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In press., Crimean-Congo haemorrhagic fever virus (CCHFV) continues to cause new human cases in Iberia while its spatial distribution and ecological determinants remain unknown. The virus remains active in a silent tick-animal cycle to which animals contribute maintaining the tick populations and the virus itself. Wild ungulates, in particular red deer, are essential hosts for Hyalomma ticks in Iberia, which are the principal competent vector of CCHFV. Red deer could be an excellent model to understand the ecological determinants of CCHFV as well as to predict infection risks for humans because it is large, gregarious, abundant and the principal host for Hyalomma lusitanicum. We designed a cross-sectional study, analysed the presence of CCHFV antibodies in 1444 deer from 82 populations, and statistically modelled exposure risk with host and environmental predictors. The best-fitted statistical model was projected for peninsular Spain to map infection risks. Fifty out of 82 deer populations were seropositive, with individual population prevalence as high as 88%. The highest prevalence of exposure to CCHFV occurred in the southwest of the Iberian Peninsula. Climate and ungulate abundance were the most influential predictors of the risk of exposure to the virus. The highest risk regions were those where H. lusitanicum is most abundant. Eight of the nine primary human cases occurred in or bordering these regions, demonstrating that the model predicts human infection risk accurately. A recent human case of CCHF occurred in northwestern Spain, a region that the model predicted as low risk, pointing out that it needs improvement to capture all determinants of the CCHFV infection risk. In this study, we have been able to identify the main ecological determinants of CCHFV, and we have also managed to create an accurate model to assess the risk of CCHFV infection., This study has been funded by the Spanish Ministry for the Science and Innovation (MCI) through projects CGL2017-89866-R & E-RTA2015-0002-C02-02, and by the Regional Government of Castilla-La Mancha and the European Social Fund (ESF) through project SBPLY/19/180501/000321. RC-M, LC-M, SJ-R and JM-G would like to thank the MCI, ESF and the University of Castilla-La Mancha for their support through contracts PRE2018-083801, PEJ2018-003155-A, 2018/12504 and BES-2015-072206, respectively. BC was supported by FCT-Fundação para a Ciência e Tecnologia (2020.04872.BD). DG-B would like to thank MCI and Instituto de Salud Carlos III (CD19CIII/00011) for funding him.

Crimean-Congo hemorrhagic fever virus (CCHFV) circulates in many countries of Asia, Africa, and Europe. CCHFV can cause a severe hemorrhagic fever in humans with case-fatality rates of up to 80%. CCHF is considered to be one of the major emerging diseases spreading to and within Europe. Ticks of the genus Hyalomma function as vector as well as natural reservoir of CCHFV. Ticks feed on various domestic animals (e.g. cattle, sheep, goats) and on wildlife (e.g. hares, hedgehogs). Those animal species play an important role in the life cycle of the ticks as well as in amplification of CCHFV. Here we present a competitive ELISA (cELISA) for the species-independent detection of CCHFV-specific antibodies. For this purpose nucleocapsid (N) protein specific monoclonal antibodies (mAbs) were generated against an Escherichia coli (E. coli) expressed CCHFV N-protein. Thirty-three mAbs reacted with homologous and heterologous recombinant CCHFV antigens in ELISA and Western blot test and 20 of those 33 mAbs reacted additionally in an immunofluorescence assay with eukaryotic cells expressing the N-protein. Ten mAbs were further characterized in a prototype of the cELISA and nine of them competed with positive control sera of bovine origin. The cELISA was established by using the mAb with the strongest competition. For the validation, 833 sera from 12 animal species and from humans were used. The diagnostic sensitivity and specificity of the cELISA was determined to be 95% and 99%, respectively, and 2% of the sera gave inconclusive results. This cELISA offers the possibility for future large-scale screening approaches in various animal species to evaluate their susceptibility to CCHFV infection and to identify and monitor the occurrence of CCHFV.

