Flu Found Resistant to Main Antiviral Drug

[HenryN]

Re: Flu Found Resistant to Main Antiviral Drug

Commentary

http://www.recombinomics.com/News/03180901/H274Y_Predict_Evolution.html

[HenryN]

Re: Flu Found Resistant to Main Antiviral Drug

More new comments

http://precedings.nature.com/documents/2832/version/1

[miso]

Re: Flu Found Resistant to Main Antiviral Drug

Quote:

"CDC Update

http://www.fda.gov/ohrms/dockets/ac/09/briefing/2009-4416B1-1.pdf"


Check out the CDC Update. The antiviral resistance table comparing Tamiflu with Relenza. Spot the antiviral that took 15 years of research and had over 12 published studies exploring resistance before it was on the market, and the other one (with no independent resistance studies) that was rushed to market in seven years, to beat it.
With a little flag waving, and some helpful nudges, it became the "Main Antiviral Drug."

Let's imagine a scenario in which Tamiflu was developed with as much care (and time) as Relenza.

Firstly Tamiflu would not have survived resistance trials, they would have mirrored what we are seeing now on a larger scale, and it would have been abandoned. Even if greed, or the need for a pill, overcame the evidence, Relenza would still have been on the market for eight years before Tamiflu.

In eight years Relenza would have made money to reward GSK for the effort of methodically bringing the first NI antiviral to market. It would have encouraged GSK to continue the lines of development already started in 1999. They would have,
-improved the inhaler for the original Relenza product.
-finished clinical trials, and had iv Relenza on the market.
-trialled the first and arguably best second generation LANI candidates, care of Biota. The best one of these could have been on the market now.


HOWEVER,


the reality is, some corners were cut, some men got rich, and the world stockpiled snake oil against a potent influenza pandemic, instead.

[HenryN]

Re: Flu Found Resistant to Main Antiviral Drug

Quote:

"CDC Update

http://www.fda.gov/ohrms/dockets/ac/09/briefing/2009-4416B1-1.pdf"


Check out the CDC Update. The antiviral resistance table comparing Tamiflu with Relenza. Spot the antiviral that took 15 years of research and had over 12 published studies exploring resistance before it was on the market, and the other one (with no independent resistance studies) that was rushed to market in seven years, to beat it.
With a little flag waving, and some helpful nudges, it became the "Main Antiviral Drug."

Let's imagine a scenario in which Tamiflu was developed with as much care (and time) as Relenza.

Firstly Tamiflu would not have survived resistance trials, they would have mirrored what we are seeing now on a larger scale, and it would have been abandoned. Even if greed, or the need for a pill, overcame the evidence, Relenza would still have been on the market for eight years before Tamiflu.

In eight years Relenza would have made money to reward GSK for the effort of methodically bringing the first NI antiviral to market. It would have encouraged GSK to continue the lines of development already started in 1999. They would have,
-improved the inhaler for the original Relenza product.
-finished clinical trials, and had iv Relenza on the market.
-trialled the first and arguably best second generation LANI candidates, care of Biota. The best one of these could have been on the market now.


HOWEVER,


the reality is, some corners were cut, some men got rich, and the world stockpiled snake oil against a potent influenza pandemic, instead.

The H1N1 Tamiflu resitance was not linked to Tamiflu treatment of seasonal flu.

[AlaskaDenise]

Re: Flu Found Resistant to Main Antiviral Drug

Commentary

http://www.recombinomics.com/News/03...Evolution.html

Commentary

Predictable Evolution in Tamiflu Resistant H1N1

Recombinomics Commentary 14:18
March 18, 2009

Recently released H1N1 sequences at Genbank and GISAID include multiple receptor binding domain changes in emerging Tamiflu resistant H1N1. The predictability of these changes is quite remarkable. The hitch-hiking paper is being updated with the more recently released sequences from this season, which describes the importance of the receptor binding domain changes. Some of these changes are detailed in the HA tree (slide 14) from the CDC, but that tree is not fully annotated. The emerging H1N1 isolates all have A193T, but each of the 5 newly formed branches is rooted in addition receptor binding changes, and all are at three flanking positions (187, 189, 196). There are two polymorphisms at 187 (N187S and N187D) and 196 (H196R and H196N), as well as four at position 189 (G189A, G189N, G189S, G189V).

