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Plant viral pathogens cause damaging diseases in many agriculture systems, and emerging viral infections are a serious threat for providing adequate food to a continuously growing population. Recent studies of biogenic substances have provided new opportunities for producing novel antiviral agents. The present work has been conducted to evaluate the antiviral activity of quinoa (Chenopodium quinoa Willd.) seeds crude extract. The antiviral activity was retained in different buffer solutions of various pH ranges (5.2–8.5) and remained after the diafiltration process. The putative virus inhibitor was sensitive to treatment with sodium dodecyl sulfate and trichloroacetic acid. An antiviral protein with ~ 25 kDa molecular weight was isolated from the seed quinoa extract using ammonium sulfate precipitation, anion and cation exchange chromatography. The purified protein (Quinoin-I) significantly inhibited TMV on tobacco leaves with an IC50 value at a 6.81 μg/ml concentration. Enzyme activity assay revealed the RNase activity of Quinoin-I, and this feature was retained in the presence of β-mercaptoethanol and ethylene diamine tetraacetic acid. This antiviral protein has been shown as a promising leading molecule for further development as a novel antiviral agent. [ABSTRACT FROM AUTHOR]