Global Influenza Surveillance Network Research Articles

65years of influenza surveillance by a World Health Organization-coordinated global network.

The 1918 devastating influenza pandemic left a lasting impact on influenza experts and the public, and the importance of global influenza surveillance was soon recognized. The World Health Organization (WHO) Global Influenza Surveillance Network (GISN) was founded in 1952 and renamed to Global Influenza Surveillance and Response System in 2011 upon the adoption by the World Health Assembly, of the Pandemic Influenza Preparedness Framework for the Sharing of Influenza Viruses and Access to Vaccines and Other Benefits (“PIP Framework”). The importance of influenza surveillance had been recognized and promoted by experts prior to the years leading up to the establishment of WHO. In the 65 years of its existence, the Network has grown to comprise 143 National Influenza Centers recognized by WHO, 6 WHO Collaborating Centers, 4 Essential Regulatory Laboratories, and 13 H5 Reference Laboratories. The Network has proven its excellence throughout these 65 years, providing detailed information on circulating seasonal influenza viruses, as well as immediate response to the influenza pandemics in 1957, 1968, and 2009, and to threats caused by animal influenza viruses and by zoonotic transmission of coronaviruses. For its central role in global public health, the Network has been highly recognized by its many partners and by international bodies. Several generations of world‐renowned influenza scientists have brought the Network to where it is now and they will take it forward to the future, as influenza will remain a preeminent threat to humans and to animals.

MATERIAL TRANSFER AGREEMENT SEBAGAI SARANA PERLINDUNGAN HUKUM PARA PIHAK DALAM PERJANJIAN PENGALIHAN MATERI BIOLOGI

Biological material is largely a free component, but the development of these materials can be objects that have commercial high value. Thus, the biological material is also associated with the ownership and control should be regulated by the misuse of biological materials hukum. According to the characteristics of MTA as a civil agreement, the parties will be decisive in the formulation of the agreement, but the characteristics of biological materials, including viruses, which can endanger the safety and threaten public health requires the use of surveillance capabilities sampled by the competent authority. Thus, the protection of the right to material biologidan implementation monitoring system redirects biological materials need to be studied in detail for the purpose of sharing microbiological material for the benefit of mankind could reached . Indonesia's cases refusal to carry through the mechanism of virus sharing GISN (Global Influenza Surveillance Network) in the system of the World Health Organization (wHO) in 2007, the result of a lack of regulations concerning the transfer of biological material not truly perfect

History and current trends in influenza virus infections with special reference to Sri Lanka.

The World Health Organization (WHO) estimates that approximately one billion people are infected and up to 500,000 people die from influenza each year in the world. Influenza is considered to be the greatest killer of the human populations, due to the 1918 Spanish flu, which killed millions around the world. Despite the effective treatment available against influenza, it still contributes to significant morbidity and mortality. Currently circulating influenza strains in humans include influenza A (H1N1)pdm09, influenza A (H3N2) and influenza B viruses, (B/Victoria and B/Yamagata). Influenza has been prevalent in Sri Lanka from 1969, since then it continued to cause morbidity and mortality in children and adults. The current global influenza surveillance network monitors the global influenza activity through WHO collaborating centres. The Medical Research Institute monitors and diagnoses influenza cases in the country as part of the WHO network laboratories. Vaccinations to high risk groups and antiviral therapy for the successful prevention of influenza have been practiced in Sri Lanka. This review highlights the impact of influenza on public health in Sri Lanka including the historical aspects, current diagnostic practices and prevention approaches in high risk individuals in the country.

Global influenza surveillance with Laplacian multidimensional scaling

The Global Influenza Surveillance Network is crucial for monitoring epidemic risk in participating countries. However, at present, the network has notable gaps in the developing world, principally in Africa and Asia where laboratory capabilities are limited. Moreover, for the last few years, various influenza viruses have been continuously emerging in the resource-limited countries, making these surveillance gaps a more imminent challenge. We present a spatial-transmission model to estimate epidemic risks in the countries where only partial or even no surveillance data are available. Motivated by the observation that countries in the same influenza transmission zone divided by the World Health Organization had similar transmission patterns, we propose to estimate the influenza epidemic risk of an unmonitored country by incorporating the surveillance data reported by countries of the same transmission zone. Experiments show that the risk estimates are highly correlated with the actual influenza morbidity trends for African and Asian countries. The proposed method may provide the much-needed capability to detect, assess, and notify potential influenza epidemics to the developing world.

