Immune defence White Spot Syndrome Virus infected shrimp, Penaeus monodon

White spot syndrome virus (WSSV) is the most important viral pathogen of cultured penaeid shrimp worldwide. Since the initial discovery of the virus inTaiwanin 1992, it has spread to shrimp farming regions in Southeast Asia, theAmericas, Europe and theMiddle Eastcausing major economic losses. The virus has a wide host range among crustaceans and induces distinctive clinical signs (white spots)on the inner surface of the exoskeletonof penaeid shrimps.Limited data is available about the immune response genes of P. monodon upon a WSSV infection. This thesis describes the results of our study into the generation of tools, like the generation of a dedicated microarray enabling the analysis of induction and regulation of (innate) immune defence genes in the host that are activated upon infection. Moreover, a putative vaccination strategy to protect shrimp against lethal WSSV infections has been developed previously. We have also analysed the induction of protective vaccination for induction and regulation of gene expression using this microarray.

The first focus had been on the haemocyte response of the shrimp upon an immersion infection (chapter 2). Immunocytochemistry and electron microscopy has been used to study the infection route of WSSV in gills and gut up to 3 days after immersion infection. Using a mouse haemocyte specific monoclonal antibody (WSH8) and a rabbit VP28 polyclonal antibody, double immunoreactivity could be observed. Differential haemocyte characteristics in the gills and the midgut of P. monodon were determined.An invasion of haemocytes in the gills was observed in Penaeus monodon upon WSSV-infection, possibly caused by the adherence of haemocytes to the haemolymph vessels. Although many infected cells were found in the gills, haemocytes were not WSSV-infected in this organ. Gills appear to be an important site of haemocyte invasion after immersion infection. In the midgut, uptake of WSSV in the epithelium could be detected, however, infected nuclei of epithelial cells were not observed. In contrast to the gills, the gut connective tissue shows a clear increase in degranulation of haemocytes, resulting in the appearance of WSH8-immunoreactive thread-like material at later time points during the infection. The significance for the different reaction of haemocytes in both organs studied remains to be investigated further. The observation that haemocytes are not the main target for WSSV suggests that free virions circulating in the haemolymph lead to systemic infection in vivo.We conclude that the most likely natural infection route for WSSV is through the gills rather than through themidgut,and that the shrimp have an evolutionary deficit in killing the virus or virus-infected cells effectively.

A combination of suppression subtractive hydridisation (SSH) and cDNA microarray analysis was used to enrich for those genes that are differentially expressed upon a WSSV infection (chapter 3). The construction of SSH libraries and subsequent selection of differentially expressed genes is described in detail. The selected clones were used to generate a dedicated WSSV infection-related cDNA microarray comprising 750 differentially expressed genes.The approach to combine suppressive subtraction hybridisation with microarray analysis has resulted in a read-out system for the detection of shrimp genes involved in the defence reaction upon a WSSV-infection. This approach has good potential for identifying genes involved in shrimp defences in the future. Further studies on these gene transcripts involved in the defence mechanism have to be initiated.

The focus of chapter 4 is to determine the expression profile of the genes selected in chapter 3. By using the generated microarray it was possible to follow a few hundred clones during the first day of infection. In addition, the immune response of the shrimp upon "vaccination" was studied with the microarray. The results obtained in this investigation provide insight into the previously unknown complexities of host-WSSV molecular interactions. The discovery of differential expression of genes in WSSV infected shrimp allowed the visualisation of several pathways and potential mechanisms that may play a role in WSSV pathogenesis. Identification of regulated genes in WSSV infected shrimp enabled the development of a model depicting several ways in which host cell responds to infection. Gene expression changes also provided clues about the possible mechanisms involved in the development of pathological changes that are characteristic of the disease. Most importantly, the data obtained in this study identifies several genes whose mRNA is regulated on virus infection suggesting an array of hypotheses which could be tested to reveal their role in WSSV molecular pathogenesis. This study also provides insight in "vaccine"-host interactions. Microarray studies coupled with in vivo experiments obtain relevant data about the functionality of "vaccines" in shrimp and invertebrates in general. The combination of host immune response genes and "vaccination" can reveal the route of WSSV infection and may unravel the immune system of the giant tiger shrimp. Taken together, the present investigation demonstrates the application of a powerful approach of combining the high throughput technologies of SSH and microarray to study differential expression of genes in response to virus infection. SSH could be used for initial isolation of differentially expressed transcripts, a large-scale confirmation of which can be accomplished very efficiently by microarray analysis. The detailed methods described herein could be potentially applied to any biological system.

With information available of Drosophila it is possible to look more thoroughly into immune related genes. Toll receptors are known to play a substantial role in detecting pathogens, both in invertebrates as well as in vertebrates, where they are called Toll-like receptors (TLR). Therefore, in chapter 5, a new Toll receptor was identified and described and expression studies upon WSSV infection were performed. The absence of regulation in different organs upon a viral challenge suggests that PmToll is not directly involved in the defense against WSSV. However, the Toll pathway can be regulated at a higher level (PGRPs and GNBPs; extra cellular) and regulation of PmToll might not be necessary. Currently we are investigating the effect of bacterial challenges on the regulation of PmToll.

Finally, the results presented, are summarised and discussed in chapter 6. We provide an evolutionary framework for the virus-host response and describe the relevance of differentially expressed and regulated innate immune response genes. We integrate the characterisation of a shrimp-specific Toll receptor with results from the microarray analysis and provideaintegrative immune defence of P. monodon to exposure with WSSV. Moreover, we describe the immunological background known so far with respect to the vaccination strategy for WSSV infection.