Xi-Lei Zeng

Replication of human noroviruses in stem cell–derived human enteroids

The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report the successful cultivation of multiple HuNoV strains in enterocytes in stem cell–derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for strain-dependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections.

Stem Cell-Derived Human Intestinal Organoids as an Infection Model for Rotaviruses

ABSTRACT Directed differentiation of stem cell lines into intestine-like tissue called induced human intestinal organoids (iHIOs) is now possible (J. R. Spence, C. N. Mayhew, S. A. Rankin, M. F. Kuhar, J. E. Vallance, K. Tolle, E. E. Hoskins, V. V. Kalinichenko, S. I. Wells, A. M. Zorn, N. F. Shroyer, and J. M. Wells, Nature 470:105-109, 2011). We tested iHIOs as a new model to cultivate and study fecal viruses. Protocols for infection of iHIOs with a laboratory strain of rotavirus, simian SA11, were developed. Proof-of-principle analyses showed that iHIOs support replication of a gastrointestinal virus, rotavirus, on the basis of detection of nonstructural viral proteins (nonstructural protein 4 [NSP4] and NSP2) by immunofluorescence, increased levels of viral RNA by quantitative reverse transcription-PCR (qRT-PCR), and production of infectious progeny virus. iHIOs were also shown to support replication of 12/13 clinical rotavirus isolates directly from stool samples. An unexpected finding was the detection of rotavirus infection not only in the epithelial cells but also in the mesenchymal cell population of the iHIOs. This work demonstrates that iHIOs offer a promising new model to study rotaviruses and other gastrointestinal viruses. IMPORTANCE Gastrointestinal viral infections are a major cause of illness and death in children and adults. The ability to fully understand how viruses interact with human intestinal cells in order to cause disease has been hampered by insufficient methods for growing many gastrointestinal viruses in the laboratory. Induced human intestinal organoids (iHIOs) are a promising new model for generating intestine-like tissue. This is the first report of a study using iHIOs to cultivate any microorganism, in this case, an enteric virus. The evidence that both laboratory and clinical rotavirus isolates can replicate in iHIOs suggests that this model would be useful not only for studies of rotaviruses but also potentially of other infectious agents. Furthermore, detection of rotavirus proteins in unexpected cell types highlights the promise of this system to reveal new questions about pathogenesis that have not been previously recognized or investigated in other intestinal cell culture models. Gastrointestinal viral infections are a major cause of illness and death in children and adults. The ability to fully understand how viruses interact with human intestinal cells in order to cause disease has been hampered by insufficient methods for growing many gastrointestinal viruses in the laboratory. Induced human intestinal organoids (iHIOs) are a promising new model for generating intestine-like tissue. This is the first report of a study using iHIOs to cultivate any microorganism, in this case, an enteric virus. The evidence that both laboratory and clinical rotavirus isolates can replicate in iHIOs suggests that this model would be useful not only for studies of rotaviruses but also potentially of other infectious agents. Furthermore, detection of rotavirus proteins in unexpected cell types highlights the promise of this system to reveal new questions about pathogenesis that have not been previously recognized or investigated in other intestinal cell culture models.

Human Intestinal Enteroids: New Models to Study Gastrointestinal Virus Infections

Human rotavirus (HRV) and human norovirus (HuNoV) infections are recognized as the most common causes of epidemic and sporadic cases of gastroenteritis worldwide. The study of these two human gastrointestinal viruses is important for understanding basic virus-host interactions and mechanisms of pathogenesis and to establish models to evaluate vaccines and treatments. Despite the introduction of live-attenuated vaccines to prevent life-threatening HRV-induced disease, the burden of HRV illness remains significant in low-income and less-industrialized countries, and small animal models or ex vivo models to study HRV infections efficiently are lacking. Similarly, HuNoVs remained non-cultivatable until recently. With the advent of non-transformed human intestinal enteroid (HIE) cultures, we are now able to culture and study both clinically relevant HRV and HuNoV in a biologically relevant human system. Methods described here will allow investigators to use these new culture techniques to grow HRV and HuNoV and analyze new aspects of virus replication and pathogenesis.

Human Norovirus Replication in Human Intestinal Enteroids as Model to Evaluate Virus Inactivation

Human noroviruses are a leading cause of epidemic and endemic acute gastroenteritis worldwide and a leading cause of foodborne illness in the United States. Recently, human intestinal enteroids (HIEs) derived from human small intestinal tissue have been shown to support human norovirus replication. We implemented the HIE system in our laboratory and tested the effect of chlorine and alcohols on human norovirus infectivity. Successful replication was observed for 6 norovirus GII genotypes and was dependent on viral load and genotype of the inoculum. GII.4 viruses had higher replication levels than other genotypes. Regardless of concentration or exposure time, alcohols slightly reduced, but did not completely inactivate, human norovirus. In contrast, complete inactivation of the 3 GII.4 viruses occurred at concentrations as low as 50 ppm of chlorine. Taken together, our data confirm the successful replication of human noroviruses in HIEs and their utility as tools to study norovirus inactivation strategies.

Role for FimH in Extraintestinal Pathogenic Escherichia coli Invasion and Translocation through the Intestinal Epithelium

ABSTRACT The translocation of bacteria across the intestinal epithelium of immunocompromised patients can lead to bacteremia and life-threatening sepsis. Extraintestinal pathogenic Escherichia coli (ExPEC), so named because this pathotype infects tissues distal to the intestinal tract, is a frequent cause of such infections, is often multidrug resistant, and chronically colonizes a sizable portion of the healthy population. Although several virulence factors and their roles in pathogenesis are well described for ExPEC strains that cause urinary tract infections and meningitis, they have not been linked to translocation through intestinal barriers, a fundamentally distant yet important clinical phenomenon. Using untransformed ex situ human intestinal enteroids and transformed Caco-2 cells, we report that ExPEC strain CP9 binds to and invades the intestinal epithelium. ExPEC harboring a deletion of the gene encoding the mannose-binding type 1 pilus tip protein FimH demonstrated reduced binding and invasion compared to strains lacking known E. coli virulence factors. Furthermore, in a murine model of chemotherapy-induced translocation, ExPEC lacking fimH colonized at levels comparable to that of the wild type but demonstrated a statistically significant reduction in translocation to the kidneys, spleen, and lungs. Collectively, this study indicates that FimH is important for ExPEC translocation, suggesting that the type 1 pilus is a therapeutic target for the prevention of this process. Our study also highlights the use of human intestinal enteroids in the study of enteric diseases.

A time-resolved immunoassay to measure serum antibodies to the rotavirus VP6 capsid protein

Highlights ► Two recombinant VP6 capsid proteins from an Indian birth cohort were expressed. ► The proteins were used to establish a time-resolved fluorescence DELFIA. ► DELFIA was compared to a gold standard ELISA using cohort serum samples. ► A significant association was observed between the two assays (p < 0.05). ► This demonstrates recombinant proteins can be used to study RV antibody responses.