Novel Serological Assays for Determination of the Distribution and of Hosts of Crimean-Congo Hemorrhagic Fever Virus

University of Veterinary Medicine Hannover

Novel serological assays for determination of the distribution and of hosts of Crimean-Congo hemorrhagic fever virus

Inaugural-Dissertation to obtain the academic degree Doctor medicinae veterinariae (Dr. med. vet.)

submitted by Isolde Ursula Elisabeth Schuster Frankfurt am Main

Hannover 2017

Academic Supervision: Professor Dr. Martin H. Groschup Institute of Novel and Emerging Infectious Diseases Friedrich-Loeffler-Institut Greifswald-Insel Riems

1. Referee: Professor Dr. Martin H. Groschup Institute of Novel and Emerging Infectious Diseases Friedrich-Loeffler-Institut Greifswald-Insel Riems

2. Referee: Professor Dr. Paul Becher Department of Virology University of Veterinary Medicine Hannover Hannover

Day of oral examination: 9th of November, 2017

To my family

Parts of this work have been published in the following journals:

Schuster I, Mertens M, Mrenoshki S, Staubach C, Mertens C, Brüning F, Wernike K, Hechinger S, Berxholi K, Mitrov D, Groschup MH. Sheep and goats as indicator animals for the circulation of CCHFV in the environment. Exp Appl Acar, 2016, 68(3): 337–346.

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 RG, Groschup MH. A competitive ELISA for species-independent detection of Crimean-Congo hemorrhagic fever virus specific antibodies. Antiviral Research, 2016, 134: 161-166.

Mertens M, Schuster I, Sas M, Vatansever Z, Hubalek Z, Georgiev G, Peshev R, Groschup MH. Crimean-Congo hemorrhagic fever virus in Bulgaria and Turkey. Vector Borne Zoonotic Dis, 2016, 16(9): 619-623.

Conference papers and presentations:

Schuster I, Korytar T, Kunze M, Köllner M, Mertens M, Groschup MH. Generation of monoclonal antibodies against the nucleocapsid protein of the Crimean-Congo hemorrhagic fever virus for diagnostic purposes. Poster presentation at the First Junior Scientist-Symposium of the Friedrich-Loeffler-Institut, Isle Vilm, Germany, 2012

Schuster I, Mertens M, Groschup MH. Development of serological assays for the detection of CCHFV-specific antibodies in sheep. Poster presentation at the Junior Scientist Zoonoses Meeting, Leipzig, Germany, 2013

Schuster I, Mertens M, Groschup MH. Development and validation of an indirect ELISA for the detection of Crimean-Congo hemorrhagic fever virus specific antibodies in sheep. Oral presentation at the Junior Scientist-Symposium of the Friedrich-Loeffler-Institut, Jena, Germany, 2013

Schuster I, Mertens M, Köllner B, Knittler M, Keller M, Hammerschmidt B, Groschup MH. Development of a competitive ELISA for the detection of CCHFV-specific antibodies. Poster presentation at the Junior Scientist Zoonoses Meeting, Munich, Germany, 2015 Table of contents

Chapter 1 Introduction 1

Chapter 2 Sheep and goats as indicator animals for the circulation of CCHFV in the environment 11

Chapter 3 Crimean-Congo Hemorrhagic Fever Virus in Bulgaria and Turkey 14

Chapter 4 A competitive ELISA for species-independent detection of Crimean-Congo Hemorrhagic fever virus specific antibodies 17

Chapter 5 Discussion 20

Chapter 6 Summary 44

Chapter 7 Zusammenfassung 47

Chapter 8 References 51

Chapter 9 Acknowledgement 69

List of abbreviations

BSL 2 Biosafety Level 2

BSL 4 Biosafety Level 4

CCHFV Crimean-Congo hemorrhagic fever virus

CDC Centre for Disease Control and Prevention

cELISA competitive Enzyme-linked Immunosorbent Assay

E. coli Escherichia coli

ELISA Enzyme-linked Immunosorbent Assay

Gc-protein Glycoprotein C (-terminal)

IFA Immunofluorescence Assay

IgG Immunoglobulin G

IgM Immunoglobulin M

L-segment Large segment

mAb monoclonal antibody

M-segment Medium segment

N-protein Nucleocapsid protein

NSS non-structural protein

S-segment Small segment

WHO World Health Organization Chapter 1: Introduction

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus, which belongs to the family Nairovirdae (Adams et al., 2017; Hoogstraal, 1979). It is endemic in many countries of Europe, Africa and Asia (Hoogstraal, 1979).

In 2015, the WHO listed CCHFV as one of the top pathogens “most likely to cause major epidemics” in future (WHO, 2015). The Centre for Disease Control and

Prevention (CDC) classified CCHFV as Risk Group 4 pathogen (CDC, 2009;

Keshtkar-Jahromi et al., 2011).

Virions of CCHFV are enveloped spherical particles of 80-100 nm in diameter with a single-stranded segmented RNA-genome of negative polarity (Clerx et al., 1981;

Donets et al., 1977; Ellis et al., 1981). The large (L)-segment encodes for the RNA- dependent RNA-Polymerase, the medium (M)-segment encodes for a membrane glycoprotein precursor, and the small (S)-segment encodes for the Nucleocapsid (N) protein (Hewson et al., 2004; Schmaljohn and Hooper, 2001).

Based on their nucleic acid sequence, the CCHFV strains are divided in up to seven clades, depending on the classification system (Atkinson et al., 2012a; Atkinson et al., 2012b; Bente et al., 2013; Carroll et al., 2010; Deyde et al., 2006; Mild et al.,

2010). Following the classification system designed by Bente et al. and focusing on the genetic information on the S-segment, CCHFV strains of clade I, II and III are circulating in Africa, strains of clade IV in Asia and the Middle East and CCHFV 1

strains of clades V and VI in Europe (Bente et al., 2013). Recent analysis of CCHFV genomes from humans and ticks further revealed the circulation of clade III CCHFV strains in Spain (Cajimat et al., 2017; Estrada-Pena et al., 2012; Negredo et al.,

2017).

CCHFV is transmitted by ixodid ticks (Hoogstraal, 1979). Since ticks of the species

Hyalomma marginatum have been demonstrated to be a competent vector and reservoir for CCHFV and the spread of CCHFV closely correlates with their distribution, they are considered to be the main vector in the transmission cycle of the virus inside Europe (Ergonul, 2006; Maltezou et al., 2010).

Within the tick population, CCHFV can be transmitted venerally, transstadially, and/or vertically by transovarial transmission as well as by co-feeding (Gonzalez et al.,

1992; Logan et al., 1989). Ticks need to take blood meals from vertebrate hosts for their maturation and reproduction. Hyalomma marginatum ticks are two-host ticks

(Hoogstraal, 1979). The immature stages prefer to infest small animals like hares, hedgehogs and ground-feeding birds, while the adult stages parasitize large animal hosts like cattle, sheep and goats (Apanaskevich, 2004; Hoogstraal, 1979).

Depending on the status of infection, CCHFV can be transmitted from ticks to vertebrate hosts or vice versa during the blood meal (Gonzalez et al., 1998;

Hoogstraal, 1979).

The CCHFV infected animals do not show clinical signs, but they can develop a viremia lasting up to 15 days (Gunes et al., 2011b). Further, infected animals develop

IgM and IgG antibody responses (Gonzalez et al., 1998). While CCHFV-specific IgM

antibodies are detectable for approximately one to two months, the IgG antibodies persist for approximately three years (Burt et al., 1993; Gonzalez et al., 1998; Zeller et al., 1997). Within our studies, we found CCHFV-specific IgG antibodies in a goat, which had been infected at least four years before our serological testing (Schuster and Mertens, data not shown). In general, the antibody response seems to be a limited protection in animals, since experiments have shown the ability of CCHFV reinfection in domestic animals (Burt et al., 1993; Gonzalez et al., 1998; Wilson et al.,

1991; Zeller et al., 1997).

Human infections mostly occur by tick bite, crushing of ticks, or unprotected contact with blood, body fluids or tissues of viremic animals or humans (Hoogstraal, 1979). In humans, CCHFV can cause a severe disease with case-fatality rates ranging from

5% to 80% (Yen et al., 1985; Yilmaz et al., 2008). Neither vaccination nor specific treatment is available for CCHF (Ergonul, 2006). The usage of Ribavirin is controversially discussed, since all data are based on observational studies which may not give representative results (Ceylan and Turhan, 2014; Mertens et al., 2013;

Tignor and Hanham, 1993; van Eeden et al., 1985; Watts et al., 1989). Protection measures to avoid CCHFV infections are: not visiting tick habitats in the active season; the usage of repellents as well as wearing long light clothes; checking the body for ticks regularly; removing ticks attached to the body as fast as possible and using personal protective equipment while handling blood or other body fluids of potentially infected humans or animals (Aidaros, 2001; Ergonul et al., 2007; Mertens et al., 2013).

Before 2002 in Europe, CCHF cases were reported from Albania, Bulgaria, the

Republic of Kosovo, the Ukraine and the Former Yugoslav Republic of Macedonia

(Christova et al., 2009; Papa et al., 2002b; Papa et al., 2004; Vesenjak-Hirjan et al.,

1991). In 1944, an unknown febrile illness appeared among military personal on the

Crimean Peninsula. It affected two hundred people and led to the discovery of the

Crimean-Congo hemorrhagic fever virus (Chumakov, 1974; Whitehouse, 2004).

