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10.14457/MU.The.2004.5 09/08/2564 21:44:31 SURVEY AND CHARACTERIZATION OF MOSQUITO-BORNE FLAVIVIRUSES IN NATURAL POPULATIONS OF MOSQUITOES IN THAILAND 10.14457/MU.the.2004.5 เมื่อ 06/10/2564 10:29:05 WORAWIT UAMCHAROEN A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE (ENVIRONMENTAL BIOLOGY) FACULTY OF GRADUATE STUDIES MAHIDOL UNIVERSITY 2004 ISBN 974-04-5121-7 COPYRIGHT OF MAHIDOL UNIVERSITY Thesis Entitled SURVEY AND CHARACTERIZATION OF MOSQUITO-BORNE FLAVIVIRUSES IN NATURAL POPULATIONS OF MOSQUITOES IN THAILAND ……………………………………… Mr. Worawit Uamcharoen Candidate 10.14457/MU.the.2004.5 เมื่อ 06/10/2564 10:29:05 ……………………………………… Assoc. Prof. Pattamaporn Kittayapong, Ph.D. Major advisor ……………………………………… Prof. Jean-Paul Gonzalez, M.D., Ph.D. Co-advisor ..………………………………… Lect. Pahol Kosiyachinda, Ph.D. Co-advisor …………………………………… …………………………………… Assoc. Prof. Rassmidara Hoonsawat Assoc. Prof. Prayad Pokethitiyook Ph.D Ph.D. Dean Chair Faculty of Graduate Studies Master of Science Programme Mahidol University in Environmental Biology Faculty of Science Thesis Entitled SURVEY AND CHARACTERIZATION OF MOSQUITO-BORNE FLAVIVIRUSES IN NATURAL POPULATIONS OF MOSQUITOES IN THAILAND was submitted to the Faculty of Graduate Studies, Mahidol University For the degree of Master of Science (Environmental Biology) On August 3, 2004 ……………………………………… 10.14457/MU.the.2004.5 Mr. Worawit Uamcharoen เมื่อ 06/10/2564 10:29:05 Candidate ……………………………………… Assoc. Prof. Pattamaporn Kittayapong, Ph.D. Chair ……………………………………… Prof. Jean-Paul Gonzalez, M.D., Ph.D. Thesis Defense Committee ……………………………………. ……………………………………… Assoc. Prof. Vijitta Lerdkamolkarn Lect. John R. Milne, Ph.D. Ph.D. Thesis Defense Committee Thesis Defense Committee …………………………………… …………………………………… Assoc. Prof. Rassamidara Hoonsawat Assoc. Prof. Prasert Sobhon Ph.D Ph.D. Dean Dean Faculty of Graduate Studies Faculty of Science Mahidol University Mahidol University ACKNOWLEDGEMENTS I would like to express my sincere gratitude and deepest appreciation to my supervisor, Assoc. Prof. Kittayapong, for her guidance, encouragement and kind support, and for reviewing the thesis and making many useful suggestions; without her this work could not have been completed. 10.14457/MU.the.2004.5I also express my sincere gratitude to Dr. Jean-Paul Gonzalez, Director of the เมื่อResearcher 06/10/2564 Center for Emerging 10:29:05 Viral Diseases, IRD, for his guidance, encouragement and many useful suggestions. I am also grateful to Dr. Uruyakorn Chansaeng, Entomology Department, Ministry of Public Health, for mosquito identification. I am indebted Dr. John Milne for reviewing and editing the thesis and for Dr. Vijittra Lerdkamolkarn for reviewing the thesis. Thanks are also due to many other people without whose help this work would not have been possible. I thank Mr. Tada Juthayothin, Miss Supanee Hirunkanokphun and Mr Toon Rueng-areerate for providing useful suggestions and for helping with mosquito collection. I would also like to express my thanks to other colleagues both at Mahidol University and IRD for their help and collaboration during my study. This investigation received financial support from