1 Department of Parasitic Agents, Kobe Institute of Health, 4-6, Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan
2 Division of Virology, Department of Public Health, Osaka Prefectural Institute of Public Health, 3-69, 1-Chome, Nakamichi, Higashinari-ku, Osaka 537-0025, Japan
3 Department of Clinical Pathology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686, Japan
Correspondence
Naoko Nakagawa
nknkgw{at}h2.dion.ne.jp
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ABSTRACT |
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DDBJ accession numbers of the nucleotide sequences reported in this paper are as follows: AB036446 for B/Kadoma/122/1999; AB036451 for B/Kadoma/506/1999; AB045009 for B/Kadoma/409/2000; AB071521for B/Kobe/69/2001-clone 1; AB083404 for B/Kobe/5/2002; AB083405 for B/Kobe/69/2001-clone 1-variant 1; AB036452 and AB036453 for B/Kadoma/122/1999-V1 and -V2; AB071525 to -33 for B/Kadoma/122/1999-V3 to -V11.
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MAIN TEXT |
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We studied the neutralizing epitope sites specific for B/Yamagata group strains.
Virus strains B/Kadoma/122/1999, B/Kadoma/409/2000, B/Kobe/69/2001 and B/Kobe/5/2002 were isolated from clinical specimens. mAb 3A12 was obtained by immunizing mice with the 5H4-nonreacting strain B/Kadoma/506/1999. Ascites fluid of mice injected with hybridoma cells were used as the source of all mAbs. The standard ferret polyclonal antibody was provided by the National Institute of Infectious Diseases, Japan: the sera against B/Yamanashi/166/1998 for B/Yamagata strains. Human post-vaccination sera were collected at Baba Children's Clinic in Kadoma City, Osaka Prefecture, Japan. Results of HI and NT tests are expressed as the reciprocal of antibody dilution (Okuno et al., 1990). Competitive assays were performed by modifying the method reported previously (Waxham & Wolinsky, 1985
). Escape mutants were induced by incubating B/Kadoma/122/1999 with mAb 3A12 by modifying the method described previously (Berton et al., 1984
; Lambkin et al., 1994
; Nakagawa et al., 2001a
). Briefly, 1x105 f.f.u. ml-1 virus were incubated for 1 h at 30 °C in the presence of 10 µl mAb 3A12. The virus/mAb mixture was inoculated to MadinDarby canine kidney cells in 24-well plates and incubated at 35 °C for 3 days. HI and NT tests on each well were performed separately with mAb in order to identify escape mutants. Nucleotide sequences of the escape mutants were analysed as described previously (Nakagawa et al., 2000
, 2001b
). Briefly, RNA was obtained from the virus and influenza B virus-specific DNA was amplified by RT-PCR. Then the products were sequenced using the DYEnamic ET Terminator Cycle Sequencing kit (Amersham Pharmacia) and the ABI Prism 310 automatic sequencer (Perkin Elmer). As sequencing of the HA1 regions was carried out in two sections, two primer sets were prepared: 5'-CTACTCATGGTAGTAACATCC-3' (nt 5272) and 3'-TGGGAAGCCACCAATCTGAGAAAC-5' (nt 774751) for the first half and 5'-ACCTCAGGATCTTGCCCTAACG-3' (positions 493514) and 3'-TGTGTATCCGTGCCAACCTGCAAT-5' (positions 11941171) for the second half.
mAb 3A12 was obtained by immunizing mice with a 5H4-nonreacting strain, as described above. Therefore, the epitopes of mAbs 3A12 and 5H4 should be distinct. However, in the competitive assay, excess amount of one mAb blocked almost 50 % of the binding of the other mAb to the virus (data not shown). To analyse the epitope sites precisely, further experiments with escape mutants were performed. We reported previously that two escape mutants (B/Kadoma/122/1999-V1 and -V2) were induced with mAb 5H4 and that they showed a single amino acid substitution at residue 149 (ArgGly) or 141 (Gly
Arg) of the HA1 protein, respectively (Nakagawa et al., 2001b
). In addition, nine mutants (B/Kadoma/122/1999-V3 to -V11) were induced with mAb 3A12 by incubating the same strain, B/Kadoma/122/1999. Table 1
shows the results of the HI and NT tests. 5H4-escape mutants did not react with either 5H4 or 3A12. Among 3A12-escape mutants, six (122-V4, -V7, -V8, -V9, -V10 and -V11) did not react with mAb 5H4. In contrast, three 3A12-escape mutants (122-V3, -V5 and -V6) reacted well with mAb 5H4. Analysis of nucleotide sequences clarified that all escape mutants had single point mutations in the HA gene that correspond to single amino acid substitutions. All of the 5H4-nonreacting mutants showed the same substitution observed with B/Kadoma/122/1999-V2 (Gly141
Arg). Two of the 5H4-reacting mutants (B/Kadoma/122/1999-V3 and -V6) showed the substitution at residue 148 (Ser
Gly) and the other (B/Kadoma/122/1999-V5) at residue 147 (Thr
Ile) (Table 1
).
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Amino acid sequences of the HA1 polypeptides of the influenza B virus strains were compared to those of A/Aichi/2/68 and numbered according to the A/Aichi sequence for ease of reference to the structure of the H3 HA molecule of influenza A virus (Berton et al., 1984; Krystal et al., 1982
). With this method, amino acid 141 is referred to as 137, 147 as 142, 148 as 143 and 149 as 144. Fig. 1
shows the three-dimensional structure of the influenza A virus H3 HA molecule, as determined by Wilson et al. (1981)
. Though the identified amino acid substitutions of influenza B virus are only predicted on the basis of sequence alignments, all of the amino acid residues above are suggested to be close to each other in the HA loop. The neutralizing epitopes of mAbs 5H4 and 3A12 are either overlapping or situated close enough to affect each other. The data of the competitive assay that the binding of one mAb was partially blocked by the other supported this idea. The loop, as well as the tip, has been reported as one of the most antigenic sites for the influenza virus HA protein. The variation in these regions is often observed in naturally occurring or laboratory-induced antigenic variants (Berton et al., 1984
; Berton & Webster, 1985
; Cleveland et al., 1997
; Daniels et al., 1987
; Lambkin et al., 1994
; Luoh et al., 1992
; Wiley et al., 1981
). In contrast to the B/Victoria group-specific epitope site being situated in the tip (Nakagawa et al., 2001a
), those of B/Yamagata strains are located in the loop. In addition, there exist more than two independent neutralizing sites in the loop. This is based on the fact that 5H4-nonreacting strains isolated from clinical specimens reacted well with mAb 3A12, as did three of the 3A12-escape mutants react with mAb 5H4 (Table 1
). Finally, it is of note that most 5H4- or 3A12-escape mutants failed to react well with polyclonal ferret antibody. Therefore, the loop plays an important role(s) in the neutralization of B/Yamagata group strains.
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ACKNOWLEDGEMENTS |
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Received 6 August 2002;
accepted 3 January 2003.