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.

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.

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.

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.

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).

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.

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).

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.

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.

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%).

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).

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).

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.