BACKGROUND
ostreid herpesvirus type  <dig> , the causative agent of major economic losses in the pacific oyster industry, is a member of the family malacoherpesviridae from the order herpesvirales
<cit> .
it was in particular identified as the main cause of the massive mortality outbreaks of c. gigas spat recently reported in europe, australia and new zealand  <cit> .
within this context, the control of oshv- <dig> infection is considered as a key element to maintain the competitiveness and to increase the sustainability of oyster industry.
however, pacific oysters as well as all other marine molluscs have unique features in terms of health management  <cit> .
not only do they live in open marine waters, directly exposed to pathogen infection but they also lack an adaptive immune system.
vaccination is therefore not an option to protect them against virus infection.
as a consequence, most strategies currently used for other farmed animal species  cannot be directly applied to pacific oysters.
one of the few applicable and promising approaches to limit the harmful effect of pathogens in oyster production is, selective breeding  <cit> .
in animal populations, susceptibility to virus infection and the modulation of disease progression display inter-individual variability .
the study of host factors that control disease susceptibility relies on the multi-approach analysis of individuals demonstrating a differential response to oshv- <dig> exposure/infection.
this type of data will facilitate the identification of new targets with the potential to be integrated in innovative selection strategies.
genes that confer resistance to oshv- <dig> may indeed be revealed by combining the “top-down” qtl approach to identify target genomic regions, with “bottom-up” transcript profiling.
a qtl analysis for oshv- <dig> dna detection and quantification that was previously performed on five f <dig> full-sib families highlighted the relevance of further studying their respective genetic controls  <cit> .
oyster gene expression has already been studied , particularly since the oyster genome was sequenced  <cit> .
however, most studies relied on the use of oysters collected in the field which are subjected to multiple stressors and pathogens  <cit> .
in addition, only three studies reported oshv- <dig> gene expression while the oshv-1 cycle has never been described at a molecular level .
the aim of our study was to determine the genetic basis of differential susceptibility to virus infection among pacific oysters.
to overcome the variability of environmental factors, we reproduced the viral infection under controlled laboratory conditions.
sixteen pacific oyster families were initially challenged with the virulent variant of oshv- <dig>  μvar  <cit> .
survival rates were evaluated five days post-injection and used as a measure of their susceptibility to oshv- <dig>  in a second step, two pacific oyster families with contrasted susceptibility were selected for transcript profiling of  <dig> virus and five oyster immune related genes.
RESULTS
screening pacific oyster families for oshv- <dig> susceptibility
two independent experimental infection trials were performed to strengthen the results in terms of survival rates of the  <dig> bi-parental families of pacific oysters that were challenged.
after infection, mortality was monitored daily for a period of 120 hours.
control batches systematically displayed 100% survival rates.
results showed that susceptibility to oshv- <dig> was different among the tested oyster families .
survival rates were 2 ±  <dig> % for family “a” and 90 ±  <dig> % for family “p” 120 hours post infection  .
based on their survival rates and wilcoxon test  , families a and p were selected to represent the most extreme performances in terms of susceptibility to oshv- <dig>  a typifies the extremely sensitive family and p the less sensitive family with regard to infection by oshv- <dig> .figure  <dig>
survival of  <dig> families of
crassostrea gigas
oysters at 120 hours post infection during oshv- <dig> challenges.
two assays were conducted in triplicates for each family .
for all  <dig> families, 100% of survival was reported among negative controls injected with 100 μl of artificial seawater.
survival rates of the challenged families and identification of groups of families with significantly distinct performances
groups based on the daily monitoring of mortality during 120 hours post infection .
lowercase letters design a family group significantly different from all the others.
all families were tested by pairs and several groups were identified.
comparing two families with contrasted susceptibility to oshv- <dig> infection
the respective mean survival rates of families a and p were 37% and 95% at  <dig> hpi and 0% and 90% at  <dig> hpi .
at later time points, family a was entirely dead whereas the survival rate was stable  for family p .
as indicated previously, the survival rate was 100% for oyster controls.figure  <dig>
kaplan-meier survival curves of two families of
crassostrea gigas
during infection with oshv- <dig>  for the third experiment, one tank was used .
for the two families, 100% of survival was reported among negative controls injected with 100 μl of artificial seawater.
virus dna was initially detected at  <dig> hpi for family a and  <dig> hpi for family p .
