Ferret animal model of severe fever with thrombocytopenia syndrome phlebovirus for human lethal infection and pathogenesis

Abstract

Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV), listed in the most dangerous pathogens by the World Health Organization, has 12-30% fatality rates with a characteristic thrombocytopenia syndrome. With a majority of clinically diagnosed SFTSV patients older than ~50 years of age, age is a critical risk factor for SFTSV morbidity and mortality. Here, we report an age-dependent ferret model of SFTSV infection and pathogenesis that fully recapitulates the clinical manifestations of human infections. Whereas young adult ferrets (≤2 years of age) did not show any clinical symptoms and mortality, SFTSV-infected aged ferrets (≥4 years of age) demonstrated severe thrombocytopenia, reduced white blood cell counts and high fever with 93% mortality rate. Moreover, a significantly higher viral load was observed in aged ferrets. Transcriptome analysis of SFTSV-infected young ferrets revealed strong interferon-mediated anti-viral signalling, whereas inflammatory immune responses were markedly upregulated and persisted in aged ferrets. Thus, this immunocompetent age-dependent ferret model should be useful for anti-SFTSV therapy and vaccine development.

Fig. 1 |. Survival and body weight of young adult and aged ferrets following inoculation with SFTSV.

a-c, Nine ferrets in each group were i.m. inoculated with 10^7.6 TCID₅₀ of virus. Survival (a), relative weight (b) and temperature (c) were assessed and are shown as standard error of the mean. Data (mean ± s.e.m.) are presented in b and c. The two-tailed Mantel-Cox method (a) or the two-tailed, unpaired t-test (b, c) was used to assess P values. *P<0.0001(a), *P = 0.0331; **P = 0.006 9 and *** P < 0.0001 (b); and *P = 0.0258, **P = 0.0062 and ***P < 0.0709 (c). The experiment was performed with three independent trials.

Fig. 2 |. Haematological analysis of SFTSV-inoculated ferrets (n = 21 per group).

a-d, The blood from infected ferrets was collected every other day and haematological examination was performed using a Celltac hematology analyzer. Platelet counts from young adult ferrets (a), platelet counts from aged ferrets (b), WBC counts from young adult ferrets (c) and WBC counts from aged ferrets (d) are shown. Normal ranges of platelet and WBC counts in ferrets are 171.7–1,280.6×10³ per μl and 2.5–16.7 × 10⁶ per μl, respectively^,. The dashed lines indicate the normal values of platelet or WBC counts. The sharp (#) indicates no samples collected because the ferrets in this group died. Data are presented in box plots, with the box showing the upper (75%) and lower (25%) quartiles, the horizontal line as the median and the whiskers as the maximum and minimum values observed. The asterisks and daggers indicate significance between non-infected and infected ferrets per dpi as determined by a two-tailed unpaired t-test. *P = 0.0783, **P = 0.0091, ***P = 0.0086, ****p = 0.0065, ^†P = 0.0014, ^††P = 0.0006, ^†††P = 0.0001 and ^††††P<0.0001 (a); * P < 0.0001 (b); *P = 0.4263, **P = 0.2951, ***P = 0.2373, ****P = 0.1614, ^†p = 0.0738, ^††P = 0.0397 and ^†††P<0.0001 (c); and *P = 0.5176, **P = 0.0014 and ***P = 0.0007 (d).

Fig. 3 |. Concentration of liver enzymes in the blood of SFTSV-inoculated ferrets (n = 21 per group).

