Analyses and Insights into Genetic Reassortment and Natural Selection as Key Drivers of Piscine orthoreovirus Evolution.

Piscine orthoreovirus (PRV) is a pathogen that causes heart and skeletal muscle inflammation in Salmo salar and has also been linked to circulatory disorders in other farmed salmonids, such as Oncorhynchus kisutch and Oncorhynchus mykiss. The virus has a segmented, double-stranded RNA genome, which makes it possible to undergo genetic reassortment and increase its genomic diversity through point mutations. In this study, genetic reassortment in PRV was assessed using the full genome sequences available in public databases. This study used full genome sequences that were concatenated and genome-wide reassortment events, and phylogenetic analyses were performed using the recombination/reassortment detection program version 5 (RDP5 V 5.5) software. Additionally, each segment was aligned codon by codon, and overall mean distance and selection was tested using the Molecular Evolutionary Genetics Analysis X software, version 10.2 (MEGA X version 10.2). The results showed that there were 17 significant reassortment events in 12 reassortant sequences, involving genome exchange between low and highly virulent genotypes. PRV sequences from different salmonid host species did not appear to limit the reassortment. This study found that PRV frequently undergoes reassortment events to increase the diversity of its segmented genome, leading to antigenic variation and increased virulence. This study also noted that to date, no reassortment events have been described between PRV-1 and PRV-3 genotypes. However, the number of complete genomic sequences within each genotype is uneven. This is important because PRV-3 induces cross-protection against PRV-1, making it a potential vaccine candidate.

Effect of a Diet-Induced Obesity on the Progeny Response in a Murine Model.

Diet-induced obesity could have detrimental effects on adults and their progeny. The aim of this study was to determine the effect of a high-energy diet on both F1 mice body weight and tissue/organ weight and F2 offspring growth. A simple murine model for obesity was developed using a high-energy diet and mice reared in litters of five or ten, from 30 dams receiving a cafeteria diet of either commercial chow (low energy), or a mixture of commercial chow, chocolate (50% cacao), and salty peanuts (high energy). This diet continued from mating until weaning, when the pups were allocated according to sex into eight groups based on maternal diet, litter size, and post-weaning diet. On day 74, the males were slaughtered, and the females were bred then slaughtered after lactation. As a result, the high-energy maternal diet increased the F1 offspring growth during lactation, while the high-energy post-weaning diet increased the F1 adult body weight and tissue/organ weight. The high-energy maternal diet could negatively affect the onset of the F1 but not the maintenance of breastfeeding of F1 and F2 offspring. For F2 offspring growth, the high energy overlapped the low-energy post-weaning diet, due to problems of gaining weight during lactation.

Epigenetic reprogramming around IFN1 and IFNy2 promoters in rainbow trout cells inoculated with infectious pancreatic necrosis virus (IPNV).

Infectious pancreatic necrosis virus (IPNV) has proven to effectively evade the host antiviral responses. This study clarifies whether the modulation of the antiviral immune response exerted by IPNV involves epigenetic mechanisms. An in-silico characterization of the rainbow trout IFN1 and IFNγ2 promoters was performed, identifying the islands or sequences rich in CpG dinucleotides and the putative transcription factor binding sites (TBS) for both gene promoters. RTS11 cells (rainbow trout monocyte/macrophage) were infected with IPNV, and the course of viral infection was followed up to 48 h post infection (hpi). Infected cells showed increased IFN1 and IFNγ2 transcriptional expression at 6 and 24 hpi, respectively. IPNV infection caused increases and decreases in global IFNγ2 promoter methylation at 6 and 24 hpi, respectively. The CpG dinucleotides at positions -392 and + 38 of this promoter were the most sensitive to methylation changes. The IFN1 promoter remained fully unmethylated during the course of the infection, similar to the control. The changes in the methylation pattern observed for the IFNγ2 promoter were coincident with the changes in DNA methyltransferase (DNMT) expression levels, increasing at 6 hpi and decreasing below basal level at 24 hpi. Similarly, the H4 histones associated with the IFN1 and IFNγ2 promoters were hyperacetylated at 6 hpi, subsequently decreasing their acetylation below basal levels at 24 hpi, in both promoters. Coincidentally with the above, overexpression of histone acetyltransferase (HAT) was observed at 6 hpi and of histone deacetylase (HDAC) at 24 hpi, with return to baseline of HAT. These results suggest that IPNV would epigenetically modulate the expression of IFN1 by changing acetylation levels of the histones H4 associated with its promoter. Also, the modulation of the expression of IFNy2 would be by switching methylation/demethylation levels of its promoter, in addition to changes in acetylation levels of histones H4 associated with this promoter. This study is the first to demonstrate the effect of epigenetic reprogramming after IPNV infection in salmonid cells, demonstrating that promoter methylation/demethylation level and changes in the histone code associated with promoters may play a role in the modulation of the immune response induced by the virus.