the mean amounts of viral dna increased gradually during the course of infection in family a until all oysters died, whereas it increased up to  <dig> hpi and decreased until  <dig> hpi in family p .
viral dna amounts were overall lower in family p than in family a .
in family a, individual amounts of oshv- <dig> dna ranged from  <dig> to  <dig>  × 10°,  <dig> to  <dig>  ×  <dig>   <dig> to  <dig>  ×  <dig>  and  <dig>  ×  <dig> to  <dig>  ×  <dig> viral dna copies per ng of total dna at  <dig> hpi,  <dig> hpi,  <dig> hpi and  <dig> hpi, respectively.
in family p, individual amounts of oshv- <dig> dna ranged from  <dig> to  <dig>  ×  <dig>   <dig> to  <dig>  ×  <dig>   <dig> to  <dig>  ×  <dig> and  <dig> to  <dig>  ×  <dig> viral dna copies per ng of total dna at  <dig> hpi,  <dig> hpi,  <dig> hpi and  <dig> hpi, respectively.
no virus dna was detected in oysters injected with artificial seawater.figure  <dig>
virus dna detection curves by real time quantitative pcr in oyster from families a and p after injection oshv- <dig> .
in both families, a result of  <dig> dna copies/ng of total dna was reported for negative controls injected with 100 μl of artificial seawater.
error bars represent ± standard deviation.
detection and expression of oshv- <dig> genes in a and p families
the level of expression in mantle tissue of  <dig> oshv- <dig> genes was studied in both families  by real-time pcr.
these particular genes were selected based on literature data and/or their specific function  <cit> .
the full list of oshv- <dig> genes is available in segarra et al.
<cit> .
normalized expression levels were calculated for each individual at each time point in both families.
two heatmaps were generated .
the most striking finding was the existence of different levels of gene expression between both families.
all  <dig> orfs were significantly over-expressed  in family a in comparison to family p, particularly at  <dig> hpi .
moreover, initial detection of virus transcripts was performed as early as  <dig> hpi in family a but did not occur until  <dig> hpi in family p. in family p, expression levels peaked at  <dig> and  <dig> hpi before and decreased at  <dig> hpi.
differences in terms of expression levels were observed between individuals belonging to the same family and collected at identical time points.
no viral transcript was detected at  <dig> hpi in either family.virus transcripts were grouped by the euclidean method to produce a dendrogram comparing the expression levels of all  <dig> orfs .
three clusters were observed for each family.
cluster  <dig> corresponds to high expression levels, regrouped  <dig> genes in family a and  <dig> genes in family p .
cluster  <dig>  with medium expression levels, comprised  <dig> genes in family a and  <dig> genes in family p, .
the third and last cluster included three genes in family a and two genes in family p and was characterized by low expression levels .
significant differences were observed between the three clusters  in family a whereas no difference was detected in family p for these clusters  .
however, 82% of the analyzed orfs  were classified in the same cluster in both families:  <dig> genes belonged to cluster  <dig> ;  <dig> genes to cluster  <dig>  and two genes to cluster  <dig> .
in both families, three viral inhibitor of apoptosis  genes  out of the four ipas that were analysed pertained to cluster  <dig> .figure  <dig>
heatmap illustrating the level of expression of  <dig> oshv- <dig> genes at different post infection time points in  family a and  family p. colors represent the fold change.
blue color  was used to represent the lack of virus detection, whereas red color  was employed for highly expressed genes.
each row corresponds to one of  <dig> viral genes and each column represents an individual .
the five groups/families of genes were: Ο: putative protein; □: enzyme; ■ ring finger protein; ★: inhibitor apoptosis and ♦: membrane protein.
the full list of genes is available in segarra et al.
