Detection and Identification of the Atypical Bovine Pestiviruses in Commercial Foetal Bovine Serum Batches

The recently emerging atypical bovine pestiviruses have been detected in commercial foetal bovine serum (FBS) of mainly South American origin so far. It is unclear how widely the viruses are presented in commercial FBS of different geographic origins. To further investigate the possible pestivirus contamination of commercially available FBS batches, 33 batches of FBS were obtained from ten suppliers and analysed in this study for the presence of both the recognised and the atypical bovine pestiviruses. All 33 batches of FBS were positive by real-time RT-PCR assays for at least one species of bovine pestiviruses. According to the certificate of analysis that the suppliers claimed for each batch of FBS, BVDV-1 was detected in all 11 countries and BVDV-2 was detected exclusively in the America Continent. The atypical pestiviruses were detected in 13 batches claimed to originate from five countries. Analysis of partial 5′UTR sequences showed a high similarity among these atypical bovine pestiviruses. This study has demonstrated, for the first time that commercial FBS batches of different geographic origins are contaminated not only with the recognised species BVDV-1 and BVDV-2, but also with the emerging atypical bovine pestiviruses

Identification and characterization of protein-protein interactions in the nuclear envelope

The nuclear envelope forms the interface between the nucleus and the cytoplasm. The nuclear envelope consists of the two concentric lipid membranes, the nuclear pores and the nuclear lamina. The inner nuclear membrane contains hundreds of unique transmembrane proteins showing high tissue diversity. Mutations of some proteins in the nuclear envelope give rise to a broad spectrum of diseases called envelopathies or laminopathies. In this thesis, I aimed to study the functional organization of the nuclear envelope by identifying and characterizing interactions between the nuclear envelope proteins. For this, we developed a novel method called the Membrane Protein Crosslink Immuno-Precipitation, which enable identification of protein-protein interactions in the nuclear envelope in live cells. We identified several novel interactions of the inner nuclear membrane protein, Samp1, and studied the interaction between the Samp1 and the nuclear GTPase, Ran in detail. Samp1 can bind to Ran and is thus the first known transmembrane Ran binding protein and Samp1 might provide a local binding site for Ran in the inner nuclear membrane. We found that Samp1 also binds to the inner nuclear membrane protein, Emerin and Ran can regulate the Samp1-Emerin interaction in the nuclear envelope. During mitosis, Samp1 distributes in the mitotic spindle. Therefore, we investigated a possible functional role of Samp1 in the mitotic machinery. Samp1 depletion resulted in aneuploid phenotypes, metaphase prolongation and decreased distribution of γ-tubulin and β-tubulin in the mitotic spindle. We found that Samp1 can bind to γ-tubulin, which is essential for the microtubule nucleation and hence for the spindle stability. The new interesting features of Samp1 provide insights on the unforeseen functions of the nuclear envelope proteins.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p

The batches of foetal bovine serum tested by real-time RT-PCR.

a<p>Sequence has not been determined.</p

Neighbor-Joining analysis of a 249-bp fragment of the 5′UTR sequences under Kimura 3-parameter model.

<p>The sequences are labelled with sample ID (-number of clone)/country or region of origin. AU, Australia; BR, Brazil; CA, Canada; CO, Colombia; DK, Denmark; DR, Dominican Republic; EU, European Union; FR, France; MX, Mexico; NI, not identified by supplier; NZ, New Zealand; SA, South American; US, USA; ZA, South Africa. Numbers are percentage of bootstrap values (1000 replicates) for major clades. Bar indicates changes per site. The GenBank accession numbers for reference strains are DQ075210, NC_001461, U97481, AF026781, AB359927, AF049221, AY363096 and FJ493479 for BVDV-1, AY763053 and NC_002032 for BVDV-2, and NC_012812 for BVDV-3.</p