DISEASES OF AQUATIC ORGANISMS Vol. 29: 205-211.1997 Published June 19 Dis Aquat Org

Ultrastructure and morphogenesis of Baculovirus (WSSV)

Stephanie ~urand'l*,Donald V. ~ightner~,Rita M. ~edman~,Jean-Robert ~onami'

'UMR 219 CNRS-IFREMER-UM 11, Place E. Bataillon, CP 80, F-34095 Montpellier Cedex 5, France 'Departement of Veterinary Science, University of Arizona, Tucson, Arizona 85721, USA

ABSTRACT: Since 1993 non-occluded baculoviruses, associated with a syndrome with high mortalities, have been reported in cultured penaeid from Asia and the Indo-Pacific region. are typically accompanied by the presence of white spots on the cuticle. Numerous names were given to the (es) in early reports on the disease, but the syndrome is increasingly known as White Spot Syndrome (\YSS) and its viral agent(s) as White Spot Syndrome Baculovirus (WSSV).The \YSS virion is a stocky rod-shaped particle w~than apical envelope extension. The nucleocdpsid is cylindr~calwith asymmetric ends, and has a superfic~alsegmented appearance. The pattern of degradat~onconfirms that the nucleocapsid is a cylinder formed by stacks of rlngs, which are in turn composed of 2 robvs of regularly spaced subunits. WSSV replication takes place in the nucleus and is f~rstindicated by chromatin margination and nuclear hypertrophy. Viral morphogenesis begins by the formation of membranes de novo in the nucleoplasm and by the elaboration of segmented, empty, long tubules. These tubules break into fragments to form naked empty nucleocapsids. After that, membranes envelop the leavlng an open extremity. The nucleoproteins, which have a filamentous appear- ance, enter the through this open end. When the core is completely formed, the envelope nar- rows at the open end and forms the apical tail of the mature virion.

KEY WORDS: Baculovirus . White Spot Syndrome Ultrastructure

INTRODUCTION The clinical signs of the syndrome include lethargy, anorexia, the presence of white spots on the cuticle Since 1993, associated with a syndrome and, often, a generalized reddish to pink discoloration. with high mortalities have been reported in cultured The syndrome is increasingly known as White Spot penaeid shrimp from Asia and the Indo-Pacific region Syndrome (WSS) and its agent(s) as White Spot Syn- (Huang et al. 1994, 1995, Inouye et al. 1994, Takahashi drome Baculovirus (WSSV). et al. 1994, Wang et al. 1995, Wongteerasupaya et al. Histologically, is characterrzed by eosino- 1995). These authors related the viruses to the Bac- philic to progressively more basophilic inclusion bodies uloviridae and to the subfamily of the non-occluded in the hypertrophied nuclei of infected cells. The target baculoviruses: the Nudibaculoviridae (Francki et al. tissues are generally of mesodermal and ectodermal 1991). However, in the most recent sixth report of origin including connective and epithelia1 tissues, the ICTV (Murphy et al. 1995), the previous classifica- hematopoietic nodules, hemocytes, the gills, epi- tion of these viruses as baculoviruses was canceled dermis, foregut (stomach), striated muscles, and nerves and the viruses were left unassigned. For clarity, we (Wongteerasupaya et al. 1995, Durand et al. 1996). will maintain in this paper the old designation Electron-microscopic observations of sections and (Francki et al. 1991) for WSSV as a non-occluded viral suspensions revealed rod-shaped, enveloped, baculovirus. non-occluded virions. The nucleocapsid seemed to be composed of rings of subunits in a stacked series (Wang et al. 1995). During morphogenesis, the capsid was formed from a 'capsid originator' and the mem-

0 Inter-Research 1997 Resale of full article not permitted Dis Aquat Org 29: 205-21 1, 1997

brane was formed de novo in the nucleoplasm (Inouye ded in LR white (The London Resin Co. Ltd.). Ultrathin et al. 1994). This disease could be diagnosed by histo- sections were stained with uranyl acetate and lead cit- logical observations (Wongteerasupaya et al. 1995), by rate according to Reynolds (1963) dot-blot and in situ hybridization using labelled gene The viral suspensions used for negative staining con- probes (Durand et al. 1996) or polymerase chain reac- sisted of virions isolated from hemolymph according to tion (Lo et al. 1996). the purification assays of Durand et al. (1996).The viral In this paper, we report the ultrastructure of the suspensions were placed on carbon-collodium coated agent after purification and negative staining, and its grids and negatively stained with phosphotungstic morphogenesis as studied in ultrathin sections of ex- acid (PTA2 %, pH 7). All observations were made with perimentally infected tissues. a Hitachi HUllC transmission electron microscope.

