IMMUNE RESPONSES TO CRIMEAN-CONGO HAEMORRHAGIC FEVER VIRUS AND MOLECULAR CHARACTERIZATION OF VIRAL ISOLATES by Dominique Goedhals Thesis submitted in fulfilment of the requirements for the degree Ph.D. Virology in the Department of Medical Microbiology and Virology, Faculty of Health Sciences, University of the Free State, Bloemfontein Promoter: Prof FJ Burt, Department of Medical Microbiology and Virology, University of the Free State, Bloemfontein Co-promoter: Prof J Paweska, Center for Emerging and Zoonotic Pathogens, National Institute for Communicable Diseases, Johannesburg February 2014 TABLE OF CONTENTS Page DECLARATION i ACKNOWLEDGEMENTS ii PUBLICATIONS iii LIST OF ABBREVIATIONS iv LIST OF FIGURES vi LIST OF TABLES viii SUMMARY x OPSOMMING xiii CHAPTER 1 Orientation, rationale and aims of the study 1 Introduction 1 Problem identification 2 Aims and objectives 5 Structure of the thesis 7 CHAPTER 2 Crimean-Congo haemorrhagic fever virus: review of a virus endemic in South Africa 8 Historical background 8 The virus 9 Epidemiology and geographic distribution 11 Genetic diversity 14 CCHFV in South Africa 15 Clinical features 16 Pathogenesis and immune responses 20 Laboratory diagnosis 22 Differential diagnosis 25 Treatment 26 Prevention and Control 27 References 29 CHAPTER 3 Identification of novel T cell epitopic regions on Crimean-Congo haemorrhagic fever virus and confirmation of long-lived memory T cell responses 52 Abstract 53 Introduction 54 Methods 56 Study subjects 56 Synthetic peptides 57 ELISPOT assays 57 Subset depletion studies 58 Epitope conservation 59 Results 59 Discussion 64 References 67 CHAPTER 4 Identification of human linear B cell epitope sites on the envelope glycoproteins of Crimean-Congo haemorrhagic fever virus 73 Abstract 74 Introduction 75 Methods 76 Results 77 Discussion 84 References 86 CHAPTER 5 Next generation sequencing of southern African Crimean-Congo haemorrhagic fever virus isolates reveals a high frequency of M segment reassortment 90 Abstract 91 Introduction 92 Methods 93 Viral isolates 93 Reverse transcription polymerase chain reaction and sequencing 93 Data analysis 97 Results 98 Sequencing data 98 Genetic analysis 99 Discussion 107 References 111 CHAPTER 6 Comparative analysis of the L, M and S RNA segments of Crimean-Congo haemorrhagic fever virus isolates from southern Africa 117 Abstract 118 Introduction 119 Methods 120 Sequence data set 120 Sequence analysis 121 Protein analysis 121 Results 122 Complete genome sequences 122 5’ and 3’ NCR cyclization analysis 123 Molecular characterization of the L segment ORF 124 Molecular characterization of the M segment 126 Discussion 131 References 135 CHAPTER 7 Conclusions and future perspectives 140 References 146 APPENDIX A: Ethics Committee letter, project approval (extension to December 2014) ECUFS NO 152/06 148 APPENDIX B: Ethics Committee letter, addition of student 150 APPENDIX C: Informed consent document and information letter study 1 (T cell immune response study) 151 APPENDIX D: Informed consent document and information letter study 2 (antibody study) 154 APPENDIX E: Informed consent document and information letter study 3 (negative control samples) 156 APPENDIX F: Permission to include article in thesis, “Next generation sequencing of southern African Crimean-Congo haemorrhagic fever virus iaolates reveals a high frequency of M segment reassortment”, Epidemiology and Infection 158 DECLARATION I certify that the thesis hereby submitted by me for the degree PhD in Virology at the University of the Free State is my independent effort and had not previously been submitted for a degree at another university/faculty. I furthermore waive copyright of the thesis in favour of the University of the Free State. Dominique Goedhals Date i ACKNOWLEDGEMENTS I would like to thank the following: • The Department of Medical Microbiology and Virology, National Health Laboratory Service and University of the Free State for providing the facilities to complete the laboratory work. • My promoter, Professor Felicity Burt for your insight, encouragement, motivation, and patience. • My co-promoter, Professor Janusz Paweska for providing the viral isolates used in the study and for constructive comments on the manuscript. • My family, friends and colleagues for support and encouragement throughout this process. • The patients who participated in the study. Financial support • This research was funded by the Medical Research Council of South Africa, the National Health Laboratory Service Research Trust Fund, and the Poliomyelitis Research Foundation ii PUBLICATIONS Goedhals D, Paweska JT, Burt FJ. Crimean-Congo haemorrhagic fever virus: review of a virus endemic in South Africa. (Manuscript in preparation) Goedhals D, Paweska JT, Burt FJ. Identification of novel T cell epitopic regions on Crimean- Congo haemorrhagic fever virus and confirmation of long-lived memory T cell responses. (Manuscript in preparation) Goedhals D, Paweska JT, Burt FJ. Identification of human linear B cell epitope sites on the envelope glycoproteins of Crimean-Congo haemorrhagic fever virus. (Manuscript in preparation) Goedhals D, Bester PA, Paweska JT, Swanepoel R, Burt FJ. Next generation sequencing of southern African Crimean-Congo haemorrhagic fever virus isolates reveals a high frequency of M segment reassortment. (In press, Epidemiology and Infection) Goedhals D, Bester PA, Paweska JT, Swanepoel R, Burt FJ. Comparative analysis of the L, M and S RNA segments of Crimean-Congo haemorrhagic fever virus isolates from southern Africa. (Manuscript in preparation) iii LIST OF ABBREVIATIONS ABTS azino di-ethyl-benzothiazoline-sulfonic acid BLAST Basic Local Alignment Search Tool BSL biosafety level CCHF Crimean-Congo haemorrhagic fever CCHFV Crimean-Congo haemorrhagic fever virus CFR case fatality rate CHF Crimean haemorrhagic fever cRNA complementary ribonucleic acid DIC disseminated intravascular coagulopathy ELISA enzyme-linked immunosorbent assay ELISPOT enzyme-linked immunospot HIV human immunodeficiency virus HRPO horse-radish peroxidase IEDB Immune Epitope Database IFN-α interferon alpha IFN-γ interferon gamma IHRQL impaired health related quality of life ISG15 interferon-stimulated gene product 15 L large M medium MEGA Molecular Evolutionary Genetics Analysis MHC major histocompatibility complex iv NCR non-coding region NGS next generation sequencing NICD National Institute for Communicable Diseases NK cells natural killer cells OD optical density ORF open reading frame OTU ovarian tumour PBMC peripheral blood mononuclear cells PBS phosphate buffered saline PTSD post-traumatic stress disorder RdRp RNA-dependent RNA-polymerase RNA ribonucleic acid RT-LAMP reverse transcription loop-mediated isothermal amplification RT-PCR reverse transcription polymerase chain reaction RVF Rift Valley fever S small SFC/106 spot forming cells per million STAT-1 signal transducer and activator of transcription-1 TNF-α tumour necrosis factor alpha vRNA viral RNA v LIST OF FIGURES CHAPTER 3 Figure 1 Detection of virus-specific T cell responses by interferon gamma ELISPOT assay. The magnitude of responses in spot forming cells per million (SFC/106) are indicated for each of the peptides to which study subjects showed a positive response. Figure 2 T cell responses to peptides N262-280 and N298-316 as determined by interferon gamma ELISPOT assay. CHAPTER 4 Figure 1 IgG-specific peptide ELISA illustrating OD values obtained for each peptide using pooled human sera. The horizontal axis denotes the peptide number and the vertical axis denotes the absorbance measured at 405nm/620nm. Peptides 1 – 32 represent the mature GN protein and peptides 33 – 103 represent the mature GC protein. Figure 2 Alignment of amino acid sequences of reference SPU103/87 with 14 southern African CCHFV isolates illustrating conservation of peptides G669-687, G1172-1190, G1181-1199, G1451-1469, G1613-1631 and G1622-1640. CHAPTER 5 Figure 1 Phylogenetic analysis of complete coding regions of (a) S segments, (b) M segments, and (c) L segments of CCHFV using a bootstrap maximum- likelihood method with 1000 replicates, with bootstrap values greater than vi 50% indicated at the relevant nodes. Each sequence is designated by the isolate name and isolates sequenced in the current study are marked (•). Figure 2 Geographical distribution of CCHFV groups for the (a) S segment, (b) M segment and (c) L segment. CHAPTER 6 Figure 1 5’ and 3’ NCR complementary regions forming panhandle structures as modelled using Mfold Web Server. (a) L segment, dG = -40.48. (b) M segment, dG = -51.91. (c) S segment, dG = -37.17. Figure 2 Schematic representation of the protein analysis of the complete genome of southern African CCHFV isolates (not drawn to scale). (a) L segment. (b) M segment. Cleavage sites are indicated by arrows with the amino acid residues and positions indicated below. Transmembrane helices are indicated as black lines in the relevant ORFs. N-glycosylation sites are indicated by stars. Figure 3 The amino acid sequences of the dual CCHC-type zinc finger motifs in the GN tail. Conserved residues are indicated by a dot, while substitutions are indicated by the relevant amino acid. The conserved C-X2-C-X11-12-H-X3-C motifs of each zinc finger are outlined. vii LIST OF TABLES CHAPTER 3 Table 1 Summary of the subjects with previous CCHFV infection from whom peripheral blood mononuclear cells were extracted for interferon gamma ELISPOT assays. Table 2 Details of the peptides representing potential epitopic regions identified by ELISPOT assay. Peptide names are derived from the relevant protein and the amino acid position relative to the coding regions of SPU 103/87 (DQ211647 and DQ211634). Six adjacent overlapping peptides were reactive, with each pair likely representing a single epitopic region as indicated with a superscript numeric and amino acid residues in the overlapping region are coloured red. CHAPTER 4 Table 1 Details of the six reactive peptides selected for further testing based on OD values obtained in the peptide ELISA using one negative and two positive serum pools. Two adjacent overlapping peptide pairs are noted with each pair likely representing a single epitopic region as indicated with a superscript numeric. Amino acid residues in the overlapping region are coloured red. Supplementary data Results of six reactive peptides tested with sera from 15 patients with previous CCHFV infection indicating OD values obtained, as well as the approximate duration from time of illness to sample collection. All OD values viii for CCHF survivors represent the mean of values obtained in duplicate testing. OD values above the relevant cut-off are indicated in red. CHAPTER 5 Table 1 PCR and sequencing primers utilized for preparation of amplicons for next generation sequencing. Table 2 Summary of data concerning the CCHFV sequences retrieved from the GenBank database and used in the study. Table 3 Summary of southern African CCHFV isolates for which complete genome sequences were obtained by next generation sequencing. CHAPTER 6 Table 1 Southern African CCHFV genome characteristics. Table 2 N-linked glycosylation sites on the M segment ORF as predicted by N- Glycosite. Amino acid residue positions are based on the aligned ORF sequences to allow uniformity of numbering. Position 760 forms part of a predicted transmembrane helix and is therefore unlikely to be N- glycosylated. ix SUMMARY Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne virus belonging to the family Bunyaviridae, genus Nairovirus. The distribution of the virus correlates with that of the principal vector, ticks belonging to the genus Hyalomma. This includes areas in Africa, Asia, Eastern Europe and the Middle East, with recent emergence in Turkey, Greece and India. CCHFV is associated with haemorrhagic fever in humans, with a case fatality rate of up to 30%. Current patient management relies on supportive therapy and administration of ribavirin, but the efficacy of this antiviral drug is controversial. Although an inactivated vaccine has been used in Eastern Europe and the former Soviet Union, it has not been accepted for widespread use. An understanding of immune correlates is therefore needed to guide further development of therapeutic and preventative interventions. This study aimed to investigate immune responses in survivors of CCHF in South Africa, focusing on the presence of detectable memory T lymphocyte responses and the identification of epitopic regions within the nucleoprotein and glycoproteins. In order to ensure applicability of identified epitopes to geographically distinct isolates, viral sequence diversity was also investigated by means of next generation sequencing and phylogenetic studies. A synthetic overlapping peptide library was used to screen for interferon gamma production by peripheral blood mononuclear cells from survivors of CCHFV infection in ELISPOT assays. Ten potential epitopic regions were identified, the majority of which were located on the nucleoprotein with only two regions identified on the glycoprotein GC in a single patient. Long-lived memory CD8+ T cell responses were detected in survivors of CCHF up to 13 years after infection. These findings indicate the presence of effective long term cellular immune responses which could be modulated through vaccination and gives an indication of epitopic x regions that should be considered in candidate vaccines and testing vaccine immunogenicity. The presence of detectable memory responses in the absence of re- exposure or chronic infection will allow future studies to fully characterize T cell responses in survivors. With an expanding area of CCHFV endemicity, safe, sensitive and specific serological assays are required for diagnostic and serosurveillance purposes. As the biosafety level 4 facilities required to culture the virus are lacking in many endemic areas, alternative means of producing reagents for diagnostic assays are needed which will not pose a safety risk to laboratory workers. The use of synthetic peptides in serological assays is one such alternative approach. In addition, identification of immunodominant epitopic regions may have application in vaccine development if they induce protective immunity. The peptide library was used to screen for antibodies recognizing human defined linear B cell epitopic regions in survivors of CCHFV infection by means of an enzyme-linked immunosorbent assay (ELISA). Two potential epitopic regions were identified on the GC glycoprotein with reactivity in 13 – 14 of 15 patients tested. Further investigation will be required to determine whether these epitopic regions also correlate with immune protection and to identify non-contiguous B cell epitopes which are likely to play an important role in antibody induction during natural infection with CCHFV. With new foci of CCHFV infections emerging in recent years, it is important to ascertain whether genomic variation will influence applicability of vaccine candidates and diagnostic assays in distinct geographic areas. Next generation sequencing techniques were used to obtain complete genome sequences for ten southern African CCHFV isolates. This is the first application of next generation sequencing technology to CCHFV isolates and proved to be a xi rapid and cost effective alternative to standard Sanger sequencing which can be effectively applied to the approximately 20kb CCHFV genome. The phylogenetic results confirmed that although there is extensive variability among geographically distinct CCHFV isolates at a genomic level, conserved areas are present which could be targeted for vaccine development and diagnostic purposes. The genetic variability seen results from point mutations and segment reassortment, which was shown to occur commonly in southern African CCHFV isolates. Despite the extensive variation in primary sequence, at a protein level, the motifs involved in protein function are well conserved. Prediction software analysis confirmed the presence of conserved OTU-like cysteine protease and RNA dependent RNA polymerase (RdRp) domains in the L segment of diverse southern African CCHV isolates. The RdRp is essential for viral replication while the OTU-like protease likely plays a role in immune evasion and therefore affects viral pathogenicity. Analysis of the M segment showed conservation of the basic protein coding strategy, with two structural and three non-structural glycoproteins. However, amino acid variation was notable across all predicted proteins but particularly in the variable mucin-like domain which is thought to play a role in viral pathogenicity. This study identifies targets for further investigation of viral pathogenicity which may include in vivo studies in animal models and mutagenicity assays. xii OPSOMMING Krimeaanse-Kongo hemorragiese koors virus (KKHKV) is ‘n bosluisoorgedraagde virus wat tot die familie Bunyaviridae en genus Nairovirus behoort. Daar is ‘n positiewe korrelasie tussen die verspreiding van die virus en die voorkoms van die hoof vektor wat bosluise in die genus Hyalomma is. Endemiese areas sluit areas in Afrika, Asië, Oos-Europa en die Midde- Ooste in met die onlangse verskyning in Turkye, Griekeland en Indië. KKHKV word geassosieer met hemorragiese koors in mense met ‘n sterftekoers van tot 30%. Huidiglik bestaan die hantering van pasiënte uit ondersteuningsterapie en die toediening van ribavirin, maar die effektiwiteit van hierdie middel is nog kontroversieel. Daar is ‘n geïnaktiveerde entstof wat in Oos-Europa en in die voormalige Sowjet-Unie gebruik word, maar nog nie aanvaar is vir wydverspreide gebruik nie. Begrip van die immuun korrelate word benodig vir die verdere ontwikkeling van terapeutiese en voorkomende intervensies. Die doelstelling van hierdie studie was om die immuunrespons in KKHKV oorlewendes te ondersoek in Suid-Afrika met die fokus op die teenwoordigheid van ‘n opspoorbare geheue T-limfosiet reaksie en die identifikasie van epitopiese areas in die nukleoproteïen en glikoproteïene. Ten einde die toepaslikheid van die geïdentifiseerde epitope in geografies geskeide isolate te verseker was virale volgorde diversiteit ook ondersoek deur volgende generasie volgordebepaling en filogenetiese studies. ‘n Sintetiese oorvleuelende peptied versameling was gebruik om die periferale bloed mononukleêre selle van KKHKV te ondersoek vir die produksie van interferon gamma in ELISPOT toetse. Sodoende was tien potensiële epitopiese areas geïdentifiseer, waarvan die meerderheid op die nukleoproteïen geleë was. In ‘n enkele pasiënt was twee ander areas geïdentifiseer op die glikoproteïen GC. ‘n Langdurige geheue CD8+ T-sel reaksie in KKHK xiii oorlewendes van tot 13 jaar na infeksie was geïdentifiseer. Die bevindinge dui op die teenwoordigheid van ‘n effektiewe langdurige sellulêre immuunrepons wat deur inenting gemoduleer kan word en bied ‘n goeie aanduiding vir die epitopiese areas wat oorweeg moet word vir kandidaat-entstowwe en toetsing vir entstof immunogenisiteit. Die teenwoordigheid van ‘n opspoorbare geheue reaksie in die afwesigheid van herblootstelling of chroniese infeksie sal toekomstige studies instaat stel om die volledige T-sel reaksie van oorlewendes te karakteriseer. As gevolg van die groeiende area van KKHKV endemisiteit word veilige, sensitiewe en spesifieke serologiese toetse benodig vir diagnostiese en serum-waarnemende doeleindes. Aangesien die bioveiligheids vlak 4 fasiliteite wat benodig word vir die kweeking van die virus in baie endemiese areas ontbreek, moet daar alternatiewe maniere vir die voorbereiding van reagense vir diagnostiese toets gevind word, wat nie ‘n veiligheidsrisiko vir die laboratorium werkers inhou nie. Een van die alternatiewe is die gebruik van sintetiese peptiede in serologiese toetse. Daarbenewens kan die identifikasie van immunodominante epitopiese gebiede, indien ‘n beskermende immuunrespons ontlok word, van toepassing wees vir entstof ontwikkeling. Teenliggame gerig teen menslike gedefinieerde linieêre B-sel epitopiese gebiede was opgespoor deur die gebruik van ‘n peptied versameling in ‘n ensiem-gebonde immunosorbent toets. Twee potensiële epitopiese gebiede was geïdentifiseer op die GC glikoproteïen met reaktiwiteit in 13 - 14 van die 15 getoetste pasiënte. Verdere ondersoek sal benodig word om te bepaal of die epitopiese gebiede ook korreleer met ‘n beskermende immuunrespons, asook vir die identifikasie van nie-aangrensende B-sel epitope wat moontlik ‘n belangrike rol in teenliggaam induksie in ‘n natuurlike infeksie met KKHKV speel. xiv Met die opkoms van nuwe foci van KKHKV infeksie meer onlangs is dit belangrik om te bepaal of die genomiese variasie die toepaslikheid van entstof kandidate en diagnostiese toetse in geografies geskeide areas sal beïnvloed. Volgende generasie volgordebepalings tegnieke was gebruik om die volledige genoom volgordes te bepaal vir tien suider- Afrikaanse KKHKV isolate. Hierdie is die eerste toepassing van volgende generasie volgordebepaling tegnologie vir KKHKV isolate en het bewys dat dit ‘n vinnige en koste- effektiewe alternatief tot standard Sanger volgordebepaling is en wat effektief was vir die ongeveerd 20kb KKHKV genoom. Die filogenetiese resultate het bevestig dat daar groot variasie tussen geografies geskeie KKHKV isolate op ‘n genomiese vlak is en dat gekonserveerde gebiede teenwoordig is en geteiken kan word vir entstof ontwikkeling en diagnostiese doeleindes. Die genetiese variasie was as gevolg van puntmutasies en segment herrangskikking wat algemeen in die suider-Afrikaanse KKHKV isolate voorkom. Ten spyte van die groot variasie in die primêre volgorde, by proteïen vlak, is die motiewe wat ‘n rol in die funksie van proteïene speel gekonserveer. Voorspellings sagteware analise het die teenwoordigheid van OTU-agtige sisteïen protease en RNA-afhanklike-RNA-polimerase (RaRp) domeine in die L segment in die diverse suider-Afrikaanse KKHKV isolate bevestig. RaRp is noodsaaklik vir virale replikasie, terwyl die OTU-agtige protease waarskynlik ‘n rol speel in immuunstelsel ontduiking en affekteer dus virale patogenese. Ontleding van die M segment dui die behoud van die basiese proteïen koderings strategie met twee strukturele en drie nie-stukturele glikoproteïene aan. Die aminosuur variasie was waarneembaar oor al die voorspelde proteïene, maar veral in die veranderlike mucin-agtige domein wat moontlik ‘n rol in virale patogenisiteit speel. Hierdie study bied teikens vir verdere ondersoek in virale patogenisiteit wat in vivo studies in diere en mutageniese toetse kan insluit. xv CHAPTER 1 Orientation, rationale and aims of the study Introduction Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne virus belonging to the family Bunyaviridae and genus Nairovirus. It occurs widely in Africa, Eastern Europe, Asia and the Balkans, and the geographic distribution correlates with the distribution range of ticks of the genus Hyalomma. Descriptions of a disease likely to be Crimean-Congo haemorrhagic fever (CCHF) date back to the twelfth century, however reports in which the disease was first given the name Crimean haemorrhagic fever (CHF) occurred in peasants harvesting crops on the Crimean peninsula in 1944. The causative agent was isolated in 1967 when suckling mice were used as laboratory hosts. In 1969, CHF was shown to be identical to Congo haemorrhagic fever isolated from a febrile child in the Belgian Congo (now Democratic Republic of the Congo) and the names were combined. The virus is now referred to as Crimean-Congo haemorrhagic fever virus. Initial observations suggested that the African strain was less pathogenic than the Asian strain. However this assumption was based on a limited numbers of cases. Prior to 1979 the virus had only been identified in Africa in countries to the north of, and including Tanzania, and whereas fatality rates up to 40% had been noted in eastern Europe and Asia from numerous outbreaks and cases, only 1 fatality out of 15 cases had been reported in Africa. In February 1981, the first case of CCHF was confirmed in South Africa leading to debate regarding the recent introduction of the virus or previous existence of the virus that had been undetected. Serological surveys of stored livestock sera from South Africa, Zimbabwe 1 and Namibia were instrumental in establishing that the virus had pre-existed in the country and that it was present throughout the country with higher frequency in areas where the species of Hyalomma tick were present. Increased awareness of the virus and the establishment of a diagnostic facility for laboratory confirmation of CCHFV infections facilitated further recognition of the incidence of cases and it was established that on average there are about 1-20 cases occurring annually. Current figures indicate a fatality rate of approximately 30% and it is widely accepted that African strains are no less pathogenic than Asian. There is now debate regarding the presence of less pathogenic strains from Greece and Turkey, especially with regard to the Greek strain initially isolated from a tick in 1982, strain AP92. Further studies are required to confirm or refute the existence of less pathogenic strains. Problem identification Transmission of the virus to humans occurs through tick-bites, crushing of ticks with bare hands, contact with blood or tissues of infected animals, and contact with blood or tissues of infected patients. Infection in humans is characterized by fever, headache, myalgia, rash and often a haemorrhagic state, and as mentioned above, with a case fatality rate of approximately 30%. No vaccine is available and treatment consists of supportive therapy and administration of the antiviral drug ribavirin although the efficacy of antiviral treatment is still controversial. CCHFV has a single-stranded, segmented RNA genome. There is limited information available regarding immune responses in patients infected with CCHFV however cellular responses to date have not been elucidated. These studies are hindered by the 2 requirement of a biosafety level (BSL) 4 containment laboratory and until recently, the lack of an animal model. It has not yet been determined what facilitates clearance of the virus. The kinetics of antibody responses have been determined and despite appearance of a detectable humoral antibody response, IgG and IgM, the antibody response does not always correlate with clearance of the virus. It has been shown that IgM antibody has no significant influence on outcome or decrease in viral load. Similarly although IgG antibody levels are inversely related to viral load, the viral titers in survivors appear to decrease independent of detectable antibodies. The observations indicate a role for innate or cellular immune responses in viral clearance. Investigation of cellular responses during the acute phase of illness will necessitate use of BSL 4 containment due to the biohazardous nature of the virus and investigation of T cell responses in survivors will be dependent on a detectable cellular immune response. As the frequencies of T cells in blood recognizing specific viral proteins or antigens is low, and has been noted to be less than 1:10 000 in peripheral blood mononuclear cells, the techniques that measure T cell immunity need to be highly sensitive. In addition there is debate regarding the ability to detect T cell responses against acute infections that are not boosted by re-exposure or persistence of the virus. Patients that have CCHF infections are seldom likely to be re-exposed to the virus and the virus does not persist. Hence it was deemed necessary to determine if survivors have a T cell memory response that could be detected irrespective of the time after illness. The subsequent identification of specific CCHFV T cell epitopes could therefore aid the development of effective protective or therapeutic vaccines by providing information as to which part of the viral genome should be targeted. 