- Email: [email protected]
A competitive ELISA for species-independent detection of Crimean-Congo hemorrhagic fever virus specific antibodies
Accepted Manuscript A competitive ELISA for species-independent detection of Crimean-Congo hemorrhagic fever virus specific antibodies Isolde Schuster, Marc Mertens, Bernd Köllner, Tomáš Korytář, Markus Keller, Bärbel Hammerschmidt, Thomas Müller, Noël Tordo, Philippe Marianneau, Claudia Mroz, Melanie Rissmann, Eileen Stroh, Lisa Dähnert, Felicitas Hammerschmidt, Rainer G. Ulrich, Martin H. Groschup PII:
S0166-3542(16)30427-2
DOI:
10.1016/j.antiviral.2016.09.004
Reference:
AVR 3902
To appear in:
Antiviral Research
Received Date: 4 August 2016 Revised Date:
8 September 2016
Accepted Date: 9 September 2016
Please cite this article as: Schuster, I., Mertens, M., Köllner, B., Korytář, T., Keller, M., Hammerschmidt, B., Müller, T., Tordo, N., Marianneau, P., Mroz, C., Rissmann, M., Stroh, E., Dähnert, L., Hammerschmidt, F., Ulrich, R.G., Groschup, M.H., A competitive ELISA for species-independent detection of Crimean-Congo hemorrhagic fever virus specific antibodies, Antiviral Research (2016), doi: 10.1016/j.antiviral.2016.09.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Isolde Schuster1, Marc Mertens1, Bernd Köllner2, Tomáš Korytář2, Markus Keller1, Bärbel Hammerschmidt3, Thomas Müller4, Noël Tordo5, Philippe Marianneau6, Claudia Mroz1, Melanie Rissmann1, Eileen Stroh1, Lisa Dähnert1, Felicitas Hammerschmidt7, Rainer G. Ulrich1, Martin H. Groschup1*
Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany 2
Institute of Immunology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany
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Department of Experimental Animal Facilities and Biorisk Management, FriedrichLoeffler-Institut, Greifswald-Insel Riems, Germany 4
Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany 5
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Unit Antiviral Strategies Antivirales, WHO collaborative Centre for Viral Haemorrhagic Fevers and Arboviruses, OIE Reference Laboratory for RVFV and CCHFV, Institut Pasteur, Paris, France 6
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Unit Virology; ANSES-Laboratoire de Lyon, Lyon, France
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A competitive ELISA for species-independent detection of Crimean-Congo Hemorrhagic fever virus specific antibodies
Chair of Food Safety, Faculty of Veterinary Medicine, Ludwig-MaximiliansUniversity Munich (LMU), Oberschleissheim, Germany
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*Corresponding author: Prof. Dr. Martin H. Groschup, Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Suedufer 10, Germany, [email protected]
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Abstract
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Crimean-Congo hemorrhagic fever virus (CCHFV) circulates in many countries of
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Asia, Africa, and Europe. CCHFV can cause a severe hemorrhagic fever in humans
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with case-fatality rates of up to 80%. CCHF is considered to be one of the major
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emerging diseases spreading to and within Europe. Ticks of the genus Hyalomma
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function as vector as well as natural reservoir of CCHFV. Ticks feed on various
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domestic animals (e.g. cattle, sheep, goats) and on wildlife (e.g. hares, hedgehogs).
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Those animal species play an important role in the life cycle of the ticks as well as in
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amplification of CCHFV.
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Here we present a competitive ELISA (cELISA) for the species-independent
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detection of CCHFV-specific antibodies. For this purpose nucleocapsid (N) protein
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specific monoclonal antibodies (mAbs) were generated against an Escherichia coli
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(E. coli) expressed CCHFV N-protein. Thirty-three mAbs reacted with homologous
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and heterologous recombinant CCHFV antigens in ELISA and Western blot test and
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20 of those 33 mAbs reacted additionally in an immunofluorescence assay with
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eukaryotic cells expressing the N-protein. Ten mAbs were further characterized in a
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prototype of the cELISA and nine of them competed with positive control sera of
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bovine origin. The cELISA was established by using the mAb with the strongest
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competition. For the validation, 833 sera from 12 animal species and from humans
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were used. The diagnostic sensitivity and specificity of the cELISA was determined to
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be 95% and 99%, respectively, and 2% of the sera gave inconclusive results.
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This cELISA offers the possibility for future large-scale screening approaches in
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various animal species to evaluate their susceptibility to CCHFV infection and to
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identify and monitor the occurrence of CCHFV.
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Highlights:
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• Monoclonal
antibodies
(mAbs)
recognizing
native
Crimean-Congo
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Hemorrhagic Fever Virus (CCHFV) antigen were generated
• Antigen binding of several mAbs is outcompeted by anti-CCHFV positive sera
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• The diagnostic sensitivity and specificity of the recombinant N-protein based CCHFV competitive ELISA were 95% and 99%
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Keywords:
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CCHFV, Crimean-Congo Hemorrhagic Fever, competitive ELISA, amplifying hosts,
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monoclonal antibody, nucleocapsid protein
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1. Introduction
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Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus, belonging to
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the genus Nairovirus in the family Bunyaviridae. CCHFV is endemic in many
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countries of Africa, Asia and South-Eastern Europe (Hoogstraal, 1979). It is
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considered to be one of the major zoonotic pathogens, spreading to and within
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Europe (Mertens et al., 2013). Inside Europe, its distribution closely correlates with
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the distribution of ticks of the species Hyalomma marginatum, which are both vector
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and reservoir of CCHFV (Whitehouse, 2004).
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In nature, the virus circulates in a tick-vertebrate-tick cycle, whereby the vertebrates
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play an important role in the life cycle of the ticks as well as in amplification of the
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virus (Whitehouse, 2004; Zeller et al., 1994). Within the tick population CCHFV can
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be transmitted horizontally by co-feeding, by venereal or transstadial transmission,
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and vertically by transovarial transmission (Gonzalez et al., 1992; Logan et al., 1989).
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Hyalomma marginatum ticks feed on various domestic animals (e.g. cattle, sheep,
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goats) and on wildlife (e.g. hares, hedgehogs). Although, there is no evidence that
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CCHFV infections causes clinical signs in animals (Ergonul, 2006; Whitehouse,
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2004), they can develop a viremia lasting for up to 15 days with a subsequent
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seroconversion (Causey et al., 1970; Gonzalez et al., 1998; Zeller et al., 1994).
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In humans, CCHFV infections can cause a severe hemorrhagic disease with case
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fatality rates of up to 80% (Ergonul, 2006; Whitehouse, 2004; Yen et al., 1985).
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Infection routes are tick-bites, crushing of infected ticks, and contact with body fluids
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or tissues of infected animals or humans. Neither vaccination nor specific treatment is
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currently available (Ergonul, 2006).
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ACCEPTED MANUSCRIPT In Europe, human Crimean-Congo hemorrhagic fever (CCHF) cases are regularly
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reported from Albania, Bulgaria and the Republic of Kosovo (Avšič-Županc, 2008;
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Papa et al., 2004; Papa et al., 2010; Papa et al., 2011). In Turkey, 9069 CCHF cases
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were reported between 2002 and 2014 (Mertens et al., 2016). Isolated CCHF cases
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have previously been reported from Hungary and Greece (Hornok and Horvath,
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2012; Papa et al., 2008). Furthermore, CCHF cases have just recently been reported
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from Spain (The Spain Report, 2016). Next to the countries with endemic areas,
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almost no information is available about the distribution of CCHFV in Europe
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(Mertens et al., 2013; Mertens et al., 2016).
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Because of its impact on human health, the distribution of CCHFV should be
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investigated in detail and potential spread should be monitored to increase the
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awareness of people at risk and for the implementation of adequate protection
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measures (Mertens et al., 2013; Spengler et al., 2016). Therefore, amongst others,
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an appropriate knowledge on amplifying hosts of the virus is necessary (Spengler et
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al., 2016).
