European Management Platform for Emerging and Re-emerging Infectious disease Entities

Project Information

EMPERIE

Grant agreement ID: 223498

Project closed

Start date 1 May 2009

End date 30 April 2014

Funded under

• Specific Programme "Cooperation": Health

Total cost € 15 967 125,20

EU contribution € 11 909 560,00

Coordinated by

ERASMUS UNIVERSITAIR MEDISCH CENTRUM ROTTERDAM
Netherlands

Final Report Summary - EMPERIE (European Management Platform for Emerging and Re-emerging Infectious disease Entities)

Executive Summary:
“It would be extremely naïve and complacent to assume that there will not be another disease like AIDS, another Ebola, or another SARS, sooner or later”. This quote from the WHO annual World Health Report 2007, summarised the main problem addressed by EMPERIE, back in 2009 at the start of its activities. Indeed, since its start we have seen the emergence of the influenza H1N1 pandemic, the outbreak of a novel SARS like coronavirus in the Middle-East (MERS-CoV), amongst several other outbreaks such as the influenza H7N9 and Ebola outbreak.
EMPERIE set out to integrate European scientific excellence in the area of emerging epidemics research, making Europe better prepared to these new infectious diseases outbreaks, by improving early detection, identification and characterisation of new pathogens. Such rapid identification and characterisation provides scientists and public health authorities valuable knowledge and time to prepare and implement containment and emergency measures. In five years time, EMPERIE has built a virus discovery pipeline comprising advanced technologies and methodologies for rapid detection and characterisation of novel viruses. This virus discovery pipeline has been at the basis of the discovery of over 40 new viruses. Both the Schmallenberg virus in 2012 and the Middle-East-Respiratory-Syndrome (MERS) Coronavirus were discovered by EMPERIE invesitigators, using the EMPERIE virus discovery pipeline. EMPERIE identified a plethora of other novel viruses in mammals, insectivores and arthropods including bat- and hedgehog-related MERS-CoV in Europe and Africa, novel European SARS-related bat CoV, bat-related paramyxo-, hepadna- and hepeviruses, ancestral hepaciviruses in rodents, parvoviruses in humans and bats as well as novel insect viruses (Flavi-, Bunya-, Nidoviruses), calicivirus, anelloviruses, and a cyclovirus in humans, picobirnaviruses in humans and pigs, an astrovirus in humans and deer, an enteric coronavirus and hepatitis E virus in ferrets, arenaviruses in boid snakes, an adenovirus in gulls, a novel b19-like parvovirus in a harbor seal, novel hepaciviruses in rodents, parvovirus and a hepevirus in red foxes, and papillomavirus and a betaherpesvirus in reindeer. During the course of EMPERIE, the NGS virus discovery protocols and the bioinformatics pipelines continued to be optimized, ensuring its continued operations as the worlds most succesful virus discovery network, that may be activated whenever a novel virus challenge emerges.
EMPERIE’s contribution to Europe’s scientific excellence in emerging epidemic research, is furthermore witnessed by over 240 peer reviewed publications, averaging almost one EMPERIE acknowledged publication per week. Scientific accomplishments published in these papers included our rapid research reponses to H1N1, H7N9, MERS-CoV (e.g laboratory assays, tropism studies, pathogenesis, epidemiological models, genetic characteristics, host susceptibility, reservoir hosts, transmission dynamics). These papers provided pivotal scientific background and evidence underpinning further research and public health responses to these outbreaks. In addition, important work has been done on the identification of genes involved in SARS-CoV and MERS-CoV virulence, leading to the development of novel vaccine candidates for these viruses. Novel computational methods and epidemiological models and software for analyzing emerging outbreaks were developed in EMPERIE, generating important inputs for the development of risk mitigation strategies. Indeed, these and other tools have been extensively deployed to assist public health partners (e.g. WHO, ECDC, US CDC, UK HPA, China CDC and a number of governments (US, UK, KSA, China) in responding to the H1N1 pandemic and the ongoing MERS-CoV outbreak. Throughout the project EMPERIE has built and maintained close working relationships with international partners sites, notably in Vietnam, Nepal, Indonesia and Ghana providing training to clinical, support and lab staff, in particular for good clinical practice/research ethics, sample processing, implementation of molecular diagnostics, development of diagnostic algorithms, QA/QC, biostatistics, data entry and database management, laboratory (bio) safety.

Project Context and Objectives:
“It would be extremely naïve and complacent to assume that there will not be another disease like AIDS, another Ebola, or another SARS, sooner or later”. This quote from the WHO annual World Health Report 2007, summarised the main problem addressed by EMPERIE, back in 2009 at the start of its activities. Indeed, since its start we have seen the emergence of influenza H1N1 into the first influenza pandemic of the 21st century in 2009, and more recently the MERS-CoV outbreak, the influenza H7N9 and Ebola outbreak in 2013 and 2014 amongst many others. EMPERIE is rooted in the lessons learned by the majority of the partners in this EMPERIE network during the SARS and influenza H5N1 outbreaks. Most of the members of the consortium participated during the SARS outbreak and HPAI H5N1 outbreaks in studying the aetiology, diagnostics, the epidemiology and intervention strategies of the outbreak. Together with other leading research groups they formed the EMPERIE network (European management platform for emerging and re-emerging infectious disease entities) that had the following mission:
“To contribute to effectively countering the potential public health threat caused by new and emerging infectious diseases in Europe by establishing a powerful network capable of structural and systematic prediction, identification, modelling and surveillance of infectious diseases health threats and pathogens”.
In pursuit of this mission EMPERIE formulated the following overall project objectives, clustered into six workpackages

WP1. Specimen Collection:
EMPERIE foresaw three levels of specimen collection:
• Baseline information on pathogen-derived nucleic acid sequences that can be expected when metagenomics approaches are applied to clinical specimens from humans that are NOT suffering from disease, and to establish the sensitivity and specificity of the metagenomics technology using clinical specimens from humans with CONFIRMED INFECTION with important emerging pathogens.
• Generate a metagenomics inventory of the pathogen diversity in risk reservoir species in Europe.
• Use metagenomics technology for the initial detection of UNKNOWN pathogens in humans and animals.
The specific objectives for this activity were:
I.1 Human baseline: Complete a metagenomics inventory of pathogen diversity in pools of human specimens Sample collections include individual samples from humans (blood, respiratory, faecal, CSF), proven to be negative for all known pathogens, humans proven to be positive for key known emerging viral pathogens, and population-based sample pools.
I.2 To complete a Metagenomic inventory of RNA viruses in key risk hosts: Secondly, we will complete a metagenomics inventory of the “pathogen diversity” in pools of specimens collected from risk reservoir species specimens (blood, respiratory, faecal, salivary glands) in Europe. Data will be compared to similar data collections from elsewhere that include positive specimens from the same reservoir species (Objective I. 3).
I.3 Pathogen sampling from America, Asia and Africa: Samples from key risk host species, focusing on RNA viruses will be processed and analysed using the pathogen discovery pipeline detailed under objectives II and III. Proof of concept will be demonstrated by sampling a combination of key-hosts, viruses and geographic regions. EMPERIE focused on those pathogens which arguably are of chief interest: new zoonotic viruses that may cause epidemics and viruses already present – but yet unrecognised- in humans, specifically (i) new viruses from animals, especially those in key reservoir species that have previously shown to represent an imminent health threat to humans and (ii) Unrecognized viruses in humans in different geographical locations.



WP2: Pathogen isolation and identification:
During the course of an outbreak, it is crucial to rapidly identify the causative agent, so that containment strategies can be developed and implemented and specific diagnostic techniques and treatments can be developed. This requires (i) amplification of pathogens, (ii) High throughput prescreening of samples for the presence of known pathogens, (iii) isolation of pathogen from samples and (iv) identification of pathogens.
Objective II: The overall objective in this activity area is to provide an integrated set of laboratory methods, covering the above steps and which, in the manner of a virtual technology platform, facilitate the identification of novel viruses. It will also characterize crucial traits of identified novel agents.

WP3: Metagenomic Sequencing and Analysis:
EMPERIE will use ultra deep sequencing to characterise the "ZOONOTIC METAVIROME" of certain samples collected in Activity I. As part of this, EMPERIE set out to gain proof of principle that this technology can be used to identify an agent in the course of an outbreak, by using samples from humans, animals and insects infected with Dengue virus, West Nile virus and/or Chikungunya virus. To this purpose, samples of potential source animals (e.g. birds, bats, pigs, rodents, arthropods) will be pooled and analysed on a metagenomic scale by ultra deep sequencing. Key to this activity will be developing methods for evaluating homologies in viral repertoires between species – since finding similar viruses in multiple animal species may be viewed as a risk factor for that virus having the potential to become a human pathogen. To identify and genetically characterise pathogens, the following strategy will be adopted:
Objective III.1 To identify a baseline of DNA and RNA sequences present in various physiological compartments of the chosen populations. This will be done using high-throughput sequencing techniques on pooled samples from different populations.
Objective III.2 To test, using samples with known infectious agents, the ability to detect novel agents against this defined background.
Objective III.3 To generate baseline databases, and the bioinformatics tools to query them using test samples.
Objective III.4 To develop novel algorithms for assessing the zoonotic potential of novel viruses by quantifying homologies with viruses in other species, plus downstream analyses of the molecular basis of species specificity.

