Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 47

White Spot Syndrome Virus Interaction with a Freshwater Crayfish

PIKUL JIRAVANICHPAISAL

ISSN 1651-6214ISBN 91-554-6235-9urn:nbn:se:uu:diva-5776

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2005

List of Papers

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals:

I Jiravanichpaisal, P., Bangyeekhun, E., Söderhäll, K. and Sö-derhäll, I. (2001). Experimental infection of white spot syndrome virus in freshwater crayfish Pacifastacus leniusculus. Dis Aquat Org, 47:151-157.

II Jiravanichpaisal, P., Sricharoen, S., Söderhäll, I. and Söderhäll, K. WSSV interaction with crayfish hemocytes. (Manuscript)

III Jiravanichpaisal, P., Söderhäll, K. and Söderhäll, I. (2004). Effect of water temperature on the immune response and infec-tivity pattern of white spot syndrome virus (WSSV) in freshwater crayfish. Fish & Shellfish Immunol 17, 265-275.

IV Söderhäll I., Kim, Y. A., Lee, S. Y., Jiravanichpaisal, P. and Söderhäll, K. (2005). An ancient role for a prokineticin domain in invertebrate hematopoiesis. J. Immunol (in press).

V Jiravanichpaisal, P., Söderhäll, K. and Söderhäll, I. Replication of white spot syndrome virus (WSSV) in in vitro cultured hema-topoietic cells of freshwater crayfish, Pacifastacus leniusculus. (Manuscript).

Reprints were made with permission from the publishers.

© Inter-Research, Oldendorf/Luhe, 2001 © 2004 Elsevier Ltd.

Contents

INTRODUCTION ....................................................................................................7

THE DEFENCE MECHANISM OF CRUSTACEANS .........................................................7Hemocytes.........................................................................................................8The prophenoloxidase activating system (proPO system)...............................10The coagulation system...................................................................................12Proteinase inhibitors ........................................................................................13The non-self recognition system .....................................................................14Antimicrobial peptides ....................................................................................16

AN OVERVIEW OF CRUSTACEAN VIRUSES .............................................................17Shrimp and crayfish viruses ............................................................................17White Spot Syndrome Virus (WSSV).............................................................17Enveloped virus entry......................................................................................19Antiviral defence .............................................................................................21Viral mechanisms of immune evasion.............................................................23

CRAYFISH HEMATOPOIESIS ...................................................................................25Hematopoiesis .................................................................................................25Regulation .......................................................................................................26

CYTOKINES IN INVERTEBRATES ............................................................................27CRUSTACEAN PRIMARY CELL CULTURE ................................................................28

AIM OF THE PRESENT STUDY ........................................................................30

RESULTS AND DISCUSSION ....................................................................................30Experimental infection of white spot syndrome virus in freshwater crayfish Pacifastacus leniusculus (Paper I)...................................................................30White spot syndrome virus (WSSV) interaction with crayfish hemocytes (Paper II) .........................................................................................................31Effect of water temperature on the immune response and infectivity pattern of white spot syndrome virus (WSSV) in freshwater crayfish (Paper III)...........32An ancient role for a prokineticin domain in invertebrate hematopoiesis (Paper IV) ...................................................................................................................34Replication of white spot syndrome virus (WSSV) in vitro culture hematopoietic (hpt) cells of freshwater, Pacifastacus leniusculus (Paper V)..35

CONCLUDING REMARKS........................................................................................36

SVENSK SAMMANFATTNING (SUMMARY IN SWEDISH) .......................37

ACKNOWLEDGEMENTS ...................................................................................39

REFERENCES........................................................................................................40

Abbreviations

AFALFsAML1AMPsbFGFCCFdsRNAFOGgcm/lz/srp/Ush hptIFN- /ILsJAK/STAT

LPS/PGNMHCPAMPPPR(s)/PRP(s)proPORNAiSDS-PAGE

SGC/GC/HC SODTLR(s)VAPsVPWSSVYHV

Astakine factor Anti-lipopolysaccharide factors Acute Myeloid leukemia Antimicrobial peptides Basic Fibroblast growth factor Coelomic cytolytic factor Double stranded RNA Friend of GATA Glia cell missing/serpent/lozenge/ U-shape Hematopoietic tissue Interferon alpha/beta InterleukinsJanus kinase/signal transducer and activator of tran-scriptionLipopolysaccharide/ Peptidoglycan Major histocompatibility complex Pathogen-associated molecular patterns Pattern recognition receptor(s)/protein(s) Prophenoloxidase RNA interference Sodium dodecylsulphate-polyacryamid gel electro-phoresisSemigrnaular cell/granular cell/hyaline cell Superoxide dismutase Toll-like receptor(s) Viral attachment proteins Viral protein White spot syndrome virus Yellow head virus

7

Introduction

Viruses are the most common microorganisms in the marine environment. Viral diseases are therefore common in marine crustaceans. Some viruses such as the white spot syndrome virus (WSSV) have become a major cause of shrimp mortality in aquaculture. This virus has a wide geographic distri-bution and host range. Crustaceans, unlike vertebrates, lack a true adaptive immune system and have to depend entirely on innate immunity. Therefore, an urgent study is needed to understand the immune response of crustaceans against this virus, which may be helpful for disease control and for sustain-able aquaculture. To date, very little is known about virus-host interactions in crustaceans as well as in other invertebrates though some progress is be-ing made with WSSV and crustaceans. Freshwater crayfish which is suscep-tible to WSSV is also an appropriate invertebrate model to study innate im-munity against WSSV.

The defence mechanism of crustaceans The immune system is commonly divided into two major branches: innate and adaptive immunity. Since crayfish, like other invertebrates, lack an adaptive immune system in which memory is the hallmark, their defence mechanisms solely rely on innate immune responses. Recently, however, cumulative experimental data from invertebrates provide some evidence that specificity and memory might exist in invertebrates (reviewed by Kurtz, 2005).

Aquatic crustaceans are in intimate contact with their environment par-ticularly in intensive culture systems, which are enriched in bacteria, viruses. Some of these are pathogenic and many are saprophytic. However, under normal conditions animals maintain a healthy state by defending itself against potential pathogens. The external cuticle is a first line of defence to provide an effective physical and chemical barrier against the attachment and penetration of pathogens. The digestive tract, which is a main route of invasion, is partly lined with chitinous membranes and its hostile environ-ment of acids and enzymes is able to inactivate and digest many viruses and bacteria. In most cases the cuticular defences are sufficient to protect against even quite virulent pathogens, which often only produce disease when the integument has been physically damaged. Once pathogens gain entry into the

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hemocoel of the host, they encounter a complex system of innate defence mechanisms involving cellular and humoral responses. First, the prophenoloxidase activating system (proPO) is induced which will lead to melanization and generation of factors which are involved in immune reac-tions such as for example peroxinectin. Next, a cellular immune response that involves different types of hemocytes, which participate in pathogen clearance by phagocytosing microorganisms, or trapping them in hemocyte aggregates or nodules, or by encapsulation of larger microorganisms and cytotoxic reactions are also triggered. Nodules and encapsulated material are often observed to become melanized, through the action of phenoloxidase (PO). Finally, during systemic infection, a wide range of inducible effector molecules, such as antimicrobial peptides (AMPs), factors required for opsonization, are produced (reviewed in Cerenius and Söderhäll, 2004). An illustration of the innate immune reactions of crayfish is shown in Figure 1. The main component of the innate immunity of crustaceans and some other invertebrates are summarized below.

HemocytesIn mammals, various types of blood cells have specific functions that are crucial for the survival of an individual, such as oxygen transport and de-fence against infection. In invertebrates, the most important role of the circu-lating hemocyte is the protection of the animal against invading microorgan-isms (Cerenius and Söderhäll, 2004; Tzou et al. 2002). Decapod crustaceans generally contain three types of circulating hemocytes; the hyaline (H), semigranular (SG) and granular (G) cells which can be distinguished by their distinct appearance (reviewed in Johansson et al. 2000), although some syn-onymous hemocyte terms have been reported by others. This classification was mostly based on morphology.

In freshwater crayfish, Procambrus clarki (Lanz et al. 1993), HCs with small, spherical and no or few granules was reported to be the most abundant type of hemocyte, whereas they are the least abundant in Astacus astacusand Pacifastacus leniusculus (Smith and Söderhäll, 1983a; Söderhäll et al. 1986).

The use of isolated cells provides a way to study the characteristics of the individual cells types in detail (Söderhäll and Smith, 1983). In freshwater crayfish, the HCs are small, spherical and contain no or few granules, and are capable of phagocytosis (Smith and Söderhäll, 1983a, b). The SGCs have variable number of small eosinophilic granules which contain the prophenoloxidase activating system (proPO system). The SGCs are respon-sible for encapsulation and also have a limited function in phagocytosis. The GCs are filled with a large number of large eosinophilic secretory granules and are the major storage cell for the proPO system but no phagocytosis function is observed (Kobayashi et al. 1990; Persson et al. 1987b; Smith and

9

ProPOppA

GBPPeroxinectinReceptorClotting protein

TransglutaminaseProteinase inhibitors (e.g. pacifascin, 2-macrogobulin)Antibacterial peptidesMas-like protein

-1, 3-glucan

a

Ca2+

?

?Clotting

Phagocytosis

Nodulation/encapsulation

Antibacterial peptides

Degranulation

Cell adhesion opsonization

Melanization

Virus evasion

proppA

ppA

proPO

PO Quinones

Melanin

Bacteria

Fungi

Virus

O2

Phenols

?

Figure 1. Schematic view of innate immune system in crayfish. (a) GBP is acti-vated by -1, 3 glucan then the degranulation of hemocytes is induced and several proteins are released. (b) Initiation of several immune responses by pattern recogni-tion proteins in crayfish.

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Söderhäll, 1983a). Both SGCs and GCs can be induced to degranulate by foreign molecules such as lipopolysaccharides (LPS) or -1, 3 glucans. The SGC is the first hemocyte type to react to foreign particles in vivo and they respond by degranulation (Johansson and Söderhäll, 1985), releasing the proPO system including the cell adhesion/degranulating factor peroxinectin from their granules into the plasma (Johansson et al. 1995). Crayfish SG and G cells can also be cytotoxic and lyse foreign eukaryotic cells (Söderhäll et al. 1985).

