Viruses in the upper respiratory tract of individuals at risk of zoonotic infection and their animals in Vietnam: follow-up and virus discovery Nguyen Thi Kha Tu Doctoral Program in Population Health, Faculty of Medicine, University of Helsinki, Finland Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Medicine, University of Helsinki, for public examination in Haartman Institute Lecture Room 2, Haartmaninkatu 3, Helsinki, on March 18th, 2021, at 11 am, Helsinki, 2021 1 Supervisors Professor. MD. Olli Vapalahti Department of Virology, Faculty of Medicine and Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland. Virology and Immunology, HUSLAB, Helsinki University Hospital, Helsinki, Finland. Le Van Tan, PhD Wellcome Trust Intermediate Fellow, Head of Emerging Infections Group, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam. Docent Anna-Maija K. Virtala Department of Veterinary Biosciences, Faculty of Veterinary Medicine University of Helsinki, Helsinki, Finland. Reviewers Docent Tytti Vuorinen Department of Clinical Microbiology, Turku University Hospital, Turku, Finland. Institute of Biomedicine, University of Turku, Turku, Finland. John H.-O. Pettersson, PhD Department of Medical Biochemistry and Microbiology (IMBIM), Zoonosis Science Center, Uppsala University, Uppsala, Sweden. Official opponent Prof. Dr. Jan Felix Drexler Charité-Universitätsmedizin Berlin; German Centre for Infection Research (DZIF), Berlin, Germany. ISBN 978-951-51-7183-2 (paperback) ISBN 978-951-51-7184-9 (PDF) Unigrafia Helsinki 2021 2 CONTENT 1. LIST OF ORIGINAL PUBLICATIONS .............................................................................. 4 2. ABSTRACT ............................................................................................................................. 5 3. ABBREVIATIONS ................................................................................................................. 8 4. LITERATURE REVIEW ...................................................................................................... 9 4.1. Human respiratory tract ................................................................................................................... 9 4.2. Acute respiratory infections ........................................................................................................... 10 4.3. Etiology of acute respiratory infections ......................................................................................... 11 4.4. Viruses detected in human respiratory tract with known human tropism or unknown tropism .... 14 4.5. Interactions of pathogens ............................................................................................................... 18 4.6. Laboratory diagnosis of respiratory viruses ................................................................................... 20 4.7. Establishment of etiological causation of a microbe and a disease ............................................... 26 4.8. Zoonotic diseases ........................................................................................................................... 29 4.9. High-risk sentinel cohort study ...................................................................................................... 32 5. AIMS OF THE STUDY........................................................................................................ 33 6. MATERIAL AND METHODS ........................................................................................... 33 7. RESULTS AND DISCUSSION ........................................................................................... 39 7.1. General data of study participants, interviews and samples (Publications i, ii, iii, iv) .................. 40 7.2. Exposure to potential zoonotic sources of individuals with sustained occupational contact with animals (Goal I; Publication i) ................................................................................................................ 41 7.3. Common respiratory viruses in respiratory infections in individuals with sustained occupational contact with animals (Goal II; Publication ii) ......................................................................................... 45 7.4. Virome of acute respiratory infections in individuals with high risk of zoonotic infections (Goal III; Publication iii) ................................................................................................................................... 47 7.5. Exploring novel viruses in upper respiratory tract of individual with sustained occupational contact with animals (Goal III; Publications iii, iv) ............................................................................................ 