| Arbovirus is a term used to refer to a group of viruses that are transmitted by arthropod vectors. It is mainly composed by genus Flavivirus in Flaviridae, genus Alphavirus in Togoviridae and Bunyavirudae arbovirus.In recent years, a variety of Bunyaviridae arbovirus have been found in China, they are Severe fever with thrombocy-topenia syndrome bunyavirus(SFTSV), Tahyna virus(TAHV) and Batai virus(BATV). Furthermore, there are many other viruses including Chaoyang virus(CV, genus Flavivirus) and Getah virus(GETV, genus Alphavirus). There is enough proof that the arbovirus were likely to enter the country through various channels. In terms of the popular trend of TAHV(family Bunyaviridae, genus Orthobunyavirus, California serogroup) was first isolated in the former Czechoslovakia in 1958 from a pool of Aedes caspius mosquitoes. TAHV is widely distributed in central Europe and isolated in the Soviet Union. The presence of TAHV in China was postulated after antibodies were detected by serum specimens collected from healthy adults in Xinjiang Province in 1985. In 2006, Tahyna virus was isolated from Culex spp. mosquitoes collected in Xinjiang. Bunyaviruses has existed China, in addition to its popular ecological conditions, it cannot be ruled out that there is a possibility of existence of other California serogoup virus in our country.All the California serogroup viruses cause a similar encephalitic disease in humans. It includes California encephalitis, guaroa, Inkoo, Jamestown Canyon, La Crosse, Snowshoe hare and Tahyna viruses.For those not found in Chinese arbovirus, enhancement of monitoring for arbovirus in our country to ensure strict control and prevention of the virus from entering China,, especially for some serological survey results which showed antibody positive but not isolated arboviruses should be the top priority of monitoring.Otherwise if there is new arbovirus diseases found or discovered, we will not be able to determine whether it is a local disease or not. Major arbovirus monitoring for our country which have been found arbovirus is another important task. It is effective to prevent the irreparable loss of public property from epidemic.Dengue fever is now the fastest spreading insect-borne virus in the world and has reached "epidemic potential," the World Health Organization said, the number of cases increased by 30 times in the past 50 years. Dengue fever has now spread to every continent and has infected around 25 million people. Therefore, port personnel should improve the surveillance of infectious diseases in the area, strengthen information exchange and cooperation of the port inspection and quarantine institutions with Vietnam, Laos and other foreign sanitation quarantine authority, and discovery of intrusion of major arboviruses by the local population. Part One Development of TaqMan real-time RT-PCR for detection of LaCrosse virus and Snowshoe hare virusObjective:This study intends to establish new methods for the viruses which have not existed in our country, especially for the arboviruses rapid detection technology research. In this study, LACV and SSHV RT-PCR method for detection were established and applied for arboviruses carried detection in mosquitoes.Methods:The twin primers and the TaqMan probe were designed and synthesized based on the S gene segment of LACV and SSHV by using Designer Beacon 7 software. The reaction conditions and primer concentrations were optimized by fluorescence RT-PCR, and the annealing temperature was set at 45℃, 50℃, 55 ℃and 60℃, the final probe concentration was set at 100 nM, 200 nM, 400nM; upstream and downstream primers concentrations were set at 200 nM, 400 nM and 800 n M. The best primers and probe concentrations were ensured through the amplification curve and the Ct value, sensitivity and specificity of the method was verified.Results:1. LACV fluorescence RT-PCR detection method was established, the optimal annealing temperature was 55℃, the final concentration of primers were 200nmol/L, the final concentration of the probe was 200nmol/L. The sensitivity of the method was 1.41×103copies/μL. There was no cross reaction with Alphavirus, Flavivirus, Bunyavirus and Seadornavirus.2. SSHV fluorescence RT-PCR detection method was established, the optimal annealing temperature was 55℃, the final concentration of primers and the final concentration of the probe were 200nmol/L. The sensitivity of the method was 5.6×102copies/μL. There was no cross reaction with Alphavirus, Flavivirus, Bunyavirus and Seadornavirus.3. Using the newly established LACV and SSHV fluorescent RT-PCR method to detect the 338 batch of mosquito samples of RNA, positive results were not found.Conclusion:Fluorescence RT-PCR detection methods for LACV and SSHV detection were established and characterized by rapidity and sensitivity. Part Two Seroepidemiological survey of important mosquito-borne diseases among China- Laos borderObjective:To provide data support for the prevention and control of disease by selecting the serum of the health population in border area, screening epidemic situation of CHIKV and DENV infection.Methods:The samples were collected from people travelling among Yunnan Mohan port. IgG antibody of CHIKV and DENV were detected by ELISA. Using Excel and SPSS17.0 statistical analysis, the composition ratio, positive rate,properties of occupation, age and gender were analyzed.Results:A total of 182 serum samples were detected by Serological survey, 42 of them were positive. Positive infections in CHIKV were 39(21.42%), DENV had 3 positive(1.65%), the total positive rate was 24.08%. There was no significant difference in the positive rates between the gender and ages of the two viruses. In occupational classification, DENV antibody positive compared with other occupations have significant differences.Conclusion:Seroepidemiological survey shows CHIKV antibody positive rate was 21.42%, positive Business of DENV antibody had significant difference compared with other professional(staff, students, support staff and crew). |
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