We have previously obtained four transfectant influenza A viruses containing neuraminidase (NA) genes with mutated base pairs in the conserved double-stranded RNA region of the viral promoter by using a ribonucleoprotein transfection system. Two mutant viruses (D2 and D1/2) which share a C-G→A-U mutation at positions 11 and 12 of the 3′ and 5′ ends, respectively, of the NA gene, showed an approximate 10-fold reduction of NA-specific mRNA and protein levels (Fodor 72, 6283–6290, 1998). These viruses have now allowed us to determine the effects of decreased NA levels on virus pathogenicity. Both D2 and D1/2 viruses were highly attenuated in mice, and their replication in mouse lungs was highly compromised as compared with wild-type influenza A/WSN/33 virus. The results highlight the importance of the level of NA activity in the biological cycle and virulence of influenza viruses. Importantly, mice immunized by a single intranasal administration of 10 infectious units of D2 or D1/2 viruses were protected against challenge with a lethal dose of wild-type influenza virus. Attenuation of influenza viruses by mutations resulting in the decreased expression of a viral protein represents a novel strategy which could be considered for the generation of live attenuated influenza virus vaccines.


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  1. Baker, A. T., Varghese, J. N., Laver, W. G., Air, G. M. & Colman, P. M. (1987). Three-dimensional structure of neuraminidase of subtype N9 from an avian influenza virus. Proteins 2, 111-117.[CrossRef] [Google Scholar]
  2. Bilsel, P. & Kawaoka, Y. (1998). New approaches to vaccination. In Textbook of Influenza, pp. 422-434. Edited by K. G. Nicholson, R. G. Webster & A. J. Hay. Oxford: Blackwell Science.
  3. Bot, A., Antohi, S., Bot, S., Garcı́a-Sastre, A. & Bona, C. (1997). Induction of humoral and cellular immunity against influenza virus by immunization of newborn mice with a plasmid bearing a hemagglutinin gene. International Immunology 9, 1641-1650.[CrossRef] [Google Scholar]
  4. Both, G. W., Sleigh, M. J., Cox, N. J. & Kendal, A. P. (1983). Antigenic drift in influenza virus H3 hemagglutinin from 1968 to 1980: multiple evolutionary pathways and sequential amino acid changes at key antigenic sites. Journal of Virology 48, 52-60. [Google Scholar]
  5. Burnet, F. M. & Stone, J. D. (1947). The receptor-destroying enzyme of V. cholerae. Australian Journal of Experimental Medical Sciences 25, 227-233.[CrossRef] [Google Scholar]
  6. Calfee, D. P. & Hayden, F. G. (1998). New approaches to influenza chemotherapy. Neuraminidase inhibitors. Drugs 56, 537-553.[CrossRef] [Google Scholar]
  7. Castrucci, M. R., Bilsel, P. & Kawaoka, Y. (1992). Attenuation of influenza A virus by insertion of a foreign epitope into the neuraminidase. Journal of Virology 66, 4647-4653. [Google Scholar]
  8. Enami, M., Luytjes, W., Krystal, M. & Palese, P. (1990). Introduction of site-specific mutations into the genome of influenza virus. Proceedings of the National Academy of Sciences, USA 87, 3802-3805.[CrossRef] [Google Scholar]
  9. Fleming, D. M. (1999). Treating influenza with zanamivir. Lancet 353, 668-669. [Google Scholar]
  10. Fodor, E., Pritlove, D. C. & Brownlee, G. G. (1995). Characterization of the RNA-fork model of virion RNA in the initiation of transcription in influenza A virus. Journal of Virology 69, 4012-4019. [Google Scholar]
  11. Fodor, E., Palese, P., Brownlee, G. G. & Garcı́a-Sastre, A. (1998). Attenuation of influenza A virus mRNA levels by promoter mutations. Journal of Virology 72, 6283-6290. [Google Scholar]
  12. Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G. & Garcı́a-Sastre, A. (1999). Rescue of influenza A virus from recombinant DNA. Journal of Virology 73, 9679-9682. [Google Scholar]
