Ross River virus: Molecular and cellular aspects of disease pathogenesis

Pharmacology & Therapeutics 107 (2005) 329 – 342 www.elsevier.com/locate/pharmthera

Associate editor: P.S. Foster

Ross River virus: molecular and cellular aspects of disease pathogenesis Nestor E. Rullia, Andreas Suhrbierb, Linda Huestonc, Mark T. Heised, Daniela Tupanceskaa, Ali Zaida, Anja Wilmese, Kerry Gilmoree, Brett A. Lidburya, Surendran Mahalingama,* b

a School of Health Sciences, University of Canberra, Kirinari Street, Canberra ACT 2601, Australia Queensland Institute of Medical Research, Australian Centre for International and Tropical Health and Nutrition, Brisbane, Australia c Arbovirus Emerging Disease Unit-ICPMR, Westmead Hospital, Westmead NSW 2145, Australia d Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7290, USA e School of Biological Sciences, University of Wollongong, Wollongong NSW 2522, Australia

Abstract Ross River virus (RRV) is a mosquito-borne alphavirus indigenous to Australia and the Western Pacific region and is responsible for several thousand cases of human RRV disease (RRVD) per annum. The disease primarily involves polyarthritis/arthralgia, with many patients also presenting with rash, myalgia, fever, and/or lethargy. The symptoms can be debilitating at onset, but they usually resolve within 3 – 6 months. Recent insights into the RRV – host relationship, associated pathology, and molecular biology of infection have generated a number of potential avenues for improved treatment. Although vaccine development has been proposed, the small market size and potential for antibody-dependent enhancement (ADE) of disease make this approach unattractive. Recent insights into the molecular basis of RRV – ADE and the virus’s ability to manipulate host inflammatory and immune responses create potential new opportunities for therapeutic invention. Such interventions should overcome virus-induced dysregulation of protective host responses to promote viral clearance and/or ameliorate inflammatory immunopathology. D 2005 Elsevier Inc. All rights reserved. Keywords: Ross River virus; Cytokines; Chemokines; Macrophage; Antibody dependent enhancement of infection; Inflammation; Viral persistence Abbreviations: ADE, antibody-dependent enhancement of infection; CCL, CC chemokine ligand; CXCL, CXC chemokine ligand; CPE, cytophatic effect; EPA, epidemic polyarthritis; IFN, interferon; IL, interleukin; IP-10, interferon inducible protein-10; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein 1; NF-nB, nuclear factor-Kappa B; NO, nitric oxide; NOS2, nitric oxide synthase 2; NSAIDs, nonsteroidal antiinflammatory drugs; RANTES, regulated on activation normal T-cell expressed and secreted; RRV, Ross River virus; RRVD, Ross River virus disease; STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor.

Contents 1.

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Introduction. . . . . . . . . . . . . . . . . . . . . . . . 1.1. Ross River virus is an alphavirus . . . . . . . . . 1.2. Ross River virus virion and genome . . . . . . . 1.3. Epidemiology of Ross River virus infection. . . . 1.3.1. Geographic distribution and case numbers Ross River virus disease . . . . . . . . . . . . . . . . . 2.1. Historical perspective of the disease. . . . . . . . 2.2. Clinical aspects of Ross River virus disease . . . 2.3. Laboratory diagnosis Ross River virus infection .

* Corresponding author. Tel.: +61 2 6201 5364; fax: +61 2 6201 57257. E-mail address: [email protected] (S. Mahalingam). 0163-7258/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2005.03.006

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3.

Dissecting the immunopathology of Ross River virus infection . . . . . . . . . . . 3.1. Mouse model of Ross River virus disease . . . . . . . . . . . . . . . . . . 3.2. Macrophages in Ross River virus disease . . . . . . . . . . . . . . . . . . . 3.3. The role for soluble mediators in Ross River virus disease . . . . . . . . . . 3.4. T-cells in Ross River virus disease . . . . . . . . . . . . . . . . . . . . . . 4. Persistence of Ross River virus disease . . . . . . . . . . . . . . . . . . . . . . . 4.1. Viral persistence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Mechanisms of persistence . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Antibody-dependent enhancement of infection. . . . . . . . . . . . . . . . . . . . 5.1. Antibody-dependent enhancement of Ross River virus infection . . . . . . . 5.2. Suppression of antiviral pathways in antibody-dependent enhancement – Ross infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Treatment and prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 1.1. Ross River virus is an alphavirus Mosquito borne arthritogenic alphaviruses, such as Sindbis-group viruses, Scandinavian Ockelbo virus, the African/Asian Chikungunya virus (CHIK), the African O’nyong-nyong virus (ONN), the South American Mayaro virus (MAY), the Australian Barmah Forest virus (BFV), and Ross River virus (RRV), are associated with outbreaks of polyarthritis/arthralgia in humans (Johnston & Peters, 1996; Lidbury & Mahalingam, 2004; Suhrbier & Linn, 2004; Ytterberg, 1999). Increases in tourism and associated increased international movement, global climate change, and emergence of insecticide resistant mosquitoes may all

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333 333 334 334 335 336 336 336 336 337 337 338 339 339 339

contribute to increasing numbers of alphaviral arthritis cases. The CHIK, ONN, and MAY diseases have recently emerged or reemerged in several countries, and a number of tourist cases have also recently been reported (Suhrbier & Linn, 2004). RRV belongs to the family Togaviridae, which comprises the genera Alphavirus and Rubivirus (Johnston & Peters, 1996). A large number of viruses constitute the genera Alphavirus (Table 1), and in terms of human disease, can be broadly divided into the American encephalitis alphaviruses and the globally distributed arthritogenic alphaviruses (Strauss & Strauss, 1994). Alphaviruses are maintained in nature by a biological transmission cycle between susceptible vertebrates and hematophagous arthropods, usually ticks or mosquitoes. Ross River virus’s enzootic vertebrate

Table 1 Alphaviruses with recognized human diseases (Johnston & Peters, 1996) Alphaviruses

