INTRODUCTION
Crimean-Congo hemorrhagic fever virus (CCHFV), a orthonairovirus of theNairoviridae family is the causative agent of a severe human hemorrhagic fever disease characterized by fever, weakness, myalgia, and hemorrhagic signs (Bente et al., 2013). The disease, named Crimean hemorrhagic fever was first observed in the Crimean Peninsula in 1944, and the causative agent which was isolated in 1967, was found to be identical to Congo virus isolated in 1956 from a febrile child in the Belgian Congo hence the names Crimean and Congo are used in combination (Casals, 1969). The mean of CCHF fatality rates in Africa (22.0%) is lower than Asia (33.5%) and Europe (33.8%) (Nasirian, 2020). The natural vector and reservoir have been identified as Hyalomma sp. ticks, and the distribution of human cases closely mirrors vector distribution CCHFV has been detected in more than 30 species of ticks, including Dermacentor sp . andRiphicephalus sp . ticks (Mehravaran et al., 2013). Studies conducted since then have consistently found a relationship between various tick species (Hyalomma, Rhipicephalus, Boophilus, Dermacentor and Ixodes ) and the presence of CCHFV in ticks are believed to be the principal means of viral transmission and persistence in nature (Drosten, Kummerer, Schmitz, & Gunther, 2003; Wolfel et al., 2007). Nevertheless, the ability to transmit infection has been demonstrated for ixodid ticks of several genera, and transovarial transmission of the virus from adult females to the succeeding generation of larval ticks has been shown to occur in a few members of the Hyalomma, Dermacentor, and Rhipicephalus genera (Zeller, Cornet, Diop, & Camicas, 1997).
In south-eastern Romania, the presence of Hyalomma was confirmed in 2014 (Dumitrescu et al., 2014). The coincidence in distribution of CCHF virus and Hyalomma ticks implies that members of this genus are important vectors of the virus (Shepherd, Swanepoel, Cornel, & Mathee, 1989). Arboviruses usually show low levels of genome diversity, perhaps since they have to be adapted both to an arthropod vector and a vertebrate host species, but CCHFV does not follow this rule and shows a high nucleic acid diversity (Weaver, 2006). This virus is a negative-sense, single-stranded RNA virus with a genome of approximative 19.2 kb in length. The genome contains three segments: small (S), medium (M) and large (L). The L segment encodes the RNA-dependent RNA polymerase, the M segment encodes the precursor of the two envelope glycoproteins Gn and Gc, and the S segment encodes the nucleocapsid protein (Aitichou, Saleh, McElroy, Schmaljohn, & Ibrahim, 2005; Estrada & De Guzman, 2011; Honig, Osborne, & Nichol, 2004). CCHFV is an enveloped virus characterized by a degree of sequence diversity of 20%, 22%, and 31% among the S-, L-, and M-segments of the virus genome (Bente et al., 2013). M and L RNA segments of nairoviruses are larger than other bunyaviruses (hantaviruses and phleboviruses) (Estrada-Pena et al., 2007; Hoogstraal, 1979). As other members of the family Bunyaviridae , CCHFV glycoproteins target the Golgi apparatus, where most viral assembly takes place. Recent studies have revealed a new strain which shows unique S-segment phylogenies and which constitutes a unique clade (provisionally identified as clade VII) (Chinikar et al., 2010). Crimean-Congo hemorrhagic virus circulates in nature in unnoticed enzootic tick–vertebrate–tick cycles. Asymptomatic CCHFV infection has been reported in numerous vertebrate species and appears to be pervasive in both wild and domestic animals. Asymptomatic viremia last up to 7–15 days in several vertebrate animal species, and CCHFV has been isolated from livestock and small mammals (horses, donkeys, goats, cattle, sheep, and pigs (Ergonul, 2006).
In Romania there is still not sufficient data concerning CCHFV and the virus has never been detected in ticks. Virus detection by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) is essential to prove an actual circulation of CCHFV in a country and can be used to identify animals and humans CCHFV infections, together with viral detection in ticks. The predominant problem in CCHFV RT-qPCR development has always been the high genetic diversity of the virus (Deyde, Khristova, Rollin, Ksiazek, & Nichol, 2006).
The aim of this study was to assess the prevalence of CCHFV inDermacentor sp . and Rhipicephalus sp . ticks collected from Romania, more precisely in Tulcea county, where in previous studies IgG antibodies anti-CCHFV were found in sheep and goats (Ceianu, Panculescu-Gatej, Coudrier, & Bouloy, 2012; Răileanu, Anită, Porea, & Savuta, 2015).