CCHF outbreaks have further been reported from Bulgaria and the Republic of

Kosovo since the 1950s (Bente et al., 2013; Mertens et al., 2013). Whereas in

Albania CCHF was first diagnosed in 1986 (Bente et al., 2013; Mertens et al., 2013).

From the then Socialist Republic of Macedonia, ten CCHF cases were reported in

1976 (Vesenjak-Hirjan et al., 1991).

From Albania, Bulgaria and the Republic of Kosovo, sporadic cases have been reported regularly within the last decade (Christova et al., 2009; Christova et al.,

2013; Fajs et al., 2014; Krasniqi and Bino, 2016; Mertens et al., 2013; Papa et al.,

2002a; Papa et al., 2002b; Papa et al., 2004). The average case-fatality rate reported from Albania and Bulgaria was 17%, while the case-fatality rate reported from the

Republic of Kosovo ranged from 0% to 67% (Avšič-Županc, 2008; Papa et al., 2004;

Papa et al., 2011a).

Within the last fifteen years, with Turkey, Hungary, Greece and Spain four additional

European countries reported CCHF cases (Ergonul et al., 2004; Hornok and Horvath,

2012; Karti et al., 2004; Negredo et al., 2017; Papa et al., 2008). While the CCHF cases reported from Greece and Hungary were isolated incidents, Turkey has since

become a “hot-spot” for CCHF with up to 1,400 cases reported annually and an average case-fatality rate of 5% (Bente et al., 2013; Mertens et al., 2013; Turkey,

2015; Yilmaz et al., 2009).

In August 2016, the first two autochthonous CCHF cases have been reported from

Western Europe (Spain) (Negredo et al., 2017). Further, evidence for the presence of

CCHFV vector-competent tick populations in Southern France, Italy and Portugal have been found in recent studies (Dantas-Torres and Otranto, 2013; Santos-Silva et al., 2011; Vial et al., 2016). From Southern France and Portugal, the detection of

CCHFV-specific antibodies in bats and humans has been reported some time ago

(Filipe et al., 1985; Hoogstraal, 1979; Spengler et al., 2016).

In nature, CCHFV may often circulate “silently” in a tick-vertebrate-tick cycle, especially in regions with a balance between the population of small animal hosts and the large animal hosts of the tick vectors (Bente et al., 2013). In this scenario, human cases occur only, when ticks are accidently biting humans, e.g. farmers, which are in close contact to the large animal hosts (Bente et al., 2013). An imbalance of the animal composition in a region, or changes in the ecological pattern might support outbreaks in humans (Bente et al., 2013). This was the case in the worldwide first detected outbreaks of CCHF on the Crimean Peninsula in 1944 and in

Bulgaria in the 1950s (Bente et al., 2013; Mertens et al., 2013). In both cases, the wild hare population grew dramatically in the years before the corresponding outbreak (Bente et al., 2013; Ergonul, 2006). Equally, a connection between the increase of the hare respective the hare and wild boar population and the emergence

of human CCHF cases in Albania and Turkey have been observed (Bente et al.,

2013; Ergonul, 2006; Papa et al., 2009).

The distribution of CCHFV in a region can be very well determined by testing the animal population of the respective area for CCHFV-specific IgG-antibodies, which are persisting for at least three years after infection (Gonzalez et al., 1998;

Hoogstraal, 1979; Zeller et al., 1994). Since this prevalence is an indicator for the

CCHFV circulation, it can help to assess the risk of human infections (Mertens et al.,

2013). Within more than hundred-fifty seroepidemiological studies, cattle, sheep and goats have been used as indicators for CCHFV distribution (Spengler et al., 2016).

Further, those species were revealed as amplifying hosts of CCHFV in animal experiments (Causey et al., 1970; Gonzalez et al., 1998; Spengler et al., 2016;

Wilson et al., 1991). Apart from the above mentioned species, camels are considered to be important hosts of CCHFV in regions, where they displace cattle (Spengler et al., 2016). Antibodies specific for CCHFV have been detected in large mammals like rhinoceroses and giraffes in a seroepidemiological study conducted in the Kruger

National Park in South Africa (Burt et al., 1993). Recently, CCHFV-specific antibodies have been found in twelve out of sixteen African bat species tested (Muller et al.,

2016).

In contrast to mammalian species, many bird species are considered to be refractory to CCHFV infection (Swanepoel et al., 1987). Nevertheless, CCHFV-specific antibodies have been detected in some bird species, e.g. blue-helmeted Guinea fowls (Burt et al., 1993; Shepherd et al., 1987a; Zeller et al., 1994). Moreover, non

viremic CCHFV transmission from a red-billed hornbill to Hyalomma rufipes ticks could be observed in an animal experiment (Shepherd et al., 1987a). In South Africa, ostriches have been identified as important part in the life cycle of CCHFV and further as potential vector for human infection (Shepherd et al., 1987a; Swanepoel et al., 1998).

The role of different amplifying hosts may vary. While some amplifying hosts like cattle, sheep, goats and ostriches are thought to be involved in the transmission of

CCHFV to humans, others, such as hares, hedgehogs and ground-feeding birds, may support the preservation of the virus circulation in an area (Spengler et al.,

2016).

However, the role of species already identified as amplifying hosts and especially the impact of other species in the ecology of CCHFV is still only partially understood.

Studying their impact on CCHFV spread is essential for risk assessment and the general understanding of the CCHFV infection cycle. Further, it is mandatory for the development of control and containment strategies, like the approach to control

CCHFV maintenance and circulation by vaccination of important amplifying hosts of the virus in combination with acaricide treatment (EFSA, 2010; Mertens et al., 2013;

Spengler and Bente, 2015).

Despite the need of studies regarding the distribution of CCHFV and on amplifying hosts of the virus, there are no commercial tests available for CCHFV serology in animals (Mertens et al., 2013). Therefore, in several laboratories in-house tests have been developed and commercial tests for serology in humans have been adapted for

testing animal sera (Burt et al., 1993; Garcia et al., 2006; Mertens et al., 2013; OIE,

2014; Qing et al., 2003; Spengler et al., 2016).

The assay formats used were: complement fixation, reverse passive hemagglutination inhibition, virus neutralization, immunodiffusion, immunofluorescence (IFA) and enzyme-linked immunosorbent (ELISA) (Hoogstraal,

1979; Mertens et al., 2013; OIE, 2014; Spengler et al., 2016; Whitehouse, 2004).

Today, complement fixation, reverse passive hemagglutination inhibition and immunodiffusion tests are considered as outdated since they lack both sensitivity and reproducibility (Hoogstraal, 1979; Spengler et al., 2016; Whitehouse, 2004). Instead,

IFA, virus neutralization and ELISA are the assay formats commonly used in CCHFV serology (OIE, 2014; Spengler et al., 2016). While the virus neutralization test is considered as the gold standard in the diagnosis of many other viruses in the order

Bunyavirales, its use is not recommended for CCHFV serology (Burt et al., 1994;

OIE, 2014; Yilmaz et al., 2009). For CCHFV it lacks sensitivity, since members of the family Nairoviridae generally induce a weaker neutralizing antibody response than other virus genera within the order Bunyavirales (OIE, 2014; Peters and LeDuc,

1991). For detection of the general IgG population IFAs for CCHFV are less sensitive than the available ELISAs (OIE, 2014; Whitehouse, 2004).

However, many of the published assays were either not validated or the validation data was not published (Mertens et al., 2013). This might be due to the lack of an accepted gold standard in CCHFV serology as well as the need of a well characterized panel of serum samples from the animal species the test should be used for (Mertens et al., 2013; OIE, 2014).

The aim of the work presented here was the establishment and validation of species- specific as well as species-independent tests for the detection of CCHFV-specific IgG antibodies. The tests should be produced and performed in a BSL2 environment.

Therefore, ELISAs were developed which are based on recombinant N-protein, which was expressed in E. coli system. The N-protein is considered to be the most conserved structure protein within the order Bunyavirales (Pepin et al., 2010) and has been used successfully for the development of several ELISAs for the detection of CCHFV-specific antibodies (Atkinson et al., 2016b; Dowall et al., 2012; Garcia et al., 2006; Qing et al., 2003; Rangunwala et al., 2014; Saijo et al., 2002; Saijo et al.,

2005a, b; Samudzi et al., 2012; Tang et al., 2003).

Initially, two commercial tests (ELISA, IFA) originally designed for CCHFV serology in humans, were adapted for testing serum samples from sheep and goats. Therewith, a reference serum panel was established which was applied in the development of new in-house ELISAs. Further, the adapted commercial assays were used as confirmation tests in seroepidemiological studies.

In a second step, indirect in-house ELISAs for the detection of CCHFV-specific IgG antibodies in sheep and goats were developed. Sheep and goats are considered to be important amplifying hosts of CCHFV and a source of human infections (Spengler et al., 2016). Next to the species-specific indirect ELISAs, a competitive ELISA

(cELISA) for the species-independent detection of CCHFV-specific antibodies was developed. For this purpose, monoclonal antibodies (mAbs) against recombinant N- protein were generated in mice and characterized in different assays for their

reactivity and their ability to compete with CCHFV-specific serum antibodies. Finally, the mAb which achieved the best results in all tests was chosen for the cELISA development. The cELISA was validated using serum samples from twelve different animal species and humans, to verify that it can be used as a species-independent test. However, the cELISA produced within this work is the second cELISA for the detection of CCHFV-specific antibodies described so far (Burt et al., 1993) and the first which was produced based on a recombinant antigen. While the indirect ELISAs functioned as low-cost screening tests in seroepidemiological studies, the cELISA should be used for studying potential hosts of the virus. In addition, the cELISA can be used to identify and monitor CCHFV foci.

