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Change in receptor-binding specificity of recent human influenza A viruses (H3N2) affects recognition of the receptor on MDCK cells
International Congress Series 1263 (2004) 472 – 475
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Change in receptor-binding specificity of recent human influenza A viruses (H3N2) affects recognition of the receptor on MDCK cells E. Nobusawa *, K. Nakajima, S. Kozuka, H. Ishihara Department of Virology, Nagoya City University, Medical School, Mizuho-cho, Mizuho-ku, Kawasumi 1, Nagoya City, 467-8601, Japan
Abstract. Influenza A viruses (H3N2) isolated in MDCK cells after 1992/1993 influenza season have changed in that these viruses agglutinate human red blood cells (HRBC) but not chicken RBC (CRBC) (Ch virus). The yield of Ch virus in MDCK cells was comparable with that of the virus that could agglutinate CRBC (Ch+ virus); however, binding assays revealed reduced binding of the Ch virus to MDCK cells. Replacement of the HA gene of A/Aichi/2/68 (Ch+) with that of A/Aichi/30/97 (Ch ) led to a reduction in binding of the resultant virus to MDCK cells. Ch viruses, however, efficiently bound to derivative MDCK cells resialylated with SAa2,6Galh1,4GlcNAc, indicating that HA of Ch viruses distinguished the sialyloligosaccharides on native MDCK cells from those on resialylated MDCK cells. Ch viruses exhibited limited sensitivity to NA-specific inhibitor in plaque reduction assays using MDCK cells, despite the fact that NA activity of the Ch viruses was susceptible to the drug. These findings showed that change in the receptor-binding specificity of Ch viruses affected the NA-dependent virus released from MDCK cells. D 2003 Elsevier B.V. All rights reserved. Keywords: Hemagglutinin; Receptor binding; NA inhibitor
1. Introduction The receptor-binding specificity of human influenza A viruses (H3N2) isolated in MDCK cells after the 1992/1993 influenza season has changed in that these viruses (Ch ) agglutinate human red blood cells (HRBC) but not chicken RBC (CRBC) [1,2]. This change in receptor-binding specificity of Ch viruses has been observed among the circulating human H3N2 viruses up to this time. After the development of a potent NA inhibitor in 1993 [3], several resistant variants against the inhibitors were obtained in vitro. Some of the resistant viruses with amino acid
* Corresponding author. Tel.: +81-52-853-8190; fax: +81-52-853-3638. E-mail address: [email protected] (E. Nobusawa). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.148
E. Nobusawa et al. / International Congress Series 1263 (2004) 472–475
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changes in HA could release from the infected cells in the presence of the inhibitor [4 –7]. Moreover, several authors demonstrated a reduced sensitivity of recent clinical isolates to NA inhibitors in MDCK cells, even though their NA activity was susceptible to the drug [8– 12]. The aim of this study was (i) to elucidate how changing the receptor-binding specificity of Ch viruses would affect their binding efficiency to MDCK cells and (ii) to examine the effect of this change on NA dependency at virus release. 2. Virus growth in MDCK cells To examine the effect of the change in receptor-binding specificity of Ch viruses on the virus growth, growth ability of seven Ch viruses isolated during 1992 –1999 was compared with that of the viruses, which were isolated before 1992 and agglutinate CRBC (Ch+ virus), using MDCK cells. The plaque formation ability of the Ch viruses was comparable with that of Ch+ viruses. Furthermore, two or three passages of the viruses in MDCK cells did not affect the agglutination ability of Ch viruses against CRBC; that is, Ch viruses could not agglutinate CRBC. Recently, Romanova et al. [13] reported that recent human influenza viruses (H3N2) isolated in MDCK cells cultivated in the medium with 2% fetal calf serum (FCS) were able to agglutinate CRBC. In our study, Ch viruses were grown in MDCK cells cultivated without FCS. The discrepancy of the hemagglutination ability against CRBC between their viruses and Ch viruses might be ascribed to the different cultivation condition of MDCK cells. Although the growth ability of Ch viruses in MDCK cells was similar to that of Ch+ viruses, we have compared the binding efficiency of these viruses with MDCK cells. 3. Binding efficiency of viruses to native and derivative MDCK cells We have examined the binding efficiency of the viruses to native and derivative MDCK cells. AI/68 and 30/97, respective representatives of Ch+ and Ch viruses, and cl-236, a reassortant virus having HA gene of 30/97 and all other genes of AI/68, were examined in the immunofluorescence assay. After the adsorption of each of the viruses to MDCK cells on ice for 1 h, removal of unadsorbed viruses was followed by 6 h incubation at 37 jC. After the incubation, virus NP was immunostained. Among native cells, the number of NP positive cells infected with 30/97 and cl-236 was about 20% that of cells infected with AI/68. The other binding assay using radiolabeled viruses also showed that the percentage of cells with Ch viruses on their surface was approximately 26% of the amount of AI/68. These results indicated that the binding efficiency of Ch virus to native MDCK cells was lower than that of Ch+ virus. If sialic acids on native MDCK cells were digested by sialidase from Arthrobacter ureafaciens, both types of viruses hardly bound to the cells. However, if the cells were resialylated by a2,6-sialyltransferase after the desialydation, both Ch+ and Ch viruses efficiently bound to the cells, despite the amount of sialic acids on the resialylated cells being smaller than those on native cells. These facts suggested that Ch viruses preferred sialic acids on the cells resialylated with SAa2,6Galh1,4GlcNAc sequence but not those on native MDCK cells.