<cit> .
three clusters were generated for each family: c <dig>  c <dig> and c <dig>
means of viral gene expression by clusters for each family.
different lowercase letters indicate significant differences  between clusters.
error bars represent ± standard deviation.
relative quantification of host gene expression in a and p families
in the third experiment, a significant increase or decrease in mrna levels was reported for ifi <dig>  myd <dig>  iap and ikb <dig> at several times post injection in challenged oysters from both families as compared with negative controls .
in family a, ifi <dig> gene expression was up-regulated at  <dig> hpi in infected oysters  compared to control oysters  .
in family p, ifi <dig> transcript level  was up-regulated from  <dig> hpi to  <dig> hpi and  <dig> hpi  comparatively to the control  .
in family a, the expression level of myd <dig> increased significantly  at  <dig> hpi and  <dig> hpi in infected oysters  comparatively to its level of expression in negative control oysters  .
in family p, myd <dig> transcript levels decreased at  <dig> hpi  and subsequently increased at  <dig> hpi  as compared with their level in negative control oysters .
however, myd <dig> transcript levels were similar in infected and negative control oysters at  <dig> and  <dig> hpi.
for the iap oyster gene, transcript levels increased significantly  at  <dig> hpi in infected oysters  versus controls  of family a. in family p, this level was also up-regulated at  <dig> and  <dig> hpi in infected oysters  versus control oysters   .
for ikb <dig> gene, the number of transcripts increased at  <dig> hpi in infected oysters in both a and p families  .for the gly gene, no significant change was observed between infected and control oysters in family a. in family p, the gly transcript level  was down-regulated at  <dig> hpi and  <dig> hpi  in infected oysters compared to controls .
then gly transcript levels were up-regulated at  <dig> hpi in infected oysters versus controls .figure  <dig>
relative expression of three selected genes  in family a and family p, determined by real time pcr.
myd88: myeloid differentiation factor  <dig>  ikb2: inhibitor of nuclear factor kappab kinase beta, and ifi44: interferon induced protein  <dig>  results at  <dig>   <dig>   <dig>   <dig>   <dig> and 144 hours post-infection.
expression levels were normalized to ef .
a value =  <dig> is arbitrarily assigned to controls.
*significant difference of gene expression compared to controls by mann–whitney test .
relative expression of two selected genes  in family a and family p, determined by real time pcr.
gly: glypican and iap: inhibitor of apoptosis.
results at  <dig>   <dig>   <dig>   <dig>   <dig> and 144 hours post-infection.
expression levels were normalized to ef .
a value =  <dig> is arbitrarily assigned to controls.
*significant difference of gene expression compared to controls by mann–whitney test .
throughout the experiment, the relative expression of these genes did not follow a consistent pattern as they were alternatively up-regulated or down-regulated.
this is particularly noticeable in the case of myd <dig>  ifi <dig> and gly.
using spearman’s test, a rank correlation was determined between the expression of oyster genes and viral dna amounts.
expression levels of myd <dig> , ikb <dig> , ifi <dig>  and iap  were positively correlated with viral dna amount in family a. in family p, a positive correlation was observed only for ifi <dig>  and iap  .table  <dig>
viral load and correlation with the levels of expression of four selected in family a and family p
p value
p value
in order to compare oyster gene expression levels between families a and p, an rf ratio was calculated .
our results show differences concerning the expression level of each gene during experimental challenge in controls in families a and p. nevertheless, at  <dig> hpi down-regulation of five genes was reported in infected oysters from family a in comparison with family p. at  <dig> hpi, expression levels were similar in both families.
at  <dig> hpi myd <dig> were strongly up-regulated and gly was down-regulated in infected animals belonging to family a compared to family p. finally at  <dig> hpi, myd <dig> and gly were up-regulated in family a as compared with family p .table  <dig>
transcript ratios for each studied gene in families a and p  at different time points prior to the onset of mortality
discussion
the objective of the present study was to compare the susceptibility of pacific oyster families towards experimental infection by oshv- <dig> and to further analyze the basis for contrasted susceptibility in two particular families.
in this study, the injection protocol was selected in order to obtain a synchronous infection in all animals at the same time.
in our experiments, survival at 120 hours post injection  was highly variable among the  <dig> tested families and remained diverse at  <dig> hpi between families a and p. this result suggests great variability of susceptibility to the viral infection in experimental conditions  <cit> .
sauvage et al.
<cit>  previously suggested that a significant genetic component exists for susceptibility to the viral infection.
oshv- <dig> dna amounts increased sharply until  <dig> hpi in family a and to a lesser extent in family p until  <dig> hpi.
the amount of detected oshv- <dig> dna decreased at  <dig> hpi in family p. the peak of viral dna copy number preceded the peak of mortality for both families.
these results suggest that the virus replicated better in family a, which was affected by the highest mortality rates.
in family p, mortality rates remained low but oshv- <dig> was able to replicate.
notwithstanding the development of an active infection some individuals belonging to this family appeared to recover from the infection.
conversely, verrier et al.
