Viral diseases in commercially exploited crabs: A review

Journal of Invertebrate Pathology 106 (2011) 6–17

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Viral diseases in commercially exploited crabs: A review Jean-Robert Bonami a,⇑, Shuyong Zhang b a b

Pathogens and Environment, UMR 5119 EcoLag cc 093, CNRS/UM2, Place E.Bataillon, 34095 Montpellier Cedex 5, France Key Laboratory of Molecular Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, PR China

a r t i c l e

i n f o

Keywords: Marine crabs Viral diseases Reoviridae Genome structure Bunyaviridae Roniviridae Envelopped bacilliform nuclear viruses Tau virus WSSV Nimaviridae

a b s t r a c t Viruses and viral diseases of crabs were observed and investigated earlier than the first observation of viruses in shrimp. In fact, crabs were used as biological models to investigate crustacean virology at the beginning of shrimp aquaculture development. More than 30 viruses have been reported in crabs, including those related to the known virus families Reoviridae, Bunyaviridae, Roniviridae and a group of Bacilliform enveloped nuclear viruses. This review reports data on several important viral diseases of crabs, particularly those associated with pathology of organs and tissues of commercially and ecologically significant host species. Ó 2010 Published by Elsevier Inc.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viruses related to Reoviridae . . . . . . . . . . . . . . . . . . . Geographic distribution and hosts . . . . . . . . . . . . . . . Tissue tropism and lesions . . . . . . . . . . . . . . . . . . . . . Biochemical characteristics and genomic structure . . Viral genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prevalence and commercial impact . . . . . . . . . . . . . . Viruses related to Bunyaviridae . . . . . . . . . . . . . . . . . Viruses related to Roniviridae . . . . . . . . . . . . . . . . . . . Enveloped bacilliform nuclear viruses . . . . . . . . . . . . Enveloped bacilliform nuclear viruses with digestive Systemic enveloped bacilliform nuclear viruses. . . . . Prevalence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Crabs and WSSV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Among crustacean viruses, those of crabs are the oldest reported in decapods. The first crustacean virus was reported by Vago in 1966 in the Mediterranean crab Portunus depurator. Since this date, numerous descriptions of virus diseases were made in different crab species particularly in the 1970s and the first half

⇑ Corresponding author. E-mail address: [email protected] (J.-R. Bonami). 0022-2011/$ - see front matter Ó 2010 Published by Elsevier Inc. doi:10.1016/j.jip.2010.09.009

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of the 1980s. Associated or not with mortalities, these descriptions were based essentially on morphological data when reported biochemical properties were scarce. To date, the listed viruses in crabs are at least as numerous as shrimp/prawn viruses and are closely related to known virus families (Table 1). Of course, as only few of them were characterized at the biochemical level, most were related to virus families only on the basis of their morphological features, i.e. structure and size of the viral particle, cytoplasmic or nuclear location and tissue affinity of the virions. On the other hand, shrimp viruses were genetically characterized and consequently detection methods were well

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J.-R. Bonami, S. Zhang / Journal of Invertebrate Pathology 106 (2011) 6–17 Table 1 Viruses of crabs.**.

dsRNA

ssRNA

Virus family

Hosts

Virus names

References

Reoviridae

Macropipus depurator

P virus

Carcinus maenas C. mediterraneus Callinectes sapidus Eriocheir sinensis Scylla serrata

W virus W2, RC84 RLV EsRV905 EsRV806 MCRV IPN-like virus

Vago, 1966; Bonami, 1973, 1980; Montanié et al., 1993a; Bonami and Comps, 1976; Mari and Bonami, 1986, 1987, 1988a,b. Johnson, 1977, 1983 Zhang, 2006 Zhang, 2006; Zhang et al., 2002 Weng et al., 2007; Zhang et al., 2007

Birnaviridae

M. depurator

Bunyaviridae

M. depurator C. mediterraneus C. maenas Cancer pagurus E. sinensis C. sapidus Hemigrapsus oregonensis E. sinensis M. depurator E. sinensis C. mediterraneus C. sapidus

C. maenas

RhLV EGV-1 EGV-2 EHV Y organ virus

Iridoviridae

C. sapidus C. mediterraneus C. maenas Paralithodes platypus Scylla serrata C. sapidus Rhithropanopaeus harrisii Paralithodes platypus M. depurator

Baculo A and B s virus, B2 RV-CM, B Baculo PP SsBV HLV RhHLV PpHLV MdIV

Johnson, 1976a, 1977, 1978 Pappalardo et al., 1986; Mari, 1987 Johnson and Lightner, 1988; Bazin et al., 1974 Johnson and Lightner, 1988 Anderson and Prior, 1992 Johnson, 1976b, 1978, 1984 Payen and Bonami, 1979 Sparks and Morado, 1986 Montanié et al., 1993b

