| Summary: | 碩士 === 國立臺灣大學 === 獸醫學研究所 === 98 === Bluetongue is an infectious, noncontagious, arthropod-borne viral disease of ruminants caused by bluetongue virus (BTV). BTV belongs to the Orbivirus genus of the family Reoviridae, and there are 24 recognized BTV serotypes. In Taiwan, one BTV strain of serotype 2 (BTV2/KM/2003) was first isolated from a clinically healthy goat in Kinmen in 2003, and this virus strain was defined as low virulent to experimental sheep. However the seroprevalence rates were high in ruminants, 32.7% in cattle and 8.2% in goat. That in sheep was highly variable by the locations of flocks ranged from 0 to 40%.
BTV is a non-enveloped, double-stranded RNA (dsRNA) virus composed of seven structural proteins (VP1-VP7) and four non-structural proteins (NS1-NS3/NS3A). In a previous study, Huismans and Verwoerd (1973) found that the genome segments of BTV are not all transcribed at a constant rate in vitro, resulting in different amounts of BTV mRNA in infected BHK21. However the mRNAs are translated and transcribed at almost the same frequency.
The purpose of this study was to establish a method of quantitating BTV mRNA expression in vitro and then applied this method to quantitate BTV mRNA expression in formalin-fixed paraffin-embedded (FFPE) tissues of experimentally infected sheep. To avoid the interference of second round virus infection, the BHK-21 cells were infected at a low MOI of 0.1 and the time point to harvest were set a 8 hr postinfection (POI).
Expressions of the 10 segments of BTV genome were successfully quantitatated in vitro, wherein NS2, VP4 and VP7 mRNA were highly expressed. The expression ratio of NS3/NS1 was 1.7. Previously this large ratio is linked to a shift of virus release from lysis of cells to budding. When applied to FFPE tissues, the most highly expressed mRNA were NS3, NS1 and NS2. The different results obtained from in vitro versus in vivo models were likely due to the differences in infection duration (8 hr POI vs DPI 7), the quality of RNA from archive tissues and other factors. Further studies are needed to relate BTV mRNA expression with pathogenesis.
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