title: Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus.
abstract: To obtain direct evidence for a relationship between hemagglutinin (HA) cleavability and the virulence of avian influenza A viruses, we generated a series of HA cleavage mutants from a virulent virus, A/turkey/Ontario/7732/66 (H5N9), by reverse genetics. A transfectant virus containing the wild-type HA with R-R-R-K-K-R at the cleavage site, which was readily cleaved by endogenous proteases in chicken embryo fibroblasts (CEF), was highly virulent in intramuscularly or intranasally/orally inoculated chickens. By contrast, a mutant containing the HA with an avirulent-like sequence (R-E-T-R) at the cleavage site, which was not cleaved by the proteases in CEF, was avirulent in chickens, indicating that a genetic alteration confined to the HA cleavage site can affect cleavability and virulence. Mutant viruses with HA cleavage site sequences of T-R-R-K-K-R or T-T-R-K-K-R were as virulent as viruses with the wild-type HA, whereas a mutant with a two-amino-acid deletion but retention of four consecutive basic residues (R-K-K-R) was as avirulent as a virus with the avirulent-type HA. Interestingly, although a mutant containing an HA with R-R-R-K-T-R, which has reduced cleavability in CEF, was as virulent as viruses with high HA cleavability when given intramuscularly, it was less virulent when given intranasally/orally. We conclude that the degree of HA cleavability in CEF predicts the virulence of avian influenza viruses.
authors.affiliation: Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
authors.forename: T
authors.initials: T
authors.lastname: Horimoto
authors.forename: Y
authors.initials: Y
authors.lastname: Kawaoka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics* / Q000378:metabolism; D009980:Influenza A virus / Q000235:genetics* / Q000472:pathogenicity*; D008969:Molecular Sequence Data; D009976:Orthomyxoviridae Infections / Q000382:microbiology; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014018:Tissue Distribution; D014162:Transfection; D014774:Virulence / Q000235:genetics
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D011994:Recombinant Proteins; D010450:Endopeptidases