Parvoviridae

C. mediterraneus

PC84

Mari and Bonami, 1988a

Roniviridae Rhabdoviridae

dsDNA

Enveloped bacilliform viruses

Herpesviridae

ssDNA

Clotilde, 1984 Bonami et al., 1975; Bonami, 1980 Mari, 1987 Bang, 1971, 1974, 1983; Hoover and Bang, 1978 Corbel et al., 2003 Zhang, 2006 Johnson, 1978 Kuris, 1979 Lu et al., 1999 Bonami, 1980 Zhang and Bonami, 2007 Mari, 1987 Jahromi, 1977 Yudin and Clark, 1978, 1979 Johnson and Farley, 1980 Chassard-Bouchaud et al., 1976

Picornaviridae

S virus CHV CpSBV EsBV CBV – – F–N virus EsRNV

Table 2 Some properties of crab reoviruses. Virus

Host

Tissue tropism

Cytoplasmic lesions

ds RNA segments

Electro-phore type

sequence

P RLV W W2 RC84 EsRV905 EsRV806 MCRV

M. depurator C. sapidus C. maenas C. mediterraneus C. mediterraneus E. sinensis E. sinensis Scylla serrata

Systemic Systemic Systemic Systemic Digestive epithelium Systemic Systemic Systemic

Crystals Crystals Rosettes Rosettes nsa nsa nsa Some crystals

12 – 12 12 – 12 10 12

1-5-6 – 1-5-6 1-5-6 – 3-4-5 1-5-4 1-5-6

Partial sequence of three segments – – – – Full sequence of RNA-1: 3722 nt – Partial sequence of RdRp gene

‘‘nsa”: No specific association; (–): no data.

Fig. 1. P virus infection in the crab Macropipus depurator. Lesions in connective cells of the hepatopancreas. (a) Cytoplasmic crystals of P virus (ds RNA) fluoresce in orange after acridine orange staining. A cytoplasmic yellow fluorescence is caused by a simultaneous presence of S virus (ssRNA), a Bunya-like virus (Bar = 50 lm); (b) semi-thin section in same tissue. Note the presence of numerous dark crystals in cytoplasm of infected cells. Toluidine blue (Bar = 50 lm).

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developed due to economical pressure of shrimp farming. As crabs represent a low economical value compared to shrimp and as they are most often caught in natural environment and not farmed, scientific efforts were less important as underlined by a slow scientific progress in viral pathology. Moreover, if quite all the crabs are edible crabs, they do not have all the same commercial value, as for example the blue crab Callinectes sapidus, the king crab Paralithodes platypus and the shore crab Carcinus sp. or the swimming crab Macropipus depurator. Moreover, the commercial value is largely dependant on the country: for example, the Chinese mitten crab Eriocheir sinensis has no commercial value in western countries compared to Asian countries, particularly in China, where it is farmed and sold in local markets. We report here only important virus families or group of viruses of interest in the field or viral diseases and viruses of crabs with a real commercial impact. 2. Viruses related to Reoviridae Viruses related to the Reoviridae family include the first virus described in a crustacean (Vago, 1966) some other uncharacterized viruses that share common morphological and biological features of the family and some other viruses that were sufficiently well characterized to be considered as members of the family (Hukuhara and Bonami, 1991). This family is the most representative among crab viruses by the number of different viruses described, by the knowledge we have on their structure, morphogenesis and genomic data. Moreover, unusual and peculiar features make interesting some of these viruses at the level of comparative virology. Except for the virus reported by Vago (1966), of which only the shape and size are known, all other viruses (Table 2) exhibit their common ability to develop in cytoplasm of infected cells and to have in common an icosahedral shape associated with a double shelled capsid and a closely related size (60–80 nm in diameter). All exhibit a multi-segmented genome (10–12 pieces) of dsRNA.

cinus maenas) in Carcinus mediterraneus (Mari and Bonami, 1986, 1987, 1988a,b; Mari, 1987), all these three species were caught in the French Mediterranean coast near Sète. W virus was noted in C. maenas caught in front of the Weymouth laboratory (MAFF, Dorset) of the English Channel (Bonami & Hill, unpublished data). RLV (for Reo-like virus) was isolated from the blue crab C. sapidus from the Chincoteague Bay, VA, USA (Johnson and Bodammer, 1975; Johnson, 1977, 1983, 1984). EsRV905 and EsRV806 (for E. sinensis reovirus 905 and 806, respectively) were isolated in the PR China from the Chinese mitten crab E. sinensis, in Hubei and Jiansu provinces, respectively (Zhang, 2006; Zhang et al., 2002, 2004). MCRV (for mud crab reovirus) was found associated with mortalities in the mud crab Scylla serrata in Guandong and Fujian provinces in southern China (Weng et al., 2007). In the same moment Zhang et al. (2007) reported a similar virus from same area named SsRV (S. serrata Reovirus). Except for RC84 (Mari and Bonami, 1988a), diseases were experimentally reproduced by injection or per os contamination in their original host, including virus development and associated

3. Geographic distribution and hosts As indicated in Table 1, eight different reovirus or reo-like particles have been reported in crabs. P virus (from Paralysis virus) was first observed in M. depurator (Bonami, 1973, 1980; Bonami and Comps, 1976), RC84 (for Reovirus of crabs, isolate 1984) and W2 (name derived from W virus, found in Weymouth, but in Car-

Fig. 3. W2 virus infection in connective cells of the crab Carcinus mediterraneus. The cytoplasm exhibit typical rosette formations (arrow) (Bar = 300 nm). Inset: detail of a rosette in section formed with virus particles. TEM (Bar = 50 nm).