WP4: pathogen Containment: intervention options and diagnostics:
As soon as a newly emerging pathogen has been identified, containment and intervention strategies should be developed. These include the establishment of diagnostic and epidemiological tools, vaccines and antiviral or antimicrobial agents. Diagnostic tools can be used to study the natural history of the infection in human and (laboratory) animal hosts. Knowledge about excretion patterns will largely influence the way in which intervention strategies (including non-pharmaceutical interventions such as case isolation and quarantine) may be implemented. Subsequently the use of broadly reactive antivirals, antimicrobials or biological response modifiers may be tested in vitro, in animal models and eventually in humans. In the meantime the rapid development of vaccines should be started as soon as possible. Depending on the nature of the agent, vaccine development may profit from existing strategies for related infections (e.g. influenza virus versus lentivirus: existing vaccine experience versus non-existing experience). The specific objective in this activity area of EMPERIE was:
Objective IV: To use information from virus identification studies, in order to facilitate early intervention in terms of diagnostics, vaccines, and antiviral agents

WP5: Modeling, Prediction, Public Health risk assessment and emergency preparedness:
To make optimal use of new technologies for rapid pathogen identification, characterization and development of diagnostics and countermeasures, an analytical framework is needed for integrating the data collected in the early phases of an emerging outbreak and translating the resulting knowledge into effective response strategies. Quantitative statistical and mathematical models are powerful tools to assist in developing such a framework, as they enable heterogeneous (e.g. clinical, virological, antigenic, genetic and epidemiological) data sources to be integrated, with proper attention to levels of uncertainty, to draw conclusions about likely trajectories of epidemic growth and potentially effective counter-measures. The power and sophistication of modelling has also been growing, but to date there has been a lack of integration with basic research on viral discovery, characterization, diagnostics and vaccine/antiviral development. This was seen in the SARS epidemic, where the virological and epidemiological networks run by the WHO were only loosely connected. EMPERIE aims to rectify this deficiency, both by bringing the full range of disciplines together, but also via a carefully constructed work package which will leverage the data and tools resulting from the experimental work to deliver improvements in models and analytical tools useful for response operations – with the ultimate goal of improving our ability to respond in real-time to rapidly moving emerging epidemics.
Objective V.1. Modeling pathogen emergence and establishment, and predicting risk factors for zoonotic transfer
Objective V.2. Real-time analysis of outbreaks of poorly characterized emerging pathogens
Objective V.3. Optimising syndromic surveillance in resource poor settings for detection of case clusters of emerging infections
Objective V.4. Developing generic modelling toolkits for modelling emerging pathogens
Objective V.5. Using novel diagnostics to optimise public health interventions for containment of emerging pathogens
Objective V.6. Optimising intervention strategies to minimize adaptation of emerging pathogens

WP6: Training, capacity building and technology transfer local and regional sites:
As it has done in the past, it is not unlikely that future outbreaks with novel or (re-) emerging zoonotic pathogens will originate from tropical regions in Asia, Africa, or the Americas, where resources and expertise to investigate outbreaks are often limited. Enhancing grass-root laboratory and scientific capacity in these regions s key and for this reason, EMPERIE has set the following objectives:
Objective VI.1: To build research and laboratory capacity at participating network surveillance sites in resource -poor regions
Objective VI.2 To achieve uniformity and quality across all network surveillance sites with regard to collection and processing of data and specimens, and diagnosis of known pathogens
Objective VI.3 To implement adjustments of laboratory procedures based on work in other work packages

Redesign into three main platforms:
During the course of EMPERIE it became apparent that the originally planned centralized baseline-sequencing strategy of EMPERIE (summarized in objectives I.1 I.2. III.1 and III.2) had become out-dated. These objectives were therefore abandoned and replaced for year 3 to 5 by an ‘intelligent sampling’ strategy, also based on the comments of the external reviewers. The sampling strategy of EMPERIE will be based on the concept of ‘smart sampling’ in which – in addition to the abovementioned samples –, EMPERIE would collect samples from other animals and/or humans in case a novel or unexpected pathogen is identified. In support of investigating possible animal-to-animal-, animal to human- and human-to-human exposure of (novel identified) pathogens and establishing risks of zoonotic transmission and spread ‘smart sampling’ can include the collection of specimens from large animals and potentially exposed humans at the same location where, or – if a novel pathogen is identified in humans – household members and animals (wild, domestic, food).
In Period 3 EMPERIE was therefore redesigned into three main platforms (see next page):

• Platform I Virus Discovery and Sequencing encompasses the actual analyses of the samples collected in WP1, using the technologies and tools established in WPs 2, 3 and 4;
• Platform II Technology and Knowledge Development encompasses with the development and validation of novel technologies in WP2, 3 and 4 and the generation of new knowledge, models and recommendations in WPs 2, 3 , 4 and 5.
• Platform III Training and Technology Exchange encompasses WP6, aimed at dissemination (within and outside of EMPERIE) of the technologies, methods and tools developed in Platform II.

Project Results:
EMPERIE’s response to the influenza A/H1N1 2009 pandemic:
At the start of EMPERIE in May 2009, the world was confronted with the first influenza pandemic of the 21st century, caused by the novel Influenza A/H1N1. Annex I of EMPERIE stated “(…) EMPERIE will be fully committed to dedicate all its resources, research potential and other capabilities during the entire project time to address newly emerging infectious disease threats whenever they would occur, to the extent that such an incident will be brought under control as soon as possible”. With the “Mexican Flu” pandemic clearly qualifying as a ‘newly emerged infectious disease threat’, EMPERIE started with refocusing a significant part of its resources in period 1 to influenza A/H1N1 response, in close collaboration with the European Commission. During the first months of EMPERIE, the principal investigators of the partners in EMPERIE identified a range of urgent research needs and associated research studies in response to the influenza pandemic that could be included in the EMPERIE framework that:
• had a clear added value to the H1N1 pandemic response from a scientific and public health perspective;
• Were complementary to other ongoing initiatives by other networks and organizations (such as WHO);
• Were in line with the overall scope and objectives of the EMPERIE project.

After feedback of the EMPERIE Advisory Board, the Influenza A/H1N1v contingency plans were allocated to the most appropriate work packages and the inclusion of over a dozen new deliverables, specifically tailored to the influenza A/H1N1 pandemic research response. Below a summary overview is given of the results of the work performed on the novel influenza H1N1 virus in the first year of EMPERIE.

Laboratory methods and new technology to study the influenza A/H1N1 pandemic virus:
With the novel virus sweeping the globe, laboratories all over the world were in need of new tools, procedures and methods to enable them to study the new virus. EMPERIE has contributed to the development of novel assays - investigative/analytical procedures to test certain aspects of the virus - specifically for the novel pandemic H1N1 virus. These include real time RT-PCR assays specific for the new pandemic H1N1 virus and a pan-influenza real-time RT-PCR assay (see publication 16). We have provided ready-to-use RT-PCR reagents and controls and training to Dr G Wickramasinghe at the Medical Research Institute, Colombo, Sri Lanka. This allowed them to initiate and provide a service in the early stages of the pandemic, demonstrating the contribution of EMPERIE to rapid response to set up diagnostic support in a developing country setting. Real time RT-PCR assays have been also been provided to all the laboratories in France and overseas territories involved in the detection of the pandemic H1N1 2009 virus as well as to all the laboratories of the network of Pasteur Institutes along with synthetic RNA transcripts corresponding to the M and H1pdm genes as controls. EMPERIE provided a real-time RT-PCR assay for the detection of H1N1 that was provided to ROCHE in an emergency measure. The assay was published online in EUROSURVEILLANCE (for more details we refer to publication 5 in section 5) and on the homepage of the German National Reference Centre at the Robert Koch Institute. It became the technical basis for the current influenza test kit after FDA licensing by ROCHE (we do not generate any income from this because the method was provided to public in an emergency measure). Moreover we characterised the utility of rapid antigen tests for H1N1.

Pyrosequencing methods for the specific detection of the polymorphism at position 222 of the HA (D,E,G) of the H1N1pdm virus have been developed which allows to screen for the presence of the D222G mutation potentially related with increased virulence of the virus. Third, a pyrosequencing method was also developed for the specific detection of the 1223R mutation in the NA of the H1N1pdm associated with reduced sensitivity to both oseltamivir and zanamivir and also shown to potentiate viral resistance to oseltamivir linked to the H275Y mutation in the NA , (see publication 68).

Moreover, in its first year EMPERIE established an indexed (multiplex) 454 whole influenza genome sequencing platform which was used for various genetic studies of the new virus (See publication 35), quantification of 454 sequencing erros rated for influenza, development of data resources for whole genome influenza sequencing and computational methods for analysis of 454 deep sequence read data. Also, an innovative ex-vivo human airway epithelial cell culture system was developed that can be used to to study the evolution of influenza H1N1 in detail.

Insights into the pathogenesis and transmission dynamics:
The basic requirement of effectively combating a new virus such as the pandemic H1N1 virus is to understand its pathogenesis, virulence, tropism and how fast and efficient it adapts to the human immune system. At the beginning of EMPERIEseveral basic studies were initiated in reponse to the unfolding pandemic, in pursuit of developing our understanding of these key traits of the novel pandemic virus. This has led to the following results, summarised below.

Pandemic H1N1 is more pathogenic than seasonal H1N1 in ferrets:
In a ferret pathogenesis and transmission study the 2009 A(H1N1) influenza virus was found to be more pathogenic than a seasonal A(H1N1) virus, with more extensive virus replication occurring in the respiratory tract. Furthermore data resulting from the study suggest that the 2009 A(H1N1) influenza virus has the ability to persist in the human population, potentially with more severe clinical consequences. For more details on the results of this study we refer to publication 2.

Pandemic H1N1 virus causes pneumonia in ferrets intermediate in severity between that caused by seasonal H1N1 virus and by the H5N1 virus
In another study in ferrets the ability of Influenza A/H1N1v to cause pneumonia was compared with that of seasonal influenza H1N1 virus and highly pathogenic H5N1 birdflu virus. Our results showed that the new H1N1 virus causes pneumonia in ferrets intermediate in severity between that caused by seasonal H1N1 virus and by the H5N1 virus. The new H1N1 virus may be intrinsically more pathogenic for humans than is seasonal H1N1 virus. For more details on the results of this study we refer to publication 3.

