Oseltamivir-Resistant Influenza Virus A (H1N1), Europe, 2007–08 Season

A high level of virus circulation and introduction of an antigenic drift variant in a susceptible population contributed to the spread of resistant virus.

I nfection with infl uenza viruses A (H1N1), A (H3N2), or B causes substantial human illness and excess deaths each year (1,2). Vaccination against seasonal infl uenza 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 infl uenza. 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 infl uenza B have limited their usefulness (3). The introduction of neuraminidase inhibitors (NAIs), oral oseltamivir and inhaled zanamivir, which are active against both infl uenza type A and B viruses, was a major breakthrough in treatment and prophylaxis of infl uenza 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).
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)(6)(7)(8)(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 infl uenza virus A (H3N2) infection and 16% (7/43) of treated Japanese children with infl uenza virus A (H1N1) infection, also with no evidence that these viruses transmitted effi ciently (12,13).
In late January 2008, we reported an unexpected high level and unexpected spread of oseltamivir-resistant infl uenza 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 infl uenza viruses A (H1N1) were the predominant circulating viruses in European countries (Table).

Clinical Infl uenza Activity
The European Infl uenza Surveillance Scheme (EISS) actively monitored infl uenza 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 infl uenza-like illness (ILI) or acute respiratory infection (ARI) (15)(16)(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 Infl uenza Centers (NICs) for virus detection and characterization by using a variety of genetic or phenotypic methods (18)(19)(20). The NICs also analyzed specimens and infl uenza 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 (IC 50 ) of virus isolates was determined by using fl uorescent or chemiluminescent enzyme assays (23,24). ORVs were defi ned as infl uenza viruses A (H1N1) with an IC 50 >100 nmol/L for oseltamivir. Susceptibility to zanamivir was determined by using the same enzymatic method. Susceptibility to M2Is was determined by cyclesequencing 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.  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 infl uenza virus A or ORV detection was allowed in estimating the fi rst week of continuous detection. The average European delay between the fi rst week of continuous detection of infl uenza 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 ap-proach (26,27) was used, which allows modeling of binomial proportions, i.e., a numerator and a denominator as a function of time, where the coeffi cients 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 fi t will shrink to its overall mean, and uncertainties increase. Three fractions were modeled: "ILI per population covered," "infl uenza A virus detections per specimens tested," and "A (H1N1) resistant per A (H1N1) tested." By multiplying the fi rst 2 fractions by the total population, we obtained the number of patients with ILI who had infl uenza A in a country. By dividing this number by the sum of the number of patients with ILI who had infl uenza 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  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 IC 50 values for further analysis (7,29). For oseltamivir outlier identifi cation, all viruses defi ned as resistant for oseltamivir (IC 50 >100 nmol/L) were fi rst removed. Minor outliers were defi ned as values lying between the upper quartile (UQ) + 1.5 × interquartile region (IQR) and UQ + 3 × IQR; major outliers were defi ned as values lying above UQ + 3 × IQR, based on analysis of all viruses in a particular subtype over a particular winter season.
The fi rst countries in Europe where infl uenza viruses A started to circulate continuously were France, Spain, Switzerland, and the United Kingdom in week 40. Spatial analysis of the timing of the fi rst week of continuous detection of infl uenza 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% confi dence 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 R 2 = 0.32 for the linear regression fi t.
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 fi rst 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 fi rst 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 R 2 = 0.36 for the linear regression fi t. The average delay between the fi rst week of continuous detection of infl uenza 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 refl ected 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 (online Appendix Figure, 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-specifi c 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.

Discussion
Unexpectedly, infl uenza 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 IC 50 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, infl uenza viruses A (H3N2) and B circulating in Europe remained sensitive to NAI drugs.
This  (20). In the other 10 seasons, infl uenza viruses A (H1N1) played a minor role, with infl uenza 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 signifi cantly 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 infl uenza, 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 infl uenza viruses A (H5N1) and A (H1N1) after introduction of tyrosine at position 275 (33). Furthermore, other previously analyzed infl uenza 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 (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 fi nding of a high prevalence of ORVs in the community and the overall temporal increase in resistance demonstrates that the previously documented reduced fi tness of viruses bearing the H275Y mutation, ostensibly caused by structural and functional constraints (10), has been overcome in currently circulating infl uenza viruses A (H1N1). The results of Rameix-Welti et al. (32) suggest that a combination of specifi c amino acid substitutions have increased the affi nity of the NA of recent infl uenza 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 fi tness 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 fi tness of ORVs, for which whole genome sequencing is necessary.
For Europe, no focal point of initiation of spread could be identifi ed. The spread of ORV from west to east paral-  lated 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 infl uenza 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 infl uenza viruses A (H1N1) in Europe; the introduction of a new antigenic drift variant in a susceptible population, partly related to the lack of substantial infl uenza 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 infl uenza 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 fi ght against seasonal and pandemic infl uenza.