Virion composition andgenomic s ofWhit e Spot SyndromeViru so fshrim p M.C .W .va nHulte n Promotoren: prof.dr .J .M . Vlak Persoonlijk Hoogleraar bij deLeerstoelgroe p Virologie prof.dr .R .W .Goldbac h Hoogleraar ind eVirologi e Samenstellingpromotiecommissie : dr.J .R .Bonam i(Universit y ofMontpellie r II, France) dr.E .O .Rijk e (Intervet International, Nederland) prof.dr .W .J . Stiekema(Plan t Research International, Wageningen) prof.dr .J .A .Verret h (Wageningen Universiteit) /vr-oT^ ' ; ^ > Virion composition and genomics ofWhit e Spot Syndrome Virus of shrimp M. C.W .va nHulte n Proefschrift ter verkrijging van de graad van doctor op gezagva n derecto r magnificus vanWageninge n Universiteit, prof.dr . ir.L .Speelman , inhe topenbaa rt e verdedigen opvrijda g 16novembe r200 1 desnamiddag st e 13.30uu r in deAula . The work presented in this thesis was carried out at the Laboratory of Virology of Wageningen University, The Netherlands. It was financially supported by Intervet International BV, Boxmeer, TheNetherlands . Van Hulten, M. C.W . Virion composition and genomics ofWhit e Spot Syndrome Virus of shrimp Thesis Wageningen University- with ref. - with summary inDutc h ISBN 90-5808-516-3 Subject headings:Nimaviridae ,Penaeus monodon, WSS V genome Aanmijn ouders Contents Chapter 1 General introduction 1 Chapter 2 Analysis of a genomic segment of white spot syndrome 11 virus of shrimp containing ribonucleotide reductase genes andrepea t regions Chapter3 Identification and phylogeny oftw oprotei n kinase genes of 23 white spot syndrome virus Chapter4 Identification of two major virion protein genes of white 31 spot syndrome virus of shrimp Chapter 5 Three functionally diverged major structural proteins of 43 white spot syndromeviru s evolvedb y gene duplication Chapter 6 Identification of VP19 and VP15 and glycosylation status 49 ofth ewhit e spot syndrome virus major structural proteins Chapter 7 White spot syndrome virus envelope protein VP28 is 59 involved inth e systemic infection of shrimp Chapter 8 Thewhit e spot syndrome virus genome sequence 67 Chapter 9 General discussion 89 References 99 Summary 109 Samenvatting 112 Nawoord 115 Curriculum Vitae 117 Account 119 General introduction Chapter 1 General introduction Penaeusmonodon The black tiger shrimp, Penaeus monodon is the largest and commercially most important species among penaeids, reaching 300 mm in body length (Fig. 1.1). P. monodon occurs mainly in Southeast Asian waters. Its carapace and abdomen are transversely banded with red and white. However, when cultured in ponds, the color changes to blackish, hence this shrimp species is often referred to as black tiger shrimp (Motoh, 1985). Mating and spawning of P. monodon generally takes place at night. The maximum number of eggs spawned at a time can be up to 800,000 per individual. The eggs hatch within 12hour s into nauplii, the first larval stage. The complete larval stage ofP . monodonconsist s of 6nauplius , 3 zoea, 3 mysis, and 3 or 4 postlarva substages (Fig. 1.2) and the time required for each of these substages is about 1.5 days, 5 days, 5 days and 6 to 15 days, respectively. The next stage is the juvenile stage, where brackish water areas serve as nursery grounds and the shrimp develop in subadults in about 4 months. The subadult stage starts at the onset of sexual maturity while the shrimpmigrat e from nursery to spawning grounds (Fig 1.2).Afte r 4 months the adult stage is reached, which is characterized by the completion of sexual maturity (Motoh, 1985). The food of P. monodon consists mainly of Crustacea (small crabs and shrimps) and molluscs. Other commercially cultured shrimp species are P. vannamei (Pacific white shrimp), P.japonicus (kuruma shrimp), P. stylirostris (blue shrimp), and P. chinensis (Chinese white shrimp) of which the first two species are mainly cultured on the Pacific coast ofCentra l and South America. Figure 1.1 Drawing of Penaeus monodon, the Figure 1.2 Schematic representation of the life cycle of "black tiger shrimp". Penaeid shrimp (Rosenberry, 2000). Shrimp culture andvira l diseases Shrimp farming has its origins in Southeast Asia where for centuries farmers have raised incidental crops of wild shrimp in tidal fish ponds (Rosenberry, 1994). The collection of wild seed in the 1970's and the advances in shrimp reproduction and hatchery technologies in the Chapter1 Shrimp production worldwide (1975-2000) Figure1. 3Shrim pproductio nfrom 197 5t o2000 . 