Genomics and transcriptomics of White spot syndrome virus Hendrik Marks Promotoren: Prof. dr. J. M. Vlak Persoonlijk hoogleraar bij de leerstoelgroep Virologie Prof. dr. R. W. Goldbach Hoogleraar in de Virologie Co-promotor: Dr. ir. M. C. W. van Hulten Wetenschappelijk medewerker bij de Commonwealth Scientific and Industrial Research Organisation (Brisbane, Australië) Samenstelling promotiecommissie: Prof. Dr. H. J. Nauwynck (Universiteit Gent, België) Prof. Dr. V. E. J. C. Schijns (Intervet International B.V., Boxmeer) Dr. O. L. M. Haenen (Centraal Instituut voor DierziekteControle, Lelystad) Prof. Dr. W. J. Stiekema (Plant Research International, Wageningen) Dit onderzoek is uitgevoerd binnen de C. T. de Wit Onderzoeksschool: Production Ecology and Resource Conservation (PE&RC) Genomics and transcriptomics of White spot syndrome virus Hendrik Marks Proefschrift ter verkrijging van de graad van doctor op het gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. M.J. Kropff, in het openbaar te verdedigen op maandag 28 november 2005 des namiddags te 13.30 uur in de Aula Marks, H. (2005) Genomics and transcriptomics of White spot syndrome virus PhD thesis Wageningen University – with references – with summary in Dutch ISBN: 90-8504-318-2 Subject headings: Nimavirus, WSSV, genomics, transcription regulation “…, mijn moeder was verbonden aan een dagverblijf voor psychiatrische patiënten. Haar beroemdste patiënt was een man die tegen een muur praatte in de veronderstelling dat de geheime dienst hem kon verstaan. Toen de geheime dienst niet op zijn berichten inging, werd hij destructief. Zo belandde hij bij mijn moeder. Op deze man is ze gepromoveerd. Dankzij mijn moeder kwam hij erachter dat hij twintig jaar voor niets tegen een muur had gepraat. Deze wetenschap was te veel voor hem en hij stortte zich in een liftschacht. Dat wierp een smet op het promotiefeestje van mijn moeder, want het was de bedoeling dat hij daar de eregast zou zijn. Na dit incident heeft ze een paar artikelen geschreven waarin ze zich afvroeg of het wel verstandig is mensen volledig te genezen van hun waanideeën.” Arnon Grunberg in “Fantoompijn” (2000) Contents Chapter 1 General Introduction 1 Chapter 2 Genetic variation among isolates of White spot syndrome virus 17 Chapter 3 Molecular epidemiology of White spot syndrome virus within 37 Vietnam Chapter 4 Fitness and virulence of an ancestral White spot syndrome virus 53 isolate from shrimp Chapter 5 Gene expression profiling of White spot syndrome virus in vivo 71 Chapter 6 Transcriptional analysis of the White spot syndrome virus major 95 virion protein genes Chapter 7 In silico identification of putative promoter motifs of White spot 105 syndrome virus Chapter 8 General Discussion 123 References 131 Summary 139 Samenvatting 142 Nawoord 145 Curriculum Vitae 148 Account 149 Persbericht 150 PE&RC PhD Education Statement Form 151 General Introduction Chapter 1 General Introduction Shrimp culture Shrimp appear in many sea food dishes and are considered a delicacy in countries around the world. To meet consumers’ demands, and stimulated by advances in shrimp reproduction and hatchery technologies, an enormous growth in shrimp production was observed from the Figure 1.1. Shrimp production from 1970 to 2002 (source: Food and Agriculture Organization of the United Nations (FAO); FAO website: http://www.fao.org/fi/figis/). 1980s onwards (Fig. 1.1). Shrimp culture developed from extensive in the 1970s (50-500 kg ha-1 yr-1) using tidal zones to super-intensive in the 2000s (20,000-100,000 kg ha-1 yr-1) using ponds more inland (Rosenberry, 2004). Traditionally, the majority of the shrimp farming takes place in Asia (especially in China, Thailand, Vietnam, Indonesia and India), where it is an important source of income for people in coastal areas. Nowadays also South- and Latin-America (predominantly Mexico, Ecuador and Brazil) are responsible for a considerable amount of the worldwide production of shrimp. Furthermore, Figure 1.2. Drawing of P. monodon shrimp the shrimp farming industry is increasing in other (source: FAO). areas in the world such as Australia and the 1 Chapter 1 Middle East (Saudi Arabia and Iran) (Rosenberry, 2004). All farmed shrimp species belong to the Penaeidae family of decapod crustaceans (genus Penaeus). The largest, most expensive and commercially most important species is Penaeus monodon (giant black tiger shrimp) (Fig. 1.2), while other important cultured species include P. chinensis (Chinese white shrimp) and P. vannamei (Western white shrimp). P. monodon is named giant black tiger shrimp after its striped tail and its big size (maximum length of 363 millimeters). This species originates from the Indian Ocean and the southwestern Pacific Ocean (Japan to Australia) and is mainly cultured in Asia (Rosenberry, 2004). The species P. vannamei originates from the Pacific coast of Central- and South- America (from Mexico to Peru) and is the leading farm-raised