The number of circulating hemocytes obviously varies and this is affected by various factors. In crayfish and other invertebrates, the number of hemo-cytes is decreased upon infection by microorganisms but after some time the number is back to normal. In contrast, the number of hemocytes was not decreased during a WSSV infection (Paper I). Injection of saline causes crayfish hemocyte number to dramatically increase, whereas injection of -1,3-glucans rapidly reduces the number of hemocytes followed by a slow re-covery to the same level after 4-6 hours (Persson et al. 1987a; Smith and Söderhäll, 1983b). The loss of circulating hemocytes after -1, 3 glucan injection is probably due to cell aggregation, indicating an important role of the hemocytes in defence. A dramatic effect of the hemocyte number reduc-tion was demonstrated by a study of the crayfish, P. leniusculus. This species is a carrier of the crayfish plague, the fungus Aphanomyces astaci, which normally is present only in the cuticle and does not cause mortality of the crayfish. After a decrease in the number of hemocytes by an injection of a -1, 3 glucan, the fungus can grow rapidly within the body cavity and kills the crayfish a few days afterwards (Persson et al. 1987b). This is probably a result of that a low number of hemocytes produces a lower capacity of the crayfish to defend itself to the crayfish plague.

The prophenoloxidase activating system (proPO system) The proPO system is an efficient innate immune response for non-self rec-ognition. It is initiated by recognition of lipopolysaccharides and peptidogly-cans from bacteria and -1, 3 glucans from fungi in minute amounts. This ensures that the system will become active in the presence of potential pathogens. This system contains pattern-recognition proteins (PRPs), several zymogenic proteinases, and prophenoloxidase (proPO) (Cerenius and Söderhäll, 2004).

Since the first cloning of an invertebrate proPO from the freshwater cray-fish P. leniusculus (Aspán et al. 1995), such enzymes have been reported from about 20 different arthropod species (reviewed in Cerenius and Söderhäll, 2004). In crayfish, proPO is synthesized in hemocytes and local-ized in granules. As shown in Figure 1a when GBP binds to -1, 3glucans it becomes activated and binds specifically to a cell-surface associated protein, a superoxide dismutase (SOD) (Johansson et al. 1999) or binds to a -

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integrin on hemocyte surface through its RGD motif (Arg-Gly-Asp) (Holm-blad and Söderhäll, 1999). The recognition of non-self triggers degranulation of SGC and GC. Among the released protein is prophenoloxidase activating enzyme (proppA), which is further activated to ppA by the presence of pathogen associated molecular pattern (PAMPs) (Aspán et al. 1990b). Active ppA converts the proPO to phenoloxidase (PO; monophenol, dihydroxy-phenylalanine: oxidoreductase; (EC1.14.18.1). PO is a bifunctional copper containing enzyme, which is known as a tyrosinase and will catalyze the early steps in the pathway to melanin formation (Ashida and Brey, 1998; Söderhäll and Cerenius, 1998).

Recently, hemocyanins were reported to have PO activity after proteolytic cleavage in the spider, Eurypelma californicum (Decker and Rimke, 1998; Decker and Tuczek, 2000) horseshoe crab, Tachypleus tridentatus (Nagai and Kawabata, 2000) and crayfish, P. leniusculus (Lee et al. 2004). Al-though the hemocyanin of chelicerates has a function as oxygen transporters under normal conditions, it may be converted to phenoloxidase after injury to prevent microbial invasion. A phylogenetic analysis shows that the proPO subfamily has much higher evolutionary relationship with the chelicerate hemocyanins than to the branchiopod hemocyanins (Burmester, 2001; Hughes, 1999). While proPO is synthesized in hemocytes, hemocyanin is produced in the hepatopancreas (Aspán et al. 1995; Söderhäll and Cerenius, 1998). Likewise, proPOs in the penaeid shrimp are localized in the SGCs and GCs (Perazzolo and Barracco, 1997; Vargas-Albores et al. 1993, 1996), and proPO mRNA was shown to be expressed only in the hemocytes (Sri-tunyalucksana et al. 1999). In insects some proPOs exist in the plasma (Sugumaran and Kanost, 1993) and several isoforms encoded by different genes have been found (Ashida and Brey, 1998; Müller et al. 1999), but the physiological significance of the presence of the different proPO polypep-tides is unknown. Two isoforms of cuticle proPOs from the silkworm, Bom-byx mori have been characterized and one of them is found to be transported from the hemolymph to the cuticle. The transported cuticle proPO has dif-ferent molecular masses than the hemolymph proPO because of the modifi-cation of one up to six methionine residues (Asano and Ashida, 2001).

ProPO is proteolytically converted to phenoloxidase by an endogenous trypsin-like serine proteinase, the so-called prophenoloxidase activating en-zyme (ppA). So far, four ppAs and one cofactor have been characterized and cloned from different animals; a beetle, Holotrichia diomphalia (Lee et al. 1998), a silkworm, Bombyx mori (Satoh et al. 1999), a tobacco horn worm, Manduca sexta (Jiang et al. 1998), and crayfish, P. leniusculus (Wang et al. 2001a). Their primary structure show that they all exist as zymogens of typi-cal serine proteinases and are similar to Drosophila serine proteinases in-volved in the organization of the developing embryo (Chasan and Anderson 1989; Jiang and Kanost, 2000; Jin and Anderson, 1990). They also contain one or two clip-domains, which have homologous amino acid sequences to

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the horseshoe crab big defensin (Saito et al. 1995) and mammalian -defensin (Bensch et al. 1995; Selsted et al. 1993). In crayfish the recombi-nant clip-domain of ppA has antibacterial activity in vitro against gram-positive bacteria suggesting a dual function of crayfish ppA and maybe also for other ppAs (Wang et al. 2001a). Interestingly, a new type of prophenoloxidase activating factor has been characterized from the coleop-teran, Holotrichia diomphalia (Kwon et al. 2000). This protein has no pro-teinase activity but is an essential factor for the activation of the proPO sys-tem in this insect. The primary structure of the protein is similar to serine proteinase homologues lacking the complete catalytic triad necessary for serine proteinase activity, such as a crayfish masquerade-like protein (Huang et al. 2000; Lee and Söderhäll, 2001), a Drosophila masquerade-like protein (Murugasu et al. 1995), horseshoe crab factor D (Kawabata et al. 1996), and mosquito infection-responsive serine protease-like protein (ispl5) (Dimopou-los et al. 1997).

During the activation of the proPO system, other proteins will also gain their biological activity in the system. One such molecule is a 76 kDa cell adhesion protein, peroxinectin, which has been purified, characterized and cloned from crayfish, P. leniusculus (Johansson, 1999) and shrimp (Liu et al. 2004; Sritunyalucksana et al. 2001). Peroxinectin is a multifunctional protein that is released by exocytosis from the hemocytes. It has two different func-tions, one is peroxidase and the other is cell-adhesion function. The likely production of antimicrobial substances by the peroxidase activity of the pro-tein might help to kill invading microorganisms, while the cell-adhesion activity can lead to phagocytosis, degranulation, and capsule formation.

The coagulation systemOne of the principal differences between vertebrates and arthropods is the fact that body fluids in vertebrates are mostly confined to blood and lym-phatic vessels, whereas arthropods have an open circulatory system. There-fore, after wounding, arthropods must produce a matrix that quickly stops the loss of hemolymph, but also aids in trapping of foreign organisms to preclude spreading throughout the hemocoel. Hemolymph clotting is thus an important part of innate immunity and is regulated in many cases by micro-bial elicitors. In horseshoe crabs hemolymph is extremely sensitive to minute amounts of bacterial lipopolysaccharide (LPS), which induces conversion of coagulogen (a soluble protein) into insoluble coagulin. The extreme sensitiv-ity of the clotting cascade for LPS is used in the Limulus test, an assay for the detection of bacterial endotoxins or LPS (Tanaka and Iwanaga, 1993). Most components of the hemolymph clotting system are stored in different types of hemocyte granules and are released on activation (Iwanaga et al. 1998). The protein components are the serine proteinase zymogen factors C,

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B, G, the proclotting enzyme and the proclottable protein coagulation. The factor C and G are highly sensitive to LPS and -glucan, respectively. Crosslinking of the clot might depend on hemocyanin, which can obtain PO activity through interaction with clotting factors (Nagai and Kawabata, 2000; Theopold et al. 2002).

In crustaceans, clotting occurs through polymerization of a clotting pro-tein in plasma and is catalyzed by a calcium ion dependent transglutaminase, which is released from hemocytes or other tissues (Hall et al. 1999; Wang et al. 2001b; Yeh et al. 1999). The clotting protein was first cloned and fully characterized in crayfish (Hall et al. 1999; Kopácek et al. 1993) and later in shrimp (Yeh et al. 1999). The primary structure of both crayfish and shrimp clotting proteins shows that they are lipoproteins and belong to the vitel-logenin superfamily and share similarity to the D domain of mammalian von Willebrand factor (Hall et al. 1999; Yeh et al. 1999).

Proteinase inhibitorsProteinase cascades, such as the clotting cascade and the proPO system, need to be carefully regulated to prevent excessive activation of endogenous cas-cades and damage to host tissue. Proteinase inhibitor activities have been detected in several invertebrates and an increasing number of proteins are being purified and their primary structure deduced. Most of them fall into well-established families of proteinase inhibitors such as Kasal, Kunitz, ser-pins, -macroglobulins and metalloproteinase inhibitors (Kanost, 1999; Wedde et al. 1998).

Proteinase inhibitors play a key role to control and regulate the proPO system to avoid the deleterious effects of its active components, particularly PO, which can produce highly toxic intermediates. Several proteinase inhibi-tors for preventing over-activating of ppA (Aspán et al. 1990a; Hergenhahn et al. 1987) and a phenoloxidase inhibitor (POI), which can directly inhibit the activity of phenoloxidase (Daquing et al. 1995, 1999; Sugumaran and Nellaiappan, 2000) have been reported from several arthropod species (re-viewed in Cerenius and Söderhäll 2004). The most efficient inhibitor of crayfish ppA is pacifastin, a 155-kDa heterodimeric protein with an unique structure. It is composed of a light chain with nine proteinase inhibitor sub-units and a heavy chain containing three transferrin lobes (Liang et al. 1997). The molecule is held together by a peptide bond and forms a new class of proteinase inhibitors named pacifastin-like serine proteinase inhibitors which are present in, and can inhibit the proPO system in many insects (Simonet et al. 2002).

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The non-self recognition system The pattern-recognition concept postulated by Janeway (1989) that there are pathogen-associated molecular patterns (PAMPs) present on pathogenic microbes. Since these molecules are absent on host cells, they can serve as discriminators between self and non-self. The receptors that bind these con-served structures are called pattern recognition receptors (proteins) (PRRs, PRPs). Candidates for such PAMPs are molecules that are considered to be immunostimulatory in a rather unspecific way in host immunity such as lipopolysaccharides (LPSs) or peptidoglycans (PGNs) of bacterial cell walls,

-1, 3 glucan of fungal cell walls and double stranded RNA (dsRNA) of replicating viruses. These molecules contain repetitive patterns and are shared among large groups of bacteria. PAMPs are often essential for sur-vival of the microbes, and this ensures that the targets of innate immunity can not be discarded by microbes in an effort to evade recognition by the host. Innate immunity uses a set of sensors, PRPs, to recognize PAMPs. These PRPs can be localized on the surface of cells or be secreted into the hemolymph, ready to signal the presence of invading microbes in every compartment. Because of this specificity for microbial structures, the innate immune system, like the adaptive immune system, is able to distinguish self from non-self.

