Reverse genetics studies of attenuation of the ca A/AA/6/60 influenza virus: the role of the matrix gene

The matrix (M) gene of influenza virus has been implicated in the attenuation phenotype of the cold adapted (ca) A/AA/6/60 vaccine. Previous studies have evaluated the ca M from A/AA/6/60 in different wild type (wt) virus backgrounds with varying results. In experiments described here, the ca M gene was transfected into the background of its own wt A/AA/6/60 to eliminate the possibility of confounding gene constellation effects. Comparison of the sequence of the wt and the ca A/AA/6/60 revealed one substitution in the nucleotide sequence of M. The molecular techniques of reverse genetics were used to rescue the ca M gene into the virulent wt A/AA/6/60 virus. The selection system used to identify the desired transfectant virus was amantadine resistance, which was introduced into the M2 gene using mutagenesis. The ca A/AA/6/60, the wt A/AA/6/60, a virus which contained wt M and was wt in the remaining seven genes and amantadine resistant (wt/969), a virus which contained the ca M but wt in the other seven genes (ca/969) were all evaluated in mice determine the effect of the ca M. The ca/969 virus was not attenuated in the mouse model when compared to the wt/969 virus, indicating 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.