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Generation of Recombinant Influenza Virus from Plasmid DNA
Published on: August 3, 2010
Generation and characterization of recombinant influenza A (H1N1) viruses harboring amantadine resistance mutations
Yacine Abed1, Nathalie Goyette, Guy Boivin
1Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Québec City, Canada.
Abstract:
The emergence of resistance to amantadine in influenza A viruses has been shown to occur rapidly during treatment as a result of single-amino-acid substitutions at position 26, 27, 30, 31, or 34 within the transmembrane domain of the matrix-(M)-2 protein. In this study, reverse genetics was used to generate and characterize recombinant influenza A (H1N1) viruses harboring L26F, V27A, A30T, S31N, G34E, and V27A/S31N mutations in the M2 gene. In plaque reduction assays, all mutations conferred amantadine resistance, with drug concentrations resulting in reduction of plaque number by 50% (IC(50)s) 154- to 3,300-fold higher than those seen for the wild type (WT). M2 mutants had no impairment in their replicative capacities in vitro on the basis of plaque size and replication kinetics experiments. In addition, all mutants were at least as virulent as the WT in experimentally infected mice, with the highest mortality rate being obtained with the recombinant harboring a double V27A/S31N mutation. These findings could help explain the frequent emergence and transmission of amantadine-resistant influenza viruses during antiviral pressure in the clinical setting.
Insights
Amantadine resistance in influenza A viruses emerges rapidly due to mutations in the M2 protein. These resistant strains show no loss in replication or virulence, explaining their prevalence.
Area of Science:
- Virology
- Antimicrobial Resistance
Background:
- Influenza A virus resistance to amantadine is a significant clinical challenge.
- Resistance arises from specific amino acid substitutions in the M2 protein transmembrane domain.
Purpose of the Study:
- To investigate the impact of specific M2 protein mutations on amantadine resistance in influenza A (H1N1) viruses.
- To characterize the in vitro and in vivo properties of amantadine-resistant influenza A strains.
Main Methods:
- Reverse genetics was employed to create recombinant influenza A (H1N1) viruses with defined M2 gene mutations (L26F, V27A, A30T, S31N, G34E, V27A/S31N).
- Plaque reduction assays were used to determine amantadine resistance levels (IC50 values).
- In vitro replication kinetics and in vivo mouse models were used to assess viral fitness and virulence.
Main Results:
- All generated M2 mutations conferred significant amantadine resistance, with IC50 values 154- to 3,300-fold higher than wild type.
- M2 mutants exhibited unimpaired in vitro replicative capacity.
- Mutant viruses demonstrated equal or greater virulence than wild type in mice, with the V27A/S31N double mutant showing the highest mortality rate.
Conclusions:
- Single amino acid substitutions in the M2 protein are sufficient to confer amantadine resistance.
- Amantadine-resistant influenza A viruses maintain their replicative fitness and virulence.
- These findings provide insights into the rapid emergence and transmission of amantadine resistance under antiviral pressure.
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