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Infection with influenza viruses A (H1N1), A (H3N2), or B causes substantial human illness and excess deaths each year (1,2). Vaccination against seasonal influenza is the key control measure used in Europe to minimize illness and death. Antigenic mismatch between vaccine components and circulating viruses occurs every few years, requiring reformulation of the vaccine (1). In addition, suboptimal immunization in patient groups for which vaccine is recommended provides the rationale for use of antiviral drugs in the prophylaxis and treatment of influenza. M2 ion channel inhibitors (M2Is), amantadine and rimantadine, have been available since 1964, but adverse effects, rapid development of resistance, and lack of activity against influenza B have limited their usefulness (3). The introduction of neuraminidase inhibitors (NAIs), oral oseltamivir and inhaled zanamivir, which are active against both influenza type A and B viruses, was a major breakthrough in treatment and prophylaxis of influenza using antiviral drugs (4). However, prescription data indicate that they are not widely used in Europe (Figure 1); by contrast, in Japan during the 2003–04 season alone, ≈6 million NAI treatment courses were prescribed (5). Figure 1 Prescription data of oseltamivir treatment courses for Western Europe (in thousands); 12 months of data for each year 2002–2007 and through September for 2008. Data from the United Kingdom, the Netherlands, Switzerland, and Portugal are excluded because of negligible values. Data provided by IMS Health (www.imshealth.com), London, UK.

a of oseltamivir treatment courses for Western Europe (in thousands); 12 months of data for each year 2002–2007 and through September for 2008. Data from the United Kingdom, the Netherlands, Switzerland, and Portugal are excluded because of negligible values. Data provided by IMS Health (www.imshealth.com), London, UK. Before the introduction of NAIs in 1999, and until 2007, <1% of viruses tested from unselected surveillance studies in a number of countries demonstrated natural resistance to NAIs (5–9). Limited development of resistance to oseltamivir has been observed in persons treated, with little evidence of onward transmission of resistant viruses (10), although low-level transmission of resistant variants cannot be discounted (11). However, oseltamivir-resistant viruses emerged in 18% (9/50) of treated Japanese children with influenza virus A (H3N2) infection and 16% (7/43) of treated Japanese children with influenza virus A (H1N1) infection, also with no evidence that these viruses transmitted efficiently (12,13). In late January 2008, we reported an unexpected high level and unexpected spread of oseltamivir-resistant influenza viruses A (H1N1) (ORVs) in Europe caused by a H275Y (H274Y in N2 numbering) amino acid substitution in the neuraminidase (NA) of these viruses (14). Here, we analyze the distribution and transmission of ORVs in Europe during the winter of 2007–08, when influenza viruses A (H1N1) were the predominant circulating viruses in European countries (Table).

pe caused by a H275Y (H274Y in N2 numbering) amino acid substitution in the neuraminidase (NA) of these viruses (14). Here, we analyze the distribution and transmission of ORVs in Europe during the winter of 2007–08, when influenza viruses A (H1N1) were the predominant circulating viruses in European countries (Table). Table Peak incidence rates of ILI or ARI infection for countries for which data were available, Europe, 2000–01 through 2007–08 influenza seasons* Country ILI/ ARI† Peak incidence rate/10,000 population during influenza season Peak incidence rates¶ 2000–01 through 2007–08 Dominant virus§ Median Range Consecutive no. seasons‡ Incidence rate ratio p value 2000–01 2007–08 Austria# ILI 168.1 108.0–263.2 4 NA 186.1 (H1) Belgium ILI 51.9 30.3–95.1 8 30.3 (H1) 38.0 (H1) 1.3 0.004 Bulgaria ARI NA NA 186.0 (H1) Czech Republic ARI 188.1 134.5–320.0 8 310.2 (H1) 144.4 (H1) 0.5 1.000 Denmark ILI 30.7 13.8–47.8 8 44.5 (H1) 13.8 (H1) 0.3 1.000 Estonia ILI 2.9 0.6–4.9 3 NA 2.9 (H1) France ARI 336.1 279.7–448.8 7 NA 279.7 (H1) Germany ARI 185.6 136.9–256.5 8 247.3 (H1) 136.9 (H1/B) 0.6 1.000 Greece ILI 27.7 23.1–42.1 3 NA 23.1 (H1) Hungary ILI 50.1 21.0–54.6 3 NA 54.6 (H1) Ireland ILI 7.5 2.9–12.1 8 12.1 (H1) 4.9 (H1/B) 0.4 1.000 Italy ILI 79.5 27.6–428.2 8 56.7 (H1) 72.1 (H1/B) 1.3 0.0001 Latvia ILI 45.6 25.1–93.3 5 NA 26.6 (H1) Lithuania ILI 34.9 13.3–47.2 7 NA 13.3 (H1) Luxembourg ILI 72.6 32.7–79.1 5 NA 67.4 (H1) The Netherlands ILI 10.3 6.6–24.0 8 6.9 (H1) 7.2 (H1/B) 1.0 0.400 Norway ILI 18.5 10.9–31.7 3 NA 10.9 (H1/B) Poland ILI 23.0 6.2–66.7 7 NA 16.6 (H1) Portugal ILI 8.1 3.0–17.4 8 3.8 (H1) 6.2 (H1/B) 1.6 0.016 Romania ILI 1.2 0.4–3.7 4 NA 1.4 (H1) Serbia ILI 37.8 30.6–44.9 2 NA 30.6 (H1) Slovakia ILI 136.3 49.5–337.3 8 337.3 (H1) 49.5 (H1) 0.1 1.000 Slovenia ILI 15.2 4.5–39.2 8 14.1 (H1) 20.4 (H1) 1.5 0.001 Spain ILI 21.2 4.2–54.1 8 4.2 (H1) 20.3 (H1/B) 4.8 0.0001 Sweden# ILI 2.0 1.6–5.8 5 5.8 (H1) 1.8 (B) 0.3 1.000 Switzerland ILI 39.8 19.4–53.2 7 NA 29.7 (H1) United Kingdom ILI 3.8 2.7–8.4 8 5.5 (H1) 2.7 (H1/B) 0.5 1.000 *ILI, influenza-like illness; ARI, acute respiratory infection; NA, data not available. †For countries where both ILI and ARI data were available, only the ILI data are shown. ‡Leading up to 2007–08. §Dominant virus estimated on the basis of combined sentinel and nonsentinel data. The limits for codominant virus types/subtypes were 45%:55%. ¶2007–08 compared with 2000–01.

nfection; NA, data not available. †For countries where both ILI and ARI data were available, only the ILI data are shown. ‡Leading up to 2007–08. §Dominant virus estimated on the basis of combined sentinel and nonsentinel data. The limits for codominant virus types/subtypes were 45%:55%. ¶2007–08 compared with 2000–01. The incidence rate ratio was calculated by dividing the peak incidence rate for 2007–08 by the peak incidence rate for 2000–01. If the p value estimated using z-statistics is <0.05, the incidence rate ratio is significantly >1, and therefore the peak incidence rate for 2007–08 is significantly higher than that for 2000–01. #Data for seasons 2002–03, 2003–04, and 2004–05 were missing. Methods Clinical Influenza Activity The European Influenza Surveillance Scheme (EISS) actively monitored influenza activity from week 40 (October 1–7) of 2007 through week 19 (May 5–11) of 2008. EISS covers all 27 European Union countries plus Croatia, Norway, Serbia, Switzerland, Turkey, and Ukraine. In each country each week, 1 or several networks of sentinel general practitioners (GPs) reported rates of consultation for influenza-like illness (ILI) or acute respiratory infection (ARI) (15–17). ARI includes ILI and all other acute respiratory infections. For Croatia, Finland, Turkey, and Ukraine, no consultation data were available.

h week, 1 or several networks of sentinel general practitioners (GPs) reported rates of consultation for influenza-like illness (ILI) or acute respiratory infection (ARI) (15–17). ARI includes ILI and all other acute respiratory infections. For Croatia, Finland, Turkey, and Ukraine, no consultation data were available. Virologic Analysis Sentinel GPs involved in clinical data recording of ILI or ARI also send nasal, pharyngeal, or nasopharyngeal specimens from a subset of their patients to the National Influenza Centers (NICs) for virus detection and characterization by using a variety of genetic or phenotypic methods (18–20). The NICs also analyzed specimens and influenza viruses obtained from other sources (e.g., from nonsentinel GPs, hospitals, or institutions). For Cyprus and Turkey, no virus detection data were available.

Centers (NICs) for virus detection and characterization by using a variety of genetic or phenotypic methods (18–20). The NICs also analyzed specimens and influenza viruses obtained from other sources (e.g., from nonsentinel GPs, hospitals, or institutions). For Cyprus and Turkey, no virus detection data were available. Antiviral Drug Susceptibility Monitoring Antiviral susceptibility data were generated either through the European Surveillance Network for Vigilance against Viral Resistance (VIRGIL) project at a single laboratory in London (UK Health Protection Agency) or directly by individual NICs by using methods described previously (14,21). Genetic analysis of virus isolates or clinical specimens was performed by using cycle-sequencing or pyrosequencing the NA gene, targeting the H275Y amino acid substitution in the N1 NA (22). The 50% inhibitory NAI concentration (IC50) of virus isolates was determined by using fluorescent or chemiluminescent enzyme assays (23,24). ORVs were defined as influenza viruses A (H1N1) with an IC50 >100 nmol/L for oseltamivir. Susceptibility to zanamivir was determined by using the same enzymatic method. Susceptibility to M2Is was determined by cycle-sequencing or pyrosequencing the M2 protein gene, targeting known resistance markers. Antiviral susceptibility data were not available for Cyprus, Lithuania, and Malta.

0 nmol/L for oseltamivir. Susceptibility to zanamivir was determined by using the same enzymatic method. Susceptibility to M2Is was determined by cycle-sequencing or pyrosequencing the M2 protein gene, targeting known resistance markers. Antiviral susceptibility data were not available for Cyprus, Lithuania, and Malta. Data Analysis To obtain United Kingdom estimates, clinical and virologic surveillance data and antiviral susceptibility data were totaled for England, Northern Ireland, Scotland, and Wales. A single web-based European database at the EISS password-protected website (www.eiss.org) was used to collect antiviral susceptibility data and linked patient demographic and clinical data (25). Updates on possible resistant viruses were provided at regular intervals to EISS members, the World Health Organization, and the European Centre for Disease Prevention and Control. The timing of the first week of continuous detection of influenza virus A and ORVs across Europe, both based on date of specimen collection, were analyzed by linear regression analysis using center longitude and center latitude of a country as explanatory variables. A maximum interruption of 1 week with no influenza virus A or ORV detection was allowed in estimating the first week of continuous detection. The average European delay between the first week of continuous detection of influenza virus A and of ORV was calculated as the average of the differences in number of weeks between both, by country.

n of 1 week with no influenza virus A or ORV detection was allowed in estimating the first week of continuous detection. The average European delay between the first week of continuous detection of influenza virus A and of ORV was calculated as the average of the differences in number of weeks between both, by country. The analysis of temporal trends in the prevalence of ORVs in countries and for Europe was confounded by different levels of sampling in different countries (18), enhanced antiviral susceptibility testing in some countries, and lack of data on the proportion of ORVs for some or most weeks for several other countries. To ensure a more representative picture of temporal trends in the proportion of ORVs, a mixed effect logistic regression modeling approach (26,27) was used, which allows modeling of binomial proportions, i.e., a numerator and a denominator as a function of time, where the coefficients of this function are allowed to vary for each country around a mean value, combining data from all countries. If there are no observations or the denominator is small, the fit will shrink to its overall mean, and uncertainties increase. Three fractions were modeled: “ILI per population covered,” “influenza A virus detections per specimens tested,” and “A (H1N1) resistant per A (H1N1) tested.” By multiplying the first 2 fractions by the total population, we obtained the number of patients with ILI who had influenza A in a country. By dividing this number by the sum of the number of patients with ILI who had influenza A for all countries, we obtained the relative weights. By multiplying the weights with the prevalences of ORVs summed over all countries, we obtained the weekly European prevalences of ORVs. The modeled weekly prevalences of ORVs were subsequently used to calculate the average prevalence of ORVs by country and for Europe (Technical Appendix).

obtained the relative weights. By multiplying the weights with the prevalences of ORVs summed over all countries, we obtained the weekly European prevalences of ORVs. The modeled weekly prevalences of ORVs were subsequently used to calculate the average prevalence of ORVs by country and for Europe (Technical Appendix). We performed all statistical analyses by using the software package R version 2.8.0 (28). Box-and-whisker plot analysis was used to select viruses with outlying high IC50 values for further analysis (7,29). For oseltamivir outlier identification, all viruses defined as resistant for oseltamivir (IC50 >100 nmol/L) were first removed. Minor outliers were defined as values lying between the upper quartile (UQ) + 1.5 × interquartile region (IQR) and UQ + 3 × IQR; major outliers were defined as values lying above UQ + 3 × IQR, based on analysis of all viruses in a particular subtype over a particular winter season. Phylogenetic analysis of NA and hemagglutinin (HA) gene sequences used maximum parsimony (PAUP* version 4.0; Sinauer Associates, Sunderland, MA, USA). Sequences of ORVs and oseltamivir-sensitive influenza A (H1N1) viruses (OSVs) were chosen as representative of influenza viruses A (H1N1) isolated during the 2007–08 influenza season (i.e., weeks 40–52 of 2007 and weeks 1–19 of 2008) in different European countries and a few from other regions of the world and were compared with those of a few influenza viruses A (H1N1) isolated before the 2007–08 season, including sporadically isolated ORVs. GenBank accession numbers are listed in the Appendix Table.

e., weeks 40–52 of 2007 and weeks 1–19 of 2008) in different European countries and a few from other regions of the world and were compared with those of a few influenza viruses A (H1N1) isolated before the 2007–08 season, including sporadically isolated ORVs. GenBank accession numbers are listed in the Appendix Table. Results Seasonal Surveillance The 2007–08 influenza season in Europe was initially dominated by influenza viruses A (n = 10,720; 60% of all influenza virus detections). Influenza viruses B (n = 7,150; 40% of all influenza virus detections) became dominant in week 8 (Figure 2). Of the 5,984 (56%) influenza viruses A subtyped, 5,748 (96%) were H1, and 236 (4%) were H3. Overall, influenza virus detections peaked in week 6, in week 4 for influenza viruses A (H1N1), and in week 8 for influenza viruses B. Of the 2,136 influenza viruses A (H1N1) characterized antigenically, 97% were reported to be closely related to the vaccine strain A/Solomon Islands/3/2006, although half of these viruses were reported to be more closely related to A/Brisbane/59/2007, the vaccine strain recommended for the 2008–09 season (30). Figure 2 Total number of influenza virus detections, by type and subtype and by week, Europe, winter 2007–08.

