Variation of the Conserved Neutralizing Epitope in Influenza B Virus Victoria Group Isolates in Japan
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微生物临床杂志 2005年第8期
Department of Microbiology, Kobe Institute of Health, 4-6, Minatojima-nakamachi, Chuo-ku, Kobe, 650-0046 Japan
Department of Infectious Diseases, Osaka Prefectural Institute of Public Health, 1-3-69, Nakamichi, Higashinari-ku, Osaka, 537-0025 Japan
ABSTRACT
For almost 20 years, the neutralizing-epitope site specific for influenza B virus Victoria group isolates was conserved at the "tip" of the hemagglutinin molecule; however, it was not detected in half of the isolates from the 2002-2003 epidemic in Japan. Amino acid substitutions (D164E or N165K) were observed at the "tip," and the epitope was altered. The viral antigenicities were affected, and human antibodies did not substantially inhibit the hemagglutination in the hemagglutination inhibition tests. It is suspected that such variants will be important in future epidemics.
TEXT
There are influenza epidemics every winter in Japan, as in European and North American countries. Over the past 20 years, influenza B virus has caused epidemics in humans, as have the H1 and H3 subtypes of influenza A virus. Recent isolates of influenza B virus strains are divided into two large lineages in a phylogenetic tree: one group is represented by B/Victoria/2/87 and the other by B/Yamagata/16/88 (2). B/Victoria group strains were dominant in the 1980s, while B/Yamagata strains became dominant in the early 1990s (2, 4, 5, 12, 14, 15). In Japan, B/Victoria reemerged and was epidemic in the 1996-1997 season and then again in the 2002-2003 season. In hemagglutination inhibition (HI) tests, all of the 2002-2003 isolates reacted poorly to the standard immune ferret serum prepared against the 1997 isolate. The new isolates obtained oligosaccharide chains near the receptor binding region (11). In addition, half of the isolates did not react to B/Victoria-specific neutralizing monoclonal antibody (MAb), which suggested the alternation of the long-conserved neutralizing epitope. We investigated the variation of the amino acid sequence of the hemagglutinin (HA) molecule, along with the influence on viral antigenicities.
MAbs 10B8 and 10D7 were obtained from mice immunized with B/Victoria group strains B/Nagasaki/1/87 (6-8) and B/Kobe/1/2002, respectively. Ascitic fluids of mice injected with hybridoma cells were used as sources of MAbs. Every year, standard sera are provided by the National Institute of Infectious Diseases, Tokyo, Japan, including the immune ferret serum against B/Shangdong/7/97 for the 2002-2003 and 2003-2004 seasons and the hyperimmune sheep serum against B/Brisbane/32/2002 for the 2004-2005 season. Human sera, collected from six individual adults before the beginning of the 2002-2003 season, were utilized. Escape mutants were induced by incubating the strains with MAbs, by modifying the method previously described (1, 3, 8, 10). Briefly, 1 x 105 focus-forming units/ml virus was incubated for 1 h at 30°C in the presence of 10 μl of MAb. The virus-MAb mixture was inoculated to MDCK cells in 24-well plates and incubated at 35°C for 3 days. HI tests with MAb on each well were performed separately to identify the escape mutants. The results of the HI tests are expressed as the reciprocals of the antibody dilutions (13). Direct sequencing of the viral nucleotide was performed as described previously (7-11). Briefly, reverse transcriptase PCR products were sequenced with a DYEnamic ET terminator cycle sequencing kit (Amersham Pharmacia, Piscataway, NJ) and were analyzed by an ABI Prism 310 automatic sequencer (Perkin Elmer, Foster City, CA).
