作者：Yacine Abed,Nathalie Goyette, andGuy Boivin 作者单位：Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Québec City, Canada
The emergence of resistance to amantadine in influenza A viruses has been shown to occur rapidly during treatment as a result of single-amino-acid substitutions at position 26, 27, 30, 31, or 34 within the transmembrane domain of the matrix-(M)-2 protein. In this study, reverse genetics was used to generate and characterize recombinant influenza A (H1N1) viruses harboring L26F, V27A, A30T, S31N, G34E, and V27A/S31N mutations in the M2 gene. In plaque reduction assays, all mutations conferred amantadine resistance, with drug concentrations resulting in reduction of plaque number by 50% (IC 50 s) 154- to 3,300-fold higher than those seen for the wild type (WT). M2 mutants had no impairment in their replicative capacities in vitro on the basis of plaque size and replication kinetics experiments. In addition, all mutants were at least as virulent as the WT in experimentally infected mice, with the highest mortality rate being obtained with the recombinant harboring a double V27A/S31N mutation. These findings could help explain the frequent emergence and transmission of amantadine-resistant influenza viruses during antiviral pressure in the clinical setting.
【关键词】 Generation Characterization Recombinant Influenza Harboring Amantadine Resistance Mutations
Influenza A viruses are still associated with significant rates of morbidity and mortality during winter epidemics in both industrial and developing countries. These viruses are also responsible for occasional pandemics with a devastating impact on human health and global economy ( 6 ). Therefore, the control of influenza A virus infections remains a major international clinical goal. The adamantane compound amantadine was the first antiviral shown to inhibit the replication of influenza A viruses by blocking the ion channel activity of the matrix (M)-2 protein and preventing viral uncoating ( 14 ). Amantadine has been used for over 3 decades in the treatment and prevention of influenza A virus infections. Its use is associated with some benefits in terms of duration of illness and severity of symptoms ( 6 ). However, the emergence of drug-resistant viruses has been shown to occur rapidly during treatment as a result of single-amino-acid substitutions at position 26, 27, 30, 31, or 34 within the transmembrane domain of the M2 protein ( 9, 12 ). It has been also reported that two or more M2 mutant virus variants could be recovered from amantadine-treated children and immunocompromised patients ( 2, 9, 12 ). A recent report showed that H3N2-infected individuals shed more amantadine-resistant variants than their H1N1 counterparts ( 11 ). Moreover, specific amantadine-resistance mutations seem to be subtype dependent, with the S31N and V27A mutations predominating in H3N2 and H1N1 viruses, respectively ( 11 ).
Drug-resistant M2 mutants of the H3N2 subtype do not show impaired growth characteristics or altered virulence in ferrets ( 13 ). However, to our knowledge, the effects of M2 mutations on the growth properties and virulence of H1N1 viruses have not been studied. Furthermore, a systematic evaluation of various M2 mutations by use of a uniform genetic background (i.e., in recombinant viruses) has not been performed.
The aim of the present study was to use a reverse genetics system to assess the effects of various single and double mutations within the transmembrane domain of the M2 protein of influenza A (H1N1) viruses on the resistance phenotype to amantadine, in vitro replication kinetics, and virulence in mice.
MATERIALS AND METHODS
Plasmids. A set of plasmids allowing the rescue of the recombinant influenza A/WSN/33 (H1N1) virus was used for generating all M2 mutants ( 3 ). The reverse genetics system includes eight influenza virus RNA-coding transcription plasmids (pPOLI-PA, -PB1, -PB2, -NP, -HA, -NA, -M, and -NS) and polymerase and nucleoprotein expression plasmids (pCAGGS-PA, -PB1, -PB2, -NP) kindly provided by Peter Palese (Mount Sinai School of Medicine, New York, N.Y.). The M2 protein of influenza A/WSN/33 virus already contains an asparagine residue at position 31 conferring natural resistance to amantadine ( 5 ). Thus, the original pPOL-M plasmid was first mutated to a susceptible variant (the so-called wild type ) by incorporating an N31S substitution by use of appropriate primers and a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). The resulted pPOLM 31S was then used for incorporation of single mutations, i.e., L26F, V27A, A30T, and G34E, whereas the original (pPOL-M 31N ) plasmid was used for incorporation of the additional V27A mutation to generate the double mutant V27A/S31N.
