Geographic summary of countries represented in CCHFV seroepidemiological surveys.

<p>Countries with evidence of seroprevalence in animals represented in blue, countries with absence of seroprevalence represented in green, and countries without reported serosurveys represented in grey.</p

Total international CCHFV seroprevalence reported in domestic animals by species.

<p>Seroprevalence determined by sum of seropositive animals over the sum of total animals, sampled internationally. Studies that did not report sample numbers or differentiate between types of animal were excluded.</p

Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin

<div><p>Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family <i>Bunyaviridae</i> (genus: <i>Nairovirus</i>). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV’s high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in <i>trans</i>, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor’s maturation to GP38, and Gn precursor’s maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public health threat.</p></div

Optimization of support plasmid ratios for CCHFV rescue in BSR-T7/5.

<p>(A) BSR-T7/5 cells were transfected with 1 μg pT7-S, 2.5 μg pT7-M, 1 μg pT7-L, 0.66 μg pC-N, and 0.33 μg pC-L opti. Cell supernatants were collected and viral titers measured by determining TCID₅₀ at the indicated times post transfection. (B) In the experiments using 2:1 ratio of pC-N to pC-L opti, cells were transfected as in panel A except that 1 μg of pC-T7 was added to the transfection mix. In the experiment using a 19:1 pC-N:pC-L opti ratio, the same plasmid mix was used as for the 2:1 ratio, but with 0.95 μg of pC-N and 0.05 μg of pC-L opti. Error bars indicate means ± standard deviation. Statistical significance was evaluated using Student’s unpaired <i>t</i> test. Asterisk (*) indicates P < 0.05 at 3 days post transfection (2:1 versus 19:1). Dashed line indicates the limit of detection.</p

Furin effect on CCHFV-WT and-ASKA growth.

<p>FD11 and FD11-Fur cells were infected with CCHFV-WT or CCHFV-ASKA (MOI = 0.1). Cell supernatants were collected daily, and RNA S-segment copy numbers and infectious virus titers were measured by qRT-PCR and TCID₅₀ determination, respectively. Means ± standard deviation (n = 3) are plotted. Statistical significance was evaluated using Student’s unpaired <i>t</i> test. Asterisk (*) indicates P < 0.05.</p

Growth kinetics of CCHFV derived from cDNA.

<p>(A) BSR-T7/5 and (B) A549 cells were infected with 0.001 of 50% tissue culture infective dose (TCID₅₀)/cell of cDNA-derived CCHFV (circles) or parental virus isolate from Nigeria (squares). Viral titers were measured daily. Dashed line indicates the limit of detection.</p

L-RdRp gene codon optimization and recovery of CCHFV from DNA.

<p>(A) Reporter minigenome luciferase activity was measured 48 h after transfecting BSR-T7/5 cells seeded in 10 cm² wells. Cells were transfected with 250 ng of pC-L or pC-L opti, together with 500 ng of pC-N, 50 ng of pT7-M-Renilla [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004879#ppat.1004879.ref019" target="_blank">19</a>], and 30 ng of internal control pGL3 per well. Data are represented as fold increase in <i>Renilla</i> luciferase expression over control transfections in which pC-L was omitted. Error bars indicate means ± standard deviation (n = 3) (B) V5-tagged L-RdRp and N protein levels in cell lysates from panel A as described before [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004879#ppat.1004879.ref009" target="_blank">9</a>]. (C) BSR-T7/5 cells were transfected as presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004879#ppat.1004879.s001" target="_blank">S1C Fig</a> (pC-L support, upper panel), and also with pC-L opti in place of WT pC-L (lower panel). Four days post transfection, BSR-T7/5 cell supernatants were passaged onto SW13 cells, and viral antigens were detected with a GP38 domain-specific mAb (BSR-T7/5 IFA). Three days after passaging, viral cytopathic effect and CCHFV were visualized by bright field (SW13) or immunofluorescence (SW13 IFA) microscopy with anti-CCHFV hyperimmune mouse ascetic fluid (HMAF).</p

Furin enhances CCHFV propagation.

<p>CHO-derived cell lines used were parental clone 6 (Par6), furin-deficient (FD11), and FD11 stably expressing furin (FD11-Fur). Each cell line was infected with CCHFV at multiplicity of infection (MOI) = 1 or with Rift Valley fever virus expressing EGFP in place of NSs (RVFV-EGFP; MOI = 0.1). Percentage of infected cells was determined by immunostaining for CCHFV, or by EGFP detection for RVFV at 24 h (A) and 48 h (B) post infection. Black bars represent CCHFV-infected cells; white bars represent RVFV-infected cells. Error bars indicate means ± standard deviation (n = 3). Statistical significance was evaluated using Student’s unpaired <i>t</i> test. Asterisk (*) indicates P < 0.01.</p

Exchanging surface glycoproteins has a greater impact on tecVLP titer and NanoLuc signal than exchanging minigenomes.

<p>(A) TCID₅₀ titers/mL and NanoLuc signals in relative light units (RLU) measured in SW-13 cells treated with tecVLPs with Afg09 GPC and with L segment NCR minigenomes from indicated CCHFV strains. NanoLuc data are presented as standard error of the mean (average of 3 experiments), and results of 3 TCID₅₀ experiments are shown. (B) TCID₅₀ titers/mL and NanoLuc signals measured in SW-13 cells treated with tecVLPs with Oman GPC and L segment NCR minigenomes from indicated CCHFV strains. NanoLuc signal data in tecVLP-treated cells are presented as absolute RLU values which are calculated as signal in SW-13 cells treated with entry-competent VLPs (i.e., containing NP, L, and GPC) minus signal in SW-13 cells treated with VLPs containing only NP, L and the corresponding minigenome. NanoLuc data are reported as standard error of the mean (average of 3 experiments), and results of 3 TCID₅₀ experiments are shown.</p