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Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

ABSTRACT: Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.

SUBMITTER: Kolawole AO

PROVIDER: S-EPMC6957189 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

Kolawole Abimbola O AO Mirabelli Carmen C Hill David R DR Svoboda Sophia A SA Janowski Andrew B AB Passalacqua Karla D KD Rodriguez Benancio N BN Dame Michael K MK Freiden Pamela P Berger Ryan P RP Vu Diem-Lan DL Hosmillo Myra M O'Riordan Mary X D MXD Schultz-Cherry Stacey S Guix Susana S Spence Jason R JR Wang David D Wobus Christiane E CE

PLoS pathogens 20191031 10

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only h ...[more]

PMID: 31671153

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Project description:The host response of the primary intestinal epithelium to human astrovirus (HAstV infection has not been elucidated to date. In order to characterize the global effects of AstV infection on human intestinal tissue, we performed transcriptional profiling of VA1-infected human intestinal enteroids (HIE) by RNAseq. We used the D124 line for our studies since AstV infections are typically symptomatic in very young children, and our prior infection studies indicated rapid infection in D124. D124 HIE were mock-infected or infected with VA1 (MOI = 1) and harvested at 0 hpi (i.e., 1h post-adsorption), 12 hpi, and 24 hpi (Fig 4A). To monitor viral replication, VA1 genome copies were detected by RT-qPCR, revealing an approximately 1 and 2 log increase at 12 and 24 hpi, respectively (Fig 4B). Cellular RNA was extracted and analyzed by RNAseq. Genome copies as determined by RT-qPCR were correlated to the proportion of viral transcripts in the pool of sequenced RNA collected from the same HIE cultures, revealing an exceptionally strong correlation between the two measures of viral replication (r2 = 0.98, P = 2.5 x 10^-14. VA1 genome reads contributed 0.05 ± 0.02 x% of total RNAseq reads at 24 hpi, further confirming robust infection. Differential expression analysis was performed to identify genes associated with the HIE host response to VA1 infection. A total of 23,220 genes were detected. Differential expression of genes at each timepoint was graphed in a volcano plot. The log2 fold change in normalized expression (transcripts per million reads [TPM]) of all expressed host genes in VA1-infected HIEs relative to mock-infected HIE is shown on the x-axis. The -log10 transformed P-value is given on the y-axis. Genes that are significantly up-regulated (adjusted P < 0.05) in VA1-infected D124 HIE relative to mock-infected HIE are colored red, while significantly down-regulated genes are colored blue. Overall, after a 1 hour adsorption (0 hpi), 110 genes were significantly upregulated and 136 genes were downregulated , indicating changes due to viral attachment to cells. This number was reduced at 12 hpi, with 8 significantly upregulated and 5 downregulated genes. At 24 hpi, 154 upregulated and 49 downregulated genes compared to the mock-infected control were identified. We next identified the top 15 significantly up- and down-regulated genes at 24 hpi. This group of genes was used to generated a heatmap of the mean scaled fold-change (Z-score) in expression of each of them in virus-infected HIE relative to mock-infected HIE at each timepoint (Fig 4D). Most of the upregulated genes at 24 hpi were involved in type I and type III interferon (IFN) signaling. Of the IFN genes, IFNL1 was highly upregulated, with IFNA1 and IFNB1 upregulation being slightly lower (Fig S4B). No upregulation was observed for the genes encoding IFN-γ, or the type I and III IFN receptors (data not shown). The top 12 IFN-stimulated genes (ISGs) also positively correlated with VA1 infection (Fig S4C). Conversely, the top 12 downregulated genes, including fermitin family member 1 (FERMT1), signal peptide peptidase like 3 (SPPL3), and tetratricopeptide repeat domain 19 (TTC19) negatively correlated with VA1 infection (Fig S4D). Next, we evaluated lists of the top 100 up- and down-regulated genes at 24 hpi using an over-abundance test to identify significantly over-represented REACTOME pathways in these lists. These data revealed that the top four significantly enriched pathways among upregulated genes were all related to innate antiviral signaling (Fig 4E), which will be investigated in more detail below. For downregulated genes, the top two pathways were “neurexins and neuroligins”, which play signaling roles in synapse development, and “protein-protein interactions at synapses”. The biological significance of synapses during astrovirus infection remains to be elucidated. In order to evaluate the potential for coordinated and directional activation of genes in known signaling pathways, we applied gene set enrichment analysis (GSEA) to our RNAseq differential expression data. Based on the strong dominance of IFN signaling pathways, we focused our GSEA analysis on immune signaling (Fig 4F). During the adsorption phase, nucleic acid pattern recognition receptor signaling pathways (TLRs, STING) were upregulated, consistent with their early role in virus recognition and induction of IFN signaling. At 24 hpi, these early signaling events had been largely replaced by the later phase of IFN signaling and expression of ISGs. Taken together, these data indicate that VA1 infection predominantly elicited antiviral IFN signaling in HIE-derived fetal duodenum at the transcript level.

