Virology 279, 27–37 (2001) doi:10.1006/viro.2000.0723, available online at http://www.idealibrary.com on CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector A Novel Model for the Study of the Therapy of Flavivirus Infections Using the Modoc Virus Pieter Leyssen,* Alfons Van Lommel,† Christian Drosten,‡ Herbert Schmitz,‡ Erik De Clercq,* and Johan Neyts*,1 *Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven B-3000, Belgium; †Division of Histopathology, University Hospitals, Leuven B-3000, Belgium; and ‡Bernhard Nocht Institute of Tropical Medicine, Hamburg, Germany Received May 31, 2000; returned to author for revision July 21, 2000; accepted October 26, 2000 The murine Flavivirus Modoc replicates well in Vero cells and appears to be as equally sensitive as both yellow fever and dengue fever virus to a selection of antiviral agents. Infection of SCID mice, by either the intracerebral, intraperitoneal, or intranasal route, results in 100% mortality. Immunocompetent mice and hamsters proved to be susceptible to the virus only when inoculated via the intranasal or intracerebral route. Animals ultimately die of (histologically proven) encephalitis with features similar to Flavivirus encephalitis in man. Viral RNA was detected in the brain, spleen, and salivary glands of infected SCID mice and the brain, lung, kidney, and salivary glands of infected hamsters. In SCID mice, the interferon inducer poly IC protected against Modoc virus-induced morbidity and mortality and this protection was associated with a reduction in infectious virus content and viral RNA load. Infected hamsters shed the virus in the urine. This allows daily monitoring of (inhibition of) viral replication, by means of a noninvasive method and in the same animal. The Modoc virus model appears attractive for the study of chemoprophylactic or chemotherapeutic strategies against Flavivirus infections. © 2001 Academic Press Key Words: Flavivirus; Modoc; antiviral therapy; West Nile virus; tick-borne encephalitis virus; Japanese encephalitis virus. INTRODUCTION an estimated 30,000 people a year become infected, despite the availability of a vaccine. The mortality rate Flaviviruses are enveloped, positive single-stranded RNA viruses that belong, together with the Hepaci- and associated with JEV infections is 30% and an additional Pestiviruses, to the family of the Flaviviridae (Trepo et al., 30% of the survivors suffer from long-lasting neurological 1997; Rice, 1996). Based on phylogenetic analysis of 58 sequelae (Schneider et al., 1974; Kalita and Misra, 1998). members, the genus Flavivirus was subdivided into three There are two subtypes of tick-borne encephalitis virus clusters: the mosquito-borne, the tick-borne, and the (TBEV): the Far Eastern, also referred to as Russian no-vector clusters (Kuno et al., 1998). Viruses belonging spring-summer encephalitis virus (RSSEV), and the Eu- to the first two cluster have a complex natural transmis- ropean, referred to as central European encephalitis sion cycle, which involves several natural hosts (mostly virus (CEEV). TBEV is transmitted to humans usually by mammals or birds) and vector(s), the latter being blood- the bite of a tick or occasionally following consumption sucking insects such as ticks or mosquitoes (Monath of unpasteurized milk. The mortality rates associated and Heinz, 1996). with the Far Eastern subtype and the European subtype Flaviviruses have a worldwide distribution and several are about 30 and 1–2%, respectively (Heinz and Mandl, of them have a major impact on human health. The WHO 1993; Dumpis et al., 1999; Ecker et al., 1999). estimates that each year about 50 million people be- Although the last large epidemic of Murray Valley come infected with the dengue fever virus (DENV) (http:// encephalitis (MVEV) occurred in 1974, new cases of www.who.int/inf-fs/en/fact117.html). At least 500,000 MVEV are regularly reported, especially in Western Aus- cases of dengue hemorrhagic fever (DHF) or dengue tralia (Mackenzie and Broom, 1995). The West Nile virus shock syndrome (DSS) are reported annually, of which (WNV) is mainly endemic around the Mediterranean Sea, Ϯ5% may have a fatal outcome, mainly in children under Africa, and the Middle East. In 1996, an outbreak of WNV the age of 15 (Halstead, 1992). Despite the existence of occurred in Romania with 393 cases, of which 352 were an effective vaccine, the yellow fever virus (YFV) causes with acute CNS involvement and 17 with fatal outcome more than 5000 cases a year with a mortality of 50% (Tsai et al., 1998; Han et al., 1999). More recently (late (Monath, 1987). For the Japanese encephalitis virus (JEV), August–September 1999), an outbreak of WNV caused over 1900 serologically proven infections in the New York 1 metropolitan area, of whom 77 had to be hospitalized To whom correspondence and reprint requests should be ad- with signs of encephalitis and of whom 7 died (Briese et dressed at Rega Institute for Medical Research, Minderbroedersstraat 10, Leuven B-3000, Belgium. Fax: (32) 16-33.73.40. E-mail: al., 1999; Lanciotti et al., 1999). During the late summer of [email protected]. 