Classical swine fever (CSF) is one of the most important infectious diseases causing significant economic losses. Its causal agent, CSF virus (CSFV), is a member of the Pestivirus genus included into the Flaviviridae family. Previous molecular epidemiology studies have revealed the CSFV diversity is divided into three main genotypes and different subgenotypes. However, the classification system for CSFV has not yet been harmonized internationally. Similarly, the phylogeny and evolutionary dynamics of CSFV remain unclear. The current study provides novel and significant insights into the origin, diversification and evolutionary process of CSFV. In addition, the best phylogenetic marker for CSFV capable of reproducing the same phylogenetic and evolutionary information as the complete viral genome is characterized. Also, a reliable cut-off to accurately classify CSFV at genotype and subgenotype levels is established. Based on the time for the most recent common ancestor (tMRCA) reconstruction and cophylogenetic analysis, it was determined that CSFV emerged around 225 years ago when the Tunisian Sheep Virus jumped from its natural host to swine. CSFV emergence was followed by a genetic expansion in three main lineages, driven by the action of positive selection pressure and functional divergence, as main natural forces.

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Gallinamide A, a metabolite of the marine cyanobacterium Schizothrix sp., selectively inhibits cathepsin L-like cysteine proteases. We evaluated the potency of gallinamide A and 23 synthetic analogues against intracellular Trypanosoma cruzi amastigotes and the cysteine protease, cruzain. We determined the co-crystal structures of cruzain with gallinamide A and two synthetic analogues at ∼2 Å. SAR data revealed that the N-terminal end of gallinamide A is loosely bound and weakly contributes in drug–target interactions. At the C-terminus, the intramolecular π–π stacking interactions between the aromatic substituents at P1′ and P1 restrict the bioactive conformation of the inhibitors, thus minimizing the entropic loss associated with target binding. Molecular dynamics simulations showed that in the absence of an aromatic group at P1, the substituent at P1′ interacts with tryptophan-184. The P1–P1′ interactions had no effect on anti-cruzain activity, whereas anti-T. cruzi potency increased by ∼fivefold, likely due to an increase in solubility/permeability of the analogues.