Isolation, Identification And Sequence Analyses Of Dengue Virus Type I For Suspected Dengue Infection Patients From Guangzhou, Guangdong Province In 2003

Objective: Comparing the sensitivity, the specificity, and the valuation of three methods of detecting Dengue virus antibody, Dengue virus IgM/IgG Rapid immununochromatographic test (ICT), Dengue virus IgM and IgG enzyme linked immunosorbent assay (ELISA), Dengue virus IgM and IgG blot immunobinding assay (DIBA).To develop a reverse transcriptase -polymerase chain reaction - restriction fragment length polymorphism (RT-PCR-RFLP) technique for the rapid and early diagnosis of dengue. To identify the virus isolated from Guangzhou, Guangdong province in 2003 and to discuss the possible origin.Method: To detect Dengue virus IgM and IgG of serum from dengue fever (DF), epidemic hemorrhagic fever (EHF), Malaria, epidemic encephalitis B, influenza and leptospirosis patients with ICT, ELISA, DIBA, the comparations of the results.Laboratory diagnosis involved three testing methodologies: virus isolation by cell culture, RT-PCR-RFLP assay, and serologic assays using ICT technique.Results: By IgM-ICT (Australia, PANBIO Limited), there are 32 positive in 53 serums from Dengue virus infection patients, none were positive by IgG-ICT; there are 5 positive in 53 serums from other patients, none were positive by IgG-ICT. By IgM-ELISA (Germang, NovaTec Immundiagnostica GMBH), there are 35 positive in 53 serums from Dengue virus infection patients, 40 positive in 57 serums by IgG-ELISA; there are 12 positive in 53 serums from other patients, 3 positive in 57 serums by IgG-ELISA. By IgM-ELISA (Australia, PANBIO Limited), there are 40 positive in 53 serums from Dengue virus infection patients, 28 positive in 57 serums by IgG-ELISA; there are 4 positive in 53 serums from other patients, none were positive by IgG-ELISA. By IgM-DIBA (Singapore, Genelabs Diagnostics), there are 45 positive in 53 serums from Dengue virus infection patients, 37 positive in 57 serums by IgG- DIBA; there are 16 positive in 53 serums from other patients, nonewere positive by IgG- DIBA.56.5% (13/23 ) were positive by IgM-ICT in 5 day, 66.7% (14/24) were positive by IgM-ICT in 5 to 10 day; 21.7% (5/23 ) were positive by IgG-DIBA in 5 day, 70.8% (17/24) were positive by IgG-DIBA in 5 to 10 day. virus were isolated from seven of 18 serum samples of patients in 5 day and were identified as dengue virus type I by RT-PCR assay and DNA sequence analyses; 83.3%(25/30) were positive by RT-PCR assay, including DV-IgM (-) patients are 93.8% (15/16), DV-IgM (+) patients are 71.4% (10/14). Data of DNA sequence analyses on nucleotide homology were 97%, 97% and 98% compared with those of dengue virus type I strain of dengue hemorrhagic. fever outbreak in Cambodia, in 1997 and in 1999 in china. Two months before attack all patients live in Guangzhou and have no recent travel history outside the area.Conclusion:By comparing the three methods of detecting Dengue virus antibody, it is rational using DIBA as primary method, using the other tow as the secondary methods. ICT is a simple and rapid method which can be carried out with a single sample without special instrument, so it is applicable to different laboratories.These results suggested we could apply RT-PCR-RFLP as a rapid, specific, and highly sensitive tool for the early detection of dengue. The isolated virus from Guangzhou, Guangdong province in 2003 belonged to dengue virus type I, which might exited a plague spot in Guangdong.