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Antibody Responses and Viral Load in Patients with with Crimean-CongoHemorrhagic Fever: A Comprehensive Analysis During the Early Stages ofthe Infection

Koray Ergunay, Zeliha Kocak Tufan, Cemal Bulut, Sami Kinikli, AliPekcan Demiroz, Aykut Ozkul

PII: S0732-8893(14)00005-4DOI: doi: 10.1016/j.diagmicrobio.2013.12.015Reference: DMB 13506

To appear in: Diagnostic Microbiology and Infectious Disease

Received date: 7 October 2013Revised date: 23 December 2013Accepted date: 26 December 2013

Please cite this article as: Ergunay Koray, Tufan Zeliha Kocak, Bulut Cemal, KinikliSami, Demiroz Ali Pekcan, Ozkul Aykut, Antibody Responses and Viral Load in Patientswith with Crimean-Congo Hemorrhagic Fever: A Comprehensive Analysis During theEarly Stages of the Infection, Diagnostic Microbiology and Infectious Disease (2014), doi:10.1016/j.diagmicrobio.2013.12.015

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Antibody Responses and Viral Load in Patients with with Crimean-Congo Hemorrhagic Fever:

A Comprehensive Analysis During the Early Stages of the Infection

Running Title: Antibody response and viral load in CCHF

Koray Ergunay1 , Zeliha Kocak Tufan2, Cemal Bulut3, Sami Kinikli3, Ali Pekcan Demiroz3, Aykut Ozkul4

1Hacettepe University; Faculty of Medicine, Department of Medical Microbiology, Virology Unit, Ankara, TURKEY

2Yıldırım Beyazıt University, Ankara Ataturk Training and Research

Hospital, Infectious Diseases and Clinical Microbiology Department, Ankara, TURKEY

3 MOH Ankara Training and Research Hospital, Infectious

Diseases and Clinical Microbiology Department, Ankara, TURKEY 4 Ankara University; Faculty of Veterinary Medicine, Department of

Virology, Ankara, TURKEY

Author for Correspondence:

Koray Ergunay MD PhD

Hacettepe University Faculty of Medicine Department of Medical Microbiology, Virology Unit

Morphology Building 3rd floor 06100 Sihhiye Ankara TURKEY Phone: 90 312 305 15 60-131

Fax: 90 312 311 52 50 Email:ekoray@hacettepe.edu.tr

The findings of this study have been submitted as an abstract to the

24th ECCMID Congress to be held in 10-13th May, 2014 in Barcelona, Spain.

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Abstract

This study was performed to assess viral load, viral nucleocapsid (N) and

glycoprotein precursor (GPC) antibodies in consecutive samples obtained

from Crimean Congo hemorrhagic fever (CCHF) patients to reveal viral

replication kinetics and antiviral immune responses during the early stages of

the infection. Among 116 samples from 20 individuals, 43.9% and 76.7% were

positive for viral RNA and IgM/IgG antibodies, respectively, whereas both

markers could be detected in 22.4%. Mean duration of viremia was 3 days

(range:1-6 days). N-IgM antibodies were identified as the initial serological

marker during the infection, becoming detectable in a median of 2-3 days after

disease onset, followed by GPC-IgM (4-6 days) and IgG antibodies (5-6

days). Clearance of viremia followed or coincided N-IgM response. Partial S

gene sequences amplified in viremic patients were identical or closely-related

to previously-characterized strains and grouped within European Lineage I

group II viruses via neighbor-joining analysis without significant aminoacid

substitutions.

Keywords: Crimean Congo Hemorrhagic Fever, CCHF, antibody, viral load,

immune response

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1.Introduction

Crimean Congo hemorrhagic fever (CCHF) is a severe human

infection, characterized by a febrile disease with headache, myalgia/arthralgia

and petechial rash, which may be followed by signs of hemorrhagic diathesis,

disseminated intravascular coagulation and circulatory shock due to

microvascular instability and impaired hemostasis (Akıncı et al, 2013). The

case fatality rates in CCHF demonstrate a notable geographical variation but

can be as high as 50% (Whitehouse, 2004; Ergonul, 2012). The causative

agent, Crimean Congo hemorrhagic fever virus (CCHFV), classified in the

Nairovirus genus of Bunyaviridae family, is an enveloped virus with a

segmented negative sense RNA genome, encoding the viral nucleocapsid,

envelope glycoproteins and replicase, along with accessory proteins (Plyusnin

et al, 2011). In nature, CCHFV circulates in an enzootic tick-vertebrate-tick

cycle. The major transmission route of CCHFV to susceptible vertebrates is

via bites of infected ticks, usually belonging to the Hyalomma genus.

