RESUMO
UNLABELLED: Infectious salmon anemia (ISA) is a severe disease that affects farmed Atlantic salmon (Salmo salar), causing outbreaks in seawater in most salmon-producing countries worldwide, with particular aggressiveness in southern Chile. The etiological agent of this disease is a virus belonging to the Orthomyxoviridae family, named infectious salmon anemia virus (ISAV). Although it has been suggested that this virus can be vertically transmitted, even in freshwater, there is a lack of compelling experimental evidence to confirm this. Here we demonstrate significant putative viral loads in the ovarian fluid as well as in the eggs of two brood stock female adult specimens that harbored the virus systemically but without clinical signs. The target virus corresponded to a highly polymorphic region 3 (HPR-3) variant, which is known to be virulent in seawater and responsible for recent and past outbreaks of this disease in Chile. Additionally, the virus recovered from the fluid as well as from the interior of the eggs was fully infective to a susceptible fish cell line. To our knowledge, this is the first robust evidence demonstrating mother-to-offspring vertical transmission of the infective virus on the one hand and the asymptomatic transmission of a virulent form of the virus in freshwater fish on the other hand. IMPORTANCE: The robustness of the data presented here will contribute to a better understanding of the biology of the virus but most importantly will constitute a key management tool in the control of an aggressive agent constantly threatening the sustainability of the global salmon industry.
Assuntos
Doenças dos Peixes/transmissão , Doenças dos Peixes/virologia , Transmissão Vertical de Doenças Infecciosas/veterinária , Isavirus/patogenicidade , Infecções por Orthomyxoviridae/veterinária , Salmo salar , Animais , Aquicultura , Chile , Eletroforese em Gel de Gradiente Desnaturante/veterinária , Feminino , Água Doce , Isavirus/genética , Microscopia de Fluorescência/veterinária , Infecções por Orthomyxoviridae/transmissão , Ovário/virologia , Óvulo/ultraestrutura , Óvulo/virologia , Carga Viral , VirulênciaRESUMO
Viral infections in salmonids represent an ongoing challenge for the aquaculture industry. Two RNA viruses, the infectious pancreatic necrosis virus (IPNV) and the infectious salmon anemia virus (ISAV), have become a latent risk without healing therapies available for either. In this context, antiviral peptides emerge as effective and relatively safe therapeutic molecules. Based on in silico analysis of VP2 protein from IPNV and the RNA-dependent RNA polymerase from ISAV, a set of peptides was designed and were chemically synthesized to block selected key events in their corresponding infectivity processes. The peptides were tested in fish cell lines in vitro, and four were selected for decreasing the viral load: peptide GIM182 for IPNV, and peptides GIM535, GIM538 and GIM539 for ISAV. In vivo tests with the IPNV GIM 182 peptide were carried out using Salmo salar fish, showing a significant decrease of viral load, and proving the safety of the peptide for fish. The results indicate that the use of peptides as antiviral agents in disease control might be a viable alternative to explore in aquaculture.
RESUMO
BACKGROUND: The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin, a carotenoid with high commercial interest. The proposed biosynthetic route in this organism is isopentenyl-pyrophosphate (IPP) --> geranyleranyl pyrophosphate (GGPP) --> phytoene --> lycopene --> beta-carotene --> astaxanthin. Recently, it has been published that the conversion of beta-carotene into astaxanthin requires only one enzyme, astaxanthin synthase or CrtS, encoded by crtS gene. This enzyme belongs to the cytochrome P450 protein family. RESULTS: In this work, a crtR gene was isolated from X. dendrorhous yeast, which encodes a cytochrome P450 reductase (CPR) that provides CrtS with the necessary electrons for substrate oxygenation. We determined the structural organization of the crtR gene and its location in the yeast electrophoretic karyotype. Two transformants, CBSTr and T13, were obtained by deleting the crtR gene and inserting a hygromycin B resistance cassette. The carotenoid composition of the transformants was altered in relation to the wild type strain. CBSTr forms yellow colonies because it is unable to produce astaxanthin, hence accumulating beta-carotene. T13 forms pale colonies because its astaxanthin content is reduced and its beta-carotene content is increased. CONCLUSION: In addition to the crtS gene, X. dendrorhous requires a novel gene, crtR, for the conversion of beta-carotene to astaxanthin.
Assuntos
Basidiomycota/enzimologia , Basidiomycota/genética , Proteínas Fúngicas/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Fúngico/genética , Proteínas Fúngicas/genética , Deleção de Genes , Biblioteca Gênica , Genes Fúngicos , Cariotipagem , Dados de Sequência Molecular , Mutação , NADPH-Ferri-Hemoproteína Redutase/genética , Filogenia , Plasmídeos , RNA Fúngico/genética , Análise de Sequência de DNA , Transformação Genética , Xantofilas/biossínteseRESUMO
The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+/idi-::hph), crtE (crtE+/crtE-::hph), crtYB (crtYB+/crtYB-::hph), crtI (crtI+/crtI-::hph) and crtS (crtS+/crtS-::hph) and homozygote mutants crtYB (crtYB-::hph/crtYB-::hph), crtI (crtI-::hph/crtI-::hph) and crtS (crtS-::hph/crtS-::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.