  13. Garcı́a-Sastre, A. (1998). Negative-strand RNA viruses: applications to biotechnology. Trends in Biotechnology 16, 230-235.[CrossRef] [Google Scholar]
  14. Garcı́a-Sastre, A., Villar, E., Hannoun, C. & Cabezas, J. A. (1990). Sialidase activity in rimantadine-resistant and -sensitive influenza A viruses. Enzyme 43, 207-211. [Google Scholar]
  15. Gutierrez, A. L., Denova-Ocampo, M., Racaniello, V. R. & del Angel, R. M. (1997). Attenuating mutations in the poliovirus 5′ untranslated region alter its interaction with polypyrimidine tract-binding protein. Journal of Virology 71, 3826-3833. [Google Scholar]
  16. Haller, A. A., Stewart, S. R. & Semler, B. L. (1996). Attenuation stem–loop lesions in the 5′ noncoding region of poliovirus RNA: neuronal cell-specific translation defects. Journal of Virology 70, 1467-1474. [Google Scholar]
  17. Hayden, F. G., Osterhaus, A. D., Treanor, J. J., Fleming, D. M., Aoki, F. Y., Nicholson, K. G., Bohnen, A. M., Hirst, H. M., Keene, O. & Wightman, K. (1997). Efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenzavirus infections. GG167 Influenza Study Group. New England Journal of Medicine 337, 874-880.[CrossRef] [Google Scholar]
  18. Hsu, M. T., Parvin, J. D., Gupta, S., Krystal, M. & Palese, P. (1987). Genomic RNAs of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle. Proceedings of the National Academy of Sciences, USA 84, 8140-8144.[CrossRef] [Google Scholar]
  19. Kawaoka, Y., Krauss, S. & Webster, R. G. (1989). Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. Journal of Virology 63, 4603-4608. [Google Scholar]
  20. Kim, H. J., Fodor, E., Brownlee, G. G. & Seong, B. L. (1997). Mutational analysis of the RNA- fork model of the influenza A virus vRNA promoter in vivo. Journal of General Virology 78, 353-357. [Google Scholar]
  21. Lamb, R. A. & Krug, R. M. (1996).Orthomyxoviridae. The viruses and their replication. In Fields Virology, pp. 1353-1395. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.
  22. La Monica, N. & Racaniello, V. R. (1989). Differences in replication of attenuated and neurovirulent polioviruses in human neuroblastoma cell line SH-SY5Y. Journal of Virology 63, 2357-2360. [Google Scholar]
  23. Li, S., Liu, C., Klimov, A., Subbarao, K., Perdue, M. L., Mo, D., Ji, Y., Woods, L., Hietala, S. & Bryant, M. (1999). Recombinant influenza A virus vaccines for the pathogenic human A/Hong Kong/97 (H5N1) viruses. Journal of Infectious Diseases 179, 1132-1138.[CrossRef] [Google Scholar]
  24. Liu, C. & Air, G. M. (1993). Selection and characterization of a neuraminidase-minus mutant of influenza virus and its rescue by cloned neuraminidase genes. Virology 194, 403-407.[CrossRef] [Google Scholar]
  25. Liu, C., Eichelberger, M. C., Compans, R. W. & Air, G. M. (1995). Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly, or budding. Journal of Virology 69, 1099-1106. [Google Scholar]
  26. Luo, G. X., Luytjes, W., Enami, M. & Palese, P. (1991). The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure. Journal of Virology 65, 2861-2867. [Google Scholar]
  27. Luo, G., Bergmann, M., Garcı́a-Sastre, A. & Palese, P. (1992). Mechanism of attenuation of a chimeric influenza A/B transfectant virus. Journal of Virology 66, 4679-4685. [Google Scholar]
  28. Luo, G., Chung, J. & Palese, P. (1993). Alterations of the stalk of the influenza virus neuraminidase: deletions and insertions. Virus Research 29, 141-153.[CrossRef] [Google Scholar]
  29. Maassab, H. F., LaMontagne, J. R. & DeBorde, D. C. (1998). Live influenza virus vaccines. In Vaccines, pp. 435-457. Edited by S. A. Plotkin & E. A. Mortimer. Philadelphia: Saunders.
  30. Murphy, B. R. & Webster, R. G. (1996). Orthomyxoviruses. In Fields Virology, pp. 1397-1445. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.
