Date Published: August 29, 2019
Publisher: Public Library of Science
Author(s): Alissa R. Young, Marissa C. Locke, Lindsey E. Cook, Bradley E. Hiller, Rong Zhang, Matthew L. Hedberg, Kristen J. Monte, Deborah J. Veis, Michael S. Diamond, Deborah J. Lenschow, Janko Nikolich-Žugich.
Chikungunya virus (CHIKV) is an arthritogenic alphavirus that acutely causes fever as well as severe joint and muscle pain. Chronic musculoskeletal pain persists in a substantial fraction of patients for months to years after the initial infection, yet we still have a poor understanding of what promotes chronic disease. While replicating virus has not been detected in joint-associated tissues of patients with persistent arthritis nor in various animal models at convalescent time points, viral RNA is detected months after acute infection. To identify the cells that might contribute to pathogenesis during this chronic phase, we developed a recombinant CHIKV that expresses Cre recombinase (CHIKV-3ʹ-Cre). CHIKV-3ʹ-Cre replicated in myoblasts and fibroblasts, and it induced arthritis during the acute phase in mice. Importantly, it also induced chronic disease, including persistent viral RNA and chronic myositis and synovitis similar to wild-type virus. CHIKV-3ʹ-Cre infection of tdTomato reporter mice resulted in a population of tdTomato+ cells that persisted for at least 112 days. Immunofluorescence and flow cytometric profiling revealed that these tdTomato+ cells predominantly were myofibers and dermal and muscle fibroblasts. Treatment with an antibody against Mxra8, a recently defined host receptor for CHIKV, reduced the number of tdTomato+ cells in the chronic phase and diminished the levels of chronic viral RNA, implicating these tdTomato+ cells as the reservoir of chronic viral RNA. Finally, isolation and flow cytometry-based sorting of the tdTomato+ fibroblasts from the skin and ankle and analysis for viral RNA revealed that the tdTomato+ cells harbor most of the persistent CHIKV RNA at chronic time points. Therefore, this CHIKV-3ʹ-Cre and tdTomato reporter mouse system identifies the cells that survive CHIKV infection in vivo and are enriched for persistent CHIKV RNA. This model represents a useful tool for studying CHIKV pathogenesis in the acute and chronic stages of disease.
Chikungunya virus (CHIKV) is a globally re-emerging arthropod-transmitted virus that originally was identified in Tanzania in 1952 [1–3]. Up until the 2000s, CHIKV was considered a self-limiting virus of relatively minimal concern; however, within the last 15 years, CHIKV has reemerged with increased virulence and range. In 2004, an East/Central/South African (ECSA) lineage  of CHIKV caused an epidemic in Kenya with over 13,000 cases, the first large epidemic in decades . By 2005, the virus spread to La Réunion Island off the east coast of Madagascar, where it infected over 200,000 people [6,7]. The La Réunion epidemic included the first reports of increased pathogenicity, including neurological symptoms, intrapartum transmission, and approximately 250 deaths [8–10]. The virus subsequently established endemic infection cycles in tropical regions including India and the South Pacific and also caused isolated outbreaks in Europe . In 2013, an Asian lineage strain of CHIKV spread to the Americas and has caused nearly 2 million suspected cases in the Caribbean, Central America, and South America [11–14].
CHIKV causes a debilitating acute disease that results in chronic arthralgia and myalgia in a substantial proportion of patients. The mechanism of this chronic CHIKV pathogenesis is unclear but may be related to persistence of viral RNA in musculoskeletal tissues. To begin to identify the cells that contribute to chronic pathogenesis, we created a recombinant CHIKV that expresses Cre recombinase and permanently marks infected cells in reporter mice. Using this tool, we provide evidence that a subset of cells survive CHIKV infection, are present during the chronic stages of disease, and harbor persistent CHIKV RNA. Previous reports have used immunofluorescence microscopy, immunohistochemistry, or RNA quantification to detect CHIKV antigen or RNA in animal and patient cells during the chronic phase of disease [18,33–35]. However, CHIKV antigen-positive cells are reportedly rare in samples from the subacute or convalescent phase, likely owing to the insensitivity of these techniques. We used our system to elucidate the cells that survive acute infection and determine whether they are reservoirs of chronic CHIKV RNA.