Chimeric swine vesicular disease viruses produced by fusion PCR: a new method for epitope mapping
Dekker, A. ; Leendertse, C.H. ; Poelwijk, F. van; Rebel, J.M.J. ; Moormann, R.J.M. - \ 2000
Journal of Virological Methods 86 (2000). - ISSN 0166-0934 - p. 131 - 141.
Construction of a full-length infectious cDNA clone of swine vesicular disease virus strain NET/1/92 and analysis of new antigenic variants derived from it
Rebel, J.M.J. ; Leendertse, C.H. ; Dekker, A. ; Poelwijk, F. van; Moormann, R.J.M. - \ 2000
Journal of General Virology 81 (2000). - ISSN 0022-1317 - p. 2763 - 2769.
|Molecular approaches for analyzing the functionality of probiotic lactic acid bacteria in the gastrointestinal tract.
Vos, W.M. de; Poelwijk, E. ; Sessitsch, A. ; Zoetendal, E.G. ; Overbeek, L.S. van; Akkermans, A.D.L. - \ 1998
In: Lactic Acid Bacteria: Actes de Collogue LACTIC 97, Adria Normandie, Villers-Bocage, France - p. 51 - 57.
Detection of early infection of swine vesicular disease virus in porcine cells and skin sections. A comparison of immunohistochemistry and in-situ hybridization
Mulder, W.A.M. ; Poelwijk, F. van; Moormann, R.J.M. ; Reus, B. ; Kok, G.L. ; Pol, J.M.A. ; Dekker, A. - \ 1997
Journal of Virological Methods 68 (1997)2. - ISSN 0166-0934 - p. 169 - 175.
Sensitive methods are required to study the early pathogenesis of swine vesicular diseases (SVD). Therefore, two new methods, immunohistochemistry (IHC) and in-situ hybridization (ISH), were developed and tested for their specificity and sensitivity. With these methods the SVD virus (SVDV) infection in cytospins of primary porcine kidney cells and in frozen skin sections was investigated. Both IHC and the ISH showed a specific cytoplasmic staining, but the IHC detected more infected cells than the ISH. Furthermore, both IHC and ISH were able to detect SVDV in skin sections 4.5 h after infection. It is concluded that IHC is the most suitable and simplest method to identify cells and tissues that support the initial replication of swine vesicular disease virus. However, IHC can only be applied to frozen sections, whereas ISH can also be used in paraformaldehyde-fixed tissues.
|Molecular methods for GI-tract ecology.
Vos, W.M. de; Poelwijk, E.S. ; Zoetendal, E.G. ; Overbeek, L.S. van; Akkermans, A.D.L. - \ 1997
In: Novel Methods for probiotic research. BTT Symp., 173 (T. Mattila-Sandholm Ed.) - p. 21 - 24.
|Completion of the genome sequence of impatiens necrotic spot virus and genetic comparison of the L proteins within the family Bunyaviridae.
Poelwijk, F. van; Prins, M. ; Goldbach, R. - \ 1997
Journal of General Virology 78 (1997). - ISSN 0022-1317 - p. 543 - 546.
A protoplast system for studying tomato spotted wilt virus infection.
Kikkert, M. ; Poelwijk, F. van; Karssies, W. ; Bloksma, H. ; Storms, M. ; Lent, J. van; Kormelink, R. ; Goldbach, R. - \ 1997
Journal of General Virology 78 (1997). - ISSN 0022-1317 - p. 1755 - 1763.
|Studying tomato spotted wilt virus maturation: exploitation of plant and animal single-cell systems.
Kikkert, M. ; Poelwijk, F. van; Storms, M. ; Bodegom, P. ; Woensel, C. van; Lent, J. van; Kormelink, R. ; Goldbach, R. - \ 1997
In: EMBO Workshop Molecular mechanisms in the replicative cycle of viruses in plants, Las Navas del Marques, Spain - p. 28 - 28.
Replication and expression of the tospoviral genome.
Poelwijk, F. van; Haan, P. de; Kikkert, M. ; Prins, M. ; Kormelink, R. ; Storms, M. ; Lent, J. van; Peters, D. ; Goldbach, R. - \ 1996
Acta Horticulturae 431 (1996). - ISSN 0567-7572 - p. 201 - 208.
