Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice.
The natural reservoir for influenza viruses is waterfowl, and from there they succeeded in crossing the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precision-cut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1 to P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized α2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with α2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190+HA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. In contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs.IMPORTANCESwine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but have not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by passaging the virus three times in differentiated porcine respiratory epithelial cells. Among the four mutations detected, the two HA mutations were analyzed by generating recombinant viruses. Depending on the infection system used, the mutations differed in their phenotypic expression, e.g., sialic acid binding activity, replication kinetics, plaque size, and pathogenicity in inbred mice. However, none of the mutations affected the ciliary activity which serves as a virulence marker. Thus, early adaptive mutation enhances the replication kinetics, but more mutations are required for IAV of the H9N2 subtype to become virulent.
Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany.
W
W
Yang
Virology and Immunology Department, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok, Thailand.
D
D
Punyadarsaniya
Department of Infection Genetics, Helmholtz Centre for Infection Research and University of Veterinary Medicine Hannover, Braunschweig, Germany.
R L O
RLO
Lambertz
Genomics Research Center, Academia Sinica, Taipei, Taiwan.
D C C
DCC
Lee
Genomics Research Center, Academia Sinica, Taipei, Taiwan.
C H
CH
Liang
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
D
D
Höper
Department of Infection Genetics, Helmholtz Centre for Infection Research and University of Veterinary Medicine Hannover, Braunschweig, Germany.
S R
SR
Leist
Department of Infection Genetics, Helmholtz Centre for Infection Research and University of Veterinary Medicine Hannover, Braunschweig, Germany.
A
A
Hernández-Cáceres
Institute for Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
J
J
Stech
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
M
M
Beer
Genomics Research Center, Academia Sinica, Taipei, Taiwan.
C Y
CY
Wu
Genomics Research Center, Academia Sinica, Taipei, Taiwan.
C H
CH
Wong
Department of Infection Genetics, Helmholtz Centre for Infection Research and University of Veterinary Medicine Hannover, Braunschweig, Germany.|University of Tennessee Health Science Center, Department of Microbiology, Immunology and Biochemistry, Memphis, Tennessee, USA.
K
K
Schughart
Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany Fandan.Meng@tiho-hannover.de Georg.Herrler@tiho-hannover.de.
F
F
Meng
Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany Fandan.Meng@tiho-hannover.de Georg.Herrler@tiho-hannover.de.
G
G
Herrler
D000220:Adaptation, Biological; D000818:Animals; D004252:DNA Mutational Analysis; D004847:Epithelial Cells / Q000821:virology*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D053127:Influenza A Virus, H9N2 Subtype / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity*; D051379:Mice; D020125:Mutation, Missense; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D020545:Respiratory Mucosa / Q000821:virology*; D059386:Reverse Genetics; D012692:Serial Passage; D013552:Swine; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D014774:Virulence; D053585:Virus Attachment
D019267:Hemagglutinin Glycoproteins, Influenza Virus; C085820:INS1 protein, influenza virus; C057027:PB2 protein, Influenzavirus A; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase; D019158:N-Acetylneuraminic Acid
H9N2; adaptation; avian viruses; precision-cut lung slices; sialic acid