Moreover, infections may occur by contact with blood, blood-containing body

fluids or tissues of viremic livestock or humans during the acute phase of the

infection (Whitehouse, 2004; Ergonul, 2012). CCHF is the most widespread

infection among medically-important tick-borne viral infections and cases as

well as outbreaks have been described in parts of Africa, Asia, Eastern

Europe and Middle East; in accordance with the distribution of tick species

acting as vectors (Ergonul, 2012). Although viremia with subsequent

seroconversion can be detected in animals, there is no evidence that the

infection manifests with clinical disease in vertebrates other than humans.

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Subclinical infections are also common in humans and may constitute over

80% of the virus exposures in endemic regions (Bodur et al, 2012)

A rapid diagnosis of CCHF is essential to initiate the appropriate

treatment protocol and employ management precautions including patient

isolation, which is especially pertinent as CCHF virus has a propensity to

cause severe nosocomial outbreaks (van Eeden et al, 1985; Mardani et al,

2009). Like in many viral infections, virus isolation is considered as the gold

standard diagnostic method in CCHF (Ergonul, 2012; Vanhomwegen et al,

2012). However, the requirement of a high-containment biosafety level 4

facility often renders this approach unpractical for routine use. Moreover, a

high level of viremia, usually encountered during the first 5 days of infection,

required for a successful cell culture or suckling mice inoculation further

restricts the use of virus isolation techniques (Vanhomwegen et al, 2012).

Therefore, detection viral nucleic acids or immune response is often employed

for the specific diagnosis of CCHV. The methods include conventional and

real-time quantitative reverse transcription PCR (RT-PCR and qRT-PCR) for

detection of the viral genome (Drosten et al, 2003; Cevik et al, 2007; Duh et

al, 2007) and indirect immunofluorescence assays (IFAs) or enzyme-linked

immonusorbent assays (ELISAs) for detection of antibodies (Saijo et al, 2002;

Emmerich et al, 2010; Dowall et al, 2012). In this study, viral load and IgM-IgG

antibodies were investigated in consecutive samples obtained from CCHF

patients with acute CCHF infection to reveal viral replication dynamics and

antiviral immune responses during the early stages of the infection.

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2. Materials and Methods

2.1. Setting and Clinical Samples:

The study was conducted in the Ankara Training and Research

Hospital, a 670-bed tertiary care teaching hospital which serves as one of the

reference centers for CCHF patients in Central Anatolia. The study protocol

was approved by the local ethics board (13.07.11/0426).

During May-August 2012, individuals referred to the institution with a

preliminary diagnosis of CCHF were evaluated for the study. All patients

originated from different locations of Kelkit valley, established as a CCHF

endemic region in Turkey (Karti et al, 2004). Criteria for inclusion were the

presence of epidemiological risk factors for CCHF, which comprise recent

history of tick bites, tick contact and/or working in animal husbandry or farm,

or a recent travel to the endemic region, plus clinical and laboratory findings

suggestive of CCHF including fever, headache, myalgia/arthralgia, lethargy,

nausea/vomiting, abdominal pain/diarrhea, and/or hemorrhages along with

thrombocytopenia, leucopenia, elevated liver enzymes (mainly alanine

aminotransferase and aspartate aminotransferase), elevated alkaline

phospatase, gamma glutamyl transferase and/or creatinine kinase. Individuals

suffering from peripheral or cerebral vascular disease, hematological

disorders, cirrhosis, portal hypertension, and malignancies were excluded.

A total of 20 adult patients (10 males and 10 females) with enrolled in

the study with informed consent. The individuals were admitted to the hospital

within 1-10 days of the tick bite and 24-72 hours of disease onset. Starting

with hospital admission, daily serum samples were obtained each day from

the patients, aliquoted and stored in -80ºC for future analyses.

