Abstract: 【Objective】In order to make researches on the RNA location and distribution of Classical Swine Fever Virus （CSFV）in infected PK15 cells, a rapid, accurate and sensitive in situ hybridization (ISH) technology was established. 【Method】After comparison with the complete sequences of CSFV, BVDV and BDV to avoid the homology regions, a set of specific probes of CSFV RNA andβ-actin were designed and synthesized. A reference strain CSFV (HeBHH1/95) was used to optimize the ISH technology through comparison several parameters such as fluorescence intensity, repeatability, protease K concentration and formalin fixation time. After these parameters were determined, an ISH technology was established. Fluorescent antibody test (FAT) was used to compare the sensitivity with the ISH technology. All the sub genotypes of CSFV (sub genotype 1.1, 2.1,2.2 and 2.3) which are present in China and other normal pig infectious virus (BVDV, PPV, PRV，PCV-2) were used to detect specificity of this ISH technology. A high virulent strain of CSFV (SM) was inoculated in PK15 cells. Infected cells were sampled at 0.5 hours post inoculation (hpi)、1, 3, 6, 8, 10, 14, 18, 24, 36, 48, 72, and 96hpi. Then FAT was performed in parallel to detect the expression and location of CSFV E2 protein. 【Result】CSFV RNA were detected in infected PK15 cells by using the ISH technology. The optimal concentration of protease K was 1:1 000 and the optimal time of formalin fixation was 30 minutes. The minimum of detection is 10-8/200μL which is 3.5 orders of magnitude higher than FAT. The specific tests showed that the ISH technology could react with sub genotypes 1.1, 2.1,2.2 and 2.3 of CSFV in China and has no cross reaction with BVDV, PPV, PRV，PCV-2 viruses. CSFV RNA ISH test results showed that CSFV RNA were firstly detected in nucleus at 0.5 hpi and gathered in nucleus from 0.5hpi to 6hpi; at 10hpi, there were more CSFV RNA gathered in cytolymph than before and less CSFV RNA were detected than before in nucleus；at 24hpi, CSFV RNA mainly gathered in cytolymph around nucleus; at 36hpi, more and more CSFV RNA gathered in cytolymph and got maximum at 72hpi; at 96hpi, the amount of CSFV RNA declined. The results of FAT showed that little E2 protein was detected in cytolymph at 8hpi. From 10hpi to 24 hpi, only few cells were detected positive. After 36hpi, the expression of E2 protein increased gradually and got to the maximum at 72hpi. But after 96hpi, the amount of E2 protein declined. The trend of E2 protein expression was in accord with CSFV RNA. 【Conclusion】 A visualization in situ hybridization technology was firstly established to detect CSFV RNA. The location and distribution of CSFV RNA was studied by using this technology. The results proved that CSFV RNA enters cell early than 0.5hpi and CSFV RNA has ever existed in nucleus.

Key words: Classical Swine Fever Virus (CSFV), location, distribution, in situ hybridization (ISH), Fluorescent antibody test (FAT)