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. 2020 Oct 21;9:e50999. doi: 10.7554/eLife.50999

Figure 1. Identification of a tick-derived Europe 2 CCHFV genotype that poorly infects human cells.

(A) Phylogenetic relationship of Malko Tarnovo strains to other CCHFV strains. Maximum likelihood phylogeny was inferred for the complete coding region of the L-segment. RAxML tree was performed with the GTR substitution model and 1000 bootstrap replicates. Only bootstrap values > 50 are shown. The tree was rooted to Dugbe virus. For each sequence, country, accession number, year of sampling, and host are shown. Sequences derived from virus growing in cell culture are marked by black triangles; sequences derived directly from infected ticks are marked by red circles. Novel sequences are marked in red. (B) Nanoluciferase (nLuc) reporter activity in Huh7 or A549 cells treated with VLPs generated using indicated combinations of viral protein components (L, GPC, and NP) from the IbAr10200 and MT-1303 strains. Error bars represent standard deviation of the mean of two independent biological replicates.

Figure 1.

Figure 1—figure supplement 1. Phylogenetic relationship of Malko Tarnovo strains to other CCHFV strains.

Figure 1—figure supplement 1.

Maximum likelihood phylogenies were inferred for the complete coding regions of the M-segment (A) and the S-segment (B), as well as for partial S-segment sequences (C). RAxML trees were performed with the GTR substitution model and 1000 bootstrap replicates. Only bootstrap values > 50 are shown. Trees were rooted to Dugbe virus. For each sequence, country, accession number, year of sampling, and host are shown. Sequences derived from virus growing in cell culture are marked by black triangles; sequences derived directly from infected ticks are marked by circles; asterisk marks partial segment sequences. Novel sequences are marked in red.
Figure 1—figure supplement 2. CCHFV Malko Tarnovo strains cannot be maintained through serial passaging in virus isolation attempts.

Figure 1—figure supplement 2.

Freshly seeded Vero E6/7 (African green monkey kidney), SW13 (Human Adrenal gland cortical small cell carcinoma), HAE/CTVM8 (Hyalomma anatolicum embryo) and BDE/CTVM16 (Rhipicephalus (Boophilus) decoloratus embryo) cells were inoculated with the supernatant of CCHFV-positive ticks and CCHFV-positive pools (MT-P-1302–1311 and MT-P-1358–1367). Eight days post-infection, supernatants of cell lines were passaged onto fresh cells. Before passaging, CCHFV RNA was measured in supernatants by qRT-PCR. Infected tick cells were observed for a period of 70 days.
Figure 1—figure supplement 3. MT-1303 L drives replication of minigenomes derived from divergent CCHFV strains.

Figure 1—figure supplement 3.

nLuc reporter activity levels from various combinations of minigenomes (nLuc flanked by L UTRs), L, and NP. Error bars represent standard deviation of the mean of two independent biological replicates.
Figure 1—figure supplement 4. Attenuation of VLP activity is attributed to MT-1303 GPC.

Figure 1—figure supplement 4.

nLuc reporter activity from Huh7 cells treated with VLPs generated using GPCs from diverse strains of CCHFV, as indicated. Error bars represent standard deviation of the mean of three technical replicate experiments.
Figure 1—figure supplement 5. Expression and processing of MT-1303 GPC.

Figure 1—figure supplement 5.

(A) Immunoblot analysis of Huh7 cell lysates transfected with N-terminal FLAG-tagged and C-terminal V5-tagged constructs of IbAr10200 or MT-1303 strain GPCs. (B) Immunofluorescence images of Huh7 cells transfected with expression constructs of wild-type GPC protein from the IbAr10200 or MT-1303 strains. Giantin was used to mark the Golgi apparatus. The mouse monoclonal 11E7 antibody was used to detect the Gc glycoprotein.