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Some mammal and avian species are already identified to be an important part in the
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ecology of the tick vectors, such as cattle, sheep, goats, hares, hedgehogs, camels
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and ostriches. However, their role in the infection cycle of CCHFV, and especially the
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impact of other species remains only partially understood. To explore the role of
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different species, animal experiments can be used. Since CCHFV is a biosafety level
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(BSL) 4 pathogen, they are difficult to conduct, because they require high
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biocontainment facilities (Keshtkar-Jahromi et al., 2011; Mertens et al., 2013).
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Alternatively, large-scale seroepidemiological studies on different animal species can
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be used to figure out which species are involved in the infection cycle of CCHFV.
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ACCEPTED MANUSCRIPT Since the antibody prevalence in animals is a good indicator for the presence or
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absence of CCHFV in the regarding region, those studies can further be used for the
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investigation of the distribution of CCHFV (Hoogstraal, 1979).
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Several seroepidemiological studies about the prevalence of CCHFV-specific
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antibodies in cattle, sheep and goats are published (Spengler et al., 2016). Different
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assay formats, based on complement fixation, immunofluorescence (IFA), reverse
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passive hemagglutination inhibition, neutralization, immunodiffusion and ELISA, were
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used in those studies (Spengler et al., 2016; Whitehouse, 2004). Since no assays are
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commercially available for CCHFV serology in animals, those assays are either in-
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house assays or commercial tests for CCHFV serology in humans, which were
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adapted for testing animals (Mertens et al., 2013). The applicability of those assays
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for different animal species is limited, either because the assay formats are
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considered as outdated or because species-specific monoclonal or polyclonal
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antibody conjugates are required, which are difficult to obtain (OIE, 2014; Spengler et
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al., 2016). Besides, species-dependent adaptation or assay development and
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validation are time-consuming. Additional obstacles are the need for corresponding
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reference serum panels from the species the assay should be used for (OIE, 2014).
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Next to the lack of an accepted gold standard for CCHFV serology in animals, this
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might possibly be one of the reasons why most of the published tests were either not
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validated or the data of their validation were not published (Mertens et al., 2013).
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The aim of the work presented here was the development and validation of a
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competitive ELISA (cELISA) for the species-independent detection of CCHFV-
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specific antibodies. This cELISA can be used for large-scale screening approaches
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for identification of amplifying hosts as well as for monitoring of CCHFV foci.
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2. Materials and methods
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2.1 Recombinant antigens
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Recombinant nucleocapsid (N) proteins of CCHFV strains Kosovo Hoti (GenBank
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accession no. DQ133507) and Turkey-Kelkit06 (GenBank accession no. GQ337053)
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were expressed in Escherichia coli (E. coli) with an N-terminal deca-His-tag and
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purified by nickel-chelate affinity chromatography (Qiagen, Hilden, Germany). The
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divergence in the aminoacid sequence between both CCHFV strains is 1.2%.
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Puumala hantavirus (PUUV) N-protein expressed in yeast Saccharomyces cerevisiae
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was used as a negative control antigen (Mertens et al., 2011).
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2.2 Generation and characterization of monoclonal antibodies
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Monoclonal antibodies (mAbs) were generated by intramuscular immunization of
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BALB/c mice three times in intervals of ten days. As antigen 50 µg of purified and in
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Aqua dest dialyzed recombinant E. coli expressed N-protein of CCHFV strain Kosovo
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Hoti were used.
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The recombinant N-protein was mixed with complete Freund's adjuvant for the initial
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immunization (Sigma, Deisenhofen, Germany), and for the second and third
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immunization the protein was mixed with incomplete Freund's adjuvant (Sigma). Four
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days prior fusion the mice were boosted intraperitoneally with 50 µg of the N-protein
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without adjuvant.
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Hybridoma supernatants were screened by an in-house ELISA and Western blot test
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based on the homologous and heterologous (N-protein of CCHFV-strain Turkey-
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Kelkit06) antigen and by an adapted commercial IFA basing on N-protein of CCHFV
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strain
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supernatants were also tested in an ELISA using PUUV N-protein. The mAbs
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showing the highest reactivity with all used CCHFV antigens were additionally tested
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in a prototype of the cELISA regarding their ability to compete with anti-CCHFV
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positive bovine sera.
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Isotyping of the finally chosen mAb was performed using the Pierce Rapid ELISA
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Mouse mAb Isotyping Kit (Thermo Scientific, Waltham, MA, USA). Afterwards, the
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mAb was purified using fast protein liquid chromatography (FPLC) and adjusted to a
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concentration of 1 µg/µl in 0.1% glycine, neutralized with a 1/10 volume of 1 M Tris
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pH 8.0. Finally, 0.01% sodium azide was added and aliquots of the mAb were stored
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either at 4°C (short-term storage) or -20°C (long-t erm storage).
(Euroimmun,
Lübeck,
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As
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hybridoma
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2.3 Sera
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Development and validation of the cELISA was conducted with sera from various
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animal species as well as from humans (Table 1). Many of the sera used for
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validation were already used in previously published studies (Lugaj et al., 2014a;
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Lugaj et al., 2014b; Lugaj et al., 2014c; Mertens et al., 2011; Mertens et al., 2015;
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Mertens et al., 2016; Schuster et al., 2016).
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As positive reference, sera from Albania, the Republic of Kosovo, Turkey, and from
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the Former Yugoslav Republic of Macedonia were used for test development and
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1).
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Additionally, sera of two rhesus macaques (Macaca mulata), which had been
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immunized with purified inactivated full virus of CCHFV strain IbAr 10200, were used
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as positive reference sera, and a serum from a non-immunized rhesus macaque was
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used as negative reference serum (Table 1).
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2.4 Development and validation of a cELISA for the detection of CCHFV-
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specific antibodies
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2.4.1 Development of a cELISA
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The cELISA was developed using a His-tagged recombinant N-protein of CCHFV
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strain Kosovo Hoti as antigen. The establishment of the test was conducted by
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variation of plate type, coating conditions, antigen amount, incubation temperatures
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and times, dilution buffers, washing buffers, washing frequency, serum dilution, mAb
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dilution, conjugate dilution, substrate incubation time, as well as the procedure of
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application of the mAb. The optimal ratio between serum-, mAb- and conjugate-
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dilution was evaluated by checkerboard titrations. Finally, the protocol was chosen,
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which best distinguished between the positive and negative reference serum
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samples.
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Prior to the coating step, the recombinant protein was spiked 1:1 with glycerin and
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incubated for 1 h at 4°C. 96-well PolySorp immuno-p lates (Nunc, Roskilde, Denmark)
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were coated with 2 µg antigen, diluted in PBS pH 10 containing 10% glycerin. Plates
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were sealed with a foil and subsequently incubated over night at 4°C.
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plates and were incubated for 1 h at 37°C. Afterwar ds, the plates were washed four
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times using 250 µl/well PBS-Tween20 1%.
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Sera were diluted 1:4 in 1% BSA in PBS-Tween20 0.1% and 95 µl of the serum
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dilution were added to each well. As controls, dilutions of a positive cattle serum from
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Turkey and of a negative sheep serum sample from Germany, as well as only buffer
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were added to duplicate wells on the plate.
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Plates were incubated for 1 h at 37°C. The mAb was diluted 1:50 in 1% BSA in PBS-
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Tween20 0.5% and 5 µl of the dilution were added to each well containing 95 µl of
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the serum dilution or buffer. The plates were incubated for 1 h at 37°C.
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After washing, 100 µl/well of anti-mouse horse-radish peroxidase (HRP) conjugate
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(Bio-Rad, Munich, Germany), diluted 1:1,000 in 1% BSA in PBS-Tween20 0.05%,
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were added and plates were incubated for 2 h at 37°C.
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Plates were washed and 100 µl/well TMB solution (Bio-Rad) were added and
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incubated for 10 min at room temperature. The reaction was stopped using H2SO4.
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The optical density (OD) was measured at 450 nm (reference wavelength 620 nm).
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Results were calculated as percent inhibition (PI) of the OD-value of each serum
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sample compared to the OD-value of the negative control serum (PI = 100-(OD-value
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serum sample/OD-value negative control sample) x 100).
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2.4.2 Validation of the cELISA
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ACCEPTED MANUSCRIPT Since no gold standard exists in CCHFV serology, the cELISA was validated by
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comparative analysis.
