Sensitive and specific detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-Specific IgM and IgG antibodies in human sera using recombinant CCHFV nucleoprotein as antigen in μ-capture and IgG immune complex (IC) ELISA tests.

As the most widespread tick-borne arbovirus causing infections in numerous countries in Asia, Africa and Europe, Crimean-Congo Hemorrhagic Fever Virus (CCHFV, family Nairoviridae) was included in the WHO priority list of emerging pathogens needing urgent Research & Development attention. To ensure preparedness for potential future outbreak scenarios, reliable diagnostic tools for identification of acute cases as well as for performance of seroprevalence studies are necessary. Here, the CCHFV ortholog of the major bunyavirus antigen, the nucleoprotein (NP), was recombinantly expressed in E.coli, purified and directly labeled with horseradish peroxidase (HRP). Employing this antigen, two serological tests, a μ-capture ELISA for the detection of CCHFV-specific IgM antibodies (BLACKBOX CCHFV IgM) and an IgG immune complex (IC) ELISA for the detection of CCHFV-specific IgG antibodies (BLACKBOX CCHFV IgG), were developed. Test performance was evaluated and compared with both in-house gold standard testing by IgM/IgG indirect immunofluorescence (IIF) and commercially available ELISA tests (VectoCrimean-CHF-IgM/IgG, Vector-Best, Russia) using a serum panel comprising paired samples collected in Kosovo during the years 2013-2016 from 15 patients with an acute, RT-PCR-confirmed CCHFV infection, and 12 follow-up sera of the same patients collected approximately one year after having overcome the infection. Reliably detecting IgM antibodies in all acute phase sera collected later than day 4 after onset of symptoms, both IgM ELISAs displayed excellent diagnostic and analytical sensitivity (100%, 95% confidence interval (CI): 85.2%-100.0%). While both IgG ELISAs readily detected the high IgG titers present in convalescent patients approximately one year after having overcome the infection (sensitivity 100%, 95% CI: 73.5%-100.0%), the newly developed BLACKBOX CCHFV IgG ELISA was superior to the commercial IgG ELISA in detecting the rising IgG titers during the acute phase of the disease. While all samples collected between day 11 and 19 after onset of symptoms tested positive in both the in-house gold standard IIFT and the BLACKBOX CCHFV IgG ELISA (sensitivity 100%, 95% CI: 71.5%-100.0%), only 27% (95% CI: 6.0%-61.0%) of those samples were tested positive in the commercial IgG ELISA. No false positive signals were observed in either IgM/IgG ELISA when analyzing a priori CCHFV IgM/IgG negative serum samples from healthy blood donors, malaria patients and flavivirus infected patients as well as CCHFV IgM/IgG IIFT negative serum samples from healthy Kosovar blood donors (for BLACKBOX CCHFV IgM/IgG: n = 218, 100% specificity, 95% CI: 98.3%-100.0%, for VectoCrimean-CHF-IgM/IgG: n = 113, 100% specificity, 95% CI: 96.8%-100.0%).

An infection with this zoonotic virus occurs either by tick bite or contact with body fluids or tissues of viremic humans or animals [5].After an incubation time of 1-9 days, unspecific flulike symptoms develop during the first week of disease including high fever, myalgia, arthralgia and headache [5].Following this prehemorrhagic phase, the disease may progress to the hemorrhagic phase manifesting in petechiae, hematomas, gastrointestinal bleeding (hematemesis, melena), urinary tract bleeding (hematuria) and/or respiratory tract bleeding (hemoptysis) [6].Case fatality rates (CFRs) range widely between 5% and more than 50% [6] with decreased platelet counts, elevated aspartate transferase and alanine transferase and decreased fribrinogen levels being predictive of fatal outcome [7].In addition, no CCHFV-specific humoral immune response is observable in the majority of fatal cases [7,8].
CCHF affects residents of endemic countries but can also be imported into non-endemic areas by travelers [1].Furthermore, the geographic distribution of the disease is expanding as evidenced by the prevalence of CCHFV-infected ticks [9] and the recent occurrence of autochthonous CCHF in Spain [10].Up to now, neither a vaccine with the potential to gain widespread international regulatory approval nor a specific treatment is available [4], which restricts prophylaxis to exposure prevention and therapy to mainly supportive measures.
Due to the severity of the disease, the lack of specific prophylactic and therapeutic options and its epidemic potential, CCHFV was included in the 2015 WHO priority list of diseases needing urgent R&D attention [11], in particular the development and improvement of diagnostic tools.Although the specificity of commercially available serological assays for the detection of CCHFV-specific antibodies in human sera was found to be excellent in a collaborative evaluation of CCHF diagnostic tests [3], the reported assay sensitivities of 87.8% (range 75.2%-95.3%)for IgM ELISA testing and 80% (range 66.9%-90.2%)for IgG ELISA testing [3] left room for improvements.
Therefore, we developed and validated both a CCHFV μ-capture ELISA (BLACKBOX CCHFV IgM ELISA) and a CCHFV IgG immune complex (IC) ELISA (BLACKBOX CCHFV IgG ELISA) based on the directly horseradish-peroxidase (HRP) labeled, recombinantly produced CCHFV ortholog of the major bunyavirus antigen [12], the nucleoprotein (NP).Assay performance was evaluated in comparison with in-house gold standard testing by IgM/IgG indirect immunofluorescence (IIF) and commercially available IgM/IgG ELISA tests (Vecto-Crimean-CHF-IgM/IgG, Vector-Best, Russia) using a serum panel comprising paired samples from 15 Kosovar patients with an RT-PCR-confirmed acute CCHFV-infection and 12 followup samples from the same group of patients taken approximately one year after they had overcome the disease.

