RESUMEN
Tiger frog virus (TFV), a species of genus Ranavirus in the family Iridoviridae, is a nuclear cytoplasmic large DNA virus that infects aquatic vertebrates such as tiger frog (Rana tigrina rugulosa) and Chinese soft-shelled turtle (Trionyx sinensis). Based on the available genome sequences of TFV, the well-developed RNA interference (RNAi) technique, and the reliable cell line for infection model, we decided to analyze the functional importance of all predicted genes. Firstly, a relative quantitative cytopathogenic effect (Q-CPE) assay was established to monitor the viral proliferation in fish cells. Then, genome-wide RNAi screens of 95 small interference (si) RNAs against TFV were performed to characterize the functional importance of nearly all (95%) predicted TFV genes by Q-CPE scaling system. We identified 32 (33.7%) genes as essential, 50 (52.6%) genes as semi-essential and 13 (13.7%) genes as nonessential for TFV proliferation. Quantitative RT-PCR and titer assays of selected genes were performed to verify the screen results. Furthermore, the screened essential genes were analyzed for their genome distribution and conservative comparison within Ranavirus. Such a systematic screen for viral functional genes by cell phenotypes should provide further insights into understanding of the information in antiviral targets, and in viral replication and pathogenesis of iridovirus.
Asunto(s)
Genes Virales , Iridovirus/fisiología , Replicación Viral , Animales , Línea Celular , Peces , Orden Génico , Silenciador del Gen , Genes Esenciales , Iridovirus/genética , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Carga Viral , Ensayo de Placa ViralRESUMEN
Betanodaviruses are the causative agents of viral nervous necrosis (VNN), a serious disease of cultured marine fish worldwide. Virus-like particles (VLPs) are one of the good novel vaccine candidates to control this disease. Until now, betanodavirus vaccine studies mainly focused on the humoral immune response and mortality after virus challenge. However, little is known about the activation of genes responsible for cellular and innate immunity by vaccines. In the present study, VLPs of orange-spotted grouper nervous necrosis virus (OGNNV) were produced in prokaryotes and their ability to enter Asian sea bass cells was the same as native virus, suggesting that they possess a similar structure to OGNNV. VLPs immunogenicity was then determined by intramuscularly vaccinating Epinephelus coioides at different concentrations (1.5 or 15 µg g(-1) fish body weight, FBW) and immunizing frequencies (administration once, twice and thrice). A single vaccination with the dosage of 1.5 µg g(-1) FBW is enough to provoke high titer antibodies (average 3 fold higher than that of negative control) with strong neutralizing antibody titer as early as 1 week post immunization. Furthermore, quantitative PCR analysis revealed that eleven genes associated with humoral, cellular and innate immunities were up-regulated in the liver, spleen and head kidney at 12h post immunization, correlating with the early antibody response. In conclusion, we demonstrated that VLP vaccination induced humoral immune responses and activated genes associated with cellular and innate immunity against betanodavirus infection in orange-spotted grouper.
Asunto(s)
Enfermedades de los Peces/virología , Nodaviridae/inmunología , Perciformes , Infecciones por Virus ARN/veterinaria , Vacunación/veterinaria , Vacunas de Partículas Similares a Virus/inmunología , Animales , Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática/veterinaria , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/prevención & control , Pruebas de Neutralización/veterinaria , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/prevención & control , Infecciones por Virus ARN/virología , ARN Viral/química , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Vacunación/métodos , Vacunación/normas , Vacunas de Partículas Similares a Virus/administración & dosificaciónRESUMEN
OBJECTIVE: To test the accuracy of real-time three-dimensional echocardiography (RT3DE) imaging system for evaluating left ventricular mass (LVM) in phantom and excised canine heart. METHODS: Ten left ventricular (LV) wall phantoms made of two rubber-bursas, ten excised canine hearts underwent RT3DE and two-dimensional echocardiography (2DE). In RT3DE "full volume" imaging, the myocardial volume was measured using 2, 4, 8, and 16-plane method with the analysis software of RT3DE. Mass was then calculated by multiplying the resulting myocardial volume by specific density of myocardial tissue. In 2DE the masses were measured by area-length method. The true LV wall phantom mass was measured by water displacement and the canine LVM was weighed by anatomy, which served as a reference standard. We compared RT3DE or 2DE with true mass. RESULTS: In LV wall phantoms, RT3DE correlated with true masses strongly (r = 0.813-0.994) and weakly correlated between 2DE and true masses (r = 0.628). In excised canine hearts, there is an excellent correlation between RT3DE and true masses (r = 0.764-0.991), while 2DE value showed a lesser correlation (r = 0.514). There are no difference between RT-3DE and true masses (P > 0.05) but different between 2DE and true masses (P < 0.05). In different planes, there was no difference between 8-plane and 16-plane (P > 0.05) but different between 8-plane and 2, 4-plane (P < 0.05). CONCLUSION: RT3DE can accurately quantify LVM and provide a new tool to evaluate LV function. For LVM by RT3DE, 8-plane measurement method is the best choice for accuracy and convenience.
