Increased Proinflammatory Cytokine Levels in Prolonged Arthralgia in Ross River Virus Infection

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Technical Appendix

Dennis Tappe

, José Vicente Pérez-Girón1, Sergio Gómez-Medina, Stephan Günther, César Muñoz-Fontela, and Jonas Schmidt-Chanasit

Author affiliations: Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany (D. Tappe, S. Günther, C. Muñoz-Fontela, J. Schmidt-Chanasit); German Centre for Infection Research, Hamburg (D. Tappe, S. Günther, C. Muñoz-Fontela, J. Schmidt-Chanasit); Leibniz Institute for Experimental Virology, Hamburg (J.V. Pérez-Girón, S. Gómez-Medina, C. Muñoz-Fontela)

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Abstract

Ross River virus, a mosquitoborne alphavirus, causes epidemic polyarthritis in Australia and the Pacific region. We analyzed serum cytokine, chemokine, and growth factor levels in travelers returning to Germany from Australia. Serum samples showed elevated concentrations in the acute phase of the illness and, more pronounced, in the long-lasting convalescent phase.

Ross River virus (RRV) is a mosquitoborne alphavirus endemic to Australia and the Pacific region. The virus is transmitted by various Aedes and Culex spp. mosquito species; macropods are the natural reservoir (1). RRV causes epidemic polyarthritis, with several thousand cases noted annually in Australia (1). Large outbreaks occurred during 1979–1980 on South Pacific islands. Infections in travelers, however, are rare (2). Epidemic polyarthritis is a self-limiting febrile arthralgia syndrome that closely resembles chikungunya and Mayaro fever. Acute-onset symmetric polyarthritis, most often affecting the fingers, wrists, ankles, and knees, is the predominant clinical presentation. Rash, myalgia, fatigue, and fever are present in half of patients (1). Joint effusions are common, and arthralgia can be long-lasting (months to years), and recurring.

The clinical disease, diagnostic procedures, and epidemiology of RRV have been well described, but immunologic parameters in humans and their usefulness in the clinical follow-up of patients warrant further investigation. Therefore, we investigated travelers returning to Germany from Australia with epidemic polyarthritis resulting from RRV infection.

We analyzed 20 serum samples from 16 patients (7 men, 9 women; age range 20–67 years [median 38 years]) who had serologic evidence for acute or recent RRV infection, as confirmed by indirect immunofluorescence assay and virus neutralization assay (2) (Table). All patients had acquired the infection during travel in Australia. We obtained serum samples at different times after symptom onset (3 days–16 weeks) and classified them as either acute (taken <30 days after symptom onset; n = 7) or convalescent with symptomatic arthralgia (taken >30 days after disease onset; n = 13). After we obtained written consent from the patients, we subjected the serum samples to multiplex cytokine analyses. In parallel with the RRV patients, we tested 20 serum samples from healthy blood donors.

Compared with concentrations in samples from healthy controls, concentrations in samples from patients in the acute phase of RRV infection showed noticeably elevated concentrations of serum interleukin (IL) 4 and 7; granulocyte-macrophage colony stimulating factor (GM-CSF); regulated on activation, normal T cell expressed and secreted (RANTES); interferon-γ–induced protein 10 (IP-10); and vascular endothelial growth factor (VEGF). We also saw a notable decrease in eotaxin levels in acute-phase samples. In samples from patients in the arthralgic convalescent phase, we noted increases in IL-1β, IL-4, IL-6, IL-8, IL-9, IL-13, IL-15, GM-CSF, interferon-γ, tumor necrosis factor-α (TNF-α), RANTES, basic fibroblast growth factor (bFGF), macrophage inflammatory protein 1α (MIP1α), and VEGF in comparison to healthy controls. Substantial elevations in the convalescent phase when compared with the acute phase were recorded for levels of bFGF and MIP1α, and a marked decrease was seen in IP-10 concentrations.

Cytokine and chemokine levels were generally higher in the convalescent phase than in the acute phase, with individual exceptions (Technical Appendix[PDF - 403 KB - 3 pages]). No notable changes in either phase were seen for IL-1RA, IL-2, IL-5, IL-10, IL-12p70, IL-17, granulocyte colony-stimulating factor, monocyte chemotactic protein 1, and platelet-derived growth factor β polypeptide levels (data not shown), and macrophage inflammatory protein 1β concentrations.

