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Home » Archive » 2018

TDK conference 2018

Detection of PRRSV with a novel RNA in situ hybridization method
Horváth Gergő Károly - year 5
University of Veterinary Medicine Budapest, Department of Pathology
Supervisors: Dr. Gyula Balka, Lilla Dénes

Abstract:

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases around the world in the swine industry. The syndrome widespread worldwide, causing enormous economic impact on the most important pig producing countries. The causative agent of the disease is PRRS-virus 1 (PRRSV-1), and PRRS-virus 2 (PRRSV-2) classified in the order Nidovirales, family Arteriviridae, genus Porarterivirus. The virus is characterized by the high level of genetic diversity, which affects the epidemiology, the immunology and the control of the disease.

The clinical signs associated with PRRSV infection could be really various depending on the age of the affected animals. In sows, reproductive failure is the most important: late-term abortion, premature farrowing, increase in number of dead-born piglets and irregular returns to estrus. The infected new-born piglets are less viable, weak and the preweaning mortality is increased due to lethargy, weakness and respiratory disorders. In weaners and growers, respiratory symptoms (dyspnoea, coughing, sneezing) are the most relevant, moreover, pneumonia could appear as a par of the porcine respiratory disease complex. The severity of the clinical signs depends mostly on the virulence of the virus strain, the immunological condition of the infected pigs, and the viral/bacterial coinfections.

In our study, we examined the lungs of 25 pigs, which have been inoculated with a wild-type PRRSV-1 strain under experimental conditions. For the visualization of the virus, 4 µm thick slides were cut from formalin-fixed, paraffin-embedded (FFPE) tissue blocks, prepared from the left medial lobes of each animal. Thereafter a new, RNA-based in situ hybridization technology (RNAscope) was applied to detect PRRSV in the samples. The oligos, servings as probes were designed based on the sequence of the strain used in the challenge model.

The presence of the virus was shown by intense red signal. The intensity of the signal was then compared to the real-time qPCR results performed on the parallel samples, showing a positive correlation. Non-infected control animals served as negative controls and the total lack of positive staining confirmed the specificity of the assay.

For the first time, our research group implemented an insitu hybridization assay for the detection of PRRSV in challenged animals. Previous attempts to use immunohistochemistry for the same purposes were unsuccessful due to the high genetic and antigenic variability of the virus and the lack of broadly reacting antibodies available on the market.



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