A comprehensive transcriptome analysis of an expressed sequence tag (EST) database of the spider Dolomedes fimbriatus venom glands using single-residue distribution analysis (SRDA) identified 7,169 unique sequences. Mature chains of 163 different toxin-like polypeptides were predicted on the basis of well-established methodology. The number of protein precursors of these polypeptides was appreciably numerous than the number of mature polypeptides. A total of 451 different polypeptide precursors, translated from 795 unique nucleotide sequences, were deduced. A homology search divided the 163 mature polypeptide sequences into 16 superfamilies and 19 singletons. The number of mature toxins in a superfamily ranged from 2 to 49, whereas the diversity of the original nucleotide sequences was greater (2-261 variants). We observed a predominance of inhibitor cysteine knot toxin-like polypeptides among the cysteine-containing structures in the analyzed transcriptome bank. Uncommon spatial folds were also found.
Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.