Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Cross-Validation of Generic Risk Assessment Tools for Animal Disease Incursion Based on a Case Study for African Swine Fever
    Vos, Clazien J. de; Taylor, Rachel A. ; Simons, Robin R.L. ; Roberts, Helen ; Hultén, Cecilia ; Koeijer, Aline A. de; Lyytikäinen, Tapani ; Napp, Sebastian ; Boklund, Anette ; Petie, Ronald ; Sörén, Kaisa ; Swanenburg, Manon ; Comin, Arianna ; Seppä-Lassila, Leena ; Cabral, Maria ; Snary, Emma L. - \ 2020
    Frontiers in Veterinary Science 7 (2020). - ISSN 2297-1769
    African swine fever - cross-validation - generic model - introduction risk - livestock diseases - model uncertainty - risk assessment

    In recent years, several generic risk assessment (RA) tools have been developed that can be applied to assess the incursion risk of multiple infectious animal diseases allowing for a rapid response to a variety of newly emerging or re-emerging diseases. Although these tools were originally developed for different purposes, they can be used to answer similar or even identical risk questions. To explore the opportunities for cross-validation, seven generic RA tools were used to assess the incursion risk of African swine fever (ASF) to the Netherlands and Finland for the 2017 situation and for two hypothetical scenarios in which ASF cases were reported in wild boar and/or domestic pigs in Germany. The generic tools ranged from qualitative risk assessment tools to stochastic spatial risk models but were all parameterized using the same global databases for disease occurrence and trade in live animals and animal products. A comparison of absolute results was not possible, because output parameters represented different endpoints, varied from qualitative probability levels to quantitative numbers, and were expressed in different units. Therefore, relative risks across countries and scenarios were calculated for each tool, for the three pathways most in common (trade in live animals, trade in animal products, and wild boar movements) and compared. For the 2017 situation, all tools evaluated the risk to the Netherlands to be higher than Finland for the live animal trade pathway, the risk to Finland the same or higher as the Netherlands for the wild boar pathway, while the tools were inconclusive on the animal products pathway. All tools agreed that the hypothetical presence of ASF in Germany increased the risk to the Netherlands, but not to Finland. The ultimate aim of generic RA tools is to provide risk-based evidence to support risk managers in making informed decisions to mitigate the incursion risk of infectious animal diseases. The case study illustrated that conclusions on the ASF risk were similar across the generic RA tools, despite differences observed in calculated risks. Hence, it was concluded that the cross-validation contributed to the credibility of their results.

    G-RAID: Assessing the incursion risk of African swine fever virus using generic risk assessment tools
    Vos-de Jong, C.J. de; Taylor, R.A. ; Simons, R.R.I. ; Roberts, H. ; Hultén, C. ; Koeijer, A.A. de; Lyytikäinen, T. ; Napp, S. ; Boklund, A. ; Petie, R. ; Sörén, K. ; Swanenburg, M. ; Comin, A. ; Seppä-Lassila, L. ; Cabral, M. ; Snary, E. - \ 2019
    Wageningen : Wageningen University & Research
    Generic approaches for Risk Assessment of Infectious animal Disease introduction (G-RAID)
    Vos-de Jong, C.J. de; Taylor, R.A. ; Simons, R.R.L. ; Roberts, H. ; Hultén, C. ; Koeijer, A.A. de; Lyytikänen, T. ; Napp, S. ; Boklund, A. ; Petie, R. ; Sörén, K. ; Swanenburg, M. ; Comin, A. ; Seppä-Lassila, L. ; Labato Guimarães Ferraira Cabral, Maria ; Snary, E.L. - \ 2019
    EFSA Supporting Publications 16 (2019)11. - ISSN 2397-8325
    The objective of the G‐RAID project was the mutual exchange of knowledge between the consortium members on the development of generic risk assessment (RA) tools for animal disease incursion. Seven generic RA tools were compared considering objectives, inputs, algorithms and outputs. All tools were designed for rapid risk assessment and could assess the incursion risk for multiple diseases and pathways. Specific objectives of the tools, however, varied from immediate response to new disease events to prioritization of diseases and horizon scanning, resulting in different approaches to evaluate the incursion risk of infectious animal diseases. Cross‐validation was explored as a method to validate the generic RA tools. All tools were applied to a case study for African swine fever (ASF) in which the incursion risk for the Netherlands and Finland was assessed for the 2017 situation and two hypothetical scenarios with ASF cases reported in Germany. The generic RA tools were parameterized using the same global databases for disease occurrence and trade in live animals and animal products. Disease‐related parameters, however, could not be standardized because of the different levels of detail included in the model calculations. A comparison of absolute results of the tools was not possible, because output parameters represented different endpoints, varied from qualitative probability levels to quantitative numbers, and were expressed in different units. Therefore, relative risks across countries and scenarios were calculated for each tool and compared. The risk assessment tools largely agreed upon the ranking of countries and scenarios based on relative risks and would thus indicate similar priorities for risk management As such, the cross‐validation increased the credibility of results obtained with the generic RA tools. The cross‐validation also contributed to the internal validation and further development of the tools. Results from the G‐RAID project were disseminated to risk assessors and risk managers at a one‐day symposium.
    Assessment of heterosis in two arabidopsis thaliana common-reference mapping populations
    Hulten, Marieke H.A. van; Paulo, Maria Joāo ; Kruijer, Willem ; Blankestijn-de Vries, Hetty ; Kemperman, Brend ; Becker, Frank F.M. ; Yang, Jiaming ; Lauss, Kathrin ; Stam, Maike E. ; Eeuwijk, Fred A. van; Keurentjes, Joost J.B. - \ 2018
    PLoS ONE 13 (2018)10. - ISSN 1932-6203

