Dicentrarchus labrax) Sea Bass (Dicentrarchus labrax) Sea Bass (Dicentrarchus labrax) Sea Bass (Dicentrarchus labrax) Sea Bass (Dicentrarchus labrax) Shi Drum (Umbrina cirrosa) Sea Bass (Dicentrarchus labrax) Striped Jack (Pseudocaranx dentex) Rock Porgy (Oplegnathus punctatus) Brownspotted Grouper (Epinephelus malabaricus) Barramundi (Lates calcarifer) Sea Bass (Dicentrarchus labrax) Sea Bass (Dicentrarchus labrax)
Malta Greece Greece Portugal Italy Italy Italy Japan Japan Thailand Singapore Spain Greece
1995 1995 1995 1996 1995 1995 1997 1995 1994 1995 1995 1998 1996
AF175512 AF175509 AF175510 AF175511 AF175513 AF175517 AF175514 AF175519 AF176620 AF175518 AF175516 AF175515 Y08700
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fects, and clarified by centrifugation at 1500× g for 15 min followed by 28 000×g for 35 min.
2.2. Virus neutralization Two polyclonal antisera were raised in rabbits against the Maltese (Mt/01/Sba) and Japanese (JP/06/SJ) nodavirusues, isolated from juvenile sea bass D. labrax and striped jack Pseudocaranx dentex, respectively (Frerichs, unpublished data, Institute of Aquaculture). Both antisera were used in neutralization assays which were performed using a modification of the method described by Rovozzo and Burke (1973). Briefly, 90 ml of nodavirus isolates were titrated (triplicate) in 96 well microtitre plates, prior to the addition of an equal volume of antiserum or maintenance medium (control). Plates were incubated for 1 h, then 100 ml of SSN-1 cells was added to wells, and plates were sealed with Nescofilm (Nesco, Kobe, Japan) and incubated at 25°C. Plates were examined for the presence/absence of neutralization for up to 12 days. The neutralization index (NI) was calculated as the ratio of the virus titre without antiserum to the virus titre in the presence of antiserum. NI less than 10 (B 1 log) were considered insignificant, between 10 and 50 (1.0 – 1.7 logs) were equivocal and over 50 (\1.7 logs) were considered significant.
2.3. Isolation of noda6irus RNA A total of 800 ml of clarified nodavirus supernatant was digested with 40 ml proteinase-K (Boehringer Mannheim, Germany) at a concentration of 20 mg/ml in the presence of 10 ml of 10% SDS and incubated at 37°C for 30 min. RNA was isolated using standard methods (Sambrook et al., 1989).
2.4. Oligonucleotide primers The design of oligonucleotide primers FG and R3 was based on the published nucleotide sequence of the coat protein gene of SJNNV (Nishizawa et al., 1995). Forward primer FG consisted of 17 nucleotides (5%-GAATCTTCCAGCGATAC-3%) designed to be complementary to nt
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306 –322 of the SJNNV coat protein gene. This region has previously been shown to be conserved amongst nodavirus isolates (Nishizawa et al., 1995). The reverse primer R3 consisted of 20 nucleotides (5%-CGAGTCAACACG GGTGAAGA-3%) corresponding to nt 1011 –1030 of the SJNNV coat protein.
2.5. Re6erse transcription and PCR amplification RNA samples were redissolved in diethyl pyrocarbonate treated water (DEPC), preheated at 90°C for 5 min and immediately placed on ice for 1 min. Reverse transcription was initiated by adding 200 units (1 ml) of M-MLV reverse transcriptase (Gibco BRL, UK) to a reaction mixture of 20 ml, containing 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 20 units of RNasin, 1 mM of each dNTP and 400 pM of the R3 reverse primer at 42°C for 40 min. Samples were then incubated at 95°C for 15 min to inactivate reverse transcriptase, then 2 ml was used as template for PCR amplification in a 50 ml reaction mixture, containing 10 mM Tris-HCl pH 8.8, 2 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 1.25 units Taq DNA polymerase (Biotools, S.A), 0.4 mM each of dNTP, 0.5 mM of each primer and overlaid with 30 ml of mineral oil. Amplification was performed using a PTC-150 Minicycler (MJ Research). Cycling conditions were: 10 min at 72°C, 2 min at 95°C, followed by 34 cycles of 40 s at 95°C, 50 s at 53°C, 120 s at 72°C, and a final extension of 5 min at 72°C. Negative controls included the substitution of RNA or cDNA with distilled water. Reaction products were analysed using 1% agarose gels.
2.6. Cloning and DNA sequencing PCR reaction products were extracted from agarose gels and purified using the QIAEX II (Qiagen, USA) extraction kit as described in the manufacturer’s instructions. Purified amplification products were ligated into the vector pT-Adv (Clontech, USA), and sequenced using the ABI Prism Big Dye Terminator cycle sequencing kit and M13 forward (-40) and reverse (-21) primers. Cycle sequencing conditions were: 10 s at 96°C, 5
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s at 50°C, 4 min at 60°C for 25 cycles, 4°C hold. Reaction products were analysed on ABI PRISM 377 autosequencer (Perkin Elmer, USA). All the sequences determined were deposited in the GenBank database and the accession numbers allocated are shown in Table 1. Sequences were analysed using MT Navigator software. Nucleotide sequence numbering was taken from Nishizawa et al. (1995). Protein sequences were compared using the DNA Strider sequence analysis software.