Following a flow-chart, the newly developed and adapted assays were used to perform seroepidemiological studies in Albania, Bulgaria, the Former Yugoslav

Republic of Macedonia and Turkey (Mertens et al., 2016; Mertens et al., 2009;

Schuster et al., 2016b). The results of the studies from Albania and the Former

Yugoslav Republic of Macedonia were further used to compare the prevalence found in cattle, sheep and goats to identify which species is most suited for the role of indicator for CCHFV circulation in an area (Schuster et al., 2016b).

Chapter 2: Sheep and goats as indicator animals for the circulation of CCHFV in the environment

Isolde Schuster, Marc Mertens, Slavcho Mrenoshki, Christoph Staubach, Corinna Mertens, Franziska Brüning, Kerstin Wernike, Silke Hechinger, Kristaq Berxholi, Dine Mitrov, Martin H. Groschup

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus, which causes a serious illness with case-fatality rates of up to 80 % in humans. CCHFV is endemic in many countries of Africa, Asia and Southeastern Europe. Next to the countries with endemic areas, the distribution of CCHFV is unknown in Southeastern Europe. As the antibody prevalence in animals is a good indicator for the presence or absence of the virus in a region, seroepidemiological studies can be used for the definition of risk areas for CCHFV. The aim of the present study was to reveal which ruminant species is best suited as indicator for the detection of a CCHFV circulation in an area.

Therefore, the prevalence rates in sheep, goats and cattle in different regions of

Albania and Former Yugoslav Republic of Macedonia were investigated. As there are no commercial tests available for the detection of CCHFV-specific antibodies in animals, two commercial tests for testing human sera were adapted for the investigation of sera from sheep and goats, and new in-house ELISAs were developed. The investigation of serum samples with these highly sensitive and specific assays (94–100 %) resulted in an overall prevalence rate of 23 % for Albania and of 49 % for Former Yugoslav Republic of Macedonia. Significant lower

seroprevalence rates for CCHFV were found in cattle than in small ruminants in given areas. These results indicate that small ruminants are more suitable indicator animals for CCHFV infections and should therefore be tested preferentially, when risk areas are to be identified.

Experimental and Applied Acarology

http://link.springer.com/article/10.1007%2Fs10493-015-9996-y

March 2016, Volume 68, Issue 3, pp 337–346.

Authors Contributions:

Generation of diagnostic materials and development of diagnostic methods: Isolde Schuster (sheep and goat specific tests), Marc Mertens (cattle specific tests)

Exploitation of the serum samples: Slavcho Mrenoshki, Corinna Mertens, Franziska Brüning, Kerstin Wernike, Silke Hechinger, Kristaq Berxholi, Dine Mitrov

Testing of the serum samples: Isolde Schuster (sheep and goat sera), Marc Mertens (cattle sera)

Evaluation and interpretation of the data: Isolde Schuster (data from sheep and goats), Marc Mertens (data from cattle), Christoph Staubach (statistical analysis)

Wrote the paper: Marc Mertens, Isolde Schuster and Martin H. Groschup

Conceived and designed the experiments: Marc Mertens, Isolde Schuster, Martin H. Groschup

Chapter 3: Crimean-Congo Hemorrhagic Fever Virus in Bulgaria and Turkey

Marc Mertens, Isolde Schuster, Miriam Andrada Sas, Zati Vatansever , Zdenek Hubalek , Esin Güven , Ahmet Deniz , Georgi Georgiev , Raiko Peshev , Martin H. Groschup.

Abstract

Infections of humans with the tick-borne Crimean-Congo hemorrhagic fever virus

(CCHFV) can cause a severe hemorrhagic fever with case fatality rates of up to 80%.

Most humans are infected by tick bite, crushing infected ticks by hand or by unprotected contact with blood of viremic mammals. Next to the notified human

CCHF cases, the real distribution and the situation in animals in Southeastern

Europe are nearly unknown. Since domestic ruminants play a crucial role in the life cycle of the vector ticks and the transmission and amplification of the virus, the antibody prevalence in those animals is a good indicator for the presence of CCHFV in a region. Therefore, the prevalence of CCHFV-specific antibodies was investigated in domestic ruminants of different regions of Bulgaria and Turkey. Sera of 1165 ruminants were tested and a prevalence of up to 90% was identified. The overall prevalence for Bulgaria was 26% and for Turkey 57%. The results highlight the risk of human infections in those regions and the importance of the investigation of the prevalence in animals for identification of risk areas. This article provides a unique overview about published CCHFV antibody prevalence in animals in comparison to human incidences in different areas of Bulgaria and Turkey. Although it will help to

complete the understanding of the CCHFV situation in these countries, it also demonstrates the lack of unpublished and published data even in these highly endemic areas.

Vector-Borne and Zoonotic Diseases

http://online.liebertpub.com/doi/10.1089/vbz.2016.1944

September 2016, Volume 16, Issue 9, pp. 619-623.

Authors Contributions:

Generation of diagnostic materials and development of diagnostic methods: Marc Mertens (tests for serum samples from cattle) and Isolde Schuster (tests for serum samples from sheep and goats)

Exploitation of the serum samples: Zati Vatansever, Zdenek Hubalek, Esin Güven, Ahmet Deniz, Georgi Georgiev, Raiko Peshev

Testing of the serum samples: Marc Mertens and Miriam Andrada Sas (serum samples from cattle) and Isolde Schuster (serum samples from sheep and goats)

Evaluation and interpretation of the data: Marc Mertens (all data) and Isolde Schuster (data from sheep and goats)

Wrote the paper: Marc Mertens, Zati Vatansever, Martin H. Groschup

Conceived and designed the experiments: Marc Mertens, Martin H. Groschup

Chapter 4: 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

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) circulates in many countries of

Asia, Africa, and Europe. CCHFV can cause a severe hemorrhagic fever in humans with case-fatality rates of up to 80%. CCHF is considered to be one of the major emerging diseases spreading to and within Europe. Ticks of the genus Hyalomma function as vector as well as natural reservoir of CCHFV. Ticks feed on various domestic animals (e.g. cattle, sheep, goats) and on wildlife (e.g. hares, hedgehogs).

Those animal species play an important role in the life cycle of the ticks as well as in amplification of CCHFV.

Here we present a competitive ELISA (cELISA) for the species-independent detection of CCHFV-specific antibodies. For this purpose, Nucleocapsid (N) protein specific monoclonal antibodies (mAbs) were generated against an Escherichia coli

(E. coli) expressed CCHFV N-protein. Thirty-three mAbs reacted with homologous and heterologous recombinant CCHFV antigens in ELISA and Western blot test and

20 of those 33 mAbs reacted additionally in an immunofluorescence assay with eukaryotic cells expressing the N-protein. Ten mAbs were further characterized in a prototype of the cELISA and nine of them competed with positive control sera of 17

bovine origin. The cELISA was established by using the mAb with the strongest competition. For the validation, 833 sera from 12 animal species and from humans were used. The diagnostic sensitivity and specificity of the cELISA was determined to be 95% and 99%, respectively, and 2% of the sera gave inconclusive results.

This cELISA offers the possibility for future large-scale screening approaches in various animal species to evaluate their susceptibility to CCHFV infection and to identify and monitor the occurrence of CCHFV.

Antiviral Research

http://www.sciencedirect.com/science/article/pii/S0166354216304272

October 2016, Volume 134, pp.161-166.

Authors Contributions:

Generation of diagnostic materials and development of diagnostic methods: Isolde Schuster (cELISA development and validation), Bernd Köllner and Tomáš Korytář (generation of the mAbs), Marc Mertens (development of the reference tests for serum samples from cattle), Markus Keller (purification of mAb)

Exploitation of the serum samples: Bärbel Hammerschmidt, Thomas Müller, Noël Tordo, Philippe Marianneau, Claudia Mroz, Melanie Rissmann, Eileen Stroh, Lisa Dähnert, Felicitas Hammerschmidt, Rainer G. Ulrich

Testing of the serum samples: Isolde Schuster

Evaluation and interpretation of the data: Isolde Schuster (main part) and Marc Mertens

Wrote the paper: Isolde Schuster, Marc Mertens, Rainer G. Ulrich, Martin H. Groschup

Conceived and designed the experiments: Marc Mertens, Isolde Schuster, Martin H. Groschup

Chapter 5: Discussion

CCHFV is a tick-borne virus which can cause a severe hemorrhagic disease in humans with high case-fatality rates and the risk of human-to-human transmission

(Hoogstraal, 1979; Schwarz et al., 1997; Yen et al., 1985). Due to changes in climate, land use, hunting activities as well as for economic reasons, CCHFV is currently spreading further north in Europe (Estrada-Pena et al., 2013; Maltezou et al., 2010; Mertens et al., 2013).