Sequence analysis of the complete, tripartite RNA genomes of two tospoviruses, tomato spotted wilt virus (TSWV) and impatients necrotic spot virus (INSV), demonstrated that they possess five genes that specify six functional proteins. The negative-stranded L RNA encodes the putative polymerase (TSWV 331.5 kDa; INSV 330.3 kDa), the ambisense M RNA encodes a common precursor to the two glycoproteins (G1 and G2) and a nonstructural protein (NSm), and the likewise ambisense S RNA encodes the nucleocapsid (N) protein and a second nonstructural protein (NSs). These viral proteins are expressed from mRNAs that contain 12–20 nontemplated nucleotides at the 5' ends. This indicates that “cap-snatching” is the mechanism used by the viral polymerase to initiate transcription. Sequence analysis revealed that there was no strict base preference at the endonucleolytic site of the cellular leaders. Whereas the function of NSs, the least conserved tospoviral protein, has remained enigmatic, evidence is accumulating that NSm represents the viral movement protein that is involved in a tubule-guided cell-to-cell movement of nonenveloped nucleocapsids. Protoplasts infected with TSWV or transfected with the NSm gene solely, develop long, NSm-containing tubules that extend from the plasma membrane into the culture medium and are similar to tubules found in plasmodesmata of infected plant tissues. Experiments with transgenic plants confirm that NSm is a plasmodesma-associated protein that can modify intercellular communication in plants.
|A protoplast system for studying tomato spotted wilt virus infection.
Kikkert, M. ; Poelwijk, F. van; Storms, M. ; Bloksma, H. ; Kormelink, R. ; Goldbach, R. - \ 1996
In: Abstract 10th Int. Congr. of Virology. Jerusalem, Israel (1996) 83. Ook: Abstract NWO-SON bijeenkomst, Lunteren - p. 55 - 55.
|Exploitation of plant and animal single-cell systems for studying tomato spotted wilt virus gene expression.
Kikkert, M. ; Poelwijk, F. van; Storms, M. ; Prins, M. ; Bodegom, P. ; Woensel, C. van; Lent, J. van; Kormelink, R. ; Goldbach, R. - \ 1996
In: Najaarsbijeenkomst Nederlandse Kring voor Plantenvirologie, Wageningen (1996) 2. Ook: Abstract Virologie Symposium, Utrecht - p. 4 - 4.
|Sequence analysis of the 5' ends of tomato spotted wilt virus N mRNAs.
Poelwijk, F. van; Kolkman, J. ; Goldbach, R. - \ 1996
Archives of Virology 141 (1996). - ISSN 0304-8608 - p. 177 - 184.
On the expression strategy of the tospoviral genome
Poelwijk, F. van - \ 1996
Agricultural University. Promotor(en): R.W. Goldbach; D. Peters. - S.l. : Van Poelwijk - ISBN 9789054855828 - 99
tomatenbronsvlekkenvirus - virussen - genetica - virologie - rna-virussen - tomato spotted wilt virus - viruses - genetics - virology - rna viruses
The work described in this thesis was aimed at the unravelling of the molecular biology of tospoviruses, with special emphasis on the process of replication of the tripartite RNA genome.
At the onset of the research the complete genome sequence of tomato spotted wilt virus (TSWV), type species of the genus Tospovirus, became available. These sequence data indicated that the tospoviruses represent plant-infecting members of the large family of the arthropod-born Bunyaviridae. Genome sequence comparisons indicated however that the L RNA segment of TSWV would encode a much larger viral polymerase (331.5 kDa) than, as far as known, its animal-infecting counterparts (reported sizes of 241 to 259 kDa). To verify whether a large polymerase represents a characteristic i.e. genus-specific property of tospoviruses the complete sequence of the L RNA segment of a second tospovirus, impatiens necrotis spot virus (INSV), was elucidated (Chapter 2). These sequence data revealed that the L RNA of INSV appeared to be comparable in size to that of TSWV (8675 nucleotides versus 8897 for TSWV), containing an open reading frame with a predicted size of 330.3 kDa of the INSV polymerase. Therefore the next question to be answered was whether the large primary translation product of the tospoviral L RNA acts as an unprocessed polymerase or whether this protein would undergo some cleavages to obtain smaller, functional replication proteins. Answering this question was even more necessary since the theoretical size of the TSWV L RNA ORF greatly exceeded previously determined sizes (110 to 220 kDa) for a large protein reported to copurify with TSWV particles. To this end both the 5'-terminal and 3'-terminal parts of the ORF in the TSWV L RNA were expressed in Escherichia coli and antibodies raised against these regions. Using these tools it could be established that the polymerase (L protein) of TSWV, though significantly larger than that of other bunyaviruses, is present in virus particles (10 to 20 copies per virion) in an unprocessed, full length form (Chapter 3). To allow further analyses of the TSWV polymerase, attempts were made to clone and express the complete L RNA ORF in the baculovirus/insect cell system. In spite of all efforts, only a shorter translation product of 67 kDa was obtained from a baculovirus recombinant containing a complete DNA copy of the TSWV L RNA (Chapter 3). Sequence analysis of the cloned copy revealed a 80 basepairs deletion, resulting in two premature stop codons, which most likely have led to the resulting truncated L protein.