Several PRPs have been isolated and characterized such as LPS or/and -1, 3 glucan binding proteins (LBP, GBP, or LGBP), peptidoglycan recogni-tion protein (PGRP), lectins and hemolin have been found in a variety of invertebrates with different biological functions after binding to their targets.

Lectins or agglutinins are glycoproteins normally without catalytic activ-ity. They exist in almost all living organisms, and have the ability to bind to specific carbohydrates. They can bind cells and an agglutination reaction occurs. Interaction between lectins and carbohydrates is involved in various biological activities, for instance the cellular and tissue transport of carbohy-drates glycoproteins, cell adhesion, opsonization and nodule formation (Marques and Barracco, 2000). Particularly, C-type lectins, calcium depend-ent lectins are reported to be involved in immune recognition in invertebrates (Vasta et al. 1999; Weis et al. 1998). Constitutively expressed lectins that have an LPS-binding property have been characterized from the silkworm, Bombyx mori (Koizumi et al. 1999) and the American cockroach, Pe-riplaneta americana (Jomori and Natori, 1992; Kawazaki et al. 1996; Natori et al. 1999). The biological function of these LPS binding proteins was shown to have bacterial clearance activity and an opsonic effect, respec-tively. Two kinds of inducible lectins, immulectins, were characterized and cloned in the tobacco hornworm, Manduca sexta, and were both found to stimulate the proPO system (Yu et al. 1999; Yu and Kanost, 2000). Many different lectins, for example tachylectins, in horseshoe crab (Tachypleus tridentatus) were found to have hemagglutinating and antibacterial activities

15

which are important in the immune system. (Kawabata and Iwanaga, 1999) Therefore, it is most likely that the immune system of horseshoe crab recog-nizes the pathogens through lectins with various specificities against carbo-hydrate present on pathogens.

So far, GBPs have been characterized from horseshoe crab (T. triden-tatus) (Seki et al. 1994), crayfish (P. leniusculus) (Cerenius et al. 1994), shrimp (P. californiensis) (Vargas-Albores et al. 1996) and two insects, Bla-berus craniifer (Söderhäll et al. 1988) and B. mori (Ochiai and Ashida, 1988). LGBPs and GBPs have a similar primary sequence to bacterial glu-canases, but they do not exhibit any glucanase activity and instead enhance or mediate activation of the proPO system (Beschin et al. 1998; Cerenius et al. 1994; Kim et al. 2000; Lee et al 2000; Ma and Kanost, 2000; Ochiai and Ashida, 2000). Crayfish GBP is present in plasma and interact with -1, 3 glucan to enhance the activation of the proPO system as well as to act as an opsonin to increase the capacity of phagocytosis (Cerenius et al. 1994; Duvic and Söderhäll, 1990, 1992, 1993; Thörnqvist et al. 1994). The GBP and glucan complex can bind to GC surface via its RGD (Arg-Gly-Asp) motif, which may indicate binding to an integrin-like protein and then induce spreading and degranulation of GCs. In addition, a crayfish LGBP has been isolated, cloned, and characterized from hemocytes and it has binding activ-ity to both LPS and -1, 3 glucans, but not to peptidoglycans (Lee et al. 2000). The primary structure and cloning studies showed that LGBP has significant homology with several gram-negative bacteria binding proteins and bacterial glucanases. LGBPs function is to bind to LPS or -1, 3 glucans and then activate the proPO system in crayfish.

Interestingly, the crayfish masquerade-like protein (mas-like protein) in hemocytes is known as a PRR. It can bind to LPS, -1, 3 glucans, gram-negative bacteria, and yeast. In addition, this protein acts as an opsonin as well as having cell adhesion activity (Huang et al. 2000; Lee and Söderhäll, 2001). Thus, the mas-like protein is a multifunctional protein. Its amino acid sequence shows homology to serine proteinases except for a substitution within the catalytic triad, which will render it without enzyme activity. Sev-eral serine proteinase homologous proteins have recently been characterized from various animals and their biological function can be to have cell adhe-sion activity (Barthalay et al. 1990; Huang et al. 2000), antimicrobial activity (Almeida et al. 1996; Kawabata et al. 1996), LPS binding activity (Gabay et al. 1990; Lee and Söderhäll, 2001), and being a component of the proPO system (Kwon et al. 2000). Taken together, the biological functions suggest that serine proteinase homologous proteins are important in immune re-sponses (Nakamura et al. 1989; Olson et al. 1990)

Hemolin belongs to the immunoglobulin (Ig) superfamily containing Ig-like domains and binds to the lipid A part of LPS and gram-negative bacte-ria. It is the major inducible immune protein identified in the hemolmph, up-

16

regulated about 18 fold upon immune challenge of Hyalophora cecropiapupae (Lanz-Mendoza et al. 1996; Lanz-Mendoza and Faye 1999).

Antimicrobial peptides The gene-encoded anti-microbial peptides/polypeptides (AMPs) are widely distributed among living organisms from microorganisms to plants and the animal kingdom. One important part of innate immunity is the production of anti-microbial substances, which are mainly peptides. Normally, AMPs con-tain less than 150-200 amino acids and are produced by different types of cells. Depending on their tissue distribution, AMPs ensure either a systemic or a local protection of the host against pathogens.

Many antimicrobial peptides have been characterized from insects and chelicerates, but a few peptides have been found in crustaceans. In the last decade, several AMPs have been isolated from crab, shrimp and crayfish. A 6.5-kDa and 3.7-kDa peptides (callinectin) were partially characterized from the hemocytes of the crabs Carcinus maenas (Schnapp et al. 1996) and Callinectes sapidus (Khoo et al. 1999). A family of AMPs displaying both antifungal and antibacterial activities was isolated from hemolymph of the shrimp Litopenaeus vannamei, and was named penaeidins (Destoumieux et al. 1997). Very recently, Histone H2A was cloned and characterized from the shrimp, L. vannamei and its N-terminus was found to resemble the known antimicrobial histone peptide buforin I, parasin, and hipposin (Patat et al. 2004). Anti-lipopolysaccharide factors (ALFs) have been first isolated from hemocytes of the horseshoe crab (Morita et al. 1985; Aketagawa et al. 1986) and have been recently identified from hemocytes of P. monodon by a genomic approach. Recombinant ALF has a broad spectrum of antifungal properties and antibacterial activities against both Gram-positive and Gram-negative bacteria (Somboonwiwat et al. 2005). Two other AMPs, crustin and hemocyanin-derived peptides have been described in shrimp and crayfish. Crustin, an 11.5 kDa antimicrobial peptide is also found in C. maenas (Gross et al. 2001; Relf et al. 1999). C-terminal hemocyanin fragments are active against fungi but the mechanism by which hemocyanin is cleaved and acti-vated in shrimp is still unclear (Destoumieux et al. 2001). Finally, two anti-bacterial peptides with low molecular masses named astacidin 1 and 2 were purified and characterized from crayfish P. leniuscus, hemolymph (Lee et al. 2003; Lee et al. unpublished). Astacidin 1 with 16 amino acid residues is generated by proteolytic cleavage of hemocyanin (Lee et al. 2003) whereas astacidin 2 with 14 amino acid residues has high similarity to the proline rich-peptide, metalnikowin 1 purified from the hemipteran insect, Palomenaprasina (Chernysh et al. 1996).

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An overview of Crustacean virusesViruses are a group of organisms which must enter a host cell to replicate, since they lack the necessary biochemical machinery to manufacture proteins and metabolize sugars. Some viruses also lack the enzymes required for nu-cleic acid replication, and are dependent on the host cell for these functions. The number of genes carried by different viruses may be as few as 3 or as many as 250, but their genomes are still much smaller than that of even the smallest bacteria.

Shrimp and crayfish viruses Viruses are the most common biological agent in the sea. They number up to ten billion per litre and probably infect many species (Fuhrman 1999). Hence, viral infections are common in crustaceans for example; penaeid shrimp are infected by approximately 20 viruses (Johnson et al. 1999; Loh et al. 1997; Nadala et al. 1998). Since the first report on the occurrence of a virus in the crab Macropipusdepurator by Vago (1966) research of viruses in the crustacean order Deca-poda has expanded considerably. The viruses described belong to or are re-lated to a wide variety of families such as the Baculoviridae, Birnavirus,Bunyaviridae, Herpesviridae, Picornaviridae, Parvoviridae, Reoviridae,Rhabdoviridae, Togaviridae, Iridoviridae or a new virus family, the Ni-maviridiae, (Adams and Bonami, 1991; Brock and Lightner, 1990; Edgerton et al. 2002; Lightner, 1993; Van Hulten et al. 2001a; Yang et al. 2001; Vogt, 1996). The list of decapods infected with viruses includes shrimps, crabs and freshwater crayfish. Over the past decades the extensive and intensive shrimp culture has been established and as a result more significant knowl-edge of viral pathogens has been generated. Therefore, the most intensively investigated viruses have been isolated from cultured penaeids. Much less is known about viruses obtained from wild decapods (Bonami and Lightner, 1991; Johnson, 1984).

White Spot Syndrome Virus (WSSV) White spot disease is one of the most disastrous in the shrimp culture, having reduced shrimp production and caused serious economic losses worldwide, Mortality rates are usually very high and cumulative mortality can reach 100 % within 3 to 10 days from the onset of visible gross signs (reviewed by Flegel, 1997). White spot syndrome virus (WSSV) has a wide host range and can naturally affect almost all species shrimp, other crustaceans and even insects. In addition to penaeid shrimp species, both natural and experimental infections have been reported in freshwater prawn (Macrobrachium rosen-bergii) crayfish, crabs, lobster, palaemonidian pest shrimp, krill (Acetes sp.),

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planktonic copepods, Artemia sp. and pupae of an ephydridian insect (Chang et al. 1998; Kanchanaphum et al. 1998; Li et al. 2003; Peng et al. 1998; Su-pamattaya et al. 1998; Wang et al. 1998). This virus can be transmitted from brood stock to their offspring (Lo et al. 1997). WSSV is a large enveloped, a circular double-stranded DNA virus. The enveloped virions are symmetrical particles, ellipsoid to bacilliform in shape resembling those seen in baculovi-ruses (Durand et al. 1997). The size is about 120-150 nm in diameter by 270-290 nm in length. Most notable is a tail-like appendage at one end of the virion. The isolated nucleocapsids measure 65-70 nm in diameter and 300-350 nm in length and have a cross-hatched appearance. The morphological characteristics and general biological properties of the virus demonstrated its uniqueness (Lo et al. 1997; Wang et al. 1995; Wongteerasupaya et al. 1995). Based on DNA polymerase and protein kinase genome sequences of differ-ent isolates, phylogenetic analysis suggests that WSSV is a new virus family, the Nimaviridiae, genus Whispovirus (Van Hulten et al. 2001a; Yang et al. 2001).