Fig. 2. P virus infection in connective cell of the crab Macropipus depurator. The arrangement of virions in the cytoplasm in regular parallel rows exhibited a crystallike appearance of the lesion. TEM (Bar = 100 nm).

Fig. 4. Purified W2 virus. Note presence of empty-like particles. Inset: higher magnification of virions; superficial sub-units are evidenced. 2% PTA (Bar = 50 nm).

J.-R. Bonami, S. Zhang / Journal of Invertebrate Pathology 106 (2011) 6–17

mortalities. Attempting cross infections, a strict specificity for their own host was demonstrated for P and W2 viruses; i.e. P virus was unable to develop in Carcinus sp. and W2 in M. depurator (Bonami, 1980; Mari, 1987). 4. Tissue tropism and lesions RC84 exhibits a tissue tropism different from all other viruses of this group (Mari and Bonami, 1988a). It is strictly located at the level of the hepatopancreatic epithelium, particularly in cytoplasm of B-cells where it forms large and electron-dense virus areas with

Table 3 Genomic structure of the segmented genome of some crab reoviruses. Values of the full length genomes are indicated as well as their electrophoretic pattern. ds RNA segments

P virus

W–W2

1

3.9

1

3.8

1

3722 nt

2 3 4 5 6

2.8 2.6 2.3 2.1 2.0

5

2.8 2.7 2.4 2.3 2.3

5

3.2 2.8 1.9 1.6 1.6

7 8 9 10 11 12 13

1.45 1.2 1.15 1.1 1.08 1.04

6

1.7 1.5 1.25 1.12 1.1 1.08

6

Total

23 kbp

24 kbp

EsRV905

1.4 1.2 1.1 0.95 0.9 0.75 23 kbp

EsRV806

MCRV/SsRVa

3

4.1

1

4.69–3.8

1

5

2.85–2.5 2.85–2.4 2.53–2.0 2.53–1.9 2.29–1.8

5

4

3.8 3.5 3.4 2.7 2.0 1.8 1.7 1.5 1.25

4

1.57–1.3 1.35–1.2 1.29–1.1 1.29–1.1 1.29–1.0 1.19–1.0 1.19

6–7

5

23 kbp

9

generally no specific association of particles. However, few crystalline arrays of virions can be observed. All the other viruses are systemic, carried in the hemolymph and develop most often in connective cells of all the organs. P virus and RLV form in cytoplasm large crystalline arrays of virions easily to evidence by UV microscopy (Figs. 1 and 2) with acridine orange staining (Bonami, 1973, 1980; Johnson and Bodammer, 1975; Johnson, 1983). W and W2 form unusual cytoplasmic structures called ‘‘rosettes” (Mari, 1987; Mari and Bonami, 1988b; Montanié, 1992; Montanié et al., 1993a). The rosettes are formed by 5–7 particles in section (Fig. 3). In volume they are organized in small spheres of arranged virions, delimiting a central empty volume. EsRV905 and EsRV806 do not exhibit any particular association of virions. 5. Biochemical characteristics and genomic structure For most viruses in this group, the double layered capsid has been demonstrated. It is particularly well evidenced by TEM in purified W2 virions (Fig. 4). Data on protein composition of the capsid are scarce and available only for P and W2 virions (Bonami, 1980; Mari, 1987; Montanié, 1992). P virus exhibits four polypeptides while W2 contains 6, all ranging from 24 to 120 kDa. Concerning the other crab reoviruses, data on the structure and biochemical composition of the capsid are lacking.

26.9–21.1 kbp

a

Data for the same virus named MCRV or SsRV with different dsRNA size reported; the largest values for a total of 26.9 kbp and 13 segments (Weng et al., 2007), the smallest for a total of 21.1 kbp and 12 segments (Zhang et al., 2007), respectively.

Fig. 5. Electrophoretic mobility of P and W2 virus in 10% poly-acrylamide gel electro-phoresis. RNA pieces were numbered. Markers are Reo3 and the Bombyx mori Cypovirus (CPV1). Ethidium bromide.

Fig. 6. Comparison of electrophoretic mobility of W2 and EsRV905 RNA genomes on a 10% poly-acrylamide gel. Left: W2; right (905): EsRV905. The gel was silver stained. The 12 double-stranded RNA bands of EsRV905 are well evidenced. Size of W2 segments expressed in kbp is indicated at left. The values were calculated from previously published data for RNA segments (Montanié et al., 1993b).

