| BackgroundDengue Fever(DF) is an acute mosquito-borne disease caused by four serotypes of dengue viruses,DENV1-4. Dengue Virus (DV) is primarily transmitted by Aedes albopictus and A. aegypti. Dengue virus infected mosquitoes can be proliferated massively in salivary glands of the mosquitoes and transmitted to humans by biting people. People infected by any one of the serotypes of dengue viruses, presented the characteristic clinical symptoms of dengue fever such as high fever, headache, muscle and joint pains, skin rash, enlargement of lymph nodes and low white blood cell count, or even worse to be dengue hemorrhagic fever and dengue shock syndrome which have high death rate. Dengue fever is widespread in more than 100 countries and regions in global tropics and subtropics, especially in Southeast Asia and West Pacific regions like Indonesia, Singapore, Thailand, Vietnam, Burma, India, Bhutan, SriLanka, Maldives, Bangladesh etc. The most serious epidemic situation is in the Southeast Asia countries which are beside our country,70% of the population is likely at risk of infecting dengue virus, and these countries have cases of dengue fever every year. The incidence of dengue fever has increased considerably in recent decades. Statistics from WHO displayed that over 40% of population (approximately 2.5 billion) in the world is facing the risk of infecting the DF and severe dengue fever (dengue hemorrhagic fever and dengue shock syndrome), there are about 50 million to 100 million new cases every year in the world. In 2013 the DF cases reach 96 million-three times the number for all of 2012. So prevention and control dengue fever has been an international public health issue to be solved urgently.China is also the high incidence of Dengue fever, from a sudden outbreak of Dengue fever in Foshan on May,1975,Guangdong province, case reports about Dengue fever can be found every year(except for 1983,1984 and 1996).Dengue fever is mainly epidemic in southern places like Guangdong, Hainan, Guangxi, Fujian, Taiwan and Southeast Coastal Areas. Among these places, the epidemic in Guangdong is the most severe. Four kinds of serum type were popular, and dengue virus type 1 was the predominant one. Since entered this century, there were case reports about Dengue fever almost every year in Guangdong province, demonstrated interval epidemic, every four or seven years, would be seen a trend of epidemic, and spread to a wider and wider range. After twice relatively larger scale of outbreak in 2002 and 2006, epidemic was controlled effectively. However, a large-scale outbreak has been occurred in China in 2013, especially the pandemic in 2014, the number of cases and epidemic areas reached a new high of nearly thirty years, much attention to the severity of the epidemic has been paid.With domestic and international exchanges and frequent population movements, the probability of occurrence and spread of dengue has increased considerably since Special Economic Zone was established in Shenzhen. After the first imported dengue fever case report in 2001, there are constantly imported cases reported, the increasing of epidemic in Southeast Asia is a big threat to Shenzhen in DF control. The overseas imported cases of DF in Shenzhen are all from Southeast Asia for years. In 2010, several suspected cases of DF on a building site in Shenzhen Futian district had been found and they were confirmed to be DENV-1 infected. Yangfan et al. inferred that they were local cases, and there may be a DENV-1 plague focus in Shenzhen, but this result has not been confirmed further. A survey on Antibody levels of DENV in healthy people in Shenzhen 2011 showed that inapparent infection in healthy people in Shenzhen is common. From September to December in 2014, DF outbroke and spread in Shenzhen.454 confirmed cases were reported. The number of cases is obviously bigger than the previous years, which is a major characteristic of this pandemic. Banan district and Futian district are the worst-hit areas. And the local infection reported earlier than last year. In 2013 the first local dengue case reported in late October but in 2014 report time is more than a month in advance than last year. Shenzhen is located in the south of Guangdong, and in the majority of the floating population; as subtropical Marine climate, the average climate is 22 ℃, with abundant rainfall, and is suitable for mosquito breeding. So the breakout of epidemic in 2014 Shenzhen does not exclude it caused locally. The virus may survive through "aedes-humans-aedes" villiage cycle and latent infection flow city cycle model, and breakout under a proper condition. However, this conclusion needs further study.ObjectThe cases of dengue fever in Shenzhen Baoan People’s Hospital occurring from the September to December 2014.ObjectiveTo determine the serotype of isolated dengue fever virus strain occurring from the September to December 2014 in Shenzhen and understand predominant serotype. Through conducting the homology and phylogenetic analysis of the E gene and genome sequence of the isolated dengue fever virus strains, in order to analyze the molecular