Replication and transcriptionomic analysis of human noroviruses in human intestinal enteroids

HIEs cultures

Wnt3a-producing cells (CRL-2647 cells) were purchased from ATCC company. R-spondin-producing cells (R-spondin1) were purchased from Trevigen company. Recombinant human Noggin were purchased from Peprotech company. Complete media with and without growth factors (CMGF⁺ and CMGF^-, respectively), differentiation media, and Wnt3a- R-spondin- and Noggin-conditioned media were prepared as previously studies [11,12].

Three-dimensional (3D) cultures of HIEs were generated and cultured as detailed by previous studies [8,9]. Briefly, HIE cultures were derived from biopsies from surgical specimens. HIEs were grown and maintained as multilobular, 3D cultures in Matrigel (BD). For all infections, single cell suspensions were prepared from the 3D HIEs by trypsin digestion, and then seeded on a 96-well plate using CMGF⁺ media. After 24 hours of growth, the culture media was changed to differentiation medium to differentiate the cells for 5 days.

Stool filtrates

To prepare 10% stool suspensions, sterile PBS was added to HuNoV-positive stools, which were then homogenized by vortexing and sonicated three times for 1 min. The sonicated suspensions were centrifuged at 1,500 × g, 10 min, at 4°C. The supernatant was passed serially through 5 μm, 1.2 μm, 0.8 μm, 0.45 μm and 0.22 μm filters. The filtered samples were aliquoted and frozen at -80°C until used.

HuNoV infection of HIE monolayers

For inoculation, the HIE cell monolayers were washed once with CMGF^- media and inoculated with HuNoV diluted in CMGF^- media containing or not containing bile substitute glycyrene deoxycholic acid (GCDCA) (Sigma, Cat. G0759) for 1 hour at 37°C. The inoculum was removed and monolayers were washed twice with CMGF^- media to remove unbound virus. Differentiation media was then added and the cultures were incubated at 37°C for the indicated time points.

RNA extraction and real-time PCR

We isolated total RNA from HIE monolayers using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. For each sample, 1 ug of RNA was reverse transcribed using the reverse transcription kit (Takara). We performed real-time PCR amplification using SYBR Green Master Mix kit (Takara) in an Applied Biosystems Q6 platform. GAPDH was used as the internal control for mRNA quantification, and the fold change was calculated using the 2^-ΔΔCt method. The primer sequences used in this study was provided in Table 1.

RNA sequencing in BGISEQ-500 platform

18 total RNA was extracted using Trizol (Invitrogen), and the quantity and quality of mRNA were assayed with an Agilent 2100 Bioanalyzer and NanoDrop. Approximately 1 μg total RNA was initially used for BGISEQ-500 library construction, and samples were then sequenced on the BGISEQ-500 platform [13]. Raw sequencing reads were filtered to get clean reads by using SOAPnuke, and HISAT pipeline was applied to align reads against reference genome. The identification of differentially expressed genes (DEGs) was based on the negative binomial distribution of DEseq2 package, and the cut-off of DEGs was Fold Change ≥ 2 and adjusted P value ≤ 0.05.

Bioinformatics analysis

Gene ontology (GO) enrichment analysis was performed using the GOseq R package, in which gene length bias was corrected [14]. For DEGs, GO terms with corrected P < 0.05 were considered significantly enriched. Additionally, the statistical enrichments of DEGs in the Kyoto encyclopedia of genes and genomes (KEGG) related pathways were implemented by the KEGG orthology based annotation system [15].

Statistical analysis

Each experiment was performed two or more times, with three technical replicates of each culture, condition and time point in each experiment. All results are reported as means ± SEM. Statistical evaluation between groups was performed by two-tailed Student’s t-test or one-way ANOVA and is presented in the Figures. P-values < 0.05 were considered statistically significant.

Establishment of human intestinal enteroid model

Intestinal crypts were isolated from fresh adult jejunal tissue specimens, then seeded on Matrigel-coated plates in the presence of growth factors, including Wnt-3A, R-Spondin, Noggin, etc. Microscopic observation showed that the cells in HIE were similar to the cells in human intestinal tissue with polarized growth (Figure 1A). Transmission electron microscopic observation revealed that the villus faced the cavity surface, the complete tight connection was present between cells, the typical vesicle bodies of goblet cells accumulated above the nucleus, and hormone-rich vesicles in endocrine cells accumulated under the nucleus (Figure 1B). The result of immunofluorescence showed that the goblet cells were dispersed in the monolayer cells after differentiation (Figure 1C).