    Hybrid vigour, or heterosis, has been of tremendous importance in agriculture for the improvement of both crops and livestock. Notwithstanding large efforts to study the phenomenon of heterosis in the last decades, the identification of common molecular mechanisms underlying hybrid vigour remain rare. Here, we conducted a systematic survey of the degree of heterosis in Arabidopsis thaliana hybrids. For this purpose, two overlapping Arabidopsis hybrid populations were generated by crossing a large collection of naturally occurring accessions to two common reference lines. In these Arabidopsis hybrid populations the range of heterosis for several developmental and yield related traits was examined, and the relationship between them was studied. The traits under study were projected leaf area at 17 days after sowing, flowering time, height of the main inflorescence, number of side branches from the main stem or from the rosette base, total seed yield, seed weight, seed size and the estimated number of seeds per plant. Predominantly positive heterosis was observed for leaf area and height of the main inflorescence, whereas mainly negative heterosis was observed for rosette branching. For the other traits both positive and negative heterosis was observed in roughly equal amounts. For flowering time and seed size only low levels of heterosis were detected. In general the observed heterosis levels were highly trait specific. Furthermore, no correlation was observed between heterosis levels and the genetic distance between the parental lines. Since all selected lines were a part of the Arabidopsis genome wide association (GWA) mapping panel, a genetic mapping approach was applied to identify possible regions harbouring genetic factors causal for heterosis, with separate calculations for additive and dominance effects. Our study showed that the genetic mechanisms underlying heterosis were highly trait specific in our hybrid populations and greatly depended on the genetic background, confirming the elusive character of heterosis.