Results Seasonal Surveillance The 2007–08 influenza season in Europe was initially dominated by influenza viruses A (n = 10,720; 60% of all influenza virus detections). Influenza viruses B (n = 7,150; 40% of all influenza virus detections) became dominant in week 8 (Figure 2). Of the 5,984 (56%) influenza viruses A subtyped, 5,748 (96%) were H1, and 236 (4%) were H3. Overall, influenza virus detections peaked in week 6, in week 4 for influenza viruses A (H1N1), and in week 8 for influenza viruses B. Of the 2,136 influenza viruses A (H1N1) characterized antigenically, 97% were reported to be closely related to the vaccine strain A/Solomon Islands/3/2006, although half of these viruses were reported to be more closely related to A/Brisbane/59/2007, the vaccine strain recommended for the 2008–09 season (30). Figure 2 Total number of influenza virus detections, by type and subtype and by week, Europe, winter 2007–08. The first countries in Europe where influenza viruses A started to circulate continuously were France, Spain, Switzerland, and the United Kingdom in week 40. Spatial analysis of the timing of the first week of continuous detection of influenza viruses A across Europe (n = 30 countries) showed a west-to-east pattern: estimated parameter for longitude was 0.261 weeks per degree longitude (95% confidence interval [CI] 0.138–0.385, p = 0.001), and for latitude –0.108 weeks per degree latitude (95% CI –0.324 through 0.108, p = 0.366), with R2 = 0.32 for the linear regression fit.

Europe (n = 30 countries) showed a west-to-east pattern: estimated parameter for longitude was 0.261 weeks per degree longitude (95% confidence interval [CI] 0.138–0.385, p = 0.001), and for latitude –0.108 weeks per degree latitude (95% CI –0.324 through 0.108, p = 0.366), with R2 = 0.32 for the linear regression fit. Antiviral Drug Susceptibility The estimated number of influenza viruses A (H1N1) among all detected influenza viruses A (n = 10,720) was 10,291 following extrapolation from the proportion of 96% influenza viruses A (H1N1) among all 5,984 subtyped influenza viruses A. Of the 10,291 influenza viruses A (H1N1), 2,949 (29%) were tested for antiviral susceptibility, 1,080 by both phenotypic assay (IC50) and sequencing, 601 by phenotypic assay alone, and 1,268 by sequencing alone. Of the 2,949 viruses tested, 712 (24%) were oseltamivir resistant either by presence of the H275Y substitution (n = 548) or an IC50 >100 nmol/L for oseltamivir (n = 463) (Figure 3). Correlation was 100% between sensitive phenotype (IC50 <100 nmol/L) and the presence of H275 (n = 781) and between resistant phenotype (IC50 >100 nmol/L) and the presence of Y275 (n = 299). OSVs (n = 1,218) had a median IC50 of 1.7 nmol/L for oseltamivir (range 0.1 nmol/L–23.2 nmol/L) and only 9 minor outliers (thresholds IC50 >12.0 nmol/L and <53.1 nmol/L) were identified. ORVs (n = 463) had a median IC50 of 653 nmol/L (range 140 nmol/L–4,000 nmol/L). None of the 429 phenotypically characterized ORVs showed evidence of resistance to zanamivir (median IC50 1.8 nmol/L, range 0.2 nmol/L–25.8 nmol/L), and only 17 minor outliers (thresholds IC50 >8.5 nmol/L and <27.5 nmol/L) were identified. None of 237 ORVs tested for M2I sensitivity had any of the common resistance substitutions in the M2 protein.

y characterized ORVs showed evidence of resistance to zanamivir (median IC50 1.8 nmol/L, range 0.2 nmol/L–25.8 nmol/L), and only 17 minor outliers (thresholds IC50 >8.5 nmol/L and <27.5 nmol/L) were identified. None of 237 ORVs tested for M2I sensitivity had any of the common resistance substitutions in the M2 protein. Figure 3 Total influenza A viruses subtyped as H1N1 and number of oseltamivir-resistant or oseltamivir-sensitive viruses among the subset of influenza viruses A (H1N1) for which oseltamivir susceptibility was determined, by week, Europe, winter 2007–08. ORVs were detected in 22 of the 30 countries for which susceptibility data were available, with Norway having the highest proportion of ORVs (Figure 4). Modeling showed the overall average prevalence of ORVs by country ranged from 8.3% (95% CI 1.3%–21%) in Italy to 65.0% (95% CI 58.2%–71.3%) in Norway; for Europe, the average prevalence of ORVs was 20.1% (95% CI 15.2%–24.6%). Figure 4 Modeled average prevalence of oseltamivir-resistant influenza viruses A (H1N1), with 95% confidence intervals (error bars), ranked by country, Europe, winter 2007–08. Text columns on the right list the absolute cumulative number of oseltamivir-resistant influenza viruses A (H1N1) and number of influenza viruses A (H1N1) tested for oseltamivir susceptibility per country.

influenza viruses A (H1N1), with 95% confidence intervals (error bars), ranked by country, Europe, winter 2007–08. Text columns on the right list the absolute cumulative number of oseltamivir-resistant influenza viruses A (H1N1) and number of influenza viruses A (H1N1) tested for oseltamivir susceptibility per country. The earliest detection of ORVs was in France and the United Kingdom in week 46 and in Norway in week 47. Countries where continuous detection of ORVs first began included Norway in week 47, France in week 49, the United Kingdom in week 51, and the Netherlands in week 52. Spatial analysis of the timing of the first week of continuous ORV detection across Europe (n = 14 countries) showed a west-to-east trend pattern: estimated parameter for longitude was 0.156 weeks per degree longitude (95% CI 0.033–0.280, p = 0.031), and for latitude 0.007 weeks per degree latitude (95% CI –0.209 through 0.223, p = 0.953), with R2 = 0.36 for the linear regression fit. The average delay between the first week of continuous detection of influenza virus A and continuous detection of ORV was 5.7 weeks (range 0–15, 95% CI 2.8–8.4).

33–0.280, p = 0.031), and for latitude 0.007 weeks per degree latitude (95% CI –0.209 through 0.223, p = 0.953), with R2 = 0.36 for the linear regression fit. The average delay between the first week of continuous detection of influenza virus A and continuous detection of ORV was 5.7 weeks (range 0–15, 95% CI 2.8–8.4). Modeling showed a gradual increase for Europe in prevalence of ORVs over time, from close to 0 in week 40 to ≈56% in week 19 (Figure 5). This overall increase reflected prevalence increases in most individual countries in addition to Norway where the modeled prevalence started high at ≈60% and remained so throughout the period of virus circulation (Appendix Figure. Outside the main influenza virus A (H1N1) outbreak period, from week 51 to week 10 (Figure 2), the CIs for the prevalence of ORVs by country and for Europe were wide (Figure 5; Appendix Figure) because of the low numbers of influenza virus A (H1N1) detected or analyzed for antiviral resistance (Technical Appendix). Figure 5 Weighted average prevalence of oseltamvir-resistant influenza viruses A (H1N1), Europe, winter 2007–08. The light gray region indicates the 95% confidence interval.

Modeling showed a gradual increase for Europe in prevalence of ORVs over time, from close to 0 in week 40 to ≈56% in week 19 (Figure 5). This overall increase reflected prevalence increases in most individual countries in addition to Norway where the modeled prevalence started high at ≈60% and remained so throughout the period of virus circulation (Appendix Figure. Outside the main influenza virus A (H1N1) outbreak period, from week 51 to week 10 (Figure 2), the CIs for the prevalence of ORVs by country and for Europe were wide (Figure 5; Appendix Figure) because of the low numbers of influenza virus A (H1N1) detected or analyzed for antiviral resistance (Technical Appendix). Figure 5 Weighted average prevalence of oseltamvir-resistant influenza viruses A (H1N1), Europe, winter 2007–08. The light gray region indicates the 95% confidence interval. Phylogenetic Analysis Phylogenetic comparisons of HA and NA genes showed that the sequences of most recent European influenza viruses A (H1N1) fell within clade 2B, represented by A/Brisbane/59/2007, the recently recommended vaccine virus for 2008–09 (Figure 6). The NA sequences of most European ORVs form a cluster, characterized by a difference in amino acid residue 354 (D354G), as well as 275 (H275Y) compared with OSVs, including some ORVs from the United States and Japan (30,31). A degree of heterogeneity was observed, especially among ORVs from the United Kingdom; however, the NA sequences in these smaller clusters, represented by, for example, A/Scotland/5/2008 (and A/Hawaii/21/2007) or A/England/654/2007, are not distinguished from those of OSVs by any common amino acid differences other than H275Y. Some of these sequences fall close to those of ORVs recently isolated in Japan (31). The corresponding HA gene sequences within clade 2B, however, did not exhibit segregation complementary to that for NA gene sequences and no common amino acid changes distinguished ORVs and OSVs (Figure 6). Although the D344N substitution in NA has been associated with increases in the enzyme activity (32), this amino acid is common to both clades 2B and 2C, and none of the clade-specific differences between the NA (13 amino acids) or HA (6 amino acids) can readily account for the greater proportion of ORVs in clade 2B over clade 2C viruses.

ubstitution in NA has been associated with increases in the enzyme activity (32), this amino acid is common to both clades 2B and 2C, and none of the clade-specific differences between the NA (13 amino acids) or HA (6 amino acids) can readily account for the greater proportion of ORVs in clade 2B over clade 2C viruses. Figure 6 Phylogenetic comparisons of the hemagglutinin (A) and neuraminidase (B) genes of influenza viruses A (H1N1). Sequences of oseltamivir-resistant viruses, possessing the H275Y (H274Y in N2 numbering) mutation are in boldface; vaccine strains are in italics. Common amino acid changes that distinguish clades 1 and 2 and subgroups of clade 2 are shown. Scale bars indicate 0.01 nucleotide substitutions per site. Discussion Unexpectedly, influenza viruses A (H1N1) with a single amino acid substitution H275Y in the NA, which caused a several hundred-fold selective reduction in susceptibility to oseltamivir, emerged and were sustained in circulation in Europe during 2007–08, despite low antivirual drug use (Figure 1). Before the 2007–08 season, <1% of viruses tested since the start of European antiviral surveillance in 2004 had IC50 values >100 nmol/L for NAI drugs (A. Lackenby et al., unpub. data), in concordance with results from worldwide surveillance (8,9). In 2007–08, influenza viruses A (H3N2) and B circulating in Europe remained sensitive to NAI drugs.

<1% of viruses tested since the start of European antiviral surveillance in 2004 had IC50 values >100 nmol/L for NAI drugs (A. Lackenby et al., unpub. data), in concordance with results from worldwide surveillance (8,9). In 2007–08, influenza viruses A (H3N2) and B circulating in Europe remained sensitive to NAI drugs. This emergence of oseltamivir-resistant influenza virus A (H1N1) in Europe coincided with the dominant circulation of this virus subtype during the 2007–08 winter in Europe and the emergence of a new drift variant, A/Brisbane/59/2007 (30). Of the last 12 influenza seasons, influenza viruses A (H1N1) were dominant only in 2000–01, which included a new drift variant, A/New Caledonia/20/99 (20). In the other 10 seasons, influenza viruses A (H1N1) played a minor role, with influenza viruses A (H3N2) dominant in 9 seasons. Compared with 2000–01, peak incidence rates for ILI or ARI in 7 of 13 countries were similar or lower in 2007–08 (Table). In 6 countries, the peak incidence rates were significantly higher in 2007–08 than in 2000–01, but with a <2-fold difference in 5 countries and, in Spain only, a 4.8-fold difference. Both the 2000–01 and 2007–08 seasons were unremarkable in the overall clinical impact of influenza, with normal seasonal activity as measured by comparison of peak incidence rates for all seasons since 2000–01.

7–08 than in 2000–01, but with a <2-fold difference in 5 countries and, in Spain only, a 4.8-fold difference. Both the 2000–01 and 2007–08 seasons were unremarkable in the overall clinical impact of influenza, with normal seasonal activity as measured by comparison of peak incidence rates for all seasons since 2000–01. Sporadically occurring A/New Caledonia/20/99-like ORVs with H275Y were detected during the 2006–07 season in the United Kingdom and United States but did not become epidemiologically important. Indeed, the genetic background plays a role in retaining the replication efficiency and pathogenicity of recombinant influenza viruses A (H5N1) and A (H1N1) after introduction of tyrosine at position 275 (33). Furthermore, other previously analyzed influenza viruses A (H1N1) with the H275Y mutation showed impaired replicative ability in cell culture and reduced infectivity and substantially compromised pathogenicity in animal models, compared with the corresponding wild-type virus (34,35). The coincidental emergence of H275Y with the circulation of the A/Brisbane/59/2007 drift variant may have favored the emergence of fit transmissible ORVs. This point is also illustrated by the emergence of A/Brisbane/59/2007-like ORVs in other parts of the Northern Hemisphere and their continued circulation during the 2008 Southern Hemisphere influenza epidemic season (36–38). Since the last quarter of 2007, ORVs have been detected in continents other than Europe, with proportions of ORVs varying from 100% in South Africa and Australia to <5% in Japan. Trend data are limited: a slight monthly increase was noted in China/Hong Kong and Japan; in Canada, the increase was similar to that in Europe, from 0% ORVs in November 2007 to 86% ORVs in April 2008 (36).

ents other than Europe, with proportions of ORVs varying from 100% in South Africa and Australia to <5% in Japan. Trend data are limited: a slight monthly increase was noted in China/Hong Kong and Japan; in Canada, the increase was similar to that in Europe, from 0% ORVs in November 2007 to 86% ORVs in April 2008 (36). Using modeling, we showed that the prevalence of ORVs increased in the European region from ≈0% at the start to 56% at the end of the season. The finding of a high prevalence of ORVs in the community and the overall temporal increase in resistance demonstrates that the previously documented reduced fitness of viruses bearing the H275Y mutation, ostensibly caused by structural and functional constraints (10), has been overcome in currently circulating influenza viruses A (H1N1). The results of Rameix-Welti et al. (32) suggest that a combination of specific amino acid substitutions have increased the affinity of the NA of recent influenza viruses A (H1N1) (ORVs and OSVs) for substrate. A better balance of NA and HA activities in ORVs compared with OSVs may have contributed to the overall fitness and transmissibility of ORVs. However, growth curves conducted in tissue culture of pairs of ORVs and OSVs demonstrated no differences in growth kinetics or final virus yields. Therefore, changes in other genes also may be involved in the overall impact on the fitness of ORVs, for which whole genome sequencing is necessary.

ransmissibility of ORVs. However, growth curves conducted in tissue culture of pairs of ORVs and OSVs demonstrated no differences in growth kinetics or final virus yields. Therefore, changes in other genes also may be involved in the overall impact on the fitness of ORVs, for which whole genome sequencing is necessary. For Europe, no focal point of initiation of spread could be identified. The spread of ORV from west to east paralleled that of influenza virus A in Europe, and there was an average delay of 5.7 weeks for the appearance of ORVs after the start of influenza virus A circulation. However, the low R2 values for both patterns make definitive conclusions difficult to draw about the spatial spread of either influenza viruses A or ORVs. Several independent introductions into European countries of a sensitive and a resistant strain might explain the low R2 values. Estimating whether a global focal point exists from which ORVs emerged to spread to the rest of the world is not possible, but the fact that Japan, the country with the highest per capita use of oseltamivir (5), had relatively low levels of circulating ORVs during the 2007–08 influenza season is relevant and reflects the limited circulation of the clade 2B A/Brisbane/59/2007-like viruses belonging to the European cluster in this region (31,36).

