GAPDH Pseudogenes and the Quantification of Feline Genomic DNA Equivalents

Helfer-Hungerbuehler, A. Katrin; Widmer, Stefan; Hofmann‐Lehmann, Regina

doi:10.1155/2013/587680

Cited by 20 publications

(33 citation statements)

References 34 publications

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“…Of the candidate reference genes evaluated for the diagnostic assay, YWHAZ was the most stably expressed in unstimulated and antigen‐stimulated whole blood and was selected as the optimal gene for qPCR normalization. This is in agreement with previous findings in whole blood for feline (Helfer‐Hungerbuehler et al., ) and other species (Peletto et al., ). Of the candidate target genes evaluated, CXCL9 showed the greatest upregulation in response to antigen stimulation and may therefore be a more sensitive measure of bioactive IFN‐γ than the direct detection of this cytokine itself (Brice et al., ; Kasprowicz et al., ).…”

Section: Discussionsupporting

confidence: 94%

Development of a Gene Expression Assay for the Diagnosis ofMycobacterium bovisInfection in African Lions (Panthera leo)

Olivier

Viljoen

Hofmeyr

et al. 2015

Transbound Emerg Dis

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Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON -TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions.

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Section: Discussionsupporting

confidence: 94%

Development of a Gene Expression Assay for the Diagnosis ofMycobacterium bovisInfection in African Lions (Panthera leo)

Olivier

Viljoen

Hofmeyr

et al. 2015

Transbound Emerg Dis

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“…of enFeLV copies, while others have used the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as a reference (10). Multiple copies of GAPDH pseudogene sequences have been found in the feline genome, indicating that GAPDH may not be the best reference gene for estimating cell copy number (42); in contrast, CCR5 is present as a diploid gene (2 copies/cell [43]). These differences likely account for the higher number of enFeLV copies than that estimated by fluorescent in situ hybridization (FISH) (9) or qPCR detecting the U3 region in the enFeLV LTR (the same region as detected in this study) and the enFeLV env gene (10).…”

Section: Discussionmentioning

confidence: 99%

Feline Leukemia Virus (FeLV) Disease Outcomes in a Domestic Cat Breeding Colony: Relationship to Endogenous FeLV and Other Chronic Viral Infections

Powers

Chiu

Kraberger

et al. 2018

J Virol

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Exogenous feline leukemia virus (FeLV) is a feline gammaretrovirus that results in a variety of disease outcomes. Endogenous FeLV (enFeLV) is a replication-defective provirus found in species belonging to the genus, which includes the domestic cat (). There have been few studies examining interaction between enFeLV genotype and FeLV progression. We examined point-in-time enFeLV and FeLV viral loads, as well as occurrence of FeLV/enFeLV recombinants (FeLV-B), to determine factors relating to clinical disease in a closed breeding colony of cats during a natural infection of FeLV. Coinfections with feline foamy virus (FFV), feline gammaherpesvirus 1 (FcaGHV-1), and feline coronavirus (FCoV) were also documented and analyzed for impact on cat health and FeLV disease. Correlation analysis and structural equation modeling techniques were used to measure interactions among disease parameters. Progressive FeLV disease and FeLV-B presence were associated with higher FeLV proviral and plasma viral loads. Female cats were more likely to have progressive disease and FeLV-B. Conversely, enFeLV copy number was higher in male cats and negatively associated with progressive FeLV disease. Males were more likely to have abortive FeLV disease. FFV proviral load was found to correlate positively with higher FeLV proviral and plasma viral load, detection of FeLV-B, and FCoV status. Male cats were much more likely to be infected with FcaGHV-1 than female cats. This analysis provides insights into the interplay between endogenous and exogenous FeLV during naturally occurring disease and reveals striking variation in the infection patterns among four chronic viral infections of domestic cats. Endogenous retroviruses are harbored by many animals, and their interactions with exogenous retroviral infections have not been widely studied. Feline leukemia virus (FeLV) is a relevant model system to examine this question, as endogenous and exogenous forms of the virus exist. In this analysis of a large domestic cat breeding colony naturally infected with FeLV, we documented that enFeLV copy number was higher in males and inversely related to FeLV viral load and associated with better FeLV disease outcomes. Females had lower enFeLV copy numbers and were more likely to have progressive FeLV disease and FeLV-B subtypes. FFV viral load was correlated with FeLV progression. FFV, FcaGHV-1, and FeLV displayed markedly different patterns of infection with respect to host demographics. This investigation revealed complex coinfection outcomes and viral ecology of chronic infections in a closed population.

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“…Genomic DNA (gDNA) and RNA were extracted from tissues as previously described 7 [47,48]. For all RNA and DNA extractions, negative controls consisting of 100 µl of 8 phosphate buffered saline were prepared for each batch to monitor cross-9 contamination.…”

Section: Nucleic Acid Extractions and Cdna Productionmentioning

confidence: 99%

“…determined by real-time PCR, by two. This number of provirus copies was then 8 divided by the number of feline albumin (fALB) copies measured by real-time PCR, 9 as described previously [48], to determine the provirus copy numbers per cell.…”

Section: Taqman Fluorogenic Real-time Pcr Assaymentioning

confidence: 99%

“…Analogous to the quantification of FeLV viral RNA loads in cats with different FeLV infection outcomes, the provirus loads were also quantified. To determine the cell 16 number of the input gDNA, FeLV provirus copy numbers were normalized to fALB [48]. In contrast to the results of the FeLV viral RNA loads, FeLV provirus was detectable in some samples from each investigated organ, although no provirus was found in a few single samples (22 of 459 measurements), which was indicative of provirus numbers around the detection limit.…”

Section: Felv Provirus Loads In Tissues Of Cats With Different Outcomementioning

confidence: 99%

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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

et al. 2015

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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 nonregenerative 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. DOI: https://doi.org/10. 1016/j.virusres.2014.12.025 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-105626 Accepted Version Originally published at: Helfer-Hungerbuehler, A Katrin; Widmer, Stefan; Kessler, Yvonne; Riond, Barbara; Boretti, Felicitas S; Grest, Paula; Lutz, Hans; Hofmann-Lehmann, Regina (2015). 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. Virus Research,

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GAPDH Pseudogenes and the Quantification of Feline Genomic DNA Equivalents

Cited by 20 publications

References 34 publications

Development of a Gene Expression Assay for the Diagnosis ofMycobacterium bovisInfection in African Lions (Panthera leo)

Development of a Gene Expression Assay for the Diagnosis ofMycobacterium bovisInfection in African Lions (Panthera leo)

Feline Leukemia Virus (FeLV) Disease Outcomes in a Domestic Cat Breeding Colony: Relationship to Endogenous FeLV and Other Chronic Viral Infections

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

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