3 No effective vaccine is available at present. A formalin-inactivated, suckling mouse brain vaccine was used in parts of Eastern Europe and the former Soviet Union but has not been accepted for widespread use. More recently, a DNA vaccine which expresses the glycoproteins GC and GN was developed and was shown to elicit neutralizing antibodies in mice. A number of challenges exist in the development of an effective vaccine including the limited knowledge regarding what comprises a protective immune response. The immune correlates of protection against CCHF are unknown and studies to date are limited probably due to the biohazardous nature of the virus and dependence on current infections that occur sporadically and unpredictably. Hence, we have selected to begin to determine the role of T lymphocytes in infection by investigating the memory T cell responses of survivors and to use the memory response to identify epitopic regions and to determine whether the T cell epitopes reside predominantly within the CCHFV nucleoprotein or glycoproteins. Current treatment of the disease consists of supportive and replacement therapy with blood products. Vaccines that promote cellular immunity may have a role in protection and/or treatment. The disease remains a significant public health concern hence it is important to investigate immune correlates that could contribute to development of novel candidate vaccines. In addition, the identification of human defined immunodominant epitopic regions that induce detectable antibody responses could play a role in development of safe recombinant reagents for diagnosis and for use as tools for surveillance studies. The recent emergence of CCHFV in previously non-endemic regions in Eastern Europe and the presence of the vector of this virus in Southern Europe emphasizes the need for improved diagnostic capacity and increased surveillance capacity. Currently diagnostic reagents are dependent on culturing the virus within BSL4 facilities which limit 4 the number of laboratories that can prepare their own reagents. In addition standardization and validation of reagents will play an important role in commercialization of assays. Identifying immunodominant epitopes may have application in development of recombinant or alternative reagents. Antigenic and serological cross reactivity exists between geographically distinct isolates and to date there is no indication that there are antigenically distinct strains. However genetically distinct isolates do circulate within geographically distinct regions and within similar regions. Hence the development of vaccines and both serological and molecular tools for detection that are based on subunits, specific epitopic regions or targeting of specific regions of the genome respectively must take into consideration genetic diversity. To date only 31 complete genome sequences were available on GenBank for geographically distinct isolates of CCHFV. It was deemed necessary to determine additional sequence data for southern African isolates to determine if epitopic regions were conserved, to confirm reassortment events and to identify and determine if protein motifs were conserved with particular respect to southern African isolates. Aims and objectives The aims of the study were to : 1. identify cellular and humoral immune responses in survivors of CCHFV infection 2. investigate sequence diversity among geographically distinct CCHFV isolates 3. characterize sequence data for conservation of epitopic regions and protein motifs. 5 Specific objectives: 1. To ascertain if there were long lived memory T cell responses that can be used to further study the role of T lymphocytes in CCHFV infection. To identify if there were T cell epitopic regions on the nucleoprotein and/or structural glycoproteins of CCHFV by means of interferon gamma ELISPOT assays using a library of overlapping peptides and peripheral blood mononuclear cells collected from survivors of CCHFV infection. 2. To identify human defined linear B cell epitopes by means of enzyme linked immunosorbent assay (ELISA) screening of the overlapping peptide library. 3. To determine sequence diversity among CCHFV isolates with an emphasis on southern African isolates, and evaluate the effect of genome reassortment on sequence diversity and viral pathogenicity. 4. To identify conserved motifs at the amino acid level to determine targets for further studies into protein function and disease pathogenesis. In recent years, CCHFV has emerged as an important tick borne pathogen with a growing number of cases and endemicity expanding into Europe and Asia. Current management relies chiefly on supportive therapy and an effective vaccine is lacking. Due to the significant public health concern and high mortality associated with CCHFV, the identification of immune correlates which may provide targets for novel vaccine candidates or therapeutic interventions are required. To ensure that diagnostic assays and therapeutic or preventative interventions will be broadly applicable against CCHFV isolates with diverse geographic origins, availability of complete genome sequence data will have application. 6 Structure of the thesis This thesis is presented as a series of research articles which will be submitted for publication in various scientific journals. The first article, presented in Chapter 2, is a review article. As there are a plethora of review articles on CCHFV that have been published recently in the international literature, this article will be submitted to a South African journal to increase awareness of the disease locally and provide an update on current knowledge of the virus. The next two articles, presented as Chapters 3 and 4, investigate immune responses in survivors of CCHFV infection. Chapter 3 specifically investigates long- lived memory T cell responses in a cohort of South African CCHFV survivors and aims to identify if human defined T cell epitopes are present on the nucleoprotein or glycoproteins, while Chapter 4 focuses on the identification of human defined linear B cell epitopic regions using a peptide based ELISA. The next two chapters investigate sequence diversity among CCHFV isolates. In Chapter 5, next generation sequencing techniques are used to sequence the complete genomes of ten southern African CCHFV isolates. These are analysed along with available complete sequence data from the GenBank database. This manuscript has been submitted to Epidemiology and Infection and has been provisionally accepted pending minor revisions. In Chapter 6, the sequences are analysed at amino acid level using predictive software to identify conserved protein domains and potential protein functions. Finally, in Chapter 7, the overall conclusions of the study and future perspectives for further research are provided. 7 CHAPTER 2 Crimean-Congo haemorrhagic fever virus: review of a virus endemic in South Africa Historical background The clinical features of Crimean haemorrhagic fever (CHF) were first described following an outbreak in Crimea in 1944 (Hoogstraal, 1979). During this outbreak approximately 200 cases of the disease were reported in peasants involved in harvesting activities. The aetiological agent of CHF was shown to be a virus following experimental inoculation of psychiatric patients undergoing pyrogenic therapy with filtered blood collected from acutely ill CHF patients. The virus was also detected in Hyalomma ticks which were collected in the area and which were later shown to be the principal vector responsible for virus transmission (Chumakov, 1965; Chumakov et al., 1968a). The virus was first propagated in the laboratory in 1967, following intracerebral inoculation of newborn white mice with the blood of CHF patients (Chumakov et al., 1968a). CHF was subsequently shown to be antigenically indistinguishable from isolates of Congo virus which had been recovered from patients in the Congo and Uganda with a similar clinical picture (Chumakov et al., 1968b). Casals et al. (1970) therefore suggested the name CHF-Congo virus, but finally the name Crimean-Congo haemorrhagic fever virus (CCHFV) was adopted (Hoogstraal, 1979). 8 The virus CCHFV belongs to the family Bunyaviridae, genus Nairovirus (Calisher, 1991; Casals, 1969; Karabatsos, 1985). This family includes four other genera, namely Orthobunyavirus, Hantavirus, Phlebovirus, and Tospovirus. Each of these, with the exception of the Tospovirus genus, includes viruses of medical importance in humans. The Nairovirus genus is comprised of at least 34 viruses that have been grouped into seven serogroups (also referred to as species, http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fs_index.htm). The serogroups were initially defined according to serological cross reactivity using complement fixation, haemagglutination, neutralization, immunoprecipitation and immunofluorescence techniques (Calisher and Karabatsos, 1989; Casals and Tignor, 1980). CCHFV belongs to the serogroup of the same name. Other members of the CCHFV serogroup include Hazara virus, the closest known antigenic relative, and Khasan virus (Nichol et al., 2006). CCHFV is the only member of the group known to be medically significant. Hazara virus was isolated from Ixodes redikorzevi ticks in Pakistan and Khasan virus was isolated from Haemaphysalis longicornis ticks from the former USSR (Begum, 1970; Smirnova, 1979). CCHF virions are spherical and approximately 100 nm in diameter. The host cell derived lipid bilayered envelope contains surface projections of 5-10 nm in length which consist of the viral glycoproteins GC and GN. The virions contain three structural proteins, namely the glycoproteins (GC and GN) and a nucleocapsid protein, and one non-structural protein which is the viral RNA-dependent RNA-polymerase (Schmaljohn and Hooper, 2001; Whitehouse, 2004). The negative sense, single-stranded RNA genome consists of three segments designated large (L), medium (M) and small (S). The highly conserved complementary terminal nucleotide sequences of each segment result in loosely circular RNAs which 9 together with the nucleocapsid protein, make up the three helical nucleocapsids. These base-paired ends likely act as a functional promoter region which interacts with the viral RNA-dependent RNA-polymerase (RdRp) (Flick et al., 2002). The L, M and S segments encode the viral RdRp, the viral glycoproteins and the viral nucleocapsid protein respectively (Schmaljohn and Hooper, 2001). The L segment is approximately 12 000 bases in length and has a single open reading frame which contains two conserved protein motifs, namely the OTU cysteine protease-like domain and the RdRp domain (Duh et al., 2008; Honig et al., 2004; Kinsella et al., 2004; Ozdarendeli et al., 2010; Yadav et al., 2013). The OTU cysteine protease domain facilitates immune evasion by hydrolysing ubiquitin and interferon-stimulated gene 15 (ISG15) (Frias- Staheli et al., 2007), while the RdRp is responsible for mRNA synthesis and genome replication (Honig et al., 2004). The M segment is approximately 5 400 bases in length and encodes a single precursor polypeptide including two structural glycoproteins, GN and GC, and a number of non-structural glycoproteins, namely the amino-terminal mucin-like domain, GP38, NSM, GP85 and GP160 (Altamura et al., 2007; Sanchez et al., 2006). Only the structural glycoproteins have been identified as virion components, while the remaining CCHFV glycoproteins are likely soluble proteins (Sanchez et al., 2006). The function of these non-structural glycoproteins is unclear although, by analogy with Ebola virus, the mucin-like variable domain may play an important role in viral pathogenesis (Yang et al., 2000). GN and GC are responsible for attachment to cell surface receptors which allows entry into the host cell cytoplasm by endocytosis, where replication takes place. The viral envelope is acquired by budding through the endoplasmic reticulum into the Golgi cisternae. The virions are then transported in vesicles to the cell membrane and released by fusion of the vesicles and 10 plasma membranes (Schmaljohn and Hooper, 2001; Whitehouse, 2004). The S segment is approximately 1 600 bases in length and encodes the viral nucleoprotein which binds the viral RNA to form ribonucleoprotein complexes (Schmaljohn and Hooper, 2001). Epidemiology and geographic distribution CCHFV has been documented in more than 30 countries of Africa, Asia, Europe and the Middle East, with a distribution following that of its vectors, ticks belonging to the genus Hyalomma (Flick and Whitehouse, 2005). Cases of naturally acquired human infection have been documented in the former Soviet Union, China, Bulgaria, Yugoslavia, Albania, Kosovo, Greece, Pakistan, Iran, Iraq, United Arab Emirates, Saudi Arabia, Oman, Tanzania, Central African Republic, Democratic Republic of Congo, Uganda, Kenya, Mauritania, Burkina Faso, South Africa and Namibia (Al Tikriti et al., 1981; Burney et al., 1980; Dunster et al., 2002; El Azazy and Scrimgeour, 1997; Gear et al., 1982; Hassanein et al., 1997; Hoogstraal, 1979; Msimang et al., 2013; NICD Comm Dis Surveill Bull, 2011; Papa et al., 2002; Papa et al., 2008; Saluzzo et al., 1984; Saluzzo et al., 1985; Schwarz et al., 1995; Suleiman et al., 1980; Tantawi et al., 1980; Watts et al., 1989). Virus has also been isolated from ticks or non- human mammals in Madagascar, Senegal, Nigeria, Central African Republic, Ethiopia, Afghanistan, Hungary, Morocco and Spain (Estrada-Pena et al., 2012; Németh et al., 2013; Palomar et al., 2013; Watts et al., 1989). Serological evidence has been reported from Zimbabwe and Benin although no clinical cases have been documented, and limited serological observations have been reported from Portugal, France, Egypt and Kuwait (Watts et al., 1989). 11 The recent emergence of CCHFV in Turkey, with more than 7000 human cases reported since 2002, has highlighted the importance of active surveillance and vigilance for cases of CCHF even in areas not previously considered to be endemic for the virus (Maltezou et al., 2010). Other areas of recent emergence include human cases in Greece and India (Mishra et al., 2011; Papa et al., 2008), serological evidence of CCHFV in sheep in Romania (Ceianu et al., 2012), and detection of CCHFV in Hyalomma ticks in Spain (Estrada-Peña et al., 2012). CCHFV is transmitted to humans by tick bite or squashing of infected ticks with bare fingers, or by contact with blood or tissues of infected animals or humans. High risk occupations therefore include those with frequent contact with livestock, particularly those performing procedures such as castration or slaughtering such as farmers, farm workers, abattoir workers, and veterinarians (Hoogstraal, 1979; Swanepoel et al., 1998). Nosocomial transmission has also been documented in a number of countries, including Turkey, Albania, Iran, United Arab Emirates, Pakistan, Sudan and South Africa, resulting in infection of health care workers and patients exposed to cases of CCHF (Elata et al., 2011; Gürbüz et al., 2009; Harxhi et al., 2005; Hasan et al., 2013; Naderi et al., 2013; Van Eeden et al., 1985). The virus has been isolated from numerous ixodid tick species, as well as two argasid species (Camicas et al., 1991; Hoogstraal, 1979; Swanepoel et al., 1983; Watts et al., 1989; Zeller et al., 1994). For the majority of these species, evidence that they could serve as vectors is lacking and it seems likely that the virus detected was present in a blood meal taken from a viraemic host. However, ixodid ticks of the genus Hyalomma appear to be competent vectors (Turrel, 2007). In addition, the distribution of CCHFV cases correlates with that of the distribution of ticks belonging to the genus Hyalomma. Hyalomma ticks are two-host ticks, with immature forms (larvae and nymphs) feeding on birds and small mammals, and 12 adult ticks feeding on large wild and domestic animals and sometimes large birds. Humans are incidental hosts and do not play a role in the maintenance of the virus in nature (Hoogstraal, 1979). Transovarial transmission of CCHFV from infected female ticks to their progeny has been reported (Wilson, 1991), as well as transstadial transmission of the virus from tick larva to nymph to adult (Shepherd et al., 1989; Shepherd et al., 1991). Ticks have also been shown to become infected when co-feeding with infected ticks on a vertebrate host, even in the absence of a detectable viraemia in the vertebrate host (Gordon et al., 1993). Venereal transmission from infected male to uninfected female ticks also occurs (Gonzalez et al., 1992). Viraemic infection of various small mammals has been documented, including hedgehogs, hares, susliks and rodents such as ground squirrels, veld rats and gerbils (Hoogstraal, 1979; Shepherd et al., 1989). Small mammals have previously been identified as reservoir hosts for CCHFV. Serological evidence of CCHFV infection has been detected in a wide range of larger wild animals including buffalo and various antelope species. Large domestic animals including cattle, horses, donkeys, sheep, goats and pigs have also been implicated as being capable of transmitting the virus (Hoogstraal, 1979; Shepherd et al., 1987). Although these mammals have detectable viraemias and show seroconversion following infection with CCHFV, they do not develop clinical disease. To serve as reservoirs or amplifying hosts of CCHFV, viremia must reach a threshold level that allows transmission of the virus, hence the exact role of many large mammals in the natural cycle remains undetermined. Most birds have been shown to be refractory to CCHFV infection with no detectable viraemia and an absence of or short-lived seroconversion following exposure to the virus. 13 Ostriches appear to be the exception and have been implicated in more than one outbreak of CCHF in abattoir workers. This was supported by the demonstration of high-level viraemia and antibody responses in ostriches following experimental infection (Shepherd et al., 1987; Swanepoel et al., 1998). Although most birds do not act as amplifying hosts, ticks may become infected by cofeeding or venereal transmission while feeding on birds, and non-viremic transmission has been reported (Jones et al., 1987). In addition, migratory birds transport infected larval and nymphal ticks over large distances (Berezin et al., 1971; Zeller, 1994). Genetic diversity Genetic diversity in arboviruses with RNA genomes is a complex interaction between the need to maintain fitness in both vertebrate and arthropod hosts which favours genome conservation, and the RdRp which lack proof-reading mechanisms and therefore show high error frequencies during replication thus favouring genetic diversity (Coffey et al., 2008; Steinhauer et al., 1992). However, CCHFV shows a surprisingly high level of genetic diversity, especially in the M segment with nucleotide variation of 20%, 31% and 22%, for the S, M and L segments and amino acid variation of 8%, 27% and 10% for the respective proteins (Deyde et al., 2006). Phylogenetic analysis of complete genomes has identified groupings of CCHFV isolates into seven lineages which reflect the geographic origin of the viral isolates, namely group I (West Africa), group II (Democratic Republic of Congo), group III (South Africa and West Africa), group IV (Asia and the Middle East), group V (Europe and Turkey), group VI (Greece), and group VII (Mauritania). More recent isolates from China do not cluster within the existing groups (Zhou et al., 2013). 