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Sera from cattle, sheep and goats from countries with areas endemic for CCHFV
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were prior characterized in an adapted commercial ELISA (Vector Best, Novosibirsk,
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Russia), and IFA (Euroimmun, Lübeck, Germany) (Mertens et al., 2015; Schuster et
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al., 2016). Sera from rhesus macaques were characterized in the commercial ELISA
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and IFA according to the protocols given by the manufacturers. Samples, which were
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tested positive in both assays, were defined as positive reference sera.
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Because there is no evidence for circulation of CCHFV in Germany, sera from
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Germany were used as negative reference sera.
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The cut-off values for the cELISA were determined by receiver-operator
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characteristics (ROC) analysis. The diagnostic sensitivity was determined as ratio of
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the number of true positive samples divided by the number of true positive plus false
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negative samples and given as percentage. The diagnostic specificity was
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determined as ratio of the number of true negative samples divided by the number of
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true negative plus the number of false positive samples and given as percentage.
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3. Results
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3.1 Generation and Characterization of mAbs
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Thirty three hybridoma supernatants reacted in Western blot test and in-house ELISA
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with the homologous recombinant N-protein of CCHFV strain Kosovo Hoti and the
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three supernatants were also tested positive in the commercial IFA, while four were
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inconclusive and nine were negative. None of the hybridoma supernatants reacted
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with the PUUV N-protein.
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Ten of the twenty mAbs, giving a positive signal in the initial CCHFV screening tests,
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were randomly chosen for examination of their ability to compete with CCHFV-
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specific bovine serum antibodies in a prototype of the cELISA. Except one, all of the
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ten selected mAbs were able to compete.
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Since mAb 13-1 showed the highest level of competition with anti-CCHFV positive
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sera, it was finally selected for the development of the cELISA. This mAb was
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classified as subclass IgG1 antibody with kappa light chains.
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3.2 Validation of the cELISA
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The majority of positive reference sera showed PI-values of >50, whereas PI-values
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of <41 were found in the majority of negative reference sera (Figure 1). Based on the
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results of a ROC analysis, upper and lower cut-off values for the cELISA were
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defined: sera with PI-values of >49 were classified as ”positive”, sera with PI-values
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of <37 as “negative”, and sera with PI-values between 37 and 49 as “inconclusive”.
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Analysis of 833 serum samples resulted in correct positive and negative results for
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143 and 659 sera, respectively (Table 2). A few false negative sera were found in the
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group of cattle, sheep and goats, but not from monkeys (Table 2). False positive
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reactions were also observed for singleton sera from three of the 13 species
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investigated (i.e. sheep, raccoon dogs and foxes). The diagnostic sensitivity of the
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cELISA was 95% (confidence interval (95%): 91-98%), the diagnostic specificity was
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99% (confidence interval (95%): 96-100%), and 2% of the sera were classified as
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“inconclusive”.
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4. Discussion
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Here we describe the establishment and validation of a cELISA for the detection of
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CCHFV-specific antibodies. ELISAs are generally considered to be the preferred
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method in CCHFV serology (Spengler et al., 2016). The benefit of a cELISA is its
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capacity to test species-independently (Meyer, 2010).
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Only one in-house cELISA for the detection of CCHFV-specific antibodies has been
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described and used so far. It was based on full virus antigen and therefore needed to
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be produced in BSL4 laboratories (Burt et al., 1993). The cELISA presented here was
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developed using an E. coli expressed recombinant N-protein, allowing its production
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under BSL2 conditions. The N-protein is considered to be the most conserved
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structural protein within all bunyaviruses (Pepin et al., 2010).
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Besides the safe use under BSL2 conditions, using recombinant antigens is also
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beneficial because of the high reproducibility of the antigen batches, which enable
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the establishment of standardized assays (Buffolano et al., 2005).
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All CCHFV strains are forming one serogroup (Bente et al., 2013). Nevertheless,
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because of their remarkable genetic diversity CCHFV strains are divided in up to 7
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clades depending on the classification system (Bente et al., 2013). Using the
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ACCEPTED MANUSCRIPT designation by Bente et al. and focusing on the genetic information of the S-segment,
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CCHFV strains of the clades V and VI are circulating within Europe. Clade V
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summarizes all European CCHFV strains except strain AP-92 (clade VI) (Bente et al.,
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2013).
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Since originally the cELISA was designed for testing serum samples from Europe,
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the N-protein of CCHFV strain Kosovo Hoti was chosen. The strain belongs to clade
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V and was isolated from the blood of a fatal human case from the epidemic in
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Republic of Kosovo in 2001 (Bente et al., 2013; Duh et al., 2008).
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The aim of the development of the cELISA was to produce a sensitive test, which can
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be applied in seroepidemiological studies on various animal species by producing a
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minimum of false positive results. Therefore, the main focus was laid on the
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specificity of the assay. Thirty three murine mAbs were generated against the N-
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protein of CCHFV strain Kosovo Hoti. The majority of the mAbs reacted in ELISA and
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Western blot test with homologous and heterologous E.coli expressed CCHFV N-
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proteins and in the IFA with eukaryotic cells expressing the N-protein. Further, they
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competed with anti-CCHFV sera in a cELISA prototype test. This indicates, that the
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E. coli expressed recombinant N-protein of CCHFV presented the same confirmation
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of epitope(s) to the murine immune system like the native viral antigen. Using the
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mAb, which gave the best results in all tests used for characterization, a cELISA was
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developed.
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The validation of the cELISA using 833 sera from 12 different animal species and
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humans resulted in a diagnostic sensitivity of 95% and a diagnostic specificity of
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99%.
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ACCEPTED MANUSCRIPT In addition to its reactivity with serum samples from Europe, the cELISA was proven
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to react with serum samples from two western and one central African country, where
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CCHFV strains of clade III are assumed to circulate (data not shown). Therefore, it
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can be concluded that the combination of the recombinant antigen and the mAb has
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the capacity to detect antibodies against different CCHFV clades (III and V) and it
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can be expected that the cELISA is able to detect antibodies specific for other
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CCHFV clades as well.
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Nevertheless, as the positive reference sera were exclusively derived from cattle,
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sheep, goats and monkeys, no predictions can yet be made for other animal species.
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Furthermore, the new test does not have the capacity to test serum samples from
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mice, since the mAb was generated in mice and accordingly an anti-mouse conjugate
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is used for its detection.
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The volume required for the cELISA is relatively high (27.5 µl), but lower than needed
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for some commercial cELISAs, e.g. for the detection of Rift-Valley-Fever-Virus and
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Hepatitis E virus specific antibodies, which require approximately twice as much
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serum. Anyhow, the still comparatively large volume may be a problem when small
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animal species are to be tested.
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In the past, more than 150 seroepidemiological studies for CCHFV have been
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conducted with sera from cattle, sheep and goats (Spengler et al., 2016). These
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studies indicated a strong association between the appearance of human CCHF
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cases and seropositivity in the above mentioned species, revealing them as
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amplifying hosts of CCHFV (Spengler et al., 2016). Rodents, hares and hedgehogs
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are classical hosts of the main vector of CCHFV, which are ticks of the species
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ACCEPTED MANUSCRIPT Hyalomma marginatum, and therefore changes of these populations may affect also
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the CCHFV ecology. An increase in the wild hare population preceded the first
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outbreak of CCHFV in 1944 on the Crimean peninsula and also the occurrence of
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CCHFV in Bulgaria in the 1950s (Bente et al., 2013). In Turkey, a positive association
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between the increase of the hare and wild boar population and the emergence of
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CCHF in humans has been observed (Bente et al., 2013; Ergonul, 2006). In South-
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Africa, ostriches are considered to be important amplifying hosts of CCHFV, and
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camels may serve as amplifying hosts of the virus in regions, were they replace cattle
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(Spengler et al., 2016). For many other species, their role in the infection cycle of
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CCHFV is fairly unknown.
358
The identification of further amplifying hosts of CCHFV is crucial for CCHFV risk
359
assessment and for the development of control strategies (Spengler et al., 2016).
360
One approach for a control strategy for CCHFV circulation in nature is the vaccination
361
of key amplifying hosts of the virus in combination with effective acaricide treatment
362
(Spengler and Bente, 2015).
