RESUMO
Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor that induces genes involved in glucose metabolism. HIF-1 is formed by a regulatory α-subunit (HIF-1α) and a constitutive ß-subunit (HIF-1ß). The white spot syndrome virus (WSSV) induces a shift in glucose metabolism and oxidative stress. HIF-1α is associated with the induction of metabolic changes in tissues of WSSV-infected shrimp. However, the contributions of HIF-1 to viral load and antioxidant responses in WSSV-infected shrimp have been not examined. In this study, the effect of HIF-1 silencing on viral load and the expression and activity of antioxidant enzymes (superoxide dismutase-SOD, glutathione S-transferase-GST, and catalase) along with oxidative damage (lipid peroxidation and protein carbonyl) in tissues of white shrimp infected with the WSSV were studied. The viral load increased in hepatopancreas and muscle after WSSV infection, and the accumulative mortality was of 100% at 72â¯h post-infection. The expression and activity of SOD, catalase, and GST decreased in each tissue evaluated after WSSV infection. Protein carbonyl concentrations increased in each tissue after WSSV infection, while lipid peroxidation increased in hepatopancreas, but not in muscle. Silencing of HIF-1α decreased the WSSV viral load in hepatopancreas and muscle of infected shrimp along with shrimp mortality. Silencing of HIF-1α ameliorated the antioxidant response in a tissue-specific manner, which translated to a decrease in oxidative damage. These results suggest that HIF-1 is essential for restoring the antioxidant response, which counters the oxidative injury associated with WSSV infection.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/patogenicidade , Animais , Aquicultura , DNA Viral/isolamento & purificação , Inativação Gênica , Hepatopâncreas/crescimento & desenvolvimento , Hepatopâncreas/metabolismo , Hepatopâncreas/virologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Injeções Intramusculares , Peroxidação de Lipídeos , México , Músculos/metabolismo , Músculos/virologia , Especificidade de Órgãos , Estresse Oxidativo , Oxirredutases/genética , Oxirredutases/metabolismo , Penaeidae/crescimento & desenvolvimento , Penaeidae/metabolismo , Carbonilação Proteica , Interferência de RNA , RNA de Cadeia Dupla/administração & dosagem , RNA de Cadeia Dupla/metabolismo , Carga Viral , Vírus da Síndrome da Mancha Branca 1/isolamento & purificação , Vírus da Síndrome da Mancha Branca 1/fisiologiaRESUMO
The Pacific white shrimp, Litopenaeus vannamei, is the most important shrimp species in volume in world aquaculture. However, in recent decades, outbreaks of diseases, especially viral diseases, have led to significant economic losses, threatening the sustainability of shrimp farming worldwide. In 2004, Brazilian shrimp farming was seriously affected by a new disease caused by the Infectious myonecrosis virus (IMNV). Thus, disease control based on rapid and sensitive pathogen detection methods has become a priority. In this study, a specific quantitation method for IMNV was developed using real-time PCR with SYBR Green chemistry and viral load of the principal target tissues of chronically infected animals was quantified. The quantitative analysis revealed that mean viral load ranged from 5.08×10(8) to 1.33×10(6)copies/µg of total RNA in the hemolymph, 5.096×10(5) to 1.26×10(3)copies/µg in the pleopods, 6.85×10(8) to 3.09×10(4)copies/µg in muscle and 8.15×10(6) to 3.90×10(3)copies/µg in gills. Different viral loads of IMNV were found with greater values in the hemolymph and muscle, followed by the pleopods and gills.