the research pour le development (IRD) and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR/RCS/A00786). Finally, I would like to express my deep gratitude to my parents, my family and my friends for their support and encouragement. Worawit Uamcharoen Fac. of Grad. Studies, Mahidol Univ. Thesis/ iv SURVEY AND CHARACTERIZATION OF MOSQUITO-BORNE FLAVIVIRUSES IN NATURAL POPULATIONS OF MOSQUITOES IN THAILAND WORAWIT UAMCHAROEN 4237510 SCEB/M M.Sc. (ENVIRONMENTAL BIOLOGY) THESIS ADVISORS: PATTAMAPORN KITTAYAPONG, Ph.D., JEAN-PAUL GONZALEZ, M.D., Ph.D., PAHOL KOSIYAJINDA, Ph.D. ABSTRACT 10.14457/MU.the.2004.5 Mosquitoes are of considerable medical and veterinary importance because they harm the host through their feeding action and vector many pathogens. In particular, เมื่อthere has06/10/2564 been no comprehensive 10:29:05survey on mosquito-borne flaviviruses, which are implicated in dengue fever, Japanese encephalitis and other diseases. Mosquito-borne flaviviruses in Thailand were surveyed in this research. A total of 1,257 individuals representing 40 species of mosquitoes in 6 genera collected from 8 provinces of Thailand were examined for flavivirus by RT-PCR assay using universal flavivirus primers. One mosquito species, Culex fuscocephala, collected from Wang Thong District, Kampaengphet Province, Thailand, was flavivirus positive. Flavivirus infection rate in this species was 11.1% (2/18). Phylogenetic relationships among this virus and other flaviviruses were inferred from comparison of sequences of the partial NS5 gene. Results of phylogenetic analysis using maximum parsimony, neighbor- joining and maximum likelihood methods indicated that this virus represented a new flavivirus genotype closely related to The Cell Fusion Agent Virus (CFAV), found in Aedes aegypti mosquito cell line and The Kamiti River Virus (KRV) detected in the mosquito Aedes macintoshi from Kenya, respectively. This new flavivirus was designated as the Wang Thong Virus (WTV) KEY WORDS: FLAVIVIRUS / CELL FUSION AGENT (CFAV) / CULEX / MOSQUITO / PHYLOGENY 115 pp. ISBN 974-04-5121-7 Fac. of Grad. Studies, Mahidol Univ. Thesis/ v การสํารวจและการศึกษาคุณลักษณะของ Flavivirus ที่นําโดยยุงพาหะในประเทศไทย (SURVEY AND CHARACTERIZATION OF MOSQUITO-BORNE FLAVIVIRUSES IN NATURAL POPULATIONS OF MOSQUITOES IN THAILAND) วรวิทย อวมเจริญ 4237510 SCEB/M วท. ม. (ชีววิทยาสภาวะแวดลอม) คณะกรรมการควบคุมวิทยานิพนธ: ปทมาภรณ กฤตยพงษ, JEAN-PAUL GONZALEZ, MD., Ph.D., พหล โกสิยจินดา, Ph.D. บทคัดยอ 10.14457/MU.the.2004.5 ยุงจัดเปนแมลงพาหะนําโรคที่สําคัญทางการแพทย เปนสาเหตุสําคัญที่ทําใหสุขภาพของคนและสัตว เมื่อเสือมโทรมลง 06/10/2564 ไมวาจะเปนการกอใหเกิดความร 10:29:05ําคาญ การเกิดอาการแพ เปนตุมคัน การสูญเสียเลือด หรือการเปน โรคตางๆ อันเนื่องมาจากยุงเปนพาหะ ซึ่งอาจรุนแรงจนถึงขั้นเสียชีวิตได แมวาในประเทศไทยจะมีการศึกษาการ ระบาดของโรค และการควบคุมโรคเปนจํานวนมาก แตยังไมมีประสิทธิภาพดีพอ ดังนั้นการศึกษาไวรัสที่ กอใหเกิดโรคในยุงธรรมชาติ จึงเปนเรื่องจําเปน เพื่อเฝาระวังและปองกันโรค อันอาจเกิดการระบาดไดในอนาคต ดังนั้นการวิจัยนี้ไดทําการศึกษาเชื้อไวรัสในตระกูลฟลาวิไวรัสในยุง โดยทําการเก็บตัวอยางจากยุงจํานวน 1,257 ตัว 40 สปชีส จาก 8 จังหวัดในประเทศไทย มาสุมตรวจหาเชื้อไวรัส ดวยเทคนิค RT-PCR โดยใช primer ที่ จําเพาะตอไวรัสเหลานั้น