DISEASES OF AQUATIC ORGANISMS Vol. 29: 197-204,1997 Published June 19 Dis Aquat Org

Virus-like particles associated with cytopathology in the digestive gland epithelium of novaezelandiae and toheroa ventricosum

P. M. Hine*,B. Wesney

National Institute of Water and Atmospheric Research, PO Box 14-901, Kilbirnie, Wellington.

ABSTRACT: Apparent replication of small DNA-negative vlrus-like particles (VLPs) is described from digestive and secretory (= basiphil) cells of scallops , Reeve, 1853 and tohcroa Paphies ventricosunl (Gray, 1843) sampled during mass rnortalities, and compared w~thapparently healthy individuals. In digestive cells with putative VLPs, endocytotic and smooth membrane vesicles increased, endoplasmic reticulum (ER)proliferated, and VLPs 22 to 30 nrn across were seen in an orderly array on the surfaces of the outer nuclear membrane and along ER. Proliferating ER mem- branes, lined with VLPs and enclosing a dense matnx, were arranged in a reticulated configuratlon. The ER cisternae dilated to form vacuolar inclusions (VI) containing elongated bodles, spherical in sec- tion, in a flocculent matrix which were ornated with VLPs arrays on the external membrane. Enclosed bodies also formed by budding of cytoplasm into the VI. In scallop secretory cells VLPs replaced ribo- somes on ER,and ER cisternae dilated, but V1 seldom formed Toheroa diverticular epithelium showed similar changes, but secretory cells differed in that the outel- membrane of the nucleus and Golgi cisternae, rather than ER,proliferated. In addit~on,complete V1 were apparently not formed. The cyto- logical changes observed in both bivalves are similar to those associated with enteroviruses (Picorna- viridae) and caliciviruses. The possible role of VLPs In bivalve pathology is discussed.

KEYWORDS: Scallops . - Diverticular disease . Virus-like particles Picornaviruses . Caliciviruses

INTRODUCTION diverticular lesions, similar to those reported as part of the normal cycle of degeneration and renewal of diver- The New Zealand scallop Pecten novaezelandiae ticular epithelium reported by Henry et al. (1991) from dredge produces 3000 to 4000 t wet weight of Pecten maxirnus, and associated with algal blooms in scallops per annum (Bull 1991), but intermittent mor- Pecten alba in Australia (Parry et al. 1989). talities of up to 39% per annum have been reported The diverticular lesions in scallops and toheroa among wild stocks (Bull 1976). Sporadic population resemble those lesions in the moribund Perna crashes have made the fishery very difficult to man- canaliculus and Mytilus galloprovincialis that have age, and have resulted in requests to re-stock areas been associated with the presence of small RNA with scallops from other regions. Attempts at growing viruses in diverticular ep~thelial cells (Jones et al. scallops under culture conditions have also resulted in 1996). In view of the similarities in scallop and toheroa mass mortalities. Similarly, toheroa Paphies ventnco- epizootics and diverticular pathology to those associ- sum, a large collected recreationally, also experi- ated with RNA viruses in mussels, scallops and toheroa ence population crashes. Subsequent examination of from mortalities and from apparently healthy indi- moribund scallops and toheroa has only revealed viduals were examined by transmission electron micro- scopy for virus-like particles similar to those reported from mussels. The possible identity and role of these small RNA viruses is considered.