    Parental DNA methylation states are associated with heterosis in epigenetic hybrids
    Lauss, Kathrin ; Wardenaar, R. ; Oka, Rurika ; Hulten, M.H.A. ; Guryev, Victor ; Keurentjes, J.J.B. ; Stam, Maike ; Johannes, Frank - \ 2018
    Plant Physiology 176 (2018)2. - ISSN 0032-0889 - p. 1627 - 1645.
    Despite the importance and wide exploitation of heterosis in commercial crop breeding, the molecular mechanisms behind this phenomenon are not completely understood. Recent studies have implicated changes in DNA methylation and small RNAs in hybrid performance, however, it remains unclear whether epigenetic changes are a cause or consequence of heterosis. Here, we analyze a large panel of over 500 A. thaliana epigenetic hybrid plants (epiHybrids), which we derived from near-isogenic but epigenetically divergent parents. This proof-of-principle experimental system allowed us to quantify the contribution of parental methylation differences to heterosis. We measured traits such as leaf area (LA), growth rate (GR), flowering time (FT), main stem branching (MSB), rosette branching (RB) and final plant height (HT) and observed several strong positive and negative heterotic phenotypes among the epiHybrids. Using an epigenetic quantitative trait locus mapping approach, we were able to identify specific differentially methylated regions (DMRs) in the parental genomes that are associated with hybrid performance. Sequencing of methylomes, transcriptomes and genomes of selected parent-epiHybrid combinations further showed that these parental DMRs most likely mediate remodeling of methylation and transcriptional states at specific loci in the hybrids. Taken together, our data suggest that locus-specific epigenetic divergence between the parental lines can directly or indirectly trigger heterosis in Arabidopsis hybrids independent of genetic changes. These results add to a growing body of evidence that points to epigenetic factors as one of the key determinants of hybrid performance.
    Salmonid alphavirus envelope glycoprotein processing determines virus-like particle production in insect cells
    Metz, S.W.H. ; Feenstra, F. ; Villoing, S. ; Hulten, M.C.W. van; Lent, J.W.M. van; Koumans, J. ; Vlak, J.M. ; Pijlman, G.P. - \ 2011
    Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation
    Metz, S.W.H. ; Feenstra, F. ; Villoing, S. ; Hulten, M.C. van; Lent, J.W.M. van; Koumans, J. ; Vlak, J.M. ; Pijlman, G.P. - \ 2011
    PLoS ONE 6 (2011)10. - ISSN 1932-6203 - 10 p.
    semliki-forest-virus - equine encephalitis-virus - nuclear polyhedrosis-virus - swine-fever virus - pancreas disease - atlantic salmon - spodoptera-frugiperda - nucleocapsid protein - baculovirus vectors - sleeping-disease
    Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ~17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production
    Molecular mechanisms involved in induced resistance signaling in Arabidopsis
    Pieterse, C.M.J. ; Pelt, J.A. van; Verhagen, B.W.M. ; Vos, M. de; Oosten, V.R. van; Ent, S. van der; Koornneef, A. ; Hulten, M.H.A. ; Pozo, M.J. ; Ton, J. ; Dicke, M. ; Loon, L.C. van - \ 2006
    In: Biology of Molecular Plant-Microbe Interactions / Sánchez, F., Quinto, C., López-Lara, I.M., Geiger, O., St. Paul, MN : The Int. Society for Molecular Plant-Microbe Interactions - ISBN 9780965462549 - p. 188 - 194.
    Mechanisms involved in inducible and constitutive resistance to pests and diseases
    Ton, J. ; Ent, S. van der; Hulten, M.C.W. van; Pozo, M.J. ; Oosten, V.R. van; Loon, L.C. van; Mauch-Mani, B. ; Turlings, T.C.J. ; Pieterse, C.M.J. - \ 2006
    In: Abstracts Breeding for inducible resistance against pests and diseases, Heraklio, Crete, Greece, 27 - 29 April, 2006. - Heraklio, Crete, Greece : - p. 2 - 2.
    On the vaccination of shrimp against white spot syndrome virus
    Witteveldt, J. - \ 2006
    Wageningen University. Promotor(en): Just Vlak; R.W. Goldbach, co-promotor(en): M.C.W. van Hulten. - [S.l. ] : S.n. - ISBN 9789085043317 - 132
    garnalen - dierenvirussen - virusziekten - dna-bindende eiwitten - vaccinatie - immuniteitsreactie - virologie - immunologie - schaal- en schelpdierenteelt - shrimps - animal viruses - viral diseases - dna binding proteins - vaccination - immune response - virology - immunology - shellfish culture
    More than a decade after its discovery inSouth-East Asia, White Spot Syndrome Virus (WSSV) is still the most important (viral) pathogen in the shrimp culture industry. Despite the shift from culturingPenaeusmonodon towards the presumed less susceptibleLitopenaeusvannamei , the use of specific pathogen free shrimp and the development of more advanced shrimp culturing techniques, WSSV continues to scourge shrimp farms. Therefore there is an urgent need for effective intervention strategies. Vaccination is the generally used method to prevent viral infections in vertebrates. The success of this method depends on the immunological memory generated by the adaptive immune system. Unfortunately, shrimps, as any other arthropod, do not have such an adaptive immune system implying that vaccination would never work. However, some phenomenological observations have been made, indicating that there might be an analogous defense system present in shrimp. With this in mind experiments in this thesis are presented to determine if and how shrimp can be protected against WSSV via vaccination.