species in South- and Latin- America. Because P. vannamei feeds on organisms which grow naturally in a pond, it is cheaper to feed than P. monodon. P. vannamei has a uniform growth rate and reaches a maximum length of 230 millimeters (Rosenberry, 2004). P. chinensis is native to the coast of China and the west coast of the Korean peninsula. The disadvantages of culturing P. chinensis for production for economy are their high protein requirement and their small size (maximum length of 183 millimeters). P. vannamei and P. chinensis, unlike P. monodon, readily mature and spawn in ponds, and are therefore easy to culture (Rosenberry, 2004). Shrimp diseases In the 1990s, the annual growth of shrimp production decelerated reaching a plateau level of around 900,000 tons per year (Fig. 1.1: 1991-1997; FAO), even though the total area used for shrimp culturing increased. This relative decline was due to mass mortalities in shrimp ponds, predominantly caused by viruses (Lotz, 1997). Whereas the various non-viral infectious agents, such as bacteria, fungi and protozoa, can be controlled by antibiotics and vaccination (Teunissen et al., 1998), efficient control of viral pathogens in the field has not been achieved thus far. More than 20 viruses have been reported to infect shrimp (Lightner, 1996) and the list is growing. The economical most damaging shrimp viruses are White spot syndrome virus (WSSV), Yellow head virus (YHV) and Taura syndrome virus (TSV), which are discussed below. The diseases caused by these viruses are notifiable to the Office International des Epizooties (OIE; World Organisation for Animal Health). The next paragraphs will focus on the large double-stranded DNA virus WSSV, the subject of this thesis. YHV is a positive single stranded RNA virus and it is, together with the closely related shrimp virus Gill-associated virus (GAV), classified in a new taxon (family Roniviridae, genus Okavirus) within the order Nidovirales (Cowley et al., 1999; Jitrapakdee et al., 2003). Signs associated with YHV infection include cessation of feeding, swimming near the water surface or near edges of a pond, and the development of a yellow coloration of the cephalothorax and gills (Chantanacookin et al., 1993). TSV is a positive single stranded RNA virus belonging to the virusfamily Dicistroviridae (Mari et al., 2002). Gross signs associated with TSV infection include the appearance of a distinct blue or red hue on the shell 2 General Introduction and tail. Infected shrimp usually have empty digestive tracts (Lightner, 1996). For a recent review on YHV and TSV see Dhar et al. (2004). As P. monodon is very susceptible to WSSV and YHV, the emergence of these two viruses in the 1990s was one of the main causes of the reduction in productivity. Therefore, farmers in Southeast-Asia often switched from P. monodon to culture P. vannamei or P. chinensis, as these species are considered to be less vulnerable for these viruses (Fig. 1.1) (Briggs et al., 2004; Rosenberry, 2004; Wyban and Sweeney, 1991). Moreover, the breeding techniques for these species are further developed than for P. monodon (Rosenberry, 2004), enabling selection of virus resistant shrimp. However, especially P. vannamei is very sensitive to TSV (Lightner et al., 1995). White spot syndrome virus Of all shrimp viruses, White spot syndrome virus (WSSV) has had the largest impact on shrimp culture and remains a major problem up to the present day (Rosenberry, 2004). In cultured shrimp, WSSV infection can reach a cumulative mortality of up to 100% within 3-10 days (Lightner, 1996). Infected animals show lethargic behavior, such as lack of appetite and slow movement, and a reddish to pink body discoloration. Characteristic for WSSV infected shrimp are white spots on the exoskeleton (Fig. 1.3). These spots are the result of calcified deposits that range in size from a few mm to 1 cm or more in diameter (Chou et al., 1995). However, in case of acute (experimental) infections the only signs of WSSV infection observed are lethargy and lack of appetite. a b Figure 1.3. Photograph of a WSSV infected P. monodon showing white spots on the exoskeleton (a). Close-up of the carapace of a WSSV infected P. monodon, emphasizing the white spots (b). (Source: C.F. Lo, National Taiwan University, Taiwan) WSSV can be transmitted horizontally either per os by predation on diseased individuals, or by virus particles in the water. Infection by the latter is thought to occur primarily through the gills, but may occur via other body surfaces as well (Chang et al., 1996; Chou et al., 1995; 1998). The virus is also transmitted from mother to offspring, although it is not clear whether the WSSV virions are present inside the shrimp eggs (Hsu et al., 1999; Lo et al., 1997; Peng et al., 2001; Tsai et al., 1999). No penaeid shrimp species is known to be resistant to WSSV infection (Lightner, 1996; Lotz, 1997). 3 Chapter 1 WSSV was first reported in the Chinese province Fujian in 1991.