To gain more insight in the "cap-snatching" event which takes place during initiation of tospovirus transcription, nucleoprotein (N) mRNAs were partially purified from TSWV-infected N.rustica leaves and cloned (Chapter 4). Sequence analysis of the cloned, 5'-proximate regions of 20 cloned mRNAs showed the presence of extra, non-templated sequences, ranging in length from 12 to 21 nucleotides, confirming our earlier primer extension studies. As these sequences were of non-viral origin a cap-snatching mechanism for tospoviral transcription initiation could thus be definitively identified. None of the hostderived leader sequences analyzed were identical and only limited sequence specificity at the endonucleolytic site was observed (some preference for cleavage at a U residue). During the course of this Ph.D. research, Adkins et al. (1995) reported that in vitro transcriptase activity was associated with freshly isolated TSWV particles. It was investigated (Chapter 5) whether the reported levels of in vitro activity could be further improved and whether this system would lend itself for analysis of the viral proteins involved by e.g. inhibition studies using specific antibodies. Trichloroacetic acid-precipitable products could consistently be obtained after incubation of detergent-disrupted TSWV virions under the assay conditions reported by Adkins et al. (1995) and using (α- 32P)CTP. No significant improvement in CMP incorporation levels could be achieved by testing variable conditions and varying concentrations of assay components. The reaction products obtained hybridized with clones from all three genomic RNA segments. No discrimination between transcription and replication could be made however, and since none of the available specific antibodies directed against any viral protein had an inhibitory effect, it was concluded that the current in vitro system will be of limited value for unravelling the RNA synthesizing process and the role of the individual viral proteins therein.
As a first step towards a manipulatable transcription/replication system, a hybrid baculovirus/bacteriophage T7 vector system was developed for transient expression in insect cells of all factors involved in TSWV genome transcription and replication. The results obtained (Chapter 6) illustrate the potential of the system. Although various foreign genes could successfully be expressed to measurable amounts, the reconstitution of a TSWV transcription/replication complex was hampered due to the apparent impossibility (Chapter 3) to clone the complete polymerase gene. Finally, in Chapter 7 (General discussion and concluding remarks), the results obtained are compared with the data reported for animalinfecting bunyaviruses, leading to a discussion of some evolutionary aspects. Furthermore, suggestions are made to circumvent some of the problems encountered during the course of the studies presented in this thesis.
|A hybrid baculovirus-bacteriophage T7 transient expression system.
Poelwijk, F. van; Broer, R. ; Belsham, G.J. ; Oudshoorn, P. ; Vlak, J.M. ; Goldbach, R.W. - \ 1995
Bio/Technology 13 (1995). - ISSN 0733-222X - p. 261 - 264.
|Tospoviral genome replication and expression.
Goldbach, R. ; Poelwijk, F. van; Kormelink, R. ; Storms, M. ; Prins, M. ; Peters, D. ; Haan, P. de; Lent, J. van - \ 1995
In: Int. Symp. on Tospoviruses and thrips of floral and vegetable crops. Taichung, Taiwan (1995)
|Tomato spotted wilt virus: genome organization, transmission, and symptom induction.
Goldbach, R. ; Kormelink, R. ; O. Resende, R. de; Avila, A.C. de; Poelwijk, F. van; Lent, J. van; Wijkamp, I. ; Prins, M. ; Peters, D. - \ 1995
In: Biotechnology and plant protection / Bills, D.D., Kung, Shain-dow, - p. 297 - 311.
|Tomato spotted wilt virus: genome structure, classification, detection and genetically engineered resistence.
Avila, A.C. de; Haan, P. de; Kormelink, R. ; Oliveira Resende, R. de; Poelwijk, F. van; Wijkamp, M.G. ; Goldbach, R.W. ; Peters, D. - \ 1993
In: Virology in the tropics / Rishi, N., - p. 90 - 100.
|Expression of the genome of tomato spotted wilt virus, a bunyavirus invading the plant kingdom.
Goldbach, R. ; Kormelink, R. ; Poelwijk, F. van; Wijkamp, I. ; Prins, M. ; Peters, D. - \ 1993
In: Abstract 9th Int. Congr. Virology, Glasgow, UK - p. 7 - 7.
|Detection of the L protein of tomato spotted wilt virus.
Poelwijk, F. van; Boye, K. ; Oosterling, R. ; Peters, D. ; Goldbach, R.W. - \ 1993
Virology 197 (1993). - ISSN 0042-6822 - p. 468 - 470.
|Characterization of the RNA polymerase of tomato spotted wilt virus.
Boye, K. ; Poelwijk, F. van; Peters, D. ; Goldbach, R.W. - \ 1993
In: Abstract 9th Int. Congr. Virology, Glasgow, UK - p. 137 - 137.