    At the start of this research project several studies were available describing various major structural proteins present in the WSSVvirionincluding the majorvirionenvelope proteins VP28 and VP19 andvirionstructural proteins VP26, VP24 and VP15 (see thesis vanHulten, 2001). In this research a number of these proteins were investigated in more detail as potential vaccine candidates. For one of these, the majornucleocapsidprotein VP15, it was determined that it was probably (one of) the DNA-binding protein(s) of WSSV (Chapter 2). Experiments revealed that VP15 binds non-specifically to double-stranded DNA, but has a strong preference tosupercoiledDNA, suggesting a possible role in the packaging of the WSSV genome. Furthermore, VP15 formshomomultimersbut does not interact with any of the other major WSSV structural proteins and unlike other basic DNA-binding proteins VP15 was notphosphorylated.

    The next structural protein studied in more detail was VP28 which, because of its abundance and location in the envelope of the WSSVvirion, was another potential candidate for use as a WSSV vaccine. The involvement of the VP28 protein in the infection process of WSSV was studied in virus neutralization experiments using polyclonal antibodies generated against the VP28 protein in rabbit (Chapter 3). The antiserum neutralized WSSV infection of P.monodon in a dose-dependent manner, whereas the pre-immune rabbit serum did not. These results suggested that VP28 is located on the surface of the virus particle and is likely to play a key role in the initial steps of the systemic WSSV infection in shrimp. Although the results from the neutralization experiments seemed conclusive, further research revealed that the observed neutralization is probably notIgG-based. Experiments showed that some rabbit pre-immune sera are already able to neutralize WSSV and furthermore, purifiedIgGfrom sera that neutralized WSSV was not able to neutralize the virus (Chapter 4). Therefore, it could be concluded that in most cases the neutralization is not antibody based, but caused by unidentified serum components.

    VP28 and the other major envelope protein VP19 were tested in vaccination and challenge experiments. The first experiments were performed via injection of the antigens and virus as this guaranteed a controlled and reproducible application. Injection with recombinant MBP-VP19 or a mixture of MBP-VP19 and His 6 -VP28 significantly reduced and delayed mortality upon WSSV challenge, suggesting a specific role of VP19 in the systemic defense response of shrimp (Chapter 5). To study the onset and duration of the vaccination, groups of shrimp were challenged two or twenty-five days after vaccination. After the challenge, VP19-vaccinated shrimp showed a significant better survival compared to the controls with a Relative Percent Survival (RPS) of 33% and 57% at two and 25 days after vaccination, respectively. Also the groups vaccinated with VP28 and a mixture of VP19 and VP28 showed a significantly better survival challenged two days after vaccination (RPS of 44% and 33% respectively), but no longer after twenty-five days (Chapter 6).

    Although these injection experiments clearly showed that shrimp are indeed capable of specifically recognizing foreign proteins and exhibit a kind of adaptive memory, the injection vaccination technique is far from suited for use under shrimp farming conditions. Therefore the potential of oral vaccination of shrimp using the same viral envelope proteins was investigated (Chapter 7). In this setup P.monodon shrimp were fed commercial food pellets coated with inactivated bacteria thatoverexpressedboth envelope proteins VP19 and VP28. In order to approach the natural route of WSSV infection and subject the virus to the full array of immunological responses of the shrimp, the challenge was performed via immersion of the shrimp in WSSV containing seawater. When the challenge was performed three days after a seven-day vaccination period, VP28 vaccinated shrimp showed a significant lower cumulative mortality compared to shrimp vaccinated with bacteria containing empty vectors (RPS of 61%), while vaccination with VP19 provided no protection. To determine the onset and duration of protection of VP28, challenges were performed three, seven and twenty-one days after the seven-day vaccination period. A significantly higher survival was observed both three and seven days post vaccination (RPS of 64% and 77%, respectively), but the protection was reduced twenty-one days after the vaccination (RPS of 29%). These results strongly suggest that a specific immune response and ultimately protection can be generated in an invertebrate species like shrimp.

    In an effort to investigate whether the oral vaccination effects were limited to P.monodon or based on more universal mechanisms, the vaccination experiments were applied to an alternative host for WSSV, the Pacific White shrimpLitopenaeusvannamei(Chapter 8). Also this species showed a significantly lower cumulative mortality upon VP28 vaccination compared to the control groups. This outcome points to a shared and therefore universal adaptive response mechanism present in crustaceans. It is still not clear whether this response is WSSV specific or more generally directed against viruses.

