Generation and Characterization of Recombinant Influenza A(H1N1) Viruses Resistant to Neuraminidase Inhibitors

2.1 Cells and viruses

Madin Darby canine kidney (MDCK) cells and human embryonic kidney cells transformed with large T antigen (293T cells) were obtained from American Type Culture Collection (Manassas, VA, USA). A/Chungbuk/4448/2008(H1N1) virus isolated in South Korea during the 2008–2009 season was used for the generation of recombinant viruses.

2.2 Antiviral compounds

Tartrate salt of oseltamivir carboxylate (active form of Tamiflu) was provided generously by F. Hoffmann-La Roche, Inc. (Basel, Switzerland); it was dissolved in distilled water such that the final oseltamivir carboxylate concentration was 10 mM. Zanamivir (Relenza), which was provided by GlaxoSmithKline (Stevenage, UK), was dissolved in distilled water to a final zanamivir concentration of 10 mM. Aliquots were stored at −20 °C until use.

2.3 Generation of recombinant viruses and site-directed mutagenesis

Recombinant viruses were generated using the eight-plasmid reverse genetics system with pHW2000 (the vector was provided kindly by Robert Webster, St. Jude Children's Research Hospital, Memphis, TN, USA) [12]. The eight genes from the cultured A/Chungbuk/4448/2008(H1N1) virus were amplified by reverse transcription polymerase chain reaction (RT-PCR) and incorporated into the pHW2000 plasmid. All eight plasmids were transfected into a coculture of 293T and MDCK cells for 3 days, and then further prepared in 10-day-old embryonated chicken eggs. The collected supernatant from the transfected cells was injected into the allantoic cavities of the eggs and incubated at 37 °C for 3 days to amplify the rescued viruses. The viruses generated in eggs were stored at −80 °C until use.

To investigate mutations at NA key residues (N1 numbering: I117V, I117M, E119V, I223V, Y275H, R293K, N295S, and S334N), site-directed mutagenesis was conducted on the NA gene cloned plasmid using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA, USA). All recombinant plasmids were sequenced to ensure the absence of undesired mutations. The eight plasmids were then cotransfected to 293T mixed with MDCK cells. Supernatants were collected at 3 days post-transfection and used to inoculate specific pathogen-free (SPF) eggs. Introduced mutations of the recombinant wild type virus as well as the NA mutants were confirmed by sequencing.

2.4 NA activity assay and NA inhibition assay

The NA activity of each virus sample was determined by a modified fluorometric assay that measured 4-methylumbelliferone released from the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA; Sigma-Aldrich, St. Louis, MO, USA) by the enzymatic activity of the influenza virus NA [13,14].

The drug resistance phenotype was determined by NA inhibition assays using the MUNANA (Sigma) substrate, with minor modifications [13–15]. The viruses were tested for susceptibility to oseltamivir and zanamivir. To determine the drug concentration required to inhibit 50% of the NA activity [50% inhibitory concentration (IC₅₀)], 50 μL of virus, diluted according to the NA activity assay, was mixed with various concentrations of inhibitor in microtiter plates (FluoroNunc plates; Nalge Nunc International, Penfield, NY, USA). The final reaction mixture concentration of the NAIs ranged from 0.01 nM to 10,000 nM. The virus–inhibitor mixture was incubated at room temperature for 45 minutes prior to the addition of 50 μL of the MUNANA substrate (0.3 mM) and then incubated at 37 °C for 60 minutes. The reaction was terminated by the addition of 100 μL of the stop solution. The IC₅₀ for each drug was calculated from the dose–response curve using GraphPad Prism software, version 5 (San Diego, CA, USA). The sensitive viruses belonged to an IC₅₀ range of 0.001–15 nM for zanamivir and 0.001–25 nM for oseltamivir carboxylate. The resistant viruses belonged to both inhibitors in the IC₅₀ range from 43 nM to as high as 8020 nM [15].

3.1 Generation of recombinant wild type and NA mutant influenza viruses by reverse genetics system

The recombinant wild-type [A/Chungbuk/4448/2008(H1N1)] virus was generated by reverse genetics. That is, the eight genes of A/Chungbuk/4448/2008(H1N1) virus (PB2, PB1, PA, HA, NP, NA, M, and NS) were amplified by RT-PCR (data not shown). The PCR products were then inserted into pHW2000 vector. After the cell transfection with these eight plasmids, supernatants were collected at 3 days post-transfection and used to inoculate SPF eggs. The generation of recombinant wild-type virus was confirmed by sequencing.

