AIX-MARSEILLE UNIVERSITE FACULTE DE MEDECINE DE MARSEILLE ECOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE THESE DE DOCTORAT Présentée par NAZLI AYHAN Née le 1er Juillet 1988 née en Turquie En vue de l’obtention du grade de DOCTEUR d’AIX-MARSEILLE UNIVERSITE Mention : Pathologie Humaine Spécialité : Maladies Infectieuses Emerging Sandfly-borne Phleboviruses in Balkan Countries: Virus isolation, Characterization, Evolution and Seroepidemiology Soutenue publiquement le 26 Septembre 2017 Composition du Jury : Monsieur le Docteur Christophe PAUPY Rapporteur Monsieur le Professeur Christophe PEYREFITTE Rapporteur Madame le Docteur Dorothée MISSE Examinateur Monsieur le Professeur Remi N CHARREL Directeur de Thése UMR "Emergence des Pathologies Virales" (EPV : Aix-Marseille Univ – IRD 190 – Inserm 1207 – EHESP – IHU Méditerranée Infection), Marseille, France ACKNOWLEDGEMENTS I would like to express my very great appreciation to Dr. Xavier de Lamballerie for giving me the opportunity to do my Ph.D. in his laboratory. His knowledge and enthusiasm on virology always encourage me to be a good scientist. I would like to express my deep gratitude to Dr. Remi N. Charrel, my research supervisor, for accepting me as a Ph.D. student, his guidance, enthusiastic encouragement and useful critiques of this research. He is the best supervisor ever. Big thanks to Dr. Christophe Paupy, Dr. Christophe Peyrefitte and Dr. Dorothée Misse for accepting to be in the thesis committee and their valuable comments. I would like to offer my special thanks to Dr. Bulent Alten for his precious support and contributions to this thesis. I thank Dr. Bulent Alten, Dr. Vladimir Ivovic and Dr. Petr Volf for organizing the sand fly field collection campaign in Balkan countries, it was an incredible experience for me. I also would like to thank all the colleagues and local contacts who worked on sand fly collection. I would like to thank Cigdem Alkan for guiding me in the laboratory and Geraldine Piorkowski for technical support on sequencing. Special thanks to everyone who works in the UMR 190 laboratory for their collaboration and friendship. Without your help, advises and jokes (however some of them are not funny) life would be much harder and boring. I would also like to thank all the anonymous reviewers for their constructive critiques on the articles in the scope of the thesis. The biggest supporter of my thesis and my life, Emrah, without you I couldn’t be in here. We have a long way to walk together. Thank you for everything. I love you. My precious family; my mother Selma and my father Yuksel, I love you so much thank you for all the support. Finally, I wish to thank the Balkan people for their hospitability and kindness. i "Doing what little one can to increase the general stock of knowledge is as respectable an object of life, as one can in any likelihood pursue." Charles Darwin To Balkan People… ii TABLE OF CONTENTS Acknowledgements…………………………………………………………………………………………………………………..i Dedication……………………………………………………………………………………………………………………………….ii PREAMBLE OF DISSERTATION…………………………………………………………………………………………….…1-6 CHAPTER 1: INTRODUCTION……………………………………………………………………………………………………7 Review 1: Of Phlebotomines (Sandflies) and Viruses: A Comprehensive Perspective on a Complex Situation…………………………………………………………………………….………………….8-16 Review 2: Novel and Emergent Sandfly-borne Phleboviruses in Asia Minor: A Systematic Review…………………………………………………………………………………….……….….17-29 Review 3: A systematic review: Novel and Emergent Sandfly-Borne Phleboviruses in Balkan Region…………………………………………………………………………...….……………….……..30-47 CHAPTER 2: GUIDELINES…………………………………………………………………………………………….………….48 Guidelines: European Network for Neglected Vectors and Vector-Borne Infections COST Action Guidelines: Guideline 1: Practical Guidelines for Studies on Sandfly-Borne Phleboviruses: Part I: Important Points to Consider Ante Field Work…………………………………………………49-57 Guideline 2: Practical Guidelines for Studies on Sandfly-Borne Phleboviruses: Part II: Important Points to Consider for Fieldwork and Subsequent Virological Screening………………………………………………………………………………………………………...58-68 CHAPTER 3: RESEARCH ARTICLES……………………………………………………………………………………….….69 Article 1: Detection of Leishaia ifatu and a Novel Phlebovirus (Balkan Virus) from Sand Flies in Albania…………………………………………………………….…70-75 Article 2: Direct Evidence for an Expanded Circulation Area of the Recently Identified Balkan virus (Sandfly fever Naples virus species) in Several Countries of the Balkan Archipelago………………………………………………………………………………………………………...…76-82 Article 3: High Rates of Neutralizing Antibodies to Toscana and Sandfly Fever Sicilian Viruses in Livestock, Kosovo……………………………………………………………….………………….83-87 Article 4: Co-circulation of Two Lineages of Toscana Virus in Croatia………………...