Herpes-like virus associated with eroded gills of the Pacific oyster Crassostrea gigas in Mexico.

ABSTRACT Since 1997, high mortality episodes of cultured oysters have occurred in Bahia Falsa, Mexico. Studies on the possible association of these mortalities with pathogens have showed some similar characteristics to those found in the Portuguese oyster Crassostrea angulata and the Japanese oyster Crassostrea gigas infected by an iridoviridae-like particles in the lately 1960s. This infection was named gill necroses virus infection (GNV). In a recent study, we could not find any virus using transmission electron microscopy (TEM) in adult oysters with clinical and histologic signs similar to those described for GNV. However, new TEM images showed the presence of viral particles in eroded gills of oysters. Morphologic characteristics, such as thin walled icosahedric shape, the presence of capsids in an extension of the nucleus or in a vacuole and size varying from 80 to 90 nm suggest that the viruses belong to the Herpes viridae family, This virus could be involved in the mortality episodes in the Bay and it is different to those described as causal agent of GNV.

KEY WORDS: Crassostrea gigas, herpes-like virus, mortality, gill erosion, oyster disease

INTRODUCTION

In a recent study on the possible association of high mortality outbreaks of the Pacific oyster C. gigas cultured in Baja California and other states of NW Mexico with a pathogenic agent (Caceres-Martinez & Vasquez Yeomans 2003), we found some clinical and histopathologic evidence similar to those described for gill necrosis viral infection (GNV) recorded in the Portuguese oyster C. angulata and to a lesser degree in the Pacific oyster cultured in France, Great Britain, Spain, and Portugal between 1966 and 1967 (Marteil 1969, Comps 1988). Previously, we did not detect the iridovirus-like the causal agent of GNV, using transmission electron microscopy (TEM), possibly because the sample process, the status of the gill tissues of surviving oysters (wound repair), and or difficulties in finding the viral agent in fixed tissues (Caceres-Martinez & Vasquez-Yeomans 2003). However, new TEM analyses of more samples have detected herpes-like virus particles.

MATERIALS AND METHODS

A sampling carried out in November 2000, of 10 adult oysters (Mean 89.3 [+ or -] 7.7 mm shell length), from an area of the Bahia Falsa, Baja California (116[degrees]00'W, 30[degrees]25'N) where mortalities were common, were reviewed and those showing highly eroded gill tissues (Caceres-Martinez & Vasquez-Yeomans 2003) were selected for TEM examination. Small pieces of tissue were cut from the eroded area of the gill and fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.8, for 4 h at 4[degrees]C. Fixed tissues were washed for 12 hours at 4[degrees]C in the same bullet and cut into 1-[mm.sup.3] pieces. These pieces were then postfixed in buffered 1% Os[O.sub.4] for 4 hours at 4[degrees]C, dehydrated through a graded series of ethanols and embedded in epoxy resin. Sections (90 nm thickhess) were cut and stained with 5% uranyl acetate for 30 minutes. then observed with TEM operated at 75 kV in the Instituto de Investigaciones Marinas from Vigo, Spain.

RESULTS AND DISCUSSION

Viral particles were found in all samples (n = 10) of eroded gill analyzed in this study. The morphologic characteristics of the viral particles including capside shape, presence of a thin wall, size range (Fig. 1), and presence in an extension of the nucleus or in a vacuole of the infected cell (Fig. 2) suggest that they are closely related to Herpes virus (virions enveloped, slightly pleomorphic, spherical, 120-200 nm in diameter). Surface projections of envelope distinct spikes were dispersed evenly over all the surface.

[FIGURES 1-2 OMITTED]

Nucleocapsid surrounded by the tegument that consists of globular material, which is frequently asymmetrically distributed and may be variable in amount. Nucleocapsids were sometimes penetrated by stain (although the intact envelope was impermeable to stain) 100-110 nm in diameter.

Nucleocapsids appeared to be angular and surface capsomer arrangement obvious. There were 162 capsomers per nucleocapsid (capsomeres hexagonal in cross-section with a hole atoning halfway down the long axis). The core consisted of a fibrillar spool on which the DNA is wrapped, and the ends of the fibers are anchored to the underside of the capsid shell. Incomplete virus particles often present, are capsids lacking the envelope. Virions contain one molecule of linear double stranded DNA (van Regenmortel et al. 2000). Based on this description of the Herpes viruses, the particles shown in Figures 1 and 2 can be described as herpes-like. There were no particles in the main part of the nucleus and no virions in the cytoplasm. Some capsides were variable in size and thin-walled.