Recombinant expression, purification and HRP-labeling of CCHFV-NP
Expression in E.coli: The plasmid pOPINJ-CCHFV-NP was transformed into E.coli pAPlacI Q cells.Cells were grown at 37˚C to an optical density at 600 nm of 0.5-0.6 in Luria-Bertani (LB) medium supplemented with kanamycin and ampicillin, then expression of the CCHFV-NP fusion protein was induced by the addition of isopropyl-β-D-thiogalactosid (IPTG).Bacteria were further incubated over night at 18˚C and then harvested by centrifugation.Cell lysis: Cells were resuspended in lysis buffer (50 mM NaH 2 PO 4 pH 8.0, 300 mM NaCl, 10 mM imidazole) supplemented with phenylmethylsulfonylfluorid (PMSF) and lysozyme and incubated at 4˚C for 30 min before sonification.After sonification, DNAse was added to a final concentration of 10 μg/ml.The lysate was incubated for 15 min on ice and then centrifuged at 10000 x g for 20 min at 4˚C.Ni-NTA chromatography (native conditions): After centrifugation of the lysate, the resulting supernatant was incubated with pre-equilibrated Ni-NTA-agarose (Qiagen) for 1 h at 4˚C.The matrix was transferred to a column and the flow-through was discarded.After washing the column, bound protein was eluted with Elution Buffer (50 mM NaH 2 PO 4 pH 8.0, 300 mM NaCl, 250 mM imidazole).On-column-cleavage of His 6 -GST tandem tag: The main fractions were pooled and the buffer was exchanged to Binding Buffer (50 mM NaH 2 PO 4 pH 7.2, 300 mM NaCl, 5 mM dithiothreitol (DTT)) using Zeba Spin Desalting Columns (Thermo Scientific).Subsequently, tagged recombinant CCHFV-NP was coupled to pre-equilibrated Glutathione HiCap matrix (Qiagen).Unbound material was removed from the column by washing with Binding Buffer.Recombinant GST-tagged 3C-protease was homogenously added to the column matrix and cleavage was performed over night at 4˚C with intermittent gentle shaking.Subsequently, CCHFV-NP was eluted from the column and concentrated using Amicon Ultra 15 micro concentrators (Merck Millipore, cut-off: 10k).Size exclusion chromatography (SEC): The eluted, concentrated protein was further purified by SEC using a Superdex 200 10/300 GL-column on an A ¨KTA pure chromatography system (GE Healthcare).Peak fractions containing recombinant CCHFV-NP were pooled and concentrated using Amicon Ultra 15 micro concentrators (cut-off: 10k).HRP-labeling: For labeling recombinant CCHFV-NP with HRP, HRP (Sigma) was activated for coupling with sodium periodate (final concentration 3.5 mg/ml) for 20 min at room temperature (RT).Subsequently, dialysis against 1 mM acetate buffer pH 4.4 was performed at 4˚C over night.Activated HRP was incubated with recombinant CCHFV-NP for 4 h at 4˚C and sodium borhydride was added at a final concentration of 0.2 mg/ml.After a 5 min incubation at RT, the labeling reaction mix was diluted 1:10 with Freezing Buffer (0.5 x phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA), 0.5% fetal calf serum (FCS), 1% Nonidet P40, 50% glycerol) and stored at -20˚C.

