The recently reported novel human astroviruses are more closely related to animals than the eight classic Human astrovirus (HAstV) serotypes, which could have resulted from cross-species transmission events. For human beings, AstVs mainly cause diarrhea and gastroenteritis, and they are second only to rotavirus as the leading cause of diarrhea in children. Astroviruses (AstVs) can infect most mammals and avian and are divided into two different genera, Mamastrovirus and Avastrovirus, depending on their source. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.Īstroviruses were firstly detected by Appleton and Higgins in the feces of children suffering from diarrhea in 1975. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. Biological analysis of the reporter virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Incorporation of the commonly used epitope tags, His, Flag, and HA, into four of the five insertion sites permitted the production of infectious viruses and allowed recognition by specifically tagged monoclonal antibodies. Five sites in the predicted coiled-coil structures (CC), genome-linked protein (VPg), and hypervariable region (HVR) in ORF1a of the PAstV genome were identified that could tolerate random 15 nt insertions. Here, transposon-mediated insertion mutagenesis was used to insert a 15-nucleotide (nt) sequence into random sites of open reading frame 1a (ORF1a) based on an infectious full-length cDNA clone of porcine astrovirus (PAstV). A tagged or reporter astrovirus can be a valuable tool for the analysis of various aspects of the virus life cycle, and to aid in the development of genetically engineered astroviruses as vectors.