    In recent years more evidence has become available suggesting the presence of a specific immune response and adaptive memory in invertebrates. The results presented in this thesis support this view by showing that the shrimp's immune system is able to specifically recognize and react upon WSSV structural proteins or more in general, proteins lacking known pathogen associated molecular patterns. Furthermore, the studies described in this thesis have shown that vaccination of shrimp against WSSV can be successful, which opens the way to the design of new strategies to control WSSV and other invertebrate pathogens.

    Increased tolerance of Litopenaeus vannamei to white spot syndrome virus (WSSV) infection after oral application of the viral envelope protein VP28
    Witteveldt, J. ; Vlak, J.M. ; Hulten, M.C.W. van - \ 2006
    Diseases of Aquatic Organisms 70 (2006)1/2. - ISSN 0177-5103 - p. 167 - 170.
    penaeus-monodon - invertebrate - protection - immunity - memory
    It has been generally accepted that invertebrates such as shrimp do not have an adaptive immune response system comparable to that of vertebrates. However, in the last few years, several studies have suggested the existence of such a response in invertebrates. In one of these studies, the shrimp Penaeus monodon showed increased protection against white spot syndrome virus (WSSV) using a recombinant VP28 envelope protein of WSSV. In an effort to further investigate whether this increased protection is limited to P. monodon or can be extended to other penaeid shrimp, experiments were performed using the Pacific white shrimp Litopenaeus vannamei. As found with P. monodon, a significantly lower cumulative mortality for VP28-fed shrimp was found compared to the controls. These experiments demonstrate that there is potential to use oral application of specific proteins to protect the 2 most important cultured shrimp species, P. monodon and L. vannamei, against WSSV. Most likely, this increased protection is based on a shared and, therefore, general defence mechanism present in all shrimp species. This makes the design of intervention strategies against pathogens based on defined proteins a viable option for shrimp culture
    In silico identification of putative promoter motifs of White Spot syndrome virus
    Marks, H. ; Ren, X.Y. ; Sandbrink, H. ; Hulten, M.C.W. van; Vlak, J.M. - \ 2006
    BMC Bioinformatics 7 (2006)309. - ISSN 1471-2105 - 13 p.
    swine-fever virus - nuclear polyhedrosis-virus - virion protein genes - transcriptional analysis - penaeus-monodon - structural proteins - proteomic analysis - genome sequence - shrimp - baculovirus
    Background: White Spot Syndrome Virus, a member of the virus family Nimaviridae, is a large dsDNA virus infecting shrimp and other crustacean species. Although limited information is available on the mode of transcription, previous data suggest that WSSV gene expression occurs in a coordinated and cascaded fashion. To search in silico for conserved promoter motifs (i) the abundance of all 4 through 8 nucleotide motifs in the upstream sequences of WSSV genes relative to the complete genome was determined, and (ii) a MEME search was performed in the upstream sequences of either early or late WSSV genes, as assigned by microarray analysis. Both methods were validated by alignments of empirically determined 5' ends of various WSSV mRNAs. Results: The collective information shows that the upstream region of early WSSV genes, containing a TATA box and an initiator, is similar to Drosophila RNA polymerase II core promoter sequences, suggesting utilization of the cellular transcription machinery for generating early transcripts. The alignment of the 5' ends of known well-established late genes, including all major structural protein genes, identified a degenerate motif (ATNAC) which could be involved in WSSV late transcription. For these genes, only one contained a functional TATA box. However, almost half of the WSSV late genes, as previously assigned by microarray analysis, did contain a TATA box in their upstream region. Conclusion: The data may suggest the presence of two separate classes of late WSSV genes, one exploiting the cellular RNA polymerase II system for mRNA synthesis and the other generating messengers by a new virus-induced transcription mechanism
    Transcription regulation of White Spot Syndrome Virus in shrimp
    Marks, H. ; Hulten, M.C.W. van; Goldbach, R.W. ; Zuidema, D. ; Vlak, J.M. - \ 2005
    Global analysis of White Spot Syndrome Virus transcription using DNA microarrays
    Marks, H. ; Vorst, O.F.J. ; Houwelingen, A.M.M.L. van; Goldbach, R.W. ; Hulten, M.C.W. van - \ 2005
    Increased protection of shrimp against white spot syndrome virus by oral administration of a major envelope protein: is vaccination reality?
    Witteveldt, J. ; Vlak, J.M. ; Hulten, M.C.W. van - \ 2005