Data analysis
Statistical analysis (One-way ANOVA/Tukey's multiple comparison test, Fisher's exact test, calculation of 95% confidence intervals) was performed with GraphPad Prism.Receiver operating characteristic (ROC) curves were generated using MedCalc.

Sequence analyses
S segment nucleotide sequences of CCHFV strains were downloaded from the National Center for Biotechnology Information (NCBI) Nucleotide database.Alignments of derived protein sequences and generation of identity/similarity matrices were performed with the ClustalW alignment tool of the MacVector (version 12.7.5)software.

Human sera
CCHF patient sera: Paired serum samples were obtained from 15 Kosovar patients with a PCR-confirmed CCHFV-infection (13/15 (87%) male; 2/15 (13%) female; median age: 40 years (range 10-75) between 2013 and 2015 (2013: n = 10, 2014: n = 4, 2015: n = 1) in the course of the joint research project (BNITM, University of "Hasan Prishtina" and National Institute of Public Health, Pristhina, Kosovo) "Diagnosis and surveillance of Crimean-Congo hemorrhagic fever (CCHF) in Kosovo", funded by the German Ministry of Foreign Affairs (Project-No 68777 EN 02761868).Eight of 15 (53%) patients had a known history of tick bite (onset of symptoms after tick bite median 2.5 days (range 1-5 days)).From each of the 15 patients, one "early" sample collected between day 2 and day 14 after onset of symptoms (median: 5 days) and one "late" sample collected 3 to 23 days after the "early" sample (median: 10 days) between day 8 and day 36 after onset of symptoms (median: 17 days) were analyzed.CCHFVinfection has been proven by RT-PCR (RealStar CCHFV RT PCR Kit, Altona Diagnostics) for all 15 patients in a serum sample collected directly after admission to the hospital.Nevertheless, for five patients this sample was not available for ELISA testing due to scarcity of material.Thus, for all patients, the earliest available serum sample (which may or may not be PCR positive) was chosen as "early" sample for the ELISA analysis.In addition, "convalescent" samples were obtained from 12 patients approximately one year after recovery from CCHFV infection.IgM/IgG status of patient samples was characterized by in-house IgM/IgG IIFT using acetone-fixed Vero cells infected with CCHFV strain ArD39554 as described previously [16].Sera were inactivated by the addition of Triton X-100 to a final concentration of 1% prior to serological testing.
TBEV: 2 TBEV IgM/IgG positive sera were obtained from Biomex (Germany).Malaria: 27 sera from patients with an acute P. falciparum malaria (confirmed by IIFT) and 6 sera from patients with an acute P. malariae infection (confirmed by IIFT) were obtained from the BNITM diagnostics department (section parasitology).
Healthy blood donors (HD): HD Europe: 49 sera from healthy blood donors from Europe were taken at the BNITM between the years 2012 and 2016.HD Asia/HD South America: Sera of healthy blood donors from Lao PDR (n = 14) and Colombia (n = 14) were collected during the projects "Savannakhet-Hamburg Research Program on Neglected Diseases" and "Valledupar-Hamburg research programme on diagnostics and research on tropical and emerging infections" in 2012 and 2013, respectively.Healthy blood donors from Colombia had been vaccinated against YF.

Ethics statement
The study complies with the Declaration of Helsinki.Written informed consent was obtained from all individuals or, in case of minors, from parents or legal guardians before enrollment.Data privacy protection was guaranteed by anonymization of serum samples.
Collection of serum samples was approved by the Ethics Committee of the University of Prishtina "Hasan Pristhina" (sera from CCHF patients and healthy blood donors from Kosovo), the Ethics Committee of the Lao People's Democratic Republic (sera from healthy blood donors from Lao PDR, no.030/NECHR), the Ethics Committee of the Hospital Rosario Pumarejo de Lopez of Valledupar/Colombia (sera from healthy blood donors from Colombia) and the Ethics Committee of the A ¨rztekammer Hamburg (sera from Dengue fever patients, malaria patients and healthy blood donors from EU, no.PV4608).

