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Combination of RT-PCR and proteomics for the identification of Crimean-Congo hemorrhagic fever virus in ticks.

Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne zoonotic disease caused by the CCHF virus (CCHFV). In this study, an experimental approach combining RT-PCR and proteomics was used for the identification and characterization of CCHFV in 106 ticks from 7 species that were collected from small ruminants in Greece. The methodological approach included an initial screening for CCHFV by RT-PCR followed by proteomics analysis of positive and control negative tick samples. This novel approach allowed the identification of CCHFV-positive ticks and provided additional information to corroborate the RT-PCR findings using a different approach. Two ticks, Dermacentor marginatus and Haemaphysalis parva collected from a goat and a sheep, respectively were positive for CCHFV. The sequences for CCHFV RNA segments S and L were characterized by RT-PCR and proteomics analysis of tick samples, respectively. These results showed the possibility of combining analyses at the RNA and protein levels using RT-PCR and proteomics for the characterization of CCHFV in ticks. The results supported that the CCHFV identified in ticks are genetic variants of the AP92 strain. Although the AP92-like strains probably do not represent a high risk of CCHF to the population, the circulation of genetically diverse CCHFV strains could potentially result in the appearance of novel viral genotypes with increased pathogenicity and fitness.


Introduction
Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne zoonotic disease causing sporadic cases or outbreaks of severe illness in humans (Ergonul, 2006;Bente et al., 2013;Papa et al., 2015a;Martina et al., 2017). CCHF is caused by the CCHF virus (CCHFV), which is distributed across a large geographic area from western China to the Middle East and Southeastern Europe and throughout most of Africa (Ergonul, 2006;Bente et al., 2013).
CCHFV is maintained in vertical and horizontal transmission cycles involving ixodid ticks and a variety of wild and domestic vertebrates, which do not show signs of illness but serve as reservoirs (Bente et al., 2013). The maintenance of active foci of CCHFV in the field may depend on tick survival, requiring favorable climatic conditions and high numbers of suitable hosts for adult ticks (Estrada-Peña et al., 2013). The virus circulates in a number of tick genera, but Hyalomma species are considered the principal source of human infection (Bente et al., 2013;Papa et al., 2015a). Humans could also be infected by contact with body fluids from infected viremic animals or patients (Bente et al., 2013;Papa et al., 2015a).
CCHFV is considered the most genetically diverse of the arboviruses because it shows differences among genotypes ranging from 20% for the viral S segment to 31% for the M segment nucleotide sequences (Bente et al., 2013). Furthermore, different genotypes can be found within the same geographic area, while closely related genotypes have been isolated in geographically distant regions (Bente et al., 2013). Phylogenetic analysis of CCHFV from different locations suggests that the virus dispersed a long time ago, possibly by ticks carried on migratory birds or Article No~e00353 through the international livestock trade (Anagnostou and Papa, 2009;Bente et al., 2013;Papa et al., 2015a).
Currently, CCHFV is diagnosed by virus isolation, serology and molecular-based techniques. Virus isolation requires laboratory biosafety level (BSL-3/4), which is not available in many institutions. Serology is useful in epidemiological studies of infected hosts by detection of CCHFV-specific IgM or IgG antibodies, but detection of anti-CCHFV IgM in infected patients requires at least 3-5 days post infection, which may account for false negative results during early acute phase of infection (Aradaib et al., 2011). Molecular techniques based on RT-PCR are used for the amplification of viral genome segments (Midilli et al., 2009;Osman et al., 2013;Bente et al., 2013;Papa et al., 2014;2015a). Most of the RT-PCR assays use a secondary nested amplification or nucleic acid hybridization to increase the sensitivity and to confirm the identity of the amplified PCR product (Aradaib et al., 2011;Osman et al., 2013). However, despite virus amplification after multiplication in ticks feeding on a susceptible host (Dickson and Turell, 1992;Bente et al., 2013;de la Fuente et al., 2017), CCHFV identification in individual ticks may be difficult due to low infection levels and the presence of other Nairoviruses (Walker et al., 2015). In this regard, the proteomics approach using matrix assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS) would provide additional information to corroborate the RT-PCR findings using a different approach, and assist in the differentiation between different CCHFV genotypes and Nairoviruses (Singhal et al., 2015) and the characterization of tick-virus molecular interactions .
In this study, a novel approach using the combination of RT-PCR and MALDI-TOF MS was used for the molecular identification and characterization of CCHFV in ticks collected from domestic ruminants in endemic areas of Greece.

Materials and methods
Partially fed ticks from 7 species (N = 106) were collected in 10 Greek counties from sheep and goats, which are considered the most suitable indicator animals for the circulation of CCHFV (Schuster et al., 2016) (Fig. 1 and Table 1). Collected ticks were classified (Manilla, 1998) and frozen until dissection of internal organs for analysis. Ticks were divided in two similar vertical halves to dissect internal organs for RNA and protein studies. Total RNA was extracted from one half ticks using TriReagent (Sigma, St. Louis, MO, USA) and following manufacturer recommendations. CCHFV RNA was amplified using a nested RT-PCR targeting virus S segment as previously described (Midilli et al., 2009). The amplicons were cloned and at least 3 clones were sequenced for each amplicon.

