PDF(Pubmed)
Abstract：
Ostreid herpes virus 1 (OsHV-1) has been classified within the Malacoherpesviridae family from the Herpesvirales order. OsHV-1 is the etiological agent of a contagious viral disease of Pacific oysters, C. gigas, affecting also other bivalve species. Mortality rates reported associated with the viral infection vary considerably between sites and countries and depend on the age of affected stocks. A variant called μVar has been reported since 2008 in Europe and other variants in Australia and in New Zealand last decade. These variants are considered as the main causative agents of mass mortality events affecting C. gigas. Presently there is no established cell line that allows for the detection of infectious OsHV-1. In this context, a technique of propidium monoazide (PMA) PCR was developed in order to quantify \"undamaged\" capsids. This methodology is of interest to explore the virus infectivity. Being able to quantify viral particles getting an undamaged capsid (not only an amount of viral DNA) in tissue homogenates prepared from infected oysters or in seawater samples can assist in the definition of a Lethal Dose (LD) 50 and gain information in the experiments conducted to reproduce the viral infection. The main objectives of the present study were (i) the development/optimization of a PMA PCR technique for OsHV-1 detection using the best quantity of PMA and verifying its effectiveness through heat treatment, (ii) the definition of the percentage of undamaged capsids in four different tissue homogenates prepared from infected Pacific oysters and (iii) the approach of a LD50 during experimental viral infection assays on the basis of a number of undamaged capsids. Although the developped PMA PCR technique was unable to determine OsHV-1 infectivity in viral supensions, it could greatly improve interpretation of virus positive results obtained by qPCR. This technique is not intended to replace the quantification of viral DNA by qPCR, but it does make it possible to give a form of biological meaning to the detection of this DNA.
摘要：
流疱疹病毒1（OsHV-1）已从疱疹病毒科分类为Malacoherpesviridae科。OsHV-1是太平洋牡蛎传染性病毒性疾病的病原体，C.gigas,也影响其他双壳类动物。报告的与病毒感染相关的死亡率在不同地点和国家之间差异很大，取决于受影响种群的年龄。自2008年以来，欧洲和澳大利亚和新西兰的其他变体已经报道了一种称为μVar的变体。这些变体被认为是影响C.gigas的大规模死亡事件的主要病原体。目前还没有确定的细胞系允许检测感染性OsHV-1。在这种情况下，为了定量“未受损”衣壳，开发了一种单叠氮丙啶（PMA）PCR技术。该方法对于探索病毒感染性是有意义的。能够量化从感染的牡蛎或海水样品中制备的组织匀浆中获得未损坏的衣壳（不仅是病毒DNA的量）的病毒颗粒，可以帮助定义致死剂量（LD）50，并在进行的实验中获得信息重现病毒感染。本研究的主要目标是（i）开发/优化PMAPCR技术，用于使用最佳量的PMA检测OsHV-1，并通过热处理验证其有效性，（ii）定义了由受感染的太平洋牡蛎制备的四种不同组织匀浆中未受损衣壳的百分比，以及（iii）在实验病毒感染测定过程中基于许多未受损衣壳的LD50方法。尽管开发的PMAPCR技术无法确定OsHV-1在病毒抑制中的感染性，它可以大大改善qPCR获得的病毒阳性结果的解释。该技术不旨在通过qPCR代替病毒DNA的定量，但它确实可以为这种DNA的检测提供一种生物学意义。