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As for regular members of the Reoviridae family (Fauquet et al., 2005) the genome was demonstrated to be a segmented dsRNA, at least for five of the crab viruses (see Table 3). P virus, W-W2 and EsRV905 possess 12 genomic segments with size ranging from 0.75 to 3.9 kbp (Mari, 1987; Mari and Bonami, 1988b; Montanié, 1992), while EsRV806 (Zhang, 2006) and MCRV (Weng et al., 2007) have 10 and 13 segments, respectively. The size of the W2 genomic segments estimated by gel electrophoresis (Fig. 5) was correlated with the length of the different dsRNA pieces as measured by TEM after shadowing (Montanié et al., 1993b). This accurate size constitutes a reference marker for measurement of other genomic dsRNA pieces (Fig. 6). Except for MCRV genome which is estimated to weight 26.9 kbp according to Weng et al. (2007), the four other have a genome of an estimated size of 23–24 kbp. But, as reported by Zhang et al. (2007), SsRV (=MCRV) possesses a 12-segmented genome – which is more in agreement with Reovirus known data – with a total estimated size of 21.1 kbp.

obtained were used, after labelling, in investigations by in situ hybridization (ISH) (Montanié, 1992; Walton et al., 1999). Among the genomes of the different reoviruses listed in Table 3, only the first RNA piece (RNA-1) of EsRV905 from E. sinensis has been to date fully sequenced (Zhang et al., 2004; Zhang, 2006). Its length is 3722 nucleotides exhibiting a single long ORF (23– 3676 nt) coding for a putative RdRp (RNA-dependant RNA-polymerase). The deduced polypeptide from the ORF is formed of 1217 amino acids with a calculated mass of 139 kDa. Sequence homologies were found with members of the genus seadornavirus particularly with Kadipiro and Banna virus (Zhang, 2006). Phylogenetic analysis based on sequence alignment of RdRp from members of different genera in the Reoviridae family exhibit close relationships with viruses of the genus seadornavirus and some rotavirus (Zhang, 2006) (Fig. 7). But differences in hosts (insects and crabs), electrophoretypes and genome size indicate most probably that EsRV905 could constitute the model for a new genus in the family.

6. Viral genes 7. Prevalence and commercial impact Very little is known about genes in this group of virus, as their location in the different RNA pieces as well as their structure and function. In terms of genomic sequences, partial sequences of three different segments are to date available from P virus. They were obtained by cloning genomic RNA pieces and the different cDNA

Impact and prevalence of RLV in C. sapidus from its main fishery on the US East coast (especially Cheasapeake and Chincoteague Bays) are not well documented. In fact, percentage loss has not been estimated for the diseases (or viral diseases) of the blue crab.

Fig. 7. Phylogenetic comparison between EsRV905 viral polymerase and members of other genera within the Reoviridae family. Analysis (presented as a radial tree) was performed with TreeView. Accession numbers and further details of the sequences and viruses used are: 1. Seadornavirus (12 segments), species Liao ning virus, Banna virus (isolate BAV-In6423; AF133430) and Kadipiro virus (isolate KDV-Ja7075; AF133429); 2. Coltivirus (12 segments), species Colorado tick fever virus (isolate CTFVFl; AF134529); 3. Orthoreovirus (10 segments), species Mammalian orthoreovirus serotypes 1 (MRV-1; M24734), 2 (MRV-2; M31057) and 3 (MRV-3; M31058); 4. Aquareovirus (11 segments), species GCHV (isolate 873;); 5. Orbivirus (10 segments), species African horse sickness virus serotype 9 (AHSV-9; U94887), Bluetongue virus serotypes 10 (BTV-10; X12819); 6. Rotavirus (11 segments), species Rotavirus A (RV-A), Rotavirus B strain Hu/MuRV-B/IDIR (M97203) and Rotavirus C strain PoRV-C/Co (M74216); 7. Fijivirus (10 segments), species Nilaparvata lugens reovirus strain NLRV-Iz (D49693); 8. Phytoreovirus (12 segments), species Rice dwarf virus isolates RDV-Ch (U73201); 9. Oryzavirus (10 segments), species Rice ragged stunt virus strain RRSV-Th (U66714); 10. Cypovirus (10 segments), species Bombyx mori cytoplasmic polyhedrosis virus-1; 11. strain Bm-1 CPV (AF323781), species Bombyx mori cytoplasmic polyhedrosis virus-14 strain Bm-14 CPV (AF323781); 12. Mycoreovirus (12 segments), species Mycoreovirus 1/Cp9B21 (AAP45577).

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Equally, data on the impact of EsRV905 that infects the Chinese Mitten crab are scarce. During the period between 2000 and 2007, EsRV905 was detected in the main crab culture areas in China, including Jiangsu, Shanghai, Anhui and Hubei provinces (Zhang, unpublished data). The virus can be detected in all the seasons, but the infection rate is much higher in summer. EsRV905 is often found associated with other pathogens in wild crabs. Experimental infection of pre-purified EsRV905 produced only 30% cumulative mortality in E. sinensis, but the agent can be detected in surviving crabs.

