| BackgroundDengue fever is an infectious tropical disease and now widespread in the world. Many countries had experienced a dengue fever pandemic several times. Millions of people suffer from dengue fever during a pandemic period. It has been become a serious threat to the healthy of about25billions people in over100countries and major public health problem we are now facing. In the high prevalence countries and regions in southeast Asia and south America, the epidemiological history of dengue fever mostly begin from some imported cases, after years of development and the imported cases keep coming, the situation evolved into several dengue fever virus types coexist, finally, dengue fever localized and become endemic, while the prevalent district and region will keep enlarging.In China, no district has reported to be a natural epidemic focus of dengue fever expect Yunan province. In recent years, Guangdong province have reported dengue fever cases every year, Fujian and Zhejiang province also arose dengue fever outbreak which were infected locally. Although other provinces in china haven’t report such cases, the number of province that reported imported dengue cases were increasing constantly, this number reach20in2008.Guangzhou city was always considered to be the key region of dengue fever prevention in Guangdong province and even Chinese mainland. There are13dengue fever prevalent years from1978to2010in Guangzhou. The prevalent-intensity of dengue fever stay low before1995, after that, there have been several large-scale dengue fever epidemic, which occurred in the year of1995,2002,2003,2006and2010. Between1990and2010, the proportion of the dengue fever cases in Guangzhou to Guangdong province was63.29%, and to the nation was49.15%. Hence, Guangzhou will always be a significant source of dengue fever contagious.The changing of the epidemiology features of dengue fever carry out the question that whether Guangzhou has become a natural epidemiology focus. From1980s to1990s, Guangzhou’s dengue fever epidemic situation was primary cause by imported cases and existed all the four type dengue virus. Furthermore, DENV-1and DENV-4erupted alternately in1991. Luo Huiming analyzed the dengue fever epidemic between1990and2000, which supposed that Guangzhou’ epidemic dengue virus may come from the nations and areas of Southeast Asia and Western Pacific. But the epidemiological research of dengue cases in Guangzhou shows that some of the cases are without travel history and no afferent cases have been reported in that area. In2006, the strain of dengue which cause local prevalence was confirmed DENV-1while the afferent cases were DENV-3and most dengue outbreak point had been found no epidemiological connection of the imported cases. In addiction, the prevalent virus in Guangzhou is DENV-1after1990s, which was high homology with the virus found between2002and2003and between2006and2007. In2007, Aedes albopictus larva insects of dengue fever outbreak in2006from Conghua district was tested dengue virus positive and confirmed dengue DENV-1 after gene sequencing. In2009, local infected cases caused by DENV-3dengue virus erupted for the first time in30years. In2010, the distribution of dengue virus of local infected cases is contrary to the way it usually behaves,4types dengue virus prevalent jointly in the same year.Therefore, there comes questions. The prevalence of dengue fever in Guangzhou comes from the circulate of the local strain or come from the imported strain? Is dengue fever in Guangzhou has the trend of changing from based on imported cases to localization? Is Guangzhou have any natural epidemiology focus? What’s the regular pattern of the prevalence and dissemination of Guangzhou’s dengue fever? All the questions above are waiting to be solved.PurposeInquiring the existence of the natural epidemiology focus in Guangzhou and analyzing the regular prevalent pattern and evolution model of dengue fever, then analyzing the scope and mold of distribution to answer the questions that whether the prevalence of dengue fever in Guangzhou comes from the circulate of the local strain or come from the imported strain. After that we could provide a scientific reference for setting up a risk evaluation and reliable prediction and warring system. To understand the epidemiology trend and evolution law then provide a scientific reference to adjust and establish proper prevention and control strategy of dengue fever. Establish risk evaluation and prediction system to improve the ability of prevention and help to control dengue fever more effectively, furthermore, guide the development and implementation of targeted control measures. Only in this way can we enhance the prevention effectiveness, save economize resource, safeguard the labor forces and improve health conditions of all multitude.Content(1) Collect the prevalence data of dengue fever from1978to2010of Guangzhou, analyze the epidemic feature, trend and dynamic changes of dengue fever from different periods in Guangzhou.