Insights into surveillance from the influenza virus and benefit sharing controversy

For more than fifty years, the World Health Organization has helped monitor flu viruses and manufacture vaccines through the Global Influenza Surveillance Network (GISN). However, GISN became a flashpoint for conflict when Indonesia refused to share its samples of avian influenza until drugs and other benefits were shared in return. Years of controversial negotiations were required to agree on a framework for virus and benefit sharing, providing important insights into disease surveillance, global norms, and international law. First, this controversy suggests that surveillance behaves like a luxury good, which complicates the conventional wisdom that it is a global public good. Second, even well-established norms were not immune to challenge, thereby limiting the significance of ‘tipping points’ and ‘normative cascades’. Finally, legal arguments were common in this controversy but equivocal and inconclusive, so international law is unlikely to affect important outcomes relating to outbreak response.

Detection of Oseltamivir-Resistant Influenza A (H1N1) Viruses with H274Y Mutation during the 2007 - 2008 Influenza Season in Turkey

Detection of Oseltamivir-Resistant Influenza A (H1N1) Viruses with H274Y Mutation during the 2007 - 2008 Influenza Season in Turkey

The 2011 Pandemic Influenza Preparedness Framework: Global Health Secured or a Missed Opportunity?

In early 2007 the Indonesian government announced that it would cease sharing H5N1 influenza virus samples with the World Health Organization's Global Influenza Surveillance Network. At the heart of the government's complaint was the fact that samples were being passed by the WHO to pharmaceutical companies which developed, and patented, influenza vaccines that the Indonesian authorities could not purchase. The decision gained widespread support among advocates of greater equity of access to medicines, and in response the WHO established an intergovernmental process to agree a framework for influenza virus sharing. The process officially concluded in April 2011 and a new Pandemic Influenza Preparedness Framework (PIPF) was agreed at the 64th World Health Assembly in May 2011. This article investigates the events that prompted the re-examination of a technical cooperation system that has provided effective global health security on influenza for 60 years, and evaluates the framework that has now been agreed. Drawing the distinction between functional and moral-political benefits, the article argues that PIPF more accurately represents a diplomatic stand-off – one that has now been effectively sidelined with the passage of the agreement – rather than genuine reform. In fact, the PIPF papers over fundamental disagreements regarding authority in global health governance, the relationship between the WHO and governments, and the role of private industry. The article concludes by examining an alternative mechanism that would arguably better address the inherent tensions between national and collective interests, and accomplish the functional and moral-political benefits that the negotiations set out to achieve.

Re: Nicoll A, Sprenger M. Learning lessons from the 2009 pandemic: putting infections in their proper place. Eur J Epidemiol 2011; 26:191–194

The article by Nicoll et al. [1] claims that learning lessons from the 2009 swine-flu pandemic means placing infectious diseases in proper perspective. We do not find a single proof for that. Moreover, the article misinterprets our paper [2]. The authors [1] agree that ‘‘sound infectious disease epidemiology must be applied to the surveillance of influenza epidemics’’ and that ‘‘data currently provided by the Global Influenza Surveillance Network are insufficient.’’ However, they do not respond to the cited work by Jefferson [3] that ‘‘the data on the seasonal influenza are similarly weak and the estimates of disease frequency, mortality, and case fatality are vague. Consequently, the effectiveness of seasonal influenza vaccination campaigns and of antiviral medications is more than questionable [3]’’. WHO’s changing of the definition or description of a pandemic in May 2009, removing the phrase ‘‘enormous numbers of deaths and illnesses’’ from WHO’s website, is not a myth [1] but has been documented [4]. To imply the invention of a new myth by proposing that the improvement of social conditions prevents epidemics of non-communicable (NCD) and communicable diseases (CD) alike, is remarkable, but contradicts the literature [5, 6, 7]. Tuberculosis is an example; although no effective drug or vaccination against tuberculosis was available in the nineteenth and first half of the twentieth century, this major scourge declined dramatically with the improvement of social conditions. Life expectancy ‘‘in the record holding countries’’ increased linearly from 1840 to 2000 by 40 years, i.e. 2.5 years per decade [8]. The life expectancy increase of 20 years from 1840 to 1920 cannot be linked to the invention of effective drugs or vaccinations but to the improvements in social conditions such as income, housing, working conditions, nutrition, education and sanitation. Nowhere did we criticise ‘‘the modern public health approach to tuberculosis control such as case finding and ensuring completion of proper antimicrobial treatment’’; nowhere did we question the present strategies of public health bodies to combat tuberculosis or vaccinations against diseases such as measles, mumps, whooping cough, etc. The attempt to label us vaccination opponents is unfounded. On the other hand, side effects of vaccinations must be monitored and investigated: Why do the authors not mention two epidemiological studies from Sweden and Finland indicating a fourto nine-fold increased risk of narcolepsy in children vaccinated with adjuvant containing Pandemrix as compared to unvaccinated children [9]. Deaths associated with Pandemrix vaccination have been reported from Sweden [10]. The willingness of the public to accept influenza vaccination depends primarily on the credibility, quality, and independence of advising institutions [11, 12]. Fear-mongering such as performed by industry-dependent marketing U. Keil (&) Institute of Epidemiology and Social Medicine, University of Munster, Domagkstrasse 3, 48149 Munster, Germany e-mail: keilu@uni-muenster.de