8. Viruses related to Bunyaviridae Bunyavirus-like particles were reported in some of the early literature in the crabs C. maenas, C. mediterraneus and M. depurator (Bang, 1971, 1974, 1983; Hoover and Bang, 1978; Bonami and Vago, 1971; Bonami et al., 1971, 1975; Bonami, 1980) (see Table 1). More recently Bunyavirus-like particles were evidenced in two commercially important crabs: the sleeping crab, Cancer pagurus (Corbel et al., 2003) and the Chinese mitten crab E. sinensis (Zhang, 2006). A Bunya-like virus reported in C. pagurus (CpBV) was found by chance during investigations on the possible hosts of WSSV from penaeid shrimp (Corbel et al., 2001). CpBV was found associated with WSSV in co-developing infections, it was later observed in naturally infected crabs caught in Brittany, France (Fig. 8). The second virus (EsBV) was reported in E. chinensis, and this occurrence was associated with mortalities. CpBV and EsBV exhibited a systemic location, i.e. infecting hemocytes and connective cells of all the organs. They developed in cytoplasmic Golgi-like vesicles where they were accumulated until they were released in circulating hemolymph by the opening of virus containing vesicles at the membrane surface. Virions were ovoid in shape, enveloped, about 100 nm in length and 70 nm in diameter (Fig. 8). Spikes were located on the envelope surface. A tail-like structure 70–110 nm length and 25–35 nm in diameter was reported extruding from particles (Corbel et al., 2003; Zhang, 2006). Few data on the physico-chemical properties of these

Fig. 8. Haemolymph of a moribund crab Cancer pagurus from Brittany infected with CpBV. Note the numerous free enveloped viral particles. 2% PTA. Inset: higher magnification of CpBV in negative staining. Virions exhibit a small tail-like structure. 2% PTA (bar = 100 nm).

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viruses (number, structure and size of nucleocapsids or polypeptide composition) are available. Only the RNA nature of their multi-segmented genome is known: three pieces of ssRNA as demonstrated by gel electrophoresis and determined to be 6.80 kb, 3.60 kb and 1.60 kb in size (Corbel et al., 2003) for CpBV (Fig. 9) and 7, 3.7 and 1.7 kb for EsBV (Zhang, 2006). The Bunya-like virus of the Chinese mitten crab E. sinensis was first observed and isolated in 2000 (Zhang, unpublished data) in diseased crabs with tremor syndrome in Jiangsu province of PR China, and it was isolated again in the nearby ponds in the same region in May 2005 (Zhang, 2006). In crabs from a diseased pond, prevalence of tremor syndrome was about 90% and the cumulative mortality of E. sinensis was about 70%. The mortality rapidly diminished when the water temperature was lower than 20 °C.

Fig. 9. CpBV. Agarose gel electrophoresis (1%) of viral nucleic acid. 3 segments are observed: large (L) about 6.80 kb, medium (M) of about 3.60 kb and small (S) of about 1.60 kb. M: ssRNA markers (9.46, 7.46, 4.40, 2.37, 1.35 and 0.24 kb).

Fig. 10. Section of gills in the Chinese mitten crab Eriocheir sinensis suffering of ‘‘sigh disease”. Infected cytoplasms fluoresce in orange-yellow. Acridine orange staining (Bar = 50 lm).

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Fig. 12. Sections of EsRNV infected cells: (a) presence of filamentous nucleocapsids and enveloped particles. TEM (Bar = 100 nm); (b) arrangement of nucleocapsids in cytoplasmic vesicles. TEM (Bar = 200 nm).

9. Viruses related to Roniviridae A crab disease, called ‘‘sigh disease” by the farmers because of the apparent ‘‘respiratory” troubles that are clearly audible at night and associated with black gill syndrome (BGS) was recently reported in the Chinese Mitten crab E. sinensis in a farm of Hubei province in PR China. Anorexia and sluggishness were non-specific associated symptoms. The disease was reproduced experimentally by inoculation of infected hemolymph into healthy crabs. A virus morphologically similar to members of the Roniviridae and called EsRNV (E. sinensis Ronivirus) was reported in experimentally infected animals (Zhang, 2006; Zhang and Bonami, 2007). Signs of infection by EsRNV were noted in numerous organs: lymphoid organ, connective tissue of gills, hepatopancreas, heart and gut. In gills, numerous deposits of debris (fouling) were located

Fig. 11. Enveloped EsRNV particles. PTA 2%.(Bar = 40 nm).

between the different gill lamellae. The infection corresponded to a multifocal to generalized necrosis, with nuclei of connective cells showing pyknosis and karyorrhexis and forming inclusion-like structures. When observed with UV microscopy after acridine orange staining, prominent masses exhibiting a strong yellow-orange fluorescence were easily evidenced (Fig. 10). Compared to the green fluorescence of normal nuclei, the yellow-orange fluorescence indicated the presence of a large amount of ssRNA in cytoplasmic inclusions (Zhang, 2006). In the supernatant prepared from homogenized infected tissues and observed by TEM after negative staining (PTA), EsRNV viral particles were 60–170 nm long and 25–45 nm in diameter. Virions were enveloped but nucleocapsids were difficult to observe. The envelope was covered with organized surface spikes (Fig. 11). In sections (Zhang, 2006; Zhang and Bonami, 2007), the cytoplasm of EsRNV infected connective cells contained rod-shaped, enveloped viral particles and non-enveloped filamentous forms (Fig. 12). Nucleocapsids were 150–250 nm long and 16–18 nm in

Fig. 13. Hepatopancreas of the crab Carcinus mediterraneus infected with s virus. Numerous nuclei of the digestive epithelium of the tubule were hypertrophied and exhibited chromatin margination. Some nuclei were in the process of release in the lumen of the tubule. Mann-Dominici staining (Bar = 75 lm).

