Natural pentacyclic triterpenoid as allosteric modulators of human 5-lipoxygenase with potential anti-inflammatory activity.

This study explored new methods to inhibit human 5-lipoxygenase (5-hLOX) by analyzing natural terpenes that share structural similarities with acetoxyboswellic acid (AKBA). Enzymatic assays were used to evaluate the terpene's ability to inhibit the enzyme, potentially providing anti-inflammatory benefits. Our research focused on how certain types of triterpenes can inhibit 5-hLOX allosterically via a newly discovered allosteric site identified by enzyme crystallization. To determine whether natural boswellic acid analogs mimicked the allosteric known inhibitor AKBA, we combined 5-hLOX inhibition with in silico modeling. Our research has discovered that certain amino acids, specifically Arg 138, Arg 101, Arg 68, and Gln129, located in the allosteric 5-hLOX pocket, play a critical role in stabilizing glycyrrhetinic isomers. These amino acids form hydrogen bonds and hydrophobic interactions that contribute to the inhibitory potency of boswellic acid derivatives. We have found that α and ß glycyrrhetinic acid isomers, carbenoxolone, and to a minor extent, prednisolone, have a potent inhibitory effect against 5-hLOX with IC50 values of 8.64, 3.94, 52.98, and 291.20 µM, respectively. These values are in line with our calculated in silico allosteric site binding energy estimations. In contrast, other steroidal or non-steroidal anti-inflammatory agents exhibited inhibitory potencies larger than 500 µM. However, the specific pharmacodynamic mechanisms are currently unknown. We propose that AKBA analogs may lead to the future development of novel anti-inflammatory agents.Communicated by Ramaswamy H. Sarma.

Evaluating the inhibitory activity of ferrocenyl Schiff bases derivatives on 5-lipoxygenase: Computational and biological studies.

In the search for new 5-LOX inhibitors, two ferrocenyl Schiff base complexes functionalized with catechol ((ƞ5-(E)-C5H4-NCH-3,4-benzodiol)Fe(ƞ5-C5H5) (3a)) and vanillin ((ƞ5-(E)-C5H4-NCH-3-methoxy-4-phenol)Fe(ƞ5-C5H5) (3b)) were obtained. Complexes 3a and 3b were biologically evaluated as 5-LOX inhibitors showed potent inhibition compared to their organic analogs (2a and 2b) and known commercial inhibitors, with IC50 = 0.17 ± 0.05 µM for (3a) and 0.73 ± 0.06 µM for (3b) demonstrated a highly inhibitory and potent effect against 5-LOX due to the incorporation of the ferrocenyl fragment. Molecular dynamic studies showed a preferential orientation of the ferrocenyl fragment toward the non-heme iron of 5-LOX, which, together with electrochemical and in-vitro studies, allowed us to propose a competitive redox deactivation mechanism mediated by water, where Fe(III)-enzyme can be reduced by the ferrocenyl fragment. An Epa/IC50 relationship was observed, and the stability of the Schiff bases was evaluated by SWV in the biological medium, observing that the hydrolysis does not affect the high potency of the complexes, making them interesting alternatives for pharmacological applications.

Prenylated Flavonoids with Potential Antimicrobial Activity: Synthesis, Biological Activity, and In Silico Study.

Prenylated flavonoids are an important class of naturally occurring flavonoids with important biological activity, but their low abundance in nature limits their application in medicines. Here, we showed the hemisynthesis and the determination of various biological activities of seven prenylated flavonoids, named 7-13, with an emphasis on antimicrobial ones. Compounds 9, 11, and 12 showed inhibitory activity against human pathogenic fungi. Compounds 11, 12 (flavanones) and 13 (isoflavone) were the most active against clinical isolated Staphylococcus aureus MRSA, showing that structural requirements as prenylation at position C-6 or C-8 and OH at positions C-5, 7, and 4' are key to the antibacterial activity. The combination of 11 or 12 with commercial antibiotics synergistically enhanced the antibacterial activity of vancomycin, ciprofloxacin, and methicillin in a factor of 10 to 100 times against drug-resistant bacteria. Compound 11 combined with ciprofloxacin was able to decrease the levels of ROS generated by ciprofloxacin. According to docking results of S enantiomer of 11 with ATP-binding cassette transporter showed the most favorable binding energy; however, more studies are needed to support this result.

