Abstract
We and others have previously isolated influenza B viruses with reduced sensitivity to neuraminidase (NA) inhibitors (oseltamivir and zanamivir) from patients who were never exposed to these drugs. It was unclear whether the NA substitutions found in these influenza B isolates arose spontaneously or were due to selective pressure. Here, we obtained influenza B viruses with reduced neuraminidase inhibitor sensitivity by in vitro selection with NA inhibitors. We found that these viruses possessed the same NA substitutions as those previously found in viruses isolated from untreated patients. These results suggest that these NA substitutions were selected in patients who were treated with an NA inhibitor and the resistant variants were then transmitted to others.
Keywords: Influenza B virus, neuraminidase, NA inhibitor, drug-resistant mutation, in vitro selection
Although neuraminidase (NA) inhibitors, oseltamivir and zanamivir, are globally used to treat influenza, they will become obsolete once influenza viruses develop resistance to them. For influenza B viruses isolated from patients treated with NA inhibitors, several amino acid substitutions in NA have been identified that confer NA inhibitor-resistance: arginine-to-lysine, aspartic acid-to-asparagine/tyrosine, and glycine-to-serine substitutions at positions 152 (Arg152Lys), 198 (Asp198Asn/Tyr), and 402 (Gly402Ser), respectively [1–4]. However, several influenza B viruses with reduced NA inhibitor sensitivity have been isolated from patients who were not treated with NA inhibitors. These viruses possess the following amino acid substitutions in NA that were not found in concurrently circulating viruses: aspartic acid-to-asparagine/glutamine, isoleucine-to-threonine, serine-to-glycine, histidine-to-tyrosine, and arginine-to-lysine at positions 198 (Asp198Asn/Glu), 222 (Ile222Thr), 250 (Ser250Gly), 274 (His274Tyr), and 371 (Arg371Lys) [3, 5, 6]. The sensitivity of some of these viruses to NA inhibitors was not dramatically reduced compared with that of isolates from NA inhibitor-treated patients. For example, NA Ile222Thr-possessing isolates from patients who were not drug-treated exhibited only 6- to 7-fold and 2- to 3-fold reduced sensitivity to oseltamivir and zanamivir, respectively, compared with the median 50% inhibitory concentration (IC50) values for type B viruses [3], whereas the zanamivir-sensitivity of an NA Arg152Lys-possessing virus that was isolated from an immunocompromised influenza patient treated with zanamivir was 1000-fold lower than that of the pretreated isolate [2]. Therefore, it was unclear whether the NA substitutions found in the influenza B isolates from patients who were not treated with the drugs arose spontaneously. Interestingly, there have been no reports of in vitro selection of influenza B viruses resistant to oseltamivir, which is the most extensively used NA inhibitor in clinical practice. In fact, only a glutamine-to-glycine/aspartic acid substitution at position 119 (Glu119Gly/Asp) and a His274Tyr substitution were detected in the NA of viruses passaged in cell culture experiments with zanamivir or peramivir [7–11]. To determine if the NA substitutions found in the influenza B isolates from patients who were not treated with the drugs arose due to selective pressure by the drugs, we attempted to select NA inhibitor-resistant viruses in vitro.
We serially passaged B/Yokohama/UT2119/2005 virus in Madin-Darby canine kidney (MDCK) cells and MDCK cells expressing the human β-galactoside a2,6-sialyltransferase I gene (MDCK-ST6 cells) [12] with 1 μmol/l of either oseltamivir carboxylate (Roche Products, Basel, Switzerland), which is the active metabolite of oseltamivir, or zanamivir (GlaxoSmithKline, Brentford, UK). Confluent monolayers of these cells were infected with 10-fold dilutions of virus (10−3 to 10−8) and the supernatants of the cytopathic effect-positive wells that were infected with the second-highest dilution of virus were subjected to subsequent passages. After 20 passages, IC50 values of viruses for oseltamivir and zanamivir were determined by using a fluorescence-based sialidase inhibition assay [3, 12]. In this way, we could select viruses with reduced sensitivity to NA inhibitors (see below for detail): P20-CK-ZV being passaged in MDCK cells with zanamivir and P20-ST6-OV being passaged in MDCK-ST6 cells with oseltamivir (data not shown).
In addition to NA, hemagglutinin (HA) is also known to affect the sensitivity of viruses to NA inhibitors [13, 14]. Therefore, to identify amino acid substitution(s) responsible for conferring NA inhibitor-resistance, we cloned the NA and HA genes of P20-CK-ZV into a cloning vector as described previously [3]. Sequence analysis of at least five cDNA clones of each gene revealed that two-thirds (6/9 clones) of P20-CK-ZV possessed NA Asp198Asn and all had HA2 Arg65Gly (7/7 clones) substitutions (Table 1). By retrospective analyses, we found that the NA Asp198Asn and HA2 Arg65Gly substitutions emerged at passages 12 and 18, respectively. Similarly, we sequenced the NA and HA genes of P20-ST6-OV and found NA Ile222Thr (5/8 clones) and HA1 Ile307Thr (5/7 clones) substitutions (Table 1): the NA Ile222Thr and HA1 Ile307Thr substitutions were first detected at passages 18 and 17, respectively. When B/Yokohama/UT2119/2005 virus was passaged 20 times without drugs, we detected only a lysine-to-arginine substitution at position 382 in HA, which is structurally far from the receptor-binding site and, therefore, unlikely to affect receptor binding or NA inhibitor susceptibility. We then plaque-purified P15-CK-ZV, P20-CK-ZV and P20-ST6-OV and obtained three cloned viruses; PP-P15-CK-ZV with the NA Asp198Asn substitution, PP-P20-CK-ZV with the NA Asp198Asn and HA2 Arg65Gly substitutions, and PP-P20-ST6-OV with the NA Ile222Thr and HA1 Ile307Thr substitutions (Table 2). Despite the extensive plaque purification of P20-ST6-OV, we could not obtain viruses possessing only the NA Ile222Thr substitution. These purified viruses were subjected to a sialidase inhibition assay to determine the IC50 values for oseltamivir and zanamivir (Table 2). As compared with the parent virus, the NA Asp198Asn substitution conferred 3.0–3.2 and 4.9–5.8 -fold higher resistance against oseltamivir and zanamivir, respectively, and the NA Ile222Thr substitution conferred 7.2 and 2.1-fold higher resistance against oseltamivir and zanamivir, respectively.