Infections of humans with the tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV) can cause a severe hemorrhagic fever with case fatality rates of up to 80%. Most humans are infected by tick bite, crushing infected ticks by hand or by unprotected contact with blood of viremic mammals. Next to the notified human CCHF cases, the real distribution and the situation in animals in Southeastern Europe are nearly unknown. Since domestic ruminants play a crucial role in the life cycle of the vector ticks and the transmission and amplification of the virus, the antibody prevalence in those animals is a good indicator for the presence of CCHFV in a region. Therefore, the prevalence of CCHFV-specific antibodies was investigated in domestic ruminants of different regions of Bulgaria and Turkey. Sera of 1165 ruminants were tested and a prevalence of up to 90% was identified. The overall prevalence for Bulgaria was 26% and for Turkey 57%. The results highlight the risk of human infections in those regions and the importance of the investigation of the prevalence in animals for identification of risk areas. This article provides a unique overview about published CCHFV antibody prevalence in animals in comparison to human incidences in different areas of Bulgaria and Turkey. Although it will help to complete the understanding of the CCHFV situation in these countries, it also demonstrates the lack of unpublished and published data even in these highly endemic areas.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus which causes lethal hemorrhagic fever in humans. Although, several reports regarding CCHFV antibody prevalence in humans exist in Greece, information about the current distribution is limited. The aim of the present study is to investigate the prevalence of CCHFV-specific IgG antibodies in cattle and sheep in Macedonia-Greece. The samplings were performed during spring 2013, in 5 regional units of Central Macedonia (Chalkidiki, Imathia, Kilkis, Pella and Thessaloniki) and in the 4 regional units of Western Macedonia (Grevena, Florina, Kastoria and Kozani). Specifically, sera from 538 cattle and 81 sheep underwent testing against CCHFV-specific IgG antibodies. Antiviral immune responses were observed in 31 cattle (6%, 95% CI: 4-8%) and in one sheep (1%, 95% CI: 0-8%). The total seroprevalence in the cattle sampled in Central Macedonia was 7% (28 out of 396, 95% CI: 5-10%). Within Central Macedonia, the highest seroprevalence was detected in Chalkidiki (38%, 95% CI: 23-56%), which was significantly higher (p

Background There are only few assays available for the detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-specific antibodies in animals, and data about diagnostic sensitivity and specificity are incompletely documented for most of these tests. This is unfortunate since CCHFV antibodies in animals can be used as indicator for virus circulation in a geographic area and therewith potential risk of human exposure. This paper therefore reports on a novel ELISA for the detection of CCHFV-specific antibodies in cattle and on its application for testing ruminant sera from the Former Yugoslav Republic of Macedonia. Principal Findings A highly sensitive and specific ELISA was developed to detect CCHFV-specific IgG antibodies in cattle. The assay was validated by using 503 negative serum samples from a country where CCHFV has never been detected until now, and by using 54 positive serum samples. The positive sera were verified by using two commercially available assays (for testing human serum) which we have adapted for use in animals. The sensitivity of the novel ELISA was 98% and its specificity 99%. The presence of Hyalomma ticks was demonstrated in the Former Yugoslav Republic of Macedonia and depending on the region antibody prevalence rates up to 80% were detected in the cattle population. Conclusion This article describes a fully validated, highly sensitive and specific ELISA for the detection of CCHFV-specific IgG antibodies in cattle. Using this assay, CCHFV-specific antibodies were detected for the first time in cattle in the Former Yugoslav Republic of Macedonia, giving evidence for an active circulation of this virus in the country. Supporting this conclusion, the occurrence of the main vector of CCHFV was demonstrated in the present work for the first time in Former Yugoslav Republic of Macedonia., Author Summary Crimean-Congo hemorrhagic fever virus (CCHFV) infections in humans can cause a deadly hemorrhagic fever with high fatality rates. Most humans are infected by ticks or by contact with blood. No vaccine prophylaxis or therapeutic interventions are available. Ticks of the genus Hyalomma function as vector of CCHFV. Domestic ruminants play a crucial role in the transmission and amplification of the virus. Since infected ruminants do not develop clinical signs, CCHFV infections can in most cases only be detected by serology. There are only few assays available for the detection of CCHFV-specific antibodies in animals. In general, the real distribution of CCHFV is fairly unknown. Here we present a novel, highly sensitive and specific ELISA, and the adaptations for two commercially available assays for the detection of CCHFV-specific antibodies in cattle. With these assays, we detected CCHFV-specific antibodies in cattle in the Former Yugoslav Republic of Macedonia. This is the first indication of an active circulation of CCHFV in this country. This knowledge will assist decision makers and public health authorities in deciding on effective countermeasures and can help to prevent outbreaks.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus, which causes a serious illness with case-fatality rates of up to 80 % in humans. CCHFV is endemic in many countries of Africa, Asia and Southeastern Europe. Next to the countries with endemic areas, the distribution of CCHFV is unknown in Southeastern Europe. As the antibody prevalence in animals is a good indicator for the presence or absence of the virus in a region, seroepidemiological studies can be used for the definition of risk areas for CCHFV. The aim of the present study was to reveal which ruminant species is best suited as indicator for the detection of a CCHFV circulation in an area. Therefore, the prevalence rates in sheep, goats and cattle in different regions of Albania and Former Yugoslav Republic of Macedonia were investigated. As there are no commercial tests available for the detection of CCHFV-specific antibodies in animals, two commercial tests for testing human sera were adapted for the investigation of sera from sheep and goats, and new in-house ELISAs were developed. The investigation of serum samples with these highly sensitive and specific assays (94–100 %) resulted in an overall prevalence rate of 23 % for Albania and of 49 % for Former Yugoslav Republic of Macedonia. Significant lower seroprevalence rates for CCHFV were found in cattle than in small ruminants in given areas. These results indicate that small ruminants are more suitable indicator animals for CCHFV infections and should therefore be tested preferentially, when risk areas are to be identified.