Abstract: Ovine Aichivirus D is an emerging virus in sheep in China, the aim of this study was to design primers targeting 3D gene of ovine Aichivirus D and optimize the reaction system and conditions, a TB Green Fluorescent RT-PCR method was successfully established; the assay has characteristics of good specificity and stability, high sensitivity. Total 253 sheep fecal samples (133 non-diarrheic and 120 diarrheic) were collected from 6 sheep farms in Sichuan province from April 2020 to May 2021, this virus detection rate was 8.7%, and the farm positive rate was 66.7%(4/6). Notably, the ovine Aichivirus D positive rate for the diarrheic feces (17.5%) was significantly higher than that for non-diarrheic feces (0.75%, P<0.001), suggesting that ovine Aichivirus D may be a pathogen of sheep diarrhea. In addition, 13 complete 3D genes in 1 422 bp in length were successfully cloned from ovine Aichivirus D positive samples in this study, which shared 99.4%-100.0% nt homology with each other. Evolutionary analysis showed that these 13 3D genes together with the ovine Aichivirus D 3D gene uploaded by our previous research clustered into a large independent branch. This study provides a new molecular tool for detecting ovine Aichivirus D and basic epidemiological data of ovine Aichivirus D.

Key words: ovine Aichivirus D, fluorescent RT-PCR method, sheep, diarrhea