Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

ABSTRACT Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct.

Growth hormone therapy in patients with dilated cardiomyopathy: preliminary observations of a pilot study.

The effects of growth hormone in six patients with dilated cardiomyopathy were evaluated in this study. The patients were studied at baseline, after six months of therapy and at six months after stopping the treatment. They were given two units of growth hormone on alternate days by subcutaneous injection. There was marked improvement in the symptomatic class with treatment (NYHA class 3.4 +/- 0.5 vs 2 +/- 0; p = 0.04). There was also significant increase in the inteventricular septal wall thickness (6.4 +/- 1.5 mm vs 10.4 +/- 0.5 mm; p = 0.04). Left ventricular posterior wall thickness also increased significantly (7.2 +/- 1.3 mm vs 10.2 +/- 0.8 mm; p = 0.04). These changes were partially reversed by the end of six months of treatment but the symptomatic status of these patients was better than before. The administration of growth hormone for six months in patients with dilated cardiomyopathy results in significant improvement in the symptomatic class, which could be considered as an additional line of management in patients with heart failure in dilated cardiomyopathy.

Automated on-line quantification of left ventricular ejection fraction by echo.

An automated border detection system based on the acoustic back scatter property of ultrasound has been recently developed. This provides beat-to-beat values for the left ventricular ejection fraction (EF). This study assesses the reliability of this method when compared with the angiocardiographic EF, and manual 2D-echo-derived EF based on the modified Simpson method. Angiocardiographically-derived EF was used as the gold standard. In a total of 49 patients the angiocardiographic EF was found to be 64.49 +/- 15%. It showed excellent correlation with the automated border detection-derived EF (r = 0.91). The automated border detection-derived EF also showed excellent correlation with the manual 2D-echo method (r = 0.9). In addition, automated border detection requires less time than the manually drawn method (40.9 +/- 7.7 sec versus 58.6 +/- 7.5 sec; p < 0.001). We conclude that the acoustic quantification of the EF correlates well with the angiocardiographically-derived EF and is less time-consuming.

Serum troponin-T in acute myocardial infarction.

This study was carried out to assess the utility of serum troponin-T estimation in the diagnosis of acute myocardial infarction (AMI). One hundred and twenty two consecutive patients presenting with Q wave myocardial infarction were included in the study. In patients with AMI, serum troponin-T level was significantly elevated (4.21 +/- 3.49 ng/ml) on the first day of AMI (normal = 0.1 ng/ml). The elevated serum levels were detected even on the second (4.82 +/- 3.01 ng/ml) and the 3rd day (5.83 +/- 3.83 ng/ml) of AMI. Our results thus indicate that serum troponin-T is elevated twenty eight folds on the first day of AMI and the levels remain elevated as long as the third day. Hence, troponin-T can be used as sensitive biochemical marker of AMI, both in the immediate as well as in the early phase of myocardial infarction.

Left ventricular diastolic function--pulsed Doppler echocardiography versus radionuclide angiography.

Fifty five consecutive patients diagnosed to have coronary artery disease by coronary angiography had their left ventricular (LV) diastolic functions evaluated by pulsed doppler (PD) methods and radionuclide angiography (RNA). Using PD, the peak velocities of the early filling wave 'E' and the late filling wave 'A' of mitral inflow were measured. LV diastolic dysfunction, defined as E/A ratio less than 1.0, was present in 31 of 38 patients with low RNA peak filling rates (PFR) of 2.3 EDV/sec or less (sensitivity 81.6%). Normal E/A ratios (> 1.0) were seen in 13 of 17 patients with normal RNA PFR of > 2.3 EDV/sec (specificity 76.5%). Both methods were in agreement in 44 of 55 patients (accuracy 80%). There was good direct correlation between RNA PFR and PD E/A ratio (correlation coefficient r = 0.51, P < 0.01). It is concluded that PD echocardiography is a simple and reliable method of identifying diastolic dysfunction in patients with ischaemic heart disease.

Neonatal pulmonary flow pattern--a pulsed Doppler echo study.

The pulmonary flow pattern was analysed by pulsed Doppler in 77 normal neonates, at < 48 hours (36 subjects), 49 to 96 hours (25 subjects) and 97 to 148 hours (16 subjects). From the flow velocity spectrum, using an built in computer system, the acceleration time and the ejection time were measured, and the ratio was calculated. This ratio was 0.24 +/- 0.08 in the subjects within 48 hours whereas this ratio was 0.53 +/- 0.11 in the subjects above 97 hours; thus the ratio increased with age. We noticed that the ratio obtained from the pulsed Doppler is useful in assessing the pulmonary vascular status, even in neonates.

Right ventricular filling abnormalities in acute inferior wall myocardial infarction--a pulsed Doppler study.

Pulsed Doppler derived velocity profile of right ventricular filling was used to assess right ventricular diastolic function in 29 patients with acute inferior wall myocardial infarction. The peak velocities of the early filling wave 'E' and the atrial wave 'A' were measured. Diastolic dysfunction, defined as E/A ratio less than 1.0, was seen in 15 patients. Of these 15 patients, 9 had electrocardiographic evidence of right ventricular infarction and only 6 had clinical evidence of right ventricular failure. None of the 14 patients without right ventricular diastolic dysfunction (E/A ratio more than 1.0) had electrocardiographic evidence of right ventricular infarction or clinical evidence of right ventricular failure. Pulsed Doppler appears to be a sensitive technique in identifying hemodynamic derangements induced by right ventricular infarction.

Severity of mitral regurgitation in mitral valve prolapse syndrome. A Doppler echocardiographic study.

Thirty-three consecutive patients with mitral valve prolapse with a systolic murmur were evaluated using pulsed doppler echocardiography to quantify the severity of mitral regurgitation. There were thirteen (39%) patients with mild regurgitation, twelve patients (36%) with moderate regurgitation and eight patients (24%) with severe regurgitation. It was noticed that, all the patients with severe regurgitation had posterior leaflet prolapse. In contrast, patients with anterior leaflet prolapse had either mild or moderate regurgitation only. Our results suggest that the degree of mitral regurgitation differs depending on the leaflet that shows the prolapse, which may be of importance in the followup of patients with mitral valve prolapse.

Electrocardiographic left atrial enlargement--correlation with echo.

To assess the utility of electrocardiogram in identifying left atrial enlargement, electrocardiogram of 600 consecutive patients were correlated with their M-mode echocardiographic findings. Left atrial enlargement, as reflected by P terminal force in V1 had sensitivity of 79%, specificity 91%, predictive value 85% and accuracy of 86%. Patients older than 30 years with large left atrium (greater than 5.0 cm) had atrial fibrillation more frequently than younger patients (P less than 0.001). It is concluded that P terminal force in V1 is a reliable indicator of left atrial enlargement.