Article Figures & Data
Figures
- FIG 1
Tree shrew primary cells are susceptible to ZIKV infection. All primary cells were isolated from a 5-month-old tree shrew and cultured according to the standard protocol. All primary cells were infected with ZIKV at a multiplicity of infection (MOI) of 1. (A) The cytopathic effects (CPEs) in primary kidney cells (PKCs) and primary testis cells (PTCs) were assessed by imaging at 48 h postinfection (hpi). (B) Immunostaining assays were performed at 48 hpi with ZIKV-specific antibodies. Positive viral antigens are shown in green, and 4′,6-diamidino-2-phenylindole (DAPI) is shown in blue. (C) The viral loads at the indicated time points were determined by reverse transcription quantitative PCR (RT-qPCR). The dotted line indicates the detection limit of the RT-qPCR assay. (D) The mRNA levels of selected genes in infected PBMCs at the indicated time points were determined using RT-qPCR. Each assay was performed at least two times with experimental triplicates. Bar, 50 μm.
- FIG 2
ZIKV infection causes dermatological manifestations in adult tree shrews. (A) Study design and experimental parameters. Twenty-six tree shrews were inoculated via the s.c. route with 106 PFU of ZIKV strain GZ01. The yellow box indicates the time course for clinical observations and virological assays. (B) Representative images of dermatological manifestations in ZIKV-inoculated tree shrews. Heat-inactivated ZIKV and PBS were used as the controls. Red arrows denote the injection sites.
- FIG 3
Histopathological and virological characterization assays in the skin of tree shrews. Skin tissues were collected from ZIKV-infected tree shrews at the indicated time points. (A) Histopathological characterization was performed by hematoxylin and eosin (H&E) staining. The arrows denote areas of hemorrhage, and the triangles denote inflammatory cell infiltration. (B) ZIKV genome RNA ISH was performed with a ZIKV-specific probe. Brown-colored staining indicates positive results (arrows). Bar, 50 μm.
- FIG 4
ZIKV establishes systemic infection in tree shrews. Viral loads in serum and saliva from ZIKV-infected tree shrews were determined by RT-qPCR. (A and B) The kinetics and time courses of viremia in all ZIKV-infected animals. (C and D) The kinetics and time courses of the viral load in saliva in all infected animals. The detection limit is indicated by the dotted line.
- FIG 5
Tissue distribution of ZIKV in tree shrews. (A) ZIKV RNA loads in the indicated tissues from ZIKV-infected animals (n = 4) at 2 and 6 dpi were determined by RT-qPCR. (B) Histopathological changes in the testes were examined by H&E staining at 2 dpi. (C) Immunostaining of testis tissue harvested at 2 dpi was performed with anti-ZIKV antibodies (green) and DAPI (blue). Bar, 50 μm.
- FIG 6
Global transcriptomic analysis in PBMCs from ZIKV-infected tree shrews. (A) RNA-seq analysis of antiviral ISGs and inflammatory genes in ZIKV-infected PBMCs isolated from two tree shrews at −2, 2, and 6 dpi. (B) RT-qPCR validation of the expression of antiviral genes, IFN-inducible receptors and transcription factors, as well as inflammatory genes, in ZIKV-infected PBMCs described in panel A. The data are shown as the means ± standard deviations (SDs) (n = 3).
- FIG 7
ZIKV infection triggers protective immunity against secondary homologous infection. (A) Neutralizing antibody kinetics in ZIKV-infected animals. Dotted lines represent the limits of detection. (B) Passive transfer protection experiments were performed in 1-day-old suckling BALB/c mice using antisera and 10-fold dilutions (n = 5) of antisera from ZIKV-infected tree shrews (n = 5). PBS was included as the mock control (n = 5). The Kaplan-Meier survival curves were analyzed by the log-rank test. *** denotes a P value of less than 0.01. (C and D) All tree shrews that received 105 or 106 PFU of ZIKV were rechallenged with the same dose of ZIKV at 21 days post primary infection. Viremia was monitored for 7 days after each challenge. The limit of detection is indicated by the dotted line.
- FIG 8
Neurovirulence of ZIKV in neonatal tree shrews. (A) Survival curve for ZIKV-infected 1-day-old tree shrews. Groups of neonatal tree shrews (n = 8) were inoculated with 105 PFU of ZIKV by the intracerebral route. PBS was used as the mock control. The Kaplan-Meier survival curves were analyzed by the log-rank test. * denotes a P value of less than 0.05. (B) Viral RNA loads in brain of ZIKV-infected neonatal tree shrews at 3 and 6 dpi were determined by RT-qPCR. (C and D) Histopathological and ISH assays of brain sections obtained from ZIKV-infected neonatal tree shrews at 3 and 6 dpi. The red arrows indicate positive results. Bar, 50 μm.
Tables
- TABLE 1
Virus isolation results using serum samples from ZIKV-infected tree shrews
Sample no. Initial viral loads After passage in C6/36 cells Result assessmenta CT value Log RNA copies/ml CT value Log RNA copies/ml 01 28.94 6.37 34.83 4.62 Negative 13 31.22 5.74 37.48 3.89 Negative 14 30.87 5.84 33.23 5.07 Negative 17 31.92 5.55 32.98 5.14 Negative 20 30.45 5.95 22.59 8.03 Positive 21 29.92 6.10 16.85 9.63 Positive 22 28.74 6.42 16.83 9.63 Positive 23 28.98 6.35 19.66 8.85 Positive 24 30.02 6.07 17.61 9.42 Positive 25 30.31 5.99 33.39 5.02 Negative ↵a An increase in the RNA copy number of at least 100-fold was defined as positive for virus isolation (in bold).