The top branch in the CDC tree is labeled with G189A (as well as S145N). These acquisitions are widespread in Asia (including most isolates in Japan, South Korea, and Taiwan) and are associated with vaccine resistance. All five of the first H1N1 isolates from Italy had these acquisitions, and three of the isolates were from patients who had been vaccinated this season. Similarly, Taiwan reported that 70% of the H1N1 isolates there were vaccine resistant. These changes are also in US isolates, but are fairly rare.

The second branch in the CDC tree is labeled with G189V and H196R. All isolates are from the US and this is the dominant strain in the US. However, this sub-clade is also in Japan and was linked to school closings in Sendai last fall, as described earlier.

The third branch in the CDC tree is labeled N187S, but there are actually two sub-clades in this branch. One was detailed in the hitch-hiking paper and has N187S and G189N. These isolates were in South Africa and Australia in the summer of 2008 and are in Kenya in the fall. There are a few isolates in the US. However, a more common sub-clade in the US has N187S and G189S.

The fourth branch in the CDC tree only has two US isolates, and is not annotated. However, this sub-clade is fairly widespread in the US and is also in Argentina. These isolates have N187D.

The fifth branch in the CDC tree has one isolate from China (Jilin), but there are now multiple isolates from Jilin, as well as isolates from South Australia, which fall on this branch and these isolates have H196N.

Thus, every major branch that has emerged this season has A193T (and hitch-hiking H274Y on NA) as well as at least one adjacent HA change, which is limited to three positions (187, 189, 196).

As described in the paper, the changes for the acquisitions can be found in other H1N1 isolates that are co-circulating with the emerging H1N1, providing additional support for acquisitions via recombination, and for predictions based on such circulating polymorphisms.

.

[miso]

Re: Flu Found Resistant to Main Antiviral Drug

Dr Niman,

With respect, I never said it was.

But everyone knows, when Tamiflu isn't being useless against a wild strain of influenza A with H274Y, it's generating it's own during treatment.
And not just H1N1 but H5N1 too.

Any further developments in Relenza resistance?

[Giuseppe]

Re: Flu Found Resistant to Main Antiviral Drug

The problem with H1N1 with H274Y mutation is that its spread can be tracked enough well to define a clear track of evolution, without any noticeable connection with antiviral drug oseltamivir usage during seasonal influenza epidemics.

So, the stability and fintness of H1N1 with this mutation could have been taken into account by national and supernational health agencies BEFORE this virus strain would become dominant, in order to update trivalent inactivated vaccines and treatment protocols for high risk population.

It isn't happened. So, for many patients - immunocomprised or critically ill with underlying conditions - very few alternatives have remained to protect from influenza H1N1 infections.

These way of tracking viruses could be useful although may not improve our ability to understand HOW this particular strain has overcome structural hurdles that previously impaired the stability.

Some researchers have also suggested to decode entire viral genome to explore other possible changes or acquisitions.

Nonetheless, I would express my admiration for this theory of ''elegant evolution'' suggested by dr Niman.

[Vibrant62]

Re: Flu Found Resistant to Main Antiviral Drug

For me the key question about the potential for Relenza resistance to develop is whether there are any studies that show a treated patient generates resitant virus over the course of treatment, (as does tamiflu on treatment, occasionally) even if the resulting variant is not a 'fit' virus for onwards transmission

[Giuseppe]

Re: Flu Found Resistant to Main Antiviral Drug

Zanamivir intermediate resistant viruses or with reduces sensitivity were surely isolated in the past, mainly H3N2 and B. I think in Scientific Library should be present some abstract, but I don't know for sure at the moment.

[Giuseppe]

Re: Flu Found Resistant to Main Antiviral Drug

Found!

Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibitors

Context
Very little is known about the frequency of generation and transmissibility of influenza B viruses with reduced sensitivity to neuraminidase inhibitors. Furthermore, transmission of resistant virus, whether influenza A or B, has not been recognized to date.

Objective
To assess the prevalence and transmissibility of influenza B viruses with reduced sensitivity to neuraminidase inhibitors.

Design, Setting, and Patients
Investigation of the neuraminidase inhibitor sensitivity of influenza B isolates from 74 children before and after oseltamivir therapy and from 348 untreated patients with influenza (including 66 adults) seen at 4 community hospitals in Japan during the 2004-2005 influenza season. Four hundred twentytwo viruses from untreated patients and 74 samples from patients after oseltamivir therapy were analyzed.