Challenges of global surveillance during an influenza pandemic

SummarySurveillance is an essential foundation for monitoring and evaluating any disease process, and is especially critical when new disease agents appear. The H1N1 influenza pandemic of 2009 tested the capacities of countries to detect, assess, notify and report events as required by the 2005 International Health Regulations (IHR). As detailed in the IHR, the World Health Organization drew on official reports from Member States as well as unofficial sources (e.g. media alerts) to quickly report and disseminate information about the appearance of the novel influenza virus. The pre-existing Global Influenza Surveillance Network for virological surveillance also provided crucial information for rapid development of a vaccine and for detection of changes in the virus. However, the pandemic also highlighted a number of shortcomings in global epidemiological surveillance for respiratory disease. These included the lack of standards for reporting illness, risk factor and mortality data, and a mechanism for systematic reporting of epidemiological data. Such measures would have facilitated direct comparison of data between countries and improved timely understanding of the characteristics and impact of the pandemic. This paper describes the surveillance strategies in place before the pandemic and the methods that were used at global level to monitor the pandemic. Enhancements of global surveillance are proposed to improve preparedness and response for similar events in the future.

Indonesia’s Refusal to Share Influenza Virus Specimens with the World: Reviving the Arguments for Justice in Influenza Pandemic Preparedness

Indonesia’s December 2006 decision to stop sending influenza virus specimens to the World Health Organization’s Global Influenza Surveillance Network (GISN) captured international attention. At the time, the H5N1 subtype of influenza A virus was predicted to be the basis for the next pandemic. While many accused Indonesia - the country most afflicted by the virus - of putting the rest of the world in peril by withholding virus samples, Indonesia maintained that GISN was unjust for failing to ensure equitable access to vaccines developed using those samples. The H5N1 pandemic threat eventually waned, yet international negotiations to create a just framework for “sharing influenza viruses and other benefits” are ongoing.In this paper, we critically evaluate Indonesia’s claims about the unjustness of GISN. We argue in favour of Indonesia’s position and conclude that GISN must be significantly altered so that it properly recognizes the values of ownership, contribution, reciprocity, and human rights.

Symposium on viral respiratory disease surveillance

Recognition of the need for respiratory disease surveillance for informing prevention strategies dates back to the late 1940’s. Sir Christopher Andrews realized that the development of a successful influenza vaccination programme would be hindered by the unpredictable nature of the influenza virus and proposed the formation of a world influenza centre and a network of linked national laboratories which tracked influenza epidemics, to the World Health Organization (WHO). This network, now the WHO Global Influenza Surveillance Network (GISN) remains in place today and, although it’s primary goal is still to inform seasonal influenza vaccine strain selection, it has had to widen its activities to address emerging public health needs. There has been a renewed focus on understanding the overall burden of disease caused by influenza, especially in countries with no seasonal influenza prevention programmes, as a strategy to increase influenza awareness. While the zoonotic and pandemic threats from the highly pathogenic avian influenza (HPAI) H5N1 and H7N7 viruses and the realization that pandemic influenza arises from avian influenza viruses have stimulated global and national pandemic preparedness activities. Nevertheless, improved surveillance systems for both animal (avian and pig) and human influenza viruses are needed for informing and refining prevention strategies. Although the public health focus globally has been on influenza, the burden of disease caused by other respiratory viruses is also substantial and is largely under-recognised. A range of respiratory viruses are implicated in pneumonia, a disease recognised as a major killer of young children in developing countries and of the elderly in developed countries. There is also evidence that some respiratory viruses may be evolving allowing virulent variants to cause outbreaks in certain at-risk populations presenting another possible pandemic risk, highlighting the need to monitor unusual outbreaks of respiratory disease and the importance of the surveillance of respiratory viruses in general. Overall, prevention strategies for respiratory viruses other than influenza are less well defined, undoubtedly contributing to their perceived lower public health priority. Major advances in the diagnosis of respiratory virus infections have been made through the wider use of molecular diagnostic techniques which are now enabling rapid and sensitive respiratory pathogen surveillance. It is now possible to identify a wide range of respiratory pathogens in a higher proportion of respiratory infections, along with multiple pathogen infections. The application of these molecular techniques has also lead to the discovery of a number of new respiratory viruses; the clinical importance of the human metapneumovirus (hMPV) is now well established, while with others such as the human bocavirus (HBoV) and human rhinovirus type C (HRV-C), their clinical correlates are only just being elucidated. The knowledge base on these viruses urgently needs expanding through well designed controlled studies and other activities, including widened surveillance. The future challenge is to build on what has been achieved for the global surveillance of influenza by establishing standardized surveillance protocols which incorporate a widening range of respiratory viruses. The purpose being to inform and refine respiratory disease prevention strategies. These strategies must then be communicated to governments and public health messages developed for the education of the general public. DOI:10.1111/j.1750-2659.2010.00140.x www.influenzajournal.com