    Since the 1990s shrimp culture has been hampered by mass mortalities in ponds throughout the world. Penaeid shrimp are affected by many infectious agents, mainly of bacterial and viral origin, and especially by white spot syndrome virus (WSSV). The lattar has had a major impact on shrimp culture since its discovery in 1992 and it is still a major problem. The only measures presently used to control WSSV are rigorous sanitation and adequate health management practices. Alternative intervention strategies such as vaccination should be and are currently being explored. Recent experiments in the shrimp Penaeus monodon revealed that oral administration of the major envelope protein VP28 overexpressed in bacteria results in high protection levels compared to control groups (Fig. 1). Though these results are promising for the further development of a practical disease prevention strategy, the mechanism of the protection is unclear and it is important to understand it from a biological viewpoint. Shrimp and crustaceans in general are thought to lack a speciimmune system and thus to be unable to recognize, let alone respond specito, foreign proteins. Nevertheless, increased protection against disease has been observed by oral administration and intramuscular injection of WSSV structural proteins and this has been called a quasi-immune response. As long as the nature of these observed responses is unknown, we can only speculate on the processes involved. Is it a speciimmune response or is it the result of something different like competition for receptors? Another question is whether the protection is durable. Experiments designed to tackle these questions and answers to them will be discussed.
    Nucleocapsid protein VP15 is the basic DNA binding protein of white spot syndrome virus of shrimp
    Witteveldt, J. ; Vermeesch, A.M.G. ; Langenhof, M. ; Lang, A. de; Vlak, J.M. ; Hulten, M.C.W. van - \ 2005
    Archives of Virology 150 (2005)6. - ISSN 0304-8608 - p. 1121 - 1133.
    nuclear polyhedrosis-virus - major structural proteins - histone modifications - penaeus-monodon - genome sequence - supercoiled dna - j-gene - wssv - p53 - identification
    White spot syndrome virus (WSSV) is type species of the genus Whispovirus of the new family Nimaviridae. Despite the elucidation of its genomic sequence, very little is known about the virus as only 6% of its ORFs show homology to known genes. One of the structural virion proteins, VP15, is part of the nucleocapsid of the virus and shows homology to some putative baculovirus DNA binding proteins. These DNA-binding or histone-like proteins are thought to be involved in the condensation and packaging of the genome in the nucleocapsid. Using bacterially expressed VP15 fusion proteins in ELISA and Far-Western experiments showed that VP15 interacts with itself, forming homomultimers, but not with the other major structural proteins of the WSSV virion. Antibodies against phosphorylated proteins revealed that VP15 originating from different sources was not phosphorylated. WSSV VP15 binds non-specifically to double-stranded DNA, but has a clear preference to supercoiled DNA suggesting that VP15 is involved in the packaging of the WSSV genome in the nucleocapsid. This research shed further light on the composition of WSSV virions and the function of one of its nucleocapsid proteins.
    Vaccination of Penaeus monodin against White Spot Syndrome Virus using structural virion proteins
    Witteveldt, J. ; Jolink, F. ; Espita Cifuentes, C. ; Vlak, J.M. ; Hulten, M.C.W. van - \ 2005
    In: Diseases in Asian Aquaculture V : Proceedings of the 5th Symposium on Diseases in Asian Aquaculture. Fish Health Section, Asian Fisheries Society / Walker, P.J., Lester, R.G., Bondad-Reantaso, M.G., Manila : Fish Health Section, Asian Fisheries Society - p. 513 - 522.
    Molecular genetics of White Spot Syndrome Virus
    Marks, H. ; Ren, X. ; Witteveldt, J. ; Sandbrink, H. ; Vlak, J.M. ; Hulten, M.C.W. van - \ 2005
    In: Diseases in Asian Aquaculture / Walker, P.J., Lester, R.G., Bondad-Reantaso, M.G., Manila : Asian Fisheries Society - p. 363 - 378.
    Genomics and transcriptomics of White spot syndrome virus
    Marks, H. - \ 2005
    Wageningen University. Promotor(en): Just Vlak; R.W. Goldbach, co-promotor(en): M.C.W. van Hulten. - Wageningen : s.n. - ISBN 9789085043188 - 152
    garnalen - penaeus monodon - dierenvirussen - transcriptie - genomen - genexpressieanalyse - shrimps - penaeus monodon - animal viruses - transcription - genomes - genomics
    White Spot Syndrome Virus (WSSV) is a large enveloped DNA virus that infects shrimp and other crustaceans. The virions are approximately 275 x 120 nm in size and have an ovoid to bacilliform shape and a tail-like appendage at one end. Sequencing revealed that the circular, double stranded (ds) DNA genome of WSSV ranges between 293 and 307 kb in size depending on the WSSV isolate. For a sequenced isolate originating fromThailand(WSSV-TH) 184 putative open reading frames (ORFs) were identified on the genome, most of which are unassigned as they lack homology to known genes in public databases. Based on its unique morphological and genetic features, WSSV has been accommodated in the new virus family Nimaviridae (genus Whispovirus ).