2.7. Phylogenetic analysis Nucleotide sequences were aligned using the CLUSTAL-X programme version 1.5b. Phylogenetic analysis was performed using the Neighbour Joining Method (Saitou and Nei, 1987) as implemented in the phyologeny inference package (PHYLIP; Felsenstein, 1993). Phylogenetic trees were constructed using Treeview 1 (Page, 1996). Bootstrap analysis was performed using 1000 data resamplings.
3. Results
3.1. Nucleotide sequence analysis Partial sequences (nucleotides 306 – 1030) of the fish nodavirus coat protein gene from twelve isolates from Europe, Asia, and Japan were determined in this study. The pairwise percentage identity analysis between these sequences together with the corresponding sequences of Gr/16/Sba and SJOri nodavirus isolates (Sideris, 1997; Nishizawa et al., 1995) are presented in Table 2. Sequence identity between isolates varied from 75 to 99.9%. All the nodaviruses isolated from Sea Bass (Dicentrarchus labrax), and those isolated from Shi Drum (Umbrina cirrosa), Rock Porgy (Oplegnathus punctatus), Brownspotted Grouper (Epinephelus malabaricus), and Barramundi (Lates calcarifer) exhibited nucleotide sequence identities \91.0%. The greatest sequence divergence was exhibited by the Japanese nodavirus isolates from Striped Jack (JP/06/SJ, and SJOri), which demonstrated only a 75–79.3% sequence identity to the other species studied.
Fig. 1. Unrooted phylogenetic tree deduced from analysis of the nucleotide sequence (688 bases) of 17 fish nodaviruses. Refer to Table 1 for details on new isolates and abbreviations used in this study, and to Nishizawa et al. (1997) for isolates marked by asterisks. The lengths of horizontal branches are proportional to the number of nucleotide substitutions and the numerals indicate bootstrap support values.
3.2. Phylogenetic analysis Phylogenetic analysis based on the sequence of nucleotides 323 –1010 of the coat protein gene using the Neighbour Joining method was performed, in order to examine the relationship between the nodavirus isolates determined in this study and previously determined isolates. The isolates of fish nodaviruses SJOri (DDBJ accession number, D30814), TP93Kag (D38637), BF93Hok (D38635) and RG91Tok (D38636), were used as representatives of the four different genotypes, described in the introduction, namely SJNNV, TPNNV, BFNNV and RGNNV respectively. The resulting dendrogram (Fig. 1) shows that among
Gr/02/Sba Gr/12/Sba Gr/16/Sba Pt/08/Sba Mt/01/Sba It/23Sba It/19/Sba Sp/20/Sba Sg/14/Bar It/24/Sdr Th/07/Bgr Jp/15/Rp Jp/06/SJ SJOri
*** 96.0 99.1 99.1 98.2 98.2 98.7 98.7 99.1 93.0 98.7 98.2 79.3 80.6
Gr/02/Sba 96.2 *** 96.0 96.0 96.0 96.0 95.6 95.6 96.0 92.1 95.6 96.0 78.9 80.2
Gr/12/Sba 98.4 96.1 *** 99.1 99.1 99.1 99.6 99.6 99.1 93.0 98.7 98.2 79.3 80.6
Gr/16/Sba 98.3 96.2 98.7 *** 99.1 98.2 99.6 99.6 99.1 93.0 98.7 98.2 80.2 81.5
Pt/08/Sba 98.1 96.1 99.7 98.7 *** 98.2 99.6 99.6 98.2 93.0 97.8 98.2 79.3 80.6
Mt/01/Sba 98.1 96.1 99.6 98.3 99.3 *** 98.7 98.7 98.2 92.1 97.8 97.4 78.4 79.7
It/23/Sba 98.3 95.9 99.9 98.8 99.9 99.4 *** 100.0 98.7 92.5 98.2 97.8 79.7 81.1
It/19/Sba 95.8 93.5 97.4 96.4 97.1 96.9 97.2 *** 98.7 92.5 98.2 97.8 79.7 81.1
Sp/20/Sba
Table 2 Nt (upper half) and aa (lower half) sequence identities of the coat protein gene partial sequences of 14 fish nodavirus isolates
98.7 96.4 99.1 99.0 98.8 98.7 99.0 96.5 *** 93.0 98.7 98.2 79.3 80.6
Sg/14/Bar 93.8 93.8 93.3 93.5 93.3 93.0 93.2 91.0 93.6 *** 92.5 93.0 79.3 80.6
It/24/Sdr
97.7 95.6 98.1 99.1 97.8 97.7 98.0 95.8 98.4 92.9 *** 97.8 79.3 80.6
Th/07/Bgr
96.8 96.1 96.9 96.8 96.9 96.7 96.8 94.8 96.9 93.3 96.2 *** 77.0 80.6
Jp/15/Rp
77.5 77.2 77.2 77.8 77.2 76.9 77.3 75.0 77.3 77.2 77.5 79.3 *** 97.4
Jp/06SJ
77.6 77.3 77.6 78.2 77.6 77.2 77.8 75.4 77.8 77.3 77.9 77.2 98.5 ***
SJOri
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the 13 isolates classified in this study, the majority (12/13) were found to belong to the RGNNV genotype (all the nodavirus isolates from Sea Bass and the isolates from Brownspotted Grouper, Barramundi, Rock Porgy, and Shi Drum), while only the isolate from Striped Jack was found to belong to the SJNNV genotype. Additionally, these results indicated that the RGNNV genotype contains nodaviruses isolated from eight countries across Europe, Asia and Japan. Essentially the same topology was obtained in dendrograms constructed using DNA-ML and DNA-PARS phylogenetic analysis programmes (data not shown).