14 Segment reassortment can occur when dual infection of a cell takes place. This phenomenon has been described for each of the CCHFV segments but appears to be more common for the M segment. The reason for the more frequent reassortment of the M segment is not clear (Burt et al., 2009; Deyde et al., 2006; Hewson et al., 2004; Morikawa et al., 2007). Segment reassortment between African and Asian isolates indicates that the virus circulates between the two continents, probably dispersed by movement of ticks on migrant birds and/or livestock trade. Evidence of recombination has also been found in the S RNA segment (Deyde et al., 2006; Lukashev, 2005). The exchange of genetic material by reassortment or recombination is thought to occur within the tick vectors due to the longer duration of infection and potential exposure to multiple infected vertebrate hosts (Deyde et al., 2006; Hewson et al., 2004; Morikawa et al., 2007). Molecular epidemiology studies have shown that genetically related isolates have been identified circulating within the same region as well as in geographically distinct regions and genetically distinct isolates have also been shown to circulate within the same geographic area. These studies support the movement of CCHFV within and between continents. It is likely that genetic diversity within regions has resulted from movement and trade in livestock and bird migration with consequent movement of infected ticks and introduction of genetically distinct isolates. Generation of genetic diversity could also be a consequence of reassortment and recombination events. CCHFV in South Africa 15 The first case of CCHF was recognized in South Africa in 1981, when a 13 year old boy contracted the disease following a tick bite while attending “veldschool” in Bloemhof. This prompted a number of serological surveys which showed that the virus was widely distributed in South Africa and had been present for many years before the first clinical case was recognized (Swanepoel et al., 1983; Swanepoel et al., 1985). Since then, 194 cases of CCHFV have been recorded in South Africa, with a case fatality rate of 24% and more than half of the cases occurring in the Free State and Northern Cape provinces. The vast majority of infections have occurred in males (91%), particularly farmers and other agricultural workers, with patients reporting either tick bites or exposure to infected animals as likely routes of transmission (personal correspondence, Prof JT Paweska). Three species of Hyalomma ticks are found in South Africa, namely H. marginatum rufipes, H. glabrum, and H. truncatum and act as the local vectors. Immature ticks feed largely on hares and ground feeding birds, with adult ticks feeding on a variety of larger wild and domestic herbivores (Shepherd et al., 1987). These ticks are found most commonly in the central and western areas of South Africa and are rare in the eastern and southern coastal areas, corresponding to the occurrence of CCHFV cases in humans. Nosocomial transmission has been reported in an outbreak at Tygerberg Hospital in the Western Cape, involving an index case followed by seven secondary cases of CCHF (van Eeden et al., 1985). The infection was fatal in two of these patients. Clinical features The incubation period following a tick bite is approximately 1-3 days, while it is slightly longer following exposure to infected blood or tissues of animals or patients at 16 approximately 5-6 days (Swanepoel et al., 1987; Whitehouse, 2004). The onset of symptoms is often sudden with non-specific symptoms including fever, rigors, chills, headache, sore throat, dizziness, malaise and myalgia. In addition, backache, nausea and vomiting, abdominal pain, neck stiffness and photophobia may occur early in the illness. Fever is a common feature, but may be intermittent. During the early stages of illness, lassitude, depression and somnolence may be apparent as well as neuropsychiatric changes such as confusion and aggression. Hyperaemia of the face, neck and chest, injected conjunctivae and chemosis may also be present. By day 3-6 of illness, a petechial rash may be seen and is often followed by larger ecchymoses and bruising (Hoogstraal, 1979; Swanepoel et al., 1987). Less commonly, a macular or maculopapular rash may be present (Akyol et al., 2010; Ergonul et al., 2004). When present, the onset of haemorrhagic manifestations occurs on day 4 to 5. The severity of the bleeding tendency varies from leakage or oozing of blood from injection or venipuncture sites to epistaxis, haematemesis, haematuria, melaena, gingival bleeding, and bleeding from other orifices. Hepatomegaly and right hypochondrial pain may be present early in the course of infection and may progress to jaundice during the second week of illness (Hoogstraal, 1979; Swanepoel et al., 1987). Ocular findings are usually related to the haemorrhagic state with subconjunctival and retinal haemorrhages occurring (Engin et al., 2009). Pulmonary parenchymal haemorrhage presenting with haemoptysis, chest pain and dyspnoea has been described (Dogan et al., 2011). Infrequent presentations during the acute stage of illness include epididymo-orchitis, parotitis, peritoneal and pleural effusions, acalculous cholecystitis and intraabdominal abscesses (Aksoy et al., 2010; Guner et al., 2011; Kaya et al., 2012; Şensoy et al., 2011; Tanir et al., 2009). Cardiac involvement in the form of depressed cardiac function and pericardial effusion have also been documented (Engin et al., 2009). 17 Fatalities occur mostly from days 5 to 14 of illness due to multi-organ failure or haemorrhagic complications such as intracranial haemorrhage. The reported fatality rate varies from 5-50% and is higher in nosocomial outbreaks than in sporadic cases (Gozalan et al., 2007; Hoogstraal, 1979; Swanepoel et al., 1989). Case fatality rates (CFR) vary significantly between distinct geographic regions, with an average fatality rate of 24% in South Africa and up to 5% in Turkey (Ergonul et al., 2006a; Swanepoel et al., 1987). The considerable differences in CFR have not yet been definitively linked to differences in CCHFV strain pathogenicity, but serosurveys in Turkey and Greece have shown that a large proportion of infections in these areas appear to be subclinical. In Greece, only a single case of CCHFV infection has been reported (Papa et al., 2008) but serosurveys revealed approximately 4% seroprevalence of antibodies to CCHFV antibodies (Papa et al., 2011; Papa et al., 2013; Sidira et al., 2012). These findings led Papa et al. (2013) to hypothesize that non-pathogenic or low-pathogenicity strains may be circulating in these areas. Similarly, a large serosurvey in Turkey revealed a seroprevalence of about 10% resulting in estimated subclinical infections in 88% of cases (Bodur et al., 2012). AP92 and AP92-like strains may represent such a low-pathogenic strain of the virus. AP92 was originally isolated from a tick in Greece in 1972 (Deyde et al., 2060) but the strain was not isolated in humans until 2007. Only a single case of documented clinical disease linked to an AP92-like strain was identified in a 6 year-old boy from Turkey who was infected with CCHFV following a tick bite. The child presented with fever, raised liver enzymes, and prolonged prothrombin time and activated partial thromboplastin time but made a full recovery. By day 10 of illness, the haemorrhagic manifestations had cleared and laboratory findings had returned to normal (Elevli et al., 2009; Midilli et al., 2009). 18 Patients who recover usually begin to improve by day 9 to 10 although the convalescent period may be prolonged. Residual symptoms during this time may include conjunctivitis, weakness, confusion, amnesia, polyneuritis, headache, dizziness, nausea, anorexia, alopecia, vision and hearing loss, and poor memory (Hoogstraal, 1979; Swanepoel et al., 1987). Long term follow up of CCHF survivors have identified post-traumatic stress disorder (PTSD) and impaired health related quality of life (IHRQL) in nearly half of patients at 12 months post- infection. Rates of PTSD and IHRQL were significantly higher in patients requiring intensive care admission or the administration of blood products, and in those with bleeding (Gul et al., 2012). Similar findings have been described in survivors of other acute, life-threatening conditions such as myocardial infarction and strokes and therefore likely relate to the severity of the illness and not CCHFV itself. Although the clinical and laboratory features of CCHFV infection in children appear similar to those in adults, tonsillopharyngitis, rash and gastro-intestinal symptoms such as nausea, vomiting, and diarrhoea are more common. A tendency towards milder disease has also been noted, with a reported mortality rate below 5% (Dilber et al., 2009; Tezer et al., 2010). Maternal CCHFV infection during pregnancy may result in intrauterine or perinatal CCHFV infection in infants, with resultant abortion or haemorrhagic manifestations at birth (Ergonul et al., 2010). However, delivery of a healthy infant with no evidence of transplacental transmission following maternal infection at 30 weeks gestation has also been documented (Aydemir et al., 2010). There is currently insufficient data to determine the incidence of congenital infections following maternal infection or whether factors such as gestational age influence transmission and outcome, as has been described with other 19 viral infections. Only two cases of CCHFV infection have been described in breastfeeding women and transmission to their infants was not detected in either case (Erbay et al., 2008). Pathogenesis and immune responses A clear understanding of the pathogenesis of CCHFV infection is lacking but it appears to involve a combination of viral and host immune-mediated mechanisms. Following inoculation, the virus is released from the basolateral membrane of epithelial cells facilitating haematogenous dissemination. Local amplification in tissue macrophages and dendritic cells may allow spread of the virus to lymph nodes and spleen further facilitating haematogenous dissemination (Akinci et al., 2013). Replication in the blood, liver and spleen amplifies the viraemia allowing spread to other organs including lungs, kidneys, and brain, as demonstrated in a STAT-1 knockout mouse model (Bente et al., 2010). Viral replication in the liver and adrenal glands may result in a decrease in coagulation and plasma protein synthesis as well as dysregulation of blood pressure homeostasis (Geisbert and Jahrling, 2004). Histopathological findings in CCHFV infected tissues of the liver, kidneys and adrenal glands include coagulative necrosis of these organs which supports this hypothesis (Burt et al., 1997). The haemorrhagic manifestations and increased vascular permeability with capillary leakage resulting in the clinical features of CCHF are primarily due to endothelial damage by direct viral replication and immune mediated mechanisms such as immune complex deposition and complement activation (Connolly-Andersen et al., 2011; Joubert et al., 1985). Proinflammatory cytokines interleukin (IL)-6, IL-8, IL-10 and tumour necrosis factor alpha 20 (TNF-α) are released during CCHFV infection and play a role in endothelial permeability. Increased levels of these cytokines have been linked with disease severity (Bente et al., 2010; Ergonul et al., 2006b; Papa et al., 2006) potentially resulting in immune dysregulation and a so-called cytokine storm (Akinci et al., 2013). TNF-α and IL-6 also stimulate monocyte activation which may result in haemophagocytosis. This phenomenon has been described both in adults and children with CCHFV infection and may contribute to the cytopaenias observed by phagocytosis of blood cells (Dilber et al., 2009; Fisgin et al., 2008; Karti et al., 2004). A possible pathogenic role for TNF-α has been suggested in other viral haemorrhagic fevers (Kanerva et al, 1998; Linderholm et al, 1996). Known functions of TNF-α include activation of macrophages, stimulation of production of vasodilating substances, and antifibrinolytic activity. IL-6 is produced by a variety of cell types including Kupfer cells and is released following liver injury, which may account for the raised levels in CCHFV infected patients (Papa et al., 2006). The haemorrhagic manifestations of CCHF also involve thrombocytopaenia, liver dysfunction, decreased levels of coagulation factors and disseminated intravascular coagulopathy (DIC) (Akinci et al., 2013; Burt et al., 1997; Swanepoel et al., 1989). Innate immune responses often play an important role in protection against and recovery from viral infections but limited information is available for their role in CCHFV infection. High natural killer (NK) cell counts have been identified as a possible prognostic marker linked to high fatality rates. It is thought that the high NK cell counts result from a strong response to a high viral load, or due to excessive cytokine release (Yilmaz et al., 2008). In the latter case, the immune response itself may play a role in the pathogenesis of the disease. Interferon alpha (IFN-α) has antiviral activity and inhibits CCHFV replication 21 through interferon-induced proteins such as MxA (Andersson et al., 2004; Andersson et al., 2006). Although CCHFV replication is inhibited by IFN-α, the virus is able to evade IFN by delaying induction of IFN synthesis and counteracting IFN signalling (Akinci et al., 2013; Weber and Mirazimi, 2008). Although the dynamics of IgG and IgM responses have been well defined, little information is available about the epitopes against which these antibodies are directed. Despite the genetic variation in the M segment of the genome and the associated antigenic variation, cell culture studies have demonstrated cross-reactive, neutralizing antibodies against a conserved GC glycoprotein epitope (Ahmed et al., 2005). Although a decline in CCHF viral load has been correlated with the appearance of antibody in clinical infections, antibody production does not always correlate with viral clearance (Wӧlfel et al., 2007). Data is lacking regarding the role of T cell responses in the protection or recovery from CCHFV infection. A study of Puumala virus, another member of the Bunyaviridae family, has shown long-lasting memory CD8+ cell responses following acute infection which greatly exceeds the responses previously documented following acute viral infections such as influenza (Van Epps et al., 2002). Laboratory diagnosis Routine chemistry and haematology tests can assist in the identification of suspected CCHF cases. Either leukocytosis or leukopaenia may be present, while raised liver enzymes and thrombocytopaenia are consistently found and associated with decreased plasma fibrinogen, raised fibrin degradation products, and increased thrombin and partial 22 thromboplastin times (Swanepoel et al., 1989). Marked elevation in transaminases and early thrombocytopaenia as well as other signs of coagulation abnormalities are associated with an increased mortality (Ergonul et al., 2006a; Swanepoel et al., 1989). Although the clinical features, chemistry and haematology tests may alert clinicians to the possibility of CCHFV infection, laboratory confirmation is required to distinguish CCHF from clinically similar conditions. Due to its biohazardous nature, CCHFV requires manipulation in a biosafely level 4 environment. Viraemia is present during the acute stage of illness and may be detectable up to day 13 of illness, during which time the virus can be detected in clinical samples of blood or tissues. The virus can be isolated in cell cultures or laboratory animals. Susceptible cell lines include a variety of mammalian cell cultures such as Vero (African Green monkey kidney), BHK-21 (Syrian hamster kidney), and SW-13 (human adrenal carcinoma) cells. Isolation in cell cultures takes 1-7 days and must be confirmed by immunofluorescence as cytopathic effects are usually not seen. Intracranially inoculated suckling mice are also susceptible to CCHFV infection and usually succumb to infection within 5 - 10 days (Hoogstraal 1979; Shepherd et al., 1986; Swanepoel et al., 1989). Mouse inoculation has been shown to be more sensitive than cell culture methods (Shepherd et al., 1986). Reverse-transcription polymerase chain reaction (RT-PCR) is a rapid and sensitive means of diagnosis in the early stages of illness as well as in fatal cases where antibodies are not produced. Both conventional and real-time RT-PCR methods have been developed for the detection of CCHFV RNA targeting conserved regions of the S and L segments (Atkinson et al., 2012; Burt et al., 1998; Duh et al., 2006). Another method for the detection of CCHFV RNA is reverse transcription loop-mediated isothermal amplification (RT-LAMP) which has 23 the advantage of isothermal amplification and the potential for naked eye turbidity read out of results which would be beneficial in resource-limited settings (Osman et al., 2013). Enzyme linked immunosorbent assay (ELISA) detection of viral antigen has also been investigated but has not been widely adopted for diagnostic use due to a lower sensitivity than viral isolation and PCR methods (Saijo et al., 2005b; Shepherd et al., 1988). The serological diagnosis of CCHFV infection relies on demonstration of seroconversion or a four-fold increase in IgG on paired serum samples, or detection of IgM in a single serum sample. IgG and IgM become detectable from approximately day 7 and are detectable in almost all survivors by day 9. IgM generally remains detectable for up to 4 months, while IgG usually remains detectable for a number of years post-infection. Cases in which antibody remains undetectable beyond day 9 are invariably fatal (Shepherd et al., 1989). Historically, methods such as complement fixation, immunodiffusion and haemagglutination inhibition were employed, but have now largely been replaced by indirect immunofluorescent assays and ELISA which can distinguish IgG and IgM. Both in-house and commercial ELISA assays are available using cell lysate and recombinant antigens (Burt et al., 1994; Dowall et al., 2012; Emmerich et al., 2010; Marriott et al., 1994; Saijo et al., 2002; Saijo et al., 2005a; Samudzi et al., 2012; Tang et al., 2003; Vanhomwegen et al., 2012). Histopathological features include hepatocellular necrosis, haemorrhage, fatty change, Kupffer cell hyperplasia and mononuclear portal inflammatory infiltrates, but are not pathognomonic for CCHFV infection. Immunohistochemistry for detection of CCHFV antigens can be used to confirm the diagnosis on tissue samples (Burt et al., 1997). 24 Quantitative real time PCR has been used to determine CCHFV viral load in clinical samples as a predictor of disease severity and prognosis. Higher viral loads have been associated with a fatal outcome, although a definite cut-off for determining severe disease and poor prognosis have not yet been defined (Ҫevik et al., 2007; Duh et al., 2007; Papa et al., 2007; Saksida et al., 2010; Wӧlfel et al., 2007; Yapar et al., 2005). Differential diagnosis The differential diagnosis for CCHFV infection includes various bacterial, parasitic and viral infections as well as a number of non-infectious conditions. The geographic location, travel history and possible exposures, such as occupation and recreational activites, can assist in refining the differential diagnosis for individual patients. Infectious diseases which should be considered in the differential diagnosis include rickettsiosis (in South Africa, Rickettsia conori and Rickettsia africae), leptospirosis, borreliosis, malaria, Q fever (Coxiella burnetti), brucellosis, systemic anthrax, meningococcal infection, bacterial septicaemia, and causes of hepatitis including viral hepatitis and disseminated herpes simplex virus infection (Burt 2011; Flick and Whitehouse, 2005; Whitehouse, 2004). Other viral haemorrhagic fevers can be considered based on geographic location and travel history such as Ebola virus, Marburg virus, Lassa virus and Lujo virus in Africa, Rift Valley fever in Africa and the Middle East, yellow fever virus in Africa, South and Central America, and dengue virus in tropical and sub- tropical regions. Although CCHFV and Rift Valley fever virus are the only viral haemorrhagic fever viruses occurring endemically in South Africa, nosocomial transmission of Marburg virus (Gear et al., 1975), Ebola virus (Richards et al., 2000) and Lujo virus (Briese et al., 2009) have been described via patients admitted for treatment to hospitals in Johannesburg. Non- 25 infectious conditions which should be considered include leukaemia, lymphoma, drug- induced hepatitis and auto-immune diseases (van Eeden et al., 1985). Treatment Supportive therapy is essential to decrease mortality and should include maintenance of fluid and electrolyte balance, and aggressive administration of blood products including platelets, fresh frozen plasma and red cell preparations (Ergonul, 2008). Haemodialysis and respiratory support in the form of oxygen or mechanical ventilation should be administered as required (Leblebicioglu et al., 2012). Although the administration of immune serum prepared from survivors of CCHFV infection has been attempted, the efficacy of this intervention is not clear and further studies are required to evaluate the use of specific immunoglobulin or monoclonal antibody preparations as a therapeutic intervention and for post-exposure prophylaxis (Keshtkar-Jahromi et al., 2011; Kubar et al., 2011). Additional difficulties with this approach include the limited availability of the product and lack of standardization. The use of corticosteroids may be beneficial in patients with severe disease and haemophagocytic syndrome (Dokuzoguz et al., 2013; Erduran et al., 2013). Ribavirin is a broad spectrum antiviral agent which resulted in the inhibition of CCHFV replication in cell culture and reduced lethality in suckling mice (Paragas et al., 2004; Tignor and Hanham, 1993). Although a number of case studies indicated improved outcome following the administration of oral ribavirin particularly within 4-5 days following onset of illness (Ergonul et al., 2004; Izadi and Salehi, 2009; Mardani et al., 2003; Ozkurt et al., 2006), larger studies including two systematic reviews and meta-analyses evaluating a randomized 26 controlled trial and observational studies did not show a clear benefit to the use of ribavirin for the treatment of CCHFV infection (Ascioglu et al., 2011; Ceylan et al., 2013; Duygu et al., 2012; Soares-Weiser et al., 2010). Ribavirin therapy is reported to be well tolerated in patients with CCHFV infection. Adverse effects are reported only rarely, including mild anaemia, thrombocytosis and bradycardia (Ergonul et al., 2004; Fisher-Hoch et al., 1995; Ozkurt et al., 2006; Uysal and Metan, 2012). Prevention and control Measures to minimize exposure to the virus may be effective in preventing CCHFV infection. General measures would include the use of acaricides for livestock, applying insect repellents to exposed skin, making use of clothing impregnated with permethrin, and avoiding direct exposure to potentially infected blood and tissues of animals for example, by wearing gloves. However, practical implementation of these measures is difficult. As evidenced by a number of nosocomial outbreaks of CCHFV infection, infection of health care workers and patients in a hospital environment and laboratory workers can occur if appropriate precautions are not taken (Elata et al., 2011; Gürbüz et al., 2009; Harxhi et al., 2005; Hasan et al., 2013; Naderi et al., 2013; Van Eeden et al., 1985). Treatment of CCHF patients requires the application of strict barrier-nursing techniques including isolation of the patient and the use of appropriate personal protective equipment. Although the efficacy of oral ribavirin for post-exposure prophylaxis has not been proven, it has been used for high-risk exposures such as needle-stick injuries and mucous membrane exposures in a nosocomial setting in South Africa. 27 An inactivated, suckling mouse brain vaccine has been in use in Bulgaria since 1974 but has not been accepted for widespread use (Papa et al., 2011; Whitehouse, 2004). This vaccine elicited both T-cell responses to nucleoprotein and neutralizing antibodies in healthy volunteers, which were significantly increased by administering several doses of the vaccine (Mousavi-Jazi et al., 2012). More recently, a recombinant DNA vaccine expressing the M segment coding region was developed and was shown to elicit neutralizing antibodies in approximately half of the immunized mice (Spik et al., 2006). A transgenic plant vaccine expressing GN and GC glycoproteins was administered both orally and by injection and resulted in IgG and IgA responses in immunized mice (Ghiasi et al., 2011). Neutralizing antibodies were not investigated for this vaccine and challenge studies were not performed for either vaccine. A number of challenges exist in the development of an effective vaccine including the previous lack of a laboratory animal model, the limited target population for such a vaccine and the limited knowledge regarding what comprises a protective immune response. CCHFV has not been shown to cause disease in vertebrates other than humans, despite inducing a viraemia (Hoogstraal, 1979). Even non-human primates failed to show signs of clinical disease following CCHFV infection (Hoogstraal et al. 1979; Smirnova, 1979). Although newborn mice can be used for virus isolation using intracerebral or intraperitoneal inoculation (Bente et al., 2013; Smirnova, 1979), they are not a suitable model for studies of pathogenesis, immunity or vaccine efficacy. The use of knockout mice lacking either the type 1 interferon receptor (Bereczky et al., 2010) or the signal transducer and activator of transcription 1 (STAT-1) protein (Bente et al., 2010) represent the first laboratory animal model which could be used for studies of pathogenesis and immunity and for development of effective antiviral and vaccine strategies. 28 The availability of an animal model will aid future studies on pathogenesis and vaccine development. Further studies on cellular immune responses in patients are required in order to better understand and possibly modulate the immune correlates of protection. 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Genome Announc 2013;1:pii. 51 CHAPTER 3 Identification of novel T cell epitopic regions on Crimean-Congo haemorrhagic fever virus and confirmation of long-lived memory T cell responses 52 Abstract Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the Nairovirus genus of the Bunyaviridae family and is associated with haemorrhagic fever in humans. Although T lymphocyte responses are known to play a role in protection from and clearance of viral infections, specific T cell epitopes have yet to be identified for CCHFV. A panel of overlapping peptides covering the CCHFV nucleoprotein and the structural glycoproteins, GN and GC, were screened by ELISPOT assay to detect interferon gamma (IFN-γ) production in vitro by peripheral blood mononuclear cells from subjects with previous CCHFV infection. Sixteen peptides reacted and taking into account overlapping peptides this likely represented ten potential T cell epitopic regions. These regions were located predominantly on the nucleoprotein, with only a single subject reacting to two peptides from the glycoprotein GC. No single epitope was immunodominant, but all subjects showed reactivity to at least one T cell epitope. These responses were present at high frequency and detectable several years after the acute infection despite the absence of continued antigenic stimulation. Depletion studies confirmed that IFN-γ production was mediated chiefly by CD8+ cytotoxic T cells. This is the first description of cytotoxic T cell epitopic regions for CCHFV and provides confirmation of long-lived memory T cell responses in survivors of CCHFV infection. 