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

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

OBJECTIVETo isolate and identify the pathogen of Dengue fever from Shenzhen city in 2005 - 2006, and to analyze the molecular characteristics of the isolated Dengue virus strain as well as to explore its possible origin.

METHODSIgM and IgG of serum samples taken from 60 suspected Dengue fever patients were detected by ELISA and immunochromatography, and 9 specimens were positive. Nine samples from patients with early stage Dengue fever were used to isolate virus with C6/36 cell line and the positive cell cultures were identified by MGB fluorescent PCR. The type of isolated virus strain was determined by RT-semi-nested-PCR and fluorescent PCR. E gene of isolated virus strain was amplified by RT-PCR and sequenced. Homology and phylogenetic tree of E gene of Shenzhen Dengue virus with the strains isolated from other areas were constructed.

RESULTSOf nine antibody-positive serum samples, one strain of Dengue virus was successfully isolated. The isolated virus strain was confirmed as Dengue virus type 2 and designated as DEN2-SZ0521. The homology of nucleotide sequence and the deduced amino acid sequence of E gene of SZ0521 with standard type 2 Dengue virus NGC strain was 94.2% and 98.2%, but the homology with standard Dengue virus 1, 3, 4 in the same fragment were 59.1%, 57.2%, 58.5% and 68.1%, 66.7%, 63.2%, respectively. The phylogenetic tree indicated that SZ0521 had the greatest similarity with the Malay0412a/Tw strain and they lied in the same branch of the phylogenetic tree. The corresponding homology of nucleotide sequence and amino acid sequence was 99.8% and 100%, respectively. The isolated Dengue virus type 2 belonged to genotype IV with Indonesia-76, Somalia-84 and Sri Lanka-90.

CONCLUSIONDengue virus was isolated from Shenzhen for the first time, and it was classified as type 2. It was confirmed that the type 2 Dengue virus may come from the epidemic area in Malaysia.

Aedes ; virology ; Animals ; China ; Dengue ; virology ; Dengue Virus ; classification ; genetics ; isolation & purification ; Genes, Viral ; Humans ; Phylogeny ; Sequence Analysis, Protein ; Sequence Analysis, RNA