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diameter; some reached 400 nm in length; some accumulated in vesicles forming para-crystalline arrays. When enveloped, virions were 60–110 nm in length and 24–42 nm in diameter with an envelope of 8 nm in thickness. Mature EsRNV virions were found dispersed in cytoplasm or packaged in cytoplasmic inclusions of 200–800 nm in size and corresponding to the inclusions noted in light microscopy. Non-mature particles were found associated with the endoplasmic reticulum; similar observations were reported for GAV (Gill associated virus), a Ronivirus from penaeid shrimp (Spann et al., 1997). Mitochondria, in close vicinity to the virions, appeared to have lost their internal organization. In the

gills, numerous tiny particles, 8–10 nm in diameter accumulated in perinuclear area forming para-crystalline rows. Due to the yellow-orange fluorescence in UV microscopy after acridine orange staining, the viral genome of EsRNV was thought to be composed of ssRNA. After extraction from infected tissues and agarose gel electrophoresis, its size was determined as slightly longer than 22 kb, and its ssRNA nature was confirmed by RNase digestion (Zhang, 2006). Although the size of EsRNV was very close to that of YHV and GAV, it appeared a little smaller, particularly in length. The cytopathology and pathogenicity were similar to that of YHV and GAV, particularly the formation of basophilic inclusions in the lymphoid organ and gut of shrimp, and by its ability to cause apoptosis in these two organs. Its genome is obviously RNA, confirmed by TRIZol extraction and RNase sensitivity. It could be interpreted as ssRNA due to its fluorescence in orange after orange acridine staining in UV microscopy. Its genome size is in agreement with values obtained for GAV and YHV (Zhang, 2006; Zhang and Bonami, 2007). Although small differences in size, another crab virus previously reported as a rhabdovirus, the RhVA (or EGV2) of C. sapidus (Yudin and Clark, 1978, 1979) could be considered by its shape and general structure as a Ronivirus (Spann et al., 1997). Experimentally, the mortalities of E. sinensis reached 100% (28 crabs in two tanks) after 17 days p.i. with EsRNV, and crabs began to die 13 days p.i. To date, it has not been demonstrated that EsRNV is the etiological agent of BGS (black gill syndrome), but the virus is undoubtedly very common in cultured crabs in inland China. In August 2002, the disease caused losses of about 3,000,000 euros to local economy with 28,000 hectares affected in Hubei province, PR China (Zhang, unpublished data).

10. Enveloped bacilliform nuclear viruses

Fig. 14. s virus infected nucleus released in the lumen of hepatopancreatic tubule. Nucleus remains were filled up with virus particles. TEM (Bar = 500 nm).

According to the number of representatives, the enveloped bacilliform nuclear viruses constitutes the largest virus group in crabs. More than a decade ago, they had been considered as

Table 4 Enveloped bacilliform nuclear viruses of crabs: main properties. HP: hepatopancreas; NC: nucleocapsids; Env: envelope; nd: no data available. Sizes are expressed in nm. Virus

Host

Tau (s)

Carcinus HP midgut 68  310 mediterraneus

65–70  330– NC/Env 345 Budding (?)

Subapical

Baculo-A

Callinectes sapidus

HP

43  240– 254

60  60–300

Subapical

Baculo-PP

Paralithodes platypus Scylla serrata

HP

70  30–265

HP

44  253

37–40  190– NC/Env 210 24  205 NC/Env budding

SsBV

Baculo-B

B virus

B2 virus

RV-CM WSSV

a

Callinectes sapidus

Target

Virus size section

Hemocytic 85– 100  370– 390 Carcinus Hemocytic 90– maenas 100  300– 320 Carcinus Hemocytic 90– mediterraneus 110  340– 380 Carcinus Hemocytic ? maenas Cancer Hemocytic 100  350 pagurus Cuticular epithelium

NC size section

Morpho- Tail-like genesisa structure

NC/Env

Origine

Virus size (PTA)

References

Pappalardo and 80–90  340– 65– French 70  300– Bonami, 1979; Mediterranean 380 Pappalardo et al., 320 coast 1986; Pappalardo, 1981 US Atlantic nd Nd Johnson, 1976a, 1983, Bays 1984; Johnson and Lightner, 1988 Kodiak and nd nd Johnson and Lightner, Pribilof Islands 1988 Australia nd nd Anderson and Prior, 1992

