1 LIST OF ACRONYMS GH General hospital AES Acute encephalitis syndromee MAC-ELISA IgM antibody capture – enzyme linked immunosorbent assay RT-PCR Reverse Transcription Polymerase Chain Reaction JE Japanese Encephalitis NIHE National Institute of Hygiene and Epidemiology 2 INTRODUCTION Acute encephalitis syndromee (AES) suspected to be caused by viruses has many different causes. There is no specific treatment for this disease (except for Herpes simplex virus), with high mortality and severe neurological sequela. Currently, there are about 100 virus types identified causing AES. In 1987 and 1992, Banna virus was isolated from AES and unidentified fever patients’ serum in China. Banna virus was isolated from Aedes dorsalis mosquitoes in China. According to some studies in Indonesia, Banna virus was transmitted by two types of mosquitoes: Anopheles and Culex. In Vietnam, in 2003 and 2005, viruses of the same group with Banna virus were isolated from patients in Thanh Hoa and Gia Lai provinces. Besides, Banna virus was isolated from Culex mosquitoes from Ha Tay (now part of Ha Noi) and Quang Binh provinces in 2002. Participating in monitoring, diagnosis, treatment and prevention of AES suspected to be caused by Banna virus, the study “Some epidemiological characteristics of acute encephalitis syndrome suspected to be caused by Banna virus in some provinces of Vietnam” was conducted with three following specific goals: 1. Describe some epidemiological characteristics, clinical syndrome of acute encephalitis syndrome suspected to be caused by Banna virus in some provinces of Vietnam, 2002- 2012. 2. Determine infection ratio of Banna virus in mosquito population collected in some provinces of Vietnam. 3. Identify some bio-molecular characteristics of Banna virus isolated in Vietnam. 3 THESIS’ PRACTICAL IMPLICATIONS AND NEW CONTRIBUTIONS - New contributions:This is the first study in Vietnam identifying incidence rate, clinical charateristics of patients with AES caused by Banna virus, infection rate of Banna virus among mosquito populations in some provinces and bio-molecular characteristics of Banna virus identified in Vietnam. - Practical implications: The research provides completely new characteristics of AES caused by Banna virus data for scientific communities of Vietnam and the world. Research results can be applied in monitoring, diagnosis and prevention of AES caused by Banna virus, with strong implication in teaching as well as research and production. THESIS STRUCTURE The thesis is 113 pages long (not including references and appendixes), including 4 chapters, 30 tables, 13 figures, 1 picture. Introduction is 2 pages long, Chapter 1: Overview (28 pages); Chapter 2: Subject, material and study methodologies (22 pages); Chapter 3: Study results (33 pages); Chapter 4: Discussions (22 pages); Conclusion is 3 pages long; Suggestions/proposals is 1 page; List of published work is 2 page. References: 102 referenced work; 2 appendixes. 4 Chapter I. OVERVIEW 1.1. Characteristics of Banna virus. Banna virus, belonging to Seadornavirus genus, Reoviridae family, has genetic material as 12 double-stranded RNA segments. The first Banna virus strain was isolated from cerebrospinal fluid of AES patients and blood samples of unidentified fever patients in Yunnan province, China; and later, isolated in different provinces from patients and mosquitoes in China, Indonesia and Vietnam. 1.2. Clinical characteristics of AES caused by Banna virus. Banna virus causing acute infectious disease damaging central nervous system or unidentified fever was recorded. Typical cases can be described as following: Onset period: lasts 1-2 days but difficult to identify when patients do not remember sudden high fever, chills, headaches, arthralgia, and anorexia symptoms. Full-fledge period: after 3-6 days, patients showing symptoms of high fever, derilium, autonomic disorder, indifference to surrounding including coma, dyspnea, photophobia, loss of appetite, nausea. Symptoms of peripheral nerve injuries include paralysis, chorea Sub-acute progression period: from days 7-9 of the disease, symptoms reduced such as milder fever, stable pulse temperature, reduced central and peripheral nervous syndromee. However, in this period, there are notable complications from laying for extended period such as pneumonia, sores, constipation Recovery period: patient only has mild fever, regaining conciousness, recovered appetite, remaining only sequelae depending on the severity of the disease such as paralysis, hemorrhage, myocarditis, pericarditis, reduced memory. 