0 Inter-Research 1997 Resale of full article not permitted 198 Dis AquatOrg

MATERIALS AND METHODS At the TEM level, diverticular epithelium of healthy scallops comprised digesti.ve cells and secretory Between December 1990 and April 1993, 264 (= basiphil) cells. Digestive cells (DC), which were dredged wild scallops and 12 moribund scallops held most abundant on the lumina1 surface, had an irregular under culture conditions, 23 to 110 mm shell height, nucleus with a prominent nucleolus and sparse mar- were examined for disease. Whole body sections ginated heterochromatin. Coated endocytotic pits be- through the mantle, gills, gonad, and digestive organ tween the microvilli of the brush border appeared to were fixed in Davidson's fixative and stalned routinely form smooth-membraned endocytotic vesicles (EV) in with haematoxylin and eosin (H&E).Sections were the apical cytoplasm. Several Golgi profiles with elec- also stained with Feulgen-picro-methyl-blue for DNA tron-dense content in the cisternae, short strands of (Farley 1969), Oil Red 0 for lipids (Lynch et al. 1969), smooth endoplasmlc reticulum (ER), a few vacuoles Perl's stain for iron, Periodic Acid-Schiff (PAS) for and lipid droplets, residual bodies, secondary lyso- glycosaminoglycans, Schmorl's stain for lipofuchsin, somes and elongated mitochondria with a dense and were bleached for melanin using 0.25 % aqueous matrix were also present. potassium permanganate (Luna 1968). Secretory cells (SC) were more common near the basal For transmission electron microscopy (TEM),excised lamina, and had a nucleus similar to DC. However, the digestive tissues were fixed in 2.5 % glutaraldehyde In cytoplasm possessed long meandering strands of 0.22 pm filtered seawater (FSW) for 1 h, washed twice rough endoplasmic reticulum (rER), a few dense peri- ir, FSW, post-fixed in 1 % OsO, for 1 hi dehydrated in nuclear Golgi arrays, membrane-bound spherical ascending (50 to 100%) ethyl alcohol, embedded in secretory granules with a finely granular moderately Araldite, thick-sectioned and stained with 1 % tolui- dense homogenous content, and large numbers of dine blue in 1 % borax solution, or ultrathin-sectioned ribosomes giving the SC a dense appearance. and stained with 5 % uranyl acetate for 10 min and 5 % In light, posslbly early, lesions, the brush border of lead citrate for 5 to 6 min, and examined on a Phillps DC was ragged, DC protruded into the diverticular 420ST TEM. lumen and detached (Fig. 3) and disintegrated, leaving Toheroa Paphies vent~icosumfrom mortalities among necrotic cellular debris and clusters of residual bodies wild stocks in April 1991 (n = 6), January 1993 (n = 6) in the lumen (Fig. 4). Sometimes the DC disintegrated and November 1994 (n = 4), and healthy toheroa col- in situin the epithelium to leave a compartment with lected in April 1991 (n = 12) were flxed and prepared clustered residual bodies. Detachment exposed the for TEM as described above. underlying basement membrane (Fig. 4) and, as the Cell organelle terminology follo~vsHenry et al. (1991). tubule became progressively affected, remnant secre- tory cells became isolated and detached, until the whole tubule lacked an epithelium. RESULTS Also in apparently early lesions, DC Golgi became less apparent, the number and size of EV increased, a Scallops Pecten novaezelandiae few more smooth ER profiles were present, and the brush border became fragmented and disorganized. In No lesions were apparent macroscopica.l.ly, but under possibly later lesions, ER and arrayed virus-like par- the light microscope all scallops had lesions in the ticles (VLPs) proliferated. Many smooth-membraned epithelia1 cells of the distal sections of digestive diver- vesicles (SMVs) (Fig. 5), some with duplicated or ticulae. In H&E sections the shape of most diverticulae multiple membranes (Fig. 6), accumulated in the cyto- appeared normal, but much of the diverticular epithe- plasm. VLPs, usually 22 to 30 nm but <36 nm in dia- 1.iu.m resembled empty compartments containing yeI- meter, were arrayed along the cytoplasmic surface of lowish irregular material (Fig. 1) In the most affected the nuclear membrane and along ER (Fig 7), often in scallops the entire tu.bule was composed of these cell proximity to mltochondria (Fig. 8). VLPs were also 'ghosts' (Fig. l), or the compartments had broken down observed free in the cytoplasm, sometimes in cyto- exposing the underlying basement membrane (Fig. 2). plasmic projections from the surface (Figs. 9 & 10). Damage was extensive and severe in many of the scal- Dense flocculent material occurred in the cisternae of lops examined and larger scallops tended to possess ER lined with VLPs, and ER membranes appeared to a higher proportion of affected cells. Feulgen-picro- rearrange to form a reticulated structure containing a methyl blue failed to reveal inclusions that might sug- dense matrix (Fig. 11). Dilated matrix-filled cisternae gest a DNA-posltive aetiolog~calagent. Th.e yellowish appeared as vacuoles with a floccular content and with amorphous material was negative with other stains, VLPs arrayed on the cytoplasmic surface (Fig. 12). The except for moderate amounts of lipid and PAS-positive surrounding cytoplasm appeared to bud into the vacuole substance. to form spheres and elongated bodies of cytoplasm with Hine & Wesney: VLPs in scallops and toheroa

