Table 1.
KFDV and AHFV Necropsy findings.
Fig 1.
KFDV causes moderate to severe clinical illness in pigtailed macaques, and virus is present in multiple tissues.
Six PTMs (KFDV 3–8) were infected with 2 x 105 pfu KFDV P9605 sc/iv (105 pfu, per route). A. Exam schedule following infection with KFDV. B. Animals were scored daily for clinical signs following infection. The dotted line indicates a score of 35. C. KFDV viremia was measured by limiting dilution plaque assay from plasma on the indicated days post infection. D. Neutralization assays were performed using heat-inactivated serum collected at 0, 6, 7, and 8 dpi by a focus reduction neutralization assay. FRNT50 values are reported. Error bars represent the standard deviation (SD) across four animals. Statistics were performed using an ordinary one-way ANOVA, comparing the FRNT50 values at D0 to D8 (N = 4; **P < 0.005). E-I. At terminal endpoints, or 9 dpi, animals were euthanized and virus was isolated from various tissues by plaque assay. Plaque counts were normalized to 1 gram of tissue. Data from each individual animal are plotted.
Table 2.
KFDV and AHFV clinical observations.
Pigtailed macaques were observed for clinical signs following sc/iv inoculation of KFDV or AHFV. Some parameters (mucous membrane color, dehydration, breathing quality, CRT, and temperature) were only measured during exams. The presence or absence of a clinical sign are reported as Y (yes) or N (no).
Fig 2.
Pigtailed macaques are susceptible to disease caused by AHFV.
Four pigtailed macaques (AHFV 1–4) were infected with 2 x 105 pfu AHFV 200300001 sc/iv and monitored for clinical illness. A. Schedule for blood draws and exams in AHFV-infected animals. B. Clinical scores in four pigtailed macaques following infection with AHFV. Dotted line represents a score of 35. C. Viremia was measured in plasma over the first 8 days of infection by plaque assay. D. Neutralization assays were performed using a focus reduction neutralization assay. FRNT50 values are reported. Error bars show the SD across four animals. Statistics were performed using an ordinary one-way ANOVA (N = 4, *P < 0.05). E-I. At day 8 or 9 post infection, virus present in the tissue homogenate was measured by limiting dilution plaque assay, and values were normalized to 1 gram of tissue. Data from each individual animal are plotted.
Fig 3.
Cytokine and chemokine analysis in serum of KFDV and AHFV-infected animals.
Pigtailed macaques were infected with KFDV by the sc or sc/iv route, or AHFV by the sc/iv route. The number of animals in each group is indicated in the legend. Serum was isolated from blood samples obtained during clinical exams at the indicated days post infection. Cytokine and chemokines were measured from sera using a 29-plex Monkey cytokine detection kit. The following parameters are included: A. IL-6, B. MCP-1, C. IL-1RA, D. IL-12, E. I-TAC, F. IFNγ, G. FGF-Basic, H. HGF, I. MIG, J. Eotaxin. Statistics were performed by comparing the change in the parameter compared to the D0 measurement using Sidaks multiple comparison test. Significance symbols are represented as follows: KFDV sc/iv *, AHFV sc/iv #, KFDV sc +. (KFDV sc/iv N = 6, AHFV sc/iv N = 4, KFDV sc N = 2; *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.
Fig 4.
Blood parameters in pigtailed macaques infected with KFDV or AHFV.
Blood was collected during exams as indicated. Complete blood counts were analyzed from EDTA blood (A-G), and blood chemistries were measured from sera (H-M). A. Hematocrit (HCT), B. Hemoglobin (HGB), C. Red blood cells (RBC), D. White blood cells (WBC), E. Neutrophils, F. Lymphocytes, G. Platelet counts, H. Albumin, I. Total protein, J. Alanine aminotransferase (ALT), K. Alanine phosphatase (ALP), L. Aspartate transaminase (AST), M. Globulin. Statistics were performed by comparing the change in the parameter at day 2, 4, and 6 compared to the day 0 measurement using Dunnett’s multiple comparison test. Significance symbols are represented as follows: KFDV sc/iv *, AHFV sc/iv #, KFDV sc +. (KFDV sc/iv N = 6, AHFV sc/iv N = 4, KFDV sc N = 2; *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001).
Fig 5.
KFDV and AHFV RNA detection by ISH in the lymph node, intestine, and spleen of infected animals.
A KFDV RNAscope probe was used to detect viral RNA from tissue sections derived from either KFDV or AHFV-infected PTMs. Tissues from KFDV sc/iv infected animals (A, C, E) or AHFV sc/iv infected animals (B, D, F) were examined. Representative images are displayed for A, B. mesenteric lymph node (40X, bar = 200 μm); C. cecum (100X, bar = 50 μm), D. ileum (100X, bar = 50 μm), and E, F. spleen (100X, bar = 50 μm).
Fig 6.
Mesenteric lymph node double staining with KFDV and CD3, CD20, CD21, CD35, or CD68.
Mesenteric lymph node sections from an animal infected sc/iv with KFDV was subjected to RNAscope and IHC for CD3, CD20, CD68, CD21, and CD35 using the RNAscope VS Universal ISH-IHC HRP fluorescent assay. KFDV (red) and cell markers (green). Scale bar for low-magnification CD3/KFDV panel, 500 μm; for high-magnification images of CD3, CD20, CD68, CD21, and CD35, 20 μm.
Fig 7.
Transcriptional dynamics in blood of PTMs inoculated with KFDV and AHFV.
Whole blood from PTMs inoculated sc/iv with KFDV or AHFV was subject to RNAseq at 0, 2, and 6 dpi. A. Differentially expressed genes (DEG) at 2 and 6 dpi with fold change > |1.5| relative 0 dpi and P < 0.05. B. Heat map of z-scores depicting changes in cellular immune response pathways following inoculation with KFDV or AHFV. C. Heat map of z-scores showing significantly enriched and differentially activated pathways between KFDV and AHFV- inoculated PTMs. D. Predicted function of the intrinsic prothrombin activation pathway at 2 dpi using IPA following inoculation with KFDV (left) and AHFV (right). (Predicted activation (orange), predicted inhibition (blue), observed downregulation (green).