Influenza and respiratory disease surveillance: the US military’s global laboratory‐based network

Please cite this paper as: Jeremy Sueker et al. (2010) Influenza and respiratory disease surveillance: the US military’s global laboratory‐based network. Influenza and Other Respiratory Viruses 4(3), 155–161.The US Department of Defense influenza surveillance system now spans nearly 500 sites in 75 countries, including active duty US military and dependent populations as well as host‐country civilian and military personnel. This system represents a major part of the US Government’s contributions to the World Health Organization’s Global Influenza Surveillance Network and addresses Presidential Directive NSTC‐7 to expand global surveillance, training, research and response to emerging infectious disease threats. Since 2006, the system has expanded significantly in response to rising pandemic influenza concerns. The expanded system has played a critical role in the detection and monitoring of ongoing H5N1 outbreaks worldwide as well as in the initial detection of, and response to, the current (H1N1) 2009 influenza pandemic. This article describes the system, details its contributions and the critical gaps that it is filling, and discusses future plans.

The role of laboratory diagnosis of influenza

Since 1948 the World Health Organization (WHO) has established an international network of laboratories for the surveillance of influenza viruses. Currently, the Global Influenza Surveillance Network (GISN) includes 128 National Influenza Centers (NICs) distributed in 89 countries. Among their attributions the NICs are responsible for collecting and receiving specimens and virus isolates from patients suspected of being infected with influenza viruses and conducting preliminary laboratory analysis. Representative virus isolates are then selected and shipped to one of four specialized WHO Collaborating Centers (WHOCCs) for reference purposes and for advanced antigenic and genetic influenza analysis. Based on the results of this, the WHO makes an annual recommendation on influenza vaccine composition. NICs also alert the WHO to unusual outbreaks of influenza or influenza-like illness, and they detect non-subtypable and low-reacting virus isolates using the WHO diagnostic reagents provided through the GISN. Under the agreed terms of reference (www.who.int/csr/disease/ influenza/TORNICs.pdf), NICs must disseminate the generated data in FluNet (www.who.int/flunet), a webbased tool for the support and coordination of national and global influenza surveillance and reporting.

Use of Antigenic Cartography in Vaccine Seed Strain Selection

Human influenza A viruses are classic examples of antigenically variable pathogens that have a seemingly endless capacity to evade the host's immune response. The viral hemagglutinin (HA) and neuraminidase (NA) proteins are the main targets of our antibody response to combat infections. HA and NA continuously change to escape from humoral immunity, a process known as antigenic drift. As a result of antigenic drift, the human influenza vaccine is updated frequently. The World Health Organization (WHO) coordinates a global influenza surveillance network that, by the hemagglutination inhibition (HI) assay, routinely characterizes the antigenic properties of circulating strains in order to select new seed viruses for such vaccine updates. To facilitate a quantitative interpretation and easy visualization of HI data, a new computational technique called "antigenic cartography" was developed. Since its development, antigenic cartography has been applied routinely to assist the WHO with influenza surveillance activities. Until recently, antigenic variation was not considered a serious issue with influenza vaccines for poultry. However, because of the diversification of the Asian H5N1 lineage since 1996 into multiple genetic clades and subclades, and because of the long-term use of poultry vaccines against H5 in some parts of the world, this issue needs to be re-addressed. The antigenic properties of panels of avian H5N1 viruses were characterized by HI assay, using mammalian or avian antisera, and analyzed using antigenic cartography methods. These analyses revealed antigenic differences between circulating H5N1 viruses and the H5 viruses used in poultry vaccines. Considerable antigenic variation was also observed within and between H5N1 clades. These observations have important implications for the efficacy and long-term use of poultry vaccines.