Microcystin-LR modulates multixenobiotic resistance proteins in the middle intestine of rainbow trout, Oncorhynchus mykiss.

Global climate change favors explosive population growth events (blooms) of phytoplanktonic species, often producing toxic products, e.g., several genera of cyanobacteria synthesize a family of cyanotoxins called microcystins (MCs). Freshwater fish such as the rainbow trout Oncorhynchus mykiss can uptake MCs accumulated in the food chain. We studied the toxic effects and modulation of the activity and expression of multixenobiotic resistance proteins (ABCC transporters and the enzyme glutathione S-transferase (GST) in the O. mykiss middle intestine by microcystin-LR (MCLR). Juvenile fish were fed with MCLR incorporated in the food every 12 h and euthanized at 12, 24, or 48 h. We estimated the ABCC-mediated transport in ex vivo intestinal strips to estimate ABCC-mediated transport activity. We measured total and reduced (GSH) glutathione contents and GST and glutathione reductase (GR) activities. We studied MCLR cytotoxicity by measuring protein phosphatase 1 (PP1) activity and lysosomal membrane stability. Finally, we examined the relationship between ROS production and lysosomal membrane stability through in vitro experiments. Dietary MCLR had a time-dependent effect on ABCC-mediated transport, from inhibition at 12 h to a significant increase after 48 h. GST activity decreased only at 12 h, and GR activity only increased at 48 h. There were no effects on GSH or total glutathione contents. MCLR inhibited PP1 activity and diminished the lysosomal membrane stability at the three experimental times. In the in vitro study, the lysosomal membrane stability decreased in a concentration-dependent fashion from 0 to 5 µmol L - 1 MCLR, while ROS production increased only at 5 µmol L - 1 MCLR. MCLR did not affect mRNA expression of abcc2 or gst-π. We conclude that MCLR modulates ABCC-mediated transport activity in O. mykiss's middle intestine in a time-dependent manner. The transport rate increase does not impair MCLR cytotoxic effects.

Chlorpyrifos stimulates ABCC-mediated transport in the intestine of the rainbow trout Oncorhynchus mykiss.