Asunto(s)
Ecocardiografía Tridimensional , Ventrículos Cardíacos/diagnóstico por imagen , Animales , Perros , Ecocardiografía Tridimensional/instrumentación , Ventrículos Cardíacos/anatomía & histología , Fantasmas de ImagenRESUMEN
BACKGROUND: Assessment of the left ventricular (LV) and the right ventricular (RV) volumes and their functions is important for prognostic prediction and clinical decision making. We compared the accuracy for quantifying the LV and the RV volumes in vitro between conventional two-dimensional echocardiography (2DE) and real-time three-dimensional echocardiography (RT3DE). METHODS: The volumes of 37 rubber-models (10 regularly shaped to simulate normal LV, 7 shaped to simulate LV with symmetric aneurysm, 8 shaped to simulate LV with asymmetric aneurysm, and 12 irregularly shaped to simulate normal RV) and 10 excised canine hearts were measured by RT3DE and 2DE. On RT3DE "full volume" imaging, the inner-surfaces of the rubber-models and canine LV and RV were outlined and the volumes were measured using 2-, 4-, 8- and 16-plane methods with the RT3DE analysis software. On 2DE imaging, the volumes were measured by the Simpson method. The LV and RV volumes measured by drained water were served as reference values, with which we compared RT3DE and 2DE data. RESULTS: In rubber models mimicking normal LV and LV with symmetric aneurysms, RT3DE results were strongly correlated with reference values (r = 0.795 - 0.998) and there was a good correlation between 2DE estimates and reference values (r = 0.715 - 0.729). There were no significant differences between RT3DE estimates, 2DE results and reference values (P > 0.05). In rubber models mimicking the RV and LV with asymmetric aneurysm, RT3DE strongly correlated with reference values (r = 0.765 - 0.988), but 2DE weakly correlated with reference values (r = 0.518 - 0.592). There were no differences between RT3DE and reference values (P > 0.05), but a significant difference between 2DE and reference values occurred (P < 0.05). For excised canine hearts, there was a strong correlation between RT3DE and reference values (r = 0.728 - 0.914), while 2DE showed a less obvious correlation (r = 0.502 - 0.615). Again, there were no significant differences between RT3DE and reference values (P > 0.05), but there was a significant difference between 2DE and reference values (P < 0.05). CONCLUSIONS: RT3DE can accurately quantify LV and RV volumes and provides a new tool to evaluate LV and RV function. For LV and RV measurements by RT3DE, 8-plane strategy is the optimum choice for accuracy and convenience.
