title: Reverse genetics studies of attenuation of the ca A/AA/6/60 influenza virus: the role of the matrix gene.
abstract: 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.
authors.affiliation: Center for NeuroVirology and Cancer Biology, Temple University, College of Science and Technology, Biology Life Sciences Building (015-96), 2400 North 12th Street, Philadelphia, PA 19122, USA. sweet@temple.edu
authors.forename: T M
authors.initials: TM
authors.lastname: Sweet
authors.forename: H F
authors.initials: HF
authors.lastname: Maassab
authors.forename: M L
authors.initials: ML
authors.lastname: Herlocher
mesh_terms: D000818:Animals; D002460:Cell Line; D003080:Cold Temperature; D004285:Dogs; D005814:Genes, Viral / Q000235:genetics*; D009980:Influenza A virus / Q000187:drug effects / Q000235:genetics*; D007252:Influenza Vaccines / Q000235:genetics* / Q000494:pharmacology / Q000627:therapeutic use; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D009976:Orthomyxoviridae Infections / Q000235:genetics* / Q000517:prevention & control; D014613:Vaccines, Attenuated / Q000235:genetics / Q000494:pharmacology / Q000627:therapeutic use; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000502:physiology
chemical_list: D007252:Influenza Vaccines; D014613:Vaccines, Attenuated; D014763:Viral Matrix Proteins