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Characterization of Ross River Virus Tropism and Virus-Induced Inflammation in a Mouse Model of Viral Arthritis and Myositis

Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Journal of Virology (Impact Factor: 4.65). 02/2006; 80(2):737-49. DOI: 10.1128/JVI.80.2.737-749.2006
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ABSTRACT Mosquito-borne alphaviruses are a significant cause of both encephalitic and arthritic disease in humans worldwide. In contrast to the encephalitic alphaviruses, the pathogenesis of alphavirus-induced arthritic disease is not well understood. Utilizing a mouse model of Ross River virus (RRV) disease, we found that the primary targets of RRV infection are bone, joint, and skeletal muscle tissues of the hind limbs in both outbred CD-1 mice and adult C57BL/6J mice. Moreover, histological analyses demonstrated that RRV infection resulted in severe inflammation of these tissues. Characterization of the inflammatory infiltrate within the skeletal muscle tissue identified inflammatory macrophages, NK cells, and CD4+ and CD8+ T lymphocytes. To determine the contribution of the adaptive immune system, the outcome of RRV-induced disease was examined in C57BL/6J RAG-1(-/-) mice, which lack functional T and B lymphocytes. RAG-1(-/-) and wild-type mice developed similar disease signs, infiltration of inflammatory macrophages and NK cells, and muscle pathology, suggesting that the adaptive immune response does not play a critical role in the development of disease. These results establish the mouse model of RRV disease as a useful system for the identification of viral and host factors that contribute to alphavirus-induced arthritis and myositis.

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We first developed SINV IgM and IgG enzyme immunoassays (EIA), based on highly purified SINV, to be used in serodiagnostics. The EIAs correlated well with the hemagglutination inhibition (HI) test, and all individuals showed neutralizing antibodies. The sensitivities of the IgM and IgG EIAs were 97.6% and 100%, and specificities 95.2% and 97.6%, respectively. E1 and E2 envelope glycoproteins of SINV were shown to be recognized by IgM and IgG in the immunoblot early in infection. We isolated SINV from five patients with acute Pogosta disease; one virus strain was recovered from whole blood, and four other strains from skin lesions. The etiology of Pogosta disease was confirmed by these first Finnish SINV strains, also representing the first human SINV isolates from Europe. Phylogenetic analysis indicated that the Finnish SINV strains clustered with the strains previously isolated from mosquitoes in Sweden and Russia, and seemed to have a common ancestor with South-African strains. Northern European SINV strains could be maintained locally in disease-endemic regions, but the phylogenetic analysis also suggests that redistribution of SINV tends to occur in a longitudinal direction, possibly with migratory birds. We searched for SINV antibodies in resident grouse (N=621), whose population crashes have previously coincided with human SINV outbreaks, and in migratory birds (N=836). SINV HI antibodies were found for the first time in birds during their spring migration to Northern Europe, from three individuals: red-backed shrike, robin, and song thrush. Of the grouse, 27.4% were seropositive in 2003, one year after a human outbreak, but only 1.4% of the grouse were seropositive in 2004. Thus, grouse might contribute to the human epidemiology of SINV. A total of 86 patients with verified SINV infection were recruited to the study in 2002. SINV RNA detection or virus isolation from blood and/or skin lesions was successful in eight patients. IgM antibodies became detectable within the first eight days of illness, and IgG within 11 days. The acute phase of Pogosta disease was characterized by arthritis, itching rash, fatigue, mild fever, headache, and muscle pain. Half of the patients reported in self-administered questionnaires joint symptoms to last > 12 months. Physical examination in 49 of these patients three years after infection revealed