Development And Application In Situ Hybridization With Oligonucleotide Probe And In Situ RT-PCR To Detect PRRSV In Panaffin-embedded Tissues

According to ORF6 and 0RF7 gene sequence of ATCC VR-2332 strain in GenBank, we designed a set of primers with the oligo software . The amplification product is a 298bp fragment as expected with RT-PCR. We also relied on oligo software to select a 37bp oligonucleodide probe which was labelled with biotin. With the primers and the probe, we developed in situ hybridization and indirect in situ RT-PCR successfully to specifically detect PRRSV nucleic acids in paraffin-embedded tissues of infected pigs.Application of in situ hybridization to detect the distribution of virulent PRRSV SC-1 in organs of infected pigs at different times postinfection show that PRRSV SC-1 nucleic acids were found in tonsil, lymphonodus as early as day 3 postinfection. And then lung, thymus were observed positive cells at 5 days postinfection. PRRSV nucleic acids emerged in kidney, brain, intestine, spleen, liver until 7 days postinfection, but were very less in lung, kidney, brain. Sometimes positive cells could be sporadically present in testis, epididymis. Uterus, heart and pancreas were negative after detection by in situ hybridization.In lymph nodes, stained cells were scattered throughout the nodes. In lung, the cytoplasm of alveoliar macrophages in alveoliar septa and endothelial cells in the capillary contained PRRS virus. In tonsil, labelled cells were often localized in the centre of follicles and interstitium. In thymus, the PRRSV positive cells most often were seen in the medulla, but few positive cells were present in the cortex. Macrophages and lymphocytes cells were found in spleen. Positive cells were readily detected in the sinusoidal of liver. A few hepatocytes showed labelling for PRRSV nucleic acid. PRRSV-positive cells observed in the kidney were found predominantly in glomerulus and it's surrounding. In intestine, some stained cells were localized in the germinal center of lymphatic nodules forming the peyer's patches and epithetlial cells of intestineal villi.We detected tonsil, spleen, hilar node, thymus by in situ RT-PCR. PRRSV SC-1 nucleic acids were found in tonsil as early as day 1 postinfection. At 3 days postinfection with PRRSV SC-1, the signal by in situ RT-PCR was detected in hilar node, thymus, spleen.We compared the two molecular methods mentioned above after detection tonsil, spleen, thymus, hilar node. The results reveal that in situ RT-PCR is more sensitive than ISH, exactly 4d earlier than ISH to detect viral nucleic acids in spleen tissues. On the other hand, stronger positive signals could be obtained by in situ RT-PCR when the same tonsil, spleen tissues detected by in situ RT-PCR and ISH.