MATERIAL AND METHODS RESULTS

The shrimp used for this study were Penaeus van- Ultrastructure namei and P. stylirostris, obtained from the University of Arizona, Tucson, Pathology Laboratory Negative staining (USA).They were experimentally infected by an inocu- hm prepared from an homogenate of infected P. mon- The morphometry and description of virion and odon from Thailand in TN buffer (0.02 M Tris-HC1, nucleocapsid were described in Durand et al. (1996) 0.4 M NaC1, pH 7.4). Th.e homogenate was centrifuged and only new data are reported here to complete the at 2500 rpm (1020 X g) for 10 min and the supernatant virus ultrastructure. fluid was diluted 1:20in 2 % NaCl and filtered (0.45 pm The complete virus is typically characterized by an filter). Experimental infection was induced by injecting apical envelope extension (Fig. 1) sometimes observed 0.2 m1 of the filtered suspension abdominally under the in one side of the particle (Fig. 2). The nucleocapsid fourth tergal plate and into the third segment. displays a superficially segmented appearance. Each Descriptions of the virus are based on transmision segment seems to be formed of 8 nm diameter subunits electron microscopy (TEM) of sectioned tissues and of which are arranged In 2 parallel rows (Fig. 3). The negatively stained viral suspensions. cylinder representing the nucleocapsid is closed at one Tissues used for the ultrathin sections were foregut extremity by a smaller segment that forms a slightly and stomach . The tissue was diced in 3% rounded end while the opposite extremity is squared cacodylate buffered glutaraldehyde and post fixed in (Fig. 3). Some degraded nucleocapsids were noted 1 % Os04. Fixed tissues were processed and embed- with cross-hatched appearance and a clear area (about

Figs. 1 to 6. Suspension of WSSV, neg- atively stained with PTA. Scale bars are 100 nm. Fig. 1. Virus showing the charactenstic envelope extension (arrow) at the apex of the particle. P?q.. Unusual location of the enve- lope extension in the middle of the particle. One cylindrical nucle- ocapsid presents a rounded and a squared extremity and has a seg- mented appearance. Segments are formed by 2 serles of queued units arranged in regular rows (arrows). The second nucleocapsid is partially swollen (arrowhead). Fig. 4. De- graded nucleocapsid exhibiting a cross-hatched appearance formed by regularly disposed units. A clear area (arrow) at one extremity represents the orientat~onof the degraded nucle- ocapsid. W. The degraded nucleo- capsid is fractured at the level of the segmentation (arrow). Nucleo- capsid with a detached segment showing its ringed shape Durand et al.: Ultrastructure and morphogenesis of WSSV 207

L 24 nm thick) at one extremity (Fig 4). Some degraded nucleocapsids were fractured at the level of the segments- . -. tion (Fig. 5). Segments were partially or completely separated from the re- mains of nucleocapsid sometimes form- .. ing a ring at one extremity (Fig. 6).The content of the capsid seems to be a filamentous material released during degradation of the nucleocapsid. b.

Thin sections

The virion is rod-shaped and about 250 X 100 nm (Fig. 7). The envelope is 6 to 7 nm thick and has the structure of % - a trilaminar unit membrane (Fig. 8). The area between the nucleocapsid and the envelope varies from about 2 to 7.5 nm. The nucleocapsid is a cylinder about 200 X 65 nm with a 6 nm thick external wall. The core of the nucleo- capsid is highly electron dense.

