Detection of E119V and E119I Mutations in Influenza A (H3N2) Viruses Isolated from an Immunocompromised Patient: Challenges in Diagnosis of Oseltamivir Resistance

Abstract

The clinical use of the neuraminidase inhibitor (NAI) oseltamivir is associated with the emergence of drug resistance resulting from subtype-specific neuraminidase (NA) mutations. The influenza A/Texas/12/2007 (H3N2) virus isolated from an oseltamivir-treated immunocompromised patient exhibited reduced susceptibility to oseltamivir in the chemiluminescent neuraminidase inhibition (NI) assay (ϳ60-fold increase in its 50% inhibitory concentration [IC 50 ] compared to that for a control virus). When further propagated in cell culture, the isolate maintained reduced susceptibility to oseltamivir in both chemiluminescent and fluorescent NI assays (ϳ50-and 350-fold increases in IC 50 , respectively). Sequencing analysis of the isolate revealed a mix of nucleotides coding for amino acids at position 119 of the NA [E119(V/I)]. Plaque purification of the isolate yielded E119V and E119I variants, both exhibiting reduced susceptibility to oseltamivir. The E119I variant also showed decreased susceptibility to zanamivir and the investigational NAIs peramivir and A-315675. The emergence of E119V variants in oseltamivir-treated patients has been previously reported; however, the E119I mutation detected here is a novel one which reduces susceptibility to several NAIs. Both mutations were not detected in unpropagated original clinical specimens using either conventional sequencing or pyrosequencing, suggesting that these variants were present in very low proportions (<10%) in clinical specimens and gained dominance after virus propagation in MDCK cells. All virus isolates recovered from the patient were resistant to adamantanes. Our findings highlight the potential for emergence and persistence of multidrug-resistant influenza viruses in oseltamivir-treated immunocompromised subjects and also highlight challenges for drug resistance diagnosis due to the genetic instability of the virus population upon propagation in cell culture.

Five years of monitoring for the emergence of oseltamivir resistance in patients with influenza A infections in the Influenza Resistance Information Study

“…This detection of the R292K substitution was performed by a specific snip RT‐PCR, a sensitive method that can detect down to 5% of a minority species 15. It confirmed also that in most cases, a mixed population is detected, supporting the hypothesis that impaired NA activity of 292K viruses may be trans‐complemented by the NA activity of R292 bystander viruses, as it has been reported for mutations at position 119 23. The mutation‐specific real‐time RT‐PCR used in the current study and HA or NA sequencing were performed on the original clinical specimen and not culture based or genetically assessed by sequencing on culture material.…”

Unique Determinants of Neuraminidase Inhibitor Resistance among N3, N7, and N9 Avian Influenza Viruses

“…In addition to E119V, the E119G/D mutations were identified in N7 and/or N3 subtypes in the present study and tended to have more effect on ZA susceptibility than on the other NAIs. As in other reports (13,(62)(63)(64), we observed that mutations at E119 also showed NAI-specific susceptibility in avian N3, N7, and N9 subtypes. Interestingly, all reported NAI resistance mutations at residue E119 (D, G, and V) were found in N7 subtype, suggesting that the residue in this subtype has more flexibility to switch amino acid sequence by OS-or ZA-selective pressure than do the other subtypes.…”

Point Mutations and Antiviral Drug Resistance

“…Mutations at catalytic (R292K) and framework (E119V and N294S) NA residues have been detected in the N2 viral subtype in oseltamivir-treated patients. A four-amino-acid deletion mutation (deletion of residues 245 to 248) was reported to confer oseltamivir resistance in an influenza A (H3N2) virus isolated from an immunocompromised patient treated with the drug (Okomo-Adhiambo et al, 2010). This shows that the clinical use of oseltamivir is associated with the emergence of drug resistance resulting from subtype-specific NA mutations (Janies et al, 2010;Okomo-Adhiambo et al, 2010).…”

“…Zanamivir-resistant mutants are less common than oseltamivir-resistant mutants, partly because of differences between how the two drugs bind to the NA active site and possibly because of lower frequency of the prescription and use of zanamivir (Okomo-Adhiambo et al, 2010).…”

“…The M2 blockers (amantadine and rimantadine) are effective against influenza A viruses, but not influenza B viruses. However, their effectiveness has been compromised by drugresistant mutants that are frequently isolated from patients within a few days of therapy (Okomo-Adhiambo et al, 2010).…”