2000, 17 more cases (1 death) were reported in New 0042-6822/01 $35.00 27 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. 28 LEYSSEN ET AL. York. From July to September 1999, an outbreak of me- and Alberta (Canada) (Zarnke and Yuill, 1985). Neutral- ningoencephalitis associated with WNV occurred in ization tests using blood samples isolated from mam- Southern Russia with hundreds of cases and dozens of mals trapped in Alberta as well as from humans indicate deaths (Lvov et al., 2000). In the late summer and early the appearance of natural infection without disease fall of 2000, an outbreak in Israel was responsible for (Zarnke and Yuill, 1985). No arthropod vector was previ- more than 169 cases and 12 deaths (Siegel, 2000). ously demonstrated (Johnson, 1967). Based on cross- Although there are no recent reports of outbreaks or serological reactivity, the virus was classified as a Flavi- epidemics of Saint Louis encephalitis virus (SLEV), the virus (Casals, 1960; Varelas and Calisher, 1982; Calisher virus causing this disease is endemic in the western et al., 1989). A phylogenetic analysis based on an ϳ1-kb region of the United States and is responsible for severe fragment of 58 flaviviruses showed that MODV belongs disease (Kramer et al., 1997). The Powassan encephalitis to the no-vector cluster of flaviviruses. The no-vector and virus (POWV), transmitted by ticks, is endemic in Ontario the vector-transmitted viruses have a common ancestor and in the Southern Far East of Russia and may cause (Kuno et al., 1998). encephalitis after a period of fever and nonspecific We determined the complete genomic sequence of the symptoms (Mandl et al., 1993; Kolski et al., 1998). Thou- Modoc virus (to be published elsewhere). An overall sands of cases of infections with the Kyasanur Forest sequence similarity with the tick-borne and mosquito- disease virus (KFDV, endemic in India) have been re- borne flaviviruses of 54.3 and 52.3%, respectively, was ported since the discovery of the virus in 1957. The calculated. This is comparable to the sequence similarity annual incidence of virologically diagnosed cases is between mosquito-borne (including YFV, DENV, JEV, 400–500, with a case fatality rate of 3–5% (Pavri, 1989; WNV, MVEV, KUNV) and tick-borne (TBEV, POWV, LIV) Banerjee, 1996; Monath and Heinz, 1996). A small num- Flaviviruses, which is 53.7%. Furthermore, based on our ber of cases of Omsk hemorrhagic fever (OHFV) occur sequence data, the Modoc virus has the same genomic each year among rural residents in the Omsk region in organization and conserved motifs for enzymatic activity Russia (Monath and Heinz, 1996). Louping ill virus (LIV) is as the human flaviviruses. In the present study, we es- primarly a disease in sheep, although there have been at tablished an infection model for MODV in SCID mice and least 37 documented cases in man. The infection was hamsters. This model may be attractive for the study of probably acquired during laboratory experiments or di- antiviral strategies against Flavivirus infections, particu- agnosis (Davidson et al., 1991; McGuire et al., 1998). larly Flavivirus encephalitis. Despite the clinical impact of Flavivirus infections, there is as yet no effective therapy. The study of antiviral RESULTS chemoprophylaxis or chemotherapy is hindered by the lack of a convenient small animal model. Antiviral susceptibility Experimental infection of mice with JEV, YFV, or TBEV MODV produces a clear cytopathic effect in Vero cells. by intracerebral or peripheral inoculation was reported to To study whether the replication of MODV is equally cause morbidity and mortality. However, use of these susceptible to a selection of antiviral agents as the highly pathogenic viruses (BSL-3 or BSL-4), for man, replication of human flaviviruses, we compared the in- requires special laboratory facilities (Holzmann et al., hibitory effects of ribavirin, EICAR (the 5Ј-ethynyl deriva- 1990; Higgs and Gould, 1991; Kaluzova et al., 1994). tive of ribavirin) (De Clercq et al., 1991), tiazofurin, selena- Attenuated strains of YFV or DENV (BSL-2) may cause zofurin (an oncolytic C-nucleoside), and mycophenolic morbidity and even mortality in small laboratory animals, acid (MPA, an inhibitor of IMP-dehydrogenase) on the but only when the virus is inoculated directly into the replication of YFV, DENV, and MODV. As can be derived brain of newborn or suckling mice (Kawano et al., 1993; from Table 1, the antiviral efficacy of the different com- Bray et al., 1998). Several models were previously de- pounds against YFV, DENV, and MODV ranked as fol- scribed for DENV infection in mice, although most of lows: mycophenolic acid Ͼ EICAR Ͼ selenazofurin Ͼ these are labor intensive because of the use of human ribavirin Ͼ tiazofurin.