We previously detected CCHFV in serum samples from the 2 patients in Spain with autochthonous CCHV (12); the index case-patient died. For this study, we obtained viral RNA (3.6 × 10⁷ copies/mL) from the secondary case-patient in a sample taken 4 days after onset of symptoms by using the QIAamp viral RNA Mini kit (QIAGEN, Hilden, Germany). We then amplified the virus in a single-step reverse-transcription PCR by using the SuperScript III One-Step RT-PCR system with the Platinum Taq High Fidelity DNA Polymerase kit (Invitrogen Life Technologies, Barcelona, Spain) with overlapping primers throughout the complete genome (Table). PCR conditions were amplification at 52.5°C for 30 min, 94°C for 2 min, and then 40 cycles at 94°C for 15 s, 48°C –55°C for 30 s, and 68°C for 1 min/kb, with a final extension cycle at 68°C for 5 min. We designed primers for each segment in most conserved regions after aligning available CCHFV sequences from all genotypes retrieved from GenBank (Table). We directly sequenced purified amplicons by using additional internal primers (data not shown). We assembled and analyzed the consensus sequence of each segment by using SeqMan Pro from the Lasergene Suite 12 (DNASTAR Inc., Madison, WI, USA). To get complete finished genomes, we used a hybrid-capture method as described by Blackley et al. (13) by using probes designed against the CCHFV sequences obtained from amplicon sequencing.

Figure

Figure. Phylogenetic analysis of Crimean-Congo hemorrhagic fever virus from patient in Spain, 2016, compared with reference sequences. A) Small segment (1,450 bp); B) medium segment (4,497 bp); C) large segment (11,829 bp)....

To characterize the complete CCHFV genome, we performed a phylogenetic analysis of the full S, M, and L segments (Figure). The 3 segments were aligned by using ClustalW in MEGA 5.2 (http://www.megasoftware.net) and representative available CCHFV sequences from GenBank of all genotypes. We generated a phylogenetic tree by using neighbor-joining algorithms and analyzed 1,000 replicates for bootstrap testing. GenBank accession numbers for sequences used in this study are MF287636 for the S fragment, MF287637 for the M fragment, and MF287638 for the L fragment.

The nucleotide sequence of the different CCHFV segments from the infected patient we analyzed in this study showed 99% identity with the Sudan AB1–2009 CCHFV strain (5) in S, M, and L segments (GenBank accession nos. HQ378179.1, HQ378187.1, and HQ378183.1, respectively) (Figure) and were grouped within genotype III (Africa 3). CCHFV found in ticks from Spain in 2010 and from birds from Morocco in 2011 also clustered in this group (10,11). In addition, no reassortant segment has been found in the analysis of the full genome, even though reassortant strains have been described in this genotype (2).

The results of the sequence analysis we describe corroborate our previous results (12), obtained by analyzing a small fragment in the S segment, showing that CCHFV from genotype III (Africa III) is circulating in southwestern Europe. CCHFV circulating in Spain caused 2 autochthonous cases that resulted in the death of the index case-patient and a serious illness in the second case-patient, providing evidence of its pathogenicity. The risk for infection in Spain is considered low, but human infection caused by the bite of an infected tick has occurred 6 years after the virus was discovered in ticks (10). Because the virus is circulating in Spain, additional studies will be required to establish the distribution of the virus in this country.