1. IntroductionInfluenza spreads around the world in seasonal epidemics, resulting in the deaths of 250,000-500,000 people every year . Especially since April 2009, 18,449 deaths from the laboratory-confirmed pandemic (H1N1) 2009 were reported from 214 countries world-wide (as of August 1, 2010) . Vaccination and antiviral treatments are essential for the prevention and control of influenza infection.Recently, the oseltamivir-resistant seasonal influenza A(H1N1) virus was first reported to the World Health Organization by Norway in late January 2008 . However, the earliest detection of oseltamivir-resistant seasonal influenza A(H1N1) virus was in France and the United Kingdom on Week 46 of 2007 and in Norway on Week 47 of 2007 , and then an increased number of oseltamivir-resistant seasonal influenza A (H1N1) viruses has been spread worldwide [5-9]. According to the World Health Organization (WHO) report (as of March 18, 2009) , the prevalence of oseltamivir resistance in seasonal influenza A (H1N1) virus was 95% worldwide. All these oseltamivir-resistant isolates have the same mutation in the neuraminidase gene (H275Y in N1 numbering), conferring resistance to oseltamivir but not to zanamivir.The predominant influenza subtype circulating in Korea during 2008-2009 season was seasonal influenza A (H1N1). To determine whether the oseltamivir-resistant viruses have spread to Korea, we examined seasonal influenza A (H1N1) isolated in Korea from Week 36 of 2008 to Week 35 of 2009 for antiviral susceptibility.In this study, we tested 534 (18.5%, 534/2,880) influenza A (H1N1) viruses among 2880 influenza A(H1N1) viruses isolated throughout the country by the Korean Influenza Surveillance Scheme (KISS) during the 2008-2009 influenza season. For genetic analysis of M2 gene, we analyzed the amino acid substitutions at positions 26, 27, 30, 31, or 34 within the transmembrane domain of the M2 protein, known to confer resistance to amantadine . For genetic analysis of NA gene, we also analyzed influenza A (H1N1) virus for amino acid substitutions at positions 119, 136, 223, 275, 293, and 295 of the NA gene (N1 numbering), known to confer resistance to neuraminidase inhibitor drugs [12,13]. We also phylogenetically analyzed both HA1 and NA sequences in the influenza A (H1N1) viruses.Here we described the high rate of oseltamivirresistant influenza A (H1N1) viruses identified from influenza surveillance in Korea during the 2008-2009 influenza season and the circulation of these viruses globally based on the sequence data.
2.1. VirusesInfluenza isolates obtained by KISS from Week 36 of 2008 to Week 35 of 2009 were used in this study. Viruses were randomly selected by region and date, and 534 A (H1N1) viruses were tested for genotype and phenotype resistant to amantadine and neuraminidase inhibitors (oseltamivir and zanamivir).Viruses were propagated in Madin-Darby Canine Kidney (MDCK) cells in Dulbecco’s modified essential medium (DMEM) supplemented with 5% fetal bovine serum at 35℃ and with 5% CO2. Viral stocks were stored at -70℃.
2.2. Extraction of viral RNAViral RNA was extracted from 140 μL of virus culture using a total RNA isolation kit and a QuickGene-810 instrument (Fujifilm, Tokyo, Japan) according to the manufacturer’s instructions. RNA was eluted in 50 μL of the elution buffer provided in the kit and either used directly in reverse transcription polymerase chain reaction (RT-PCR) or stored at -70℃.
2.4. Sequence analysisThe PCR products were purified for sequencing by using PCR product purification kit (Qiagen, Hilden, Germany). Both strands were sequenced by an automated method with fluorescent dideoxy-chain terminators (Applied Biosystems, Foster City, USA). At least two corresponding electropherograms for the M2, NA, and HA regions were analyzed. The sequences were aligned with the MegAlign program (DNASTAR Inc. Software, Madison, WI, USA). We analyzed the established M2 inhibitor resistance markers (26, 27, 30, 31, or 34 of M2 protein) and NA inhibitor resistance markers (119, 136, 223, 275, 293, and 295 of the NA protein in N1 numbering) in influenza viruses. We also phylogenetically analyzed the HA1 and NA coding regions in the influenza A (H1N1) virus by using MEGA with the neighbor-joining method . The nucleotide sequences of the Korean isolates used in this study were compared with those of the WHO recommended vaccine strains and other reference strains from GenBank (National Center for Biotechnology Information, NCBI).
2.5. Neuraminidase inhibition assayFluorescence-based neuraminidase inhibition (NI) assay according to the WHO Collaborating Centre for Reference and Research on Influenza manual was conducted using 405 influenza A (H1N1) viruses isolated in Korea during the 2008-2009 season. A detailed method of the neuraminidase inhibition assay is described in the Supplementary Methods online.
