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.

Novel technologies applied to the nucleotide sequencing and comparative sequence analysis of the genomes of infectious agents in veterinary medicine.

Next-generation sequencing (NGS), also referred to as deep, high-throughput or massively parallel sequencing, is a powerful new tool that can be used for the complex diagnosis and intensive monitoring of infectious disease in veterinary medicine. NGS technologies are also being increasingly used to study the aetiology, genomics, evolution and epidemiology of infectious disease, as well as host-pathogen interactions and other aspects of infection biology. This review briefly summarises recent progress and achievements in this field by first introducing a range of novel techniques and then presenting examples of NGS applications in veterinary infection biology. Various work steps and processes for sampling and sample preparation, sequence analysis and comparative genomics, and improving the accuracy of genomic prediction are discussed, as are bioinformatics requirements. Examples of sequencing-based applications and comparative genomics in veterinary medicine are then provided. This review is based on novel references selected from the literature and on experiences of the World Organisation for Animal Health (OIE) Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine, Uppsala, Sweden.

Le séquençage de nouvelle génération (également désigné « séquençage à très haut débit ¼ ou « séquençage massivement parallèle ¼) est un nouvel outil extrêmement puissant permettant de procéder à des diagnostics sophistiqués et d'assurer un contrôle vétérinaire intensif de maladies infectieuses complexes. Les technologies du séquençage de nouvelle génération sont également d'un grand secours pour étudier l'étiologie, la génomique, l'évolution et l'épidémiologie des maladies infectieuses ainsi que les interactions hôtes­agents pathogènes et bien d'autres aspects de la biologie des maladies infectieuses. Pour présenter les progrès et les accomplissements les plus récents dans ce domaine, les auteurs décrivent d'abord une série de techniques innovantes puis quelques exemples d'applications du séquençage de nouvelle génération dans le champ de la biologie des maladies animales infectieuses. Ils exposent un certain nombre d'étapes et de processus opérationnels régissant la sélection et la préparation des échantillons, l'analyse séquentielle et les études de génomique comparative, ainsi que ceux qui permettent d'améliorer la justesse prédictive de la génomique ; les exigences particulières de la bio-informatique sont également évoquées. Cette analyse est complétée par quelques exemples d'applications de l'analyse séquentielle et de la génomique comparative en médecine vétérinaire. Cette synthèse est basée sur une sélection de références bibliographiques récentes ainsi que sur l'expérience acquise par le Centre collaborateur de l'Organisation mondiale de la santé animale (OIE) pour le diagnostic basé sur la biotechnologie des maladies infectieuses en médecine vétérinaire, situé à Uppsala (Suède).

La secuenciación de próxima generación, también denominada secuenciación profunda, de alto rendimiento o masivamente paralela, es una nueva y poderosa herramienta para efectuar diagnósticos complejos y vigilar muy de cerca enfermedades infecciosas complejas en el ámbito de la medicina veterinaria. Estas técnicas también se utilizan cada vez más para estudiar la etiología, genómica, evolución y epidemiología de las enfermedades infecciosas, así como las interacciones entre patógeno y anfitrión y otros aspectos de la biología de las infecciones. Los autores resumen brevemente una serie de logros y adelantos obtenidos últimamente en este ámbito, presentando en primer lugar un conjunto de técnicas novedosas y ofreciendo después ejemplos de aplicaciones de la secuenciación de próxima generación a la biología de las infecciones veterinarias. También describen varios protocolos y procesos de trabajo para la obtención y preparación de muestras, el análisis de secuencias y las labores de genómica comparada, explican cómo mejorar la exactitud de la predicción genómica y examinan las herramientas bioinformáticas necesarias para ello. A continuación presentan ejemplos de aplicaciones basadas en técnicas de secuenciación y en la genómica comparada en medicina veterinaria. Este artículo está basado en referencias muy recientes, tomadas de publicaciones científicas, y en la experiencia del Centro Colaborador de la Organización Mundial de Sanidad Animal (OIE) para el Diagnóstico de las enfermedades infecciosas de la medicina veterinaria basado en la biotecnología, sito en Upsala (Suecia).

Chlamydia trachomatis prevalence, genotype distribution and identification of the new Swedish variant in Southern Germany.