Epidemic

Disease caused

Chikunguya Mayaro O’nyong-nyong Igbo Ora Ross River Sindbis Ockelbo Babanki Barmah Forest Semliki Forest Venezuelan equine Encephalitis Everglades Mucambo Tonate Eastern equine Encephalitis Western equine Encephalitis Highlands J

Yes Yes Yes Yes Yes

+ + + + +

Africa South America Africa Australia, Oceania Australia, Oceania

Yes No No No

+ + +

Scandinavia West Africa Australia Africa, Eurasia

Acute arthropathy

Yes No No No

Geographic distribution Systemic febrile illness

Primarily encephalitis

+ + + + +

South and Central America Florida Brazil, Peru

Yes

+

North and South America

Yes No

+ +

North and South America Eastern United States

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hosts are largely limited to marsupial mammals, however RRV shows a wide vector range with respect to the mosquito species involved in transmission (Harley et al., 2001). 1.2. Ross River virus virion and genome The structure of RRV has been established by the use of cryoelectron microscopy and image reconstruction (Tellinghuisen et al., 1999). The virion has 2 glycosylated envelope proteins: E1 (52 kDa) and E2 (49 kDa). The viral genome encodes a third glycoprotein (E3), which is not incorporated in the virion (Faragher et al., 1988). The nucleocapsid protein C (32 kDa) is 40 nm in diameter with icosahedral symmetry and is surrounded by a lipid bilayer (4.8 nm thick). The genomic RNA is found at the core of the virion. There are 240 heterodimers of E1 and E2, which form 80 trimeric spikes on the surface of the virion (Strauss & Strauss, 1994). The heterodimers associate one-to-one with nucleocapsid monomers in the lipid bilayer (Cheng et al., 1995). RRV has an 11.7 kb single-stranded positive sense RNA genome. The 5V end encodes proteins involved in genomic replication and mRNA synthesis, and the 3V end encodes the envelope glycoproteins and the 6K protein (recently associated with formation of ion channel; Melton et al., 2002). There are a number of topotypes of RRV with nucleotide sequences diverging by up to 6% at the nucleotide level (Lindsay et al., 1993). Studies to date support the role of viral E2 in virulence. For instance, Vrati et al. (1986) isolated an RRV mutant with a 21-nucleotide deletion in the gene encoding for the envelope glycoprotein E2 (RRV dE2). Day-old mice infected with RRV dE2 when compared with wild-type RRV showed less severe symptoms of hind leg paralysis, together with a small increase in LD50 and average survival time. When the mutant was inoculated in week-old mice, the animals showed no symptoms, even at high doses. Virus titres were found to be 2 to 5 log units less in RRV dE2infected mice compared with wild-type RRV-infected animals. Subsequently, it was reported that the E2 glycoprotein contains a major antigenic domain involved in the neutralisation of the virus (Vrati et al., 1988). Interestingly, a recent study demonstrated that a single amino acid substitution in E2 could expand the host range of RRV, allowing the virus to infect cells of avian origin (Heil et al., 2001). The reported substitution at residue 218 created a heparan sulfate binding site that allowed the mutant virus to infect chicken cells. Recently, studies have shown that the small, hydrophobic 6K protein forms cation-selective ion channels in planar lipid bilayers (Melton et al., 2002). This observation could account for the increased permeability of alphavirusinfected cells to monovalent cations, which is followed by increased virion budding. Mutations in the 6K protein of other related viruses such as Sindbis (SIN) and Semliki Forest virus (SFV) have been shown to result in defects in

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the processing and trafficking of viral glycoproteins, virus assembly, and budding (Loewy et al., 1995; Sanz & Carrasco, 2001). In addition, a 6K deleted RRV (RRV d6K) was recently shown to be unable to establish a persistent infection in a mouse macrophage cell line (Dr. G. Ewart, personal communication). 1.3. Epidemiology of Ross River virus infection The virus is sustained primarily by mosquito– mammal cycles. Based on serological evidence and experimental infection studies, the main vertebrate hosts are believed to be nonmigratory native macropods, such as kangaroos and wallabies. Other native reservoir hosts, such as the New Holland mouse (Gard et al., 1973) and flying foxes (Ryan et al., 1997; Harley, 2000), have also been implicated in the natural cycle of the virus. Horses are suspected to be amplifying hosts and may transport the virus over wide areas (Mackenzie et al., 1994; Amin et al., 1998; Azoulas, 1998). Possums have also been shown to be efficient reservoirs and may be involved in urban transmission cycles (Azoulas, 1997). Although the viraemia in humans is thought to be short lived, a man – mosquito –man cycle has been described during explosive epidemics, such as those seen in the Western Pacific in 1979 (Marshall & Miles, 1984), and was suspected in Perth, Western Australia, in 1988/89 and 1991/ 92 (Lindsay et al., 1992) and in Brisbane, Queensland, in 1992 and 1994 (Ritchie et al., 1997). While the known vertebrate host range for RRV is relatively small, it has an unusually broad range of known mosquito vectors. The virus has been recorded in 42 species of mosquito representing 7 genera (Russell, 2000). It is not surprising, then, that the vector ecology of RRV is complex. Different mosquito species are involved in different regions and under varying seasonal and environmental conditions. In coastal regions, Aedes vigilax and A. camptorhynchus are the main vectors, while Culex annulirostris is the main vector further inland, and A. notoscriptus is the predominant urban vector (Russell, 2000). Evidence suggests that the virus survives over winter and between epidemics by vertical transmission (Broom et al., 1989; Russell et al., 1992). 1.3.1. Geographic distribution and case numbers Ross River is endemic throughout Australia and Papua New Guinea (Karabatsos, 1985; Russell, 2000; Harley et al., 2001). The virus has also been the cause of a large epidemic in 1979 in the Western Pacific, involving Fiji, New Caledonia, Samoa, and the Cook Islands (Aaskov et al., 1981; Fauran et al., 1984; Mackenzie et al., 1994). As RRV has such a broad vector base, the epidemiology of the disease may vary both within and between areas. In general, in Australia, RRV disease (RRVD) epidemics are associated with the summer/autumn rainfall and with elevated summer temperatures and/or tidal inundation of marshes along the coast; however, a large number of specific local factors