Recombinant expression and purification of CCHFV-NP
In order to generate highly purified recombinant CCHFV-NP for use as an antigen in CCHFV ELISA applications a fusion protein consisting of an N-terminal His/GST-tandem tag and the

Development of IgM/IgG ELISA protocols using HRP-labeled recombinant CCHFV-NP as antigen
Using the HRP-labeled recombinant CCHFV-NP (CCHFV-NP HRP ) as antigen, both a μ-capture ELISA protocol for the detection of human anti-CCHFV IgM antibodies (designated as "BLACKBOX CCHFV IgM") and an IgG IC ELISA protocol for the detection of human anti-CCHFV IgG antibodies (designated as "BLACKBOX CCHFV IgG") were developed.
For the BLACKBOX CCHFV IgM ELISA, plates were coated with anti-human IgM antibodies and subsequently incubated for 1 h at RT with human serum and CCHFV-NP HRP .After washing, binding was quantified by measuring optical density after incubation with a chromogenic HRP-substrate.
For the BLACKBOX CCHFV IgG ELISA, plates were coated with a recombinant fragment of the human FcγR CD32 [15,17].Human serum and CCHFV-NP HRP were co-incubated for 24 h at 4˚C on the plates.After washing, binding of immune complexes was quantified by measuring optical density after incubation with a chromogenic HRP-substrate.

Detection range and reproducibility of BLACKBOX CCHFV IgM/IgG ELISAs
Titration of an IgM/IgG IIFT positive patient serum and analysis of a panel of a priori CCHFV IgM/IgG negative sera from healthy human blood donors and malaria patients indicated a broad detection range and low background signal of the assays (Fig 2).Furthermore, intraand inter-assay variation (Table 1, Table 2) as well as inter-laboratory variation (Table 3, Table 4) were analyzed, revealing a high reproducibility of both assay systems (mean intra- https://doi.org/10.1371/journal.pntd.0006366.g002assay CV for positive sera < 5%; mean inter-assay CV for positive sera < 10%, inter-laboratory CV for positive serum < 10%).

Determination of optimal assay cut-offs for the BLACKBOX CCHFV IgM ELISA and the BLACKBOX CCHFV IgG ELISA, assay sensitivities and specificities
For assay validation, a serum panel comprising serum samples from 15 CCHF patients from Kosovo (Table 5) was analyzed with the BLACKBOX CCHFV IgM ELISA (Fig 3A ) and the BLACKBOX CCHFV IgG ELISA (Fig 3B).From each of the 15 patients, one "early" sample collected between day 2 and day 14 after onset of symptoms (median: 5 days) and one "late" To determine the intra-and inter-assay variation of the BLACKBOX CCHFV IgG ELISA, the absolute OD values (OD450 -OD620) for three anti-CCHFV-NP IgG positive samples (pos1, pos2, pos3) and three anti-CCHFV-NP IgG negative samples (neg1, neg2, neg3) were measured in triplicate on three different days (day 1, day 2, day 3).Triplicates were used to calculate the intra-assay coefficient of variation for each individual experiment (CV intra , in %), mean CV intra (CV intra,av , in %) was calculated by averaging the three obtained CV intra values.The inter-assay coefficient of variation (CV inter , in %) was calculated according to the formula CV inter = 100 Ã (sd of mean av )/mean av with mean av being the mean of the mean OD values obtained for the respective sample on day 1, day 2 and day 3 and sd of mean av being the standard deviation of those three values. https://doi.org/10.1371/journal.pntd.0006366.t002 sample collected 3 to 23 days after the "early" sample (median: 10 days) between day 8 and day 36 after onset of symptoms (median: 17 days) were tested.In addition, "convalescent" samples taken from 12 patients approximately one year after recovery from CCHFV infection were analyzed.To evaluate assay specificity, 120 a priori CCHFV IgM/IgG negative sera originating from either healthy blood donors from Germany (Europe), Colombia (South America For determination of the optimal assay cut-offs, ROC analysis was performed using specific subsets of CCHF patient sera corresponding to the respective assay's intended diagnostic purpose (S2 Fig) .The main intention of performing an IgM ELISA is the diagnosis of acute infections as early in the course of disease as possible, whereas IgG testing rather becomes relevant at later stages of acute disease progression and for seroprevalence studies on healthy individuals.Therefore, assay cut-offs were optimized for optimal differentiation of PCR negative "early" and "late" samples from the a priori negative control samples for the BLACKBOX CCHFV IgM ELISA (panel  To determine the inter-laboratory variation of the BLACKBOX CCHFV IgM ELISA, n independent measurements of the absolute OD values (OD450 -OD620) for an anti-CCHFV-NP IgM positive sample and an anti-CCHFV-NP IgM negative sample were performed in three different laboratories.The inter-laboratory coefficient of variation (CV inter-lab , in %) was calculated according to the formula CV inter-lab = 100 Ã sd/mean with mean being the mean of the OD values obtained for the respective sample in the three different laboratories (each as a mean of n independent measurements) and sd being the standard deviation of those three values.
https://doi.org/10.1371/journal.pntd.0006366.t003To determine the inter-laboratory variation of the BLACKBOX CCHFV IgG ELISA, n independent measurements of the absolute OD values (OD450 -OD620) for an anti-CCHFV-NP IgG positive sample and an anti-CCHFV-NP IgG negative sample were performed in three different laboratories.The inter-laboratory coefficient of variation (CV inter-lab , in %) was calculated according to the formula CV inter-lab = 100 Ã sd/mean with mean being the mean of the OD values obtained for the respective sample in the three different laboratories (each as a mean of n independent measurements) and sd being the standard deviation of those three values.
https://doi.org/10.1371/journal.pntd.0006366.t0047) display a 100% specificity (95% CI: 98.3%-100.0%)and a 100% sensitivity (95% CI: 78.2%-100.0%)when testing "late" serum samples.While all five PCR negative "early" samples tested positive in the BLACKBOX CCHFV IgM ELISA, CCHFV-specific IgM antibodies could only be detected in 40% of the PCR positive "early" samples (Fig 3A, Table 6).The BLACK-BOX CCHFV IgG ELISA detected CCHFV-specific antibodies weakly in 2 out of 10 PCR positive "early" samples, but generated a clearly positive result in four out of five PCR negative   7).For all 6 patients whose PCR positive "early" samples tested negative in both the BLACKBOX CCHFV IgM ELISA and the BLACKBOX CCHFV IgG ELISA, clear IgM and IgG seroconversion was observed from the "early" to the "late" sample (panels A and B in S3 Fig).
Surprisingly, 9 out of 12 "convalescent" serum samples taken from CCHF patients more than one year after having overcome the disease tested weakly positive in the BLACKBOX IgM ELISA (Fig 3A , Table 6).Nevertheless, a clear differentiation between an acute CCHFV infection and the convalescent state is possible by calculating the IgG/IgM ratio defined as the quotient of the optical densities obtained when performing the BLACKBOX CCHFV IgG ELISA and the BLACKBOX CCHFV IgM ELISA, respectively (Fig 4A).While the IgG/IgM ratio in the patient samples taken during the acute phase of the disease ranges from 0 to < 3.0, the "convalescent" patient samples display a significantly higher IgG/IgM ratio (Fig 4A).

