Detection and genetic characterisation of dengue virus among patients in Dar es salaam, Tanzania during the 2013-2014 outbreak

MSc ThesisThe present study was carried out to confirm and genetically characterize dengue virus (DENV) present in sera collected from patients with dengue fever during the 2013 and 2014 dengue outbreaks in Tanzania. Sera were collected from patients who met the clinical case definition for dengue fever. Dengue fever was diagnosed in patients included in this study by detection of IgM and IgG DENV antibodies using a combination of a rapid test and enzyme-linked immunosorbent assay (ELISA). DENV detection in sera samples positive for anti-DENV IgM and IgG was performed using conventional and real-time reverse transcription polymerase chain reaction (RT-PCR). All sera (n = 23) tested positive for anti-DENV IgG and IgM antibodies and for the presence of DENV genomes using RT-PCR. In addition, both conventional and real- time RT-PCR showed the presence of DENV serotype 2 (DENV-2). Nucleotide sequencing of RT-PCR products after amplification of the core-pre-membrane (CprM) region of DENV using conventional RT-PCR produced a 500 bp fragment. BLASTn and phylogenetic analysis of the DENV nucleotide sequence obtained from this study clustered DENV-2 confirming the results obtained during conventional and real-time RT-PCR. The DENV-2 involved during the dengue fever outbreaks in 2013 and 2014 had 100% nucleotide and amino acid identity. This indicated that the same DENV-2 was responsible for the dengue fever outbreaks of 2013 and 2014.The phylogenetic analysis and BLASTn results of the DENV-2 CprM junction region obtained in the present study indicated that it has 98.2% nucleotide identity to SG/D2Y98P-PP1/2009 (Accession number JF327392). The SG/D2Y98P-PP1/2009 is a DENV isolate with a Phe-to-Leu alteration at position 52 in the NS4B protein of the original D2Y98P virus that was isolated in 1998 from a DENV-infected patient in Singapore. This mutation completely abolished the pathogenicity of the D2Y98P virus, as evidenced by a lack of lethality and the absence of histological signs of disease, which correlated with reduced viral titers and intact vascular permeability. Future studies are recommended in order to fully sequence the DENV-2 isolate obtained in the present study in order to determine the presence or absence of this mutation. In addition, it is recommended that the control of Aedes mosquitoes in Dar es Salaam be performed in order to avoid maintenance of DENV in mosquitoes that may lead to future outbreaks.Southern African Centre for Infectious Disease Surveillance (SACIDS