53 Introduction Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the Bunyaviridae family and has a tripartite, single-stranded, negative sense RNA genome (Calisher, 1991; Casals, 1969; Karabatsos, 1985). The three segments are referred to as the large (L), medium (M) and small (S) segments (Clerx et al., 1981). The L segment encodes the viral RNA dependant RNA polymerase which is responsible for mRNA synthesis and replication of the RNA genome (Honig et al., 2004). The M segment encodes a number of non-structural glycoproteins and two structural glycoproteins, GN and GC (Altamura et al., 2007; Sanchez et al., 2006). The structural glycoproteins are responsible for attachment to host cell surface receptors and therefore determine the host range and cell tropism and are also the targets for neutralizing antibodies. The viral nucleoprotein, encoded by the S segment, binds the RNA segments for the formation of ribonucleoprotein complexes and shows endonuclease activity, although the role of this activity in CCHFV infection is not yet clear (Guo et al., 2012; Schmaljohn and Hooper, 2001). CCHFV is the only member of the CCHFV serogroup of medical importance as the other members of the group, Hazara virus and Khasan virus, have not been associated with disease in humans (Begum, 1970; Nichol et al., 2006; Smirnova, 1979). CCHFV infection in humans is associated with haemorrhagic fever and is fatal in up to 30% of cases (Bente et al., 2013). The principal vectors of the virus are ticks belonging to the genus Hyalomma (Chumakov et al., 1968). The distribution of disease follows that of the principal vector of the virus (Bente et al., 2013; Whitehouse, 2004). Clinical disease is well described in Africa, Asia, Eastern Europe and the Middle East and has recently emerged in Turkey, Greece and India (Maltezou et al., 2010; Mishra et al., 2011; Papa et al., 2008). With over 7000 human 54 cases reported in Turkey since 2002, the development of effective preventative and therapeutic measures have now become a priority (Maltezou et al., 2010). To this end, the correlates of protection against CCHFV need to be determined. Antibody responses develop within 7-9 days of infection and include an IgM response which is present for up to 4 months following infection and has been detected in a small number of cases a year following onset of illness, and an IgG response which likely persists for life (Burt et al., 2013; Shepherd et al., 1989). The antibody response likely plays a role in clearance of CCHFV. In cases where an antibody response is not detectable by day 9, a fatal outcome is almost invariably seen (Shepherd et al., 1989). However studies looking at viral load and antibody titre deduced that the detection of IgM had no influence on clearance of the virus or outcome, and viral load decreased independently of IgG. Hence, antibody production does not always correlate with viral clearance, implying that innate and T cell immunity likely also play an important role in viral clearance (Wӧlfel et al., 2007). In addition, neutralizing antibodies identified in cell-culture assays do not always confer protection in vivo, while non-neutralizing antibodies may confer protection through mechanisms such as antibody-dependent cell-mediated cytotoxicity (Bertolotti-Ciarlet et al., 2005). The nucleoprotein also induces antibody production and this region is widely used as the antigenic target in enzyme-linked immune-sorbent assays (ELISA) due to the robust nature of the antibody response to this protein following natural infection with CCHFV (Burt et al., 1994; Dowall et al., 2012; Emmerich et al., 2010; Marriott et al., 1994; Saijo et al., 2002; Saijo et al., 2005; Samudzi et al., 2012; Tang et al., 2003; Vanhomwegen et al., 2012). The Bulgarian CCHFV vaccine, which is an inactivated vaccine that was associated with a four-fold reduction in the number of CCHF cases following its introduction, has been shown 55 to induce both neutralizing antibody responses and T cell responses identified by interferon gamma (IFN-γ) enzyme-linked immunospot (ELISPOT) assays (Mousavi-Jazi et al., 2012). Ideally, an effective vaccine will therefore likely require the induction of both B and T cell responses. The Bulgarian vaccine was shown to induce T cell responses to the nucleoprotein, but there are currently no data available evaluating T cell responses following natural infection with CCHFV in human cases. To further understand the role of T lymphocytes in the immune response to CCHFV infection, we aimed to determine if it was possible to identify a memory T cell response in survivors of CCHFV infection and to identify T cell epitopes. An overlapping peptide library was used to screen for T cell responses by IFN-γ ELISPOT assay using peripheral blood mononuclear cells (PBMC) derived from subjects with previous CCHFV infection. The novel epitopic regions which were identified represent the first such T cell epitopes to be described following natural infection with CCHFV and may play an important role in vaccine development and evaluation of vaccine immunogenicity. Methods Study subjects Ten patients with a history of laboratory confirmed CCHFV infection were included in the study. Laboratory confirmation of CCHFV infection was performed at the National Institute of Communicable Diseases, Johannesburg at the time of the acute illness by means of viral nucleic acid detection by reverse transcription polymerase chain reaction (RT-PCR) or virus isolation, or detection of CCHFV specific antibodies. Sodium heparin blood samples were 56 transported to the laboratory for processing within 4 to 6 hours after collection. Two additional subjects were included in the study as negative controls and were selected as they had no history of CCHFV infection, exposure to CCHFV or risk factors for such exposure. Informed consent was obtained from all subjects and approval was obtained from the institutional Ethics Committee (ECUFS NR 152/06, renewed to Dec 2014). Synthetic peptides An overlapping peptide library containing 156 peptides consisting of 19-mers with a 9-mer overlap and spanning the nucleoprotein (482 amino acids) and the mature glycoproteins, GN (292 amino acids) and GC (648 amino acids), were synthesized (Mimotopes) based on the deduced amino acid sequences of CCHFV isolate SPU103/87 (GenBank accession numbers DQ211647 and DQ211634). The peptide length was selected to reduce the cost of the library production and to decrease the number of cells required to perform the ELISPOT assays. ELISPOT assays Fresh PBMC were isolated from whole blood using Ficoll-Hypaque density gradient centrifugation. IFN-γ ELISPOT assays were performed using 96 well plates (MultiScreen-IP, Millipore) pre-coated with anti-IFN-γ antibody (clone 1-D1K; Mabtech) at 2μg/ml at 4°C overnight. After washing with sterile phosphate buffered saline (PBS), the plate was blocked with R10 (RPMI 1640 medium plus 1% penicillin-streptomycin, 1% L-glutamine and 10% foetal calf serum) for 2 hours at room temperature. Screening of the peptide library was performed by means of a matrix of 36 peptide pools containing 7 to 9 peptides each so that each peptide is included in two separate pools. Peptides were added at a final 57 concentration of 5μg/ml for each peptide. PBMC were then seeded at 1x105 to 2x105 cells per well in 100 μl final volume. For positive controls, PBMC were stimulated with phytohaemaglutinin (Sigma-Aldrich) and 0.1μg/ml monoclonal antibody to human CD3 (clone CD3-2, Mabtech) in separate wells. For negative controls, a well containing only media and duplicate wells of PBMC incubated with media only were included. Plates were incubated for 16 hours at 37°C and 5% CO2. The plates were then washed 6 times with PBS and 2μg/ml of biotinylated anti-IFN-γ antibody (clone 7-B6-1, Mabtech) was added and incubated for 3 hours at room temperature. The plates were again washed with PBS and streptavidin-alkaline phosphatase conjugated antibody (Mabtech) was added and the plate incubated for 1 hour at room temperature in the dark. After a final wash, the alkaline phosphatase conjugate substrate kit (Bio-Rad) was used according to manufacturer’s instructions to detect IFN-γ producing cells and the reaction was stopped using distilled water. After drying, the number of spots were counted manually and results were expressed as the number of spot forming cells per million cells (SFC/106 cells). A response was considered positive if it exceeded 50 SFC/106 cells while negative controls were consistently below 20 SFC/106. The peptides showing a positive response using the pool screening method were then tested individually using the same method and with a final peptide concentration of 5 μg/ml per well. Subset depletion studies To determine whether the positive responses obtained were predominantly due to CD4+ or CD8+ T cells, PBMC from patient VBD 02/13 were depleted of CD8+ T cells using Dynabeads® CD8 (Invitrogen) according to the manufacturer’s instructions. The ELISPOT 58 assay was then performed using depleted and undepeleted PBMC with duplicate wells for each peptide previously shown to be reactive for this patient. Epitope conservation To determine whether the identified T cell epitopes are conserved amongst geographically distinct CCHFV isolates, available sequences were retrieved from the GenBank database. Results Ten subjects were included in the final analysis, all of whom were Caucasian males residing in the Free State, Northern Cape, and North West provinces of South Africa. The interval between CCHFV infection and sample collection for this study ranged from 10 months to 13 years. The details of the subjects are summarized in Table 1. IFN-γ responses were detected against 16 peptides using the ELISPOT assays, with each subject responding to at least one peptide (Figure 1). As responses were detected to some overlapping peptides, a total of ten epitopic regions where identified and further studies using nonamers to determine the optimal epitopes are required. The details of the peptides showing positive responses are summarized in Table 2. The majority of the potential epitopes were located on the nucleoprotein, with only two located on the glycoprotein GC. PBMC from two negative controls were tested against the 16 reactive peptides and showed no responses, confirming that the responses detected in the study subjects likely result from previous CCHFV exposure and were not due to non-specific reactivity. 59 Table 1 Summary of the subjects with previous CCHFV infection from whom peripheral blood mononuclear cells were extracted for interferon gamma ELISPOT assays. Study number Gender Time since infection VBD 07/13 Male 13 years VBD 05/14 Male 12 years VBD 05/13 Male 5 years VBD 13/13 Male 5 years VBD 04/13 Male 5 years VBD 02/13 Male 4 years VBD 10/13 Male 3 years VBD 16/13 Male 3 years VBD 04/14 Male 10 months VBD 06/13 Male 13 years VBD 14/13 Male 5 years VBD 15/13 Male 8,5 years VBD 19/13 Male 7 years 60 Figure 1 Detection of virus-specific T cell responses by interferon gamma ELISPOT assay. The magnitude of responses in spot forming cells per million (SFC/106) are indicated for each of the peptides to which study subjects showed a positive response. 61 Table 2 Details of the peptides representing potential epitopic regions identified by ELISPOT assay. Peptide names are derived from the relevant protein and the amino acid position relative to the coding regions of SPU 103/87 (DQ211647 and DQ211634). Six adjacent overlapping peptide pairs were reactive, with each pair likely representing a single epitopic region as indicated with a superscript numeric. Amino acid residues in the overlapping region are coloured red. 6 Peptide Amino acid sequence Number of positive responses Range of magnitude of responses (SFC/10 ) G1280-1298 TLHPRIEEGFFDLMHVQKV 1 115 G1352-1370 DGCDLDYYCNMGDWPSCTY 1 110 N 1 19-37 EFKKGNGLVDTFTNSYSFC 3 55 – 110 N 1 28-46 DTFTNSYSFCESVPNLDRF 3 55 – 65 N 2 136-154 DIGFRVNANTAALSNKVLA 1 65 N 2 145-163 TAALSNKVLAEYKVPGEIV 1 >500 N 3 262-280 DKHKDEVDRASADSMITNL 3 65 - >500 N 3 271-289 ASADSMITNLLHKIAKAQE 1 235 N298-316 RAQGAQIDTAFSSYYWLYK 5 55 - >500 N343-361 KMKKALLSTPMKWGKKLYE 1 >500 N 4 397-415 VANPDDAAQGSGHTKSILN 2 65 – 250 N 4 406-424 GSGHTKSILNLRTNTETNN 1 85 N 5 442-460 NIQDMDIVASEHLLHQSLV 1 >500 N 5 451-469 SEHLLHQSLVGKQSPFQNA 2 70 - >500 N 6 460-478 VGKQSPFQNAYNVKGNATS 1 >500 N 6 464-482 SPFQNAYNVKGNATSANII 1 60 62 No clear conclusion could be reached on the effect of the time interval between CCHFV infection and sampling with regard to the magnitude and range of T cell responses. Although the two subjects with intervals of 12 and 13 years respectively showed only low level responses (55 – 70 SFC/106) to one or two peptides, the subject with the most recent history of infection only 10 months prior to sampling also showed a low level response (65 SFC/106) to a single peptide. This variation likely is a consequence of individual patient responses rather than duration after infection. Due to the number of patients showing IFN-γ responses and the magnitude of these responses (Figure 2), peptides N262-280 and N298-316 were selected for CD8+ depletion studies. Subject VBD 02/13 was selected due to strong responses to both of these peptides, with 290 and >500 SFC/106 detected respectively. Following CD8+ T cell depletion, the number of SFC/106 dropped to 5 and 25 respectively indicating a predominantly CD8+ T cell response. Figure 2 T cell responses to peptides N262-280 and N298-316 as determined by interferon gamma ELISPOT assay. The vertical axis is capped at 500 spot forming cells per million (SFC/106). 63 Comparison of the amino acid residues in the epitopic regions identified revealed some diversity among geographically distinct CCHFV isolates with a maximum of three amino acid differences compared to SPU103/87, with the exception of isolate AP92 which showed four amino acid differences for peptides N262-280 and N271-289. Amino acid sequence conservation was higher among the southern African isolates with a maximum of one amino acid difference across each 19mer. Discussion A library of overlapping peptides covering the nucleoprotein, GN and GC proteins of CCHFV were screened by ELISPOT assay to determine in vitro induction of IFN-γ production. The present study is the first description of T cell epitopes in survivors of CCHFV infection. Ten probable epitopic regions were identified in this way, residing predominantly on the nucleoprotein with only two epitopes on glycoprotein GC. The predominance of nucleoprotein epitopes is similar to findings on Hantaan virus and Rift Valley fever (RVF) T cell epitopes (Wang et al., 2011; Xu et al., 2013). The reason for this is unclear but may result from the abundance of nucleoprotein production during CCHFV replication in vivo. RVF nucleoprotein derived subunit vaccines have been shown to protect against challenge with a lethal dose of virus using a mouse model. As antibodies directed against nucleoprotein are not neutralizing, it is likely that the protective mechanism is not dependent on induction of neutralizing antibodies but rather on a cellular immune response. 64 No single epitope was immunodominant, rather a variety of epitopes were identified in different patients. This likely represents the HLA diversity within the population studied and the differing HLA restrictions of the peptides, however HLA typing of patients and HLA restriction of epitopes was not determined in the study and will be included in future studies. A range of epitopes will most probably need to be included in any prospective vaccine candidates in order to induce protective T cell and antibody responses in all vaccine recipients. The high level of amino acid sequence conservation amongst the available southern African CCHFV isolates implies that variable responses to epitopes did not result from sequence variation which may result in an inability of T cells to recognise the synthetic peptides. The strength of the T cell responses identified differed between peptides and between patients responding to the same peptide. The magnitude of T cell responses induced by longer peptides such as the 19mers used in this study, may be underestimated in comparison to the more effective responses induced by optimal nonamers. This increased efficiency of T cell response induction may result from direct binding of antigen presenting cells to the nonamers in the peripheral blood (Wang et al., 2011). This phenomenon would not have influenced the outcomes of this study which aimed to identify the presence of T cell epitopes on viral proteins rather than to accurately quantify the magnitude of these responses. Further studies using nonamers of the epitopes identified will investigate this possibility and confirm the optimal epitopes. Depletion studies confirmed that the T cell responses observed result from cytotoxic CD8+ cells rather than CD4+ helper cells. Further studies making use of flow cytometry are 65 planned to determine which lymphocyte subsets are responsible for the IFN-γ production observed and to evaluate functionality of the cells which respond to peptide stimulation. CCHFV is transmitted to humans by tick bites, squashing of ticks with bare hands and contact with blood or tissues of infected humans and animals. Persons at high risk of exposure to CCHFV therefore include those likely to be exposed to ticks and those performing procedures resulting in exposure to blood or tissues of infected animals such as castration or slaughtering including farmers, farm workers, abattoir workers and veterinarians (Hoogstraal, 1979; Swanepoel et al., 1998). Since the first recognised case of CCHFV infection in South Africa in 1981, a total of 194 cases have been recorded in this country. Of these cases, 91% have occurred in males and more than 50% originated in the Free State and Northern Cape Provinces (personal communication, Prof JT Paweska). The exclusively male cohort analyzed in this study is therefore representative of the gender distribution of cases in South Africa. T cell responses to CCHFV peptides were detected in this study population up 13 years after acute CCHFV infection. The longevity of the responses points towards memory T cells playing an important role and support findings of the long-term presence of memory CD8+ T cells following infection with another bunyavirus, Puumala virus. This virus causes an acute infection with no known latent or chronic phase in humans, however Puumala virus specific memory T cell responses were present up to 15 years after infection despite the absence of continued antigenic stimulation or reexposure (van Epps et al., 2002). 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PLoS One 2013;8:e59210. 72 CHAPTER 4 Identification of human linear B-cell epitope sites on the envelope glycoproteins of Crimean-Congo haemorrhagic fever virus 73 Abstract A peptide library was used to screen for regions containing potential linear B-cell epitope sites in the glycoproteins and nucleoprotein of Crimean-Congo haemorrhagic fever virus (CCHFV) in an enzyme-linked immunosorbent assay (ELISA). The library consisted of 156 peptides, spanning the nucleoprotein and mature GN and GC proteins in a 19-mer with 9-mer overlap format. Using pooled serum samples from convalescent patients to screen the library, six peptides were identified as potential epitope sites. Further testing of these six peptides with individual patient sera identified two of these peptides as probable epitopic regions, with peptide G1451-1469 reacting to 13/15 and peptide G1613-1631 to 14/15 human sera. These peptides are situated on the GC protein at amino acid positions 1451-1469 (relative to CCHFV isolate SPU103/97) (TCTGCYACSSGISCKVRIH) and 1613-1631 (FMFGWRILFCFKCCRRTRG). Identified peptides may have application in ELISA for diagnostic or serosurveillance purposes. 74 Introduction Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne virus belonging to the family Bunyaviridae (Clerx et al., 1981). The negative-stranded RNA genome consists of three segments (S, M and L) coding for the nucleoprotein, the envelope glycoproteins (GN and GC) and the viral polymerase respectively (Clerx et al., 1981). The emergence of CCHFV in recent years in Southeast Europe and Southwest Asia highlighted the importance of developing serological assays for both diagnostic and serosurveillance purposes (Maltezou et al., 2010). Although the preparation of CCHFV antigens from cell cultures or inoculation of suckling mice have been used, these techniques require the use of a biosafety level 4 laboratory which is not widely available and therefore limits the diagnostic and surveillance capabilities of many laboratories in endemic areas. Expression of recombinant CCHFV nucleoprotein antigens in baculovirus, mammalian and bacterial systems have also been utilized and have potential for application in detection assays (Samudzi et al., 2012). Glycoproteins are more difficult to express, although by analogy with other bunyaviruses the glycoproteins should have application in detection assays. Peptide libraries have been used to identify antigenic regions of CCHFV nucleoprotein, but epitopic regions of the glycoproteins have not previously been defined. In addition to their utility in diagnostic assays, the identification of antigenic regions or B-cell epitopes also play an important role in vaccine development, both in selecting appropriate peptides for subunit vaccines and in the evaluation of vaccine immunogenicity, as well as in elucidating virus-antibody interactions (Wu et al., 2003). B-cell epitopes are classified as either conformational (discontinuous) or non-conformational (linear/continuous). Linear epitopes consist of short peptide fragments which are contiguous in the primary amino acid 75 sequence, while conformational epitopes are a set of amino acids that are brought into proximity by protein folding but are not contiguous in the primary sequence (Bottino et al., 2013; Peters et al., 2005). As antigen processing is not involved in immune recognition by B cells, these epitopes are often located on surface structures, are generally hydrophilic and are usually 4-8 amino acids in length (Bottino et al., 2013). In the present study, we screened for linear B-cell epitope regions in the nucleoprotein and envelope glycoproteins of CCHFV using a synthetic peptide library and sera from CCHF survivors in an enzyme-linked immunosorbent assay (ELISA) format. Methods An overlapping peptide library consisting of 156 peptides (19-mers with a 9-mer overlap) spanning the 482 amino acids of the nucleoprotein, the 292 amino acids of the mature GN and the 648 amino acids of the mature GC were synthesized (Mimotopes) based on the deduced amino acid sequences of CCHFV isolate SPU103/87 (GenBank accession numbers DQ211647 and DQ211634). The peptide length was selected to reduce the cost of the library production while allowing coverage of the complete genes for the major structural proteins of CCHFV. A peptide ELISA was developed using the synthetic peptides to screen for linear B-cell epitope regions using the serum of survivors of CCHF infection. Polysorb plates (Nunc) were coated with 100µl per well of 20µg/ml peptide in 0.2M carbonate buffer (pH 9.6) overnight at 4⁰C. The following day, plates were blocked with 10% skimmed milk in carbonate buffer for 2 hours at room temperature (22-24⁰C). The plates were then washed using 0.05% Tween 20 in phosphate buffered saline (PBS, pH 7.2). Human sera diluted 1:100 in 10% skimmed milk in PBS were then added and incubated for 1 hour at 37⁰C. After 76 washing, 100 µl of anti-human IgG horse radish peroxidase (HRPO) conjugate (Zymed) at a dilution of 1:1000 in 10% skimmed milk in PBS was added to each well and incubated for 1 hour at 37⁰C. After further washing, plates were incubated with azino di-ethyl- benzothiazoline-sulfonic acid (ABTS®) peroxidase substrate (Kirkegaard and Perry Laboratories, KPL) for 40 minutes at room temperature in the dark and optical density (OD) values were read at 405nm/620nm. All assays were performed in duplicate. For screening the full peptide library of 156 peptides, three pools of sera were generated to yield two positive and one negative pool each comprised of nine sera. The positive sera derived from survivors of previous infection with CCHFV as confirmed by ELISA or reverse transcription polymerase chain reaction testing performed at the National Institute for Communicable Diseases, Johannesburg, while the negative sera derived from volunteers with no history of CCHFV infection, exposure to CCHFV or risk factors for such exposure. Results The OD values obtained for each peptide of the envelope glycoproteins are illustrated in Figure 1. Six peptides derived from the envelope glycoproteins reacted to both positive pools but not the negative pool, however, no peptides in the nucleoprotein were found to be reactive to the pooled sera (data not shown). The details of the reactive peptides, as well as specific OD values obtained with each pool are shown in Table 1. Five peptides were located on the GC protein, with one reactive peptide on the GN protein. 