Dengue ; epidemiology ; Dengue Virus ; genetics ; isolation & purification ; Evolution, Molecular ; Genes, Viral ; Humans ; Viral Envelope Proteins ; genetics

OBJECTIVE: To investigate the rapid and accurate method for the detection of dengue virus (DENV) by using nicking enzyme assisted strand-displacement amplification (SDA) combined with gold nanoparticles-based lateral flow strip. METHODS: Total RNA of the virus was extracted by using magnetic beads method and transcribed to cDNA for SDA detection system. Nicking enzyme-assisted method was used for detecting DENV, and agarose gel electrophoresis was used for analyzing the sensitivity of SDA amplification products. A gold nanoparticles-based lateral flow strip was developed based on the principle of nucleic acid base complementary pairing to design the test line and control line. The gold particles were prepared by using sodium citrate reduction method for gold nanoparticles-based lateral flow strip construction. RESULTS: The sensitivity of the SDA method was 10 fmol/L, and the sensitivity of gold nanoparticles-based lateral flow strip based on SDA method was also 10 fmol/L. In a linear range from 10 fmol/L to 10 fmol/L, the corresponding linear correlation coefficient () of DENV was 0.98. The specificity of nanoparticles-based lateral flow strip based on SDA for DENV detection was high, which was no crossing with other control groups. CONCLUSIONS A gold nanoparticles-based lateral flow strip based on SDA method for DENV detection has been established, which is convenient, fast, and the result is visible to naked eyes.
Dengue ; diagnosis ; Dengue Virus ; isolation & purification ; Gold ; Humans ; Metal Nanoparticles ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVETo screen the molecules binding dengue II virus expressed in Aedes albopictus C6/36 cells and characterize their biological functions.

METHODSAedes albopictus C6/36 cells were infected with dengue II virus, and the virus were collected and purified. The total and membrane proteins of C6/36 cells were extracted and analyzed using 12% SDS-polyacrylamide gel (PAGE). After electrophoresis, the proteins were transferred to a nitrocellulose membrane, and virus overlay protein-binding assay (VOPBA) was carried out using an anti-dengue virus 1-4 monoclonal antibody.

RESULTSTwo specific bands of 67 000 and 30 000 occurred after VOPBA of the proteins from the cells incubated with the virus, while the negative control group did not show these specific bands.

CONCLUSIONTwo putative dengue virus receptor molecules of 67 000 and 30000 have been obtained from C6/36 cells using VOPBA, and their functional identification is in progress.

Aedes ; cytology ; virology ; Animals ; Cells, Cultured ; Dengue Virus ; isolation & purification ; Membrane Proteins ; Receptors, Virus ; isolation & purification ; metabolism ; Virus Attachment

Dengue fever is an acute febrile disease caused by the dengue virus, which belongs to the flaviviridae family, and this virus is transmitted by the bite of the mosquito Aedes aegypti. It occurs in the tropical climates of the South Pacific, Southeast Asia, India, Africa and the subtropical zone of America. Imported cases of Dengue fever and Dengue hemorrhagic fever are rapidly increasing as many Koreans are now traveling abroad. Liver injury is usually detected by laboratory investigation according to a surveillance protocol. Although liver injury by dengue virus has been described in Asia and the Pacific islands, the pathogenic mechanisms are not yet fully clarified. It is usually expressed in a self-limiting pattern and the patient has a complete recovery. We report here on a case of a young woman who presented with general weakness, nausea and significant elevation of the aminotransferase levels, and she was diagnosed with dengue fever.
Acute Disease ; Adult ; Dengue Hemorrhagic Fever/complications/*diagnosis/virology ; Dengue Virus/*isolation & purification ; Female ; Hepatitis, Viral, Human/*diagnosis/virology ; Humans

OBJECTIVETo study the epidemiology and etiologic characteristics of a Dengue fever outbreak in Fuzhou from the beginning of September to the end of October in 2004 in order to understand the source of infection.