Four previously unaffected European countries reported CCHF cases within the last fifteen years (Garcia Rada, 2016; Hornok and Horvath, 2012; Karti et al., 2004; Papa et al., 2010). In August 2016, the first autochthonous CCHF cases in Western Europe were reported from Spain (Negredo et al., 2017). The detection of Hyalomma marginatum populations in Southern France, Italy and Portugal as well as reports of

CCHFV-specific antibodies in humans and bats in Portugal and France raised concerns regarding a further spread of CCHFV in Western Europe (Dantas-Torres and Otranto, 2013; Filipe et al., 1985; Hoogstraal, 1979; Santos-Silva et al., 2011;

Vial et al., 2016).

Despite those developments, little is known regarding the CCHFV situation in countries next to those with confirmed human cases (Mertens et al., 2013), albeit the knowledge about the spread of the virus is essential for the prevention of human

CCHF cases (Mertens et al., 2013). Therefore, studies on the distribution and the ecology of CCHFV within Europe are required urgently (Mertens et al., 2013).

The aim of this thesis was the development of highly sensitive and specific CCHFV

ELISAs for testing serum samples from sheep and goats on the basis of a protocol for testing serum samples from cattle (Mertens et al., 2015). For species-independent testing, a competitive ELISA for the detection of CCHFV-specific antibodies should be developed. Those tests should be used as low-cost screening tests in affected and endangered areas and further be applied in partner laboratories for conducting seroepidemiological studies to create a comprehensive map of the distribution of

CCHFV in Europe. The competitive ELISA should additionally be used for studying the involvement of different animal species in the infection cycle of CCHFV.

Presumed to be the most appropriate test among the three assay formats commonly used in CCHFV serology (Enzyme-linked immunosorbent, Immunofluorescence,

Virus Neutralization), the ELISA was chosen, being a fast, reproducible, objective and reliable technique that can be used to process a high throughput of samples

(Dowall et al., 2012) Spengler et al. 2016, OIE 2014). In contrast, the IFA has been shown to be less sensitive and more time-consuming. Further, the interpretation of results depends very much on the experience of the evaluating person (Dowall et al.,

2012; Spengler et al., 2016; Whitehouse, 2004). The Virus Neutralization Test (VNT) is not presumed to be a sensitive test for the virus as well, since CCHFV and other viruses in the family Nairoviridae, only cause a weak neutralizing antibody response in their mammalian host (Mertens et al., 2013; OIE, 2014; Peters and LeDuc, 1991).

In order to develop new ELISA tests, recombinant N-protein of a CCHFV strain from

Kosovo was expressed with a His-tag in E.coli system and purified using Ni-NTA

system (Duh et al., 2008). The N-protein has been shown to be the most conserved structure protein within the order Bunyavirales and induces a strong antibody response in the mammalian hosts of the virus (Bertolotti-Ciarlet et al., 2005; Burt et al., 2013; Burt et al., 1993; Dowall et al., 2012; Mousavi-Jazi et al., 2012; Pepin et al.,

2010; Wei et al., 2010; Zivcec et al., 2016).

Since CCHFV is a Risk Group 4 pathogen, recombinant CCHFV antigens are preferred for developing safe serological tests under BSL2 conditions (Dowall et al.,

2012). Further, they are highly reproducible and highly immunogenic (Buffolano et al., 2005; Meyer, 2010). Recombinant N-proteins, expressed in mammalian cells via the Semliki Forest alphavirus replicon, by baculovirus system in insect cells and in plant and E. coli expression systems have previously been published as antigens for development of different ELISAs for the detection of CCHFV-specific IgG and IgM antibodies in humans and animals (Atkinson et al., 2016a; Dowall et al., 2012; Garcia et al., 2006; Qing et al., 2003; Rangunwala et al., 2014; Saijo et al., 2002; Saijo et al.,

2005a, b; Samudzi et al., 2012; Tang et al., 2003).

The indirect in-house ELISAs for testing sheep and goat sera developed within this work were established by variation of various parameters. Thereby, the indirect

ELISAs for testing serum samples from sheep and goats are slightly different, with variations in the blocking buffers, conjugates and conjugate dilutions.

For setting up a reference serum panel for the validation of the new in-house ELISAs and for confirming positive results of sera in seroepidemiological studies, two commercially available tests for serology in humans (ELISA, IFA) have been adapted

for the detection of CCHFV-specific IgG antibodies in sheep and goats. The commercial ELISAs based on inactivated whole-virus from a CCHFV strain from

Uzbekistan (clade IV, dependent on a nucleic acid sequences of the S-segment) as antigen, the IFAs based on eukaryotic cells expressing N-protein and Gc-protein of a

CCHFV strain from Nigeria (clade III). In contrast, the recombinant N-protein used for the development of the in-house ELISAs based on a CCHFV strain (clade V) from

Kosovo (Bente et al., 2013; Duh et al., 2008).

To overcome the lack of a gold standard in CCHFV serology, the in-house ELISAs were validated by comparative analysis with the reference assays and the reference tests were validated in comparison with the respective indirect in-house ELISA and the second reference test.

The validation revealed high diagnostic sensitivities and specificities for the in-house and the adapted commercial tests. The diagnostic sensitivities of the adapted commercial tests for sheep and goats were >97% and the diagnostic specificities were >93%. The diagnostic sensitivity of the indirect in-house ELISA for testing serum samples from sheep was 96% and the diagnostic specificity was 98%. The validation of the in-house ELISA for testing serum samples from goats revealed a diagnostic sensitivity of 98% and a diagnostic specificity of 96%. In both assays, only

5% of the tested serum samples were inconclusive.

The in-house ELISA for testing serum samples from goats developed within this work is the first ELISA described so far specifically designed for the detection of CCHFV- specific antibodies in this species.

For testing sera from sheep, a few CCHFV ELISAs have been described, but only one of them has been validated (Burt et al., 1993; Garcia et al., 2006; Qing et al.,

2003). The test was based on a recombinant antigen and was validated utilizing an

IFA based on native and recombinant antigens of a CCHFV strain assigned to the same clade, resulting in diagnostic sensitivities of 90-95% and diagnostic specificities of 95-100%, depending on the cut-off value chosen respectively (Qing et al., 2003).

For the validation of the here introduced new ELISAs, a more reliable validation system was used, in which the ELISAs were not only compared to a different test system (ELISA vs. IFA) or different types of antigens (recombinant N-protein expressed in E.coli system vs. whole-virus antigens vs. eukaryotic cells expressing

CCHFV proteins), but also to antigens of different strains and of different clades

(CCHFV strain from Kosovo, clade V vs. CCHFV strain from Uzbekistan, clade IV vs.

CCHFV strain from Nigeria, clade III; clades depending on the genetic information on the s-segment, following the designation by Bente et al. 2013) (Bente et al., 2013).

Further, in contrast to the test developed by Quing et al., the here presented ELISAs base on two cut-offs, one upper and one lower cut-off with an inconclusive zone in- between (Qing et al., 2003). Thereby testing of samples can lead to an inconclusive result instead of giving a wrong positive or wrong negative result. It reflects the reality of virological serology that every test has its limitations and some samples cannot be tested with every test. Therefore, the use of a 3-zone model including an inconclusive zone is recommended by the OIE (Feinstein, 1990; OIE, 2009).

Both for the development of the ELISA published by Qing et al. and the ELISAs developed within this work, the negative reference serum samples have been

obtained from a region or country considered to be free of CCHFV (Qing et al.,

2003). This method is disputed since sera obtained from animals originated from a disease-free area might not necessarily represent the population to which the serological test should be applied (Mboloi et al., 1999; OIE, 2009). On the other hand, without having a gold standard test, this approach offers the highest likelihood to work with real negative samples and to be sure, that positive results are really wrong positive in the new assay and not wrong negative in the reference test (Mboloi et al., 1999).

Unfortunately, potential cross-reactivity of the here presented new ELISAs with antibodies derived against other viruses in the order Bunyavirales could only partially be examined, due to the lack of serum samples confirmed positive for those viruses.

In general, CCHFV and Hazara virus should be cross-reactive with each other, since they are both assigned to one serogroup (Hoogstraal, 1979). Further, cross-reactivity between CCHFV and Dugbe, Qalyub and Nairobi sheep disease virus are considered possible, but results of studies on that topic are contradictory (2006; Burt et al., 1996; Marriott et al., 1994; Spengler et al., 2016; Ward et al., 1992). However, since those viruses have never been detected in Europe, potential cross-reactivities with those viruses should not have an impact on the tests developed within this work, as they were primarily created to be used for seroepidemiological studies within

Europe. A cross-reactivity with Rift Valley fever virus could be excluded (Mertens et al., data not shown), as well as a cross-reaction with Schmallenberg virus, a virus in the order Bunyavirales circulating in Europe (Schuster et al., data not shown).

In a next step, the new species-specific ELISAs for the detection of CCHFV-specific antibodies in sheep and goats as well as a cattle-specific ELISA were applied in two seroepidemiological studies in four different CCHFV affected European countries

(Mertens et al., 2016; Mertens et al., 2015; Schuster et al., 2016b), using a defined diagnostic hierarchy based on the different test systems (ELISAs and IFA) in order to obtain valid results.

