Mots-clé
(Allemand)
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REO Virus, Pharmakopoe, Fremdvirusprüfung, Qualitätskontrolle, Vakzinen, Geflügelimpfstoffe, Polymerasekettenreaktion (PCR)
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Description succincte
(Allemand)
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Rechtliche Grundlagen: Alle immunbiologischen Erzeugnisse (insbesondere Geflügelimpfstoffe), die registriert und deren Chargen in den Verkehr gebracht werden sollen, haben bezüglich Reinheit den Anforderungen der Europäischen Pharmakopoe (Ph. Eur.) zu genügen. Die Prüfung auf Fremdviren in Geflügelimpfstoffen umfasst u.a. das Untersuchen des Produktes auf das Vorhandensein von kontaminierenden REO Viren. Die Ph.Eur. schreibt vor, dass in der Fremsvirusprüfung Impfstoffe auf deren Kontamintation überprüft wersen. Es ist Aufgabe der Kontrollstelle für immunbiologische Erzeugnisse (IVI) solche Qualitätskontrollen durchzuführen.
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Objectifs du projet
(Allemand)
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Entwicklung einer PCR Methode zum Nachweis von REO Viren, die schnell, empfindliche und zuverlässig ist. Die Validierung erfolgt gemäss den Anforderungen in der Prüfstelle IVI für akkreditierte Labormethoden. Die etablierte und validierte Methode soll möglichst rasch in die Routineuntersuchung integriert werden.
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Résumé des résultats (Abstract)
(Anglais)
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The object of this work was to establish and validate an in vitro method to detect contaminations with avian reoviruses (ARV) in the quality control of biologicals. The European Pharmacopoeia (Ph. Eur.) allows the use of alternative methods if they are fully validated and if the competent authority agrees.
Viral safety of biologicals includes certification of freedom from extraneous agents. In particular poultry vaccines - where embryonated hen eggs and primary cell cultures are used for vaccine production - impose the risk of contamination with microorganisms. The current requirements involve testing in animals. To replace the use of animals for the quality control of batches of avian viral vaccines (The Three Rs approach) an alternative testing scheme using the reverse transcriptase polymerase chain reaction (RT-PCR) was developed.
Two independent RT-PCR assays were designed to amplify either parts of the ARV S2 or S4 genome segment. Four ARV vaccine strains (1133, 1733, 2408 and strain Olson), two ATCC strains (VR826 and VR856) as well as several field isolates could be easily detected whereas various other avian pathogenic viruses (i.e. infectious bronchitis virus, infectious bursal disease virus, Newcastle disease virus, avian encephalomyelitis virus, avian leucosis virus) and the equine and mammalian reoviruses were not. The identity of the amplified fragments was confirmed by restriction endonuclease analysis and/or sequence analysis. With the obtained S4 fragment sequences of the field isolates, a phylogenetic tree was constructed.
The senstivities were shown by serial dilution tests with the four vaccine virus strains 1133, 1733, 2408 and Olson in commercial available vaccine preparations (with two exceptions).
Thus, the single-tube RT-PCR has been shown to be a rapid, sensitive and specific alternative testing method for extraneous agent testing in the quality control of avian viral vaccines.
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Publications / Résultats
(Anglais)
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Bruhn, Silke, Tierärztin von Kluis, Deutschland (2003): Detection of Avian Reovirus Contaminations in Poultry Vaccines using RP-PCR. Dissertation, Institut für Viruskrankheiten und Immunprophylaxe, Mittelhäusern und Veterinär-Medizinische Fakultät der Universität Bern.
Bruhn, S. 2003: How to develop a PCR. Laboratory Training Course organized by VACTRAIN, a project sponsored by the European Commission, IVI .
In preparation:
Bruhn, S., Bruckner L., Ottiger, H-P.: Detection of Avian Reovirus Contaminations in Poultry Vaccines using RT-PCR
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