Long-term follow up of feline leukemia virus infection and characterization of viral RNA loads using molecular methods in tissues of cats with different infection outcomes.

It is a remarkable feature for a retrovirus that an infection with feline leukemia virus (FeLV) can result in various outcomes. Whereas some cats contain the infection and show a regressive course, others stay viremic and succumb to the infection within a few years. We hypothesized, that differences in the infection outcome might be causally linked to the viral RNA and provirus loads within the host and these loads therefore may give additional insight into the pathogenesis of the virus. Thus, the goals of the present study were to follow-up on experimentally infected cats and investigate tissues from cats with different infection outcomes using sensitive, specific TaqMan real-time PCR and reverse transcriptase (RT)-PCR. Nineteen experimentally FeLV-A/Glasgow-1-infected cats were categorized into having regressive, progressive or reactivated FeLV infection according to follow-up of FeLV p27 antigen detection in the blood. Remarkably, regressively infected cats showed detectable provirus and viral RNA loads in almost all of the 27 tested tissues, even many years after virus exposure. Moreover, some regressively infected cats reactivated the infection, and these cats had intermediate to high viral RNA and provirus tissue loads. The highest loads were found in viremic cats, independent of their health status. Tissues that represented sites of virus replication and shedding revealed the highest viral RNA and provirus loads, while the lowest loads were present in muscle and nerve tissues. A supplementary analysis of 20 experimentally infected cats with progressive infection revealed a median survival time of 3.1 years (range from 0.6 to 6.5 years); ∼70% (n=14) of these cats developed lymphoma, while leukemia and non-regenerative anemia were observed less frequently. Our results demonstrate that the different infection outcomes are associated with differences in viral RNA and provirus tissue loads. Remarkably, no complete clearance of FeLV viral RNA or provirus was detected in cats with regressive infection, even up to 12 years after exposure. In several cases FeLV reactivation could be observed. Thus, retroviruses integrated as provirus into the host's genome, could not be eliminated completely by the host and maintained their full potential for replication and reactivation.

GAPDH Pseudogenes and the Quantification of Feline Genomic DNA Equivalents.

Quantitative real-time PCR (qPCR) is broadly used to detect and quantify nucleic acid targets. In order to determine cell copy number and genome equivalents, a suitable reference gene that is present in a defined number in the genome is needed, preferably as a single copy gene. For most organisms, a variable number of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pseudogenes have been reported. However, it has been suggested that a single-copy of the GAPDH pseudogene is present in the feline genome and that a GAPDH assay can therefore be used to quantify feline genomic DNA (gDNA). The aim of this study was to determine whether one or more GAPDH pseudogenes are present in the feline genome and to provide a suitable alternative qPCR system for the quantification of feline cell copy number and genome equivalents. Bioinformatics and sequencing results revealed that not just one but several closely related GAPDH-like sequences were present in the cat genome. We thus identified, developed, optimized, and validated an alternative reference gene assay using feline albumin (fALB). Our data emphasize the need for an alternative reference gene, apart from the GAPDH pseudogene, for the normalization of gDNA levels. We recommend using the fALB qPCR assay for future studies.

Tele-echocardiography in paediatrics.

UNLABELLED: Echocardiographic images can be transmitted over increasing distances with less cost and better quality thanks to advances in the field of telecommunications. This technological support can be used to detect heart defects in newborns and children in remote situations. The intent of this study was to confirm the feasibility and usefulness of telemedical communication for echocardiographic evaluation of paediatric cardiovascular disease. A total of 214 echocardiographs were performed in 194 children at a remote hospital by an experienced sonographer in paediatric echocardiography. These echocardiograms were transmitted to a distant tertiary care paediatric cardiology centre using a telemedicine link across three ISDN lines. There an experienced paediatric cardiologist interpreted the tele-echocardiograms. Tele-distant diagnoses were prospectively documented and compared with the diagnoses made subsequently on direct consultation and echocardiography. The quality of transmitted echocardiographic images was sufficient for evaluation except for one case. In 191 children (98%), the remote echocardiographic diagnosis was correct as confirmed by follow-up face to face consultations. Three cases were diagnosed incorrectly. CONCLUSION: our results confirm that accurate and rapid diagnosis can be provided by tele-echocardiography in neonates and children. This facilitates the appropriate care of these patients as expensive and potentially dangerous long-distance transfers can be avoided.