Viral gametocytic hypertrophy caused by a papova-like virus infection in the Pacific oyster Crassostrea gigas in Korea

Abstract

During a routine survey of the Pacific oyster Crassostrea gigas in Tongyoung (previously Chungmu) on the southern coast of Korea, basophilic inclusions were observed in the gonadal tissues. They were detected from March to May at a prevalence rate of 3.3 to 7.1%. The inclusion bodies were Feulgen-positive and stained orange-red with phloxine tartrazine. Electron microscopic observation revealed non-enveloped, icosahedral particles 40 to 45 nm in diameter. These morphological characteristics resemble those of papova virus-like inclusions previously described from Pacific and eastern (American) oysters C. virginica in North America. Although many mitochondrial bodies and intact sperm cells were observed around the inclusion body, no host reaction, such as hemocytic infiltration, was detected. KEY WORDS: Pacific oyster · Crassostrea gigas · Viral gametocytic hypertrophy · Papova-like virus · Seminal glandResale or republication not permitted without written consent of the publisher embedding and cut into 4 µm thick sections. Some sections were stained with Harris's hematoxylin and eosin (H&E), some with Feulgen stain and some with phloxine tartrazine, for bright field light microscopy (Olympus Vanox AHBS3) examination.Ultrastructural examination. Samples showing inclusion bodies within the gonadal epithelia were selected for processing for transmission electron microscopy. Tissue specimens from the same oysters, preserved in Carson's fixative solution were rinsed in 0.2 M cacodylate buffer at pH 7.2 for 48 h at 4°C before post-fixing in 2.5% glutaraldehyde solution, and 1% osmium tetroxide. After dehydration through graded alcohols, the tissues were embedded in Epon resin compound (Ouken, Japan). Hardening was carried out at 35°C for 12 h, 45°C for 12 h, and 60°C for 48 h. Semi-thin sections (200 nm) were stained with toluidine blue, and ultra-thin sections (60 nm) were stained with uranyl acetate and lead citrate. Ultra-thin sections were examined with a JEOL 1200 EX-2 transmission electron microscope (TEM) at 80 kV. RESULTSBasophilic inclusions in gonoducts were observed in specimens from all sites except Stn 4. Infections were observed from March to May at prevalences of 3.3 to 7.1%. Prevalence at Stn 3 was higher than at the other sites ( Table 1). The heaviest infection included 62 inclusion bodies in a single tissue section; however, no host reaction was observed. The longest axis of the inclusion bodies varied from 15 to 60 µm, and the shape was oval to spherical. Some inclusion bodies had dense staining margins (Figs. 2 & 3). Feulgen staining confirmed the presence of dense nucleic acid (Fig. 4), and orange-red staining with phloxine tartrazine (Fig. 5) demonstrated a deoxyribonucleic acid (DNA) composition. Advanced infections led to disruption of the nuclear membrane and putative release of the viral particles (Fig. 4, arrowhead).Non-enveloped, icosahedral viral particles, 40 to 45 nm in diameter, filled the inclusion bodies (Figs. 6 & 7). These morphological characteristics resemble those of Pa...

Viral gametocytic hypertrophy of the Pacific oyster Crassostrea gigas in Ireland

“…In the present study we observed between 3 and 14 infected cells per section in 3 female oysters; however, the number of oysters examined is too low to establish infection rate or infection intensity. In common with other studies (Choi et al 2004, Garcia et al 2006, no haemocytic reaction was observed in the present study, suggesting limited health implications for the infected oysters. However, Garcia et al (2006) comment that gamete viability and consequently oyster fecundity could be altered by VGH.…”

“…Farley (1976Farley ( , 1985 described a papova-like virus in hypertrophied gametocytes of the eastern oyster Crassostrea virginica; since then other authors have reported similar conditions in various oyster species in North America, Asia and Europe (McGladdery & Stephenson 1994, Elston 1997, Choi et al 2004, Garcia et al 2006. This is the first report of VGH in C. gigas in Ireland.…”

“…In Site B, 4 operators noted mortalities of approximately 30%. From a total of 77 oysters examined, only 3 female oysters were found to have basophilic inclusions, with the number of infected cells ranging from 3 to 14 per section.Based on the reprocessed TEM material, although of reduced quality, these viral particles appear similar to those described by Winstead & Courtney (2003), Choi et al (2004) and Garcia et al (2006). Farley (1976Farley ( , 1985 described a papova-like virus in hypertrophied gametocytes of the eastern oyster Crassostrea virginica; since then other authors have reported similar conditions in various oyster species in North America, Asia and Europe (McGladdery & Stephenson 1994, Elston 1997, Choi et al 2004, Garcia et al 2006.…”

Detection of viruses and virus-like particles in four species of wild and farmed bivalve molluscs in Alaska, USA, from 1987 to 2009

“…Ultrastructural examination of inclusion body material from one affected male showed arrays of intranuclear icosahedral virus-like particles (Fig. 5C) 38 to 40 nm in diameter, consistent with a virus previously described as causing hypertrophied gametocytes (ovacystis) in eastern Crassostrea virginica (Farley 1976, Winstead & Courtney 2003 and Pacific (Choi et al 2004, Garcia et al 2006 oysters. The same inclusion bodies or other known types of virus-associated cytopathology were not observed (Table 2) in any of the juvenile or adult oysters examined from the other farm sites listed in Table 1.…”

“…The condition was first described in ova of Crassostrea virginica from the Piscataqua River, Maine, USA (Farley 1976(Farley , 1978. Papovavirus, causing gametogenic or other cellular hypertrophy, has subsequently been reported in C. virginica from the Gulf of Mexico (Winstead & Courtney 2003) and Atlantic Canada (McGladdery & Stephenson 1994), in C. gigas from Korea (Choi et al 2004), France (Garcia et al 2006), Germany (Watermann et al 2008) and Ireland (Cheslett et al 2009), in the golden-lipped pearl oyster Pinctada maxima from Torres Strait, Australia (Norton et al 1993), in the softshell clam Mya arenaria in Massachusetts, USA, and Ruditapes philippinarum from Spain (Elston 1997, Montes et al 2001. Similar examples of unconfirmed virus-associated gametogenic hypertrophy have been reported in other ostreid species including Saccostrea glomerata, C. rhizophorae, Ostrea edulis and Ostreola conchaphila (Farley 1978, Bower et al 1994.…”