Attenuated Duck Hepatitis A Virus Infection Is Associated With High mRNA Maintenance in Duckling Liver via m6A Modification

Figures

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  Figure 1. DHAV virulence contributes to the clinical manifestations of infected ducklings. (A) Representative images of liver lesions in ducklings. Significant liver haemorrhage is observed after infection with virulent DHAV (CH strain) and corresponding attenuated DHAV (CH60 strain). Scale bar is equal to 1 cm. (B) The survival probability of virulent DHAV-attenuated DHAV-infected ducklings (n = 10). Ducklings injected with physiological normal saline served as a control group. Virulent DHAV significantly reduces the survival probability. (C) The relative virus titer, CH strain, and CH60 strain in the liver are quantified by RT–qPCR. The liver viral load in ducklings infected with virulent DHAV is significantly increased compared with ducklings infected with attenuated DHAV. Student’s unpaired t test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001. Data are displayed as the mean ± SD with 3 biological replicates and 3 technical replicates.

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  Figure 2. Attenuated DHAV maintains high m6A levels in duckling liver. (A) Quantification of m6A modification in total RNAs and mRNAs from duckling livers using m6A antibody-based immunoblotting. Methylene blue staining served as the loading control. Representative dot blot images at 12 hpi and 36 hpi are displayed. m6A-modified mRNA is present at greater levels in the CH60-infected group compared with the CH-infected group (middle panel). (B) A more sensitive LC–MS/MS approach was used to further quantify m6A RNA (m6A/A) and m6A mRNA (m6Am/A) in duckling livers infected by a virulent CH strain and attenuated CH60 strain. The virulent CH strain significantly reduces m6A modification levels in the poly-A tailed mRNA pool. However, m6Am (an m6A-resembling modification) modification levels remain similarly low in both groups. Data are displayed as the mean ± SD with 4 replicates.

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  Figure 3. Dynamic density change, distribution, and modification motif of m6A mRNA during infection. (A) Enrichment analysis of IP reads around start and stop codons in duckling liver infected with virulent CH and attenuated CH60 strains. During infection, the m6A peak read density in livers infected with the attenuated CH60 strain is much higher than that in livers infected with the virulent CH strain. Each time point after infection is indicated by a different colour. (B–C) This m6A modification in attenuated CH60-infected livers is dominantly distributed near the start codon position and near the 5’UTR. (C) The m6A distribution of input and IP samples among all samples is analyzed. Inputs and IPs of CH60- or CH-infected livers are displayed in different colours. The m6A density distribution at different infection times is displayed. (D) Gene region analysis indicated that the 5’UTR and antisense region are highly modified by m6A (lower panel). Three biological replicates of both m6A IP samples and Input samples were used to calculate m6A IP/Input ratio. The P values were determined using Student’s unpaired t test. *P < 0.05, **P < 0.01. (E) The number of peaks and genes with m6A…

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  Figure 4. Functional analysis of coding genes with altered m6A peaks between virulent (CH) and attenuated (CH60) DHAV-1 duckling livers. (A) The number of hypermethylated (up, red colour) and hypomethylated (down, blue colour) m6A peaks between the DHAV CH60 and CH strains at 12 h, 24 h, 36 h, and 48 h postinfection with DHAV-1. (B) Venn diagrams showing the number of altered m6A peaks between DHAV CH60- and CH-stained livers at each time point. Some m6A-modified genes are further validated by qRT–PCR ( Supplementary Figures 5–7). (C) Venn diagrams showing the number and relationship of m6A peaks and mRNA expression between CH60 and CH at each time point. (D, upregulated genes and E, downregulated genes) GO biological terms of genes with altered m6A peaks and mRNA expression levels between the DHAV CH60 and CH strains at each time point.

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  Figure 5. m6A-related RNA–protein biology was coordinately and dynamically regulated by DHAV virulence. (A) Commonly regulated GO biological processes in both CH60- and CH strain-infected livers. Basic biological processes, such as mRNA translation, positive regulation of GTP activity, and protein stabilization, are concurrently regulated by both groups. (B) A similar analysis was used to identify commonly regulated KEGG pathways in which proteolysis, ribosome, and aminoacyl-tRNA biosynthesis were enriched. This is partially in line with certain m6A modification on tRNA related fragments in sense and antisense, despite lacking statistical significance between both two groups ( Supplementary Figure 3 ). (C) The dynamic regulation of biological processes of m6A-modified genes was analyzed. During infection, virulence-related mRNA translation and protein metabolism-related pathways were differentially regulated at different infection stages, such as nucleotide metabolism, ribosome, and RNA degradation for mRNA translation, amino acid metabolism, ribosome biosynthesis, and protein processing for protein metabolism.