Propidium monoazide PCR, a method to determine OsHV-1 undamaged capsids and to estimate virus Lethal Dose 50

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.

Keywords: Lethal Dose 50; Mortality; OsHV-1; PCR; Pacific oyster; Propidium monoazide; Undamaged capsids.

Fig. 1

Optimizing PMA concentration. The 1e+10⁵ value on the X axis corresponds to the nondiluted viral suspension and the 1e+10¹ value to the 1/10 000 dilution. 1A-Test of 3 concentrations of PMA on the virus suspension A at different dilutions (nondiluted to 1/10 000). 1B- Test of different concentrations of PMA and different dilutions of the virus suspension A expressed in delta Ct (Ct with PMA - Ct without PMA).

Fig. 2

Viral DNA detection in the suspension A at different dilutions with or without PMA (500 μM) and with or without exposure to 97 °C for 45 min. The 1e+10⁵ value on the X axis corresponds to the nondiluted viral suspension and the 1e+10¹ value to the 1/10 000 dilution.

Fig. 3

3A-Viral DNA detecton in viral suspensions A–D undiluted with or without PMA (500 µM) and with or without heat treated. 3B- Calculation of the number of intact capsids (expressed as a percentage) in the four tested viral suspension.

Fig. 4

Kaplan-Meier survival curves of Pacific oysters injected with the OsHV-1 suspension A from three different experiments with Log-rank (p = 0.00018). The amount of estimated undamaged viral capsids injected to each series of 10 oysters was declined as follows: absence of undamaged capsids (1/10 000 dilution), 1 to 10 (1/1000 ditution), 11 to 200 (1/100 dilution), 201 to 1000 (1/10) and more than 6000 undamaged capsids (undiluted).