The organophosphorus pesticide chlorpyrifos, detected in water and food worldwide, has also been found in the Río Negro and Neuquén Valley, North Patagonia, Argentina, where the rainbow trout, Oncorhynchus mykiss, is one of the most abundant fish species. We analyzed whether chlorpyrifos affects the transport activity of the ATP-binding cassette protein transporters from the subfamily C (ABCC), which are critical components of multixenobiotic resistance. We exposed ex vivo O. mykiss middle intestine strips (non-polarized) and segments (polarized) for one hour to 0 (solvent control), 3, 10, and 20 µg L-1 and to 0, 10, and 20 µg L-1 chlorpyrifos, respectively. We estimated the Abcc-mediated transport rate by measuring the transport rate of the specific Abcc substrate 2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, we measured the enzymatic activity of cholinesterase, carboxylesterase, glutathione-S-transferase, and 7-ethoxyresorufin-O-deethylase (EROD, indicative of the activity of cytochrome P450 monooxygenase 1A, CYP1A). We also measured lipid peroxidation using the thiobarbituric acid reactive substances method and the gene expression of Abcc2 and genes of the AhR pathway, AhR, ARNT, and cyp1a, by qRT-PCR. Chlorpyrifos induced the DNP-SG transport rate in middle intestine strips in a concentration-dependent manner (49-71%). In polarized preparations, the induction of the DNP-SG transport rate was observed only in everted segments exposed to 20 µg L-1 chlorpyrifos (40%), indicating that CPF only stimulated the apical (luminal) transport flux. Exposure to chlorpyrifos increased GST activity by 42% in intestine strips and inhibited EROD activity (47.5%). In addition, chlorpyrifos exposure inhibited cholinesterase (34-55%) and carboxylesterase (33-42.5%) activities at all the concentrations assayed and increased TBARS levels in a concentration-dependent manner (71-123%). Exposure to 20 µgL-1 chlorpyrifos did not affect the mRNA expression of the studied genes. The lack of inhibition of DNP-SG transport suggests that chlorpyrifos is not an Abcc substrate. Instead, CPF induces the activity of Abcc proteins in the apical membrane of enterocytes, likely through a post-translational pathway.

Synthesis, antitumor activity, 3D-QSAR and molecular docking studies of new iodinated 4-(3H)-quinazolinones 3N-substituted.

A novel series of 6-iodo-2-methylquinazolin-4-(3H)-one derivatives, 3a-n, were synthesized and evaluated for their in vitro cytotoxic activity. Compounds 3a, 3b, 3d, 3e, and 3h showed remarkable cytotoxic activity on specific human cancer cell lines when compared to the anti-cancer drug, paclitaxel. Compound 3a was found to be particularly effective on promyelocytic leukaemia HL60 and non-Hodgkin lymphoma U937, with IC50 values of 21 and 30 µM, respectively. Compound 3d showed significant activity against cervical cancer HeLa (IC50 = 10 µM). The compounds 3e and 3h were strongly active against glioblastoma multiform tumour T98G, with IC50 values of 12 and 22 µM, respectively. These five compounds showed an interesting cytotoxic activity on four human cancer cell types of high incidence. The molecular docking results reveal a good correlation between experimental activity and calculated binding affinity on dihydrofolate reductase (DHFR). Docking studies proved 3d as the most potent compound. In addition, the three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis exhibited activities that may indicate the existence of electron-withdrawing and lipophilic groups at the para-position of the phenyl ring and hydrophobic interactions of the quinazolinic ring in the DHFR active site.

The insecticide chlorpyrifos modifies the expression of genes involved in the PXR and AhR pathways in the rainbow trout, Oncorhynchus mykiss.

Chlorpyrifos (CPF) is an organophosphate pesticide, commonly detected in water and food. Despite CPF toxicity on aquatic species has been extensively studied, few studies analyze the effects of CPF on fish transcriptional pathways. The Pregnane X receptor (PXR) is a nuclear receptor that is activated by binding to a wide variety of ligands and regulates the transcription of enzymes involved in the metabolism and transport of many endogenous and exogenous compounds. We evaluated the mRNA expression of PXR-regulated-genes (PXR, CYP3A27, CYP2K1, ABCB1, UGT, and ABCC2) in intestine and liver of the rainbow trout, Oncorhynchus mykiss, exposed in vivo to an environmentally relevant CPF concentration. Our results demonstrate that the expression of PXR and PXR-regulated genes is increased in O. mykiss liver and intestine upon exposure to CPF. Additionally, we evaluated the impact of CPF on other cellular pathway involved in xenobiotic metabolism, the Aryl Hydrocarbon Receptor (AhR) pathway, and on the expression and activity of different biotransformation enzymes (CYP2M1, GST, FMO1, or cholinesterases (ChEs)). In contrast to PXR, the expression of AhR, and its target gene CYP1A, are reduced upon CPF exposure. Furthermore, ChE and CYP1A activities are significantly inhibited by CPF, in both the intestine and the liver. CPF activates the PXR pathway in O. mykiss in the intestine and liver, with a more profound effect in the intestine. Likewise, our results support regulatory crosstalk between PXR and AhR pathways, where the induction of PXR coincides with the downregulation of AhR-mediated CYP1A mRNA expression and activity in the intestine.

