An Animal Model for Studying the Pathogenesis of Chikungunya Virus Infection Sarah A. Ziegler, Liang Lu, Amelia P. A. Travassos da Rosa, Shu-Yuan Xiao, and Robert B. Tesh* Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas Abstract. Newborn and 14-day-old mice inoculated subcutaneously with chikungunya virus (CHIKV) developed lethargy, difficulty walking, dragging of hind limbs, and reduced weight gain within 7–10 days after infection (PI). During the initial 6–7 days PI, the animals had viremia; high levels (10 6 –10 8 PFU) of CHIKV were also present in leg muscle. The virus persisted in muscle for several days after viremia disappeared. The major histopathologic changes were in skeletal muscle, which were focal necrosis and inflammation, followed by fibrosis and dystrophic calcification. Some mice also showed dystrophic calcification in the joint cartilage, but there were few deaths, and most of the animals eventually recovered. CHIKV antigen was shown by immunohistochemistry in the muscle for several weeks after infection. Based on the clinical and pathologic similarities with CHIKV infection in humans, young ICR and CD-1 mice offer a useful and realistic model for further study of the pathogenesis and treatment of CHIKV infection. INTRODUCTION Chikungunya virus (CHIKV), the etiologic agent of chikungunya fever (CHIKF), is a mosquito-transmitted al- phavirus belonging to the family Togaviridae. It is included within the Semliki Forest complex of alphaviruses, based on its antigenic and phylogenetic relationships. 1 CHIKV was first isolated during an epidemic of dengue-like disease in Tanganyika (Tanzania) in 1952. 2 The word “chikungunya” is Swahili and means “that which bends up”; it was the name used by indigenous people of the region to describe the char- acteristic posture assumed by patients afflicted with the se- vere muscle and joint pains associated with this illness. 3,4 The current known geographic distribution of CHIKV includes sub-Saharan Africa, India, islands in the Indian Ocean, Southeast Asia, and the Philippines. 3–5 Carey 6 presented his- toric evidence suggesting that CHIKF probably has occurred sporadically in India and Southeast Asia for at least 200 years, although the clinical and epidemiologic similarities of CHIKF and dengue fever make precise differentiation of the two dis- eases difficult without laboratory confirmation. During the past several years, there has been renewed in- terest in CHIKV because of large outbreaks of the disease in India, Sri Lanka, and a number of smaller islands in the In- dian Ocean 4,5 and by the appearance of cases of CHIKF in Europe and the Americas among tourists and other travelers returning from the affected regions. 4,7 During the summer of 2007, a small outbreak occurred in Italy, apparently the result of an introduction from a traveler visiting from India. 8 These recent outbreaks illustrate the ability of certain vector-borne viral diseases to be introduced into non-epidemic regions, when the appropriate ecologic conditions for their transmis- sion exist. 9 A number of alphaviruses within the Semliki Forest com- plex produce an illness in humans or equines characterized by fever, arthralgia, and rash. This group includes chikungunya, o’nyong-nyong, Getah (horses), Ross River, Mayaro, and Barmah Forest viruses. 3,10 Each of these viruses has a unique geographic distribution and mosquito vector(s), but the ill- nesses associated with them are clinically very similar. The myalgia and arthralgia associated with these alphavirus infec- tions tends to be transient (5–7 days), but in some patients, it persists longer or is recurrent. 4,10 The pathogenesis of CHIKV in humans and the mechanism by which it causes arthritic disease is poorly understood. Ross River virus (RRV) is the best studied of the arthritogenic alphaviruses; and a mouse model of RRV-induced arthritis/ arthralgia has been used to study the pathology and immu- nology of the disease. 11–16 In the RRV mouse model, infec- tion results in severe inflammation and necrosis of skeletal muscle. To determine whether the pathogenesis of CHIKV is similar and to gain insight into the cause of the severe muscle and joint pain observed in humans with the infection, a series of experiments was carried out in mice experimentally in- fected with a recent epidemic strain of CHIKV. This report describes our results. MATERIALS AND METHODS Animals. Two outbred mouse lines were used in this study: the Institute for Cancer Research (ICR) strain obtained from Harlan Sprague-Dawley (Indianapolis, IN) and the CD-1 strain obtained from Charles River Laboratories (Wilming- ton, MA). Mice were cared for in accordance with guidelines of the Committee on Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources National Re- search Council, Washington, DC). All experiments were con- duced in an animal biosafety level 3 (ABSL-3) facility under a protocol approved by the University of Texas Medical Branch (UTMB) Institutional Animal Care and Use Com- mittee (IACUC). Virus. CHIKV strain LR 2006-OPY1 was used to infect the mice. The virus isolate was obtained from a CHIKF patient during an outbreak on La Reunion Island in 2006. 7 The virus was kindly provided to us by Dr. Remi Charrel, Emerging Virus Unit, Faculty of Medicine, University of the Mediter- ranean, Marseilles, France; it had been passed five times in Vero cell cultures. Virus assay. Samples of mouse blood, brain, and skeletal muscle were titrated by plaque assay in monolayer cultures of Vero cells. 17 Tissues were first triturated in individual sterile 2-mL glass TenBroeck tissue grinders (Kimble/Kontes, Vine- land, NJ) in 1.0 mL of diluent (phosphate-buffered saline with 10% fetal bovine serum) to prepare ∼20% (wt/vol) suspen- sions. After centrifugation, serial 10-fold dilutions from 10 -1 to 10 -6 of the tissue supernatants and of the blood samples * Address correspondence to Robert B. Tesh, Department of Pathol- ogy, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609. E-mail: rtesh@utmb.edu Am. J. Trop. Med. Hyg., 79(1), 2008, pp. 133–139 Copyright © 2008 by The American Society of Tropical Medicine and Hygiene 133