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 (approximately 60-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 (approximately 50- 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.

FIG. 1.

Pyrograms showing sequence data for detection of base substitutions in the codon of the NA gene for residue E119 of A/Texas/12/2007 (H3N2) (passage X/C3) and its plaque-purified variants. Reverse primer HuN2NA-119-R377-seq (Table 1) was used for sequencing the region containing residue 119; hence, the sequence displayed on the pyrograms is a reverse complement to the original sequence. The panels represent pyrograms for virus isolate A/Texas/12/2007 (H3N2) with the mixed sequence TA(T/C), complementary to (G/A)TA (GTA, valine; ATA, isoleucine) (A), a plaque-purified variant possessing the sequence TAC, complementary to GTA, which codes for valine (B), a plaque-purified variant with the sequence TAT, complementary to ATA, which encodes isoleucine (C), and sensitive wild-type A/Illinois/01/2007 (H3N2) with the sequence TTC, complementary to GAA, which encodes glutamic acid (E119) (D).