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Table 2 Number of Ross River virus infections reported in the Australian states/ territories (1992 – 2004a) Year

ACT

NSW

NT

Qld

SA

Tas

Vic

WA

Total

1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004b

1 4 1 2 1 9 6 7 16 9 0 1 6

324 599 332 236 1032 1597 581 939 751 703 178 488 590

239 264 312 369 137 218 127 132 145 220 55 116 206

4183 2263 3002 1650 4885 2366 1946 2277 1444 1560 882 2513 1908

105 773 28 21 55 635 66 36 415 134 41 23 31

1 0 0 28 76 12 9 67 8 13 117 4 20

162 1198 58 35 152 1042 128 222 324 346 37 11 80

686 153 95 303 1445 717 288 486 1087 197 124 392 1034

5701 5254 3828 2644 7783 6596 3151 4166 4190 3182 1434 3548 3875

ACT: Australian Capital Territory; NSW: New South Wales; NT: Northern Territory; Qld: Queensland; SA: South Australia; Tas: Tasmania; Vic: Victoria; WA: Western Australia. a Data courtesy of the Commonwealth Department of Health, Communicable Diseases Network, Australia, New Zealand National Notifiable Diseases Surveillance System (correct as of September 2004). b Data for 2004 are provisional.

contribute to a rise in RRVD incidence (Kelly-Hope et al., 2004; Gatton et al., in press). Between 1992 and 2004, more than 45,000 notifications of RRV infection were recorded by the National Notifiable Diseases Surveillance System (Table 2). Generally, the prevalence of RRV infection is higher in northern Australia than in the south. One interesting feature of notifications in the last 10 years is the spread of RRV into urban areas. Locally acquired cases have been detected in Perth (Lindsay et al., 1992, 1996), Brisbane (Ritchie et al., 1997), Sydney (Amin et al., 1998), Adelaide (Selden & Cameron, 1996), Melbourne, and Hobart (Russell, 2000). There are many contributing factors involved in this changing pattern of virus distribution, including increasing urbanisation, changes in agricultural practices, and expanding residential and industrial developments in coastal areas, all of which increase the exposure risk of humans to infected mosquitoes (Kay et al., 1996; Russell, 1998; Ryan et al., 2001; Tong, 2004; Tong et al., 2000).

2. Ross River virus disease

vigilax mosquitoes near the Ross River at Townsville, north Queensland (Harley et al., 2001). In 1979, there was a large epidemic involving Fiji, the Cook Islands, and Samoa, with more than 60,000 cases reported (Aaskov et al., 1981; Harley et al., 2001). Since then, the virus has been isolated from patients all over Australia. 2.2. Clinical aspects of Ross River virus disease There are 3 major characteristics of RRVD, namely, rheumatic symptoms, rash, and constitutional effects, such as myalgia, low-grade fever, fatigue, and headache (Harley et al., 2001). The onset of disease is rather sudden, with the first symptom usually being joint pain (arthralgia) involving the wrists, knees, ankles, fingers, elbows, toes and tarsal joints (Johnston & Peters, 1996; Dalgarno & Marshall, 1999; Bossingham et al., 2002). These manifestations can range from tenderness with minor restriction on movement to severe redness and swelling. Rash (maculopapular, vesicular, or purpuric) is observed in 50 –75% of patients and affects mainly the torso and the limbs, but usually does not last for more than 10 days (Fraser, 1986; Dalgarno & Marshall, 1999). Myalgia (muscle pain) affects around 60% of patients. An infected individual experiences pain on movement and, in some cases, is unable to perform simple tasks such as lifting a cup (Johnston & Peters, 1996). Fatigue is the most consistent constitutional effect and is independent of any other manifestation (Dalgarno & Marshall, 1999). Fever is also very common and does not necessarily occur at the onset of symptoms (Harley et al., 2001). The reported duration of the disease varies in the literature, with conflicting survey data reporting symptoms persisting from 3 months to a year or more (Fraser, 1986; Condon & Rous, 1995; Westley-Wise et al., 1996). However, recent studies (Harley et al., 2002; Mylonas et al., 2002) have shown that the disease usually resolves within 3 to 6 months, and that additional conditions are usually responsible for the ongoing chronic symptoms observed in some patients. In monetary terms, the direct cost to the community estimated by Mylonas et al. (2002) was ¨ $A1018 per patient per annum, with diagnostic costs and lost productivity contributing most to this cost. In Australia, up to 8000 cases of RRVD are reported annually, however, the incidence of cases is decreasing, especially in urban areas, due to better mosquito control.

2.1. Historical perspective of the disease 2.3. Laboratory diagnosis Ross River virus infection The first reports of an unusual epidemic appeared in 1928, in Narrandera and Hay, New South Wales, and described a condition of temporary arthritis and rash (Harley et al., 2001). Fifteen years later, similar symptoms were reported in the Northern Territory, Queensland, and the northern coast of Papua New Guinea. In 1946, the disease was named Fepidemic polyarthritis_ (EPA) because of the general arthritis observed in patients (Harley et al., 2001). The causative agent was first isolated from a pool of A.

Diagnosis is usually made serologically. The reliability of serodiagnosis depends on the antibody response and the sensitivity and specificity of the test used. Techniques used in the past included haemagglutination inhibition (HI) and neutralisation (NT). NT is more sensitive and type specific, but it requires the use of live virus and considerable technical skill to perform. It is also relatively time consuming, taking 3 days to perform in the case of RRV.