Human influenza viruses attach better to cells in the upper respiratory tract than avian influenza viruses:
In our studies of three human and three avian influenza viruses in respiratory cells we found that the human influenza viruses—two seasonal influenza viruses and pandemic H1N1 virus—attached abundantly to ciliated epithelial cells and goblet cells throughout the upper respiratory tract. These and other findings lead to the conclusion that influenza viruses that are transmitted efficiently among humans attach abundantly to human upper respiratory tract, whereas inefficiently transmitted influenza viruses attach rarely, suggesting that the ability of an influenza virus to attach to human upper respiratory tract is a critical factor for efficient transmission in the human population. For more details on the results of this study we refer to publication 4.

Pathogenicity of the pandemic H1N1 compared to the seasonal H1N1:
In a fourth study we demonstrated that the H1N1v virus infects alveolar epithelial cells and causes diffuse alveolar damage in a non-human primate model. Its higher pathogenicity compared to a seasonal H1N1 virus may be explained in part by higher replication in the lower respiratory tract. For more details on the results of this study we refer to publication 19.

Genetic studies of the influenza A/H1N1 pandemic virus:
EMPERIE contributed to a study unravelling the antigenic and genetic characteristics of the novel Influenza A/H1N1v and to a study on the impact of immune escape on transmission dynamics of Influenza. Both studies provided important data in light of pandemic response measure. For more details on the results of this study we refer to publications 11 and 12.

Effect of D222G mutation on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission
We investigated the effect of amino acid substitution D222G in influenza A/H1N1v on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models. It was shown that mutation D222G in f influenza A/H1N1v affects the binding of the virus to receptors on host cells. Viruses with D222G displayed increased binding to cells in the human lower respiratory tract. This change in binding might explain why this virus causes more severe disease in humans. For more details on the results of this study we refer to publication 22.

Potential of reassortment (mixing of genetic material) of Influenza A/H1N1v with seasonal influenza viruses:
Using different laboratory generated reassortants of seasonal and pandemic viruses by means of reverse genetics and in vitro selection we investigated the effect of different mutations on the virulence and transmissibility of the influenza viruses. Our studies demonstrate that the 2009 pandemic H1N1 virus has the potential to reassort with seasonal influenza viruses, possibly resulting in increased virulence while maintaining the capacity of transmission via aerosols or respiratory droplets. For more details on the results of this study we refer to publication 21.

Susceptibility of poultry to Influenza A/H1N1:
Pandemic (H1N1) 2009 virus caused only mild disease compared with pandemic influenza viruses from the 20th century. However, if the H1N1 pandemic virus were to acquire virulence markers by reassorting with influenza viruses that cause severe disease in humans, such as highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype, the H1N1 virus would become more dangerous to humans too. The chances of these happening increases if poultry can be infected with the pandemic (H1N1) 2009 virus. Therefore, we determined the susceptibility of chickens, turkeys, and mice to pandemic (H1N1) 2009 virus. Our results demonstrate lack of susceptibility of chickens and minor susceptibility of turkeys for infection by pandemic (H1N1) 2009 virus. For more details on this study we refer to publication 15.

Other influenza A/H1H1 related studies with support of EMPERIE:

Infection attack rate and severity of the novel pandemic virus in Hong Kong.
Serial cross-sectional data on antibody levels to the 2009 pandemic H1N1 influenza A virus from a population were used to estimate the infection attack rates and immunity against future infection in the community. For more details on this study we refer to publication 31 and 32.

Insights from modeling on the studies needed to Address Public Health Challenges.
EMPERIE contributed to a publication on the public health challenges and identification of data that are required for public health decision making: Measuring age-specific immunity to infection; accurately quantifying severity; improving treatment outcomes for severe cases; quantifying the effectiveness of interventions; capturing the full impact of the pandemic on mortality; and rapidly identifying and responding to antigenic variants. For more details on the results of this study we refer to publication 20.

Study on the Early Transmission Dynamics of H1N1pdm Influenza in the United Kingdom.
EMPERIE contributed to a study that analyzed data on all laboratory-confirmed cases of H1N1pdm influenza in the UK to 10th June 2009 to estimate epidemiological characteristics. For more details on the results of this study we refer to publication 17.

Study on the sensitivity of the influenza A/H1N1 virus to temperature?
Different influenza viruses, including the influenza A/H1N1 pandemic virus, have been compared for their temperature sensitivity and the effect on their virulence. For more details on the results of this study we refer to publication 18 in section 5.

Epidemiological analyses
The envisaged real-time analysis of emerging outbreaks took a more practical and topical slant than expected with the advent of the 2009 H1N1 pandemic. EMPERIE supported 2 analyses relevant to the pandemic: the first a collaboration with the UK Health Protection Agency of the early data on confirmed cases and their contacts. This analysis allowed the incubation period, generation time and household transmissibility of the pandemic virus to be estimated (see publication 17). The second study estimated the extent to which influenza transmissibility varies seasonally in temperate countries – relevant to trying to estimate how much more transmissible the pandemic virus was likely to be in the autumn/winter of 2009 compared with the spring and summer (see publication 18).

3.2 Results generated by the Virus Discovery Platform: novel viruses identified

Within weeks of the first reported cases of the SARS outbreak in 2003, an international network of laboratories identified a novel coronavirus - currently known as SARS-CoV - as the causative agent of SARS. This rapid identification was crucial in containing the further spread of SARS. The successful international collaboration formed the basis of establishing EMPERIE as a network capable of rapidly identifying the causative agent of newly emerging or re-emerging infectious disease outbreaks. To this aim, EMPERIE has established a common virus discovery platform used by the network partners to do just that: identifying viruses in animals and humans from existing samples and samples from humans and high risk animal species - such as bats and birds - collected in key regions across the globe. Testimony to the functioning of this virus discovery platform are the novel viruses discovered in EMPERIE. The platform will be instrumental in the discovery of novel pathogens threatening animals and humans alike. The most well known examples are the identification of the Schmallenberg virus and the Middle East Respiratory Syndrome (MERS) Coronavirus using the EMPERIE virus discovery platform. The attached pdf “Virus Discovery in EMPERIE” gives an overview of the novel viruses discovered.

The results emanating from the Technology and Knowledge Development platform can be divided into three main categories of results as depicted below:
Virus discovery technology, procedures, tools and methods
These results encompassed the following:

SOPs for generic PCR assays
Early in the project, EMPERIE completed a consortium-wide survey of generic PCR assays. These assays are taxon-wide either on genus or even family level. In year 2, assays for papilloma-, astro- and anelloviridae were implemented in addition to the generic PCRs that were already available/developed within the consortium in year 1. The SOPs for the generic PCR assays were made available to the different partners within the consortium upon request.

Suite of SOPS for prescreening for known pathogens
To exclude known pathogens in diseases/outbreaks samples should be carefully tested for known pathogens. Therefore an inventory of routine diagnostics was made and processed into a database with lists of all routine diagnostics was generated and made available for all EMPERIE partners.

Banks of immortalized primary cells
The following cell banks have been developed an/or made available trhough EMPERIE in support of the virus discovery and characterisation studies of (re) emerging viruses:
• Ex-vivo lung culture system comprised of 3mm human lung biopsies that are cultured up to 72 hours in 95% O2 at 37oC, obtained from surgically removed non-malignant leftover lung tissue of patients having undergone surgical resection of respiratory tissue.
• Ex-vivo ferret trachea culture, in which trachea rings can be cultured in 95% O2 at 37oC up to 72 hours.
• Primary mammalian kidney fibroblast cultures of multiple mammalian species such as Harbour seal (Phoca vitulina) Harbour porpoise (Phoceana phocoena), Bottlenose dolphin (Tursiops truncates), California sea lion (Zalophus californianus), Grey seal (Halichoerus grypus), Striped dophin (Stenalla Coeruleoalba), Sheep (Ovis Aries). These three culture systems can be used for virusdiscovery since these culture systems might mimic the natural host more closely and will enhance the ability to isolate a (new) virus.
• 25 different primary bat cell cultures from 11 bat species, producing 73 different immortalized clonal bat cell lines;
• A bank of about 1.280 cell lines of 79 different species (insects, fish, reptils, birds, mammals) are available. In addition, cells from exotic animal species are also generated and /or collected (e.g. Sturgeon, Gilt-head bream, Grass carp, Blackbird, Cliff swallow, Magpie, Swan, Serotine bat, Northern white rhinoceros, Pigmy hippopotamus, Asian elephant,Yak, Giant Panda, Leopard, Saiga antelope). From the Serotine bat (Eptesicus serotinus) both kidney and brain cell cultures were established (CCLV- RIE 1091/1093) which can be 20 to 40 times passaged. In addition, the cells were immortalized using SV40 large T antigen. The different cell lines, and especially the bat cell lines, are available (or were transferred) for the partners of the project for further studies like virus isolation, virus growth analysis or in vitro tropism.