Evaluation of the Antiviral Activity against Infectious Pancreatic Necrosis Virus (IPNV) of a Copper (I) Homoleptic Complex with a Coumarin as Ligand.

The aquatic infectious pancreatic necrosis virus (IPNV) causes a severe disease in farmed salmonid fish that generates great economic losses in the aquaculture industry. In the search for new tools to control the disease, in this paper we show the results obtained from the evaluation of the antiviral effect of [Cu(NN1)2](ClO4) Cu(I) complex, synthesized in our laboratory, where the NN1 ligand is a synthetic derivate of the natural compound coumarin. This complex demonstrated antiviral activity against IPNV at 5.0 and 15.0 µg/mL causing a decrease viral load 99.0% and 99.5%, respectively. The Molecular Docking studies carried out showed that the copper complex would interact with the VP2 protein, specifically in the S domain, altering the process of entry of the virus into the host cell.

Development of Inclusion Complexes With Relative Humidity Responsive Capacity as Novel Antifungal Agents for Active Food Packaging.

Background: Allyl isothiocyanate is an excellent antimicrobial compound that has been applied in the development of active food packaging materials in the last years. However, the high volatility of this compound could prevent a lasting effect over time. In order to avoid this problem, cyclodextrin inclusion complexes have been proposed as an alternative, being beta-cyclodextrin (ß-CD) as the main candidate. In addition, ß-CD could act as a relative humidity-responsive nanoparticle. In this regard, the aim of this study was to develop inclusion complexes based on ß-CD and AITC as relative humidity-responsive agents, which can be used in the design of active food packaging materials. Methods: Two different ß-CD:AITC inclusion complexes (2:1 and 1:1 molar ratios) were obtained by the co-precipitation method. Entrapment efficiency was determined by gas chromatography, while inclusion complexes were characterized through thermal, structural, and physicochemical techniques. Antifungal capacity of inclusion complexes was determined in a headspace system. Furthermore, the AITC release from inclusion complexes to headspace at different percentages of relative humidity was evaluated by gas chromatography, and this behavior was related with molecular dynamic studies. Key Findings and Conclusions: The entrapment efficiency of inclusion complexes was over to 60%. Two coexisting structures were proposed for inclusion complexes through spectroscopic analyses and molecular dynamic simulation. The water sorption capacity of inclusion complexes depended on relative humidity, and they exhibited a strong fungicide activity against Botrytis cinerea. Furthermore, the AITC release to headspace occurred in three stages, which were related with changes in ß-CD conformational structure by water sorption and the presence of the different coexisting structures. In addition, a strong influence of relative humidity on AITC release was evidenced. These findings demonstrate that ß-CD:AITC inclusion complexes could be used as potential antifungal agents for the design of food packaging materials, whose activity would be able to respond to relative humidity changes.

Biological Properties and Absolute Configuration of Flavanones From Calceolaria thyrsiflora Graham.

Flavanones (-)-(2S)-5,4'-dihydroxy-7-methoxyflavanone (1) and (-)-(2S)-5,3',4'-trihydroxy-7-methoxyflavanone (2) were isolated from the extracts of Calceolaria thyrsiflora Graham, an endemic perennial small shrub growing in the central zone of Chile. The absolute configuration of these compounds was resolved by optical rotation experiments and in silico calculations. Three analogs (3, 4, and 5) were synthesized to do structure-activity relationships with the biological assays studied. Biological tests revealed that only flavanone 2 exhibited a moderate inhibitory activity against the methicillin-resistant strain S. aureus MRSA 97-77 (MIC value of 50 µg/ml). In addition, flavanone 2 showed a potent, selective, and competitive inhibition of 5-hLOX, which supports the traditional use of this plant as an anti-inflammatory in diseases of the respiratory tract. Also, 2 exhibited cytotoxic and selective effects against B16-F10 (8.07 ± 1.61 µM) but 4.6- and 17-fold lesser activity than etoposide and taxol.

A Heterologous Viral Protein Scaffold for Chimeric Antigen Design: An Example PCV2 Virus Vaccine Candidate.