We used mutagenesis and plasmid-based reverse genetics to generate recombinant A/Chungbuk/4448/2008(H1N1) viruses containing either the wild-type NA (Y275) or a single amino acid change at NA residue 117 (I→V), 117 (I→M), 119 (E→V), 223 (I→V), 275 (Y→H), 293 (R→K), 295 (N→S), or 334 (S→N). For example, the recombinant Chungbuk-Y275-I117V virus contained an I→V amino acid change at residue 117 of NA in the wild-type virus [A/Chungbuk/4448/2008(H1N1)] and the recombinant Chungbuk-Y275H-I117V virus contained I→V and Y→H amino acid changes at residues 117 and 275 of NA in the wild-type virus, respectively (numbering is based on the N1 NA protein throughout). Totally, eight single mutants (Y275-I117V, Y275-I117M, Y275-E119V, Y275-I223V, Y275H, Y275-R293K, Y275-N295S, and Y275-S334N), 10 double mutants (Y275H-I117V, Y275H-I117M, Y275H-E119V, Y275H-I223V, Y275H-R293K, Y275H-N295S, Y275H-S334N, Y275-N295S-I117V, Y275-N295S-I117M, and Y275-N295S-I223V), and five triple mutants (Y275H-E119V-I117V, Y275H-E119V-I117M, Y275H-N295S-I117V, Y275H-N295S-I117M, and Y275H-N295S-I223V) were generated (Table 1). Introduced mutations of the recombinant NA mutants were confirmed by sequencing.

Table 1

Generation of the recombinant influenza A(H1N1) viruses.

3.2 Susceptibility of recombinant wild-type and NA mutant influenza viruses to NAIs in vitro

An enzymatic NA inhibition assay was used to characterize the susceptibilities of recombinant A/Chungbuk/4448/2008(H1N1) virus and each of the 23 mutants to oseltamivir and zanamivir (Table 2). NA-inhibition assays showed that E119V mutation of the wild-type virus containing tyrosine (Y) at NA residue 275 (Chungbuk-Y275-E119V) and E119V mutation of the mutant virus containing histidine (H) at NA residue 275 (Chungbuk-Y275H-E119V) conferred important levels of resistance to zanamivir, with 202.5-fold and 135.7-fold increases in IC₅₀ values compared with that of the wild-type virus (Chungbuk-Y275) and Chungbuk-Y275H mutant virus, respectively, whereas these mutants were susceptible to oseltamivir. Compared with the single Chungbuk-Y274H mutant, triple Chungbuk-Y275H-E119V-I117V and Y275H-E119V-I117M mutants had increased IC₅₀ values for zanamivir (390.7- and 181.0-fold), whereas these mutants were susceptible to oseltamivir. All the single, double, and triple mutants containing the E119V mutation were associated with the resistance to zanamivir.

Table 2

Susceptibility of the recombinant influenza A(H1N1) viruses to NAIs.

All the single and double mutants containing tyrosine (Y) at NA residue 275 of the wild-type virus except the single mutant (Chungbuk-Y275-E119V) conferred important levels of resistance to oseltamivir, whereas the wild-type virus and these mutants were associated with the susceptibility to zanamivir.

The N295S mutation in the wild-type virus (Chungbuk-Y275-N295S) was associated with a reduction in oseltamivir resistance (0.2-fold decrease in IC₅₀ compared with that of the wild-type virus), whereas N295S mutation of the mutant virus containing histidine (H) at NA residue 275 (Chungbuk-Y275H-N295S) was associated with an increase in oseltamivir resistance (40.3-fold increase in IC₅₀ compared with that of Chungbuk-Y275H mutant virus). Compared with the single Chungbuk-Y274-N295S mutant, double Chungbuk-Y275-N295S-I117V, Y275-N295S-I117M, and Y275-N295S-I223V mutants had slightly increased IC₅₀ values for oseltamivir (3.1-fold, 1.1-fold, and 2.8-fold), whereas these mutants were susceptible to zanamivir. Compared with the double Chungbuk-Y274H-N295S mutant, triple Chungbuk-Y275H-N295S-I117V, Y275H-N295S-I117M, and Y275H-N295S-I223V mutants also had slightly increased IC₅₀ values for oseltamivir (1.5-fold, 1.3-fold, and 2.4-fold), whereas these mutants were susceptible to zanamivir.

The single I117V, I117M, I223V, R293K, N295S, or S334N mutation did not seem to contribute significantly to a phenotype resistant to oseltamivir as well as to zanamivir.