PURPOSE: In Germany, reliable data about the prevalence of urogenital Chlamydia trachomatis infections, causative genotypes, as well as corresponding clinical, demographic and behavioural information are sparse. We, therefore, performed a prospective prevalence study including 1,003 sexually active volunteers of a Southern German city. METHODS: Study participants completed a standardised questionnaire and provided first void urine samples for analysis. Our screening strategy included the performance of two nucleic acid amplification tests with different target genes, enabling the detection of the new Swedish variant of C. trachomatis (nvCT). Direct genotyping of positive specimens was performed by sequence analysis of the ompA gene. RESULTS AND CONCLUSION: The overall prevalence of C. trachomatis infection was 4.2 % in women and 4.6 % in men. A relatively high prevalence of 8.3 % was found in men older than 25 years. Never using condoms was an independent risk factor for infection. The most common symptom was discharge; however, 64.5 % of infected females and all of the infected men were asymptomatic, supporting the need for screening programmes. The most frequently encountered genotypes were E (46.5 %), F (20.9 %) and K (14.0 %). Since the nvCT was detected in one female student, this is one of the rare studies that reports on the molecular identification of nvCT apart from Sweden.

High-throughput sequencing in veterinary infection biology and diagnostics.

Sequencing methods have improved rapidly since the first versions of the Sanger techniques, facilitating the development of very powerful tools for detecting and identifying various pathogens, such as viruses, bacteria and other microbes. The ongoing development of high-throughput sequencing (HTS; also known as next-generation sequencing) technologies has resulted in a dramatic reduction in DNA sequencing costs, making the technology more accessible to the average laboratory. In this White Paper of the World Organisation for Animal Health (OIE) Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine (Uppsala, Sweden), several approaches and examples of HTS are summarised, and their diagnostic applicability is briefly discussed. Selected future aspects of HTS are outlined, including the need for bioinformatic resources, with a focus on improving the diagnosis and control of infectious diseases in veterinary medicine.

Development of improved analytical methods for use in animal health and in foodborne disease surveillance for source attribution.

Considering the 'One Health' principles, the links between animal and human health are very strong. Both domestic and wild animals are sources of infectious agents that cause diseases in humans. Poor animal health may also indirectly affect human health, through reduced access to food. A large number of infectious diseases of animals, the transboundary animal diseases, spread rapidly across borders. Robust and accurate diagnostic assays are needed to detect the infectious agents rapidly and to limit their spread. A large arsenal of novel assays has been developed during the last three decades, with a tremendous impact on the detection of infectious agents. The new diagnostic methods are mostly laboratory-based and expensive, requiring sophisticated equipment and special skills. However, rapid and cheap field-based assays have also been developed. Herein, the authors give several examples of the development of novel assays, with special focus on the 'One Health' principles.

Molecular epidemiology of hepatitis E virus in humans, pigs and wild boars in Sweden.

Hepatitis E infections in humans are usually acquired in endemic countries in Asia or Africa. In Sweden 17 cases infected in Europe, between 1993 and 2009, were identified. All had clinical hepatitis E with unknown source of infection. Hepatitis E virus (HEV) was identified in faecal samples from 63 piglets in 12 pig farms in Sweden. HEV was also identified in blood from 13 out of 159 investigated Swedish wild boars from nine counties. Partial HEV genomes from humans, pigs and wild boars were sequenced and compared by phylogeny. The results showed close relatedness between HEV strains from piglets from the same farm and from wild boars from the same county. HEV strains from humans showed relatedness with strains from pigs and wild boars from the same county. This study showed that HEV strains form geographical clusters in the phylogenetic tree. The methods used in this study may thus be used for tracing the origin of an infecting strain. Furthermore, this study indicated that there are endemic sources of human HEV infections in Sweden.

Real-time PCR-based pathotyping of Newcastle disease virus by use of TaqMan minor groove binder probes.

A real-time reverse-transcription PCR was developed to detect and pathotype Newcastle disease viruses (NDV) in clinical samples. Degenerate oligonucleotide primers and TaqMan probes with nonfluorescent minor groove binder (MGB) quencher amplified and hybridized to a region in the fusion protein (F) gene that corresponds to the cleavage site of the F0 precursor, which is a key determinant of NDV pathogenicity. The application of degenerate primers and TaqMan MGB probes provided high specificity to the assay, as was shown by the successful and rapid pathotype determination of 39 NDV strains representing all the known genotypes (I to VIII) and pathotypes (lentogens/mesogens/velogens). The PCR assays specific for lentogenic and velogenic/mesogenic strains had high analytical sensitivity, detecting approximately 10 and 20 copies of the target molecule per reaction, respectively. The detection limit was also determined in terms of 50% egg infective dose (EID(50)) by using dilution series of virus stock solutions to be approximately 10(1.0) and 10(-1.3) EID(50)/ml for lentogens and velogens/mesogens, respectively. Organ, swab, and stool specimens from experimentally infected animals were tested to prove the clinical suitability of the method. The results of this study suggest that the described real-time PCR assay has the potential to be used for the rapid detection/pathotyping of NDV isolates and qualitative/quantitative measurement of the virus load.