(2) Survey the dengue virus antibody level of people from outbreak areas and not outbreak areas, analyze the recessive infection level different groups of people and assess their susceptibility to dengue fever.(3) Launch field epidemiology investigation of dengue fever outbreak to chase infection sources.(4) Sequence the gene of dengue fever epidemic strains from past years, then build phylogenetic tree of dengue virus and do bioinformatics analysis to determine the source and variation of dengue fever epidemic strains in Guangzhou, finally establish dengue virus molecular biology information library of Guangzhou region.(5) Research the dengue virus carrying status of main vector in natural environment and analyze the homologous relationship between dengue virus from main vector and from epidemic strains.(6) Investigate the dengue virus infection status of main host animal.Method(1) Field epidemiology investigation:Establish representative surveillance spot all over Guangzhou, not only in previous dengue fever outbreak areas, to capture sufficient Aedes adults and larva worms, then analyze the species of dengue fever host animal and their dengue virus carrying status in the infected mosquito-bone’s maximum active areas.(2) Geographic epidemiology:Survey and mark the type of breeding ground for the dengue virus spreading mosquito-borne, analyze its geographical distribution and seasonal fluctuation associated with local climate.(3) Molecular epidemiology:use C6/36cells to culture dengue virus and extract dengue viral RNA, then launch RT-PCR test for the amplification of E gene, then sequence it to establish dengue virus gene sequences database in Guangzhou. Compare the sequencing results to the gene of virus reference strains from GenBank, use DNAstar software package to analyze gene sequence, use BLASTN gene bank to compare different gene, use CLUSTALX1.83to sequence gene queuing and use MEGA4.0to draw evolutionary tree. Analyze the homology among local dengue virus strains from different year, between local strains and imported strains, between virus strains from mosquito or host animals and virus strains from infected people.Result1. Collect the prevalence data of dengue fever from1978to2010of Guangzhou analyze the epidemic feature of dengue fever from different periods.(1) In Guangzhou, the occurrence of dengue fever has gone through three prevalence periods and experienced12high prevalence years, the incidence rate intermittent increase periodically and experienced an incidence rate peak every3to5years.(2) All four types dengue virus have been occurred in Guangzhou, outbreak in every year was caused by a single type of dengue virus excepting1991, which experience two types alternately.(3) From May to December, there was always having dengue cases occurred in Guangzhou. From July to October was the significant incidence peak of dengue fever.(4) Urban areas experienced a higher incidence rate, followed by the outskirts areas; the incidence rate of rural areas was lowest. From1978to2007, the top three districts which experienced more cumulative dengue cases were Panyu District, Haizhu and Yuexiu District.(5) The incidence rate of dengue fever in Guangzhou is low, the year recorded a highest incidence rate was1995, reached83/100000per year, which was caused by a serious outbreak in Shawan town, Panyu district. The deaths surveillance system shows that there was a total of12deaths cases due to dengue fever from1978to2007and the last deaths cases reported in1991, there is no deaths case reported since then.(6) The prevalence feature of dengue fever in Guangzhou still characterized by imported cases to cause local outbreak. Despite lack of sufficient molecular evidence, but the epidemiological history of the index local cases and the relationship between the onset time of local cases and imported cases show that local dengue fever epidemic was mainly triggered by imported cases.(7) Age and gender distribution:the incidence rate of male and female are basically the same and with no significant different (P>0.05). All the people from different age groups have cases reported, besides, the age group of25-50reported most of cases. Occupation distribution was housework and unemployed, workers and retired employees based.