Exposure to hydrocarbons and chlorpyrifos alters the expression of nuclear receptors and antioxidant, detoxifying, and immune response proteins in the liver of the rainbow trout, Oncorhynchus mykiss.

The development of oil and gas production together with the fruit production in nearby areas of North Patagonia, Argentina, suggests aquatic pollution scenarios which include permanent oil pollution combined with short events of pesticides application. It has been reported that oil hydrocarbons activate the aryl hydrocarbon receptor (AhR) pathway in the rainbow trout, Oncorhynchus mykiss, and that the insecticide Chlorpyrifos (CPF) interacts with these effects. Thus, it is interesting to investigate whether hydrocarbons and insecticides, applied by separate or combined, can affect fish health and reproductive signaling by acting on different nuclear receptors' regulatory pathways. To study this kind of interactions, we exposed juvenile rainbow trout to water accommodated fraction (WAF) of crude oil (62 µg L-1 TPH) for 48 h and subsequently exposed the livers ex vivo to the insecticide Chlorpyrifos (CPF) (20 µg L-1) for 1 h. We analyzed the mRNA expression of nuclear receptors and proteins involved in detoxifying, antioxidant, immune and apoptosis responses by qRT-PCR. We also performed histopathological analysis. WAF induced the expression of the androgen (AR) and the Liver X receptor (LXR) by 8- and 3-fold, respectively. AR induction was reversed by subsequent exposure to CPF. The progesterone receptor (PR) and glucocorticoid receptor (GR) were increased 2-fold and 3-fold by WAF respectively, while estrogen and mineralocorticoid receptors were not affected. GR was also induced by CPF with an additive effect in the WAF-CPF treatment. The antioxidant genes, gamma glutamyl transferase (GGT), superoxide dismutase (SOD1) were induced by WAF (2-3-fold). WAF upregulated the ATP Binding Cassette Subfamily C Member 2 (ABCC2, MRP2) (4-fold) and downregulated alkaline phosphatase. WAF also induced the inflammatory interleukins (IL) IL-8, and IL-6 and the anti-inflammatory IL-10, while CPF induced the inflammatory tumor necrosis factor (-α) and IL-6, and activated the intrinsic apoptotic pathway through the induction of caspases 3 and 9. Both, WAF and CPF downregulated the expression of the extrinsic apoptosis initiator caspase 8 and the inflammatory caspase 1. In conclusion, WAF hydrocarbons alter O. mykiss endocrine regulation by inducing AR, PR and GR. The subsequent exposure to CPF reverses AR, suggesting a complex interaction of different pollutants in contaminated environments, WAF hydrocarbons alter liver metabolism by inducing the expression of LXR, GR, antioxidant and detoxifying enzymes, and both inflammatory and anti-inflammatory cytokines, and causing mild hepatic steatosis. CPF activates inflammatory and stress responses associated with the induction of inflammatory cytokines together with apoptosis initiator and executioner caspases.

Effect of Type of Pregnancy on Transcriptional and Plasma Metabolic Response in Sheep and Its Further Effect on Progeny Lambs.

