Multidrug Resistant 2009 A/H1N1 Influenza Clinical Isolate with a Neuraminidase I223R Mutation Retains Its Virulence and Transmissibility in Ferrets

Abstract

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza.

Author Summary

Recently, a 2009 pandemic A/H1N1 influenza virus was isolated from an immune compromised patient, with antiviral resistance to the neuraminidase inhibitor class of drugs. This virus had an amino acid change in the viral neuraminidase enzyme; an isoleucine at position 223 was substituted for an arginine (I223R). Patients infected with a pandemic virus that is resistant to all neuraminidase inhibitors, would leave physicians without antiviral treatment options, since these viruses are naturally resistant to the other class of antivirals, the adamantanes. To date, it is unknown if this I223R mutant virus is affected in its ability to cause severe disease and to transmit to other humans. Therefore, we have addressed this question by comparing the I223R mutant virus with a wild type reference virus in a ferret pathogenicity and transmission model. We found that the I223R mutant virus was not severely affected in its pathogenicity, although fewer lung lesions and alveolitis scores were found for the I223R mutant virus. In addition, we demonstrated that this virus transmitted efficiently to naïve ferrets. Consequently, we conclude that this I223R mutant virus has the potential to cause disease and may spread among humans. Therefore, influenza surveillance for this resistance pattern is advised.