  31. Muster, T., Subbarao, E. K., Enami, M., Murphy, B. R. & Palese, P. (1991). An influenza A virus containing influenza B virus 5′ and 3′ noncoding regions on the neuraminidase gene is attenuated in mice. Proceedings of the National Academy of Sciences, USA 88, 5177-5181.[CrossRef] [Google Scholar]
  32. Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M., Perez, D. R., Donis, R., Hoffmann, E., Hobom, G. & Kawaoka, Y. (1999). Generation of influenza A viruses entirely from cloned cDNAs. Proceedings of the National Academy of Sciences, USA 96, 9345-9350.[CrossRef] [Google Scholar]
  33. Palese, P. & Compans, R. W. (1976). Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA): mechanism of action. Journal of General Virology 33, 159-163.[CrossRef] [Google Scholar]
  34. Palese, P., Schulman, J. L., Bodo, G. & Meindl, P. (1974). Inhibition of influenza and parainfluenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA). Virology 59, 490-498.[CrossRef] [Google Scholar]
  35. Palese, P., Muster, T., Zheng, H., O’Neill, R. & Garcı́a-Sastre, A. (1999). Learning from our foes: a novel vaccine concept for influenza virus. Archives of Virology 15, 1-8. [Google Scholar]
  36. Parkin, N. T., Chiu, P. & Coelingh, K. (1997). Genetically engineered live attenuated influenza A virus vaccine candidates. Journal of Virology 71, 2772-2778. [Google Scholar]
  37. Pleschka, S., Jaskunas, R., Engelhardt, O. G., Zürcher, T., Palese, P. & Garcı́a-Sastre, A. (1996). A plasmid-based reverse genetics system for influenza A virus. Journal of Virology 70, 4188-4192. [Google Scholar]
  38. Pritlove, D. C., Fodor, E., Seong, B. L. & Brownlee, G. G. (1995). In vitro transcription and polymerase binding studies of the termini of influenza A virus cRNA: evidence for a cRNA panhandle. Journal of General Virology 76, 2205-2213.[CrossRef] [Google Scholar]
  39. Scholtissek, C., Rohde, W., Von Hoyningen, V. & Rott, R. (1978). On the origin of the human influenza virus subtypes H2N2 and H3N2. Virology 87, 13-20.[CrossRef] [Google Scholar]
  40. Subbarao, E. K., Park, E. J., Lawson, C. M., Chen, A. Y. & Murphy, B. R. (1995). Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine. Journal of Virology 69, 5969-5977. [Google Scholar]
  41. Tulip, W. R., Varghese, J. N., Baker, A. T., van Donkelaar, A., Laver, W. G., Webster, R. G. & Colman, P. M. (1991). Refined atomic structures of N9 subtype influenza virus neuraminidase and escape mutants. Journal of Molecular Biology 221, 487-497.[CrossRef] [Google Scholar]
  42. Varghese, J. N., Laver, W. G. & Colman, P. M. (1983). Structure of the influenza virus glycoprotein antigen neuraminidase at 2·9 Å resolution. Nature 303, 35-40.[CrossRef] [Google Scholar]
  43. Varghese, J. N., McKimm-Breschkin, J. L., Caldwell, J. B., Kortt, A. A. & Colman, P. M. (1992). The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor. Proteins 14, 327-332.[CrossRef] [Google Scholar]
  44. von Itzstein, M., Wu, W. Y., Kok, G. B., Pegg, M. S., Dyason, J. C., Jin, B., Van Phan, T., Smythe, M. L., White, H. F., Oliver, S. W., Colman, P. M., Varghese, J. N., Ryan, D. M., Woods, J. M., Bethell, R. C., Hotham, V. J., Cameron, J. M. & Penn, C. R. (1993). Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 363, 418-423.[CrossRef] [Google Scholar]
  45. Webster, R. G., Laver, W. G., Air, G. M. & Schild, G. C. (1982). Molecular mechanisms of variation in influenza viruses. Nature 296, 115-121.[CrossRef] [Google Scholar]
  46. Webster, R. G., Bean, W. J., Gorman, O. T., Chambers, T. M. & Kawaoka, Y. (1992). Evolution and ecology of influenza A viruses. Microbiological Reviews 56, 152-179. [Google Scholar]

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