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Type of publication: Baccalaureate Thesis
Type of document:

Year: 2012

Authors: Hagen, Benedikt

Title: Analysis of retroviral gene expression in feline lymphoma using qPCR and next-generation-sequencing technology.

Other title: Analyse retroviraler Gen-Expression im felinen Lymphom mithilfe von qPCR und next-generation-sequencing Technologie

Source: Bakkalaureatsarbeit, Vet. Med. Univ. Wien, pp. 32.


Advisor(s):

Klein Dieter

Reviewer(s):
Schlötterer Christian

Vetmed Research Units:
VetCore


Graduation date: 26.09.12


Abstract:
Endogenous as well as exogenous sequences of feline leukemia virus (FeLV) can be found in domestic cats and various wild cat species. Both virus forms are related to lymphomagenesis. Due to vaccination programs, infection rates of exogenous feline leukemia virus (exFeLV) have decreased significantly. However, the number of lymphomas in virus-negative cats increased. As a result, understanding the development of lymphomas in exFeLV negative cats has gained importance. The endogenous virus forms (enFeLV) are passed on via vertical gene transfer and have lost the ability to produce functioning virus particles. These sequences possibly play a role in lymphomagenesis as well. Endogenous forms of FeLV can be distinguished from exogenous FeLV using already established qPCR assays and by Next-Generation- Sequencing based on known genetic differences. Here, we apply next generation sequencing using the Ilumina technology and reverse transcription quantitative real time PCR (RT-qPCR) to investigate the expression levels of retroviral FeLV genes in five lymphoma samples and three non-cancerous lymph-node tissues. All samples were previously tested negative for infection with exFeLV and feline immunodeficiency virus (FIV). Next-generation-sequencing reveals a higher expression of enFeLV genes in lymph nodes compared to lymphoma samples, which was also confirmed by RT-qPCR. RNA-copy numbers and sequencing reads were normalized to the expression levels of GAPDH as a reference gene. This was a standardized normalization strategy to maintain comparability of the two methods. NGS results and RT-qPCR show a 4.5 and 4.6 fold change in enFeLV expression levels respectively. These results indicating lower transcription of endogenous retrovirus sequences in lymphomas contrast findings in humans and mice, where higher expression levels of endogenous retroviruses are related to lymphatic malignancies. This may suggest a yet unrevealed role of endogenous virus sequences in the formation of lymphatic malignancies in cats.


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