Table 1.
Properties of influenza B viruses after passage in the presence of NA inhibitors.
| Virus passage condition | Virus | Number of molecular clones possessing amino acid substitutiona in: | |||
|---|---|---|---|---|---|
| NA | HA2 | HA1 | |||
| Asp198Asn | Ile222Thr | Arg65Gly | Ile307Thr | ||
| In MDCK cells with zanamivir | P10b-CK-ZV | 0/5 clone | NDc | 0/6 clone | ND |
| P12-CK-ZV | 1/8 clone | ND | NTd | ND | |
| P14-CK-ZV | 2/7 clone | ND | NT | ND | |
| P15-CK-ZV | 7/7 clone | ND | 0/7 clone | ND | |
| P16-CK-ZV | 4/5 clone | ND | 0/5 clone | ND | |
| P18-CK-ZV | 5/6 clone | ND | 3/8 clone | ND | |
| P20-CK-ZV | 6/9 clone | ND | 7/7 clone | ND | |
| In MDCK-ST6 cells with oseltamivir | P16-ST6-OV | ND | 0/8 clone | ND | 0/8 clone |
| P17-ST6-OV | ND | 0/8 clone | ND | 3/7 clone | |
| P18-ST6-OV | ND | 5/7 clone | ND | 8/8 clone | |
| P20-ST6-OV | ND | 5/8 clone | ND | 5/7 clone | |
Table 2.
IC50 values of plaque-purified variants with an NA substitution
| Virus | Amino acid substitution in: | IC50 value, nmol/l (fold-changea) | |||
|---|---|---|---|---|---|
| NA | HA1 | HA2 | Oseltamivir | Zanamivir | |
| B/Yokohama/UT2119/2005 | -b | - | - | 72.5 | 10.3 |
| PP-P15-CK-ZV | Asp198Asn | - | - | 218.0 (3.0-fold) | 50.5 (4.9-fold) |
| PP-P20-CK-ZV | Asp198Asn | - | Arg65Gly | 235.4 (3.2-fold) | 59.4 (5.8-fold) |
| PP-P20-ST6-OV | Ile222Thr | Ile307Thr | - | 523.3 (7.2-fold) | 21.6 (2.1-fold) |
Fold-change was calculated based on the IC50 value of the parent B/Yokohama/UT2119/2005 virus.
not applicable.
We have reported that several influenza B viruses with reduced sensitivity to oseltamivir and/or zanamivir were isolated from untreated patients in Japan, where these drugs are extensively used [3]. The IC50 values of the clinical isolates with the NA Asp198Asn (237.3 and 48.9 nmol/l for oseltamivir and zanamivir, respectively) and NA Ile222Thr substitution (479.9 and 23.3 nmol/l for oseltamivir and zanamivir, respectively) were comparable to those of the corresponding in vitro isolates in this study (Table 2). These results suggest that influenza B viruses isolated from patients who were not treated with NA inhibitors must have been selected in patients who were treated with an NA inhibitor and then transmitted to others.
In addition to the NA substitutions, HA2 Arg65Gly and HA1 Ile307Thr substitutions were detected in viruses passaged with NA inhibitors (Table 1). HA with decreased affinity for receptor binding may partly contribute to the reduced NA inhibitor-sensitivity of viruses [14]. In fact, the amino acid residue at position 65 in HA2 maps close to the second ligand binding site [14], whose significance for HA-receptor binding is as yet unclear. Although the amino acid residue at position 307 in HA1 is distant from the primary receptor binding site [14, 15], the HA1 Ile307Thr substitution may also play a role in the emergence of NA inhibitor-resistant viruses.
Acknowledgments
We thank Larisa Gubareva (Department of Internal Medicine, University of Virginia Health Sciences Center, VA) for providing us with a protocol for the sialidase inhibition assay, Susan Watson for editing this manuscript, and Krisna Wells for technical assistance. This work was supported, in part, by Grants-in-Aid for Specially Promoted Research and for Scientific Research, by a Contract Research Fund for the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, by ERATO (Japan Science and Technology Agency), by the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by National Institute of Allergy and Infectious Diseases Public Health Service research grants, USA. None of the funding sources had any role in the design or conduct of the study, in the collection, management, analysis, or interpretation of the data, or in the preparation, review, or approval of the manuscript.
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