JVI Accepts, published online ahead of print on 4 November 2009

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J. Virol. doi:10.1128/JVI.01581-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Increased virulence for chickens of a genetically engineered waterfowl influenza virus with a deletion in the stalk of the neuraminidase.

S. Munier, T. Larcher, F. Cormier-Aline, D. Soubieux, B. Su, L. Guigand, B. Labrosse, Y. Cherel, P. Quéré, D. Marc^*, and N. Naffakh^*

Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, CNRS URA 3015, Université Paris Diderot-Paris 7, 25-28 rue du Dr Roux 75724 Paris cedex 15, France; INRA UR1282, Infectiologie Animale et Santé Publique, IASP, 37380 Nouzilly, France; INRA UMR 703, APEX, Ecole Nationale Vétérinaire, B.P. 40706, 44307 Nantes, France; INSERM U941, Unité de Génétique et Ecologie des Virus, IUH, Hôpital Saint-Louis, 1 rue Claude Vellefaux, 75475 Paris Cedex 10, France

^* To whom correspondence should be addressed. Email: daniel.marc{at}tours.inra.fr. nadia.naffakh{at}pasteur.fr.

A deletion of about 20 amino acids in the stalk of the neuraminidase (NA) is frequently detected upon transmission of influenza A viruses from waterfowl to domestic poultry. Using reverse genetics, a recombinant virus derived from a wild duck influenza isolate, A/Mallard/Marquenterre/Z237/83 (MZ), and an NA stalk-deletion variant (MZ-delNA), were produced. When compared to the wild-type, the MZ-delNA virus showed a moderate growth advantage on avian cultured cells. In 4-week old chickens inoculated intra-tracheally with the MZ-delNA virus, viral replication in the lungs, liver and kidneys was enhanced and interstitial pneumonia lesions were more severe than with the wild-type virus. The MZ-delNA-inoculated chickens showed significantly increased levels of mRNAs encoding IL-6, TGF-ß4 and CCL5 in the lungs and a higher frequency of apoptotic cells in the liver as compared to their MZ-inoculated counterparts. Molecular mechanisms possibly underlying the growth advantage of the MZ-delNA virus were explored. The enzymatic activity measured towards a small substrate was similar for the wild-type and deleted NA, but the MZ-delNA virus eluted from chicken erythrocytes at reduced rates. Pseudoviral particles expressing the MZ hemagglutinin in combination with the MZ-NA or MZ-delNA protein were produced from avian cultured cells with similar efficiencies, suggesting that the deletion in the NA stalk does not enhance the release of progeny virions and probably affects an earlier step of the viral cycle. Overall, our data indicate that a shortened NA stalk is a strong determinant of adaptation and virulence of waterfowl influenza viruses in chickens.