Faculty of Applied Biological Science, Hiroshima University, Higashi-hiroshima 739-8528, Japan1
Author for correspondence: Toyohiko Nishizawa.Fax +81 824 22 7059. e-mail jjnishi{at}ipc.hiroshima-u.ac.jp
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Abstract |
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The isolates of fish nodaviruses, SJOri (DDBJ accession number, D30814), TP93Kag (D38637), BF93Hok (D38635) and RG91Tok (D38636), were used as representatives of four different genotypes, SJNNV, TPNNV, BFNNV and RGNNV types, respectively. Partial coat protein genes (T2 and T4 regions) of these isolates were sequenced in our previous work (Nishizawa et al., 1995b , 1997
). The PCR primers, F1exp (5' aaacatatgGGATTTGGACGTGCGACCAA 3'), F2exp (5' aaacatatgGTGTCAGTCATGTGTCGCTG 3') and R3exp (5' gctaagcttcaCGAGTCAACACGGGTGAAGA 3') were used for PCR amplification of the T2 and T4 regions from the above four virus samples. The sense primers, F1exp (nt 155174) and F2exp (nt 605624), included an additional 9 bases of linker sequence as an NdeI recognition site. The antisense primer, R3exp (complementary to nt 10111030), has an additional 11 bases of linker sequence, representing a HindIII recognition site and termination codon. PCR amplification was performed under the conditions previously described (Nishizawa et al., 1994
, 1995
b). After digestion with NdeI and Hin dIII, the PCR products were ligated into an expression vector plasmid, pET-25b (+) (Novagen) and used to transform Escherichia coli (BL21). Eight clones of recombinant plasmids, pET- S261/pET-S403 (from SJOri), pET-T201/pET-T402 (from TP93Kag), pET- B203/pET-B404 (from BF93Hok) and pET-R283/pET-R483 (from RG91Tok) were obtained. Agarose gel electrophoresis following digestion with Nde I and HindIII confirmed that approximately 900-base-long DNAs were inserted into the recombinant plasmids, pET-S261, pET-T201, pET-B203 and pET-R283, whereas approximately 450-base-long DNAs were found in pET-S403, pET-T402, pET-B404 and pET-R483 (Fig. 1A
). The length of each inserted DNA was consistent with that of the T2 or T4 region of the fish nodavirus coat protein gene (Nishizawa et al., 1995b
). The DNAs were blotted onto a nylon membrane (Hybond-N+; Amersham) and Southern blot hybridization with digoxigenin (DIG)-labelled DNA probes was carried out at 45 °C for 2 h in the presence of 50% (v/v) formamide (Fig. 1B
). The DIG probes were prepared from the cloned cDNAs of the T4 region of each fish nodavirus genotype with a PCR DIG labelling kit (Boehringer Mannheim) and PCR primers F2 and R3 (Nishizawa et al., 1994
) according to the manufacturer's instructions. The DIG-labelled SJNNV, TPNNV, BFNNV and RGNNV T4 probes showed clear signals with the inserted DNAs in the clones obtained from respective homologous viruses, whereas very weak reactions were shown for heterologous virus DNA. These results confirmed that the T2 and T4 regions of each of the four different virus genotypes were cloned into the respective expression vector plasmids. Additionally, it was shown that rapid genotyping of fish nodaviruses was possible by Southern hybridization with the DNA probes against the T4 region of the viral coat protein gene.
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SDSPAGE analysis of the SJNNV coat protein and the induced products was performed according to Laemmli (1970) and proteins were electroblotted onto a nitrocellulose membrane by the procedure of Towbin et al. (1979)
. Proteins on the membrane were immunostained with an anti-SJNNV serum (A/S SJNNV) and two specific SJNNV MAbs, 102B and 204D (Nishizawa et al., 1995 a
), and then visualized with an immunoblot kit (Bio-Rad) according to the manufacturer's instructions (Fig. 2
). The coat protein of SJNNV had an apparent molecular mass of 40 kDa, which was in agreement with that estimated by Mori et al. (1992)
. The expressed proteins from the T2 region of SJNNV, TPNNV and BFNNV showed the same relative mobility in the gel, the apparent molecular mass being 32 kDa. The expressed product from the RGNNV T2 region also showed the same molecular mass although the amount of the product was lower (data not shown). In contrast, different mobilities were observed among the expressed products from the T4 regions of the four virus genomes. All of them ranged between 15·5 and 16·0 kDa (Fig. 2A
, B
). The sizes of the expressed proteins encoded by the T2 and T4 regions of the viral coat protein genes were in accordance with those estimated from the deduced amino acid sequences. In the immunoblot analyses, the A/S SJNNV showed positive cross-reactions with all of expressed proteins, although differences in the intensity of the antibody reaction were observed among them. In particular, the intensities with the products of homologous virus were much stronger than those exhibited by heterologous viruses, indicating that the four different fish nodavirus genotypes share a significant number of antigenic determinants, although they are not identical to each other. This is in agreement with previous findings (Nguyen et al., 1994
; Nakai et al., 1994
; Grotmol et al., 1997
). On the other hand, MAbs 102B and 204D reacted not only with a native coat protein and the expressed T2 and T4 proteins of SJNNV, but also with the expressed T2 proteins of TPNNV and BFNNV. However, no cross-reactions were observed with those from T4 regions of heterologous viruses (Fig. 2C
, D
). Both MAbs have a neutralizing activity against SJNNV (Nishizawa et al., 1995a
), suggesting that SJNNV could be distinguishable serologically from the other genotypes of fish nodaviruses, and also that the antigenic determinant recognized by both MAbs has to be a linear epitope on the SJNNV coat protein.
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A matrix plot analysis of amino acid sequences encoded in the T2 and T4 regions of the SJNNV coat protein was performed with a window of three residues and 100% for a minimum score (Fig. 3A). Four different repeated amino acid sequences were found on the SJNNV coat protein as follows: PAG (aa 6971, 294296 and 328330); AGT (aa 7072 and 329331); LLP (aa 8890 and 326328); and PAN (aa 116118 and 254256). These elements, together with their flanking sequences, are displayed, after alignment, in Fig. 3(B)
(Thompson et al., 1994
). As described above, the epitope for the MAbs should be present within aa 54203 as a common sequence among the four genotype viruses and also present within aa 204331 as a specific sequence to SJNNV. Among the observed amino acid sequences, only AGT (aa 7072 and 329331) and PAN (aa 116118 and 254256) satisfied these conditions. However, an additional AGT sequence existed at aa 291293 within the RGNNV T4 protein (Fig. 3B
). Therefore, we conclude that the repeated sequence, PAN, is most likely a potential neutralizing epitope for both MAbs 102B and 204D. It was confirmed that a synthetic peptide with eight residues containing the PAN sequence reacts with A/S SJNNV and both MAbs 102B and 204D (data not shown). It is not clear from our results which PAN sequence, at aa 116118 or aa 254256, exists on the surface of SJNNV particle and takes part in the neutralizing reaction.
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References |
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Received 14 April 1999;
accepted 23 July 1999.