he fact that Japan, the country with the highest per capita use of oseltamivir (5), had relatively low levels of circulating ORVs during the 2007–08 influenza season is relevant and reflects the limited circulation of the clade 2B A/Brisbane/59/2007-like viruses belonging to the European cluster in this region (31,36). The close relationships between the NA sequences of most of the 2007–08 European ORVs and their segregation from those of OSVs suggest that resistance results in large part from the spread of a single variant. Phylogenetic analyses show that this is a property of clade 2B A/Brisbane/59/2007-like viruses and is not associated with emergence of another antigenic variant. However, identification of other resistant variants in the United Kingdom, some of which are more closely related to OSVs than to most ORVs (e.g., A/England/654/2007) indicates the independent parallel emergence of multiple resistant variants. This is emphasized by small distinct clusters of closely related ORVs in Japan that are related to European OSVs, whereas only a few of the Japanese ORVs belonged to the large European ORVs cluster (31). Resolution of the origin and frequency of emergence of ORVs and association with drug use clearly require substantially more intimate knowledge of the genetic relationships among OSVs and ORVs worldwide. Our observations suggest that the new genetic background of influenza viruses A (H1N1) that appeared in 2007 enabled the virus to develop oseltamivir resistance independently at several locations in the world.

arly require substantially more intimate knowledge of the genetic relationships among OSVs and ORVs worldwide. Our observations suggest that the new genetic background of influenza viruses A (H1N1) that appeared in 2007 enabled the virus to develop oseltamivir resistance independently at several locations in the world. The combined effect of the relatively high level of circulation of influenza viruses A (H1N1) in Europe; the introduction of a new antigenic drift variant in a susceptible population, partly related to the lack of substantial influenza virus A (H1N1) circulation since the 2000–01 season; and the uncompromised transmissibility of the ORVs contributed to the epidemiologic success of the ORVs during the 2007–08 season. This phenomenon shows clearly that continuation of antiviral susceptibility monitoring and increasing capacity for timely response are essential (21,39). In addition, the appearance of viable transmitting ORVs is a reminder that the level of resistance to oseltamivir of seasonal or pandemic virus cannot be predicted, and therefore antiviral strategies should not rely on single drugs (40). Although oseltamivir remains a valuable influenza antiviral agent, the emergence of natural resistance shifts attention from oseltamivir to other antiviral agents and to improved vaccination (e.g., greater vaccination coverage, more immunogenic and broadly reacting vaccines) in the fight against seasonal and pandemic influenza.

oseltamivir remains a valuable influenza antiviral agent, the emergence of natural resistance shifts attention from oseltamivir to other antiviral agents and to improved vaccination (e.g., greater vaccination coverage, more immunogenic and broadly reacting vaccines) in the fight against seasonal and pandemic influenza. Supplementary Material Appendix Figure Fitted curves to the proportion oseltamivir-resistant viruses among influenza viruses A (H1N1) tested for resistance (both sentinel and nonsentinel) for all countries for which data were available for inclusion in modeling the European trend (see Figures 4 and 5). The x axes display the week in which the clinical specimens were collected (weeks 40-52 of 2007 and weeks 1-19 of 2008). The y axes display the percentage oseltamivir resistant influenza viruses A (H1N1). Plus signs indicate the actual determined proportions resistant A (H1N1) viruses; light gray region is the 95% confidence interval of the model. Appendix Table GenBank accession numbers of hemagglutinin and neuraminidase sequences used in the phylogenetic Analyses. Technical Appendix Statistical Analysis of Temporal Trends of Resistant Influenza A (H1N1) Viruses, Europe. Suggested citation for this article: Meijer A, Lackenby A, Hungnes O, Lina B, van der Werf S, Schweiger B, et al. Oseltamivir-resistant influenza A (H1N1) virus, Europe, 2007–08 season. Emerg Infect Dis [serial on the Internet]. 2009 April [date cited]. Available from http://www.cdc.gov/EID/content/15/4/552.htm

Technical Appendix Statistical Analysis of Temporal Trends of Resistant Influenza A (H1N1) Viruses, Europe. Suggested citation for this article: Meijer A, Lackenby A, Hungnes O, Lina B, van der Werf S, Schweiger B, et al. Oseltamivir-resistant influenza A (H1N1) virus, Europe, 2007–08 season. Emerg Infect Dis [serial on the Internet]. 2009 April [date cited]. Available from http://www.cdc.gov/EID/content/15/4/552.htm 1 European Influenza Surveillance Scheme members, 2007–08 season: P. Lachner, T. Popow-Kraupp, R. Strauss (Austria); B. Brochier, M. Sabbe, I. Thomas, V. Casteren, F. Yane (Belgium); T. Georgieva, M. Kojouharova, R. Kotseva, A. Kurchatova (Bulgaria); B. Aleraj, V. Drazenovic (Croatia); D. Bagatzouni-Pieridou, A. Elia (Cyprus); M. Havlickova, J. Kyncl (Czech Republic); S. Glismann, A. Mazick, L. Nielsen (Denmark); D.M. Fleming, A. Lackenby, J. Watson, M. Zambon (England); O. Sadikova, I. Sarv (Estonia); T. Ziegler (Finland); J.-M. Cohen, V. Enouf, B. Lina, A. Mosnier, M. Valette, S. van der Werf (France); U. Buchholz, W. Haas, B. Schweiger (Germany); A.G. Kossivakis, V. Kyriazopoulou-Dalaina, A. Mentis, G. Spala (Greece); G. Berencsi, A. Csohán, I. Jankovics (Hungary); S. Coughlan, L. Domegan, M. Duffy, M. Joyce, J. O’Donnell, D. O'Flanagan (Ireland); F. Ansaldi, P. Crovari, I. Donatelli, F. Pregliasco (Italy); R. Nikiforova, I. Van Velicko, N. Zamjatina (Latvia); A. Griskevicius, N. Kupreviciene, G. Rimseliene (Lithuania); J. Mossong, M. Opp (Luxembourg); C. Barbara, T. Melillo (Malta); A. Arkema, T. Meerhoff, W.J. Paget, K. van der Velden, (EISS-CC, the Netherlands); F. Dijkstra, G. Donker, J.C. de Jong, A. Meijer, G. Rimmelzwaan, M. van der Sande, B. Wilbrink (the Netherlands); P. Coyle, H. Kennedy, H. O’Neill (Northern Ireland); O. Hungnes, B. Iversen (Norway); L. Brydak, M. Romanowska (Poland); I.M. Falcão, J.M. Falcão, H. Rebelo de Andrade (Portugal); V. Alexandrescu, E. Lupulescu (Romania); W. Carman, R. Gunson, J. Kean, J. McMenamin (Scotland); N. Milic, J. Nedeljkovic (Serbia); H. Blaskovicova, Z. Kristufkova, M. Sláciková (Slovakia); K. Prosenc, M. Socan (Slovenia); I. Casas, A. Larrrauri, S. de Mateo, R. Ortiz de Lejarazu, P. Pérez-Breña, T. Pumarola Suñé, T. Vega Alonso (Spain); M. Brytting, A. Linde, P. Penttinen, S. Rubinova (Sweden); Y. Thomas, M. Witschi (Switzerland); N. Yilmaz (Turkey); M. Aranova, A. Mironenko (Ukraine); A. Hay (United Kingdom); and R. Jones, D. Thomas (Wales).

as, A. Larrrauri, S. de Mateo, R. Ortiz de Lejarazu, P. Pérez-Breña, T. Pumarola Suñé, T. Vega Alonso (Spain); M. Brytting, A. Linde, P. Penttinen, S. Rubinova (Sweden); Y. Thomas, M. Witschi (Switzerland); N. Yilmaz (Turkey); M. Aranova, A. Mironenko (Ukraine); A. Hay (United Kingdom); and R. Jones, D. Thomas (Wales). Acknowledgments We thank all EISS members and sentinel GPs in the national surveillance networks for seasonal surveillance data, the EISS colleagues in the National Influenza Centre laboratories, and virologists of hospital and peripheral laboratories for contributing viruses for testing at the UK Health Protection Agency. In particular, we thank Theresia Popow-Kraupp, Lars Nielsen, Inna Sarv, Thedi Ziegler, Andreas Mentis, Margaret Duffy, Isabella Donatell, Guus Rimmelzwaan, Helena Rebelo de Andrade, Pilar Pérez-Breña, Mia Brytting, and Yves Thomas for providing national antiviral susceptibility data. We also thank Vicki Gregory for assistance in the phylogenetic analyses, Rianne van Gageldonk and Berry Wilbrink for providing the Dutch ARI-EL study data, Paul Taylor for development and programming of the seasonal and antiviral databases and the internet interface for data entry and automated data upload, and Angus Nicoll and Fred Hayden for helpful comments on the manuscript. Funding support for this research came from the European Union FP6 Programme for VIRGIL contract no. 503359 and from European Centre for Disease Prevention and Control for EISS Contract No ECD.604.

Acknowledgments We thank all EISS members and sentinel GPs in the national surveillance networks for seasonal surveillance data, the EISS colleagues in the National Influenza Centre laboratories, and virologists of hospital and peripheral laboratories for contributing viruses for testing at the UK Health Protection Agency. In particular, we thank Theresia Popow-Kraupp, Lars Nielsen, Inna Sarv, Thedi Ziegler, Andreas Mentis, Margaret Duffy, Isabella Donatell, Guus Rimmelzwaan, Helena Rebelo de Andrade, Pilar Pérez-Breña, Mia Brytting, and Yves Thomas for providing national antiviral susceptibility data. We also thank Vicki Gregory for assistance in the phylogenetic analyses, Rianne van Gageldonk and Berry Wilbrink for providing the Dutch ARI-EL study data, Paul Taylor for development and programming of the seasonal and antiviral databases and the internet interface for data entry and automated data upload, and Angus Nicoll and Fred Hayden for helpful comments on the manuscript. Funding support for this research came from the European Union FP6 Programme for VIRGIL contract no. 503359 and from European Centre for Disease Prevention and Control for EISS Contract No ECD.604. Dr Meijer is a virologist and the head of the Respiratory Viruses section of the Virology Laboratory of the Centre for Disease Control at the National Institute for Public Health and the Environment, Bilthoven, the Netherlands. His research interests are the virology and epidemiology of viral respiratory infections, with a focus on influenza virus infections.

Seasonal influenza, caused by influenza A subtypes H3N2 and H1N1 and influenza B viruses, occurs as annual epidemics. Although vaccination remains the primary measure for prevention, antiviral drugs are available for prevention and treatment of influenza. The influenza virus neuraminidase inhibitors zanamivir and oseltamivir were introduced into clinical practice in various parts of the world from 1999 through 2002 (1). Oseltamivir limits replication of both influenza A and B viruses (1). In most European countries, neuraminidase inhibitors are not widely used to treat seasonal influenza, but they are being stockpiled in many countries as part of their pandemic influenza preparedness. In Norway, oseltamivir is registered for prophylactic and therapeutic use in persons >1 year of age; however, it is not available without a prescription and it is rarely prescribed (2).

ly used to treat seasonal influenza, but they are being stockpiled in many countries as part of their pandemic influenza preparedness. In Norway, oseltamivir is registered for prophylactic and therapeutic use in persons >1 year of age; however, it is not available without a prescription and it is rarely prescribed (2). Until 2007, resistance against neuraminidase inhibitors was rarely observed (1,3,4). Nevertheless, to better understand the potential for development of resistance against neuraminidase inhibitors, surveillance of antiviral susceptibility in influenza virus in Europe has been ongoing since 2004 (5). As part of the World Health Organization (WHO) Global Influenza Surveillance Network, the national influenza centers in Europe submit influenza viruses to the WHO Influenza Collaborating Centre in the United Kingdom each influenza season. Within the framework of the European Surveillance Network for Vigilance against Viral Resistance (VIRGIL), these viruses are also tested for drug susceptibility at the Health Protection Agency in London.

rope submit influenza viruses to the WHO Influenza Collaborating Centre in the United Kingdom each influenza season. Within the framework of the European Surveillance Network for Vigilance against Viral Resistance (VIRGIL), these viruses are also tested for drug susceptibility at the Health Protection Agency in London. In mid-January 2008, antiviral susceptibility testing (enzyme inhibition assays) of the first shipment of influenza viruses from Norway for the 2007–08 season showed an unusually large proportion (5/7) of influenza viruses A (H1N1) with high-level resistance to oseltamivir. In subsequent days, testing of additional viruses from Norway at the Norwegian national influenza center and at the Health Protection Agency confirmed the high proportion of oseltamivir resistance. This unexpected and unprecedented discovery had possible public health implications of international concern. On January 25, 2008, the Norwegian Institute of Public Health notified WHO of these findings under the International Health Regulations (6) and notified the European Commission through the Early Warning and Response System. The Institute also informed hospitals and physicians in Norway about a possible lack of therapeutic effect when treating patients with oseltamivir. By the end of January, oseltamivir-resistant viruses had been reported from several European countries (7).

ropean Commission through the Early Warning and Response System. The Institute also informed hospitals and physicians in Norway about a possible lack of therapeutic effect when treating patients with oseltamivir. By the end of January, oseltamivir-resistant viruses had been reported from several European countries (7). The oseltamivir-resistance trait is caused by a previously described point mutation in the virus neuraminidase gene (histidine to tyrosine at position 275 of the N1 neuraminidase, commonly referred to as H274Y in N2 numbering), which is known to confer high-level resistance to oseltamivir while retaining susceptibility to zanamivir (8). Influenza viruses A (H1N1) carrying the H274Y mutation have reduced ability to replicate and transmit efficiently when compared with parental, susceptible virus, but the clinical implications of infection with these viruses have been largely unknown (9). Consequently, we undertook studies to determine whether the emergence and spread of the resistant viruses were associated with exposure to oseltamivir, whether resistant viruses would continue to circulate in similar proportions into the epidemic phase of the season, and whether the new resistant viruses differed from their susceptible counterparts in their ability to cause disease. To do so, we tested all influenza viruses A (H1N1) available from the 2007–08 outbreak for oseltamivir susceptibility. We furthermore enhanced surveillance by collecting an extended set of data regarding clinical symptoms, complications, and prior exposure to oseltamivir for all laboratory-verified cases of influenza viruses A (H1N1) infection.

fluenza viruses A (H1N1) available from the 2007–08 outbreak for oseltamivir susceptibility. We furthermore enhanced surveillance by collecting an extended set of data regarding clinical symptoms, complications, and prior exposure to oseltamivir for all laboratory-verified cases of influenza viruses A (H1N1) infection. Methods The influenza viruses A (H1N1) included in this study were obtained from the sentinel and nonsentinel collaborators as part of routine national virologic influenza surveillance. From all 19 counties, 71 sentinel practices collect samples from patients with influenza-like illness and send them to the national influenza center for diagnostic testing. From all parts of the country, 15 medical microbiology laboratories submit materials containing influenza A or B materials (original specimens, nucleic acid preparations from original specimens, or viral isolates) to the national influenza center for further characterization. Most of these samples originate from primary care clinics; the rest, from hospitals.

