Experimental cross-species infection of donkeys with equine hepacivirus and analysis of host immune signatures

Abstract

Background: The Equine Hepacivirus (EqHV) is an equine-specific and liver-tropic virus belonging to the diverse genus of Hepaciviruses. It was recently found in a large donkey (Equus asinus) cohort with a similar seroprevalence (30%), but lower rate of RNA-positive animals (0.3%) compared to horses. These rare infection events indicate either a lack of adaptation to the new host or a predominantly acute course of infection.

Methods: In order to analyze the susceptibility and the course of EqHV infection in donkeys, we inoculated two adult female donkeys and one control horse intravenously with purified EqHV from a naturally infected horse. Liver biopsies were taken before and after inoculation to study changes in the transcriptome.

Results: Infection kinetics were similar between the equids. All animals were EqHV PCR-positive from day three. EqHV RNA-levels declined when the animals seroconverted and both donkeys cleared the virus from the blood by week 12. Infection did not have an impact on the clinical findings and no significant histopathological differences were seen. Blood biochemistry revealed a mild increase in GLDH at the time of seroconversion in horses, which was less pronounced in donkeys. Transcriptomic analysis revealed a distinct set of differentially expressed genes, including viral host factors and immune genes.

Conclusion: To summarize, our findings indicate that donkeys are a natural host of EqHV, due to the almost identical infection kinetics. The different immune responses do however suggest different mechanisms in reacting to hepaciviral infections.

Fig. 1

Study design and course of disease. A Study design: Two donkeys and one horse were intravenously inoculated with purified EqHV-positive serum. B EqHV genome equivalents (black line) and anti-NS3 antibody course (red line) during the observation period. C Plasma levels of fibrinogen (G/L), aspartate transaminase (AST, U/L), gamma-glutamyl transferase (GGT, U/L), glutamatdehydrogenase (GLDH, U/L), hematocrit (Hkt, I/L), total protein (TP, g/L), and white blood cell counts (WBC, G/L). Reference intervals are indicated in grey

Fig. 2

Morphological findings in liver biopsies from donkeys pre- and post- infection with EqHV. A-D Histologically, a mild periportally-accentuated, lympho-histiocytic inflammation was detected within liver biopsies with a similar appearance in samples taken pre- (A, C) and post- (B, D) infection. Hematoxylin and eosin, scale bar = 100 μm. E-P Inflammatory cell infiltrates consisted mainly of T-lymphocytes (E-H, asterisk), fewer macrophages (I-L, asterisk) and single B-lymphocytes (M-P, asterisk) pre- and post-infection. Immunohistochemistry, scale bar = 100 μm. Q-T Fluorescence in situ-hybridization lacked a positive reaction pre-infection (Q, S). Post-infection, EqHV specific RNA sequences were detected within the cytoplasm (R, T). Scale bar = 100 μm

Fig. 3

Analysis of deregulated genes. The threshold for deregulated genes (DREG) was set at a fold change above or below 2 or − 2, a significant p-value (FDR < = 0.05) and reads per kilobase million mapped reads (RPKM) of at least 1. A Number of up- and down-regulated genes per sample. Saturated area corresponds to immune-associated genes. B Overview of DREGs within each sample. Yellow dots indicate immune-associated genes. C Overlap of genes between the donkey and horse reference genome (upper Venn diagram). Overlap of all up- and down-regulated genes (indicated by arrows)

Fig. 4

Analysis of immune signatures in donkeys and the horse. A Chord plots depicting the 250 most de-regulated genes for each animal and their contribution to the (de-)activation of immune system processes. B Regulation of selected GO-terms. The dot color reflects whether a term is up- or down-regulated (z-score). The size ratio indicates the number of deregulated genes per term and the dot border is a binary indicator for a significant (FDR < = 0.05) deregulation

Fig. 5

Disease ontology classification. A Gene set enrichment analysis (GSEA) for terms associated with viral liver hepatitis. B Network analysis of the same terms showing the interaction between terms and involved genes