Serologic Evidence of Crimean-Congo Hemorrhagic Fever Virus Infection in Hungary

Introduction

C rimean-Congo hemorrhagic fever virus (CCHFV) (Nairovirus genus, Bunyaviridae family) is a tick-borne pathogen, transmitted primarily by Hyalomma spp. The spectrum of natural hosts for CCHFV is broad and includes wild and domestic animals. Humans are infected through tick bites or contact with the viremic blood of patients or livestock. CCHFV is widely distributed in Africa, Asia, the Middle East, and the Balkans, but CCHFV infection has also been reported from many other parts of Europe (Ergönül 2006). The main clinical symptoms of infection in humans are fever, myalgia, neck pain, headache, photophobia, and various hemorrhagic manifestations, ranging from petechiae to large hematomas appearing on the mucous membranes and skin. Severely ill persons may develop extensive bleeding and multiorgan failure with a case fatality rate of 3–30% (Ergönül 2006).

Our knowledge regarding the occurrence of CCHFV in Hungary is limited and based on historical observations. A serological investigation conducted in late 1960s identified sheep, cattle, and human sera positive for CCHFV in 31%, 0.9%, and 2.9%, respectively. Authors used the agar gel diffusion precipitation method in these studies (Horváth 1975, Horváth 1976). Although, Ixodes ricinus ticks are an unusual species for CCHFV, two strains were isolated in suckling mice as a part of a national survey for endemic foci of Arboviruses during the early 1970s in Hungary (Molnár 1982). Since then, no systematic surveillance has been conducted for CCHFV, raising questions of whether these viruses still circulate in Hungary and could be considered as a potential public health threat. Recently, active surveillance has been initiated in Hungary to understand the epidemiology and ecology of CCHFV in human diseases.

To support these surveys, we have developed two validated antibody detection assays, an enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence assay (IFA), both using recombinant CCHFV nucleoprotein (rNP) as antigen. A 1448-bp-long fragment of the nucleocapsid protein (NP) of the IbAr 10200 Nigerian strain of CCHF virus was produced with an Escherichia coli protein expression system, as described previously (Németh et al. 2011). Hare blood samples were screened for the presence of immunoglobulin G (IgG) antibodies against CCHFV using arecombinant antigen-based ELISA. Microtiter plates (Maxisorp Nunc-Immuno Plate) were coated with 0.5 μg of rNP antigen per well, followed by incubation for 2 h at 37°C. After further washing steps, wells were blocked with 5% phosphate-buffered saline–bovine serum albumin (PBS-BSA) for 1 h at 37°C. Hare serum (100 μL, diluted 1:100 in PBS+Tween 20 (PBS-T) with 1% BSA was added, and plates were incubated at 37°C for 1 h. Goat anti-rabbit horseradish peroxidase (HRP, 1:3000) was used as secondary antibody, with incubation at 37°C for 1 h. After 5 washing steps, 100 μL of 3,3′,5,5′-tetreamethylbenzidine (TMB) was added to the ELISA plates. Color development was quantified by reading the optical density at 450 nm. Specimens were considered positive if the absorbance of samples was equal to or greater than three times the mean value of a panel of negative control sera derived form the same species.

A transgenic Chinese hamster ovary (CHO) cell line was developed as described previously (Kvell et al. 2005). Transgenic CHO cells were also used for detection of IgG antibodies against CCHFV in Lepus europeus. Briefly, a confluent CHO monolayer, which was grown in 96-well tissue culture plate, was fixed in ice-cold acetone and ethanol (1:1) after air drying. Hare serum samples were incubated in 1:100 dilutions for 1 h at room temperature with the transgenic cells. After the incubation, the plate was washed three times with PBS-T, and fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG was added at dilution of 1:200. The plate was washed again three times after 1 h of incubation and was observed under a fluorescence microscope at 20× magnification. Cells showing strong green fluorescence were recorded as positive. Sera from uninfected hares were used as a negative control. In both assays, reference human sera containing IgG antibody against CCHFV were used as positive controls.

A total of 198 serum samples collected from European brown hares (L. europeus) in the surrounding area of Dévaványa village (47°05′N, 20°56′E) between 2008 and 2009 were tested in parallel with ELISA and IFA (Table 1). Out of the 198 samples, 12 (6%) were positive for IgG antibody against CCHF virus by both methods. Four samples were positive with IFA, and 1 serum was positive with ELISA only. A total of 181 samples were negative; therefore, the homology was 71% between the 2 tests. We note that ELISA is an objective, measurable detection method, whereas IFA is more subjective, being highly dependent on the person evaluating the assay, and requires a long-term procedure. Hares have been identified as a common reservoir for CCHFV in the former U.S.S.R., Bulgaria, and Africa; however, a study from Albania has reported failure to detect antibodies against CCHFV in this host (Hoogstraal 1979; Papa et el. 2009). Our findings nicely complement historic observations that endemic foci of CCHFV are present in Hungary, although the full spectra of reservoirs, vectors, and amplifying hosts await further investigation.

Collectively, serologic evidence for CCHFV infections has been provided; however, there is much left to learn about the public health implication of these findings. The annual number of reported viral hemorrhagic fever cases from 2000 onward averaged 10 (range, 3–21) in Hungary. Most of these episodes were associated with hantavirus infections, but some remained undiagnosed, raising the possibility that additional causes of viral hemorrhagic fever may be important in Hungary. Nevertheless, suspected cases were tested for CCHFV, but serological examinations in conjunction with the diseases remained negative. No infection showed etiological correlation with the virus so far (data not shown).

The failure to detect CCHFV cases in the clinical setting may be explained by the following. (1) One theoretic possibility is that no highly virulent CCHFV strain circulates in Hungary and thus human CCHFV infections may be less severe or may be associated with altered disease manifestations. A particular genetic clade of CCHFV represented by strain AP92 was detected in Rhipicephalus bursa ticks, which was considered apathogenic in Greece and showed low pathogenecity in a reported case in Turkey (Papadopoulos et al. 1980, Midilli et el. 2009). Although, Hyaloma spp. as the primary viral vector are not present in Hungary, other tick species such as Rhipicephalus spp. and Ixodes spp. may serve as putative vectors in the country. The question remains whether I. ricinus species could play a role in the transmission of AP92-like strains or another apathogenic clade of CCHFV in Hungary. Therefore, surveys are needed to detect and characterize the genetic clade(s) of CCHFV strains endemic in Hungary. (2) Alternatively, it is possible that there is no human exposure to CCHFV. However, European brown hares as well as cattle and sheep are important export items for Hungary; thus, the lack of exposure is unlikely and appears reasonable only if CCHFV viremia does not coincide with slaughter activity or hunting season of the amplifying hosts. The antibody detection methods we have developed will enable us to perform large-scale surveillance that should help us to gain a clearer picture of the epidemiology and ecology of CCHFV infections in Hungary.