1980s resulted in a rapid development of shrimp farming. In the 1990s, the world's production of cultured shrimp increased less dramatically and plateaud at around 700,000 tonnes per year (Fig. 1.3) (Rosenberry, 1995, 1996, 1997, 1999,2000) .Th e growth stagnated despite an increasing number of hectares used for production. This was caused by mass mortalities in pond culture throughout the world, predominantly caused by viruses (Lotz, 1997). In particular in Asia shrimp culture was heavily afflicted, resulting in a decrease of shrimpproductio n threeyear s ina ro w from 1995t o 1997(Rosenberry , 1997). Penaeid shrimp canb e affected byman y different infectious agents, including viruses, rickettsiae, gram-negative and gram-positive bacteria, fungi and protozoa. The most important diseases of cultured penaeid shrimp have viral or bacterial etiologies. Since 1995 viruses have caused the greatest impact on shrimp culture, as no treatment for these agents is available yet,wherea s bacteria canb e controlled by antibiotics andvaccine s (Teunissen et al, 1998). The major diseases of cultured shrimp caused by viruses are described in table 1.1. Baculovirus penaei (BP) and Monodonbaculoviru s (MBV) are rod-shaped viruses, which are occluded in large proteinaceous capsules (polyhedra) and are therefore thought to be members of theBaculoviridae (Bonami et al, 1995a; Mari etal, 1993). MBV is enzootic in Southeast Asia in both wild and farmed P. monodon populations, and has been reported to cause major mortalities in Asia in the late 1980s (Mari et al, 1993). Baculovirus midgut gland necrosis virus (BMNV) has mainly been reported in P. japonicus in Japan and generally infects shrimp larvae (Sano et al, 1981;Momoyam a and Sano, 1996). Not much research has been performed on this virus and therefore it is not clear what virus family this virus belongs to (Table 1.1). Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a highly contagious and infectious virus for many penaeid species (Bonami et al, 1990). As the overall genomic organization is similar to that of the mosquito brevidensoviruses, it was concluded that IHHNV is closely related to densoviruses of the genus Brevidensovirus in the family Parvoviridae (Shike et al, 2000). Hepatopancreatic Generalintroduction parvovirus (HPV) causes disease in several species of penaeid shrimp andanalysi s ofit s genome indicated that this virus is most likely a member of the family Parvoviridae (Sukhumsirichart etal., 1999). The two RNA viruses indicated intabl e 1.1, Taura syndrome virus (TSV) and Yellow head virus (YHV) both have a great impact onshrim p culture and havebee n studied extensively. TSVha s caused seriousmortalitie s in shrimp (P. vannamei) in the Americas (Lightner etal., 1995).Researc h on the TSV genome organization showed that TSV is similar to insect picornaviruses (Robles-Sikisaka etal., 2001). YHV hadi t greatest impact in Asian countries (Boonyaratpalin etal., 1993).I nAustrali a the closely related Gill- associated virus (GAV), and its minor variant Lymphoid organ virus (LOV) have been identified (Cowley etal., 1999). Research on the ssRNA genome revealed that these viruses are members of the order Nidovirales andfurthe r research will be needed to resolve their taxonomic classification within this order (Cowley etal., 2000) . Virus name Virus family Reference Detection DNA viruses Baculovirus penaei (BP) Baculoviridae Bonami era/., 1995 Bonami et al, 1995; Wang et al, 1996 Monodon baculovirus (MBV) Baculoviridae Vickers etal, 2000; Mari etal., Belcher andYoung , 1998; Hsuet al, 1993 2000 Baculovirus midgut gland Unclassified Momoyama and Sano, 1996 necrosis virus (BMNV) Infectious hypodermal and Parvoviridae Shike etal, 2000 Tang and Lightner, 2001; Nunan et hematopoietic necrosis virus al, 2000 (IHHNV) Hepatopancreatic parvovirus Parvoviridae Sukhumsirichart et al, 1999; Pantoja and Lightner, 2001; Pantoja (HPV) Bonami e;a/. , 1995 and Lightner, 2000 White spot syndrome virus New virus family van Hulten etal, 2001a; Wang Lo et al, 1996b; Tapay et al, 1999; (WSSV) et al, 1995 Tang and Lightner, 2000; Shih et al, 2001 RNA viruses Taura syndrome virus (TSV) Picornaviridae Robles-Sikisaka et al, 2001; Nunan etal, 1998;Mar i etal, 1998 Bonami etal, 1997 Yellow head virus (YHV) Coronaviridae or Cowley etal, 2000; Cowley et Tang and Lightner, 1999; Arteriviridae (order: al, 1999 Sithigorngul etal, 2000; Nadala and Nidovirales) Loh, 2000 Table 1.1 Major viral diseases of cultured Penaeid shrimp. In the first column, the virus name. In the second column, the (putative) virus family and in the third column the reference for this classification. References describing detection techniques in the last column. White Spot Syndrome Virus Fromth e viruses mentioned intabl e 1.1,Whit e spot syndrome virus (WSSV) has had the greatest impact on shrimp culture and remains a major problem up to the present day (Rosenberry, 2000). In cultured shrimp WSSV infection can reach acumulativ e mortalityo f up to 100%withi n 3-10 days (Lightner, 1996). The virus was first discovered inTaiwa ni n 1992, from where itquickl y spread toothe r shrimp farming areas in Southeast Asia and the Chapter 1 Indian subcontinents (Cai etal, 1995).WSS V initially appeared to be limited to Asia until it was found in Texas in 1995 (Rosenberry, 1996) (Fig. 1.4). The contamination of the Texas shrimp farm is believed to be caused by untreated wastes from plants processing imported shrimp from Asia (Naylor et al, 2000). In 1997 the virus was detected in South Carolina.