Comparison to in-house gold standard serological testing (CCHFV IgM/ IgG IIFT)
All CCHF patient samples had previously been analyzed with CCHFV IgM and CCHFV IgG IIFT (Table 5).While the BLACKBOX CCHFV IgM ELISA displayed a comparable sensitivity  as the IgM IIFT for all sample types tested (Table 6), the CCHFV IgG IIFT detected low IgG titers in 5 of the 10 PCR positive "early" samples while only 2 of those samples were recognized as IgG positive by the BLACKBOX CCHFV IgG ELISA (Table 7).Nevertheless, 4 of 5 PCR negative "early" samples as well as all "late" (15/15) and "convalescent" (12/12) samples tested positive in both the CCHFV IgG IIFT and the BLACKBOX CCHFV IgG ELISA (Table 7).Thus, the performance of both ELISAs is comparable to gold standard testing when applied according to their respective intended diagnostic purpose (BLACKBOX CCHFV IgM ELISA for acute diagnostics, BLACKBOX CCHFV IgG ELISA for later stages of disease progression and seroprevalence studies).

Comparison to commercially available ELISA tests (VectoCrimean-CHF-IgM, VectoCrimean-CHF-IgG; Vector-Best)
The same CCHFV serum panel which had been analyzed with the BLACKBOX CCHFV IgM ELISA and the BLACKBOX CCHFV IgG ELISA (15 serum pairs from CCHF patients, 12 sera from convalescent patients collected approximately one year after the infection), 15 a priori CCHFV-IgM/IgG negative control sera and 98 CCHFV IgM/IgG IIFT negative sera from healthy Kosovar blood donors were tested with both the VectoCrimean-CHF-IgM ELISA (Fig  6), the BLACKBOX https://doi.org/10.1371/journal.pntd.0006366.g005 CCHFV IgG ELISA was found to be more efficient than the VectoCrimean-CHF-IgG ELISA (Table 7) in detecting IgG antibodies in the "late" serum samples collected between day 8 and day 36 (median: day 17) after onset of symptoms.To dissect this observation more thoroughly, the 30 paired "early" and "late" serum samples were classified according to the day after onset on which they were collected (Fig 5

