ArticleViruses2021
Insertion of Exogenous Genes within the
Article in Viruses, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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0 citing papers in PubMed, 3 citations in OpenAlex.
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Authors and funding
8 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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. 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). 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. 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. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. 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. 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. 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.
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