A Novel I221L Substitution in Neuraminidase Confers High-Level Resistance to Oseltamivir in Influenza B Viruses

Abstract

Influenza B viruses with a novel I221L substitution in neuraminidase (NA) conferring high-level resistance to oseltamivir were isolated from an immunocompromised patient after prolonged oseltamivir treatment.Methods. Enzymatic characterization of the NAs (Km, Ki) and the in vitro fitness of viruses carrying wild-type or mutated (I221L) NA genes were evaluated. Proportions of wild-type and mutated NA genes were directly quantified in the patient samples. Structural characterizations by X-ray crystallography of a wild-type and I221L variant NA were performed.Results. The Km and Ki revealed that the I221L variant NA had approximately 84 and 51 times lower affinity for oseltamivir carboxylate and zanamivir, respectively, compared with wild-type NA. Viruses with a wild-type or I221L variant NA had similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, and 5 passages in MDCK cells revealed no reversion of the I221L substitution. The crystal structure of the I221L NA and oseltamivir complex showed that the leucine side chain protrudes into the hydrophobic pocket of the active site that accommodates the pentyloxy substituent of oseltamivir.Conclusions. Enzyme kinetic and NA structural analyses provide an explanation for the high level of resistance to oseltamivir while retaining good fitness of viruses carrying I221L variant NA.

Genetic characterization of influenza B virus in Cameroon and high frequency of reassortant strains

“…The following substitutions have been reported to be responsible for resistance to neuraminidase inhibitors in Influenza B viruses: E105K, G109E, E117V/A, D197N/E/Y, I221T/V/L, H273Y, R292K, N294S, and R374K . None of these resistance‐inducing mutations were observed in our sequences as reported previously.…”

“…There was no amino acid substitution characteristic of resistance to neuraminidase inhibitors among Cameroonian strains, as reported by previous studies . This result is not surprising because a majority of patients with flu‐like symptoms from Cameroon do not take any anti‐influenza therapy to control the infection.…”

Zanamivir-resistant influenza viruses with Q136K or Q136R neuraminidase residue mutations can arise during MDCK cell culture creating challenges for antiviral susceptibility monitoring

“…In addition to the Q136K/R variants described here, there are many other NA mutations that alter NAI susceptibility and also appear to be selected during MDCK cell culture [19,23]. Interestingly these seem to be increasingly reported for influenza B viruses [23][24][25].…”

Mycophenolic acid, an immunomodulator, has potent and broad-spectrum in vitro antiviral activity against pandemic, seasonal and avian influenza viruses affecting humans

“…In vitro, the IC 50 of zanamivir is generally higher than that of influenza A virus (Sheu et al, 2008). Recent influenza B virus isolates showed reduced susceptibility to neuraminidase inhibitors due to the emergence of N294S and I221V mutation of the neuraminidase (Carr et al, 2011;Garg et al, 2013), and high-level resistance, due to I221L mutation, has been reported (Escuret et al, 2014). Clinical studies have shown that neuraminidase inhibitors are less effective against influenza A viruses ( Burnham et al, 2013).…”