363
The here described highly sensitive and specific cELISA can be used for large-scale
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screening approaches with sera from numerous animal species and therefore it will
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help to identify yet unknown amplifying hosts in different countries of the world.
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Acknowledgments
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ACCEPTED MANUSCRIPT This study was funded in parts by the EU grant FP7-261504
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EDENext(http://www.edenext.eu) catalogued by the EDENext Steering Committee as
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EDENext465 (http://www.edenext.eu), and by the German Office for Foreign Affairs
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in the frame of the German Partnership Program for Excellence in Biological and
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Health Security. The content of this publication is the sole responsibility of the
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authors and does not necessarily reflect the views of the European Commission and
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the German Office for Foreign Affairs. The funders had no role in study design, data
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collection and analysis, decision to publish, or preparation of the manuscript.
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Compliance with ethical standards
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Conflict of interest
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The authors have declared that no competing interests exist.
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Ethics statement
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The sample collection was performed in compliance with fundamental ethical
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principles. The human sera were collected from forestry worker volunteers, who gave
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a written consent (Mertens et al., 2011).
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Avšič-Županc, T., 2008. Epidemiology and Current Geographical Distribution of Crimean-Congo Haemorrhagic Fever, http://www.arbo-zoo.net/_data/arbozoonetnews_No2.pdf.
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Bente, D.A., Forrester, N.L., Watts, D.M., McAuley, A.J., Whitehouse, C.A., Bray, M., 2013. Crimean-Congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity. Antiviral Res 100, 159-189.
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Burt, F.J., Swanepoel, R., Braack, L.E., 1993. Enzyme-linked immunosorbent assays for the detection of antibody to Crimean-Congo haemorrhagic fever virus in the sera of livestock and wild vertebrates. Epidemiol Infect 111, 547-557.
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Causey, O.R., Kemp, G.E., Madbouly, M.H., David-West, T.S., 1970. Congo virus from domestic livestock, African hedgehog, and arthropods in Nigeria. Am J Trop Med Hyg 19, 846-850.
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Gonzalez, J.P., Camicas, J.L., Cornet, J.P., Faye, O., Wilson, M.L., 1992. Sexual and transovarian transmission of Crimean-Congo haemorrhagic fever virus in Hyalomma truncatum ticks. Res Virol 143, 23-28.
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Lugaj, A., Koni, M., Mertens, M., Groschup, M., Berxholi, K., 2014a. Serological survey of Crimean-Congo hemorrhagic fever virus in cattle in Berat and Kolonje, Albania. Albanian J Agric Sci 13(Special Edition), 325–328.
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Lugaj, A., Koni, M., Schuster, I., Mertens, M., Groschup, M., Berxholi, K., 2014b. A seroepidemiological survey of Crimean-Congo hemorrhagic fever virus among goats and sheep in Lezhe-Torovica Province, Albania. Albanian J Agric Sci 13, 28–31.
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Lugaj A, M.M., Groschup MH, Berxholi K, 2014c. Serological survey of CCHFV in cattle in 10 regions of Albania. Int J Res Appl Nat Soc Sci 2, 55–60.
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Mertens, M., Hofmann, J., Petraityte-Burneikiene, R., Ziller, M., Sasnauskas, K., Friedrich, R., Niederstrasser, O., Kruger, D.H., Groschup, M.H., Petri, E., Werdermann, S., Ulrich, R.G., 2011. Seroprevalence study in forestry workers of a non-endemic region in eastern Germany reveals infections by Tula and DobravaBelgrade hantaviruses. Med Microbiol Immunol 200, 263-268.
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Mertens, M., Schmidt, K., Ozkul, A., Groschup, M.H., 2013. The impact of CrimeanCongo hemorrhagic fever virus on public health. Antiviral Res 98, 248-260.
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Mertens, M., Vatansever, Z., Mrenoshki, S., Krstevski, K., Stefanovska, J., Djadjovski, I., Cvetkovikj, I., Farkas, R., Schuster, I., Donnet, F., Comtet, L., Tordo, N., Ben Mechlia, M., Balkema-Buschmann, A., Mitrov, D., Groschup, M.H., 2015. Circulation of Crimean-Congo Hemorrhagic Fever Virus in the former Yugoslav Republic of Macedonia revealed by screening of cattle sera using a novel enzymelinked immunosorbent assay. PLoS Negl Trop Dis 9, e0003519.
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Mertens, M., Schuster, I., Sas, M.A., Vatansever, Z., Hubalek, Z., Guven, E., Deniz, A., Georgiev, G., Peshev, R., Groschup, M.H., 2016. Crimean-Congo Hemorrhagic Fever Virus in Bulgaria and Turkey. Vector Borne Zoonotic Dis 16, 619-623.
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Meyer, T., 2010. Competitive ELISA, Second Edition ed. Springer.
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OIE, 2014. Terrestrial Manual 1-8, http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.05_CCHF.pdf.
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Papa, A., Christova, I., Papadimitriou, E., Antoniadis, A., 2004. Crimean-Congo hemorrhagic fever in Bulgaria. Emerg Infect Dis 10, 1465-1467.
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Papa, A., Dalla, V., Papadimitriou, E., Kartalis, G.N., Antoniadis, A., 2010. Emergence of Crimean-Congo haemorrhagic fever in Greece. Clin Microbiol Infect 16, 843-847.
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Pepin, M., Bouloy, M., Bird, B.H., Kemp, A., Paweska, J., 2010. Rift Valley fever virus(Bunyaviridae: Phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention. Vet Res 41, 61.
460 461 462 463
Schuster, I., Mertens, M., Mrenoshki, S., Staubach, C., Mertens, C., Bruning, F., Wernike, K., Hechinger, S., Berxholi, K., Mitrov, D., Groschup, M.H., 2016. Sheep and goats as indicator animals for the circulation of CCHFV in the environment. Exp Appl Acarol 68, 337-346.
464 465
Spengler, J.R., Bente, D.A., 2015. Therapeutic intervention in Crimean-Congo hemorrhagic fever: where are we now? Future Virol 10, 203-206.
466 467 468
Spengler, J.R., Bergeron, E., Rollin, P.E., 2016. Seroepidemiological Studies of Crimean-Congo Hemorrhagic Fever Virus in Domestic and Wild Animals. PLoS Negl Trop Dis 10, e0004210.
469 470 471
The Spain Report, 2016. https://www.thespainreport.com/articles/883-160901142820-one-dead-one-inisolation-after-two-cases-of-crimean-congo-hemorrhagic-fever-confirmed-in-madrid
472 473
Whitehouse, C.A., 2004. Crimean-Congo hemorrhagic fever. Antiviral Res 64, 145160.
474 475 476
Yen, Y.C., Kong, L.X., Lee, L., Zhang, Y.Q., Li, F., Cai, B.J., Gao, S.Y., 1985. Characteristics of Crimean-Congo hemorrhagic fever virus (Xinjiang strain) in China. Am J Trop Med Hyg 34, 1179-1182.
477 478 479
Zeller, H.G., Cornet, J.P., Camicas, J.L., 1994. Experimental transmission of Crimean-Congo hemorrhagic fever virus by west African wild ground-feeding birds to Hyalomma marginatum rufipes ticks. Am J Trop Med Hyg 50, 676-681.