The WSSV genome is different in size between different isolates: 305107 bp (AX151396), 292967 bp (Van Hulten et al. 2001a) and 307287 bp (AF440570) for viruses isolated from China, Thailand, and Taiwan, respec-tively. This is mostly due to several small insertions and one large, approxi-mate 12 kb, deletion. In total, 684 open reading frames (ORFs) starting with an ATG initiation codon and 50 amino acids or larger are located on both strands of the WSSV genome. From these ORFs, 184 ORFs of 51-6077 amino acids in size with minimum overlap were predicted. Some of the ORFs are similar to known viral genes or eukaryotic genes but most of the encoded putative proteins have no homology to known genes in the Gen-Bank (Van Hulten et al. 2001a; Yang et al. 2001).

The virions contain nine structural proteins so far and they have been named according to their sizes in SDS-PAGE. Viral protein (VP) 19, VP22, VP28, VP281, and VP466 are located in the envelope, whereas VP15, VP24, VP26 and VP35 are located in the nucleocapsid (Chen et al. 2002; Huang et al. 2002a,b; Van Hulten et al. 2000 a,b; 2002; Zhang et al. 2002a,b). Tran-scription analysis of the major WSSV structural virion protein genes, vp28, vp26, vp24, vp19 and vp15, shows that all five genes are expressed late in infection compared to the early ribonucleotide reductase large subunit gene (Marks et al. 2003). Following other genes, such as ribonucleotide reductase, collagen-like protein, and protein kinase have also been identified and stud-ied (Li et al. 2004; S. T. Lin et al. 2002; Liu et al. 2001). VP28, VP26 and VP24 may have evolved by gene duplication (Van Hulten et al. 2000b). They are all encoded by ORFs of roughly the same size approximately 206 amino acids, but their proteins have distinct electrophoretic mobilities. Fur-ther studies of the major envelope protein VP28 have shown that this protein plays a key role in the systemic infection of shrimp using an in vivo neutrali-zation strategy (Van Hulten et al. 2001b). The putative function for VP15, a

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highly basic histone-like protein, is as a DNA-binding protein in the WSSV nucleocapsid (Van Hulten et al. 2002; Zhang et al. 2001). Most recently, Witteveldt et al. (2005) demonstrated that VP15 had homology to some pu-tative baculovirus DNA binding proteins. Thus, they suggest that VP15 is involved in the packaging of the WSSV genome in the nucleocapsid. Envel-oped viruses of vertebrates and invertebrates contain glycoproteins in their viral envelopes and these often play important roles in the interaction be-tween virus and host, such as attachment to receptors and fusion with cell membranes (Granof and Webster, 1999; Van Regenmortel et al. 2000). However, none of these five major structural WSSV proteins appear to be glycosylated, which is an unusual feature among enveloped animal viruses (Van Hulten et al. 2002). Very recently, VP 28 was shown to be involved in the attachment and penetration on to shrimp cells (Yi et al. 2004). VP 35 was shown to have a nuclear localization signal (NLS) which may play a role in mediating the import of WSSV DNA into the nuclei of infected cells (Chen et al. 2002).

It has been shown that WSSV is relatively susceptible to inactivation by heating, within 20 min at 50 ºC, 1 min at 60 ºC and 0.2 min at 70 ºC. WSSV remains viable in frozen shrimp tissue for an extended period of time. WSSV in sterile seawater kept at 30 ºC and in the dark remains viable for up to 30 days, but it is believed to be inactivated in about 3 days in shrimp ponds due to UV radiation and heating. Treatments to achieve complete inactivation of chlorine compounds, formalin, providone iodine, ethyl alcohol, ozone, UV and pH have been determined (reviewed in Edgerton et al. 2002). Tempera-ture is one of the most important environmental factors because it can di-rectly affect an aquatic animals metabolism, O2-consumption, moulting and also indirectly, when combined with other environmental factors such as salinity and dissolved O2. A few studies of the effect of temperature on im-munity of shrimp have been studied (Cheng and Chen, 2000; LeMoullac and Haffner, 2000) as well as on the development of WSSV in shrimp (Guan et al. 2003; Vidal et al. 2001) and (Paper III).

Enveloped virus entry The entry strategies used by different viruses are determined by their struc-ture. Based on structure, viruses are divided into two main groups, envel-oped and non-enveloped viruses (Klasse et al. 1998). Enveloped viruses contain the viral genome and core proteins wrapped within one or more membranes. These membranes are acquired from the host cell during virus assembly and budding (Pettersson, 1991). To infect, a virus must first attach itself to the surface of an appropriate cell and delivery of the viral nucleic acid. Many viruses with a DNA genome must enter the nucleus, whereas RNA viruses, with a few exceptions, replicate in the cytosol. The molecules to which viruses bind comprise a diverse collection of cellular proteins, car-

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bohydrates, and lipids. Some of them merely serve as attachment factors that concentrate viruses on the cell’s surface. Others are true receptors in that they not only bind viruses but are also responsible for guiding the bound viruses into endocytic pathways and for transmitting signals to cytoplasm. Moreover, receptors can serve as cues that induce conformational changes that lead to membrane fusion and penetration. The identity and distribution of attachment factors determines to a large extent which cell types, tissues, and organism a virus can infect. Some viruses use multiple attachment fac-tors and receptors in parallel. Carbohydrate/protein interactions have long been known to play an important role in viral invasion. Some viruses bind specifically to sialic acid-containing groups, and others bind to glycosami-noglycans or glycolipids. In some systems, the lectin is located on the cell surface and the carbohydrate ligand is located on the virus (reviewed in Smith and Helenius, 2004). Enveloped viruses such as myxo- and paramyxo-viruses that bind to sialic acid groups have glycoproteins with neuraminidase activity (Skehel and Wiley, 2000). They are often multifunctional proteins serving additionally as membrane fusion factors and/or receptors-destroying enzymes. Unlike enveloped viruses, non-enveloped viruses do not contain membranes such as rotaviruses (Taylor et al. 1993) and they must rely on strategies other than fusion for getting into cells such as by lysing a mem-brane or creating a porelike structure in a membrane.

Many animal viruses rely on the cell’s endocytic machinery for produc-tive infection. Most cells have the capacity for endocytosis through several distinct mechanisms including clathrin-coated vesicles, phagocytosis, macropinocytosis and caveolae (Klasse et al. 1998). In this step viruses are taken up into the cytoplasm. Depending on virus, incoming virus particles can be seen entering endosomal structures, lysosomes, the endoplasmic re-ticulum (ER), and occasionally the Golgi complex. The mildly acidic pH in endosome provides an essential cue that triggers penetration and uncoating. For example entry of an eveloped negative-stranded RNA virus, influenza virus A, proceeds through endosomes by means of the following steps: Viral hemagglutinin (HA) binds to sialic acid-containing glycoproteins or glycol-ipids. Viruses internalized by cathrin-coated pits are transported by way of the early endosmes to the late endosomes. Low pH activates the M2 protein ion channel in the viral membrane, allowing the internal capsid to be acidi-fied. HA-mediated fusion occurs between the viral enveloped and the en-dosomal membrane triggered by low pH (~5.5). Viral nucleoproteins sepa-rate from each other, bind importin , and move through the nuclear pore complex (NPC) and into the nucleus (reviewed in Smith and Helenius, 2004). After penetration, the genome of most viruses must be transported either to the nucleus or to specific cytosolic membranes. To move inside the cell, incoming viruses often exploit the cytoskeleton and cellular proteins.

The nucleus provides excellent functions for virus replication, ranging from DNA and RNA polymerases to RNA-splicing and –modifying en-

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zymes. However, the nucleus is difficult to entry and exit, and virus must again rely on cellular mechanisms (Whittaker, 2003). In interphase cells, the import of virus and viral capsids occurs through the NPCs. For targeting, viruses use nuclear localization signals and cytosolic import receptors. HIV-1 and adenovirus bind to importin 7, whereas hepatitis B and influenza virus nucleoproteins bind to importins and (Smith and Helenius, 2004). The upper limit in particle diameter for transport through the NPC is 39 nm (Pante and Kann, 2002). The smallest viruses and capsids, as well as helical capsids in extended form, can therefore be imported into the nucleus without disassembly or deformation. Larger viruses and capsids must either deforme or disassemble to allow the genome to pass through the NPC. Interaction of the bound virus with histone H1 and importin and 7 induce disassembly of the virus capsid. Thus, the viral DNA is imported into the nucleoplasm. With the exception of lentiviruses, retroviruses do not use the NPCs for nuclear entry. Preintegration complexes can only enter the nucleus during mitosis when the nuclear envelope is temporarily absent, limiting their infectivity to dividing cells. How invertebrate viruses enter host cells is so far unknown.

Antiviral defenceIn the past decade, a number of proteins involved in innate immunity in crus-taceans have been characterized at both the protein and molecular level such as members of the proPO activating system, antimicrobial peptides and lectins (Cerenius and Söderhäll, 2004; Cuthbertson et al. 2002; Gross et al., 2001; Rojtinnakorn et al. 2002; Roux, et al. 2002). However, these factors are aimed at bacteria, fungi or parasites rather than viruses. So far little is known about the possible innate antiviral factor generated by the interaction between host and virus.

Toll-like receptors (TLR) play a critical role in innate immune responses. TLRs share structural features with the mammalian interleukin-1 receptor and function as activators of intracellular signalling in response to injury and infection. So far, ten family members have been identified in mammals (Akira and Hemmi, 2003). For example, TLR4, 5 and 9 are essential for recognition of LPS, bacterial flagellin and bacterial DNA containing un-methylated CpG motifs (CpG DNA), respectively. TLR2 is implicated in the recognition of peptidoglycan and lipopeptides. TLR6 can associate with TLR2 and recognize PGN and lipopeptides derived from Mycoplasma. TLR3 has been shown to activate immune cells in response to viral-derived dsRNA. Recently, small anti-viral compounds called imidazoquinolines (imiquimod) and R-848, have been shown to activate immune cells via the TLR7MyD88-dependent signalling pathway. Therefore, TLRs discriminate between specific compounds derived from pathogens. Upon their activation, TLRs engage a variety of intracellular adaptor molecules, leading to signal transduction events that regulate the expression of immune response genes.