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2.2. Detection of CCHFV RNA and Viral Load

All samples were subjected to nucleic acid purification and reverse

transcription using random hexamers via commercial assays (High Pure Viral

Nucleic Acid Kit, Roche Diagnostics, Germany; RevertAid First Strand cDNA

Synthesis Kit, Thermo Scientific, Japan) as directed by the manufacturers. For

the quantitative determination of CCHFV RNA, a previously-described

TaqMan-based single-step qRT-PCR was employed with minor modifications

(Yapar et al. 2005). Briefly, sense and antisense primers (50-TCT TYG CHG

ATG AYT CHT TYC-30 and 50-GGG ATK GTY CCR AAG CA-30) and

labeled probe (50-FAM-ACA SRA TCT AYA TGC AYC CTG C-TAMRA-30)

(H: A/C/T, K: G/T, R: A/G, S: G/C, Y: C/T), targeting the S segment of the viral

genome were used for reaction mixture containing 5 l extracted RNA, 5 pmol

of each primer, 4 pmol of labeled probe, 0.2 mM of each deoxyribonucleotide

triphosphate, 6 mM MgCl2 and a mixture of reverse transcriptase (Fermentas,

Vilnius, Lithuania) plus hot start Taq polymerase (Bioron GmBH, Munchen,

Germany) in a total volume of 25 l. Cycling conditions were set as follows: a

single cycle of 30 min at 42 C for reverse transcription followed by 5 min. at

95 C and 45 cycles of 15 s at 95 C and 60 s at 60 C, performed in a Rotor-

Gene 6000 instrument (Corbett Research, Australia) for a 103 bp amplicon.

In each run, plasmid standards with 4x102 to 4x109 copies/ml per reaction

were included for quantitation.

For the characterization of CCHFV partial S gene sequences in

patients, an in house PCR was performed as described previously (Rodriguez

et al. 1997), using a PTC-200 Thermal Cycler (MJ Research, Massachusetts,

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USA). Amplicons of approximately 520 basepairs from the first PCR round

were visualized by ethidium bromide staining after 1.5% agarose gel

electrophoresis and further characterized by sequencing. For this purpose,

PCR products were cleaned up via High Pure PCR Product Purification Kit

(Roche Diagnostics, Mannheim, Germany), and sequenced using forward and

reverse primers in an ABI Prism 310 Genetic Analyzer (Applied Biosystems,

CA, USA). Obtained sequences were aligned and analyzed using CLC Main

Workbench v5.5 (CLCBio, Aarhus, Denmark).

2.3. Detection of CCHFV Antibodies

Samples were tested for anti-CCHFV IgM and IgG antibodies by a

commercial IFA (Crimean-Congo fever virus mosaic 2, IgM and IgG,

Euroimmun, Luebeck, Germany), performed according to the manufacturer's

recommendations. The IFAs utilize EU90 cells transfected with CCHFV

nucleocapsid (N) and glycoprotein precursor (GPC) separately, as well as

non-transfected cells to differentiate non-specific reactions. For the IgM

detection, interfence in the assay was prevented using IgG-rheumatoid factor

absorbant in the sample dilution buffer as directed by the manufacturer. The

assays were performed at the serum dilution of 1:10 for IgM, 1:100 for IgG

and interpreted via fluorescence microscopy. The positive results were

evaluated as equivalent (+), moderate (++) and strong (+++), according to

visual intensity of fluorescence compared to control sera. The sensitivity and

specificity of the IgM assay is reported as 97.2% and 97.5%, respectively

whereas they are 89.5% and 100% for IgG, respectively. An independent

comparison of various assays have also revealed an overall sensitivity and

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specificity of 93.9% and 98.9% for IgM and 86.1% and 100% for IgG assays,

respectively (Vanhomwegen et al, 2012).

2.4. Statistical Analysis

Descriptive parameters of the study group, viral load levels,

fluorescence intensities and seroconversion times were assessed via

statistical tests. Student's t, Mann-Whitney U and Fisher's Exact tests were

employed for univariate comparisons whereever appropriate. Statistical

significance was considered as p<0.05 level. All statistical analyses were

performed by SPSS package version 15.0 (SPSS Inc.,USA).