3.3. Comparison of deduced amino acid sequences Deduced amino acid sequence of the coat protein region (aa 103 – 331) were compared among 14 isolates and the results are summarized in Table 2. The degree of coat protein sequence identity between isolates belonging to the same group was 92.1 –100% (RGNNV genotype) or 97.4% (SJNNV genotype), while the identity percentage between isolates belonging to different genotypes ranged from 77 to 81.1%. All the isolates from Sea Bass were highly conserved, with homologies ranging between 95.6 and 100%. Among the 229 amino acid residues of the SJOri isolate and the corresponding residues in the other isolates, which were analysed in this study, substitutions at 45 positions were observed, including two amino acid deletions at positions 236 and 237. (Fig. 2). Multiple alignment analysis of the deduced coat protein amino acid sequences indicated a highly conserved region at aa 103 – 222 (more than 95% sequence identity) and a variable region at aa 223 –331 (63.3% sequence identity). These results are in accord with previous observations (Nishizawa et al., 1995). Furthermore, this analysis also indicated that the cysteine residues at positions 187 and 201, were completely conserved among all the isolates analysed, while the cysteine residue at position 115 has been substituted to glycine in the JP/15/Rp isolate. This finding suggests that the cysteine residues at positions 187 and 201 may be more significant for the structure stabilisation of the coat protein.
3.4. Viral neutralization The viral neutralization assay was employed to determine the antigenic relationship of the different viral isolates, as well as for the identification of neutralizing epitopes among viruses belonging to different genotypes. Neutralization tests which were carried out using the Maltese (Mt/01/Sba) and Japanese (Jp/06/SJ) viruses, showed that both isolates were strongly neutralized with the homologous antisera, while no neutralization was evident with the heterologous antisera. Cross neutralizing activity of the remaining isolates was investigated against the two polyclonal antisera. All isolates tested with the exception of isolate Jp/06/SJ, were significantly neutralized by the Maltese antiserum (NI\ 4 log 10), while none was neutralized by the Japanese antiserum (Table 3). These results appear to be in good agreement with the obtained data from the phylogenetic analysis.
4. Discussion In this report, we present evidence on the classification of 12 new nodavirus isolates from Europe and Asia. For this work, phylogenetic analysis based on 688 nucleotide sequence of the coat protein was employed. The analyzed cDNA segment was larger than segments of the coat protein gene examined in previous studies (Nishizawa et al., 1997). Efforts to isolate the entire cDNA sequences for the coat protein for use in genotyping were not successful due to the possible nucleotide variability in the 5% region of the gene in the species studied. Specifically, when the forward primer Fo (Sideris, 1997), that corresponds to positions 17–38 of the SJOri isolate was used in amplification experiments no PCR products were generated in all of the isolates tested. For this reason an alternative forward primer, FG, corresponding to a highly conserved area of the gene was used instead of Fo. Our results of the nucleotide sequence and phylogenetic analyses showed that the majority of the isolates tested were classified to the RGNNV genotype. It is noted that the isolate from the
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species Rock Porgy has not been classified before. These data are in agreement with the findings of Nishizawa et al. (1997). A high level of nucleotide sequence identity between nodaviruses isolated from Sea Bass and Redspotted Grouper has also been reported by Sideris (1997), and clustering of nodaviruses isolated from Tiger Puffer, Striped Jack, and Barfin Flounder into separate lineages was reported by Nishizawa et al. (1997). Bootstrap support values for the phylogram clustering pattern in the present report varied from 89 to
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100%; support values \ 70% have been estimated to be statistically significant (Hillis and Bull, 1993). The RGNNV lineage identified in this report contained isolates from Spain, Portugal, Malta, Italy, France, Thailand, Singapore, and Japan. This finding indicates that nodavirus lineages are intercontinentally distributed, and that different lineages co-circulate within geographic regions. This evolutionary pattern contrasts with that exhibited by other viral fish pathogens including
Fig. 2. Multiple alignment of the deduced amino acid sequences of 14 fish nodavirus isolates. Upper line position are numbered according to the residue number of SJOri (Nishizawa et al., 1997).Symbols: dot, amino acid residue identical to that at the same position: asterisk, cysteine residues: arrow, hypothetical neutralizing epitopes.