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In Drosophila, recognition of bacteria and fungi triggers immune responses through activation of the Toll and immune deficiency (Imd) pathways (Hetru et al. 2003). Although, the invertebrate immune system has been well stud-ied in the context of antibacterial and antifungal responses, there is little published data on the possible involvement of the TLR family in viral infec-tion (Bowie et al. 2000; Kurt-lones et al. 2000).

In vertebrates, it has long been known that most cell types in the body re-spond to incoming viral infection by rapidly secreting antiviral cytokines such as interferon alpha/beta (IFN- / ). IFN- / has the potential to trigger the activation of multiple noncytolytic intracellular antiviral pathways that can target many steps in the viral life cycles, thereby limiting the amplifica-tion and spread of the virus and attenuating the infection (reviewed in Gui-dotti and Chisari, 2001). Upon induction of cells by circulating IFNs, signal transduction through the Janus kinase/signal transducer and activator of tran-scription (JAK/STAT) system results in the induction of hundreds of genes (de Veer et al. 2001; Ehrt et al. 2001). The most intensely studied type I IFN-induced gene is the dsRNA-activated serine/threonin protein kinase (PKR), the myxovirus-resistance (Mx) proteins, oligoadenylate synthetase, RNase L, RNA-specific adenosine deaminase ADAR and IFNs themselves (Katze et al. 2002). This self-amplifying system can be triggered not only by IFN, but also directly by viral components. A potent inducer of the IFN re-sponse is dsRNA, a molecule that often occurs during viral infection as a result of viral genomic replication and viral RNAs with extensive secondary structure (reviewed by Jacobs and Langland, 996). In mammal dsRNA is recognized by TLR3, which activates myeloid differentiation factor 88 (Myd88)-dependent and an independent signal transduction cascade, leading to expression of IFN- (Alexopoulou et al. 2001; Oshiumi et al. 2003). DsRNA also induces antiviral responses intracellularly, by directly activat-ing PKR, which leads to inhibition of cellular and viral protein synthesis via phosphorylation of eukaryotic translation initiation factor 2 (eIF2 ) (Meurs et al. 1990). It has been generally accepted that these dsRNA-induced im-mune responses are absent from invertebrates (Adams et al. 2000; Caenor-habditis elegans Sequencing Consortium, 1998; Dehal et al. 2002; Holt et al. 2002). The complexity of the biological properties of dsRNA in vivo became more apparent with the discovery of dsRNA-mediated posttranscriptional gene silencing (PTGS) or RNA interference (RNAi) (Hammond et al. 2000, 2001). It has been proposed that RNAi represents an ancient antiviral mechanism used to inhibit the expression of viral gene products in infected cells due to the occurrence of viral dsRNA in the process of infection.

Most recently, induction of antiviral immunity by dsRNA was discovered in the crustacean, L. vannamei. Shrimp showed increased resistance to infec-tion by two unrelated viruses, WSSV and Taura syndrome virus (TSV) when treated with dsRNA. Induction of this antiviral state is independent of the sequence of the dsRNA used and therefore distinct from the sequence-

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specific dsRNA-mediated genetic interference phenomenon. This provides the first evidence that an inducible, general antiviral immune response may be present in an invertebrate (Robalino et al. 2004). Other studies have re-ported that microbial components such as LPS, -glucans as well as certain viral proteins, can enhance antiviral resistance in shrimp (Chang et al. 2003; Huang et al. 1999; Song et al. 1997; Witteveldt et al. 2004). It was demon-strated that the LGBP gene is upregulated in WSSV-infected shrimp and the consequent downregulation of proPO with disease progression (Roux et al. 2002). A new gene named PmAV which appears to be involved in virus re-sistance was cloned from shrimp, P. monodon. It encodes a 170 amino acid peptide with a C-type lectin-like domain (CTLC). Recombinant PmAV pro-tein showed a strong antiviral activity in inhibiting virus-induced cytopathic effect in fish cell in vitro. Immunohistological study showed that this protein was located mainly in the cytoplasm, and not bound to the WSSV. It sug-gests that the antiviral mechanism of the PmAV protein is not by inhibiting the attachment of virus to the host cell and some unknown mechanism is responsible for the antivival activity of this protein (Luo et al. 2003).

Antiviral substances from tissue extracts of shrimp (Penaeus setiferus), blue crab, and crayfish that bind to a variety of DNA and RNA viruses, Sindbis virus, vaccinia virus, vesicular stomatitis virus, mengovirus, Banzi virus, and poliomyelitis virus, have been isolated. The inhibitory activity of these antiviral substances is still unknown (Pan et al. 2000). Very recently, hemocyanin with molecular masses of 73 and 75 kDa were isolated from P. monodon and were shown to have non-specific antiviral properties and no cytotoxicity against host cells (Zhang et al. 2004).

Likewise, nothing is known about the response to virus infection in Dro-sophila. Recently, the Drosophila host-defence against viral infection was studied by developing a model based on Drosophila C virus (DCV). DCV is a non-enveloped small ssRNA virus and has Drosophila as its natural host. Using MALDI-TOF mass spectrometry differential analysis there was only one peptide which was induced upon virus infection. This peptide, pherokine-2 (Phk-2), is related to OS-D/A10 (Phk-1), which was previously characterized as a putative odour/pheromone binding protein. The virus-induced molecule is also similar to the product of the gene CG9358 (Phk-3), which is induced by septic injury. Both Phk-2 and Phk-3 are highly ex-pressed during metamorphosis, therefore it is suggested that they are in-volved in tissue-remodelling rather than directly involved in the antiviral response (Sabatier et al. 2003).

Viral mechanisms of immune evasion After entry of viruses, generally the host recognizes these viruses as foreign particles and initiates an efficient immune response to prevent entry of the virus into cells, their subsequent replication and spread. The initial phase of

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this antiviral response in vertebrates is recognition of viral components, par-ticularly peptides, as foreign in the context of the major histocompatibility complex (MHC) antigens on the surface of the antigen-presenting cells. Once this recognition event has occurred, a cascade of cellular and humoral responses to distinct viral determinants is triggered. For instance, antibodies can neutralize free circulating virus and also destroy virus-infected cells. The cellular responses can lyse virus-infected cells as well as help initiate anti-body synthesis. Soluble factors, such as tumour necrosis factors (TNFs), interleukins (ILs) and interferons (IFNs), interact with lymphocytes, either suppressing or expanding their immune functions. The interaction of the virus with the immune system is very critical in determining the result for the host to clear viruses, host recovery and survival. Many problems con-cerning virus-immune system interactions have been resolved over the last decade. However, it has not been fully answered how pathogenic viruses are able to avoid the host immune responses and establish a persistent infection. Different mechanisms that viruses use to evade host immune responses in-clude inhibition of humoral responses, interference with IFNs, inhibition and modulation of cytokines and chemokines, inhibitors of apoptosis and evad-ing CTLs and NKs, and modulating MHC function (reviewed by Alcami and Koszinowski, 2000). Apoptosis can be considered an innate cellular response to limit viral replication; the induction of early cell death would severely limit virus production and reduce or eliminate spread of progeny virus in the host (Aubert and Jerome, 2003). Most viruses have evolved strategies to evade or delay early apoptosis to allow production of high yields of progeny virus. Many viruses have evolved genes encoding proteins, which effectively suppress or delay apoptosis long enough for production of sufficient quanti-ties of progeny. For example, Baculovirus contains p35 and IAP proteins to inhibit multiple caspases (Everett and McFadden, 1999; Kalvakolanu, 1999; Tortorella et al. 2000). In addition, a growing number of viruses are now known to induce apoptosis actively at late stages of infection. This process may represent a final and important step in the spread of progeny to neighbouring cells and also to evade host immune inflammatory responses and protect progeny virus from host enzymes and antibodies (reviewed in Teodoro and Branton, 1997).

Research in naturally virus-infected shrimp indicated that the mechanism responsible for eliminating the virus-infected cells is apoptosis (Anggraeni and Owens, 2000). However, Wu and Moroga (2004) suggest that apoptosis can not be considered as an effective protection against WSSV in kuruma shrimp. Sahtout et al (2001) found that shrimp with gross signs of WSSV-infection from shrimp farms had up to 40 % apoptotic cells and it was sug-gested that apoptosis might be implicated in shrimp death. Similarly, the wide spread and progressive occurrence of apoptosis in P. monodon infectedwith yellow head virus (YHV) is a major cause of dysfunction and death of the host (Khanobdee et al. 2002). However, the degree of apoptosis in vari-

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ous tissues of P. monodon infected with WSSV strongly suggests that apop-tosis occurs following WSSV infection, but the extent to which it causes shrimp mortality requires further study (Wongprasert et al. 2003).

Crayfish hematopoiesis HematopoiesisAn efficient immune system depends upon the interaction of many cellular and humoral components which develop at different rates during foetal and early postnatal life. Mostly the cells involved in the immune response are derived from undifferentiated hematopoietic cells (hpt cells) and are thought to differentiate into the various cell lineages under the influence of different microenvironmental factors.

Hematopoiesis is the process by which hemocytes mature and subse-quently enter the circulation. The formation and development of mature blood cells involve proliferation, commitment and differentiation from un-differentiated haematopoietic cells. Hematopoiesis provides a mechanism by which hemocytes that have expired or are damaged can be replaced by newly synthesized cells (Barreda and Belosevic, 2001; Medwinsky and Dzierzak, 1999). Hemocytes are constantly produced, although the rate by which this process occurs can be altered rapidly under the influence of dif-ferent microenvironmental factors.

The hematopoietic tissue (hpt) has been detected and its morphology studied in several crustaceans, but the mechanisms behind the release of blood cells into circulation in most invertebrates are still unknown. Hpt is believed to contain precursor cells of the different hemocyte types. In crus-taceans such as crayfish, crabs, lobsters and shrimp, the sheet-like hpt is located dorsally on the stomach and is particularly abundant in the hollow between cardiac and pyloric stomach. The hpt comprises small lobules con-taining differentiating and maturing hemocytes, is surrounded by thin sheath of collagenous connective tissue and is in close contact with the blood si-nuses (reviewed in Johansson et al. 2000). The hpt has different cell types based on their morphology. Hence, in crayfish P. leniusculus, hpt has at least five different types of cells which might correspond to developmental stages of GCs and SGCs. Type 1 was considered the least differentiated precursor cell while Type 2, 3 and 4 were speculated to be different stages of GCs development due to the presence of granules. Type 5, cells differ to Type 2-4 and was suggested to be a precursor of SGCs. Moreover, some cells in the lobules of hpt show similar morphology with the circulating hemocytes. This suggests that the hemocytes are mature when they are released to the circula-tion (Chaga et al. 1995). Similar cell types have been described in the hpt of lobster, Homarus americanus (Martin et al. 1993), blue crab, Carcinus

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sapidus (Johnson, 1984) and shrimp, ridgeback prawn, S. ingentis (Hose et al. 1992) and black tiger shrimp, P. monodon (Van de Braak, 2002b) al-though different synonyms were named for the different cell types.