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Figure legends
484 485
Figure 1: Distribution of percent inhibition values determined by the cELISA
487 488 489
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Table 1: Sera used for cELISA development and validation Species (Taxonomy)
Country (number of samples)
Cattle (Bos taurus)
Albania (23),Turkey (13), adapted commercial ELISA Former Yoguslav Republic of and IFA1, Macedonia (9),Germany** negative by origin** (100)
Sheep (Ovinae)
Albania (52),Republic of Kosovo (4),Germany** (106)
adapted commercial ELISA and IFA2, negative by origin**
Goats (Caprinae)
Albania (41), Former Yoguslav Republic of Macedonia (13),Germany** (108)
adapted commercial ELISA and IFA2, negative by origin**
France (3)
commercial ELISA and IFA*
Germany (4)**
negative by origin**
Germany (6)**
negative by origin**
Germany (4)**
negative by origin**
Germany (47)**
negative by origin**
Germany (45)**
negative by origin**
Germany (110)**
negative by origin**
Germany (51)**
negative by origin**
Germany (52)**
negative by origin**
Germany (42)**
negative by origin**
Characterized by
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Rhesus Macaques (Macaca mulata) Camels (Camelus dromedarius) Rats (Rattus norvegicus) Ferrets (Mustela putorius furo) Raccoon dogs (Nyctereutes procyonoides) Raccoons (Procyon lotor) Foxes (Vulpes vulpes) Hares (Lepus europaeus) Pigs (Sus scrofa domestica) Humans (Homo sapiens)
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ACCEPTED MANUSCRIPT Total
833
Protocols published in: 1 Mertens et al. 2015, 2 Schuster et a. 2016
492 493
*protocols by manufacturer (IgG-ELISA Vector Best, Novosibirsk, Russia; IgG-IFA Euroimmun, Lübeck, Germany)
494 495
**no evidence for CCHFV circulation in Germany, therefore sera from Germany were defined as negative reference samples
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Table 2: Results of the cELISA for sera from twelve different animal species and humans
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Negative Reference Sera
38 3 4
0 97 3
Sheep (Ovinae) Positive Negative Inconclusive
54 1 1
2 102 2
Goats (Caprinae) Positive Negative Inconclusive
49 3 2
0 108 0
Rhesus Macaque (Macaca mulata) Positive Negative Inconclusive
2 0 0
0 1 0
Camels (Camelus dromedarius) Positive Negative Inconclusive
0 0 0
0 3 1
Rats (Rattus norvegicus) Positive
0
0
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Cattle (Bos taurus) Positive Negative Inconclusive
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Positive Reference Sera
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6 0
Ferrets (Mustela putorius furo) Positive Negative Inconclusive
0 0 0
0 3 1
Raccoon Dogs (Nyctereutes procyonoides) Positive Negative Inconclusive
0 0 0
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Raccoons (Procyon lotor) Positive Negative Inconclusive
1 42 4
0 45 0
0 0 0
3 107 0
Hares (Lepus europaeus) Positive Negative Inconclusive
0 0 0
0 51 0
Pigs (Sus scrofa domestica) Positive Negative Inconclusive
0 0 0
0 52 0
Humans (Homo sapiens) Positive Negative Inconclusive
0 0 0
0 42 0
All Species Positive Negative Inconclusive
143 7 7
7 659 10
Total
157
676
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0 0 0
Foxes (Vulpes vulpes) Positive Negative Inconclusive
500
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Negative Inconclusive
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights:
• Monoclonal
antibodies
(mAbs)
recognizing
native
Crimean-Congo
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Hemorrhagic Fever Virus (CCHFV) antigen were generated • Antigen binding of several mAbs is outcompeted by anti-CCHFV positive sera
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• The diagnostic sensitivity and specificity of the recombinant N-protein based
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CCHFV competitive ELISA were 95% and 99%
S0166-3542(16)30427-2
DOI:
10.1016/j.antiviral.2016.09.004
Reference:
AVR 3902
To appear in:
Antiviral Research
Received Date: 4 August 2016 Revised Date:
8 September 2016
Accepted Date: 9 September 2016
Please cite this article as: Schuster, I., Mertens, M., Köllner, B., Korytář, T., Keller, M., Hammerschmidt, B., Müller, T., Tordo, N., Marianneau, P., Mroz, C., Rissmann, M., Stroh, E., Dähnert, L., Hammerschmidt, F., Ulrich, R.G., Groschup, M.H., A competitive ELISA for species-independent detection of Crimean-Congo hemorrhagic fever virus specific antibodies, Antiviral Research (2016), doi: 10.1016/j.antiviral.2016.09.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT
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Isolde Schuster1, Marc Mertens1, Bernd Köllner2, Tomáš Korytář2, Markus Keller1, Bärbel Hammerschmidt3, Thomas Müller4, Noël Tordo5, Philippe Marianneau6, Claudia Mroz1, Melanie Rissmann1, Eileen Stroh1, Lisa Dähnert1, Felicitas Hammerschmidt7, Rainer G. Ulrich1, Martin H. Groschup1*
Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany 2
Institute of Immunology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany
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3
Department of Experimental Animal Facilities and Biorisk Management, FriedrichLoeffler-Institut, Greifswald-Insel Riems, Germany 4
Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany 5
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Unit Antiviral Strategies Antivirales, WHO collaborative Centre for Viral Haemorrhagic Fevers and Arboviruses, OIE Reference Laboratory for RVFV and CCHFV, Institut Pasteur, Paris, France 6
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Unit Virology; ANSES-Laboratoire de Lyon, Lyon, France
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A competitive ELISA for species-independent detection of Crimean-Congo Hemorrhagic fever virus specific antibodies
Chair of Food Safety, Faculty of Veterinary Medicine, Ludwig-MaximiliansUniversity Munich (LMU), Oberschleissheim, Germany
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*Corresponding author: Prof. Dr. Martin H. Groschup, Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Suedufer 10, Germany, [email protected]
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ACCEPTED MANUSCRIPT 34
Abstract
35
Crimean-Congo hemorrhagic fever virus (CCHFV) circulates in many countries of
37
Asia, Africa, and Europe. CCHFV can cause a severe hemorrhagic fever in humans
38
with case-fatality rates of up to 80%. CCHF is considered to be one of the major
39
emerging diseases spreading to and within Europe. Ticks of the genus Hyalomma
40
function as vector as well as natural reservoir of CCHFV. Ticks feed on various
41
domestic animals (e.g. cattle, sheep, goats) and on wildlife (e.g. hares, hedgehogs).
42
Those animal species play an important role in the life cycle of the ticks as well as in
43
amplification of CCHFV.
44
Here we present a competitive ELISA (cELISA) for the species-independent
45
detection of CCHFV-specific antibodies. For this purpose nucleocapsid (N) protein
46
specific monoclonal antibodies (mAbs) were generated against an Escherichia coli
47
(E. coli) expressed CCHFV N-protein. Thirty-three mAbs reacted with homologous
48
and heterologous recombinant CCHFV antigens in ELISA and Western blot test and
49
20 of those 33 mAbs reacted additionally in an immunofluorescence assay with
50
eukaryotic cells expressing the N-protein. Ten mAbs were further characterized in a
51
prototype of the cELISA and nine of them competed with positive control sera of
52
bovine origin. The cELISA was established by using the mAb with the strongest
53
competition. For the validation, 833 sera from 12 animal species and from humans
54
were used. The diagnostic sensitivity and specificity of the cELISA was determined to
55
be 95% and 99%, respectively, and 2% of the sera gave inconclusive results.
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This cELISA offers the possibility for future large-scale screening approaches in
57
various animal species to evaluate their susceptibility to CCHFV infection and to
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identify and monitor the occurrence of CCHFV.
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Highlights:
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• Monoclonal
antibodies
(mAbs)
recognizing
native
Crimean-Congo
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Hemorrhagic Fever Virus (CCHFV) antigen were generated
• Antigen binding of several mAbs is outcompeted by anti-CCHFV positive sera
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• The diagnostic sensitivity and specificity of the recombinant N-protein based CCHFV competitive ELISA were 95% and 99%
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Keywords:
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CCHFV, Crimean-Congo Hemorrhagic Fever, competitive ELISA, amplifying hosts,
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monoclonal antibody, nucleocapsid protein
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1. Introduction
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Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus, belonging to
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the genus Nairovirus in the family Bunyaviridae. CCHFV is endemic in many
77
countries of Africa, Asia and South-Eastern Europe (Hoogstraal, 1979). It is
78
considered to be one of the major zoonotic pathogens, spreading to and within
79
Europe (Mertens et al., 2013). Inside Europe, its distribution closely correlates with
80
the distribution of ticks of the species Hyalomma marginatum, which are both vector
81
and reservoir of CCHFV (Whitehouse, 2004).
82
In nature, the virus circulates in a tick-vertebrate-tick cycle, whereby the vertebrates
83
play an important role in the life cycle of the ticks as well as in amplification of the
84
virus (Whitehouse, 2004; Zeller et al., 1994). Within the tick population CCHFV can
85
be transmitted horizontally by co-feeding, by venereal or transstadial transmission,
86
and vertically by transovarial transmission (Gonzalez et al., 1992; Logan et al., 1989).