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HI is less time consuming to perform and also detects antibodies directed against other closely related alphaviruses (e.g., BFV). Both of these techniques detect total antibody, although both can be modified to distinguish IgG from IgM. Another less commonly used technique is complement fixation (CF). This technique can be useful in arboviral serology, as the antibody used is produced later in an infection than NT and HI, is relatively type specific, and short lived. With the advent of ELISA technology came the ability to easily determine specific IgG and IgM antibodies accurately, making it easier to distinguish recent infections from infections in the distant past. In many infectious diseases, IgM is used as a marker of recent infection, as it is believed to be type specific and short lived (8 to 12 weeks). An IgG seropositive result alone does not indicate a recent infection for people living in endemic areas (parts of Queensland, Australia) where up to 30% of the population are RRV IgM /IgG+ due to past asymptomatic and symptomatic infections (Ryan et al., 2003). Paired serology obtained from samples taken around 2 weeks apart showing immunoglobulin change from IgM+/IgG to IgM+/IgG+, IgM /IgG to IgM+/IgG+, or IgM+/IgG to IgM /IgG+ are indicative of recent infection. Persistent IgM+ (i.e., paired tests both IgM+/IgG+) does not represent a reliable serodiagnosis, as such, data are occasionally found in both asymptomatic seroconversions and healthy individuals with past RRVD (Suhrbier & Linn, 2004). In addition, serology results should not be used in isolation, and the date of onset of symptoms, the clinical presentation, and the travel history of a patient should also be considered.

3. Dissecting the immunopathology of Ross River virus infection The rheumatic synovial exudates from EPA patients are predominantly composed of monocytes, vacuolated and phagocytic macrophages, T-cells, B-cells, and some natural killer (NK) cells (Linn et al., 1996; Flexman et al., 1998;

333

Soden et al., 2000). High numbers of CD4+ T-cells are found in mononuclear synovial effusions of patients with RRVD (Fraser, 1984). Unlike other arthritic conditions, such as rheumatoid arthritis (RA), neutrophils only appear episodically and in low numbers (Soden et al., 2000). Immune complexes, which are usually involved in the pathogenesis of viral arthritis, are not found in the serum or synovial fluid of EPA patients (Harley et al., 2001). A soluble mediator, such as interferon (IFN)-g, produced by T lymphocytes is elevated in synovial effusions of patients with RRVD (Cunningham, personal communication). The role that these cellular subsets and host soluble protein/s play in RRVD is not clear. To dissect the mechanism/s of disease, studies in a small animal model for RRV infection will advance our understanding of the pathophysiology associated with RRV infection, as well as the mechanisms that contribute to immunity and disease pathogenesis. 3.1. Mouse model of Ross River virus disease Although small animal models exist for studying alphavirus pathogenesis, these models have almost exclusively focused on virus-induced neurologic disease, and relatively little is known about the pathogenesis of alphavirus-induced arthritides. Early studies with RRV infection in mice utilized neonatal animals, where the virus exhibited a virulent replication pattern involving a number of tissues, including skeletal muscle, periosteum, and brown fat (Mims et al., 1973; Murphy et al., 1973). However, these mouse infections did not appear to provide a model for studying virus-induced arthritis/arthralgia. In a study by Lidbury et al. (2000), older mice (14 to 21 days old) infected with RRV developed hind limb dysfunction characterized by limb weakness, muscle wasting, and mononuclear infiltrates into the striated skeletal muscle (Lidbury et al., 2000; Fig. 1). Further analysis demonstrated that these inflammatory cells were monocytes/macrophages, and the administration of macrophage toxic agents to the infected mice significantly reduced the hind limb dysfunction (Lidbury et al., 2000). Additional studies have

Fig. 1. Pathological differences between normal and RRV-infected tissue. The infected tissue exhibits a massive monocytic infiltration (brown stained cells) and an abnormal morphology. There is a general cell lysis, and muscle fibers are completely destroyed. The mechanism of tissue damage by macrophages is not known. The secretion of toxic factors and induction of apoptosis (cell death) may be involved. Reproduced with permission from Lidbury et al. (2000). Macrophage-induced muscle pathology results in morbidity and mortality for Ross River virus-infected mice (Lidbury et al., 2000).

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demonstrated that following subcutaneous inoculation, RRV was readily detectable in the synovial joints of infected mice by plaque assay as early as 12 hr post-infection (M.T. Heise, unpublished data; S. Mahalingam, unpublished data). The virus was readily detectable in multiple joint associated tissues, including the ligaments and periosteum. In addition, RRV infection also induced inflammatory infiltrates into the infected joints (M.T. Heise, unpublished data; S. Mahalingam, unpublished data). These findings are consistent with observations seen in RRV patients, in particular the detection of RRV antigen in joint effusions of patients with chronic joint symptoms and the associated joint inflammation. 3.2. Macrophages in Ross River virus disease Although the pathogenesis of RRVD is not fully understood, there is sufficient evidence to implicate macrophages in the pathogenesis of the disease. RRV can persist in macrophages for long periods after infection (Way et al., 2002), suggesting a role for macrophages in the maintenance of infectious virus. Early studies of RRVD in mice noted the presence of a monocytic infiltration into various tissues but failed to associate this with the observed necrosis of muscle tissue (Mims et al., 1973; Murphy et al., 1973; Seay et al., 1981). It was then suggested that myositis was due to viral lysis of muscle cells and a possible role for immune-mediated pathology was disregarded (Seay et al., 1981). In opposition to a cytolytic hypothesis of muscle damage was the finding that mouse muscle cell cultures showed virus production for a period of 42 days postinfection, with little or no cytophatic effect (CPE) detected (Eaton & Hapel, 1976). The discrepancy between these later findings and the total muscle destruction observed in vivo by Murphy et al. (1973) suggested a host-mediated response as the cause of pathological changes in muscle tissue. Lidbury et al. (2000) finally confirmed macrophages as the cellular perpetrator of muscle damage in mice. They showed that at the height of clinical disease, the predominant muscle-infiltrating cells were positive for F4/80, a cell surface marker for certain subpopulations of monocytes and macrophages. More importantly, they reported that treatment of mice with silica or carrageenan (macrophage-toxic agents) prior to infection completely abrogated disease symptoms. Moreover, it was also found that virus titres were either falling or not detected at the peak of clinical disease and that, as reported by early studies, the gradual clearance of macrophages from damaged tissue was a feature associated with recovery from disease symptoms (Lidbury et al., 2000). 3.3. The role for soluble mediators in Ross River virus disease The observation that monocytes/macrophages are involved in RRV muscle pathology of mice suggests a role