(8) Imported cases epidemic characteristics:Between2006and2010, except January, the rest of the year all have imported cases reported, which were from dengue endemic areas around the world, especially the Southeast Asian countries and regions nearby Guangzhou. The age group which reported most imported cases of was20-40, accounting for83.3%of total cases. Imported cases pathogen surveillance results show that four types of dengue virus were all isolated. All the cases confess a travel history and all onsets in a maximum incubation period.2. Dengue fever serological antibody level survey of people in Guangzhou.(1) The dengue virus antibody level of people in Guangzhou is low. A total of904serum samples were collected from outbreak spot, only7of which tested for IgG antibody positive, with the positive rate0.77%(7/904). A total of1347serum samples were collected from normal area, only3of which tested for IgG antibody positive, with the positive rate0.22%(3/1347). The positive rate between outbreak spot and normal area were no significant different (P>0.05). Both in outbreak spot and normal area, the positive rate of age groups were significant different (P<0.05).(2) From2009to2010, the investigation of latent infection status of people from four outbreak spots shows that the latent infection rate was low and between0.0% and2.0%.(3) A total10samples were tested for IgG antibody positive,7of them were from outbreak spot and3of them from normal areas. The positive rate between outbreak spot and normal area were no significant different (P>0.05).3. Molecular epidemiology and virus gene tracing research of GuangzhouTotally130dengue virus strains were isolated from2000to2010. All the strains were classificated by years, types and sources and launch gene sequence. After that, a dengue virus molecular biology information database was established and successfully used for gene tracing research of Guangzhou, which finally help to confirm the source and molecular biology characteristics of dengue virus in Guangzhou and the nature feature and spread rule of dengue fever in Guangzhou.(1) In2006, two dengue virus strains from imported cases of Indonesia and the Dominican Republic was confirmed to be nothing to do with local virus strains in Guangzhou. Virus isolation test shows that59serum samples of local infected person were all confirmed to be DENV-1virus. Evolutionary tree analyze results that the gene sequence of DENV-1virus strain GZ06/1707was similar with the strains from Cambodia (1998), Thailand (2001), Myanmar (2001), Zhejiang province (2001) and a strain from Guangzhou in2005.(2) Gene sequence analyze of DENV-1E gene from Guangzhou in2009shows that the nucleotide sequence homology of four DENV-1strains was between90.5%to100%. Evolutionary tree homology analyze results that strains09/GZ/11562and09/GZ/11534were highly homology with GZ061707(2006)、FJ231/04(2004),09/GZ/11562and GZ061707even share the same Amino acid sequence.(4) Gene sequence analyze of seven DENV-3virus strains E gene from Guangzhou in2009shows that the nucleotide sequence homology of strains1081、1483、10806was between99.8%to100%and strains10616、11144、11194、13105was between99.9%to100%, the nucleotide sequence also shows that homology of strains1081、1483、10806and Guangxi province80-2was between99.5%t o99.6%. Evolutionary tree homology analyze compare those strains to28strains, E gene from other countries and results that strains1081、1483、10806was belong to Southeast Asia/South Pacific type and strains10616、1114、11194、13105was belong to Indian sub-continent type.(5) Gene sequence analyze of five DENV-4virus strains from a outbreak in Jingtai street, Guangzhou in2009shows that the nucleotide sequence homology of those five strains was between99.9%to100%. The nucleotide sequence also shows that homology of strain CH/Guangzhou10660/2010and a strain02-12-1HuNIID which imported from Thailand and isolated from Japan was98.9%. Compare to strain SW36i from Indonesia, strain Guangzhou B5from Guangzhou in1990and strain CN78-56from Foshan city, Guangdong province, the nucleotide sequence homology result was98.8%,93.9%,96.7%respectively. Evolutionary tree homology analyze compare the DENV-4strain E gene of CH/Guangzhou10660/2010to other strains from other countries and different years, the result shows that strain CH/Guangzhou10660/2010have a closest relationship with strain02-12-1HuNIID isolated from Japan and imported from Thailand, strain0712aTw isolated from Taiwan and imported from Indonesia, strain SW36i isolated from Indonesia. Strain CH/Guangzhou10660/2010also share the same DENV-4/GII gene with strain ThD4-0734/00isolated from Thailand and imported from Thailand, strain0108aTw isolated from Taiwan and imported from Singapore, strain CN78-56isolated from Foshan city and strain Dominica/81. Compare to strain ThD4-0087/77, ThD4-0485/01, ThD4-0348/91from Thailand and Guangzhou B5from Guangzhou, CH/Guangzhou10660/2010was distantly related with them. The most distantly related strains of CH/Guangzhou10660/2010were strain ThD4-0017/97and strain ThD4-0476/97from Thailand. 