microbiology laboratories submit materials containing influenza A or B materials (original specimens, nucleic acid preparations from original specimens, or viral isolates) to the national influenza center for further characterization. Most of these samples originate from primary care clinics; the rest, from hospitals. Viruses confirmed as influenza A (H1), by either reverse transcription–PCR (RT-PCR) or virus isolation in MDCK cells and subsequent subtyping by immunofluorescence, were included in the study. In-country susceptibility testing was performed by detecting the H274Y mutation by sequence analysis, through either a pyrosequencing assay targeting the single relevant point mutation (10) or through full- or partial-length cycle sequencing of the coding region for the viral neuraminidase. These analyses were mostly performed on RNA prepared from the original patient specimen. A large proportion of the isolated viruses were sent to the WHO Collaborative Centre for Influenza Research and Reference in the National Institute of Medical Research, Mill Hill, UK, for further characterization. Within the framework of VIRGIL, these viruses were forwarded to the Health Protection Agency for phenotypic antiviral susceptibility testing and more extensive genotypic analyses. To determine neuraminidase susceptibility, assays to determine the drug concentration that provides 50% inhibition (IC50) were performed by using the fluorescent substrate methylumbelliferyl N-acetylneuraminic acid based on the method described by Wetherall et al. (11) with minor modifications.

sive genotypic analyses. To determine neuraminidase susceptibility, assays to determine the drug concentration that provides 50% inhibition (IC50) were performed by using the fluorescent substrate methylumbelliferyl N-acetylneuraminic acid based on the method described by Wetherall et al. (11) with minor modifications. Relative quantitative data on virus shedding, i.e., virus RNA content in the patient specimens, were obtained through a real-time RT-PCR targeting a conserved part of the matrix protein (M1) gene of influenza A virus. Two microliters of nucleic acid prepared from specimens (MagNApure LC Total Nucleic Acid Isolation Kit; Roche Diagnostics, Mannheim, Germany) was added to a 23-μL reaction mixture containing 0.3 μM forward primer M52c (5′-CTT CTA ACC GAG GTC GAA ACG-3′); 0.3 μM reverse primer M149r (5′-CTT GTC TTT AGC CAT TCC ATG AG-3′); 0.15 μM probe M93c (FAM-5′ CCG TCA GGC CCC CTC AAA GCC GA 3′-Black Hole Quencher 1); and 5× QIAGEN OneStep RT-PCR buffer, dNTP mixture, and enzyme mixture according to the manufacturer’s instructions (QIAGEN OneStep RT PCR Kit; QIAGEN, Hilden, Germany). Forward primer and probe sequences were as described by Fouchier et al. (12), and the reverse primer was designed by Tom Øystein Jonassen (Akershus University Hospital, Lørenskog, Norway). Reactions were run in a Corbett Rotorgene RG-3000 or RG-6000 thermocycler (Corbett Research Pty Ltd, Sydney, New South Wales, Australia) with the following cycling conditions: reverse trancription for 30 min at 50°C, then 15 min at 95°C, followed by 50 cycles at 95°C for 10 sec, 54°C for 30 sec, and 72°C for 20 sec.

). Reactions were run in a Corbett Rotorgene RG-3000 or RG-6000 thermocycler (Corbett Research Pty Ltd, Sydney, New South Wales, Australia) with the following cycling conditions: reverse trancription for 30 min at 50°C, then 15 min at 95°C, followed by 50 cycles at 95°C for 10 sec, 54°C for 30 sec, and 72°C for 20 sec. Participants and Study Design We included all patients with a diagnosis of influenza virus A (H1N1) infection made by national influenza center during the 2007–08 influenza season. For the 72 patients who received this diagnosis before the end of January 2008, data were collected retrospectively. From February on, data were collected as soon as possible after laboratory confirmation of influenza virus A (H1N1) infection. Structured questionnaires returned from consulting physicians provided auxiliary information about clinical signs and symptoms, complications, predisposing diseases for severe outcome of influenza (diabetes, cardiac disease, lung disease, and immunodeficiency), use of oseltamivir, and influenza vaccination status. If the questionnaire was not returned by mail within 3 weeks, a reminder call was made. When available, relevant clinical information on the original referral sample form was used to supplement the data from the written questionnaire. The consulting physician, usually the primary care physician, was not informed about the result of the susceptibility testing when the information was collected. Information for the first 12 patients infected with a resistant virus was collected from the consulting physicians by telephone.

om the written questionnaire. The consulting physician, usually the primary care physician, was not informed about the result of the susceptibility testing when the information was collected. Information for the first 12 patients infected with a resistant virus was collected from the consulting physicians by telephone. Statistical Analysis Data from the questionnaires and selected laboratory testing outcomes were merged, checked for quality, and analyzed by using Stata version 9.0 (StataCorp LP, College Station, TX, USA). We used the Fisher exact test to compare the proportions of possible confounders among those infected with a resistant and a susceptible virus. To estimate the association between exposure (resistant virus infection) and outcome (subsequent clinical findings and complications), we calculated crude risk ratios (RRs) and 95% confidence intervals (CIs). We used binomial regression to calculate RRs adjusted for possible confounders. For each variable, we used the number of respondents as the denominator, except for predisposing disease, for which missing values were coded as “no.”

complications), we calculated crude risk ratios (RRs) and 95% confidence intervals (CIs). We used binomial regression to calculate RRs adjusted for possible confounders. For each variable, we used the number of respondents as the denominator, except for predisposing disease, for which missing values were coded as “no.” Results The overall influenza activity in Norway was low in 2007–08 compared with that of previous years. Virologic surveillance showed most influenza virus A and 95% of subtyped viruses to be subtype H1N1 (13). From the sentinel practices, the national influenza center received 229 specimens for influenza testing. Of the 108 that were positive for influenza virus, 61 were type A, subtype H1N1, and most of the rest were type B. In total, 297 patients had an influenza virus A (H1N1) infection confirmed by the national influenza center in Norway from week 47 in 2007 until the end of week 20 in 2008. We obtained a resistance profile for 272 of the 297 viruses. We could not determine the resistance profile for the remaining 25 because of low virus content and consequently excluded them from analysis.

infection confirmed by the national influenza center in Norway from week 47 in 2007 until the end of week 20 in 2008. We obtained a resistance profile for 272 of the 297 viruses. We could not determine the resistance profile for the remaining 25 because of low virus content and consequently excluded them from analysis. A total of 196 viral isolates were available (133 carried the resistance mutation); of these, 113 (79 with the resistant genotype) were reference tested by the VIRGIL laboratory. Phenotypic and genotypic reference analysis results agreed completely with the in-country genotypic testing results; all mutant viruses showed large reductions in susceptibility to oseltamivir when compared with non-H274Y viruses (IC50 260–2,161 nM, mean 673 nM, for the 274Y mutant and 0.4–5.6 nM, mean 2.6 nM, for the nonmutant viruses). No evidence of mixed genotype or phenotype was observed. In phylogenetic analysis of the H1 gene, all viruses tested grouped together in subclade 2B (Figure 1). In the phylogenetic tree, the resistant viruses from Norway all formed a single branch that was distinct, but closely related, to the susceptible viruses from Norway.

ence of mixed genotype or phenotype was observed. In phylogenetic analysis of the H1 gene, all viruses tested grouped together in subclade 2B (Figure 1). In the phylogenetic tree, the resistant viruses from Norway all formed a single branch that was distinct, but closely related, to the susceptible viruses from Norway. Figure 1 Phylogenetic reconstruction of the H1 genes of influenza viruses A (H1N1) in Norway, 2007–08 season. The analysis was performed on an alignment spanning positions 84–1054 of viral RNA segment 4. Pairwise distances were calculated by using the Kimura 2-parameter model with a transition:transversion ratio of 2.0; the phylogenetic tree was constructed by the neighbor-joining method, as implemented in the programs DNADIST and NEIGHBOR in the PHYLIP package (14 15,). Published sequences were obtained from the Influenza Sequence Database, Los Alamos National Laboratory (16). Boldface indicates viruses from the 2007–08 influenza season in Norway; red indicates oseltamivir-resistant viruses; blue, susceptible viruses. New sequences presented in this analysis have been deposited in GenBank (accession nos. CY036664–CY036694).

he Influenza Sequence Database, Los Alamos National Laboratory (16). Boldface indicates viruses from the 2007–08 influenza season in Norway; red indicates oseltamivir-resistant viruses; blue, susceptible viruses. New sequences presented in this analysis have been deposited in GenBank (accession nos. CY036664–CY036694). Of the 272 influenza viruses A (H1N1), 183 (67.3%) were oseltamivir resistant (Table 1). The proportion of resistant viruses did not differ between samples from sentinel 67.9% (38/56) and nonsentinel 67.1% (145/216) practices and persisted throughout the season (Figure 2). No difference in virus shedding, as quantified by real-time RT-PCR of available patient specimens, was observed between susceptible and resistant viruses (Figure 3). From the original sample form, we obtained demographic information for all 272 patients. Returned questionnaires provided information for 265 patients (97.4%), but the response rate on individual questions varied. Of the 272 patients infected with influenza viruses A (H1N1), 132 (48.5%) were male (Table 1), and slightly more than half (50.7%) were 29–64 years of age (median 27 years, range 2 months–71 years); median ages of those infected with a resistant and a susceptible virus were 31 and 21 years, respectively. The highest proportion of resistant virus infection was found for those 25–59 years of age (102/138, 73.9%) and differed significantly from the proportion for only those 5–14 years of age (25/45, 55.6%) (Fisher exact p = 0.03). We obtained influenza viruses A (H1N1) from 18/19 counties (Figure 4).The oseltamivir resistance proportion was >80% in 8 counties in southern Norway, compared with 63.5% in the rest of the country (Fisher exact p = 0.001).

tly from the proportion for only those 5–14 years of age (25/45, 55.6%) (Fisher exact p = 0.03). We obtained influenza viruses A (H1N1) from 18/19 counties (Figure 4).The oseltamivir resistance proportion was >80% in 8 counties in southern Norway, compared with 63.5% in the rest of the country (Fisher exact p = 0.001). Table 1 Proportion of oseltamivir-resistant and oseltamivir-susceptible influenza viruses A (H1N1), 2007–08 influenza season, Norway Sample source Total no. samples No. (%) resistant samples No. (%) susceptible samples All 272 183 (67.3) 89 (32.7) Type of practice Sentinel 56 38 (67.8) 18 (32.1) Nonsentinel 216 145 (67.1) 71 (32.9) Patient gender Male 132 85 (64.4) 47 (35.6) Female 140 98 (70.0) 42 (30.0) Patient age group, y 0–4 45 27 (60.0) 18 (40.0) 5–14 45 25 (55.6) 20 (44.4) 15–24 31 20 (64.5) 11 (35.5) 25–59 138 102 (73.9) 36 (26.1) 60–99 13 9 (69.2) 4 (30.8) Patient with predisposing disease Diabetes 10 9 (90.0) 1 (10.0) Lung disease 11 8 (72.7) 3 (27.3) Cardiac disease 5 2 (40.0) 3 (60.0) Immunodeficiency 5 3 (60.0) 2 (40.0) Any 27 20 (74.1) 7 (25.9) Figure 2 Oseltamivir-resistant (n = 183) and oseltamivir-susceptible (n = 89) influenza viruses A (H1N1) in the 2007–08 influenza season in Norway, by week of sampling.

9 (90.0) 1 (10.0) Lung disease 11 8 (72.7) 3 (27.3) Cardiac disease 5 2 (40.0) 3 (60.0) Immunodeficiency 5 3 (60.0) 2 (40.0) Any 27 20 (74.1) 7 (25.9) Figure 2 Oseltamivir-resistant (n = 183) and oseltamivir-susceptible (n = 89) influenza viruses A (H1N1) in the 2007–08 influenza season in Norway, by week of sampling. Figure 3 Comparison of virus shedding, measured as relative viral RNA content, in respiratory specimens taken from patients infected with oseltamivir-susceptible and oseltamivir-resistant influenza viruses A (H1N1), respectively, during the 2007–08 influenza season in Norway. Viral RNA content is expressed as the reverse-transcription–PCR cycle number (Ct) during which the fluorescence threshold was exceeded. Figure 4 Proportion of oseltamivir-resistant influenza viruses A (H1N1) in the 2007–08 influenza season in Norway, by county of sampling. The total number of samples analyzed for each county is given inside each county. Information about use of antiviral drugs was obtained for 237 patients. No patients had received antiviral treatment in the 14 days before the onset of symptoms, and none had been in close contact with others known to have used antiviral drugs. Oseltamivir was received after sampling by 7 patients, 5 of whom were infected with an oseltamivir-resistant virus. Of 225 patients, 9 had traveled abroad in the week before symptom onset; 4 were infected with a resistant virus. Of all 272 patients, 2 had been vaccinated against influenza and were both infected with a resistant virus.

received after sampling by 7 patients, 5 of whom were infected with an oseltamivir-resistant virus. Of 225 patients, 9 had traveled abroad in the week before symptom onset; 4 were infected with a resistant virus. Of all 272 patients, 2 had been vaccinated against influenza and were both infected with a resistant virus. We received information about predisposing disease for 213 patients. Having a predisposing disease more than doubled the risk for complications (RR 2.5, 95% CI 1.2–5.4) but was not clearly associated with being infected with a resistant virus (RR 1.4, 95% CI 0.6–3.2). Information about clinical symptoms was obtained for 252/272 patients; most frequently reported were fever (229/242) and dry cough (182/218). Resistant virus infection was not associated with any particular symptom (Table 2). Of 241 patients, 58 (24.1%) had >1 complications recorded, but no difference was observed between those infected with a resistant virus and those infected with a susceptible virus (Table 2). Bronchitis and pneumonia were the most frequent complications, reported for 22 and 17 patients, respectively. The age of the 17 patients who had pneumonia ranged from 8 months to 65 years (mean 29 years): 2 (12.5%) were 0–4 years of age, 5 (31.3%) were 5–14 years of age, 2 (12.5%) were 15–24 years of age, 4 (25.0%) were 25–59 years of age, and 3 (18.8%) were >59 years of age. Of the 17 patients with pneumonia, 15 were infected with a resistant virus. The attack rates of pneumonia and of sinusitis were higher for those infected with a resistant virus than for those infected with a susceptible virus, although the risk ratios were not statistically significant after adjusting for age, gender, and predisposing disease (pneumonia RR 3.2, 95% CI 0.7–13.7; sinusitis RR 1.7, 95% CI 0.4–7.5) (Table 2). Of 264 patients, 45 had been hospitalized, 28 and 17 infected with a resistant and a susceptible virus, respectively. No deaths were reported for patients included in the study.

ant after adjusting for age, gender, and predisposing disease (pneumonia RR 3.2, 95% CI 0.7–13.7; sinusitis RR 1.7, 95% CI 0.4–7.5) (Table 2). Of 264 patients, 45 had been hospitalized, 28 and 17 infected with a resistant and a susceptible virus, respectively. No deaths were reported for patients included in the study. Table 2 Reported associations for patients infected with oseltamivir-resistant or oseltamivir-susceptible influenza virus A (H1N1), 2007–08 influenza season, Norway Association Resistant, n = 183 Susceptible, n = 89 Crude associations Adjusted associations* Attack rate, % No. responses Attack rate, % No. responses Risk ratio (95% CI)† Risk ratio (95% CI) Sign or symptom Productive cough 38.4 125 31.9 47 1.2 (0.8–1.9) Fever 94.4 162 95.0 80 1.0 (0.9–1.1) Myalgia 72.9 129 73.3 60 1.0 (0.8–1.2) Dry cough 82.1 145 86.3 73 1.0 (0.8–1.1) Headache 63.4 131 67.2 58 0.9 (0.8–1.2) Sore throat 57.5 134 67.2 58 0.9 (0.7–1.1) Runny nose 62.2 127 66.1 56 0.9 (0.8–1.2) Complication Pneumonia 9.2 153 2.9 69 3.2 (0.7–13.5) 3.2 (0.7–13.7) Sinusitis 6.2 145 3.0 67 2.1 (0.5–9.4) 1.7 (0.4–7.5) Otitis media 4.8 145 4.4 69 1.1 (0.3–4.2) 1.3 (0.4–4.8) Bronchitis 8.7 149 11.8 76 0.7 (0.3–1.7) 0.8 (0.4–1.8) Any 24.4 164 22.1 77 1.1 (0.7–1.8) 1.1 (0.7–1.8) Hospitalization 15.8 177 19.5 87 0.8 (0.5–1.4) 0.8 (0.5–1.3) *Risk ratio adjusted for age, gender, and predisposing disease. †CI, confidence interval.