Software tools to store and analyse DNA and RNA metagenomic sequences, and allow querying with novel metagenomic sequences
EMPERIE has contributed to the development and publishing of the software tool QUASR – a lightweight pipeline written to process and analyse next-generation sequencing (NGS) data from Illumina, 454, and Ion Torrent platforms. A portable JAVA version is freely available on Sourceforge (http://sourceforge.net/projects/quasr).
We have developed a method for iterative search-classification-removal algorithm, called SLIM for analysis of virus discovery meta-genome sequencing. For a detailed description of the methods we refer to publication 188 in section 5.
Thirdly we have developed a Compare Script for antibody-capture-VIDISCA-454. A script was written to compare sequence reads obtained from the input (untreated sample) and the harvest after enrichment by autologous antibodies. For a detailed description of the script we refer to publication 186;

Continuous updating
During the course of EMPERIE the abovenetioned laboratory methods and sequencing technology have been continuously upgraded and expanded, in order for the EMPERIE platform as a whole to reamin state-of-the-art. Examples of virus discovery and characterisation tools and methods that have bee resulting from the EMPERIE consortium are:
• Pan-virological microarray (PathogenID v3.0)
• Novel serological assays for seroepidemiological studies of MERS coronavirus by HKU;
• New method Antibody-capture-VIDISCA-454;
• PCRs for EV71;
• Method of high res. melting analysis for verification of reverse genetically generated recombinant influenza A viruses;
• Updates on pipelines for detection of pathogen related sequences in metagenomic sequence datasets;
• Optimized microarray based viral pathogen detection methods;
• Diagnostic real time RT-PCR assays for influenza H7N9;
• Antibody-capture-VIDISCA-454 by P8 AMC and P11 WTSI
• High throughput MERS-CoV full genome sequencing
• Influenza A virus (H5N1, H7N7, H7N9) deep sequencing
• Norovirus full genome deep sequencing
• Human respiratory syncytial full genome deep sequencing;
• Full genome deep sequencing methods useful for small amounts of clinical materials;

Novel diagnostic assays for important new viruses discovered:
Besides the development and distribution of novel diagnostic assays for the influenza A/H1N1 pandemic virus, EMPERIE also developed real-time RT-PCR assays for:
• Gouléako virus, which is the prototype species of a putative novel bunyavirus genus. See publication 56 for more details on this assay;
• Cavally virus, which is most likely a member of a novel virus family within the order Nidovirales. See publication 44 for more details on this assay;
• Newly identified bat coronaviruses, bat adenoviruses and bat astroviruses. See publications 27 and 30 for more details on these assays;
• The entire genus Alphavirus that allows for the first time a clinical-grade testing for this highly diversified Arbovirus genus. See publications 26 and 65 for more detailed information on this assay;
• Pan-paramyxovirus detection: see publication 49.

The pan-bat real-time RT-PCR assays and two different bioassays were used to characterize several bat cell lines from frugivorous bats (Eidolon helvum and Rousettus aegyptiacus) to prove their IFN competence. Their potential to induce and react on IFN was confirmed, and biological assays were adapted to application in bat cell cultures, enabling comparison of landmark IFN properties with that of common mammalian cell lines. These novel IFN-competent cell lines will allow comparative research on zoonotic, bat-borne viruses in order to model mechanisms of viral maintenance and emergence in bat reservoirs. See publication 59 for more details.

New knowledge on the mechanisms involving host-switch of viruses, using Coronaviruses as a model:
EMPERIE contributed to the generation of a wealth of new knowledge on Coronaviruses, that collectively has enhanced our understanding of the factors promoting zoonotic viruses crossing the interspecies barrier. Studies of EMPERIE included:
• Findings regarding virus persistence and underlying mechanisms of host innate immunity, in reservoir host. See publications 14, 46 and 47;
• Ecological study on the role of antibodies, organ tropism and population behaviour in coronavirus (re) emergence. See publication 30;
• New knowledge on the critical protein domains constituting the host tropism and species barrier of bat coronaviruses. See publications 14, 37, 38, 45, 47 and 91;
• Identification of genes involved in SARS-Cov and MERS-CoV virulence. In order to identify SARS-COV and MERS-CoV genes involved in virulence, reverse genetics systems were developed by P13 CNB-CSIC for these two coronaviruses using the bacterial artificial chromosome (BAC) system. By generating a comprehensive collection of virus mutants missing 1, 2, 3, 6, or 7 genes, we have identified that genes E and 3a are essential virulence genes. It was shown that both SARS-CoV and MERS-CoV missing gene E were attenuated and excellent virus candidates, as they provided protection against the challenge by the homologous virulent viruses. In addition, studies directed to the identification of host cell signaling pathways affecting virus replication in cell in culture and in vivo, led to the identification of the NF-kB pathway as an essential one in the induction of inflammation, acute respiratory disease and death. Interestingly, we have proven that inhibition of NF-kB activation by drugs significantly increased the survival of mice infected by SARS-CoV. Therefore, we have identified useful antivirals providing protection against SARS-COV infection. These results have been provided publications 92, 204, 205, 206, 207, 208 , 209 and 210.


EMPERIE’s response to the Middle-East-Respiratory-Syndrome (MERS) Coronavirus outbreak
In July 2012 Prof. Ron Fouchier was contacted by Dr. A.M. Zaki of Dr. Soliman Fakeeh Hospital in Jeddah, Kingdom of Saudi Arabia to assist in the characterization of what appeared to be a viral agent isolated from a man with acute pneumonia and renal failure, who died as the consequence of this disease. While initial contact was made to test for paramyxoviruses using EMPERIE technology (see publication 49 in section 5), it was agreed to include testing for coronaviruses. After positive detection of coronavirus by generic RT-PCR and the identification of a potential novel coronavirus based on sequence analyses of the RT-PCR product, technology developed in Platform 1 was used for further virus characterization. The full genome sequence of what is now known as the MERS-Coronavirus was generated within a day and shared rapidly with EMPERIE partners and numerous external laboratories. This positive identification of a novel coronavirus from a man, along with clinical data and basic clinical microbiology data was reported through the scientific literature (See publication 100) and PromedMail.

This marked the start of a series of studies conducted by EMPERIE laboratories (in collaboration with the ANTIGONE FP7 project also led by Erasmus MC) and made possible by EMPERIE funding. These EMPERIE funded studies put Europe, EMPERIE partners in particular, in the frontline of the international research response to this important new emerging virus, providing crucial scientific input to the international outbreak response to MERS-CoV. EMPERIE partners were involved in numerous teleconference calls, meetings, risk assessments, and research prioritization to assist public health agencies such as WHO and ECDC and laboratories in countries where new human cases were detected. Studies on MERS-CoV have continued after EMPERIE, within ANTIGONE or other networks. However, given that EMPERIE funding has ended, the sustainability of the research effort on MERS-CoV is limited if no additional new funding can be secured. The results of the EMPERIE funded research are summarised below.

Comprehensive annotation of the virus genome
Shortly after the virus was discovered bioinformatics approaches were used in collaboration with external experts to provide a comprehensive annotation of the virus genome, and the formal classification of the novel coronavirus as a new species belonging to lineage C of the genus Betacoronavirus (see publication 103).

Development and distribution of diagnostic tests
After notification of a second case of human infection with the same coronavirus in London, EMPERIE labs worked closely with other laboratories towards the development of diagnostic tests specifically targeted at the novel coronavirus (see publications 104 and 105). EMPERIE characterized cell types expressing MERS-CoV antigen and the associated histopathological changes in postmortem specimens from a fatal human case of HCoV-EMC infection.
Development of serological tests was started to serve in epidemiological investigations (publication 116). EMPERIE also contributed to the development of novel serological assays (microneutralisation and pseudoparticle virus neutralisation assays) for seroepidemiological studies of MERS coronavirus. These assays were used to study the prevalence of MERS coronavirus infection in animals (see below).

Identification of the human receptor in search for novel treatment options
Also studies on receptor use and host-range were initiated (see publications 101 and 121 in section 5). We subsequently identified dipeptidyl peptidase 4 (DPP4) - also known as CD26 - as a functional receptor for MERS-CoV. The use of the evolutionary conserved DPP4 protein from different species as a functional receptor provides clues about MERS-CoVs host range potential. In addition, it contributes critically to our understanding of the pathogenesis and epidemiology of this emerging virus, and may facilitate the development of intervention strategies (See publication 102).
In subsequent studies we showed the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus-binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection. (See publications 182, 183 en 231).

P4 HKU received MERS-CoV from P1 EMC and studied the tropism of this novel pathogens using ex vivo cultures of human bronchial and lung tissue specimens, revealing that MERS-CoV infected nonciliated bronchial epithelium, bronchiolar epithelial cells, alveolar epithelial cells, and endothelial cells that interferon alpha and beta treatments could reduce the replication of this virus, suggesting a potential therapeutic use of IFNs for treatment of human infection (see publication 126). Together with researchers from St. Gallen, Switzerland, EMPERIE further analysed the virus host tropism and the interferon response of MERS-CoV where we demonstrated that both type I and type III IFN can efficiently reduce MERS-CoV replication in HAE cultures, providing a possible treatment option in cases of suspected MERS-CoV infection. See publication 121 in section 5. Collaborations were initiated with other consortia on basic science and risk assessments (e.g. NIAID/NIH, FP7 programs ANTIGONE and PREDEMICS) and evaluation of treatment options (e.g. FP7 program SILVER; see publication 137).

Development of sequencing technology and identification of evolutionary patterns
EMPERIE also contributed to the development of a novel deep genome sequencing process for generating full genomes of the MERS coronavirus directly from a small amount of patient derived material. Molecular clock analysis showed that all human infections of MERS-CoV share a common virus ancestor most likely considerably before early 2012, suggesting the human diversity is the result of multiple zoonotic events (see publication 154 ).

In collaboration with the Kingdom of Saudi Arabia Ministry of Health, the method was employed to examine MERS-CoV evolution during a hospital outbreak in Al-Hasa KSA. Four complete MERS-CoV genome sequences were determined directly from <25 µl of sputum derived nucleic acid (see publication 155). These genome sequence data were important in defining the transmission patterns of MERS-Cov and the method will be important in following the evolution of the virus, defining an animal reservoir and informing public health measures for controlling this virus.

Other studes providing a detailed description of the changes in the MERS-CoV occurring over the initial course of the MERS epidemic included publications 191 and 192. These sequence data have been used by other groups for calculating an Ro for the virus in publication 197.
Studies in search of the animal reservoir of MERS-CoV

EMPERIE also contributed to a range of studies initiated in search of the animal reservoir of MERS-CoV, which is a crucial piece of the puzzle in the design and implementation of effective interventions against the (further) spread of MERS-CoV.

Although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of MERS-CoV. As anecdotal exposure histories suggested that patients had been in contact with dromedary camels or goats, we investigated possible animal reservoirs of MERS-CoV by assessing specific serum antibodies in livestock. 50 of 50 (100%) sera from Omani camels and 15 of 105 (14%) from Spanish camels had protein-specific antibodies against MERS-CoV spike. Sera from European sheep, goats, cattle, and other camelids had no such antibodies. MERS-CoV or a related virus has infected camel populations. Both titres and seroprevalences in sera from different locations in Oman suggest widespread infection. (See publications 163, 164 and 165).

Based on these findings EMPERIE partners collected and screened samples through 2013 and 2014 from dromedary camels from the Middle East and the Greater Horn of Africa). Analysis of respiratory camel samples is ongoing and will be completed after the end of EMPERIE.

EMPERIE partners also conducted MERS coronavirus surveillance in animals samples collected from Egypt and the Kingdom of Saudi Arabia, respectively. Data suggests that dromedary camels might be a source for human MERS cases and that the genetic diversity of MERS coronavirus might be greater than previously appreciated. EMPERIE contributed to a longitudinal study in a dromedary farm in Al-Hasa, in the Kingdom of Saudi-Arabia during the period November 2013-February 2014. Virological and serological evidence of MERS-CoV infection was documented in association with sero-conversion in calves over a one month period. Comparison of the full genome sequences of the Saudi dromedary viruses compared with related human MERS-CoV isolates indicate that the dromedary viruses are likely to retain full replication competence for the human respiratory tract. See publications 170, 171, 172 and 173)

Studies in response to human MERS-CoV cases in Europe
Two cases of infection with the novel A MERS coronavirus were detected in France. One imported case and one case resulting from nosocomial transmisison. A variety of sequential samples were obtained, that allowed to highlight the importance of lower respiratory tract samples for MERS-CoV diagnostics, to document the presence of the virus in blood and urine and determine the kinetics of virus excretion in the respiratory tract which could last for over 20 days. Sequence determination of the sequence of the virus isolate from the first french case contributed to the evaluation of the transmissibility of this virus (See publication 243). MERS-CoV sequences (1x full genome, 1x partial genome) from MERS-CoV cases imported to Munich and Essen, Germany could be amplified and sequenced by 454 next-generation sequencing and conventional sequencing. The sequence information was published as a part of a case report published. (See publication 168).
EMPERIE agent-based simulation software and methods were employed in the last period to analyse the transmission of MERS-CoV in the Middle East, which provided the first robust estimates of the transmissibility, incubation period, generation time and case fatality ratio of the virus (see publications 226 and 227).


Analytical tools for rapid epidemiological characterization:

Extending agent-based models to model emerging or endemic vector-borne diseases
A paper presenting the result on EMPERIE suporoted work on an agent-based model of malaria was close to submission at the time of writing of the final report. A paper on spatially targeted elimination strategies has been submitted. Work on vector borne disease modelling was reprioritised to focus on dengue (an emerging threat to the south of the EU) in the last period of EMPERIE, with work being pursued in 3 areas:
o collaborative work with Sanofi Pasteur plc to analyse all their phase I and II data for their live attenuated tetravalent dengue vaccine. Papers modelling the immunogenicity of the vaccine and potential usage scenarios are close to submission;
o collaborative work with the Eliminate Dengue consortium (http://www.eliminatedengue.com/ ) on modelling the effectiveness and potential use of the insect bacterial symbiont Wolbachia as a control measure for dengue (1 paper submitted, 1 nearing submission);
o underpinning research to understand the dynamics of dengue pathogenesis in the human host (see publication 230)

Integrated analysis of multiple epidemiological data streams in a pandemic and on inferring epidemic dynamics from sequence data
EMPERIE contributed to the integrated analysis of influenza-like-illness and virological surveillance data during the influenza A/H1N1 pandemic. A new approach to characterising disease transmission dynamics was published in 2011. For details s we refer to publication 72.

EMPERIE has also contributed to developing methods for real-time phylodynamic analysis during epidemics, with application to H1N1 influenza. A scientific paper on this work was published in February 2012. See publication 73.

Bioinformatic (phylogenetic/antigenic) methods for predicting pathogen emergence
EMPERIE contributed to research on predicting the probability of avian A/H5N1 influenza virus evolving to become transmissible between humans (see publication 135). In addition to its scientific contribution the paper was also influential in the WHO and NSABB decisions on publication of the Imai et al and Herfst et al manuscripts on A/H5N1. There has been substantial follow up work on analyses of deleterious substitutions, and collaborations set up on deep sequence analyses to test the theory in this publication. Two review manuscripts were produced with support of EMPERIE to summarize all major progress on the topic of avian influenza virus adaptation to acquire increased airborne transmissibility between ferrets and humans. See publications 236 and 237 for more details.

EMPERIE research groups further performed studies to determine the molecular basis of antigenic drift for the hemagglutinin (HA) of human influenza A/H3N2 virus. From 1968 to 2003, antigenic change was shown to be caused mainly by single amino acid substitutions, which occurred at only seven positions in HA immediately adjacent to the receptor binding site. Most of these substitutions were involved in antigenic change more than once. Equivalent positions were responsible for the recent antigenic changes of influenza B and A/H1N1 viruses. Substitution of a single amino acid at one of these positions substantially changed the virus-specific antibody response in infected ferrets. These findings have potentially far-reaching consequences for understanding the evolutionary mechanisms that govern influenza viruses, making influenza virus antigenic evolution more predictable than thought previously. See publication 238 for more details.

Strategies to minimise the antigenic evolution of an emerging pathogen
EMPERIE submitted a paper on this work to Science on 14th March 2014; and a revised version; following review has been submitted on 3rd June 2014. At the time of writing of this final report, the paper was not yet pubished. The details of the publication are as follows:
Title: Dengue viruses cluster antigenically but not as discrete serotypes
Authors: Leah C. Katzelnick, Judith M. Fonville, Gregory D. Gromowski, Sarah L. James, Theodore C. Pierson, Philippe Buchy, Eva Harris, John G. Aaskov, Jorge L. Muñoz-Jordán, Nikos Vasilakis, Robert V. Gibbons, Robert B. Tesh, Laura A. VanBlargan, Colin A. Russell, Albert D.M.E. Osterhaus, Ron A.M. Fouchier, Cameron P. Simmons, Edward C. Holmes, Stephen S. Whitehead, Derek J. Smith.

Agent based simulation modelling software for modelling emerging pathogen outbreaks, surveillance and control policies.
EMPERIE has contributed to the development and launch of open-source software to model and analyse emerging infectious disease outbreaks:
1. The Global Epidemic Simulator (http://sourceforge.net/projects/globalepidemics/ ) – an agent based simulation model capable of modelling the global spread of an emerging respiratory pathogen and a range of potential control measures (e.g. vaccines, antivirals, social distancing, travel restrictions).
2. Outbreaker (https://sites.google.com/site/therepiproject/r-pac/outbreaker) – a tool for jointly analyzing epidemiological data and pathogen sequence data collected early in an outbreak to infer transmission pathways and estimate key epidemiological parameters. For a detailed description of the tool (see publication 228);
3. EpiEstim (https://sites.google.com/site/therepiproject/r-pac/epiestim ) – a user-friendly analysis package to infer pathogen transmissibility from time series data on disease incidence. The tool is described in an American Journal of Epidemiology paper (see publication 152)


3.4 Results generated by the Training and technology Exchange Platform
Throughout its five year duration, EMPERIE has contributed to training of laboratory staff in Asia and Africa and of technology transfer (both internally and externally).

Training of Asian sites by UOXF
The OUXF traning programme focused on support of staff in partnering sites, including clinical, support and lab staff, in particular for good clinical practice/research ethics, sample processing, implementation of molecular diagnostics, development of diagnostic algorithms, QA/QC, biostatistics, data entry and database management, laboratory (bio) safety. Training records are kept from all staff involved in our studies (several hundreds) and update GCP and biosafety training every two years and annually, respectively. Below a list of the training programmes is presented:

Vietnam (Hospital for Tropical Diseases (HTD) , Ho Chi Minh City (HCMC); National Hospital for Tropical Diseases (NHTD) Hanoi
• Laboratories in Vietnam Ha Noi have been upgraded to include separate extraction and mix rooms with air flow control. Staff have been trained in various molecular diagnostic procedures and have demonstrated proficiency in external QA schemes;
• Molecular diagnostic labs were established in HTD, NHTD, the National Hospital for Pediatrics (Ha Noi) and the children's hospitals 1 and 2 in HCMC. Staff were inititally trained to perform influenza diagnostics and have received continued support from OUCRU to implement molecular diagnostics for other pathogens including HBV, HCV, HIV, HSV/VZV, EV71, CMV, EBV;
• On site Quality Assurance training in Ha Noi and HCMC;
• UOXF Viet Nam staff attended training in specimen labeling, tracking, & management using Freezer works™ in Bangkok;
• NHTD (Ha Noi) lab staff trained on specimen handling and processing for diagnosis of respiratory pathogens and dengue;
• All lab staff at UOXF/HTD HCMC participated in a 2 day BSL3 training workshop.
• Staff in Buon Me Thuot, Dong Thap and Children's hospital II trained on bacteriology diagnostics and susceptibility testing on blood, CSF, and respiratory samples;
• Masters students have been trained on JEV detection and isolation in suckling-mouse-brain preparations at HTD, HCMC;
• Additional research assistants, including staff from Ha Noi have been trained in the virus culture and serology at HTD HCMC;
• Staff from UOXF Viet Nam, HTD and NHTD attended the 7th Hong Kong University Pasteur Virology Course in July 2010;
• Staff of UOXF HCMC and Ha Noi and collaborating hospitals attended Sanger Centre training on pathogen genome sequencing at HTD in HCMC, Feb-March 2010;
• Training for international PhD focusing on pathogen discovery in collaboration with AMC;
• Staff from Shantou University Medical College, China attended training and were provided technical support for PCR of respiratory samples in Shantou, China.

Indonesia
• Clinical microbiological training in the isolation of pathogens from acutely ill febrile patients conducted at Sulianti Soeroso Infectious Diseases Hospital (Jakarta), Friendship Hospital (Jakarta), Cipto Mangunkusumo Hospital (Jakarta), Hasan Sadikin Hospital (Bandung), and the National Police Hospital (Jakarta)
• Clinical microbiological training in BACTEC screening of blood for blood-borne pathogens, including the isolation of Salmonella typhi and interstate shipment of specimens to reference laboratories conducted at Adam Malik Hospital (Medan, Sumatra) and Hasanuddin University (Makassar, Sulawesi);
• Study design, protocol development, clinical research team training and clinical chemistry & microbiology quality assurance training conducted in support of initial execution of 2-year systematic survey of etiologies among acutely ill febrile patients at Cipto Mangunkusumo Hospital (Jakarta) and Hasan Sadikin Hospital (Bandung);
• Laboratory training in automated extraction of DNA from clinical specimens conducted at the Eijkman Institute for Molecular Biology (Jakarta);
• Staff at the National Institute of Health Research and Development (NIHRD) in Jakarta trained on qualitative and quantitative influenza PCR in September 2010;
• Indonesian scientist trained in Mahidol-Oxford Research Unit in Bangkok, Thailand on systematic laboratory analyses of clinical specimens collected in 2008 and 2009 from patients with acute febrile illness from Jayapura, Papua, Indonesia. These patients were predominantly suspect malaria cases but many were (co) infected with dengue or scrub typhus (O. tsutsugamushi);
• Technicians from Jakarta (Cipto Mangungkusumo Hospital) and Bandung (Hasan Sadikin Hospital) were supported to develop a clinical and laboratory algorithm for the efficient application of diagnostics in febrile patients. The research teams also received training in collection, storage and analysis of clinical and laboratory specimens and data;
• Research teams received GCP and GLP training and certifications.
Nepal (Patan Hospital and Patan Academy of Health Sciences, Kathmandu)
• Training of Microbiologists including technicians and PhD students (Abhilasha Karkey, Amit Arjyal, Sabina Dongol) from the Patan Academy of Health Sciences in Kathmandu on site and in HCMC.
• Workshop on Ethics and Research Governance in Patan Academy of Health Sciences Kathmandu Nepal (run by OXTREC and Laura Merson) November 2010;
• Workshops on Clinical Trials and Clinical Research Methodology;
• Training for PhD, Masters and Technical staff in country and internationally – including 2 Masters in Oxford, one in Novartis Institute of Vaccines in Sienna, 4 PhD students;

Staff exchanges
• 1 PhD student from Vietnam was invited to Mill Hill, UK to learn reverse engineering of influenza viruses and to the AMC to learn virus culture on differentiated human airway epithelium.
• 1 MSc student from Vietnam was trained in application and analysis of 454-VIDISCA at AMC on samples from patients with encephalitis of unknown origin from Vietnam, Nepal, Cambodia and Bangladesh.

Training of Kumasi site personnel by University Bon Medical Center
UBMC trained the technicians at the Kumasi Centre for Collaborative Research at Kumasi, Ghana in cell culture, virus isolation, PCR-technologies and serology. PhD students received in-depth training with emphasis on PCR technologies. At the institute, assistance was given in logistics, structuring of the laboratories and maintenance of laboratory equipment especially towards the recently opened BSL-3 facility. Partner 2 UBMC (University Bonn Mecdical Center) arranged a transfer of knowledge by implementing diagnostic real-time PCRs at the University Hospital of Kumasi. In addition two Ghanaian students (Michael Owusu and Richard Larbi) visited UBMC for several months to learn PCR-technologies and serology.

Technology exchange

VIDISCA-454 training by 8-AMC
The VIDISCA-454 training at AMC included technicians, no training on samples collection was performed, but only on techniques (VIDISCA, VIDISCA pretreatment, 454 sequencing), and one training on site specifically addressed techniques to investigate animal (wild life) samples. Training into VIDISCA-454 covered amplification via VIDISCA, a subsequent 454 run and introduction into data analysis in case the trainee is at the AMC for one month. A one week training includes VIDISCA-amplification (3 days) and data analysis (2 days). The VIDISCA-amplified samples are 454-sequenced at a later date by the AMC. This course is practical for those labs that have 454 sequencing up and running in their laboratory. We performed 2 one week courses and partner FLI and partner WTSI joined the courses. A one month training was provided for partner UOXF. The courses were attended by bioinformaticians, technicians, senior scientists and master students (see table 1). The pre-treatment of samples prior to VIDISCA amplification is crucial; therefore AMC trained technicians on site to perform proper sample handling and preparation for VIDISCA-454 (North Carolina College of Veterinary Medicine, USA). In the fourth year of EMPERIE one extended course was given to Dipartimento di Scienze biomediche per la salute, Università degli Studi di Milano, Milan, Italy. The course covered sample pre-treatment, VIDISCA amplification, next generation sequencing via Roche 454 FLX sequencing and data analysis. In the fifth year of EMPERIE one extended course was given to two post docs from Pakistan: Department of Virology, National Institute of Health, Islamabad, Pakistan. The course covered sample pre-treatment, VIDISCA amplification, next generation sequencing via Roche 454 FLX sequencing, data analysis, and Rolling Circle amplification.

Training on 454 NGS Optimization by FLI
In February 2012, Marta Canuti, Bas Oude Munnink, and Martin Deijs from AMC were trained for one week at the FLI in the laboratory for Next Generation Sequencing and Microarray diagnostics in optimization of 454 sequencing. Topics of the training included preparation of cDNA libraries, shotgun library preparation from DNA and RNA, and reliable library quantification using qPCR. Moreover, possible targets for optimization of VIDISCA-454 were identified which will allow for further optimization of this procedure. In addition, the trainees were introduced in-depth into the software pipeline for rapid taxonomic individual read classification developed at the FLI in the course of another project. Building on this training and the previous training of FLI colleagues (M. Scheuch and M. Reimann) at the AMC we established a very fruitful ongoing collaboration.

Ad-hoc Training on Schmallenberg and MERS CoV diagnostics
• FLI provided training to Chinese delegates Prof. Wu Shaoqiang and Prof. Zhang Yongning, Chinese Academy of Inspection and Quarantine (CAIQ) FLI Insel Riems from 11th to 22nd March 2013 about Schmallenberg virus diagnostics.
• A team of three UBMC people (Dr. Marcel Müller, Benjamin Meyer, Sebastian Brünink) visited the Regional lab in Riyadh, Kingdom of Saudi-Arabia to implement and establish serological assays for MERS-CoV antibody detection. Christian Drosten gave counsel to the Ministry of Health in Saudi Arabia and validated the local PCR diagnostic laboratories.
• A research scientist from College of Public Health and Health Professions, and Emerging Pathogens Institute, University of Florida, visited HKU within the period 5. He was trained to conduct pseudoparticle virus neutralisation assays for serosurveillance of MERS coronavirus.
• Similarly, a research scientist from the National Research Centre, Giza, Cario, Egypt visited HKU and was trained in molecular detection and serological assays for MERS-CoV.




Potential Impact:
The call topic HEALTH-2007-2.3.3-9. to which EMPERIE responded back in 2007, stated the following expected impact:

“Expected impact: The results of research in this area will integrate European scientific excellence and make Europe better prepared for emerging epidemics: Earlier detection and identification of new pathogens will give scientists and public health authorities precious time to prepare and implement containment and emergency measures”

At the outset of EMPERIE, its main envisaged impact was that it would establish a network of centres of excellence with the capability to anticipate on potential infectious threats and to identify and characterize existing, unrecognized and emerging pathogens responsible for outbreaks in a significantly shorter time than before. The HEALTH work programme 2007 justifiably emphasized that “….while the current situation justifies a strong focus on the threat of pandemic influenza, efforts will also be made to strengthen the European research capacity for other potential causes of infectious epidemics and to foster networking of scientists with expertise in the early identification of new pathogens – where appropriate in collaboration with scientists from affected third countries.”

Now, at the end of the project period, EMPERIE can state with confidence that is has indeed achieved this desired impact on the European research capacity to rapidly respond to emerging epidemics, creating a internationally embedded network of research groups that collaborate and share their technology and expertise in pursuit of early identification of new pathogens. This EMPERIE network has a major impact at the scientific/technological level as well as on Europe’s research response capacity to emerging infectious disease outbreaks of (potential) public health concern.
Scientific and Technological impact
The impact of EMPERIE on the scientific and technological (S&T) aspects of emerging and re-emerging infectious diseases has been considerable and remains to be made in the coming years. Testimony to this S&T impact are the 240+ scientific publications in high raking journals (including Nature, Science, Cell, Nature communication, The Lancet Infectious Diseases, New England Journal of Medicine, PLoS Pathogens and PNAS) to which EMPERIE has made a contribution.

In general, EMPERIE has made an S&T impact in three areas:
- Virus discovery: Advancing the methods and tools for rapid detection and characterization of novel viruses (ranging from first discovery (e.g. SBV and MERS-CoV) to known viruses in new hosts);
- Containment strategy: Advancing our understanding of the complex molecual mechanisms involving host-switch of zoonotic viruses (using Coronaviruses as a model);
- Computational tools and Epidemiological models: Novel mathematical/epidemiological models for emerging outbreks research.

Virus discovery
The major impact of EMPERIE is the development of a powerful pipeline for optimized sample preparation, examination, and, very importantly, data processing for the screening of samples from cases of undiagnosed illness for potential disease entities and their subsequent in-depth characterization. Various sample collections from humans and animals, healthy and sick, were used for virus discovery programs. Within the framework of EMPERIE, we were able to set up routine diagnostics using NGS for the fast and reliable detection and characterization of unexpected and/or novel pathogens. The detection and characterization of Schmallenberg virus in 2011 and MERS-CoV in 2012 were both proof of the usefulness of NGS to detect novel or unexpected pathogens and simultaneously enable their basic characterization as well as to provide a foundation to set up targeted diagnostics tackling the novel pathogen. EMPERIE identified a plethora of novel viruses in mammals, insectivores and arthropods including bat- and hedgehog-related MERS-CoV in Europe and Africa, novel European SARS-related bat CoV, bat-related paramyxo-, hepadna- and hepeviruses, ancestral hepaciviruses in rodents, parvoviruses in humans and bats as well as novel insect viruses (Flavi-, Bunya-, Nidoviruses) including a sylvatic ancestor of St. Louis encephalitis virus (SLEV), calicivirus, anelloviruses, and a cyclovirus in humans, picobirnaviruses in humans and pigs, an astrovirus in humans and deer, an enteric coronavirus and hepatitis E virus in ferrets, arenaviruses in boid snakes, an adenovirus in gulls, a novel b19-like parvovirus in a harbor seal, novel hepaciviruses in rodents, hepadnaviruses in bats, parvovirus and a hepevirus in red foxes, and papillomavirus and a betaherpesvirus in reindeer. The full genome of a previously identified dolphin rhabdovirus was sequenced, and Merkel cell polyomavirus was for the first time described in follicular spicules in Multiple Myeloma. During the course of EMPERIE, the NGS virus discovery protocols and the bioinformatics pipelines continued to be optimized, such that they are now operating smoothly and efficiently.

The workflows that were put in place with the aid of EMPERIE are also the basis for further optimization and sustainable use of NGS and microarrays for pathogen detection. Over the course of the 5 years we have established the following tools and technologies:
• Optimized conventional and 454-based sequencing pipelines and bioinformatics pipelines;
• Antibody-capture techniques;
• Methods for appropriate sample collection and handling for deep sequencing applications. We defined functional methods full genome sequencing from blood samples, fecal samples and respiratory samples.
• Methods for computationally-intensive de novo assembly of the resulting short read data into properly annotated consensus genomes suitable for public database deposition. De novo assembly is essential for generating full virus genomes from short read data in the absence of a close reference genome.
• Computational algorithms to detect unknown virus sequences amidst large sequence data sets (Cotten et al. 2014 PLOS One). These methods are required for processing the large sequence sets generated by by Illumina methods.
• Computational algorithms for assessing the validity of genome assembly from short read data, especially open reading analysis and Computational tools for extracting specific coding regions from large numbers of related genomes.
• Computational algorithms for designing primers sets for full genome sequencing and for assessing the function of these primers sets by virtual PCR. These tools were used for all of the MERS-CoV full genome sequencing, for norovirus full genome sequencing (Cotten et al., manuscript submitted) and respiratory syncytial virus full genome sequencing (Naigoti et al., manuscript in preparation).
• Computational algorithms for assessing the statistical supported virus transmission events based on full genomes, dated samples and an estimated evolutionary rate for the virus. These tools were used to assess MERS-CoV transmission in a large hospital outbreak (Assiri et al. 2013 NEJM) and to assess likely tranmsision patterns of the Hafr-Al-Batin clade of MERS-CoV (Memish et al., 2014 IJID).

Coronavirus research to novel vaccine candidates
The impact on the scientific state of the art in this area has been significant, as we have demonstrated that it was possible to generate recombinant vaccine candidates for both SARS-CoV and MERS-CoV in three months, using synthetic biology and reverse genetics technology, based on the assembly of infectious cDNA clones of these coronaviruses using bacterial artificial chromosomes and the deletion of genes essential for virus virulence. EMPERIE researchers have shown that the vaccine candidates engineered provided homologous and heterologous protection against the challenge with virulent SARS-CoV in three animal model systems. Therefore the consortium capacity to respond to public health events is very high, taking into account the difficulties in the generation of infectious cDNA clones for coronaviruses with the largest RNA genome know for an RNA virus. The impact capabilities and skills at low resource countries is also high as we have also developed antivirals to increase the survival of SARS-CoV patients by the administration of inhibitors of NF-kB signaling pathway, which inhibited the virus induced exacerbated inflammation and death.

Computational software tools for emerging outbreaks research:
EMPERIE research has contributed to the development and launch of a range of software tools to improve capacity to plan for and respond to emerging infectious disease outbreaks. The Global Epidemic Simulator provides a platform for pandemic scenario analysis and the modelling of potential mitigation strategies. The Outbreaker and EpiEstim tools allow for data collected early in outbreaks to be analysed rapidly to enhance situational awareness and to provide short term projections of outbreak trajectory.
These and other methods were deployed extensively during the EMPERIE project to assist public health partners (including WHO, US CDC, UK HPA/PHE, ECDC, China CDC and a number of governments – UK, US, China, KSA) in responding to the 2009 H1N1 influenza pandemic and the ongoing MERS-CoV outbreak. Staff were heavily involved in providing scientific advice for both outbreaks.

In 2012, the University of Cambridge received designation as a World Health Organization (WHO) Collaborating Centre for Modelling Evolution and Control of Emerging Infectious Diseases. This designation was primarily in recognition and support of the contribution to public health by the Centre for Pathogen Evolution, in the Department of Zoology.Our Centre was recruited to support WHO programs in Epidemic and Pandemic Alert and Response by providing a suite of computational tools for analyzing the evolution of re-emerging and emerging pathogens. Such tools are applicable to all pathogens and are of high value for surveillance, vaccine strain selection, and support of decision-making. Our Centre will provide services and training that will enhance the value of existing surveillance, show how to improve current surveillance, and allow data to be shared and combined with WHO, its collaborating centers and other parties identified by WHO.

The tools provided by our Centre are state-of-the-art, at the forefront of research, and they have proven to be of high value to WHO and laboratories identified by WHO worldwide. In addition, our Centre will provide WHO with expertise in pandemic response, clinical management, immunopathology, international/travel medicine, and enteroviral and other emerging encephalitides. Our Centre will also provide expertise on modelling of infectious and vector-born diseases. Additionally, our Centre will develop software for clinical care and data collection, particularly in low-resource settings, and for the development of models for natural ventilation in healthcare facility design. Software, other tools, and expertise will be provided to support activities in other Collaborating Centers and to build capacity in developing countries. Specific research and development and service projects will be undertaken as directed by WHO and the Global Alert Response Network. Drawing on research groups and institutes within the University’s Veterinary, Biological and Clinical Schools, our Centre also provides the WHO with expertise in pandemic response, modelling of infectious and vector-borne diseases, clinical management, immunopathology, international/ travel medicine, enteroviruses and other emerging encephalitides.

To date, antigenic cartography has been used for human swine, equine and avian influenza, rabies viruses, foot and mouth disease viruses, and most recently dengue and enterovirus -71.
We have founded the Antigenic Cartography Project, an international collaborative consortium to systematically characterise antigenic variation in dengue viruses. One thousand dengue viruses are being tested with non-human primate antisera to generate a large dataset of neutralisation titres, and antigenic cartography used to probe the forces underlying antigenic evolution. The antigenic map will allow us to test: (1) how well serotypes as currently defined match observed antigenic clusters on the map, (2) if antigenic subtypes (antigenic clusters) exist within serotypes and (3) how quantitatively different antigenic clusters are.

In Taiwan, the reemergence of EV-71 in 2008 resulted in the largest outbreak in the country for the past 11 years. Several molecular epidemiology studies of EV-71 have revealed the genetic evolution of EV-71 isolates by using partial- or whole-genome sequencing. However, little had been reported about the antigenic evolution of EV-71. In a 2009 study in collaboration with colleagues in Taiwan, we used antigenic cartography to analyse samples from over 15 years of outbreaks. This analysis revealed that the re-emerging EV-71 strains formed a separate cluster which was antigenically distinct from previous strains. Vaccines against EV-71 are now in development, and we are involved in the evaluation of EV-71 vaccine clinical trials and selection of vaccine strains.
Socio-economic impact and wider societal implications of the project
The wider socio-economic and societal impact of EMPERIE stems from its impact on Europe’s research preparedness and response to emerging outbreaks. This impact has been shown in several occasions during the course of EMPERIE, notably in response to the influenza H1N1 pandemic in 2009, in response to the MERS-CoV outbreak in 2012 and in response to zoonotic inflrunza outbreaks (H5N1, H7N9)

Response to H1N1 pandemic:
In response to the 2009 influenza A/H1N1 virus pandemic, P1 EMC worked closely with the US CDC and WHO network and EMPERIE partners on the initial genetic, antigenic, and further phenotypic characterization of the pandemic virus. Within weeks, ferret infection models were developed, to describe the viral properties with respect to virulence and airborne transmission and to evaluate influenza vaccine candidates. Using a macaque model, it was demonstrated that the “cytokine storms” that were associated with severe disease during the 1918 H1N1 pandemic, did not apply to 2009 H1N1 virus. Virus attachment studies were conducted to explain why the 2009 pandemic viruses replicated more efficiently in the lower airways of mammals as compared to seasonal influenza viruses. Further studies were conducted to show that the H1N1 virus could acquire increased virulence upon reassortment or acquisition of mutations in the HA and PB2 genes. One of these mutations (D222G in HA) was subsequently identified in infected patients with severe lower airway disease, the explanation for which was already available from our studies. Thus, EMPERIE succeeded in providing support to national and international organizations throughout the first year of the pandemic, in particular being able to generate solid scientific data to answer the questions that were raised by authorities, the public, and medical and scientific communities.