RegulationIt is very likely there is no, or very few, hemocytes dividing after entering the circulation. The presence of dividing cells in the circulatory system was monitored, but the proportion of these dividing cells is extremely low around 1 % or less (Gargioni and Barracco, 1998; Sequeira et al. 1996). The number of hemocytes in the circulating system, thus, is regulated by the hematopoi-esis in hpt or by storage at other sites. Surprisingly, few studies have been performed to understand hematopoiesis in invertebrates, although the blood cells are important for the immune responses.

Transcription factors are key elements in lineage determination during blood cell formation and several factors are conserved in Drosophila (Denk et al. 2000; Fossett and Schulz, 2001; Lebetsky et al. 2000) and mammals (Busslinger et al. 2000; Cumano and Godin, 2001). Recently, four genes; serpent (srp), lozenge (lz), U-shape (Ush) and glial cell missing (gcm) have been found to be involved in Drosophila hematopoietic lineage commitment (Fossett et al. 2001; Lebetsky et al. 2000; Rehorn et al. 1996). These genes, except gcm, have similarities to mammalian hematopoietic factors; GATA, Acute Myeloid Leukemia 1 (AML1) or Runx and Friend of GATA (FOG), respectively. In Drosophila, the srp gene is earliest expressed in the larval hemocyte precursor pool while Ush is expressed in hemocyte precursors and plasmatocytes during embryo and larval development. A small subset of srp-expressing cells then express lz gene which commits the cells to crystal cells. Most of the srp-expressing cells begin to express gcm and differentiate into plasmatocytes. Ush also functions to block crystal cell production (Fossett et al. 2001).

It is generally agreed that hemocytes in the circulatory system of most crustacean do not divide (Söderhäll and Cerenius, 1992) and thus it was sug-gested that old cells must be replenished continuously by cells released into the hemolymph. Recently, the hpt of crayfish was found to be actively pro-liferating. Injection of a 1, 3glucan caused a severe loss of hemocytes re-sulting in an accelerated maturation of hemocyte precursors in the hpt fol-lowed by release into the circulation of new cells, which developed into functional SGCs and GCs expressing the proPO transcript. It was also con-firmed that very little cell proliferation, if any occurs in the circulation of crayfish. A gene coding a Runt-domain protein similar to Drosophila loz-enge was found to be involved in hematopoiesis and was upregulated prior to release of hemocytes into the blood circulation (Söderhäll et al. 2003).

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Cytokines in invertebrates In mammals, cytokines are proteins that serve many functions in host de-fence against pathogens and provide links between innate and adaptive im-mune immunity. The same cytokines can be produced by many cell types, and individual cytokines often act on various cell types. Cytokines also regu-late the magnitude and nature of immune responses by influencing the growth and differentiation of different blood cell types.

Earlier search for invertebrate cytokines, mainly based on immunohisto-chemical methods have revealed cross reactivity of invertebrate molecules with antibodies against vertebrate interleukins (Beck et al. 1996). However, recent analysis of genome sequences among representatives from different invertebrate phyla did not uncover the presence of any interleukine-like genes, whereas TNF-like genes were shown to be present in the ascidian Ciona intestinalis genome, and IL1-receptor like genes were found in the ascidian as well as in the Drosophila genome (Azumi et al. 2003). Morover, a receptor tyrosine kinase (RTK) belonging to the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) subfamily of RTKs has been identified in D. melanogaster, and overexpression of one of its ligands (PVF2) has revealed cytokine activity in inducing proliferation and migration of hemocytes in vivo (Munier et al. 2003).

Furthermore invertebrate proteins or peptides lacking any sequence homologue to vertebrate proteins have recently been assigned cytokine-like activity, suggesting also a convergent evolution of these regulatory mole-cules. These molecules are the CCF (coelomic cytolytic factor) found in earthworm (Eisenia foetida) showing TNF-like activity (Beschin et al. 2001), the ENF-peptides found in lepidopteran insects (Matsumoto et al. 2003) and the upd’s found in D. melanogaster which are upregulated and released from hemocytes upon septic injury, and then triggering the JAK/STAT pathway in the fat body by binding to its receptor Dome (Agaisse et al. 2003).

In the present study a homologue of Bv8, a member of the prokineticin family of secreted cysteine rich proteins, was found to have cytokine activity in stimulating crustacean hematopoiesis (Paper IV). The prokineticins origi-nally isolated from snake venom (Joubert and Strydom 1980, Schweitz et al. 1999) and frog skin secretions (Mollay et al. 1999) are found in many verte-brates such as mouse, human, bull and fish (Kaser et al. 2003; LeCouter et al. 2001; Li et al. 2001; Wechselberger et al. 1999). Prokineticins have been named to reflect their specific activity in stimulating intestinal smooth mus-cle contraction (Li et al. 2001), but several additional biological activities have been assigned for this protein family, including neuronal survival (Mel-chiorri et al. 2001, pain sensation (Mollay et al. 1999), circadian rhythm (Cheng et al. 2002), stimulation of proliferation of adrenal cortex endothelial cells, and angiogenesis in ovary and testis (LeCouter et al. 2001, 2003). The

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common structural motifs of the prokineticins covers the N-terminal se-quence of 20 amino acids and the C-terminal ten cysteine residues that in the black mamba venom protein are formed into five disulphide bonds (Bois-bouvier et al. 1998). This structure shares some similarities with the Dick-kopf (Dkk) family of regulatory proteins as well as with colipases (Aravind and Koonin 1998; Boisbouvier et al. 1998; Brott and Sokol, 2002; Glinka et al. 1998; Van Tilbeurgh et al. 1992).

Receptors for PK-1 and PK-2, named prokineticin receptor 1 and 2 (PKR-1 and PKR-2), have been identified in specific endothelial cells (R. Lin et al. 2002), neurons (Melchiorri et al. 2001) and transfected cell lines (D. C.-H Lin et al. 2002; Masuda et al. 2002; Soga et al. 2002). These receptors be-long to the family of G-protein-coupled receptors and were recently shown to be expressed in mouse and human hematopoietic stem cells and specific mature blood cells, including lymohocytes (LeCouter et al. 2004). Addition-ally, these studies demonstrate that Bv8 functions as a monocyte chemoat-tractant and a hematopoietic cytokine, since activation of EG-VEGFRs by Bv8 or EG-VEGF stimulates production of granulocytes and monocytes. Based on the ability of Bv8 to induce monocyte migration and the expres-sion of receptors on lymphocytes suggests that Bv8 may directly contribute to the immune response by recruiting additional effectors and mediating B and T cell responses in vivo (LeCouter et al. 2004) and also Lai et al. (2003) suggest that the Bv8 peptides are potential mediators of amphibian innate immune system.

Crustacean primary cell culture Two different ways are often used to obtain cells from tissue explants either by dissociation techniques using enzymes, or letting the cells migrate out into the surrounding medium from the explant. Explanted or dissociated cell cultures have been obtained from various tissues and organs of penaeid shrimp, including the lymphoid (Oka) organ (Chen and Wang, 1999; Itami et al. 1999; Kasornchandra et al. 1999; Lang, et al. 2002; Owens and Smith 1999; Wang et al. 2000; West et al. 1999), the heart (Chen et al. 1986; Chen and Wang 1999; Owens and Smith, 1999), nerve cord (Nadala et al. 1993; Owens and Smith, 1999), hepatopancreas and gut (Chen et al. 1986; Ke et al. 1990; Nadala et al. 1993; Owens and Smith, 1999) and ovary (Chen et al. 1986; Chen and Wang, 1999; Fraser and Hall, 1999; Lang, et al. 2002; Luedeman and Lightner, 1992; Maeda et al. 2003; Nadala et al. 1993; Owens and Smith, 1999; Rosenthal and Diamant, 1990). Only one report (West et al. 1999) used a mixture of hematopoietic tissues and lymphoid cells from P. monodon to develope primary cell cultures. Cells derived from the ovary and lymphoid organ have been most attractive especially for virology studies. There are some reports of primary cell cultures of the lymphoid organs

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which are susceptible to WSSV from P. monodon (Kasornchandra et al., 1999; Wang et al., 2000), from the blue shrimp, L. stylirostris (Tapay et al., 1995) and from the kuruma shrimp, Marsupenaeus japonicus (Itami et al., 1999) as well as ovarian primary cultures from kuruma shrimp (Maeda et al. 2004). Primary cell cultures of lymphoid organ were also reported to be sus-ceptible to YHV (Assavalapsakul et al. 2003).

Most commonly used media are modifications of commercially available media, M199 and L-15 often at double strength (Toullec, 1999; Wang et al. 2000) to maintain cell culture for long-term periods. A variety of supple-ments and concentrations have been used. However, the addition of hemo-lymph or tissue extract appears to be more controversial. Adding hemo-lymph appears to be harmful to cell survival (Fraser and Hall, 1999; Tong and Miao, 1996), while the study of Najafabadi et al (1992) and Chen and Wang (1999) revealed that shrimp hemolymph and lobster hemolymph en-hanced the survival of the cultured cells of penaeid shrimp, respectively. Addition of muscle extract was not necessary for confluent growth (Fraser and Hall, 1999; Luedeman and Lightner, 1992). However, Tong and Miao (1996) found that adding 10 % muscle extract enhanced cell growth which is in agreement with the study of Chen and Wang (1999). Cell derived from lymphoid tissue treated with basic fibroblast growth factor (bFGF) at 20 ng ml-1 could be subcultured for more than 90 passages (Hsu et al. 1995).

Very few studies on crustacean cell cultures report quantitative data on cell proliferation and growth rate although the microscopic appearance (i.e. cell adhesion, attachment or cell confluency) indicates cell growth. How-ever, one study by Maeda et al. (2003) showed that ovarian cells and their proliferation was 11.5 and 35.0 % at 2 and 24 h, respectively, measured as BrdU incorporation.

An attempt to formulate a culture medium for shrimp cells using data de-rived from biochemical analysis of hemolymph from blue shrimp, L. styli-rostris was first reported by Shimizu et al. (2001). To mimic more closely the hemolymph composition, they created two new media based on either the L-15 or the M199 medium and ovary cells formed monolayers in either of the new media. However, there was no evidence of sustained DNA or protein synthesis, which is a key factor to establish a cell line.

So far, primary cell cultures of several crustacean species can be obtained using a wide range of media. In most cases although these cultured cells can survive well, the rate of cell multiplication is low. However, these primary cell cultures have exhibited normal cell functions such as vitellogenin pro-duction (Fraser and Hall, 1999) and susceptibility to virus infection and can be used to study virus-host interaction.