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Table 3 Cross-neutralizing activity between fish nodavirus isolates, using polyclonal antisera raised against the Maltese Sea Bass (Mt/01/Sba) and Japanese Striped Jack (Jp/06/SJ) isolates Isolates
Genotype
Anti-Mt/01/Sba Anti-Jp/06/SJ
Mt/01/Sba Gr/02/Sba Gr/12/Sba Pt/08/Sba It/23/Sba It/24/Sdr It/19/Sba Jp/06/SJ Jp/15/Rp Th/07/Bgr Sg/14/Bar Sp/20/Sba Gr/16/Sba
RGNNV RGNNV RGNNV RGNNV RGNNV RGNNV RGNNV SJNNV RGNNV RGNNV RGNNV RGNNV RGNNV
+ + + + + + + − + + + + +
− − − − − − − + − − − − −
walleye dermal sarcoma virus (Zhang et al., 1996); viral haemorrhagic septicaemia virus (Benmansour et al., 1997); and infectious hematopoietic necrosis virus (Nichol et al., 1995) where correlation between phylogenetic relationships and geographic origin has been reported. However, the majority of the nodaviruses studied in this report were isolated from commercial fish farms, and consequently it is possible that trade in live fish and eggs has influenced the distribution of nodavirus lineages. Nishizawa et al. (1997) suggested that the Japanese Flounder may have played an important role in the dissemination of VNN in Japanese waters, and effects of fish transportation between Europe and the Pacific on the geographic distribution of nodavirus lineages have also been reported by Aspehaug et al. (1999). Information about serological relationships and neutralizing epitopes among fish nodaviruses is currently limited. All of the nodaviruses studied in this report were neutralized by a polyclonal antiserum raised against a nodavirus isolated from Sea Bass, with the exception of JP/06/SJ, which was isolated from Striped Jack. Therefore, the serological grouping data are in agreement with the genotyping results. Similar findings have been reported for enzyme linked immunosorbent assays using rabbit polyclonal sera raised against Striped
Jack, which were unable to detect viruses isolated from Sea Bass, Barramundi, Japanese parrotfish and Redspotted Grouper (Munday and Nakai, 1997). Differences between neutralizing epitopes on the structural protein of a nodavirus isolated from Striped Jack and other fish nodaviruses have been described by Nishizawa et al. (1999). However, immunofluorescent antibody based methods have identified antigenic similarities between nodaviruses isolated from Striped Jack and other fish nodaviruses (Mori et al., 1992), suggesting that these viruses exhibit close, but not identical antigenic relationships (Munday and Nakai, 1997). Neutralizing epitope identification (Nishizawa et al., 1999) have shown the existence of two PAN epitopes in SJNNV type isolates, at positions 116 –118 and 254 –256 and suggested that the second PAN sequence is most likely the major neutralizing epitope. Our sequence alignment analysis data indicates that the first PAN sequence remains constant in all of the isolates in this study, with the exception of JP/06/SJ which contains a PAD sequence instead of PAN at positions 116 –118. Our serological analysis results reflect this difference. The antiserum raised against the JP/06/SJ isolate does not recognize all isolates that contain a PAN sequence at that position. Additionally, it must be taken into consideration that the antiserum raised against the Mt/01/Sba that is not able to recognise the JP/06/ SJ isolate, can neutralize the Th/07/Bgr isolate which bears a PGG sequence at positions 254 – 256, as well as the rest of the studied isolates which contain a PDG sequence at the same position. These observations could suggest that an additional neutralizing epitope may be located at amino acid residues 116 –118 of the viral coat protein. Further analysis of fish nodavirus nucleotide sequence variation and detailed antigenic epitope characterization are required to facilitate the development of vaccines and diagnostic reagents effective against circulating strains of this virus.
Acknowledgements We thank Dr F. Chua, Dr Bovo and Dr A.
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Prappas for providing nodavirus isolates; Dr G.N. Frerichs for providing nodavirus antisera; S. Powell for technical assistance with DNA sequencing and all the PhD students at the Molecular Biology Laboratory, University of Athens for their help and support. This work was supported by a grant from the British Council in Greece. G.P.S. was a recipient of grants from the State Scholarships Foundation of Greece and the A.G. Leventis Foundation of France.
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Phylogenetic and antigenic characterization of new fish nodavirus isolates from Europe and Asia George P. Skliris a, John V. Krondiris b, Diamantis C. Sideris b, Andrew P. Shinn a, William G. Starkey a, Randolph H. Richards a,* b
a Virology Department, The Institute of Aquaculture, Uni6ersity of Stirling, Scotland FK9 4LA, UK Uni6ersity of Athens, School of Biology, Department of Biochemistry and Molecular Biology, Panepistimioupolis, 15701 Athens, Greece
Received 19 September 2000; received in revised form 29 December 2000; accepted 5 January 2001
Abstract Nodaviruses are widespread causative agents of viral nervous necrosis in fish. Based on the coat protein sequence, fish nodaviruses are categorized into four different genotypes. In this study, we present data on the phylogenetic and antigenic characterization of 12 new isolates, eight European and four of Asian origin, from farmed and wild species of fish. Phylogenetic analysis based on the nucleotide sequence (688 bases) of the coat protein classified the majority of these new isolates to the RGNNV genotype. Geographic or host-species specificities were not revealed by this study. Neutralizing assay experiments, further confirmed the genotypic classification, supporting the possibility that the different nodavirus genotypes can also be serologically distinguishable. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Fish nodavirus; Nervous necrosis virus; Viral encephalopathy
1. Introduction The nodaviridae is a family of small, non-enveloped, icosahedral RNA viruses, which infect a wide range of insects and fish (Kaesbergh, 1987; Mori et al., 1992). Fish nodaviruses belonging to the genus betanodavirus are the causative agents of viral nervous necrosis (VNN), also referred to
* Corresponding author. Tel.: + 44-1786-467873; fax: +441786-472133. E-mail address: [email protected] (R.H. Richards).
as viral encephalopathy and retinopathy (VER), and fish encephalitis. This disease is characterised by abnormal swimming behaviour, and vacuolating lesions within the brain and retinal tissue of affected fish. VNN has been associated with high mortalities in several marine fish species of economic importance to the aquaculture industry in Europe, Asia, Japan, and Australia (Nakai et al., 1994; Munday and Nakai 1997; Skliris and Richards 1999). European sea bass production has been seriously affected by VNN, with significant losses occurring in stocks of juvenile and ongrowing fish (Le Breton et al., 1996).
