A 3739 nucleotide fragment of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) from Brazil was amplified and sequenced. This fragment contains the entire coding sequences of viral proteins, the full 3' untranslated region (3'UTR) and a partial sequence of 5' untranslated region (5'UTR). The genome organization of IHHNV revealed the three typical major coding domains: a left ORF1 of 2001 bp that codes NS1, a left ORF2 (NS2) of 1091 bp that codes NS2 and a right ORF3 of 990 bp that codes VP. Nucleotide and amino acid sequences of the three viral proteins were compared with putative amino acid sequences of viruses reported from different regions. Comparisons among genomes from different geographic locations reveal 31 nucleotide regions that are 100% similar, distributed throughout the genome. An analysis of secondary structure of UTR regions, revealed regions with high probability to form hairpins, that may be involved in mechanisms of viral replication. Additionally, a maximum likelihood analysis indicates that Brazilian IHHNV belongs to lineage III, in the infectious IHHNV group, and is clustered with IHHNV isolates from Hawaii, China, Taiwan, Vietnam and South Korea. A new nested PCR targeting conserved nucleotide regions is proposed to detect IHHNV.
BACKGROUND The genus Flavivirus includes a variety of medically
important viruses, including dengue virus (DENV) and Zika virus (ZIKV),
which are most prevalent in Brazil. Because the clinical profile of patients
affected by different DENV serotypes or ZIKV may be similar, the development
of new methods that facilitate a rapid and accurate diagnosis is
crucial.OBJECTIVES The current study aimed to develop an improved reverse
transcription-polymerase chain reaction (RT-PCR) protocol for universal
detection of flaviviruses by using semi-nested primers that discriminate
between DENV serotypes and ZIKV.METHODS The bioinformatics workflow adopted for primer design included: (1)
alignment of 1,442 flavivirus genome sequences, (2) characterisation of 27
conserved regions, (3) generation of a primer set comprising 77 universal
primers, and (4) selection of primer pairs with greatest coverage and
specificity. Following primer design, the reaction was validated in
vitro. The same approach was applied to the design of primers
specific for DENV and ZIKV, using a species-specific sequence database.FINDINGS The new assay amplified an 800-806 nt variable region of the NS5 gene and
allowed discrimination of virtually all flavivirus species using
reference-sequence comparison. The 800-806 nt fragment was validated as a
template for a semi-nested multiplex PCR using five additional primers for
the detection of DENV and ZIKV. These primers were designed to generate
amplicons of different sizes, allowing differentiation of the four serotypes
of DENV, and ZIKV using agarose gel electrophoresis.MAIN CONCLUSIONS The bioinformatics pipeline allowed efficient primer design, making it
possible to identify the best targets within the coding region of the NS5
protein. The multiplex system proved effective in differentiation of DENV1-4
and ZIKV on a 2% agarose gel. The possibility of discriminating DENV
serotypes and ZIKV in the same reaction provided a faster result consuming
less sample. In addition, this simplified approach ensured the reduction of
the cost per analysis.
The most widely used technique for the production of DNA aptamers/oligonucleotides is chemical synthesis. Despite its effectiveness, this technique cannot be performed "in house", making the user fully dependent on a supplier. In this work, we present a simplified method by which it is possible to enzymatically produce DNA aptamers "in house". This new method uses the rolling circle replication followed by a unique cleavage step using the SchI endonuclease. Potentially, any oligonucleotide can be produced by the enzymatic method proposed in this study. To illustrate, we present the production of three variations of the 31-TBA aptamer, a single stranded DNA which has anticoagulant action.
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