characteristics of the epidemic dengue virus strain as well as explore the possible origin in Shenzhen.Methods1. Collecting the data of 35 confirmed cases of dengue fever which include the epidemiologic data, clinical feature and Laboratory tests. Serum IgM of Dengue virus and viral nucleic acid is confirmed by Centers for Disease Control of Shenzhen2. Extracting the early serum viral RNA from patients in acute phase of dengue fever, and synthesizing cDNA by a Reverse Transcriphase(RT) to polymerase chain reaction(PCR) amplification with universal primer in the first round, identifying the serotypes of the dengue virus isolates by the size of products yielded in the second round PCR amplification with specific primers.3. Carrying out the culture and isolation of the dengue virus with BHK-21 cell from the acute serum of six DENV-1 infectious cases.4.Using the software Primer 3.0 and DNAMAN to design two pairs of DENV-1 E gene primers to amplify and sequence the whole length E gene by RT-PCR. Homology and phylogenetic analysis were also constructed.5. Using the software of PCR DESIGN and DNAMAN to design 11 pairs of PCR primers, RT-PCR amplification of the whole genome sequence of one DENV-1 virus strains and then sequenced. The bioinformatics analysis was performed.Results1. In the 35 confirmed cases of dengue fever, local cases accounted for 80.0%, while imported cases accounted for only 20.0%.2. The results of RT-nPCR of 21 patients were positive. For the agarose gel electrophoresis analysis,20 cases yielded a sized 482bp type-specific fragment and identified DENV-1, only one cases yielded an 119bp type-specific fragment and identified DENV-2.16(80.0%)DENV-1 cases were local ones and 4 (20.0%)DENV-1 cases were imported, while DENV-2 case was imported.3. Serum samples from 6 patients whose PCR positive with early stage dengue fever were used to culture and isolate dengue virus by BHK-21 cells,6 specimens cell swelling appears rounded vacuoles.4. The complete coding region of E genes from 6 DENV-1 strains was all 1485 nucleotides and encoded 495 amino acids. Nucleotide sequence homology of 6 strains DENV-1 was 100.0% identical, and their homology was very similar with DENV-1 virus epidemic strains in 2010 in Shenzhen. Nucleotide sequence homology was between 99.5%, and the deduced amino acids sequence homology was 99.8%. Compared with epidemic DENV-1 strains isolated from other country, E gene of six strains DENV-1 from Shenzhen in 2014 was close to the strains isolated from Singapore and Japan, and the highest homology of their E genes and deduced amino acids were 99.7% and 100%. The phylogenetic tree of E genes indicated that six strains DENV-1 had the greater similarity with Shenzhen2010, Singapore2009 and Japan2004 and they all belonged to the genotype Ⅰ.5. Genome sequence of DENV-1 strain isolated from two patients,shows homology of two strains of shenzhen DENV-1 was 100.0%. According to whole genome sequencing determination, the DENV-1 strain reaches 10735bp in lengthen and encodes 3329 amino acids. The phylogenetic tree of whole genome sequence also indicated that the isolated dengue virus type 1 belonged to gene subtype Ⅰ, that was highest homologous with the strain of the virus isolated from Southeast Asia countries. Compared with the GZ/80 which also belonged to dengue virus type 1 gene subtypes G I, DENV-1 isolation from Shenzhen appeared 430 nucleotide variations, and 41 amino acid variations. There were 94bp in 5’ non-coding region and formed the same secondary structure of seven stem-loop although they had one base difference; while there were obvious differences in 31 noncoding regions,there were 495bp and had a total of eight sites of nucleotide variations, those variations caused the former formed 21 stem-loop secondary structures, and the later formed 24 stem-loop secondary structures.Conclusions1. The serotypes of dengue fever in Shenzhen from September to December 2014 appeared as DENV-1 and DENV-2. The dominant serotypes was DENV-1 and DENV-1 infection majorly came from the local.2. The phylogenetic tree based on E gene and whole genome sequence both indicated that DENV-1 isolates from Shenzhen in 2014 had the greatest similarity with isolates from Singapore, Malaysia and other Southeast Asian, suggested that DENV-1 isolates from Shenzhen in 2014 may be transferred from Southeast Asian region.3. Compared the six DENV-1 isolates in Shenzhen in 2014 with the local isolates in Shenzhen in 2010, we found out they were highly homology and had close evolutionary distance, those strains were isolated only after a lapse of four years, this suggested that the DENV-1 isolates in Shenzhen in 2014 were closed to the strains in 2010. And belongs to natural and social factors, we assumed that these epidemic dengue virus strains had been "settled down" in Shenzhen in 2010 and then caused 2014 dengue fever outbreak. There maybe exists a plague focus of DENV-1 in Shenzhen.4. DENV-1 isolates in Shenzhen in 2014 were not highly homologous with the GZ/80 strain which belonging to the same genotype, and they had a certain variation. |
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