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The genome of fish nodaviruses is bipartite, comprising two molecules of single stranded, positive polarity RNA (Mori et al., 1992; Comps et al., 1994). RNA 1 (1.01 ×106 Da) encodes a non-structural protein of approximately 110 kDa (Nagai and Nishizawa 1999), while RNA 2 ( 0.49 ×106 Da) encodes the 42 kDa coat protein precursor. Both RNA molecules are nonpolyadenylated (Mori et al., 1992). The coat protein gene of a striped jack nervous necrosis virus (SJNNV) is 1410 nucleotides in length, containing an open reading frame of 1023 nucleotides, encoding a protein of 340 amino acids, and 5% and 3%-non-coding regions of 16 and 371 nucleotides respectively (Nishizawa et al., 1995). A potential neutralizing determinant has been mapped to amino acid residues 254 – 256 of the coat protein of SJNNV (Nishizawa et al., 1999). Analysis of the genetic variation exhibited by fish nodaviruses is required for the rational development of effective vaccines and diagnostic reagents. The nucleotide sequence homology between the coat protein genes of insect and fish nodaviruses have previously been investigated (Nishizawa et al., 1995). Based on partial sequences of the coat protein gene fish nodaviruses have been classified into four clusters: striped jack nervous necrosis virus (SJNNV), tiger puffer jack nervous necrosis virus (TPNNV), barfin flounder nervous necrosis virus (BFNNV) and red-spotted
grouper nervous necrosis virus (RGNNV) (Nishizawa et al., 1997). However, these studies have predominantly focused on Japanese nodavirus isolates. In this report, we have studied nucleotide sequence and serological relationships of a collection of nodaviruses isolated from Europe, Asia, and Japan. This collection included eight isolates from Sea Bass obtained from European fish farms. Phylogenetic and serological analysis was performed to characterise the relationships between the nodavirus isolates studied in this report, and other previously characterised betanodaviruses.
2. Materials and methods
2.1. Virus isolation and propagation The source and date of isolation of the nodaviruses studied in this report are listed in Table 1. All viruses were isolated from farmed species, with the exception of It/24/Sdr, isolated from Shi Drum (Umbrina cirrosa), which is a wild species. Viruses were propagated in SSN-1 cells, (derived from Striped Snakehead, Channa striatus), grown in Leibovitz L-15 medium (Gibco BRL, UK) containing 5% foetal bovine serum (Gibco BRL) and incubated at 25°C. Culture fluids were harvested from monolayers exhibiting cytopathic ef-
Table 1 Sources of fish nodavirus isolates used in the present study and GenBank accession number for the coat protein sequences Virus isolate
Host species
Country of origin
Year of isolation
Accession number
Mt/01/Sba Gr/02/Sba Gr/12/Sba Pt/08/Sba It/23/Sba It/24/Sdr It/19/Sba Jp/06/SJ Jp/15/Rp Th/07/Bgr Sg/14/Bar Sp/20/Sba Gr/16/Sba
Sea Bass (Dicentrarchus labrax) Sea Bass (Dicentrarchus labrax) Sea Bass (Dicentrarchus labrax) Sea Bass (Dicentrarchus labrax) Sea Bass (Dicentrarchus labrax) Shi Drum (Umbrina cirrosa) Sea Bass (Dicentrarchus labrax) Striped Jack (Pseudocaranx dentex) Rock Porgy (Oplegnathus punctatus) Brownspotted Grouper (Epinephelus malabaricus) Barramundi (Lates calcarifer) Sea Bass (Dicentrarchus labrax) Sea Bass (Dicentrarchus labrax)
Malta Greece Greece Portugal Italy Italy Italy Japan Japan Thailand Singapore Spain Greece
1995 1995 1995 1996 1995 1995 1997 1995 1994 1995 1995 1998 1996
AF175512 AF175509 AF175510 AF175511 AF175513 AF175517 AF175514 AF175519 AF176620 AF175518 AF175516 AF175515 Y08700
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fects, and clarified by centrifugation at 1500× g for 15 min followed by 28 000×g for 35 min.
2.2. Virus neutralization Two polyclonal antisera were raised in rabbits against the Maltese (Mt/01/Sba) and Japanese (JP/06/SJ) nodavirusues, isolated from juvenile sea bass D. labrax and striped jack Pseudocaranx dentex, respectively (Frerichs, unpublished data, Institute of Aquaculture). Both antisera were used in neutralization assays which were performed using a modification of the method described by Rovozzo and Burke (1973). Briefly, 90 ml of nodavirus isolates were titrated (triplicate) in 96 well microtitre plates, prior to the addition of an equal volume of antiserum or maintenance medium (control). Plates were incubated for 1 h, then 100 ml of SSN-1 cells was added to wells, and plates were sealed with Nescofilm (Nesco, Kobe, Japan) and incubated at 25°C. Plates were examined for the presence/absence of neutralization for up to 12 days. The neutralization index (NI) was calculated as the ratio of the virus titre without antiserum to the virus titre in the presence of antiserum. NI less than 10 (B 1 log) were considered insignificant, between 10 and 50 (1.0 – 1.7 logs) were equivocal and over 50 (\1.7 logs) were considered significant.
2.3. Isolation of noda6irus RNA A total of 800 ml of clarified nodavirus supernatant was digested with 40 ml proteinase-K (Boehringer Mannheim, Germany) at a concentration of 20 mg/ml in the presence of 10 ml of 10% SDS and incubated at 37°C for 30 min. RNA was isolated using standard methods (Sambrook et al., 1989).
2.4. Oligonucleotide primers The design of oligonucleotide primers FG and R3 was based on the published nucleotide sequence of the coat protein gene of SJNNV (Nishizawa et al., 1995). Forward primer FG consisted of 17 nucleotides (5%-GAATCTTCCAGCGATAC-3%) designed to be complementary to nt
61
306 –322 of the SJNNV coat protein gene. This region has previously been shown to be conserved amongst nodavirus isolates (Nishizawa et al., 1995). The reverse primer R3 consisted of 20 nucleotides (5%-CGAGTCAACACG GGTGAAGA-3%) corresponding to nt 1011 –1030 of the SJNNV coat protein.