Within the first seroprevalence study, 534 serum sample from cattle, sheep and goats from five regions of Albania as well as 330 serum samples from cattle, sheep and goats from three regions of the Former Yugoslav Republic of Macedonia were tested (Schuster et al., 2016b). Sporadic CCHF cases have been reported from

Albania since 1986, the majority of them from the regions of Kukes and Has in the north-eastern part of the country (Papa et al., 2009). Further, a spread of CCHFV through out whole of Albania was discussed, as sporadic cases had been reported from several regions within the country (Papa et al., 2002a).

In a previous study, a prevalence of CCHFV-specific antibodies of 20% was found in sera collected from ten goats from a farm in Has, which was associated with a CCHF outbreak in 2003 (Papa et al., 2009). In the here presented study, a prevalence of

24% was found in the goat sera collected from that region, which perfectly matches the result of the study conducted by Papa et al. (Papa et al., 2009). The overall prevalence for CCHFV-specific antibodies in the animal population from Albania tested within the here presented study was 23% (Schuster et al., 2016b). The detection of CCHFV-specific antibodies in at least one of three animal species tested from each of the five regions included in the study presented here supports the

theory of a spread of CCHFV throughout Albania (Papa et al., 2002a; Schuster et al.,

2016b).

From the Former Yugoslav Republic of Macedonia, few CCHF cases have been reported so far (Vesenjak-Hirjan et al., 1991). Within the study presented here, an overall prevalence for CCHFV-specific antibodies of 49% was found in the animal population tested. Except of goats from the region of Vardar and cattle from the southeastern region, CCHFV-specific antibodies were found in all species from all regions, indicating a wide spread of the virus throughout the Former Yugoslav

Republic of Macedonia (Schuster et al., 2016b).

For some regions in Albania and the Former Yugoslav Republic of Macedonia, significant differences have been found between the three ruminant species tested.

Those differences may be due to factors which will be discussed in detail later on in this text. Further, they may be artifacts owed the sampling.

The high prevalence found in the animal population tested from the Former Yugoslav

Republic of Macedonia within the study was unexpected and surprising, considering the low number of CCHF cases reported so far from this country and even more so when compared to the prevalence found in the animal population from Albania, a country from which CCHF cases are reported regularly (Mertens et al., 2015; Papa et al., 2002a).

The incongruence of the number of reported CCHF cases and the seroprevalence in the animal population in the Former Yugoslav Republic of Macedonia may be

explained by a “silent” circulation of the virus in nature, due to a balance between the population of the first and the second feeding hosts of the tick vectors of the virus

(Bente et al., 2013). From Albania, an imbalance in both populations was described for the years with CCHF epidemics (Papa et al., 2009). Further, the low number of notified CCHF cases from the Former Yugoslav Republic of Macedonia may be the consequence of the circulation of (a) low-pathogenic or apathogenic CCHFV strain(s) as it is considered for Greece (Antoniadis and Casals, 1982; Bente et al., 2013;

Mertens et al., 2013; O. Papadopoulos and Koptopoulos, 1980; Papa et al., 2010;

Papa et al., 2011b). In Greece, seropositivity in animals and humans has been described over a few decades, while the only CCHF case reported from Greece so far was caused by a CCHFV strain more likely of Bulgarian origin (Antoniadis and

Casals, 1982; Papa et al., 2010). Likewise, the low number of CCHF cases notified from the Former Yugoslav Republic of Macedonia could be explained by a lack of awareness of the local medical community for CCHF and the consequent lack of detection of the virus (Mertens et al., 2013; Mertens et al., 2015). In a reverse conclusion, the high number of CCHF cases reported from Albania could be due to a sensitive reporting system for CCHF cases owed to a high awareness of the medical system, as considered for Turkey (Burki, 2012), or a virus strain with much higher pathogenicity as highlighted by the high case fatality rate. Since from both countries not only the positive but also the negative samples were confirmed in the reference

ELISA which were based on an antigen of a highly different CCHFV strain than the antigen used in the screening ELISAs (Schuster et al., data not shown), the incongruence of the prevalence and the reported CCHF cases can also not be

explained by a better cross-reactivity of the local CCHFV strains with the antigen used in the screening ELISA.

The second seroepidemiological study was conducted in Turkey and Bulgaria using

497 serum samples from cattle and sheep and 619 serum samples from cattle, sheep and goats respectively (Mertens et al., 2016).

From Turkey, the first CCHF case was reported in 2002 (Karti et al., 2004).

Subsequently, Turkey developed into a high-endemic country, with up to 1,400

CCHF cases reported annually (Mertens et al., 2016; Yilmaz et al., 2009; Public

Health Agency Turkey, 2015). The high-risk areas are central and eastern Anatolia

(Karti et al., 2004; Maltezou et al., 2010; Yilmaz et al., 2008). Despite the high number of CCHF cases reported from this country, only three seroepidemiological studies in animals have been published from Turkey so far. Those studies revealed a high prevalence in the ruminant population tested (Albayrak et al., 2012; Kirbas et al.,

2010; Tuncer et al., 2014).

The 497 serum samples from cattle and sheep tested within the present study were collected in five provinces in the central north, from one province in the western and one in the eastern part of the country. The overall prevalence found in animals from

Turkey was 57%. In animals from the central northern part a prevalence of up to 87% was found, highlighting a high-level of CCHFV circulation in this area. In contrast, no

CCHFV-specific antibodies have been detected in animals from the eastern and western province.

A comparison of the seroprevalence found in the animals tested and the human

CCHF cases notified from that particular region revealed that, from all provinces in which CCHFV-specific antibodies have been found in the animal population tested, high numbers of CCHF cases in humans have also been notified (Public Health

Agency Turkey, 2015). However, inconsistency between the seroprevalence in animals and the notified human CCHF cases could be seen for the regions of

Eskisehir and Erzurum, where no CCHFV-specific antibodies have been detected in the animal population, although high numbers of CCHF cases have been reported from those regions (Yagci-Caglayik et al., 2014; Public Health Agency Turkey, 2015).

The lack of detection of CCHFV-specific antibodies in the animals from the region of

Eskisehir is most likely owed the low number of samples collected from that region, while the inconsistency reported from the region of Erzurum can be explained by taking a closer look at the particular sampling side. The province of Erzurum consists of twenty districts, eight of whom in the northern part of the province human CCHF cases have been reported (Public Health Agency Turkey, 2015). Other districts of the province not even consist of suitable habitats to harbor stable populations of

Hyalomma marginatum ticks. The side from where the sera tested within the here presented study have been obtained falls into the latter category. It is therefore not surprising, that no CCHFV-specific antibodies have been found in the animal population from that province (Public Health Agency Turkey, 2015). Those results point out the impact of sampling sides as well as the sampling size on the results in seroepidemiological studies and confirm that negative results in seroepidemiological

studies should never be taken as definitive proof of the absence of the virus in that particular region.

From Bulgaria, CCHF cases were reported since the 1950s (Escadafal et al., 2012;

Papa et al., 2004). Within the last decade, sporadic cases have been reported regularly, mainly from the regions of Burgas, Haskovo, Kardgali, Plovdiv, Pazardgik and Shumen (Mertens et al., 2013; Papa et al., 2004). In a prior study conducted in the region of Burgas with serum samples from sheep, cattle, goats and donkeys, an overall seroprevalence of 72% had been found, but this varied depending on the species and age of the animals from 28% up to 90% (Barthel et al., 2014).

Within the study presented here, an overall prevalence of 26% was found in the animal population tested in Bulgaria. Further, the prevalence found within this study revealed a south-to-north gradient of CCHFV circulation within the country and matched quite well with the CCHF cases reported from the areas the serum samples originated from (Avsic-Zupanc, 2007; Christova et al., 2013; Mertens et al., 2016;

Vescio et al., 2012). From one province (Burgas) serum samples were available from

1974 and 2014. In the animal samples collected in 1974, a prevalence of 86% of

CCHFV-specific antibodies was found, whereas the prevalence in the serum samples collected in 2014 was 50%. The high prevalence found in the samples from both sampling dates indicates the persistence of CCHFV in this region over the last four decades.

Further, the results are comparable to those of the study conducted by Barthel et al. in that region, when taken in consideration that the species-composition used by

Barthel et al. was different than that used within the here presented study and within the here presented work the age of the tested animals had not been taken into account except of that they had been at least 18 month old when tested (Barthel et al., 2014).

In total, the results of the seroprevalence study in Turkey and Bulgaria verify those countries as high-endemic countries for CCHFV, whereby for Bulgaria the south could be confirmed as high endemic region, whereas in Turkey eastern and central

Anatolia were confirmed as high-risk areas (Mertens et al., 2016).

In conclusion, the species-specific tests developed within this work have been proven to have the capacity to be applied in seroepidemiological studies. In combination with the diagnostic scheme implemented in those studies, the tests have been shown to gain highly valid and reliable results, which matched those obtained in prior studies and with reports of human CCHF cases (Mertens et al., 2009). Unfortunately, for the animals from Albania and the Former Yugoslav Republic of Macedonia, except of the species and of their origin, no further information was available about the animals and their further conditions. For the animals from Turkey and Bulgaria, at least information regarding the age (all animals tested were at least 18 month old) and the conditions (the tested animals were fed predominantly on common grazing grounds and had been born and raised locally) was available. As different factors could have had an influence on the antibody prevalence found in particular areas or individual animals (e.g. the use of repellents or the husbandry conditions) data obtained should be interpreted cautiously (Mertens et al., 2015; Schuster et al., 2016a; Schuster et

al., 2016b; Tuncer et al., 2014). Furthermore, as the detected CCHFV-specific IgG antibodies can persist for at least three years after a CCHFV infection, no conclusions can be made regarding the CCHFV situation in the region at the time the serum samples were taken (Gonzalez et al., 1998; Zeller et al., 1997).

