Development of in house assays for detection of Sindbis virus infections

Abstract

Sindbis virus is a mosquito-borne virus associated with chronic arthritis and is endemic in South Africa. It is the prototype virus for the genus Alphavirus in the family Togaviridae. Sporadic outbreaks occur naturally, often related to heavy rainfall and an increase in mosquito populations. The virus causes a mild disease and hence the exact prevalence in South Africa is not known. In addition, the association of Sindbis virus disease and arthritis is not well documented in South Africa. The aim of this study was to investigate Sindbis virus prevalence and develop serological assays for detection of Sindbis virus infection. An in-house ELISA was developed and optimized. The ELISA was used to screen a total of 165 stored serum samples collected from patients attending a local arthritis clinic in Universitas Hospital, 266 stored serum samples from patients with acute febrile illness, suspected of tickbite fever and with no diagnosis, as well as 136 serum samples from high risk populations (horse and stable workers in Bainsvlei). Production of a recombinant antigen of the Sindbis virus E2 protein for use in immunofluourescence assays (IFA) was attempted. An in-house IFA, prepared with Sindbis virus infected cells, was developed. The positive samples were tested using a commercial immunofluorescence assay (IFA), a neutralisation assay and the in-house IFA. The results indicated that 31/165 samples from patients attending arthritis clinic, 13/136 samples from high risk populations, and 25/266 samples from acute febrile illness patients with no diagnosis tested positive for immunoglobulin G (IgG) Sindbis virus antibodies using the in-house ELISA. Commercial IFA results were as follows: 46/69 samples tested positive, 15/69 samples tested negative and 8/69 samples were indeterminate. A total of 65/69 samples tested positive using the neutralisation assay. Sensitivity for the ELISA and commercial IFA was determined and found to be 100% for the ELISA and 70.7% for the commercial IFA. Unfortunately, the recombinant protein could not be transiently expressed in mammalian cells and used to develop an in-house IFA. In-house antigen slides were prepared for in-house IFA tests. Using the in-house slides, a total of 50/68 samples tested positive for anti-Sindbis virus IgG antibodies and 8/56 samples tested positive for anti-Sindbis virus IgM antibodies. The ELISA and in house IFA were shown to be more sensitive than the commercial IFA. The prevalence of IgG antibody in targeted populations suggests a higher occurrence of Sindbis virus infections and that Sindbis virus infection should be considered in patients with joint pain.