1.3. Epidemiological characteristics of AES caused by Banna virus Some studies in Vietnam and around the world showed that Banna virus exist in mosquitoes, there is clear evidence of Banna virus transmission among animals (pigs) from virus isolation results. Moreover, Banna virus exist in some migratory birds and the migration of these birds enables the virus to spread to other areas. Mosquito has been confirmed as Banna virus transmission vector in a number of Asian countries from isolation results of Culex tritaeniorhynchus, Culex 5 vishnui, Culex fuscocephalus, Anopheles vagus, Aedes albopictus and Aedes dorsalis mosquitoes. Human is the infection target of Banna virus, the study of Liu, et al (2010) of Banna virus in China from 1987 to 2007 showed that Banna virus appeared in areas with Japanese Encephalitis (JE) outbreak and where Culex tritaeniorhynchus mosquito act as the main vector. The virus is infected through the skin from mosquito bite, once infected, the virus multiplies in the lymphatic system, virions are passively transmitted via vascular endothelia or choroid plexus, then to the central nervous system and remain in the cerebrospinal fluid. Virus effectiveness peaked in the early days of the onset and decreases rapidly when neutralizing antibodies apprear. After virus infection, the body may have immunizing response, neutralizing antibodies increases from day 12 of the infection, IgM antibodies have higher neutralizing effect than neutralizing antibodies. IgG antibodies appear from week 3 and lower than IgM antibodies but remain for the whole lifetime. 1.4. Treatment and prevention of AES caused by Banna virus Treatment: Currently, there is no specific medical treatment for AES caused by Banna virus, mainly treatment of symptoms and complications. Prevention: Banna virus is a mosquito-borne virus, a new virus discovered in the past few decades mainly in Asian region, studies of disease burden have not been mentioned, so far, there is no vaccine for the disease, the most effective prevention method to this day is to prevent mosquito as transmission vector. Chapter II. SUBJECT AND STUDY METHODS 2.1. Regions and time of study Retrospective study of mosquito samples and specimens were collected from 01/2002 to 12/2008 and prospectively from 01/2009 to 12/2012. 6 Research locations: Northern region (area previously Ha Tay, Bac Giang, Thanh Hoa); Central region (Quang Binh); Tay Nguyen region (Gia Lai, Kon Tum, Dak Lak, Dak Nong); Southern region (Long An, Can Tho) 2.2. Study subjects: Patients:  Patients clinically diagnosed with AES suspected to be caused by viruses according to standards of the World Health Organization: - Sudden high fever > 38 o C, accompanied by one of following symptoms: - Change of mental state, or - Nervous symptoms as meningeal signs, movement disorders  Patients diagnosed with AES suspected to be caused by Banna virus: Cases of AES suspected to be caused by viruses detected Banna IgM antibodies from cerebrospinal fuild from positive ELISA technique. Mosquito species: Study subjects are mosquito samples collected at study sites in the Northern, Central, Southern and Tay Nguyen regions from 2001 to 2011. 2.3. Contents of research Study cases of AES: Collect samples of cerebrospinal fuilds from patients with AES suspected to be caused by viruses in Infectious diseases department in provincial hospitals. Test for Banna virus IgM antibodies. Investigate epidemiological characteristics, clinical symptoms of AES cases identified (+) with Banna virus antibodies, JE, ECHO30 positive isolated from retrospective medical record. Study Culex mosquito vector: Collect mosquito samples in provinces with high number of patients with AES suspected to be caused by viruses in Northern, Central, Southern and Tay Nguyen regions, once per year in the period from March to December. Mosquitoes are categorized and identified specie compositions, isolated to identify Banna virus. 7 Banna virus trains isolated from AES patients, from pigs and mosquitoes are genotype identified based on nucleotide gene sequence No. 12. 2.4. Study methods 2.4.1. Structure of the study: The struture of the study is cross-sectional, retrospective and prosprective epidemiologically combined with laboratory analysis. 2.4.2. Determining investigation of epidemiological characteristics of AES patients Study method and sample taking Sample size: Take cerebrospinal fuild samples of all patients with AES suspected to be caused by viruses based on diagnostic standards above when hospitalized. Choose sample based on convenient method, samples are taken in accordance with regular protocol and surveyed with pre-designed questionnaire. Study method for vector mosquito: Sample size: Sample size is calculated based on regular protocol of NIHE; 30 household/night x 2 nights x 1 site (district/province) x 1 time/year = 60 household turn/site. Mosquito investigation is conducted at night according to regular procedure of NIHE (capture mosquitoes with CDC traps, capture female mosquitoes resting in the house and barn, from 18h00 to 22h00 in the winter and 19h00 to 23h00 in the summer. Laboratory material and testing techniques Samples including patients' ceresbrospinal fluid samples and mosquito samples collected at study sites are analyzed in the laboratory. With cerebrospinal fluid samples, use indirect ELISA technique detecting specific Banna virus antibodies. With mosquito samples, use isolation technique detecting mosquito types carrying Banna virus. 