for chemokines in RRVD (Lidbury et al., 2000; Mahalingam et al., 2003; Lidbury & Mahalingam, 2004). Chemokines are major regulators of leukocyte traffic, as they selectively and specifically control the adhesion, chemotaxis, and activation of many types of leukocyte subpopulations. Compelling evidence came from an in vitro study by Mateo et al. (2000), which showed the up-regulation of CXC chemokine ligand (CXCL) 8 (IL-8) and CCL2 (monocyte chemoattractant protein [MCP-1]) following RRV infection of human fibroblasts. CC chemokine ligand (CCL) 2 is a potent chemoattractant and activating agent for monocytes and macrophages (Jiang et al., 1992), and its overexpression by RRV-infected cells may explain the observed monocytic infiltration in vivo. Mateo et al. (2000) also observed altered chemokine expression in acute and chronically infected murine macrophages (RAW 264.7). Acute infection of naive RAW 264.7 cells showed elevated levels of CXCL1 (KC) and CXCL8, whereas chronically infected macrophages showed overexpression of CXCL1, CXCL8, CXCL10 (IP10), and CCL2. This differential chemokine expression in acutely and chronically infected cells is in agreement with the observation of EPA being episodic in some patients. RRV may persist in the joints of patients through periods of clinical symptoms and subsequently re-infect naive macrophages, repeating a cycle of local chemokine/cytokine activity correlating with disease (Soden et al., 2000). Further studies in our laboratory have found that several chemokines were overexpressed in the muscle tissue of RRVinfected mice (S. Mahalingam, unpublished data). It was found that CCL2, CCL5 (regulated on activation normal Tcell expressed and secreted [RANTES]), and CXCL10 were up-regulated on days 5, 8, and 11 postinfection. This timing correlates with a massive mononuclear infiltrate and associated muscle damage (Murphy et al., 1973; Seay et al., 1981; Lidbury et al., 2000; Fig. 2). Chemokines can also play an important role in the resolution of viral infections. They can inhibit viral infection by blocking the receptor used by the virus to infect cells, activate cytotoxic functions, and, more importantly, trigger the recruitment of activated leukocytes to the site of infection to mediate viral clearance (Mahalingam et al., 2001). Chemokines and their receptors, however, have also been found to mediate the pathogenesis of many viral infections, such as human immunodeficiency virus (HIV; Feng et al., 1996), human cytomegalovirus (HCMV; Craigen et al., 1997), and hepatitis B virus (Kakimi et al., 2001). A disease model of potential relevance to the study of RRV pathogenesis in humans is the manipulation of the chemokine system by caprine arthritis encephalitis virus (CAEV). CAEV is a lentivirus that infects goats, and, like RRV, CAEV can establish a persistent infection in macrophages, which chronically infiltrate various tissues, particularly radiocarpal joints, resulting in the development of arthritis (Lechner et al., 1997a). Lechner et al. (1997b) found that CAEV infection leads to the overexpression of CXCL8 and CCL2. Elevated levels of CCL2 and CCL5

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335

Endothelium Muscle Tissue Blood Vessel Monocytes Key RRV Antibody-RRV complex Chemokines (e.g. CCL2

and

Macrophages

CCL5

Fig. 2. Proposed model for RRV mechanism of disease. When resident macrophages are infected with RRV, they secrete CCL2, CCL3, and CCL5. These chemokines mediate the migration of more macrophages to the site of inflammation, where they are susceptible to infection. RRV can also complex with specific antibodies and infect monocytes and macrophages by binding to the Fc receptors on the surface of these cells. This results in a further increase in the amount of chemokines secreted. Activated macrophages can also secrete factors such as reactive oxygen and nitrogen intermediates that have the potential to damage tissue.

have also been detected in the synovial tissues of rheumatoid arthritis (RA) patients. The presence of these chemokines could explain the high numbers of macrophages and T lymphocytes found in RA synovial tissues. IFN-g may also be involved in RRVD, as the cytokine has been detected in the synovial exudate from patients (T. Cunningham, personal communication). In addition, IFNg-secreting RRV-specific T-cells have been isolated from humans (A. Suhrbier, unpublished data) and mice (Linn et al., 1998). Furthermore, we have also shown that mice depleted of IFN-g did not develop hind limb dysfunction characterized by limb weakness and muscle wasting (S. Mahalingam, unpublished data; M. Heise, unpublished data). IFN-g is known to mediate differentiation of monocytes to macrophages as well as their activation. It has also been demonstrated that this activation process may include the up-regulation of a collagen IV-binding receptor (a1h1 integrin), which was recently identified as a cellular receptor for RRV (A. Suhrbier, unpublished data). 3.4. T-cells in Ross River virus disease Both CD4+ T-cell and CD8+ T-cell responses are critical in the resolution of primary viral infections. In particular, CD8+ T-cells play a primary role by directly killing infected cells through the induction of cytolytic mechanisms and the production of antiviral factors (Kagi & Hengartner, 1996). Impaired CD8+ T-cell activity has been associated with

ineffective clearance and persistence of a number of Togaviruses and Lentiviruses, including caprine arthritis virus (Lichtensteiger et al., 1993), measles virus (Niewiesk et al., 1993), and rubella virus (Verder et al., 1986). With regards to RRVD, CD8+ T-cells predominate in the skin of EPA patients who recover rapidly from the rash (Fraser, 1984). In contrast, CD4+ T-cells are most numerous in the synovial effusions of chronic patients (Fraser, 1984). Interestingly, CD8+ T-cells raised by the vaccination of mice with RRV capsid proteins are capable of completely clearing infection from persistent and productively infected RAW 264.7 macrophages in vitro (Linn et al., 1998). This suggests that defective cell mediated immunity may be, in part, responsible for viral persistence and the development of chronic athralgia in EPA patients (Fraser, 1986). In accordance with this is the observation that macrophages infected with RRV in vitro showed a deregulation of CD80 costimulatory molecule (Way et al., 2002). On the surface of macrophages, CD80 interacts with CD28 on T helper cells and cytotoxic T-lymphocyte-associated-4 (CTLA-4) on CTL cells, and this interaction is crucial for the proper antigen presentation and activation of CTL. Way et al. (2002), working with murine macrophages, showed that in persistent infections, CD80 is expressed below the basal level found in noninfected cells. Moreover, it has been shown that blocking CD80 in mixed lymphocyte reaction cultures results in the generation of alternative activated macrophages (AAM), which suppress T-cell responses (Tzachanis et al., 2002). AAM display an enhanced capacity