The following study was performed in order to determine the effect of type of pregnancy on the transcriptional expression of genes that are engaged in angiogenesis and cell turnover/lactogenesis in the ewe mammary gland, evaluating its impact on the plasma metabolic response. In addition, an assessment of its further influence on plasma metabolic response, performance, and muscle transcriptional expression of lipogenic enzymes in progeny lambs was made. Thirteen Ile de France sheep (six twin- and seven single-bearing ewes) were allocated to graze ad libitum naturalized pasture from d 45 pre-partum to day 70 post-partum, while keeping their lambs on the same diet until day 60 after weaning. The samples were collected at different times and analyzed by qRT-PCR and plasma metabolic indicators. The data were processed using SPSS package. The results showed that twin-bearing ewes overexpressed VEGFR1 at birth, and BCL2 at birth and day 35 post-partum; however, single-bearing ewes overexpressed CAIV and IGF1 at day 35 post-partum. Similar metabolite concentrations in blood plasma were found between groups of ewes. The plasma metabolic response in lambs was similar between groups and it did not influence their performance, where a similar transcriptional expression of lipogenic enzymes in muscle was observed. Therefore, the type of pregnancy can explain the slight differences in mRNA expression that were found in angiogenesis and cell turnover/lactogenesis in mammary gland, although these differences not only did not affect the plasma metabolic response in ewes, but they also had no influence on plasma metabolic response, performance, and muscle transcriptional expression of their lambs.

Effect of Diet and Type of Pregnancy on Transcriptional Expression of Selected Genes in Sheep Mammary Gland.

These trials were carried out to determine firstly the effect of diet and type of pregnancy on the transcriptional expression of genes involved in angiogenesis and cell turnover/lactogenesis inside the sheep mammary gland from late gestation to late lactation. Eighteen Ile de France sheep, 8 twin- and 10 single-bearing ewes were alloted into two groups according to their diet, either based on ad libitum naturalized pasture or red clover hay plus lupine from day -45 pre-partum until day +60 post-partum. Samples from diets and mammary glands were collected at day -10 pre partum (time 1), day +30 (time 2) and day +60 post-partum (time 3) and analyzed by qRT-PCR. Additionally, samples from longissimus dorsi muscle were taken from lambs twice, at weaning and 45 days later, to determine the effect of the maternal treatment with regard to diet and type of pregnancy, on the mRNA expression of genes involved in lipid metabolism. The data was processed using the lme4 package for R, and SPSS Statistics 23.0 for Windows®. The results showed that the group of twin-bearing ewes fed red clover showed a higher expression of genes involved in angiogenesis before lambing and in cell turnover/lactogenesis during late lactation, explained by a lamb survival mechanism to delay apoptosis as a way to keep a secretory cells population and boosted by the diet quality, assuring a longer milk production potential during late lactation. Regarding lambs, apparently the maternal diet would influence the transcriptional expression of lipogenic enzymes in the longissimus dorsi muscle after weaning, but further studies are necessary to validate these results. In summary, Twin-bearing ewes fed red clover performed best at increasing the expression of genes associated with angiogenesis and cell turnover/lactogenesis in the mammary gland.

Effect of Breed on Transcriptional and Protein Expression of Lipogenic Enzymes in Tail and Subcutaneous Adipose Tissue from Two Grazing Breeds of Lambs.

This experiment was carried out to determine the effect of breed on mRNA and protein expression levels of lipogenic enzymes acetyl-CoA carboxylase α (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1) plus sterol regulatory element binding transcription factor 1c (SREBP1c) in the subcutaneous fat (SCF) from the back of the animal, and tail fat (TF) of both Chilota and Suffolk Down lambs grazing Calafatal. Eight Chilota and six Suffolk Down 2-month-old male lambs were allocated to graze a "Calafatal", a typical secondary succession of Chiloé Archipelago, Chile. After 62 d, lambs were slaughtered according to Chile's meat industry standards. Fatty acid profile, RT-qPCR, and Western blot analyses from SCF and TF samples were performed. Although the mRNA expression levels of ACC, FAS, SCD1 and SREBP1c in SCF did not differ significantly between breeds (p > 0.05), a trend to higher mRNA expression of FAS and SREBP1c in TF from Chilota lambs was observed (p = 0.06). On the other hand, FAS levels in SCF were higher in Chilota than in Suffolk Down lambs (p < 0.02), although Suffolk Down showed higher fat contents and saturated fatty acid (SFA) proportions than Chilota lambs (p < 0.01). The FAS protein expression in TF was similar in both breeds (p > 0.05). Although the fat content was higher in Suffolk Down than in Chilota lambs (p < 0.01), the SFA proportions were similar in both breeds. Finally, it can be concluded that although mRNA expression of enzymes was similar in both breeds, there were differences in some protein levels in the SCF, partially related with the fatty acid profiles, thus affecting the selection of lamb breed either for human consumption or experimental purposes.

