Tissue tropism and target cells of NSs-deleted rift valley fever virus in live immunodeficient mice

Abstract

Background: Rift Valley fever virus (RVFV) causes disease in livestock and humans. It can be transmitted by mosquitoes, inhalation or physical contact with the body fluids of infected animals. Severe clinical cases are characterized by acute hepatitis with hemorrhage, meningoencephalitis and/or retinitis. The dynamics of RVFV infection and the cell types infected in vivo are poorly understood.

Methodology/principal findings: RVFV strains expressing humanized Renilla luciferase (hRLuc) or green fluorescent protein (GFP) were generated and inoculated to susceptible Ifnar1-deficient mice. We investigated the tissue tropism in these mice and the nature of the target cells in vivo using whole-organ imaging and flow cytometry. After intraperitoneal inoculation, hRLuc signal was observed primarily in the thymus, spleen and liver. Macrophages infiltrating various tissues, in particular the adipose tissue surrounding the pancreas also expressed the virus. The liver rapidly turned into the major luminescent organ and the mice succumbed to severe hepatitis. The brain remained weakly luminescent throughout infection. FACS analysis in RVFV-GFP-infected mice showed that the macrophages, dendritic cells and granulocytes were main target cells for RVFV. The crucial role of cells of the monocyte/macrophage/dendritic lineage during RVFV infection was confirmed by the slower viral dissemination, decrease in RVFV titers in blood, and prolonged survival of macrophage- and dendritic cell-depleted mice following treatment with clodronate liposomes. Upon dermal and nasal inoculations, the viral dissemination was primarily observed in the lymph node draining the injected ear and in the lungs respectively, with a significant increase in survival time.

Conclusions/significance: These findings reveal the high levels of phagocytic cells harboring RVFV during viral infection in Ifnar1-deficient mice. They demonstrate that bioluminescent and fluorescent viruses can shed new light into the pathogenesis of RVFV infection.

Figure 1. Survival curve of mice challenged with rZHΔNSs-hRLuc RVFV.

Ifnar1-deficient and wild-type mice were infected i.p. with 10⁴ PFU rZHΔNSs-hRLuc virus. Mice were monitored twice daily for 4 days and, later, once daily for up to 13 days post-infection. Ifnar1-deficient mice succumbed to disease between 40 and 50 hours post-infection (N = 10). Wild-type 129S2/SvPas mice challenged with rZHΔNSs-hRLuc did not succumbed to infection (N = 5).

Figure 2. Replication sites of rZHΔNSs-RLuc RVFV following inoculation via the intraperitoneal route.

(A) Imaging of an Ifnar1-deficient mouse after infection i.p. with 10⁴ PFU rZHΔNSs-hRluc. Imaging was performed at the indicated times post-infection. This image is representative of thirteen mice. (B) Imaging of the left profile of a mouse at 8 h post-infection showing a bioluminescent signal in the spleen. This image is representative of ten mice. (C) Imaging of the thorax at 16 h post-infection of the same mouse as in (A). Occulting the abdominal cavity revealed bioluminescent signal in the thymus. (D) Ex vivo imaging after dissection at 8 and 16 h post-infection (T, thymus; Lg, lungs; P, pancreas; S, spleen; L, liver; B, brain; I, intestines). Photographs were overlaid with false-color representations of bioluminescence intensity, measured in photons/s/cm²/sr and indicated on the scales.

Figure 3. Luciferase levels of the liver, thymus and pancreas during the time course of infection.

Five Ifnar1-deficient mice were infected i.p. with 10⁴ PFU rZHΔNSs-hRLuc. Imaging of the liver (A), thymus (B) and pancreas (C) was performed in living mice at 8, 16 and 34 h post-infection by defining regions-of-interests (ROIs). Results are given as photon/s/cm²/steradian and plotted as the mean and standard error of the mean of photon counts over time.

Figure 4. Emitted photons and viral RNA copy number in the pancreas, spleen and liver.

Mice were infected i.p with 10⁴ PFU rZHΔNSs-hRLuc. Bioluminescence imaging was performed at 8, 16 and 34 h post-infection. (A) Bioluminescence imaging of the pancreas was performed in anesthetized mice. Subsequently, mice were euthanized and pancreas were harvested and further processed for determination of the viral RNA copy number. (B–D) Mice were euthanized to harvest the pancreas, spleen and liver. The light emission was measured in the harvested pancreas (B), spleen (C) and liver (D) by imaging. Each cross represents an individual organ. Results are given as the number of RNA copies per gram of organ (x axis) with the y axis showing the flux expressed in p/s/cm²/sr. R² and P indicate the correlation coefficient and the significance level, respectively.

