Acute gastroenteritis is caused by various etiological agents, including viruses, bacteria, and parasitic protozoa; however, among these, acute gastroenteritis is most frequently caused by viruses (1). Together with rotavirus, human norovirus (HuNoV) is the main causative viral agent in epidemic and endemic acute gastroenteritis globally (2). Norovirus was previously called the Norwalk virus and was first identified in an outbreak of gastroenteritis in Norwalk, Ohio, USA in 1968. Noroviruses are non-enveloped, positive-sense, single-stranded RNA viruses that belong to the family Caliciviridae. Their viral genome consists of approximately 7.5–7.7. kb, which are organized into 3 open reading frames (ORFs) (ORF1, ORF2, and ORF3). There is significant genetic diversity among noroviruses owing to specific mutations and genetic recombination. Based on major and minor differences in the genetic sequence, the norovirus genus is subdivided into 10 genogroups (GI–GX) (3, 4). Currently, 41 genotypes belonging to 3 genogroups (GI, GII, GIII and GIV) have been associated with acute gastroenteritis in humans (5). HuNoVs are highly contagious viruses that cause vomiting and diarrhea within 24–48 hour (h) of the incubation period. These viruses can pass through the fecal-oral route by direct person-to-person contact or through feces-contaminated food or water (6). Although the infection is self-limiting within a few days, viral shedding continues in the feces for the next few weeks in immunocompromised patients (6). Susceptibility to norovirus infection at all ages leads to high rates of hospitalization and mortality. Therefore, norovirus gastroenteritis is recognized as a considerable societal and economic burden worldwide. Children under the age of 5 years, adults older than 65 years, and immunocompromised individuals are particularly vulnerable and more likely to develop severe disease. In children <5 years of age, there are currently more than 50,000 norovirus-associated deaths annually (7).

Despite the global burden of human norovirus infections on the economy and healthcare, developing effective control measures to prevent and treat norovirus gastroenteritis remains a challenge. Over the past several decades, despite many attempts to solve these problems, no fundamental changes have been made to date. The lack of an affordable small animal model and a robust and reproducible in vitro cell culture system for HuNoV propagation has been a significant obstacle for the successful development of live attenuated vaccines for this poorly characterized RNA virus. In particular, to understand the HuNoV-host interaction that underlies the high virus infectivity and volatile illness, it is necessary to develop an in vitro culture system. Therefore, developing a robust and reproducible culture system for HuNoV propagation can help to identify ways to prevent the transmission and treat infection and illness. Since the discovery of the Norwalk virus in 1972, many experiments toward the development of a cultivation system for HuNoV in vitro propagation have been performed using typical simple transformed cell lines. However, most of these laboratory efforts have not produced highly satisfactory results (8, 9, 10, 11, 12). Therefore, up to now, the development of multigenerational passaging systems of HuNoVs remain as a conundrum to be solved. However, struggles to cultivate this virus in vitro eventually led to the development of new strategies for HuNoV in vitro culture. These include the development of an infection-and-replication model using either primary stem cells or pluripotent stem cells to better mimic the native small intestine. This strategy is also called ‘organoid technology’, which bridges the gap between conventional two-dimensional cell line culture and the in vivo model. Cultivation of microorganisms in human intestinal organoid models has led to a new era for studying infectious diseases, such as those caused by noroviruses. Various intestinal infectious disease processes have been modeled using stem cell-derived organoids. Recently, it has been shown that many infectious pathogens can infect organoids, including parasites, rotavirus, enterohemorrhagic Escherichia coli, and Salmonella (13). In this review, we describe the key advances that led to the development of the currently available HuNoV in vitro culture systems that support replication of the virus.

HuNoV remains a difficult problem to solve globally. The availability of the classical infection models, including various 2-D cultures of immortalized cells and animal models, has not produced highly satisfactory results with regard to the understanding of how pathogens modulate host functions during infection and the development of successful live attenuated vaccines. Clearly, organoid technology has facilitated studies to understand the interactions between the gastrointestinal tract and enteric viruses and has proven to be a good model for the replication of non-cultivable viruses, such as the norovirus. Despite the progress in the development of the HuNoV culture systems, the field still faces several challenges that need to be solved in order to further enhance these culture systems, including the expense and complexity of culture media and lack of sustained passaging of the HuNoV strains. And also, Low viral replication yields and reproducibility and labor intensive process is problems to be solved. Fortunately, biological and bioengineering solutions are being developed at a rapid pace to solve these problems. Compared with two-dimensional cultures, three-dimensional organoid culture systems can provide more fundamental insights into the pathogenesis of HuNoV and may further enhance new translational approaches for the treatment and prevention of this enteric virus.