Enhanced GII.4 human norovirus infection in gnotobiotic pigs transplanted with a human gut microbiota

Abstract

The role of commensal microbiota in enteric viral infections has been explored extensively, but the interaction between human gut microbiota (HGM) and human norovirus (HuNoV) is poorly understood. In this study, we established an HGM-Transplanted gnotobiotic (Gn) pig model of HuNoV infection and disease, using an infant stool as HGM transplant and a HuNoV GII.4/2006b strain for virus inoculation. Compared to germ-free Gn pigs, HuNoV inoculation in HGMT Gn pigs resulted in increased HuNoV shedding, characterized by significantly higher shedding titres on post inoculation day (PID) 3, 4, 6, 8 and 9, and significantly longer mean duration of virus shedding. In addition, virus titres were significantly higher in duodenum and distal ileum of HGMT Gn pigs on PID10, while comparable and transient HuNoV viremia was detected in both groups. 16S rRNA gene sequencing demonstrated that HuNoV infection dramatically altered intestinal microbiota in HGMT Gn pigs at the phylum (Proteobacteria, Firmicutes and Bacteroidetes) and genus (Enterococcus, Bifidobacterium, Clostridium, Ruminococcus, Anaerococcus, Bacteroides and Lactobacillus) levels. In summary, enhanced GII.4 HuNoV infection was observed in the presence of HGM, and host microbiota was susceptible to disruption upon HuNoV infection.

Keywords: Human norovirus; diarrhea; faecal microbiota transplantation; gnotobiotic pig; human gut microbiota.

Fig. 1.

Experimental design and faecal bacteria shedding. (a) Schematic representation of Gn pig study. HGM, human gut microbiota; PPD, post-partum day; PID, post inoculation day. (b) HGM colonization in Gn pigs. Concentrations of culturable aerobic bacteria were measured in serial dilution of pig faeces and enumeration of colony-forming unit (c.f.u.) grown on lysogeny broth (LB) media agar plates. Data were combined from four independent experiments and presented as individual animal data points. Sample sizes are shown in Table 1.

Fig. 2.

Increased faecal HuNoV shedding in HGMT pigs. (a) Daily virus shedding was measured from PID1 to PID10 by quantitative reverse transcription (qRT) PCR to quantify HuNoV genomes in faeces. (b) Peak shedding titres during PID1 to PID3 and PID4 to PID10 in individual pigs were present. (c) Individual pigs’ cumulative shedding was shown as the area under curve based on daily virus shedding in (a). Sample sizes are indicated in Table 1. Dashed line shows the limit of detection. Data were combined from four independent experiments and presented as individual animal data points with mean±sem. Statistical significance was determined by Mann–Whitney test. NS, not significant, *P<0.05, **P<0.01.

Fig. 3.

Faecal consistency scores. (a) Daily faecal consistency scores after HuNoV inoculation. Faecal consistency was scored as follows: 0, solid; 1, semisolid; 2, pasty; 3, semiliquid; and 4, liquid. Dashed line shows the minimal value to be considered as diarrhea. (b) Individual pigs’ cumulative faecal scores were shown as the area under curve based on daily faecal consistency scores in (a). Data were combined from ten independent measurements and presented as individual animal data points with mean±sem. Statistical significance was determined by Mann–Whitney test, *P<0.05.

Fig. 4.

HuNoV distribution in gut tissues, blood and MNCs. HuNoV genomes in gut tissues from pigs euthanized on PID3 (a) and PID10 (b) were quantified by qRT-PCR. P-ileum, proximal ileum; d-ileum, distal ileum. HuNoV genomes in plasma (c), whole blood cells (d), and mononuclear cells (MNCs) were quantified by qRT-PCR. (a, b) HuNoV group size: PID3 n=7, PID10 n=6. HGM+HuNoV group size: PID3 n=5, PID10 n=6. (c–e) Group sizes were shown in Table 1. Dashed line shows the limit of detection. Data were combined from four to five independent experiments and presented as individual animal data points with mean±sem. Statistical significance was determined by Mann–Whitney test. NS, not significant, *P<0.05, **P<0.01.

Fig. 5.

Microbiome composition analysis of HGMT Gn pigs. (a) Bacterial taxonomic summary showing relative abundance at the genus level. Unassigned and minor group includes genus less than 0.1 % of total community in each sample. (b) Principal coordinate analysis (PCoA) of beta diversity based on weighted UniFrac distances among HGMT Gn pigs. The first two axes that explain largest variations (PC1 and PC2) are plotted. Significantly different taxa at the phylum level (c) and at the genus level (d) between the HGM group and HGM+HuNoV group. (c, d) Data are presented as individual animal data points with mean±sem. Statistical significance was determined by Mann–Whitney test. *P<0.05, **P<0.01.