Herpes and herpes-like viruses have been recorded in turtles, carps, ictalurids, frogs, and other aquatic organisms (Buchanan & Richards 1982). In oysters there are several records of herpes or herpes-like viruses around the world (Table 1), and these virus infections in bivalves seem to be ubiquitous (Le Deuff & Renault 1999). All these herpes-like viruses have been described in larval stages and young spat with the exception of adult Ostrea angasi in Australia (Hine & Thorne 1997) and possibly adult Crassostrea virginica in Maine USA (Farley et al. 1972). However, Farley et al. did not mention the age or size of the infected oysters. Studies on the development of molecular analysis to detect the herpes-like virus in oyster larvae and juveniles have been carried out (Le Deuff & Renault 1999, Renault et al. 2000, Renault & Arzul 2001). Interspecies transmission also has been studied (Arzul et al. 2001a, Arzul et al. 2001b). More recently, Arzul et al. (2002) detected the herpes-like virus in a symptomatic C. gigas adult using molecular techniques, including PCR and in situ hybridization (ISH). These authors suggest that this virus may persist in its host after primary infection, and a comparison between adult oysters and infected spat indicated that the spat contain higher amounts of virus DNA. They concluded that viral replication appears less common in adults than in spat. In contrast, our results (Caceres-Martinez & Vasquez-Yeomans 2003) and those shown in this study, suggest the connection of a herpes-like virus in gill tissue of adults C. gigas with clinical signs, histologic alterations, and high mortality episodes. To our knowledge, this is the first record of herpes-like viruses by TEM in adult oysters.

More research is being conducted to farther characterize the virus and to determine the influence of environmental factors on the development of the disease in larvae, juveniles, and adults. Experimental infections by cohabitation and by injection of a homogenate of eroded gill tissues from symptomatic oysters are being carried out to confirm the role of these herpes virus-like particles as the cause of these mortalities.

TABLE 1.
Herpes-like virus infection in oysters.

        Host             Growth Stage           Tissue

Crassostrea virginica   Not mentioned,    Haemocytes
                        possibly adult

Crassostrea gigas       Larvae            Connective and
                                          mantle epithelium

Crassostrea gigas       Larvae            Entire larvae

Ostrea edulis           Young spat        Connective, around
                        (5 months old)    digestive tubules

Crassostrea gigas       Young spat (3-7   Connective in the
                        months old)       gills, mantle and
                                          around digestive
                                          nibules

Ostrea angasi           Adults

                                      Name of
        Host            Mortality   the Disease

Crassostrea virginica               Unnamed

Crassostrea gigas                   Unnamed

Crassostrea gigas        60-100%    Unnamed

Ostrea edulis              90%      Herpes-like
                                    infection

Crassostrea gigas        80-90%     Unnamed

Ostrea angasi                       Unnamed

        Host                Locality            Reference

Crassostrea virginica   Maine, USA         Farley et al. 1972

Crassostrea gigas       Northern France    Nicolas et al. 1992

Crassostrea gigas       Auckland,          Hine et al. 1992
                        New Zealand

Ostrea edulis           Brittany, France   Comps & Cochennec
                                           1993

Crassostrea gigas       France, Atlantic   Renault et al. 1994
                        Coast

Ostrea angasi           Australia          Hine and Thorne 1997

AKNOWLEDGEMENTS

The authors thank anonymous herpesvirus structural expert for his help in the ultrastructural identification of the virus and Dra. Carmen Paniagua Chavez for suggestions to the manuscript and help in translation. This work was supported by Centro de Investigacion Cientifica y de Educacion Superior de Ensenada, Mexico throughout the project number 623106, the project from Consejo Nacional de Ciencia y Tecnologia CONACyT 225080-5-3933PB and Instituto de Investigaciones Marinas de Vigo, Spain. R. V.-Y. was supported by a grant of the Consejo Nacional de Ciencia y Tecnologia (CONACyT) from Mexico.

LITERATURE CITED

Arzul. I., T. M. Renault & C. Lipart. 2001a. Experimental herpes-like viral infections in marine bivalves: demonstration of interspecies transmission. Dis. Aquat. Org. 46:1-6.

