A reporter Oropouche virus expressing ZsGreen from the M segment enables pathogenesis studies in mice

Abstract

Oropouche fever caused by Oropouche virus (OROV) is a significant zoonosis in Central and South America. Despite its public health significance, we lack high-throughput diagnostics, therapeutics, and a comprehensive knowledge of OROV biology. Reporter viruses are valuable tools to rapidly study virus dynamics and develop neutralization and antiviral screening assays. OROV is a tri-segmented bunyavirus, which makes generating a reporter virus challenging, as introducing foreign elements into the viral genome typically affects fitness. We previously demonstrated that the non-structural gene NSm on the OROV medium (M) segment is non-essential for replication in vitro. Taking advantage of this, we have now generated a recombinant OROV expressing fluorescent protein ZsGreen in place of NSm. This reporter OROV is both stable and pathogenic in IFNAR^-/- mice and provides a powerful tool for OROV pathogenesis studies and assay development.IMPORTANCEEmerging and reemerging infectious agents such as zoonotic bunyaviruses are of global health concern. Oropouche virus (OROV) causes recurring outbreaks of acute febrile illness in the Central and South American human populations. Biting midges are the primary transmission vectors, whereas sloths and non-human primates are their reservoir hosts. As global temperatures increase, we will likely see an expansion in arthropod-borne pathogens such as OROV. Therefore, developing reagents to study pathogen biology to aid in identifying druggable targets is essential. Here, we demonstrate the feasibility and use of a fluorescent OROV reporter in mice to study viral dynamics and pathogenesis. We show that this reporter OROV maintains characteristics such as growth and pathogenicity similar to the wild-type virus. Using this reporter virus, we can now develop methods to assist OROV studies and establish various high-throughput assays.

Keywords: Oropouche virus; bunyavirus; fluorescent virus; pathogenesis; reporter virus; tropism.

Fig 1

Design and characterization of rOROVMZsG. (A) Schematic representation of the antigenome wild-type rOROV M segment and the reporter rOROV encoding ZsG in place of NSm. (B) Western blot analysis of OROV N protein expression. Vero E6 cells were mock-, rOROV-, or rOROVMZsG-infected. N protein was detected with anti-OROV mouse ascetic fluid (green; 27 kDa), and an anti-Beta actin antibody (red, 50 kDa) was used as a loading control. (C) Viral plaque phenotype comparison between rOROV and rOROVMZsG. Plaque assays in Vero E6 cells were fixed at 3 d.p.i. and stained with crystal violet. (D and E) Viral growth curves in Vero E6 (D) and A549 (E) cells infected with either rOROV or rOROVMZsG at MOI 0.1. Infectious viral titers were determined at 18, 24, and 48 h.p.i. and represented as TCID₅₀/mL. Error bars represent standard deviations (n  =  3), and the dotted line represents the limit of detection.

Fig 2

Stability of rOROVMZsG. (A) Representative fluorescence microscopy images of Vero E6 cells infected with rOROV or rOROVMZsG at 24 h.p.i. OROV proteins were detected using indirect immunofluorescence with anti-OROV mouse ascetic fluid. Scale bar, 100 µm; 20× magnification. (B) Passage experiment. Representative fluorescence microscopy images of Vero E6 cells infected with serially passaged rOROVMZsG at 24 h.p.i. Scale bars, 75 µm; 40× magnification. (C) Fluorescence intensity of rOROVMZsG. Relative fluorescence from five passages (B) was normalized to the number of nuclei (I), and fluorescence intensity was quantified and expressed as arbitrary units (AU; ii).

Fig 3

rOROV infection in IFNAR^-/- mice. (A) Experimental outline of mice infection. Mice were SC infected with either rOROV, rOROVMZsG, or Opti-MEM. Survival curves of IFNAR^-/- mice infected with either 10 or 25000 TCID₅₀ rOROV ( = 5; B) or with 10, 25, 2,500, or 25,000 TCID₅₀ of rOROVMZsG (n = 5; C). (D-J) Percent weight change of mice from baseline during the course of infection. (K) Area under the curve (AUC) analysis. Weight changes compared with the baseline weight of each animal were determined, and AUC was calculated in GraphPad Prism 9.

Fig 4

vRNA load and serum cytokine levels. Viral load per gram of tissue in the liver, spleen, heart, lung, and brain of mock- (A), rOROV- (B), and rOROVMZsG-infected (C) mice as measured by RT-qPCR (QuantStudio 5 Applied Biosystems). Survivors are indicated as open symbols. Mice serum cytokine levels at euthanasia. (D-I) Cytokines IFNα, IFNβ, IFNγ, IL-10, TFNα, and Rantes were quantified in the serum of individual mice using a custom murine cytokine ProcartaPlex Simplex kit (25 µL of serum; Thermo Fisher Scientific) per manufacturer’s instructions using a 2 h incubation and read on a Luminex 200 platform. Data points represent the cytokine expression of each mouse per group. Survivors are indicated as open symbols.

Fig 5

rOROVMZsG infection in male and female IFNAR^-/- mice. (A-C) Percent weight change from baseline of mice during the course of infection. (D) Survival curves of IFNAR^-/- mice. Male and female mice infected with either 25,000 TCID₅₀ rOROVMZsG or UV-inactivated rOROVMZsG (n = 5). (E) Viral load per gram of tissue in the liver, spleen, heart, lung, and brain of rOROVMZsG infected mice as measured by qRT-PCR (QuantStudio 5 Applied Biosystems). (F) Number of mice from which infectious virus was isolated.

Fig 6

ZsG expression in rOROVMZsG-infected mice tissue. Gross microscopy images of freshly harvested whole tissues at necropsy as visualized under a fluorescence microscope (EVOS M5000 imagining system, ThermoFisher). Scale bar, 300 µm. (A) Liver, spleen, heart, lung, and brain from female and male mice infected with 25,000 TCID₅₀ rOROVMZsG, 5 dpi. (B) Enlarged images of the heart, spleen, and (C) liver from a UV-inactivated rOROVMZsG control mouse.

Fig 7

Histopathology of rOROV- and rOROVMZsG-infected mice. Liver sections (A) depicting severe multifocal hepatic necrosis (rOROV, rOROVMZsG) and minimal multifocal lymphocytic infiltrate (mock) of mice infected at a viral dose of 25000 TCID₅₀. The violin plot represents the distribution of liver histopathology scores in each group. Brain sections (B) depicting mild focal lymphocytic infiltration near the cerebellum (rOROV; yellow rectangle), mild focal lymphocytic infiltration in the meninges (rOROVMZsG; yellow oval), and a normal brain section (mock) from mice infected at a viral dose of 25,000 TCID₅₀. The violin plot represents the distribution of brain histopathology scores in each group. Histopathological score used an ordinal method (0, negative; 1, minimal; 2, moderate; 3, severe) where distribution was scored as focal (localized lesion), multifocal (several scattered focal lesions), and diffuse (throughout the liver). Sections were H&E stained, visualized under a light field microscopy, and images captured using an Aperio whole slide imaging system.