Epidemiological, antigenic and genetic characteristics of seasonal influenza A(H1N1), A(H3N2) and B influenza viruses: Basis for the WHO recommendation on the composition of influenza vaccines for use in the 2009–2010 Northern Hemisphere season

Influenza vaccines form an important component of the global response against infections and subsequent illness caused in man by influenza viruses. Twice a year, in February and September, the World Health Organisation through its Global Influenza Surveillance Network (GISN), recommends appropriate influenza viruses to be included in the seasonal influenza vaccine for the upcoming Northern and Southern Hemisphere winters. This recommendation is based on the latest data generated from many sources and the availability of viruses that are suitable for vaccine manufacture. This article gives a summary of the data and background to the recommendations for the 2009–2010 Northern Hemisphere influenza vaccine formulation.

Vaccines against Influenza A (H1N1) Pandemic

The World Health Organization (WHO) has reported, as of September 2009, that the influenza A (H1N1) influenza pandemic has originated >300,000 laboratory-confirmed cases and 3917 deaths in 191 countries. It is recognized that pandemic vaccines have their greatest impact as a preventive strategy when administered before or near the peak incidence of cases in an outbreak. Therefore, vaccination campaigns should be in place when influenza A (H1N1) 2009 vaccines are available. We undertook this study to provide updated information on clinical evaluation of influenza A (H1N1) vaccines and review recommendations for influenza A (H1N1) vaccination campaigns and public health policy. The following methods were used: 1) review of registry at ClinicalTrials.gov. 2) search of PubMed Central (PMC) for influenza A (H1N1) vaccine. 3) review of recommendations of WHO, Mexican Health Secretariat (SSA) and Advisory Committee on Immunization Practices (ACIP) on influenza A (H1N1) vaccination campaigns. Until October 1, 2009 there were 11 available influenza A (H1N1) candidate strains provided by WHO Global Influenza Surveillance Network. ClinicalTrials.gov registers 45 phase I and II clinical trials evaluating immunogenicity and safety of influenza A (H1N1) vaccines. Preliminary results support administration of a single dose and use of adjuvants. Main recommendations of WHO, SSA and ACIP include epidemiologic considerations, objectives, definition of target groups and reinforcement of other mitigation measures. The present pandemic of influenza A (H1N1) has shown mild to moderate severity. Vaccination strategies in Mexico will have the objective of decreasing severe outcomes, slowing transmission, protecting groups at increased risk of infection, complications, or death, and preventing overload of health services. Control of the pandemic should include reinforcement of other non-pharmacologic measures of mitigation and, importantly, an adequate strategy of social communication.

Diagnostic Assay Recommended by the World Health Organization for Swine Origin Influenza A (H1N1) Virus Cross-Reacts with H5N1 Influenza Virus

