Cloning and Characterization of the Dengue Virus Envelope Gene from Clinical Sample.
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Date
2022
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Department of Genetics and Plant Breeding, Banaras Hindu University, Varanasi.
Abstract
Dengue feveris one of the most significant arboviral infections that affect people. It is principally transmitted by the mosquitoes Aedes aegypti and rarely by Aedes albopictus. It is brought on by the dengue virus, a member of the flavivirus genus that belongs to the Flaviviridae family. Dengue virus is enveloped,single-stranded RNA genomes of positive polarity. The genome is ~11kb in length, capped at 5’end but lacks poly-A tail at 3’ end. The ORF region of the genome is flanked by the two untranslated regions (UTR’s) at the 5’end and 3’ end.The genomic organization consists of three structural proteins (capsid, C; membrane, M and envelope, E) and seven non-structural proteins (NS1, NS2, NS3, NS4, NS4A, NS4B, and NS5). The dengue virus exists as four antigenically distinct serotypes,DENV1, DENV2, DENV3, and DENV4 which diverge ~30% in their genomic similarity.The E protein present on the outer surface is the first point of attachment for the viral entry and contains the epitope-specific for dengue serotypes.The pathogenicity of viruses can be affected by mutations in this protein. Thus, this study targeted the E gene of dengue for the characterization of the dengue virus.Reverse transcription-polymerase chain reaction (RT-PCR) was used to establish the diagnosis of dengue from the viral RNA recovered from the suspected dengue clinical sample. DENV-specific gene primers amplified the 1700 bp of the targeted E-gene. With the help of restriction enzyme analysis and nucleotide sequencing of genomic fragments, the desired amplified product was further examined for serotype identity. The restriction enzyme Eco RI was used to carry out the digestion. A T/A cloning vector was used to ligate the digested product. The ligated product was cloned into competent cells DH5α of E.coli plated on an LB-AIX plate. The study based on cloning the gene fragments of DENV1 and DENV2 is useful to diagnose dengue fever and study the structure and function of E-genes in detail. In addition, the selection of the E coding sequence as a sequencing target provides a sufficient phylogenetic signal to distinguish DENV from other flaviviruses and between serotypes.
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Dengue virus