Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en

Abstract

The compounds 4-amino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-amino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid) and 4-guanidino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-guanidino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid), which selectively inhibit the influenza virus neuraminidase, have been tested in vitro for their ability to generate drug-resistant variants. NWS/G70C virus (H1N9) was cultured in each drug by limiting-dilution passaging. After five or six passages in either compound, there emerged viruses which had a reduced sensitivity to the inhibitors in cell culture. Variant viruses were up to 1,000-fold less sensitive in plaque assays, liquid culture, and a hemagglutination-elution assay. In addition, cross-resistance to both compounds was seen in all three assays. Some isolates demonstrated drug dependence with an increase in both size and number of plaques in a plaque assay and an increase in virus yield in liquid culture in the presence of inhibitors. No significant difference in neuraminidase enzyme activity was detected in vitro, and no sequence changes in the conserved sites of the neuraminidase were found. However, changes in conserved amino acids in the hemagglutinin were detected. These amino acids were associated with either the hemagglutinin receptor binding site, Thr-155, or the left edge of the receptor binding pocket, Val-223 and Arg-229. Hence, mutations at these sites could be expected to affect the affinity or specificity of the hemagglutinin binding. Compensating mutations resulting in a weakly binding hemagglutinin thus seem to be circumventing the inhibition of the neuraminidase by allowing the virus to be released from cells with less dependence on the neuraminidase.

Characterization of 2 Influenza A(H3N2) Clinical Isolates with Reduced Susceptibility to Neuraminidase Inhibitors Due to Mutations in the Hemagglutinin Gene

“…In one of these studies, it was demonstrated that substitution of the HA1 Arg229 residue by a Ser or an Ile resulted in a significant reduction of virus susceptibility to NAIs in cell-based assays [10]. The Arg229, which is part of the left edge of the HA receptor-binding site, is conserved in 12 (H1-H12) influenza A subtypes [14] and seems to be important for HA stability [10]. We recently isolated 2 influenza A(H3N2) viruses with an Ile at position 229 of the HA1 subunit from untreated patients.…”

“…In addition to the NA mutations, most resistant influenza viruses generated in vitro also contained mutations in or near the HA receptor-binding site [10,12,13]. In one of these studies, it was demonstrated that substitution of the HA1 Arg229 residue by a Ser or an Ile resulted in a significant reduction of virus susceptibility to NAIs in cell-based assays [10]. The Arg229, which is part of the left edge of the HA receptor-binding site, is conserved in 12 (H1-H12) influenza A subtypes [14] and seems to be important for HA stability [10].…”

“…NA changes have been predominantly ascribed to aa 119 and 292, which are part of the framework and catalytic residues, respectively [4,9,[11][12][13]. In addition to the NA mutations, most resistant influenza viruses generated in vitro also contained mutations in or near the HA receptor-binding site [10,12,13]. In one of these studies, it was demonstrated that substitution of the HA1 Arg229 residue by a Ser or an Ile resulted in a significant reduction of virus susceptibility to NAIs in cell-based assays [10].…”

“…However, in light of the experience with other anti-influenza compounds, such as the adamantanes rimantadine and amantadine, some concerns have been raised about the potential for the development of resistance to NAIs [7]. Several in vitro studies have shown that resistance to NAIs may result from amino acid substitutions in NA, HA, or both [8][9][10][11][12][13]. NA changes have been predominantly ascribed to aa 119 and 292, which are part of the framework and catalytic residues, respectively [4,9,[11][12][13].…”

Difluorosialic acids, potent novel influenza virus neuraminidase inhibitors, induce fewer drug resistance-associated neuraminidase mutations than does oseltamivir

“…This is achieved by reducing the affinity of HA to the sialic acid, therefore the progeny virus particles rely less on NA activity when leaving the cell surface (Bantia et al, 1998;Blick et al, 1998;Ginting et al, 2012;McKimm-Breschkin et al, 1996;Molla et al, 2002). Indeed, sequence analyses revealed Fig.…”