2.5. Re6erse transcription and PCR amplification RNA samples were redissolved in diethyl pyrocarbonate treated water (DEPC), preheated at 90°C for 5 min and immediately placed on ice for 1 min. Reverse transcription was initiated by adding 200 units (1 ml) of M-MLV reverse transcriptase (Gibco BRL, UK) to a reaction mixture of 20 ml, containing 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 20 units of RNasin, 1 mM of each dNTP and 400 pM of the R3 reverse primer at 42°C for 40 min. Samples were then incubated at 95°C for 15 min to inactivate reverse transcriptase, then 2 ml was used as template for PCR amplification in a 50 ml reaction mixture, containing 10 mM Tris-HCl pH 8.8, 2 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 1.25 units Taq DNA polymerase (Biotools, S.A), 0.4 mM each of dNTP, 0.5 mM of each primer and overlaid with 30 ml of mineral oil. Amplification was performed using a PTC-150 Minicycler (MJ Research). Cycling conditions were: 10 min at 72°C, 2 min at 95°C, followed by 34 cycles of 40 s at 95°C, 50 s at 53°C, 120 s at 72°C, and a final extension of 5 min at 72°C. Negative controls included the substitution of RNA or cDNA with distilled water. Reaction products were analysed using 1% agarose gels.
2.6. Cloning and DNA sequencing PCR reaction products were extracted from agarose gels and purified using the QIAEX II (Qiagen, USA) extraction kit as described in the manufacturer’s instructions. Purified amplification products were ligated into the vector pT-Adv (Clontech, USA), and sequenced using the ABI Prism Big Dye Terminator cycle sequencing kit and M13 forward (-40) and reverse (-21) primers. Cycle sequencing conditions were: 10 s at 96°C, 5
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s at 50°C, 4 min at 60°C for 25 cycles, 4°C hold. Reaction products were analysed on ABI PRISM 377 autosequencer (Perkin Elmer, USA). All the sequences determined were deposited in the GenBank database and the accession numbers allocated are shown in Table 1. Sequences were analysed using MT Navigator software. Nucleotide sequence numbering was taken from Nishizawa et al. (1995). Protein sequences were compared using the DNA Strider sequence analysis software.
2.7. Phylogenetic analysis Nucleotide sequences were aligned using the CLUSTAL-X programme version 1.5b. Phylogenetic analysis was performed using the Neighbour Joining Method (Saitou and Nei, 1987) as implemented in the phyologeny inference package (PHYLIP; Felsenstein, 1993). Phylogenetic trees were constructed using Treeview 1 (Page, 1996). Bootstrap analysis was performed using 1000 data resamplings.
3. Results
3.1. Nucleotide sequence analysis Partial sequences (nucleotides 306 – 1030) of the fish nodavirus coat protein gene from twelve isolates from Europe, Asia, and Japan were determined in this study. The pairwise percentage identity analysis between these sequences together with the corresponding sequences of Gr/16/Sba and SJOri nodavirus isolates (Sideris, 1997; Nishizawa et al., 1995) are presented in Table 2. Sequence identity between isolates varied from 75 to 99.9%. All the nodaviruses isolated from Sea Bass (Dicentrarchus labrax), and those isolated from Shi Drum (Umbrina cirrosa), Rock Porgy (Oplegnathus punctatus), Brownspotted Grouper (Epinephelus malabaricus), and Barramundi (Lates calcarifer) exhibited nucleotide sequence identities \91.0%. The greatest sequence divergence was exhibited by the Japanese nodavirus isolates from Striped Jack (JP/06/SJ, and SJOri), which demonstrated only a 75–79.3% sequence identity to the other species studied.
Fig. 1. Unrooted phylogenetic tree deduced from analysis of the nucleotide sequence (688 bases) of 17 fish nodaviruses. Refer to Table 1 for details on new isolates and abbreviations used in this study, and to Nishizawa et al. (1997) for isolates marked by asterisks. The lengths of horizontal branches are proportional to the number of nucleotide substitutions and the numerals indicate bootstrap support values.