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E. Nobusawa et al. / International Congress Series 1263 (2004) 472–475
4. Virus growth in MDCK cells in the presence of zanamivir To elucidate how the weak binding of Ch virus to MDCK cells relates to NA dependency at virus release from host cells, we examined the effect of an NA inhibitor, zanamivir (kindly provided by Glaxo Welcome Research and Development), on the growth of viruses in MDCK cells. Ch+, Ch and cl-236 were grown in MDCK cells in the presence of wide range of zanamivir. Ch+ viruses were sensitive to zanamivir in their growth in MDCK cells, while Ch and cl-236 viruses provided similar HA titer and plaque formation ability irrespective of the presence or absence of the inhibitor. However, NA activity of both types of viruses was sensitive to the drug. When NA inhibition assay was performed using fetuin as a substrate, 0.01 AM was a high-enough concentration to reduce NA activity to less than 50% of that without the drug. When small substrates, 3V-sialyllactose and 6V-sialyllactose, were used, 0.1-AM inhibitor was high enough to inhibit the NA activity of both types of viruses by 90% of that of the control. These results indicated that Ch viruses were resistant to the NA inhibitor in their growth in MDCK cells due to their decreased binding efficiency to MDCK cells. Acknowledgements We thank Dr. K. Shimizu for providing rabbit anti-A/USSR/72 serum. A Scientific Research Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan supported this work. References [1] E. Nobusawa, et al., Change in receptor-binding specificity of recent human influenza viruses (H3N2): a single amino acid change in hemagglutinin altered its recognition of sialyloligosaccharides, Virology 278 (2000) 587 – 596. [2] R. Medeiros, et al., Hemagglutinin residues of recent human A (H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes, Virology 289 (2001) 74 – 85. [3] M. von Itzstein, et al., Rational design of potent sialidase-based inhibitors of influenza virus replication, Nature 363 (1993) 418 – 423. [4] J.L. McKimm-Breschkin, et al., Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and zanamivir, Antimicrobial Agents and Chemotherapy 40 (1996) 40 – 46. [5] L.V. Gubareva, et al., Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor zanamivir, Journal of Virology 70 (1996) 1818 – 1827. [6] T.J. Blick, et al., The interaction of neuraminidase and hemagglutinin mutations in influenza virus in resistance to zanamivir, Virology 246 (1998) 95 – 103. [7] D.F. Smee, et al., Characterization of an influenza A (H3N2) virus resistant to the cyclopentane neuraminidase inhibitor RWJ-270201, Antiviral Research 52 (2001) 251 – 259. [8] F.G. Hayden, et al., Efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenza virus infections, The New England Journal of Medicine 337 (1997) 874 – 880. [9] A.S. Monto, et al., Efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenza A and B virus infections, Journal of Infectious Diseases 180 (1999) 254 – 261. [10] J.M. Barnett, et al., Zanamivir susceptibility monitoring and characterization of influenza virus clinical isolates obtained during phase II clinical efficacy studies, Antimicrobial Agents and Chemotherapy 44 (2000) 78 – 87.
