New insights into the biology of torque teno sus viruses
- Autores: Laura Martínez Guinó
- Directores de la Tesis: Joaquim Segalés Coma (codir. tes.), Tuija Kekarainen (codir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2011
- Idioma: inglés
- Tribunal Calificador de la Tesis: Erwin van den Born (presid.), Fernando Rodríguez (secret.), Laura Maria Kakkola (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
Toque teno sus viruses (TTSuV) are swine infecting, non-enveloped, circular, single-stranded DNA viruses belonging to the family Anelloviridae. Anelloviruses are currently considered non-pathogenic although they have been linked to pathological conditions in human and swine. Two different viral species have been described in swine so far, Torque teno sus virus 1 (TTSuV1) and Torque teno sus virus 2 (TTSuV2). Both TTSuV species are widespread, with high prevalence in domestic pig and wild boar population. After years of research, little is known about the mechanisms of TTSuVs infection and in vitro propagation of them has been never achieved so far. In addition, all diagnostic tools available for TTSuV detection are based on viral DNA detection by PCR methods. The present thesis aimed to elucidate biological aspects of TTSuV species, develop diagnostic tools and evaluate their in vitro propagation in cell cultures.
In the study I, TTSuV1 open reading frames (ORF) 1 (ORF1) and ORF2 proteins were expressed in a prokaryotic (Escherichia coli (E. coli)) expression system and used to produce monoclonal antibodies (mAbs) against viral proteins. Specific TTSuV1 ORF2 monoclonal antibodies obtained and confirmed their specificity by western blot (WB) were applied in an immunoflorescence (IF) assay on transfected cells. The expressed TTSuV1 ORF2 protein was distributed in the cytoplasm and nucleus of the cells. Specific mAbs against TTSuV1 ORF1 protein were also generated but were not applicable in IF technique.
In the study II, the gene expression and subcellular localization of TTSuV proteins from distinct species were characterized in vitro. Following transfection of three TTSuV1 and TTSuV2 full-length ORF (ORF1, ORF2 and ORF3) expression constructs into porcine kidney cells, alternative spliced variants encoding new TTSuV protein isoforms were identified. Proteins encoded from ORF1 and ORF3 were localized in the nucleoli of porcine kidney cells and that of ORF2 througout the cells (excluding the nucleoli). The subcellular localization of the different protein isoforms was not only similar between distinct TTSuV species but also to the ones described in human Torque teno virus. Results of the present in vitro study were not based on full-length viral clones but suggested that alternative splicing strategy to generate TTSuV protein isoforms likely occurs in vivo.
In the study III, to attempt in vitro propagation of TTSuV species, cell lines of porcine origin including newborn porcine trachea (NPT), newborn swine kidney (NSK), porcine aortic endothelial (PEDSV 15), porcine kidney (PK-15), swine testis (ST) and macrophage-like cells were inoculated with a TTSuV positive sample (spleen) or transfected with a plasmid containing two copies of the TTSuV1 genome (pTTSuV1-dimer). In PK-15 and ST cells inoculated with the tissue suspension, specific mRNAs corresponding to the TTSuV2 ORF1 transcript were detected at 48 and 72 h after inoculation. However, when cells were subcultured, viral transcription was not confirmed. Results obtained from the pTTSuV1-dimer transfected cell lines demonstrated that the constructed clone was active and able to synthesize specific mRNAs. In these cells the expression of TTSuV1 ORF2 protein was also confirmed by an IF assay using specific mAbs produced against ORF2 in the study I. However, after passages of inoculated cells, propagation of TTSuV1 was not achieved as well.
Finally, to explore the impact of TTSuV on immune defences, in the study IV the modulation of interferon responses was assessed by TTSuV and Porcine circovirus type-2 (PCV2) DNA interaction with porcine dendritic cells (DCs). Data obtained demonstrated that viral DNA from TTSuV species and two of the PCV2 genotypes (PCV2a and PCV2b) were able to impair the porcine DC function by interferon (IFN)-¿ inhibition in a dose-dependent manner. It was also found that the inhibitory effect was different between TTSuV species. TTSuV2 DNA was more efficient in inhibition than that of TTSuV1, with a similar level of inhibition to the ones observed for both PCV2 genotypes.
Overall, obtained results contribute to elucidate some basic aspects of the biology and host immune responses to TTSuV species. Moreover, methods developed can be applied in further studies to address important unanswered questions regarding epidemiology and pathogenesis of anelloviruses.