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For proteomics analysis, internal tissues were dissected from the another half of CCHV-infected (n = 2) and uninfected (n = 4) ticks, lysed with phosphate buffered saline (PBS) supplemented with 1% Triton X-100 and complete protease inhibitor cocktail (Roche, Basel, Switzerland), and homogenized by passing through a needle (27G). Samples were sonicated for 1 min in an ultrasonic cooled bath, followed by vortexing for 10 sec. After three cycles of sonication-vortex, tick lysates were centrifuged at 200 x g for 5 min to remove cell debris. The supernatants were collected and protein concentration was determined using the BCA Protein Assay (Life Technologies, Carlsbad, CA) with BSA as standard.

Results and discussion
The results showed that 2 of the 106 sampled ticks were positive for CCHFV by RT-PCR (Table 1). The positive ticks corresponded to Dermacentor marginatus and Haemaphysalis parva collected from a goat and a sheep at Korinthos and Fokida counties, respectively ( Fig. 1 and Table 1). These tick species were previously reported to be infected with CCHFV (Albayrak et al., 2010;Hekimoglu et al., 2012). The sequences of the S segment (GenBank accession numbers KU365757 and KU365758) were 99% identical between them at the nucleotide and amino acid levels. Furthermore, these sequences showed a 98% and 99-100% identity at the nucleotide and amino acid levels, respectively to CCHFV strain AP92 (DQ211638) previously isolated from a Rhipicephalus bursa tick. To confirm the identity of amplified and sequenced PCR products, a proteomics analysis was conducted using tissues from infected and control uninfected ticks.
Tick-and host-derived proteins were identified in both infected and uninfected ticks, and were excluded from the analysis. Nairovirus-derived peptides corresponding to the L segment containing a putative RNA-dependent RNA polymerase (Bente et al., 2013), and unique to CCHFV were identified in infected D. marginatus and H. parva ticks ( Table 2). The results confirmed CCHFV infection in these ticks with an alternative method and a different genome region (L segment) from that targeted by RT-PCR (S segment). The proteomics analysis was targeted at the L segment because this segment showed a higher peptide identification among different Nairoviruses in this study. The sequences for L segment peptides No. 1, 2 and 5 (Table 2) were 100% identical to the corresponding sequence of the prototype AP92 strain (ABB30012).
The results suggested that the CCHFV identified in the present study in ticks are genetic variants of the AP92 strain, but are closer to the old AP92 strain (98% nucleotide sequence identity) than to the novel AP92-like strain (91% nucleotide sequence identity) (Fig. 2). Greece is a Mediterranean country where currently only one CCHF case has been reported (Papa et al., 2015a). The absence of CCHF cases in Greece, together with the high seroprevalence in the human population, and the fact that one of the veterinarians who isolated the AP92 strain  The identified Nairovirus-derived peptides corresponding to the L segment were aligned to viral amino acid sequences available at the GanBank using BLAST. Conserved amino acids when compared to the CCHFV sequence are shown underlined. Only virus sequences for which a hit was found after alignment are shown. Sequence identity among CCHFV genotypes (in parenthesis) was obtained after alignment with all available CCHFV sequences.

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demonstrated very high titres of anti-CCHFV antibodies, led to the suggestion that the AP92 strain is not pathogenic for humans (Papa et al., 2015b). Besides the prototype AP92 strain, a novel AP92-like strain (KF146306) has been recently detected in Greece in R. bursa collected from sheep in an area with 6% CCHFV seroprevalence (Papa et al., 2014). Currently, one human case with mild symptoms has been associated with the AP92-like strain in Turkey (Midilli et al., 2009), and [ ( F i g . _ 2 ) T D $ F I G ] These results showed the possibility of using this experimental approach for the identification and characterization of CCHFV in ticks, and confirmed the presence of genetically diverse CCHFV strains in different tick species collected from goats and sheep in Greece. The combination of RT-PCR with MALDI-TOF MS increases the possibility of characterizing CCHFV genetic variants circulating in different regions, and differentiating CCHFV from other Nairoviruses (Table 2).
CCHFV shows a higher genetic diversity when compared to other arboviruses, a finding that has been linked to increased virulence and emergence in new geographic locations (Xia et al., 2016). These results also supported the increasing evidence that the circulation of the low or not pathogenic AP92-like strains probably does not represent a high risk of CCHF to the population. However, the circulation of genetically diverse CCHFV strains could potentially result in the appearance of novel viral genotypes with increased pathogenicity and fitness (Bente et al., 2013).

Declarations
Author contribution statement