Abstract
Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 5′ and 3′ ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 5′ and 3′ ends of dengue virus types 1 to 4. The 5′ and 3′ ends of twenty-nine dengue virus isolates from acute infections were amplified through a modified protocol of the rapid amplification cDNA ends approach. For the 5′ end cDNA synthesis, specific anti-sense primers for each serotype were used, followed by polyadenylation of the cDNA using a terminal transferase and subsequent PCR amplification with oligo(dT) and internal specific reverse primer. At the 3′ end of the positive-sense viral RNA, an adenine tail was directly synthetized using an Escherichia coli poly(A) polymerase, allowing subsequent hybridization of the oligo(dT) during cDNA synthesis. The incorporation of the poly(A) tail at the 5′ and 3′ ends of the dengue virus cDNA and RNA, respectively, allowed for successful primer hybridization, PCR amplification and direct sequencing. This approach can be used for completing dengue virus genomes obtained through direct and next-generation sequencing methods.
Highlights
Dengue virus (DENV), the etiological agent of dengue fever, has been considered one of the most important vector-borne viruses in the tropical world, where an estimated of 390 million infections occur each year [1,2,3]
The 50 end of Colombian strains corresponding to the four DENV serotypes was successfully amplified by using the standard RACE-PCR approach, that involved the polyadenylation of the cDNA
The amplification of the DENV 30 end was attempted by a similar RACE-PCR strategy, using total RNA extracts from DENV-infected C6/36 cells and forward serotype-specific SP4 primers, which were expected to hybridize the minus strand viral RNA for the cDNA synthesis
Summary
Dengue virus (DENV), the etiological agent of dengue fever, has been considered one of the most important vector-borne viruses in the tropical world, where an estimated of 390 million infections occur each year [1,2,3]. A high proportion (around 75%) of DENV infections are asymptomatic, with the remaining 50–100 million infections ranging from dengue without/with warning signs to severe dengue, a life-threatening condition requiring precise clinical management [2]. The genome consists of a positive-sense single-stranded RNA of approximately 11 kb, which encodes for three structural (capsid, pre-membrane and envelope) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. At the 50 and 30 untranslated regions (UTR), DENV contains several evolutionarily conserved structural RNA elements involved in viral protein synthesis, genome replication, the regulation of viral and host processes and pathogenesis [6].
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