87
Hyalomma marginatum ticks feed on various domestic animals (e.g. cattle, sheep,
88
goats) and on wildlife (e.g. hares, hedgehogs). Although, there is no evidence that
89
CCHFV infections causes clinical signs in animals (Ergonul, 2006; Whitehouse,
90
2004), they can develop a viremia lasting for up to 15 days with a subsequent
91
seroconversion (Causey et al., 1970; Gonzalez et al., 1998; Zeller et al., 1994).
92
In humans, CCHFV infections can cause a severe hemorrhagic disease with case
93
fatality rates of up to 80% (Ergonul, 2006; Whitehouse, 2004; Yen et al., 1985).
94
Infection routes are tick-bites, crushing of infected ticks, and contact with body fluids
95
or tissues of infected animals or humans. Neither vaccination nor specific treatment is
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currently available (Ergonul, 2006).
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ACCEPTED MANUSCRIPT In Europe, human Crimean-Congo hemorrhagic fever (CCHF) cases are regularly
98
reported from Albania, Bulgaria and the Republic of Kosovo (Avšič-Županc, 2008;
99
Papa et al., 2004; Papa et al., 2010; Papa et al., 2011). In Turkey, 9069 CCHF cases
100
were reported between 2002 and 2014 (Mertens et al., 2016). Isolated CCHF cases
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have previously been reported from Hungary and Greece (Hornok and Horvath,
102
2012; Papa et al., 2008). Furthermore, CCHF cases have just recently been reported
103
from Spain (The Spain Report, 2016). Next to the countries with endemic areas,
104
almost no information is available about the distribution of CCHFV in Europe
105
(Mertens et al., 2013; Mertens et al., 2016).
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Because of its impact on human health, the distribution of CCHFV should be
107
investigated in detail and potential spread should be monitored to increase the
108
awareness of people at risk and for the implementation of adequate protection
109
measures (Mertens et al., 2013; Spengler et al., 2016). Therefore, amongst others,
110
an appropriate knowledge on amplifying hosts of the virus is necessary (Spengler et
111
al., 2016).
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Some mammal and avian species are already identified to be an important part in the
113
ecology of the tick vectors, such as cattle, sheep, goats, hares, hedgehogs, camels
114
and ostriches. However, their role in the infection cycle of CCHFV, and especially the
115
impact of other species remains only partially understood. To explore the role of
116
different species, animal experiments can be used. Since CCHFV is a biosafety level
117
(BSL) 4 pathogen, they are difficult to conduct, because they require high
118
biocontainment facilities (Keshtkar-Jahromi et al., 2011; Mertens et al., 2013).
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Alternatively, large-scale seroepidemiological studies on different animal species can
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be used to figure out which species are involved in the infection cycle of CCHFV.
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absence of CCHFV in the regarding region, those studies can further be used for the
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investigation of the distribution of CCHFV (Hoogstraal, 1979).
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Several seroepidemiological studies about the prevalence of CCHFV-specific
125
antibodies in cattle, sheep and goats are published (Spengler et al., 2016). Different
126
assay formats, based on complement fixation, immunofluorescence (IFA), reverse
127
passive hemagglutination inhibition, neutralization, immunodiffusion and ELISA, were
128
used in those studies (Spengler et al., 2016; Whitehouse, 2004). Since no assays are
129
commercially available for CCHFV serology in animals, those assays are either in-
130
house assays or commercial tests for CCHFV serology in humans, which were
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adapted for testing animals (Mertens et al., 2013). The applicability of those assays
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for different animal species is limited, either because the assay formats are
133
considered as outdated or because species-specific monoclonal or polyclonal
134
antibody conjugates are required, which are difficult to obtain (OIE, 2014; Spengler et
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al., 2016). Besides, species-dependent adaptation or assay development and
136
validation are time-consuming. Additional obstacles are the need for corresponding
137
reference serum panels from the species the assay should be used for (OIE, 2014).
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Next to the lack of an accepted gold standard for CCHFV serology in animals, this
139
might possibly be one of the reasons why most of the published tests were either not
140
validated or the data of their validation were not published (Mertens et al., 2013).
141
The aim of the work presented here was the development and validation of a
142
competitive ELISA (cELISA) for the species-independent detection of CCHFV-
143
specific antibodies. This cELISA can be used for large-scale screening approaches
144
for identification of amplifying hosts as well as for monitoring of CCHFV foci.
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2. Materials and methods
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2.1 Recombinant antigens
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Recombinant nucleocapsid (N) proteins of CCHFV strains Kosovo Hoti (GenBank
150
accession no. DQ133507) and Turkey-Kelkit06 (GenBank accession no. GQ337053)
151
were expressed in Escherichia coli (E. coli) with an N-terminal deca-His-tag and
152
purified by nickel-chelate affinity chromatography (Qiagen, Hilden, Germany). The
153
divergence in the aminoacid sequence between both CCHFV strains is 1.2%.
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Puumala hantavirus (PUUV) N-protein expressed in yeast Saccharomyces cerevisiae
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was used as a negative control antigen (Mertens et al., 2011).
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2.2 Generation and characterization of monoclonal antibodies
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Monoclonal antibodies (mAbs) were generated by intramuscular immunization of
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BALB/c mice three times in intervals of ten days. As antigen 50 µg of purified and in
160
Aqua dest dialyzed recombinant E. coli expressed N-protein of CCHFV strain Kosovo
161
Hoti were used.
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The recombinant N-protein was mixed with complete Freund's adjuvant for the initial
163
immunization (Sigma, Deisenhofen, Germany), and for the second and third
164
immunization the protein was mixed with incomplete Freund's adjuvant (Sigma). Four
165
days prior fusion the mice were boosted intraperitoneally with 50 µg of the N-protein
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without adjuvant.
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Hybridoma supernatants were screened by an in-house ELISA and Western blot test
168
based on the homologous and heterologous (N-protein of CCHFV-strain Turkey-
169
Kelkit06) antigen and by an adapted commercial IFA basing on N-protein of CCHFV
170
strain
171
supernatants were also tested in an ELISA using PUUV N-protein. The mAbs
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showing the highest reactivity with all used CCHFV antigens were additionally tested
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in a prototype of the cELISA regarding their ability to compete with anti-CCHFV
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positive bovine sera.
175
Isotyping of the finally chosen mAb was performed using the Pierce Rapid ELISA
176
Mouse mAb Isotyping Kit (Thermo Scientific, Waltham, MA, USA). Afterwards, the
177
mAb was purified using fast protein liquid chromatography (FPLC) and adjusted to a
178
concentration of 1 µg/µl in 0.1% glycine, neutralized with a 1/10 volume of 1 M Tris
179
pH 8.0. Finally, 0.01% sodium azide was added and aliquots of the mAb were stored
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either at 4°C (short-term storage) or -20°C (long-t erm storage).
(Euroimmun,
Lübeck,
Germany).
As
control,
hybridoma
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2.3 Sera
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Development and validation of the cELISA was conducted with sera from various
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animal species as well as from humans (Table 1). Many of the sera used for
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validation were already used in previously published studies (Lugaj et al., 2014a;
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Lugaj et al., 2014b; Lugaj et al., 2014c; Mertens et al., 2011; Mertens et al., 2015;
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Mertens et al., 2016; Schuster et al., 2016).
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As positive reference, sera from Albania, the Republic of Kosovo, Turkey, and from
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the Former Yugoslav Republic of Macedonia were used for test development and
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191
1).
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Additionally, sera of two rhesus macaques (Macaca mulata), which had been
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immunized with purified inactivated full virus of CCHFV strain IbAr 10200, were used
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as positive reference sera, and a serum from a non-immunized rhesus macaque was
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used as negative reference serum (Table 1).