The rapid molecular subtyping and pathotyping of avian influenza viruses.

Highly conserved nucleotide stretches flanking the cleavage site of the haemagglutinin (HA) gene of influenza type A viruses were utilised for generating PCR amplicons from a broad range of avian influenza viruses (AIV) in a one-step real-time SYBR Green RT-PCR assay. The nucleotide sequencing of the amplified PCR products simultaneously reveals both the HA subtype and the pathotype of the AIV isolates, as we demonstrated in case of H5 subtype viruses. The specificity of the assay was confirmed by investigating 66 strains of AIV and nine heterologous pathogens, including influenza B, C and various avian pathogenic viruses. This assay enables a general HA subtype identification and pathotype determination of AIV isolates providing a useful alternative tool for avian influenza diagnosis.

New developments in the diagnosis of avian influenza.

Avian influenza has become a serious concern from both veterinary and public health points of view. National and international organisations, veterinary health authorities, research institutions, diagnostic laboratories and field services make enormous efforts worldwide to detect, combat and prevent this important disease. Accordingly, the standard diagnostic protocols are being supported by a wide variety of molecular detection techniques, including improved polymerase chain reaction assays, microarray-based detection and characterisation methods, very rapid sequencing, simple pen-side tests and other on-site approaches. These recently developed 'closer to the field' methods allow rapid detection of influenza viruses and the identification of pathogenicity variants. However, in order to harmonise the diagnosis worldwide, attention has to be paid to the validation and standardisation of these technologies, to avoid erroneous interpretation of assay results, and, consequently, inappropriate epidemiological measures. This review gives an overview of the current and potential future developments related to avian influenza diagnostics.

Experiences of an OIE Collaborating Centre in molecular diagnosis of transboundary animal diseases: a review.

The highly contagious transboundary animal diseases (TADs), e.g., foot-and-mouth disease (FMD), classical swine fever (CSF), African swine fever (ASF) and highly pathogenic avian influenza (HPAI) are regularly occurring and re-occurring on various continents, causing severe losses. This epidemiological situation indicates the urgent need for the development of powerful, robust and high capacity new diagnostic methods in order to detect and identify the causative agents very rapidly. This report is on the experiences of an OIE Collaborating Centre and those of the MULTIPLEX-PCR and the LAB-ON-SITE EU project consortia with the deveopment of novel methods for the improved molecular diagnosis of a range of viral diseases. Thermal amplification based real-time PCR methods (e.g.,TaqMan, Molecular Beacons, Primer-Probe Energy Transfer, and Light Upon eXtension (LUX) fluorogenic primers), and amplification without thermocycling have been elaborated for the improved diagnosis of TADs, such as FMD, swine vesicular disease, vesicular stomatitis, CSF, ASF, HPAI and Newcastle disease (ND). The simultaneous detection of various pathogens in a disease complex is facilitated by the development of multiplex PCR packages. By introducing nucleic acid extraction and pipetting robotics, together with the multi-channel real-time PCR machines, the molecular diagnostic procedures have become rapid, robust and automated. Quality control is strengthened by special precautions to avoid false positive and false negative results. By following the steps of OIE standardisation and validation, the diagnostic PCR assays have become nationally and nternationally standardised and harmonised. The development of additional methods, like padlock probes and microarrays, is further improving the arsenal of nucleic acid based novel molec ular diagnostic tests for TADs.

Simultaneous detection of three porcine viruses by multiplex PCR.

Specific oligonucleotide primers were selected and combined in a multiplex arrangement, in order to detect simultaneously three economically important porcine viruses by polymerase chain reaction (PCR). The pathogen panel was comprised of viruses that cause reproductive failure in infected herds: Aujeszky's disease virus (ADV), porcine parvovirus (PPV) and porcine respiratory and reproductive syndrome virus (PRRSV). In order to reduce the time required for the detection of the pathogens, the assay was optimised to a RapidCycler PCR instrument. The multiplex PCR assay was shown to be specific, sensitive and rapid, because the results were read in less than 60 min after sample preparation. Due to its speed, efficiency and sensitivity, the described rapid multiplex PCR assay serves as a useful novel tool in the veterinary diagnostic laboratories for the quick and complex detection of these important porcine pathogens.