Pandemic influenza poses a significant risk to global human health. The unpredictable nature of the influenza virus necessitates accurate, reliable, and rapid diagnostic assays for the detection of, and characterization of the current pandemic caused by, swine origin influenza virus (SOIV) in order to better understand the nature of the pandemic and implement appropriate health interventions. SOIV emerged in mid-April 2009 (1), leaving laboratories around the world scrambling to establish a diagnostic test to detect this novel influenza virus. The World Health Organization (WHO) responded with remarkable speed by releasing guidelines and protocols for a real-time reverse transcription (RT)-PCR assay 15 days after the reported identification of SOIV (2). This provided a rapid and sensitive assay for the detection of SOIV that was critical for the determination of the spread and extent of SOIV infections. These guidelines recommend three primer-and-probe sets: InfA, amplifying a conserved region of the matrix gene from all influenza A viruses; SW H1, designed to specifically detect the hemagglutinin gene segment (subtype H1) from SOIV; and SW InfA, designed to specifically detect the nucleoprotein (NP) gene segment from all swine influenza viruses. As part of the WHO Global Influenza Surveillance Network, the New Zealand National Influenza Centre regularly participates in WHO external quality assurance panels of 10 RNA samples that are H1N1, H3N2, or H5N1 influenza A virus or, recently, SOIV. In following the WHO recommended guidelines exactly, it was found that the SW InfA primer-and-probe set amplified all six H5N1 RNA samples. This is clinically relevant, as the proficiency panel RNA returned threshold cycle (CT) values (InfA assay, 23.44 to 28.67) well within the range of what we regularly observe with clinical samples. In addition, the SW InfA primer-and-probe set detected two additional H5N1 viruses not provided in the proficiency panel, the human H5N1 strain A/Vietnam/3028/2004 and an avian H5N1 strain, A/Mallard/New Zealand/272-73/2008. The efficiency of this reaction was 79% (three replicates) compared to 94% (five replicates) for the amplification of SOIV RNA, suggesting that mismatches within the primer target region reduced assay efficiency. To prove that the H5N1 NP gene sequence was amplified by the SOIV SW InfA assay, all real-time RT-PCR amplicons were sequenced. The two SOIV RNA samples from the proficiency panel and SOIV-positive control showed 100% identity to the consensus sequence of 394 NP full-length gene segments available as of 29 July 2009. In contrast, all H5N1 RNA samples from the proficiency panel contained at least 31 nucleotide differences from the SOIV consensus sequence but showed between 93.3% and 96.8% identity to A/Vietnam/1203/2004. Alignment of all SOIV and human H5N1 NP gene segments available in GenBank showed that 391 SOIV viruses have one mismatch in the SW InfA forward primer (Table ​(Table1).1). The remaining three SOIV sequences have a further mismatch in either the forward primer or the probe. The majority (74.6%, or 91 sequences) of available human H5N1 sequences are identical within the SW InfA primer-and-probe regions, having two mismatches in the forward primer, two in the probe, and four in the reverse primer region. None of the mismatches occurred within 9 nucleotides of the 3′ end, an important determinant for primer specificity. TABLE 1. Comparison of SOIV and human H5N1 NP gene sequences with SW infA primer and probe sequences The speed at which these assays were developed and deployed to the WHO Global Influenza Surveillance Network was remarkable, particularly with the limited SOIV sequence information available at the time of development. Other factors which constrained the development of RT-PCR primer targets include the origin and genetic rate of change for each gene segment; only five of the eight gene segments are of swine origin (3), while the rapidly evolving HA and NA genes have a propensity to drift at a much higher rate than conserved segments such as NP (5). This report demonstrates that the SW InfA assay is not specific to SOIV and is able to detect both human and avian (H5N1) influenza A viruses and so there is the potential for misidentification. Following WHO guidelines, the utilization of all primer-and-probe sets for the detection of SOIV should provide an accurate diagnosis. However, we have observed that for the same SOIV RNA sample, the SW InfA assay consistently returns lower CT values than the SW H1 assay and so clinical samples with higher CT values can yield an SW H1-negative, SW InfA-positive result. In these cases, we suggest sequencing of real-time SW InfA PCR amplicons to ensure the amplification of SOIV (4). Alternatively, and especially for countries in which H5N1 is endemic, an H5N1-specific assay could be performed to exclude this diagnosis. However, a negative H5N1 result would not exclude other influenza virus subtypes that may cross-react with the SOIV assay.

Rapid Method To Support Diagnosis of Swine Origin Influenza Virus Infection by Sequencing of Real-Time PCR Amplicons from Diagnostic Assays