51 7.6. Comparison of detection of novel viruses by mNGS versus PCRs (Goal V; Publications iii, iv) . 57 7.7. Characterization of the detected novel viruses (Goal III; Publications iii, iv) ............................... 58 7.8. Assessment of zoonotic potential of the viruses detected in respiratory tract of the individuals (Goal IV; Publication iv) ................................................................................................................................... 61 8. CONCLUDING REMARKS ............................................................................................... 64 9. ACKNOWLEDGEMENTS ................................................................................................. 68 10. REFERENCES ...................................................................................................................... 69 ORIGINAL PUBLICATIONS .................................................................................................. 91 3 1. LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following publications: i. Nguyen Thi Kha Tu, Ngo Tri Tue, Olli Vapalahti, Anna-Maija K. Virtala, Le Van Tan, Maia A. Rabaa, Juan Carrique-Mas, Guy E. Thwaites, Stephen Baker. Occupational Animal Contact in Southern and Central Vietnam. EcoHealth. 2019. doi:10.1007/s10393-019-01444-0. ii. Nguyen Thi Kha Tu, Ngo Tri Tue, Tran My Phuc, Pham Thi Thanh Tam, Vu Thi Ty Hang, Nguyen Thi Han Ny, Guy Thwaites, Anna-Maija K Virtala, Olli Vapalahti, Stephen Baker and Le Van Tan. Respiratory viruses in individuals with high frequency of animal exposure in southern and highland Vietnam. J Med Virol. 2019. doi:10.1002/jmv.25640. iii. Nguyen Thi Kha Tu, Nguyen Thi Thu Hong, Nguyen Thi Han Ny, Tran My Phuc, Pham Thi Thanh Tam, H. Rogier van Doorn, Ho Dang Trung Nghia, Dang Thao Huong, Duong An Han, Luu Thi Thu Ha, Xutao Deng, Guy Thwaites, Eric Delwart^, Stephen Baker^, Anna-Maija K Virtala^, Olli Vapalahti^ and Le Van Tan. The Virome of Acute Respiratory Diseases in Individuals at Risk of Zoonotic Infections. Viruses. 2020;12:960. doi:10.3390/v12090960. iv. Nguyen Thi Kha Tu, Xutao Deng, Nguyen Thi Thu Hong, Nguyen Thi Han Ny, Tran My Phuc, Pham Thi Thanh Tam, Duong An Han, Luu Thi Thu Ha, Guy Thwaites, H. Rogier van Doorn, Anna-Maija K. Virtala^, Eric Delwart^, Stephen Baker^, Olli Vapalahti and Le Van Tan. Redondoviridae: High Prevalence and Possibly Chronic Shedding in Human Respiratory Tract, But No Zoonotic Transmission. Viruses. 2021; Vol. 13, Issue 4. doi.org/10.3390/v13040533. ^ equal contribution 4 2. ABSTRACT Historically, the majority of emerging infections of serious public health threat have been caused by respiratory viruses of zoonotic origin. Notably, Southeast Asia (including Vietnam) is considered as one of the hotspots of emerging infections. The ongoing coronavirus disease 2019 (COVID-19) pandemic emphasizes the importance of understanding the nature of human exposures to zoonotic reservoirs, conducting active surveillance for pathogens and novel viruses, especially at the human-animal interface, as well as having feasible laboratory testing available for quick identification of emerging viruses in future outbreaks. This thesis therefore aims to 1) to characterize the nature of exposures to potential zoonotic sources, 2) to determine the spectrum of respiratory viral viruses detected by PCR, 3) to gain insights into the human virome of acute respiratory infections by metagenomic analysis, 4) to assess zoonotic potential of novel viruses discovered by metagenomics in respiratory tract of individuals with sustained occupational contact with animals, and 5) to evaluate viral detection of metagenomic next generation sequencing (mNGS) and compare viral detection of mNGS versus PCR. As a foundation for answering the proposed research questions, a three-year cohort of 581 individuals working with animals in Vietnam was established during 2013–2016. To establish the baseline data, each of the study participants had nasal-throat swabs collected at the beginning of each year when absence of any respiratory symptoms. During follow-up, whenever the participants reported having any respiratory symptom and fever, their nasal-throat swabs were collected. In parallel, nasal-throat swabs of their animals were also collected. To characterize the nature of human exposures to animals, the baseline data from the three years were combined to analyze exposure as cross-sectional data. The data show that the participants were frequently exposed to zoonotic sources by both occupational activities (as animal raising farmers, animal health workers, slaughter-house workers and rat traders) and non-occupational