….88-94 Article 5: Isolation and Genetic Characterization of Two Novel Viruses Belong to the Salehabad Virus Complex from Croatia and Macedonia……………………………..…...….95-114 iii CONCLUSION AND DISCUSSION …………………………………………………………………………………...115-122 REFERENCES………………………………………………..……………………………………………………….……….123-127 APPENDIXES………………………………………..……………………………………………………………….……….……..128 APPENDIX 1: SUPPLEMENTARY TABLE 1…………………………………………………………………..129 APPENDIX 1: SUPPLEMENTARY TABLE 2…………………………………………………………………..130 Abstract……………………………………………………………………………………………………………………………….131 Résumé…………………………………………………………………………………………………………………………….….132 iv PREAMBLE OF DISSERTATION Vector borne agents cause many important and emerging diseases on humans, livestock and wildlife animals. However, different definitions of a vector are currently being used in different fields. For World Health Organization (WHO), vectors are living organisms that can transmit infectious diseases between humans or from animals to humans (WHO, 2004). Most of the defined vector species are haematophagous arthropods such as ticks, mosquitos, sandflies and biting midges. Vectors can spread a variety of agents like viruses, bacteria and parasites within and among vertebrates (Lemon, 2008). For instance, Anopheles mosquitoes that transmit Plasmodium parasites, Aedes mosquitoes transmitting a variety of viruses like Dengue, Yellow fever, Chikungunya, West Nile and Zika virus, ticks transmitting Crimean- Congo haemorrhagic fever and tick-borne encephalitis virus, sandflies transmitting Leishmania parasites and Phleboviruses. More than half of the world’s population under risk of the vector-borne diseases and all the one sixth of the diseases caused by vector transmitted agents (aWHO, 2014). Within the vector-borne human agents, arthropod-borne viruses (arboviruses) consist of the largest class; over 500 arboviruses have been characterized, 20 percent of the recorded arboviruses described as human pathogens (Gray and Banerjee, 1999; Gubler, 1998, 2001). Arboviruses cause human and animal stock emerging infectious diseases by blood feeding from an infected human or animal host and transmit into a new host (Weaver, 2017). The arthropod-borne diseases affect both urban, peri-urban and rural population but mostly the communities with poor living conditions. Economic, social and ecological conditions have a huge impact on arthropod-borne diseases (Teutsch, 2000; Lemon, 2008). The factors that described as associated with arbovirus emergence or invasion are (i) competent vector and vertebrate host population repeatedly contact within an appropriate environment, (ii) viral genetic changes, (iii) vertebrate or vector host species composition changes and (iv) environmental or niche changes (Weaver, 2010). The genus Phlebovirus belongs to the Phenuiviridae family within the Bunyavirales order (Adams, 2017). Phleboviruses are enveloped viruses with a negative sense single–stranded tri- segmented RNA (Elliott, 1991). They contain three genomics segments: L (Large) segment encodes the viral RNA polymerase (RdRp), M (medium) segment encodes envelope glycoproteins (Gn and Gc) and non-structural protein m (NSm) and S (small) segment 1 encodes nucleocapsid protein (N) and non-structural protein s (NSs) (Figure1-2) (Elliott, 1990; International Committee on Taxonomy of Viruses, 2012). Figure1. Schematic representation of Phlebovirus Figure2. Coding strategies of genome segments of Phleboviruses. Genomic RNAs are represented by thin lines (the number of nucleotides is given above the line) and mRNAs are shown as arrows Phleboviruses are globally distributed agents which use ticks, mosquitos and sand flies as vector. The majority use sand flies as vectors to transmitted vertebrate hosts with blood- feeding (Tesh, 1988). Sand flies are small (1.5-3mm), delicate, hairy insects; adult females need at least one blood meal to allow egg development (Figure3) (Maroli, 2013). 2 Figure3. Blood feeding female sandfly. Sandfly distribution covers a vast of geographical area from Europe to Asia, Africa, Australia, Central and South America (Killick-Kendrick, 1999). Two genera (Phlebotomus and Sergentomyia) of Phlebotominae recorded in the Old World warmer temperature zones and the other genera Lutzomyia exist in New World (Tesh, 1988). For Mediterranean region sandflies have been assumed as natural faunal elements (Aspöck, 2008). Old World sandfly-borne phleboviruses can be classified using their antigenic properties into 3 serological complexes, namely Sandfly fever Sicilian complex (grouping Sand fly fever Sicilian virus and Corfou Virus), Sandfly fever Naples complex and Salehabad complex. Acronyms designate viruses, not species which are italicized. Human infections with phleboviruses transmitted