77 Figure 1 IgG-specific peptide ELISA illustrating OD values obtained for each peptide using pooled human sera. The horizontal axis denotes the peptide number and the vertical axis denotes the absorbance measured at 405nm/620nm. Peptides 1 – 32 represent the mature GN protein and peptides 33 – 103 represent the mature GC protein. 78 Table 1 Details of the six reactive peptides selected for further testing based on OD values obtained in the peptide ELISA using one negative and two positive serum pools. Two adjacent overlapping peptide pairs are noted with each pair likely representing a single epitopic region as indicated with a superscript numeric. Amino acid residues in the overlapping region are coloured red. Peptide Peptide Amino acid position relative Protein Positive pool 1 Positive pool 2 Negative pool number to strain DQ211634 OD values OD values OD values Start End G669-687 KIPLLGKMAIYICRMSNHP 669 687 GN 0.845 0.407 0.152 G 1 1172-1190 RCGCTSSTCLHKEWPHSRN 1172 1190 GC 1.038 0.583 0.241 G 1 1181-1199 LHKEWPHSRNWRCNPTWCW 1181 1199 GC 0.803 1.073 0.170 G1451-1469 TCTGCYACSSGISCKVRIH 1451 1469 GC 1.434 0.811 0.305 G 2 1613-1631 FMFGWRILFCFKCCRRTRG 1613 1631 GC 0.871 0.574 0.174 G 2 1622-1640 CFKCCRRTRGLFKYRHLKD 1622 1640 GC 0.874 0.544 0.189 79 The six reactive peptides were then screened using individual sera from 15 survivors of previous CCHFV infection and three negative volunteers. The cut-off for each peptide was calculated using the mean of the negative sera plus two standard deviations. Peptides G1451- 1469 and G1613-1631 were identified as including a probable linear B-cell epitope site with 13/15 and 14/15 sera showing positive OD results respectively (Supplementary data). Both of these peptides are located on the GC envelope glycoprotein. The OD values of the positive sera obtained with peptide G1451-1469 ranged from 0.385 – 0.931 (cut-off 0.352), while the OD values for peptide G1613-1631 ranged from 0.408 – 1.257 (cut-off 0.317). Peptides G1181-1199 and G1622-1640 each reacted with 7/15 sera. These peptides showed lower reactivity in comparison to peptides G1451-1469 and G1613-1631, with OD values for positive sera obtained with peptide G1181-1199 ranging from 0.318 – 0.576 (cut-off 0.317) and with peptide G1622-1640 from 0.474 – 0.720 (cut-off 0.381). None of the sera reacted to peptide G1172-1190 and only two sera had OD values above the cut-off determined for peptide G669-687. The two sera (VBD 49/10 and VBD10/09) which did not react to peptides G1451-1469 and G1613- 1631 also failed to react to any of the other four peptides tested against the individual sera. As no further samples were available from these two patients at other time points, it could not be confirmed whether this reflects true lack of reactivity to all six peptides or whether sample integrity was compromised. However, sample VBD42/10 showed a low OD value on antibody ELISA testing in the laboratory, with an antibody titre of 1:100. The lack of reactivity to the peptides tested could likely be based on a low titred antibody rather than compromised sample integrity. Sample VBD10/09 was collected from a patient approximately three months after the onset of illness. The only other patient sampled at a similar time point showed relatively weak reactivity for peptides G1451-1469 and G1613-1631, and 80 negative results for the remaining peptides. The remainder of the samples in the panel were obtained between 11 months and 11 years post infection. It is known that epitope recognition varies with IgM and IgG class switching and with affinity maturation of the IgG response to viral infections (Gao et al., 2012; Lenova et al., 1998). As the majority of the serum samples included in the pools used to screen the peptide library were obtained at least one year post infection, it is possible that the six peptides identified in the initial ELISA screen are better recognized by antibodies in the later convalescent phase when extensive affinity maturation of IgG responses has occurred. Although acute phase and early convalescent phase serum panels were not available in the current study, it would be of interest to evaluate differences in peptide recognition at these time points after infection with CCHFV, particularly if the peptide ELISA were to be developed as a diagnostic assay for use in the acute phase of infection. Sequence data for CCHFV isolates from patients sampled in this study were unavailable. Conservation of predicted epitopic regions was determined using complete genome sequence data for southern African isolates. Complete genome sequence data determined in our laboratory for 10 southern African isolates as well as four complete genomes from South African CCHFV isolates available on GenBank were used to determine the conservation of the epitopic regions identified (Figure 2). The deduced amino acid sequences for peptide G1613-1631 show that this region is highly conserved with one isolate showing a single amino acid difference. Similarly, four isolates showed a single amino acid difference for peptide G1451-1469 compared to isolate SPU103/87 which was used as the reference sequence for the construction of the peptide library. Peptides G1172-1190 and G1181- 1199 showed 100% conservation in amino acid sequence, while peptides G669-687 and G1622-1640 81 Figure 2 Alignment of amino acid sequences of reference SPU103/87 with 14 southern African CCHFV isolates illustrating conservation of peptides G669- 687, G1172-1190, G1181-1199, G1451-1469, G1613-1631 and G1622-1640. 82 showed a maximum of two amino acid differences between isolates. All amino acid substitutions were conserved in polarity. The lack of reactivity to the individual patient sera by these peptides therefore cannot be explained by amino acid diversity, as all of the peptides found to be reactive in the screening ELISA were well conserved. In order to predict potential cross-reactivity of peptides G1451-1469 and G1613-1631 against other viruses, the peptide sequences were subjected to Basic Local Alignment Search Tool (BLAST) analysis using the protein-protein BLAST search (available at http://blast.ncbi.nlm.nih.gov/Blast.cgi). The BLAST search against peptide G1451-1469 recognized only CCHFV glycoprotein with query coverage and identity greater than 80%, implying that the specificity of this peptide is likely to be high. The search against peptide G1613-1631 returned hits for Hazara virus, Dugbe virus and Nairobi sheep disease virus glycoproteins although the coverage and identity were all below 80%. Further laboratory testing is required to determine whether this would lead to significant cross-reactivity and therefore lower the sensitivity of an assay based on this peptide. The predicted amino acid sequences used to generate the peptide library were analysed using the Immune Epitope Database (IEDB) Analysis Resource B Cell Epitope Prediction Tools and compared to the epitopes identified by the screening ELISA. Although peptide G669-687, located on the GN protein, was identified by the Kolaskar and Tongaonkar Antigenicity software as a predicted protein antigen (threshold setting = 1.000) (Kolaskar and Tongaonkar, 1990), only four amino acids in the peptide were identified as a possible epitope by the Bepipred Linear Epitope Prediction software (threshold setting = 0.350) (Larsen et al., 2006). Regions of peptides G1172-1190, G1181-1199, G1451-1469, G1613-1631 and G1622- 1640 on the GC protein were all identified as potential antigens by the Kolaskar and 83 Tongaonkar Antigenicity software, while Bepipred Linear Epitope Prediction software identified only peptides G1172-1190 and G1181-1199. Parker Hydrophilicity Prediction was also performed and identified peptides G1172-1190 and G1451-1469 as hydrophilic peptides and therefore potentially epitopic with scores of 2.937 and 2.074 respectively (threshold setting 1.290) (Parker et al., 1986). Peptides G669-687, G1181-1199, G1613-1631 and G1622-1640 were identified as hydrophobic, with scores of -0.426, 0.505, -1.311 and 0.179. As linear B-cell epitopes are mostly hydrophilic, the strongly hydrophobic score of peptide G1613-1631 emphasizes the necessity of functional laboratory testing to support predictive software. As the precise epitopic region of peptide G1613-1631 was not determined within the 19-mer, the true hydrophilicity score of this epitope could not be determined and may differ from the overall score for the peptide. Discussion As the aim of this study was to identify widely recognized epitopes for possible use in serological assays rather than the complete elucidation of linear B-cell epitope sites of CCHFV, the use of serum pools to screen the complete peptide library represented a practical approach given the size of the peptide library. Epitopes recognized at a low frequency in survivors of CCHFV infection may, however, not have been detected. The efficacy of synthetic peptides in immunoassays is influenced by their ability to bind to solid surfaces, the fact that antigenicity may be lost when binding to solid surfaces due to antigenic side chains not being accessible, and differing solubility (Tam, 1996). In a previous study, an epitopic site in the CCHFV nucleoprotein was identified using 8-mer overlapping peptide libraries (Burt et al., 2013). The peptide used in the previous study was of higher 84 purity (95%) and was biotinylated and therefore bound to the surface via neutravidin. This technique significantly enhances binding properties and site accessibility which can be hidden when peptides are bound directly to a solid phase. As these characteristics cannot be reliably predicted, laboratory testing of peptide libraries or peptides based on prediction software is required to identify peptides with good binding ability which retain antigenicity in an immunoassay, such as the two peptides identified in this study. Unbiotinylated 19- mer peptides were used in the current study as they were primarily selected for ELISPOT assays for the study of T-cell responses in CCHFV survivors, for which biotinylated peptides are not used. The ease with which peptides are manufactured and stability are advantageous in a diagnostic setting. A combination of peptides may be required to ensure adequate sensitivity for the detection of IgM and IgG. Humoral antibody responses to the nucleoprotein and glycoproteins can be detected following infection with CCHFV from approximately day 7 onwards after disease onset. While IgM responses generally decline to undetectable levels by 4-6 months post infection, IgG responses are detectable for at least 10 years (Burt et al., 2013). In the current study, IgG responses to epitopes on the GC glycoprotein were confirmed at least 11 years after infection. The envelope glycoproteins are responsible for binding of CCHFV to cell receptors and are targets for neutralizing antibodies which have been shown to protect mice in passive-immunization experiments (Bertolotti-Ciarlet et al., 2005). Recombinant nucleoproteins are usually targeted for development of detection assays because they are more conserved and recombinant glycoproteins are difficult to express. However peptides mimicking epitopic or immunodominant regions from conserved regions of the glycoprotein may be useful for serological assay development particularly for serosurveillance which is 85 important for monitoring disease endemnicity. We identified two potential linear B-cell epitope sites on the GC protein. Further investigation using shorter peptides would enable identification of specific epitopic sites within the peptides. Validation of the two peptides using larger numbers of serum samples would determine the usefulness of these peptides in serological assays. 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New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and X-ray-derived accessible sites. Biochemistry 1986;25:5425-5432. Peters B, Sidney J, Bourne P, Bui HH, Buus S, Doh G, Fleri W, Kronenberg M, Kubo R, Lund O, Nemazee D, Ponomarenko JV, Sathiamurthy M, Schoenberger SP, Stewart S, Surko P, Way S, Wilson S, Sette A. The design and implementation of the immune epitope database and analysis resource. Immunogenetics 2005;57:326-336. Samudzi RR, Leman PA, Paweska JT, Swanepoel R, Burt FJ. Bacterial expression of Crimean- Congo hemorrhagic fever virus nucleoprotein and its evaluation as a diagnostic reagent in an indirect ELISA. J Virol Methods 2012;179:70-76. Tam JP. Recent advances in multiple antigen peptides. JImmunol Methods 1996;196:17-32. 87 Wu HC, Jung MY, Chiu CY, Chao TT, Lai SC, Jan JT, Shaio MF. Identification of a dengue virus type 2 (DEN-2) serotype-specific B-cell epitope and detection of DEN-2-immunized animal serum samples using an epitope-based peptide antigen. J Gen Virol 2003;84:2771-2779. 88 Supplementary data Results of six reactive peptides tested with sera from 15 patients with previous CCHFV infection indicating OD values obtained, as well as the approximate duration from time of illness to sample collection. All OD values for CCHF survivors represent the mean of values obtained in duplicate testing. OD values above the relevant cut-off are indicated in red. Laboratory Approximate duration Peptide 18 Peptide 48 Peptide 49 Peptide 79 Peptide 97 Peptide 98 number since illness OD value OD value OD value OD value OD value OD value 52/10 10 years 0.271 0.227 0.315 0.846 1.257 0.625 10/09 3 months 0.083 0.100 0.112 0.092 0.115 0.088 41/10 8.5 years 0.324 0.261 0.279 0.717 0.868 0.654 06/11 11 months 0.242 0.204 0.318 0.632 0.970 0.474 38/11 8 months 0.180 0.151 0.332 0.499 0.733 0.422 51/10 2 years 0.277 0.391 0.373 0.714 1.013 0.503 30/10 10 years 0.502 0.338 0.455 0.931 1.210 0.720 42/10 2.5 years 0.154 0.156 0.231 0.200 0.324 0.156 39/11 7 years 0.201 0.192 0.576 0.636 1.173 0.270 40/11 8 months 0.215 0.167 0.287 0.478 0.720 0.394 43/11 11 years 0.160 0.180 0.412 0.525 1.100 0.277 49/11 3 years 0.182 0.157 0.354 0.428 0.812 0.258 51/11 5.5 years 0.183 0.162 0.232 0.411 0.591 0.343 54/11 2 years 0.209 0.208 0.266 0.457 0.671 0.347 20/13 3 months 0.170 0.199 0.168 0.385 0.408 0.338 Negative N/A 0.169 0.214 0.133 0.184 0.189 0.218 Cut-off N/A 0.290 0.508 0.317 0.352 0.317 0.381 89 CHAPTER 5 Next generation sequencing of southern African Crimean-Congo haemorrhagic fever virus isolates reveals a high frequency of M segment reassortment 90 Abstract Crimean-Congo haemorrhagic fever virus (CCHFV) is a bunyavirus with a single-stranded RNA genome consisting of 3 segments (S, M and L), coding for the nucleocapsid protein, envelope glycoproteins and RNA polymerase respectively. To date only five complete genome sequences are available from southern African isolates. Complete genome sequences were generated for 10 southern African CCHFV isolates using next generation sequencing techniques. The maximum likelihood method was used to generate tree topologies for 15 southern African plus 26 geographically distinct complete sequences from GenBank. M segment reassortment was identified in 10 of 15 southern African isolates by incongruencies in grouping compared to the S and L segments. These reassortant M segments cluster with isolates from Asia/Middle East, while the S and L segments cluster with strains from South/West Africa. The CCHFV M segment shows a high level of genetic diversity, while the S and L segments appear to co-evolve. The reason why M segment reassortment occurs is not known. It has previously been suggested that M segment reassortment results in a virus with higher fitness but a clear role in increased pathogenicity has yet to be shown. 91 Introduction Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the family Bunyaviridae, genus Nairovirus (Nichol et al., 2006). This tick-borne zoonotic virus is associated with clinical disease ranging from a non-specific febrile illness to severe disease manifesting as haemorrhagic fever. CCHFV has a negative sense, single-stranded RNA genome consisting of three segments designated large (L), medium (M) and small (S). The highly conserved complementary terminal nucleotide sequences result in loosely circular RNAs which together with the nucleocapsid protein, make up the three helical nucleocapsids. The L segment encodes the viral RNA polymerase while the S segment encodes the viral nucleocapsid protein. The M segment encodes a polyprotein which undergoes proteolytic processing to yield the viral glycoproteins, GC and GN (Schmaljohn and Hooper, 2001). CCHFV has been documented in more than 30 countries of Africa, Asia, Europe and the Middle East, with a distribution following that of Hyalomma ticks, the principal vector of the virus (Bente et al., 2013; Whitehouse, 2004). Since the first description of clinical disease due to CCHFV infection in South Africa in 1981, 192 cases have been confirmed (Msimang et al., 2013). However, complete S, M and L sequences are available for only four isolates collected from humans and one tick isolate from South Africa. These, as well as other published CCHFV sequences, were determined by Sanger sequencing using primer walking (Aradaib et al., 2011; Chamberlain et al., 2005; Deyde et al., 2006; Duh et al., 2008; Hewson et al., 2004; Kinsella et al., 2004; Meissner et al., 2006; Olschläger et al., 2011; Ozdarendeli et al., 2010; Papa et al., 2002; Seregin et al., 2004; Yadav et al., 2013; Zhou et al.2013). In recent years, a number of next generation sequencing (NGS) methods have been developed which yield large amounts of sequencing data at relatively low cost. Although the specifics 92 differ, these methods all make use of three steps, namely, library preparation, DNA capture and enrichment, and sequencing or detection. In the field of virology, these techniques have been employed for various purposes including the discovery of novel viruses, whole viral genome sequencing, and “deep” sequencing to determine viral quasispecies or genome variability (Capobianchi et al., 2013). This study aimed to make use of NGS techniques to obtain whole genome sequences of southern African CCHFV isolates in order to perform genetic analysis including identification of reassortment events. Methods Viral isolates Ten CCHFV isolates obtained from patients in southern Africa between 1985 and 2008 were sequenced retrospectively. RNA was extracted and supplied by Prof JT Paweska, Centre for Emerging and Zoonotic Pathogens, National Institute for Communicable Diseases (NICD), Johannesburg from cell culture preparations or mouse brain suspensions. Total RNA was extracted for isolate SPU44/08 using TRIzol® reagent according to the manufacturer’s instructions. The remainder of the isolates were stored at -70°C as freeze-dried 10% suckling mouse brain suspensions at the level of mouse brain passage 2-3. The suspensions were inoculated into Vero cell cultures and total RNA extracted from the infected cells using the acid guanidium thiocyanate-phenol-chloroform method as described previously (Burt and Swanepoel, 2005). The extracted RNA was stored at -70°C until use. Reverse transcription polymerase chain reaction and sequencing 93 The complete L segment was amplified using two previously described primer pairs that generated overlapping amplicons (Deyde et al., 2006). The complete M segment was amplified using primers designed by Deyde et al. (2006). Existing sequences on GenBank were used to design primers SF1 and SR3 (Table 1) for amplification of the S segment. The respective forward primers were used to perform the reverse transcription step for each amplicon using SuperScript™ III Reverse Transcriptase (Invitrogen). PCR was performed with the Expand Long Template PCR System (Roche Diagnostics GmbH) using standard cycling conditions according to the manufacturer’s instructions and an annealing temperature of 48°C. The PCR products were gel extracted and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega). The complete genomes for 3 of 10 isolates (SPU431/85, SPU383/87 and SPU 130/89) were determined using the Ion Torrent PGM™ sequencer by the Central Analytical Facility, Stellenbosch University. For the remaining 7 isolates, the S segment data was previously determined in the laboratory using the Big Dye™ Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) according to the manufacturer’s instructions and three overlapping primer sets as described in Table 2. The L and M segments of these isolates were sequenced at the NICD, Johannesburg using the Roche 454 GS Junior™ sequencing system. Additional sequencing of all isolates for incomplete coverage and clarification of ambiguities including mixed bases and base insertions or deletions was performed as required using the Big Dye™ Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). Details regarding the additional primers used are available from the corresponding author upon request. 94 Table 1 PCR and sequencing primers utilized. Approximate length of amplicons using primers LF1 and LR1 is 7800 bp, LF2 and LR2 is 6000 bp, MF and MR is 5350 bp and SF1 and SF3 1650 bp. Primer 5’-3’ position relative 5’-3’ sequence Reference name to SPU415/85 L1F 1 – 22 TCT CAA AGA TAT CAA TCC CCC C Deyde 2006 L1R 7773 – 7752 TTG GCA CTA TCT TTC ATT TGA C Deyde 2006 L2F 6137 - 6158 GAA GAG CTA TAT GAC ATA AGG C Deyde 2006 L2R 12157 - 12134 TCT CAA AGA AAT CGT TCC CCC CAC Deyde 2006 MF 1 – 18 TCT CAA AGA AAT ACT TGC Deyde 2006 MR 5354 - 5337 TCT CAA AGA TAT AGT GGC Deyde 2006 SF1 1 – 21 TCT CAA AGA AAC ACG TGC CGC In house SR1 * 590 – 569 GGT TCC TTC TCC TAA TCA TGT C In house SF2 * 467 – 487 GGT TTC CGT GTC AAT GCA AAC In house SR2 * 1114 – 1095 CAT TGG GGT GCT CAG CAG AG In house SF3 * 1029 - 1048 CGA CGG TGT CAC AGT TCC TC In house SR3 1673 - 1651 TCT CAA AGA TAT CGT TGC CGC AC In house *Primers designed for sequence determination 95 Table 2 Summary of data concerning the CCHFV sequences retrieved from the GenBank database and used in the study Strain Accession number Year and location Source L M S of isolation of isolate C-68031 DQ211616 DQ211629 DQ211641 1968 China Sheep Oman DQ211619 DQ211632 DQ211645 1997 Oman Human ArD15786 DQ211614 DQ211627 DQ211640 1972 Senegal Goat ArD8194 DQ211613 DQ211626 DQ211639 1969 Senegal Tick ArD39554 DQ211615 DQ211628 DQ211641 1984 Mauritania Tick SPU415/85 DQ211622 DQ211635 DQ211648 1985 South Africa Human SPU97/85 DQ211620 DQ211633 DQ211646 1985 South Africa Human SPU103/87 DQ211621 DQ211634 DQ211647 1987 South Africa Human Drosdov DQ211617 DQ211630 DQ211643 1967 Russia Human Kashmanov DQ211618 DQ211631 DQ211644 1967 Russia Human Baghdad-12 AY947890 AJ538197 AJ538196 1979 Iraq Human Matin AY422208 AF467769 AF527810 1976 Pakistan Human Kosova Hoti EU044832 EU037902 DQ133507 2001 Kosovo Human Turkey 200310849 DQ211623 DQ211636 DQ211649 2003 Turkey Human UG3010 DQ211624 DQ211637 DQ211650 1956 DRC Human AP92 DQ211612 DQ211625 DQ211638 1975 Greece Tick TADJ/HU8966 AY720893 AY179962 AY049083 1990 Tajikistan Human ROS/HUVLV-100 AY995166 DQ206448 DQ206447 2003 Russian Human Federation Ibar10200 AY947891 AF467768 U88410 1966 Nigeria Tick Semunya DQ076412 DQ094832 DQ076413 1958 Uganda Human Turkey-Kelkit06 GQ337055 GQ337054 GQ337053 2006 Turkey Human SPU128/81/7 DQ076414 DQ157174 DQ076415 1981 South Africa Tick SPU4/81 DQ076417 DQ157175 DQ076416 1981 South Africa Human Afg09-2990 HM452307 HM452306 HM452305 2009 Afghanistan Human NIVA 118594 JN572092 JN572084 JN572087 2011 India Tick NIV 112143 JN572091 JN572085 JN572089 2011 India Human NIVA 118595 JN572090 JN572083 JN572088 2011 India Tick YL04057 FJ562095 FJ562094 FJ562093 2004 China Tick Sudan AB1-2009 HQ378183 HQ378187 HQ378179 2009 Sudan Human Sudan Al-Fulah 3- HQ378180 HQ378184 GQ862371 2008 Sudan Human 2008 79121M18 GU477492 GU477493 GU477494 1979 China Rodent 96 Data analysis Raw sequencing data was converted from SFF format to FASTQ files using the sff_extract script (available as part of seq_crumbs at http://bioinf.comav.upv.es/) and trimming and filtering of reads based on length and quality scores was then performed using PRINSEQ (Schmieder and Edwards, 2011). Sequences for the L, M and S segments available on Genbank were used to compile databases and separate the reads into L, M and S segment related data using filter_by_blast (available at http://bioinf.comav.upv.es/seq_crumbs/available_crumbs.html). De novo assembly of the blast-filtered and unfiltered reads was performed using MIRA (Chevreux et al., 1999). The resulting contigs from both methods were assembled in Geneious (Geneious version 4.8.5 created by Biomatters, available from http://www.geneious.com/) and compared to known complete CCHFV sequences to identify areas of incomplete coverage or ambiguities which required further investigation. Ambiguities and homopolymers were investigated using Gap5 (Bonfield et al., 1995) and the majority could be clarified by evaluation of the quality scores at each relevant position. The additional Sanger sequences were incorporated into the assembly using Geneious and ChromasPro version 1.42. Alignments were confirmed by visual inspection with reference sequences and comparison with previously published partial sequences. Complete genome sequence data for 31 isolates were retrieved from GenBank as summarized in Table 2. The sequences were aligned using Clustal X version 2.0 (Larkin et al., 2007) and analysed using Molecular Evolutionary Genetics Analysis (MEGA) version 5 (Tamura et al., 2011) with the bootstrap maximum likelihood method with 1000 replicates. Sequence divergence was also determined using MEGA by calculating the average P 97 distances within and between sequence groups as well as pairwise distances for nucleotide and deduced amino acid sequences. Results Sequencing data Full coverage of complete L, M and S segments was obtained for 10 southern African CCHFV isolates. The details of the 10 isolates are summarized in Table 3. Ion Torrent™ PGM sequencing yielded raw data of >80 000 reads including >13 million bases for each of the 3 isolates. Following stringent filtering and trimming of reads based on length as well as quality scores, >35 000 reads amounting to approximately 7 million bases were included in the final analysis of each of these isolates. The 454 GS Junior™ sequencing data were more variable and duplicate runs were performed for four of the isolates in order to obtain complete coverage. The raw data for these 7 isolates yielded between 2404 and 17 315 reads including 1-8 million bases per isolate. Following filtering and trimming, the final alignments were performed on between 1105 and 7042 reads with approximately 500 000 – 3.5 million bases per isolate. The alignments generated by MIRA de novo assembly of both blast-filtered and unfiltered data corresponded, although improved contiguous quality was achieved with some isolates following the application of filter_by_blast. A comprehensive sequence database and optimization of blast parameters were required to ensure effective filtering of reads without loss of coverage, particularly of the highly variable M segment. 