Fig 1.
An illustrative summary of WNV infection kinetics experiment in saltwater crocodile hatchlings.
Samples, including blood, tissues, cloacal swabs (collected at necropsy) and water were collected daily for the first 7 dpi and every second day from day 9 dpi. Created with BioRender.com.
Fig 2.
Viremia and viral load in selected tissues of infected saltwater crocodiles in relation to viremia measured by RT-qPCR at various timepoints.
A. Viremia per timepoints expressed in log10 TCID50 equivalent per mL of blood. The number of animals euthanized per time point is summarized in S1 Table. B. Viral load in liver, spleen, kidney and cloacal mucosa in relation to viremia in each timepoint. Only tissues from animals with viremia were examined. The horizontal bars indicate median values.
Fig 3.
Proportion of WNV positive among infected, in-contact control and mock-infected crocodiles.
A. Proportion of cloacal swabs positive for WNV by RT-qPCR at different time points post experimental WNV challenge. B. The proportion (%) of crocodile hatchlings positive for WNV by RT-qPCR in blood at various time points post-infection.
Fig 4.
Lymphoid aggregates in different tissue sections.
A. Macroscopic “pix” lesions (red arrow heads) in the skin of an infected crocodile. B. Lymphoid aggregates in a skin from an infected crocodile. C. Periportal lymphoid aggregates in the liver with mononuclear infiltration in periportal zones. D. Interstitial lymphoid aggregates the in kidney. E and F. Exocrine pancreatic cell degeneration with interstitial mononuclear infiltration. Scale bars: A and C 625 µm; D and E 125 µm; F 65 µm.
Fig 5.
Histopathological lesions in infected saltwater crocodiles.
A. Mild intraepithelial and epidermal infiltration of mononuclear cells (lymphocytes and monocytes) (red arrow), mild perivascular lymphocyte infiltration in the dermis (blue arrow), scale bar 1250 µm. B. Immunohistochemical detection of WNV NS1, using mAb 4G4, in spleen from a crocodile infected with WNVKUN. There are viral protein-positive monocytes (red arrow), scale bar 125 µm. Note that the brown round oval cells in panel B are nucleated erythrocytes. C. and D. Immunohistochemical detection of WNV NS1 (red signals) in exocrine pancreas from animals terminated 13 and 15 dpi, scale bars 250 and 65 µm, respectively.
Fig 6.
Neutralizing antibody titers in individual experimentally infected and in-pen contact hatchling saltwater crocodiles. The dotted line represents the limit of detection at a titer of 1:20 dilution.
Fig 7.
Principal component analysis (PCA) plots for RNA-seq data of the kidney and liver.
A, B. PCA plots for RNA-seq data of the kidney at various time points during early and late response to infection. C. PCA plots for RNA-seq data of the liver at different timepoints of late response to infection.
Fig 8.
Distinct transcriptional signatures in the kidney during early and late response to infection.
(A and B) Volcano plot for early and late response to infection in kidney. Blue dots indicate downregulated genes, red dots indicate upregulated genes, grey dots indicate non-significantly expressed genes. (C) Venn diagram of genes differentially expressed during early (blue area, n = 54) and late (yellow area, n = 3141) response to infection. The dark blue overlap corresponds to the genes (n = 49) that were expressed during both phases of infection.
Fig 9.
Enriched gene ontology associated with transcriptional response in kidney during early and late response to infection.
(A and B) Heatmaps of all significant differentially expressed genes in the kidney (early and late response to infection). (C) Gene Ontology (GO) enrichment analysis of genes differentially expressed in the kidney during the late response to infection. TP means timepoint.
Fig 10.
Distinct transcriptional signatures in liver during early and late response to infection.
(A and B) Volcano plot for early and late response to infection in liver. Blue dots indicate downregulated genes, red dots indicate upregulated genes, grey dots indicate non-significantly expressed genes. (C) Venn diagram of genes differentially expressed during early (blue area, n = 20) and late (yellow area, n = 125) response to infection. The dark blue overlap corresponds to the genes that are expressed during both phases of infection (n = 12).
Fig 11.
Enriched gene ontology associated with transcriptional response in liver during early and late response to infection.
(A – D) Heatmaps of all significant differentially expressed genes in the liver (early and late response to infection). (A) Heatmap of all genes differentially expressed during early response to infection, (B) Heatmap of all genes differentially expressed during late response to infection, (C) Heatmap of genes upregulated during late response to infection, (d) Heatmap of genes downregulated during late response to infection. (E and F) Gene Ontology (GO) enrichment analysis of genes differentially expressed in the liver during early and late phase of infection.TP means timepoint.