Establishment And Preliminary Application Of CSFV QuantiGene ViewRNA In Situ Hybridization Technique

Classical Swine Fever(CSF) caused by Classical Swine Fever Virus(CSFV) is a highly contagiousand hemorrhagic disease of pigs and has caused significant economical losses in pig industry worldwide.In this study, CSFV QuantiGene ViewRNA in situ hybridization technique was established; thenresearched on the proliferation dynamics and distribution regularity of CSFV RNA in infected cells invitro and detection CSFV RNA in vivo with the established method.In order to detect and localize CSFV accurately and explore the proliferation dynamics anddistribution regularity of CSFV RNA after infecting PK15in vitro, CSFV specific probes and swinehouse-keeping gene β-actin probes were designed and conducted research on CSFV moderate strainHeBHH1/95(TCID50equals10-4.5/200μL) cultured in PK15cells. In consideration of detection results,fluorescent intensity and repeatability, the hybridization effects could be best when the Protease Kconcentration was1:1000and Formaldehyde fixed time was30min. Under the fluorescent confocalmicroscopy, the positive cells could be detected with bright red fluorescence. Compared with CSFVimmunohistochemical technique and CSFV monoclonal antibody immunofluorescent method, CSFVQuantiGene ViewRNA in situ hybridization technique was the most sensitive whose detection limit canbe up to10-8dilution, higher4and3degrees, respectively. Other viruses and other CSFV strains wereused to test for specificity. CSFV specific probes could bond to different CSFV strains rather than otherviruses which indicated the good specificity.In order to study the accurate localization and proliferation dynamics of CSFV RNA in infectedcells in vitro, infected PK15cells with CSFV moderate virulent strain HeBHH1/95and HCLV, detectedCSFV RNA with established CSFV QuantiGene ViewRNA in situ hybridization technique at differenttimes such as0.5hpi,1hpi,1.5hpi,2hpi,2.5hpi,3hpi,3.5hpi,4hpi,4.5hpi,5hpi,5.5hpi,6hpi,12hpi,18hpi,24hpi,36hpi,48hpi,72hpi and96hpi under fluorescent confocal microscopy. The results showedthat after infection by HeBHH1/95and HCLV, with the time going, virus replicated and proliferated incells until72hpi; CSFV RNA could be detected0.5hpi, and during0.5hpi and12hpi, CSFV RNA was innucleus, while after18hpi, CSFV RNA was mainly around the nucleus until96hpi. HeBHH1/95andHCLV has the similar proliferation regularity. In a word, this study clarify the proliferation dynamicsand distribution regularity of CSFV in vitro firstly.In order to visually detect CSFV RNA in cells in vivo accurately, CSFV infected animal model wasestablished and tonsil, peripheral lymph nodes and kidney were sampled to make Formalin FixedParaffin Embedded(FFPE), then used QuantiGene ViewRNA technique to detect CSFV RN A in variousinfected organs and targeted cells. The results showed that red fluorescent dots could be seen under thefluorescence microscope which indicated that the established CSFV QuantiGene ViewRNA in s ituhybridization technique could visually detect CSFV RNA in FFPE accurately. Therefore, this methodcould be helpful to explore the difference between acute and chronic CSFV infected cells type, in the meantime, to analyse the relevance between replication and distribution dynamics with the course ofdisease and pathological damage.In conclusion, the successfully established CSFV QuantiGene ViewRNA in situ hybridizationtechnique has the advantage of high sensitivity, good specificity, good stability and fast speed which canabsolutely supply a high sensitive and specific method for accurate diagnostic against CSFV. In themeantime, it will offer significant technical support for CSFV RNA proliferative and replicativedynamics study in targeted cells in vitro and in vivo and offer instructive suggestion for clinical CSFVRNA samples collection.