98 Table 3 Summary of southern African CCHFV isolates for which complete genome sequences were obtained by next generation sequencing. Strain GenBank accession numbers Year and location of Source of Outcome isolation human L M S infection SPU431/85 KJ682799 KJ682812 KJ682815 1985 Northern Cape Nosocomial Fatal SPU383/87 KJ682801 KJ682806 KJ682816 1987 Free State Tick Survived SPU556/87 KJ682798 KJ682811 KJ682817 1987 Northern Cape Tick Fatal SPU18/88 KJ682803 KJ682810 KJ682818 1988 Northern Cape Tick Fatal SPU45/88 KJ682796 KJ682809 KJ682819 1988 Free State Tick Fatal SPU497/88 KJ682804 KJ682808 KJ682820 1988 Namibia Livestock/ tick Fatal SPU130/89 KJ682802 KJ682807 KJ682821 1989 Northern Cape Tick Survived SPU48/90 KJ682797 KJ682813 KJ682822 1990 North West Unknown Survived Province SPU187/90 KJ682795 KJ682814 KJ682823 1990 North West Abbatoir Fatal Province SPU44/08 KJ682800 KJ682805 KJ682824 2008 Free State Livestock/ tick Survived Genetic analysis The genetic relationship of isolates as determined by the maximum-likelihood method are shown for the S (Figure 1(a)), M (Figure 1(b)) and L (Figure 1(c)) segments. The phylogenetic groups were designated I-VII as defined previously (Deyde et al., 2006; Carroll et al., 2010). 99 The complete sequences confirm the phylogenetic grouping of southern African isolates SPU497/88, SPU130/89, SPU383/87, SPU18/88 and SPU45/88 and the occurrence of reassortment of the M segment of these isolates as suggested by partial sequencing (Burt et al., 2009). The complete sequences also confirm that the incongruencies in M segment grouping were due to reassortment rather than recombination events. This pattern of M segment reassortment, showing clustering of the S and L segments with group III strains from South and West Africa while the M segments cluster with group IV isolates from Asia and the Middle East, was also noted for newly sequenced isolates SPU556/87, SPU44/08 and SPU431/85. Isolates SPU48/90 and SPU187/90 showed no evidence of reassortment, with all 3 segments falling within the group III South/West Africa lineage. Further evidence of M segment reassortment was seen in southern African isolates SPU97/85 and SPU415/85, as described previously (Deyde et al., 2006). No evidence of S segment reassortment was seen in the available complete sequences, but L segment reassortment was seen in two CCHFV isolates from Senegal, ArD15786 and ArD8194 (Deyde et al., 2006). The Chinese isolates YL04057 and 79121M18 appear to represent a new group as suggested by Zhou et al (2013), with S segments related to group IV Asia and Middle East, while the M and L segments cluster separately from other known groups. Isolate SPU431/85 was obtained from the same patient but from a sample collected 3 days subsequent to previously published SPU415/85. The sequences correlated well with only a single non- synonymous mutation in the coding region of the L segment and 4 non-synonymous mutations in the coding region of the M segment, as confirmed by Sanger sequencing. RNA from both of these isolates was obtained from cell cultures and these additional passages may have contributed to the accumulated mutations noted. 100 Figure 1(a) 101 Figure 1(b) 102 Figure 1 (c) Phylogenetic analysis of complete coding regions of (a) S segments, (b) M segments, and (c) L segments of CCHFV using a bootstrap maximum-likelihood method with 1000 replicates, with bootstrap values greater than 50% indicated at the relevant nodes. Each sequence is designated by the isolate name and isolates sequenced in the current study are marked (•). 103 The geographical distribution of CCHFV groups is illustrated in Figure 2. The S and L segments show a strong correlation in the distinct geographical grouping. The map displaying M segment group distribution clearly illustrates the blending of Asian and African strains, while the remaining groups correlate with S and L segment distributions. The mean P distances within groups and between groups as calculated with MEGA support the phylogenetic groupings. The S segment nucleotide distances within groups were generally low at between 0.1 – 1.4% and similar to the amino acid distances within groups which ranged from 0.1 - 1.8%. The nucleotide and amino acid distances between S segment groups were also similar at 2.5 – 6.8% and 2.8 – 8.4% respectively. The L segment showed a similar degree of diversity within groups at both nucleotide and amino acid levels, but increased diversity between groups. Nucleotide distances within groups ranged from 0.4 – 2.5 % and between groups from 3.8 – 11.3%, while amino acid distances within groups were between 0.5 – 1.8% and amino acid distances between groups were 2.9 – 10.1%. The M segment showed the greatest genetic diversity, particularly between groups, including numerous non-synonymous mutations. The nucleotide distances within groups ranged from 0.4 – 5.5%, while the nucleotide distances between groups were between 10.9 – 26.8%. At the amino acid level, the distances within groups were between 1.1 – 8.3% and the distances between groups were between 15.1 – 30.1%. Among the southern African isolates, the S segment was highly conserved with both nucleotide and amino acid mean distances of <1% (range 0 - 1.3% and 0 - 1.7% respectively). The diversity was also low for the L group with mean distances of 1.7% at the nucleotide level (range 0 - 2.8%) and 1.2% at the amino acid level (range 0 - 2.1%). 104 Figure 2(a) Figure 2(b) 105 Figure 2(c) Figure 2 Geographical distribution of CCHFV groups for the (a) S segment, (b) M segment and (c) L segment. The diversity of M segments was higher among group IV isolates than group III, with mean nucleotide distances of 5% and 2.8% respectively and mean amino acid distances of 7.5% and 4.1% respectively. Overall, the southern African M segments showed distances of 0 - 12.4% at the nucleotide level and 0 - 15.6% at the amino acid level. Although no association between M segment reassortment and pathogenicity was noted in the southern African isolates included in the current study, the number of both non-fatal 106 infections and non-reassortants included was small. Furthermore, no temporal association was seen with the isolation of reassortants. Discussion The S segment of CCHFV was previously investigated as a surrogate for complete sequencing due to its length and the fact that it is relatively conserved in comparison to the M and L segments thereby simplifying the sequencing process. However, the demonstration of both reassortment (Burt et al., 2005; Burt et al., 2009; Deyde et al., 2006; Grard et al., 2011) and, less commonly, recombination (Lukashev, 2005) among CCHFV genomes confirmed the necessity of at least partial sequencing of all three segments in order to perform accurate genetic analyses. The present study made use of two NGS platforms, namely the Ion Torrent PGM™ and the Roche 454 GS Junior™ sequencing systems, to obtain complete CCHFV sequences for ten southern African isolates. The largest collection of complete CCHFV genome sequences to date made use of 25 S, 40 M and 84 L primers in order to obtain complete sequence data for 13 geographically distinct isolates (Deyde et al., 2006). NGS methods present a relatively simple alternative, requiring only appropriate primers for the generation of amplicons by RT-PCR and potentially a limited number of primers to verify isolated regions or bases. This process could be simplified further by performing NGS of CCHFV directly from clinical samples without prior amplification. This would not only negate the need for specific primers and therefore allow sequencing of diverse CCHFV isolates but would also remove bias introduced by PCR errors (Bracho et al., 1998) as well as multiple passages sometimes required to generate adequate viral titres. This method has been 107 successfully used to sequence RNA viruses such as lyssaviruses directly from tissue samples and cell culture lysates (Marston et al., 2013). The introduction of errors during PCR can largely be overcome by using high fidelity enzymes. Although the assembly of raw data generated by NGS platforms can be complex, the workflow described in the present study made use almost exclusively of open source software and could be applied to a range of datasets from various sources. Confirmation of the validity of the assemblies obtained was made possible by comparison with a number of partial sequences which were available from a previous study (Burt et al., 2009), confirming the accuracy of the methods used. Genetic evolution of arboviruses, including CCHFV, is a complex process influenced by multiple factors. As with other RNA viruses, the RNA-dependent RNA-polymerases lack proof-reading activity and show error frequencies of approximately 10-4 (Steinhauer et al., 1992). This is offset by the effect of alternating infections of arthropods and vertebrates which constrains virus adaptation of arboviruses in comparison to other RNA viruses (Coffey et al., 2008). Despite this, CCHFV shows a high level of genetic variability. Inclusion of diverse isolates in the current study, particularly from China and central and West Africa, led to nucleotide variation of up to 7, 27 and 11% for the S, M and L segments respectively and amino acid variation of up to 8, 30 and 10%. This is similar to previous studies with greater variability of the M and L segments in comparison to the S segment, which is contrary to expectation as the viral RNA polymerase is usually highly conserved (Anagnostou et al., 2009; Deyde et al., 2006). Tree topologies for southern African isolates based on partial S, M and L sequence data correlated with topologies constructed using complete genome data (data available from corresponding author on request), providing further evidence that incongruencies in 108 grouping are likely reassortant events and not recombination (Burt et al., 2009). Genetic relationships indicate movement of CCHFV isolates within and between continents. Isolates from the same group can be found in geographically distinct locations and isolates from different groups can be found to cocirculate in similar regions. Genetic diversity within regions supports movement of the virus by bird migration and livestock trade. Reassortment events have occurred between West African and southern African isolates and between southern African and Asian isolates. Interestingly, the reassortment events between West and southern Africa involved the L segment, whereas reassortment events between southern Africa and Asia involved the M segment. This may be related to the tick species found in these geographical areas. The mechanism of reassortment is unclear however, it is assumed to occur in the tick host where dual persistent infections are more likely. Few complete sequences are available from West Africa but as 3 of the 4 available isolates show either L or M segment reassortment, it appears that reassortant viruses may occur frequently in this area. The two novel Chinese isolates described by Zhou et al (2013) cluster independently in an as yet unnamed group, suggesting the occurrence of further genetic groups. Additional complete genome sequence data from geographically distinct isolates are required to corroborate and expand these findings. Reassortment is widely described for members of the Bunyaviridae family (Briese et al., 2013). Both homologous and heterologous reassortment may occur and may result in altered viral phenotypes. Ngari virus is a reassortant of Bunyamwera and Batai viruses and is associated with haemorrhagic fever, in contrast to the mild disease associated with the parent viruses (Bowen et al., 2001). Both increased neuroinvasiveness and enhanced transmission by insect vectors have been specifically linked to the M segment of La Crosse 109 virus, another member of the Bunyaviridae (Beaty et al., 1981; Gonzalez-Scarano et al., 1992). Burt et al (2009) suggested a possible association between M segment reassortment of CCHFV and pathogenicity although this finding was not statistically significant. The current study could not confirm this association but given the small number of isolates included, further investigation is warranted. As 10 of the 15 complete sequences of CCHFV from southern Africa show M segment reassortment, it would be of interest to determine whether these M segments provide a competitive advantage such as the increased transmissibility demonstrated in La Crosse virus reassortants. The postulated co-evolution of S and L segments, possibly related to functional interdependency (Chamberlain et al., 2005), may account for the relative scarcity of reassortment of these segments. It is likely that both genetic drift and shift of CCHFV genomes occur chiefly during infection of insect vectors rather than in mammalian hosts due to the longer period of infection and increased likelihood of superinfection with more than one strain (Hoogstraal, 1979; Kinsella et al., 2004). In contrast to the short period of viraemia seen in humans (Shepherd et al., 1986), ticks remain infected over much longer periods with transstadial and, more rarely, transovarial transmission playing an important role in virus perpetuation and therefore also virus evolution (Gonzalez et al., 1992; Gordon et al., 1993; Hoogstraal, 1979). In conclusion, next generation sequencing methods provide a simple and effective alternative to Sanger sequencing for the determination of complete CCHFV genomes. The application of these methods to clinical samples without prior PCR amplification would provide further advantages and should be validated on CCHFV isolates. 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Seregin SV, Samokhvalov EI, Petrova ID, Vyshemirskii OI, Samokhvalova EG, Lvov DK, Gutorov VV, Tyunnikov GI, Shchelkunov SN, Netesov SV, Petrov SV. Genetic characterization of the M RNA segment of Crimean-Congo hemorrhagic fever virus strains isolated in Russia and Tajikistan. Virus Genes 2004;28:187-193. Shepherd AJ, Swanepoel R, Leman PA, Shepherd SP. Comparison of methods for isolation and titration of Crimean-Congo hemorrhagic fever virus. J Clin Microbiol 1986;24:654-656. Schmieder R, Edwards R. Quality control and preprocessing of metagenomic datasets. Bioinformatics 2011;27:863-864. 115 Steinhauer DA, Domingo E, Holland JJ. Lack of evidence of proofreading mechanisms associated with an RNA virus polymerase. Gene 1992;122:281-288. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731-2739. Whitehouse CA. Crimean-Congo hemorrhagic fever. Antiviral Res 2004;64:145-160. Yadav PD, Cherian SS, Zawar D, Kokate P, Gunjikar R, Jadhav S, Mishra AC, Mourya DT. Genetic characterization and molecular clock analyses of the Crimean-Congo hemorrhagic fever virus from human and ticks in India, 2010-2011. Infect Genet Evol 2013;14:223-231. Zhou Z, Meng W, Deng F, Zia H, Li T, Sun S, Wang M, Wang H, Zhang Y, Hu Z. Complete genome sequences of two Crimean-Congo hemorrhagic fever viruses isolated in China. Genome Announc 2013;1:pii. 116 CHAPTER 6 Comparative analysis of the L, M and S RNA segments of Crimean-Congo haemorrhagic fever virus isolates from southern Africa 117 Abstract Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the Bunyaviridae family with a tripartite, negative sense RNA genome. This study used predictive software to analyse the L (large), M (medium) and S (small) segments of 14 southern African CCHFV isolates. The L segment’s two domains, namely an OTU-like cysteine protease domain and the RdRp domain, are highly conserved among southern African CCHFV isolates. The M segment encodes the structural glycoproteins, GN and GC, and the non-structural glycoproteins which are post-translationally cleaved at highly conserved furin and subtilase SKI-1 cleavage sites. All of the sites previously identified were shown to be conserved among southern African CCHFV isolates. The N-terminal variable mucin-like domain of the M segment is heavily O-glycosylated and shows the highest sequence variability of the CCHFV proteins. Five transmembrane domains are predicted in the M segment polyprotein resulting in three regions internal to and three regions external to the membrane across the GN, NSM and GC glycoproteins. Eleven highly conserved N-glycosylation sites are predicted on the M protein. The corroboration of conserved genome domains and sequence identity among geographically diverse isolates may assist in the identification of protein function and pathogenic mechanisms, as well as the identification of potential targets for antiviral therapy and vaccine design. 118 Introduction Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne virus belonging to the genus Nairovirus in the family Bunyaviridae. Other genera in this family are Orthobunyavirus, Hantavirus, Phlebovirus and Tospovirus (Nichol et al., 2006). The single- stranded, negative sense RNA genomes of the Bunyaviridae consist of three segments namely the small (S), medium (M) and large (L) segments (Clerx et al., 1981). The CCHFV S segment encodes the nucleocapsid protein which encapsidates both viral RNA (vRNA) and complementary RNA (cRNA) to form ribonucleoprotein complexes. The vRNA serves as a template for both mRNA and cRNA, while the cRNA then serves as template for further vRNA (Bergeron et al., 2010). The nucleocapsid protein has a racket-shaped structure consisting of a head or globular domain and a stalk domain which are both composed predominantly of α-helices (Carter et al., 2012; Guo et al., 2012). In addition to binding RNA for the formation of the ribonucleoprotein complexes, the nucleocapsid protein also shows endonuclease activity attributed to the head domain (Guo et al., 2012). The polyprotein encoded by the M segment is co- and post-translationally cleaved into the two structural glycoproteins, GN and GC, and three non-structural proteins, NSM, a mucin-like domain and GP38 (Altamura et al., 2007, Sanchez et al., 2006). Two further non-structural glycoproteins, GP85 and GP160, contain GP38 and the mucin-like variable domain. These four non-structural glycoproteins are likely released from infected cells as secretory products (Sanchez et al., 2006). The L segment encodes the RNA-dependent RNA polymerase (RdRp) which is responsible for the synthesis of both mRNA for translation into proteins and cRNA for genome replication (Honig et al., 2004). 119 CCHFV infection in humans can result in haemorrhagic fever with a fatality rate of up to 30%. In recent years, the distribution of disease has expanded to numerous countries in Africa, Asia, the Middle East and Europe (Bente et al., 2013). The expanding endemicity in the absence of an effective vaccine or antiviral therapy has prioritized CCHFV research to identify possible conserved targets for such interventions. Antiviral drugs targeting enzymes involved in viral replication are now available for viral infections such as human immunodeficiency virus (HIV), the herpes viruses and hepatitis C. Molecules inhibiting viral adsorption and entry into host cells, such as the CCR-5 inhibitors used for the treatment of HIV, and immunotherapy for pre- and post-exposure prophylaxis are other approaches which have been shown to be effective for managing diverse viral infections. If similar approaches are to be followed for the management of CCHFV infection, then detailed information regarding viral proteins and their functions in geographically distinct isolates will be required. A thorough understanding of the pathogenesis of CCHFV disease is also paramount and this may, in part, be elucidated by the comparison of gene products and protein functions of other viral haemorrhagic fevers such as Ebola and Lassa viruses. In this study, nucleotide and deduced amino acid sequences were analysed using a range of predictive software to identify conserved domains, O- and N-linked glycosylation sites, transmembrane helices, cleavage sites and genetic distances among southern African CCHFV isolates. Methods Sequence data set 120 Complete genome sequences for 14 southern African CCHFV isolates were included in the analysis. The sequence data for ten of the CCHFV isolates were determined in a previous study (Goedhals et al., submitted for review), namely SPU431/85, SPU383/87, SPU556/87, SPU18/88, SPU45/88, SPU497/88, SPU130/89, SPU48/90, SPU187/90 and SPU44/08. A further four complete sequences were retrieved from GenBank, namely SPU415/85, SPU97/85, SPU103/87 and SPU4/81. All of the CCHFV isolates were from humans, with one isolate originating in Namibia while the remaining isolates were from South Africa including the Free State, Northern Cape and North West Provinces. Sequences were aligned using ClustalX version 2.0 (Larkin et al., 2007) and manually edited using BioEdit version 7.2.3 (available at http://www.mbio.ncsu.edu/bioedit/bioedit.html). Sequence analysis Genome cyclization due to interactions between the complementary 5’ and 3’ non-coding regions (NCR) was investigated by joining the 5’ NCR with an approximately equivalent number of bases from the relevant 3’ NCR using a 50 base poly-A spacer (Khromykh et al., 2001). The poly-A linked construct for each segment was analyzed using the Mfold Web Server (available at http://mfold.rna.albany.edu/?q=mfold) with default parameters and folding predictions at 37⁰C (Zuker, 2003). Sequence diversity within specific motif regions was calculated with Molecular Evolutionary Genetics Analysis v5 (MEGA5) using the p- distance option (Tamura et al., 2011). Protein analysis The deduced amino acid sequences of the L segment were submitted to InterProScan 4 (Zdobnov and Apweiler, 2001) and PSI-BLAST (Altschul et al., 1997) to identify functional 121 sites and conserved protein domains. Prediction of transmembrane helices was performed on the M segment amino acid sequences using TMHMM 2.0 (Krogh et al., 2001) and signal sequence cleavage sites were predicted using SignalP version 4.1 (Petersen et al., 2011). Potential sites of N-linked and O-linked glycosylation sites were determined using N- Glycosite (available at www.hiv.lanl.gov/sequence/GLYCOSITE/glycosite.html) (Zhang et al., 2004) and NetOGlyc 4.0 (Steentoft et al., 2013) respectively. Visual inspection of sequences was performed using BioEdit. Results Complete genome sequences The characteristics of the southern African CCHFV complete genome sequences are summarized in Table 1. The complete L segment, including the 5’ and 3’ NCR, ranged from 12157 – 12170 nucleotides in length for the southern African CCHFV isolates. The open reading frame (ORF) was 11838 nucleotides in length therefore encoding a protein of 3945 amino acid residues (approximate nucleotide position 77 – 11911). The variability in length of the L segment was chiefly due to variations in size of the 3’ NCR (243 – 256 nucleotides) while the 5’ NCR (76 nucleotides) and ORF were conserved in length. The 3945 amino acid ORF of the L segment had a GC content of 41%. 