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2.4 Development and validation of a cELISA for the detection of CCHFV-
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specific antibodies
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2.4.1 Development of a cELISA
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The cELISA was developed using a His-tagged recombinant N-protein of CCHFV
201
strain Kosovo Hoti as antigen. The establishment of the test was conducted by
202
variation of plate type, coating conditions, antigen amount, incubation temperatures
203
and times, dilution buffers, washing buffers, washing frequency, serum dilution, mAb
204
dilution, conjugate dilution, substrate incubation time, as well as the procedure of
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application of the mAb. The optimal ratio between serum-, mAb- and conjugate-
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dilution was evaluated by checkerboard titrations. Finally, the protocol was chosen,
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which best distinguished between the positive and negative reference serum
208
samples.
209
Prior to the coating step, the recombinant protein was spiked 1:1 with glycerin and
210
incubated for 1 h at 4°C. 96-well PolySorp immuno-p lates (Nunc, Roskilde, Denmark)
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were coated with 2 µg antigen, diluted in PBS pH 10 containing 10% glycerin. Plates
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were sealed with a foil and subsequently incubated over night at 4°C.
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plates and were incubated for 1 h at 37°C. Afterwar ds, the plates were washed four
215
times using 250 µl/well PBS-Tween20 1%.
216
Sera were diluted 1:4 in 1% BSA in PBS-Tween20 0.1% and 95 µl of the serum
217
dilution were added to each well. As controls, dilutions of a positive cattle serum from
218
Turkey and of a negative sheep serum sample from Germany, as well as only buffer
219
were added to duplicate wells on the plate.
220
Plates were incubated for 1 h at 37°C. The mAb was diluted 1:50 in 1% BSA in PBS-
221
Tween20 0.5% and 5 µl of the dilution were added to each well containing 95 µl of
222
the serum dilution or buffer. The plates were incubated for 1 h at 37°C.
223
After washing, 100 µl/well of anti-mouse horse-radish peroxidase (HRP) conjugate
224
(Bio-Rad, Munich, Germany), diluted 1:1,000 in 1% BSA in PBS-Tween20 0.05%,
225
were added and plates were incubated for 2 h at 37°C.
226
Plates were washed and 100 µl/well TMB solution (Bio-Rad) were added and
227
incubated for 10 min at room temperature. The reaction was stopped using H2SO4.
228
The optical density (OD) was measured at 450 nm (reference wavelength 620 nm).
229
Results were calculated as percent inhibition (PI) of the OD-value of each serum
230
sample compared to the OD-value of the negative control serum (PI = 100-(OD-value
231
serum sample/OD-value negative control sample) x 100).
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2.4.2 Validation of the cELISA
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235
comparative analysis.
236
Sera from cattle, sheep and goats from countries with areas endemic for CCHFV
237
were prior characterized in an adapted commercial ELISA (Vector Best, Novosibirsk,
238
Russia), and IFA (Euroimmun, Lübeck, Germany) (Mertens et al., 2015; Schuster et
239
al., 2016). Sera from rhesus macaques were characterized in the commercial ELISA
240
and IFA according to the protocols given by the manufacturers. Samples, which were
241
tested positive in both assays, were defined as positive reference sera.
242
Because there is no evidence for circulation of CCHFV in Germany, sera from
243
Germany were used as negative reference sera.
244
The cut-off values for the cELISA were determined by receiver-operator
245
characteristics (ROC) analysis. The diagnostic sensitivity was determined as ratio of
246
the number of true positive samples divided by the number of true positive plus false
247
negative samples and given as percentage. The diagnostic specificity was
248
determined as ratio of the number of true negative samples divided by the number of
249
true negative plus the number of false positive samples and given as percentage.
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3. Results
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3.1 Generation and Characterization of mAbs
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Thirty three hybridoma supernatants reacted in Western blot test and in-house ELISA
255
with the homologous recombinant N-protein of CCHFV strain Kosovo Hoti and the
13
ACCEPTED MANUSCRIPT heterologous N-protein of CCHFV strain Turkey-Kelkit06. Twenty out of the thirty
257
three supernatants were also tested positive in the commercial IFA, while four were
258
inconclusive and nine were negative. None of the hybridoma supernatants reacted
259
with the PUUV N-protein.
260
Ten of the twenty mAbs, giving a positive signal in the initial CCHFV screening tests,
261
were randomly chosen for examination of their ability to compete with CCHFV-
262
specific bovine serum antibodies in a prototype of the cELISA. Except one, all of the
263
ten selected mAbs were able to compete.
264
Since mAb 13-1 showed the highest level of competition with anti-CCHFV positive
265
sera, it was finally selected for the development of the cELISA. This mAb was
266
classified as subclass IgG1 antibody with kappa light chains.
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3.2 Validation of the cELISA
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The majority of positive reference sera showed PI-values of >50, whereas PI-values
270
of <41 were found in the majority of negative reference sera (Figure 1). Based on the
271
results of a ROC analysis, upper and lower cut-off values for the cELISA were
272
defined: sera with PI-values of >49 were classified as ”positive”, sera with PI-values
273
of <37 as “negative”, and sera with PI-values between 37 and 49 as “inconclusive”.
274
Analysis of 833 serum samples resulted in correct positive and negative results for
275
143 and 659 sera, respectively (Table 2). A few false negative sera were found in the
276
group of cattle, sheep and goats, but not from monkeys (Table 2). False positive
277
reactions were also observed for singleton sera from three of the 13 species
278
investigated (i.e. sheep, raccoon dogs and foxes). The diagnostic sensitivity of the
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cELISA was 95% (confidence interval (95%): 91-98%), the diagnostic specificity was
280
99% (confidence interval (95%): 96-100%), and 2% of the sera were classified as
281
“inconclusive”.
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4. Discussion
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Here we describe the establishment and validation of a cELISA for the detection of
286
CCHFV-specific antibodies. ELISAs are generally considered to be the preferred
287
method in CCHFV serology (Spengler et al., 2016). The benefit of a cELISA is its
288
capacity to test species-independently (Meyer, 2010).
289
Only one in-house cELISA for the detection of CCHFV-specific antibodies has been
290
described and used so far. It was based on full virus antigen and therefore needed to
291
be produced in BSL4 laboratories (Burt et al., 1993). The cELISA presented here was
292
developed using an E. coli expressed recombinant N-protein, allowing its production
293
under BSL2 conditions. The N-protein is considered to be the most conserved
294
structural protein within all bunyaviruses (Pepin et al., 2010).
295
Besides the safe use under BSL2 conditions, using recombinant antigens is also
296
beneficial because of the high reproducibility of the antigen batches, which enable
297
the establishment of standardized assays (Buffolano et al., 2005).
298
All CCHFV strains are forming one serogroup (Bente et al., 2013). Nevertheless,
299
because of their remarkable genetic diversity CCHFV strains are divided in up to 7
300
clades depending on the classification system (Bente et al., 2013). Using the
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302
CCHFV strains of the clades V and VI are circulating within Europe. Clade V
303
summarizes all European CCHFV strains except strain AP-92 (clade VI) (Bente et al.,
304
2013).
305
Since originally the cELISA was designed for testing serum samples from Europe,
306
the N-protein of CCHFV strain Kosovo Hoti was chosen. The strain belongs to clade
307
V and was isolated from the blood of a fatal human case from the epidemic in
308
Republic of Kosovo in 2001 (Bente et al., 2013; Duh et al., 2008).
309
The aim of the development of the cELISA was to produce a sensitive test, which can
310
be applied in seroepidemiological studies on various animal species by producing a
311
minimum of false positive results. Therefore, the main focus was laid on the
312
specificity of the assay. Thirty three murine mAbs were generated against the N-
313
protein of CCHFV strain Kosovo Hoti. The majority of the mAbs reacted in ELISA and
314
Western blot test with homologous and heterologous E.coli expressed CCHFV N-
315
proteins and in the IFA with eukaryotic cells expressing the N-protein. Further, they
316
competed with anti-CCHFV sera in a cELISA prototype test. This indicates, that the
317
E. coli expressed recombinant N-protein of CCHFV presented the same confirmation
318
of epitope(s) to the murine immune system like the native viral antigen. Using the
319
mAb, which gave the best results in all tests used for characterization, a cELISA was
320
developed.
321
The validation of the cELISA using 833 sera from 12 different animal species and
322
humans resulted in a diagnostic sensitivity of 95% and a diagnostic specificity of
323
99%.