<cit>  showed that resistant cell lines obtained from rhabdovirus-infected fish clones did not support virus production whereas strong virus production took place in a highly susceptible cell line.
on the contrary, purcell et al.
<cit>  reported results that are consistent with those observed in the present study, as resistant rainbow trout families showed lower infectious hematopoietic necrosis virus  replication, particularly at very early stages of infection.
others authors investigated the genetic basis underlying contrasted susceptibility to marek’s disease virus  in chicken through experimental trials  <cit> .
heatmap approach was selected to compare the global viral gene expression profiles for the  <dig> selected oshv- <dig> genes in infected pacific oysters from families a and p.
although mortality rates remained very low for family p, virus gene transcripts were detected.
expression levels were clearly different between family a and family p. cluster studies showed that viral orfs were over-expressed in family a as compared with family p during the course of time.
however, most of the analyzed viral genes  had a similar expression profile in both families.
three different expression profiles were observed in both families: low, moderate and high expression.
among the latter, viral iap genes  were identified, suggesting that oshv- <dig> may actively manipulate host apoptosis in order to multiply itself.
interestingly, in oysters from the family demonstrating the highest susceptibility to the viral infection  all  <dig> orfs were up expressed at  <dig> hpi in comparison to the less susceptible one .
as for virus dna detection, expression of gene transcripts confirmed that oshv- <dig> was able to replicate in oysters from family p to a lesser extent than in oysters from family a. although virus infection effectively developed in pacific oysters from family p, most animals appeared to recover from the infection.
moreover, a decrease in the amount of virus transcripts until they became undetectable at  <dig> hpi was recorded in family p. this result suggests that the virus may enter a persistence/latency phase as described in vertebrate herpesviruses.
however, 70% of oshv- <dig> genes encode putative proteins with unknown function, which makes the study of virus interactions and of the viral cycle particularly difficult.
other studies would be necessary to further unravel the development of the cycle of oshv- <dig>  including functional genomics analysis.
our data also shows high variability of virus gene expression in both oyster families.
this could be explained by the involvement of more than one oshv- <dig> genotype in the infectious process.
however, partial sequencing and genotyping of six viral microsatellite loci indicated the presence of a unique oshv- <dig> genotype , in both families after the onset of viral infection .
the observed variability may therefore be interpreted as the presence in each oyster family of individuals with different degrees of susceptibility to the same oshv- <dig> genotype.
these results confirm a genetic basis for susceptibility to oshv- <dig> infection in the pacific oyster  <cit> .
moreover, they suggest that oysters belonging to family p, the less susceptible one, could be able to circumvent infection by oshv- <dig> after experimental inoculation of the virus challenge.
in this family, a phase of active virus replication took place, as demonstrated by the initial increase of viral dna and rna.
ensuing this phase, virus dna and rna amounts decreased rapidly suggesting that the host immune defense had been activated and that it might be effective to limit virus multiplication  <cit> .
to explore this hypothesis, the expression of immune related genes  <cit>  was also monitored by real time pcr.
the expression levels of five host genes , interferon induced protein  <dig> , glypican , inhibitor of nuclear factor kappab kinase beta , inhibitor of apoptosis ) were thus analyzed in families a and p. in our study, transcript ratios shown small differences  in control oysters suggesting a family effect on the gene expression.
nevertheless, this ratio was highly different in infected oysters, especially at  <dig> and  <dig> hpi for myd <dig>  it was previously shown that the expression of three of these genes  in c. gigas haemocytes is modulated after a contact with oshv- <dig>  <cit> .
in the case of ikb <dig>  decreased abundance of its transcripts was associated with a certain level of resistance to summer mortality  <cit> .
finally, an inhibitor of apoptosis  was selected for this study due to the fact that it could play a pivotal role in the pacific oyster immune defense  <cit> .
myd <dig> transcripts were up-regulated at  <dig> hpi in infected pacific oysters from both families, although up-regulation was higher in family a .
in this family, the expression level of myd <dig> was positively correlated with viral dna amounts in family a  whereas no significant correlation was detected in family p .
myd <dig> is a universal adapter for the tlr/il-1r family .
it has been described in many species including humans  <cit> , fish  <cit> , drosophila  <cit> , and molluscs  <cit> .
