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The Investigation of Highly Pathogenic Viruses in Kazakhstan

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The Investigation of Highly Pathogenic Viruses in Kazakhstan Out of the Division of infectious Diseases and Tropical Medicine University Hospital, Ludwig- Maximilians-University (LMU) Munich The investigation of highly pathogenic viruses in Kazakhstan Doctoral Thesis for the awarding of a Doctor of Philosophy (Ph.D.) at the Medical Faculty of Ludwig-Maximilians-Universität, Munich submitted by Karlygash Abdiyeva born in Almaty, Kazakhstan submitted on April 25, 2019 i Supervisors LMU: Title, first name, last name Habilitated Supervisor Prof. Michael Hoelscher Direct Supervisor Dr. Sandra Essbauer 4th LMU Supervisor Dr. Norbert Heinrich Supervisor External: Local Supervisor Dr. Gerhard Dobler Reviewing Experts: 1st Reviewer Prof. Michael Hoelscher 2nd Reviewer Dr. Sandra Essbauer Dean: Prof. Dr. med. dent. Reinchard Hickel Date of Oral Defense: November 12, 2019 ii Abstract Background Kazakhstan consists of favorable conditions for different vector transmitted infectious diseases such as Tick-borne encephalitis (TBE) and Crimean Congo hemorrhagic fever (CCHF). Up to 50 cases of TBE and 10 cases of CCHF are registered annually. Due to the lack of diagnostics approaches in remote areas many cases remain undiagnosed and the correct case numbers could be higher. With this study project I claimed to understand the infection rate of TBEV and CCHFV in ticks, to identify the circulating genotypes and find out the role of these infections among patients who suffer from fever of unknown origin (FUO). Methods In six districts of the southern part of Kazakhstan 2341 ticks were collected. RNA was extracted from the homogenates and all samples were screened for the presence of TBEV and CCHFV by real-time reverse transcription (RT-) PCR. Positive samples were amplified in conventional RT-PCR using pathogen-specific primers. Products of conventional PCR were sequenced using Sanger sequencing. In two regions the serological and molecular investigation of human sera from two pilot regions was conducted. Results TBEV RNA was detected in 48 out of 493 investigated tick pools. No positive tick pools with CCHFV RNA were detected. The picture of serological investigation of human sera among patients with fever of unknown origin shows that the antibodies to TBEV and CCHFV were detected in serum samples of 18 (2.24%) and 102 (12.7%) out 802 tested patients respectively. Molecular investigation revealed that CCHFV subtypes Asia1 and reassortant Asia1 and Asia2 and Siberian subtype of TBEV circulate. Conclusion For the first time comprehensive data on the prevalence of TBEV and CCHF in vectors and among patients with FUO based on molecular methods of investigation, the circulating of Siberian genotype of TBEV and reassortant of Asia 1 and Asia 2 of CCHFV in Kazakhstan were described. The new data will help to improve the surveillance system of these infections in Kazakhstan. iii Key words Tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, Siberian Subtype, tick, fever of unknown origin. iv List of abbreviation PCR Polymerase chain reaction CCHFV Crimean-Congo hemorrhagic Fever virus TBEV Tick-borne encephalitis virus ELISA Enzyme-linked immunosorbent assay IFA Immunofluorescence assay FUO Fever of unknown origin WHO World Health Organization RNA Ribonucleic acid DNA Deoxyribonucleic acid RT-PCR Reverse transсriptase polymerase chain reaction Env Envelope S-segment Small segment L-segment Large segment v Declaration of Published Contents Parts of this thesis have been published in the peer-reviewed paper Abdiyeva K, Turebekov N, Dmitrovsky A, Tukhanova N, Shin A, Yeraliyeva L,Heinrich N, Hoelscher M, Yegemberdiyeva R, Shapiyeva Z, Kachiyeva Z,Zhalmagambetova A, Montag J, Dobler G, Zinner J, Wagner E, Frey S, Essbauer S. Seroepidemiological and molecular investigations of infections with Crimean-Congo haemorrhagic fever virus in Kazakhstan. Int J Infect Dis. 2019 Jan;78:121-127. vi Content Abstract ...........................................................................................................................iii Key words....................................................................................................................... iv List of abbreviation .......................................................................................................... v Declaration of Published Contents ................................................................................. vi List of tables .................................................................................................................... x List of figures .................................................................................................................. xii 1 CHAPTER ................................................................................................................. 1 1.1 Introduction ........................................................................................................ 1 2 CHAPTER ................................................................................................................. 5 2.1 Literature review ................................................................................................. 5 2.1.1 Literature review. Tick-borne encephalitis (TBE) ......................................... 5 2.1.1.1 History of TBE .......................................................................................... 5 2.1.1.2 Microbiology of TBE virus ......................................................................... 7 2.1.1.3 Clinical presence and treatment of TBE ................................................... 8 2.1.1.4 Vaccines against TBEV .......................................................................... 11 2.1.1.5 Biosafety containment of TBEV .............................................................. 12 2.1.1.6 Diagnostics of TBE ................................................................................. 13 2.1.1.7 Epidemiology and vectors of TBE .......................................................... 14 2.1.1.8 Tick-borne encephalitis in Kazakhstan ................................................... 17 2.1.2 Literature review. Congo-Crimean Hemorrhagic Fever (CCHF) ................ 20 2.1.2.1 History of CCHF ..................................................................................... 20 2.1.2.2 Microbiology of CCHF ............................................................................ 22 2.1.2.3 Clinical presence of CCHF ..................................................................... 23 2.1.2.4 Vaccines CCHF ...................................................................................... 26 2.1.2.5 Biosafety containment of CCHF ............................................................. 27 2.1.2.6 Diagnostics of CCHF .............................................................................. 27 2.1.2.7 Epidemiology and vectors of CCHFV ..................................................... 29 2.1.2.8 Crimean-Congo Hemorrhagic Fever in Kazakhstan ............................... 30 3 CHAPTER 3 ............................................................................................................ 34 3.1 Rationale and objectives .................................................................................. 34 3.1.1 Rationale of the study ................................................................................ 34 3.1.2 Objectives of the study .............................................................................. 36 4 CHAPTER 4 ............................................................................................................ 37 4.1 Methods ........................................................................................................... 37 vii 4.1.1 Tick study .................................................................................................. 37 4.1.1.1 Tick collection ......................................................................................... 37 4.1.1.2 Room regime .......................................................................................... 38 4.1.1.3 Tick homogenization............................................................................... 38 4.1.1.4 RNA extraction ....................................................................................... 39 4.1.1.5 TBEV RT –qPCRs and target E gene conventional PCR ....................... 40 4.1.2 Serological study ....................................................................................... 41 4.1.2.1 Ethical approval and informed consent administration ........................... 41 4.1.2.2 Questionnaire ......................................................................................... 41 4.1.2.3 Administration of questionnaires ............................................................ 42 4.1.2.4 Description of variables .......................................................................... 42 4.1.2.5 Sera collection ........................................................................................ 46 4.1.2.6 Sera inactivation ..................................................................................... 47 4.1.2.7 RNAs extraction ..................................................................................... 47 4.1.2.8 The Enzyme linked immunosorbent assay (ELISA) ............................... 47 4.1.2.9 TBE investigation with ELISA ................................................................. 49 4.1.2.10 Immunofluorescence assay (IFA) .......................................................
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