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Aim of the present study

The work of this thesis was aimed to investigate virus-host interaction. Stud-ies were performed on a pathogenic WSSV from shrimp versus the freshwa-ter crayfish, P. leniusculus (Paper I-II). In a subsequent study an environ-mental effect on the immune response and infectivity pattern of WSSV in crayfish was investigated (Paper III). Further expanding the study from invivo into in vitro, a growth and survival factor as well as its importance for primary hematopoietic cells was identified (Paper IV). Finally the replication of WSSV in primary hematopoietic cells of crayfish was studied (Paper V).

Results and discussion Experimental infection of white spot syndrome virus in freshwater crayfish Pacifastacus leniusculus (Paper I) Freshwater crayfish constitute an important part of the freshwater fauna and play an important ecological role as well as a useful laboratory model for the study of defence mechanisms among crustaceans. Therefore, to ascertain whether this crayfish is susceptible to WSSV, an experimental infection of WSSV in crayfish was performed. After introduction of WSSV into the crayfish P. leniusculus either by injection or oral administration, the crayfish was found to be susceptible to infection with WSSV and mortality occurred from day 7-8 and reached 100 % at day 10 post-injection. Infected crayfish showed a significantly delayed clotting reaction and a weak response to stimulation. The appearance of white spots in the cuticle, overall reddish body coloration or both, typical gross signs of WSSV infection in shrimp, were not found in infected crayfish at that time. However, some crayfish in a new batch had red spots throughout the body after infection with WSSV.

Lesions caused by viral injection were observed in various tissues such as gill, cuticular epidermis under shell and around the stomach, adipose con-nective tissue, hematopoietic tissue, heart and hemocytes. No affected cells were found in the epithelial cells of hepatopancreatic tubules, but a lot of affected cells were found in the interstitial tissue and in hemal vessels be-tween tubules. It seems likely that the epithelial cells of hepatopancreatic tubules are not susceptible to WSSV. The affected cells had enlarged nuclei that contained marginated chromatin. Since moribund crayfish showed heav-

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ily infection in various organs, this suggests that crayfish eventually die of disturbance or loss of organ functions.

In situ hybridization showed a positive reaction as indicated by a strong signal in affected cells of various target tissues. No reactions were observed in cells of sham-injected or un-injected control crayfish or in the cells from virus-infected crayfish when the in situ hybridization was performed without the WSSV probe.

The number of both total hemocytes (THC) and granular cells (GC) showed that normal hemocyte balance was rapidly restored after sham injec-tion, but not after virus injection. Whereas the THCs remain unchanged, the relative proportion of granular cells increased. There are several possible explanations for this, such as it is possible that granular cells are more resis-tant to viral infection than non-granular cells, or that WSSV may affect he-matopoietic tissue development and lead to changes in the proportion of different circulating blood cells types, or the presence of some mechanisms in GC to prevent viral infection. The results of the total hemocyte count upon viral infection in crayfish are in contrast to those in shrimp where THCs are dramatically decreased during viral infection (Guan et al. 2003; Van de Braak et al. 2002a).

White spot syndrome virus (WSSV) interaction with crayfish hemocytes (Paper II) Our previous results of hemocyte counts during viral infection in paper I showed that WSSV significantly affected the proportion of differential hemocytes of crayfish. Unlike shrimp, THC did not dramatically decrease during virus infection. This inspired us to investigated more to better under-stand the interaction of WSSV and host hemocytes. In this paper separated GC and SGC were used and special attention was focused on the GC.

First, WSSV particles were detected in separated GC and SGC by in situhybridization and the prevalence of WSSV-infected GCs was 5 % whereas it was 22 % in SGCs. This indicated that SGCs are more susceptible and that WSSV replicated more rapidly in SGC than in GCs and then the SGC gradu-ally decreased from the blood circulation.

Next, effect of HLS, containing the degranulation factor (peroxinectin), PMA, the Ca2+ ionophore A23187 on GCs from WSSV-infected and sham injected crayfish were performed and were compared to control crayfish injected with CFS.. The results showed that the percentage of degranulated GC of WSSV-infected crayfish treated with HLS or PMA was significantly higher than in the control, whereas no significant difference was at hand when treated with the Ca2+ ionophore. It was previously shown that peroxi-nectin and PMA have a degranulation effect via intracellular signalling in-volving protein kinase C (PKC) (Johansson and Söderhäll, 1993) whereas

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the Ca2+ ionophore uses an alternative pathway. HLS treatment of GCs and SGCs from WSSV infected crayfish results in three different morphological types; non-spread, spread and degranulated cells. The non-spread cell group from both GCs and SGCs had more WSSV positive cells than degranulated cells, when detected by in situ hybridization. Therefore, it is reasonable to speculate that the PKC pathway might be affected during WSSV infection.

Finally, we also investigated apoptosis in hemocytes although the per-centage of apoptotic was very low, it was significantly higher than in the control either on day 3 or 5 post-infection. Another interesting phenomenon was that GC from non-infected crayfish exhibited melanization, when incu-bated in L-15 medium, while no melanization was found in GCs of WSSV-infected crayfish. However, the phenoloxidase activities in HLS of both non- and WSSV–infected crayfish remain similar as well as proPO expression as detected by RT-PCR. Obviously thw WSSV inhibits the proPO system up-stream of phenoloxidase or simply consumes the native substrate for the enzyme so that no activity is shown. The TEM observation in hpt cells sug-gests that WSSV infect specific cell types in hematopoietic tissue and non granule-hpt cells seem more favorable to WSSV infection.

Effect of water temperature on the immune response and infectivity pattern of white spot syndrome virus (WSSV) in freshwater crayfish (Paper III) In this paper we compared the susceptibility of Astacus astacus to WSSV with that of P. leniusculus. At an early period of infection, 4-6 days post-injection, P leniusculus had a significantly higher mortality than A. astacus.On day 14 post-injection, however, WSSV caused 100 % mortality in both species. Such susceptibility of P. leniusculus to WSSV at an early stage may be correlated to the fungal parasite, Aphanomyces astaci, which always is present as a viable parasite encapsulated by melanin sheaths in the cuticle of this species. Moreover, this species was found to have a permanent high level of immune transcripts such as proPO whereas an induction of more proPO transcript was found in A. astacus following immune challenge (Cerenius et al. 2003). Hence, it is likely that the immune system of A. astacus can respond more efficiently to the WSSV infection.

Our results clearly showed that water temperature had an effect on the in-fectivity of WSSV in P. leniusculus. At 22 ºC crayfish had 100 % mortality at day 12 and day 25 after receiving WSSV by injection or oral administra-tion, respectively. At 4 and 12 ºC no moribund or death crayfish were ob-served, as well as no visible symptoms of WSSV infection. Since these cray-fish did not die of WSSV during 45 days after the onset of the experiment, they were transferred to 22 ºC. As a result, mortality commenced at day 7 and reached 100 % at day 11, whereas the crayfish groups, which received

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WSSV by oral administration had 40 % mortality at day 45 after transfer. To further confirm that water temperature affects the mortality, another experi-ment was performed. Crayfish were injected with WSSV and maintained at 22 ºC for 5 days and these crayfish showed a weak response.Then they were divided into three groups and distributed to different temperatures, 22, 16 and 12 ºC. The mortality of WSSV-infected crayfish at 22 ºC was up to 100 % within 3 days while crayfish held at 16 and 12 ºC reached 100 % mortality by 20 and 35 days, respectively. The presence of WSSV DNA was detected in all dead animals by PCR.

One aspect of concern is whether the immune system of crayfish is af-fected by temperature. To ascertain this point, THC, and PO activity as well as proPO and LGBP gene expression of normal crayfish were analyzed at different temperatures. We found that THC in crayfish held at 18 ºC was higher than those held at 22 and 12 ºC, and was significantly higher than those at 4 ºC. There was no significant variation in the numbers, proportion of GC or phenoloxidase activity irrespective of temperature exposure. No difference in the amount of proPO-mRNA could be detected in hemocytes from crayfish maintained at different temperatures, except that the LGBP-mRNAs level of crayfish at 22 ºC was remarkably lower than in crayfish at lower temperature. However, these small variations at different temperatures could probably not affect the susceptibility to WSSV.

Finally, the cell proliferation in hpt, which is one target tissue of WSSV, was studied to investigate whether temperature affects viral replication. We found that when hpt cells from BrdU-injected crayfish were analyzed, the number of cells which had incorporated BrdU 4 h after injection at 22 and 18 ºC were significantly higher than in crayfish kept at lower temperatures, 12 and 4 ºC. These findings show that most likely the pathogenicity of WSSV is dependent on actively dividing cells. It is possible that these dividing cells may provide necessary factors for WSSV replication. Viruses with dsDNA genomes often have large genomes, up to 375 kbp, allowing them to have complex virions and to produce a range of virus-specific enzymes, unlike smaller viruses that always are dependent on cellular enzymes and processes. For instance, the enzyme thymidine kinase, which supplies components of DNA synthesis and is produced by Herpesviridae and Poxviridae, is only present in actively dividing cells. Although WSSV is a large dsDNA virus, so far it is not known whether WSSV replication rely on its own enzymes, or host enzymes, or both. In turn, as mentioned above that WSSV is a large dsDNA virus and has a possibility to produce their specific enzymes, their enzymes may work more efficiently at higher temperature and that means that temperature affects WSSV replication directly.

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An ancient role for a prokineticin domain in invertebrate hematopoiesis (Paper IV) During the past decades a number of studies of invertebrate cytokines have been carried out to understand the regulation of cellular immunity and hema-topoietic development. In crayfish, Söderhäll et al. (2003) performed prolif-eration and gene expression experiments in vivo to get some understanding about the regulation of the synthesis and differentiation processes. They found that the hpt is actively proliferating and hemocytes are released to the blood circulation following a severe loss of hemocytes by introduction of a

-1, 3glucan. A PlRunt gene, encoding a Runt protein, which is involved in hematopoiesis in Drosophila and mammals, was upregulated prior to hemo-cyte release. In contrast proPO were expressed in these cells after they were released into the circulation.