Project description:Human sapoviruses (HuSaVs), like human noroviruses (HuNoV), belong to the Caliciviridae family and cause acute gastroenteritis in humans. Since their discovery in 1976, numerous attempts to grow HuSaVs in vitro were unsuccessful until 2020, when these viruses were reported to replicate in a duodenal cancer cell-derived line. Physiological cellular models allowing viral replication are essential to investigate HuSaV biology and replication mechanisms such as genetic susceptibility, restriction factors, and immune responses to infection. In this study, we demonstrate replication of two HuSaV strains in human intestinal enteroids (HIEs) known to support the replication of HuNoV and other human enteric viruses. HuSaVs replicated in differentiated HIEs originating from jejunum, duodenum and ileum, but not from the colon, and bile acids were required. Between 2h and 3 to 6 days postinfection, viral RNA levels increased up from 0.5 to 1.8 log10-fold. Importantly, HuSaVs were able to replicate in HIEs independent of their secretor status and histo-blood group antigen expression. The HIE model supports HuSaV replication and allows a better understanding of host-pathogen mechanisms such as cellular tropism and mechanisms of viral replication. IMPORTANCE Human sapoviruses (HuSaVs) are a frequent but overlooked cause of acute gastroenteritis, especially in children. Little is known about this pathogen, whose successful in vitro cultivation was reported only recently, in a cancer cell-derived line. Here, we assessed the replication of HuSaV in human intestinal enteroids (HIEs), which are nontransformed cultures originally derived from human intestinal stem cells that can be grown in vitro and are known to allow the replication of other enteric viruses. Successful infection of HIEs with two strains belonging to different genotypes of the virus allowed discovery that the tropism of these HuSaVs is restricted to the small intestine, does not occur in the colon, and replication requires bile acid but is independent of the expression of histo-blood group antigens. Thus, HIEs represent a physiologically relevant model to further investigate HuSaV biology and a suitable platform for the future development of vaccines and antivirals.

