Characterization of Ross River virus tropism and virus-induced inflammation in a mouse model of viral arthritis and myositis

Abstract

Mosquito-borne alphaviruses are a significant cause of both encephalitic and arthritic disease in humans worldwide. In contrast to the encephalitic alphaviruses, the pathogenesis of alphavirus-induced arthritic disease is not well understood. Utilizing a mouse model of Ross River virus (RRV) disease, we found that the primary targets of RRV infection are bone, joint, and skeletal muscle tissues of the hind limbs in both outbred CD-1 mice and adult C57BL/6J mice. Moreover, histological analyses demonstrated that RRV infection resulted in severe inflammation of these tissues. Characterization of the inflammatory infiltrate within the skeletal muscle tissue identified inflammatory macrophages, NK cells, and CD4+ and CD8+ T lymphocytes. To determine the contribution of the adaptive immune system, the outcome of RRV-induced disease was examined in C57BL/6J RAG-1(-/-) mice, which lack functional T and B lymphocytes. RAG-1(-/-) and wild-type mice developed similar disease signs, infiltration of inflammatory macrophages and NK cells, and muscle pathology, suggesting that the adaptive immune response does not play a critical role in the development of disease. These results establish the mouse model of RRV disease as a useful system for the identification of viral and host factors that contribute to alphavirus-induced arthritis and myositis.

FIG. 1.

Ross River virus-induced disease in 15-day-old CD-1 mice. Fifteen-day-old CD-1 mice were infected with 10³ PFU of RRV (Δ) by subcutaneous injection in the left rear footpad. Mock-infected mice (□) were injected with diluent alone. (A) Mice were scored for development of hind limb dysfunction and disease based on the following scale: 0, no disease signs; 1, ruffled fur; 2, very mild hind limb weakness; 3, mild hind limb weakness; 4, moderate hind limb weakness and dragging of hind limbs; 5, severe hind limb weakness/dragging; 6, complete loss of hind limb function; 7, moribund; and 8, death. (B) Mice were monitored for weight gain or loss at 24-h intervals. Each data point represents the arithmetic mean ± standard deviation (SD) for 7 (mock-infected) or 25 (RRV-infected) mice.

FIG. 2.

Ross River virus tissue titers in 15-day-old CD-1 mice. Fifteen-day-old CD-1 mice were infected with 10³ PFU of RRV by subcutaneous injection in the left rear footpad. At 12, 24, 48, 72, 96, and 120 hpi, ankle (A), quadriceps muscle (B), serum (C), brain (D), and spleen (E) were harvested and homogenized, and the amount of infectious virus present was quantified by plaque assay on BHK-21 cells. Each data point represents the arithmetic mean ± SD for three mice.

FIG. 3.

Ross River virus replication within bone and joint-associated connective tissues and skeletal muscle tissue. Fifteen-day-old CD-1 mice were infected with 10³ PFU of RRV by subcutaneous injection in the ventral thorax. Mock-infected mice were injected with diluent alone. Mice were sacrificed at 24 (A, B, C), 48 (D, E, F), and 72 (G, H, I) hours postinfection and perfused with 4% paraformaldehyde. Following decalcification, 5-μm-thick paraffin-embedded sections derived from the hind limbs were probed with ³⁵S-labeled riboprobes complementary for RRV (A-I) or the EBER2 gene from Epstein-Barr virus (data not shown). M, muscle; B, bone. (A) RRV-specific in situ signal in tarsal bone periosteum. (B) RRV-specific in situ signal in synovial connective tissue of the knee joint. (C) Absence of RRV-specific in situ signal in hind limb skeletal muscle. (D) RRV-specific in situ signal in tarsal bone periosteum and associated skeletal muscle. (E) RRV-specific in situ signal in synovial tissue of a tarsal joint. (F) RRV-specific in situ signal in hind limb skeletal muscle tissue. (G) RRV-specific in situ signal in metatarsal bone periosteum and associated skeletal muscle. (H) RRV-specific in situ signal in hind limb tendon. (I) RRV-specific in situ signal in hind limb skeletal muscle.

FIG. 4.

Ross River virus double-promoter virus targets cells within joint, bone, and skeletal muscle tissues. (A) Schematic diagram of Ross River virus that was engineered to express EGFP by inserting a second RRV 26S subgenomic promoter at the 3′ end of the viral genome followed by the EGFP coding sequence. (B) Ten- to 12-day-old CD-1 mice were infected with 10³ PFU of RRV-EGFP by subcutaneous injection in the ventral thorax. At 48 and 72 hpi, tissues were harvested and sections were analyzed for EGFP expression. SC, synovial cavity; M, muscle; P, periosteum. The white arrows indicate the articular surface. (Subpanel A) Synovial cavity in the foot at 48 hpi. (Subpanel B) Synovial cavity in the ankle at 48 hpi. (Subpanel C) Periosteum in the hind limb at 48 hpi. (Subpanel D) Hind limb skeletal muscle at 72 hpi.

FIG. 5.

