NSG-Mice Reveal the Importance of a Functional Innate and Adaptive Immune Response to Overcome RVFV Infection

Abstract

Rift Valley fever (RVF) is a zoonotic disease caused by RVF Phlebovirus (RVFV). The RVFV MP-12 vaccine strain is known to exhibit residual virulence in the case of a deficient interferon type 1 response. The hypothesis of this study is that virus replication and severity of lesions induced by the MP-12 strain in immunocompromised mice depend on the specific function of the disturbed pathway. Therefore, 10 strains of mice with deficient innate immunity (B6-IFNAR^tmAgt, C.129S7(B6)-Ifng^tm1Ts/J, B6-TLR3^tm1Flv, B6-TLR7^tm1Aki, NOD/ShiLtJ), helper T-cell- (CD4^tm1Mak), cytotoxic T-cell- (CD8^atm1Mak), B-cell- (Igh-J^tm1DhuN?+N2), combined T- and B-cell- (NU/J) and combined T-, B-, natural killer (NK) cell- and macrophage-mediated immunity (NOD.Cg-Prkdc^scidIl2rg^tm1WjI/SzJ (NSG) mice) were subcutaneously infected with RVFV MP-12. B6-IFNAR^tmAgt mice were the only strain to develop fatal disease due to RVFV-induced severe hepatocellular necrosis and apoptosis. Notably, no clinical disease and only mild multifocal hepatocellular necrosis and apoptosis were observed in NSG mice, while immunohistochemistry detected the RVFV antigen in the liver and the brain. No or low virus expression and no lesions were observed in the other mouse strains. Conclusively, the interferon type 1 response is essential for early control of RVFV replication and disease, whereas functional NK cells, macrophages and lymphocytes are essential for virus clearance.

Keywords: Rift Valley fever; adaptive immunity; immunodeficiency; innate immunity; virus persistence.

Figure 1

Kaplan–Meier survival curve of all mouse strains infected with RVFV and placebo groups: B6-IFNAR^tmAgt mice died or were euthanized three days after subcutaneous RVFV infection. Except for 1/12 (8%) of the NSG mouse, all other mice survived until the end of the study after subcutaneous RVFV infection. The surviving groups are summarized for better readability. RVFV: Rift Valley fever virus.

Figure 2

Comparisons of PCR results in B6-IFNAR^tmAgt and NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ mice: RT-qPCR results of both mouse strains show significant viral loads in liver, spleen and brain when compared to RVFV-infected wildtype controls (C57Bl/6 or NOD/ShiLtJ mice, respectively).

Figure 3

Serum neutralization test results of all mouse strains infected with RVFV and placebo groups: detectable antibody titers in serum samples were measured between 1:10 and 1:120 dilutions. The placebo groups of all mouse strains are summarized for better readability. The detection range is shown as punctuated lines. RVFV: Rift Valley fever virus.

Figure 4

Histology (HE staining, 200x magnification) of RVFV-infected IFNAR mice (3 dpi) and NSG mice (14 dpi): IFNAR mice exhibit severe, multifocal to coalescing hepatocellular necrosis (arrowheads, (A)) and apoptosis (arrows, (A)) as well as mild lymphocytolysis in the white pulp (arrowheads, (B)) and moderate, multifocal necrosis in the red pulp of the spleen (arrow, (B)). No lesions were observed in the brain (C) and placebo-infected control mice (D–F). Likewise, no lesions were present in the majority of RVFV-infected (G–I) or any placebo (J–L) NSG mice. RVFV: Rift Valley fever virus; dpi: days post infection; IFNAR: B6-IFNAR^tmAgt mice. NSG; NOD.Cg-Prkdc^scid Il2rg^tm1WjI/SzJ mice.

Figure 5

Immunohistochemistry (against RVFV Np, 200x magnification) of RVFV-infected IFNAR mice (3 dpi) and NSG mice (14 dpi): IFNAR mice exhibit multifocal to diffuse expression of RVFV Np in the liver (star (A)) and the spleen (star (B)) while 3/6 (50%) animals did not show any expression of RVFV Np in the brain (C). Control animals are negative for RVFV antigen (D–F). NSG mice reveal small nests of immunolabeled hepatocytes (arrowhead, (G)), no signal in the spleen (H) and diffuse expression of RVFV Np in neurons (arrowheads, (I)). Control animals are negative (J–L). RVFV: Rift Valley fever virus; Np: nucleoprotein; dpi: days post infection; IFNAR: B6-IFNAR^tmAgt mice; NSG: NOD.Cg-Prkdc^scid Il2rg^tm1WjI/SzJ mice.

Figure 6

Graphic summary of the semi-quantitative immunohistochemistry evaluation: Each point represents one individual animal and mean values are given (black bars). While B6-IFNAR^tmAgt mice exhibit high scores in liver and spleen, NOD.Cg-Prkdc^scidIl2rg^tm1WjI/SzJ mice show RVFV Np expression within the CNS in a subset of animals. Furthermore, a single C.129S7(B6)-Ifng^tm1/s/J mouse exhibited viral antigen expression in the kidney.