จากผลการทดลองพบวารอยละ 11 ของยุงชนิด Culex fuscocephala จากตําบลวัง ทอง จังหวัดกําแพงเพชร มีการติดเชื้อไวรัสใหม 1 ชนิด เมื่อศึกษาความสัมพันธทางวิวัฒนาการระหวางไวรัสอื่นๆ ในตระกูลเดียวกันพบวา ไวรัสที่พบใหมนี้มีความใกลเคียงกับไวรัส Cell Fusion Agent (CFAV) ซึ่งเปน ไวรัสที่พบในเซลลของยุง Aedes agegypti ที่เพาะเลี้ยงในหองทดลอง และ Kamiti River (KRV) จากยุง Aedes macintoshi จากปาในประเทศเคนยา และใหชื่อไวรัสที่พบใหมชนิดนี้วาไวรัสวังทอง (Wang Thong Virus (WTV)) 115 หนา ISBN 974-04-5121-7 CONTENTS ACKNOWLEDGEMENTS iii ABSTRACT iv LIST OF TABLES ix LIST OF FIGURES x 10.14457/MU.the.2004.5CHAPTER เมื่อ 06/10/25641 INTRODUCTION 10:29:05 1 OBJECTIVES 2 2 LITERATURE REVIEW 3 2.1 Mosquitoes 3 2.1.1 General characteristics 3 2.1.2 The eggs 5 2.1.3 The larvae 5 2.1.4 The pupae 6 2.1.5 The adult 7 2.2 The Flaviviruses 2.2.1 Background and classification 10 2.2.2 Structure and physical properties of the virion 12 2.2.3 Binding and entry 12 2.2.4 Genome structure 13 2.2.5 Translation and proteolytic processing 14 2.2.6 Features of the structural proteins 15 2.2.6.1 The C protein 15 2.2.6.2 The prM protein 15 2.2.6.3 The E protein 16 2.2.7 Features of the nonstructural protein (NS) 17 vii CONTENTS (CONTINUED) 2.2.7.1 The NS1 protein 17 2.2.7.2 The NS2A and NS2B proteins 18 2.2.7.3 The NS3 protein 19 2.2.7.4 The NS4A and NS4B proteins 20 2.2.7.5 The NS5 protein 20 2.2.8 RNA replication 21 10.14457/MU.the.2004.52.2.9 Membrane reorganization and 22 compartmentalization of flavivirus เมื่อ 06/10/2564 10:29:05 replication 2.2.10 Assembly and release of virus particles 23 2.3 The prevalence of flaviviruses 25 2.4 Reverse transcriptase polymerase chain reaction (RT-PCR) detection of flaviviruses in mosquitoes 32 2.5 Phylogenetic analysis 36 2.5.1 Algorithm-based vs. criterion-based 37 2.5.2 Distance-based vs. character-based 37 2.5.3 Distance Based Methods 37 2.5.3.1 Neighbor Joining (NJ) 38 2.5.4 Character-Based Methods 38 2.5.4.1 Maximum Parsimony (MP) 38 2.5.4.2 Maximum Likelihood (ML) 39 2.5.5 Bootstrapping 40 2.5.6 Phylogeny of flaviviruses 41 3 MATERIALS AND METHODS 48 3.1 Collection and identification of mosquitoes 48 3.2 Reverse transcriptase polymerase chain reaction 49 (RTPCR) viii CONTENTS (CONTINUED) 3.2.1 RNA extraction and cDNA synthesis 49 3.2.2 PCR amplification and gel electrophoresis 49 3.2.2.1 PCR amplification using FLV1/FLV2 51 3.2.2.2 Onestep RT-PCR for flaviviruses 51 3.3 Cloning and Sequencing 52 10.14457/MU.the.2004.53.3.1 Ligation 52 3.3.2 Transformation 53 เมื่อ 06/10/25643.3.3 10:29:05 Screening for recombinant colonies 53 3.3.4 DNA sequencing 54 3.4 Phylogenetic analysis 54 4 RESULTS 57 4.1 Mosquito collection sites 57 4.2 Mosquito collections 61 4.2.1 Collected mosquito in each province 61 4.3 RT-PCR detection of flaviviruses in mosquitoes 63 4.4 Flavivirus infection rates 72 4.5 Phylogenetic relationships of flaviviruses 72 5 DISCUSSION 86 6 CONCLUSIONS 90 REFERENCES 91 APPENDICES 104 BIOGRAPHY 115 LIST OF TABLES Pages 1. Vectors and potential vectors of mosquito-borne diseases in Thailand and neighboring countries. 29 10.14457/MU.the.2004.52. Oligonucleotide primers used for PCR detection of flaviviruses
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