Project description:Rotaviruses (RVs) are a significant cause of severe diarrheal illness in infants and young animals, including pigs. Group C rotavirus (RVC) is an emerging pathogen increasingly reported in pigs and humans worldwide, and is currently recognized as the major cause of gastroenteritis in neonatal piglets that results in substantial economic losses to the pork industry. However, little is known about RVC pathogenesis due to the lack of a robust cell culture system, with the exception of the RVC Cowden strain. Here, we evaluated the permissiveness of porcine crypt-derived 3D and 2D intestinal enteroid (PIE) culture systems for RVC infection. Differentiated 3D and 2D PIEs were infected with porcine RVC (PRVC) Cowden G1P[1], PRVC104 G3P[18], and PRVC143 G6P[5] virulent strains, and the virus replication was measured by qRT-PCR. Our results demonstrated that all RVC strains replicated in 2D-PIEs poorly, while 3D-PIEs supported a higher level of replication, suggesting that RVC selectively infects terminally differentiated enterocytes, which were less abundant in the 2D vs. 3D PIE cultures. While cellular receptors for RVC are unknown, target cell surface carbohydrates, including histo-blood-group antigens (HBGAs) and sialic acids (SAs), are believed to play a role in cell attachment/entry. The evaluation of the selective binding of RVCs to different HBGAs revealed that PRVC Cowden G1P[1] replicated to the highest titers in the HBGA-A PIEs, while PRVC104 or PRVC143 achieved the highest titers in the HBGA-H PIEs. Further, contrasting outcomes were observed following sialidase treatment (resulting in terminal SA removal), which significantly enhanced Cowden and RVC143 replication, but inhibited the growth of PRVC104. These observations suggest that different RVC strains may recognize terminal (PRVC104) as well as internal (Cowden and RVC143) SAs on gangliosides. Finally, several cell culture additives, such as diethylaminoethyl (DEAE)-dextran, cholesterol, and bile extract, were tested to establish if they could enhance RVC replication. We observed that only DEAE-dextran significantly enhanced RVC attachment, but it had no effect on RVC replication. Additionally, the depletion of cellular cholesterol by MβCD inhibited Cowden replication, while the restoration of the cellular cholesterol partially reversed the MβCD effects. These results suggest that cellular cholesterol plays an important role in the replication of the PRVC strain tested. Overall, our study has established a novel robust and physiologically relevant system to investigate RVC pathogenesis. We also generated novel, experimentally derived evidence regarding the role of host glycans, DEAE, and cholesterol in RVC replication, which is critical for the development of control strategies.

Project description:Porcine epidemic diarrhea virus (PEDV), a member of the group of alphacoronaviruses, is the pathogen of a highly contagious gastrointestinal swine disease. The elucidation of the events associated with the intestinal epithelial response to PEDV infection has been limited by the absence of good in vitro porcine intestinal models that recapitulate the multicellular complexity of the gastrointestinal tract. Here, we generated swine enteroids from the intestinal crypt stem cells of the duodenum, jejunum, or ileum and found that the generated enteroids are able to satisfactorily recapitulate the complicated intestinal epithelium in vivo and are susceptible to infection by PEDV. PEDV infected multiple types of cells, including enterocytes, stem cells, and goblet cells, and exhibited segmental infection discrepancies compared with ileal enteroids and colonoids, and this finding was verified in vivo Moreover, the clinical isolate PEDV-JMS propagated better in ileal enteroids than the cell-adapted isolate PEDV-CV777, and PEDV infection suppressed interferon (IFN) production early during the infection course. IFN lambda elicited a potent antiviral response and inhibited PEDV in enteroids more efficiently than IFN alpha (IFN-?). Therefore, swine enteroids provide a novel in vitro model for exploring the pathogenesis of PEDV and for the in vitro study of the interplay between a host and a variety of swine enteric viruses.IMPORTANCE PEDV is a highly contagious enteric coronavirus that causes significant economic losses, and the lack of a good in vitro model system is a major roadblock to an in-depth understanding of PEDV pathogenesis. Here, we generated a porcine intestinal enteroid model for PEDV infection. Utilizing porcine intestinal enteroids, we demonstrated that PEDV infects multiple lineages of the intestinal epithelium and preferably infects ileal enteroids over colonoids and that enteroids prefer to respond to IFN lambda 1 over IFN-?. These events recapitulate the events that occur in vivo This study constitutes the first use of a primary intestinal enteroid model to investigate the susceptibility of porcine enteroids to PEDV and to determine the antiviral response following infection. Our study provides important insights into the events associated with PEDV infection of the porcine intestine and provides a valuable in vitro model for studying not only PEDV but also other swine enteric viruses.

Dataset Information

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

Publications

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

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