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for phagocytosis but decreased capacity for nitric oxide (NO) and O2 production and reduced antigen presentation capability (Tzachanis et al., 2002). Interestingly, macrophages isolated from synovial exudates of EPA patients also show an enhanced phagocytic capability (Clarris et al., 1975; Fraser, 1986). Moreover, antiinflammatory agents such as interleukin (IL)-10 induce alternative activation of macrophages. This cytokine has been found to be overexpressed in macrophages during acute antibody-dependent RRV infection (Mahalingam & Lidbury, 2002).

4. Persistence of Ross River virus disease 4.1. Viral persistence The first report of persistent RRV infection was by Eaton and Hapel (1976), who performed experimental analyses of RRV in mouse muscle cells and found continued virus production up to 42 days postinfection. Similar viral persistence has also been reported for RRV infection of human synovial fibroblasts (Journeaux et al., 1987) and, since then, has also been identified by plaque assay in in vitro-infected cultures of macrophages (Lidbury et al., 2000). In addition, virus was also found to persist in macrophage cultures in vitro in the presence of neutralising antibodies (Linn et al., 1998). More sensitive molecular approaches, such as reverse transcriptase polymerase chain reaction (RT-PCR), have allowed for detection of virus in the synovial tissue of RRVD patients up to 5 weeks following the onset of disease (Soden et al., 2000). This study showed that RRV persisted despite the presence of neutralising antibodies. A more recent study by Way et al. (2002), examining RRV persistence in macrophages, revealed that virus persisted for as long as 170 days. RRV may remain inactive until the immune system is susceptible to infection or has become weakened by some factor, before inducing its infectious ability. In support of this, Way et al. (2002) have demonstrated that following evident biological clearance of RRV infection (but unaltered genetic presence, as shown by the detection of RRV RNA), relapse may occur either spontaneously or in response to stress. 4.2. Mechanisms of persistence Persistent RRV infection has, to date, been reported only in macrophages and not in monocytes, however, the mechanism responsible for this phenomenon is not entirely understood (Linn et al., 1996). Alphavirus infections are very sensitive to the antiviral effects of type I IFNs, and these factors are known to limit viral replication (Mahalingam & Lidbury, 2002). Despite the presence of antibodies and IFNs, RRV can persist in the host. Several possible mechanisms have recently been suggested to explain the persistent nature of RRV: (1) the

ability of RRV to inhibit antiviral activity via the induction of IL-10 (an immunosuppressive cytokine) in macrophages when infection occurs via the antibody dependent enhancement pathway (see below; Mahalingam & Lidbury, 2002); (2) the ability of alphaviruses to trigger apoptosis of cells (Li & Stollar, 2004), and the subsequent phagocytosis of these cells by macrophages might allow virus to infect phagocytes via the phagosome. This route of infection prevents contact between the virus and the circulating antibodies; (3) the presence of viral particles localized within intracellular vesicles during prolonged infection (as reported by Way and colleagues), which may offer protection to the virus, to allow evasion of the host immune response (Way et al., 2002); and (4) the possibility that RRV might induce the expression of the antiapoptotic protein Bcl-2 in a small percentage of macrophages and thus allow virus persistence in surviving cells. This possibility is supported by a recent study using a virulent strain of the alphavirus Sindbis virus (SV), which also infects cells in a persistent manner. SV has been found to induce the expression of the antiapoptotic protein Bcl-2 in glial cells (Appel et al., 2000). Studies are currently in progress to elucidate whether RRV infection could modulate Bcl-2 expression. Considering these various possibilities, it is reasonable to speculate that viral persistence may contribute to the chronic infection cycle and disease phases of RRV.

5. Antibody-dependent enhancement of infection The majority of viral infections in humans and animals are not fatal and are followed by recovery and a state of resistance against re-infection with the same or, in some cases, serologically related viruses. Much of this resistance is attributable to the development of memory B-cells, which are able to rapidly secrete highly specific antibodies when the individual or animal encounters the same pathogen. Normally, these specific antibodies reduce or neutralise viral infectivity. However, some antiviral antibodies at low or subneutralising levels might potentiate or enhance viral infectivity, resulting in what is known as antibody-dependent enhancement of infectivity (ADE). ADE has been reported in vitro for numerous virus families and genera, many of which are of increasing importance with regard to public health (Halstead & O’Rourke, 1977; Ochiai et al., 1992; Amor et al., 1996; Ponnuraj et al., 2003; Takada et al., 2003). For example, the presence of antibodies raised against any one of the dengue virus, is considered a risk factor in the development of dengue hemorrhagic fever and dengue shock syndrome, two life-threatening conditions (Kliks et al., 1989; Morens & Halstead, 1990). The mechanism of enhanced infection is believed to involve increased virus uptake into cells via Fc-g receptors (Halstead & O’Rourke, 1977; Peiris & Porterfield, 1979; Peiris et al., 1981) when

