title: The Low-pH Resistance of Neuraminidase Is Essential for the Replication of Influenza A Virus in Duck Intestine following Infection via the Oral Route.
abstract: Influenza A viruses are known to primarily replicate in duck intestine following infection via the oral route, but the specific role of neuraminidase (NA) for the intestinal tropism of influenza A viruses has been unclear. A reassortant virus (Dk78/Eng62N2) did not propagate in ducks infected via the oral route. To generate variant viruses that grow well in ducks via the oral route, we isolated viruses that effectively replicate in intestinal mucosal cells by passaging Dk78/Eng62N2 in duck via rectal-route infection. This procedure led to the isolation of a variant virus from the duck intestine. This virus was propagated using embryonated chicken eggs and inoculated into a duck via the oral route, which led to the isolation of Dk-rec6 from the duck intestine. Experimental infections with mutant viruses generated by using reverse genetics indicated that the paired mutation of residues 356 and 431 in NA was necessary for the viral replication in duck intestine. The NA assay revealed that the activity of Dk78/Eng62N2 almost disappeared after pH 3 treatment, whereas that of Dk-rec6 was maintained. Furthermore, to identify the amino acid residues associated with the low-pH resistance, we measured the activities of mutant NA proteins transiently expressed in 293 cells after pH 3 treatment. All mutant NA proteins that possessed proline at position 431 showed higher activities than NA proteins that possessed glutamine at this position. These findings indicate that the low-pH resistance of NA plays an important role in the ability of influenza A virus to replicate in duck intestine.
  
  
  OBJECTIVE
  Neuraminidase (NA) activity facilitates the release of viruses from cells and, as such, is important for the replicative efficiency of influenza A virus. Ducks are believed to serve as the principal natural reservoir for influenza A virus; however, the key properties of NA for viral infection in duck are not well understood. In this study, we identify amino acid residues in NA that contribute to viral replication in ducks via the natural route of infection and demonstrate that maintenance of NA activity under low-pH conditions is associated with the biological properties of the virus. These findings provide insights into the mechanisms of replication of influenza A virus in ducks.
authors: Avian Zoonosis Research Center, Tottori University, Tottori, Japan.|Laboratory of Veterinary Public Health, Faculty of Agriculture, Tottori University, Tottori, Japan.|Department of Biomedicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors: Yoshikazu
authors: Y
authors: Fujimoto
authors: Avian Zoonosis Research Center, Tottori University, Tottori, Japan.|Laboratory of Veterinary Public Health, Faculty of Agriculture, Tottori University, Tottori, Japan.
authors: Hiroshi
authors: H
authors: Ito
authors: Avian Zoonosis Research Center, Tottori University, Tottori, Japan.|Department of Biomedicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors: Etsuro
authors: E
authors: Ono
authors: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan.|Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan.|Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors: Yoshihiro
authors: Y
authors: Kawaoka
authors: Avian Zoonosis Research Center, Tottori University, Tottori, Japan toshiito@muses.tottori-u.ac.jp.|Laboratory of Veterinary Public Health, Faculty of Agriculture, Tottori University, Tottori, Japan.
authors: Toshihiro
authors: T
authors: Ito
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D004372:Ducks; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000502:physiology*; D005585:Influenza in Birds / Q000635:transmission / Q000821:virology*; D007422:Intestines / Q000821:virology*; D008958:Models, Molecular; D009055:Mouth / Q000821:virology; D009154:Mutation; D009439:Neuraminidase / Q000737:chemistry / Q000378:metabolism*; D016865:Reassortant Viruses; D012007:Rectum / Q000821:virology; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase