Characterization and full genome sequencing of a velogenic Newcastle disease virus (NDV) strain Ck/IR/Beh/2011 belonging to subgenotype VII(L).

Avian avulavirus 1, better known as Newcastle disease virus (NDV), causes substantial loss to the poultry industry in many developing countries. In this study we have characterized and fully sequenced the genome of a velogenic NDV strain named Beh (Ck/IR/Beh/2011) that has been used in our lab for a number of challenge and immunological studies over the last few years. This strain was isolated from poultry in the city of Behshahr, Mazandaran Province, Iran after an outbreak reported in the region in 2011. The intracerebral pathogenicity index (ICPI) was 1.8 in one-day-old chicks, characteristic of a velogenic NDV strain. Later, the virus was purified using a sucrose gradient centrifugation and used for next-generation sequencing (NGS). The results showed that the genome length was 15192 bp, similar to those of class II velogenic strains. In addition, the phylogenetic analysis based on the complete F gene showed that the NDV strain Beh has an F protein cleavage site 112RRQKR↓ F117 and belongs to the newly identified subgenotype VII(L). Based on the biological and genetic characterization, NDV strain Beh is now the best documented reference isolate representing the novel subgenotype VII(L) in Iran. Keywords: NDV; NGS; velogenic strain, subgenotype VII(L); phylogenetic analysis.

Rapid and simple detection of foot-and-mouth disease virus: Evaluation of a cartridge-based molecular detection system for use in basic laboratories.

Highly contagious transboundary animal diseases such as foot-and-mouth disease (FMD) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen-side. Here, we report on a study to evaluate a fully automated cartridge-based real-time RT-PCR diagnostic system (Enigma MiniLab® ) for the detection of FMD virus (FMDV). The modular system integrates both nucleic acid extraction and downstream real-time RT-PCR (rRT-PCR). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV, and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter-laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge-based automated real-time RT-PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world.

Foot-and-mouth disease virus serotype SAT1 in cattle, Nigeria.

The knowledge of foot-and-mouth disease virus (FMDV) dynamics and epidemiology in Nigeria and the West Africa subregion is important to support local and regional control plans and international risk assessment. Foot-and-mouth disease virus serotype South African territories (SAT)1 was isolated, identified and characterized from an FMD outbreak in cattle in Nigeria in 2015, 35 years after the last report of FMDV SAT1 in West Africa. The VP1 coding sequence of the Nigerian 2015 SAT1 isolates diverges from reported SAT1 topotypes resulting in a separate topotype. The reporting of a novel FMDV SAT1 strain in the virus pool 5 (West and Central Africa) highlights the dynamic and complex nature of FMDV in this region of Africa. Sustained surveillance is needed to understand the origin, the extent and distribution of this novel SAT1 topotype in the region as well as to detect and monitor the occurrence of (re-)emerging FMDV strains.

Detection and Molecular Characterization of Foot and Mouth Disease Viruses from Outbreaks in Some States of Northern Nigeria 2013-2015.

Control measures for foot and mouth disease (FMD) in Nigeria have not been implemented due to the absence of locally produced vaccines and risk-based analysis resulting from insufficient data on the circulating FMD virus (FMDV) serotypes/strains. In 2013-2015, blood and epithelial samples were collected from reported FMD outbreaks in four states (Kaduna, Kwara, Plateau and Bauchi) in northern Nigeria. FMDV non-structural protein (NSP) seroprevalence for the outbreaks was estimated at 80% (72 of 90) and 70% (131 of 188) post-outbreak. Antibodies against FMDV serotypes O, A, SAT1, SAT2 and SAT3 were detected across the states using solid-phase competitive ELISA. FMDV genome was detected in 99% (73 of 74) of the samples from FMD-affected animals using rRT-PCR, and cytopathic effect was found in cell culture by 59% (44 of 74) of these samples. Three FMDV serotypes O, A and SAT2 were isolated and characterized. The phylogenetic assessments of the virus isolates showed that two topotypes of FMDV serotype O, East Africa-3 (EA-3) and West Africa (WA) topotypes were circulating, as well as FMDV strains belonging to the Africa genotype (G-IV) of serotype A and FMDV SAT2 topotype VII strains. While the serotype O (EA-3) strains from Nigeria were most closely related to a 1999 virus strain from Sudan, the WA strain in Nigeria shares genetic relationship with three 1988 viruses in Niger. The FMDV serotype A strains were closely related to a known virus from Cameroon, and the SAT2 strains were most closely related to virus subtypes in Libya. This study provides evidence of co-occurrence of FMDV serotypes and topotypes in West, Central, East and North Africa, and this has implication for control. The findings help filling the knowledge gap of FMDV dynamics in Nigeria and West Africa subregion to support local and regional development of vaccination-based control plans and international risk assessment.

Technological advances in veterinary diagnostics: opportunities to deploy rapid decentralised tests to detect pathogens affecting livestock.

Sustainable food production capable of feeding a growing human population is a significant global challenge, and is a priority encompassed within the United Nations Millennium Development Goal to 'eradicate extreme poverty and hunger'. Infectious diseases reduce the productivity of farm animals, and the globalised trade of animals and their products increases the threat of disease incursion. Accurate and rapid diagnostic tests are an essential component of contingency plans to detect, control and eradicate such diseases. Diagnosis involves a 'pipeline' that normally starts with clinical suspicion, followed by collecting samples, transporting specimens to a centralised laboratory setting (e.g. national/international Reference Laboratories), analysing these samples using a range of diagnostic tests and reporting the results. However, the transport of specimens from the field to the laboratory can be a lengthy process that can delay critical decision-making and severely affect the quality of the samples. This important limitation of centralised diagnostic testing has motivated the development of tools for the rapid, simple detection of livestock pathogens. Recent advances in the development of technologies for personalised human medicine have motivated the development of prototype diagnostic tests for a wide selection of diseases of livestock. However, many of these tests are not yet routinely used or commercially available. This paper critically reviews the most promising examples of such assays, and highlights the challenges that remain to transition these tests from applied research and development into routine use.

La production durable de denrées alimentaires pour nourrir une population humaine en constante augmentation constitue un vaste enjeu planétaire ainsi que l'une des priorités définies par les Nations Unies dans le cadre des Objectifs du Millénaire pour le développement visant à « éradiquer l'extrême pauvreté et la faim dans le monde ¼. D'une part, les maladies animales réduisent la productivité des animaux d'élevage ; d'autre part, la mondialisation des échanges d'animaux et de produits d'origine animale intensifie les risques d'incursion de maladies. L'utilisation de tests de diagnostic rapides et fiables est une composante essentielle des plans d'urgence visant à détecter, contrôler et éradiquer ces maladies. Une procédure de diagnostic est généralement constituée de plusieurs opérations, depuis la suspicion clinique, la collecte d'échantillons, leur transport vers un laboratoire central (par exemple un laboratoire de référence national/international), jusqu'à l'analyse de ces échantillons au moyen d'une série de tests diagnostiques et la notification des résultats. Néanmoins, le transport des échantillons depuis le terrain jusqu'au laboratoire est parfois un processus très long qui peut retarder la prise de décisions cruciales, voire compromettre gravement la qualité des échantillons. Cette limitation importante des procédures diagnostiques centralisées a incité à mettre au point des outils permettant une détection rapide et aisée des agents pathogènes affectant le bétail. Les progrès récents accomplis dans les technologies relevant de la médecine humaine personnalisée ont encouragé le développement de prototypes d'épreuves de diagnostic pour nombre de maladies du bétail. Toutefois, plusieurs de ces tests ne sont pas encore utilisés en routine ni disponibles commercialement. Les auteurs font le point sur les exemples les plus prometteurs de ces tests et soulignent les difficultés restant à résoudre pour que ces tests puissent évoluer d'une application en recherche et développement à une utilisation en routine.

El logro de una producción sostenible de alimentos en cantidad suficiente para abastecer a una población humana cada vez más numerosa es una difícil empresa que el mundo tiene ante sí, que además entronca con una de las prioridades plasmadas en los Objetivos de Desarrollo del Milenio de las Naciones Unidas: «erradicar la pobreza extrema y el hambre¼. Las enfermedades infecciosas merman la productividad de los animales de granja, al tiempo que el comercio mundializado de animales y sus derivados amplifica la amenaza de incursiones infecciosas. La existencia de pruebas de diagnóstico rápidas y exactas es un elemento básico de todo plan de emergencia encaminado a detectar, controlar y erradicar esas enfermedades. Las labores de diagnóstico entrañan un «circuito¼ que normalmente empieza con la sospecha clínica, sigue con la obtención de muestras, su transporte a un laboratorio central (como un laboratorio de referencia nacional o internacional) y su análisis mediante diversas pruebas de diagnóstico y culmina con la notificación de los resultados. Sin embargo, el transporte hasta un laboratorio de las muestras obtenidas sobre el terreno es a veces un proceso lento, que puede retrasar la adopción de decisiones cruciales y mermar sensiblemente la calidad de las muestras. Este importante inconveniente derivado de la realización centralizada de pruebas ha llevado a concebir herramientas que permitan detectar de forma rápida y sencilla patógenos presentes en el ganado. Los avances registrados últimamente en la obtención de tecnologías destinadas a la medicina humana personalizada han propiciado también la elaboración de prototipos de pruebas para diagnosticar numerosas enfermedades del ganado, aunque muchas de ellas todavía no se utilizan sistemáticamente ni están comercializadas. Los autores, tras examinar en clave crítica los más prometedores ejemplos de estos nuevos ensayos, señalan las dificultades que aún subsisten para que estas pruebas puedan pasar del ámbito de la investigación aplicada y el desarrollo al de su utilización sistemática.

Next-generation sequencing workflows in veterinary infection biology: towards validation and quality assurance.

Recent advancements in DNA sequencing methodologies and sequence data analysis have revolutionised research in many areas of biology and medicine, including veterinary infection biology. New technology is poised to bridge the gap between the research and diagnostic laboratory. This paper defines the potential diagnostic value and purposes of next-generation sequencing (NGS) applications in veterinary infection biology and explores their compatibility with the existing validation principles and methods of the World Organisation for Animal Health. Critical parameters for validation and quality control (quality metrics) are suggested, with reference to established validation and quality assurance guidelines for NGS-based methods of diagnosing human heritable diseases. Although most currently described NGS applications in veterinary infection biology are not primary diagnostic tests that directly result in control measures, this critical reflection on the advantages and remaining challenges of NGS technology should stimulate discussion on its diagnostic value and on the potential to validate NGS methods and monitor their diagnostic performance.

Les avancées récentes enregistrées en matière de séquençage de l'ADN et d'analyse des données de séquences ont révolutionné la recherche dans de nombreux domaines de la biologie et de la médecine, notamment la biologie des maladies animales infectieuses. Ces nouvelles technologies vont permettre de combler le fossé qui séparait la recherche fondamentale du laboratoire de diagnostic. Après avoir défini l'intérêt diagnostique des applications du séquençage de nouvelle génération (SNG) ainsi que leurs finalités dans le domaine de la biologie des maladies animales infectieuses, les auteurs examinent leur compatibilité avec les méthodes et les principes actuels de validation recommandés par l'Organisation mondiale de la santé animale. Ils proposent quelques paramètres critiques de validation et de contrôle qualité (mesure de la qualité), en se référant aux lignes directrices de validation et d'assurance qualité des techniques diagnostiques basées sur le séquençage de nouvelle génération visant à détecter les maladies humaines héréditaires. Certes, la plupart des applications actuelles des méthodes de séquençage de nouvelle génération en biologie des maladies animales infectieuses ne constituent pas des tests de diagnostic primaire (dont dépendent directement les décisions de contrôle sanitaire) ; toutefois, l'analyse critique proposée par les auteurs sur les avantages de cette technologie et sur les difficultés restant à résoudre devrait ouvrir la voie à des discussions sur l'intérêt diagnostique des méthodes recourant au séquençage de nouvelle génération ainsi que sur les perspectives de validation et de contrôle de leurs performances diagnostiques.

Los recientes avances en los métodos de secuenciación del ADN y el análisis de los datos de secuencias han revolucionado la investigación en muchos ámbitos de la biología y la medicina, entre ellos la biología de las infecciones veterinarias. Las nuevas técnicas encierran la promesa de reducir la distancia entre el mundo de la investigación y los laboratorios de diagnóstico. Tras explicar el interés que pueden revestir las aplicaciones de la secuenciación de próxima generación y su posible uso con fines de diagnóstico de infecciones veterinarias, los autores examinan su compatibilidad con los principios y métodos de validación que tiene definidos la Organización Mundial de Sanidad Animal. Asimismo, proponen parámetros básicos para su validación y control de calidad (medición de la calidad), haciendo referencia a las pautas ya establecidas de validación y garantía de calidad de métodos de diagnóstico de enfermedades humanas hereditarias que reposan en técnicas de secuenciación de próxima generación. Aunque la mayoría de las aplicaciones de estas técnicas actualmente descritas en biología de las infecciones veterinarias no constituyen pruebas primarias de diagnóstico, esto es, cuyos resultados puedan inducir directamente medidas de control, esta crucial reflexión sobre las ventajas que entraña la secuenciación de próxima generación y los problemas que aún plantea debería alentar un debate sobre su interés para labores de diagnóstico y sobre la posibilidad de validar métodos basados en estas técnicas y de hacer un seguimiento de la eficacia diagnóstica que ofrezcan.

Using genomics for surveillance of veterinary infectious agents.

Factors such as globalisation, climate change and agricultural intensification can increase the risk of microbial emergence. As a result, there is a growing need for flexible laboratory-based surveillance tools to rapidly identify, characterise and monitor global (re-)emerging diseases. Although many tools are available, novel sequencing technologies have launched a new era in pathogen surveillance. Here, the authors review the potential applications of high-throughput genomic technologies for the surveillance of veterinary pathogens. They focus on the two types of surveillance that will benefit most from these new tools: hazard-specific surveillance (pathogen identification and typing) and early-warning surveillance (pathogen discovery). The paper reviews how the resulting sequencing data can be used to improve diagnosis and concludes by highlighting the major challenges that hinder the routine use of this technology in the veterinary field.

La mondialisation, le changement climatique et l'intensification de la production agricole sont des facteurs pouvant entraîner un risque accru d'émergence d'agents pathogènes infectieux. En conséquence, il est impératif de disposer d'outils de surveillance souples d'emploi au sein du laboratoire pour identifier, caractériser et contrôler rapidement les maladies émergentes et ré-émergentes au niveau mondial. Un grand nombre d'outils sont disponibles, mais ce sont les technologies de séquençage qui ont ouvert une nouvelle ère de la surveillance des agents pathogènes. Les auteurs passent en revue les différentes applications potentielles des technologies génomiques à haut débit dans le domaine de la surveillance des agents pathogènes vétérinaires. Ils examinent plus particulièrement les deux types de surveillance auxquels ces nouveaux outils vont le plus contribuer : la surveillance axée sur les dangers (identification et caractérisation des agents pathogènes) et la surveillance précoce en amont (découverte des agents pathogènes). Après avoir expliqué les perspectives d'amélioration des moyens diagnostiques en ayant recours à l'utilisation des données de séquençage, les auteurs soulignent les principales difficultés qui entravent l'utilisation systématique de cette technologie dans le domaine vétérinaire.

Factores como la mundialización, el cambio climático o la intensificación de la agricultura pueden acrecentar el riesgo de que surjan nuevos patógenos microbianos. De ahí la creciente necesidad de contar con herramientas flexibles de vigilancia en laboratorio, que permitan identificar, caracterizar y vigilar enfermedades (re)emergentes de importancia mundial. Aunque ya existen muchas herramientas, las novedosas técnicas de secuenciación han inaugurado una nueva era en la vigilancia de patógenos. Los autores examinan las posibles aplicaciones de las técnicas genómicas de alto rendimiento en el terreno de la vigilancia de patógenos veterinarios, centrándose en los dos tipos de vigilancia para los que más útiles serán estas nuevas herramientas: la vigilancia en función del riesgo específico (identificación y tipificación de patógenos) y la vigilancia de pronta alerta (descubrimiento de patógenos). Por último, tras explicar cómo se pueden utilizar los datos de secuenciación resultantes para mejorar el diagnóstico, los autores concluyen destacando los principales problemas que dificultan un uso sistemático de estas técnicas en el ámbito de la veterinaria.

Next-generation sequencing shows West Nile virus quasispecies diversification after a single passage in a carrion crow (Corvus corone) in vivo infection model.

West Nile virus (WNV) occurs as a population of genetic variants (quasispecies) infecting a single animal. Previous low-resolution viral genetic diversity estimates in sampled wild birds and mosquitoes, and in multiple-passage adaptation studies in vivo or in cell culture, suggest that WNV genetic diversification is mostly limited to the mosquito vector. This study investigated genetic diversification of WNV in avian hosts during a single passage using next-generation sequencing. Wild-captured carrion crows were subcutaneously infected using a clonal Middle-East WNV. Blood samples were collected 2 and 4 days post-infection. A reverse-transcription (RT)-PCR approach was used to amplify the WNV genome directly from serum samples prior to next-generation sequencing resulting in an average depth of at least 700 × in each sample. Appropriate controls were sequenced to discriminate biologically relevant low-frequency variants from experimentally introduced errors. The WNV populations in the wild crows showed significant diversification away from the inoculum virus quasispecies structure. By contrast, WNV populations in intracerebrally infected day-old chickens did not diversify from that of the inoculum. Where previous studies concluded that WNV genetic diversification is only experimentally demonstrated in its permissive insect vector species, we have experimentally shown significant diversification of WNV populations in a wild bird reservoir species.

Quantification of rHVT-F genome load in feather follicles by specific real-time qPCR as an indicator of NDV-specific humoral immunity induced by day-old vaccination in SPF chickens.

The purpose of this study was to look for a reliable molecular method for confirmation of uptake of recombinant turkey herpesvirus vaccine against Newcastle disease (rHVT-F) and for use as a valuable prediction tool of Newcastle disease virus (NDV)-specific immune response in chickens deprived of maternally derived antibody (MDA). A quantitative real-time polymerase chain reaction (real-time qPCR) specific to rHVT-F was developed. The method was applied to various tissue samples taken from specific pathogen free (SPF) chickens experimentally inoculated at day-old with one dose of rHVT-F vaccine over a 6-week period. Among the tested tissues, the rHVT-F vaccine was detected predominantly in the bursa of Fabricius (BF) and the lung for the first week, followed by a progressive decline from 9 days onwards. Then, an increase of genome load was observed in the feather follicles (FF) with a peak at 2 weeks, rising to a level almost 10(3)-fold greater than in the other tissues. Importantly, the rHVT-F genome load in FF appeared to be strongly correlated to the humoral immunity specific to NDV as evaluated by haemagglutination inhibition (HI) test and NDV-specific IgG, IgM and IgA ELISAs. This is the first report of quantification of rHVT-F vaccine in FF and its correlation with the induction of ND-specific immune response in chickens with no MDA. Our data indicate that the application of this real-time qPCR assay on FF samples taken from chickens in the field may be used to confirm rHVT-F vaccine administration and uptake with the important added benefit of offering a non-disruptive sampling procedure.

False-positive results in metagenomic virus discovery: a strong case for follow-up diagnosis.

A viral metagenomic approach using virion enrichment, random amplification and next-generation sequencing was used to investigate an undiagnosed cluster of dairy cattle presenting with high persistent fever, unresponsive to anti-microbial and anti-inflammatory treatment, diarrhoea and redness of nose and teat. Serum and whole blood samples were taken in the predicted hyperviraemic state of an animal that a few days later presented with these clinical signs. Bioinformatics analysis of the resulting data from the DNA virus identification workflow (a total of 32 757 sequences with average read length 335 bases) initially demonstrated the presence of parvovirus-like sequences in the tested blood sample. Thorough follow-up using specific real-time RT-PCR assays targeting the detected sequence fragments confirmed the presence of these sequences in the original sample as well as in a sample of an additional animal, but a contamination with an identical genetic signature in negative extraction controls was demonstrated. Further investigation using an alternative extraction method identified a contamination of the originally used Qiagen extraction columns with parvovirus-like nucleic acids or virus particles. Although we did not find any relevant virus that could be associated with the disease, these observations clearly illustrate the importance of using a proper control strategy and follow-up diagnostic tests in any viral metagenomic study.

Chasing notifiable avian influenza in domestic poultry: a case report of low-pathogenic avian influenza h5 viruses in two Belgian holdings.

In December 2008, bird species in two geographically distant holdings were found positive for H5 viruses following the annual Avian influenza serological screening in Belgium. The virological tests performed identified in one holding a low-pathogenic avian influenza (LPAI) virus subtype H5N2, and a H5 LPAI virus was identified by real-time PCR and direct sequencing at the second holding. The first farm was an outdoor mixed holding housing ornamental birds and poultry (n = 6000) and the second a free-range geese breeding farm (n = 1500). No clinical signs or mortalities were reported. Control measures defined by Council Directive 2005/94/EC were followed, including notification to the European Commission via the Animal Disease Notification System and to the World Organization for Animal Health, and poultry were killed, while ornamental bird species were quarantined. Partial sequencing of the H5N2 virus haemagglutinin and neuraminidase N2 gene sequences revealed a close homology to some recent LPAI isolates identified from wild birds in Germany and Italy and from wild birds in Eurasia and Africa, respectively. It is noteworthy that, these two holdings were already H5 positive based on HI test results carried out during the previous serological screening; however, no virus was detected at that time. To have a better understanding of the potential 'silent' circulation of the H5N2 isolate in the field, experimental infections of chickens and turkeys were performed. The low excretion detected might in part explain viral persistence not associated with spread between gallinaceous birds in the same holding, indicating that the H5N2 LPAI isolate was not fully adapted to these two poultry species. Our results highlighted limitations to only using serological screening for the early detection of LPAI in an 'at-risk farm', suggesting that virological and serological monitoring tests be applied simultaneously as a means of testing animals in 'at-risk farms'.

Molecular epidemiological analysis of the transboundary transmission of 2003 highly pathogenic avian influenza H7N7 outbreaks between the Netherlands and Belgium.

The 2003 outbreak of Highly pathogenic avian influenza (HPAI) A(H7N7) in the Netherlands, Belgium and Germany resulted in significant genetic diversification that proved informative for tracing transmission events. Building on previous investigations on the Dutch outbreak, we focused on the potential transnational transmissions between the Netherlands and Belgium. Although no clear epidemiological links could be identified from the tracing data, the transmission network based on concatenated HA-NA-PB2 sequences supports at least three independent introductions from the Netherlands to Belgium and suggests one possible introduction form Belgium back to the Netherlands. Two introductions in the Belgian province of Limburg occurred from nearby farms in the Dutch province of Limburg. One introduction resulted in three secondary infected farms, while a second introduction did not cause secondary infections. The third introduction into Belgium occurred in the north of the Antwerp province, very close to the national border, and originated from the North of the Dutch province Brabant (long distance transmission, >65 km). The virus spread to two additional Belgian farms, one of which may be the source of a secondarily infected farm in the Netherlands. One infected turkey farm in the province of Antwerp (Westmalle) was geographically close to the latter introduction, but genetically clustered with the first introduction event in the Limburg province. Epidemiological tracing data could neither confirm nor exclude whether this outbreak was a result from long distance contacts within Belgium or whether this farm presented a fourth independent transboundary introduction. These multiple transnational transmissions of HPAI in spite of reinforced biosecurity measures and trade restrictions illustrate the importance of international cooperation, legislation and standardization of tools to combat transboundary diseases.

Genetic characterization of low pathogenic H5N1 and co-circulating avian influenza viruses in wild mallards (Anas platyrhynchos) in Belgium, 2008.

As part of a long-term wild bird monitoring programme, five different low pathogenic (LP) avian influenza viruses (AIVs) were isolated from wild mallards (subtypes H1N1, H4N6, H5N1, H5N3, and H10N7). A LP H5N1 and two co-circulating (same location, same time period) viruses were selected for full genome sequencing. An H1N1 (A/Anas platyrhynchos/Belgium/09-762/2008) and an H5N1 virus (A/Anas platyrhynchos/Belgium/09-762-P1/2008) were isolated on the same day in November 2008, then an H5N3 virus (A/Anas platyrhynchos/09-884/2008) 5 days later in December 2008. All genes of these co-circulating viruses shared common ancestors with recent (2001 to 2007) European wild waterfowl influenza viruses. The H5N1 virus shares genome segments with both the H1N1 (PB1, NA, M) and the H5N3 (PB2, HA) viruses, and all three viruses share the same NS sequence. A double infection with two different PA segments from H5N1 and from H5N3 could be observed for the H1N1 sample. The observed gene constellations resulted from multiple reassortment events between viruses circulating in wild birds in Eurasia. Several internal gene segments from these 2008 viruses and the N3 sequence from the H5N3 show homology with sequences from 2003 H7 outbreaks in Italy (LP) and the Netherlands (highly pathogenic). These data contribute to the growing sequence evidence of the dynamic nature of the avian influenza natural reservoir in Eurasia, and underline the importance of monitoring AIV in wild birds. Genetic information of potential hazard to commercial poultry continues to circulate in this reservoir, including H5 and H7 subtype viruses and genes related to previous AIV outbreaks.

Further evidence of antigenic drift and protective efficacy afforded by a recombinant HVT-H5 vaccine against challenge with two antigenically divergent Egyptian clade 2.2.1 HPAI H5N1 strains.

In this study, we have compared the protection afforded by a recombinant turkey herpesvirus vaccine expressing the H5 gene from a clade 2.2 H5N1 strain (rHVT-H5) and a Mexican-origin H5N2 inactivated vaccine, alone or in combination, against two antigenically divergent H5N1 Egyptian strains isolated in 2007 and 2008. Our results confirm the existence of a major antigenic drift among the Egyptian H5N1 strains such that, although protection against the "classical" 2007 HPAI H5N1 Egyptian strain could be obtained with both types of vaccines, only vaccination with the rHVT-H5 vaccine protected against challenge with the "variant" 2008 HPAI H5N1 Egyptian strain.

Passive protection afforded by maternally-derived antibodies in chickens and the antibodies' interference with the protection elicited by avian influenza-inactivated vaccines in progeny.

Systematic vaccination can be applied when a disease has become enzootic in a country or region. The final goal of the approach is to control or eradicate the disease within the country. This is a long-term vaccination plan that could be applied nationwide to all commercial and backyard poultry. However, after several months of vaccination in enzootic areas, maternally derived antibody (MDA) is present in young chicks, providing some protection and/or interference with vaccination. The aim of this study was to evaluate the level of protection afforded by MDA against challenge with highly pathogenic avian influenza virus (HPAIV), and its suspected interference with current inactivated vaccines in broilers under controlled laboratory conditions. In the first set of experiments, broilers were vaccinated with inactivated vaccines containing H5N2 subtype antigens in the presence or absence of homologue MDAs and challenged with a clade 2.2 H5N1 HPAIV. In the second set of experiments, day-old broilers, either with or without avian influenza MDA, received a regular-type monovalent H5N2 AI vaccine (0.5 ml) or a concentrated (0.2 ml) AL-Newcastle disease virus combined inactivated vaccine subcutaneously. They were then challenged at 11 or 35 days of age. In conclusion, our results indicate that protection induced by day-old administration of inactivated vaccine (regular or concentrated) in the presence or absence of MDA to H5N2 AIV induces poor protection against challenge with H5N1 HPAIV and should not be recommended. Based on our results, vaccination of MDA-positive chickens at a later age (10 days) seems to be a valuable recommendation, although MDAs may still interfere with vaccination to a lesser extent because they are present up to 3 wk posthatch. Therefore, in areas with high infection pressure, when possible, two vaccinations are recommended for optimal protection. Also, it might be advisable to take into account day-old AI MDA titers when one is determining the optimal age of vaccination.

Prime-boost vaccination with a fowlpox vector and an inactivated avian influenza vaccine is highly immunogenic in Pekin ducks challenged with Asian H5N1 HPAI.

The efficacy of different vaccination schedules was evaluated in 17-day-old Pekin ducks using an experimental inactivated whole virus vaccine based on the H5N9 A/chicken/Italy/22A/98 isolate (H5N9-It) and/or a fowlpox recombinant (vFP-H5) expressing a synthetic HA gene from an Asian H5N1 isolate (A/chicken/Indonesia/7/2003). Full protection against clinical signs and shedding was induced by the different vaccination schemes. However, the broadest antibody response and the lowest antibody increase after challenge were observed in the group of ducks whose immune system was primed with the fowlpox vectored vaccine and boosted with the inactivated vaccine, suggesting that this prime-boost strategy induced optimal immunity against H5N1 and minimal viral replication after challenge in ducks. In addition, this prime-boost vaccination scheme was shown to be immunogenic in 1-day-old ducklings.

A universal avian endogenous real-time reverse transcriptase-polymerase chain reaction control and its application to avian influenza diagnosis and quantification.

Real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) is becoming an established first-line diagnostic assay as well as a precise quantification tool for avian influenza virus detection. However, there remain some limitations. First, we show that the sensitivity of RRT-PCR influenza detection can be 10- to 100-fold inhibited in oropharyngeal and cloacal swabs. Adding 0.5 U of heat-activated Taq DNA polymerase successfully reverses PCR inhibition. Second, an excellent strategy for detecting false negative samples is the coamplification of an internal control from each sample. We developed a universal avian endogenous internal control (bird beta-actin) and apply it to influenza A diagnosis. Moreover, this internal control proves useful as a normalizer control for virus quantification, because beta-actin gene expression does not change in infected vs. uninfected ducks. A combined panel of wild bird cloacal swabs, wild bird tissue samples, experimental duck swabs, and experimental duck and chicken tissue samples was used to validate the endogenous control. The application of an endogenous internal control proves an excellent strategy both for avoiding false negative diagnostic results and for standardizing virus quantification studies.

Development of an M2e-specific enzyme-linked immunosorbent assay for differentiating infected from vaccinated animals.

Vaccination programs for the control of avian influenza (AI) in birds have restrictions because of some limited efficacy and the difficulty of discriminating between vaccinated and virus-infected poultry. We studied M2e, the highly conserved external domain of the influenza A M2 protein, as a potential differential diagnostic marker for influenza virus infection. The M2 protein is an integral membrane protein, scarcely present on virus particles, but abundantly expressed on virus-infected cells. M2e-specific enzyme-linked immunosorbent assays (ELISAs) for different avian influenza strains were developed by coating the peptides corresponding to the first 18 amino acids, without the first methionine, of the universal human consensus M2e sequence and the specific M2e sequence of two highly pathogenic AI (HPAI) strains, H7N7 and H5N1. Using the M2e ELISAs, M2e-specific antibodies were observed in chickens and ducks experimentally infected with H7 or H5 HPAI, respectively, that correlated well with hemagglutination inhibition (HI) antibodies. Conversely, sera from chicken and ducks inoculated with inactivated AI vaccines were positive for HI test but negative for the M2e ELISAs. Moreover, ducks inoculated with inactivated vaccine and challenged with a HPAI H5N1 seroconverted for antibodies to the M2e peptide, with significantly different levels from those measured between the vaccinated and infected groups. These results indicate the potential benefit of a simple and specific M2e ELISA in the assessment of the efficacy of vaccination as well as for diagnostic and survey applications.

Efficacy of an inactivated and a fowlpox-vectored vaccine in Muscovy ducks against an Asian H5N1 highly pathogenic avian influenza viral challenge.

The efficacy of an inactivated vaccine containing the Eurasian isolate A/chicken/Italy/22A/98 H5N9 (H5N9-It) was compared with that of the fowlpox-vectored TROVACTM-AIV H5 (rFP-AIV-H5) vaccine against an H5N1 highly pathogenic avian influenza challenge. Five-week-old Muscovy ducks were vaccinated with either H5N9-It (0.5 ml) or rFP-AIV-H5 (5 log10 50% tissue culture infectious dose (TCID50)/dose), followed by a boost at 7 wk of age with the same vaccine (1.0 ml of H5N9-It or 5 log10 TCID50/dose rFP-AIV-H5), and a challenge at 9 wk of age with 10(7) egg infectious dose (lethality 50%) of A/crested eagle/ Belgium/01/2004 (H5N1). All unvaccinated challenged birds showed severe nervous signs (loss of balance, torticollis) starting 7 days postinfection (dpi). None of the vaccinated ducks showed these nervous signs. Shedding was measured in oropharyngeal and cloacal swabs, sampled from 3 to 19 dpi by titration in chicken embryo fibroblasts and by real-time reverse transcription-polymerase chain reaction. Virus shedding was significantly higher in oropharyngeal compared to cloacal swabs. Both vaccines reduced the percentage of positive swabs and the viral load in the swabs, but the reduction was higher with the H5N9-It vaccine. The inactivated vaccine induced hemagglutination inhibition (HI) titers (5.4 log2) that were boosted after the second administration (7.5 log2). rFP-AIV-H5-induced HI titers were lower (3 log2 only after the second administration), most probably because the fowlpox vector does not replicate in ducks. Altogether, these results indicate that significant protection from clinical signs and reduction in virus shedding may be achieved in ducks with conventional inactivated or fowlpox-vectored vaccine as compared with nonvaccinated challenged control birds.

Lethality and molecular characterization of an HPAI H5N1 virus isolated from eagles smuggled from Thailand into Europe.

On October 18, 2004, two crested hawk eagles, Spizaetus nipalensis, smuggled into Europe from Thailand were seized at Brussels International Airport. A highly pathogenic avian influenza virus, denominated A/crested eagle/Belgium/01/2004, was isolated from these birds and antigenically characterized as H5N1. Here we report on the molecular characterization of A/crested eagle/Belgium/01/2004 (H5N1). We completely sequenced all eight genome segments. The hemagglutinin (HA) and neuraminidase (NA) sequences clustered within the Z genotype and were closely related to strains circulating in Thailand during 2004, although some mutations in the HA were evident, notably a unique arginine (R) > lysine (K) replacement in the cleaving site. The HA cleavage site contained six basic amino acids, confirming its high pathogenicity (intravenous pathogenicity index = 2.94). The 20-amino acid deletion in the NA stalk region is consistent with its Thai-Viet origin. We further discuss the assembled genetic information in the light of currently known host adaptation, virulence, and antiviral resistance factors. Using infection experiments, we show that pathogenicity in chickens depends on breed, inoculation route (oculonasal vs. intramuscular), and dose. Additionally, in Muscovy ducks, pathogenicity proved to be age dependent.