In Vitro Replication of Human Norovirus

Abstract

Human norovirus (HuNoV) infection is a major cause of gastroenteritis all over the world. Despite this, these non-enveloped RNA viruses are poorly characterized due to the lack of robust and widely available HuNoV culture systems. The two published systems (B cell line and stem cell-derived enteroids) support replication of HuNoVs but the levels of replication are not sufficient for the generation of highly purified virus stocks or the development of culture-based quantification assays. Therefore, improvement of HuNoV in vitro replication is still needed. Murine norovirus and other caliciviruses have provided insights into norovirus replication that paved the way for the development of the current HuNoV culture systems and may also aid in the improvement of these systems. This review will highlight ways in which previous research guided and impacted the development of HuNoV culture systems and discuss ways in which more recent discoveries might be utilized to improve the quality of the HuNoV in vitro replication.

Keywords: human intestinal enteroids; human norovirus; in vitro culture models; murine norovirus; norovirus replication.

Figure 1

Proposed tropism model of MNV and HuNoV to various cells in the human small intestine [36,50]. These viruses may enter in free form or as clusters [51] potentially crossing through microfold (M) cells [52,53] or through intestinal epithelial cells of the villus. Tuft cells express CD300lf and have been shown to be infected by MNV [39]. Norovirus can further cross the brush border of epithelial cells to infect cells of hematopoietic origin like dendritic cells [36,43], T cells [14,36,41], B cells [36,43], and macrophages [14,36,41]. All the cell types depicted as HuNoV positive have been shown to be positive for viral antigen or genome in either animal studies or human studies.

Figure 2

Viral mechanisms to increase the cellular multiplicity of infection (MOI). Single RNA viruses lack proofreading mechanisms diminishing the ability of the virus to undergo successful replication in its host due to the production of attenuating mutations [89]. On the other hand, infection of a single cell with multiple viruses increases infection efficiency and viral fitness [51]. Two such mechanisms have been identified in enteric viruses: Attachment to commensal bacteria (poliovirus) and egress in extracellular vesicles (rotavirus) [51,90]. A similar mechanism may be employed by HuNoV, which could explain enhanced B cell replication in vitro in the presence of enteric bacteria such as Enterobacter cloacae [12]. Noroviruses are also found inside multi-vesicular body (MVB) derived exosomes and cause higher infection as compared to single viruses of same MOI [51].