    WSSV causes serious economic losses in shrimp culture, as 100% cumulative mortalities can be reached within 3-10 days under farming conditions. After its discovery in 1992 in Taiwan WSSV has quickly spread into Southeast-Asia and subsequently to shrimp farming areas all over the world. This thesis aims at obtaining fundamental insights in the genomic structure ("genomics") and transcription regulation ("transcriptomics") of WSSV. This in turn may provide better insight in the molecular basis of WSSV biology and epidemiology, which can be useful in the identification of targets for WSSV intervention strategies.

    Alignment of the complete genome sequences of the isolates WSSV-TW, WSSV-CN and WSSV-TH, originating from Taiwan, China and Thailand, respectively, revealed that the sequences were very similar (over 99% sequence identity), suggesting that the isolates are variants of the same virus species ( Whispovirus ) and probably evolved recently from a common ancestor (Chapter 2). Two major polymorphic loci were identified, variable region (VR) ORF14/15 and VR ORF23/24, and both appeared to be genomic regions where large deletions occur. Further polymorphisms included loci with variable numbers of tandem repeats (VNTR loci). Next to VR ORF14/15 and VR ORF23/24, three of these loci, located in the regions coding for ORF75, ORF94 and ORF125, were identified as useful markers in epidemiological and ecological studies. The highly conserved genomic loci, e.g. the gene encoding the major structural virion protein VP26, are useful for reliable monitoring of WSSV infections in PCR based assays.

    The observation that the isolate WSSV-TH contains a large deletion in VR ORF23/24 relative to the isolates WSSV-TW and WSSV-CN suggested the evolution and spread of WSSV from a common ancestor, provisionally located near the Southeast coast ofChina. Further support for this model was obtained by the genomic characterization of eight WSSV isolates collected in 2003 and 2004 along the central- and south-coast of Vietnam (VN) during WSSV outbreaks (Chapter 3). These WSSV-VN isolates contained deletions of intermediate size in VR ORF23/24 relative to WSSV-TW and WSSV-TH. In VR ORF14/15, the WSSV-VN isolates contained deletions of various sizes compared to WSSV-TH. These collective data suggest that the VN isolates and WSSV-TH have a common lineage, which branched off from WSSV-TW and WSSV-CN early on, and that WSSV possibly enteredVietnamby multiple introductions. Further comparisons among the WSSV-VN isolates revealed that the VNTR loci in ORF75 and ORF125, but not in ORF94, are suitable markers to study local and regional spread of WSSV.

    To study the possible effect of the genetic differences on the fitness and virulence of WSSV, two divergent WSSV isolates (TH-96-II and WSSV-TH) were compared (Chapter 4). TH-96-II was a newly characterized archival WSSV isolate from 1996, which has the largest genome size (~312 kb) of all WSSV isolates identified thus far. As TH-96-II does not contain deletions in either VR ORF14/15 or VR ORF23/24, it may be ancestral to all known WSSV isolates. WSSV-TH contains the smallest genome (~293 kb) identified at present, due to large deletions in VR ORF14/15 and VR ORF23/24. Comparison between TH-96-II and WSSV-TH, when administered to shrimp Penaeus monodon, showed a higher virulence and competitive fitness for the latter. This may suggest that the virus became more virulent over the years during the epidemic while moving south. This enhanced virulence is possibly caused by the continuous contact with susceptible animals, a behavior also seen with some other emerging viruses. Since the more virulent variant (WSSV-TH) has a smaller genome, it may replicate faster to reach a lethal dose. However, it is also possible that the observed differences in virulence are caused by other genetic polymorphisms between the two isolates.