Morphogenesis

Early infection is indicated by nuclear hypertrophy and margination of the chromatin. The nucleus contains finely fibrillar and granular material centrally located, and numerous llnear or circular membrane fragments are present (Fig. 9). Some membrane pro- files formed a series of vesicles joined together (Fig. 10). In highly infected nuclei, forming virions are located centrally, while mature virions tend to be concentrated along the nuclear margin. Mature virions may be assembled in ordered arrays or randomly scattered (Fig. 7). We observed, in several instances, structures that appeared to be empty Figs. 7 & 8. Ultrathin sections of infected stomach epithelium, TEM. Scale bars capsids about 45 nm in are 150 nm. Fig. Longitudinal section of WSSV virions arranged in parallel in which is less than the diameter of the the nucleoplasm. Fig. 8. Cross-section of WSSV virions. The envelope (E) is true nucleocapsid (with its comple- clearly evidenced, surrounding a nucleocapsid (Nc) formed by an electron- inent of DNA material). These empty dense central area enclosed in a structured layer capsids are located in the central por- tion of affected nuclei and are usually enveloped but Some unusual formations were observed in infected with an open extremity. In this case, the space between nuclei. These objects are formed by 2 electron-dense the envelope and the capsid is reduced and the open 20 nm wide longitudinal bands separated by a central end is large (Fig. 11). However, we noted some par- 6 nm wide electron-clear band, all of which are found tially enveloped capsids. side by side with another such structure. The final 208 Dis Aquat Org 29: 205-21 1, 1997 -- -

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Figs. 9 to 15. Ultrathin section of ~nfectedstomach epithelium, TEM. Scdle bars are 150 nm. Fig. 9: Pro- files of envelopes. Fig,lO. Enve- lopes forming series of vesicles jolned together. Fig_ 11. Empty nucleocapsid partially enveloped. Thv envelope is open at one ex- tremity (arrow). Fig. 12. Nucleo- capsid precursors in longitudinal section (arrowheads) and in cross- section (arrows). Fig..- 13, Nucleo- capsid precursor are formed h)- units corresponding to the segments of mature particle. ..Fiq. .-- 14. Nucleocapsid in longitu- d~nalsection showing an electron- dense central tenuous structure. Fig. 15. Nucleocapsids in cross- sectlon with the central bands of different diameters. S,,: empty enveloped nucleocapsid; S:: tenu- ous filament, S,: dugmenldlion of the d~ameter;S,: max~rnal filling of the nucleocapsid

structure of this formation that appears tubular is con- diameter of the empty capstd. Moreover, the segmen- firmed by the observation of rings close to the tubules tation of the tubule seems to correspond to that of the about 60 nm in external diameter and 30 nm in inter- nucleocapsid. nal diameter, which correspond to cross-sections of Some enveloped capsids had a fine electron-dense, the empty tubules (Fig. 12). These tubules (nucleo- central, tenuous band with an appearance of a filament capsid precursors), in longitudinal section, show a (Fig. 14). In cross-section, these bands had different segmentation that corresponds to regularlv spaced diameters (Fig. 15).Other virions were observed in the units measuring 16 nm in width (Fig 13). Usually, we cytoplasm of hemocytes (Fig 16). Viral particles were observed 2 or 3 tubules placed side by side. Their either free in the cytoplasm or contained in vacuoles lengths are not constant and vary from 90 to 650 nm (singularly or in groups), where they were often asso- The diameter of the tubules is relatively close to the ciated with membranous structures. Durantiet al.. Ultrastructure and morphogenes~sof WSSV