4. Main vector’s dengue virus carrying status research.(1) Use RT-PCR test to detect dengue virus of Aedes albopictus larvae samples, which were captured from different surveillance spot and outbreak spot in Guangzhou. There was total118batches of Aedes albopictus larvae been collected in2005and214batches collected in2006, but only one batch in august2006test for DENV-1virus positive.(2) Two mosquito samples, captured from old outbreak spot in2007, tests for dengue virus nucleic acid positive.(3) captured Aedes albopictus larvae specimens from various types of antiquated water from seven surveillance spot of natural environment (Including the external and residential indoor environment, most of places were belong to dengue fever outbreak areas) and capture Aedes albopictus adult mosquitoes by using electric mosquito suction device, a total of12348Aedes albopictus were collected and detected dengue virus, the results of those Aedes albopictus were all negative.5. Host animal dengue virus carrying status researchA total of1653animals serum or brain cell samples, included266dogs,916rats,300pigs,121cats and50bats blood samples were collected, all the positive control can be amplified a corresponding bands. During the experiment, some samples have appeared some positive bands, but after careful verification, positive results were confirmed false-positive. The detection of rats and bats blood samples was negative for dengue virus.ConclusionAccording to the epidemical features and trend analysis of dengue fever in Guangzhou from1978to2010, to the serological investigation of both people from outbreak area and normal area, to the field epidemiological investigation and routine surveillance of imported cases and outbreak events, to the investigation of dengue virus circulating in the natural environment and the main vector of the virus how to carry and pass the virus, to the research of host animal dengue virus carrying status, especially the sources tracing analysis of dengue virus strains from different years by using biological technology, indubitably indicate that the dengue fever’s prevalent feature in Guangzhou still belongs to "imported epidemic or imported cases caused local transmission", still have not become a endemic dengue fever natural epidemic focus. In accordance with the following basis:1. Over the years, Guangzhou’s epidemic situation still stay semi-natural, cases here have no distinct distributional difference in age and sexuality.2. Both people from epidemic area and normal area got a extreme low level of dengue virus antibody and most of the people were susceptible to dengue fever. The implicit rate of infection was low in epidemic area, once infected, most of people will fall ill.3.Over the past three years, author work on the specimen of aedes albopictus from six district and seven surveillance spots, come out with the result that our the all the samples tested negative and with no direct evidence of aedes albopictus carrying virus to survive for winter or transversal transmission.4. The author fined no dengue virus infection in the research of the primate host animal from natural environment.5. The gene sequencing test point out that the local strain in Guangzhou was homologous with which prevalent in Africa, America, especially Southeast Asia.However, Guangzhou do have the natural element and social condition that suit for the prevalence of dengue virus:(1) Objectively, Guangzhou located at subtropics and got a high mean temperature of air and a large amount of precipitation, the climatic conditions is appropriate for the survival of aedes albopictus.(2) More frequently the population mobility acts, more possibility the dengue fever transmits from overseas and other region. What’s more, as the epidemic area of dengue fever was keeping expanding, the epidemic of dengue fever in urban area could be prevalent in a low-intensity of mosquito density, then spread outwards rapidly when population move around frequently.(3) The population mobility is frequent and the member of vacant house is big in the old urban and urban fringe area, which led to the result that there are lots of mosquito breeding spot all over the internal and external environment. We are still lack of effective methods to prevent the breeding of mosquitoes. In this way, once dengue fever outbreak, it’s difficult to bring the epidemic under control.Consequently, if the dengue virus adapt to the main vector and host animal more well, we couldn’t exclude the probability that Guangzhou may turn into a natural epidemic focus of dengue fever and dengue fever become an endemic diseases in Guangzhou City. |
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