Figs. l to 4. Histological changes. S H&E section showing normal epithelium of the main gut, a normal digestive diverticulum (D)and affected diverticulae with empty compartments, some containing yellow material (arrows) 1x210). Section through partly affected tubule showing apparently normal digestive cells (DC) with a brush border, rounded DC and necrotic debris in the lumen, and bare basement membrane (arrow) where the epithelium has been lost (x1050).Flg.. Protrusion into the lumen (L) and sloughing by DC with prominent residual bodies (RB),vacuoles and secondary lysosoines (SL)(~6060). Fig. 4. Surface of a diverticulum devoid of epithelium, leaving a bare surface overlying the basal lamina, and clumped RB in the lumen (~3660)

VLPs arrayed along the inside surface to form vacuolar In SC the only changes observed were the replace- inclusions (VI) (Figs. 12 & 13). Mitochondria were ire- ment of 15 nm ribosomes on rER with VLPs, and dila- quently swollen, with detached cristae (Fig. 10). Mem- tion of ER cisternae containing flocculent material. brane-bound ovoid or pleomorphic bodies with a fine granular content were present in the cytoplasm (Fig. 7). Toheroa Paphies ventricosum Sloughed DC were rounded and possessed few fragmented microvilli. Their cytoplasm was densely The cytology of normal SC was similar to that of scal- packed with VIs, free VLPs, reticulated structures, sec- lops. DCs differed from scallops in the greater density ondary lysosomes, SMVs, lipid droplets, EVs, and of EVs, and the denser more granular appearance of clustered residual bodies (Fig. 14). secondary lysosomes. Dls Aquat Org 29 197-204, 1997 P

Flgs 5 to 10 Cytoplasm~cchdng~s in dlgcstiv~ccells. Fig 5 Cytoplasm of a sloutlhed d~gestlvecell IDC) tvith many cytoplasm~c vacuoles, a resldual body (RB) and lip~ddroplet 1/21 9701 Fig. 6. Detdl! of cytoplds1111c vdfuol~shnwirig t!uplicatlon of the boundlnq mpmbrdne (drrows) (xlO5 0001 Firj. 7. Endoplasm~cret~culum (ER) c~sternae w~th arrayed v~rus-l~kepart~cles (VLPs) (arrows) and a bodu contdlnlng d flne granular matrix [FC;) within the cytoplasm 1x64 800) Flg. 8. ER w~thVLP on thc surface nearest a m~tochondr~on(x82 000). Flg.~- 9.- F~nji~r-l~keproject~ons from the cc11 suriace enclos~nc[\.:LP5 (X 513 0801 FI~.10. Surface of adjolmng DCs showing cytoplasnilc prolectlons containing VLPs, and swoll~nm~tochon~lna (M) [x4i080) H~ne& Wesney: VLPs ~nscallops dnd toherod 201

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Figs. 11to 14 Retlculated structures and vacuolated inclusions In digestive cells (DC) FKJ. 11. Ret~culatedstructure (KS) wlth dense matnx in the cisternae, and smooth-membranes vesicles (SMVs) (arrows) (x54760)m0 Vacuolar lncluslon (VI),show- Ing sphencal bodies withln the vacuole containing flocculent materlal and virus-like parmw~~s)(x546801 F1 13 Cyto- plasm with VLPs (arrow) apparently buddlng Into a V1 lx64720).Fig 14. Necrotic digestive cell wlth a ~esidua body (RB) surrounded by a dense RS, Golgl (G),vacuolar lnclusion~ndswollen mitochondrla (M)(X 17 500)+