Rescue of recombinant viruses. To generate the recombinant viruses, 1 µg each of the 12 plasmids was first transfected into 293T (human embryonic kidney) cells followed by viral amplification in Madin-Darby canine kidney cells essentially as reported previously ( 3 ). The M2 genes of all recombinant viruses were sequenced after reverse transcription-PCR amplification from cell culture supernatants to verify the presence of the desired mutations.
Determination of amantadine-resistance phenotype and replication capacities. Susceptibility of recombinant viruses to amantadine was determined using a plaque reduction assay in Madin-Darby bovine kidney (MDBK) cells. The number of plaques formed at different drug concentrations was recorded, and the drug concentration resulting in reduction of plaque number by 50% (IC 50 ) was calculated. The replicative capacity of the different recombinant viruses was evaluated on the basis of the virus plaque size generated in the absence of drug and by replication kinetics experiments. Briefly, MDBK monolayers were infected with recombinant viruses at a multiplicity of infection (MOI) of 0.001 and supernatants were collected at 12, 24, 36, 48, and 72 h postinfection and assayed for numbers of PFU in MDBK cells.
Experimental infection. The virulence of the recombinant viruses was evaluated in 7- to 8-week-old female BALB/c mice (Charles River Canada, Lasalle, Quebec, Canada). The animals were housed three per cage and kept under conditions which prevented cage-to-cage infections. Groups of 16 mice were inoculated intranasally, under isoflurane anesthesia, with 10 3 PFU of WT or mutant recombinant viruses in 40 µl of phosphate-buffered saline (PBS). One group of mice was inoculated with phosphate-buffered saline and served as sham-infected negative control. Mice were monitored daily during 14 days postinoculation for clinical signs (loss of body weight, abnormal behavior, and ruffled fur) and mortality. The deaths were recorded daily and the mean day of death (MDD) was determined.
Statistical evaluations. In animal studies, mortality rates were analyzed by using the Fisher’s exact test whereas the percentages of weight loss and MDDs were analyzed by using the nonparametric Mann-Whitney U rank-sum test. Virus titers generated in the in vitro replication kinetics experiments were analyzed by Student’s t test with Satterthwaite correction. A P value of 0.05 was considered significant.
Generation of influenza A (H1N1) virus recombinant M2 mutants. As part of this study, seven recombinant influenza A viruses, including the WT, five single mutants (L26F, V27A, A30T, S31N, and G34E), and one double (V27A/S31N) mutant, were rescued in the WSN/33 (H1N1) background. Sequencing of the M genes from recovered viruses revealed that all M2 mutations were correctly incorporated and that no additional changes were present.
Susceptibility to amantadine of recombinants. As shown in Table 1, all single- and double-recombinant M2 mutants exhibited resistance to amantadine when assessed by plaque reduction assay. The IC 50 values for these mutants were 154- to 3,300-fold higher than that for the recombinant WT virus. The IC 50 values for the double (V27A/S31N)-recombinant mutant were 10-fold and 2-fold lower than those for the V27A and S31N single mutants, respectively.
TABLE 1. In vitro and in vivo characteristics of recombinant influenza A (H1N1) M2 mutants
Replicative capacity and virulence of recombinants. Recombinant mutants generated plaques of sizes similar to those seen with the recombinant WT, with the exception of L26F and A30T mutants, whose plaque size was slightly reduced (by 1.5-fold) compared to the results seen with the WT (Fig. 1 ). Also, all recombinants showed similar replication kinetics in a 72-hour in vitro experiment, with no significant differences in virus titers at each time point (Fig. 2 ).