Recombinant vaccines have low-cost manufacturing, regulatory requirements, and reduced side effects compared to attenuated or inactivated vaccines. In the porcine industry, post-weaning multisystemic disease syndrome generates economic losses, characterized by progressive weight loss and weakness in piglets, and it is caused by porcine circovirus type 2 (PCV2). We designed a chimeric antigen (Qm1) to assemble the main exposed epitopes of the Cap-PCV2 protein on the capsid protein of the tobacco necrosis virus (TNV). This design was based on the Cap-N-terminal of an isolated PCV2 virus obtained in Chile. The virus was characterized, and the sequence was clustered within the PCV2 genotype b clade. This chimeric protein was expressed as inclusion bodies in both monomeric and multimeric forms, suggesting a high-molecular-weight aggregate formation. Pigs immunized with Qm1 elicited a strong and specific antibody response, which reduced the viral loads after the PCV2 challenge. In conclusion, the implemented design allowed for the generation of an effective vaccine candidate. Our proposal could be used to express the domains or fragments of antigenic proteins, whose structural complexity does not allow for low-cost production in Escherichia coli. Hence, other antigen domains could be integrated into the TNV backbone for suitable antigenicity and immunogenicity. This work represents new biotechnological strategies, with a reduction in the costs associated with vaccine development.

Development of an Isavirus minigenome system to study the function of the pocket RNA-binding domain of the viral nucleoprotein (NP) in salmon cells.

The Isavirus is an orthomyxovirus with a genome composed of eight segments of negative single-strand RNA (-ssRNA). It has been proposed that the eight genomic segments of the Isavirus are organized as a ribonucleoprotein (RNP) complex called a minigenome, which contains all the viral RNA segments, a viral heterotrimeric polymerase and multiple copies of the viral nucleoprotein (NP). Here, we develop an Isavirus minigenome system and show the importance of the formation of active RNPs and the role of viral NP R189, R194, R302 and K325 residues in the NP RNA-binding domain in the context of RNPs. The results indicate it is possible to generate a minigenome in salmon cells, a composite ISAV RNPs with EGFP-based chimeric vRNA with heterotrimeric polymerase (PB1, PB2, PA) and NP protein using CMV-based auxiliary plasmids. It was also shown that NP R189, R194, R302 and K325 residues are important to generate viral mRNA from the constituted RNPs and a detectable reporter protein. This work is the first salmon cell-based minigenome assay for the Isavirus, which was evaluated by a bioinformatic and functional study of the NP protein in viral RNPs, which showed that correct NP-vRNA interaction is key to the functioning of RNPs.

Antifungal and physicochemical properties of inclusion complexes based on ß-cyclodextrin and essential oil derivatives.

Inclusion complexes based on ß-cyclodextrin (ß-CD) and antimicrobial compounds, were prepared by co-precipitation method, and characterized by entrapment efficiency (EE), thermal analysis, X-ray diffraction, 1H NMR spectroscopy, and water sorption. In addition, experiments associated to evaluate the effect of relative humidity on the release of active compounds and antifungal tests were performed. The analysis evidenced the encapsulation of active compounds into the ß-CD structure with EE of 91 ±â€¯4.1% and 66 ±â€¯2.1% for ß-CD/cinnamaldehyde and ß-CD/eugenol complexes, respectively. Additionally, high relative humidities favored the release of active compounds from inclusion complexes. On the other hand, inclusion complexes were able to control the growth of B. cinerea, which was evidenced by a reduction of its mycelialradial growth. Finally, specific interactions between the active compounds and ß-CD were evaluated through molecular dynamics simulation techniques. According to the obtained results, these complexes could be applied as additives in the design of antifungal packaging.

Docking, steered molecular dynamics, and QSAR studies as strategies for studying isoflavonoids as 5-, 12-, and 15-lipoxygenase inhibitors.