Since the reported emergence of swine origin influenza virus (S-OIV) A/H1N1 in April 2009, 39 countries have reported 8,480 confirmed cases (18 May 2009), and the virus appears to have the potential to cause the next influenza pandemic (2). Despite the pandemic preparedness plans in place for public health laboratories around the world and the wide-spread diagnostic capability for influenza A/H5N1 (4), there was an interim period between the emergence of the virus and the development and distribution of a rapid diagnostic test specific for S-OIV. During this interim period, a “probable” diagnosis of S-OIV infection in humans was achieved by using the CDC (Centers for Disease Control and Prevention, Atlanta, GA) 2007 real-time PCR assay for human seasonal influenza (5). Nonspecific detection of S-OIV was made with a primer/probe set that targets a highly conserved region of the matrix gene of seasonal influenza A strains. An additional primer/probe set designed to subtype H1 and H3 seasonal influenza strains was unable to detect S-OIV. Therefore, a probable diagnosis of S-OIV was based upon the detection of an influenza A virus that could not be subtyped as H1 or H3. To add confidence to this test result, we have determined that sequencing of the real-time PCR matrix gene amplicon can distinguish between human seasonal A/H1N1 and S-OIV. The CDC 2007 real-time PCR protocol for human seasonal influenza viruses is in common usage throughout the WHO Global Influenza Surveillance Network and other public health laboratories. Testing within our laboratory confirmed the detection of S-OIV from clinical samples using the seasonal matrix gene primers and probe and also confirmed that the H1 and H3 human seasonal influenza primer/probe sets do not detect S-OIV. Matrix gene real-time RT-PCR products from S-OIV and human seasonal H1N1 clinical samples were purified using a QIAquick PCR purification kit (Qiagen), including the addition of 10 μl 3 M NaCH3COO to ensure a correct pH for binding to the filter column. The purified PCR product was subjected to direct sequencing by the Sanger method on an ABI3130 sequencer, using the same forward and reverse primers described in the CDC real-time RT-PCR protocol. Informative sequence reads were consistently obtained despite the short length of the template (106 bp). An alignment of this sequenced region with all recent GenBank entries (accessed 18 May 2009) for the influenza A matrix gene (2004 to 2009) was constructed and showed a high degree of nucleotide sequence conservation within this small region both for S-OIV and for human seasonal A/H1N1 viruses. One nucleotide in the matrix gene PCR product was identified as being variable and largely discriminatory for S-OIV in comparison to human seasonal influenza A/H1N1 viruses, that being G at nucleotide (nt) 174 for seasonal H1N1 but A for S-OIV (174 G>A; Table ​Table1).1). A total of 57 S-OIV sequences were available, all having an adenine at position 174. From the remainder of the available nonredundant matrix gene sequences for influenza A viruses (all subtypes, n = 2,957), only an additional 88 deposited sequences had nt 174 G>A, and only 3 of these were A/H1N1 viruses isolated from humans (Table ​(Table1).1). Two, A/Thailand/271/2005 and A/Aragon/RR32182008, were known zoonotic transmissions of a swine virus to humans (1, 3), and only one human seasonal A/H1N1 virus showed exact sequence identity to S-OIV across the entire sequenced region of 60 bp, that being A/Tennesse/UR06-0236/2007 (Table ​(Table1)1) (GenBank accession no. {type:entrez-nucleotide,attrs:{text:CY031141,term_id:183396093,term_text:CY031141}}CY031141). The A/Tennesse/UR06-0236/2007 virus would provide a positive H1 test result for the CDC 2007 real-time RT-PCR and would thus have been excluded from identification as S-OIV. It can therefore be concluded that, for influenza A/H1N1 viruses isolated from humans, the nt 174 G>A sequence variant in the matrix gene is informative for S-OIV diagnosis. TABLE 1. Comparison of the discriminatory region in the real-time RT-PCR product of the matrix gene, including all (nonredundant) influenza A/H1N1 viruses isolated from humans between 2004 and 2009 We do not recommend that this method replace the current confirmatory testing prescribed by WHO, which requires either specific S-OIV real-time PCR, viral culture, full-length influenza gene sequencing, or a fourfold rise in S-OIV antigen-neutralizing antibodies (5). We propose that this approach could be used as a provisional diagnostic test by laboratories without access to specific S-OIV testing. We also note that this method provides a rapid validation of real-time PCR assay results, especially in the instance of unsubtypable influenza A viruses isolated from humans. If new influenza A subtypes or sequence variants were to arise in the future, sequencing of real-time PCR matrix gene products could provide an interim diagnostic test before a specific real-time PCR assay is developed.

ATIVS: analytical tool for influenza virus surveillance

The WHO Global Influenza Surveillance Network has routinely performed genetic and antigenic analyses of human influenza viruses to monitor influenza activity. Although these analyses provide supporting data for the selection of vaccine strains, it seems desirable to have user-friendly tools to visualize the antigenic evolution of influenza viruses for the purpose of surveillance. To meet this need, we have developed a web server, ATIVS (Analytical Tool for Influenza Virus Surveillance), for analyzing serological data of all influenza viruses and hemagglutinin sequence data of human influenza A/H3N2 viruses so as to generate antigenic maps for influenza surveillance and vaccine strain selection. Functionalities are described and examples are provided to illustrate its usefulness and performance. The ATIVS web server is available at http://influenza.nhri.org.tw/ATIVS/.