Correlation of IgM/IgG ELISA results with CCHFV IgM/IgG IIFT titers
Evaluation of the correlation of optical densities measured using the BLACKBOX and Vector-Best IgM/IgG tests and the antibody titers obtained by in-house CCHFV IgM/IgG IIFT (Fig 6) revealed a marked difference between serum samples collected during the acute/early convalescent phase of the disease and follow-up samples collected approximately one year after the patients having overcome the disease.All follow-up samples generated a highly positive signal in the BLACKBOX CCHFV IgG test even a titer of 1:640, the lowest CCHFV IgG IIFT titer observed in these samples.On the other hand, optical densities measured for acute/early convalescent phase sera with the identical CCHFV IgG IIFT titer varied strongly from sample to sample (Fig 6B).Thereby, later sampling correlated with higher signals (Fig 6B).(RF) was used as a capture molecule to bind immune complexes formed between pathogenspecific IgG antibodies and either native or recombinant viral antigens [16,24,[31][32][33].
Recently, we showed that the method could be further improved by replacing RF by the recombinantly produced tandem immunoglobulin-like domain of the human FcγR CD32 [17] and we have already demonstrated the potential of this by now patented assay format for the sensitive and specific detection of DENV-subtype-specific IgG antibodies [15].In nature, CCHFV circulates in an enzootic tick-vertebrate-tick cycle and can be transmitted from livestock to human by contact with blood or other body fluids during activities like slaughtering [34].Thus, ELISA tests for animal sera like the double-antigen sandwich ELISA recently developed by Sas et al. [35] are needed for surveillance purposes.The pronounced interspecies cross-reactivity of the recombinant CD32 fragment [17] may facilitate the development of such tests.
To evaluate the performance of the newly developed BLACKBOX CCHFV IgM/IgG ELISA tests, acute and convalescent phase sera from 15 patients with a PCR-confirmed CCHFV infection collected in Kosovo between 2013 and 2016 have been analyzed.Kosovo is a landlocked, southeastern European country on the central Balkan Peninsula with a population of 1.9 million.Due to a hot, dry climate favoring vector prevalence and extensive agricultural use, CCHFV is endemic in approximately half of its territory of 10,908 km 2 [36].Between 1954 and 2014, 304 confirmed cases of CCHF were reported with a case fatality rate (CFR) of 21% with the highest incidence in late spring/early summer (May-July) [37].A seroprevalence of up to 9% was found in inhabitants of hyperendemic areas, the majority (70%) of seropositive individuals being males of advanced age (median age 62) who had probably been exposed to the virus during professional activities like farming or slaughtering [36].Correspondingly, a high seroprevalence of up to 20% was observed in livestock (cows, goats, sheep) being kept in endemic areas [36].Recently, analysis of tick species revealed Hyalomma marginatum as the dominant species (90% of all collected specimen) in endemic municipalities [38].The percentage of infected H. marginatum ticks in endemic municipalities varied strongly between sampling sites (range 1.25%-29.4%,average 11%) [38].Partial S segment sequences from all CCHFV-positive ticks could be classified as two different lineages, Kosovo I and II, in CCHFV clades VI (Europe 2) and V (Europe 1), respectively.Thereby, sequences isolated from H. marginatum ticks cluster with Kosovar human-derived sequences in clade V (Europe 1), suggesting that the Kosovo outbreaks are most likely due to H. marginatum transmitted CCHFV [38].
Results obtained with the BLACKBOX IgM/IgG ELISAs were compared with the respective in-house serological gold standard (CCHFV IgM/IgG IIFT) and commercial ELISA tests (Vec-toCrimean-CHF-IgM/IgG, Vector-Best, Russia) that have been extensively used for seroprevalence studies in various countries (e.g.Turkey [39][40][41][42], Greece [43][44][45][46][47], Bulgaria [48], Afghanistan [49], Kosovo [36], Tunisia [50], Ghana [51]) and for the diagnosis of acute cases [52,53].Recently, a collaborative evaluation of both the VectoCrimean-CHF-IgG and the Vec-toCrimean-CHF-IgM test in five reference laboratories revealed a sensitivity of 87.8% (range 75.2%-95.3%)for the VectoCrimean-CHF-IgM test and a sensitivity of 80.4% (range 66.9%-90.2%)for the VectoCrimean-CHF-IgG test when compared with the respective in-house reference serological tests [3].Specificities were found to be 98.9% (range 