Oseltamivir-resistant influenza A viruses are transmitted efficiently among guinea pigs by direct contact but not by aerosol

Abstract

Influenza viruses resistant to the neuraminidase (NA) inhibitor oseltamivir arise under drug selection pressure both in vitro and in vivo. Several mutations in the active site of the viral NA are known to confer relative resistance to oseltamivir, and influenza viruses with certain oseltamivir resistance mutations have been shown to transmit efficiently among cocaged ferrets. However, it is not known whether NA mutations alter aerosol transmission of drug-resistant influenza virus. Here, we demonstrate that recombinant human influenza A/H3N2 viruses without and with oseltamivir resistance mutations (in which NA carries the mutation E119V or the double mutations E119V I222V) have similar in ovo growth kinetics and infectivity in guinea pigs. These viruses also transmit efficiently by the contact route among cocaged guinea pigs, as in the ferret model. However, in an aerosol transmission model, in which guinea pigs are caged separately, the oseltamivir-resistant viruses transmit poorly or not at all; in contrast, the oseltamivir-sensitive virus transmits efficiently even in the absence of direct contact. The present results suggest that oseltamivir resistance mutations reduce aerosol transmission of influenza virus, which could have implications for public health measures taken in the event of an influenza pandemic.

FIG. 1.

Oseltamivir-sensitive and -resistant viruses demonstrate different plaque phenotypes in vitro but have similar growth kinetics in ovo. (A) After 3 days of incubation at 37°C under an agar overlay, plaques produced in MDCK cells by rPan/99 NA-E119V and rPan/99 NA-E119V+I222V are smaller than those produced by rPan/99 NA-wt. (B) When the agar overlay is supplemented with 1 mU/ml of V. cholerae NA, the parental virus plaques are phenotypically unchanged; however, the mutant virus plaques are enlarged to wild-type size under these conditions. (C) In ovo growth of oseltamivir-sensitive and -resistant viruses is not significantly different between 12 and 72 h postinoculation. Data points represent the average log₁₀ virus titer ± SD in allantoic fluid.

FIG. 2.

Mutant viruses are less susceptible to inhibition by oseltamivir than rPan/99 NA-wt. (A) Log dose inhibition curves for the mutant viruses are shifted to the left of that of the parental virus, indicating greater oseltamivir resistance, as determined in a chemiluminescent substrate cleavage assay. Data points represent the average number of RLUs at each drug concentration, expressed as a percentage of the RLUs obtained in the absence of drug; error bars indicate SDs. (B) For the mutant viruses rPan/99 NA-E119V and rPan/99 NA-E119V+I222V, the IC₅₀s of oseltamivir are 139-fold and 224-fold (respectively) higher than the IC₅₀ for rPan/99 NA-wt. Results are expressed as average IC₅₀ (in nM) ± SD.

FIG. 3.

Aerosol transmission of rPan/99 NA-wt is more efficient than that of rPan/99 NA-E119V or rPan/99 NA-E119V+I222V. The parental virus was transmitted by aerosol among seven of eight guinea pig pairs, while the single mutant was transmitted among two of eight pairs, and the double mutant was transmitted among zero of eight pairs. Data represent two experiments (four guinea pig pairs per experiment) per virus. Series 1 was performed under ambient conditions, and series 2 was performed in an environmental chamber at 20°C and 20% RH. The time course of log₁₀ nasal wash virus titers collected from inoculated animals is represented by dotted lines, while the time course of those collected from exposed animals is represented by solid lines. Each inoculated-exposed guinea pig pair is represented by a unique symbol (square, triangle, diamond, or circle).

FIG. 4.

Contact transmission of rPan/99 NA-E119V and rPan/99 NA-E119V+I222V is highly efficient. Each mutant virus transmitted among four of four cocaged guinea pig pairs at 20°C and 20% RH. The time course of log₁₀ nasal wash virus titers collected from inoculated animals is represented by dotted lines, while the time course of those collected from exposed animals is represented by solid lines. Each inoculated-exposed guinea pig pair is represented by a unique symbol (square, triangle, diamond, or circle).