To construct a three-dimensional (3D) culture model of adenovirus in vitro using the nanoself-assembling peptide RADA16-I as a 3D cell culture scaffold combined with virology experimental technology to provide a novel research method for virus isolation and culture, pathogenesis research, antiviral drug screening and vaccine preparation. Methods: The nanoself-assembling peptide RADA16-I was used as a 3D scaffold material for 293T cell culture, and adenovirus was cultured in the cells. The growth, morphological characteristics and pathological effects of 3D-cultured 293T cells after adenovirus infection were observed with an inverted microscope and MTS. The proliferation of adenovirus in 293T cells was observed by TEM and detected by qPCR. The levels of TNF-α and IL-8 secreted by adenovirus-infected 293T cells in the RADA16-I 3D culture system were detected by ELISA. Results: The 293T cells grew well in the RADA16-I 3D culture system for a prolonged period of time. The adenovirus infection persisted for a long time with multiple proliferation peaks, which closely resembled those of in vivo infections. The adenovirus virions amplified in the 3D system remained infectious. There were multiple secretion peaks of TNF-α and IL-8 secretion levels in adenovirus-infected 293T cells cultured in 3D culture systems. Conclusion: The nanoself-assembling peptide RADA16-I can be used as a 3D scaffold for adenovirus isolation, culture and research. The 3D culture system shows more realistic in vivo effects than two-dimensional (2D) culture.

Viral infectious diseases seriously threaten human health. At present, the prevention and treatment of viral diseases depends primarily on vaccines and drugs. Commonly used research method include animal models or simple traditional two-dimensional (2D) isolation culture, but 2D cell behaviour is different from the human physiological microenvironment. Therefore, a new model that can simulate the human microenvironment is needed. Here, the nanoself-assembled peptide KLD-12 was used as a culture scaffold for 293T cells in threedimensional (3D) adenovirus culture. As a new 3D virus culture model, it simulates in vivo virus infection, and the model can produce infectious particles. In addition, the antiviral drug sensitivity between the 3D and 2D cultures was significantly different. We established a 3D adenovirus culture model that can be used for adenovirus proliferation and antiviral drug screening, as well as for gene therapy, vaccine research and other research. Additionally, it can partially replace animal models.