Arzul, I., T. M. Renault, C. Lipart & A. J. Davison. 2001b. Evidence for interspecies transmission of oyster herpesvirus in marine bivalves. J. of Gen. Vir. 82:865-870.

Arzul. I., T. M. Renault, A. Thebault & A. Gerard. 2002. Detection of oyster herpesvirus DNA and proteins in asymptomatic Crassostrea gigas adults. Virus Res. 84:151-160.

Buchanan, J. S. & R. H. Richards. 1982. Herpes-type virus diseases of marine organisms. Porc. Royal Soc. of Edimburg. 81:151-168.

Caceres-Martinez, J. & R. Vasquez-Yeomans. 2003. Presence of giant polymorphic cells in Crassostrea gigas cultured in Bahia Falsa, Baja California NW Mexico. J. Shellfish Res. 22(3):711-714.

Comps, M. 1988. Epizootic diseases of oysters associated with viral infections. In: W. S. Fisher, editor. Disease processes in marine bivalve molluscs. Bethesda, Maryland: American Fisheries Society, special publication 18. pp. 23-37.

Comps, M. & N. Cochennec. 1993. A herpes-like virus from European oyster Ostrea edulis L. J. of Inv. Patol. 62:201-203.

Farley, C. A., W. G. Banfield, G. Kasnic, Jr. & W. S. Foster. 1972. Oyster herpes-type virus. Science 178:759-760.

Hine, P. M., B. Wesney & B. E. Hay. 1992. Herpesviruses associated with mortalities among hatchery-reared larval Pacific oysters Crassostrea gigas. Dis. aquat. Org. 12:135-142.

Hine, P. M. & T. Thorne. 1997. Replication of herpes-like viruses in haemocytes of adult flat oysters Ostrea angasi (Sowerby, 1871): an ultraestructural study. Dis. aquat. Org. 29:189-196.

Le Deuff, R. M. & T. Renault, 1999, Purification and partial genome characterization of a herpes-like virus infecting the Japanese oyster, Crassostrea gigas. J. of Gen. Vir. 80:1317-1322.

Marteil, L. 1969. La maladie des branches des huitres portugaises des cotes francaises de l'altlantique. Rev. Trav. Inst. Peches Marit. 33(2):145-150.

Nicolas, J. L., M. Comps & N. Cochenec. 1992. Herpes-like virus infecting Pacific-oyster larvae, Crassostrea gigas. Bull. Eur. Ass. Fish Patol. 12(1):11.

Renault, T., N. Cochennec, R. M. Le Deuff & B. Chollet. 1994. Herpes-like virus infecting Japanese oyster (Crassostrea gigas) spat. Bull. Eur. Ass. Fish Pathol. 14(2):64-66.

Renault, T., R. M. Le Deuff, C. Lipart & C. Delsert. 2000. Development of a PCR procedure for detection of a herpes-like virus infecting oysters in France. J. Virol. Methods 88:41-50.

Renault, T. M. & l. Arzul. 2001. Herpes-like virus infections in hatchery-reared bivalve larvae in Europe: specific viral DNA detection by PCR. J. Fish. Dis. 24:161-167.

van Regenmortel, M. H. V., C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon. J. Maniloff, M. A. Mayo. D. J. McGeoch, C. R. Pringle & R. B. Wickner. 2000. Virus taxonomy: the classification and nomenclature of viruses. The seventh report of the international committee on taxonomy of viruses. San Diego: Academic Press. 1167 pp.

REBECA VASQUEZ-YEOMANS, (1), ([dagger]) JORGE CACERES-MARTINEZ, (1), *, ([dagger]) AND ANTONIO FIGUERAS HUERTA (2)

(1) Laboratorio de Biologia y Patologia de Organismos Acuaticos del Departamento de Acuicultura, Centro de Investigacion Cientifica y de Educacion Superior de Ensenada, Apdo. Postal 2732, C.P. 22860, Ensenada Baja California, Mexico and (2) Laboratorio Nacional de Referencia de enfermedades de Moluscos del Instituto de Investigaciones Marinas de Vigo. Eduardo Cabello #6, 36208, Vigo, Espana

* Corresponding author.

([dagger]) Present address: Instituto de Sanidad Acuicola. A.C. Calle 9na y Gastelum No. 468 Local 14, Zona Centro, C.P. 22800, Ensenada, Baja California, Mexico.