1. Microbiology and Infectious Disease
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A single mutation in Crimean-Congo hemorrhagic fever virus discovered in ticks impairs infectivity in human cells

  1. Brian L Hua
  2. Florine EM Scholte
  3. Valerie Ohlendorf
  4. Anne Kopp
  5. Marco Marklewitz
  6. Christian Drosten
  7. Stuart T Nichol
  8. Christina Spiropoulou
  9. Sandra Junglen  Is a corresponding author
  10. Éric Bergeron  Is a corresponding author
  1. Centers for Disease Control and Prevention, United States
  2. Institute of Virology, Charité-Universitätsmedizin Berlin, corporate member of Free University Berlin, Humboldt-University Berlin, and Berlin Institute of Health, Germany
  3. German Center for Infection Research (DZIF), Germany
Research Article
Cite this article as: eLife 2020;9:e50999 doi: 10.7554/eLife.50999
5 figures, 9 data sets and 5 additional files

Figures

Figure 1 with 5 supplements
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 supplement 1
Phylogenetic relationship of Malko Tarnovo strains to other CCHFV strains.

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.

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.

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.

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.

(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.

Figure 2 with 2 supplements
Poor infectivity of the MT-1303 strain in human cells is associated with a single amino acid variation in the Gc glycoprotein.

(A) Schematic representation of the chimeric FLAG/HA-tagged Gc constructs tested. MLD, mucin-like domain. (B) nLuc reporter activity levels of VLPs generated using the indicated chimeric Gc constructs in Huh7 cells. *p<0.05 and n.s. = not significant according to the two-tailed Student’s t-test; n = 3 independent biological replicates. (C) Region of Gc protein sequence alignment from various CCHFV strains highlighting the two amino acids unique to the MT-1303 strain. Asterisks (*) denote sequences derived directly from tick sources. Amino acid positions are denoted relative to the entire MT-1303 GPC. (D) nLuc reporter activity levels of VLPs generated using N-terminal FLAG/HA-tagged point mutant GPC constructs in Huh7 cells. *p<0.05 and n.s. = not significant according to the two-tailed Student’s t-test; n = 2 independent biological replicates. For all plots, error bars represent standard deviation of the mean. (E) Replication kinetics of recombinant viruses containing IbAr10200 S-ZsGreen and L segments and either IbAr10200 M or MT-1303 M-G1116R segment. BSR-T7/5, CHO, Huh7, A549, or ISE6 cells were infected at an MOI of 0.1, and ZsG fluorescence or TCID50 (ISE6 cells) was measured daily as an indicator of viral replication. Data represents three replicates and error bars represent standard deviation of the mean.

Figure 2—figure supplement 1
VLPs generated from MT-1303 GPC in Huh7 cells exhibit low reporter activity in tick-derived ISE6-recipient cells.

VLPs were generated in Huh7 cells from NP, L, and vL-nLuc reporter derived from IbAr10200 (A) or MT-1303 (B). These VLPs were generated with various GPC variants and then passaged onto either fresh Huh7 or ISE6 cells. Reporter activity was measured in recipient cells in technical triplicate (IbAr10200-based VLPs) or technical duplicate (MT-1303-based VLPs). Error bars represent standard deviation of the mean.

Figure 2—figure supplement 2
Activity of VLPs containing MT-1303 GPC is not restored at lower temperatures.

VLPs were generated with the given GPC in Huh7 cells incubated at either 28°C, 32°C, or 37°C. VLPs were harvested and incubated with fresh Huh7-recipient cells at either 28°C, 32°C, or 37°C. nLuc reporter activity level was measured in recipient cells relative to the mock GPC control. For all plots, error bars represent standard deviation of the mean of three technical triplicates.

The MT-1303 G1116 GPC variant promotes the formation of VLPs.