Lipoxygenases (LOX) are enzymes that catalyze polyunsaturated fatty acid peroxidation and have a non-heme iron atom located in their active site. They are implicated in the arachidonic acid pathway and involved in inflammation, fever, pain production, and in the origins of several diseases such as cancer, asthma, and psoriasis. The search for inhibitors of these enzymes has emerged in the last years, and isoflavonoids have a broad spectrum of biological activity with low cytotoxicity. Our previous results have shown that isoflavonoids inhibited different LOX isoforms in vitro. For this reason, we studied the most important interactions that govern the potency and selectivity of some isoflavones and isoflavans toward different LOX isoforms using computational methods. The docking results have shown that all the molecules can be located in different zones in the LOX active site. Steered molecular dynamics indicated that selectivity was present at the cavity entry, but not at its exit. We also observed the correlation between the potential mean force and the best (HIR-303) and worst inhibitors (IR-213) in 5-LOX. Finally, structure-activity relationship (QSAR) studies showed a good correlation between theoretical IC50 values and experimental data for 5-LOX and 12-LOX with 96 and 95%, respectively, and a lower correlation for 15-LOX (79%). Conclusively, pharmacophore analysis showed that our proposed molecules should possess a donor-acceptor and aromatic centers to encourage interactions in the active site.

New antibacterial and 5-lipoxygenase activities of synthetic benzyl phenyl ketones: Biological and docking studies.

We investigated twelve benzyl phenyl ketone derivatives which are synthetic precursors of isoflavonoids that are shown be good 5-hLOX inhibitors, especially those that have the catechol group, but these precursors never have been assayed as 5-hLOX inhibitors being a novelty as inhibitors of the enzyme, due to sharing important structural characteristics. Screening assays, half maximal inhibitory concentration (IC50) and kinetic assays of all the studied molecules (5 µg/ml in media assay) showed that 1-(2,4-dihydroxy-3-methylphenyl)-2-(3-chlorophenyl)-ethanone (K205; IC50 = 3.5 µM; Ki = 4.8 µM) and 1-(2,4-dihydroxy-3-methylphenyl)-2-(2-nitrophenyl)-ethanone (K206; IC50 = 2.3 µM; Ki = 0.7 µM) were potent, selective, competitive and nonredox inhibitors of 5-hLOX. Antioxidant behavior was also assayed by DPPH, FRAP, and assessing ROS production, and those with antibacterial and antiproliferative properties relating to 1-(2,4-dihydroxy-3-methylphenyl)-2-(2-chlorophenyl)-ethanone (K208) established it as the most interesting and relevant compound studied, as it showed nearly 100% inhibition of bacterial growth of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Finally, docking studies were done that helped to characterize how the inhibitor structures correlated to decreased 5-hLOX activity.

Metabolic Syndrome and Antipsychotics: The Role of Mitochondrial Fission/Fusion Imbalance.

Second-generation antipsychotics (SGAs) are known to increase cardiovascular risk through several physiological mechanisms, including insulin resistance, hepatic steatosis, hyperphagia, and accelerated weight gain. There are limited prophylactic interventions to prevent these side effects of SGAs, in part because the molecular mechanisms underlying SGAs toxicity are not yet completely elucidated. In this perspective article, we introduce an innovative approach to study the metabolic side effects of antipsychotics through the alterations of the mitochondrial dynamics, which leads to an imbalance in mitochondrial fusion/fission ratio and to an inefficient mitochondrial phenotype of muscle cells. We believe that this approach may offer a valuable path to explain SGAs-induced alterations in metabolic homeostasis.

Easy and rapid preparation of benzoylhydrazides and their diazene derivatives as inhibitors of 15-lipoxygenase.

Two series of diaza derivatives were prepared by solvent-free condensation of benzoic acid and 4-substituted phenylhydrazines in order to obtain phenylhydrazides (HYD series) and, by oxidation of these compounds, the corresponding benzoyldiazenes (DIA series). Both sets were evaluated as inhibitors of soybean 15-lipoxygenase activity and antioxidant capability in the FRAP and CUPRAC assays. The most potent inhibitors of both series exhibited IC50 values in the low micromolar range. Kinetic studies showed that at least the more active compounds were competitive inhibitors. Docking results indicated that the most potent inhibitor interacts strongly with Ile-839 and iron in the active site.

Structural analysis and molecular docking of trypanocidal aryloxy-quinones in trypanothione and glutathione reductases: a comparison with biochemical data.