FIG. 1. Virus plaque size of recombinant WT A/WSN/33 (H1N1) virus and various M2 mutants. Recombinant WT and M2 mutant (L26F, V27A, A30T, S31N, G34E, and V27A/S31N) viruses generated in the supernatant of transfected 293T cells were propagated in 6-well plates of MDBK cells under 0.8% agarose. Plaques were visualized after 48 h of incubation at 37°C in the absence of drug. Numbers in parentheses indicate the mean plaque diameter in millimeters ± standard deviation determined from four plaques after enlargement (magnification, x 2.5).
FIG. 2. Replication kinetics of recombinant WT A/WSN/33 (H1N1) virus and various M2 mutants. MDBK cells were infected with the recombinant WT and M2 mutant (L26F, V27A, A30T, S31N, G34E, and V27A/S31N) viruses at an MOI of 0.001. At the indicated times postinfection, supernatants were collected, and virus titers were determined in MDBK cells by use of a standard plaque assay. No significant differences were found when comparisons of mean titers of recombinant mutants with those of the WT virus were made at each time point (Student’s t test with Satterthwaite correction). The values represent the means of three experiments with similar results.
An inoculum of 10 3 PFU resulted in clinical signs of illness (body weight loss, reduced vitality, and ruffled fur) in mice; these results appeared by day 4 postinoculation with all tested recombinants. As shown in Table 1, there were no significant differences in the mortality rates of the different M2 mutants compared to the WT results, with the exception of the double mutant (V27A/S31N), which was associated with an increased mortality rate (87.5 versus 50% for the WT). Similarly, the double mutant and the S31N mutant were associated with increased weight loss on day 5 compared to the WT results. The lowest MDD (4.71 days) was obtained with the double mutant (V27A/S31N), whereas there were no significant differences between the results for the WT and those for the remaining mutant groups (Table 1 ).
Despite the development of a new class of anti-influenza virus agents, the neuraminidase inhibitors, use of amantadine and its congener rimantadine still constitutes an important antiviral strategy for the control of influenza A virus epidemics; this strategy would have even more significance in the context of an eventual pandemic. Indeed, amantadine has an excellent inhibitory effect on different subtypes of influenza A viruses in addition to its low cost, good chemical stability, and excellent bioavailability. On the other side, a major concern has been the rapid emergence of amantadine-resistant viruses. Indeed, despite the fact that naturally occurring amantadine-resistant influenza A viruses are rarely found about (0.8% in the general population [ 16 ]), rates of resistance exceeding 30% and up to 80% have been found in H3N2 viruses after only a few days of therapy in both immunocompetent and immunocompromised subjects ( 6, 9, 12 ). Moreover, drug-resistant M2 mutants (H3N2 subtype) are transmissible and appear to retain good replication capacities and virulence in ferrets ( 13 ).
For unknown reasons, amantadine resistance appears to emerge less frequently during amantadine therapy in influenza A (H1N1) viruses than in A (H3N2) strains ( 11 ). In a study in Japan, the most frequent M2 mutation conferring amantadine resistance in H1N1 viruses occurred at position 27 (V27A) whereas the S31N mutation predominated in the H3N2 subtype ( 11 ). Other drug-resistant M2 mutations at positions 26 (L26F), 31 (S31N), and 34 (G34E) were recovered from H1N1 clinical isolates at a lower frequency ( 11, 12 ). In addition, a few uncommon M2 mutations such as A29V and I43V were also detected in H1N1 viruses recovered from amantadine-treated children, although the role of these mutations in drug resistance remains to be confirmed ( 12 ).