Formerly, we reported that MAb 10B8 possessed neutralizing and HI activities against all B/Victoria isolates in the 1996-1997 season in Osaka Prefecture, Japan, along with the representative strains isolated during the previous seasons (6, 8). There are two immunodominant antigenic sites in influenza B virus HA, which correspond to antigenic site A (the "loop") and site B (the "tip") of influenza AH3 virus HA (1). The epitope site of 10B8 was at the "tip" of the HA1 molecule, which had been conserved in B/Victoria isolates since the mid-1980s (8). After the 1996-1997 season, B/Victoria was isolated sporadically. At the end of the 2001-2002 season, five B/Victoria isolates (DDBJ accession no. AB081570, AB081571, AB083182, AB083183, and AB196144) were obtained in Kobe City, Japan, which is situated 20 km to the west of Osaka Prefecture. They did not react to 10B8 in the HI tests (Fig. 1). Genetic analysis clarified a single amino acid substitution at the "tip" (D164E) (Fig. 1). To obtain MAbs that possess HI activities against the new isolates, we immunized mice with B/Kobe/1/2002. Thereafter, MAb 10D7 was established. Though HI titers of 10D7 were lower than those of 10B8, 10D7 reacted to the 1996-1997 and the 2001-2002 isolates to almost the same degree. Genetic analysis of laboratory-induced escape mutants (B/Kobe/1/2002-V1 and -V2) clarified that the epitope of 10D7 situates also at the "tip" (DDBJ accession no. AB196142 and AB196143) (Fig. 1).
In the 2002-2003 season, there was an epidemic of B/Victoria and 33 strains were isolated from clinical specimens in Kobe City. However, they were divided into three groups according to the HI titers to MAbs 10B8 and 10D7 (Fig. 1). In HI tests, hemagglutination of group 1 strains (14 strains) was inhibited by both MAbs, similarly to the 1996-1997 isolates, while hemagglutination of group 2 strains (18 strains) was inhibited only by 10D7, similarly to the 2002-2003 isolates. There was only one strain in group 3, and its hemagglutination was not inhibited by either MAb. Genetic analysis clarified that group 1 isolates (represented by B/Kobe/1/2003, B/Kobe/25/2003, and B/Kobe/26/2003 [DDBJ accession no. AB126835, AB126839, and AB126840, respectively]) had the same amino acid sequence at the "tip" as the old isolates, while group 2 isolates (represented by B/Kobe/2/2003, B/Kobe/3/2003, and B/Kobe/4/2003 [DDBJ accession no. AB126836, AB126837, and AB126838, respectively]) had the same sequence as the 2001-2002 isolates (D164E). B/Kobe/28/2003 (DDBJ accession no. AB126841) showed another amino acid substitution at the "tip" (N165K).
In contrast to the antigenicities of influenza A virus, those of influenza B virus were relatively stable (4, 5). For example, the neutralizing-epitope site at the "tip," strictly specific for B/Victoria, had been conserved since the 1980s (6). It is suggested that established and accumulated human immunity began to induce naturally occurring antigenic variants. As reported previously, the first B/Victoria variants after the reemergence appeared in the 1996-1997 season (7) and became major in the 2002-2003 season (11). One amino acid substitution in the HA1 region (A199T) created an N-linked glycosylation site near the receptor binding site, and the virus gained extra oligosaccharide chains (11). Consequently, all 2002-2003 isolates reacted poorly to the immune ferret serum against B/Shangdong/7/97. Then, the second variants appeared already in the 2002-2003 season. They were detected since they reacted heterogeneously to MAbs 10B8 and 10D7. They showed an amino acid substitution at the conserved neutralizing epitope (Fig. 1). Therefore, their antigenicities were further analyzed (Table 1). The serum against B/Brisbane/32/2002 was provided as the standard serum for the 2004-2005 season. Group 1 isolates reacted to it well. The HI titers of group 1 strains were as high as 1,280, while those of group 2 strains were 320 and that of the group 3 strain was 40. The idea was confirmed further with data from adult human sera collected before the beginning of the 2002-2003 season. In HI tests, five of six human sera (designated A through F in Table 1) showed HI titers of 40 to 160 to group 1 isolates, which were as high as those to the classical strain isolated in the 1996-1997 season. No human serum inhibited hemagglutination of group 2 and 3 isolates as much. Thus, their antigenicities are distinct from those of group 1 isolates. Single amino acid substitutions at the "tip" play important roles. It is suspected that established human antibodies, raised against B/Victoria isolates in previous seasons, are not able to prevent infection by the new variants.
The conserved neutralizing epitope of B/Victoria was finally altered, and the new variants appeared in the 2002-2003 season. These new variants may be important in B/Victoria epidemics in the near future, as the former variants from the 1996-1997 season with an extra oligosaccharide chain became major in the 2002-2003 season (11). Then, the scale of the epidemics will be positively modified. This information is expected to benefit public health management, especially from the perspective of selecting suitable strains for vaccines.
