Physiological change under OsHV-1 contamination in Pacific oyster Crassostrea gigas through massive mortality events on fields

Abstract

Background: Massive mortalities have been observed in France since 2008 on spat and juvenile Pacific oysters, Crassostrea gigas. A herpes virus called OsHV-1, easily detectable by PCR, has been implicated in the mortalities as demonstrated by the results of numerous field studies linking mortality with OsHV-1 prevalence. Moreover, experimental infections using viral particles have documented the pathogenicity of OsHV-1 but the physiological responses of host to pathogen are not well known.

Results: The aim of this study was to understand mechanisms brought into play against the virus during infection in the field. A microarray assay has been developed for a major part of the oyster genome and used for studying the host transcriptome across mortality on field. Spat with and without detectable OsHV-1 infection presenting or not mortality respectively were compared by microarray during mortality episodes. In this study, a number of genes are regulated in the response to pathogen infection on field and seems to argue to an implication of the virus in the observed mortality. The result allowed establishment of a hypothetic scheme of the host cell's infection by, and response to, the pathogen.

Conclusions: This response shows a "sensu stricto" innate immunity through genic regulation of the virus OsHV-1 life cycle, but also others biological processes resulting to complex interactions between host and pathogens in general.

Figure 1

Percentage of infected spat according to the viral load in log class. The level of virus is expressed in genomic units (GU) per nanogram of DNA and illustrate viral load at (A) the field oyster growout site and in the (B) sanctuary site (dark grey - CRIC) and in the tank (grey – CAB)) during mortality events observed in the field. The asterisks indicate the sample taken for microarray analysis. For graphic A, the first asterik corresponds to sample infected at 4 viral GU ng^-1 of DNA (BL), and the second refers to samples infected at 14 viralGU ng^-1 of f DNA ( i). Data in B are from spat considered to be uninfected.

Figure 2

Clustering of 249 ESTs obtained after ANOVA analysis with four groups (CAB, CRIC, BL and i – see legend to Figure1) with adjusted Bonferroni and p values< 0,01 made on TMeV 4.6.0 software (Saeed et al., 2003 and 2006). CRIC and i correspond, respectively, to uninfected spat from sanctuary area or from tank. A: Cluster 1 is a major cluster with 126 ESTs and cluster 3 is the smaller cluster with 7 ESTs. BL and CAB correspond to infected spat from oyster field (two levels 1 and 2 log class of viral GU ng^-1 DNA). B: Graphs represent values transcriptional variation of genes in each category.

Figure 3

Sector diagram representing the percentage of the 128 genes by gene function. The list of 128 genes was obtained by comparison between infected or uninfected spat.

Figure 4

Graph representing the profiles obtained from microarray (light grey) and qPCR (dark grey) analyses for twelve genes. Values of qPCR are expressed in reference to EF1alpha. The same spat samples were used for both microrarry and qPCR assays. Conditions are precised in abciss and left axis represent qPCR data in DCt for100 copies of EF1alpha and right axis represent microarray data in fluorescence. R² represent the correlation between microarray and qPCR data. Accession number of the genes used are BAR1: AF075691 (A); Vps53: AM866620 (B); RAC 1: CU983949 (C); PRDM5: CU989501(D); Activin receptor type-2A : AM855334 (E); RABAC: AM867263 (F); DRAP 1 : AM857148 (G); GroupeL1: CU990573 (H); ABPA2 : AM858827 (I); DRG2 : AM859481 (J); Nup54 : AM854457 (K); Cbx1: AM863306 (L).

Figure 5

Hypothetical scheme representing the different steps occuring during viral infection. A: Attachment. B: Membrane fusion with intervention of the actin cytoskeleton and cell adhesion proteins. C: Translocation of capside to the nucleus with Microtubules MT. D: Docking at the nuclear pore: release of viral genome into the nucleus. E: Concatemer. F: Transcription of the viral mRNA. G: Traduction of the viral mRNA in the RER for the capsid proteins and maturation in the Golgi for the envelope proteins. H: Replication. I: Capsid formation. J: Budding to the inner nuclear membrane followed by release of the capsid into the cytoplasm. K: Envelopment of Trans Golgi network vesicule with proteins to form the envelope. L: Fusion of the vesicule with the virion to the plasma membrane to ensure releasing of the mature virion. M and N: hijacking cellular machinery.