With the data obtained from the here presented studies, a comprehensive map of the

CCHFV distribution in those four countries can be created, which can help to assess the risk of human infection and can be used as a basis for the implementation and coordination of further local surveillance programs (Mertens et al., 2013). Those programs should incorporate more details of animals as well as additional animal species (Spengler et al., 2016; Whitehouse, 2004). In order to assess the current

CCHFV circulation, either very young animals without possible maternally acquired immunity against CCHFV should be tested or CCHFV-specific IgM-antibodies should be detected (Schuster et al., 2017). An alternative would be the detection of CCHFV in animals, but due to the short viremic period and the lack of clinical signs in animals, the chance of direct CCHFV detection is not necessarily high (Gunes et al.,

2011a; Spengler et al., 2016; Tuncer et al., 2014). It is further restricted, since reinfected animals exhibit low virus titers, which might be below the detection limit of most tests (Gonzalez et al., 1998; Wilson et al., 1991). Likewise, the success of

CCHFV antigen detection in vertebrates is dependent on the seasonal activity of the tick vectors. Therefore, next to seroepiemiological studies in animals, current virus circulation should be monitored by testing vector ticks (Mertens et al., 2015).

Most importantly, measures should be implemented to educate the public and especially the people at risk and to raise awareness in the local medical communities to prevent (further) human infections and facilitate a quick response to and treatment of new infections (Mertens et al., 2013).

In the next part of this work, the prevalence of CCHFV-specific antibodies in three different ruminant species was compared and discussed.

Due to several factors, the prevalence of CCHFV-specific antibodies might vary between animal species (Tuncer et al., 2014). First of all, the susceptibility for a

CCHFV infection might differ among species and additionally among breeds (Adam et al., 2013; Shepherd et al., 1987b; Tuncer et al., 2014). Further, host preferences of the tick vectors may play an important role (Shepherd et al., 1987b). Moreover, the host resistance to tick infestation, which was shown to differ between breeds, may have a great influence on CCHFV transmission and on the antibody response in different animal species (Ali and de Castro, 1993; Solomon and Kaaya, 1996).

Additionally, age was revealed to have an impact, since increased age of tested animals is correlated with increased antibody prevalence (Wilson et al., 1990).

Furthermore, husbandry conditions, including the usage of repellents, might have an influence on the CCHFV seroprevalence in animals as well as the habitat preferences of the different animal species (Shepherd et al., 1987b; Tuncer et al.,

2014).

Therefore, the epidemiological studies in Albania and the Former Yugoslav Republic of Macedonia were not only performed to determine the circulation of CCHFV in

those countries; they were also conducted to identify optimal indicator animals for

CCHFV circulation in those regions by comparing the prevalence for CCHFV-specific antibodies in sheep, goats and cattle.

Within the respective studies, the prevalence in sheep and goats was significantly higher than the prevalence in cattle. In contrast, no statistically relevant difference could be found between the overall prevalence found in sheep and goats.

The results were surprising, since previously published studies have demonstrated that cattle are the preferential feeding hosts of Hyalomma marginatum ticks and therefore they were considered to be the most sensitive indicator animals for low- level CCHFV circulation (Hoch et al., 2016; Spengler et al., 2016; Wilson et al.,

1990).

On the other hand, the findings of this study are in accordance with those of prior ones from Turkey, Egypt, Iraq, Iran, Oman and Saudi Arabia, in which a higher prevalence for CCHFV-specific antibodies has been found in sheep and goats compared with cattle (Kirbas et al., 2010; Mohamed et al., 2008; Tantawi et al., 1981;

Telmadarraiy et al., 2010; Tuncer et al., 2014; Williams et al., 2000). The calculation of the overall prevalence of further studies, published recently in a review on seroepidemiological studies of CCHFV in domestic and wild animal species, resulted in a higher prevalence in small ruminants as well (Spengler et al., 2016). Due to those findings, it can be speculated that cattle are the preferred hosts of Hyalomma marginatum ticks, whereby small ruminants have a higher susceptibility for CCHFV

infections. Definite prove of this theory requires animal experiments in a controlled setting.

However, on the basis of the data obtained in the study conducted here, it can be concluded that sheep and goats are the best indicator animals for CCHFV and should therefore be tested preferentially for identification of risk areas. In further studies, the role of different animal species in the ecology of CCHFV should be further examined and those species should be assessed for their suitability as additional indicators for CCHFV circulation.

To clarify the role of different animal species, as well as different breeds in CCHFV amplification and transmission, their susceptibility and finally in CCHFV ecology requires animal experiments in high biocontainment facilities. For ethical and biosafety reasons animal challenge experiments in general and under BSL4 conditions in particular should only be conducted when absolutely necessary and when no alternative is available. Only a few laboratories worldwide have the capacity to work with larger animals under BSL4 conditions (Keshtkar-Jahromi et al., 2011).

An alternative to those experiments revealing the CCHFV infection cycle could be the screening for CCHFV-specific antibodies in a broader variety of species (Schuster et al., 2016a). For this purpose, modifications of the indirect ELISA and the commercial

IFA could be used, but the species-specific adaptation of those tests for testing various animals is time-consuming, limited and sometimes even impossible (e.g. when specific antibodies or binding partners to immunoglobulins are missing or the assay cannot be adapted for unknown reasons) (OIE, 2014; Spengler et al., 2016).

Further, it requires a well characterized reference serum panel of each species the test should be used for (OIE, 2014).

For this reason, a cELISA was developed within the here presented work. cELISAs are species-independent, as the competition of the specific antibodies of the tested serum and a specific mAb is tested by detecting the reduction in binding of the specific mAb instead of the detection of the species specific antibodies derived from the sera (Meyer, 2010).

For test development, 33 mAbs were generated in mice against the recombinant N- protein of CCHFV strain Kosovo Hoti. The reaction of all mAbs with homologous and heterologous recombinant CCHFV N-proteins of the strains Kosovo Hoti and Turkey-

Kelkit06 (clade V) and in the ELISA and Western Blot tests demonstrated the high immunogenicity of the recombinant antigen as well as of the CCHFV N-protein

(Bente et al., 2013; Bertolotti-Ciarlet et al., 2005; Buffolano et al., 2005; Burt et al.,

2013; Burt et al., 1993; Meyer, 2010; Wei et al., 2010; Zivcec et al., 2016). Further, it can be assumed that they are targeting linear epitopes. A reaction of the mAbs with the His-tag could be excluded since they did not react with the recombinant His- tagged N-protein of the Puumala virus (Mertens et al., 2009).

Two-thirds of the mAbs were additionally reactive in the IFA. The other mAbs may have either been less cross-reactive with the different CCHFV strain the IFA based on or the detection limit of the IFA is lower than those of Western Blot and ELISA tests.

The ten mAbs showing the most promising results in all tests were additionally tested for their ability to compete with antibodies from sera in a first prototype of a cELISA.

Competition could be verified for nine of the ten mAbs. Finally, the mAb which exhibited the best results in all tests was chosen for the development of the cELISA.

The establishment of the test was conducted by variation of several parameters. The optimal ratio between serum-, mAb- and conjugate dilution was evaluated by checkerboard titrations. In the end, the protocol was chosen which best distinguished between the positive and negative reference serum samples. With the new cELISA, a test should be available which can be applied in large-scale approaches in various species, countries and in partner laboratories, thereby giving a minimum of false- positive results (Schuster et al., 2016a). Hence, a high specificity was emphasized in the test development (Schuster et al., 2016a).

To verify its applicability as species-independent test, the cELISA was validated using 833 serum samples from twelve different animal species and humans. The positive reference serum samples for validation originated from ruminants from south-eastern Europe, where CCHFV strains of clade V are considered to circulate.

Further, two serum samples from rhesus macaque immunized with a Nigerian

CCHFV strain (clade III) were applied for the validation.

Just like the species-specific tests, the cELISA was validated in comparison to the reference tests (ELISA, IFA) applying a two-cut-off system. The validation revealed a diagnostic sensitivity of 95% and a diagnostic specificity of 99%, while only 2% of the serum samples were inconclusive.

In addition to the sera from Europe used for validation, sera from Western and

Central Africa, where CCHFV strains of clade III are considered to circulate, reacted in the cELISA as well (Schuster et Mertens, data not shown). Based on that data, it can be assumed, that the test can be applied to various animal species and is able to detect antibodies generated against CCHFV strains from different clades (Schuster et al., 2016a).

It is the first cELISA for the detection of CCHFV-specific antibodies based on recombinant antigen and the second cELISA for CCHFV serology described so far

(Burt et al., 1993; Schuster et al., 2016a).

The first reported cELISA for the detection of CCHFV-specific antibodies was based on whole-virus antigen and therefore needed to be produced in high-biocontainment facilities, whereas the development of the cELISA presented here can be conducted in a BSL2 environment, reducing costs and expenditures (Burt et al., 1993). Further, the first reported cELISA was validated in comparison to an IFA based on the same antigen. This yields less reliable results than validating the test in comparison to reference tests based on different CCHFV strains of different clades, as done with the here introduced new cELISA (Burt et al., 1993).