3. Results

3.1. Samples and CCHFV Laboratory Follow-Up

A total of 116 samples from 20 patients (samples per patient:

mean:5.8, median:5, range:3-13) were evaluated in the study. Among the 116

samples tested, 51(43.9%) were positive for viral RNA, 89 (76.7%) were

positive for antibodies, whereas in 26 (26/116, 22.4%), both markers were

reactive (Table 1).

In 9 patients (45%), seroconversion of CCHFV antibodies and

simultaneous or subsequent elimination of viremia could be observed (Figure

1, patients 1-9). In two patients (10%), precise timing of seroconversion and

control of viral replication could not be determined due to missing days of

sampling (Figure 1, patients 10-11). In 6 patients (30%), antibodies were

present in all samples studied, with or without detectable viremia (Figure 1,

patients 12-17). All samples from 3 patients (15%) remained CCHFV RNA

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negative despite seroconversion in 2 (Figure 1, patients 18 and 19) and

IgM+IgG seroreactivity in 1(Figure 1, patient 20). All patients except patient 2

survived the disease and discharged from the hospital (19/20, 95%).

3.2. CCHFV Viral Loads and S Segment Sequences

Detectable viremia was present in 17 cases (17/20, 85%), which lasted

for a mean of 3 days (median:3, range:1-6). In patients with more than one

viremic sample, mean viral loads on the day of hospital admission and on the

last viral RNA-detectable sample were 2.4 x108 copies/ml and 6.3 x106

copies/ml, respectively (standard deviation: 1.008-1.01 log). The differences

in viral loads were statistically significant (p:0.005).

CCHFV partial S segment sequences were amplified and sequenced

in 11 cases (patients 1-11); from samples acquired on the day of hospital

admission. All samples used for sequence characterization were negative for

CCHFV antibodies. The alignment of the nucleotide sequences demonstrated

similarities ranging from 60.69% to 99.24% among isolates. All sequences

were grouped with CCHFV European Lineage I group II viruses via neighbor-

joining analysis (Figure 3). Comparison of S segment sequences identified in

various provinces of Turkey during 2006-2008 revealed identical or similar

nucleotide compositions and distribution of current sequences in subgroup 2a

and 2b within group I CCHFV strains (Figure 3). Comparison of aminoacid

sequences deduced from the nucleotide sequence demonstrated identical

sequences without aminoacid variations in 10 cases. However, two aminoacid

substitutions in the S segment (R146K and S165T) were identified in patient

6.

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3.2. Dynamics of CCHFV GPC and N Antibody Responses

Detectable immune responses to CCHFV could be observed in 89

samples (76.7%), originating from all patients studied. IgM antibodies were

present in 26 samples (26/89, 29.2%) that include N-specific responses in 20

and GPC+N responses in 6 samples. In 63 samples (70.8%), IgM and IgG

antibodies were detected simultaneously.

In patients with observed IgM and/or IgG seroconversion, average

times required for reactivity were calculated (Table 2). Overall, IgM

seroconversion was followed by IgG seroconversion in 1-2 days (mean:1.73,

median:2). Detection of N-IgM was significantly earlier than GPC-IgM,

regardless of the interpreted fluorescence intensity (Table 2). When evaluated

separately as equivalent or moderate IFA results, a statistically-significant

difference in days required for seroconversion was observed for N IgM

(p:0.013), but not for GPC (p:0.170). N-IgM response preceded or coincided

with the clearance of viremia as determined by undetectable CCHFV RNA

levels (Figure 1). For IgG detection, no differences related to viral proteins

(Table 2) or for fluorescence intensities could be revealed (p:0.261 and 0.195,

data not given).

4. Discussion

Emergence or reemergence of CCHF poses a serious public health

threat due to being a highly contagious and potentially lethal infection that is

difficult to treat, prevent, and control, with the potential to cause nosocomial

spread (Ergonul, 2012; Vanhomwegen et al, 2012). Access to early, sensitive,

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and specific laboratory diagnosis is of paramount importance for increasing

preparedness in countries at risk, as well as for enhanced surveillance and

development of therapeutics (Maltezou et al, 2010). Various in house and

commercial assays have been developed for the detection and/or quantitation

CCHFV RNA and antibodies. However, consensus on the most efficient

molecular and serologic testing method is yet to be reached and early events

during the initial phases of the human infection are relatively less known

(Vanhomwegen et al, 2012, Akinci et al, 2013). This study was performed to

elucidate viral replication kinetics associated with the patterns of antibody

responses to different viral antigens in individuals with CCHFV infection

during the early stages of the disease.