METHODSData on descriptive epidemiology was collected to study the characteristics and related factors to the epidemic. Dengue virus was isolated through the use of C6/36 cell line while viral serotypes were identified by indirect immunofluorecent assay with type-specific monoclonal antibody. The sources of infection were traced by nucleotide sequencing.

RESULTSDuring the epidemic, 93 cases occured consistently with the region entomoplily growth and decay. The viruses of 6 strains isolated from 10 patients' blood specimens were identified as dengue virus type 1. Phylogenetic evidence suggested that the viral isolate had high genetic relation with the isolates from Kampuchea (DENV-1/KHM/2001; GenBank Accession No. L0904278).

CONCLUSIONThe epidemic was caused by introduction of patients migrating into Fuzhou.

China ; epidemiology ; Dengue ; epidemiology ; Dengue Virus ; genetics ; isolation & purification ; Disease Outbreaks ; Emigration and Immigration ; Genetic Variation ; Humans ; Phylogeny

OBJECTIVETo screen DENV-2 binding proteins from Aedes albopictus and Culex. quinquefasciatus.

METHODSThe total proteins of Aedes albopictus and Culex. quinquefasciatus in different developmental stages were prepared and analyzed with SDS-12% polyacrylamide gel. After electrophoresis the proteins were transferred using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad ) to a nitrocellulose membrane. Virus overlay protein-binding assay (VOPBA) was carried out using anti-dengue virus 1-4 monoclonal antibody.

RESULTSIn Aedes albopictus, VOPBA detected DEN-2 binding molecules of 25 000, 35 000, and 50 000 in larvae samples, molecules of 35 000 and 50 000 in pupae samples, a 50 000 molecule in male mosquito samples, and molecules of 35 000 and 50 000 in female mosquito samples. DENV-2 binding protein of 35 000 was found in the larvae, pupae, and female mosquitoes, but not in male mosquitoes. In Culex. Quinquefasciatus, VOPBA detected a molecule of 100 000 in larvae samples, molecules of 40 000, 100 000, and around 50 000 (48 000 and 60 000) in pupae samples, and molecules of 40 000 and 100 000 in male mosquitoes and female mosquito samples.

CONCLUSIONSeveral proteins capable of binding DENV are found in Aedes albopictus and Culex. quinquefasciatus in different development stages. The 35 000 molecule expressed in Aedes albopictus as a putative receptor protein may be related to virus tropism in mosquito tissues.

Aedes ; virology ; Animals ; Culex ; virology ; Dengue Virus ; Female ; Insect Proteins ; isolation & purification ; Larva ; Male ; Pupa ; Receptors, Virus ; isolation & purification

OBJECTIVETo develop multiplex reverse translation-polymerase chain reaction (RT-PCR) method for detection of dengue virus type 1-4.

METHODSBased on the genomes sequence analysis of dengue virus type 1-4, four-pair of primers were designed. The specificity of the primers was primarily tested by searching the GenBank DNA sequence database. The optimal reaction conditions of the multiplex RT-PCR were then established. The specificity of RT-PCR was tested using the homologous yellow fever virus and Japanese encephalitis virus. 30 serum samples of dengue virus from suspected sufferers in the prevalence of dengue virus in 2003 were detected using the methods we developed.

RESULTSPositive segments about 295, 237, 118, 347 bp could be seen in the multiplex RT-PCR production of dengue virus type 1-4, respectively. There were no positive segments in the RT-PCR productions of Japanese encephalitis virus and yellow fever virus. 25 of the 30 serum samples showed dengue virus type 1 positive results, while the sequencing results suggesting the amplification sequence having a high homology with dengue virus type 1 strain Cambodia, GD14/97 and GD05/99 (97%, 97%, 98%, respective).

CONCLUSIONThe method of multiplex RT-PCR we established could be used for early detection and identification of dengue virus type 1-4.