Main Outcome Measure
Sialidase inhibition assay was used to test the drug sensitivities of influenza B viruses. The neuraminidase and hemagglutinin genes of viruses
showing reduced sensitivity to neuraminidase inhibitors were sequenced to identify mutations that have the potential to confer reduced sensitivity to these drugs.

Results
In 1 (1.4%) of the 74 children who had received oseltamivir, we identified
a variant with reduced drug sensitivity possessing a Gly402Ser neuraminidase substitution.
We also identified variants with reduced sensitivity carrying an Asp198Asn,
Ile222Thr, or Ser250Gly mutation in 7 (1.7%) of the 422 viruses from untreated patients.
Review of the clinical and viral genetic information available on these 7 patients indicated that 4 were likely infected in a community setting, while the remaining 3 were probably infected through contact with siblings shedding the mutant viruses.

Conclusions
In this population, influenza B viruses with reduced sensitivity to neuraminidase inhibitors do not arise as frequently as resistant influenza A viruses. However, they appear to be transmitted within communities and families, requiring continued close monitoring.

JAMA. 2007;297:1435-1442
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[gsgs]

Re: Flu Found Resistant to Main Antiviral Drug

earliest identified possible ancestor of Tamiflu resistance
Europe 2007/8 so far is :

A/Victoria/07159220/2007/10/03(H1N1)

---------edit--------------

ahh, that is the Hawaii-lineage, not really what was in Europe.
so earliest is still: A/Sydney/142/2007/11/02(H1N1)

[Giuseppe]

Re: Flu Found Resistant to Main Antiviral Drug

ECDC. Monitoring of Influenza antiviral resistance in EU during 2008-09 season (March 18, 2009)

Monitoring of Influenza antiviral resistance in EU during 2008-09 season

[Original page at this LINK. EDITED.]

Monitoring of antiviral resistance in EU for the season 2008-09 is currently ongoing. The coordinators of The Community Network of Reference Laboratories for Human Influenza in Europe (CNRL), routinely collect, analyze and disseminate information on antiviral resistance from Influenza viruses isolated from 25 European (European Union, EEA/EFTA) countries. The analysis of resistance against Neuraminidase Inhibitors and Adamantanes is done by measuring IC50 values and/or by genotyping of viruses for detection of known drug resistance mutations. Summary information on antiviral resistance in EU will be published weekly in the EISS bulletin (also featured in the weekly ECDC influenza News)


<table style="width: auto;"><tbody><tr><td></td></tr><tr><td style="font-family: arial,sans-serif; font-size: 11px; text-align: right;">From TABLES</td></tr></tbody></table>

[Giuseppe]

Re: Flu Found Resistant to Main Antiviral Drug

CIDRAP >> Signs of drug resistance found in African H5N1 viruses

Signs of drug resistance found in African H5N1 viruses

Robert Roos * News Editor
Mar 19, 2009 (CIDRAP News) ?

Scientists who analyzed 67 H5N1 avian influenza viruses from across Africa report that the viruses fall into three distinct sublineages, or families, and that some have mutations that make them resistant to antiviral drugs.


The scientists also found that some of the African viruses have genetic markers that are characteristic of human flu viruses rather than avian strains, according to their report, published yesterday in the online journal PLoS One.

"These findings raise concern for the possible human health risk presented by viruses with these genetic properties and highlight the need for increased efforts to monitor the evolution of A/H5N1 viruses across the African continent," says the report by a large international team of scientists.

The group includes several from African countries and the UN Food and Agriculture Organization.

Lethal H5N1 viruses made their African debut on Nigerian poultry farms in January 2006, the report notes. Soon afterward the virus cropped up in Egypt, Niger, and Cameroon, and in April 2006 it was found in Sudan, Burkina Faso, Djibouti, and Ivory Coast. The virus surfaced in Ghana and Togo in mid-2007 and in Benin in December 2007.

All but two human cases of H5N1 disease in Africa have occurred in Egypt, whose official case count is 58, with 23 deaths. Nigeria and Djibouti have each had one human case.

Rapid spread of 3 sublineages
The scientists looked at 494 H5N1 gene sequences from 67 African isolates, including the complete hemagglutinin and neuraminidase gene segments, all collected between February 2006 and early 2008 and representing all 11 affected countries. The analysis also included hundreds of gene sequences from European and Middle Eastern H5N1 viruses.