3.2. Phylogenetic analysis Phylogenetic analysis based on the sequence of nucleotides 323 –1010 of the coat protein gene using the Neighbour Joining method was performed, in order to examine the relationship between the nodavirus isolates determined in this study and previously determined isolates. The isolates of fish nodaviruses SJOri (DDBJ accession number, D30814), TP93Kag (D38637), BF93Hok (D38635) and RG91Tok (D38636), were used as representatives of the four different genotypes, described in the introduction, namely SJNNV, TPNNV, BFNNV and RGNNV respectively. The resulting dendrogram (Fig. 1) shows that among
Gr/02/Sba Gr/12/Sba Gr/16/Sba Pt/08/Sba Mt/01/Sba It/23Sba It/19/Sba Sp/20/Sba Sg/14/Bar It/24/Sdr Th/07/Bgr Jp/15/Rp Jp/06/SJ SJOri
*** 96.0 99.1 99.1 98.2 98.2 98.7 98.7 99.1 93.0 98.7 98.2 79.3 80.6
Gr/02/Sba 96.2 *** 96.0 96.0 96.0 96.0 95.6 95.6 96.0 92.1 95.6 96.0 78.9 80.2
Gr/12/Sba 98.4 96.1 *** 99.1 99.1 99.1 99.6 99.6 99.1 93.0 98.7 98.2 79.3 80.6
Gr/16/Sba 98.3 96.2 98.7 *** 99.1 98.2 99.6 99.6 99.1 93.0 98.7 98.2 80.2 81.5
Pt/08/Sba 98.1 96.1 99.7 98.7 *** 98.2 99.6 99.6 98.2 93.0 97.8 98.2 79.3 80.6
Mt/01/Sba 98.1 96.1 99.6 98.3 99.3 *** 98.7 98.7 98.2 92.1 97.8 97.4 78.4 79.7
It/23/Sba 98.3 95.9 99.9 98.8 99.9 99.4 *** 100.0 98.7 92.5 98.2 97.8 79.7 81.1
It/19/Sba 95.8 93.5 97.4 96.4 97.1 96.9 97.2 *** 98.7 92.5 98.2 97.8 79.7 81.1
Sp/20/Sba
Table 2 Nt (upper half) and aa (lower half) sequence identities of the coat protein gene partial sequences of 14 fish nodavirus isolates
98.7 96.4 99.1 99.0 98.8 98.7 99.0 96.5 *** 93.0 98.7 98.2 79.3 80.6
Sg/14/Bar 93.8 93.8 93.3 93.5 93.3 93.0 93.2 91.0 93.6 *** 92.5 93.0 79.3 80.6
It/24/Sdr
97.7 95.6 98.1 99.1 97.8 97.7 98.0 95.8 98.4 92.9 *** 97.8 79.3 80.6
Th/07/Bgr
96.8 96.1 96.9 96.8 96.9 96.7 96.8 94.8 96.9 93.3 96.2 *** 77.0 80.6
Jp/15/Rp
77.5 77.2 77.2 77.8 77.2 76.9 77.3 75.0 77.3 77.2 77.5 79.3 *** 97.4
Jp/06SJ
77.6 77.3 77.6 78.2 77.6 77.2 77.8 75.4 77.8 77.3 77.9 77.2 98.5 ***
SJOri
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the 13 isolates classified in this study, the majority (12/13) were found to belong to the RGNNV genotype (all the nodavirus isolates from Sea Bass and the isolates from Brownspotted Grouper, Barramundi, Rock Porgy, and Shi Drum), while only the isolate from Striped Jack was found to belong to the SJNNV genotype. Additionally, these results indicated that the RGNNV genotype contains nodaviruses isolated from eight countries across Europe, Asia and Japan. Essentially the same topology was obtained in dendrograms constructed using DNA-ML and DNA-PARS phylogenetic analysis programmes (data not shown).
3.3. Comparison of deduced amino acid sequences Deduced amino acid sequence of the coat protein region (aa 103 – 331) were compared among 14 isolates and the results are summarized in Table 2. The degree of coat protein sequence identity between isolates belonging to the same group was 92.1 –100% (RGNNV genotype) or 97.4% (SJNNV genotype), while the identity percentage between isolates belonging to different genotypes ranged from 77 to 81.1%. All the isolates from Sea Bass were highly conserved, with homologies ranging between 95.6 and 100%. Among the 229 amino acid residues of the SJOri isolate and the corresponding residues in the other isolates, which were analysed in this study, substitutions at 45 positions were observed, including two amino acid deletions at positions 236 and 237. (Fig. 2). Multiple alignment analysis of the deduced coat protein amino acid sequences indicated a highly conserved region at aa 103 – 222 (more than 95% sequence identity) and a variable region at aa 223 –331 (63.3% sequence identity). These results are in accord with previous observations (Nishizawa et al., 1995). Furthermore, this analysis also indicated that the cysteine residues at positions 187 and 201, were completely conserved among all the isolates analysed, while the cysteine residue at position 115 has been substituted to glycine in the JP/15/Rp isolate. This finding suggests that the cysteine residues at positions 187 and 201 may be more significant for the structure stabilisation of the coat protein.
3.4. Viral neutralization The viral neutralization assay was employed to determine the antigenic relationship of the different viral isolates, as well as for the identification of neutralizing epitopes among viruses belonging to different genotypes. Neutralization tests which were carried out using the Maltese (Mt/01/Sba) and Japanese (Jp/06/SJ) viruses, showed that both isolates were strongly neutralized with the homologous antisera, while no neutralization was evident with the heterologous antisera. Cross neutralizing activity of the remaining isolates was investigated against the two polyclonal antisera. All isolates tested with the exception of isolate Jp/06/SJ, were significantly neutralized by the Maltese antiserum (NI\ 4 log 10), while none was neutralized by the Japanese antiserum (Table 3). These results appear to be in good agreement with the obtained data from the phylogenetic analysis.
