Development of in house assays for detection of Sindbis virus infections

[摘要] Sindbis virus is a mosquito-borne virus associated with chronic arthritis and isendemic in South Africa. It is the prototype virus for the genus Alphavirus in thefamily Togaviridae. Sporadic outbreaks occur naturally, often related to heavyrainfall and an increase in mosquito populations. The virus causes a milddisease and hence the exact prevalence in South Africa is not known. Inaddition, the association of Sindbis virus disease and arthritis is not welldocumented in South Africa. The aim of this study was to investigate Sindbisvirus prevalence and develop serological assays for detection of Sindbis virusinfection. An in-house ELISA was developed and optimized. The ELISA wasused to screen a total of 165 stored serum samples collected from patientsattending a local arthritis clinic in Universitas Hospital, 266 stored serumsamples from patients with acute febrile illness, suspected of tickbite fever andwith no diagnosis, as well as 136 serum samples from high risk populations(horse and stable workers in Bainsvlei). Production of a recombinant antigen ofthe Sindbis virus E2 protein for use in immunofluourescence assays (IFA) wasattempted. An in-house IFA, prepared with Sindbis virus infected cells, wasdeveloped. The positive samples were tested using a commercialimmunofluorescence assay (IFA), a neutralisation assay and the in-house IFA.The results indicated that 31/165 samples from patients attending arthritisclinic, 13/136 samples from high risk populations, and 25/266 samples fromacute febrile illness patients with no diagnosis tested positive forimmunoglobulin 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 65/69samples tested positive using the neutralisation assay. Sensitivity for theELISA and commercial IFA was determined and found to be 100% for theELISA and 70.7% for the commercial IFA. Unfortunately, the recombinantprotein could not be transiently expressed in mammalian cells and used todevelop an in-house IFA. In-house antigen slides were prepared for in-houseIFA tests. Using the in-house slides, a total of 50/68 samples tested positive foranti-Sindbis virus IgG antibodies and 8/56 samples tested positive foranti-Sindbis virus IgM antibodies. The ELISA and in house IFA were shown tobe more sensitive than the commercial IFA. The prevalence of IgG antibody intargeted populations suggests a higher occurrence of Sindbis virus infectionsand that Sindbis virus infection should be considered in patients with joint pain.

[发布机构] University of the Free State