ACKNOWLEDGMENTS
We thank T. Iwamoto for his valuable advice on nucleotide sequencing.
REFERENCES
Berton, M. T., C. W. Naeve, and R. G. Webster. 1984. Antigenic structure of the influenza B virus hemagglutinin: nucleotide sequence analysis of antigenic variants selected with monoclonal antibodies. J. Virol. 52:919-927.
Kanegae, Y., S. Sugita, A. Endo, M. Ishida, S. Senya, K. Osako, K. Nerome, and A. Oya. 1990. Evolutionary pattern of the hemagglutinin gene of influenza B viruses isolated in Japan: cocirculating lineages in the same epidemic season. J. Virol. 64:2860-2865.
Lambkin, R., L. McLain, S. E. Jones, S. L. Aldridge, and N. J. Dimmock. 1994. Neutralization escape mutants of type A influenza virus are readily selected by antisera from mice immunized with whole virus: a possible mechanism for antigenic drift. J. Gen. Virol. 75:3493-3502.
Lindstrom, S. E., Y. Hiromoto, H. Nishimura, T. Saito, R. Nerome, and K. Nerome. 1999. Comparative analysis of evolutionary mechanisms of the hemagglutinin and three internal protein genes of influenza B virus: multiple cocirculating lineages and frequent reassortment of the NP, M, and NS genes. J. Virol. 73:4413-4426.
McCullers, J. A., G. C. Wang, S. He, and R. Webster. 1999. Reassortment and insertions-deletion are strategies for the evolution of influenza B viruses in nature. J. Virol. 73:7343-7348.
Nakagawa, N., A. Maeda, T. Kase, R. Kubota, and Y. Okuno. 1999. Rapid detection and identification of two lineages of influenza B strains with monoclonal antibodies. J. Virol. Methods 79:113-120.
Nakagawa, N., R. Kubota, A. Maeda, T. Nakagawa, and Y. Okuno. 2000. Heterogeneity of influenza B virus strains in one epidemic season differentiated by monoclonal antibodies and nucleotide sequences. J. Clin. Microbiol. 38:3467-3469.
Nakagawa, N., R. Kubota, T. Nakagawa, and Y. Okuno. 2001. Antigenic variants with amino acid deletions clarify a neutralizing epitope specific for influenza B virus Victoria group strains. J. Gen. Virol. 82:2169-2172.
Nakagawa, N., R. Kubota, S. Morikawa, T. Nakagawa, K. Baba, and Y. Okuno. 2001. Characterization of new epidemic strains of influenza B virus by using neutralizing monoclonal antibodies. J. Med. Virol. 65:745-750.
Nakagawa, N., R. Kubota, T. Nakagawa, and Y. Okuno. 2003. Neutralizing epitope specific for influenza B virus Yamagata group strains are in the "loop." J. Gen. Virol. 84:769-773.
Nakagawa, N., R. Kubota, A. Maeda, and Y. Okuno. 2004. Influenza B virus Victoria group with a new glycosylation site was epidemic in Japan in the 2002-2003 season. J. Clin. Microbiol. 42:3295-3297.
Nerome, R., Y. Hiromoto, S. Sugita, N. Tanabe, M. Ishida, M. Matsumoto, S. E. Lindstrom, T. Takahashi, and K. Nerome. 1998. Evolutionary characteristics of influenza B virus since its first isolation in 1940: dynamic circulation of deletion and insertion mechanism. Arch. Virol. 143:1569-1583.
Okuno, Y., K. Tanaka, K. Baba, A. Maeda, N. Kunita, and S. Ueda. 1990. Rapid focus reduction neutralization test of influenza A and B viruses in microtiter system. J. Clin. Microbiol. 28:1308-1313.
Rota, P., T. R. Wallis, M. W. Harmon, J. S. Rota, A. P. Kendal, and K. Nerome. 1990. Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983. Virology 175:59-68.