A set of aryloxy-quinones, previously synthesized and evaluated against Trypanosoma cruzi epimastigotes cultures, were found more potent and selective than nifurtimox. One of the possible mechanisms of the trypanocidal activity of these quinones could be inhibition of trypanothione reductase (TR). Considering that glutathione reductase (GR) is the equivalent of TR in humans, biochemical, kinetic, and molecular docking studies in TR and GR were envisaged and compared with the trypanocidal and cytotoxic data of a set of aryloxy-quinones. Biochemical assays indicated that three naphthoquinones (Nq-h, Nq-g, and Nq-d) selectively inhibit TR and the TR kinetic analyses indicated that Nq-h inhibit TR in a noncompetitive mechanism. Molecular dockings were performed in TR and GR in the following three putative binding sites: the catalytic site, the dimer interface, and the nicotinamide adenine dinucleotide phosphate-binding site. In TR and GR, the aryloxy-quinones were found to exhibit high affinity for a site near it cognate-binding site in a place in which the noncompetitive kinetics could be justified. Taking as examples the three compounds with TR specificity (TRS) (Nq-h, Nq-g, and Nq-d), the presence of a network of contacts with the quinonic ring sustained by the triad of Lys62, Met400', Ser464' residues, seems to contribute hardly to the TRS. Compound Nq-b, a naphthoquinone with nitrophenoxy substituent, proved to be the best scaffold for the design of trypanocidal compounds with low toxicity. However, the compound displayed only a poor and non-selective effect toward TR indicating that TR inhibition is not the main reason for the antiparasitic activity of the aryloxy-quinones.

Enzymatic Studies of Isoflavonoids as Selective and Potent Inhibitors of Human Leukocyte 5-Lipo-Oxygenase.

Continuing our search to find more potent and selective 5-LOX inhibitors, we present now the enzymatic evaluation of seventeen isoflavones (IR) and nine isoflavans (HIR), and their in vitro and in cellulo potency against human leukocyte 5-LOX. Of the 26 compounds tested, 10 isoflavones and 9 isoflavans possessed micromolar potency, but only three were selective against 5-LOX (IR-2, HIR-303, and HIR-309), with IC50 values at least 10 times lower than those of 12-LOX, 15-LOX-1, and 15-LOX-2. Of these three, IR-2 (6,7-dihydroxy-4-methoxy-isoflavone, known as texasin) was the most selective 5-LOX inhibitor, with over 80-fold potency difference compared with other isozymes; Steered Molecular Dynamics (SMD) studies supported these findings. The presence of the catechol group on ring A (6,7-dihydroxy versus 7,8-dihydroxy) correlated with their biological activity, but the reduction of ring C, converting the isoflavones to isoflavans, and the substituent positions on ring B did not affect their potency against 5-LOX. Two of the most potent/selective inhibitors (HIR-303 and HIR-309) were reductive inhibitors and were potent against 5-LOX in human whole blood, indicating that isoflavans can be potent and selective inhibitors against human leukocyte 5-LOX in vitro and in cellulo.

Development of a reverse genetic system for infectious salmon anemia virus: rescue of recombinant fluorescent virus by using salmon internal transcribed spacer region 1 as a novel promoter.

Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV(901_09) and rISAVr(S6-NotI-HPR) containing a NotI restriction site and rISAV(S6/EGFP-HPR) harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5105 PFU/ml,similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry.

Derivatives of 3H-spiro1-benzofuran-2, 1’-cyclohexanes: immunostimulants for veterinary use / Derivados de 3H-espiro1-benzofurano-2, 1’-ciclohexanos: inmunoestimulantes de uso veterinario

Four 3H-spiro1-benzofuran-2, 1’-cyclohexanes were synthesized from filifolinol, two of which are reported for the first time. Docking molecular studies were carried out to determine in silico whether these derivatives have similar immunostimulant activity to that reported for filifolinol, and its oxidation product, filifolinone. Through of the study of interactions of these compounds with the heterodimer of the protein present in teleost TLR1-TLR2, filifolinol, 3’-filifolinchloride and filifolinyl acetate shows similar interactions between them, allowing to predict that they would have similar immunostimulant activity, but different to filifolinone and filifolinane or that they would act by a different mechanisms.