Cell-free plasma from crayfish, after hemocyanin removal by ultracentri-fugation, was added to cultures of hpt-cells and was found to induce spread-ing, attachment and proliferation of the cells. The protein causing this activ-ity was further purified and identified with an apparent molecular mass of 15 kD. This protein was named astakine and was cloned, sequenced and ex-pressed. The deduced protein sequence shows homology with the proki-neticin-domain of Bv8 from the frog Bombina sp.. As mentioned above PlRunt is expressed at a low level in hpt-cells, but at a high level in mature SG and GC as well as proPO. Similarly, hpt-cells cultured in vitro without supplemented astakine did not express high levels of proPO. However, PlRunt and proPO-mRNAs could be detected by in situ hybridysation and by RT-PCR after astakine was added for 30-60 min. Semigranular cells were found to release astakine, when separated SG were incubated with modified L15-medium for 5-24 h, the spent medium could activate hpt-cells. In addi-tion, silencing of the astakine gene in SG by siRNA affected astakine secre-tion into the medium and also lowered the astakine transcript.

Plasma obtained from LPS-injected crayfish had higher astakine content compared to than in non-injected ones, but no difference was found in hemocyte astakine expression. The high content of astakine in LPS-injected plasma is due to some other mechanisms, such as increased translation, stimulation of release of release of astakine from hemocytes, decreased turn-over of the protein or a combination of these processes.

Astakine plays an important role in the hematopoiesis in vivo. When puri-fied or recombinant astakine was injected into crayfish, it could induce rapid increase in the THC, while injection of a cysteine-rich antibacterial peptide as a control did not affect the number of hemocytes. To further confirm that astakine is required for hpt-cell proliferation, astakine gene knock out ex-periment was done. SiRNA and large dsRNA were injected twice with 24 h intervals into live crayfish and the astakine gene was silent for 30-48 h. THC did not significantly change during this short period of silencing. However,

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when LPS was injected to cause severe loss of hemocytes there was no re-covery of THC in astakine silenced crayfish.

Discovery of this first invertebrate prokineticin-like protein involved in hematopoiesis may provide interesting information concerning evolution of these secreted cysteine-rich regulators.

Replication of white spot syndrome virus (WSSV) in vitro culture hematopoietic (hpt) cells of freshwater, Pacifastacusleniusculus (Paper V) In general, cell culture is a basic and powerful tool for the study of viruses. To obtain a better understanding of virus host interaction techniques for maintenance and culture of viruses and their host cells in vitro are important. No continuous cell line has been established for replication of crustacean viruses so far, even though primary cultures obtained from diverse organ sources such as lymphoid organ and ovary of shrimp are used. In this paper we attempted to optimize and culture cells from the hematopoietic tissue, which are one target of WSSV infection in vivo and then use them for WSSV replication.

Cells isolated from the hematopoietic tissue were added into culture wells, and they formed monolayers of round shaped cells of different size. When astakine factor (AF) was added regularly for 2-3 day these round cells were changed to three types including round, epithelial-like and fibroblast-like cells and all cells adhered very well to the substratum. The cells could be maintained proliferating for approximately more than one month with supplemented AF at 16 ºC but they could not be sub-cultured due to the firm attachment to the surface and their sensitivity to trypsin treatment. The pres-ence of elongated or fibroblast-like cells appeared in a dose dependent man-ner of AF. At 25 ºC, however, the cells needed lower concentration of AF for their survival, whereas at 4 ºC hpt cells survived several months with 2-3 times higher concentration of AF.

A time course study of WSSV replication in vitro of hpt was performed at 12, 16 and 25 ºC, and the relative amounts of WSSV were determined by measuring the intensity of the PCR amplification band after ethidium bro-mide staining. The intensity of WSSV bands gradually increased from day 4 to day 10 and from day 4 to day 14 at 25 and 16 ºC, respectively, but not at 12 ºC. These results are consistent with our previous study in paper III. Later we found that at 4 ºC WSSV could not replicate but they could repli-cate very efficiently at 28 ºC. Above 28 ºC, WSSV was able to replicate due to detection of WSSV at day 1 by RT-PCR of the VP28 gene, but these tem-peratures were not appropriate for culturing hpt cells.

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WSSV particles that replicated in the hpt cell culture had the same viru-lence as WSSV propagated in crayfish as demonstrated, by re-infection ex-periments.

We also found that AF was necessary for replication of this virus in vitro.In the last experiment of this paper we tried to show whether WSSV infec-tion could be affected by RGD peptides. Since these peptides block integrin receptors and therefore infection would be inhibited if WSSV utilizes in-tegrin as a receptor for entry into the host cells. However, RGD peptides did not display any effects on the infection efficiency of the WSSV. This may suggest that this virus is not using integrins to gain entry into the host cells or they used alternative receptors when these integrins were occupied. Be-sides pathological studies the use of the hpt cell culture may provide a tool for better understanding of the molecular mechanism of WSSV infection or other virus infections, In future the hpt cell culture may also be used as a homologous expression system for the development of functional genomics for invertebrate molecular biology.

Concluding remarks The results of these studies show that WSSV can infect freshwater crayfish and also can replicate in an in vitro cell culture. The pathogenicity is similar to shrimp, which the WSSV is originally isolated from, except that the pat-tern of the total number of circulating hemocytes during WSSV infection between shrimp and crayfish is significantly different. WSSV infection causes many alternations in crayfish hemocytes such as inhibition of de-granulation and melanization suppression. It is clear that temperature af-fected the WSSV replication both in vivo and in vitro. Although, much has been learnt, there are still many unanswered questions. The discovery of the factor in plasma, astakine that was found to enhance the differentiation and maturation of the hpt cell culture is important for future studies on hpt cell cultures. This cell culture can be used to study virus-host interaction in crus-taceans and also for other interactions between crustacean parasites and their host cells. A cell culture in which it is possible to perform gene silencing to ascertain the role and function of different immune factors is an important tool since at present reverse genetics can not be done in any crustacean.

More work is also required to understand viral receptor(s) and how virus evades the innate immune response.

Hopefully, studies on virus-host interactions may help to develop better methods to avoid viral diseases in shrimp culture.

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Svensk sammanfattning (Summary in Swedish)

Virus förekommer i stora mängder i hav och vattendrag, och halter på ända upp emot 10 miljarder viruspartiklar per liter vatten kan uppmätas i havet. Sjukdomar orsakade av virus är därför mycket vanliga hos marina organis-mer, och dessa sjukdomar medför stora problem i kommersiella odlingar av såväl kräftdjur som musslor. En av de sjukdomar som haft förödande konse-kvenser för vattenbruk i många länder är Vitfläcksviruset (WSSV= White Spot Syndrome Virus).

Kräftdjur liksom andra ryggradslösa djur har till skillnad från ryggrads-djuren inte tillgång till antikroppar, utan dessa djurs immunförsvar bygger enbart på medfödda (s.k. innate) försvarssystem. Dessa försvarssystem är dock mycket effektiva och det finns proteiner i djurens kroppsvätska som kan upptäcka minimala mängder av ämnen från främmande organismer. Det betyder att om bara några få bakterier eller svampsporer kommer igenom djurens yttre skal så aktiveras det inbyggda försvaret mycket snabbt. Meka-nismerna för hur kräftdjur försvarar sig mot bakterier och svamp är relativt väl undersökta. Kräftans olika blodkroppar har en central roll i försvaret, där vissa celler medverkar vid t.ex. fagocytos, medan andra typer utsöndrar an-timikrobiella peptider och koaguleringsfaktorer som snabbt förhindrar en spridning av infektionen. Vissa blodkroppar, eller hemocyter, bär också en-zymet fenoloxidas som vid en infektion utsöndras hemolymfan, och med-verkar till melanisering vilket också förhindrar vidare spridning av svampar och bakterier i kroppshålan.

När det gäller virussjukdomar är de ryggradslösa djurens försvarssystem däremot mycket lite undersökta. I den här avhandlingen användes Vitfläcks-viruset tillsammans med sötvattenskräftor för att studera de försvarsreaktio-ner som kräftdjuren använder mot virus. Först och främst studerades själva infektionsförloppet där vi fann att viruset främst infekterar den blodkropps-bildande vävnaden och blodkropparna. Infektionsförloppet är också starkt beroende av vattentemperaturen, och vid låga temperaturer kan kräftan här-bärgera viruset i latent form för att sedan när temperaturen stiger bli sjuk och dö. Detta visar att en införsel av detta virus till Europa kan ha förödande konsekvenser för den europeiska kräftfaunan. Viruset har även effekt på fördelningen i hemolymfan av olika typer av blodkroppar och våra resultat visar också att de granulära hemocyterna är infekterade i mindre utsträck-

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ning än andra typer. Samtidigt påverkar viruset hemocyternas förmåga att utsöndra försvarsrelaterade proteiner och förmågan till melaninbildning. För att mer i detalj kunna studera hur viruset interagerar med den blodkroppsbil-dande vävnaden i kräfta har vi utvecklat en ny metod för att odla dessa celler och infektera dessa med virus in vitro. Utvecklingen av denna odlingsmetod ledde till att vi upptäckte ett nytt protein, (astakine) som verkar som blod-kroppsbildande cytokin i dessa djur. Proteinet har inte tidigare hittats hos ryggradslösa djur, men har hos däggdjur visat sig ha viktiga biologiska funk-tioner bl.a. för blodkärlsbildning i vissa vävnader. Närvaron av detta cytokin visade sig nödvändigt för att virus skulle kunna infektera cellerna in vitro och metoden möjliggör därför fördjupade studier av virus-värdcell interak-tioner hos ryggradslösa djur.

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Acknowledgements

I would like to thank:

Prof. Kenneth Söderhäll, for dragging me outside the coconut shell to an amazing molecular biological world, for never ending encouragement and being patient for all aspects. Thank you indeed for your kindness and tough-ness. Since only kindness will never work out to drive me, stubborn Pikul. Dr Irene Söderhäll, for always being extremely kind whenever I need help, advice, discussion and also for wonderful Swedish dishes.

Dr Lage Cerenius, for DNA work and discussions with enthusiasm, Rag-nar Ajaxon, my labpolice chief supervisor, who gave me the first lesson of cell separation, Anbar Khodabandeh, my neighbour in the lab, who always inspired me to optimistic thinking, Stefan Gunnarsson and Annette Axénfor helping with microscope and TEM. All former and present friends inside and outside the lab during my stay in Sweden, for your try and error food, a lot of fun, dried my tears sometimes and of course interesting scientific discussions.

All Thai friends in Sweden ‘Khob Khun Mak Mak Kha’ who always take care of me, give moral support, are patient with my complaints and for ex-cellent delicious Thai food. AROY Jung!

All my former supervisors and friends in Thailand, who fulfil my success via hot lines and hot mail.

All my brothers and sisters, for their support and love.And most of all to my parents Thank you ex-tremely much for your endless love, support and never say No! whatever and wherever Pikul want to be. Just a few words that I am familiar with ‘do your best and good luck’.

Finally, Thank to Khun P. leniusculus & A. astacus, who devoted their lives to fulfill my Ph D.

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A doctoral dissertation from the Faculty of Science and Technology, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology. (Prior to January, 2005, the series was published under the title "Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology".)

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