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Updated: Jan 30, 2026

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity
Published on: June 25, 2015
[INCLUSION OF SITE-SPECIFIC MUTATIONS INTO CONSERVATIVE SEG- MENTS OF PA-GENE RESULTS IN ATTENUATION OF VIRULENT
Aim:
Study the possibility of obtaining attenuated variants of influenza virus by including specially selected site-specific mutations into a conservative sequence of PA-gene (terminal segment of COOH-domain of the PA-gene) of a virulent strain.
Materials And Methods:
A/ WSN/33 - a virulent strain of influenza virus was used in the study. Inclusion of site-specif- ic mutations into PA-gene of the A/WSN/33 virulent strain was carried out using a two-step mutation PCR. Cloning was carried out using GoldenGate reaction. 8-plasmid transfection system based on pHW2000 vector was used. Transformation was carried out in rubidium competent bacterial cells of DH5(α strain. Transfection was done using Lipofectamine LTX (Invitrogen) reagentin a 293T and MDCK cells' co-culture.
Results:
Transfectants with F658A substitution in the COOH-domain of the PA-gene were shown to acquire ts-phenotype and sharply reduce the ability to reproduce in mice lungs. Introduction of F658A substitution into COOH-domain of the PA-gene in combination with introduction of ts-mutations from ca influenzavirus strains into the genome ofthe virulent strain resulted in obtaining transfectants that have phenotypic characteristics typical for live influenza vaccine candidates.
Conclusion:
The ability to obtain attenuated variants of influenza viruses by introducing spe- cially selected site-specific mutations into conservative sequence of the PA-gene is shown.
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