A herpes-like virus infects a non-ostreid bivalve species: virus replication in Ruditapes philippinarum larvae

Abstract

Sporadic high mortalities were reported in June 1997 among hatchery-reared larval Manila clam Ruditapes philippinarum in a French commercial hatchery. Cellular abnormalities were observed in semi-thin sections in affected animals. Transmission electron microscopy revealed the presence of herpes-like virus particles in larvae. This is the first description of a herpes-like virus infection in larval R. philippinarum, a non-ostreid bivalve species. Virus particles were similar to other herpes-like viruses described from different oyster species with respect to ultrastructure and morphogenesis. Electron microscopic examination also demonstrated cells with condensed chromatin and extensive perinuclear fragmentation of chromatin. Like viruses infecting oysters, the herpes-like virus detected in clams may induce apoptosis in infected animals. KEY WORDS: Herpes-like virus · Manila clam · Ruditapes philippinarum · Mortality · Virus replication · Apoptosis Resale or republication not permitted without written consent of the publisherDis Aquat Org 45: [1][2][3][4][5][6][7] 2001 scopy. The larvae were fixed in 2.5% glutaraldehyde in 0.2 M cacodylate buffer at pH 7.2 for 1 h at 4°C. After 2 washes in cacodylate buffer, samples were post-fixed in 1% osmium tetroxide in the same buffer at 4°C. Larval samples were dehydrated by serial ethanol baths (70 to 100%), partially rehyrated by two 10 min baths in 0.2 M cacodylate buffer and de-calcified with a 2% ethylenetetra-acetic acid (EDTA) solution in 0.2 M cacodylate buffer overnight at 4°C. After fixation and post-fixation, larvae were washed in 0.2 M cacodylate buffer, dehydrated by means of an ascending ethanol series (70 to 100%), cleared twice for 15 min in propylene oxide and infiltrated for 1 h in 50:50 propylene oxide:Epon resin. After 1 h infiltration in pure Epon resin, they were embedded in resin and cured for 48 h at 60°C. Sections were cut at 1 µm for light microscopic examination which were stained in 0.5% toluidine blue in 1% sodium borate solution. Ultrathin sections were stained in 5% uranyl acetate in 50% ethanol for 20 min and in 5% lead citrate in fresh boiled distilled water for 3 min. They were examined in a JEOL JEM 1200 EX transmission electron microscope at 80 kV. RESULTS Course of the diseaseA few days after spawning, Ruditapes philippinarum larvae showed reduction in feeding and swimming activities. Mortality occurred, at the beginning of June 1997, with total mortality by Day 13. Affected larvae swam weakly in circles before dying appromimately at 100 to 150 µm in size. Histological examinationAbnormal nuclei were present throughout the connective tissue in semi-thin sections of moribund larvae. Enlarged nuclei with abnormal marginated chromatin were evident in fibroblastic-like cells (Fig. 1). Highly condensed nuclei were observed in ovoid cells interpreted as haemocytes. Some nuclei of fibroblastic-like cells and haemocytes appeared with a ring-shaped or crescent-shaped chromatin (Fig. 1). The lesions were mainly confined to connective t...

Antiviral immunity in marine molluscs

“…The first description of herpes-like virus associated with mollusc mortality was reported in 1972 in the Eastern oyster (Crassostrea virginica) from the east coast of the USA (Farley et al, 1972). Since this time, herpes-like viruses have been described in other species of oyster (Burge et al, 2006;Hine et al, 1992;Renault et al, 1994), scallops (Arzul et al, 2001a;Ren et al, 2013) and clams (Renault et al, 2001). The virus has been purified from naturally infected Crassostrea gigas larvae (Le Deuff & Renault, 1999) and its genome entirely sequenced (Davison et al, 2005).…”

Analysis of Clinical Ostreid Herpesvirus 1 (Malacoherpesviridae) Specimens by Sequencing Amplified Fragments from Three Virus Genome Areas

“…Finally, 2 French virus specimens collected in 1993 showed high homology to variant OsHV-1 Var (3). This variant was reported in 1997 during a mortality outbreak affecting both larval Pacific oysters and larval Manila clams (Ruditapes philippinarum) in a commercial hatchery (19,20). Both French samples from 1993 correspond to C. gigas larvae collected in a commercial hatchery.…”

“…Although OsHV-1 variants have already been reported (2,3,14,19,20), more work is needed to gauge the range of OsHV-1 polymorphisms. Since 2008, massive mortality outbreaks among Pacific oysters (Crassostrea gigas) have been reported in Europe (8,21) and have been associated with a virus genotype labeled Var (22).…”

Les virus infectant les mollusques marins: un exemple d'actualité, les herpèsvirus

“…Ils ont ainsi été décrits aux USA (Farley et al 1972, Friedman et al 2005, Meyers et al 2009), en Nouvelle Zélande (Hine et al 1992 ;Hine et al 1998), en France (Nicolas et al 1992 ;Comps & Cochennec, 1993 ;Renault et al 1994aRenault et al , 1994bRenault et al 2000aRenault et al , 2000bArzul et al 2001aArzul et al , 2001bArzul et al , 2001cRenault et al 2001aRenault et al , 2001b, en Australie (Hine & Thorne, 1997) (Hooper et al 2007 ;Tan et al 2008) associés à des épi-sodes de mortalité massive à Taiwan et en Australie. Un variant du virus, dénommé OsHV-1var, a été rapporté chez différentes espèces de bivalves incluant l'huître creuse C. gigas (Arzul et al 2001a ;2001b, 2001c. Les isolats OsHV-1 et OsHV-1var sont considérés comme appartenant à la même espèce.…”