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the virus is bound by nonneutralising IgG antibody, leading to increased virus titres (Morens & Halstead, 1990). An alternative hypothesis has recently been proposed suggesting that FcR engagement causes a suppression of antiviral responses leading to increased virus replication (Mahalingam & Lidbury, 2002; Suhrbier & Linn, 2003; discussed in Section 5.2). The mechanism(s) of ADE infection needs to be further explored to understand how the ADE infection pathway might suppress the host cell’s innate antiviral responses. 5.1. Antibody-dependent enhancement of Ross River virus infection ADE was first described for RRV by Linn et al. (1996). They used RRV complexed with serial dilutions of anti-RRV serum to infect human and murine monocyte/macrophage cell lines. In vitro RRV infection appears to be largely restricted to adherent cells, suggesting that the natural receptor for RRV may be a protein used by cells to adhere to the extracellular matrix (White, 1993). In this regard, adherent RAW 264.7 cells could be infected by RRV, presumably through its unidentified natural receptor, whereas the nonadherent Mono Mac 6 cells could not (Linn et al., 1996; Zielgler-Heitbrock et al., 1988). However, subneutralising levels of anti-RRV IgG antibody dramatically increased the infection of Mono Mac 6 cells. It is crucial to understand that clinical data clearly linking in vitro phenomena of ADE with disease enhancement is missing even for dengue, where the ADE concept is widely held to be relevant. ADE remains a theoretical concept, and a clear correlate between ADE in vitro and ADE-mediated immunopathogenesis in vivo has proven elusive. Therefore, it should be noted that no clear association could be established between preexisting antibodies capable of mediating ADE prior to RRV infection and symptomatic RRV infection (Linn et al., 1996). However, this does not preclude a role for antibodies raised during the primary infection in being able to mediate ADE and somehow contribute to disease pathogenesis. 5.2. Suppression of antiviral pathways in antibodydependent enhancement of Ross River virus infection New insights into the mechanism of viral ADE infection came from studies of RRV infection of macrophages in vitro. Studies in our laboratories have found that ADE – RRV infection specifically ablated the production of two antiviral mediators, tumor necrosis factor (TNF)-a and inducible nitric oxide synthase (NOS2) in response to lipopolysaccharide (LPS; Lidbury & Mahalingam, 2000). LPS is a major component of the outer membrane of Gramnegative bacteria that induces innate immunity and the expression of antimicrobial cytokines such as IFN-a/h,

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IFN-g, TNF-a and molecules such as nitric oxide (Adams & Hamilton, 1984; Palsson-McDermott & O’Neill, 2004). The RRV-mediated disruption of gene expression was specific for antiviral genes, and non-ADE-infected macrophages showed a normal increase in TNF-a and NO production upon LPS treatment. This demonstrated that ADE-mediated RRV infection allowed the virus to dysregulate the host’s antiviral response and thereby promoted the viruses’ ability to replicate. The observed RRV – ADE-mediated down-regulation of antiviral genes triggered the search for viral dysregulation of downstream transcription factors. LPS induction of antiviral genes is mediated by transcription factors signal transducer and activator of transcription-1 (STAT), nuclear factor-kappa B (NF-nB), and interferon regulatory factor-1 (IRF-1; Gao et al., 1998). NF-nB and IRF-1 can be directly activated by LPS, and the activation of STAT-1 is mediated by LPS-induced type I IFNs (Gao et al., 1998; Jacobs & Ignarro, 2001; Fig. 3). A second study in our laboratory focused on the activity of NF-nB, AAF (STAT homodimer), and ISGF3 (STAT heterodimer), as well as the expression of TNF-a, NOS2 (nitric oxide synthase), IFN inducible protein 10 (IP-10), IRF-1, and IL-10 (interleukin 10) in RRV – ADE-infected murine macrophages (Mahalingam & Lidbury, 2002). It was found that ADE infection resulted in the suppression of NF-nB, AAF, and ISGF3 activity and, consequently, in the downregulation of the above mentioned cytokines and host factors. In addition, it was also reported that, although only 42% of macrophages were ADE infected, there was a global suppression of cellular antiviral genes and proteins. These findings raised the question of whether virally induced cellular proteins are involved in the antiviral activity of neighboring uninfected macrophages. Interestingly, IL-10 was found to be elevated in RRV –ADEinfected macrophage cultures (Mahalingam & Lidbury, 2002). IL-10 is a potent immunosuppressive and antiinflammatory molecule that can inhibit the production of inflammatory cytokines such as IL-12, IFN-g, and TNF-a (Tone et al., 2000). Elevated levels of IL-10 have also been reported for Sindbis virus and Venezuelan equine encephalitis virus infections of mice (both alphaviruses; Grieder et al., 1997; Rowell & Griffin, 1999) and correlate with fatal outcomes of Ebola and dengue virus infections (Green et al., 1999; Baize et al., 2002). It is possible that the inhibition of antiviral genes and suppression of inflammatory responses through the manipulation of cellular proteins in RRV – ADE infection of cells, might contribute to viral persistence. It is obvious that the observed ADE in RRV infection represents a problem for the development of a protective vaccine intended to generate a substantial antibody response. Currently, there is no vaccine against RRV and related viruses. Although a correlation between preexisting antibodies against RRV and severe manifestation of EPA has not been reported, the ADE phenomenon cannot be

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N.E. Rulli et al. / Pharmacology & Therapeutics 107 (2005) 329 – 342 ADE-mediated suppression LPS-induced antiviral response

of antiviral response

LPS

TLR-4

Fc R

Viral entry and replication NF-κB

IFN-α/β α/β R

STAT-1

IRF-3

IFN-α/β α/β R

NF-κB

STAT-1

Sp-1

IRF-1 iNOS IFN-β ISRE

GAS

Sp-1 ISRE

GAS

IFN-α/β TNF-α IP-10 NO

ISRE

κB

SOCS-3 ISRE

κB

SOCS-3

IL-10 R

Key to Diagram: IFN- α/β

RRV

IL-10

LPS

Suppresses/downregulates

Fig. 3. A model for the suppression of antiviral cytokines by RRV-ADE infection in murine macrophages upon LPS induction. LPS activates a toll-like receptor (TLR4), which causes the activation of NF-nB and IRF-3. The genes encoding type I IFNs are transcribed and translated, and the secreted products bind to the interferon receptor (IFN-a/hR), which results in STAT-1 phosphorylation. STAT-1 promotes the transcription of IRF-1 and antiviral cytokines. NF-nB, IRF-1, and STAT-1 (AAF and ISGF3 dimers) act in concert to promote the expression of IFN-a/h, TNF-a, iNOS, IP-10, and IRF-1. RRV-ADE infection and replication results in the suppression of NF-nB, IRF-1, and STAT-1, and in the up-regulation of IL-10. The latter suppresses the kinases that activate STAT-1 and therefore inhibits signal transduction by IFNs. This model is based on studies from Mahalingam and Lidury (2002), Gao et al. (1998), Jacobs and Ignarro (2001). Key: ISRE, interferon-stimulated response element; NF-nB, nuclear factor kappa B element; GAS, IFN-g activation sequence; SOCS3, suppressor of cytokine signaling.

disregarded in the development of a vaccine for RRV and other RNA viruses.