Baculo-B and RV-CM (Johnson 1988), which have a polarized nucleocapsid due to a specialized apex, and B2 , (Mari 1987), which has a rounded end at one extremity and at the other a 2-layered cap. Ultrastructure of WSSV is similar to the non-occluded baculovirus of the Carcinus maenas (Bazin et al. 1974, Bonami 1980) and particu- larly to the baculovirus called B2 in Carcinus mediterraneus (Mari 1987). The latter viruses have in common with WSSV (1) the envelope exten- sion, (2) the development in the connective cells and hemocytes, and (3) the asymmetry of the extremities and the segmented appearance of the nucleocapsid. Spacing of the WSSV nucleocapsid segments is the same as that of B2 (20 to 22 nm) but greater than that of the baculo- virus of C, maenas (15 nm). The number of nucleocapsid segments varies from 14 in B2 (Mari 1987) to 16 in the baculovirus of C. maenas (Bonami 1980). The lack of appropriate cul- tures with which to investigate virion morphogenesis chronologically forces Fig.- 16. Ultrathin section of infected tissues, TEM. Bar is 1 pm. Phagocytosed virions in cytoplasmic vacuole of a hemocyte us to use observations of sectioned infected cells occurring at random and at an unknown infection stage. DISCUSSION In the same nucleus, the observation of structures corresponding to the different developmental stages The nucleocapsids with a different superficial leads us to interpret these as different stages of viro- appearance, with fractures in the segmentation, or a genesis. ring at the extremity, were presumably degraded by The finely fibrillar and granular material observed in osmotic shock. The pattern of degradation begins with the nucleoplasm seems to correspond to the forming distention of the nucleocapsid which reveals the regu- viral stroma. The linear and circular membranous larly spaced structural subunits. After the nucleocapsid structures present in the nucleoplasm appear to for is destroyed by separation of the segments, it then de novo because proliferation of nuclear membrane or appears as a stacked, ringed structure. Our findings points of contact between the intranuclear membranes confirm those of Wang et al. (1995), who reported that and those of nuclear envelope were not observed. The nucleocapsids were formed by a series of ringed struc- development of nucleocapsids in the nucleus appar- tures. We previously mentioned degraded ('swollen') ently begins by formation of long empty tubules capsids (Durand et al. 1996) but did not mention frac- that have a diameter relatively close to that of empty tured and separated rings. nucleocapsids and have a segmentation similar to that The nucleocapsids exhibit asymmetric extremities, of the nucleocapsid. The assembled tubules break into oneis rounded and the other squared. These structures shorter tubules to form empty naked capsids. This seem to be fragile because the capsids are usually pattern of formation could explain why mature virions observed squared at both extremities. This structural blend into ordered arrays. asymmetry not previously reported for WSS syndrome Some empty and partially enveloped capsids were viruses is a characteristic of the and usually noted. In contrast, full nucleocapsids were has been observed in the non-occluded baculoviruses never seen without an envelope. Hence, the empty 210 Dis Aquat Org