Yamashita, M., M. Krystal, W. M. Fitch, and P. Palese. 1988. Influenza B virus evolution: co-circulating lineages and comparison of evolutionary pattern with those of influenza A and C viruses. Virology 163:112-122.(Naoko Nakagawa, Ritsuko K)
Department of Infectious Diseases, Osaka Prefectural Institute of Public Health, 1-3-69, Nakamichi, Higashinari-ku, Osaka, 537-0025 Japan
ABSTRACT
For almost 20 years, the neutralizing-epitope site specific for influenza B virus Victoria group isolates was conserved at the "tip" of the hemagglutinin molecule; however, it was not detected in half of the isolates from the 2002-2003 epidemic in Japan. Amino acid substitutions (D164E or N165K) were observed at the "tip," and the epitope was altered. The viral antigenicities were affected, and human antibodies did not substantially inhibit the hemagglutination in the hemagglutination inhibition tests. It is suspected that such variants will be important in future epidemics.
TEXT
There are influenza epidemics every winter in Japan, as in European and North American countries. Over the past 20 years, influenza B virus has caused epidemics in humans, as have the H1 and H3 subtypes of influenza A virus. Recent isolates of influenza B virus strains are divided into two large lineages in a phylogenetic tree: one group is represented by B/Victoria/2/87 and the other by B/Yamagata/16/88 (2). B/Victoria group strains were dominant in the 1980s, while B/Yamagata strains became dominant in the early 1990s (2, 4, 5, 12, 14, 15). In Japan, B/Victoria reemerged and was epidemic in the 1996-1997 season and then again in the 2002-2003 season. In hemagglutination inhibition (HI) tests, all of the 2002-2003 isolates reacted poorly to the standard immune ferret serum prepared against the 1997 isolate. The new isolates obtained oligosaccharide chains near the receptor binding region (11). In addition, half of the isolates did not react to B/Victoria-specific neutralizing monoclonal antibody (MAb), which suggested the alternation of the long-conserved neutralizing epitope. We investigated the variation of the amino acid sequence of the hemagglutinin (HA) molecule, along with the influence on viral antigenicities.
MAbs 10B8 and 10D7 were obtained from mice immunized with B/Victoria group strains B/Nagasaki/1/87 (6-8) and B/Kobe/1/2002, respectively. Ascitic fluids of mice injected with hybridoma cells were used as sources of MAbs. Every year, standard sera are provided by the National Institute of Infectious Diseases, Tokyo, Japan, including the immune ferret serum against B/Shangdong/7/97 for the 2002-2003 and 2003-2004 seasons and the hyperimmune sheep serum against B/Brisbane/32/2002 for the 2004-2005 season. Human sera, collected from six individual adults before the beginning of the 2002-2003 season, were utilized. Escape mutants were induced by incubating the strains with MAbs, by modifying the method previously described (1, 3, 8, 10). Briefly, 1 x 105 focus-forming units/ml virus was incubated for 1 h at 30°C in the presence of 10 μl of MAb. The virus-MAb mixture was inoculated to MDCK cells in 24-well plates and incubated at 35°C for 3 days. HI tests with MAb on each well were performed separately to identify the escape mutants. The results of the HI tests are expressed as the reciprocals of the antibody dilutions (13). Direct sequencing of the viral nucleotide was performed as described previously (7-11). Briefly, reverse transcriptase PCR products were sequenced with a DYEnamic ET terminator cycle sequencing kit (Amersham Pharmacia, Piscataway, NJ) and were analyzed by an ABI Prism 310 automatic sequencer (Perkin Elmer, Foster City, CA).