In apparently infected DC, VLPs 22 to 36 nm across In SC containing VLPs, the outer nuclear membrane were arrayed along the outer nuclear membrane and was sometimes detached and enlarged to form a dilated ER clsternae containing flocculent matenal, nuclear extrusion (Fig 161. Large SMVs were fre- particularly In the perinuclear region (Fig. 15) How- quently observed near Golgl clsternae, and may have ever, V1 were not observed. SMVs were less common derived from them A fine granular matrix occurred in In Intact cells than in scallops, but after lysis large the centre of mitochondria in some SC wlth cytoplas- numbers of ovoid membranes remained bearing VLPs mlc VLPs (Fig l?). Cisternae of rER denuded of ribo- 20 to 24 nm across, slmllar to those observed in somes and with VLPs arrayed along them were often scallops. dilated and contained flocculent materlal (Fig If) The Flgs. 15 to 17 Toheroa infections Fig 15 Edge of nucleus 1~1thvirus-llke particles (VLPs) along the nuclear membrane (arrows) and around dilated endoplasmic ret~culum(ER] cisternae containing flocculent material (~39370).Fig. 16. Nucleus of secretory cells (SC) with prollferatlon of the outer nuclear membrane to form a nuclear extrusion (NE) (X14 820). Pig. 17. Mitochondria in SC showing the central f~negranular matrix (*) (~21580) early stages of V1 formation were occasionally ob- P-glycogen by their affinity for membranes, and ribo- served. somes on the basis of size (22 to 36 nm). More impor- In both bivalve hosts, extracellular VLPs were not tantly, many of the structures reported here resemble observed and some sloughed DC showed none of the those involved in replication of RNA viruses. signs of putative viral infection reported above. The putative virogenic inclusions (VI, reticulated structures) reported here are unlike the organelles of eukaryotic cells, and the VLP arrayed along ER could DISCUSSION also be distin.guished from rER by the greater electron denslty of VLP Si.milar non-ribosomal arrays occurred Studies on viral pathogens of molluscs are lim~tedby along the nuclear membrane, unlike ribosomes in the lack of molluscan cell-lines in wh~chto culture the eukaryotic cells The large number of cytoplasmic putatlve vlruses, necessitating other approaches, such vesicles and vacuoles in sloughing and sloughed as the use of TEM TEM has its lim~tationsas replica- digestive cells were not observed in healthy cells. tion may not involve much visual cytopathology. In None of these structures, including VI, have been addition the process of replication may not only differ reported from normal epithelium of bivalve diverticu- between and w~thingroups, but also in vivo and in lae (Owen 1970, Pal 1971,1972,Henry 1984a, b, Henry vitro or in relation to virulence Finally, small viruses et a1 1991) or other molluscs (Nelson & Morton 1979). are structurally at the limits of resolution. The latter is The Picornavuidae comprises the enteric viruses true of this study, and the putative VLP are the same (polioviruses, enteroviruses, echoviruses, coxsackie- slze as P-glycogen (30 nm], only sllghtly larger than viruses), rhinoviruses, which are a cause of the ribosomes (15 nm) and may array along ER in a common cold, Cardiovirus, which causes encephalo- manner very similar to ribosomes along rER Despite myocarditis, and aphthoviruses which cause foot-and- this, the VLPs in this study cam be distinguished from mouth disease and similar diseases (Melnick 1983). At I-line & Wesney: VLPs in scallops and toheroa 203