The researchers determined that all the African viruses belong to clade 2.2 and are related to the H5N1 viruses that have been circulating throughout Europe, Russia, and the Middle East since late 2005. Clade 2.2 traces back to the outbreak of avian flu in thousands of migratory birds at China's Qinghai Lake in the spring of 2005, the article notes.

Detailed analysis of the hemagglutinin genes showed that the viruses fall into three sublineages (labeled I, II, and IV). All three groups "had been co-circulating since the beginning of the epidemic in Africa," suggesting that all three had been introduced into Africa separately, as reported in previous studies, the report says.Just how the three groups entered Africa and spread so rapidly is still unclear. But the viruses emerged in Africa when related strains were present in European migratory birds, "and such birds may have played a significant role in the introduction of the virus," the scientists write.

The three sublineages had geographic dimensions, but the patterns were complex. All the Egyptian isolates were in sublineage IV, which they shared with isolates from Gaza and Israel. Strains from Burkina Faso, Ivory Coast, Ghana, and Cameroon formed a single cluster in sublineage I. However, the authors found all three groups in Nigeria, a finding that agreed with an earlier study.

Viruses collected in Sudan were in sublineage I and closely related to those from Nigeria, Burkina Faso, and Ivory Coast, rather than to those from nearby Egypt and Djibouti. Overall, the findings "may suggest that a certain degree of geographical segregation has occurred in Africa" since the initial viral introductions, the report states.

Antiviral resistance, markers of human flu
In searching signs of antiviral resistance, the team found four bird isolates from Egypt carrying a mutation linked with resistance to the older class of flu drugs, the adamantanes (amantadine and rimantadine). They also found viruses from two human cases in Egypt that had a mutation (known as N294S) that confers resistance to oseltamivir (Tamiflu) and slightly reduced sensitivity to zanamivir (Relenza).

However, no mutations conferring resistance to oseltamivir or zanamivir were found in any of the African viruses from birds.

The authors also found a number of isolates with genetic markers usually found in human flu viruses rather than avian strains. In particular, they checked the African viruses for 13 genetic markers consistently found in the flu viruses that caused the pandemics of 1918, 1957, and 1968.

They found two, both in the PB2 gene. One of these, known as E627K and associated with increased H5N1 virulence in mice, was found in all the African isolates. Another was found in just two bird viruses from Egypt.

In other findings, the report says that two different reassortant viruses representing combinations of two of the three sublineages were found in Nigeria in 2006 and 2007. One of these became the predominant strain in Nigeria's poultry in 2007.

Evidence of international spread
"The continued circulation of A/H5N1 viruses in the African continent not only affects the local economy but also impacts on animal and human health," the report states. It concludes with a call for constant efforts to monitor and control avian flu across Africa.

David A. Halvorson, DVM, an avian flu expert at the University of Minnesota in St. Paul, said the report appears to confirm that there were three separate introductions of H5N1 into Africa and that those strains continue to circulate.

Further, he said the study shows that genetic sequencing "shows evidence for international spread within Africa as well as evidence for local spread; and that there have been no additional introductions since the first ones."

Halvorson also commented that the findings regarding antiviral resistance are not surprising: "These mutations are typical of viruses as they circulate in a host. It seems they can mutate to resistance without any antiviral compound present."

Cattoli G, Monne I, Fusaro A, et al. Highly pathogenic avian influenza subtype H5N1 in Africa: a comprehensive phylogenetic analysis and molecular characterization of isolates. PLoS One 2009 March;4(3)
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<cite cite="http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/news/mar1909africa-jw.html">CIDRAP >> Signs of drug resistance found in African H5N1 viruses</cite>

[Giuseppe]

Flu Found Resistant to Main Antiviral Drug

WHO. Influenza A(H1N1) virus resistance to oseltamivir - 2008/2009 influenza season, northern hemisphere (March 21, 2009)

Influenza A(H1N1) virus resistance to oseltamivir - 2008/2009 influenza season, northern hemisphere

18 March 2009

[Original PDF Document at this LINK. EDITED.]


During weeks 1-4 (28 December 08 ? 24 January 09), the level of overall influenza activity in the world increased. In Europe, most countries reported regional or widespread activity with influenzaA (H3) viruses predominating.


