A simple centrifugation method for improving the detection of Ostreid herpesvirus-1

Ostreid herpesvirus-1 (OsHV-1) is one of the key pathogens causing summer mortalities in the Pacific oyster in Europe, Australia, New Zealand and the US. The virus affects oysters of all ages and size classes. Despite extensive studies on the virus itself, little information exists on the mechanisms of transmission and the spread of the disease in open marine environments. As a result there is little knowledge about methods to detect OsHV-1 in sea water.

It has been suggested that seawater may act as a medium in the horizontal transmission of the virus. A study by Paul-Pont et al. (2013) has led to a hypothesis about the attachment of the virus to plankton-like particles. Viruses, like other microbes, attach to particles in their environment and so their fate and transport is associated with those particles.

Methods such as ultrafiltration, tangential flow filtration, ultracentrifugation, precipitation, and adsorption of viruses onto charged membranes, have been used to concentrate viruses from water samples. However these methods are not practical when processing large numbers of, as is the case for epidemiological studies onOsHV-1. These methods also cannot be utilised in the assessment of potential virus association to particulates of varying sizes, as many of they require or result in virus dissociating from matter within the sample matrix. Evans et al. (2014) assessed and compared several simple centrifugation methods to detect OsHV-1 in seawater samples, in such way that a large number of samples can be processed efficiently and also assessed the potential for particulate attachment of the virus using a simple filtration methodology.

Low speed centrifugation of seawater at 1000 × g for 20 min, then testing the resulting pellet, improved OsHV-1 detection rates by two fold compared to the unprocessed seawater samples. 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. 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.

For something to pellet at such low force it means the particles have to be approximately 7-12μm in diameter or larger. This suggests that as least some of the OsHV-1 virions are attached to particles large enough to be centrifuged at low speed or are present in larger viral aggregates. Viral DNA was detected equally well in supernatant after low speed centrifugation. This suggests that OsHV-1 virions are not only attached to particles, of the size mentioned above, but are also present as free viruses, viruses associated with smaller particles, or as free DNA. Microbial communities are constantly growing, changing and cycling so it can be assumed that while some viral particles are attached others may not be intact or exist free in sea water. This supports the hypothesis that the distribution and transmission of OsHV-1 in natural sea water might reflect attachment to some form of particulate matter.

This study has shown that detection by qPCR in seawater improved two fold by testing the pellet obtained by low speed centrifugation, compared to testing unprocessed seawater samples. This provides a simple method suitable for testing large numbers of water samples that will be applicable to epidemiological studies of OsHV-1. This has further application as such a simple method can be used to increase the detection of other viruses that have levels below the limits of quantitation of the qPCR. Simple filtration of seawater samples through low protein binding filters did not prove to be an appropriate method for assessing OsHV-1 association and/or attachment to particles of different sizes, probably due to dynamic interactions of the virus within the sample matrix itself as well as the filter membranes. This finding therefore needs to be explored further to identify the true nature of the viral interaction with particles as this will be critical to understanding the distribution and transmission of OsHV-1 in natural environments. Although this method of detection provides very fruitful results for OsHV-1 there was little mention if this method can be used for the detection of other viral strains. Further experiments could test this and may help reduce costs and increase efficiency of other viral research.

Evans, O., Paul-Pont, I., Hick, P., & Whittington, R. J. (2014). 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. Journal of virological methods. Oct 5;210C:59-66. doi: 10.1016/j.jviromet.2014.09.023. [Epub ahead of print]
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