122 Table 1 Southern African CCHFV genome characteristics. Characteristic S segment M segment L segment Length (nucleotides) 1671 - 1673 5344 – 5364 12155 – 12170 % GC content 45.9 – 47.7 43.4 – 45.0 41.2 – 41.4 5’ NCR length – nucleotides 54 – 55 77 – 93 76 ORF length – nucleotides 1449 5055 - 5070 11832 - 11838 (amino acids) (482) (1684 – 1689) (3943 - 3945) 3’ NCR length – nucleotides 168 – 169 198 – 218 243 - 256 The complete M segment, including the 5’ and 3’ NCR, ranged from 5344 – 5365 nucleotides in length. The M segment 5’ and 3’ NCR were more variable than the L segment, at 77 – 91 nucleotides and 198 – 218 nucleotides in length respectively. The M segment coding region was between 5055 – 5070 nucleotides in length, encoding a polyprotein of 1684 – 1689 amino acid residues. The GC content of the M segments ranged from 43.4 – 45.0%. The S segment ORF encoded a single nucleoprotein of 482 amino acid residues (1449 nucleotides) in length for all available southern African CCHFV isolates. The complete S segment was 1671 nucleotides long, including a 5’NCR of 55 nucleotides and a 3’NCR of 169 nucleotides and with a GC content of 45.9 – 47.7%. 5’ and 3’ NCR cyclization analysis 123 Mfold analysis confirmed that the complementary 5’ and 3’ UTR of each segment allow for cyclization to form a panhandle RNA structure. Figure 1 shows representative examples of the three most stable structures according to the estimated change in Gibbs free energy predicted by Mfold and ranged from -37.17 to -51.91 using SPU97/85 as an example. Molecular characterization of the L segment ORF Conserved domains identified by InterProScan and PSI-BLAST analysis included an N- terminal cysteine-protease motif of the ovarian tumour (OTU) protein superfamily and the RNA-dependent RNA polymerase (RdRp) motif. The OTU domain was located at approximate amino acid residues 29 – 158 of the L segment ORF and showed little sequence diversity, with a maximum of two amino acid differences between isolates over the 118 amino acid region and calculated p-distances of 0 – 1.7%. The catalytic triad of the CCHFV OTU-like cysteine protease is predicted to contain cysteine, histidine and aspartic acid residues, initially identified potentially as GDGNCFYHSIAX100HFD with the position of the catalytic triad indicated in bold (Duh et al., 2008; Kinsella et al., 2004). A recent study, however, has identified the catalytic triad as GDGNCFYHSIAX100HFD by means of kinetic studies using OTU mutated at the candidate residues (Capodagli et al., 2011). The amino acid sequence of the OTU-like cysteine protease catalytic triad was conserved among the southern African CCHFV isolates. The approximate position of the catalytic domain of the RdRp was predicted to occur at amino acid residue positions 2043 – 2776 using PFAM and 2342 – 2551 using PROFILE relative to SPU97/85. The exact position of the RdRp catalytic domain has yet to be experimentally determined. Sequence diversity within the catalytic site ranged from 0 – 1.6% between isolates. 124 Figure 1 5’ and 3’ NCR complementary regions forming panhandle structures as modelled using Mfold Web Server. A) L segment, dG = -40.48. B) M segment, dG = - 51.91. C) S segment, dG = -37.17. 125 The C2H2-type zinc finger motif (amino acid residue positions 609 – 632) identified previously (Duh et al., 2008, Ozdarendeli et al., 2010, Yadav et al., 2013) was not predicted using either InterProScan or PSI-BLAST although the amino acid sequence in this region was highly conserved. In comparison to Turkey-Kelkit06, Kosova Hoti, NIVA118594, NIVA118595 and NIV112143 CCHFV isolates, a single, semi-conserved amino acid residue substitution of arginine to lysine at position 611 was present in 2/14 southern African isolates and the C2H2 motif was identified visually in all isolates. Similarly, the leucine zipper motif (amino acid residue positions 1386 – 1407) identified in strain IbAr10200 was not predicted in the southern African isolates although a maximum of two amino acid substitutions were present in the region (Honig et al., 2004, Kinsella et al., 2004). Isoleucine was substituted for leucine at position 1386 in 4/14 isolates and serine for glycine at position 1389 in 13/14 isolates in comparison with IbAr10200. The conserved protein domains in the L ORF are illustrated in Figure 2(a). Molecular characterization of the M segment Analysis of the southern African M segments using SignalP identified a signal peptide cleavage site at the N terminus for all isolates with the exception of SPU415/85. The exact position of the cleavage site differed between isolates, occurring variably between amino acid residues 20/21 (SPU187/90 and SPU48/90), 22/23 (SPU4/81 and SPU103/87), 24/25 (SPU383/87, SPU130/89, SPU497/88, SPU431/85, SPU97/85, SPU556/87, SPU45/88 and SPU18/88) or 27/28 (SPU44/08). Signal peptides are commonly found in proteins which are targeted to organelles such as the endoplasmic reticulum or Golgi, and in many membrane- bound proteins. The mucin-like variable region stretches from the signal peptide cleavage site to a furin cleavage site motif, RSKR, which was conserved among all southern African 126 isolates at either amino acid residue position 244 - 247 or 249 - 252. The calculated p- distance for the mucin-like domain of southern African isolates showed nucleotide sequence diversity of 0 – 40.7% (amino acid diversity 0 – 60.1%) for this region. The furin cleavage site is followed by GP38 which was 804 nucleotides (268 amino acid residues) in length for all southern African CCHFV isolates studied. The nucleotide and amino acid sequences in this region were more conserved than the mucin-like region with p-distances calculated at 0 – 12.7% at the nucleotide level and 0 – 16.8% at the amino acid level. The N terminus of GN is cleaved from GP38 at a protease SKI-1 cleavage site motif, RRLL, which was conserved among southern African isolates at amino acid residue position 516 – 519 or 521 – 524. The smaller of the two envelope glycoproteins, GN, was 852 nucleotides (268 amino acid residues) in length and showed nucleotide variation of 0 – 8.1% (amino acid 0 – 8.5%). The C terminus of the GN protein is cleaved at a further SKI-1 cleavage site motif, RRLL or RKLL, at amino acid residue position 804 – 807 or 809 – 812. Calculated p-distances for the 687 nucleotide (229 amino acid) long NSM protein were 0 – 10.5% at the nucleotide level and 0 – 11.5% at the amino acid level. A final cleavage site, RRPL or RKPL, was situated at amino acid residue position 1037 - 1040 or 1042 - 1045, at the GC N terminus. The 1935 nucleotide long GC protein showed the highest sequence conservation of the M segment polyprotein with amino acid divergence of 0 – 4.8% (nucleotide divergence 0 – 6.2%). The genome organization of the M segment is illustrated in Figure 2(b). Five transmembrane helices were predicted for all southern African CCHFV isolates analyzed, resulting in three regions internal to and three regions external to the membrane. Using isolate SPU97/85 as an example, the transmembrane helices were located at approximate amino acid residue positions 700 – 722, 825 – 847, 862 – 884, 974 – 996, and 127 Figure 2(a) Figure 2(b) Figure 2 Schematic representation of the protein analysis of the complete genome of southern African CCHFV isolates (not drawn to scale). (a) L segment. (b) M segment. Cleavage sites are indicated by arrows with the amino acid residues and positions indicated below. Transmembrane helices are indicated as black lines in the relevant ORFs. N-glycosylation sites are indicated by stars. 128 1600 – 1622. These regions were located within the GN, NSM, and GC proteins. Predicted mucin-type GalNAc O-glycosylation sites were identified in 5.4 – 6.5% of the M segment polyprotein, predominantly in the mucin-like variable region where 29.4 – 34.0% of amino acid residues were predicted to be O-glycosylated. In contrast, only 1.3 – 1.9% of the rest of the M segment was predicted to have O-linked glycosylation. O-linked glycosylation involves the addition of N-acetyl-galactosamine to serine or threonine residues. The serine and threonine content in the M segment ORF averaged 9.1% (range 8.9 – 9.3%) and 8.6% (8.3 – 9.1%), respectively. In comparison, the mucin-like variable region showed increased serine and threonine content at 14.6% (range 12.1 – 16.9%) and 17.4% (16.1 – 19.0%), correlating with the distribution of predicted O-linked glycosylation sites. The positions of predicted N-glycosylation sites for all the aligned M segment sequences are summarized in Table 2. Eleven of these predicted sites were conserved in 13 - 14 of the southern African isolates as indicated in Figure 2(b), excluding position 760 which was within a predicted transmembrane helix region. The M segment of the southern African CCHFV isolates contained 79 cysteine residues which were highly conserved. This may suggest the presence of a large number of disulphide linkages, however the GN cytoplasmic tail has been shown to contain two ββα-type zinc fingers with a CX2CX11-12HX3C motif of cysteine and histidine residues (Estrada and De Guzman, 2011). These motifs were conserved in the southern African isolates (Figure 3) beginning at amino acid residue positions 736/741 and 761/766 respectively and separated by a 4 amino acid linker, specifically SYNI. 129 Table 2 N-linked glycosylation sites on the M segment ORF as predicted by N-Glycosite. Amino acid residue positions are based on the aligned ORF sequences to allow uniformity of numbering. Position 760 forms part of a predicted transmembrane helix and is therefore unlikely to be N- glycosylated. 30 35 37 46 82 108 116 186 201 205 229 248 381 431 562 760 981 1059 1350 1568 SPU4/81 N N N N N N N N N N N N N N SPU187/90 N N N N N N N N N N N N N SPU48/90 N N N N N N N N N N N N N N SPU103/87 N N N N N N N N N N N N SPU44/08 N N N N N N N N N N N N SPU383/87 N N N N N N N N N N N N SPU130/89 N N N N N N N N N N N N SPU497/88 N N N N N N N N N N N N SPU18/88 N N N N N N N N N N N N SPU45/88 N N N N N N N N N N N N N SPU556/87 N N N N N N N N N N N N N N SPU97/85 N N N N N N N N N N N N N N SPU431/85 N N N N N N N N N N N N N N SPU415/85 N N N N N N N N N N N N N N 130 Figure 3 The amino acid sequences of the dual CCHC-type zinc finger motifs in the GN tail. Conserved residues are indicated by a dot, while substitutions are indicated by the relevant amino acid. The conserved C-X2-C-X11-12-H-X3-C motifs of each zinc finger are outlined. Discussion Characterization of the deduced amino acid sequences for the L segment of 14 southern African CCHFV isolates confirmed two conserved functional domains as previously identified, namely the OTU cysteine protease domain and the RdRp catalytic domain (Duh et al., 2008, Honig et al., 2004, Kinsella et al., 2004, Ozdarendeli et al., 2010, Yadav et al., 2013). Analysis of domains in southern African isolates was consistent with geographically distinct isolates. It has been shown that the OTU cysteine protease domain situated in the N terminus of the CCHFV L protein is not required for viral replication or RdRp function (Bergeron et al., 2010). Rather, these proteases hydrolyze ubiquitin and interferon- stimulated gene product 15 (ISG15) thereby allowing the virus to evade the type 1 interferon and tumour necrosis factor alpha cytokine pathways (Frias-Staheli et al., 2007). This domain is highly conserved, showing 98.3 - 100% sequence identity among southern 131 African CCHFV isolates. Similarly, the predicted catalytic domain of the RdRp is highly conserved among isolates as would be expected of the functional site of an enzyme which is essential for viral replication. Despite the sequence conservation of the previously identified C2H2-type zinc finger motif and leucine zipper among geographically diverse isolates (Duh et al., 2008; Honig et al., 2004; Kinsella et al., 2004; Ozdarendeli et al., 2010; Yadav et al., 2013), these motifs were not identified by the predictive software used in this analysis. To confirm that sequence differences were not the reason for this discrepancy, the published isolates were analysed and the zinc finger and leucine zipper were not predicted. This may be due to the dynamic nature of the hidden Markov model based databases such as Pfam (Finn et al., 2010) and the extensive variation in the primary protein structure between nucleotide polymerases and RdRp. The L segment translational product is poorly defined with regards to posttranslational modifications including proteolytic cleavage and maturation and experimental studies are required to confirm such motifs. The genome organization of CCHFV M segment follows the sequence: 5’NCR-mucin like region-GP38-GN-NSM-GC-3’NCR. Cleavage of the M polyprotein is accomplished at four cleavage sites including one furin cleavage site (RSKR) and two subtilase SKI-1 cleavage sites (RRLL, RR/KLL) (Vincent et al., 2003; Sanchez et al., 2006). The final cleavage site (RR/KPL), which separates GC from NSM, has been shown to be poorly cleaved by SKI-1 although the protease involved in this cleavage event has not been identified (Vincent et al., 2003). The four cleavage sites are conserved among all CCHFV isolates including those from southern Africa. The structural glycoproteins, GN and GC, were the most conserved of the M segment proteins with up to 91.9% nucleotide (91.5% amino acid) and 95.2% nucleotide (93.8% amino acid) sequence identity between southern African isolates respectively. The role of 132 the non-structural or secretory glycoproteins (mucin-like variable domain, GP38, NSM, GP85 and GP160) in CCHFV infection is currently unclear, but a similar mucin-like domain glycoprotein of Ebola virus plays a role in viral pathogenesis by causing endothelial cell disruption resulting in increased vascular permeability (Yang et al., 2000). Sequence diversity was highest in the mucin-like variable region with divergence of up to 40.7% at the nucleotide level and 60.1% at the amino acid level. CCHFV utilizes various vertebrate and tick hosts in distinct geographical regions. This ability may relate to the variation within the mucin-like region (Ozdarendeli et al., 2010). This is supported by grouping of isolates based on phylogenetic analysis of the mucin-like region which correlates with the complete M segment (data not shown) showing geographic linkage of isolates within groups. The mechanism underlying the variablility of mucin-like regions of negative stranded RNA viruses including CCHFV has been explained chiefly by a relaxation of purifying selection in this region. This implies that the amino acid composition of the region is not important for its function as long as the O-glycosylation is maintained (Wertheim and Worobey, 2009). Although the high cysteine content of the M protein which is conserved in all available CCHFV isolates may point to extensive disulphide bonds and a complex secondary structure, the presence of two CCHC-type zinc finger motifs in the GN cytoplasmic tail also contribute to this cysteine content. The GN tail has been shown to bind RNA and likely plays a role in RNA packaging and viral assembly by associating with the viral ribonucleoprotein complexes, as do the glycoprotein zinc fingers of hantaviruses, also belonging to the Bunyaviridae family (Estrada and De Guzman, 2011). Of the 11 conserved N-linked glycosylation sites predicted for the M segments of southern African CCHFV isolates, three have thus far been confirmed experimentally namely one site 133 located on GN (amino acid residue position 557) and two sites located on GC (amino acid residue positions 1054 and 1563). The N-linked glycosylation site on GN was shown to be important for glycoprotein localization and for transport of both GN and GC proteins from the endoplasmic reticulum to the Golgi apparatus (Erickson et al., 2007). The conserved nucleotide complementarity of the 5’ and 3’ NCR of the L, M and S segments of CCHFV were modelled using Mfold and showed cyclization to form panhandle structures. This can be compared to the complementary genomic ends of another member of the Bunyaviridae family, Bunyamwera virus. In the case of Bunyamwera virus, the cooperation of the complementary 5’ and 3’ NCR were required for RNA synthesis to generate both mRNA and cRNA (Barr and Wertz, 2004). A similar role in CCHFV replication seems likely. The first nine nucleotides of the L, M and S segments are identical in CCHF, as in other nairoviruses such as Dugbe virus and Hazara virus, and may function as an RdRp recognition site for the initiation of viral mRNA transcription and replication (Lasecka and Baron, 2013; Marriott et al., 1992). Genetic characterization of CCHFV isolates can help to identify protein functions and to suggest pathogenic mechanisms which require further examination. The development of diagnostic assays, vaccine design and identification of potential targets for antiviral interventions may also benefit, particularly when information from geographically distinct areas is available. It is clear that further experimental studies are required to investigate the significance and function of many of the CCHFV genome properties identified in this way. However, this examination of southern African CCHFV isolates provides corroboration of conserved genome domains and sequence identity which may direct further studies. 134 References Altamura LA, Bertolotti-Ciarlet A, Teigler J, Paragas J, Schmaljohn CS, Doms RW. Identification of a novel C-terminal cleavage of Crimean-Congo hemorrhagic fever virus PreGN that leads to generation of an NSM protein. J Virol 2007;81:6632-6642. Altschul SF, Madden TL, Schäffer AA, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI- BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389-3402. Barr JN, Wertz GW. Bunyamwera Bunyavirus RNA synthesis requires cooperation of =- and 5-terminal sequences. J Virol 2004;78:1129-1138. Bente DA, Forrester NL, Watts DM, McAuley AJ, Whitehouse CA, Bray M. Crimean-Congo hemorrhagic fever: History, epidemiology, pathogenesis, clinical syndrome and genetic diversity. Antiviral Research 2013;100:159-189. Bergeron E, Albariño CG, Khristova ML, Nichol ST. Crimean-Congo hemorrhagic fever virus- encoded ovarian tumor protease activity is dispensable for virus RNA polymerase function. J Virol 2010;84:216-226. Capodagli GC, McKercher MA, Baker EA, Masters EM, Brunzelle JS, Pegan SD. Structural analysis of a viral ovarian tumor domain protease from the Crimean-Congo hemorrhagic fever virus in complex with covalently bonded ubiquitin. J Virol 2011;85:3621-3630. Carter SD, Surtees R, Walter CT, Ariza A, Bergeron E, Nichol ST, Hiscox JA, Edwards TA, Barr JN. Structure, function and evoluation of the Crimean-Congo hemorrhagic fever virus nucleocapsid protein. J Virol 2012;86:10914-10923. 135 Clerx JP, Casals J, Bishop DH. Structural characteristics of nairoviruses (genus Nairovirus, Bunyaviridae). J Gen Virol 1981;55:165-178. Duh D, Nichol ST, Khristova ML, Saksida A, Hafner-Bratkovic I, Petrovec M, Dedushaj I, Ahmeti S, Avsic-Zupanc T. The complete genome sequence of a Crimean-Congo hemorrhagic fever virus isolated from an endemic region in Kosovo. Virol J 2008;5:7. Erickson BR, Deyde V, Sanchez AJ, Vincent MJ, Nichol ST. N-linked glycosylation of Gn (but not Gc) is important for Crimean Congo hemorrhagic fever virus glycoprotein localization and transport. Virology 2007;361:348-355. Estrada DF, De Guzman RN. Structural characterization of the Crimean-Congo hemorrhagic fever virus Gn tail provides insight into virus assembly. J Biol Chem 2011;286:21678-21686. Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer ELL, Eddy SR, Bateman A. The Pfam protein families database. Nucleic Acids Res 2010;38:D211-D222. Frias-Staheli N, Giannakopoulos NV, Kikkert M, Taylor SL, Bridgen A, Paragas J, Richt JA, Rowland RR, Schmaljohn CS, Lenschow DJ, Snijder EJ, Garcia-Sastre A, Virgin HW 4th. Ovarian tumor domain-containing viral proteases evade ubiquitin- and ISG 15-dependent innate immune responses. Cell Host Microbe 2007;2:404-416. Guo Y, Wang W, Ji W, Deng M, Sun Y, Zhou H, Yang C, Deng F, Wang H, Hu Z, Lou Z, Rao Z. Crimean-Congo hemorrhagic fever virus nucleoprotein reveals endonuclease activity in bunyaviruses. Proc Natl Acad Sci U S A 2012;109:5046-5051. 136 Honig JE, Osborne JC, Nichol ST. Crimean-Congo hemorrhagic fever virus genome L RNA segment and encoded protein. Virology 2004;321:29-35. Khromykh AA, Meka H, Guyatt KJ, Westaway EG. Essential role of cyclization sequences in flavivirus RNA replication. J Virol 2001;75:6719-6728. Kinsella E, Martin SG, Grolla A, Czub M, Feldmann H, Flick R. Sequence determination of the Crimean-Congo hemorrhagic fever virus L segment. Virology 2004;321:23-28. Krogh A, Larsson B, von Heijne G, Sonnhammer ELL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001;305:567-580. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez A, Thompson JD, Gibson TJ, Higgins DG. Clustal W and Clustal X version 2.0. Bioinformatics 2007;23:2947-2948. Lasecka L, Baron MD. The molecular biology of nairoviruses, an emerging group of tick- borne arboviruses. Arch Virol Dec 2013; Dec 11 [Epub ahead of print]. Marriott AC, el-Ghorr AA, Nuttall PA. Dugbe Nairovirus M RNA: nucleotide sequence and coding strategy. Virology 1992;190:606-150. Nichol ST, Beaty BJ, Elliott RM, Goldbach R, Plyusnin A, Schmaljohn CS, Tesh RB. Index of Viruses – Bunyaviridae. In: Büchen-Osmond C, editor. ICTVdB – The Universal Virus Database, 2006, version 4. New York: Columbia University. http://www.ncbi.nlm.nih.gov/ ICTVdb/Ictv/fs_index.htm 137 Ozdarendeli A, Canakoğlu N, Berber E, Aydin K, Tonbak S, Ertek M, Buzgan T, Bolat Y, Aktaș M, Kalkan A. The complete genome analysis of Crimean-Congo hemorrhagic fever virus isolated in Turkey. Virus Res 2010;147:288-293. Petersen TN, Brunak S, van Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 2011;8:785-786. Sanchez AJ, Vincent MJ, Erickson BR, Nichol ST. Crimean-Congo hemorrhagic fever virus glycoprotein precursor is cleaved by furin-like and SKI-1 proteases to generate a novel 38- kilodalton glycoprotein. J Virol 2006;80:514-525. Steentoft C, Vakhrushev SY, Joshi HJ, Kong Y, Vester-Christensen MB, Schjoldager KT, Lavrsen K, Dabelsteen S, Pedersen NB, Marcos-Silva L, Gupta R, Bennett EP, Mandel U, Brunak S, Wandall HH, Levery SB, Clausen H. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. EMBO J 2013;32:1478-1488. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731-2739. Vincent MJ, Sanchez AJ, Erickson BR, Basak A, Chretien M, Seidah NG, Nichol ST. Crimean- Congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase SKI-1. J Virol 2003;77:8640-8649. Wertheim JO, Worobey M. Relaxed selection and the evolution of RNA virus mucin-like pathogenicity factors. J Virol 2009;83:4690-4694. 138 Yadav PD, Cherian SS, Zawar D, Kokate P, Gunjikar R, Jadhav S, Mishra AC, Mourya DT. Genetic characterization and molecular clock analyses of the Crimean-Congo hemorrhagic fever virus from human and ticks in India, 2010-2011. Infect Genet Evol 2013;14:223-231. Yang ZY, Duckers HJ, Sullivan NJ, Sanchez A, Nabel EG, Nabel GJ. Identification of the Ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury. Nat Med 2000;6:886-889. Zdobnov EM, Apweiler R. InterProScan – an integration platform for the signature- recognition methods in InterPro. Bioinformatics 2001;17:847-848. Zhang M, Gaschen B, Blay W, Foley B, Haigwood N, Kuiken C, Korber B. Tracking global patterns of N-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin. Glycobiology 2004;14:1229-1246. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003;31:3406-3415. 