53– Env/NC 75  220–280

Subapical Subapical extension ? US Atlantic Bays

75–80  230– Env/NC 280

Apical French extension Atlantic coast

70–80  280– Env/NC 320

Apical French extension Mediterranean coast ? Massachusetts nd coast (USA) Apical Experimental 70– extension in crabs 167  210– 380

65–70  210– Env/NC 280 65  200 Env/NC

NC size (PTA)

nd

nd

Johnson, 1983, 1984, 1988

95– 110  300– 350 120  350

73  215

Bazin et al., 1974; Bonami, 1980

Mari and Bonami, 70– 85  320– 1986; Mari, 1987 360 nd Johnson, 1988 Corbel et al., 2001; 54– 85  180– Escobedo-Bonilla et al., 2008 420

NC first formed before envelope (NC/Env) or Env present before NC formation (Env/NC); Budding: acquisition of envelopes by budding through the nuclear membrane.

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Fig. 15. Morphogenesis of s virus: (a) in longitudinal section, long structures appeared first before envelopes were at the origin of nucleocapsids; (b) in transversal section, empty nucleocapsids (white arrowhead), dense nucleocapsids (arrows) and enveloped nucleocapsids were evidenced; (c) in longitudinal section, full enveloped virions exhibited a lateral appendage of the envelope. TEM (Bar = 200 nm).

non-occluded baculoviruses (Nudibaculovirinae) (Matthews, 1982) and classified in the C sub-group of the Baculoviridae (Francki et al., 1991). Some developed in epithelial cells of digestive tract, other exhibited a systemic location. More recently virus taxonomists (Van Regenmortel et al., 2000) added these viruses to the group of unclassified viruses according to the evolution of the Nomenclature and Classification of Viruses of the International Committee for Taxonomy of Viruses (ICTV). Recently, a new family (Nimaviridae) and a new genus (Whispovirus) were created for the White Spot Syndrome Virus (WSSV) of shrimp (Fauquet et al., 2005). WSSV exhibits numerous morphological characters that are identical to those of some enveloped bacilliform viruses of crabs. However to prevent confusion, rather than adding certain crab viruses to the genus Whispovirus in the present paper, we will use the old terminology and names previously accepted for the agents from crabs. Twenty years ago, these viruses had been reported as ‘‘gut-infecting species” and hemocyte-infecting species” by Johnson and Lightner (1988) and Johnson (1988), respectively. Since these two reports, and except for SsBV from S. serrata (Anderson and Prior, 1992), no new data were added to the knowledge base for this group of viruses.

Fig. 16. Cytoplasmic migration of s virus particles in epithelial cells of the hepatopancreas located here on the two sides of neighbouring cell membranes, indicating cell-to-cell contamination. TEM (Bar = 200 nm).

11. Enveloped bacilliform nuclear viruses with digestive location Four different enveloped bacilliform viruses exhibiting a digestive location have been reported in crabs: the Baculo-A from the blue crab C. sapidus of the Atlantic US bays (Johnson, 1976a, 1983, 1984; Johnson and Lightner, 1988), the s (Tau) virus of the shore crab C. mediterraneus from the French Mediterranean coast (Pappalardo and Bonami, 1979; Pappalardo et al., 1986; Pappalardo, 1981), the Baculo-PP of the king crab P. platypus (Johnson and Lightner, 1988) in Alaska (Pribilof and Kodiak Islands) and the SsBV of the mud crab S. serrata in Australia (Anderson and Prior, 1992). Johnson and Lightner (1988) gave a complete review on Baculo A and Baculo-PP. These data confirm previous observations on s virus particularly on ultra-structure and morphogenesis reported by Pappalardo (1981), Pappalardo and Bonami (1979) and

Fig. 17. Enriched Tau virus suspension. The particles exhibited a characteristic distal appendage in prolongation with the envelope as showed by the partially degraded particle in center of the picture. PTA 2%. (Bar = 200 nm).

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Pappalardo et al. (1986) who produced more details on ultrastructure of the virus (Figs. 13 and 14) particularly by investigations after negative staining of isolated particles. All of these results strongly suggest close relationships between these viruses (see Table 4). In viral morphogenesis, nucleocapsids (NC) pre-exist to envelopes which are tightly applied on their surface (Fig. 15). Not observed in the cases of Baculo-A and Baculo-PP, budding of particles through the nuclear membrane was clearly evidenced in SsBV. Even though not reported in s virus, a similar release of virions from the nucleus to the cytoplasm could occur because s particles were observed in cytoplasm and particularly arranged close to the plasma membrane (Fig. 16). The main morphological difference between these four viruses seems to be the size, with smaller diameter of NC in section. Data on superficial structure of isolated particles were only available with s virus. The observations indicate the presence of a lateral appendage issued from the viral envelope (Figs. 17 and 18a) and the peculiar structure of the NC (Fig. 18b), blunt ended by two sheets 15 nm thick at the extremities and exhibiting a cross-hatched superficial structure making a 38° angle with the longitudinal axis of the NC. In an isolated NC, a braid-like structure, 13–15 nm thick and 100–200 nm in length was often observed and was interpreted as the nucleoprotein.

