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MicroRNA-mediated species-specific attenuation of influenza A virus


Influenza A virus leads to yearly epidemics and sporadic pandemics. Present prophylactic strategies focus on egg-grown, live, attenuated influenza vaccines (LAIVs), in which attenuation is generated by conferring temperature sensitivity onto the virus. Here we describe an alternative approach to attenuating influenza A virus based on microRNA-mediated gene silencing. By incorporating nonavian microRNA response elements (MREs) into the open-reading frame of the viral nucleoprotein, we generate reassortant LAIVs for H1N1 and H5N1 that are attenuated in mice but not in eggs. MRE-based LAIVs show a greater than two-log reduction in mortality compared with control viruses lacking MREs and elicit a diverse antibody response. This approach might be combined with existing LAIVs to increase attenuation and improve vaccine safety.

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Figure 1: Influenza A virus infection does not disrupt cellular miRNA function.
Figure 2: Species-specific attenuation of H1N1 and H5N1 influenza A viruses.
Figure 3: Characterization of miRNA-mediated attenuation of influenza A virus.
Figure 4: MRE-containing influenza A viruses as live attenuated vaccines.

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This material is based upon work supported in part by the US Army Research Laboratory and the US Army Research Office under grant number 54677-LS-YIP. J.T.P. is supported by the NYU-MSSM Mechanisms of Virus-Host Interactions National Institutes of Health T32 training grant (no. AI007647-09). B.R.t. is supported in part by the Pew Charitable Funds. We thank A. Garcia-Sastre for giving us polyclonal A/PR/8/34 (MSSM, NY); G. Sen for ISG54 (Cleveland Clinic, OH, USA); A. Fernandez-Sesma (MSSM, NY) for primary human dendritic cell RNA; A. Tarakhovsky (Rockefeller University, NY) and D. O'Carroll (EMBL, Monterotondo, Italy) for Dicer−/− murine fibroblasts; E. Makeyev (Nanyang Technological University, Singapore) for firefly luciferase constructs containing miR-124 MREs and control SV40 3′ UTRs; and P. Palese (MSSM, NY) for pPol-I driven nucleoprotein vector, monoclonal nucleoprotein and A/PR/8/34 NS1. We thank members of the Palese and Garcia-Sastre labs for advice, reagents and comments during the course of this work.

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J.T.P. and B.R.t. designed and wrote the manuscript. Animal studies were conducted by A.M.P. and M.A.C. Experiments were done by J.T.P., A.M.P. and M.H.L. Cloning and rescue of H5N1 reassortants was done by J.S.

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Correspondence to Benjamin R tenOever.

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Perez, J., Pham, A., Lorini, M. et al. MicroRNA-mediated species-specific attenuation of influenza A virus. Nat Biotechnol 27, 572–576 (2009).

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