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[11] M. Tisdale, Monitoring of viral susceptibility: new challenges with the development of influenza NA inhibitors, Reviews in Medical Virology 10 (2000) 45 – 55. [12] M. Zambon, F. Hayden, Position statement: global neuraminidase inhibitor susceptibility network, Antiviral Research 49 (2001) 147 – 156. [13] J. Romanova, et al., Distinct host range of influenza H3N2 virus isolates inVero and MDCK cells is determined by cell specific glycosylation pattern, Virology 307 (2003) 90 – 97.
www.ics-elsevier.com
Change in receptor-binding specificity of recent human influenza A viruses (H3N2) affects recognition of the receptor on MDCK cells E. Nobusawa *, K. Nakajima, S. Kozuka, H. Ishihara Department of Virology, Nagoya City University, Medical School, Mizuho-cho, Mizuho-ku, Kawasumi 1, Nagoya City, 467-8601, Japan
Abstract. Influenza A viruses (H3N2) isolated in MDCK cells after 1992/1993 influenza season have changed in that these viruses agglutinate human red blood cells (HRBC) but not chicken RBC (CRBC) (Ch virus). The yield of Ch virus in MDCK cells was comparable with that of the virus that could agglutinate CRBC (Ch+ virus); however, binding assays revealed reduced binding of the Ch virus to MDCK cells. Replacement of the HA gene of A/Aichi/2/68 (Ch+) with that of A/Aichi/30/97 (Ch ) led to a reduction in binding of the resultant virus to MDCK cells. Ch viruses, however, efficiently bound to derivative MDCK cells resialylated with SAa2,6Galh1,4GlcNAc, indicating that HA of Ch viruses distinguished the sialyloligosaccharides on native MDCK cells from those on resialylated MDCK cells. Ch viruses exhibited limited sensitivity to NA-specific inhibitor in plaque reduction assays using MDCK cells, despite the fact that NA activity of the Ch viruses was susceptible to the drug. These findings showed that change in the receptor-binding specificity of Ch viruses affected the NA-dependent virus released from MDCK cells. D 2003 Elsevier B.V. All rights reserved. Keywords: Hemagglutinin; Receptor binding; NA inhibitor
1. Introduction The receptor-binding specificity of human influenza A viruses (H3N2) isolated in MDCK cells after the 1992/1993 influenza season has changed in that these viruses (Ch ) agglutinate human red blood cells (HRBC) but not chicken RBC (CRBC) [1,2]. This change in receptor-binding specificity of Ch viruses has been observed among the circulating human H3N2 viruses up to this time. After the development of a potent NA inhibitor in 1993 [3], several resistant variants against the inhibitors were obtained in vitro. Some of the resistant viruses with amino acid
* Corresponding author. Tel.: +81-52-853-8190; fax: +81-52-853-3638. E-mail address: [email protected] (E. Nobusawa). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.148
E. Nobusawa et al. / International Congress Series 1263 (2004) 472–475
473
changes in HA could release from the infected cells in the presence of the inhibitor [4 –7]. Moreover, several authors demonstrated a reduced sensitivity of recent clinical isolates to NA inhibitors in MDCK cells, even though their NA activity was susceptible to the drug [8– 12]. The aim of this study was (i) to elucidate how changing the receptor-binding specificity of Ch viruses would affect their binding efficiency to MDCK cells and (ii) to examine the effect of this change on NA dependency at virus release. 2. Virus growth in MDCK cells To examine the effect of the change in receptor-binding specificity of Ch viruses on the virus growth, growth ability of seven Ch viruses isolated during 1992 –1999 was compared with that of the viruses, which were isolated before 1992 and agglutinate CRBC (Ch+ virus), using MDCK cells. The plaque formation ability of the Ch viruses was comparable with that of Ch+ viruses. Furthermore, two or three passages of the viruses in MDCK cells did not affect the agglutination ability of Ch viruses against CRBC; that is, Ch viruses could not agglutinate CRBC. Recently, Romanova et al. [13] reported that recent human influenza viruses (H3N2) isolated in MDCK cells cultivated in the medium with 2% fetal calf serum (FCS) were able to agglutinate CRBC. In our study, Ch viruses were grown in MDCK cells cultivated without FCS. The discrepancy of the hemagglutination ability against CRBC between their viruses and Ch viruses might be ascribed to the different cultivation condition of MDCK cells. Although the growth ability of Ch viruses in MDCK cells was similar to that of Ch+ viruses, we have compared the binding efficiency of these viruses with MDCK cells. 3. Binding efficiency of viruses to native and derivative MDCK cells We have examined the binding efficiency of the viruses to native and derivative MDCK cells. AI/68 and 30/97, respective representatives of Ch+ and Ch viruses, and cl-236, a reassortant virus having HA gene of 30/97 and all other genes of AI/68, were examined in the immunofluorescence assay. After the adsorption of each of the viruses to MDCK cells on ice for 1 h, removal of unadsorbed viruses was followed by 6 h incubation at 37 jC. After the incubation, virus NP was immunostained. Among native cells, the number of NP positive cells infected with 30/97 and cl-236 was about 20% that of cells infected with AI/68. The other binding assay using radiolabeled viruses also showed that the percentage of cells with Ch viruses on their surface was approximately 26% of the amount of AI/68. These results indicated that the binding efficiency of Ch virus to native MDCK cells was lower than that of Ch+ virus. If sialic acids on native MDCK cells were digested by sialidase from Arthrobacter ureafaciens, both types of viruses hardly bound to the cells. However, if the cells were resialylated by a2,6-sialyltransferase after the desialydation, both Ch+ and Ch viruses efficiently bound to the cells, despite the amount of sialic acids on the resialylated cells being smaller than those on native cells. These facts suggested that Ch viruses preferred sialic acids on the cells resialylated with SAa2,6Galh1,4GlcNAc sequence but not those on native MDCK cells.