Cuatro 3H-spiro1-benzofuran-2, 1'-ciclohexanos se sintetizaron a partir de filifolinol, dos de los cuales son reportados por primera vez. Se llevaron a cabo estudios de docking molecular para determinar in silico si estos derivados tienen actividad inmunoestimulante similar a la reportada para filifolinol y su producto de oxidación, filifolinona. A través del estudio de las interacciones de estos compuestos con el heterodímero de la proteína presente en teleósteos TLR1-TLR2 se estableció que el filifolinol, 3'-cloruro de filifolinilo y acetato de filifolinilo tienen interacciones similares con el heterodímero, lo que permite predecir que entre ellos tendrían una actividad simi- lar, pero diferente a la de la filifolinona y filifolinano o que estos últimos actuarían por diferentes mecanismos.

In vitro study of isoflavones and isoflavans as potent inhibitors of human 12- and 15-lipoxygenases.

In this study, we have investigated 16 isoflavone and isoflavan derivatives as potential inhibitors of human lipoxygenase (platelet 12-lipoxygenase, reticulocyte 15-lipoxygenase-1, and epithelial 15-lipoxygenase-2). The flavonoid baicalein, a known lipoxygenase inhibitor, was used as positive control. Four compounds, 6,7-dihydroxy-3'-chloroisoflavone (1c), 7-hydroxy-8-methyl-4'-chloroisoflavan (5a), 7,8-dihydroxy-4'-methylisoflavan (5b), and 7,8-dihydroxy-3'-methylisoflavan (5c), were effective inhibitors of 12-lipoxygenases and 15-lipoxygenase-1 with IC50 's <10 µm, while 6,7-dihydroxy-4'-nitroisoflavone (1b) was a selective inhibitor of 12-lipoxygenases. Docking studies, antioxidant assays, and kinetic measurements were carried out for the three best inhibitors (1b, 5b, 5c). The results showed that a catechol group in ring A is critical for the antioxidant properties of these compounds, and probably essential for their inhibitory activity. Kinetic assays showed that compounds 1b, 5b, and 5c are competitive inhibitors with Ki values in the range of 0.3-3 µm.

Molecular phylodynamics and protein modeling of infectious salmon anemia virus (ISAV).

BACKGROUND: ISAV is a member of the Orthomyxoviridae family that affects salmonids with disastrous results. It was first detected in 1984 in Norway and from then on it has been reported in Canada, United States, Scotland and the Faroe Islands. Recently, an outbreak was recorded in Chile with negative consequences for the local fishing industry. However, few studies have examined available data to test hypotheses associated with the phylogeographic partitioning of the infecting viral population, the population dynamics, or the evolutionary rates and demographic history of ISAV. To explore these issues, we collected relevant sequences of genes coding for both surface proteins from Chile, Canada, and Norway. We addressed questions regarding their phylogenetic relationships, evolutionary rates, and demographic history using modern phylogenetic methods. RESULTS: A recombination breakpoint was consistently detected in the Hemagglutinin-Esterase (he) gene at either side of the Highly Polymorphic Region (HPR), whereas no recombination breakpoints were detected in Fusion protein (f) gene. Evolutionary relationships of ISAV revealed the 2007 Chilean outbreak group as a monophyletic clade for f that has a sister relationship to the Norwegian isolates. Their tMRCA is consistent with epidemiological data and demographic history was successfully recovered showing a profound bottleneck with further population expansion. Finally, selection analyses detected ongoing diversifying selection in f and he codons associated with protease processing and the HPR region, respectively. CONCLUSIONS: Our results are consistent with the Norwegian origin hypothesis for the Chilean outbreak clade. In particular, ISAV HPR0 genotype is not the ancestor of all ISAV strains, although SK779/06 (HPR0) shares a common ancestor with the Chilean outbreak clade. Our analyses suggest that ISAV shows hallmarks typical of RNA viruses that can be exploited in epidemiological and surveillance settings. In addition, we hypothesized that genetic diversity of the HPR region is governed by recombination, probably due to template switching and that novel fusion gene proteolytic sites confer a selective advantage for the isolates that carry them. Additionally, protein modeling allowed us to relate the results of phylogenetic studies with the predicted structures. This study demonstrates that phylogenetic methods are important tools to predict future outbreaks of ISAV and other salmon pathogens.