Pre-Clinical Evaluation of a Real-Time PCR Assay on a Portable Instrument as a Possible Field Diagnostic Tool: Experiences from the Testing of Clinical Samples for African and Classical Swine Fever Viruses.

African swine fever (ASF) and classical swine fever (CSF) are two highly infectious transboundary animal diseases (TADs) that are serious threats to the pig industry worldwide, including in China, the world's largest pork producer. In this study, a duplex real-time PCR assay was developed for the rapid detection and differentiation of African swine fever virus (ASFV) and classical swine fever virus (CSFV). The assay was performed on a portable, battery-powered PCR thermocycler with a low sample throughput (termed as 'T-COR4 assay'). The feasibility and reliability of the T-COR4 assay as a possible field method was investigated by testing clinical samples collected in China. When evaluated with reference materials or samples from experimental infections, the assay performed in a reliable manner, producing results comparable to those obtained from stationary PCR platforms. Of 59 clinical samples, 41 had results identical to a two-step CSFV real-time PCR assay. No ASFV was detected in these samples. The T-COR4 assay was technically easy to perform and produced results within 3 h, including sample preparation. In combination with a simple sample preparation method, the T-COR4 assay provides a new tool for the field diagnosis and differentiation of ASF and CSF, which could be of particular value in remote areas.

Highly diverse type of equine arteritis virus (EAV) from the semen of a South African donkey: short communication.

Equine arteritis virus (EAV) was detected by RT-nested PCR in semen samples from a naturally infected South African donkey. Sequence analysis of the amplified ORF5 fragment revealed only 60 to 70% nucleotide identity to a panel of EAV reference sequences. The unique donkey EAV sequence was also found to be stable during passage in horses. The sequence data reported in this study indicate that the South African donkey variant might represent a new genotype of EAV. The distinct genetic properties of the South African asinine strain of EAV suggest a divergent evolution of this arterivirus in various host species or, alternatively, a possible role for African donkeys in the emergence of EAV in horses.

Application of real-time RT-PCR utilising lux (light upon extension) fluorogenic primer for the rapid detection of avian influenza viruses.

A real-time RT-PCR assay utilising light upon extension fluorogenic primer (LUX RT-PCR) was developed for the rapid and efficient detection of avian influenza viruses (AIV). The assay detected each of the AIV isolates tested (16/16) and gave negative results with heterologous pathogens (17/17). The detection limit of the assay proved to be 10(-0.5) EID50/0.2 ml and 10(1.5) EID50/0.2 ml in allantoic fluid of virus-infected embryonated chicken eggs and in spiked chicken faeces samples, respectively. Based on its specificity, sensitivity and relative simplicity, the LUX RT-PCR assay provides a novel, rapid and cost-effective diagnostic tool for avian influenza surveillance and monitoring programs.

Recent achievements and trends in the molecular diagnosis of bovine viral diseases--a view from the "OIE Collaborating Centre for the Application of Polymerase Chain Reaction Methods for the Diagnosis of Viral Diseases in Veterinary Medicine".

The activities of the "OIE Collaborating Centre for the Application of Polymerase Chain Reaction Methods for the Diagnosis of Viral Diseases" and its partner laboratories are summarised, providing a view of recent achievements and trends in the field the molecular diagnosis of bovine viral diseases. Results are briefly discussed concerning the diagnostic application of various techniques and approaches, such as PCR, semi-nested and nested PCR assays, phylogeny and molecular epidemiology, real-time PCR assays, "general" PCR systems, multiplex PCR, multi PCR, robotics in nucleic acid extraction, automated diagnosis, international standardisation and validation of the PCR-based diagnostic assays, and several future trends in molecular diagnostics. These achievements and trends lead to more rapid and specific detection of various viral infections in the cattle populations. Their application will certainly contribute to the reduction of losses which are caused by bovine viral diseases worldwide.

Assessment of Preparation of Samples Under the Field Conditions and a Portable Real-Time RT-PCR Assay for the Rapid On-Site Detection of Newcastle Disease Virus.

Newcastle disease virus (NDV), also known as virulent forms of avian paramyxovirus serotype 1 (AMPV-1), is the causative agent of Newcastle disease affecting many species of birds and causing heavy losses to the poultry industry worldwide. Early, rapid and sensitive detection of the viruses or the viral nucleic acids is very important for disease diagnosis and control. This study aimed to evaluate sample preparation under field conditions and the application of a real-time RT-PCR method in the portable T-COR4 platform for the rapid, on-site detection of NDV on a farm. In the laboratory setting, the portable real-time RT-PCR assay had a similar performance compared with that obtained with a larger, stationary Rotor Gene real-time thermocycler. In the field conditions, viral nucleic acids were manually extracted just outside of animal units with minimal equipment and real-time RT-PCR detection was performed with the portable thermocycler T-COR4 placed in a nearby room. The portable assay at the farm detected viral RNA in 15 samples and reached an agreement of 83% (39/47) when the same RNA preparations were tested in the Rotor Gene thermocycler under the laboratory setting. The results demonstrated the feasibility of performing field detection but also the need to improve and further simplify sample preparation procedures.