12. Systemic enveloped bacilliform nuclear viruses Four different enveloped bacilliform viruses exhibiting a systemic development were reported in crabs: B, B2 viruses from carcinid crabs (Bazin et al., 1974; Bonami, 1980; Mari and Bonami, 1986; Mari, 1987) from the French coasts (Carcinus. maenas and C. mediterraneus, respectively), the RV-CM from C. maenas of the Atlantic USA coasts (Johnson, 1988) and Baculo-B from C. sapidus from the Atlantic USA Bays (Johnson, 1983, 1984, 1988). In this group, the review by Johnson (1988) is still of relevant and similar observations were done as well in B and B2 viruses in France as in RV-CM and Baculo-B in the USA. In this group of systemic enveloped bacilliform nuclear viruses (Fig. 19), and opposite to that of

15

the previous group, the envelopes appear during virus assembly before capsid formation and densification. Similarities between these four viruses are very high in terms of size, shape and morphogenesis (Fig. 20). In this case too, negative staining of isolated particles gave more data on the virus ultra-structure, particularly the superficial design of NC and the tail-like structure of enveloped particles (Fig. 21). The tail-like structure, variable in length is formed by the prolongation of the envelope. The NC is characterized by one rounded extremity, the other being blunt ended by a trilaminar structure 15–18 nm thick (Fig. 22). The surface appears crosshatched perpendicular to its long axis. This ornamental structure is composed of stacked rings arranged in 14 striations of about 22 nm thick. These last observations are strongly reminiscent of structures reported for NC and enveloped particles of WSSV. In fact, they appear morphologically indistinguishable. Finally, recent data on dot-blot hybridization suggest close relationships between shrimp WSSV and at least B2 virus (HernandezHerrera, Mari, and Bonami, unpublished data). According to these results, B2 virus could be considered as being a sort of WSSV ancestor.

13. Prevalence Data on the prevalence of enveloped bacilliform nuclear viruses in natural environments are scarce. However, prevalence of Baculo-A in C. sapidus collected in Chesapeake and Chincoteague Bays (MD and VA, USA) varied from 4% to 20% in different batches collected, with a mean value of 6% prevalence in a total of 1500 examined crabs (Johnson and Lightner, 1988). Baculo-PP found in two populations of the king crab P. platypus exhibited a prevalence of 40% in April 1982 at Olga Bay (Kodiak island) and 20% in June 1982 at the Pribilof islands (Johnson and Lightner, 1988). Prevalence of s virus in French Mediterranean area C. mediterraneus was very low, seasonly dependent and always less than 5% (unpublished data). RV-CV from C. maenas was found only in 1 of 29 crabs sampled at Woods Hole (MA, USA) and this crab was the only individual infected in a total of 74 crabs collected during 1982–1983

Fig. 18. Isolated Tau virus particles at high magnification: (a) full mature enveloped particle with its characteristic appendage; (b) after loosing the envelope, the nucleocapsid appears with a cross-hatched superficial structure; the treat released at one extremity corresponded to the nucleoprotein. 2% PTA (Bar = 50 nm).

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Fig. 19. B2 virus disease of Carcinus mediterraneus. An infected nucleus is shown. TEM (Bar = 200 nm).

Fig. 21. Isolated B2 virus from infected hemolymph. Full mature and enveloped particle exhibiting the characteristic tail-like structure at one extremity (arrow). 2% PTA (Bar = 100 nm).

Fig. 22. Partially degraded B2 virus. Through the envelope, penetrated with PTA, the nucleocapsid is clearly evidenced, with one rounded extremity (one arrow), the opposite blunted (2 arrows). Its superficial structure is formed with stacked rings, a feature that closely resembles the structure of WSSV nucleocapsids from penaeid shrimp. 2% PTA (Bar = 100 nm).

14. Crabs and WSSV Fig. 20. Morphogenesis of B2 virus in the nucleoplasm. TEM (Bar = 200 nm).

(Johnson, 1988). The same author indicated that only 19 crabs of a total of 1500 examined (Chesapeake and Chincoteague Bays, USA) were Baculo-B infected, moreover seven had been experimentally injected. This underlines the paucity of information on this disease in the natural environment. Similar results were obtained in France where B and B2 viruses were found in a limited number of samples, less than 1%, as well for C. maenas as for C. mediterraneus (Bonami, unpublished data).

To date, the economic impact of a possible epizooty due to WSSV in crabs has not been documented. However, as most of the crabs so far tested were found sensitive to the disease it appears necessary to increase surveillance of wild crab stocks to monitor and possibly prevent WSSV transmission into susceptible wild crabs. Crabs naturally infected with WSSV and the results of experimental infection of different crab species with WSSV have been reported. The list of possible hosts continues to expand Escobedo-Bonilla et al. (2008) reported at that time that 41 crab species were susceptible to infection by WSSV. Some 14 species were found to be naturally infected, and 27 other species were experimentally infected.

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