Assuntos
Corantes Fluorescentes , Penaeidae/virologia , Infecções por Vírus de RNA/veterinária , Vírus de RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Actinas/genética , Animais , Brasil/epidemiologia , Clonagem Molecular , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Brânquias/virologia , Hemolinfa/virologia , Músculos/virologia , Penaeidae/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Infecções por Vírus de RNA/diagnóstico , Infecções por Vírus de RNA/epidemiologia , Infecções por Vírus de RNA/virologia , Vírus de RNA/genética , Vírus de RNA/patogenicidade , RNA Viral/análise , Sensibilidade e Especificidade , Carga ViralRESUMO
Infectious myonecrosis virus (IMNV) has been causing a progressive disease in farm-reared shrimps in Brazil and Indonesia. Immunodiagnostic methods for IMNV detection, although reliable, are not employed currently because monoclonal antibodies (MAbs) against this virus are not available. In this study, a fragment of the IMNV major capsid protein gene, comprising amino acids 300-527 (IMNV(300-527)), was cloned and expressed in Escherichia coli. The nucleotide sequence of the recombinant IMNV(300-527) fragment displayed a high degree of identity to the major capsid protein of IMNV isolates from Brazil (99%) and Indonesia (98%). Ten MAbs were generated against the expressed fragment, and eight of these, mostly IgG(2a) or IgG(2b), were able to bind to IMNV in tissue extracts from shrimps infected naturally in immunodot-blot assays. Six of these MAbs recognized a approximately 100 kDa protein in a Western-blot, which is the predicted mass of IMNV major capsid protein, and also bound to viral inclusions present in muscle fibroses and in coagulative myonecrosis, as demonstrated by immunohistochemistry. Among all those MAbs created, four did not cross-react with non-infected shrimp tissues; this observation supports their applicability as a sensitive and specific immunodiagnosis of IMNV infection in shrimps.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Proteínas do Capsídeo/análise , Penaeidae/virologia , Totiviridae/isolamento & purificação , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Western Blotting , Brasil , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Imunoglobulina G/isolamento & purificação , Indonésia , Dados de Sequência Molecular , Peso Molecular , Músculos/virologia , RNA Viral/genética , Proteínas Recombinantes/imunologia , Análise de Sequência de DNARESUMO
The Caribbean spiny lobster Panulirus argus is a valuable fishing resource and the trade in frozen lobster tails is an important industry. However, the presence of the pathogenic virus Panulirus argus Virus 1 (PaV1), which causes systemic infection in P. argus and is particularly lethal to juvenile individuals, has not been previously examined in imported/exported lobster products. We used PCR assays to determine the presence of PaV1 in abdominal muscle tissue of 22 frozen P. argus tails exported from Belize to Mexico. Based on their size, the tails belonged to subadult-adult lobsters. Using specific primers targeted for PaV1 resulted in 11 tails showing a specific 499 bp band. The sequence of positive amplified fragments showed a high similarity to PaV1 (95% identity with GenBank accession no. EF206313.1). Although the pathogenicity of PaV1 was not evaluated in the present study, our results provide the first evidence of PaV1 in frozen lobster tails exported in the seafood industry as well as the first molecular evidence of PaV1 in adult lobsters.
Assuntos
Vírus de DNA/fisiologia , Palinuridae/virologia , Animais , Região do Caribe , Vírus de DNA/isolamento & purificação , Feminino , Contaminação de Alimentos , Masculino , Músculos/virologiaRESUMO
BACKGROUND: To be transmitted by its mosquito vector, dengue virus (DENV) must infect midgut epithelial cells, replicate and disseminate into the hemocoel, and finally infect the salivary glands, which is essential for transmission. The extrinsic incubation period (EIP) is very relevant epidemiologically and is the time required from the ingestion of virus until it can be transmitted to the next vertebrate host. The EIP is conditioned by the kinetics and tropisms of virus replication in its vector. Here we document the virogenesis of DENV-2 in newly-colonized Aedes aegypti mosquitoes from Chetumal, Mexico in order to understand better the effect of vector-virus interactions on dengue transmission. RESULTS: After ingestion of DENV-2, midgut infections in Chetumal mosquitoes were characterized by a peak in virus titers between 7 and 10 days post-infection (dpi). The amount of viral antigen and viral titers in the midgut then declined, but viral RNA levels remained stable. The presence of DENV-2 antigen in the trachea was positively correlated with virus dissemination from the midgut. DENV-2 antigen was found in salivary gland tissue in more than a third of mosquitoes at 4 dpi. Unlike in the midgut, the amount of viral antigen (as well as the percent of infected salivary glands) increased with time. DENV-2 antigen also accumulated and increased in neural tissue throughout the EIP. DENV-2 antigen was detected in multiple tissues of the vector, but unlike some other arboviruses, was not detected in muscle. CONCLUSION: Our results suggest that the EIP of DENV-2 in its vector may be shorter that the previously reported and that the tracheal system may facilitate DENV-2 dissemination from the midgut. Mosquito organs (e.g. midgut, neural tissue, and salivary glands) differed in their response to DENV-2 infection.