Formerly, we reported that MAb 10B8 possessed neutralizing and HI activities against all B/Victoria isolates in the 1996-1997 season in Osaka Prefecture, Japan, along with the representative strains isolated during the previous seasons (6, 8). There are two immunodominant antigenic sites in influenza B virus HA, which correspond to antigenic site A (the "loop") and site B (the "tip") of influenza AH3 virus HA (1). The epitope site of 10B8 was at the "tip" of the HA1 molecule, which had been conserved in B/Victoria isolates since the mid-1980s (8). After the 1996-1997 season, B/Victoria was isolated sporadically. At the end of the 2001-2002 season, five B/Victoria isolates (DDBJ accession no. AB081570, AB081571, AB083182, AB083183, and AB196144) were obtained in Kobe City, Japan, which is situated 20 km to the west of Osaka Prefecture. They did not react to 10B8 in the HI tests (Fig. 1). Genetic analysis clarified a single amino acid substitution at the "tip" (D164E) (Fig. 1). To obtain MAbs that possess HI activities against the new isolates, we immunized mice with B/Kobe/1/2002. Thereafter, MAb 10D7 was established. Though HI titers of 10D7 were lower than those of 10B8, 10D7 reacted to the 1996-1997 and the 2001-2002 isolates to almost the same degree. Genetic analysis of laboratory-induced escape mutants (B/Kobe/1/2002-V1 and -V2) clarified that the epitope of 10D7 situates also at the "tip" (DDBJ accession no. AB196142 and AB196143) (Fig. 1).
In the 2002-2003 season, there was an epidemic of B/Victoria and 33 strains were isolated from clinical specimens in Kobe City. However, they were divided into three groups according to the HI titers to MAbs 10B8 and 10D7 (Fig. 1). In HI tests, hemagglutination of group 1 strains (14 strains) was inhibited by both MAbs, similarly to the 1996-1997 isolates, while hemagglutination of group 2 strains (18 strains) was inhibited only by 10D7, similarly to the 2002-2003 isolates. There was only one strain in group 3, and its hemagglutination was not inhibited by either MAb. Genetic analysis clarified that group 1 isolates (represented by B/Kobe/1/2003, B/Kobe/25/2003, and B/Kobe/26/2003 [DDBJ accession no. AB126835, AB126839, and AB126840, respectively]) had the same amino acid sequence at the "tip" as the old isolates, while group 2 isolates (represented by B/Kobe/2/2003, B/Kobe/3/2003, and B/Kobe/4/2003 [DDBJ accession no. AB126836, AB126837, and AB126838, respectively]) had the same sequence as the 2001-2002 isolates (D164E). B/Kobe/28/2003 (DDBJ accession no. AB126841) showed another amino acid substitution at the "tip" (N165K).
In contrast to the antigenicities of influenza A virus, those of influenza B virus were relatively stable (4, 5). For example, the neutralizing-epitope site at the "tip," strictly specific for B/Victoria, had been conserved since the 1980s (6). It is suggested that established and accumulated human immunity began to induce naturally occurring antigenic variants. As reported previously, the first B/Victoria variants after the reemergence appeared in the 1996-1997 season (7) and became major in the 2002-2003 season (11). One amino acid substitution in the HA1 region (A199T) created an N-linked glycosylation site near the receptor binding site, and the virus gained extra oligosaccharide chains (11). Consequently, all 2002-2003 isolates reacted poorly to the immune ferret serum against B/Shangdong/7/97. Then, the second variants appeared already in the 2002-2003 season. They were detected since they reacted heterogeneously to MAbs 10B8 and 10D7. They showed an amino acid substitution at the conserved neutralizing epitope (Fig. 1). Therefore, their antigenicities were further analyzed (Table 1). The serum against B/Brisbane/32/2002 was provided as the standard serum for the 2004-2005 season. Group 1 isolates reacted to it well. The HI titers of group 1 strains were as high as 1,280, while those of group 2 strains were 320 and that of the group 3 strain was 40. The idea was confirmed further with data from adult human sera collected before the beginning of the 2002-2003 season. In HI tests, five of six human sera (designated A through F in Table 1) showed HI titers of 40 to 160 to group 1 isolates, which were as high as those to the classical strain isolated in the 1996-1997 season. No human serum inhibited hemagglutination of group 2 and 3 isolates as much. Thus, their antigenicities are distinct from those of group 1 isolates. Single amino acid substitutions at the "tip" play important roles. It is suspected that established human antibodies, raised against B/Victoria isolates in previous seasons, are not able to prevent infection by the new variants.
The conserved neutralizing epitope of B/Victoria was finally altered, and the new variants appeared in the 2002-2003 season. These new variants may be important in B/Victoria epidemics in the near future, as the former variants from the 1996-1997 season with an extra oligosaccharide chain became major in the 2002-2003 season (11). Then, the scale of the epidemics will be positively modified. This information is expected to benefit public health management, especially from the perspective of selecting suitable strains for vaccines.