Widespread Oseltamivir Resistance in Influenza A Viruses (H1N1), South Africa

To the Editor: Oseltamivir is the most widely used antiviral drug for influenza; it is a potent inhibitor of influenza virus neuraminidase (NA) protein (1). Until recently, oseltamivir resistance occurred in <1% of circulating viruses globally. An increased number of influenza A viruses (H1N1) with resistance to oseltamivir was first reported to the World Health Organization (WHO) by Norway in late January 2008. The viruses carried a specific histidine-to-tyrosine mutation at position 274 (H274Y; H275Y in N1 numbering system) in the NA protein that confers high-level resistance to oseltamivir (2). Further surveillance by the European Surveillance Network for Vigilance against Viral Resistance and the WHO Global Influenza Surveillance Network (GISN) showed that 16% of community isolates (0%–67% by country) of influenza A viruses (H1N1) circulating in the 2007–08 season in several other countries were also oseltamivir resistant (3). The predominant influenza subtype circulating in South Africa this winter season is H1N1. To determine whether oseltamivir-resistant viruses have spread to South Africa, we examined influenza A (H1N1) isolated during the 2008 winter season for resistance to this antiviral compound. Specimens were obtained mainly from the National Institute of Communicable Diseases (NICD) active sentinel surveillance program in all 9 provinces. Throat or nasopharyngeal swabs were taken from patients within 48–72 hours of onset of symptoms and sent to NICD laboratories for virus isolation as described (4). Of the H1N1 subtype viruses isolated in May and June, 23 were sent to the WHO Collaborating Centers for Reference and Research on Influenza in London and Melbourne for resistance testing (5,6). Forty-five of the viruses, which included viruses isolated in July, were tested at NICD by using a modified amplification refractory mutation system PCR (ARMS-PCR) (7). This method can simultaneously detect wild-type or mutant virus with the 274 mutation in a single PCR. Partial sequencing of the NA and hemagglutinin (HA) genes was performed to confirm the NA H274Y resistance mutation and to determine genetic drift in HA from the A/Brisbane/59/2007 virus recommended for the Northern Hemisphere 2007–08 vaccine. At the time of resistance testing, 92 H1N1 subtype viruses had been isolated. The 23 virus isolates sent to the WHO Collaborating Centers were highly resistant to oseltamivir by the NA inhibition enzyme assay, with 50% inhibitory concentration values of 554 nM to 1,485 nM (A. Hay, I. Barr, pers. comm.). All 45 isolates tested locally were positive by ARMS-PCR for oseltamivir resistance at position 274. The H274Y mutation was confirmed by sequence analysis of the N1 genes. The N1 sequences were closely related to those isolated in Europe and elsewhere in the 2007–08 winter season. However, the presence of 1 or 2 aa mutations in viruses from South Africa (M23L and N73K in the stalk region) compared with resistant European isolates indicated that some genetic drift of N1 from the older strains had occurred. Although most 2008 isolates were closely related to the A/Brisbane/59/2007 strain, several of the isolates from South Africa had mutations in an additional 2 or 3 aa residues at positions 183, 185, and 189, which mapped close to the receptor binding site of HA. (GenBank accession numbers for nucleotide sequences obtained in this study are {type:entrez-nucleotide-range,attrs:{text:EU914901-EU914916,start_term:EU914901,end_term:EU914916,start_term_id:195661082,end_term_id:195661112}}EU914901-EU914916.) Before the 2007–08 Northern Hemisphere winter, surveillance by GISN laboratories showed that oseltamivir-resistant H1N1 subtype viruses were extremely rare. Low numbers of drug-resistant viruses carrying the H274Y mutation usually followed oseltamivir treatment and showed reduced fitness with poor transmission (8). Consequently, fitter nonresistant viruses appear to have predominated. In contrast, no evidence indicated that persons from whom resistant viruses were isolated during the European 2007–08 winter season had either been treated or been in close contact with another person who had been treated with oseltamivir (2,8). We report oseltamivir-resistant H1N1 subtype viruses in Africa and the Southern Hemisphere. It appears that resistant viruses have spread from the Northern Hemisphere and have undergone extensive transmission within the population. These viruses may soon appear in other countries in the Southern Hemisphere. Ongoing monitoring is needed to understand the further evolution of oseltamivir resistance. Clinical symptoms of all patients in this study suggest an illness similar to that generally associated with seasonal influenza A virus (H1N1); no complications were reported. This finding is not unexpected because all isolates tested in the study were from outpatients. The policy in South Africa for use of oseltamivir for treatment of severe influenza remains unchanged because 2008 H3N2 subtype viruses are still drug sensitive.