(A) Schematic of C-terminal V5-tagged IbAr10200 or MT-1303 strain GPC constructs. (B) Schematic representation of how the V5-tagged endodomain of CCHFV Gc glycoprotein is protected from proteolytic degradation. (C) Immunoblot analysis of virus-like particles generated by cells transfected with the IbAr10200 or MT-1303 V5-tagged GPC constructs. Closed arrowhead denotes the expected size of the mature CCHFV Gc glycoprotein (75 kDa). Open arrowhead denotes the expected size of the theoretical minimal protected V5 fragment (13.5 kDa). α-tubulin is used as a loading control.

Figure 4 with 1 supplement
MT-1303 G1116 GPC variant impairs membrane fusion.

(A) Fluorescent images of individual Huh7 syncytia in the GFP cell-cell fusion reporter assay. Cells expressing GPC and T7 polymerase were exposed to pH 6 DMEM media before co-culture with Huh7 cells transfected with a reporter plasmid containing GFP under control of the T7 promoter. Cell nuclei were stained with DAPI. Scale bar represents 100 μm. (B) Quantification of syncytia density. Error bars represent standard deviation of the mean of three biological replicate experiments. ****p<0.0001 and n.s. = not significant according to the two-tailed Student’s t-test with n = 3. (C) Quantification of syncytium area. For each condition, the areas of all of syncytia identified across three biological replicate experiments are shown. *p<0.05, ***p<0.001, ****p<0.0001, and n.s. = not significant according to the Mann-Whitney U-test with n = 3.

Figure 4—figure supplement 1
MT-1303 GPC-mediated membrane fusion activity is not restored at lower pH.

(A) Fluorescent images of individual Huh7 syncytia in the GFP cell-cell fusion reporter assay. Cells expressing the given GPC and T7 polymerase were exposed to DMEM media at pH 4, 5, 6, or 7.4 before co-culture with Huh7 cells transfected with a reporter plasmid containing GFP under control of the T7 promoter. Scale bar represents 500 μm. (B) Syncytia number per cm2 was quantified for each image.

Author response image 1

Data availability

All sequencing data have been deposited in GB under accession codes MK299338, MK299339, MK299340, MK299341, MK299342, MK299343, MK299344, MK299345 and MK299346.

