Prevalence, Cell Tropism, and Clinical Impact of Human Parvovirus Persistence in Adenomatous, Cancerous, Inflamed, and Healthy Intestinal Mucosa

Abstract

Parvoviruses are single-stranded DNA viruses, infecting many animals from insects to humans. Human parvovirus B19 (B19V) causes erythema infectiosum, arthropathy, anemia, and fetal death, and human bocavirus (HBoV) 1 causes respiratory tract infections, while HBoV2-4 are enteric. Parvoviral genomes can persist in diverse non-permissive tissues after acute infection, but the host-cell tropism and the impact of their tissue persistence are poorly studied. We searched for parvoviral DNA in a total of 427 intestinal biopsy specimens, as paired disease-affected and healthy mucosa, obtained from 130 patients with malignancy, ulcerative colitis (UC), or adenomas, and in similar intestinal segments from 55 healthy subjects. Only three (1.6%) individuals exhibited intestinal HBoV DNA (one each of HBoV1, 2, and 3). Conversely, B19V DNA persisted frequently in the intestine, with 50, 47, 31, and 27% detection rates in the patients with malignancy, UC, or adenomas, and in the healthy subjects, respectively. Intra-individually, B19V DNA persisted significantly more often in the healthy intestinal segments than in the inflamed colons of UC patients. The highest loads of B19V DNA were seen in the ileum and colon specimens of two healthy individuals. With dual-RNAscope in situ hybridization and immunohistochemistry assays, we located the B19V persistence sites of these intestines in mucosal B cells of lymphoid follicles and vascular endothelial cells. Viral messenger RNA transcription remained, however, undetected. RNA sequencing (RNA-seq) identified 272 differentially expressed cellular genes between B19V DNA-positive and -negative healthy ileum biopsy specimens. Pathway enrichment analysis revealed that B19V persistence activated the intestinal cell viability and inhibited apoptosis. Lifelong B19V DNA persistence thus modulates host gene expression, which may lead to clinical outcomes.

Keywords: RNA-seq; RNAscope in situ hybridization; human bocavirus 1; immunohistochemistry; parvovirus B19; tissue persistence.

Figure 1

B19V viral load in the disease-affected colonic mucosal specimens (orange) and healthy surrounding colonic specimens or healthy ileum (gray) from patients diagnosed with malignancy, inactive UC, active UC, or adenomas, and in the healthy colonic specimens from healthy individuals (blue). No statistically significant difference in viral load was noted among groups (value of p >0.05; one-way ANOVA).

Figure 2

B19V nucleic acid staining of intestinal sections (ileum and colons) from the healthy individual with the highest viral loads by RNAscope ISH (RISH). Red dots represent B19V NS1 or reference gene signals, nuclei are stained blue with hematoxylin. (A,B) The high viral load intestinal sections stained with the B19V-NS1 probes showed positive B19V staining in the blood vessels in the healthy ileum (A) and lymphoid follicles in the descending colon (B), respectively. Green arrows indicate locations of the dense B19V-signal areas, and higher magnifications of areas with the strongest B19V signals in selected boxes are shown in the bottom-right corners. (C) Negative control: the B19V PCR-negative tissue sections showed no B19V staining by RISH. (D) Technical positive control: human reference gene PPIB RISH showing positive staining of the ascending colon in the above-mentioned individual. (E) Technical negative control: probes targeting the bacterial gene dapB worked accurately on a consecutive tissue section. Scale bars, 200 μm (A,B) and 20 μm (C-E).

Figure 3

Dual RISH-IHC staining of B19V nucleic acids and cellular markers in the high-load intestinal sections and removal of RNA with RNase A pretreatment. RISH signals appear in red, whereas nuclei are stained blue. (A,B) IHC staining of the vascular endothelial cells (CD31; A) in the healthy ileum and B lymphocytes (CD20; B) in the healthy sigmoid colon following B19V RISH, with cellular markers, stained green. Higher magnifications of the selected areas are shown in the bottom-right corners. (C) A RNase A-treated section was stained with the technical positive control probe targeting the human PPIB gene. (D,E) High-load intestinal sections were pretreated with RNase A before B19V RISH, B19V signals remained in the vascular endothelial cells (D) and B lymphocytes (E). Scale bars, 50 μm (A,B), 10 μm (D), and 20 μm (C,E).

Figure 4

RNA-seq analysis for intestinal mucosal specimens. (A) Neutrophil chemotaxis is elevated in the active sigmoid colon tissue compared to healthy sigmoid colon, this pathway involves 29 DEGs: 19 upregulated (red nodes) and 10 downregulated (green nodes), value of p 1.6 × 10⁻⁸, z-score 3.6. (B) Scatter plot of the gene expression (272 DEGs) comparing B19V-DNA-positive and -negative ileum, x-axis: Log₂ fold-change, y-axis: -Log₁₀ (adjusted p value). DEGs with adjusted p values of less than 6.24 × 10⁻³ are shown as orange dots. (C,D) Ingenuity pathway on IPA platform (Qiagen) identified different cellular pathways of B19V-altered ileum; 46 DEGs were involved in viral infection (C), and 38 DEGs were involved in cell viability (26 DEGs) or apoptosis (12 DEGs; D). DEGs were identified by the Wald test for DEseq2, with Benjamini–Hochberg correction. The p values were calculated with Fischer’s exact test (right-tailed), indicating the likelihood of association between a gene set with a process. Z-score indicates the directional effect of the DEGs on a process, z-score > 0 indicates activation, z-score < 0 indicates inhibition, and z-score > 2 or < −2 is considered significant for a given process. Orange to red nodes, upregulated DEGs; green nodes, downregulated DEGs; orange dashed line, DEGs leading to activation; blue dashed line, DEGs leading to inhibition; yellow dashed line, findings inconsistent; gray dashed line, effects unknown; orange center, DEGs leading to activation; and blue center, DEGs leading to inhibition.