8 Isolated Banna virus strains are categorized by RT-PCR technique, collecting PCR products for product purification techniques and sequenced by sequencing machine. Data collected from the study is analyzed by bioinfomatic software such as: GraphPad, biological software DNA Star (Lasegene), MEGA 4.0 2.4.3. Data processing: Use biostatistic software Epi-info 6.04 and Stata 10 to input and process data. Chapter III. STUDY RESULTS 3.1. Some epidemiological, clinical characteristics of AES caused by Banna virus. 3.1.1. Description of ratio of patients with AES caused by Banna virus Table 3.1. Identification result of Banna virus IgM antibodies in cerebrospinal fluid sample of patients with AES, 2002 – 2012 Region Province Number of samples Number of positive Ratio (+) % Northern Bac Giang 216 30 13,63 Ha Tay (previously) 120 43 35,83 Ha Noi 50 17 34,00 Hai Phong 48 11 22,92 Thai Binh 108 36 33,33 Thanh Hoa 65 21 32,31 Central Hue 18 4 22,22 Tay Nguyen Gia Lai 20 5 25,00 Southern Long An 72 17 23,61 Total 717 184 25,66 There are 1,285 cerebrospinal fluid samples collected from patients with AES susptected to be caused by viruses in 9 provinces/cities in the period from 2002 to 2012, excluding causes by JE virus, ECHO30 and herpes simplex virus type 1 and type 4, there are 717 cerebrospinal fluid samples with unidentified causes. Using indirect ELISA technique 9 identifying Banna virus IgM antibodies from 717 cerebrospinal fluid samples, resulting in identification of 184 (+) samples, average (+) ratio of cerebrospinal fluid samples identified with Banna virus IgM antibodies is 25.66% (184/717), and 14.32% (184/1285) when calculated on the total cerebrospinal fluid samples of patients with AES. In 9 provinces/cities with sample specimens, identified (+) ratio ranges from 13.83% to 35.83%. Province/city with highest (+) ratio is area previously Ha Tay province with 35.83%, then Ha Noi with (+) ratio of Banna virus antibodies at 34.00%; lowest identified (+) ratio with Banna virus antibodies is 13.53% in Bac Giang. According to serological surveillance, AES caused by viruses are recorded throughout the year, but cases are recorded mainly in May, June, July and August, with recorded peak of the epidemic is June with 239/717 recoded cases (33.33% of total number of cases) Table 3.2. Ratio of cases of AES caused by viruses by age group, 2002 – 2012 Age group <1 n = 61 1 - 4 n = 159 5 - 9 n = 183 10 - 14 n = 141 ≥ 15 n = 173 Total Number of samples (+) 11 35 44 42 52 184 Rate of incidence (%) 5,98 19,02 23,91 22,83 28,26 100 In 717 cases of AES suspected to be caused by viruses with unidentified cause having cerebrospinal fluid sample tested with indirect ELISA technique identifying IgM, resulted in 184 confirmed cases of Banna virus antigen, cases of confirmed (+) recorded in all age groups. Among them, incidence rate of AES caused by Banna virus in age group of <1 year old is lowest at 5.98%, while incidence rate of AES caused by Banna virus at age group ≥ is highest at 28.26%. 10 In 184 cases of AES identified to be caused by Banna virus, the ratio of cases of AES caused by Banna virus in men is higher than women in all age group/ 3.1.2. Clinical characteristics of patients with AES caused by Banna virus 3.1.2.1. Some signs, clinical symptoms at admission Table 3.3. Some clinical symptoms at admission Signs, symptoms BANNA virus n=103 (%) ECHO30 virus n=43 (%) JE virus (n=5) (%) Ratio of BANNA and ECHO30 Ratio of Banna and JE p1 p2 Headache 48,54 88,37 30,51 <0,0001 0,0252 Vomitting 32,04 86,05 28,81 <0,0001 0,6685 Seizures 32,04 2,33 61,02 0,0001 0,0003 Nausea 1,94 30,23 5,08 <0,0001 0,2659 Myalgia 0 0 0 - - Joint pain 0 2,33 0 - - Fever > 37,5 o C 78,64 74,42 81,36 0,5784 0,6791 Bulging fontanel 23,30 2,33 0 0,0022 - Stiff neck 77,45 39,53 50,85 <0,0001 0,0005 Kernig sign 67,96 34,88 38,98 0,0002 0,0003 Mental disorder 81,55 11,63 88,14 <0,0001 0,2715 Bradykinesia 18,63 4,65 23,73 0,0288 0,4390 Loss of sensation 0 0 6,78 - - Analysis of clinical symptoms of AES patients at admission shows that almost all typical clinical symptoms of AES such as headache, vomitting, seizures, high fever above 37.5 degrees, bulging fontanel, stiff neck, Kernig sign, mental disorder and bradykinesia appear in newly admitted patients with AES caused by Banna virus at high ratio from 23.3% to 78.64%. However, myalgia, joint pain and loss of [...]