ABSTRACT Vector-Borne Viral Infections in South-West
Total Page：16
File Type：pdf, Size：1020Kb
저작자표시-비영리-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게 l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다. 다음과 같은 조건을 따라야 합니다: 저작자표시. 귀하는 원저작자를 표시하여야 합니다. 비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다. 변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다. l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다. l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다. 저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다. Disclaimer August 2016 Master’s Degree Thesis Vector-Borne Viral Infections in South-West Region of Korea Graduate School of Chosun University Department of Biomedical Sciences Babita Jha August 2016 Master’s Degree Thesis Vector-Borne Viral Infections in South-West Region of Korea Graduate School of Chosun University Department of Biomedical Sciences Babita Jha Vector-Borne Viral Infections in South-West Region of Korea 한국의 남서부 지역에서 매개체 관련 바이러스 질환 August, 2016 Graduate School of Chosun University Department of Biomedical Sciences Babita Jha Vector-Borne Viral Infections in South-West Region of Korea Advisor: Prof. Dong-Min Kim, MD, PhD THESIS SUBMITTED TO THE DEPARTMENT OF BIOMEDICAL SCIENCES, CHOSUN UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF BIOMEDICAL SCIENCES April, 2016 Graduate School of Chosun University Department of Biomedical Sciences Submitted by Babita Jha August Master’s Vector-Borne Viral Infections in Babita Jha Degree 2016 Thesis South-West Region of Korea Table of Contents LIST OF TABLES……………………….................iv LIST OF FIGURES…………………………………v ABBREVIATIONS AND SYMBOLS…………….vi ABSTRACT…………………………………...…….ix 한 글 요 약……………………………………...…..xii I. Introduction………………………………...1 Vector-borne viral infection…………….1 Aim of the study…………………………1 Severe Fever with Thrombocytopenia Syndrome………………………………..2 Genome…………………………….....2 Disease distribution……………….....3 Diagnostic methods…………….…....3 Reported SFTS cases in ROK….......5 Hemorrhagic Fever with Renal Syndrome………………….….................5 Hantavirus…………………………….5 Virology………………........................6 Diagnosis…………………………...…6 Flavivirus……………………………..….7 Virology…………………………........8 Epidemiology and clinical features....8 II. Material and Methods………….…..….......9 Study sites………………………………..9 Clinical case definition……………....…..9 SFTS case definition…………….……9 HFRS case definition…..…………….9 Flavivirus case definition……............9 Extraction of viral RNA……………….10 Preparation of viral cDNA…………….10 PCR Amplification……………………..10 RT-PCR targeting M segment for SFTSV detection…………………….10 RT-N-PCR targeting L segment for Hantavirus detection………………..11 RT-PCR targeting NS5 gene for Flavivirus detection…………………11 Immunofluorescence Assay (IFA)…….12 Nucleotide sequencing……………...….12 Phylogenetic analysis…………………..12 III. Results…………………….……………….13 RT-PCR targeting M segment for SFTSV from patients’ whole blood or plasma……………………….13 RT-N-PCR targeting L segment for HFRSV from patients’ whole blood or plasma…………………….......13 RT-PCR targeting NS5 for Flavivirus from patients’ whole blood or plasma...14 RT-PCR targeting M segment for SFTSV from mouse’s organ……….14 RT-N-PCR targeting L segment for Hantavirus from mouse’s organ………14 RT-PCR targeting NS5 for Flavivirus from mouse’s organ………....................14 IFA detection of mouse’s organ………15 Phylogenetic tree analysis…………......15 IV. Discussion………………………………….16 V. Conclusions………………………………..19 VI. References………………………………....39 Acknowledgements………………………….…..48 LIST OF TABLES Table 1. Oligonucleotide primers and probes used in this study…. ……………………………………………………………………….20 Table 2. PCR Conditions used in this study……………………….21 Table 3. Sequencing results and identification of SFTSV from six patients serum samples.......................................................................22 Table 4. Sequencing results and identification of HFRSV (HTNV) from eight patients serum and one whole blood samples…………25 Table 5. Sequencing results and identification of Hantavirus from mouse organ samples……………………………….………………29 Table 6. Comparison between results of IFA and RT-N-PCR of wild mice lung samples for the month of October and November ………………………………………………………………………32 iv LIST OF FIGURES Figure 1 Schematic diagram of the structure and genome of the SFTSV………………………………………………………………. 33 Figure 2. Phylogenetic tree based on M segment (480 bp) partial sequences from GenBank and SFTS positive patients gene sequence of SFTS positive patients samples………………………………………………………………34 Figure 3. Phylogenetic tree based on L segment (380 bp) partial sequences from GenBank and HFRS positive patients specimens collected in this study..