Preparation of recombinant antigen for serological detection of African hantaviruses
Damane, Deborah Rethabile
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Unlike other members of the Bunyaviridae family, hantaviruses are transmitted to humans through direct exposure or inhalation of virus contaminated urine or droppings from their reservoir hosts. Hantaviruses were first discovered in 1976 with the identification of Hantaan virus (HNTV) from the reservoir Apodemus agarius in Asia and later in North America. In 2006, Sangassou virus (SANGV) was the first to be isolated in Africa in the African house mouse, Hylomyscus sinus and subsequently followed by the identification of ten more African hantaviruses in both rodent and insectivore hosts through reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assay (IFA). Hantaviruses are a public health concern with annual cases of disease reported to be approximately 200,000 per year, with most cases reported in Asia. In Africa, disease associated with hantaviruses is not well defined. Culturing the virus and preparing reagents using native virus requires the use of biosafety level (BSL) 3 or 4 laboratories limiting the number of facilities with capability to prepare serological assays. Hence, the use of recombinant antigens that are safe to use in a BSL 1 laboratory that have application as serological tools for surveillance are required. The aim of the study was to develop serological assays to test for antibodies against hantaviruses in human serum samples collected in the Free State, South Africa using a recombinant nucleocapsid protein (NP) of SANGV as a representative of African hantaviruses. Transiently transfected cells were used to prepare antigen slides for IFA and expressed protein was used in an in-house enzyme linked immunosorbent assay (ELISA). In-house assays and commercially available ELISA kits were used to screen human serum samples. There are limited seroprevalence studies performed in Africa to detect IgG antibodies against hantaviruses in humans and no commercial serological assays are available using an African antigen. Hence, it was considered that the preparation of a recombinant African hantavirus antigen based on SANGV could have application in serological surveillance studies. The S gene segment of the SA14 strain of SANGV was modified and codon optimized for enhanced expression and detection. The construct was sequenced and aligned to the native S gene. It was used to transfect baby hamster kidney cells (BHK-21). Expression of a 50kDA protein was confirmed by SDS-PAGE and Western blot assay. Antigen slides were prepared from transfected cells fixed on 12 well chamber slides. Positive controls from the commercially available ELISA kits were used in the IFA. Four of the 176 serum samples tested gave a positive test. For the in-house ELISA, protein was harvested from T75 culture flasks. The antigen was tested using positive and negative controls from the commercial ELISA kits. A suitable differentiation between positive and negative samples was not detectable despite attempts to optimize the in house ELISA. It is likely that the protein yield was insufficient for the ELISA and further attempts, beyond the scope of this project, to increase the protein yield will be investigated using a stable cell line. Commercially available ELISA kits comprising of HNTV, Dobrava (DOBV) and Puumala (PUUV) recombinant NP antigens for Europe and Asia group and Andes virus (ANDV) and SNV for the American group were used to screen acute human sera in the laboratory. Positive reactors were identified using both kits. The significance of the results is difficult to interpret as there was lack of concordance. However, it does suggest that hantaviruses are to be found in this area and that the use of a homologous antigen for serological surveillance is essential. Results confirmed some serological cross-reactivity between heterologous Asian, American and African hantaviruses and a potential application for an African hantavirus as a tool for surveillance.