capsid is probably enveloped by membrane after for- association with membranes (Johnson 1988). In con- mation and before densification of the core. Some trast, the WSSV capsid is enveloped only after its images suggest that the newly developed envelope is formation, leading us to assume that development of well separated from the capsid but later becomes the nucleocapsid is not dependent on association with closely applied except for a large open extremity. The membrane nucleoprotein, which has a filamentous appearance, Although structure of the nucleocapsid and method enters the capsid through this open end. At the initial of capsid development are unlike those described for insertion, the filament is tenuous but, as the filling con- other non-occluded baculoviruses, the WSSV shares tinues, an increase of the diameter of the filament is many characteristics with these viruses including its apparent until the core formation is complete. The nuclear localization, its ultrastructure (shape, size, augmentation of the diameter between the empty cap- orientation), ~tsmorphogenesis and the absence of a sid and the full nucleocapsid could be due to a slight polyhedrin occlusion. distention of the nucleocapsid near its center due to the maximal filling of the core. The ultimate step before Acknowledgements. Partial funding of this work was pro- completion of the mature virions is the narrowing and vided by the Gulf Coast Research Laboratory Consortium the extension of the envelope as a sort of tail. Marine Program, CSREES, U S. Dept of Agriculture (grant no. 95-38808-1424), the National Sea The presence of virions in cytoplasmic vacuoles of Grant Program, U.S. Dept of Commerce, NOAA (grant no. hemocytes is probably due to phagocytosis by the NA56RG0617). hcmocytes. The hypothesis of intracytoplasmic multi- plication by the virus seems to be excluded because LITERATURE CITED WSSV replicates in the nucleus. Baz~nF, Monsarrat P, Bonarni JR, Croizier G, Meynadier G, Because WSSV morphogenesis is exclusively intra- Quiot JM. Vago C (1974) Particules virales de type baculo- nuclear, it is closer to that of the nuclear polyhedrosis virus observees chez le crabe Carcinus maenas. Rev Trav viruses than that of the granulosis viruses. As in the Inst P&chesMant 38:205-208 nuclear polyhedrosis viruses, the envelope is form.ed Bonami JR (1980) Recherches sur les infect~onsvirales des crustacee manns: etude des maladies a et~ologiesimple novoand development and replication of the virions de et complexe chez les decapodes des cotes franqaises. is intranuclear. These Doct Etat. Universite des Sciences et Techniques du However, WSSV morphogenesis presents some par- Languedoc. Montpelliar ticular characteristlcs: the WSSV capsid originates Durand S, Lightner DV, Nunan LM, Redman RM, Evlari J, from long tubules which give rise to naked empty Bonami JR (1996) Application of gene prohes as diagnos- tic tool for the White Spot Baculovirus (USI3V) of penaeid nucleocapsid precursors. These tubules show a seg- . Dis Aquat Org 2759-66 mentation which is similar in size to that of the nucleo- Francki RIB, Fauquet CM, Knudson DL, Brown F (1991) capsid and have not been reported previously. Pap- Classification and nomenclature of viruses. Archives of palardo & Bonami (1979) described tubules in the virology. Springer-Verlag, Vienna Gouranton J (1972) Development of an intranuclear non- morphogenesis of the Tau-virus which form nucleo- occluded rod-shaped vi.rus In some midgut cells of an capsids after densification. Further, Gouranton (19?2), adult insect. Gyr~nusnatator L (Coleoptera) J Ultrastruct who described morphogensesis of the Cyrinus natator Res 39:281-294 virus, found cylinders with a typical unit-membrane Huang J, Song XL, Yu J, Yang CH (1994) Baculoviral hypo- structure and spiny coat on the exterior. He hypothe- dermal and hematopoietic necrosis-pathology of the shrimp explosive epidermic d~sease.k'cllow Sea Fishery sized that the cylinder formed the nucleocapsid and Research Inst~tute,Qingdao, PR China (abstract) the envelope derived from the cyllnder wall. In these Huang J, Yu J, Song XL, Kong J, Yang CH (1995) Studies 2 cases, the tubules or cylinders do not have the seg- on fine structure, nucleic acid, polypept~deand serology mentation or the subunit organization of WSSV of hypodermal and hematopoietic necrosis baculovirus of penaeid shrimp. Mar Fish Res 16:ll-23 In thin sections, the empty capsids were usually com- lnouye K, Miwa S, Oseko N, Nakano H, Kimura T, Mon~o- pletely or partially enveloped and seldom completely yama K,Hiraoka M (1994) Mass mortalities of cultured na.ked. This is also true in the hemocyte-infecting crus- Kuruma shrimp Penacus japon1cu.s in Japan In 1993: tacean viruses (Bazin et al. 1974, Mari 1987, Johnson electron microscopic evidence of the causative virus. Fish 1988). Like WSSV, empty capsids of these viruses have Path01 29:149-158 Johnson PT (1988) Rod shaped nuclear viruses of crustaceans: been observed which are partially enveloped and with hemocyte-infecting species. Dis Aquat Org 5,11-122 an open extremity. They were called 'capsid or1gin.a- Lo CF, Leu JH, Ho CH, Chcn (:H, Peng SE. Chen YT, Chou tors' by Johnson (1988).Therefore, the morphogenesis CM, Yeh PY, Huang CJ, Chou HY, il'dng CH, Kou GH of WSSV suggests that it is related to the hemocyte- (1996) Detection of hilculovirus with whlte spot syndrome (\l-SBV)in penae~dshrimps using polymerase chain reac- infecting crustacean viruses described previously. tion. Dis Aquat Org 25:133-l41 However, the nucleocapsid development of the hemo- Man J (1987) Recherches sur 1es malad~esvirales du crustace cyte-infecting crustacean viruses always occurred in decapode m.arin Carcinus medjterraneus, Czeniaski 1884. Durand et al.: Ultrastructure and morphogenesis of WSSV

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Responsible Subject Editor: J. E. Stewart, Dartmouth, Manuscript first received: Decentber 5, 1996 Nova Scotia, Canada Revised version accepted: February 27, 1997