Twenty patients with the history of tick bites and the clinical diagnosis

of CCHF were enrolled in the study during 2012. A total of 116 samples from

the patients were investigated via a well-characterized single-step qRT-PCR

for viral RNA quantitation and a commercial IFA to detect IgM-IgG antibodies

against viral GPC and N proteins. Samples from selected cases were further

amplified via an in house PCR assay for partial characterization of S segment

of the viral genome. Various phases of the disease were observed in the

patients. Detectable viremia with relatively high viral loads and subsequent

elimination of circulating viruses via specific immune response were identified

in the majority of the cases (Figure 1). Mean duration of the viremia was 3

days and statistically-significant declines in viral load levels were

demonstrated between the initial and last viremic samples. In two patients,

viral RNA could not be demonstrated despite early sampling and subsequent

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seroconversion (patients 18 and 19, Figure 1). Initial samples from these

patients might have suffered from PCR inhibitors.

In the study, clearance of viremia was associated with IgM response to

N protein, which frequently became reactive prior to the reduction of the viral

load to undetectable levels (Figure 1). Moreover, N-IgM antibodies were

identified as the initial serological marker during the infection, appearing in a

median of 2-3 days in IFA, significantly earlier than GPC-IgM antibodies. This

observation is further supported by the comparison of the results of various

fluorescence intensities (Table 2). Evidence for a robust N-IgM response is

indirectly provided by the significant increase for the time required for a

detectable elevation in fluorescence (2.63 versus 3.73 days, Table 2). IgG

seroconversion emerged after 1-2 days following the IgM responses. No

significant variations in GPC or N-IgG antibodies according to fluorescence

intensity were demonstrated. A relatively later phase of the infection were

observed in 6 patients, where IgG and/or IgM antibodies were reactive in all

evaluated samples (Figure 1). However, similar dynamics for IgG

seroconversion were noted.

Previous reports indicate that IgM and IgG antibodies are detectable in

CCHFV infection from about 7-9 days after the onset of disease (Shepherd et

al, 1989). However, IgM antibodies are reported to be present as early as day

5 and when viremia is also evident (Tang et al, 2003; Saijo et al, 2005).

Maximum antibody titers were usually attained in the 2nd to 3rd week of

illness. Specific IgM declines to undetectable levels by 4 months post-

infection, and IgG remains detectable for at least 5 years (Shepherd et al,

1989; Ergonul, 2006). Our findings demonstrate that seroconversion can be

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detected during 2nd to 6th day of hospital admission using currently-

developed assays, depending on the antigens used. Emergence of IgG also

follows a rapid course and both antibodies could be identified during the 2nd

week. In this study, a highly-sensitive and specific commercial IFA was

employed. Generally, IFA is considered to be relatively more labor-intensive

due to the requirement of the individual examination of all samples under the

fluorescent microscope and the interpretation can vary according to the

microscopist. However, it enables the interpretation of fluorescence patterns

that may be useful to differentiate positive from cross-reactive samples, a

clear advantage over ELISA (Vanhomwegen et al, 2012).

The fatality rates in individuals with CCHF range between 5% and 30%,

depending on geographic region and route of entry (Jamil et al. 2005, Akinci

et al, 2013). The outcome of the disease has been shown to be correlated

with viral load as well as certain laboratory and clinical parameters such as

platelet count, hepatic enzyme and fibrinogen levels, presence of

somnolence, gastrointestinal bleeding and others (Akinci et al, 2013). The

fatality rate in this study was 1/20 (5%), comparable to the rates reported from

Turkey during 2003-2009 (4.5-6.2%) (Ergunay et al, 2011). The fatal case

observed in the study had the highest initial viral load (2.36 x 109 copies/ml)

which remained within 108-109 range during follow-up (Figure 1A, Patient 2).