Gills CYP1A of Oncorhynchus mykiss as a sensitive biomarker of crude oil pollution in freshwater environments.

The induction of CYP1A activity (EROD) and protein expression was compared in liver and gills of rainbow trout from a stream polluted with crude oil, and through laboratory exposures to 1% and 5% of water accommodated fraction of the crude oil (WAF) for 1 and 4 days. Gills EROD increased 1.6-2.7-fold in fish from the polluted stream and during experiments, while liver EROD was induced only by 1% WAF at day 1 (1.5-fold). Contrastingly, crude oil pollution strongly induced both liver and gills CYP1A protein expression in the field (14-36-fold) and in experiments (4-25-fold). This highlights that crude oil induced CYP1A activity markedly in gills but only slightly or not at all in the liver, suggesting that differences between organ EROD activities are related to the modulation of CYP1A enzyme activity but not to the regulation at transcriptional or translational levels.

Effects of sequential exposure to water accommodated fraction of crude oil and chlorpyrifos on molecular and biochemical biomarkers in rainbow trout.

Fish can be simultaneously or sequentially exposed to various kinds of pollutants, resulting in combined effects. Polycyclic aromatic hydrocarbons induce cytochrome P450 monooxygenase 1A (CYP1A) expression, which catalyzes the conversion of the organophosphorus insecticide chlorpyrifos (CPF) into its most active derivative, CPF-oxon. CPF-oxon inhibits CYP1A and other enzymes, including carboxylesterases (CEs) and acetylcholinesterase (AChE). We studied the effects of an in vivo exposure to crude oil water accommodated fraction (WAF) followed by an ex vivo exposure of liver tissue to CPF on the expression of Cyp1a, AhR and ARNT mRNA, CYP1A protein and on the activity of biomarker enzymes in the rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout were exposed to WAF (62 µg L-1 TPH) for 48 h. Then, liver was dissected out, sliced and exposed to 20 µg L-1 CPF ex vivo for 1 h. Liver tissue was analyzed for mRNA and protein expression and for CEs, AChE, glutathione S-transferase (GST) and CYP1A (EROD) activity. WAF induced Cyp1a mRNA and CYP1A protein expression by 10-fold and 2.5-8.3-fold, respectively, with no effect of CPF. WAF induced AhR expression significantly (4-fold) in control but not in CPF treated liver tissue. ARNT mRNA expression was significantly lowered (5-fold) by WAF. CPF significantly reduced liver EROD activity, independently of WAF pre-treatment. CEs activity was significantly inhibited in an additive manner following in vivo exposure to WAF (42%) and ex vivo exposure to CPF (19%). CPF exposure inhibited AChE activity (37%) and increased GST activity (42%).

Comparative Pan-Genome Analysis of Piscirickettsia salmonis Reveals Genomic Divergences within Genogroups.

Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia, a disease that seriously affects the salmonid industry. Despite efforts to genomically characterize P. salmonis, functional information on the life cycle, pathogenesis mechanisms, diagnosis, treatment, and control of this fish pathogen remain lacking. To address this knowledge gap, the present study conducted an in silico pan-genome analysis of 19 P. salmonis strains from distinct geographic locations and genogroups. Results revealed an expected open pan-genome of 3,463 genes and a core-genome of 1,732 genes. Two marked genogroups were identified, as confirmed by phylogenetic and phylogenomic relationships to the LF-89 and EM-90 reference strains, as well as by assessments of genomic structures. Different structural configurations were found for the six identified copies of the ribosomal operon in the P. salmonis genome, indicating translocation throughout the genetic material. Chromosomal divergences in genomic localization and quantity of genetic cassettes were also found for the Dot/Icm type IVB secretion system. To determine divergences between core-genomes, additional pan-genome descriptions were compiled for the so-termed LF and EM genogroups. Open pan-genomes composed of 2,924 and 2,778 genes and core-genomes composed of 2,170 and 2,228 genes were respectively found for the LF and EM genogroups. The core-genomes were functionally annotated using the Gene Ontology, KEGG, and Virulence Factor databases, revealing the presence of several shared groups of genes related to basic function of intracellular survival and bacterial pathogenesis. Additionally, the specific pan-genomes for the LF and EM genogroups were defined, resulting in the identification of 148 and 273 exclusive proteins, respectively. Notably, specific virulence factors linked to adherence, colonization, invasion factors, and endotoxins were established. The obtained data suggest that these genes could be directly associated with inter-genogroup differences in pathogenesis and host-pathogen interactions, information that could be useful in designing novel strategies for diagnosing and controlling P. salmonis infection.

Molecular characterization of infectious pancreatic necrosis virus strains isolated from the three types of salmonids farmed in Chile.

BACKGROUND: The infectious pancreatic necrosis virus (IPNV) causes significant economic losses in Chilean salmon farming. For effective sanitary management, the IPNV strains present in Chile need to be fully studied, characterized, and constantly updated at the molecular level. METHODS: In this study, 36 Chilean IPNV isolates collected over 6 years (2006-2011) from Salmo salar, Oncorhynchus mykiss, and Oncorhynchus kisutch were genotypically characterized. Salmonid samples were obtained from freshwater, estuary, and seawater sources from central, southern, and the extreme-south of Chile (35° to 53°S). RESULTS: Sequence analysis of the VP2 gene classified 10 IPNV isolates as genogroup 1 and 26 as genogroup 5. Analyses indicated a preferential, but not obligate, relationship between genogroup 5 isolates and S. salar infection. Fifteen genogroup 5 and nine genogroup 1 isolates presented VP2 gene residues associated with high virulence (i.e. Thr, Ala, and Thr at positions 217, 221, and 247, respectively). Four genogroup 5 isolates presented an oddly long VP5 deduced amino acid sequence (29.6 kDa). Analysis of the VP2 amino acid motifs associated with clinical and subclinical infections identified the clinical fingerprint in only genogroup 5 isolates; in contrast, the genogroup 1 isolates presented sequences predominantly associated with the subclinical fingerprint. Predictive analysis of VP5 showed an absence of transmembrane domains and plasma membrane tropism signals. WebLogo analysis of the VP5 BH domains revealed high identities with the marine birnavirus Y-6 and Japanese IPNV strain E1-S. Sequence analysis for putative 25 kDa proteins, coded by the ORF between VP2 and VP4, exhibited three putative nuclear localization sequences and signals of mitochondrial tropism in two isolates. CONCLUSIONS: This study provides important advances in updating the characterizations of IPNV strains present in Chile. The results from this study will help in identifying epidemiological links and generating specific biotechnological tools for controlling IPNV outbreaks in Chilean salmon farming.

Consecutive emamectin benzoate and deltamethrin treatments affect the expressions and activities of detoxification enzymes in the rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) subjected to three consecutive, alternating treatments with emamectin benzoate (EMB) and deltamethrin (DM) during outbreaks of Caligus rogercresseyi in a farm located in southern Chile (Hornopiren, Chiloé), were studied to determine the effects of these treatments on the protein and enzymatic activity levels of cytochrome P450 1A (CYP1A), flavin-containing monooxygenase (FMO) and glutathione S-transferase (GST) in different tissues. Consecutive and alternating EMB/DM treatments resulted in a 10-fold increase and 3-fold decrease of CYP1A protein levels in the intestine and gills, respectively. Notably, CYP1A activity levels decreased in most of the analyzed tissues. FMO protein and activity levels markedly increased in the kidney and the intestine. GST was up-regulated in all tissues, either as protein or enzyme activity. When comparing consecutive EMB/DM treatments against previous studies of EMB treatment alone, CYP1A activity levels were similarly diminished, except in muscle. Likewise, FMO activity levels were increased in most of the analyzed tissues, particularly in the muscle, kidney, and intestine. The increases observed for GST were essentially unchanged between consecutive EMB/DM and EMB only treatments. These results indicate that consecutive EMB/DM treatments in rainbow trout induce the expression and activity of FMO and GST enzymes and decrease CYP1A activity. These altered activities of detoxification enzymes could generate imbalances in metabolic processes, synthesis, degradation of hormones and complications associated with drug interactions. It is especially important when analyzing possible effects of consecutive antiparasitic treatments on withholding periods and salmon farming yields.