Assuntos
Basidiomycota/genética , Regulação Fúngica da Expressão Gênica/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Genes Fúngicos/genética , Reação em Cadeia da Polimerase , RNA Fúngico/genética , Xantofilas/biossíntese , Xantofilas/genéticaRESUMO
Genetic reassortment plays an important role in the evolution of several segmented RNA viruses and in the epidemiology of their associated diseases. In particular, orthomyxoviruses show rapid fluctuation in the proportion of viral variants coexisting in an infected individual, especially under strong selective pressure. This is particularly relevant in salmon production carried out under confined and stressful conditions where one of the most feared pathogenic agents is the Infectious Salmon Anemia Virus, an orthomyxovirus family member whose biological behavior is only recently beginning to be understood. Pathogenicity of the virus has been mainly associated with deletions of the HPR region in coding segment 6 and the presence or absence of a specific insertion in a key region in coding segment 5. In this study we report, for the first time in Chile, the coexistence of two variants in fully asymptomatic fish. Of five samples analyzed, two were identified as the non-pathogenic variant, HPR0, and two as the highly pathogenic HPR7b variant, though with no clinical signs detectable in the fish. Interestingly, one of the samples unequivocally carried both variants, again without any clinical signs. Considering that in none of the samples the typical insertion in coding segment 5 was detected, it is our impression that this may represent a shift from the non-pathogenic HPR0 variant towards the highly infective HPR7b variant. If this were the case, the transition may be triggered first by deleting the corresponding sequence of the HPR region of segment 6, followed by the putative insertion in segment 5 to generate a virulent strain.
Assuntos
Doenças dos Peixes/virologia , Variação Genética/genética , Isavirus/genética , Infecções por Orthomyxoviridae/genética , Salmão/virologia , Virulência/genética , Animais , Chile , Infecções por Orthomyxoviridae/virologiaRESUMO
Genetic variability is a key problem in the prevention and therapy of RNA-based virus infections. Infectious Salmon Anemia virus (ISAv) is an RNA virus which aggressively attacks salmon producing farms worldwide and in particular in Chile. Just as with most of the Orthomyxovirus, ISAv displays high variability in its genome which is reflected by a wider infection potential, thus hampering management and prevention of the disease. Although a number of widely validated detection procedures exist, in this case there is a need of a more complex approach to the characterization of virus variability. We have adapted a procedure of High Resolution Melting (HRM) as a fine-tuning technique to fully differentiate viral variants detected in Chile and projected to other infective variants reported elsewhere. Out of the eight viral coding segments, the technique was adapted using natural Chilean variants for two of them, namely segments 5 and 6, recognized as virulence-associated factors. Our work demonstrates the versatility of the technique as well as its superior resolution capacity compared with standard techniques currently in use as key diagnostic tools.
Assuntos
Orthomyxoviridae/classificação , Salmão/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , DNA Viral/genética , Eletroforese em Gel de Ágar , Genoma Viral , Dados de Sequência Molecular , Orthomyxoviridae/genética , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Infectious Salmon Anemia is a devastating disease critically affecting world-wide salmon production. Chile has been particularly stricken by this disease which in all cases has been directly related with its causative agent, a novel orthomyxovirus which presents specific and distinctive infective features. Among these, two molecular markers have been directly associated with pathogenicity in two of the eight RNA sub genomic coding units of the virus: an insertion hot spot region present in viral segment 5 and a Highly Polymorphic Region (HPR) located in viral segment 6. Here we report the successful adaptation of a PCR-dependent denaturing gel electrophoresis technique (DGGE), which enables differentiation of selected reported HPR epizootic variants detected in Chile. At the same time, the technique allows us to distinguish one nucleotide differences in sequences associated with the intriguing, and still not well-understood, insertion events which tend to occur on RNA Segment 5. Thus, the versatility of the technique opens new opportunities for improved understanding of the complex biology of all ISA variants as well as possible applications to other highly variable pathogens.
Assuntos
Eletroforese em Gel de Gradiente Desnaturante/métodos , Doenças dos Peixes/diagnóstico , Doenças dos Peixes/virologia , Isavirus/genética , Isavirus/isolamento & purificação , Infecções por Orthomyxoviridae/veterinária , Salmo salar , Animais , Chile , Primers do DNA/genética , Infecções por Orthomyxoviridae/diagnóstico , Polimorfismo Genético/genética , Especificidade da EspécieRESUMO
The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+ / idi-::hph), crtE (crtE+ / crtE -::hph), crtYB (crtYB + / crtYB -::hph), crtI (crtI+ / crtI-::hph) and crtS (crtS +/crtS -::hph) and homozygote mutants crtYB (crtYB -::hph/crtYB -::hph), crtI (crtI -::hph/crtI -::hph) and crtS (crtS -::hph / crtS -::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.
Assuntos
Basidiomycota/genética , Regulação Fúngica da Expressão Gênica/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Genes Fúngicos/genética , Reação em Cadeia da Polimerase , RNA Fúngico/genética , Xantofilas/biossíntese , Xantofilas/genéticaRESUMO
Se realizó un estudio descriptivo retrospectivo, de corte transversal, en el área del Policlínico Docente "Wilfredo Pérez Pérez" del municipio San Miguel del Padrón, de Ciudad de La Habana, con el fin de explorar los conocimientos sobre algunos aspectos de la sexualidad, en un grupo de 100 adolescentes en edades entre 10 y 19 años y de uno y otro sexos, en el año 2001. Se apreció un elevado tanto por ciento de jóvenes que tenían conocimientos sobre la anatomía del aparato genital, la mejor edad para tener hijos y la anticoncepción. En general, no tenían conocimientos de todas las enfermedades de transmisión sexual, no así el método más eficaz para evitarlas. El grupo de edad de mayor relevancia lo constituyó el de 10 a 13 años por sus escasos conocimientos sobre sexualidad