(0.4–7.5) Otitis media 4.8 145 4.4 69 1.1 (0.3–4.2) 1.3 (0.4–4.8) Bronchitis 8.7 149 11.8 76 0.7 (0.3–1.7) 0.8 (0.4–1.8) Any 24.4 164 22.1 77 1.1 (0.7–1.8) 1.1 (0.7–1.8) Hospitalization 15.8 177 19.5 87 0.8 (0.5–1.4) 0.8 (0.5–1.3) *Risk ratio adjusted for age, gender, and predisposing disease. †CI, confidence interval. Discussion During the 2007–08 influenza season in the Northern Hemisphere, widespread circulation of oseltamivir-resistant influenza viruses A (H1N1) was observed. Percentage of resistant viruses circulating in different countries varied markedly; the highest proportion reported worldwide (67%) was in Norway (17,18).

(0.4–7.5) Otitis media 4.8 145 4.4 69 1.1 (0.3–4.2) 1.3 (0.4–4.8) Bronchitis 8.7 149 11.8 76 0.7 (0.3–1.7) 0.8 (0.4–1.8) Any 24.4 164 22.1 77 1.1 (0.7–1.8) 1.1 (0.7–1.8) Hospitalization 15.8 177 19.5 87 0.8 (0.5–1.4) 0.8 (0.5–1.3) *Risk ratio adjusted for age, gender, and predisposing disease. †CI, confidence interval. Discussion During the 2007–08 influenza season in the Northern Hemisphere, widespread circulation of oseltamivir-resistant influenza viruses A (H1N1) was observed. Percentage of resistant viruses circulating in different countries varied markedly; the highest proportion reported worldwide (67%) was in Norway (17,18). Our study did not show any association between oseltamivir use in Norway and emergence of the oseltamivir-resistant influenza viruses A (H1N1). Because only a minority of influenza cases are laboratory confirmed, oseltamivir use in nonsampled persons could have contributed to the development of resistance. However, for this suggestion to be plausible, use of oseltamivir would have to be widespread to exert substantial selective pressure on the viruses. Sales of oseltamivir in Norway have been low: 699 5-day regimens (0.15/1,000 population) were sold in 2004; 66,249 (14.4/1,000 population) in 2005; 33,573 (7.3/1,000 population) in 2006; and 4,686 (1.0/1,000 population) in 2007 (2). In countries where oseltamivir use has been high, e.g., Japan, the proportion of oseltamivir-resistant influenza viruses A (H1N1) reported during the 2007–08 season was low (18). Because influenza strains from Norway were genetically similar to resistant viruses that appeared just as early in several other European countries (A. Hay, pers. comm.), we consider it unlikely that the resistant variant originated in Norway. Conceivably, the initial emergence of a resistant virus could be associated with oseltamivir use elsewhere. Our data indicate that the viruses carrying this resistance mutation are fully capable of persistence and spread in the absence of selective pressure.

it unlikely that the resistant variant originated in Norway. Conceivably, the initial emergence of a resistant virus could be associated with oseltamivir use elsewhere. Our data indicate that the viruses carrying this resistance mutation are fully capable of persistence and spread in the absence of selective pressure. In Norway, the initially high proportion of resistant influenza viruses A (H1N1) was maintained throughout the entire 2007–08 influenza season; countrywide, 2 of 3 viruses were resistant. The reason for this exceptionally high resistance proportion is unknown. However, it likely reflects the proportion of resistant viruses introduced into Norway in the fall of 2007. Globally, the proportion of resistant influenza viruses A (H1N1) reported is highly variable between the different countries for which data are available (18). This large variation, apparently in the absence of oseltamivir selective pressure, suggests that a high level of randomness determined the frequency of resistance. In temperate countries, the influenza viruses are reintroduced each autumn after the absence of influenza during the summer. If only a limited number of viruses are reintroduced into each country and initiate virus circulation and outbreaks, the result will be considerable random variation in virus variant proportions between the different countries. Consistent with this result, almost all the characterized influenza viruses A (H1N1) in Norway could be assembled into a small number of genetically discernible groups (Figure 1). We propose that such randomness in virus introductions may be sufficient to explain the differences in the proportions of resistant viruses between the countries.

esult, almost all the characterized influenza viruses A (H1N1) in Norway could be assembled into a small number of genetically discernible groups (Figure 1). We propose that such randomness in virus introductions may be sufficient to explain the differences in the proportions of resistant viruses between the countries. Conceivably, a difference in the antigenic characteristics of the resistant and susceptible viruses could have favored one virus over the other in the face of host population immunity. Such differences might contribute to different relative effect of the 2 viruses in different populations (e.g., countries) or subpopulations (e.g., age groups). However, the resistant and susceptible viruses were closely related and were not distinguishable in hemagglutination inhibition tests (19).

opulation immunity. Such differences might contribute to different relative effect of the 2 viruses in different populations (e.g., countries) or subpopulations (e.g., age groups). However, the resistant and susceptible viruses were closely related and were not distinguishable in hemagglutination inhibition tests (19). Overall, the observed clinical manifestations associated with influenza viruses A (H1N1) in this study were as expected for seasonal influenza. No differences were noted for virus shedding, primary symptoms, or overall complication and hospitalization rates caused by oseltamivir-resistant and -susceptible viruses. We did find, although not a statistically significant finding, that patients infected with a resistant virus appeared to be more likely than those infected with a susceptible virus to have pneumonia or sinusitis. Patients with more severe illness may be more likely to be sampled; however, the resistance pattern of the virus was not known by the physician at the time of sampling and reporting. We therefore believe that these findings are not influenced by selection bias. Adjusting for possible confounders (age, sex, and predisposing disease) did not change the results. Because of our limited sample size, the precision of our estimates is low, but they do indicate findings that warrant further investigation. Our data will also be analyzed with data from other European countries, and the findings may strengthen the conclusions about the clinical implications of oseltamivir-resistant influenza viruses A (H1N1) .

ple size, the precision of our estimates is low, but they do indicate findings that warrant further investigation. Our data will also be analyzed with data from other European countries, and the findings may strengthen the conclusions about the clinical implications of oseltamivir-resistant influenza viruses A (H1N1) . The future effect of resistant influenza viruses A (H1N1) is unpredictable. In Europe, the H1N1 subtype was predominant during the 2007–08 influenza season and, according to historical patterns, is unlikely to predominate during the 2008–09 influenza season. In the following 2008–09 season in the Northern Hemisphere, influenza viruses A (H1N1) may well predominate in areas where they had not recently been present in large numbers. Early reporting from the Southern Hemisphere 2008 influenza season indicates that detection of influenza virus A (H1N1) is low (20). However, in South Africa, oseltamivir resistance has been detected in 100% of influenza viruses A (H1N1) tested (21).

e in areas where they had not recently been present in large numbers. Early reporting from the Southern Hemisphere 2008 influenza season indicates that detection of influenza virus A (H1N1) is low (20). However, in South Africa, oseltamivir resistance has been detected in 100% of influenza viruses A (H1N1) tested (21). Whether oseltamivir-resistant viruses will persist beyond 2008 depends on several factors. First, their persistence will depend on the prevalence of resistant viruses in the populations that are the source of global influenza spread. Countries in East and Southeast Asia have been proposed as the most likely source for global dissemination of new influenza virus variants (22). The prevalence of resistant influenza viruses A (H1N1) in this region may therefore be more likely to influence future occurrence of these viruses than the prevalence in Europe; resistance monitoring thus needs to be global. Second, changes in recent influenza viruses A (H1N1) may have provided a genetic background that permits H274Y mutants to replicate and transmit. Previous studies have concluded that resistant viruses are less pathogenic and less transmissible than their susceptible counterparts (9,23). In contrast, however, reverse genetics–derived mutants (A/WSN/33 or PR8 backbone) had the same phenotype as wild type viruses in vitro and in vivo (24,25). A recent study on the enzymatic properties of the N1 neuraminidase of the resistant viruses from the 2007–08 season suggested some genetic background changes that could potentially be involved (26).

tics–derived mutants (A/WSN/33 or PR8 backbone) had the same phenotype as wild type viruses in vitro and in vivo (24,25). A recent study on the enzymatic properties of the N1 neuraminidase of the resistant viruses from the 2007–08 season suggested some genetic background changes that could potentially be involved (26). As long as such a postulated permissive genetic background is common, resistant mutants may arise anew in purely oseltamivir-susceptible influenza virus A (H1N1) populations. Identification of such predisposing genetic traits and monitoring of their occurrence in influenza viruses A (H1N1) and other influenza viruses should continue. Similar resistance can arise in viruses other than the current human influenza viruses A (H1N1). Resistance in a more virulent influenza virus can have serious public health implications because of fewer therapeutic and prophylactic options, which may result in more persons being affected by influenza and more severe illness and death in those who become infected. Oseltamivir is a prime option for influenza treatment and prophylaxis and forms a substantial part of pandemic preparedness in many countries. The prevalence of oseltamivir-resistant viruses reported in Europe throughout the 2007–08 influenza season clearly shows that this resistant mutation is stable and that these viruses sustain their fitness and ability to spread among persons. These findings should be taken into consideration when shaping future strategies for treating and preventing seasonal and pandemic influenza.

ope throughout the 2007–08 influenza season clearly shows that this resistant mutation is stable and that these viruses sustain their fitness and ability to spread among persons. These findings should be taken into consideration when shaping future strategies for treating and preventing seasonal and pandemic influenza. Suggested citation for this article: Hauge SH, Dudman S, Borgen K, Lackenby A, Hungnes O. Oseltamivir-resistant influenza viruses A (H1N1), Norway, 2007–08. Emerg Infect Dis [serial on the Internet]. 2009 Feb [date cited]. Available from http://www.cdc.gov/EID/content/15/2/155.htm Acknowledgments We thank the clinicians and laboratorians who collected specimens and data. Torstein Aune is gratefully acknowledged for substantial help with data entry and management; Kirsten Konsmo, for performing the reminder calls and producing the map; and Remilyn Ramos-Ocao, Marianne Morken, Anne Marie Lund, Valentina Johansen, and Grethe H. Krogh, for expert technical laboratory assistance. We also thank Maria Zambon, Dipa Lakhman, Kameljit Bedi, and Carol Sadler for performing the extensive antiviral testing that initially uncovered the emergence of the resistant viruses, and Alan J. Hay and others for reference analyses on the viruses from Norway. Dr Hauge is a medical officer at the department of Infectious Disease Epidemiology at the Norwegian Institute of Public Health, Oslo. She is a fellow in the Norwegian Field Epidemiology Training Programme. Her research interests are influenza and other vaccine-preventable diseases.

Influenza A and B viruses are major pathogens that represent a threat to public health with subsequent economic losses worldwide (1). Vaccination is the primary method for prevention; antiviral drugs are used mainly for prophylaxis and therapy. Currently, 2 classes of drugs, matrix 2 (M2) blockers and neuraminidase inhibitors (NAIs) are available, but M2 blockers such as amantadine and rimantadine are not commonly used because of the rapid generation of resistance and lack of efficacy against influenza B virus (2–4). The NAIs zanamivir and oseltamivir are widely used because of effects against influenza A and B viruses and a low frequency of resistance. NAI virus surveillance studies by several groups have demonstrated that <1% of viruses tested show naturally occurring resistance to oseltamivir as of 2007 (5–10), indicating limited human-to-human transmission of these viruses.

sed because of effects against influenza A and B viruses and a low frequency of resistance. NAI virus surveillance studies by several groups have demonstrated that <1% of viruses tested show naturally occurring resistance to oseltamivir as of 2007 (5–10), indicating limited human-to-human transmission of these viruses. At the beginning of the 2007–08 influenza season, however, detection of a substantially increased number of oseltamivir-resistant influenza viruses A (H1N1) (ORVs) was reported, mainly in countries in Europe where the prevalence varies, with the highest levels in Norway (67%) and France (47%) (11–14). These viruses showed a specific NA mutation with a histidine-to-tyrosine substitution at the aa 275 position (N1 numbering, H275Y), conferring high-level resistance to oseltamivir. Most of these ORVs were isolated from NAI-untreated patients and retained similar ability of human-to-human transmission to oseltamivir-sensitive influenza viruses A (H1N1) (OSVs) (10,15). In response to public health concerns about ORVs, the World Health Organization (WHO) directed Global Influenza Surveillance Network laboratories to intensify NAI surveillance and announced regularly updated summaries of ORV data collected from each laboratory on its website (16). This site reported that the global frequency increased from 16% (October 2007–March 2008) to 44% (April 2008–September 2008) to 95% (October 2008–January 2009), indicating that ORVs have spread rapidly around the world.

nnounced regularly updated summaries of ORV data collected from each laboratory on its website (16). This site reported that the global frequency increased from 16% (October 2007–March 2008) to 44% (April 2008–September 2008) to 95% (October 2008–January 2009), indicating that ORVs have spread rapidly around the world. Japan has the highest annual level of oseltamivir usage per capita in the world, comprising >70% of world consumption (10). Such high use of oseltamivir has raised concerns about emergence of OSVs with increased resistance to this drug. Moreover, in Japan, 2 recent influenza seasons were dominated by influenza viruses A (H1N1) (Figure 1). If a high prevalence of ORVs is observed, primary selection of oseltamivir treatment for influenza patients should be reconsidered. Thus, monitoring ORVs is a serious public health issue.

sed resistance to this drug. Moreover, in Japan, 2 recent influenza seasons were dominated by influenza viruses A (H1N1) (Figure 1). If a high prevalence of ORVs is observed, primary selection of oseltamivir treatment for influenza patients should be reconsidered. Thus, monitoring ORVs is a serious public health issue. Figure 1 Weekly cases of influenza and isolation of influenza viruses in the 2007–08 and 2008–09 seasons in Japan (as of July 2, 2009). The National Epidemiologic Surveillance of Infectious Diseases (NESID) Network comprises the Ministry of Health, Labor and Welfare; the National Institute of Infectious Diseases; 76 local public health laboratories; ≈3,000 pediatric clinics; and 2,000 internal medical clinics. The NESID Network monitored influenza activity during the 2007–08 season (week 36, September 2007–week 35, August 2008) and 2008–09 season (week 36, September 2008–week 22, May 2009). Clinically diagnosed influenza-like cases were reported weekly by influenza sentinel clinics. Boldface line indicates weekly cases of influenza-like illness per influenza sentinel clinic (values shown in right bar). Bars indicate numbers of influenza A (H1N1) (yellow), A (H3N2) (blue), and B (red) isolates (values shown in left bar). Influenza activity started week 47 of 2007 and finished in week 14 of 2008 in the 2007–08 season and started week 49 of 2008 and finished in week 22 of 2009 in the 2008–09 season. Among all influenza isolates, influenza A (H1N1) consisted of 81% during 2007–08 and 49% during 2008–09. Seasonal influenza surveillance showed that influenza viruses A (H1N1) dominated the 2 recent influenza seasons in Japan.