Cellular composition and interferon-gamma expression of the local inflammatory response in feline infectious peritonitis (FIP).

Feline infectious peritonitis (FIP) is one of the most important viral diseases of cats. International studies estimate that approximately 80% of all purebred cats are infected with the causative agent, feline coronavirus (FCoV). Out of these, 5-12% develop clinical symptoms of FIP. The pathogenesis of the disease is complex with many unresolved issues relating to the role of the immune system. The aim of the present study was to determine the proportions of various inflammatory cell types in FIP lesions by using a panel of cat specific, thoroughly validated, monoclonal antibodies. In addition, the expression of interferon-gamma within the inflammatory lesions was examined by RT-PCR. Our results confirm the mixed nature of the inflammatory reaction in FIP, involving B cells and plasma cells as well as CD4+ and CD8+ T cells. However, one cell type stands out as being the key element in both the "wet" and "dry" forms of FIP: the macrophage. Upregulation of IFN-gamma expression within the inflammatory lesions suggests a local activation of macrophages, which might result in increased viral replication.

The molecular diagnosis of porcine viral diseases: a review.

The worldwide occurrence and re-occurrence of transboundary diseases like foot-and-mouth disease or classical swine fever indicates that there is a high need for the development of powerful, robust and high-capacity new diagnostic methods, which are able to detect the causative agents before they could spread to large populations and cause tremendous losses. This article reports the experiences of a research group on the development of molecular methods for the improved diagnosis of a range of porcine viral diseases, including diseases on List A of the Office International des Epizooties (OIE). Nucleic acid hybridisation and various polymerase chain reaction (PCR) assays have been applied for routine diagnosis of a large range of viral diseases. During the last one-and-a-half decade more than 40 nested PCR assays have been developed to detect a variety of DNA and RNA viruses. False positive and negative results are avoided by the use of special tools, practices and internal controls of amplification (mimics). Recently, real-time PCR methods (TaqMan, molecular beacons, Primer-Probe Energy Transfer system) have been developed for the diagnosis of a wide range of diseases, such as foot-and-mouth disease, swine vesicular disease and vesicular stomatitis. Multiplex PCR packages have been developed for the simultaneous detection of eight important viruses of swine. By introducing nucleic acid extraction and pipetting robotics, together with the multi-channel real-time PCR machines, the diagnostic procedures have become rapid, robust and automated. In order to standardise the real-time PCR assays, the rules of OIE are considered. By following the five steps of OIE standardisation and validation, the new diagnostic procedures are nationally and internationally standardised and harmonised. The rapid, powerful and internationally standardised molecular diagnosis contributes to the reduction of losses caused by the transboundary viral diseases in swine populations.

Cytopathogenicity markers in the genome of Hungarian cytopathic isolates of bovine viral diarrhoea virus.

Since genetic recombination is a major factor in the evolution of the cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) biotypes, in this study the cytopathogenicity markers were investigated in the genomes of two cp BVDV strains recently isolated from mucosal disease (MD) cases in Hungary. In the genome of strain H4956, a Jiv-like insertion was found similar to those described in reference strain NADL and in other BVDV 1, BVDV 2 and border disease virus (BDV) strains. The 133 amino acid Jiv-like sequence is inserted at nucleotide position 4984 (amino acid position 1533), 9 nucleotides upstream of that of strain NADL. The insertion showed 96% amino acid sequence identity with the cellular Jiv protein. In the genome of cp BVDV strain H115/PCR, an ubiquitin-containing duplication was found. The duplicated sequence started at nucleotide position 7978 (amino acid 2531) in the NS4B gene. The duplication contained a complete ubiquitin monomer of 76 amino acids and the complete NS3 gene starting at nucleotide position 5153 (amino acid 1589), which corresponds to the first N-terminal amino acid of NS3. The duplication was located further downstream of the known ubiquitin-containing genomic regions of cp BVDV strains, and it consisted of the shortest inserted nucleotide sequence. The insertions and duplication of strains H4956 and H115/PCR further confirmed that recombinations occurring at positions A and B are the most common mechanisms leading to the development of BVDV cytopathogenicity.