Abstract Title
Establishment of a novel Caco-2-based cell culture system for human sapovirus propagation
Presenter
Reiko Todaka, Kitasato University
Co-Author(s)
Reiko Todaka1, Yuya Fukuda2, Miyabi Arai1, Ryoka Ishiyama1, Kei Haga1 and Kazuhiko Katayama1
1 Kitasato University 2 Sapporo Medical University School of Medicine
Abstract Category
Entry and Replication
Abstract
Human sapovirus (HuSaV), first identified in the 1970s, is a significant cause of acute gastroenteritis. Despite its clinical importance, research has been limited by the absence of a robust cell culture system. In 2020, HuSaV propagation in HuTu80 cells with bile acids was demonstrated. However, a 2024 study revealed that efficient replication required specialized HuTu80 subclones after over 100 passages. This study aimed to establish a stable and reproducible alternative to HuTu80 cells for HuSaV propagation. Human colorectal cell lines (Caco-2, HCT15, HCT116, etc.) were infected with a HuSaV GI.1-positive stool suspension. For susceptible cell lines, single-cell cloning was performed to isolate highly responsive subclones.
In the presence of GCA, HuSaV GI.1 infection led to a ≥3.2 log₁₀-fold increase in RNA copy numbers in Caco-2 cells and a 4.8 log₁₀-fold increase in Caco-2/Cas9 cells from dpi 0 to dpi 7. Serial propagation was successfully achieved by passaging culture supernatants onto fresh cells. At dpi 4, flow cytometry revealed 19.1% VP1-positivity in Caco-2 cells and 38.8% in Caco-2/Cas9 cells. Single-cell cloning of Caco-2/Cas9 cells led to the establishment of Caco-2MC cells, with 91.5% susceptibility. Using Caco-2MC cells, serial propagation was achieved for multiple HuSaV genotypes, including GI.1, GI.2, GI.3, GII.1, GII.3, and GV.1. Furthermore, virus neutralization assay using these cells showed that the virus was neutralized by anti-HuSaV GI.1VLP serum.
In conclusion, we established a HuSaV-susceptible Caco-2-derived cell line, enabling efficient vaccine and antiviral development, receptor identification, and epidemiological studies.
In the presence of GCA, HuSaV GI.1 infection led to a ≥3.2 log₁₀-fold increase in RNA copy numbers in Caco-2 cells and a 4.8 log₁₀-fold increase in Caco-2/Cas9 cells from dpi 0 to dpi 7. Serial propagation was successfully achieved by passaging culture supernatants onto fresh cells. At dpi 4, flow cytometry revealed 19.1% VP1-positivity in Caco-2 cells and 38.8% in Caco-2/Cas9 cells. Single-cell cloning of Caco-2/Cas9 cells led to the establishment of Caco-2MC cells, with 91.5% susceptibility. Using Caco-2MC cells, serial propagation was achieved for multiple HuSaV genotypes, including GI.1, GI.2, GI.3, GII.1, GII.3, and GV.1. Furthermore, virus neutralization assay using these cells showed that the virus was neutralized by anti-HuSaV GI.1VLP serum.
In conclusion, we established a HuSaV-susceptible Caco-2-derived cell line, enabling efficient vaccine and antiviral development, receptor identification, and epidemiological studies.