A mouse model of chikungunya virus-induced musculoskeletal inflammatory disease: evidence of arthritis, tenosynovitis, myositis, and persistence

Abstract

Chikungunya virus (CHIKV), an emerging mosquito-borne Alphavirus, causes debilitating rheumatic disease in humans that can last for weeks to months. Starting in 2004, a CHIKV outbreak in the Indian Ocean region affected millions of people, and infected travelers introduced CHIKV to new regions. The pathogenesis of CHIKV is poorly understood, and no approved vaccines or specific therapies exist. A major challenge to the study of CHIKV disease is the lack of a small animal model that recapitulates the major outcomes of human infection. In this study, the pathogenesis of CHIKV in C57BL/6J mice was investigated using biological and molecular clones of CHIKV isolated from human serum (CHIKV SL15649). After 14-day-old mice were inoculated with CHIKV SL15649 in the footpad, they displayed reduced weight gain and swelling of the inoculated limb. Histologic analysis of hind limb sections revealed severe necrotizing myositis, mixed inflammatory cell arthritis, chronic active tenosynovitis, and multifocal vasculitis. Interestingly, these disease signs and viral RNA persisted in musculoskeletal tissues for at least 3 weeks after inoculation. This work demonstrates the development of a mouse model of CHIKV infection with clinical manifestations and histopathologic findings that are consistent with the disease signs of CHIKV-infected humans, providing a useful tool for studying viral and host factors that drive CHIKV pathogenesis and for evaluating potential therapeutics against this emerging viral disease.

Figure 1

Chikungunya virus (CHIKV)–induced disease signs. Fourteen-day-old C57BL/6J mice were inoculated with virus diluent only (control), 100 PFU of CHIKV SL15649, 100 PFU of UV light–inactivated CHIKV SL15649, or 100 PFU of three independent plaque-purified isolates of CHIKV SL15649 (PL1, PL2, PL3). Mice were weighed at 24-hour intervals, and percentage of starting body weight (A), actual body weights (B), and the average rate of weight gain in grams per day (C) were determined (n = three mice per group). D: Fourteen-day-old C57BL/6J mice were inoculated with virus diluent only (control) or 100 PFU of plaque-purified CHIKV SL15649. At 7 days after inoculation, a dorsoventral measurement of the left foot was performed (n = four mice per group). ***P < 0.001 as determined by 2-tailed t-test. E: Fourteen-day-old C57BL/6J mice were inoculated with virus diluent only (control) or 100 PFU of plaque-purified CHIKV SL15649. At 7 days after inoculation, mice were sacrificed and perfused by intracardial injection with 4% paraformaldehyde. Tissue sections were stained with H&E, and the footpads were assessed for evidence of inflammation and edema (asterisk). Images are representative of at least three mice per group. F: Fourteen-day-old C57BL/6J mice were inoculated with 100 PFU of plaque-purified CHIKV SL15649. At 1 day after inoculation (n = 4) and 4 days after inoculation (n = 4), mice were sacrificed and perfused by intracardial injection with 1× PBS. Left tarsus or foot (filled circles) and right tarsus or foot (open circles) were dissected, weighed, and homogenized, and the amount of infectious virus present was quantified by plaque assays. Dashed line indicates the limit of detection. ***P < 0.0001 as determined by two-tailed t-tests.

Figure 2

Chikungunya virus (CHIKV) SL15649-induced musculoskeletal disease. Fourteen-day-old C57BL/6J mice were inoculated with virus diluent only (control) or 100 PFU of plaque-purified CHIKV SL15649. At 7 days after inoculation, mice were sacrificed and perfused by intracardial injection with 4% paraformaldehyde. Tissue sections were stained with H&E and evaluated for tissue disease. Metatarsophalangeal joint of control-inoculated (A) or CHIKV-inoculated (B) inoculated mouse. Asterisks indicate bone. Pelvic limb tendon of control-inoculated (C) or CHIKV-inoculated (D) mouse. Arrows indicate tendon sheath. Gastrocnemius muscle of control-inoculated (E and G) or CHIKV-inoculated (F and H) mouse. Pelvic limb vasculitis of CHIKV-inoculated mouse (I and J). Asterisk indicates normal vessel (I). Arrowheads indicate vessels with vasculitis (I). Pelvic limb bone marrow of control-inoculated (K) or CHIKV-inoculated (L) mouse. Images are representative of at least three mice per group (M). At 3 (n = 3), 5 (n = 3), and 7 (n = 3) days after inoculation, leukocytes were isolated from hind limb tissues of control-inoculated and CHIKV SL15649–inoculated mice. Isolated cells were counted and analyzed by flow cytometry as described in the “Materials and Methods” section. Total numbers of natural killer cells (NK1.1⁺/CD3⁻), monocytes/macrophages (F4/80⁺/CD11b⁺), CD4 T lymphocytes (CD3⁺/CD8⁻/CD4⁺), and CD8 T lymphocytes (CD3⁺/CD8⁻/CD4⁺) isolated from the left and right hind limbs are shown. Each bar represents the arithmetic mean ± SEM of three mice per group.

Figure 3

A cDNA clone of chikungunya virus (CHIKV) SL15649 induces inflammation of the musculoskeletal tissues. Fourteen-day-old C57BL/6J mice were inoculated with virus diluent only (control), 100 PFU of plaque-purified CHIKV SL15649, or 100 PFU of cDNA clone–derived CHIKV SL15649 (icCHIKV SL15649). At 10 days after inoculation, mice were sacrificed and perfused by intracardial injection with 4% paraformaldehyde. Tissue sections from control-inoculated (A), CHIKV SL15649–inoculated (B), and icCHIKV SL15649–inoculated mice (C) were stained with H&E and evaluated for tissue disease. Shown are images of the left tarsus. Asterisks indicate bone. Images are representative of at least three mice per group. D: Fourteen-day-old C57BL/6J mice were inoculated with virus diluent only (control) or 100 PFU of icCHIKV SL15649. At 7 days after inoculation, dorsoventral measurements of the left and the right foot were performed (n = four mice per group). Data are representative of at least two independent experiments. ***P < 0.0001 as determined by two-tailed t-test. E: Fourteen-day-old C57BL/6J mice were inoculated with 100 PFU of icCHIKV SL15649. At 1 (n = 4), 4 (n = 4), and 7 (n = 4) days after inoculation, mice were sacrificed and perfused by intracardial injection with 1× PBS. Left tarsus or foot (filled circles) and right tarsus or foot (open circles) were dissected, weighed, and homogenized, and the amount of infectious virus present was quantified by plaque assays. Dashed line indicates the limit of detection. *P = 0.0006 as determined by two-tailed t-test. **P = 0.002 as determined by two-tailed t-test. F: Fourteen-day-old C57BL/6J mice were mock inoculated or inoculated with 100 PFU of icCHIKV SL15649. At 2 (n = 3), 14 (n = 3), and 21 (n = 3) days after inoculation, mice were sacrificed and perfused by intracardial injection with 1× PBS. Left tarsus/foot (LA), right tarsus/foot (RA), left quadriceps femoris (LQ), right quadriceps femoris (RQ), and spleens (S) were homogenized in TRIzol. Isolated RNA was analyzed for the presence of CHIKV RNA by RT-PCR. Gel images are representative of three mice per group.