6. Treatment and prevention A longitudinal prospective study of 67 RRVD patients showed that conventional treatment of RRVD was largely restricted to aspirin or paracetamol, taken by 15% of patients, and nonsteroidal antiinflammatory drugs (NSAIDs), used by 58% of patients at disease onset. NSAIDs were taken for an average of 7.6 weeks (SD = 5.9; range, 2 –22 weeks), with 70 –100% of patients reporting being satisfied with the treatment (Mylonas et al., 2002). Several NSAIDs may need to be tried before effective relief is obtained without excessive side effects. No significant differences in effectiveness were identified between the different NSAIDs or the

different classes of NSAIDs used in the study described above (unpublished observations). RRVD is currently also being treated with the new generation of Cox-2-selective NSAIDs, which promised reduced gastrointestinal (GI) side effects. However, recent evidence suggests that these drugs may not be as GI-safe as first believed (Malhotra et al., 2004). Corticosteriods are occasionally used for RRVD (4% of patients in the above study) but are probably not generally justified, although incidental findings in this small cohort found no evidence of steroid-induced disease exacerbation (Mylonas et al., in press). The inflammatory process of RRVD may involve the activation of toll-like receptor 3 and/or dsRNA-dependent protein kinase (PKR) by viral dsRNA (S. Mahalingam, unpublished data). Alphaviruses efficiently amplify their RNA (Leitner et al., 2003), and experimental arthritis induced by intraarticular injection of dsRNA shares several

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features associated with RRV arthritogenesis, primarily monocytic infiltrates and induction of the chemokines CCL2 and CXCL2 (Mateo et al., 2000; Zare et al., 2004). A number of new antiinflammatory drugs that block NF-nB and p38 mitogen-activated protein kinase (O’Neill, 2003), both transcription factors activated by dsRNA, are currently in clinical development. These new drugs may prove to be effective for the treatment of viral arthritides like RRVD. A number of antiviral compounds, including ribavirin, Ampligen, and interferons, have been shown to be effective in reducing alphaviral viraemias in murine systems (Sidwell & Smee, 2003). However, proinflammatory compounds that simply induce antiviral resistance (such as Ampligen and interferons) are unlikely to be useful in treating inflammatory viral arthritides. Whether ribavirin would be effective against arthritides caused by RNA viruses remains an open question (Olivieri et al., 2003). The inflammatory cytokine TNF-a has been successfully targeted by anti-TNF-a therapy in the treatment of autoimmune arthritides and reactive arthritis (Meador et al., 2002). Whether such treatments would also be viable for RRVD is unclear, as the disease is self-limiting and steadily improves over the average 3- to 6-month course of the disease (Mylonas et al., 2002). Furthermore, the presence of TNF-a in inflamed joints has yet to be clearly implicated as a critical component of viral arthritides, although TNF-a RNA is detected in caprine lentivirus-induced arthritis (Lechner et al., 1997b). Macrophages persistently infected with RRV in vitro do not secrete detectable TNF-a (with the presence of such macrophages in the synovial infiltrate thought to contribute to arthritogenesis; Suhrbier & Linn, 2004). The chemokines (CCL2, CXCL2, and IL-8), which are secreted by such macrophages, may prove to be more suitable as targets for therapy, and several chemokine agonists have proved effective in preventing inflammatory arthritides in murine systems (Gong et al., 2004; Maurer & Von Stebut, 2004; S. Mahalingam, unpublished data). A number of viruses are known to target macrophages and trigger IL-10 expression, as well as produce IL-10 homologues to suppress the host immune responses and avoid elimination from the host (Redpath et al., 2001). The model for ADE described earlier raises the question of possible targets for therapies. Clearly, from these, elevated IL-10 levels have been shown to correlate with enhanced virus production. Interestingly, fatal outcomes in Ebola infections (Baize et al., 2002) and disease severity in dengue infections have been associated with elevated levels of IL10 (Green et al., 1999). Therefore, the development of molecules/compounds that can dampen IL-10 activity may have potential therapeutic benefits.

7. Conclusion It is clear that virus – host interactions are exceedingly complex, even for small genome RNA viruses like RRV. The

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recognition that such viral agents have the ability to manipulate the fundamental molecular and biochemical processes that underpin cellular defences may eventually lead to a complete understanding of the basic biology of infection and its impact on host immunology. Translating such basic findings into effective therapies with few side effects remains a pharmacological goal for most viral infections. An additional challenge for geographically localized disease agents like RRV is that small market size may reduce the enthusiasm of pharmaceutical companies to invest in the necessary research, clinical trials, and regulatory clearances required to bring a new drug to the market place. These concerns do not need to be barriers, and excellent basic research should assess the relevance of discoveries relating to specific viruses to other virus families of more global health significance. Reassessment of existing drug therapies in light of the growing abundance of knowledge about both virus and host molecules influenced by infection may result in the new applications of these drugs. RRV provides an excellent model to perform basic research into viral disease involving the perturbation of inflammatory and/or early innate immune responses. As detailed by the literature cited in this review, we already have excellent animal and cell culture-based models with which to explore the pathogenesis of RRV infection. A feature of these models is that RRV-mediated disease shows phases of preclinical virus amplification and disease associated with virus clearance and recovery, demonstrating the spectrum of host responses to virus infection. Such a spectrum provides a basis for testing both new and established drug protocols. RRV can thus be commended to pharmaceutical interests as an avenue to gain insight into novel drug solutions to viral disease.

Acknowledgments This work was supported by grants from the Australian National Health and Medical Research Council (NHMRC) and the Clive and Vera Ramaciotti Foundation (to SM). SM is also the recipient of the Australian NHMRC R. Douglas Wright Fellowship.

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