Response to MERS CoV:
A family-wide RT-PCR test to detect paramyxoviruses was developed by P1 EMC, and used to screen large panels of human and avian sample collections. It was this test that prompted Prof. A.M. Zaki in Jeddah to work with P1 EMC on the characterization of what later became known as the MERS-coronavirus. At the time of the first report of MERS-COV, algorithms were in place to design primers for full genome sequencing from small amounts of clinical material and these methods were quickly applied to generate a full genome from the first UK case of MERS. Furthermore as the epidemic continued, the EMPERIE consortium provided a framework for a rapid analysis and interpretation of the resulting MERS-CoV sequence data. The first clinical description of MERS and initial virus genetic sequence data were rapidly released to the public, and the full virus genome sequence was generated and released in a matter of weeks. A diagnostic PCR was developed and published within one week. Serologic diagnostic assays could be provided within one month. Positive controls were sent to 200 laboratories world-wide via the EU-funded infrastructural program EVA.
Tropism studies made clear that the MERS-CoV replicated very well in human cells, but did not rely on ACE, the SARS-CoV receptor. Rather, we showed that DPP4 was the cellular entry receptor for MERS-CoV, and that adenosine deaminase can function as a natural entry antagonist. We showed that macaques (but not ferrets) could be used to study MERS-CoV infections, and continued to search for better animal models, guided by homology between DPP4 of humans and various animal species. In collaboration with the EU SILVER program, cyclosporin A and interferon-alpha were identified as potential drugs to inhibit MERS-CoV replication. PCR and serologic assays formed the basis for MERS-CoV case-contact and serosurvey studies, description of the clinical picture of MERS patients and enabled the rapid discovery of putative MERS-CoV hosts all within one year after emergence of MERS-CoV.
Protein micro-arrays were developed for the detection of antibodies against MERS-CoV in various human and animal serum collections. The arrays were instrumental to identify dromedary camels as a potential host for MERS-CoV in Jordan, Qatar, UAE, and elsewhere. Indeed, subsequently it was shown that dromedary camels were infected with the virus. We further showed that viruses related to MERS-CoV were present in bats in Ghana and Europe. From these studies, the working hypothesis emerged that the MERS-CoV emerged from bats to humans, with dromedary camels as intermediate host, with major implications to the outbreak response in affected countries.

Response to Other influenza outbreaks:
In response to zoonotic influenza virus outbreaks (H5N1, H7N7, H7N9), we used the NGS pipelines and bioinformatics tools developed within EMPERIE, to characterize genetic changes in zoonotic influenza A viruses that were associated with increased virulence and adaptation to humans (H7N7) and increased airborne transmission between ferrets (H5N1). With respect to the latter, it was shown that only 5 amino acid substitutions were sufficient to make an avian A/H5N1 virus airborne transmissible between ferrets, and that some of these mutations emerged rapidly within ferrets. Following the finding that avian influenza A/H5N1 might mutate and/or reassort to be transmissible between mammals via respiratory droplets, we pursued various lines of investigations pertaining to understanding, assessing, and potentially mitigating, the risk of avian A/H5N1 influenza viruses emerging in a mammalian host. Specifically we developed computational pipeline to create “Evergreen” phylogenetic trees of H5 influenza viruses tracking substitutions of concern as well as evergreen phylogenetic trees of H5, H7 and H9 subtypes.

We performed a statistical analysis of the frequency of mutations found in avian and human A/H5N1 influenza infections, we identified dozens of currently untested mutations in each gene segments which are preferentially found in human H5 influenza infections. We identified amino acid substitutions in A/H5N1 strains which may be preferentially selected within mammalian hosts and pose an increased risk to human infections, and performed computational studies to estimate the effects of mutations in HA on the binding of human-like a 2,6 glycans.

When the H7N9 virus emerged in China, this virus was rapidly shown to already contain some of the adaptive changes that were also seen in the human-adapted H7N7 viruses, and in the airborne transmissible H5N1 virus. We performed transmission studies in ferrets, to show that the H7N9 virus was transmitted via the airborne route in contrast to other avian influenza viruses, but far less efficiently than pandemic and seasonal influenza viruses. EMPERIE NGS pipelines and bioinformatics tools again demonstrated their application to characterize the emerging H7N9 viruses, and the protein micro-arrays demonstrated their usefulness to measure antibody levels against H7N9 virus in the human population.

Furthermore, novel diagnostics for the raid diagnosis of important influenza viruses (e.g. H1N1pdm and H7N9) were developed within EMPERIE, transferred as SOPs to national and international reference laboratories, and also published in peer reviewed journals. Finally, novel avian influenza viruses (HPAIV H5N2 Asia, H1N1pdm, avian influenza virus H7N9) were in vitro and in vivo characterized to allow further insights for risk analyses or for intervention strategies like novel types of vaccines.

In addition to our contribution to scientific understanding of the phylogeny and potential evolution of these pathogens, EMPERIE PIs advised several governmental agencies in Europe and the United States on the safety concerns regarding the risks of a highly pathogenic virus evolving in nature, and the risk of nefarious use of published research on these pathogens. At the National Academy Institute of Medicine in Washington DC (18th – 19th March 2013), Derek Smith spoke on: “What have we learned in the past 10 years on predicting the pandemic potential of zoonotic influenza viruses?” The talk also looked at aspects such as DURC and the relationship that must exist between science and security. At a highly-specialised, restricted-access meeting involving senior officials from several countries, and organisations such as the Department for Homeland Security, Derek Smith spoke on the subject of “Dual-Use Biology – how to balance open science and security”. The meeting at Wilton Park took place between 15th – 18th September 2013.


The 2010 Cochrane review on efficacy, effectiveness and safety of influenza vaccination in the elderly by Jefferson et al. covering dozens of clinical studies over a period of four decades, confirmed vaccine safety, but found no convincing evidence for vaccine effectiveness (VE) against disease thus challenging the ongoing efforts to vaccinate the elderly. This publication had a negative impact on the uptake of the seasonal influenza vaccine by the elderly in Europe (Jefferson T, Di Pietrantonj C, Al-Ansary LA, Ferroni E, Thorning S, Thomas RE.Vaccines for preventing influenza in the elderly. Cochrane Database Syst Rev 2010;(2), http://dx.doi.org/10.1002/ 14651858.CD004876.pub3)
In collaboration with Walter Beyer of EMC we re-arranged the very same data according to a biological and conceptual framework based on the basic sequence of events throughout the ‘patient journey’ (exposure, infection, clinical outcome, observation) and using broad outcome definitions and simple frequency distributions of VE values. This approach produced meaningful predictions for VE against influenza-related fatal and non-fatal complications (average ∼30% with large dispersion), typical influenza-like illness (∼40%), disease with confirmed virus infection (∼50%), and biological vaccine efficacy against infection (∼60%), under conditions of virus circulation. We could also demonstrate a VE average around zero in the absence of virus circulation, and decreasing VE values with decreasing virus circulation and increasing antigenic drift.
We regard these findings as substantial evidence for the ability of influenza vaccine to reduce the risk of influenza infection and influenza-related disease and death in the elderly.

Impact on local capacity levels in low resource countries
A major impact on basic science and clinical diagnostics and research has been through providing a continuous good clinical practice (GCP), clinical diagnostics and clinical research training program for all our clinical and laboratory partners in Asia including certification and follow-up/refreshment training (in Vietnam, Nepal, China and Indonesia). This has been achieved through courses on site, central courses at OUCRU, short (weeks) and long (up to 3 months) trainings at OUCRU for clinical and laboratory staff, MSc and PhD student exchanges etc. Funds were also used to build and refurbish clinical diagnostic and research laboratories in several sites in Vietnam, Nepal and Indonesia with continuing support for QA/QC further development. A serumbank was established during the H1N1pdm09 response, which is available as a resource for EMPERIE. Another longitudinal age-stratified serumbank was established in Nepal. During the H1N1pdm09 response we were able to quickly disseminate molecular diagnostics across national and regional networks of hospitals and Oxford units and to adjust ongoing community cohort studies to include epidemiological, virological and immunological H1N1pdm09 endpoints. Technologies and knowledge was shared with the sites, worldwide, of which it can be expected that they may encounter a novel outbreak of a virus. Several sites, e.g. Asia (Taiwan, China, Viet Nam), Pakistan were trained into virus detection and the next generation sequencing virus discovery technique VIDISCA-454. Besides the dissemination of knowledge, strengths of EMPERIE partners were combined by sharing details on cDNA library preparation techniques, sequencing tools, and analysis scripts.

In Africa, EMPERIE helped to establish novel BSL2 and 3 labs in Ghana and trained personnel on-site and in Bonn. UBMC is currently giving scientific counsel and support to different laboratories in the Kingdom of Saudi Arabia in order to implement on-site MERS-CoV diagnostics. In the course of the program, P11 WTSI trained and worked with visiting scientists from lower resource countries including Charles Naigoti (Kenya, RSV deep sequencing) and Phan Vu Tra My (Vietnam, norovirus rotavirus sequencing, virus discovery) and Velislava Petrova (Bulgaria, norovirus sequencing, 1 year sandwich studentship).

List of Websites:
Project Website: www.emperie.eu
Contact details: Prof. Dr. ADME Osterhaus, Department of Viroscience, Erasmus University Medical Center
Wytemaweg 80, Room Ee 17-26, 3015 CN Rotterdam, the Netherlands
Te;" [phone], E-mail: [e-mail]

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• final1-virus-discovery-in-emperie.pdf