Rapid Genotypic Assay for Detection of Oseltamivir-Resistant Influenza A (H1N1) Viruses

Two classes of drugs are currently used for the treatment of influenza virus infections: M2 inhibitors (M2Is; amantadine and rimantadine) and neuraminidase inhibitors (NAIs; oseltamivir and zanamivir). In recent years, a high percentage of circulating influenza A (H3N2) viruses have shown resistance to M2Is (6). Until recently, there was a low prevalence of NAI resistance among circulating viruses (8). However, surveillance in Europe during the 2007-2008 influenza season revealed the sudden emergence of oseltamivir resistance in influenza A (H1N1) virus isolates, and these resistant viruses spread worldwide in the following 2008-2009 season (10). Since early April 2009, a novel strain of influenza A (H1N1) virus has spread rapidly worldwide (3), and the World Health Organization declared an H1N1 pandemic in 2009. While resistance to oseltamivir is rare in novel H1N1 viruses at present (4, 5), constant surveillance is essential to monitor the emergence and spread of drug-resistant viruses. The oseltamivir-resistant viruses harbor a specific mutation in the neuraminidase (NA) protein: a substitution of Tyr for His at position 275 (N1 numbering). The current screening methods for the resistant viruses are performed to detect this mutation. Recently, new molecular detection methods involving the use of real-time PCR have been reported (1, 2, 9). Although these methods are rapid and sensitive, the probes used in real-time PCR are rather expensive. Hence, there is a need to develop a more cost-effective approach. In a previous study, we developed a mismatch amplification mutation assay-PCR (MAMA-PCR) approach to detect a gene mutation that confers amantadine resistance on influenza A (H3N2) viruses (7). The present report describes a new MAMA-PCR method for detecting the H275Y mutation in the NA genes of oseltamivir-resistant influenza A (H1N1) viruses. Throat swab specimens from patients were collected as part of a nationwide surveillance program. The specimens were inoculated onto MDCK cells, and viruses were isolated from infected cells. Viral RNA was extracted from throat swab specimens or from viral culture supernatants. cDNA was synthesized from viral RNA and used as the template for PCR. Primers with conserved sequences (NAN1F and NAN1R) (Table ​(Table1)1) were designed for the control PCR amplification of a 938-bp NA gene fragment. Mismatched reverse primers (MAMA275H and MAMA275Y) (Table ​(Table1)1) were designed for the detection of codons for His 275 (CAT) and Tyr 275 (TAT), respectively. A MAMA primer was added to each PCR mixture in order to generate a 539-bp PCR product only when the cDNA contained the corresponding gene sequence. The PCR mixture (25 μl) contained 2.5 μl of cDNA, 1 U of Ex Taq DNA polymerase (TaKaRa), 10 pmol of the forward primer, 2.5 pmol of the reverse primer, and 25 pmol of the MAMA primer. The reaction mixture was subjected to an initial denaturation step at 94°C for 3 min and then 30 cycles of PCR at 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s. TABLE 1. MAMA-PCR primers for detection of the H275Y mutation Forty influenza A (H1N1) virus isolates from the 2007-2008 season and 16 from the 2008-2009 season were analyzed. A representative result from screening for the H275Y mutation is shown in Fig. ​Fig.1.1. The 589-bp product observed in lanes 1H to 4H indicates the presence of His at position 275, which renders the strain sensitive to oseltamivir, and the absence of the mutation. In contrast, the 589-bp product observed in lanes 5Y to 8Y indicates the presence of the mutation conferring oseltamivir resistance. The nucleic acid sequences of the NA gene were verified by sequencing the 938-bp control band. The MAMA-PCR method was found to clearly distinguish the mutant type from the wild type. This method can also be performed using viral RNA directly from throat swab specimens. FIG. 1. MAMA-PCR analysis for detecting a single mutation in the NA genes of oseltamivir-resistant and oseltamivir-sensitive influenza A (H1N1) viruses. Lanes: M, molecular size marker (100-bp ladder); 1, Aichi/92/2007 virus (harboring 275H); 2, Aichi/96/2007 ... Among the 40 examined strains isolated in the 2007-2008 season, 1 (2.5%) was found to have the H275Y mutation. All (100%) of the 16 examined strains from the 2008-2009 season were found to harbor the mutation. The incidences of resistant viruses in the two seasons are consistent with reports from the nationwide surveillance (10). Twenty-four pandemic H1N1 virus strains that were isolated in June 2009 were also examined using the primers listed in Table ​Table1.1. The MAMA-PCR revealed that all these strains were 275H viruses. The results were also verified by a subsequent sequencing analysis. The assay described herein will allow for more convenient screening of the viruses, without the need for expensive real-time PCR approaches, and is useful for not only seasonal influenza A (H1N1) viruses but also pandemic 2009 influenza (H1N1) virus strains.