Previous studies have indicated viral load as a strong predictor of clinical

outcome in CCHF patients (Cevik et al, 2007; Duh et al, 2007). However,

suggested viral load thresholds vary according to the rRT-PCR set-up,

probably due to the variations of sensitivity and the lack of consensus

quantitation standards. Nevertheless, a viral load exceeding 109 copies/ml is

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widely considered as a severe infection with impending mortality (Cevik et al,

2007; Duh et al, 2007). Interestingly, in the patient with the fatal outcome,

GPC-IgM and IgG responses could be mounted during the disease course,

but viral replication could not be limited (Figure 1, Patient 2). These findings

further support the impact of N-IgM response in controlling viral replication.

Generally, antibodies against viral glycoproteins demonstrate a neutralizing

effect, preventing viral attachment and entry into uninfected cells and further

promoting immune clearance. Neverheless, data from various phleboviruses

suggest that in Bunyaviruses, neutralization is a complex phenomenon

involving a number antigenic epitopes. Even the majority of the exposed

persons develop antibodies against linear epitopes of N protein, the immune

response against viral glycoproteins, which directly affects virus neutralization

in vitro, display considerable variations (Besselaar and Blackburn 1991, Di

Bonito et al, 2002). This is consistent with the previous data that most of the

CCHF patients developed relatively low levels of neutralizing antibodies

(1:8 to 1:32 by fluorescent-focus reduction tests), while titers of 1:256 to

1:512 could be observed in some individuals (Shepherd et al, 1989).

The main limitation of this study is that relatively mild or non-fatal cases of

CCHF constitute the majority in the study group. Although this enabled

precise detection of antiviral immune responses suppressing viral replication,

GPC and N responses and their relation to viremia could not be evaluated in

cases with viral loads over 109 copies/ml. For a comprehensive understanding

of in vivo CCHFV neutralization, which will potentially influence the

therapeutic use of convescelent sera in patients, neutralizing antibody titers

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for viral glycoprotein and nucleocapsid antigens must be evaluated in cases

with higher viral loads in future efforts.

Seven lineages of CCHFV, distributed according to distinct

geographical locations exist (Deyde et al. 2006). Previous analyses of partial

CCHFV sequences in ticks and affected individuals demonstrated that the

majority of the virus strains in Turkey are closely-related and belonged to the

European lineage I (Ozkaya et al, 2010; Kalaycioglu et al, 2012). The S-

segment sequences were observed to be suitable for assessments of

genomic variations. Moreover, local viruses could be grouped in two main

clusters in which two subgroups were also distinguishable (Ozkaya et al.

2010). In Central Anatolia, both subgroups were observed to be prevalent,

regardless of the location (Ozkaya et al. 2010). Partial sequences

characterized in this study are also consistent with these findings, as

sequences identical or similar to subgroup IIa and IIb were distributed in the

cases (Figure 2). In Turkey, AP92-like strains, grouped within European

lineage II viruses, have also been identified in the Thrace region as well as

Corum province in Central Anatolia (Midilli et al, 2009; Ozkaya et al, 2010).

Originally characterized in Greece and associated with low pathogenicity,

AP92 and closely-related CCHFV strains have not been detected in Central

Anatolia during 2009-2010 (Kalaycioglu et al, 2012), as well as in this study

(Figure 2). In CCHFV, variations in the S segment of the viral genome, that

codes for the nucleocapsid protein, may affect individual immune response as

well diagnostic assays targeting the nucleocapsid (Dowall et al, 2012).

Although sequence diversity in CCHFV isolates in this study have been

observed, aminoacid substitutions (R146K and S165T) were only detected in

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one CCHFV case, with indistinguishable antibody/viral load kinetics and

clinical presentation (Figure 1, patient 6). Remarkable variations have

previously been revealed among the CCHFV isolates in Turkey and low-rate

homologous recombination events have been proposed to affect amino acids

155-200 of the S segment, according to yearly analysis of corresponding

peptide regions (Ozkaya et al, 2010). It is likely that the aminoacid

substitutions identified in this study in a single case without any impact on

diagnostics or prognosis reflect the occurance of such events.