HuNoV replication in HIE

To evaluate whether the HIE support replication of the HuNoVs, monolayers of HIEs were inoculated with HuNoVs GII.4 from stool filtrates of children or adult patients. In the absence of GCDCA, at 72 hours post-infection (hpi), the genome equivalents of viral progeny of HuNoVs from adult patients increased 3.38 folds compared with the amount of genomic RNA detected at 1 hpi; while the virus genome of HuNoVs from children patients increased 11.88 folds at the same time point. By contrast, in the presence of GCDCA, at 72 hpi, the genome equivalents of viral progeny of HuNoVs from adult patients increased 6.10 folds compared with the amount of genomic RNA detected at 1 hpi; while the virus genome of HuNoVs from children patients increased 17.23 folds at the same time point (Figure 2A). In addition, we observed the expansion rate of different GII.4 variants in HIE monolayers from the same source. Three strains of HuNoVs GII.4, including GII.4 New Orleans, GII.4 Taoyuan, and GII.4 Iwate5, were used to infected the HIE monolayers. In the presence of GCDCA, at 72 hpi, the virus genome of these three HuNoV strains increased 1.55 folds, 12.60 folds, and 17.23 folds, respectively, compared with that detected at 1 hpi (Figure 2B).

RNA-sequencing analysis of gene expression profile in HIE infected by HuNoV

We systematically investigated the mRNA transcriptome in HIE during the infection of HuNoV using RNA sequencing. To this end, monolayers of HIEs from two different donors (L and M) were inoculated with GII.4 HuNoVs. At 0, 1, and 72 hpi, cells were harvested to detect the gene expression profiles. Principal component analysis showed that the intraculture transcriptional profiles for a specific infection status clustered together, but the basal human epithelial transcriptome differs significantly between HIEs from different individuals (Figure 3A). In total, after normalization, 116 and 2,899 genes were found differentially expressed in the HIE from donor L at 1 and 72 hpi, respectively, compared with the uninfected control; while 4,327 and 5,376 genes were deregulated in the HIE from donor M at 1 and 72 hpi, respectively, compared with the uninfected control (Figure 3B).

The aberrant genes were subjected to Gene Ontology (GO) analysis for functional classification (Figure 4). Differential genes associated with biological processes were mainly enriched in “cellular process”, “biological regulation”, “metabolic process”, “regulation of biological process”, “response to stimulus”, “multicellular organismal process”, “localization”, “signaling”, “developmental process”, “cellular component organization or biogenesis”, “multi-organism process”, “immune system process”, which implied that HuNoV infection significantly activated the cellular responses, intestinal cell-related metabolic processes, and biological regulation and immune response of HIE cells. Deregulated genes related to cellular component were mainly enriched in “cell”, “cell part”, “organelle”, “membrane”, “extracellular region”, “extracellular region part”, “macromolecular complex”, “membrane-enclosed lumen”, which suggested that multiple cellular components were involved in the infection and replication of HuNoV. Differential genes associated with molecular function were primarily enriched in “binding”, “catalytic activity”, “transporter activity”, “signal transducer activity”, “molecular function regulator”, “binding transcription factor activity”, and “structural molecule activity”, which indicated that HuNoV infection resulted in complicated interaction between pathogen and host cell.

The differential genes also were subjected to pathway analysis for functional classification (Figure 5). Six significantly enriched pathway were found, including “cellular Processes”, “Environmental Information Processing”, “Genetic Information Processing”, “Human diseases”, “Metabolism”, and “Organismal systems”. These differential genes could be further allocated to 45 pathways, in which “Signal transduction” and “Immune system” were the most markedly enriched pathways.

At last, we analyzed and classified differentially expressed genes with transcription factor activity. The results showed that differential transcription factors were mainly enriched in several families, such as zf-C2H2, zf-C2HC, Homeobox, HMG and ARID, suggesting that these transcription factor families played a crucial role in the regulation of gene expression after infection (Figure 6A). In addition, cluster analysis that the expression pattern of transcription factors in different samples were similar (Figure 6B).

The expression of IFNs and IFN-stimulated genes in HIE infected by HuNoV

GO analysis showed that differentially expressed genes in HIE cells were enriched in type III interferon (IFN) signaling pathway after infection (P < 0.001, Figure 7). To verify the sequencing results, we examined the expression of IFN-β, IFN-λ, and IFN-stimulated genes (ISGs) using qPCR. Consistent with the results of RNA sequencing, the qPCR result revealed that the expression of IFN-β in HIE cells was not changed after infection, but the expression of IFN-λ, ISG-15, and ISG-45 was significantly elevated after infection.