4. Discussion In this report, we present evidence on the classification of 12 new nodavirus isolates from Europe and Asia. For this work, phylogenetic analysis based on 688 nucleotide sequence of the coat protein was employed. The analyzed cDNA segment was larger than segments of the coat protein gene examined in previous studies (Nishizawa et al., 1997). Efforts to isolate the entire cDNA sequences for the coat protein for use in genotyping were not successful due to the possible nucleotide variability in the 5% region of the gene in the species studied. Specifically, when the forward primer Fo (Sideris, 1997), that corresponds to positions 17–38 of the SJOri isolate was used in amplification experiments no PCR products were generated in all of the isolates tested. For this reason an alternative forward primer, FG, corresponding to a highly conserved area of the gene was used instead of Fo. Our results of the nucleotide sequence and phylogenetic analyses showed that the majority of the isolates tested were classified to the RGNNV genotype. It is noted that the isolate from the
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species Rock Porgy has not been classified before. These data are in agreement with the findings of Nishizawa et al. (1997). A high level of nucleotide sequence identity between nodaviruses isolated from Sea Bass and Redspotted Grouper has also been reported by Sideris (1997), and clustering of nodaviruses isolated from Tiger Puffer, Striped Jack, and Barfin Flounder into separate lineages was reported by Nishizawa et al. (1997). Bootstrap support values for the phylogram clustering pattern in the present report varied from 89 to
65
100%; support values \ 70% have been estimated to be statistically significant (Hillis and Bull, 1993). The RGNNV lineage identified in this report contained isolates from Spain, Portugal, Malta, Italy, France, Thailand, Singapore, and Japan. This finding indicates that nodavirus lineages are intercontinentally distributed, and that different lineages co-circulate within geographic regions. This evolutionary pattern contrasts with that exhibited by other viral fish pathogens including
Fig. 2. Multiple alignment of the deduced amino acid sequences of 14 fish nodavirus isolates. Upper line position are numbered according to the residue number of SJOri (Nishizawa et al., 1997).Symbols: dot, amino acid residue identical to that at the same position: asterisk, cysteine residues: arrow, hypothetical neutralizing epitopes.
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Table 3 Cross-neutralizing activity between fish nodavirus isolates, using polyclonal antisera raised against the Maltese Sea Bass (Mt/01/Sba) and Japanese Striped Jack (Jp/06/SJ) isolates Isolates
Genotype
Anti-Mt/01/Sba Anti-Jp/06/SJ
Mt/01/Sba Gr/02/Sba Gr/12/Sba Pt/08/Sba It/23/Sba It/24/Sdr It/19/Sba Jp/06/SJ Jp/15/Rp Th/07/Bgr Sg/14/Bar Sp/20/Sba Gr/16/Sba
RGNNV RGNNV RGNNV RGNNV RGNNV RGNNV RGNNV SJNNV RGNNV RGNNV RGNNV RGNNV RGNNV
+ + + + + + + − + + + + +
− − − − − − − + − − − − −
walleye dermal sarcoma virus (Zhang et al., 1996); viral haemorrhagic septicaemia virus (Benmansour et al., 1997); and infectious hematopoietic necrosis virus (Nichol et al., 1995) where correlation between phylogenetic relationships and geographic origin has been reported. However, the majority of the nodaviruses studied in this report were isolated from commercial fish farms, and consequently it is possible that trade in live fish and eggs has influenced the distribution of nodavirus lineages. Nishizawa et al. (1997) suggested that the Japanese Flounder may have played an important role in the dissemination of VNN in Japanese waters, and effects of fish transportation between Europe and the Pacific on the geographic distribution of nodavirus lineages have also been reported by Aspehaug et al. (1999). Information about serological relationships and neutralizing epitopes among fish nodaviruses is currently limited. All of the nodaviruses studied in this report were neutralized by a polyclonal antiserum raised against a nodavirus isolated from Sea Bass, with the exception of JP/06/SJ, which was isolated from Striped Jack. Therefore, the serological grouping data are in agreement with the genotyping results. Similar findings have been reported for enzyme linked immunosorbent assays using rabbit polyclonal sera raised against Striped
Jack, which were unable to detect viruses isolated from Sea Bass, Barramundi, Japanese parrotfish and Redspotted Grouper (Munday and Nakai, 1997). Differences between neutralizing epitopes on the structural protein of a nodavirus isolated from Striped Jack and other fish nodaviruses have been described by Nishizawa et al. (1999). However, immunofluorescent antibody based methods have identified antigenic similarities between nodaviruses isolated from Striped Jack and other fish nodaviruses (Mori et al., 1992), suggesting that these viruses exhibit close, but not identical antigenic relationships (Munday and Nakai, 1997). Neutralizing epitope identification (Nishizawa et al., 1999) have shown the existence of two PAN epitopes in SJNNV type isolates, at positions 116 –118 and 254 –256 and suggested that the second PAN sequence is most likely the major neutralizing epitope. Our sequence alignment analysis data indicates that the first PAN sequence remains constant in all of the isolates in this study, with the exception of JP/06/SJ which contains a PAD sequence instead of PAN at positions 116 –118. Our serological analysis results reflect this difference. The antiserum raised against the JP/06/SJ isolate does not recognize all isolates that contain a PAN sequence at that position. Additionally, it must be taken into consideration that the antiserum raised against the Mt/01/Sba that is not able to recognise the JP/06/ SJ isolate, can neutralize the Th/07/Bgr isolate which bears a PGG sequence at positions 254 – 256, as well as the rest of the studied isolates which contain a PDG sequence at the same position. These observations could suggest that an additional neutralizing epitope may be located at amino acid residues 116 –118 of the viral coat protein. Further analysis of fish nodavirus nucleotide sequence variation and detailed antigenic epitope characterization are required to facilitate the development of vaccines and diagnostic reagents effective against circulating strains of this virus.
Acknowledgements We thank Dr F. Chua, Dr Bovo and Dr A.
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Prappas for providing nodavirus isolates; Dr G.N. Frerichs for providing nodavirus antisera; S. Powell for technical assistance with DNA sequencing and all the PhD students at the Molecular Biology Laboratory, University of Athens for their help and support. This work was supported by a grant from the British Council in Greece. G.P.S. was a recipient of grants from the State Scholarships Foundation of Greece and the A.G. Leventis Foundation of France.
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