139 CHAPTER 7 Conclusions and future perspectives CCHFV is endemic in South Africa and is emerging as an important pathogen in Europe and Asia. In the absence of specific treatment or a vaccine, determination of immune correlates and a better understanding of pathogenic mechanisms are essential to guide investigation of therapeutic or preventative interventions. This study was aimed at investigating immune responses in survivors of CCHF in South Africa, with particular emphasis on investigating the presence of detectable memory T lymphocyte responses and epitopic regions within the nucleoprotein/or and glycoproteins. The human immune response to viral infections is a complex process comprised of innate and adaptive responses. The innate immune response includes factors such as interferon production and natural killer cells (Campbell and Hasegawa, 2013; Yan and Chen, 2012). In the case of CCHFV, the virus has been shown to delay interferon production, in part mediated by the OTU-like cysteine protease domain of the L segment, thereby allowing viral replication in the early stages of infection (Akinci et al., 2013; Weber and Mirazimi, 2008). The adaptive immune response is comprised of humoral and cellular components. The humoral response consists primarily of virus-specific antibody production by B cell lymphocytes. The antibodies produced can both protect from infection and assist in clearance of established infection. This is accomplished not only through direct virus neutralization but also through antibody dependent cell-mediated cytotoxicity and complement activation (Dӧrner and Radbruch, 2007). Infection with CCHFV is known to induce antibody production and this attribute is widely used to aid diagnosis by means of 140 serological assays. The majority of antibody responses that have been described previously are based on ELISA and immunofluorescent techniques rather than virus neutralization assays. Using monoclonal antibodies raised in mice, cross-reactive neutralizing antibodies have been demonstrated to a conserved epitope on the GC glycoprotein (Ahmed et al., 2005). However, antibody production does not directly correlate with viral clearance and the immune response to CCHFV is likely to also involve both innate and cellular components (Wӧlfel et al., 2007). Both humoral and cell mediated immune responses result in the production of long-lived memory cells which can recognize and respond rapidly following re- exposure to an antigen which has previously been encountered. Although related bunyaviruses such as Hantaan virus and Puumala virus have been shown to induce long-lived memory T cell responses (Van Epps et al., 2002; Wang et al., 2011), it was unknown whether acute CCHFV infection would induce memory T cells present at high enough frequencies in the peripheral blood to be detectable using ELISPOT assays in the absence of continued antigenic exposure or reinfection. No data is currently available regarding T cell responses following CCHFV infection. Using a synthetic overlapping peptide library to screen for interferon gamma production by PBMC from survivors of CCHFV infection, ten potential epitopic regions were identified. The majority of these were located on the nucleoprotein with only two regions identified on the glycoprotein GC in a single patient. T cell responses to the nucleoprotein of Rift Valley fever virus are detected in patients with a history of RVF infection and can be induced by vaccination. These vaccines can induce protective immunity in the absence of neutralizing antibody, likely through cell mediated immunity (Xu et al., 2103). This study provides the first report of long-lived memory CD8+ T cell responses in survivors of CCHF up to 13 years after infection. For 141 vaccine development, it is necessary to differentiate between the mechanisms of protection and mechanisms of recovery. Cellular responses are frequently more significant for recovery from viral infection or viral clearance (Plotkin, 2010). An effective vaccine will likely require induction of both cellular and humoral responses. If the nucleoprotein induces memory T cells then one can theorize that a vaccine without a nucleoprotein component may not induce memory T cell responses. The significance of this will depend on the role that it plays in protection against disease or in recovery. The frequency of responses detected in some CCHFV survivors is similar to those detected in Hantaan and Puumala viruses and correlate with responses induced by other acute infections such as influenza viruses (Van Epps et al., 2002; Wang et al., 2011). These data indicates that induction of effective long term cellular immune responses should be possible through vaccination and gives an indication of epitopic regions that should be included in the vaccine and applied to assays testing vaccine immunogenicity. Although the magnitude and frequency of spot forming cells in the ELISPOT assays were similar to those detected previously for Hantaan and Puumala viruses, the levels were far lower than those detected in Rift Valley fever survivors (Xu et al., 2103). It may be significant to determine whether there is a threshold level of memory response required to confer full protection or full clearance of the virus on re-exposure. Further studies are planned to confirm the optimal epitopes using peptide nonamers in an ELISPOT format and to determine the HLA restriction of these optimal epitopes. Depletion studies confirmed that CD8+ cells were likely the dominant phenotype secreting IFN-γ in the memory response. More detailed information regarding the subsets of memory T cells involved in the long-lived responses and the role of CD8+ and CD4+ cells in secretion of additional cytokines will be determined using flow cytometry. The reasons for severity of illness are not clear however cytokine activation may 142 play a role in pathogenicity during the acute phase of infection. A comparison of cytokine levels in serum samples has shown high levels of TNF-α associated with severe disease and IL-6 levels were high in both severe and mild disease (Papa et al., 2006). A comparison of cytokine secretion from PBMC collected from fatal and non-fatal cases during acute illness would be useful for further defining the role of T cell responses. In addition, functional assays including the CD107a degranulation assay will be used to confirm that the cytokine secreting cells are functional and able to induce cytolysis. As correlates of protection may be dependent on antigen presentation on MHC and therefore HLA type, it will be important to determine HLA restriction of these epitopes before making significant decisions regarding vaccine development. Rapid, sensitive and specific assays are required for increasing diagnostic and surveillance capacity. The use of synthetic peptides in ELISA provides an alternative approach for safe production of diagnostic reagents. Identification of epitopic regions on viral proteins would greatly aid the development of serological assays. If these epitopes induce protective antibody responses, then they could also find relevance in the production of subunit vaccines. Linear epitopes are short peptide fragments contiguous in the primary amino acid sequence which comprise approximately 10% of B cell epitopes. Although less frequent than discontinuous epitopes, they are potentially involved in inducing neutralizing antibodies and/or antibodies allowing detection and diagnosis. The peptide library was therefore used to screen for linear B cell epitopes in survivors of CCHFV infection. Two potential epitopic regions were identified on the GC glycoprotein with reactivity in 13 – 14 of 15 patients tested. These two epitopic regions are located within a conserved region of the genome and within regions of hydrophilicity suggesting that they are exposed and therefore 143 increasing the likelihood that they have antigenic significance. One of the reactive peptides identified, peptide G1613-1631, overlaps peptide G1622-1640 which also showed reactivity although to a lesser extent and in only 7 of 15 patients tested. This lack of reactivity against G1622-1640 in some sera could be as a result of a lower antibody titre or due to the position of the epitope which may partially overlap peptide G1613-1631. In contrast the optimal epitope of the other reactive peptide, peptide G1451-1469, is likely situated within the centre of the 19mer as no reactivity was observed against overlapping peptides. This region is also highly conserved within geographically distinct isolates. Further investigation will be required to determine whether these epitopic regions also correlate with immune protection and to identify non-contiguous B cell epitopes which are likely to play an important role in antibody induction during natural infection with CCHFV. Expanding CCHFV complete genome sequence data for isolates from geographically distinct regions will contribute to studies investigating immune correlates of protection and development of diagnostic reagents for rapid detection and identification. Complete sequence data will also have application in identifying conserved epitopic regions that play a role in inducing memory T cell responses. Development of serological and molecular diagnostic assays that can be applied to isolates distributed worldwide will depend on knowledge of cross reactivity and conservation of targeted regions of the genome. To date there were 31 complete genome sequences available on GenBank hence it was deemed necessary to determine sequence data for additional isolates from our Southern African cohort. To investigate CCHFV sequence diversity among southern African isolates and isolates from other endemic countries, ten complete CCHFV genomes were sequenced using next generation sequencing techniques. With new foci of CCHFV infection emerging over 144 recent years, it is important to ascertain whether genomic variation is likely to have an effect on vaccine efficacy and ensure that vaccine candidates and diagnostic assays will be applicable in distinct geographic areas. This paper reports the first application of next generation sequencing technology to CCHFV isolates. These techniques are rapid and cost effective alternatives to standard Sanger sequencing which can be effectively applied to the approximately 20kb CCHFV genome. The results confirmed that, at a genomic level, there is extensive variability among geographically distinct CCHFV isolates. While point mutations are common and result in increased genetic diversity over time, whole segment reassortment is also common among CCHFV isolates and contributes significantly to genetic variability. The role of reassortment in viral pathogenicity in humans is as yet unclear, but animal studies using one of the recently described mouse models may shed some light on this. Despite the extensive variability noted, at a protein level, the motifs involved in protein function are well conserved. Prediction software confirmed the presence of conserved OTU-like cysteine protease and RNA dependent RNA polymerase (RdRp) domains in the L segment of diverse southern African CCHFV isolates. The RdRp is essential for viral replication while the OTU-like protease likely plays a role in immune evasion and therefore affects viral pathogenicity. Analysis of the proteins encoded by the M gene showed conservation of the basic protein coding strategy, with two structural and three non- structural glycoproteins. However, amino acid variation was notable across all predicted proteins but particularly in the variable mucin-like domain which is thought to play a role in viral pathogenicity. This study provides targets for further investigation into viral pathogenicity which may include in vivo studies in animal models and mutagenicity assays. The identification of conserved protein motifs such as the OTU-like cysteine protease has 145 application in the development of novel anti-viral therapies targeting these proteins such as cysteine-modifying agents (Casini et al., 2002). References Ahmed AA, McFalls JM, Hoffmann C, Filone CM, Stewart SM, Paragas J, Khodjaev S, Shermukhamedova D, Schmaljohn CS, Doms RW, Bertolotti-Ciarlet A. Presence of broadly reactive and group-specific neutralizing epitopes on newly described isolates of Crimean- Congo hemorrhagic fever virus. J Gen Virol 2005;86:3327-3336. Akinci E, Bodur H, Leblebicioglu H. Pathogenesis of Crimean-Congo hemorrhagic fever. Vector Borne Zoonotic Dis 2013;13:429-437. Campbell KS, Hasegawa J. Natural killer cell biology: an update and future directions. J Allergy Clin Immunol 2013;132:536-544. Casini A, Scozzafava A, Supuran CT. Cysteine-modifying agents: a possibly approach for effective anticancer and antiviral drugs. Environ Health Perspect 2002;110:801-806. Dӧrner T, Radbruch A. Antibodies and B cell memory in viral immunity. Immunity 2007;27:384-392. Papa A, Bino S, Velo E, Harxhi A, Kota M, Antoniadis A. Cytokine levels in Crimean-Congo hemorrhagic fever. J Clin Virol 2006;36:272-276. Van Epps HL, Terajima M, Mustonen J, Arstila TP, Corey EA, Vaheri A, Ennis FA. Long-lived memory T lymphocyte responses after Hantavirus infection. J Exp Med 2002;196:579-588. 146 Wang M, Zhu Y, Wang J, Lv T, Jin B. Identification of three novel CTL epitopes within nucleocapsid protein of Hantaan virus. Viral Immunol 2011;24:449-454. Weber F, Mirazimi A. Interferon and cytokine responses to Crimean Congo hemorrhagic fever virus: an emerging and neglected viral zoonosis. Cytokine Growth Factor Rev 2008;19:395-404. Wӧlfel R, Paweska JT, Petersen N, Grobbelaar AA, Leman PA, Hewson R, Georges-Courbet MC, Papa A, Günther S, Drosten C. Virus detection and monitoring of viral load in Crimean- Congo hemorrhagic fever virus patients. Emerg Infect Dis 2007;13:1097-1100. Yan N, Chen ZJ. Intrinsic antiviral immunity. Nat Immunol 2012;13:214-222. Xu W, Watts DM, Costanzo MC, Tang X, Venegas LA, Jiao F, Sette A, Sidney J, Sewell AK, Wooldridge L, Makino S, Morrill JC, Peters CJ, Kan-Mitchell J. The nucleocapsid protein of Rift Valley fever virus is a potent human CD8+ T cell antigen and elicits memory responses. PLoS One 2013;8:e59210. 147 APPENDIX A 148 149 APPENDIX B 150 APPENDIX C CONSENT DOCUMENT 1 ETOVS no CONSENT TO PARTICIPATE IN RESEARCH You have been asked to participate in a research study. You have been informed about the study by your general practitioner or other healthcare worker. You may contact Prof F J Burt at 051 405 3173 at any time if you have questions about the research. You may contact the Secretariat of the Ethics Committee of the Faculty of Health Sciences, UFS at telephone number (051) 4052812 if you have questions about your rights as a research subject. Your participation in this research is voluntary, and you will not be penalized or lose benefits if you refuse to participate or decide to terminate participation. If you agree to participate, you will be given a signed copy of this document as well as the participant information sheet, which is a written summary of the research. The research study, including the above information has been verbally described to me. I understand what my involvement in the study means and I voluntarily agree to participate. I consent that 1. I will donate blood on 3 separate occasions as described in the information document 2. my donated blood maybe stored and analyzed as part of future research 3. research data obtained from the study may be published anonymously in a scientific journal Name of participant Telephone no Email address Approximate date of CCHF infection _____________________ __________________ Signature of Participant Date _____________________ __________________ Signature of Witness Date (Where applicable) ____________________ __________________ Signature of Translator Date (Where applicable) 151 INFORMATION DOCUMENT 1 Study title: Immune responses in survivors of Crimean-Congo haemorrhagic fever infection and evaluation of candidate vaccines. Good morning We, the Department of Virology, are doing research to identify how the human body responds to infections of Crimean-Congo haemorrhagic fever (CCHF). In this project we want to study which cells in your immune response contributed to your recovery from the infection Invitation to participate: We are inviting you to participate in this research study What is involved in the study – We are inviting patients with a confirmed history of infection with CCHF to participate in the study. We will request blood from you on 3 occasions. We require initially request 1x 10 ml blood tube and then 4 x 10 ml blood tubes on two separate occasions. Different cells in the body are activated when we have a viral infection and these cells all play a role in clearing the virus from our body. We will isolate blood cells from your sample and try to identify which cells were activated by the virus. We will use DNA from your blood to identify immune cells specific to you and we will use the blood to look at your immune response. Patients who survive infection with CCHF virus have antibodies against the virus. It is possible to isolate and immortalize (keep) the cells that are responsible for making these antibodies. We request permission to store these cells indefinitely and use them for related projects to study the CCHF virus. Risks There are no foreseeable risks of being involved in the study: Benefits There is very little information available regarding the mechanisms by which the body responds to infection against CCHF virus and we are hoping this study will lead to results that will contribute to development of a vaccine or specific treatment. Participation is voluntary, and refusal to participate will involve no penalty or loss of benefits to which the subject is otherwise entitled; the subject may discontinue participation at any time without penalty or loss of benefits to which the subject is otherwise entitled. Reimbursements We can reimburse you for any cost incurred at the pathology laboratory for collection of blood for this study. Confidentiality: Efforts will be made to keep personal information confidential. Absolute confidentiality cannot be guaranteed. Personal information may be disclosed if required by law. Organizations that may inspect and/or copy your research records for quality assurance and data analysis include groups such as the Ethics Committee for Medical Research. Contact details of researcher– Prof Felicity Burt, Department of Virology, University of the Free State, Bloemfontein. Tel 051 4053173, burtfj.md@ufs.ac.za Contact details of REC Secretariat and Chair – For reporting complaints or problems please contact 152 Ms H Strauss Research Division Faculty if Health Sciences University of the Free State E-mail address: gndkhs.md@mail.uovs.ac.za (051) 4052812 153 APPENDIX D CONSENT DOCUMENT 2 ETOVS no CONSENT TO PARTICIPATE IN RESEARCH You have been asked to participate in a research study. You have been informed about the study by your general practitioner or healthcare worker. You may contact Prof F J Burt at 051 405 3173 any time if you have questions about the research. You may contact the Secretariat of the Ethics Committee of the Faculty of Health Sciences, UFS at telephone number (051) 4052812 if you have questions about your rights as a research subject. Your participation in this research is voluntary, and you will not be penalized or lose benefits if you refuse to participate or decide to terminate participation. If you agree to participate, you will be given a signed copy of this document as well as the participant information sheet, which is a written summary of the research. The research study, including the above information has been verbally described to me. I understand what my involvement in the study means and I voluntarily agree to participate. I consent that: I will donate blood as described in the information document my donated blood may be stored and analyzed as part of future research research data obtained from the study may be published anonymously in a scientific journal Name of participant Telephone no Email address Approximate date of CCHF infection _____________________ __________________ Signature of Participant Date _____________________ __________________ Signature of Witness Date (Where applicable) ____________________ __________________ Signature of Translator Date (Where applicable) 154 INFORMATION DOCUMENT 2 Study title: Immune responses in survivors of Crimean-Congo haemorrhagic fever infection and evaluation of candidate vaccines. Good morning We, the Department of Virology, are doing research to identify how the human body responds to infections of Crimean-Congo haemorrhagic fever (CCHF). In this project we want to study which cells in your immune response contributed to your recovery from the infection Invitation to participate: We are inviting you to participate in this research study What is involved in the study – We are inviting patients with a confirmed history of infection with CCHF to participate in the study. We will request blood 2x 10 ml blood tubes. Patients who survive infection with CCHF virus have antibodies against the virus. We want to use these antibodies to study how the body reacts to different proteins in the virus. We request permission to store your blood indefinitely for related projects to study the CCHF virus. Risks There are no foreseeable risks of being involved in the study: Benefits There is very little information available regarding the mechanisms by which the body responds to infection against CCHF virus and we are hoping this study will lead to results that will contribute to development of a vaccine or specific treatment. Participation is voluntary, and refusal to participate will involve no penalty or loss of benefits to which the subject is otherwise entitled; the subject may discontinue participation at any time without penalty or loss of benefits to which the subject is otherwise entitled. Reimbursements We can reimburse you for any cost incurred at the pathology laboratory for collection of blood for this study. Confidentiality: Efforts will be made to keep personal information confidential. Absolute confidentiality cannot be guaranteed. Personal information may be disclosed if required by law. Organizations that may inspect and/or copy your research records for quality assurance and data analysis include groups such as the Ethics Committee for Medical Research. Contact details of researcher– Prof Felicity Burt, Department of Virology, University of the Free State, Bloemfontein. Tel 051 4053173, burtfj.md@ufs.ac.za Contact details of REC Secretariat and Chair – For reporting complaints or problems please contact Ms H Strauss Research Division Faculty if Health Sciences University of the Free State E-mail address: gndkhs.md@mail.uovs.ac.za 155 APPENDIX E CONSENT DOCUMENT 3 ETOVS no CONSENT TO PARTICIPATE IN RESEARCH You have been asked to participate in a research study. You have been informed about the study by your general practitioner or healthcare worker. You may contact Prof F J Burt at 051 405 3173 any time if you have questions about the research. You may contact the Secretariat of the Ethics Committee of the Faculty of Health Sciences, UFS at telephone number (051) 4052812 if you have questions about your rights as a research subject. Your participation in this research is voluntary, and you will not be penalized or lose benefits if you refuse to participate or decide to terminate participation. If you agree to participate, you will be given a signed copy of this document as well as the participant information sheet, which is a written summary of the research. The research study, including the above information has been verbally described to me. I understand what my involvement in the study means and I voluntarily agree to participate. I consent that: I will donate blood as described in the information document my donated blood may be stored and analyzed as part of future research research data obtained from the study may be published anonymously in a scientific journal Name of participant Telephone no Email address _____________________ __________________ Signature of Participant Date _____________________ __________________ Signature of Witness Date (Where applicable) ____________________ __________________ Signature of Translator Date (Where applicable) 156 INFORMATION DOCUMENT 3 Study title: Immune responses in survivors of Crimean-Congo haemorrhagic fever infection and evaluation of candidate vaccines. Good morning We, the Department of Virology, are doing research to develop new laboratory tests for detecting Crimean-Congo haemorrhagic fever (CCHF) infections. In this project we want to use blood from volunteers that have not previously had CCHF virus. The blood will be used as negative controls for development of laboratory tests. Invitation to participate: We are inviting you to participate in this research study What is involved in the study – We are inviting patients with NO history of infection with CCHF virus to participate in the study. We will request blood 2x 10 ml blood tubes. We request permission to store your blood indefinitely for related projects to study the CCHF virus. Risks There are no foreseeable risks of being involved in the study: Benefits We are developing rapid and reliable tests for detecting CCHF virus infections and request blood from healthy donors with no history of infection with the virus that will be used to determine the specificity of the assays. Participation is voluntary, and refusal to participate will involve no penalty or loss of benefits to which the subject is otherwise entitled; the subject may discontinue participation at any time without penalty or loss of benefits to which the subject is otherwise entitled. Reimbursements We can reimburse you for any cost incurred at the pathology laboratory for collection of blood for this study. Confidentiality: Efforts will be made to keep personal information confidential. Absolute confidentiality cannot be guaranteed. Personal information may be disclosed if required by law. Organizations that may inspect and/or copy your research records for quality assurance and data analysis include groups such as the Ethics Committee for Medical Research. Contact details of researcher– Prof Felicity Burt, Department of Virology, University of the Free State, Bloemfontein. Tel 051 4053162, burtfj@ufs.ac.za s Contact details of REC Secretariat and Chair – For reporting complaints or problems please contact Ms H Strauss Research Division Faculty if Health Sciences University of the Free State E-mail address: strausshs@ufs.ac.za (051) 4052812 157 APPENDIX F 158