93.9%-100.0%)for the VectoCrimean-CHF-IgM tests and 100% (range 95.8%-100%) for the VectoCrimean-CHF-IgG test [3].Here, we found that both the newly developed BLACKBOX CCHFV IgM ELISA (employing recombinant CCHFV NP) and the VectoCrimean-CHF-IgM test readily detected CCHFV specific IgM antibodies in all samples collected after day 4 after onset of symptoms corresponding to a sensitivity of 100% when compared to the in-house gold standard CCHFV IgM IIFT.Both the newly developed BLACKBOX CCHFV IgG ELISA employing recombinant CCHFV NP and the VectoCrimean-CHF-IgG test generated strong positive results with serum samples originating from convalescent patients approximately one year after having overcome the disease.However, the BLACKBOX CCHFV IgG ELISA was found to show a superior sensitivity in the early detection of the rising IgG antibody titer in acutely ill patients: the BLACKBOX CCHFV IgG ELISA recognized, like the in-house gold standard IgG IIFT, all tested patient samples which were collected after day 11 after onset of symptoms as IgG positive, whereas only 43% of those samples tested positive in the VectoCrimean-CHF-IgG test.This increased sensitivity of the BLACKBOX CCHFV IgG ELISA may also be of importance for the main intended use of an IgG assay, that is seroprevalence studies.
When analyzing the correlation between CCHFV IgG IIFT titers and the OD values obtained using the BLACKBOX CCHFV IgG ELISA, a striking difference between serum samples collected during the acute/early convalescent phase of the disease and follow-up samples collected approximately one year after the patients having overcome the disease was observed.While the latter samples stably generated high OD values in the ELISA even at a rather low CCHFV IgG IIFT titer of 1:640, the signal heights of different samples with an identical CCHFV IgG IIFT titer varied strongly for acute/early convalescent phase samples.Thereby, higher signals correlated with later sampling.Thus, the very first IgG antibodies generated during acute CCHFV infection presumably do not predominantly recognize the CCHFV nucleoprotein but rather other antigens like the virus envelope glycoprotein G c and therefore are not detectable with serological tests employing solely CCHFV NP as an antigen.A similar observation was made by Putkuri et al. [54] for a member of orthobunyavirus genus, the Inkoo virus (INKV).Here, the fraction of IgG antibodies recognizing either the nucleoprotein or the glycoprotein Gc, respectively, was found to differ considerably between serum samples from individuals with a long-standing immunity and samples collected during acute infection [54].
Both BLACKBOX CCHFV ELISAs employ the nucleoprotein of CCHFV strain Afg09-2990 (belonging to S segment lineage IV (Asia1), [55]) as antigen and were validated using CCHF serum samples originating from Kosovo.Thus, the patients' infections were most likely caused by CCHFV strains belonging to S segment lineage V (Europe 1) [38,56].To further assess the potential of the two BLACKBOX tests to detect anti-NP-antibodies induced by infections with CCHFV strains belonging to different S segment lineages, a sequence alignment of the nucleoprotein (NP) amino acid (aa) sequences of six CCHFV strains belonging to five different S segment lineages [55] was performed (panels A and B in S4 Fig) .Sequence identities between the full length NP of the Afg09-2990 strain (lineage IV, Asia 1) and strains ArD157856 (lineage I, Africa 1), UG3010 (lineage II, Africa 2), ArD39554 (lineage III, Africa 3), Kosovo Hoti (lineage V, Europe 1) and Drosdov (lineage V (Europe 1)) were found to be 94.8%, 96.9%, 97.3%, 96.9% and 96.7%, respectively (panel C in S4 Fig) .When the analysis was restricted to the NP stalk domain (aa 180-300) harboring the main antigenic region of the protein [21][22][23], even higher percentages of identity were observed with strains UG3010 (97.5%),ArD39554 (99.2%),Kosovo Hoti (99.2%), and Drosdov (99.2%) (panel C in S4 Fig) .Therefore, the BLACKBOX tests most probably allow reliable detection of anti-CCHFV NP antibodies induced by CCHFV strains from different endemic regions of the world.This assumption is further supported by recent findings of Sas et al. [35] who demonstrated cross-detection of anti-CCHFV-NP antibodies in sera of animals infected with CCHFV strains belonging to S segment lineages III (Africa 3), IV (Asia 1), and V (Europe 1) in a double-antigen sandwich ELISA based on recombinant CCHFV NP derived from strain lbAr10300 (S segment lineage III (Africa 3)).