Asunto(s)
Volumen Cardíaco , Ecocardiografía Tridimensional , Ventrículos Cardíacos/diagnóstico por imagen , Animales , Perros , Ecocardiografía , Valores de Referencia , Función Ventricular Izquierda , Función Ventricular DerechaRESUMEN
BACKGROUND: Both real-time three-dimensional echocardiography (RT3DE) and myocardial contrast echocardiography (MCE) are novel imaging techniques. The purpose of this study was to confirm the feasibility and accuracy of RT3DE combined with MCE for quantitative evaluation of myocardial perfusion defects. METHODS: Thirteen dogs underwent ligation of the left anterior descending artery (LAD, n = 6) or distal branch of the left circumflex artery (LCX, n = 7) under general anaesthesia. Three to four ml of a perfluoropropane (C3F8) microbubble contrast agent was injected intravenously to assess the resulting myocardial perfusion defects with a commercially available Philips SONOS-7500 ultrasound system. After removal of the dog hearts, Evans blue dye was injected via the left and right coronary arteries to stain the myocardium at risk. In vitro anatomic measurements of myocardial mass after removal of the animals' hearts were used as controls. RESULTS: Left ventricular (LV) mass determined by RT3DE ranged 36.7 - 68.9 g [mean, (54.6 +/- 9.6) g] before coronary artery ligation, and correlated highly (r = 0.99) with in vitro measurement of LV mass [range, 38.9 - 71.1 g; mean, (55.6 +/- 9.3) g]. There was no significant difference between RT3DE and in vitro measurements of LV mass [range, 36.7 - 68.9 g; mean, (51.3 +/- 12.5) g. Or range, 38.9 - 71.1 g; mean, (53.7 +/- 12.3) g, respectively] and under-perfused mass [range, 0 - 21.4 g; mean, (12.0 +/- 6.9) g. Or range, 0 - 19.8 g; mean, (10.8 +/- 6.3) g, respectively] after the LAD ligation (P > 0.05). Likewise, no significant difference was present between RT3DE and in vitro measurements of LV mass [range, 50.1 - 65.4 g; mean, (57.5 +/- 5.9) g. Or range, 51.5 - 65.8 g; mean, (57.3 +/- 6.4) g, respectively] and under-perfused mass [range, 0 - 25.6 g; mean, (13.3 +/- 9.6) g. Or range, 0 - 22.7 g; mean, (12.8 +/- 8.1) g, respectively] after the LCX ligation (P > 0.05). For all the animals with coronary ligation, LV mass measured by RT3DE ranged 35.9 - 68.6 g [mean, (54.8 +/- 10.0) g] and there was no significant difference between RT3DE and in vitro measurements of LV mass and under-perfused mass (P > 0.05, r = 0.99). Further, the under-perfused mass derived from RT3DE [range, 0 - 25.6 g; mean, (12.7 +/- 8.2) g] correlated strongly with the in vitro measurements [range, 0 - 22.7 g; mean, (11.9 +/- 7.2) g] (r = 0.96). CONCLUSION: RT3DE with MCE is a rapid and accurate method for estimating LV myocardial mass and quantifying perfusion defects.
Asunto(s)
Enfermedad Coronaria/diagnóstico por imagen , Ecocardiografía Tridimensional , Ecocardiografía , Animales , Perros , Estudios de Factibilidad , FluorocarburosRESUMEN
OBJECTIVES: To evaluate the feasibility and accuracy of measurement of left ventricular mass with intravenous contrast enhanced real-time three-dimensional (RT3D) echocardiography in the experimental setting. METHODS: RT3D echocardiography was performed in 13 open-chest mongrel dogs before and after intravenous infusion of a perfluorocarbon contrast agent. Left ventricular myocardium volume was measured according to the apical four-plane method provided by TomTec 4D cardio-View RT1.0 software, then the left ventricular mass was calculated as the myocardial volume multiplied by the relative density of myocardium. Correlative analysis and paired t-test were performed between left ventricular mass obtained from RT3D echocardiography and the anatomic measurements. RESULTS: Anatomic measurement of total left ventricular mass was 55.6 +/- 9.3 g, whereas RT3D echocardiographic calculation of left ventricular mass before and after intravenous perfluorocarbon contrast agent was 57.5 +/- 11.4 and 55.5 +/- 9.3 g, respectively. A significant correlation was observed between the RT3D echocardiographic estimates of total left ventricular mass and the corresponding anatomic measurements (r = 0.95). A strong correlation was found between RT3D echocardiographic estimates of left ventricular mass with perfluorocarbon contrast and the anatomic results (r = 0.99). Analysis of intraobserver and interobserver variability showed strong indexes of agreement in the measurement of left ventricular mass with pre and post-contrast RT3D echocardiography. CONCLUSIONS: Measurements of left ventricular mass derived from RT3D echocardiography with and without intravenous contrast showed a significant correlation with the anatomic results. Contrast enhanced RT3D echocardiography permitted better visualization of the endocardial border, which would provide a more accurate and reliable means of determining left ventricular myocardial mass in the experimental setting.