3.1. Seasonal surveillanceInfluenza viruses were obtained from KISS during the 2008-2009 season from influenza-like illness patients in seven cities and nine provinces, representing all regions of South Korea.Overall influenza activity in Korea was prevailed by A (H1N1) and A (H3N2) viruses during the 2008-2009 season compared with B virus during the 2007-2008 season. A total of 5025 influenza viruses were isolated and 3214 (64.0%), 1748 (34.8%), and 63 (1.3%) were identified as influenza A (H1N1), A (H3N2), and influenza B viruses, respectively, during the 2008-2009 season (Figure 1). The first virus in this season was isolated on Week 37 of 2008 and the strain was influenza A (H3N2) virus and then one more virus of A(H3N2) was isolated on Week 43. Influenza A (H1N1) virus was first observed on Week 48 of 2008, and it dramatically increased until Week 52 of 2008, and then decreased to Week 7 of 2009. Isolation of the viruses
3.2.1. Resistance of NA inhibitorsFor genetic analysis of NA gene, nucleotide sequences of the NA gene were determined for the influenza A isolates. At the end of November 2008 (Week 48), an unusual high proportion (15/16) of oseltamivir-resistant strains was detected in A (H1N1) viruses that had an amino-acid change of histidine to tyrosine at the 275th position (N1 numbering) in the NA gene. All the resistant isolates (n = 533) possessed the H275Y mutation in the NA gene. Overall, a resistance profile for 533 of the 534 A (H1N1) viruses (99.8%) was obtained through genotypic analysis of NA genes (Table 1). Influenza A (H1N1) viruses (n = 405) were also investigated by phenotypic analysis using fluorescence-based neuraminidase inhibitor (NI) assay. Among these, all the H275Y resistant isolates (n = 404) were resistant to oseltamivir (mean IC50: 643.4), while still susceptible to zanamivir (mean IC50: 0.53). The data from NI assay correlated 100% for virus genotypes and was identified by sequencing.
3.2.2. Amantadine resistanceInfluenza A viruses resistance to amantadine were analyzed by the sequencing of M2 region. We randomly
|No. of resistant/tested||Resistance rate (%)|
3.3. Phylogenetic analysis of HA and NA genesA total of 523 HA (HA1 subunit 326 amino-acids) sequences (16.3%, 523/3214) and 532 NA (full-length 470 amino-acids) sequences (16.6%, 532/3214) of influenza A (H1N1) during the 2008-2009 season were analyzed to generate phylogenetic trees. The first A (H1N1) isolate during the 2008-2009 season was oseltamivir-sensitive virus and clustered in clade 2C, represented by A/Hong Kong/2652/2006, while most of
4. DiscussionTo monitor the drug resistance of influenza A viruses, we performed genetic and phenotypic analyses of the NA and M2 genes of Korean isolates during the 2008-2009 season (Table 2). For oseltamivir and zanamivir resistance, a total of 829 influenza A viruses.To investigate whether these oseltamivir resistant viruses might be transmitted from abroad, we examined travelers who suspected influenza-like illness coming through Incheon International Airport in South Korea. Of 15 specimens collected at the airport during January to March 2009, six A (H1N1) and three A (H3N2) viruses were isolated. The A (H1N1) viruses were isolated from travelers who visited Hong Kong, Kazakhstan, the Philippines, and Thailand. Through a sequence analysis of the HA and NA genes, all A (H1N1) viruses were identified as the oseltamivir resistant viruses and all A (H3N2) viruses were not. Furthermore, the sequences of these A (H1N1) viruses were grouped together with most of Korean A (H1N1) isolates (Figure 2, arrows). It revealed that the resistant A (H1N1) viruses could be introduced into Korea by travelers coming from these foreign countries.Based on the phylogenetic analysis of the HA and NA genes, this emergence of oseltamivir-resistant influenza A (H1N1) virus in Korea coincided with the dominant circulation of this virus during the 2007-2008 season in Europe . Phylogenetic analysis of the HA1 coding region of Korean isolates during the 2008-2009 season showed most of Korean A (H1N1) viruses belonged to clade 2B, represented by A/Brisbane/10/2007, which is the 2008-2009 influenza vaccine strain (Figure 2A). Most of Korean isolates from the 2008-2009 season possessed an A189 T mutation in the HA1 coding regions (Figure 2A), as reported in Japan . Phylogenetic analysis of the NA coding region of the Korean isolates during the 2008-2009 season showed that the viruses fall into clade 2B. They clustered together with A/Norway/1736/2007, which were isolated in Norway in November 2007 (Figure 2B). It also showed that all of the oseltamivir resistant viruses since the 2007-2008 season containing the Korean resistant viruses and A/Norway/1736/2007 possessed the D354 G mutation as well as the H275Y mutation in NA genes compared with those of A/Brisbane/59/2007 (Figure 2B) [4,5]. This suggests that the oseltamivir resistant viruses first emerged in Europe in November 2007 and circulated globally to many countries,including the United States, Canada, South Africa, Australia, and Japan, and then would spread to Korea in November 2008 [4-9]. These oseltamivir-resistant viruses are easily transmissible and circulate independently of drug use. It suggests that the genetic variations of the HA and NA genes may contribute to their overall fitness and transmissibility.Here, our data provide the information on the adequate use of antiviral drugs in a rapid manner for influenza treatment through antiviral resistance monitoring. Thorough monitoring of antiviral resistance in influenza viruses is therefore an essential part of influenza virus surveillance and is needed to track the emergence and worldwide spread of drug-resistant viruses.