Similar to chikungunya and Mayaro fever, the most prominent clinical symptom of RRV infection is long-lasting arthralgia. Nearly 60% of patients reported persisting pain after 2–3 years (1). RRV RNA has been detected in synovial fluid up to 5 weeks after symptom onset (3), suggesting ongoing viral replication and inflammation (4). In the RRV-infected travelers examined in this study, the increased proinflammatory cytokine serum concentrations during the prolonged arthralgic convalescence phase strengthen the hypothesis of persisting inflammation of the joints. In patients and in a murine model, macrophage-derived TNF-α, interferon-γ, and IL-6 were elevated in synovial fluid during RRV disease (5), as we have shown here in serum.

Furthermore, increased levels of IL-1β, IL-6, IL-15, MIP1α, and GM-CSF, as seen in our study, have been described in the clinically similar chikungunya (6). Elevated levels of RANTES (and IP-10 initially) indicate T-cell activation, possibly reflecting ongoing viral replication in the joints, also as described in chikungunya (6). A similar effect could recently be demonstrated in Mayaro fever patients with prolonged arthralgia (7).

In conclusion, cytokine level testing in alphavirus infections with prolonged arthralgia may aid monitoring patient symptoms. This information is particularly valuable when clinical signs of arthritis, such as joint swelling and redness, are no longer present, and standard serum inflammatory parameters are within reference ranges. Low-grade inflammation in persistent alphavirus-induced arthritis might place patients at risk for bone loss and fractures (8). Levels of RANKL (receptor activator of nuclear factor κB ligand), which were not determined in our study, were recently shown to be elevated in RRV infection and associated with increased osteoclast formation (9).

The pattern of cytokine concentration elevations we demonstrated for patients with epidemic polyarthritis is similar to what has recently been described for patients with Mayaro fever (7) and chikungunya (6,10). Our data broaden the knowledge of alphavirus pathogenesis in arthralgia syndromes; however, more immunological investigations, including human T-cell analyses, are needed.

Dr. Tappe is a clinical microbiologist at the National Reference Center for Tropical Diseases, Bernhard Nocht Institute, Hamburg, Germany. His research interests focus on the immune response in viral and rickettsial diseases.

Acknowledgment

We thank Birgit Hüsing, Corinna Thomé, and Alexander Schlaphof for their excellent technical assistance.

References

Harley D, Sleigh A, Ritchie S. Ross River virus transmission, infection, and disease: a cross-disciplinary review. Clin Microbiol Rev. 2001;14:909–32. DOI PubMed
Tappe D, Schmidt-Chanasit J, Ries A, Ziegler U, Müller A, Stich A. Ross River virus infection in a traveller returning from northern Australia. Med Microbiol Immunol (Berl). 2009;198:271–3. DOI PubMed
Soden M, Vasudevan H, Roberts B, Coelen R, Hamlin G, Vasudevan S, et al. Detection of viral ribonucleic acid and histologic analysis of inflamed synovium in Ross River virus infection. Arthritis Rheum. 2000;43:365–9. DOI PubMed
Suhrbier A, La Linn M. Clinical and pathologic aspects of arthritis due to Ross River virus and other alphaviruses. Curr Opin Rheumatol. 2004;16:374–9. DOI PubMed
Lidbury BA, Rulli NE, Suhrbier A, Smith PN, McColl SR, Cunningham AL, et al. Macrophage-derived proinflammatory factors contribute to the development of arthritis and myositis after infection with an arthrogenic alphavirus. J Infect Dis. 2008;197:1585–93. DOI PubMed
Ng LF, Chow A, Sun YJ, Kwek DJ, Lim PL, Dimatatac F, et al. IL-1β, IL-6, and RANTES as biomarkers of chikungunya severity. PLoS One. 2009;4:e4261. DOI PubMed
Tappe D, Pérez-Girón JV, Just-Nübling G, Schuster G, Gómez-Medina S, Günther S, et al. Sustained elevated cytokine levels during recovery phase of Mayaro virus infection. Emerg Infect Dis. 2016;22:750–2. DOI PubMed
Chen W, Foo SS, Sims NA, Herrero LJ, Walsh NC, Mahalingam S. Arthritogenic alphaviruses: new insights into arthritis and bone pathology. Trends Microbiol. 2015;23:35–43. DOI PubMed
Chen W, Foo SS, Rulli NE, Taylor A, Sheng KC, Herrero LJ, et al. Arthritogenic alphaviral infection perturbs osteoblast function and triggers pathologic bone loss. Proc Natl Acad Sci U S A. 2014;111:6040–5. DOI PubMed
Chow A, Her Z, Ong EK, Chen JM, Dimatatac F, Kwek DJ, et al. Persistent arthralgia induced by chikungunya virus infection is associated with interleukin-6 and granulocyte macrophage colony-stimulating factor. J Infect Dis. 2011;203:149–57. DOI PubMed