474
E. Nobusawa et al. / International Congress Series 1263 (2004) 472–475
4. Virus growth in MDCK cells in the presence of zanamivir To elucidate how the weak binding of Ch virus to MDCK cells relates to NA dependency at virus release from host cells, we examined the effect of an NA inhibitor, zanamivir (kindly provided by Glaxo Welcome Research and Development), on the growth of viruses in MDCK cells. Ch+, Ch and cl-236 were grown in MDCK cells in the presence of wide range of zanamivir. Ch+ viruses were sensitive to zanamivir in their growth in MDCK cells, while Ch and cl-236 viruses provided similar HA titer and plaque formation ability irrespective of the presence or absence of the inhibitor. However, NA activity of both types of viruses was sensitive to the drug. When NA inhibition assay was performed using fetuin as a substrate, 0.01 AM was a high-enough concentration to reduce NA activity to less than 50% of that without the drug. When small substrates, 3V-sialyllactose and 6V-sialyllactose, were used, 0.1-AM inhibitor was high enough to inhibit the NA activity of both types of viruses by 90% of that of the control. These results indicated that Ch viruses were resistant to the NA inhibitor in their growth in MDCK cells due to their decreased binding efficiency to MDCK cells. Acknowledgements We thank Dr. K. Shimizu for providing rabbit anti-A/USSR/72 serum. A Scientific Research Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan supported this work. References [1] E. Nobusawa, et al., Change in receptor-binding specificity of recent human influenza viruses (H3N2): a single amino acid change in hemagglutinin altered its recognition of sialyloligosaccharides, Virology 278 (2000) 587 – 596. [2] R. Medeiros, et al., Hemagglutinin residues of recent human A (H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes, Virology 289 (2001) 74 – 85. [3] M. von Itzstein, et al., Rational design of potent sialidase-based inhibitors of influenza virus replication, Nature 363 (1993) 418 – 423. [4] J.L. McKimm-Breschkin, et al., Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and zanamivir, Antimicrobial Agents and Chemotherapy 40 (1996) 40 – 46. [5] L.V. Gubareva, et al., Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor zanamivir, Journal of Virology 70 (1996) 1818 – 1827. [6] T.J. Blick, et al., The interaction of neuraminidase and hemagglutinin mutations in influenza virus in resistance to zanamivir, Virology 246 (1998) 95 – 103. [7] D.F. Smee, et al., Characterization of an influenza A (H3N2) virus resistant to the cyclopentane neuraminidase inhibitor RWJ-270201, Antiviral Research 52 (2001) 251 – 259. [8] F.G. Hayden, et al., Efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenza virus infections, The New England Journal of Medicine 337 (1997) 874 – 880. [9] A.S. Monto, et al., Efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenza A and B virus infections, Journal of Infectious Diseases 180 (1999) 254 – 261. [10] J.M. Barnett, et al., Zanamivir susceptibility monitoring and characterization of influenza virus clinical isolates obtained during phase II clinical efficacy studies, Antimicrobial Agents and Chemotherapy 44 (2000) 78 – 87.
E. Nobusawa et al. / International Congress Series 1263 (2004) 472–475
475
[11] M. Tisdale, Monitoring of viral susceptibility: new challenges with the development of influenza NA inhibitors, Reviews in Medical Virology 10 (2000) 45 – 55. [12] M. Zambon, F. Hayden, Position statement: global neuraminidase inhibitor susceptibility network, Antiviral Research 49 (2001) 147 – 156. [13] J. Romanova, et al., Distinct host range of influenza H3N2 virus isolates inVero and MDCK cells is determined by cell specific glycosylation pattern, Virology 307 (2003) 90 – 97.