Table 1.
Primary and secondary antibodies used in this study for virus titration, flow cytometry, or histology staining.
Table 2.
PCR primers for amplification of USUV fragments for infectious clone generation via circular polymerase extension reaction (CPER).
Fig 1.
A genetically-defined infectious clone of USUV TC508 was generated using the circular polymerase extension reaction (CPER) and remains infectious in cell culture.
(A) Schematic representation of the workflow for generating an USUV infectious clone from an avian clinical isolate. Some figure elements (bird [64], cells [65], and DNA fragments [66]) were sourced from the public domain and are listed as references. (B) Schematic representation of the USUV genome and its division into seven fragments used for CPER. Each fragment was inserted into pCR-Blunt II-TOPO vectors. The purple arrows represent the USUV genes. Other vector components include lacZa (blue), ccdB (blue), antibiotic resistance markers (green), ori (sand), and the lac promoter (gray). The red half arrows indicate the PCR primer binding sites. (C) Replication kinetics of the USUV TC508 infectious clone in human hepatoma (Huh7) cells 1–4 days post-infection (dpi) using a multiplicity of infection (MOI) of 0.001, 0.01, or 0.1. The percentage of infected cells was quantified based on envelope (E) antigen staining detected by flow cytometry. Shading indicates standard error of the mean.
Fig 2.
USUV TC508 has variable Infection kinetics in murine, avian, and mosquito cell lines.
(A) Percent of E antigen-positive cells in mouse hepatoma (Hepa1-6), chicken hepatoma (LMH), and embryonic mosquito cells (Aag2) 1–4 days post-infection (dpi) using a multiplicity of infection (MOI) of 0.01. Shading indicates standard error of the mean. (B) Percent of E antigen-positive cells in various murine cell lines at 4 dpi using an MOI of 0.01. (C) Percent of E antigen-positive cells in mouse fibroblasts (L929) 1–4 dpi using an MOI of 0.01. Shading indicates standard error of the mean.
Fig 3.
USUV infection is lethal in mice with compromised type I interferon or STAT1-mediated antiviral defenses regardless of inoculation dose or route.
The inoculation doses tested ranged from 101 to 105 focus-forming units (FFU), all of which were administered via subcutaneous (SC) route. The inoculation routes tested were SC, intravenous (IV), intramuscular (IM), intraperitoneal (IP), and intradermal (ID) using a consistent inoculation dose of 103 FFU. (A) Survival, (B) weight changes, and (C) clinical scoring of Ifnar1-/- mice following inoculation with varying doses of USUV via SC injection (101 FFU, n = 4; 102 FFU, n = 4; 103 FFU, n = 7; 104 FFU, n = 3; 105 FFU, n = 3). (D) Survival, (E) weight changes, and (F) clinical scoring of Stat1-/- mice following inoculation with varying doses of USUV via SC injection (n = 3/group). (G) Survival, (H) weight changes, and (I) clinical scoring of Ifnar1-/- mice following inoculation with 103 FFU of USUV by various routes (n = 3/group). (J) Survival, (K) weight changes, and (L) clinical scoring of Stat1-/- mice following inoculation with 103 FFU of USUV by various routes (n = 3/group).
Fig 4.
USUV infection in tissue-specific STAT1-deficient mouse strains demonstrates a necessity for functional STAT1-mediated signaling in the hematopoietic compartment.
(A) Schematic representation of the breeding scheme used to generate whole-body, hepatocyte-specific, neuronal cell-specific, hematopoietic cell-specific, macrophage-specific, or dendritic cell-specific STAT1 KO mice. Wild-type mice harboring loxP sites within the Stat1 gene (Stat1fl/fl) were crossed with mice containing the Cre recombinase downstream of a ubiquitous (CMV) or tissue-specific (Alb, Syn, Vav, LysM, CD11c) promoter. Some figure elements (mouse [69], liver [70], neuron [71], and immune cells [72–77]) were sourced from the public domain and are listed as references. (B) Survival, (C) weight changes, and (D) clinical scoring of Stat1fl/fl (n = 11), Stat1-/- (n = 10), Vav-Cre/Stat1fl/fl (n = 7) Alb-Cre/Stat1fl/fl (n = 6), and Syn-Cre/Stat1fl/fl (n = 9) mice following inoculation with 103 focus-forming units (FFU) of USUV TC508 via SC injection. (E) Changes in viremia 1–4 days post-infection compared to a pre-bleed sample (“pre”) collected prior to infection (n = 3/mouse genotype). (F) Survival, (G) weight changes, and (H) clinical scoring of LysM-Cre/Stat1fl/fl (n = 8), CD11c-Cre/Stat1fl/fl (n = 8), and LysM-CD11c-Cre/Stat1fl/fl (n = 4) following inoculation with 103 FFU of USUV TC508 via SC injection.