... research, including 2 nucleotide genetic sequence No 12 from Banna virus isolated from AES patients in Vietnam This means the study for genome sequencing of Banna virus isolated from Vietnamese patient needs to be mentioned in following studies CONCLUSION 1 Description of some epidemiological, clinical characteristics of AES caused by Banna virus in some provinces of Vietnam 1.1 Ratio of Banna virus infection... Banna virus strains isolated from Vietnamese patient do not have any close genetic relation to the Banna virus strain isolated from Chinese patients Chapter IV DISCUSSION 4.1 Describe some epidemiology and clinical symptoms of AES suspected to be caused by Banna virus in some provinces in Vietnam, 2002-2012 In this study, there are 717 cerebrospinal fluid samples of patients with AES suspected to be. .. bio-molecular characteristics of Banna virus isolated in Vietnam Genetic sequences No 12 of Banna virus strains were selected for genotype analysis of Banna virus strains isolated in Vietnam and some geographical areas in Asia Result of the analysis identified various Banna virus strains divided into two different genotype, Chinese and Vietnamese strains belong to genotype A, while virus strains isolated in Indonesia... of AES caused by Banna virus, ECHO30 virus and JEV, there are 16 cases of death after treatment Mortality rate of AES caused by Banna virus is 14.6% (15/103), followed by mortality rate of AES caused by JEV at 1.7% (1/59) Conversely, no cases of death recorded in all cases of AES caused by ECHO30 virus 3.2 Determining Banna virus prevalence in mosquito polulation collected in some provinces of Vietnam. .. primarily in patients with AES caused by Banna virus and rarely in patients with AES caused by ECHO30 virus and particularly not in patients with AES caused by JE virus 3.1.2.2 Some signs, clinical symptoms during treatment Table 3.4 Signs, clinical symptoms after 7 days of treatment of patients infected with Banna virus comparing to infection with ECHO30 and JE viruses Symptoms Headache Vomitting Seizures... antigen in cases of AES suspected to be caused by viruses (excluding cases identified positively with JEV antigen) in 9 provinces/ cities of Vietnam ranges from 13.83% to 35.83% (averaging 25.66%), showing that in different geological separations, the ratio of AES infection could be caused by different agents When calculated on the total of 1,285 ceresbrospinal fluid samples not excluding a number of pathogens,... mosquito in all regions including Northern, Central and Tay Nguyen regions In the Southern region, Banna virus is mainly isolated from Culex pseudovishnui and Culex quinquefaciatus mosquitoes However, the isolation of Banna virus from 8 different species of mosquitoes showed that the number of mosquito species being or cound be the vector of Banna virus is bigger that for JEV vector 4.3 Determining some. .. with AES caused by Banna virus is 13.5 days and the maximum treatment time is 85 days Mortality rate of patients with AES caused by Banna virus is very high at 14.6% compared to JEV (1.7%) and ECHO30 virus (0%) 2 Determining Banna virus prevalence in mosquito polulation collected in some provinces of Vietnam Average isolation rate of Banna virus from mosquitoes is 4.22% of collected mosquito samples... regions, Banna virus isolation rate ranges from 1.61% to 16.67% Among 46 Banna virus strains isolated from mosquitoes, there are 02 strains isolated from male Culex pseudovishnui mosquitoes in Long An, 44 strains of Banna virus were isolated from female mosquitoes 3 Determining some bio-molecular characteristics of Banna virus circulating in Vietnam There are 5 Banna virus strains sequenced gene coding... Indonesia belong to genotype B Genotype A is divided into two subgroups: A1 and A2; genotype A1 is divided into 4 independent clades including Banna virus strains isolated in Northern China, Liaoning and Vietnam; genotype subgroup A2 include Banna virus strains isolated from the Central region of Vietnam and China 22 Molecular characteristic of viruses in Reoviridae family is double stranded ARN including . epidemiological characteristics, clinical syndrome of acute encephalitis syndrome suspected to be caused by Banna virus in some provinces of Vietnam, 2002- 2012. 2. Determine infection ratio of Banna virus. epidemiological characteristics of acute encephalitis syndrome suspected to be caused by Banna virus in some provinces of Vietnam was conducted with three following specific goals: 1. Describe some epidemiological. (now part of Ha Noi) and Quang Binh provinces in 2002. Participating in monitoring, diagnosis, treatment and prevention of AES suspected to be caused by Banna virus, the study Some epidemiological