Furthermore, sera obtained from experimentally infected animals were applied for the comparison of the cELISA developed by Burt et al. and the regarding IFA, which makes it difficult to make predictions regarding the fitness of the test when used with serum samples originated from naturally infected animals (Burt et al., 1993; OIE,

2009). Except for the two positive reference sera from monkeys which had been infected experimentally, all serum samples used for the development and validation

of the new cELISA presented here were derived from animals which had a natural

CCHFV infection (Schuster et al., 2016a).

The cELISA developed by Burt et al. required 50 µl of serum for each testing, whereas the cELISA developed within this work requires only 25 µl (Burt et al., 1993;

Schuster et al., 2016a). This is less than the volume required for many commercially available cELISAs for the detection of antibodies specific for other viruses like Rift

Valley Fever virus and Hepatitis E virus, but still is a large and therefore prohibitive volume especially when very small animal species are to be tested.

One possible further restriction of the applicability of the here introduced new cELISA are (cross-) reactions of antibodies of certain species with the anti-mouse IgG conjugate which detects the binding of the mAb (Schuster et al., 2016a). Yet, reactivity of the conjugate with antibodies derived from animal sera could exclusively be seen for sera from mice (Schuster et al., 2016a).

Likewise, the applicability of the cELISA for testing other species than those used for the validation has not been proven yet and could potentially restrict its use (Schuster et al., 2016a). However, the cELISA seems to be highly specific for testing other animal species, since only very few false-positive results were produced by testing

676 negative serum samples from twelve different species and from humans. This may suggest that the test is applicable for testing other species than the above mentioned as well.

Anyhow, the new cELISA can be used for large-scale approaches in various animal species to reveal their role in the infection cycle of CCHFV. Such studies are crucial

for CCHFV risk assessment and further for the development of control strategies

(Spengler et al., 2016).

Once identified, the populations of amplifying hosts of CCHFV could be monitored and controlled by forestry and agricultural agencies, to prevent increases of and imbalances between the populations which have led to CCHF outbreaks in the past

(Bente et al., 2013; EFSA, 2010; Ergonul, 2006; Papa et al., 2009). In addition to controlling the populations of amplifying hosts approaches could be taken to control the circulation of the virus in nature (Spengler et al., 2016). One promising approach of controlling the CCHFV circulation and thereby decreasing the risk of human CCHF infections could be the vaccination of important amplifying hosts against the infestation with CCHFV vector competent ticks and/or CCHFV in combination with repellents treatment (EFSA, 2010; Mertens et al., 2013; Spengler and Bente, 2015).

As such vaccines are currently unavailable, an alternative approach could be the use of animal breeds naturally (more) resistant to tick infestation or CCHFV infection in

CCHF endemic areas to lower the risk of CCHFV transmission from livestock and its ticks to humans (EFSA, 2010; Mertens et al., 2013). Such breeds could be identified using the here introduced new cELISA.

In a future study, the antigen binding side of the mAb used for the cELISA should be determined. So far, only few studies were conducted to explore the antigenic regions and conserved epitopes on the N-protein of CCHFV (Burt et al., 2013; Liu et al.,

2014; Saijo et al., 2002; Wei et al., 2010). Within those studies, computer-based analysis has been used to predict potential epitopes on the N-protein. Subsequently,

those peptide fragments were expressed in E. coli or synthetized and tested for their ability to react with CCHFV-specific antibodies derived from human and animal sera.

On basis of the data revealed in those studies, the region between amino acid 123 and amino acid 396 was considered to be the main antigenic region of CCHFV N- protein, but promising epitopes could be found only in one of these studies (Burt et al., 2013; Liu et al., 2014; Saijo et al., 2002; Wei et al., 2010). In this study only a few sera were applied for epitope-identification (Liu et al., 2014).

Using the mAb applied in the here introduced new cELISA, a reverse approach is possible. Since the cELISA was successfully used for the discrimination of negative and positive samples from different animal species from different geographical regions, the epitope targeted by the mAb seems to be conserved among species and was shown to be recognized by specific antibodies generated against different

CCHFV strains (Schuster et al., 2016a). Using an epitope mapping technique, the epitope could be identified and the respective peptide fragment could then be synthesized and used for the development of further tests for CCHFV serology.

Moreover, such subunit vaccines and antibodies against them could possibly be utilized for the prophylaxis and therapy of CCHF.

Since there are currently no tests (commercially) available for CCHFV serology in animals, both the cELISA as well as the species-specific in-house ELISAs developed within this work should be commercialized and made available for CCHFV endangered and affected countries in Europe and beyond (OIE, 2014; Schuster et al., 2016b).

Using those tests as well as the diagnostic scheme which has shown to produce highly valid and reliable results, seroepidemiological studies can be performed to assess the CCHFV distribution in countries (Mertens et al., 2009; Schuster et al.,

2017; Schuster et al., 2016a; Schuster et al., 2016b), which will allow to draw a comprehensive map of the CCHFV distribution within Europe and worldwide eventually.

Chapter 6: Summary Novel serological assays for determination of the distribution and of hosts of Crimean-Congo hemorrhagic fever virus

Isolde Schuster

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that is endemic in many countries of Africa, Asia and Europe. The vector and reservoir of

CCHFV in nature are ixodid ticks, that can transmit the virus during their blood meal to their blood hosts and vice versa. CCHFV infected animals do not show clinical signs but can develop a viremia and seroconvert subsequently.

Via tick-bite, the crushing of infected ticks and contact to blood, bodily fluids and tissue of viremic animals or humans, the virus can be transmitted to humans, in which it can cause the Crimean-Congo hemorrhagic fever (CCHF) with case-fatality rates of up to 80%. No vaccination or treatment is currently available.

Four previously unaffected European countries reported CCHF cases within the last fifteen years, among them Spain as first affected Western European country in 2016.

Despite the thread of a further spread of the virus within Europe and the impact of prevention measures against human infections, little is known regarding the CCHFV situation in countries other than those with known CCHF cases. Seroepidemiological studies in animals could provide more insight, but there are no commercial tests for

CCHFV serology in animals available and most of the published in-house tests have not been validated. Therefore, the aim of the here presented work was the

development and validation of Enzyme-linked immunosorbent assays (ELISAs) for the detection of CCHFV-specific IgG antibodies in animals.

At first, species-specific in-house ELISAs for the detection of CCHFV-specific antibodies in sheep and goats where developed on the basis of a protocol for testing sera from cattle by modification of several parameters. Hence, the in-house ELISA for testing of goats is the first CCHFV ELISA specifically designed for this species. As reference tests, immunofluorescence and ELISA tests commercially available for

CCHFV serology in humans were adapted to be used in sheep and goats. All tests where validated in comparison to each other, revealing diagnostic sensitivities and specificities of 94-100%. Further, the new tests as well as cattle-specific tests were applied implementing a specifically designed diagnostic scheme in two seroepidemiological studies in Albania and the Former Yugoslav Republic of

Macedonia as well as Bulgaria and Turkey, finding high seroprevalence in cattle, sheep and goats. The results indicated a high level of circulation of CCHFV in the

Former Yugoslav Republic of Macedonia, which was rather surprising, as only few

CCHF cases have been reported from this country so far. They also confirmed a spread of CCHFV throughout the whole of Albania, and verified the south of Bulgaria and northern and central Turkey as the main endemic areas of those countries.

To assess the species most suited as indicator of the distribution of CCHFV, the prevalence found in the animals from Albania and the Former Yugoslav Republic of

Macedonia were compared, revealing sheep and goats as more suitable indicator animals than cattle, which should therefore be used preferably for the determination of the local CCHFV distribution.

Next to the species-specific tests, the first competitive ELISA (cELISA) for CCHFV based on recombinant antigen and the second CCHFV cELISA described so far has been developed within this work. In order to find a suitable monoclonal antibody

(mAb) for the development of the test, 33 CCHFV-specific mAbs have prior been characterized in different tests.

The cELISA was validated using 833 sera from twelve different animal species and humans, revealing a diagnostic sensitivity of 95% and a diagnostic specificity of 99%.

Utilizing the new cELISA, seroprevalence studies in different animal species will be possible which in turn may help to understand their role in the ecology of CCHFV.

Moreover, species essential for the circulation of CCHFV could therewith be identified and vaccinated against CCHFV and/or tick infestation in order to control the

CCHFV circulation in the wild. The monoclonal antibody used in the new cELISA as well as its epitope may further be promising targets in the development of diagnostic tests, therapeutics and vaccines.

The new tests for the detection of CCHFV-specific antibodies developed within this work have been demonstrated to be valid and reliable and should be commercialized and made available to CCHFV affected and endangered countries to identify high risk areas and to draw a comprehensive map of the CCHFV distribution in Europe and the world eventually.

Chapter 7: Zusammenfassung

Neue serologische Tests zur Ermittlung der Verbreitung und zur Identifizierung von Wirten des Krim-Kongo-Hämorrhagischen- Fieber-Virus

Isolde Schuster

Das Krim-Kongo-Hämorrhagische-Fieber-Virus (CCHFV) ist ein Zecken-

übertragenes Virus, das in vielen Ländern Afrikas, Asiens und Europas endemisch ist. Seine Vektoren und Reservoire sind Schildzecken, die das Virus während der

Blutmahlzeit auf ihre Blutwirte übertragen und umgekehrt. CCHFV löst in Tieren keine Erkrankung aus, diese können aber eine Virämie entwickeln und serokonvertieren.

