Monoclonal antibodies developed for detection of an epizootic virus associated with mass mortalities of cultured scallop Chlamys farreri

Abstract

Recently, an enveloped, spherical RNA virus was identified as the causative agent of mass mortalities among adult scallop Chlamys farreri, which is cultured on the northern coast of China. Hybridomas were prepared from mice immunized with highly purified virions. Four stable hybridomas secreting monoclonal antibodies (MAbs) of IgG isotype were obtained after screening by means of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). The specificity of the MAbs to this virus was confirmed by immunogold electron microscopy (IEM). All the selected MAbs recognized epitopes on the envelope spikes of the virions. Subsequently, the MAbs were used for in situ immunofluorescent detection of the virus in Davidson's fixed tissue sections. The results showed that the fluorescent cells were mostly observed in epithelia of different organs, but not in the epithelium of the digestive diverticulae. Cytopathological changes and focal lesions corresponding to virus-positive cells were clearly recognized in the affected epithelia, revealing a potential role of this virus in pathogenesis. KEY WORDS: Scallop · Chlamys farreri · Acute virus necrobiotic disease · Monoclonal antibodies Resale or republication not permitted without written consent of the publisherDis Aquat Org 65: [17][18][19][20][21][22] 2005 mass mortalities in mid-July 2001 and 2002 from several private farms in Qingdao and Rizhao (Shandong Province of China). The gills and mantles were removed from the scallops and stored at -85°C before use. Virus purification by centrifugation was performed according to C. M. . Tissues were homogenized in 8 volumes of TEN buffer (50 mM Tris-HCl; 10 mM EDTA; 360 mM NaCl, pH 7.8) and clarified by 2 low speed centrifugations (3500 × g for 15 min followed by 7500 × g for 15 min). All centrifugations were carried out at 4°C. The supernatants were then concentrated by sedimentation through a 6 ml cushion of 35% sucrose (w/w) at 113 000 × g for 90 min and the resulting pellets were resuspended in TEN buffer. After centrifugation at 8000 × g for 20 min, supernatants were layered onto a 30 to 60% (w/w) continuous sucrose density gradient. After centrifugation at 113 000 × g for 2.5 h, the opalescent virus containing band was collected. The virus was washed with TN buffer (50 mM Tris-HCl; 360 mM NaCl, pH 7.8) and resuspended in a small amount of TN buffer. The purity was checked by a transmission electron microscope (TEM, JEOL JEM-1200) using 2% phosphotungustic acid (PTA, pH 7.0) as the negative stain. The concentration of viral proteins was determined using the procedure described by Bradford (1976).Hybridoma production. Eight week old BALB/c mice were immunized intraperitoneally (i.p.) with purified virus (100 µg mouse -1 ) mixed with an equal volume of Freund's complete adjuvant (Gibco). Subsequent boosters with antigen in incomplete adjuvant were given on Days 14, 28, and 42. Three days before cell fusion, mice received the last immunization via intrasplenic injection (20 µg mouse -1 ) with the puri...

Physiological and immune responses of zhikong scallop Chlamys farreri to the acute viral necrobiotic virus infection

“…In addition, Activity fluctuation of several enzymes in the hemocytes following AVNV challenge of the scallops is reported [11]. Immunodiagnostic techniques for virus detection also have been developed recently [13]. The disease is prevalent typically in summer when seawater temperature reaches 25e27 C [6,10], and up to 90% of an affected 1e2 year old scallop population can die in a few days after the diagnosis of AVNV infection [4].…”

Variations of enzyme activities in the haemocytes of scallop Chlamys farreri after infection with the acute virus necrobiotic virus (AVNV)

“…Many studies have been carried out to investigate the causative aetiology of this abnormal mortality from the field of epidemiology, pathology, genetics and breeding technology. Finally, an unknown spherical virus, named as acute virus necrobiotic virus (AVNV), was verified as one of the main pathogens [16,17]. The acute virus necrobiotic disease usually occurs in the second-year-old scallops, in early July and peaks in the end of this month, as water temperature reaches the highest period (25-27 C) of the year, then subsides in early August.…”

Antiviral immunity in marine molluscs

“…Water temperature is also a factor in the expression of disease caused by OsHV-1 infection. The water temperatures must exceed 16 uC for OsHV-1 to cause mortality of juvenile Crassostrea gigas (Petton et al, 2013) and mortality of Chlamys farreri to AVNV occurs in late summer (Fu et al, 2005). It is unknown how host physiology and the environment influences disease expression of abalone to AbHV.…”

“…The complete genome sequence of AVNV indicates it's a variant of OsHV-1 (Ren et al, 2013). Since the 1980s, AVNV has caused disease events in summer of farmed Chlamys farreri in China, with mortality reaching w90 % within 5-8 days after first appearance (Fu et al, 2005). Studies investigating the physiological and immunological responses of Chlamys farreri infected with AVNV have been undertaken (Chen et al, 2011(Chen et al, , 2014Tang et al, 2010;Xing et al, 2008).…”