Base Sequence ; China ; epidemiology ; Dengue ; epidemiology ; virology ; Dengue Virus ; classification ; isolation & purification ; Humans ; Molecular Sequence Data ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Seroepidemiologic Studies ; Severe Dengue ; virology

Aedes ; virology ; Animals ; Dengue Virus ; classification ; isolation & purification ; Genome, Viral ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo determine a dengue fever outbreak in Yiwu city, Zhejiang Province in 2009 and to trace the origin of the pathogen.

METHODSThe dengue virus IgM, IgG antibodies and viral nucleic acid were detected and virus was isolated using 40 serum samples from the suspected patients. The viral RNA of the isolated virus strains was extracted and the E gene was amplified by RT-PCR. The amplicons were sequenced and the phylogenetic and homological analyses were also constructed.

RESULTSAmong 40 serum samples from dengue fever suspected patients, 17 were positive from for dengue IgM (42.5%); 4 were IgG positive (10.0%); 34 samples were dengue virus RNA positive (85.0%), 28 dengue virus type 3 (D3) strains were isolated (70.0%). The complete coding region of envelope genes (E) from 13 D3 strains was all 1479 nt without any insertion or deletion, which encoded with 493 amino acids (aa). E gene from the 13 D3 strains from Zhejiang in 2009 (D3/ZJ/2009) was 100.0% identical. The strain from Saudi Arabia shared the highest similarity with the D3 strain, 99.3% and 100.0% of their E genes and deduced amino acids were identical, respectively, whereas they were 93.4% and 97.4% between D3/ZJ/2009 strain and its prototype strain (D3/H87/1956), and 93.6% and 97.4% between D3/ZJ/2009 and a D3 strain isolated in Guangxi Province in 1980. The phylogenetic tree of E genes also indicated that D3/ZJ/2009 had maximum similarity with the D3/Saudi Arabia/2004. They all belonged to the D3/GIII branch, which was originated from Indian Subcontinent.

CONCLUSIONThe outbreak of dengue fever in Zhejiang in 2009 was caused by type 3 dengue virus III genotype. The virus was most likely originated from Saudi Arabia.

Adolescent ; Adult ; Aged, 80 and over ; China ; epidemiology ; Dengue ; epidemiology ; virology ; Dengue Virus ; classification ; genetics ; isolation & purification ; Disease Outbreaks ; Female ; Humans ; Male ; Middle Aged

OBJECTIVEImpact of climate warming in winter on the potential epidemics of dengue fever in Hainan was assessed.

METHODSBased on historic data of mean monthly temperature in January from 8 weather observation stations, tendency and amplitude of variation were analyzed. Using 21 degrees C as lowest limit of temperature suitable for dengue fever transmission, impact caused by climate warming on dengue fever epidemic was estimated by means of geography information system (GIS), insect vector and epidemiological features.

RESULTSTemperature in winter in Hainan province had shown an obvious increase. The maximum amplitude of increase appeared in Dongfang which was 1.4 degrees C and the minimum 0.5 degrees C in Shanhudao, but the increase amplitude in the other stations was varied from 0.7 to 1.3 degrees C. By the year of 2050, 21 degrees C contour will have moved 190 km or so northward, nearly spanned 6/7 of distance from south to north in Hainan province and under the condition of daily fraction surviving of Aedes aegypti as P = 0.89, Qionghai city which stands north in Hainan province will probably have become epidemic area of dengue fever all year round.

CONCLUSIONClimate warming in winter will probably make half or more of the areas in Hainan province with temperature that permitting transmission of dengue fever by 2050. Monitoring and prevention of dengue fever in winter should be emphasized.

Aedes ; physiology ; virology ; Animals ; China ; epidemiology ; Climate ; Dengue ; epidemiology ; Dengue Virus ; isolation & purification ; Geographic Information Systems ; Humans ; Insect Vectors ; virology ; Seasons ; Temperature