In conclusion, N-IgM antibodies appear as the initial serological marker

and become detectable earlier than GPC-IgM during the course of CCHF

infection. N-IgM response also correlates with the control of viral replication.

IgM antibodies are rapidly followed by IgG responses to GPC and N. In the

single fatal case, an initial viral load exceeding 109 copies/ml and GPC-

IgM/IgG were noted, lacking N responses. Further data are required to assess

the impact of GPC/N responses during in vivo virus neutralization and in

patients with very high viral loads.

Acknowledgements

The CCHFV IFA employed in this study were kindly provided by

EuroImmun, Turkey. The company and the representatives had no

involvement in study design, performance or evaluation of the assays and

interpretation of the results. The authors declare no competing interests and

are grateful to N.Emin Guven for graphics.

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Table and Figure Legends:

Table 1. Distribution of CCHFV RNA and antibody results in patient samples

Table 2. Timing and comparison of CCHFV antibody seroconversion

according to the viral antigens and fluorescence intensity in IFA.

Figure 1: CCHFV viral load and GPC-N antibody responses according to the

sampling times. Viral loads were given as copies/ml and antibody titers in IFA

were interpreted as equivalent:+, moderate:++, strong:+++/++++) (N/A:not

available). Patient with the fatal outcome is underlined (Patient 2).

Figure 2: Phylogenetic analysis of the partial S segment sequences identified

in the study (●) with related local and global sequences. Numbers at the

nodes indicate bootstrapping values. Bars represent nucleotide substitutions

per position. Local sequences are represented with location, strain number

and year of isolation. GeneBank accession numbers of the isolates are:

OMAN: DQ211645, IRAN-52: DQ446212, CHINA: AF358784, HODZHA:

AY223475, TAJ.Hu8978: AY297691, CONGO.3010: DQ144418,

AP92:DQ211638, CORUM1.2007: FJ601872, GUMUSHANE67.2008:

FJ601848, KASTAMONU127.2008: FJ601855, CORUM95.2008: FJ601884,

KASTAMONU71.2008: FJ601850, KARABUK179.2006: FJ601866,

KAYSERI.2008: FJ601891, TOKAT92.2008: FJ601882, KARABUK70.2008:

FJ601866, KARABUK126.2008: FJ601854, ANKARA.2008: FJ601893,

CANKIRI180.2006: FJ601867, CORUM6.2007: FJ601876, SAMSUN2.2008:

FJ601861, TOKAT85.2008: FJ601880, CANKIRI.2008: FJ601890,

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TOKAT93.2008: FJ601883, SAMSUN124.2008: FJ601894, SIVAS98.2008:

FJ601885, AYDIN156.2008: FJ601896, KARABUK172.2008: FJ601860,

BOLU173.2006: FJ601863, TOKAT5.2007: FJ601878, BINGOL130.2008:

FJ601856, YOZGAT8.2007: FJ601870, CORUM11.2007: FJ601873,

YOZGAT7.2007: FJ601871, BAYBURT51.2008: FJ601847, Patient1:

KF705528, Patient2: KF705527, Patient3: KF705529, Patient4: KF705535,

Patient5: KF705531, Patient6: KF705536, Patient7: KF705533, Patient8:

KF705526, Patient9: KF705532, Patient10: KF705534, Patient11: KF705530)

Sequence from the patient with the fatal outcome is underlined (Patient 2).

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Table 1. Distribution of CCHFV RNA and antibody results in patient samples

RNA Positive Negative

# (%) # (%) Total

IgM/IgG Positive 26 (29.2) 63 (70.8) 89

Negative 25 (92.6) 2 (7.4) 27

Total 51 (43.9) 65 (56.1) 116

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Table 2. Timing and comparison of CCHFV antibody seroconversion

according to the viral antigens and fluorescence intensity in IFA.

IgM (equivalent) IgM (moderate)

GPC N p value GPC N p

value

Mean 4.45 2.63 0.005a 5.33 3.73

0.008a

Median 4 2 6 3

Time of

Detection IgG (equivalent) IgG (moderate)

(days)

GPC N p value GPC N p

value

Mean 5 5 0.898 5.78 5.67

0.903

Median 5 5 6 6

a statistically significant

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