    As WSSV differs profoundly from other large ds DNA viruses and mainly contains unique genes, the mechanism of gene expression and transcription regulation of this new virus was investigated in the second part of this thesis. To study WSSV gene expression on a genome wide scale, a WSSV DNA microarray was constructed containing probes corresponding to nearly all putative WSSV ORFs (Chapter 5). Using a WSSV infection time course we could show expression of at least 79% of the WSSV ORFs included on the microarray in gill tissue of Penaeus monodon . Clustering of the transcription profiles of the individual genes showed the presence of two major classes of genes, a putative early and a putative late class, suggesting that the WSSV genes at large are expressed in a coordinated and cascaded fashion. Five genes encoding WSSV major virion proteins (VP28, VP26, VP24, VP19 en VP15), which clustered in the late class, were further confirmed to be late by RT-PCR (Chapter 6). Furthermore, the 5' and 3' ends of the mRNA of these late genes were determined for identification of common promoter motifs.

    To search for common conserved WSSV promoter motifs associated with WSSV early or late gene expression, as determined by the microarrays, two in silico methods were employed (Chapter 7). The abundance of all 4 through 8 nucleotide motifs in the upstream sequences of WSSV genes relative to the complete genome was determined and the upstream sequences of early or late WSSV genes were analyzed for conserved sequences motifs using MEME. Both methods were complemented by alignments of empirically determined 5' ends of various WSSV mRNAs. The collective information shows that the upstream region of WSSV early genes, containing a TATA box and an initiator sequence, is reminiscent to Drosophila RNA polymerase II promoters, suggesting utilization of the cellular transcription machinery for generating early transcripts. The alignment of the 5' ends of known late genes, including the 5' ends determined in chapter 6, identified a degenerate consensus late transcription initiation motif (ATNAC). Of these genes, only one contained a functional TATA box. However, almost half of the WSSV late genes, as assigned by microarrays, did contain a TATA box in their upstream region. This may suggest the presence of two separate classes of late WSSV genes, one exploiting the cellular RNA polymerase II system for mRNA synthesis and the other generating messengers by a new virus-induced transcription mechanism.

    Alignments of the 3' ends of various WSSV mRNAs suggest that there is no difference in polyadenylation between early and late mRNAs. The WSSV polyadenylation characteristics of both classes resemble regular polyadenylation in eukaryotic mRNAs, which is typically located 10 to 25 nt downstream of the sequence AATAAA.

    In conclusion, the research performed for this thesis has led to a model on the mechanism of WSSV gene expression, and the promoter motifs involved (Chapter 8). The identification of genetic markers has led to more insight in the quick geographical spread of the virus, and the genetic characterization of WSSV isolates may add to the identification of virulence related factors on the WSSV genome. The fundamental insights obtained in the biology and epidemiology of WSSV in this thesis may help in the identification of WSSV genes which can be targets for WSSV intervention strategies.

    Fitness and virulence of an ancestral White Spot Syndrome Virus isolate from shrimp
    Marks, H. ; Duijse, J.J.A. ; Zuidema, D. ; Hulten, M.C.W. van; Vlak, J.M. - \ 2005
    Virus Research 110 (2005)1-2. - ISSN 0168-1702 - p. 9 - 20.
    experimental-infection - genome sequence - baculovirus - wssv - thailand - wsbv
    White Spot Syndrome Virus, the type species of the virus family Nimaviridae, is a large dsDNA virus infecting shrimp and other crustaceans. Genomic analysis of three completely sequenced WSSV isolates identified two major polymorphic loci, ¿variable region ORF14/15¿ and ¿variable region ORF23/24¿. Here, we characterize a WSSV isolate originating from shrimp collected in Thailand in 1996 (TH-96-II). This isolate contains the largest WSSV genome (312 kb) identified so far, mainly because of its sequences in both major polymorphic loci. Analysis of ¿variable region ORF14/15¿ suggests that TH-96-II may be ancestral to the WSSV isolates described to date. A comparison for virulence was made between TH-96-II and WSSV-TH, a well characterized isolate containing the smallest genome (293 kb) identified at present. After injection of the isolates into Penaeus monodon the mortality rates showed that the median lethal time (LT50) of TH-96-II was approximately 14 days, compared to 3.5 days for WSSV-TH. When both isolates were mixed in equal amounts and serially passaged in shrimp, WSSV-TH outcompeted TH-96-II within four passages. These data suggest a higher virulence of WSSV-TH compared to TH-96-II. The molecular basis for the difference in virulence remains unclear, but a replication advantage of the 19 kb smaller WSSV-TH genome could play a role
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