Resistance-nodulation-division efflux pump acrAB is modulated by florfenicol and contributes to drug resistance in the fish pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is a fastidious intracellular pathogen responsible for high mortality rates in farmed salmonids, with serious economic consequences for the Chilean aquaculture industry. Oxytetracycline and florfenicol are the most frequently used antibiotics against P. salmonis, but routine use could contribute to drug resistance. This study identified differentiated florfenicol susceptibilities in two P. salmonis strains, LF-89 and AUSTRAL-005. The less susceptible isolate, AUSTRAL-005, also showed a high ethidium bromide efflux rate, indicating a higher activity of general efflux pump genes than LF-89. The P. salmonis genome presented resistance nodulation division (RND) family members, a family containing typical multidrug resistance-related efflux pumps in Gram-negative bacteria. Additionally, efflux pump acrAB genes were overexpressed in AUSTRAL-005 following exposure to the tolerated maximal concentration of florfenicol, in contrast to LF-89. These results indicate that tolerated maximum concentrations of florfenicol can modulate RND gene expression and increase efflux pump activity. We propose that the acrAB efflux pump is essential for P. salmonis survival at critical florfenicol concentrations and for the generation of antibiotic-resistant bacterial strains.

Characterization and pathogenic role of outer membrane vesicles produced by the fish pathogen Piscirickettsia salmonis under in vitro conditions.

Piscirickettsia salmonis is one of the major fish pathogens affecting Chilean aquaculture. This Gram-negative bacterium is highly infectious and is the etiological agent of Piscirickettsiosis. Little is currently known about how the virulence factors expressed by P. salmonis are delivered to host cells. However, it is known that several Gram-negative microorganisms constitutively release outer membrane vesicles (OMVs), which have been implicated in the delivery of virulence factors to host cells. In this study, OMVs production by P. salmonis was observed during infection in CHSE-214 cells and during normal growth in liquid media. The OMVs were spherical vesicles ranging in size between 25 and 145 nm. SDS-PAGE analysis demonstrated that the protein profile of the OMVs was similar to the outer membrane protein profile of P. salmonis. Importantly, the bacterial chaperonin Hsp60 was found in the OMVs of P. salmonis by Western-blot and LC-MS/MS analyses. Finally, in vitro infection assays showed that purified OMVs generated a cytopathic effect on CHSE-214 cells, suggesting a role in pathogenesis. Therefore, OMVs might be an important vehicle for delivering effector molecules to host cells during P. salmonis infection.

Draft Genome Sequence of Virulent Strain AUSTRAL-005 of Piscirickettsia salmonis, the Etiological Agent of Piscirickettsiosis.

We report here the draft genome sequence of a lethal pathogen of farmed salmonids, Piscirickettsia salmonis strain AUSTRAL-005. This virulent strain was isolated in 2008 from Oncorhynchus mykiss farms, and multiple genes involved in pathogenicity, environmental adaptation, and metabolic pathways were identified.

Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: binding of substrate induces a second class of site with lowered affinity and catalytic activity.

BACKGROUND: Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P2 and by high concentrations of its substrate Fru-1,6-P2. The mechanism that produces substrate inhibition continues to be obscure. METHODS: Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P2 and Fru-1,6-P2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244. RESULTS: The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P2, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a "stapler" that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition. CONCLUSIONS: Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits. GENERAL SIGNIFICANCE: Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.