Division of Virology, Department Public Health, Osaka Prefectural Institute of Public Health, 3-69 1-Chome, Nakamichi, Higashinari-ku, Osaka 537-0025, Japan1
Department of Clinical Pathology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686, Japan2
Author for correspondence: Naoko Nakagawa. Present address: Department of Parasitic Agents, Kobe Institute of Health, 4-6 Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan. Fax +81 78 302 0894. e-mail kih-info-1{at}mse.biglobe.ne.jp
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Abstract |
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In order to study neutralizing epitopes that are specific for the B/Victoria group strains, antigenic variants were induced by incubating B/Osaka/983/97 with MAb 10B8 or 8E6. B/Osaka/983/97 is a B/Victoria group strain with which MAbs 10B8 and 8E6 show strong reactivity in HI and NT tests (Nakagawa et al., 2000 ). B/Osaka/983/97 was cloned by the plaque method and then diluted to 1x106 f.f.u./ml. As the source of MAbs, ascitic fluids of mice injected with hybridoma cells were utilized. Table 1
shows the HI and NT test results expressed as a reciprocal of the antibody dilution (Okuno et al., 1990
). A 2 ml sample of virus was incubated for 1 h at 30 °C in the presence of either 125 µl of MAb 8E6 or 62·5 µl of MAb 10B8. The virusMAb mixture was inoculated into 10-day-old embryonated chicken eggs (100 µl per egg) and the allantoic fluid was harvested after 60 h of incubation at 35 °C (Berton et al., 1984
; Lambkin et al., 1994
). HI and NT tests on the allantoic fluid of each egg were performed separately with each MAb in order to identify antigenic variants. Finally, eight variants (V18) of B/Osaka/983/97 were obtained in the presence of MAb 10B8 and two variants (M1 and M2) were obtained in the presence of MAb 8E6. Table 1
shows a summary of the three tests. None of the eight 10B8-induced variants reacted with MAb 10B8 in subsequent PAP staining, HI or NT tests. Although MAb 8E6 did not react with V3 and V8 in any of the three tests, reactivity with the other six variants was evident. The degree of the reactivity was, however, varied. Similarly, MAb 8E6 did not react with 8E6-induced variants M1 and M2, but, in contrast, MAb 10B8 did react with M1 and M2 in each of the three tests at levels similar to those found for parental strain B/Osaka/983/97 (Table 1
).
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The 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 164 is referred to as 158, 165 as 159, 170 as 160, 203 as 193 and 241 as 225, respectively. Fig. 1
shows the three-dimensional structure of the influenza A virus H3 HA molecule, as determined by Wilson et al. (1981)
. Residues 158, 159, 160, 193 and 225 are shown. In contrast to influenza A virus, influenza B virus strains possess four extra amino acids at the top of the HA molecule, which correspond to residues 166169. Therefore, residues 165 and 170 are referred to as residues 159 and 160 and are situated next to each other. In our previous study with naturally occurring antigenic variants of the B/Victoria group strains (Nakagawa et al., 2000
), we reported that substitutions of amino acids at residues 197 (Ser
Lys or Asn) and 199 (Ala
Asn or Thr) modulated the neutralizing epitope for MAb 8E6. Therefore, in Fig. 1
, we marked both amino acid residues and referred to them as residues 187 and 189, respectively. However, certain amino acid substitutions at residues 197 (Asp
Ser) and 199 (Ala
Thr or Ile) did not alter MAb 8E6 neutralization. The same phenomenon is observed with the amino acid substitution at residue 203, in that V3 and V8 (Lys203
Thr) are not recognized by MAb 8E6 in the three tests, while V5 (Lys203
Asn) is recognized and neutralized by MAb 8E6, similar to the parental strain (Table 1
). Therefore, it is unclear whether these residues constitute an epitope for the MAb or whether only some of the residues constitute an epitope and the remainder influence it. Furthermore, differences in the three-dimensional structure of the influenza A and B virus HA molecules should be considered. However, in Fig. 1(B)
, it is clear that the epitopes for the two MAbs are situated close to each other, with the amino acid residue 203 (pink) in the middle. Residues 164, 165, 170 and 203 constitute or influence the epitope for MAb 10B8 and residues 197, 199, 203 and 241 constitute or influence the epitope for MAb 8E6. The epitope for MAb 10B8 is located at the very tip of the HA molecule, while that for MAb 8E6 is in the proximity of the proposed receptor-binding pocket (Weis et al., 1988
). These sites are close enough to affect each other and, therefore, it is understandable that MAb 8E6 reacted with 10B8-induced variants in the HI and NT tests (Table 1
).
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The neutralizing epitopes of influenza B virus Victoria group strains were analysed. The fact that single amino acid deletions altered the antigenicity of the HA molecule of influenza virus clarified that the epitope for MAb 10B8 is strictly specific for the B/Victoria group strains.
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Acknowledgments |
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Footnotes |
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References |
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Received 20 February 2001;
accepted 15 May 2001.