Fig 5.
USUV replicates in STAT1-sufficient peripheral tissues leading to spleen and liver pathology.
Vav-Cre/Stat1fl/fl, Stat1-/-, and Stat1fl/fl mice (n = 3/group) were infected with 103 focus-forming units (FFU) of USUV via subcutaneous (SC) injection and euthanized at 4 days post-infection (dpi). Tissues were collected and subjected to molecular and histological analysis. For each mouse genotype, one representative image was chosen. USUV RNA was quantified in the (A) liver, (B) spleen, (C) kidney, (D) lung, and (E) brain of Vav-Cre/Stat1fl/fl, Stat1-/-, and Stat1fl/fl mice by qPCR. Significant levels of USUV RNA were detected in the liver, spleen, kidney, and lung of Vav-Cre/Stat1fl/fl and Stat1-/- mice. In some samples collected from Stat1fl/fl mice (6 out of 6 liver samples, 5 out of 6 spleen samples, and 3 out of 6 kidney samples), the amount of USUV RNA was below the limit of detection and was not plotted (nd indicates “not detected”). For each organ harvested from infected mice, two cuts were processed and used as separate sample inputs for qPCR. The dotted line indicates the limit of detection. Statistical analysis was performed using one sample t tests comparing USUV RNA detected in samples to the limit of detection. (F) H&E staining of liver tissue collected from mock or USUV-infected mice with a 200 µm scale bar. Liver necrosis was observed in Vav-Cre/Stat1fl/fl and Stat1-/- mice and is indicated by black arrows. (G) H&E staining of spleen tissue collected from mock or USUV-infected mice with a 200 µm scale bar. Decreased red pulp due to massive immune cell infiltration was observed in Vav-Cre/Stat1fl/fl and Stat1-/- mice. H&E staining of kidney, lung, and brain tissues can be found in S1 Fig. (H) Immunostaining of liver tissue harvested from a Stat1-/- mouse using the histiocyte marker CD68, anti-JEV NS3 to mark USUV infection, and DAPI with a 50 µm scale bar. (I) Liver tissue harvested from a Stat1-/- mice and stained with anti-JEV NS3 and DAPI with a 200 µm scale bar. The white asterisk indicates an inflammatory lesion.
Fig 6.
B, T, and natural killer cells are dispensable for restricting USUV infection.
(A) Survival, (B) weight changes, and (C) clinical scoring of NRG (n = 6) and wild-type C57BL/6 (n = 6) mice following inoculation with 103 focus-forming units (FFU) of USUV TC508 via subcutaneous (SC) injection. (D) Changes in viremia 1–4 days post-infection (dpi) compared to a pre-bleed sample (“pre”) collected prior to infection in NRG (n = 4) and C57BL/6 (n = 3) mice.
Fig 7.
Administration of IFN-beta (IFN-β) partially protects Vav-Cre/Stat1fl/fl mice from lethal USUV infection.
(A) Survival, (B) weight changes, and (C) clinical scoring of Vav-Cre/Stat1fl/fl mice that received 104 IU IFN-β via SC injection at two timepoints before or after infection with 103 FFU of USUV TC508. The pre-treatment group (n = 6) received IFN-β injections at 2 hours prior to USUV infection and 24 hours post-infection (hpi). One post-treatment group received IFN-β injections 6 and 24 hpi (n = 8) and the second post-treatment group received IFN-β injections at 24 and 48 hpi (n = 7).