Novel serological and molecular assays for Crimean‑Congo hemorrhagic fever virus infections and their application in prevalence studies on sub‑Saharan African countries

Crimean-Congo hemorrhagic fever (CCHF) is a fatal disease in humans with observed case fatality rates varying from 5% in Turkey to 80% in the People’s Republic of China. This variability most likely depends on general awareness of the population, quality of the public healthcare system, sensitivity of the notification system and on the individual virus strain. The primary transmission route of Crimean-Congo hemorrhagic fever virus (CCHFV) is by tick bites. Therefore, the virus distribution on the African, Asian and European continent is closely linked to the main vector, ticks of the genus Hyalomma. Hyalomma spp. generally prefer a warm and dry climate with a fragmented and less dense vegetation. Hence, these ticks are not able to create a stable and self-sustaining population further north than 46° N. Other sources of infections are contact to blood, body fluids and tissues of viremic animals or human patients and by crushing infected ticks. The detection of CCHFV-specific antibodies in ruminant sera is commonly used as indicator to reveal infection risk areas also for humans. Even though animals do not show clinical signs, they develop a stable IgG antibody titer that is detectable for many years after infection. CCHFV is best investigated in endemic areas in Europe. In contrast, research on the CCHFV prevalence on the African continent was fairly neglected for many decades and up-to-date information is only available for a few African countries. Therefore, the main goal of this thesis was to investigate the CCHFV prevalence in four different countries located in sub-Saharan Africa. For this purpose, a series of serological assays recently developed for testing ruminant sera from Southeastern Europe was employed. These assays included a commercial (species adapted) enzyme linked immunosorbent assay (ELISA) and an in-house CCHFV‑IgG-ELISA as well as a commercial (species adapted) immunofluorescence assay (IFA). The adaptation of these assays included protocol and cut-off changes to reach convincing diagnostic sensitivities (95% - 98%) and specificities (98% - 100%). However, as the assays were supposed also to be run in less well equipped laboratories in Africa, a robust and highly reproducible indirect cattle in-house CCHFV-IgG-ELISA was developed. The extensive modifications in the novel CCHFV-IgG-ELISA did not only increase the robustness of the assay and simplify the procedure but also enabled testing sera under the technical standards currently available in African laboratories. Nonetheless this new assay had an excellent diagnostic sensitivity and specificity (both 99%) and was therefore used in the seroepidemiological studies in Mali and Cameroon (sera collected in 2014). In parallel, a highly specific and sensitive multiplex CCHFV RT-qPCR using 12 genotype‑specific primers, 2 universal primers and 2 carboxyfluorescein probes for the reliable detection of all currently known CCHFV strains was newly designed and validated. This assay detects synthetic and native virus sequences of all six known genotypes and six calibrator RNAs enable the genotype-specific and precise quantification of CCHFV. Serological assays were subsequently used to screen more than 3,000 ruminant sera from Mauritania, Mali, Cameroon and the Democratic Republic of the Congo (DR Congo), countries all situated in sub-Saharan Africa, and questions about the correlation of CCHFV prevalence with vegetation, climate and animal density were addressed. Ruminant sera were considered positive for CCHFV antibodies, if they were reactive in two independent ELISAs. In case of divergent results, the IFA was used for final diagnosis. This approach revealed CCHFV infections in ruminants in all four countries Mauritania, Mali, Cameroon and DR Congo, which was a surprising result especially for Cameroon as this was the first demonstration of this infectious disease in this country. Highest prevalence rates were detected in Mauritania, Mali and North Cameroon, which is consistent with the vegetation and climatic conditions and the habitat preference of Hyalomma ticks. Conversely, medium to low prevalence rates were found in Southern Cameroon and the DR Congo, which was also in accordance with the suitability of local vegetation and clime for these arthropods. Moreover, cattle densities seemed also to correlate with the presence of CCHFV in an area. Most CCHFV-specific antibody positive cattle were detected in North Cameroon, where cattle density is much higher than in the South and a similar correlation was noticed for Mali. As this study revealed for the first time CCHFV infections in Cameroon, it was of crucial importance also to prove that the virus is circulating in this environment. For this purpose, 109 Hyalomma ticks were collected from infested bovines in a CCHFV highly endemic area in North Cameroon and were assayed for CCHFV genomes using a novel highly sensitive multiplex SYBR Green CCHFV RT-qPCR based on 14 different primers for the detection of all currently known CCHFV genotypes. Samples with a characteristic amplification were eventually reconfirmed using a multiplex CCHFV RT-qPCR, which had the same genotype specific primer sets and two additional CCHFV specific probes included. CCHFV was found in 7 of the 109 ticks and amplified genome sequences clustered phylogenetically into genotype III. In summary, the work presented here highlights the importance of seroprevalence studies in ruminants as indicator animals and molecular diagnostic studies in ticks to determine, whether CCHFV is circulating in sub-Saharan countries/regions. The current data obtained for Cameroon, DR Congo, Mali and Mauritania allow carrying out risk assessments for human infections and therefore help to define whether public health protection measures (e.g. raising awareness for risk groups and the broader public) should be applied.