title: A simple centrifugation method for improving the detection of Ostreid herpesvirus-1 (OsHV-1) in natural seawater samples with an assessment of the potential for particulate attachment.
abstract: Ostreid herpesvirus-1 (OsHV-1) is responsible for massive mortality events in commercially farmed Pacific oysters (Crassostrea gigas) in Australia, New Zealand, Europe and the USA. Economic losses have been severe in many countries since 2008, associated with a strain known as OsHV-1μ-var. Despite intensive studies of the virus itself, there is almost no information on its detection in natural seawater, how it is spread over wide geographic distance in water or on how it is transmitted from oyster to oyster via seawater. The aim of the current work was to (1) assess and compare several centrifugation methods in order to detect OsHV-1 in natural seawater samples using real-time quantitative PCR, in such a way that large numbers of samples could be processed efficiently and (2) assess the potential for particulate attachment of OsHV-1 using filtration. Compared to testing unprocessed seawater samples, centrifugation of seawater at 1000×g for 20 min with testing of the pellet improved OsHV-1 detection rates by two fold. Results suggest that OsHV-1 may be attached to particles large enough to be pelleted at low g-force, as well as in the form of small particles, free virus or free viral DNA. Filtration of seawater using low protein binding filters could not be used to assess OsHV-1 particle attachment, due to interactions between particles, free virus or free viral DNA and the membranes.
authors.affiliation: University of Sydney, Faculty of Veterinary Science, 425 Werombi Road, Camden, New South Wales 2570, Australia.
authors.forename: O
authors.initials: O
authors.lastname: Evans
authors.affiliation: University of Sydney, Faculty of Veterinary Science, 425 Werombi Road, Camden, New South Wales 2570, Australia.
authors.forename: I
authors.initials: I
authors.lastname: Paul-Pont
authors.affiliation: University of Sydney, Faculty of Veterinary Science, 425 Werombi Road, Camden, New South Wales 2570, Australia.
authors.forename: P
authors.initials: P
authors.lastname: Hick
authors.affiliation: University of Sydney, Faculty of Veterinary Science, 425 Werombi Road, Camden, New South Wales 2570, Australia. Electronic address: richard.whittington@sydney.edu.au.
authors.forename: R J
authors.initials: RJ
authors.lastname: Whittington
mesh_terms: D000818:Animals; D002498:Centrifugation / Q000379:methods / Q000662:veterinary*; D049896:Crassostrea / Q000821:virology*; D004267:DNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D012623:Seawater / Q000821:virology
chemical_list: D004279:DNA, Viral
keywords: Centrifugation; Detection; Filtration; OsHV-1; Oyster; Seawater