a–d, The blood from infected ferrets was collected every other day and haematological examination was performed using a Celltac hematology analyzer. AST concentrations in young adult ferrets (a), AST concentrations in aged ferrets (b), ALT concentrations in young adult ferrets (c) and ALT concentrations in aged ferrets (d) are shown. The normal range of AST or ALT concentration is marked by the dashed lines in each panel. The sharp (#) indicates no samples collected because the ferrets in this group died. Data are presented in box plots, with the box showing the upper (75%) and lower (25%) quartiles, the horizontal line as the median and the whiskers as the maximum and minimum values observed. The asterisks or daggers indicate significance between non-infected and infected ferrets per dpi as determined by a two-tailed, unpaired t-test. *P = 0.5246, **P = 0.3488, ***P = 0.0486, ****P = 0.0174 and ^†P <0.0001 (a); *P = 0.0003 and **P < 0.0001 (b); *P = 0.9668, **P = 0.1132, ***P = 0.0508, ****P = 0.0426, ^†P = 0.0375, ^†P = 0.0081, ^†††P = 0.0049 and ^††††P < 0.0001 (c); and *P = 0.0935 and **P<0.0001 (d).

Fig. 4 |. Distribution of the number of viral RNA copies in tissues and blood of SFTSV-infected ferrets.

a–h, Tissues (n = 3 per group) were collected at 2, 4, 6 and 8 dpi and blood samples (n = 21 per group) were collected at 0,2,4,6,8,10,12,14 or 16 dpi. The spleen (a), liver (b), kidney (c), lung (d), brain (e), spinal cord (f), intestines (g) and serum (h) were titred with real-time PCR. The sharp (#) indicates no samples collected because the ferrets in this group died. Data are shown as mean ± s.e.m. The asterisks indicate significance compared to each dpi sample by the two-tailed, unpaired t-test. *P = 0.9749, **P = 0.0299 and ***P = 0.0040 (a); *P = 0.4652, **P = 0.2919 and ***P = 0.0106 (b); *P = 0.5449, **P = 0.3324 and ***P = 0.0710 (c); *P = 0.9757, **P = 0.1344 and ***P = 0.0025 (d); *P = 0.1343 and **P = 0.0425 (e); *P = 0.3740 and **P = 0.027 0 (f); *P =0.0060 (g); and *P = 0.9170, **P = 0.5987 and ***P < 0.0001 (h). Each experiment was performed for three biological and three technical repeats.

Fig. 5 |. Immunohistochemistry of tissues from aged ferret infected with SFTSV.

a-f, Ferrets were i.m. inoculated with 10^7.6 TCID₅₀. Tissues were harvested on day 6 after inoculation. The presence of the SFTSV NP antigen was confirmed in the spleen (red pulp) (a), liver (central vein) (b), kidney (renal tubule) (c), brain (cerebellar cortex) (d), spinal cord (ventral horn) (e) and intestine (small intestinal crypts) (f) by immunohistochemistry. Magnification × 400.SFTSV-positive regions were magnified × 400 for each figure. Yellow arrows indicate SFTSV antigen-positive cells. Experiments were performed with three independent trials and similar results were reproduced. Scale bars, 20μm.

Fig. 6 |. Overview of RNA-seq data from PBMCs of young adult and aged ferrets infected with SFTSV and control ferrets at 2 and 4 dpi (n = 2 for control and 2 dpi of young adult ferrets and n = 3 for 4 dpi of young adult ferrets and aged ferrets).

Comparison of DEGs identified immune-related genes as shown by Venn diagrams and heat maps. a, Genes upregulated or downregulated by more than twofold were selected from among mock and young adult or aged ferret groups (DEGs were identified with Bonferroni-corrected P value, P < 0.05). b, Canonical signalling pathways activated by SFTSV infection. The IPA tool was used to generate a list of the significant canonical pathways and the activated networks with their respective scores. A comparison of gene expression among mock and young adult or aged ferret groups is shown. c, Canonical signalling pathways activated by SFTSV infection. A comparison of gene expression between aged and young adult ferrets is shown. The RNA-seq data were statistically analysed by the two-sided Wald test (FDR < 0.05) CCR3, C-C chemokine receptor type 3; CXCR4, C-X-C chemokine receptor type 4; iCOS, inducible T cell costimulator, iCOSL, iCOS ligand; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; NF-κB; nuclear factor-κB; PDGF, platelet-derived growth factor; STAT, signal transducer and activator of transcription; T_H, T helper; TNFR2, tumour necrosis factor receptor superfamily member 2.