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ACCEPTED MANUSCRIPT In addition to its reactivity with serum samples from Europe, the cELISA was proven
325
to react with serum samples from two western and one central African country, where
326
CCHFV strains of clade III are assumed to circulate (data not shown). Therefore, it
327
can be concluded that the combination of the recombinant antigen and the mAb has
328
the capacity to detect antibodies against different CCHFV clades (III and V) and it
329
can be expected that the cELISA is able to detect antibodies specific for other
330
CCHFV clades as well.
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Nevertheless, as the positive reference sera were exclusively derived from cattle,
333
sheep, goats and monkeys, no predictions can yet be made for other animal species.
334
Furthermore, the new test does not have the capacity to test serum samples from
335
mice, since the mAb was generated in mice and accordingly an anti-mouse conjugate
336
is used for its detection.
337
The volume required for the cELISA is relatively high (27.5 µl), but lower than needed
338
for some commercial cELISAs, e.g. for the detection of Rift-Valley-Fever-Virus and
339
Hepatitis E virus specific antibodies, which require approximately twice as much
340
serum. Anyhow, the still comparatively large volume may be a problem when small
341
animal species are to be tested.
342
In the past, more than 150 seroepidemiological studies for CCHFV have been
343
conducted with sera from cattle, sheep and goats (Spengler et al., 2016). These
344
studies indicated a strong association between the appearance of human CCHF
345
cases and seropositivity in the above mentioned species, revealing them as
346
amplifying hosts of CCHFV (Spengler et al., 2016). Rodents, hares and hedgehogs
347
are classical hosts of the main vector of CCHFV, which are ticks of the species
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ACCEPTED MANUSCRIPT Hyalomma marginatum, and therefore changes of these populations may affect also
349
the CCHFV ecology. An increase in the wild hare population preceded the first
350
outbreak of CCHFV in 1944 on the Crimean peninsula and also the occurrence of
351
CCHFV in Bulgaria in the 1950s (Bente et al., 2013). In Turkey, a positive association
352
between the increase of the hare and wild boar population and the emergence of
353
CCHF in humans has been observed (Bente et al., 2013; Ergonul, 2006). In South-
354
Africa, ostriches are considered to be important amplifying hosts of CCHFV, and
355
camels may serve as amplifying hosts of the virus in regions, were they replace cattle
356
(Spengler et al., 2016). For many other species, their role in the infection cycle of
357
CCHFV is fairly unknown.
358
The identification of further amplifying hosts of CCHFV is crucial for CCHFV risk
359
assessment and for the development of control strategies (Spengler et al., 2016).
360
One approach for a control strategy for CCHFV circulation in nature is the vaccination
361
of key amplifying hosts of the virus in combination with effective acaricide treatment
362
(Spengler and Bente, 2015).
363
The here described highly sensitive and specific cELISA can be used for large-scale
364
screening approaches with sera from numerous animal species and therefore it will
365
help to identify yet unknown amplifying hosts in different countries of the world.
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Acknowledgments
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ACCEPTED MANUSCRIPT This study was funded in parts by the EU grant FP7-261504
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EDENext(http://www.edenext.eu) catalogued by the EDENext Steering Committee as
372
EDENext465 (http://www.edenext.eu), and by the German Office for Foreign Affairs
373
in the frame of the German Partnership Program for Excellence in Biological and
374
Health Security. The content of this publication is the sole responsibility of the
375
authors and does not necessarily reflect the views of the European Commission and
376
the German Office for Foreign Affairs. The funders had no role in study design, data
377
collection and analysis, decision to publish, or preparation of the manuscript.
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Compliance with ethical standards
380
Conflict of interest
381
The authors have declared that no competing interests exist.
382
Ethics statement
383
The sample collection was performed in compliance with fundamental ethical
384
principles. The human sera were collected from forestry worker volunteers, who gave
385
a written consent (Mertens et al., 2011).
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Whitehouse, C.A., 2004. Crimean-Congo hemorrhagic fever. Antiviral Res 64, 145160.
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477 478 479
Zeller, H.G., Cornet, J.P., Camicas, J.L., 1994. Experimental transmission of Crimean-Congo hemorrhagic fever virus by west African wild ground-feeding birds to Hyalomma marginatum rufipes ticks. Am J Trop Med Hyg 50, 676-681.
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Figure legends
484 485
Figure 1: Distribution of percent inhibition values determined by the cELISA
487 488 489
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Table 1: Sera used for cELISA development and validation Species (Taxonomy)
Country (number of samples)
Cattle (Bos taurus)
Albania (23),Turkey (13), adapted commercial ELISA Former Yoguslav Republic of and IFA1, Macedonia (9),Germany** negative by origin** (100)
Sheep (Ovinae)
Albania (52),Republic of Kosovo (4),Germany** (106)
adapted commercial ELISA and IFA2, negative by origin**
Goats (Caprinae)
Albania (41), Former Yoguslav Republic of Macedonia (13),Germany** (108)
adapted commercial ELISA and IFA2, negative by origin**
France (3)
commercial ELISA and IFA*
Germany (4)**
negative by origin**
Germany (6)**
negative by origin**
Germany (4)**
negative by origin**
Germany (47)**
negative by origin**
Germany (45)**
negative by origin**
Germany (110)**
negative by origin**
Germany (51)**
negative by origin**
Germany (52)**
negative by origin**
Germany (42)**
negative by origin**
Characterized by
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Rhesus Macaques (Macaca mulata) Camels (Camelus dromedarius) Rats (Rattus norvegicus) Ferrets (Mustela putorius furo) Raccoon dogs (Nyctereutes procyonoides) Raccoons (Procyon lotor) Foxes (Vulpes vulpes) Hares (Lepus europaeus) Pigs (Sus scrofa domestica) Humans (Homo sapiens)
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486
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ACCEPTED MANUSCRIPT Total
833
Protocols published in: 1 Mertens et al. 2015, 2 Schuster et a. 2016
492 493
*protocols by manufacturer (IgG-ELISA Vector Best, Novosibirsk, Russia; IgG-IFA Euroimmun, Lübeck, Germany)
494 495
**no evidence for CCHFV circulation in Germany, therefore sera from Germany were defined as negative reference samples
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491
496
Table 2: Results of the cELISA for sera from twelve different animal species and humans
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Negative Reference Sera
38 3 4
0 97 3
Sheep (Ovinae) Positive Negative Inconclusive
54 1 1
2 102 2
Goats (Caprinae) Positive Negative Inconclusive
49 3 2
0 108 0
Rhesus Macaque (Macaca mulata) Positive Negative Inconclusive
2 0 0
0 1 0
Camels (Camelus dromedarius) Positive Negative Inconclusive
0 0 0
0 3 1
Rats (Rattus norvegicus) Positive
0
0
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Cattle (Bos taurus) Positive Negative Inconclusive
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Positive Reference Sera
24
ACCEPTED MANUSCRIPT 0 0
6 0
Ferrets (Mustela putorius furo) Positive Negative Inconclusive
0 0 0
0 3 1
Raccoon Dogs (Nyctereutes procyonoides) Positive Negative Inconclusive
0 0 0
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Raccoons (Procyon lotor) Positive Negative Inconclusive
1 42 4
0 45 0
0 0 0
3 107 0
Hares (Lepus europaeus) Positive Negative Inconclusive
0 0 0
0 51 0
Pigs (Sus scrofa domestica) Positive Negative Inconclusive
0 0 0
0 52 0
Humans (Homo sapiens) Positive Negative Inconclusive
0 0 0
0 42 0
All Species Positive Negative Inconclusive
143 7 7
7 659 10
Total
157
676
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0 0 0
Foxes (Vulpes vulpes) Positive Negative Inconclusive
500
RI PT
Negative Inconclusive
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights:
• Monoclonal
antibodies
(mAbs)
recognizing
native
Crimean-Congo
RI PT
Hemorrhagic Fever Virus (CCHFV) antigen were generated • Antigen binding of several mAbs is outcompeted by anti-CCHFV positive sera
SC
• The diagnostic sensitivity and specificity of the recombinant N-protein based
AC C
EP
TE D
M AN U
CCHFV competitive ELISA were 95% and 99%