To our knowledge no information is publicly available on the precise nature of the antigen (native or recombinant antigen, CCHFV strain) used in the Vector-Best ELISAs.Nevertheless, Vanhomwegen et al. [3] cite a personal communication stating that the manufacturer validated the tests with a serum panel from CCHF cases originating from southwestern Russia.Therefore, we included a CCHFV strain originating from that region (Drosdov) in the alignment.Pairwise comparison of the full length NP sequence of the Kosovo Hoti strain with the Afg09-2990 NP (used as antigen in the BLACKBOX tests), the ArD39554 NP (full virus used in the IIFT tests) and the Drosdov NP revealed an identity of 96.9%, 96.9%, and 99.4%.When the analysis was restricted to the NP stalk domain, percentages were even higher (99.2%, 98.3%, and 100.0%).Thus, it is unlikely that the major reason for the observed differences in sensitivity of the BLACKBOX CCHFV IgG ELISA, the VectoCrimean-CHF-IgG ELISA and the CCHFV IgG IIFT is the use of CCHFV antigens resulting from different S segment lineages.
Due to the presence of all viral antigens in their native confirmation including potential posttranslational modifications, CCHFV IgG IIFT performed on CCHFV-infected Vero-cells is still the most sensitive method for detecting low levels of CCHFV-specific IgG antibodies in patient sera taken in the early phase of the disease.In addition, the obtained staining pattern allows the internal assessment of signal specificity.Nevertheless, application of this method requires a significant effort in the generation of assay components (mainly the infection of Vero cells with CCHFV under BSL-4 conditions), expensive and sensitive equipment (i.e. a fluorescence microscope) and last but not least specialized expertise for test evaluation.In contrast, ELISA components can be produced under BSL-1 conditions and, although assays have to be performed by trained lab personnel, no specialized expertise is necessary to evaluate the result.
Beside the competitive sensitivity and specificity of the BLACKBOX CCHFV ELISA tests, there are several advantages of the newly developed tests.First of all, the virus antigen can be produced in large amounts in E.coli; no cost-efficient eukaryotic expression system or cultivation of native virus is necessary.Furthermore, the BLACKBOX CCHFV IgG ELISA requires, due to the co-incubation of recombinant antigen and the patient serum, only one washing step and thus is easy to perform with only short hands-on times.Last but not least, all assay components are routinely produced and quality controlled in a specialized laboratory of the Bernhard Nocht Institute for Tropical Medicine (Hamburg, Germany) and thus can be easily made available to the scientific community.
In summary, two new serological tests (BLACKBOX CCHFV IgM and BLACKBOX CCHFV IgG) for the identification of acute CCHF cases and the performance of seroprevalence studies have been developed.The tests employ recombinant CCHFV NP as antigen and exhibit high reproducibility (Inter-/inter-assay and inter-laboratory variation) and competitive assay performance (sensitivity, specificity) in comparison with in-house gold standard testing by IIFT and commercially available test kits.
in S2 Fig, cut-off: 0.129) and PCR negative "late" and "convalescent" samples from the a priori negative control samples for the BLACKBOX CCHFV IgG ELISA (panel B in S2 Fig, cut-off: 0.161), respectively.

Table 1 . Intra-and interassay variation of BLACKBOX CCHFV IgM ELISA. day 1 day 2 day 3 sample mean sd CV intra mean sd CV intra mean sd CV intra CV intra, av mean av sd of mean av CV inter
To determine the intra-and inter-assay variation of the BLACKBOX CCHFV IgM ELISA, the absolute OD values (OD450 -OD620) for three anti-CCHFV-NP IgM positive samples (pos1, pos2, pos3) and three anti-CCHFV-NP IgM negative samples (neg1, neg2, neg3) were measured in triplicate on three different days (day 1, day 2, day 3).Triplicates were used to calculate the intra-assay coefficient of variation for each individual experiment (CV intra , in %), mean CV intra (CV intra,av , in %) was calculated by averaging the three obtained CV intra values.The inter-assay coefficient of variation (CV inter , in %) was calculated according to the formula CV inter = 100 Ã (sd of mean av )/mean av with mean av being the mean of the mean OD values obtained for the respective sample on day 1, day 2 and day 3 and sd of mean av being the standard deviation of those three values. https://doi.org/10.1371/journal.pntd.0006366.t001