Figure 5. In vivo replication sites of rZHΔNSs-RLuc RVFV following inoculation via intradermal or intranasal route.

(A) Representative image of luminescence in the lymph node (white arrowhead) draining the right ear of Ifnar1-deficient mouse at 24 h after intradermal inoculation of 10⁴ PFU rZHΔNSs-RLuc. (B) Luminescence signal in the lungs (white arrowhead) at 48 h after intranasal inoculation of 10⁴ PFU rZHΔNSs-RLuc. Images (A) and (B) are representative of five mice. Photographs were overlaid with false colour representation of bioluminescence intensity, measured in photon/s/cm²/sr and indicated on the scales.

Figure 6. Lesions in the pancreas and expression of viral proteins in the pancreas and ovary.

A–F: Histological lesions in Ifnar1^−/− mice infected i.p. with rZHΔNSs-hRLuc RVFV. (A) Multifocal lesion centered on the mesentery, sparing the pancreatic tissue. (B) The expression of viral proteins is seen in the mesentery exclusively (arrowheads). No viral immunolabeling is detected in the endocrine and exocrine components of the pancreas (asterisks). (C) The lesion is characterized by necrosis of mesenteric adipocytes associated with infiltration of macrophages and neutrophils that could be fragmented (suppuration). Note the presence of high number of cell debris in these lesions. (D) Macrophages in the mesentery expressed viral antigens as seen by immunohistochemistry. (E) Neutrophils also expressed viral proteins. Almost all macrophages in the lesions were infected by RVFV. By contrast, neutrophils expressing viral proteins were less numerous. (F) In the ovary, infected macrophages were multifocally detected in the stroma. G–I: Lesions in wild-type 129S2/SvPas mice infected i.p. with ZH548 RVFV strain. (G–H) Multifocal macrophages expressing viral protein were detected in the pancreaticoduodenal lymph node sinus (arrowheads). (I) Some of these macrophages displayed morphological modifications suggesting irreversible injury, including a shrunken cytoplasm and a hyperbasophilic and fragmented nucleus. Hematoxylin and eosin staining (A and C). Immunohistochemistry using antibodies directed against the RVFV (B, and D to I).

Figure 7. Expression of GFP reporter protein in splenocytes from RVFV-GFP-infected mice.

Mice were either infected i.p. with 10⁴ PFU rZHΔNSs-GFP or mock-treated (control) and euthanized 24 h after infection. Splenocytes of rZHΔNSs-GFP-infected mice (lower plots) and control (upper plots) were analyzed by flow cytometry for the expression of GFP. (A) Representative flow cytometry dot plots showing that CD45.2⁺ cells (total hematopoietic cells) of an RVFV-GFP-infected mouse expressed GFP, as evaluated by fluorescence intensity (bottom plot). (B) Histogram showing GFP expression in CD11c⁺ MHCII⁺ CD11b⁺ cells (dendritic cells). (C) Histograms showing GFP expression in Ly6G⁻ CD11b⁺ CD115⁺ cells (macrophages) (left panels) and CD11b⁺ Ly6G⁺ (granulocytes) (right panels). (D) GFP expression was assessed in CD19⁺ CD3⁻ cells (B lymphocytes). (E) GFP expression in NKp46⁺ CD3⁻ cells (NK cells) (left panels) and NKp46⁻ CD3⁺ cells (T lymphocytes) (right panels). The data are representative of four independent experiments. For each sample a total number of 100,000 to 450,000 splenic cells of infected animals was collected and analyzed. The histograms show GFP expression in a total number of 1,700 dendritic cells, 1,500 macrophages, 1,600 granulocytes, 200,000 B lymphocytes, 3,000 NK cells, and 100,000 T lymphocytes. *Normalized frequency of cells is the number of cells falling within each bin of the histogram; there are 256 bins for each histogram.

Figure 8. Survival curve of mice treated with clodronate-liposomes prior to infection with RVFV-GFP.

Groups of twelve mice were injected i.p. and i.v. with either PBS-liposomes (PBSL) or clodronate-liposomes (CLL) prior to infection with 10⁴ PFU of the recombinant rZHΔNSs-GFP strain. The experiment was repeated once. Clodronate-liposome treated mice survived for a significantly longer period than mice treated with PBS-liposomes (P<0.0018).