–08 season and started week 49 of 2008 and finished in week 22 of 2009 in the 2008–09 season. Among all influenza isolates, influenza A (H1N1) consisted of 81% during 2007–08 and 49% during 2008–09. Seasonal influenza surveillance showed that influenza viruses A (H1N1) dominated the 2 recent influenza seasons in Japan. To estimate the frequency of ORVs and characterize these viruses, we analyzed 1,734 clinical samples isolated from the 2007–08 season and 1,482 isolates from the 2008–09 season by NA sequencing and/or NAI inhibition assay. The total frequencies were 2.6% in the 2007–08 season and 99.7% in the 2008–09 season, indicating that ORVs increased dramatically in Japan.

cterize these viruses, we analyzed 1,734 clinical samples isolated from the 2007–08 season and 1,482 isolates from the 2008–09 season by NA sequencing and/or NAI inhibition assay. The total frequencies were 2.6% in the 2007–08 season and 99.7% in the 2008–09 season, indicating that ORVs increased dramatically in Japan. Materials and Methods Virus Testing Influenza sentinel clinics send clinical specimens to local public health laboratories for virus isolation. Several culture tissues, including MDCK, Caco-2, and LLC-MK2, are used for virus isolation. Without successful viral isolation, clinical specimens are analyzed directly. Influenza viruses were collected from all 47 prefectures in Japan for this study; 1,734 samples of influenza A (H1N1) were isolated during the 2007–08 season (September 2007–August 2008) and 1,482 samples of influenza A (H1N1) were isolated in the 2008–09 season (September 2008–April 2009). During the 2007–08 season, viruses were isolated primarily after December 2007. All influenza viruses A (H1N1) were subjected to full or partial (nt 615–1076) NA sequencing to detect H275Y substitution on the N1 NA protein. Representative influenza viruses A (H1N1), including ORVs and OSVs, were subjected to NA inhibition assay (number of tested viruses isolated during the 2007–08 and 2008–09 seasons was 306 and 58, respectively), full NA sequencing (891 and 83), hemagglutinin (HA) 1 sequencing (299 and 83), M2 sequencing (288 and 79), and hemagglutinin inhibition (HI) test (187 and 59).

ng ORVs and OSVs, were subjected to NA inhibition assay (number of tested viruses isolated during the 2007–08 and 2008–09 seasons was 306 and 58, respectively), full NA sequencing (891 and 83), hemagglutinin (HA) 1 sequencing (299 and 83), M2 sequencing (288 and 79), and hemagglutinin inhibition (HI) test (187 and 59). Sequence Analysis The phylogenetic tree of NA and HA1 genes was constructed by neighbor-joining methods. The phylogenetic tree was described by representative ORVs and OSVs isolated from several prefectures in Japan. Sequence information for isolates from other countries was obtained from the Global Initiative on Sharing Avian Influenza Data and the Los Alamos National Laboratory database. All amino acid positions in the phylogenetic tree were described by N1 numbering. NA Inhibition Assay The chemiluminescent NA inhibition assay was performed by using the NA Star Kit (Applied Biosystems, Tokyo, Japan) with slight modifications of the instructions provided by the manufacturer. The final drug concentration ranged from 0.03 nmol/L to 6,500 nmol/L for oseltamivir and from 0.03 nmol/L to 12,500 nmol/L for zanamivir. Chemiluminescent light emission was measured by using an LB940 plate reader (Berthhold Technologies, Bad Wildbad, Germany). Drug concentrations required to inhibit NA activity by 50% (IC50) were calculated by a 4-parameter method using MikroWin 2000 version 4 software (Mikrotek Laborsysteme GmbH, Overath, Germany).

ir. Chemiluminescent light emission was measured by using an LB940 plate reader (Berthhold Technologies, Bad Wildbad, Germany). Drug concentrations required to inhibit NA activity by 50% (IC50) were calculated by a 4-parameter method using MikroWin 2000 version 4 software (Mikrotek Laborsysteme GmbH, Overath, Germany). Hemagglutination Inhibition Test The HI test was performed to evaluate the reactivity of ferret antiserum against 2008–09 vaccine strain A/Brisbane/59/2009, as described by the WHO manual (17). Antiserum was treated by receptor-destroying enzyme II (Denka Seiken, Tokyo, Japan) and adsorbed with packed turkey erythrocytes before testing to prevent nonspecific reaction. A 0.5% suspension of turkey erythrocytes was used for the HI test. Viruses with >8-fold reduced HI titer to the homologous titer of A/Brisbane/59/2009 antiserum were regarded as antigenic variants. Statistical Analysis To determine the cutoff value between NAI-resistant (outlier) and -sensitive viruses, box-and-whisker plots were used. The cutoff value was defined as upper quartile + 5.0×interquartile range from the 25th to 75th percentile. In this study, ORVs with H275Y were excluded from the overall population for statistical analysis. Outliers were excluded from the calculation of mean values and standard deviations for IC50.

sker plots were used. The cutoff value was defined as upper quartile + 5.0×interquartile range from the 25th to 75th percentile. In this study, ORVs with H275Y were excluded from the overall population for statistical analysis. Outliers were excluded from the calculation of mean values and standard deviations for IC50. Results Geographic Distribution of ORVs during the 2007–08 and 2008–09 Influenza Seasons To estimate the frequency of influenza A (H1N1) ORVs in each prefecture of Japan, 1,734 isolates during the 2007–08 season and 1,482 isolates during the 2008–09 season were collected from all prefectures and examined by NA sequencing to detect the H275Y mutation in NA protein. In the 2007–08 season, 45 viruses possessing H275Y mutation (total frequency of ORVs 2.6%; Figure 2, panel A) were observed in 10 prefectures, indicating that the frequency of ORVs was significantly lower than that in countries in Europe and the United States (8,11–14). In Tottori prefecture, however, 22 of 68 influenza viruses A (H1N1) tested possessed H275Y, showing a markedly higher frequency (32.4%) than that in other prefectures. In the 2008–09 season, however, ORVs were observed nationwide. Of 1,482 influenza viruses A (H1N1), 1,477 viruses possessed a H275Y mutation, for a total frequency of 99.7% (Figure 2, panel B). These data show that ORVs increased dramatically in Japan from the 2007–08 season to the 2008–09 season.

in other prefectures. In the 2008–09 season, however, ORVs were observed nationwide. Of 1,482 influenza viruses A (H1N1), 1,477 viruses possessed a H275Y mutation, for a total frequency of 99.7% (Figure 2, panel B). These data show that ORVs increased dramatically in Japan from the 2007–08 season to the 2008–09 season. Figure 2 Geographic distribution of oseltamivir-resistant influenza viruses A (H1N1) (ORVs) with H275Y in Japan during the 2007–08 and 2008–09 seasons. The total number of influenza A (H1N1) isolates tested is described inside each prefecture. Total frequency in Japan was 2.6% (45/1,734) during the 2007–08 season, although a high frequency (32.4%) of ORVs was observed in Tottori prefecture (A). On the other hand, total frequency was 99.7% (1,477/1,482) during the 2008–09 season (B), indicating a drastic increase in ORVs in Japan from the 2007–08 season to the 2008–09 season. *Number of ORVs/number of subtype H1N1 isolates tested.

lthough a high frequency (32.4%) of ORVs was observed in Tottori prefecture (A). On the other hand, total frequency was 99.7% (1,477/1,482) during the 2008–09 season (B), indicating a drastic increase in ORVs in Japan from the 2007–08 season to the 2008–09 season. *Number of ORVs/number of subtype H1N1 isolates tested. Genetic Analysis Influenza viruses A (H1N1) during 2007–08 fell into either clade 2B, including the current vaccine strain A/Brisbane/59/2007, or clade 2C, and almost all influenza viruses A (H1N1) during 2008–09 fell into clade 2B. Most ORVs with H275Y belong to clade 2B, which can be further divided into 2 distinct lineages by an aspartic acid to glycine substitution at aa 354: a Northern-Eu lineage sharing 354G, which was first isolated from countries in northern Europe and now represents most ORVs worldwide; and a Hawaii lineage sharing 354D, which was first detected in Hawaii and was rarely isolated in a few countries during the 2007–08 season (Appendix Figure). In the 2007–08 season, of 45 ORVs, 1 virus (A/Yokohama/91/2007) isolated in November 2007 belonged to clade 2C, and 44 viruses fell into either the Hawaii lineage or Northern-Eu lineage. Conversely, in the 2008–09 season, all ORVs belonged to the Northern-Eu lineage, indicating that ORVs of the Northern-Eu lineage dominated in the 2008–09 season in Japan.

virus (A/Yokohama/91/2007) isolated in November 2007 belonged to clade 2C, and 44 viruses fell into either the Hawaii lineage or Northern-Eu lineage. Conversely, in the 2008–09 season, all ORVs belonged to the Northern-Eu lineage, indicating that ORVs of the Northern-Eu lineage dominated in the 2008–09 season in Japan. In the Hawaii lineage, OSVs genetically close to ORVs were observed. The NA gene of some ORVs had only 1 nucleotide difference from that of OSV counterparts (i.e., A/Tochigi/8/2008 and A/Tochigi/9/2008, A/Nagano/1100/2008 and A/Nagano/1071/2008, A/Yamagata/68/2008 and A/Yamagata/41/2008, respectively), and the other ORVs are also genetically close to OSVs from Japan (Appendix Figure). These HA genes were also genetically identical or close together (Appendix Figure), suggesting that almost all ORVs from Japan with the Hawaii lineage are derived from OSVs from Japan. On the other hand, in the Northern-Eu lineage, OSV counterparts were not observed, but foreign ORVs genetically close to ORVs from Japan were observed. During the 2007–08 season, the NA gene of ORVs from Japan was close to that of ORVs isolated from countries in Europe (i.e., A/Paris/0341/2007 and A/England/26/2008). During the 2008–09 season, the ORVs from Japan, which shared A189T on HA protein, were further divided into 4 subclades (C-1 to C-4) by common amino acid changes on HA and/or NA (Appendix Figure). ORVs from Japan in C-2 and C-3 were genetically close to the ORVs isolated from North America or Hawaii (e.g., A/Memphis/03/2008 and A/Hawaii/19/2008), whereas ORVs in C-1, representing most influenza viruses A (H1N1) from the 2008–09 season in Japan, and ORVs in C-4 were close to ORVs isolated from South Africa and Australia in the Southern Hemisphere (e.g., A/Kenya/1432/2008 and A/Victoria/501/2008). All ORVs except C-3 were isolated before the emergence of ORVs from Japan in each subclade. These findings suggest that ORVs from Japan within a Northern-Eu lineage would not have emerged domestically but instead may have been introduced from various countries.

isphere (e.g., A/Kenya/1432/2008 and A/Victoria/501/2008). All ORVs except C-3 were isolated before the emergence of ORVs from Japan in each subclade. These findings suggest that ORVs from Japan within a Northern-Eu lineage would not have emerged domestically but instead may have been introduced from various countries. Antiviral Drug Susceptibility Of the 364 viruses (306 isolates in the 2007–08 season and 58 isolates in the 2008–09 season) tested by NA inhibition assay, 101 possessed a H275Y substitution. With the NA inhibition assay, although precise IC50 values were calculated from a normal sigmoid curve (Figure 3, panels A and B), some viruses generated 2 types of unusual sigmoid curves (Figure 3, panels C and D) resulting from the mixed population of NAI-resistant and -sensitive viruses, as previously reported (18). Tentative IC50 values were calculated from type A curves (Figure 3, panel C) and included in overall statistical analysis, but values could not be calculated from type B curves (Figure 3, panel D). Later viruses were regarded as resistant candidates.

of NAI-resistant and -sensitive viruses, as previously reported (18). Tentative IC50 values were calculated from type A curves (Figure 3, panel C) and included in overall statistical analysis, but values could not be calculated from type B curves (Figure 3, panel D). Later viruses were regarded as resistant candidates. Figure 3 Assessment of drug concentrations required to inhibit neuraminidase activity by 50% (IC50) for neuramindase inhibitors (NAIs). Normal sigmoid curves were generated for most tested viruses by a neuraminidase inhibition assay for oseltamivir (A) and zanamivir (B). Sensitive A/Gunma/55/2007 (blue), oseltamivir-resistant A/Kobe/49/2008 (red) with H275Y, zanamivir-resistant A/Tottori/16/2008 (green) with Q136K, and oseltamivir/zanamivir-resistant A/Tottori/44/2008 (orange) with H275Y and D151D/G are shown. Unusual sigmoid curves were sometimes generated by the mixed population of NAI-resistant and -sensitive viruses for zanamivir: A/Yokohama/74/2008 with D151D/G (C, type A curve); and A/Hiroshima/92/2008 with D151D/G (D, type B curve). Tentative IC50 values (nM), shown below each panel, were obtained from type A curves but not from type B curves. NA, not available; NC, negative control; PC, positive control.

and -sensitive viruses for zanamivir: A/Yokohama/74/2008 with D151D/G (C, type A curve); and A/Hiroshima/92/2008 with D151D/G (D, type B curve). Tentative IC50 values (nM), shown below each panel, were obtained from type A curves but not from type B curves. NA, not available; NC, negative control; PC, positive control. In the NA inhibition assay for oseltamivir, OSVs showed a mean IC50 ± SD of 0.10 ± 0.05 nmol/L (range 0.01–0.35 nmol/L), and ORVs had a mean ± SD IC50 of 67.7 ± 44.1 nmol/L (range 26.1–239.2 nmol/L), showing a reduction of >260-fold in susceptibility to oseltamivir. One OSV identified as a statistical outlier (cutoff IC50, >0.40 nmol/L; upper quartile + 5.0× interquartile range) showed a D151E substitution on the NA protein (Table 1).