Widespread influenza A activity (H1 and H3) was reported in Japan. In Canada, Hong Kong SAR and the United States, influenza activity increased but remained relatively low. Sporadic influenza activity was observed in Brazil (A), Croatia (H1,H3, B), Greece (H1, H3, B), Iran (H1, H3), Mongolia (A), Portugal (H1, H3, B), Serbia (H1, H3, B), Singapore (H1, H3, B), Slovakia (H3) and Turkey (H3, B).

During this period, a total of 30 countries from all WHO regions reported oseltamivir resistance for 1291 of 1362 A(H1N1) viruses analysed. The prevalence of oseltamivir resistance was very high in the following countries/territory: Canada (52 of 52 tested), Hong Kong SAR (72 of 80), Japan (420 of 422), the Republic of Korea (268 of 269) and the United States of America (237 of 241).

The resistance prevalence was relatively low in China (6 of 44 tested). In Europe, H1N1 circulation was low during this period while the resistance prevalence was high: France (12 of 12tested), Germany (66 of 67), Ireland (9 of 10), Italy (16 of 16), Sweden (11 of 12) and the United Kingdom (61 of 62).

WHO is collecting global data about this phenomenon from multiple laboratories participating in Global Influenza Surveillance Network. Data from European countries participating in EISS were provided by the EISS and VirGil project. This summary table will be updated regularly (every four weeks).

Oseltamivir resistance results were based on phenotypic and/or genotypic analyses. A comprehensive table of influenza A(H1N1)virus resistance to oseltamivir (Fourth quarter 2008 - 31 January 2009) can be found on the following page.


<table style="width: auto;"><tbody><tr><td></td></tr><tr><td style="font-family: arial,sans-serif; font-size: 11px; text-align: right;">From TABLES</td></tr></tbody></table>

<table style="width: auto;"><tbody><tr><td></td></tr><tr><td style="font-family: arial,sans-serif; font-size: 11px; text-align: right;">From TABLES</td></tr></tbody></table>

<table style="width: auto;"><tbody><tr><td></td></tr><tr><td style="font-family: arial,sans-serif; font-size: 11px; text-align: right;">From TABLES</td></tr></tbody></table>

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[HenryN]

Re: Flu Found Resistant to Main Antiviral Drug

EID Journal Home > Volume 15, Number 4?April 2009
<!-- InstanceEndEditable --> <!-- content_starts_here //--> <!-- InstanceBeginEditable name="Article Content" --> Volume 15, Number 4?April 2009

Research

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

Adam Meijer, Angie Lackenby, Olav Hungnes, Bruno Lina, Sylvie van der Werf, Brunhilde Schweiger, Matthias Opp, John Paget, Jan van de Kassteele, Alan Hay, and Maria Zambon, on behalf of the European Influenza Surveillance Scheme¹
Author affiliations: Netherlands Institute for Health Services Research, Utrecht, the Netherlands (A. Meijer, J. Paget); National Institute for Public Health and the Environment, Bilthoven, the Netherlands (A. Meijer, J. van de Kassteele); European Surveillance Network for Vigilance against Viral Resistance (A. Lackenby, B. Lina, S. van der Werf, A. Hay, M. Zambon); Health Protection Agency, London, UK (A. Lackenby, M. Zambon); Norwegian Institute of Public Health, Oslo, Norway (O. Hungnes); Centre National de R?f?rence des Virus Influenza (R?gion Sud), Lyon, France (B. Lina); Centre National de R?f?rence des Virus Influenza (R?gion Nord), Paris, France (S. van der Werf); Robert Koch Institute, Berlin, Germany (B. Schweiger); Laboratoire National de Sant?, Luxembourg, Luxembourg (M. Opp); and World Health Organization Collaborating Centre Medical Research Council/National Institute of Medical Research, London (A. Hay)
Suggested citation for this article
Abstract
In Europe, the 2007?08 winter season was dominated by influenza virus A (H1N1) circulation through week 7, followed by influenza B virus from week 8 onward. Oseltamivir-resistant influenza viruses A (H1N1) (ORVs) with H275Y mutation in the neuraminidase emerged independently of drug use. By country, the proportion of ORVs ranged from 0% to 68%, with the highest proportion in Norway. The average weighted prevalence of ORVs across Europe increased gradually over time, from near 0 in week 40 of 2007 to 56% in week 19 of 2008 (mean 20%). Neuraminidase genes of ORVs possessing the H275Y substitution formed a homogeneous subgroup closely related to, but distinguishable from, those of oseltamivir-sensitive influenza viruses A (H1N1). Minor variants of ORVs emerged independently, indicating multiclonal ORVs. Overall, the clinical effect of ORVs in Europe, measured by influenza-like illness or acute respiratory infection, was unremarkable and consistent with normal seasonal activity.

