Human Sapovirus Replication in Human Intestinal Enteroids

Abstract

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.

Keywords: enteric virus; enteric viruses; histo-blood group antigens; human intestinal enteroids; norovirus; sapovirus.

FIG 1

Two human sapovirus strains replicate in jejunal human intestinal enteroids (HIE). (A) Five HuSaV-positive stools filtrates were used to infect differentiated monolayers of HIE J2 cells in the presence of 500 μM bile acid GCDCA, with inocula of 1.10⁵ to 1.10⁸ HuSaV genome copies per well (horizontal axis). HuSaV genome quantities in each well (vertical axis) were measured by qRT-PCR at 1 or 2h (empty circles) and 72h (full disks) postinfection (pi). Differences between HuSaV titers at 2 and 72 hpi were not statistically significant, n = 2 to 3 experiments, P > 0.05, Wilcoxon test. (B) An inoculum of 1 × 10⁸ gc/well of HuSaV strain S611 was used in repeated experiments to infect differentiated J2 monolayers with 500 μM GCDCA and showed a mean genome fold change of 31.3 (n = 40 experiments, P < 0.0001, Wilcoxon test). (C) An inoculum of 1x10⁷ gc/well of HuSaV strain S513 was also used to infect differentiated J2 monolayers with 500 μM GCDCA and showed a geometrical mean genome amplification of 7.7 (n = 9 experiments, P = 0,012, Wilcoxon test). (D) In parallel with HuSaV infections, differentiated J2 monolayers were also infected with 1.10⁵ genome copies per well of HuNoV GII.4 TCH11-64 as a control with 500 μM GCDCA and showed efficient replication with a geometric mean fold increase of 4258 (n = 40 experiments, P < 0.0001, Wilcoxon test). (A, B, C, D) each dot is the mean of three technical replicates for one independent experiment at 2h (empty circles or pink diamonds) and 72h postinfection (plain disks and red diamonds), horizontal lines are the geometrical mean of virus genome copies per well considering all experiments, and the number above indicates the mean fold change in viral genome between the two time points.

FIG 2

Kinetics and magnitude of HuSaV S611 replication in HIE. (A) Differentiated J2 monolayers were infected with 1.10⁸ cg/well of HuSaV S611 with 500 μM GCDCA and the viral genome quantified after 2h, 72h and 144h. Each dots represents the mean of three technical replicates in an experiment (n = 7). In comparison with 2 hpi, viral replication was detected at 72 hpi (P < 0.05, Wilcoxon test) and 144 hpi (P < 0.05, Wilcoxon test) with geometrical mean fold changes in viral genome of 9.8 and 17.1, respectively. (B) HuTu80 were infected with S611 at four different concentrations from 1.10⁵ to 1.10⁹ cg/well with 500 μM GCDCA. Mean viral titers of three technical replicates were measured in the culture supernatants at 1, 3, 6, and 7 days postinfection and are plotted for each experiment (n = 2), with lines connecting the geometrical means. (C) Differentiated D109 monolayers were inoculated with 1.10⁸ cg/well native (plain black circles) or heat-inactivated (crossed circles) S611 with 500 μM GCDCA and the viral genome quantified after 2h, 24h, 48h, 72h, 96h, and 144h. The mean of technical replicates is plotted as a circle for each experiment (n = 2) and geometrical means are connected with a plain (native virus) or a dotted (inactivated virus) line. (D) The viability of the differentiated D109 monolayers either infected by native (plain black circles) or heat-inactivated (crossed circles) S611 with 500 μM GCDCA for n = 2 experiments in comparison with an uninfected control (100%, dotted line).

FIG 3

Bile acids are a necessary cofactor of HuSaV infection in HIE. (A) Differentiated J2 monolayers were infected with 1.10⁸ cg/well of S611 in the presence of the bile acid GCDCA at concentrations ranging from 50 μM to 1 mM, or in the absence of bile acid. The viral titer was measured and is depicted as the mean of three technical replicates for n = 2 to 6 experiments at 1 hpi (white circles) and 72 hpi (black circles) and the geometrical mean fold change between the two points indicated above. (B) The bile acid GlyCA (right side) was also used in comparison to GCDCA (left side) with concentrations of 500 μM on differentiated J2 monolayers infected with 1.10⁸ cg/well of S611, n = 5 experiments. (C) and (D) Experiments in (A) were also conducted with 1.10⁶ cg/well of HuNoV GII.3 (purple) or 1.10⁵ cg/well of HuNoV GII.4 (red), with GCDCA concentrations of 500 μM or without GCDCA.

FIG 4

HuSaV replicate in differentiated jejunal HIE. Comparison of the mean viral titer per well of S611 HuSaV (A) and GII.4 HuNoV (B) at 2 hpi (white bars) and 72 hpi (black bars) in J2 monolayers cultured and infected in growth or differentiation (diff.) medium with 500 μM GCDCA, for n = 3 experiments, 3 technical replicates per experiment. Geometric mean fold changes between the two time points are indicated above for each condition. (C) Fold change in relative expression levels of 5 genes (normalized on GAPDH) between HIE J2 monolayers cultured in differentiation versus growth medium in the experiments presented in (A). *, P < 0.05, ****, P < 0.0001, paired t test.

FIG 5

HuSaV replicates in human cells from the three segments of the small intestine. Differentiated HIE monolayers from different segments of the small intestine (J for jejunum, D for duodenum, IL for ileum, C for colon and TC for terminal colon) originating from two donors, 109 (A, B, C) and 2002 (D), were infected with 1.10⁸ cg/well of HuSaV S611 (A, D – white, gray, black circles), 1.10⁷ cg/well of HuSaV S513 (B – blue circles) or 1.10⁵ cg/well of HuNoV GII.4 as a control (C – pink and red diamonds) with 500 μM GCDCA. The mean viral titer per well for three technical replicates is depicted as circles for 2h, 72h or 144 hpi, for n = 2 to 6 experiments. Above each condition is displayed the corresponding viral genome fold change between the two time points.

FIG 6

HuSaV replicate in HIE independently of HBGA genotype. Differentiated monolayers of jejunal HIE from different donors were infected with 1.10⁸ cg/well of HuSaV S611 (white and black) or 1.10⁵ cg/well of HuNoV GII.4 (pink and red) with 500 μM GCDCA in each case and the mean viral titer per well measured for three technical replicates and n = 3 to 7 experiments at 2 hpi (white, pink) and 72 hpi (black, red). (A) Three jejunal HIE cells lines, J2, J10, J11 with contrasted HBGA genotype (OB Sec+, Sec-, and OO Sec+, respectively). (B) J4 cells (OO Sec- genotype) and the corresponding knock-in, J4FUT2KI (OO Sec+). (C) J2 cells (OB Sec+) and the corresponding knockout, J2FUT2KO (OB Sec-).