), and ORVs had a mean ± SD IC50 of 67.7 ± 44.1 nmol/L (range 26.1–239.2 nmol/L), showing a reduction of >260-fold in susceptibility to oseltamivir. One OSV identified as a statistical outlier (cutoff IC50, >0.40 nmol/L; upper quartile + 5.0× interquartile range) showed a D151E substitution on the NA protein (Table 1). Table 1 Influenza virus A (H1N1) outliers to oseltamivir and/or zanamivir, Japan* Strain Sequence change(s) Clinical specimen Curve fit‡ IC50, nmol/L D151 H275 Q136 D151 Oseltamivir Zanamivir Oseltamivir outlier A/YAMAGATA/28/2008 D/E† H Q NA Normal 0.55 0.60 Zanamivir outlier candidates A/TOTTORI/16/2008 D H K NA Normal 0.08 41.89 A/TOTTORI/60/2008 D Y Q NA Normal 113.86 3.64 A/KOBE/31/2008 D Y Q NA Type A 26.05 2.75 A/KOBE/32/2008 D Y Q NA Type A 135.85 3.56 A/MIE/13/2008 D/G H Q D Type A 0.18 14.80 A/YOKOHAMA/75/2007 D/G H Q NA Type A 0.13 6.53 A/HAMAMATSU/33/2008 D/G H Q NA Type A 0.13 6.15 A/TOCHIGI/30/2008 D/G H Q NA Type A 0.13 4.32 A/YOKOHAMA/74/2007 D/G H Q NA Type A 0.12 2.81 A/HIROSHIMA/92/2007 D/G H Q NA Type B 0.07 NA A/MIE/9/2008 D/G H Q D Type B 0.08 NA A/MIE/1/2008 D/G H Q D Type B 0.16 NA A/MIE/14/2008 D/G H Q D Type B 0.08 NA A/YAMAGATA/60/2008 D/G H Q NA Type B 0.19 NA A/SAPPORO/64/2008 D/G Y Q NA Type B 147.90 NA A/TOTTORI/44/2008 D/G Y Q NA Normal§ 180.06 84.42 A/HIROSHIMA/44/2008 D/N Y Q NA Type A 239.23 2.26 A/YOKOHAMA/79/2008 D/N Y Q NA Type A 167.66 2.28 A/HIROSHIMA/46/2008 D/N Y Q NA Type A 190.35 2.40 A/HIROSHIMA/45/2008 D/N Y Q NA Type A 169.92 3.34 A/MIE/1/2009 D/N Y Q NA Type A 231.78 3.55 A/HIROSHIMA/47/2008 D/N Y Q NA Type A 106.19 4.24 A/YOKOHAMA/96/2008 D/V Y Q NA Type B 126.50 NA Zanamivir sensitive A/MIE/18/2008 D/E¶ H Q D Normal 0.35 1.06 A/MIE/21/2008 D/N H Q D Normal 0.22 1.18 IC50 mean of sensitive viruses 0.10 ± 0.05 0.40 ± 0.26 Cutoff IC50 values (UQ +5.0 IQR) 0.40 1.99 *IC50, drug concentrations required to inhibit neurominidase activity by 50%; UQ, upper quartile; IQR, interquartile range; NA, not available. Oseltamivir-resistant viruses with H275Y were excluded from overall population in statistical analysis of oseltamivir. †Mixture of D151 and D151 variants. ‡Curve fit patterns were evaluated based on Figure 3, panels C (Type A) and D (Type B). Although the viruses with D151D/G tend to generate both patterns from repeat testing for the same samples, type B was selected in this case. §Although A/TOTTORI/44/2008 showed mixed population of D151D/G, it tended to show a normal curve fit (Figure 3, panel B).

ated based on Figure 3, panels C (Type A) and D (Type B). Although the viruses with D151D/G tend to generate both patterns from repeat testing for the same samples, type B was selected in this case. §Although A/TOTTORI/44/2008 showed mixed population of D151D/G, it tended to show a normal curve fit (Figure 3, panel B). ¶The IC50 values of most viruses with D151D/E tend to be higher than mean IC50 values but do not exceed the cutoff value.

ated based on Figure 3, panels C (Type A) and D (Type B). Although the viruses with D151D/G tend to generate both patterns from repeat testing for the same samples, type B was selected in this case. §Although A/TOTTORI/44/2008 showed mixed population of D151D/G, it tended to show a normal curve fit (Figure 3, panel B). ¶The IC50 values of most viruses with D151D/E tend to be higher than mean IC50 values but do not exceed the cutoff value. In the NA inhibition assay for zanamivir, statistical analysis showed that 341 viruses were regarded as the zanamivir-sensitive viruses, with a mean ± SD IC50 of 0.40 ± 0.26 nmol/L (range 0.01–1.92 nmol/L), and 16 viruses (10 ORVs and 6 OSVs) were identified as outliers (cutoff IC50, >1.99 nmol/L) (Table 1). Seven viruses (2 ORVs and 5 OSVs) were regarded as resistant candidates from curve fit patterns. NA-sequencing for these 23 viruses (12 ORVs and 11 OSVs) showed 2 types of amino acid changes on the NA protein. One virus, A/Tottori/16/2008 (OSV), possessed a Q136K substitution, showing a high IC50 (41.89 nmol/L), and 19 of the other 22 viruses displayed an amino acid change G, N, or V at the D151 position (Table 1). These data suggest that D151 changes have a substantial effect on sensitivity to zanamivir (and oseltamivir). Moreover, A/Tottori/44/2008 with H275Y and D151D/G substitutions conferred high-level resistance to both NAIs (Figure 3, panels A and B). However, a recent study reported that a D151E change was detected only after virus propagation in cell culture, but not in the original clinical specimen (19), suggesting a possible role of cell culture in selecting these D151 variant viruses. To further investigate D151 variants, available original clinical specimens of viruses with D151 variation were subjected to NA sequencing, so that all D151 variations (D151G/E/N) were not detected in the original clinical specimens (Table 1). We thus concluded that D151 variants might not have emerged as a natural occurrence and all recent ORVs would retain sensitivity to zanamivir.

specimens of viruses with D151 variation were subjected to NA sequencing, so that all D151 variations (D151G/E/N) were not detected in the original clinical specimens (Table 1). We thus concluded that D151 variants might not have emerged as a natural occurrence and all recent ORVs would retain sensitivity to zanamivir. Susceptibility to M2 inhibitors was determined to find established-resistant markers by M2-sequencing. The 367 viruses (288 isolates in the 2007–08 season; 79 isolates in the 2008–09 season) including 123 ORVs (45 and 78, respectively) and 244 OSVs (243 and 1, respectively) were tested. Viruses belonging to clade 2B were sensitive to M2 inhibitors, and viruses belonging to clade 2C were resistant to M2 inhibitors, so all ORVs except A/Yokohama/91/2007 were sensitive to M2 inhibitors. A/Yokohama/91/2007 belonged to clade 2C and was the only virus resistant to both oseltamivir and M2 inhibitors. Antigenic Characteristics To estimate the reactivity of ORVs and OSVs to ferret antiserum against 2008–09 vaccine strain A/Brisbane/59/2009, the HI test was performed. Good inhibition was achieved in 76% of OSVs and 69% of ORVs by A/Brisbane/59/2009 ferret antiserum, and 22% of OSVs and 28% of ORVs showed a 4-fold reduction in HI titer to the homologous titer, respectively (Table 2). Only 2% and 3% of OSVs and ORVs showed a >8-fold reduction in HI titer to A/Brisbane/59/2009 ferret antiserum. These data demonstrated that OSVs and ORVs were anitigenitically indistinguishable from each other and were similar to the 2008–09 vaccine strain A/Brisbane/59/2009.

e homologous titer, respectively (Table 2). Only 2% and 3% of OSVs and ORVs showed a >8-fold reduction in HI titer to A/Brisbane/59/2009 ferret antiserum. These data demonstrated that OSVs and ORVs were anitigenitically indistinguishable from each other and were similar to the 2008–09 vaccine strain A/Brisbane/59/2009. Table 2 Antigenic characterization of oseltamivir-resistant and oseltamivir-sensitive influenza virus A (H1N1), Japan, 2007–2009 Antiserum Low to homologous titer, -fold* No. (%) sensitive, n = 169 No. (%) resistant, n = 77 A/Brisbane/59/2007 <2 128 (76) 53 (69) 4 36 (22) 22 (28) >8 3 (2) 2 (3) *Viruses with >8-fold reduced hemagglutinin inhibition titer to homologous titer were regarded as an antigenic variant. High Frequency of ORVs in Tottori Prefecture during the 2007–08 Season Tottori Prefecture is located in the western part of the main island of Japan. Comprising 19 cities and geographically divided into 3 areas, this prefecture has the lowest population in Japan (Figure 4, panel B). Despite a low frequency of only 2.6% in Japan during 2007–08 season, an unexpectedly high frequency (32.4%) of ORVs was observed in Tottori prefecture (Figure 2, panel A). ORVs from Tottori were collected from 4 cities in 2 areas with no systematic bias apparent in the sampling process (Figure 4, panel B).

4, panel B). Despite a low frequency of only 2.6% in Japan during 2007–08 season, an unexpectedly high frequency (32.4%) of ORVs was observed in Tottori prefecture (Figure 2, panel A). ORVs from Tottori were collected from 4 cities in 2 areas with no systematic bias apparent in the sampling process (Figure 4, panel B). Figure 4 Phylogenetic analysis of influenza A (H1N1) neuraminidase genes (A) and geographic distribution of oseltamivir-resistant viruses (ORVs) (B) isolated from Tottori Prefecture, Japan, 2007–08. ORVs fell into either Northern-Eu lineage (red) or Hawaii lineage (blue); Tottori ORVs and current vaccine strains are indicated by black and purple, respectively. A) ORVs formed 3 subclades: T-1, sharing V75A and D354G; T-2, without common changes; and T-3, sharing M188L. Sampling dates are given after each strain name. Scale bar indicates nucleotide substitutions per site. B) Tottori Prefecture is geographically divided into 3 areas, comprising 19 cities. ORVs from Tottori were collected from 4 cities over 2 areas. The sampling month for each ORV is indicated by a triangle (January), circle (February), or square (March). *Zanamivir resistant.

indicates nucleotide substitutions per site. B) Tottori Prefecture is geographically divided into 3 areas, comprising 19 cities. ORVs from Tottori were collected from 4 cities over 2 areas. The sampling month for each ORV is indicated by a triangle (January), circle (February), or square (March). *Zanamivir resistant. Phylogenetic analysis of NA genes showed that these ORVs formed 3 subclades (Figure 4, panel A): the first with a Northern-Eu lineage sharing V75A and D354G (T-1); the second with a Hawaii lineage without common changes (T-2); and the third with a Hawaii lineage and sharing M188L (T-3). Among these, only OSVs genetically close to ORVs were observed in T-2, suggesting that ORVs in T-2 would be derived from OSVs in Tottori prefecture.

rthern-Eu lineage sharing V75A and D354G (T-1); the second with a Hawaii lineage without common changes (T-2); and the third with a Hawaii lineage and sharing M188L (T-3). Among these, only OSVs genetically close to ORVs were observed in T-2, suggesting that ORVs in T-2 would be derived from OSVs in Tottori prefecture. A mapping study for ORVs showed that all ORVs in the Hawaii lineage were collected from Tottori city only, primarily at the end of January, whereas ORVs with the Northern-Eu lineage were collected from 4 cities, including Tottori city, during February and March. Genetically diverse ORVs belonging to T1-T3 were cocirculating only in Tottori city in the eastern area (Figure 4, panel B). The Tottori case raised concern about the possibility that these Tottori ORVs could survive to become an origin ORV for the 2008–09 season in Japan. However, phylogenetic analysis showed that all ORVs isolated during the 2008–09 season were not genetically close to ORVs from Tottori (Appendix Figure). As a result, all ORVs from Tottori seemed to have been eliminated in the 2007–08 season, and ORVs that may have been introduced from other counties were circulating during 2008–09 in Japan.

nalysis showed that all ORVs isolated during the 2008–09 season were not genetically close to ORVs from Tottori (Appendix Figure). As a result, all ORVs from Tottori seemed to have been eliminated in the 2007–08 season, and ORVs that may have been introduced from other counties were circulating during 2008–09 in Japan. Discussion Our study demonstrated that ORVs dramatically increased in Japan from the 2007–08 season (2.6%) to the 2008–09 season (99.7%). All tested ORVs showed a reduction of >260-fold in susceptibility to oseltamivir by NA inhibition assay. On the other hand, almost all ORVs remained sensitive to the other antiviral-drugs, e.g., zanamivir, and M2 inhibitors. HI testing suggested that the current vaccine, A/Brisbane/59/2008, would be effective against recent ORVs. In addition, recent studies have reported that symptoms and hospitalization rates of patients infected with ORVs are no different from those seen with OSVs (14,20).

viral-drugs, e.g., zanamivir, and M2 inhibitors. HI testing suggested that the current vaccine, A/Brisbane/59/2008, would be effective against recent ORVs. In addition, recent studies have reported that symptoms and hospitalization rates of patients infected with ORVs are no different from those seen with OSVs (14,20). Japan has the largest per capita use of oseltamivir (>70%) in the world (10). Because this use could cause efficient selection of ORVs in individual patients, Japan might be the initial site of worldwide spread of ORVs. However, long-term NAI surveillance in Japan during 1996–2007 and recent surveillance showed a low frequency of NAI-resistant viruses for any strains and subtypes (10,21,22), suggesting that transmissibility of ORVs selected by drug pressure was remarkably decreased. In addition, previous NAI surveillance (5–10) and several animal studies (23–26) also suggested that NAI-resistant viruses would become defective viruses with attenuated infectivity and transmissibility to human. In contrast, despite little NAI use, a high emergence of ORVs has been detected in several countries in Europe since November 2007. These ORVs had as efficient transmissibility as OSVs in human-to-human transmission, resulting in worldwide spread in a short period of time. Although whether the initial ORV detected in Norway in the 2007–08 season appeared because of NAI drug pressure is unknown, those ORVs may have obtained amino acid changes on NA and/or other proteins to compensate for the defect, in addition to the H275Y substitution on the NA protein. Most ORVs belong genetically to the Northern-Eu lineage in clade 2B, suggesting that the gene constellation may contain a big advantage to retain infectivity and transmissibility.