<table align="right" border="0" cellpadding="5" cellspacing="0" width="150"> <tbody><tr> <td bgcolor="#d8eceb">

Figure 1

</td> </tr> <tr> <td bgcolor="#d8eceb"></td> </tr> <tr> <td bgcolor="#d8eceb">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...
</td> </tr> <tr> <td> </td> </tr> <tr> <td bgcolor="#d8eceb">

Figure 2

</td> </tr> <tr> <td bgcolor="#d8eceb"></td> </tr> <tr> <td bgcolor="#d8eceb">Figure 2. Total number of influenza virus detections, by type and subtype and by week, Europe, winter 2007?08.
</td> </tr> <tr> <td> </td> </tr> <tr> <td bgcolor="#d8eceb">

Figure 3

</td> </tr> <tr> <td bgcolor="#d8eceb"></td> </tr> <tr> <td bgcolor="#d8eceb">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.
</td> </tr> <tr> <td> </td> </tr> <tr> <td bgcolor="#d8eceb">

Figure 4

</td> </tr> <tr> <td bgcolor="#d8eceb"></td> </tr> <tr> <td bgcolor="#d8eceb">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...
</td> </tr> <tr> <td> </td> </tr> <tr> <td bgcolor="#d8eceb">

Figure 5

</td> </tr> <tr> <td bgcolor="#d8eceb"></td> </tr> <tr> <td bgcolor="#d8eceb">Figure 5. Weighted average prevalence of oseltamvir-resistant influenza viruses A (H1N1), Europe, winter 2007?08...
</td> </tr> <tr> <td> </td> </tr> <tr> <td bgcolor="#d8eceb">

Figure 6

</td> </tr> <tr> <td bgcolor="#d8eceb"></td> </tr> <tr> <td bgcolor="#d8eceb">Figure 6. Phylogenetic comparisons of the hemagglutinin (A) and neuraminidase (B) genes of influenza viruses A (H1N1)...
</td> </tr> <tr> <td> </td> </tr> <tr> <td bgcolor="#d8eceb">

Appendix Figure

</td> </tr> <tr> <td bgcolor="#d8eceb"></td> </tr> <tr> <td bgcolor="#d8eceb">Appendix Figure. Fitted curves to the proportion oseltamivir-resistant viruses among influenza viruses A (H1N1) tested for resistance...
</td> </tr> </tbody></table> 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).
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).
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.
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.
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.
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.
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 [ 211 KB, 10 pages]).
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.
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).
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.
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.
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 [ 211 KB, 10 pages]).
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.
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.
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.
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).
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.
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).
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.
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.
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 P?rez-Bre?a, 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.

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Figures

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...
Figure 2. Total number of influenza virus detections, by type and subtype and by week, Europe, winter 2007?08.
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...
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...
Figure 5. Weighted average prevalence of oseltamvir-resistant influenza viruses A (H1N1), Europe, winter 2007?08...
Figure 6. Phylogenetic comparisons of the hemagglutinin (A) and neuraminidase (B) genes of influenza viruses A (H1N1)...
Appendix Figure. Fitted curves to the proportion oseltamivir-resistant viruses among influenza viruses A (H1N1) tested for resistance...
Tables

Table. Peak incidence rates of ILI or ARI infection for countries for which data were available, Europe, 2000?01 through 2007?08 influenza seasons
Appendix Table. GenBank accession numbers of hemagglutinin and neuraminidase sequences used in the phylogenetic analyses
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
DOI: 10.3201/eid1504.081280

¹European 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. Csoh?n, 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. Falc?o, J.M. Falc?o, 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. Sl?cikov? (Slovakia); K. Prosenc, M. Socan (Slovenia); I. Casas, A. Larrrauri, S. de Mateo, R. Ortiz de Lejarazu, P. P?rez-Bre?a, T. Pumarola Su??, 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).