Ross River virus infection induces severe hind limb disease in 24-day-old C57BL/6J mice. Twenty-four-day-old C57BL/6J mice were inoculated with 10³ PFU of RRV (Δ) by subcutaneous injection in the left rear footpad. Mock-infected mice (□) were injected with diluent alone. (A) Mice were scored for development of hind limb dysfunction and disease based on the following scale: 0, no disease signs; 1, ruffled fur; 2, very mild hind limb weakness; 3, mild hind limb weakness; 4, moderate hind limb weakness and dragging of hind limbs; 5, severe hind limb weakness/dragging; 6, complete loss of hind limb function; 7, moribund; and 8, death. (B) Mice were monitored for weight gain or loss at 24-h intervals. Each data point represents the arithmetic mean ± SD for three (mock-infected) or six (RRV-infected) animals. Data are representative of four independent experiments.

FIG. 6.

Ross River virus tissue titers in 24-day-old C57BL/6J mice. Twenty-four-day-old C57BL/6J mice were infected with 10³ PFU of RRV by subcutaneous injection in the left rear footpad. At 12, 24, 48, 72, 96, and 120 hpi, the following tissues were harvested and homogenized and the amount of infectious virus present was quantified by plaque assay on BHK-21 cells. (A) □, ankle of injected leg; Δ, ankle of noninjected leg. (B) □, quadriceps muscle of injected leg; Δ, quadriceps muscle of noninjected leg. (C) Serum. (D) Spleen. (E) Brain. (F) □, lower spinal cord; Δ, upper spinal cord. Each data point represents the arithmetic mean ± SD for three mice.

FIG. 7.

Ross River virus induces inflammation in hind limb bone and joint tissues of C57BL/6J mice. Twenty-four-day-old C57BL/6J mice were infected with 10³ PFU of RRV by subcutaneous injection in the left rear footpad. Mock-infected mice were injected with diluent alone. M, muscle; B, bone; P, periosteum; ST, synovial tissue. At 5 days (A and B) and 7 days (C and D) postinfection, mice were perfused with 4% paraformaldehyde. Following decalcification, 5-μm-thick paraffin-embedded sections generated from ankle and foot tissues of mock-infected (A and C) and RRV-infected (B and D) mice were H & E stained. Images (magnification, ×200) are representative of at least six mice per group.

FIG. 8.

Ross River virus induces inflammation in hind limb skeletal muscle tissue of C57BL/6J mice. Twenty-four-day-old C57BL/6J mice were infected with 10³ PFU of RRV by subcutaneous injection in the left rear footpad. Mock-infected mice were injected with diluent alone. At 3, 5, 7, 10, and 30 dpi, mice were perfused with 4% paraformaldehyde and 5-μm-thick paraffin-embedded sections generated from the quadriceps muscle were H & E stained. (A) Mock infection. (B) RRV infection at 3 dpi. (C) RRV infection at 5 dpi. (D) RRV infection at 7 dpi. (E) RRV infection at 10 dpi. (F) RRV infection at 30 dpi. Images (magnification, ×200) are representative of three to six mice per group.

FIG. 9.

Characterization of Ross River virus-induced inflammatory infiltrates. Twenty-four-day-old C57BL/6J mice were infected with 10³ PFU of RRV by subcutaneous injection in the left rear footpad. Mock-infected mice were injected with diluent alone. (A) Cell surface staining of cells isolated from the quadriceps muscle at 5 dpi. Dot plots shown for each stain are representative of three mice. Two independent experiments gave similar results. (B) Cell surface staining of cells isolated from the quadriceps muscle at 7 dpi. Dot plots shown for each stain are representative of three mice per group. Three independent experiments gave similar results. (C) Total numbers of natural killer cells (NK1.1⁺/CD3⁺), inflammatory macrophages (F4/80⁺/Gr-1⁺), CD4 T lymphocytes (CD3⁺/CD4⁺), and CD8 T lymphocytes (CD3⁺/CD8⁺) from the quadriceps muscle of mock- and RRV-infected animals at 5 and 7 days postinfection. Data presented are the means ± standard errors of the means for three to four mice per group and are representative of at least two independent experiments.

FIG. 10.

Ross River virus-induced inflammation and disease in RAG-1^−/−. Twenty-four-day-old C57BL/6J RAG-1^−/− mice were infected with 10³ PFU of RRV (Δ) by subcutaneous injection in the left rear footpad. Mock-infected mice (□) were injected with diluent alone. (A) Mice were scored for development of hind limb dysfunction and disease based on the following scale: 0, no disease signs; 1, ruffled fur; 2, very mild hind limb weakness; 3, mild hind limb weakness; 4, moderate hind limb weakness and dragging of hind limbs; 5, severe hind limb weakness/dragging; 6, complete loss of hind limb function; 7, moribund; and 8, death. Each data point represents the arithmetic mean ± SD for two (mock-infected) or six (RRV-infected) mice and are representative of two independent experiments. (B) Mice were monitored for weight gain or loss at 24-h intervals. Each data point represents the arithmetic mean ± SD for four (mock-infected) or six (RRV-infected) mice and are representative of two independent experiments. (C) At 10 days postinfection, mice were perfused with 4% paraformaldehyde and 5-μm-thick paraffin-embedded sections generated from the quadriceps muscle were H & E stained. Images (magnification, ×200) are representative of three mice per group. (D) Total numbers and percentages of natural killer cells (NK1.1⁺/CD3⁻) and inflammatory macrophages (F4/80⁺/Gr-1⁺) from the quadriceps muscles of mock-infected (black bars) or RRV-infected (gray bars) RAG-1^−/− mice at 7 days postinfection. Data presented are the means ± standard errors of the means for three mice per group and are representative of two independent experiments.