The following data sets were generated
  1. 1
    NCBI GenBank
    1. BL Hua
    2. FE Scholte
    3. V Ohlendorf
    4. A Kopp
    5. M Marklewitz
    6. C Drosten
    7. ST Nichol
    8. C Spiropoulou
    9. S Junglen
    10. r Bergeron
    (2020)
    ID MK299338. Crimean-Congo hemorrhagic fever orthonairovirus strain Malko Tarnovo-BG2012-T1302 segment S, complete sequence.
  2. 2
    NCBI GenBank
    1. BL Hua
    2. FE Scholte
    3. V Ohlendorf
    4. A Kopp
    5. M Marklewitz
    6. C Drosten
    7. ST Nichol
    8. C Spiropoulou
    9. S Junglen
    10. r Bergeron
    (2020)
    ID MK299339. Crimean-Congo hemorrhagic fever orthonairovirus strain Malko Tarnovo-BG2012-T1302 segment M, complete sequence.
  3. 3
    NCBI GenBank
    1. BL Hua
    2. FE Scholte
    3. V Ohlendorf
    4. A Kopp
    5. M Marklewitz
    6. C Drosten
    7. ST Nichol
    8. C Spiropoulou
    9. S Junglen
    10. r Bergeron
    (2020)
    ID MK299340. Crimean-Congo hemorrhagic fever orthonairovirus strain Malko Tarnovo-BG2012-T1302 segment L, complete sequence.
  4. 4
    NCBI GenBank
    1. BL Hua
    2. FE Scholte
    3. V Ohlendorf
    4. A Kopp
    5. M Marklewitz
    6. C Drosten
    7. ST Nichol
    8. C Spiropoulou
    9. S Junglen
    10. r Bergeron
    (2020)
    ID MK299341. Crimean-Congo hemorrhagic fever orthonairovirus strain Malko Tarnovo-BG2012-T1303 segment S, complete sequence.
  5. 5
    NCBI GenBank
    1. BL Hua
    2. FE Scholte
    3. V Ohlendorf
    4. A Kopp
    5. M Marklewitz
    6. C Drosten
    7. ST Nichol
    8. C Spiropoulou
    9. S Junglen
    10. r Bergeron
    (2020)
    ID MK299342. Crimean-Congo hemorrhagic fever orthonairovirus strain Malko Tarnovo-BG2012-T1303 segment M, complete sequence.
  6. 6
    NCBI GenBank
    1. BL Hua
    2. FE Scholte
    3. V Ohlendorf
    4. A Kopp
    5. M Marklewitz
    6. C Drosten
    7. ST Nichol
    8. C Spiropoulou
    9. S Junglen
    10. r Bergeron
    (2020)
    ID MK299343. Crimean-Congo hemorrhagic fever orthonairovirus strain Malko Tarnovo-BG2012-T1303 segment L, complete sequence.
  7. 7
    NCBI GenBank
    1. BL Hua
    2. FE Scholte
    3. V Ohlendorf
    4. A Kopp
    5. M Marklewitz
    6. C Drosten
    7. ST Nichol
    8. C Spiropoulou
    9. S Junglen
    10. r Bergeron
    (2020)
    ID MK299344. Crimean-Congo hemorrhagic fever orthonairovirus strain Malko Tarnovo-BG2012-T1362 segment S, complete sequence.
  8. 8
    NCBI GenBank
    1. BL Hua
    2. FE Scholte
    3. V Ohlendorf
    4. A Kopp
    5. M Marklewitz
    6. C Drosten
    7. ST Nichol
    8. C Spiropoulou
    9. S Junglen
    10. r Bergeron
    (2020)
    ID MK299345. Crimean-Congo hemorrhagic fever orthonairovirus strain Malko Tarnovo-BG2012-T1362 segment M, complete sequence.
  9. 9
    NCBI GenBank
    1. BL Hua
    2. FE Scholte
    3. V Ohlendorf
    4. A Kopp
    5. M Marklewitz
    6. C Drosten
    7. ST Nichol
    8. C Spiropoulou
    9. S Junglen
    10. r Bergeron
    (2020)
    ID MK299346. Crimean-Congo hemorrhagic fever orthonairovirus strain Malko Tarnovo-BG2012-T1362 segment L, complete sequence.

Additional files

Supplementary file 1

Differences between strain Malko Tarnovo from tick T1303 (MTBG2012-T1303) and pathogen strains of other lineages.

In parentheses is information about the country and year of strain isolation, the clade into which the strain groups, and the host from which it was isolated. Numbers represent length of the complete nucleotide and amino acid sequences, or lengths of particular domains/proteins, positions of the domains/proteins in the complete amino acid sequence, and pairwise identity of the sequences compared to MTBG2012-T1303.

https://cdn.elifesciences.org/articles/50999/elife-50999-supp1-v2.docx
Supplementary file 2

Comparison of amino acid substitutions between protein sequences of MTBG2012-T1303 (accession numbers MK299341, MK299342, MK299343) and AP92 (accession numbers DQ211638, DQ211625, DQ211612), as well as between MT-BG2012-T1303 and strains comprising Europe one lineage (Kosovo Hoti, accession numbers DQ133507, EU037902, EU044832; Turkey200310849, accession numbers DQ211649, DQ211636, DQ211623; Turkey-Kelkit06, accession numbers GQ337053, GQ337054, GQ337055; Drosdov, accession numbers DQ211643, DQ211630, DQ211617; Kashmanov, accession numbers DQ211644, DQ211631, DQ211618).

Positions of substitutions are indicated by subscripted numbers.

https://cdn.elifesciences.org/articles/50999/elife-50999-supp2-v2.docx
Supplementary file 3

Cell culture passage history of CCHFV strains pertinent to this study.

https://cdn.elifesciences.org/articles/50999/elife-50999-supp3-v2.docx
Supplementary file 4

Primers used to generate chimeric and point mutant IbAr10200 and MT-1303 GPC expression constructs.

https://cdn.elifesciences.org/articles/50999/elife-50999-supp4-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/50999/elife-50999-transrepform-v2.docx

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