tained amino acid changes on NA and/or other proteins to compensate for the defect, in addition to the H275Y substitution on the NA protein. Most ORVs belong genetically to the Northern-Eu lineage in clade 2B, suggesting that the gene constellation may contain a big advantage to retain infectivity and transmissibility. An interesting question arose as to where the ORVs in Japan originated. In the Hawaii lineage, almost all ORVs in Japan would be derived from OSVs in Japan because the NA gene of ORVs was similar to OSV counterparts isolated at similar times or from similar regions (Appendix Figure). On the other hand, in the Northern-Eu lineage, ORVs in Japan would have been introduced from other countries. In 2007–08, almost all ORVs would be imported from countries in Europe. In 2008–09, the ORVs in C-1, which comprised most isolates in 2008–09, and ORVs in C-4 were genetically similar to ORVs isolated from the Southern Hemisphere. Because influenza activity in the Southern Hemisphere occurs half a year earlier than that in the Northern Hemisphere, most ORVs in Japan conceivably could have been imported from the Southern Hemisphere. ORVs in C-2 and C-3 were genetically similar to ORVs isolated in North America and Hawaii, but the collection month of ORVs in C-3 were similar to each other, suggesting that ORVs in C-3 might be derived from an unknown common origin ORV. The ORVs obtained during 2008–09 were not genetically similar to any ORVs isolated in Tottori during 2007–08, indicating that ORVs from Tottori had been eliminated and had not formed the origin ORVs for the 2008–09 season in Japan. As for A/Yokohama/91/2007 belonging to clade 2C, the patient from which this virus was isolated was known to have taken oseltamivir before sampling (22), indicating that selective drug pressure in this person might have selected for this ORV.

liminated and had not formed the origin ORVs for the 2008–09 season in Japan. As for A/Yokohama/91/2007 belonging to clade 2C, the patient from which this virus was isolated was known to have taken oseltamivir before sampling (22), indicating that selective drug pressure in this person might have selected for this ORV. In the NA inhibition assay for zanamivir, some viruses, including ORVs and OSVs, showed reduced sensitivity to zanamivir. NA sequencing of these viruses showed 2 types of amino acid changes. One virus, A/Tottori/16/2008 (OSV), possessed a Q136K substitution, which reportedly confers resistance to zanamivir (27,28). Conversely, most of the other viruses possessed D151 G/V/N. The amino acid changes D151 to N or E among subtype H1N1 viruses and to A, G, E, N, or V among H3N2 have been reported (7,8,19), and viruses with D151 substitutions often exhibit reduced sensitivity to NAIs (8,19,29). However, a recent study reported a possible role for cell culture in selecting these D151 variant viruses (19). In the present study, D151 variations (D151G/E/N) also were not detected from available original clinical specimens (Table 1), supporting the previous finding. We thus concluded that viruses with D151 variations would not have emerged naturally, and all ORVs would remain sensitive to zanamivir. By sequencing of M2 gene, we confirmed that almost all Japanese ORVs belonging to clade 2B retained sensitive genotype to M2 inhibitors, consistent with previously reports that recent clade 2B viruses are sensitive to M2 inhibitors, but clade 2C viruses are resistant (27).

In the NA inhibition assay for zanamivir, some viruses, including ORVs and OSVs, showed reduced sensitivity to zanamivir. NA sequencing of these viruses showed 2 types of amino acid changes. One virus, A/Tottori/16/2008 (OSV), possessed a Q136K substitution, which reportedly confers resistance to zanamivir (27,28). Conversely, most of the other viruses possessed D151 G/V/N. The amino acid changes D151 to N or E among subtype H1N1 viruses and to A, G, E, N, or V among H3N2 have been reported (7,8,19), and viruses with D151 substitutions often exhibit reduced sensitivity to NAIs (8,19,29). However, a recent study reported a possible role for cell culture in selecting these D151 variant viruses (19). In the present study, D151 variations (D151G/E/N) also were not detected from available original clinical specimens (Table 1), supporting the previous finding. We thus concluded that viruses with D151 variations would not have emerged naturally, and all ORVs would remain sensitive to zanamivir. By sequencing of M2 gene, we confirmed that almost all Japanese ORVs belonging to clade 2B retained sensitive genotype to M2 inhibitors, consistent with previously reports that recent clade 2B viruses are sensitive to M2 inhibitors, but clade 2C viruses are resistant (27). During the 2007–09 seasons, we also addressed NAI surveillance for A/H3N2 and type B circulating in Japan and identified no viruses resistant to both NAIs. Conversely, in March and early April 2009, a new swine-origin influenza virus A (H1N1) (now known as pandemic [H1N1] 2009 virus) emerged in Mexico and the United States and spread rapidly to many countries, including Japan (30–33). In June 2009, detection of pandemic (H1N1) 2009 virus with H275Y on the NA protein was reported from Denmark, Hong Kong Special Administrative Region, People’s Republic of China, and Japan, but all ORVs of pandemic (H1N1) 2009 virus emerged as sporadic cases with no evidence of efficient human-to-human transmission (34). Although oseltamivir remains a valuable drug for treatment of pandemic (H1N1) 2009, many ORVs were isolated after prophylaxis with a half dose of the drug. Therefore, prophylaxis with oseltamivir may not be recommended as stated by WHO (35). Rapid and continuous monitoring of NAI-resistant viruses, including pandemic (H1N1) 2009 virus, and dissemination of the findings in timely manner remains essential.

y ORVs were isolated after prophylaxis with a half dose of the drug. Therefore, prophylaxis with oseltamivir may not be recommended as stated by WHO (35). Rapid and continuous monitoring of NAI-resistant viruses, including pandemic (H1N1) 2009 virus, and dissemination of the findings in timely manner remains essential. Supplementary Material Appendix Figure Phylogenetic analysis of influenza A (H1N1) A) neuraminidase (NA) genes and B) hemagglutinin (HA) (HA1 region) genes. Recent influenza viruses A (H1N1) fell into either clade 2B or clade 2C. Almost all oseltamivir-resistant viruses (ORVs) with H275Y belong to clade 2B and were further divided into 2 distinct lineages: Northern-EU lineage sharing 354G (pink shading); and Hawaii lineage sharing 354D. ORVs during 2008-09 shared A189T on HA, and formed 4 subclades: C-1 (HA: G185A and S141N); C-2 (HA: G185V); C-3 (HA: G185A, NA: A86T and T339A); and C-4 (HA: G185S/N and N183S). OSVs during 2007-09, Japanese ORVs during 2007-08, Japanese ORVs during 2008-09, foreign ORVs during 2007-09 and 2008-09 current vaccine strains are indicated in black, blue, red, orange, and purple, respectively. Sampling month of each isolate is described after the strain name. Viruses resistant to zanamivir are marked with an asterisk. The phylogenetic tree of NA and HA1 genes was constructed by using neighbor-joining methods.

2008-09 current vaccine strains are indicated in black, blue, red, orange, and purple, respectively. Sampling month of each isolate is described after the strain name. Viruses resistant to zanamivir are marked with an asterisk. The phylogenetic tree of NA and HA1 genes was constructed by using neighbor-joining methods. Suggested citation for this article: Ujike M, Shimabukuro K, Mochizuki K, Obuchi M, Kageyama T, Shirakua M, et al. Oseltamivir-resistant influenza A (H1N1) viruses during 2007–2009 influenza seasons, Japan. Emerg Infect Dis [serial on the Internet]. 2010 Jun [date cited]. http://www.cdc.gov/EID/content/16/6/926.htm

2008-09 current vaccine strains are indicated in black, blue, red, orange, and purple, respectively. Sampling month of each isolate is described after the strain name. Viruses resistant to zanamivir are marked with an asterisk. The phylogenetic tree of NA and HA1 genes was constructed by using neighbor-joining methods. Suggested citation for this article: Ujike M, Shimabukuro K, Mochizuki K, Obuchi M, Kageyama T, Shirakua M, et al. Oseltamivir-resistant influenza A (H1N1) viruses during 2007–2009 influenza seasons, Japan. Emerg Infect Dis [serial on the Internet]. 2010 Jun [date cited]. http://www.cdc.gov/EID/content/16/6/926.htm 1 Members of the Working Group for Influenza Virus Surveillance in Japan: Hideki Nagano (Hokkaido Institute of Public Health), Masayuki Kikuchi (Sapporo City Institute of Public Health), Masaki Takahashi (Research Institute for Environmental Sciences and Public Health of Iwate Prefecture), Yuki Sato (Miyagi Prefectural Institute of Public Health and Environment), Masanori Katsumi (Sendai City Institute of Public Health), Hiroyuki Saito (Akita Research Center for Public Health and Environment), Katsumi Mizuta (Yamagata Prefectural Institute of Public Health), Syoko Hirose (Fukushima Prefectural Institute of Public Health), Setsuko Fukaya (Ibaraki Prefectural Institute of Public Health), Asumi Hirata(Tochigi Prefectural Institute of Public Health and Environmental Science), Hiroyuki Tsukagoshi (Gunma Prefectural Institute of Public Health and Environmental Sciences), Yuka Uno (Saitama City Institute of Health Science and Research), Hiromi Maru (Chiba Prefectural Institute of Public Health), Hajime Yokoi (Chiba City Institute of Health and Environment), Mami Nagashima (Tokyo Metropolitan Institute of Public Health), Sumi Watanabe (Kanagawa Prefectural Institute of Public Health), Hideaki Shimizu (Kawasaki City Institute of Public Health), Sumiko Ueda (Sagamihara City Laboratory of Public Health), Chika Hirokawa (Niigata Prefectural Institute of Public Health and Environmental Sciences), Gen Kobayashi and Yoko Miyajima (Niigata City Institute of Public Health and Environment), Sanae Kuramoto and Hiroe Kodama (Ishikawa Prefectural Institute of Public Health and Environmental Science), Masako Nakamura (Fukui Prefectural Institute of Public Health), Hiroyoshi Asakawa (Yamanashi Institute for Public Health), Seiko Sawatari (Gifu Prefectural Institute of Health and Enviromental Sciences), Yasunori Tanaka (Gifu Municipal Institute of Public Health), Yoshinobu Miwa (Shizuoka Institute of Environment and Hygiene), Shinobu Ide (Shizuoka City Institute of Environmental Sciences and Public Health), Yoshinori Kohno and Ryu Hibino (Hamamatsu City Health Environment Research Center), Mami Hata (Aichi Prefectural Institute of Public Health), Noriko Goto (Nagoya City Public Health

u Miwa (Shizuoka Institute of Environment and Hygiene), Shinobu Ide (Shizuoka City Institute of Environmental Sciences and Public Health), Yoshinori Kohno and Ryu Hibino (Hamamatsu City Health Environment Research Center), Mami Hata (Aichi Prefectural Institute of Public Health), Noriko Goto (Nagoya City Public Health Research Institute), Takuya Yano (Mie Prefecture Health and Environment Research Institute), Fumie Matsumoto (Shiga Prefectural Institute of Public Health), Tohru Ishizaki (Kyoto Prefectural Institute of Public Health and Environment), Satoshi Hiroi (Osaka Prefectural Institute of Public Health), Hideyuki Kubo (Osaka City Institute of Public Health and Environmental Sciences), Kiyoko Uchino (Sakai City Institute of Public Health), Tomohiro Oshibe (Hyogo Prefectural Institute of Public Health and Consumer Sciences), Ai Mori and Tomoko Suga (Kobe Institute of Health), Yoshiteru Kitahori (Nara Prefectural Institute for Hygiene and Environment), Fumio Terasoma (Wakayama Prefectural Research Center of Environment and Public Health), Yoshiaki Kimura and Naomi Matsumoto (Tottori Prefectural Institute of Public Health and Environmental Science), Tamaki Omura (Shimane Prefectural Institute of Public Health and Environmental Science), Shinichi Takao (Center for Public Health and Environment, Hiroshima Prefectural Technology Research Institute), Katsuhiko Abe (Hiroshima City Institute of Public Health), Yumiko Kawakami (Tokushima Prefectural Centre for Public Health and Environmental Sciences), Ootsuka Yuka (Ehime Prefecture Institute of Public Health and Environmental Science), Daisuke Kawamoto (Fukuoka City Institute for Hygiene and the Environment), Takayuki Hirano and Hisato Masumoto (Saga Prefectural Institute of Public Health and Pharmaceutical Research), Manabu Hirano and Akinori Yamaguchi (Nagasaki Prefectural Institute for Environment Research and Public Health), Seiya Harada and Koichi Nishimura (Kumamoto Prefectural Institute of Public Health and Environmental Science), Miki Kato (Oita Prefectural Institute of Health and Environment), Miho Miura (Miyazaki Prefectural Institute for Public Health and Environment), Kanji Ishitani and Akihide Kamimura (Kagoshima Prefectural Institute for Environmental Research and Pubulic Health), Katsuya Taira (Okinawa Prefectural Institute of Health and Environment), Mitsutaka Kuzuya (Okayama Prefectural Institute for Environmental Science and Public Health), Shoichi Toda (Yamaguchi Prefectural Institute of Public Health and Envir

(Kagoshima Prefectural Institute for Environmental Research and Pubulic Health), Katsuya Taira (Okinawa Prefectural Institute of Health and Environment), Mitsutaka Kuzuya (Okayama Prefectural Institute for Environmental Science and Public Health), Shoichi Toda (Yamaguchi Prefectural Institute of Public Health and Envir onment), Chiharu Kawakami (Yokohama City Institute of Health), Mayumi Konno (Kyoto City Institute of Health and Environmental Sciences), Hiroya Komoda (Kagawa Prefectural Research Institute for Environmental Sciences and Public Health), Tae Taniwaki (Kochi Public Health and Sanitation Institute), Toshitaka Minegishi (Saitama Institute of Public Health), Rika Tsutsui (Aomori Prefectural Institute of Public Health and Environment), Shizuko Kasuo (Nagano Environmental Conservation Research Institute), Yuichiro Okamura (Nagano City Health Center), Eiji Horimoto (Toyama Institute of Health), Nobuyuki Sera (Fukuoka Institute of Health and Environmental Sciences), and Hiromi Yoshikawa (Kitakyushu City Institute of Environmental Sciences) Acknowledgments We thank Miho Ejima and Mariko Tokunaga for the production of several figures. This study was supported by Grants-in-Aid for Seisaku-Soyaku-Sougo (H18-Iyaku- Ippan 033), and Emerging and Reemerging Infectious Diseases (H20-Shinko-Ippan 005) from the Ministry of Health, Labour and Welfare of Japan.

onment), Chiharu Kawakami (Yokohama City Institute of Health), Mayumi Konno (Kyoto City Institute of Health and Environmental Sciences), Hiroya Komoda (Kagawa Prefectural Research Institute for Environmental Sciences and Public Health), Tae Taniwaki (Kochi Public Health and Sanitation Institute), Toshitaka Minegishi (Saitama Institute of Public Health), Rika Tsutsui (Aomori Prefectural Institute of Public Health and Environment), Shizuko Kasuo (Nagano Environmental Conservation Research Institute), Yuichiro Okamura (Nagano City Health Center), Eiji Horimoto (Toyama Institute of Health), Nobuyuki Sera (Fukuoka Institute of Health and Environmental Sciences), and Hiromi Yoshikawa (Kitakyushu City Institute of Environmental Sciences) Acknowledgments We thank Miho Ejima and Mariko Tokunaga for the production of several figures. This study was supported by Grants-in-Aid for Seisaku-Soyaku-Sougo (H18-Iyaku- Ippan 033), and Emerging and Reemerging Infectious Diseases (H20-Shinko-Ippan 005) from the Ministry of Health, Labour and Welfare of Japan. Dr Ujike is a virologist and the senior researcher of Influenza Virus Research Center of the National Institute of Infectious Diseases, Tokyo, Japan. His research interests are influenza virus and severe acute respiratory syndrome–coronavirus.