ACKNOWLEDGMENTS
We thank T. Iwamoto for his valuable advice on nucleotide sequencing.
REFERENCES
Berton, M. T., C. W. Naeve, and R. G. Webster. 1984. Antigenic structure of the influenza B virus hemagglutinin: nucleotide sequence analysis of antigenic variants selected with monoclonal antibodies. J. Virol. 52:919-927.
Kanegae, Y., S. Sugita, A. Endo, M. Ishida, S. Senya, K. Osako, K. Nerome, and A. Oya. 1990. Evolutionary pattern of the hemagglutinin gene of influenza B viruses isolated in Japan: cocirculating lineages in the same epidemic season. J. Virol. 64:2860-2865.
Lambkin, R., L. McLain, S. E. Jones, S. L. Aldridge, and N. J. Dimmock. 1994. Neutralization escape mutants of type A influenza virus are readily selected by antisera from mice immunized with whole virus: a possible mechanism for antigenic drift. J. Gen. Virol. 75:3493-3502.
Lindstrom, S. E., Y. Hiromoto, H. Nishimura, T. Saito, R. Nerome, and K. Nerome. 1999. Comparative analysis of evolutionary mechanisms of the hemagglutinin and three internal protein genes of influenza B virus: multiple cocirculating lineages and frequent reassortment of the NP, M, and NS genes. J. Virol. 73:4413-4426.
McCullers, J. A., G. C. Wang, S. He, and R. Webster. 1999. Reassortment and insertions-deletion are strategies for the evolution of influenza B viruses in nature. J. Virol. 73:7343-7348.
Nakagawa, N., A. Maeda, T. Kase, R. Kubota, and Y. Okuno. 1999. Rapid detection and identification of two lineages of influenza B strains with monoclonal antibodies. J. Virol. Methods 79:113-120.
Nakagawa, N., R. Kubota, A. Maeda, T. Nakagawa, and Y. Okuno. 2000. Heterogeneity of influenza B virus strains in one epidemic season differentiated by monoclonal antibodies and nucleotide sequences. J. Clin. Microbiol. 38:3467-3469.
Nakagawa, N., R. Kubota, T. Nakagawa, and Y. Okuno. 2001. Antigenic variants with amino acid deletions clarify a neutralizing epitope specific for influenza B virus Victoria group strains. J. Gen. Virol. 82:2169-2172.
Nakagawa, N., R. Kubota, S. Morikawa, T. Nakagawa, K. Baba, and Y. Okuno. 2001. Characterization of new epidemic strains of influenza B virus by using neutralizing monoclonal antibodies. J. Med. Virol. 65:745-750.
Nakagawa, N., R. Kubota, T. Nakagawa, and Y. Okuno. 2003. Neutralizing epitope specific for influenza B virus Yamagata group strains are in the "loop." J. Gen. Virol. 84:769-773.
Nakagawa, N., R. Kubota, A. Maeda, and Y. Okuno. 2004. Influenza B virus Victoria group with a new glycosylation site was epidemic in Japan in the 2002-2003 season. J. Clin. Microbiol. 42:3295-3297.
Nerome, R., Y. Hiromoto, S. Sugita, N. Tanabe, M. Ishida, M. Matsumoto, S. E. Lindstrom, T. Takahashi, and K. Nerome. 1998. Evolutionary characteristics of influenza B virus since its first isolation in 1940: dynamic circulation of deletion and insertion mechanism. Arch. Virol. 143:1569-1583.
Okuno, Y., K. Tanaka, K. Baba, A. Maeda, N. Kunita, and S. Ueda. 1990. Rapid focus reduction neutralization test of influenza A and B viruses in microtiter system. J. Clin. Microbiol. 28:1308-1313.
Rota, P., T. R. Wallis, M. W. Harmon, J. S. Rota, A. P. Kendal, and K. Nerome. 1990. Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983. Virology 175:59-68.
Yamashita, M., M. Krystal, W. M. Fitch, and P. Palese. 1988. Influenza B virus evolution: co-circulating lineages and comparison of evolutionary pattern with those of influenza A and C viruses. Virology 163:112-122.(Naoko Nakagawa, Ritsuko K)