Durch Zeckenstiche, das Zerdrücken infizierter Zecken oder durch den Kontakt zu

Blut, Körperflüssigkeiten oder Geweben virämischer Tiere und Menschen kann das

Virus auf Menschen übertragen werden und in ihnen eine schwere Erkrankung, das

Krim-Kongo-Hämorrhagische Fieber (Crimean-Congo hemorrhagic Fever, CCHF) auslösen, das mit einer Sterblichkeitsrate von bis zu 80% einhergeht. Bisher ist weder eine Impfung noch eine spezifische Therapie verfügbar.

In den letzten fünfzehn Jahren sind CCHF Fälle in vier bis dato als CCHF-frei geltenden europäischen Ländern erstmals aufgetreten, unter ihnen Spanien als erstes betroffenes westeuropäisches Land 2016. Trotz der Angst einer weiteren

Ausbreitung in Europa und trotz der Dringlichkeit der Vorbeugung humaner

Infektionen ist wenig über die Verbreitung von CCHFV außerhalb der Länder, aus denen bereits CCHF-Fälle berichtet wurden, bekannt. Hierzu können

Seroprävalenzstudien in Tieren wesentliche Hinweise liefern, jedoch sind keine kommerziellen serologischen Tests zur Diagnostik von CCHFV in Tieren verfügbar und die bisher publizierten laborspezifischen Tests wurden nicht validiert.

Deshalb war das Ziel der hier vorgelegten Arbeit, Enzym-linked Immunosorbent

Assays (ELISA) Tests zur Detektion CCHFV-spezifischer IgG Antikörper in Tieren zu entwickeln und zu validieren.

Zunächst wurden Spezies-spezifische Tests zur Detektion CCHFV-spezifischer

Antikörper in Schafen und Ziegen auf Basis eines Protokolls zur Untersuchung von

Rindern durch Modifikation verschiedener Parameter hergestellt. Dabei ist der neue

CCHFV in-house-ELISA zur Untersuchung von Ziegenseren der erste speziell für diese Spezies hergestellte CCHFV-ELISA. Als Referenztestes wurden kommerziell erhältliche, für die Untersuchung humaner Seren hergestellte ELISAs und

Immunfluoreszenztests für die Untersuchung von Schafen und Ziegen adaptiert. Alle

Tests wurden durch vergleichende Analyse gegeneinander validiert, wobei diagnostische Sensitivitäten und Spezifitäten von 94 bis 100% ermittelt werden konnten. Außerdem wurden diese sowie Rinder-spezifische Tests in zwei

Seroprävalenzstudien in Albanien und der Ehemaligen Jugoslawischen Republik

Mazedonien beziehungsweise Bulgarien und der Türkei durchgeführt, wobei eine hohe Seroprävalenz in den aus diesen Ländern untersuchten Rindern, Schafen und

Ziegen gefunden wurde. Die Ergebnisse weisen auf eine starke Zirkulation des Virus in der Ehemaligen Jugoslawischen Republik Mazedonien hin, was überraschend ist, da von dort bisher nur wenige CCHF-Fälle berichtet wurden. Außerdem belegen sie eine Verbreitung des Virus über ganz Albanien und verifizieren den Süden

Bulgariens und die Nord- und Zentraltürkei als Hauptendemiegebiete in diesen beiden Ländern.

Um zu ermitteln, welche Tierart der geeignetste Indikator für die Verbreitung von

CCHFV ist, wurde die Seroprävalenz der drei getesteten Tierarten aus Albanien und der Ehemaligen Jugoslawischen Republik Mazedonien miteinander verglichen.

Hierbei wurde ermittelt, dass Schafe und Ziegen geeignetere Indikatoren sind als

Rinder und deshalb bevorzugt zur Ermittlung der Verbreitung von CCHFV herangezogen werden sollten.

Neben den Spezies-spezifischen Tests wurde der erste auf rekombinantem Antigen basierende kompetitive ELISA (cELISA) für CCHFV und der zweite überhaupt beschriebene CCHFV cELISA entwickelt und unter Verwendung von 833 Seren von zwölf verschiedenen Tierarten und Menschen validiert, wobei eine diagnostische

Sensitivität von 95% und eine diagnostische Spezifität von 99% ermittelt wurden.

Zuvor waren 33 CCHFV-spezifische monoklonale Antikörper (mAk) in verschiedenen

Tests charakterisiert worden, um den für die Entwicklung des Tests geeignetsten mAk zu identifizieren.

Mittels des neuen cELISA können Seroprävalenzstudien in verschiedenen Tierarten durchgeführt werden, auch um die Rolle verschiedener Tierarten im

Infektionsgeschehen des Virus zu verstehen. Außerdem können mittels des Tests

Tierarten, die essentiell zur Aufrechterhaltung der Viruszirkulation in der Natur sind, identifiziert und dann gegen CCHFV und/oder Zeckenbefall geimpft werden. Der monoklonale Antikörper, der im cELISA verwendet wird, sowie sein Epitop, könnten darüber hinaus vielversprechend für die Entwicklung diagnostischer Tests,

Therapeutika und Impfungen sein.

Die neuen Tests zur Detektion CCHFV-spezifischer Antikörper haben sich als verlässlich und einfach durchführbar erwiesen und sollten kommerzialisiert und

CCHF-betroffenen und gefährdeten Ländern zur Identifikation von Risikogebieten und Erstellung von CCHFV-Verbreitungskarten zur Verfügung gestellt werden.

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Chapter 9: Acknowledgement

First of all, I would like to thank Prof. Dr. Martin H. Groschup and Dr. Marc Mertens for giving me the opportunity to work on this fascinating topic. Thank you for the supervision of my thesis, for your outstanding support and for giving me the chance to learn so much from you. Thank you for constantly pushing me to excellence.

Moreover, for the many troubleshoots and discussions and advises for my work.

Further, I would like to thank you for your trust in my work and for your enthusiasm.

I would like to thank Carolin Rüdiger, Jaqueline Mentz and Martina Abs for your help when needed and for sharing your knowledge and experience with me. Likewise, I would like to thank you as well as Dr. Miriam A. Sas and Dr. Dorothee Zielke for the nice years we spend together in the laboratory. I very much enjoyed the fruitful and good times we had together.

Further, I would like to thank all my colleagues from our former neighbor laboratory

(the Balkema-Buschmann-Lab) for the warm atmosphere and your moral support and help.

Also, I would like to thank all my colleagues at the Institute for Novel and Emerging

Infectious Diseases and the other institutes at the Friedrich-Loeffler-Institute for your support and help and for the warm atmosphere. Especially, I would like to thank Dr.

Bernd Köllner, Sabine Weber and Dr. Tomáš Korytář for the preparation of the mAbs and for your advice, Dr. Markus Keller for the purification of the mAb, Dr. Ute Ziegler

for taking your time to help me to improve my IFAs, Dr. Katja Schmidt for teaching me how to carry out protein expressions, Dr. Martin Eiden and Birke Böttcher for your advice and help with PCR and many other things. Dr. Axel Karger and Barbara

Bettin for their help with all issues regarding recombinant proteins, Dr. Christoph

Staubach for the statistical analysis and validation of the data of the seroprevalence studies. Kathrin Schwabe, Dr. Rainer Ulrich, Dörte Kaufmann, Claudia Mroz, Dr.

Melanie Rissmann, Dr. Sandra Diederich, Eileen Stroh, Bärbel Hammerschmidt, Dr.

Corinna Mertens, Dr. Kerstin Wernicke, Dr. Silke Hechinger, Dr. Christine Fast,

Franziska Brüning, Dr. Thomas Müller, Eileen Stroh, Dr. Felicitas Hammerschmidt and Lisa Dähnert for your help and for providing serum samples for the test validations.

I would like to thank the many international collaborators I had the pleasure of working with during my thesis. Especially, I would like to thank Prof. Dr. Kristaq

Berxholi, Prof. Dr. Zdenek Hubalek, Prof. Dr. Dine Mitrov, Prof. Dr. Zati Vatansever,

Dr. Esin Güven , Prof. Dr. Ahmet Deniz , Prof. Dr. Georgi Georgiev , Prof. Dr. Raiko

Peshev, Dr.Noël Tordo and Dr. Philippe Marianneau. My deepest gratitude further to

Dr. Vasiliki Christodoulou, Prof. Dr. Maria Antoniou, Dr. Apostolos Mazeris, “the Girls” and to Dr. Samuel Abah. Thank you so much to all of you for your help.

Moreover, I would like to extend my gratefulness to my friends for always having been there for me, especially Claudia, Melanie, Ivett, Marc and Peter.

I would like to thank my family for always being there for me, especially in times of need. I owe it all to you. Thank you to my mother Hildegard, my siblings Frauke,

Arno and Thilo, grandma Anneliese, aunt Ursula, uncle Hans-Josef, aunt Marina, my cousins Andreij and Max, Jutta, Alfred, Anneliese, Walter and Andrea and her family.

Last but not least, I would like to thank my late father for guiding my path through life as long as you could.