persistent joint manifestations. Arthritis (swelling and tenderness in physical examination) was diagnosed in 4.1% (2/49) of the patients. Tenderness in palpation or in movement of a joint was found in 14.3% of the patients in the rheumatologic examination, and additional 10.2% complained persisting arthralgia at the interview. Thus, 24.5% of the patients had joint manifestations attributable to the infection three years earlier. A positive IgM antibody response persisted in 3/49 of the patients; both two patients with arthritis were in this group. Persistent symptoms of SINV infection might have considerable public health implications in areas with high seroprevalence. The age-standardized seroprevalence of SINV (1999-2003, N=2529) in the human population in Finland was 5.2%. The seroprevalence was high in North Karelia, Kainuu, and Central Ostrobothnia. The incidence was highest in North Karelia. Seroprevalence in men (6.0%) was significantly higher than in women (4.1%), however, the average annualized incidence in the non-epidemic years was higher in women than in men, possibly indicating that infected men are more frequently asymptomatic. The seroprevalence increased with age, reaching 15.4% in persons aged 60-69 years. The incidence was highest in persons aged 50-59 years. Pogostantauti on virusinfektio, joka ilmenee ihmisellä nivel- ja ihottumaoirein. Noin 5 % suomalaisista on sairastanut infektion. Pogostantautia esiintyy syyskesällä, sillä ilmeisesti loppukesän hyttyslajit siirtävät viruksen ihmiseen. Spesifiä hoitoa tai rokotetta tautiin ei ole. Tauti löydettiin vuonna 1974 Ilomantsissa ja potilaiden vasta-ainetutkimukset osoittivat, että taudinaiheuttaja on läheistä sukua Sindbis-virukselle. Sindbis-virusta esiintyy Euraasiassa, Afrikassa ja Oseaniassa, mutta ihmisten tautitapauksia lähinnä vain Pohjois-Euroopassa, etenkin Suomessa. Pogostantauti on puhjennut satojen tai tuhansien potilaiden epidemiaksi täsmälleen seitsemän vuoden välein. Viimeisin epidemia oli syyskesällä 2002, jolloin diagnosoitiin 600 tapausta. Tämä väitöskirjatutkimus pohjautuu suurelta osin vuoden 2002 pogostantautiepidemian aikana koottuun näyte- ja potilasaineistoon. Potilaita seurattiin akuutin vaiheen jälkeen kolmen vuoden ajan. Väitöstutkimuksessa kuvattiin akuutin pogostantaudin tyyppioireiksi nivelkivut/-turvotus, kutiava ihottuma, väsymys, kuume, lihaskivut ja päänsärky. Nämä oireet alkavat usein samanaikaisesti. Tyypillisesti oireilevia niveliä ovat nilkat, polvet, sormet ja ranteet. Tutkimuksessa osoitettiin, että pogostantauti aiheuttaa osalle potilaista vuosikausia kestäviä niveloireita. Kolmen vuoden kuluttua infektiosta 4 %:lla potilaista todettiin krooninen niveltulehdus, ja kaikkiaan 14 %:lla todettiin objektiivisesti havaittavia pitkittyneitä niveloireita. Tutkimuksen mukaan Pohjois-Karjalassa noin 10 % väestöstä on sairastanut pogostantaudin, joten voidaan arvioida, että maakunnan alueella on satoja potilaita, joiden krooninen niveltulehdus johtuu aiemmin saadusta Sindbis-virusinfektiosta. Pogostantaudin mahdollisuus tulisi siis huomioida epäselvien niveltulehdusten erotusdiagnostiikassa. Tutkimuksessa kehitettiin potilasdiagnostiikkaan sopivia vasta-ainetestejä sekä kuvattiin infektion jälkeistä vasta-aineiden kinetiikkaa. Lisäksi työssä eristettiin viisi uutta Sindbis-viruskantaa suoraan akuuttien potilaiden iho- ja verinäytteistä. Kyseessä ovat ensimmäiset ihmisestä eristetyt Sindbis-viruskannat Euroopassa. Viruskantojen sukupuuanalyysi osoitti niiden olevan läheistä sukua Etelä-Afrikan kannoille. Virus saattaakin levitä maailmanlaajuisesti pohjois-eteläsuunnassa muuttolintujen välityksellä. Tutkimuksessa osoitettiin, että pienessä osassa Suomeen palaavista muuttolinnuista on Sindbis-virusvasta-aineita. Lisäksi suomalaisten metsäkanalintujen Sindbis-virusvasta-aineiden esiintyvyydessä havaittiin mahdollinen yhteys pogostantaudin vuosittaiseen esiintyvyyteen. Sekä muuttolinnuilla että vuoden ympäri Suomessa olevilla metsäkanalinnuilla saattaa siis olla rooli pogostantaudin erikoisessa seitsemän vuoden epidemiasyklissä, jonka syytä ei edelleenkään tunneta.
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