In this study, we aimed to assess the effects of known amantadine resistance M2 mutations on drug resistance, in vitro replicative capacities, and virulence in an influenza A (H1N1) virus (A/WSN/33) by use of a reverse genetics system. The rescue of our mutants from the same genetic background precluded the confounding effects of gene constellation and additional changes in other genes. A total of seven recombinant viruses, including the WT and five single and one double M2 mutants, were rescued. As expected, all mutants exhibited high-level resistance to amantadine, with 100-fold reductions (range, 154- to 3,300-fold) in susceptibility compared to the WT results (Table 1 ). Interestingly, the levels of resistance of the double V27A/S31N mutant were lower than that of each of the corresponding single mutants by factors of 10- and 2-fold, respectively (Table 1 ). Thus, the combination of these two M2 mutations did not confer a synergistic effect in term of resistance. The differences seen between the different mutants in the levels of resistance could be related to the variable effects of these mutations on the M2 protein activity as previously described ( 4 ). For example, M2 substitutions at positions 26, 30, 31, and 34 were found to decrease the M2 activity of influenza A/chicken/Germany/34 (H7N1) virus whereas substitutions at position 27 increased such activity ( 4 ). Furthermore, the I27T mutation was shown to suppress the attenuating effect of the S31N mutation on M2 activity in the double (I27T/S31N) mutant ( 4 ). Thus, although the M2 activity of our recombinants was not determined, it is likely that the different mutations evaluated in this study had different effects on the levels of M2 protein activity.
There was almost no impact of the different M2 mutations on viral fitness, as assessed on the basis of the size of viral plaques generated in the absence of drug and of virus titers following MDBK infection at a low MOI (Fig. 1 and 2 ). These findings are in agreement with a previous report showing that influenza A/H3N2 recombinant viruses can undergo multiple cycles of replication without M2 ion channel activity ( 15 ). In that study, recombinants containing the M2 gene of A/Udorn/307/72 (H3N2) with V27T, A30P, and S31N mutations were found to replicate as efficiently as the recombinant WT in Madin-Darby canine kidney cells. Also, a recombinant mutant which had no detectable M2 ion channel activity due to a deletion in the transmembrane domain of the protein (M2-del 29-31 ) exhibited replication efficiency in vitro similar to that of the WT virus ( 15 ). Similarly, the virulence of our recombinant M2 mutants was at least as high as that of the WT virus when evaluated in experimentally infected BALB/c mice (Table 1 ). Of note, the inoculation of mice with viruses harboring the V27A/S31N mutations resulted in a significantly lower MDD and higher mortality rate compared to the recombinant WT virus results. In addition, both the double mutant and the single S31N mutant were associated with increased weight loss compared to the WT virus. It is therefore possible that the S31N mutation could confer an advantageous virulence impact for the A/WSN/33 virus, which is a natural S31N variant. Of interest, the same mutation has also been recently reported in highly pathogenic H5N1 viruses infecting birds and humans in Asia ( 10 ). Other amantadine- or rimantadine-resistant human and avian influenza viruses have also shown a virulence pattern similar to those seen with their respective WT viruses in various animal models ( 1, 7, 13 ).
In conclusion, the results of our study suggest that in similarity to what has been reported with viruses of other subtypes, including H3N2, H2N2, and H5N2 viruses ( 1, 7, 13 ), M2 mutations conferring resistance to amantadine do not compromise the replicative capacity or virulence of H1N1 viruses. Such results support the clinical relevance of M2 mutants, particularly for immunocompromised patients, with whom amantadine-resistant influenza viruses have been associated with fatal infections ( 2, 9 ). However, the reason(s) that such amantadine-resistant influenza A virus variants have not been associated with a major epidemic remain(s) to be clarified. Finally, the effects of M2 mutations are in sharp contrast with those of neuraminidase mutations associated with resistance to neuraminidase inhibitors, which were found to compromise more severely the viral fitness and virulence of influenza A viruses ( 8 ). Altogether, these results may explain the difference in the frequency of drug resistance between these two classes of anti-influenza virus agents in the clinic.
This study was supported by the Canadian Institutes of Health Research and GlaxoSmithKline Canada.
We are grateful to Peter Palese (Mount Sinai School of Medicine, New York, N.Y.) and Ervin Fodor (Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom) for providing viral plasmids.
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