Virulence studies associated with influenza A/Ann Arbor/6/60.

The matrix (M) gene of influenza virus has been implicated in the attenuation phenotype of the cold adapted (ca) A/AnnArbor/6/60 vaccine in reassortants containing the ca A/AA/6/60 M in different wild type (wt) virus backgrounds. In experiments described here, the ca M gene was transfected into the background of wt A/AA/6/60, to eliminate the possibility of confounding gene constellation effects. For the first time, the molecular techniques of reverse genetics were used to rescue the ca M gene into the wt A/AA/6/60 virus, using amantadine resistance as the selection system. The ca A/AA/6/60, the wtF A/AA/6/60, and two amantadine resistant viruses, the wt/969, which contained all eight wt genes, and the ca/969virus, which contained the ca M with the remaining gene from the wt, were all evaluated in mice and ferrets. The ca/969 virus was not attenuated in either animal model when compared to the wt/969 virus, showing that the ca A/AA/6/60 M does not independently contribute to the attenuation phenotype attributed to the ca A/AA/6/60 vaccine virus. wt A/AA/6/60 viral clones of varying degrees of mouse adaptation were analyzed to determine which genes were responsible for increased virulence in mice. These data were used to determine possible mechanisms of influenza pathogenesis. The influenza A/AA/6/60 viral strain used were: ca A/AA/6/60, which was not virulent in mice, wt2 A/AA/6/60, which was slightly more virulent, wt10 A/AA/6/60, which was intermediate in virulence for mice, and the wtF A/AA/6/60, which was passaged the most in mice and was the most virulent. The wt10 and wtF both contained one mutation in M compared to the wt2 and the ca, however, the ca/969 (ca M) was as virulent as wtF, which makes M unlikely to be involved in mouse adaptation. In the HA gene, wt10 had four amino acid changes from the wt2, two in receptor binding sites, while the wtF contained one additional change. The most likely mechanism for mouse adaptation for A/AA/6/60 is a change in the receptor binding site of the HA protein.PhDBiological SciencesGeneticsHealth and Environmental SciencesMicrobiologyMolecular biologyPublic healthUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131335/2/9840655.pd