22 to 30 nm they, and caliciviruses, are similar in size proliferation of the outer nuclear membrane to form a to the VLPs seen here, and some picornaviruses and nuclear extrusion (Fig. 16) is similar to nuclear mem- caliciviruses show similarities in replication to bivalve brane extrusions in calicivirus (Love & Sabine 1975), VLPs. and enterovirus (Rabin et al. 1964, Mattern & Daniel In cells infected with mengovirus (Amako & Dales 1965, Shahrabadi & Morgante 1977) infected cells. 1967) or poliovirus (Bienz et al. 1980, 1983, Hashimoto The VLPs may be mature virions that are shed in the et al. 1984) large numbers of smooth membrane vesi- spherical to elongated bodies in VI, or in surface cyto- cles develop in the central region of the infected cell, plasmic protr.usions, as in some enteroviruses (Dales et resembling the SMVs reported here. Polioviral protein al. 1965, Wroblewska et al. 1977, Yilma & Breese 1980, (Bienz et al. 1980, 1983) or VLPs (Hashimoto et al. Tucker et al. 1993) and aphthoviruses (Yilma et al. 1984) accumulate around these vesicles, which are 1978, Wool et al. 1982, Polatnick & Wool 1983). part of a poliovirus replication complex (Bienz et al. Picornaviruses differ from bivalve VLPs in usually 1987, 1992) in which viral RNA is synthesized (Bienz et forming crystalline aggregates or matrices in infected al. 1980, 1987). In poliovirus infected cells, a smooth cells (Dales et al. 1965, Yilma & Breese 1980, membranous tubular network develops that encloses Rodriguez et al. 1983).The lack of crystalline arrays in fingers of cytoplasm in which viruses subsequently the bivalves studied here may indicate lack of virion develop, resembling V1 in this study (Dales et al. 1965). maturation, or be related to the cell type infected as In poliovirus (Mattern & Daniel 1965) and coxsackie- poliovirus forms crystalline arrays in vitro, but not in virus B5 (Shahrabadi & Morgante 1977) infected cells, vivo (Hashimoto et al. 1984). Coxsackievirus infections similar configurations result from proliferation of the may also differ in the involvement of the host cell outer lamina of the nuclear membrane to form multi- nucleus in replication in some species (Rabin et al. layer membranous structures containing finger-like 1964, Jezequel & Steiner 1966). Caliciviruses may projections of cytoplasm. The subsequent accumula- differ in their association with fibrils (Love & Sabine tion of coxsackie B5 viruses along the outer membrane 1975, Studdert & O'Shea 1975), which was not ob- and development of viruses within the membrane- served here. bound vesicles results in configurations indistinguish- Despite these differences, a viral aetiology is sug- able from scallop VI (Shahrabadi & Morgante 1977; gested because the VI, RS, ovoid membrane-bound Figs. 2a & 3 therein). Duplication of SMV membranes, bodies with fine granular matrix and reduplicated similar to those in Fig 6, also occurs in mengovirus SMV membranes are not known from the digestive infection (Amako & Dales 1967). Swelling of mitochon- epithelium of scallops (Henry et al. (1991),but do occur dria has been reported in calicivirus (Studdert & during replication of several picornaviruses and some O'Shea 1975), poliovirus (Hashimoto et al. 1984), and caliciviruses. coxsackievirus (Rabin et al. 1964) infections, but, like It would be premature to conclude that cellular alter- the increase in EV, is likely to be related to cell death ations associated with the VLPs are the cause of scallop rather than the presence of an infectious agent. The or toheroa mortalities, despite the common observation SMVs may have developed from modified EVs, but of putative virogenic structures among many slough- this was not observed. ing and necrotic cells. I11 affected bivalves, the slough- Enterovirus virions may be arrayed along cisternae ing of DC lacking VLPs may result from loss of struc- to give the superficial appearance of rER (Yilma & tural integrity following loss of infected cells. However, Breese 1980), and several studies show that entero- diverticular epithelia1 cell renewal appears to be a nor- viruses may be released into spherical to ovoid or mal process in molluscs (Nelson & Morton 1979, Henry elongated structures, as seen in V1 (Dales et al. 1965, et al. 1991).VLPs may be irrelevant to degeneration, or Wroblewska et al. 1977, Yilrna & Breese 1980, Tucker they may alter the klnetics of degeneration and renewal, et al. 1993). In particular, the surface cytoplasmic pro- leading to disease. Further studies are needed to iden- jections containing VLPs (Figs. 9 & 10) resemble those tify the VLPs, to fulfil Koch's postulates and show the caused by aphthoviruses (Yilma et al. 1978, Polatnick VLPs cause diverticular disease, and show such dis- & Wool 1983), which may be formed by budding into ease is the cause of the large scale mortalities. vacuoles (Wool et al. 1982) in a manner identical to budding into VIs. During calicivirus infections, Golgi Acknowledgements. Th~sresearch was funded by the New cisternae may form fine granular dense bodies (Love & Zealand Mlnistry of through project FDI?01. Sabine 1975) similar to those observed here (Fig. 7) and in coxsackievirus infections (Jezequel & Steiner LITERATURE CITED 1966). In coxsackievirus infections, Golgi cisternae Amako K. Dales S (1967) Cytopathology of mengovirus in- proliferate to form membranous vesicles (Jezequel & fection 11.Proliferation of membranous cisternae. Virology Steiner 1966) resembling those In toheroa SC. The 32:201-215 204 Dis Aquat Org 29: 197-204, 1997

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Responsible Subject Editor. A. K. Sparks, Seattle, Manuscript first received: October 22, 1996 ~vddshlfl~t~fl,L'SA Rewsed version accepted. March 13, 1997