………………………………………………35 Figure 4. Agarose gel electrophoresis of the PCR products after SFTS M segment (560 bp) targeting RT-PCR using patients specimens……………………………………………………………36 Figure 5. Agarose gel electrophoresis of the PCR products after HFRS L segment targeting RT-nested PCR using patients specimens…………………………………………………………….36 Figure 6. Agarose gel electrophoresis of the PCR products after HFRS L segment targeting RT-nested PCR using mouse organ specimens (Collected in August)……………………………………37 Figure 7. Agarose gel electrophoresis of the PCR products after HFRS L segment targeting RT-nested PCR using mouse organ specimens (Collected in October)…………………………………..37 Figure 8. Agarose gel electrophoresis of the PCR products after HFRS L segment targeting RT-nested PCR using mouse organ specimens (Collected in November)………………………………..38 v ABBREVIATIONS AND SYMBOLS BLAST basic local alignment search tool C-RT-PCR conventional reverse transcriptase polymerase chain reaction C.I. confidence interval cDNA complementary DNA DENV dengue virus DNA deoxyribonucleic acid gDNA genomic DNA bp base pairs DOBV dobra virus e.g. example given EM electron microscope HTNV hantaan virus HFRS hemorrhagic fever with renal syndrome HCPS hemorrhagic cardio pulmonary syndrome vi JEV Japanese encephalitis virus Kb kilobase (one thousand bases) kDa kilodalton km2 kilometre squared L(K) likelihood distribution; estimates number of clusters in STRUCTURE L-segment large segment Mb megabase pairs M-segment medium segment NS5 nonstructural protein OR odds ratio ORF open reading frame PBS phosphate buffered saline PCR polymerase chain reaction PUUV puumala virus ROK Republic of Korea RNA ribonucleic acid vii spp species SEOV seoul virus SFTS severe fever with thrombocytopenic syndrome SFTSV severe fever with thrombocytopenia syndrome virus S-segment small segment Tm melting temperature VBVI vector borne viral infections VBVD vector borne viral diseases WNFV west-nile fever virus viii ABSTRACT Vector-Borne Viral Infections in South-West Region of Korea Babita Jha Supervisor: Prof. Dong-Min Kim, MD, PhD Department of Medicine Graduate School of Chosun University Background Vector-borne viral infections (VBVIs) are defined as viral infections transmitted by vectors such as mosquitoes, ticks, lice, biting flies, mites, and fleas. Vector-borne infections account for more than 17% of all infectious diseases, causing more than 1 million deaths annually. Severe fever with thrombocytopenia syndrome (SFTS) caused by severe fever with thrombocytopenia syndrome virus (SFTSV) and Hemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses from the family Bunyaviridae are two of the emerging VBVIs worldwide. Similarly, Flavivirus of the family Flaviviridae transmitted by arthropod vectors especially by ticks and mosquitoes causing various infections such as West Nile fever, Yellow ix fever, Dengue, etc are widely distributed . SFTSV, a novel bunyavirus reported to be endemic in central and northeastern China, was first detected in 2010 in China and later reported in other Asian and Mediterranean countries as well as in the USA. On the other hand Hantaviruses found global attention during the Korean War between 1951 to 1953, when more than 3000 United Nations and US soldiers experienced an acute febrile illness with acute renal failure and shock. In this study, we investigated the positive rate of SFTSV, Hantavirus and Flavivirus in humans and rodents in south-western region of ROK. Methods In 2015, all the suspected cases of SFTS and HFRS from two different University Hospitals located in Gwangju City, which is in south-west of ROK were investigated for these three clinical entities by different PCR techniques. We also collected rodents (striped field mouse; Apodemas agrarius) from the suburbs of Gwangju city to detect the presence of SFTSV and Hantavirus as wild rodents are largely considered as the natural reservoirs of these pathogenic viruses. A total of 267 patients were investigated for SFTSV by RT-PCR targeting ‘M’ segment and 295 patients for HFRS by RT-Nested PCR targeting ‘L’ segment. Similarly, we tried to investigate the presence of Flavivirus in the suspected cases by conventional RT-PCR targeting ‘NS5 ‘gene. Also, 41 wild mice captured in August, October and November were investigated for the presence of both these viral pathogens by similar molecular methods as for human samples. The amplified products were sequenced and analyzed. Results In this study, 2.2 % (6/267) of patients’ plasma sample were positive for SFTS and 3.1 % (9/295) were found positive for HFRS. On the other hand, none of the mouse samples were positive for SFTSV but a proportionally high positive rate of Hantavirus in mouse; 46.3 % (19/41) was found. This fact suggests that SFTSV x and hantavirus are prevalent in human population at a reasonably higher rate than thought previously. Conclusions SFTS and HFRS have been endemic to Korea, and these vector-borne infectious