Laboratory tests for evaluating the level of attenuation of bluetongue virus.

Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise G. Caporale, Via Campo Boario, 64100 Teramo, Italy.
Journal of Virological Methods (Impact Factor: 1.9). 10/2008; 153(2):263-5. DOI: 10.1016/j.jviromet.2008.07.007
Source: PubMed

ABSTRACT One of the most important steps when preparing a live attenuated vaccine is the assessment of the level of attenuation in target animals. It is costly and time consuming as it requires, on each occasion, a large number of susceptible animals and contained accommodation. This study assessed the consistency of the bovine foetal aorta endothelial (BFA) cell line and newborn mice for evaluating the attenuation level of BTV4, BTV9 and BTV16 Italian field isolates. Following serial passages in BHK(21c13) or Vero cell cultures, BTV attenuated clones demonstrated a reduced replication capability in the BFA cells compared to the homologous virulent strains. Similarly, following intracerebral inoculation, the attenuated clones were completely innocuous to newborn mice contrary to the homologous virulent strains which killed all animals within 10 days. Vaccines produced with the BTV9 or BTV4 attenuated clones were safe, immunogenic and capable of preventing clinical symptoms and viraemia in sheep following challenge with homologous virulent virus. The two assays may be valuable indicators of the gradual changes occurring in the BTV population leading to virus attenuation, they can predict the safety of a BTV attenuated vaccine and, in turn, reduce the number of sheep and cattle required to assess the level of attenuation attained.

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    ABSTRACT: Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77-79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell.
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    ABSTRACT: Bluetongue is an arthropod-borne disease caused by a virus of the genus Orbivirus, the bluetongue virus (BTV), which affects ruminant livestock such as cattle, sheep, and goats and wild ruminants such as deer, and camelids. Recently, adult mice with gene knockouts of the interferon α/β receptor (IFNAR-/-) have been described as a model of lethal BTV infection. IFNAR(-/-) mice are highly susceptible to BTV-1, BTV-4 and BTV-8 infection when the virus is administered intravenously or subcutaneosuly. Disease progression and pathogenesis closely mimics signs of bluetongue disease in ruminants. In the present paper we review the studies where IFNAR(-/-) mice have been used as an animal model to study BTV transmission, pathogenesis, virulence, and protective efficacy of inactivated and new recombinant marker BTV vaccines. Furthermore, we report new data on protective efficacy of different strategies of BTV vaccination and also on induction of interferon α/β and proinflammatory immune responses in IFNAR(-/-) mice infected with BTV.
    Virus Research 10/2013; · 2.75 Impact Factor
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    ABSTRACT: Experimental infection studies with bluetongue virus (BTV) in the mammalian host have a history that stretches back to the late 18(th) century. Studies in a wide range of ruminant and camelid species as well as mice have been instrumental in understanding BTV transmission, bluetongue (BT) pathogenicity/pathogenesis, viral virulence, the induced immune response, as well as reproductive failures associated with BTV infection. These studies have in many cases been complemented by in vitro studies with BTV in different cell types in tissue culture. Together these studies have formed the basis for the understanding of BTV-host interaction and have contributed to the design of successful control strategies, including the development of effective vaccines. This review describes some of the fundamental and contemporary infection studies that have been conducted with BTV in the mammalian host and provides an overview of the principal animal welfare issues that should be considered when designing experimental infection studies with BTV in in vivo infection models. Examples are provided from the authors' own laboratory where the three Rs (replacement, reduction and refinement) have been implemented in the design of experimental infection studies with BTV in mice and goats. The use of the ARRIVE guidelines for the reporting of data from animal infection studies is emphasised.
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May 14, 2014

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