several studies demonstrated that myd88-deficient mice were susceptible to hsv- <dig> infection  <cit> .
the signaling pathway via myd <dig> is initiated to activate nuclear factor-kappa b , c-jun nh <dig> terminal kinase  and mitogen-activated proteins kinase   <cit> .
ikb <dig> transcripts showed significant up-regulation only at  <dig> hpi in both families and expression levels were almost identical in both families .
nevertheless, the positive correlation of the expression level of ikb <dig> and viral dna amounts was only observed in family a .
ikb <dig> is a member of nf-kappab signal pathway and plays an important role in regulating the innate immune response of invertebrates.
zhang et al.
<cit>  described ikb <dig> mrna up-regulation in hemocytes at different time-points  after contact with vibrio alginolyticus in pearl oyster.
over-expression of both myd <dig> and ikb <dig> genes suggests a crucial role of the nf-kappab signal pathway in virus recognition and cell activation  <cit> .
some viruses, such as the african swine fever virus   <cit> , hiv  <cit>  or ebv  <cit>  have evolved strategies to interfere with nf-kb activation in order to evade the immune response.
however, viruses can also activate nf-kb to block apoptosis and prolong survival of the host cell in order to gain time for replication and increase the production of viral progeny  <cit> .
although myd <dig> is reported as a key element in the activation of immunity, over-expression of this gene appeared higher in pacific oysters belonging to family a, which is more susceptible to oshv- <dig> infection.
in this context, the over expression of myd <dig> could be interpreted more as a marker of infectious processes and viral replication than that of an effective antiviral response.
gagnaire et al.
<cit>  previously suggested that gene over-expression in pacific oysters could lead to tissue injury and thus result in higher mortality rates.
defense mechanisms may play a key role in pathogenesis as they induce cell and tissue damages  <cit> .
here, ifi <dig> was highly up-regulated in both families and continued to increase in family p at  <dig> hpi.
the detection of increasing levels of ifi <dig> transcripts in oysters from this family between  <dig> hpi and  <dig> hpi was thus concomitant with the detection of decreasing amounts of oshv- <dig> dna and rna by real-time pcr.
nevertheless, the analysis of data collected throughout the entire experiment reveals an overall positive correlation between the expression of this gene and viral dna amounts in both families .
this result suggests that ifi <dig> may be a key element for effective antiviral defense against infection by oshv- <dig> infection as it provides pacific oysters with the ability to circumvent the virus proliferation.
ifi <dig> gene is a member of the ifns  inducible gene family and may function as a mediator of antiviral activity against hepatitis c or d virus infections through interferons  <cit> .
however, the role of ifi <dig> in antiviral immune response remains unclear.
the glypican transcript level decreased significantly  <dig> hpi in family p whereas no change was observed in family a as shown by the rf value.
glypicans belong to a family of heparan sulfate proteoglycans that are linked to the cell surface by a glycosylphosphatidylinositol  anchor  <cit> .
heparan sulfate proteoglycans may be used by different viruses including herpesviruses as cellular receptors  <cit> .
binding of a viral protein to heparan sulfate is the first step in a cascade of interactions between viruses and cells that is required for viral entry into the cells and the initiation of infection.
cho cells treated with heparinases that prevent heparan sulfate biosynthesis have reduced capacity to bind hsv- <dig> and are at least partially resistant to hsv- <dig> infection  <cit> .
therefore, our results suggests that reduced glypican levels in family p may obstruct or hinder to some extent oshv- <dig> entry in host cells and contribute to this family decreased susceptibility to viral infection.
iap cellular transcripts were respectively up-regulated in families a and p between  <dig> hpi and  <dig> hpi and between  <dig> hpi and  <dig> hpi.
our study showed a positive correlation between the level of iap transcripts and the amounts of viral dna in both families .
iap proteins are conserved throughout animal evolution and can block apoptosis.
apoptosis is one of the major mechanisms of anti-viral response  <cit> .
over-expression of iap could be a reaction to the apoptotic process induced by oshv- <dig> infection.
nevertheless, some pathogens enter a cell and inhibit apoptosis to increase their life span  <cit> .
all the members of the gamma-herpesvirus family encode genes that are able to inhibit apoptosis including, one or two bcl- <dig> homologues  <cit> .
moreover, studies have shown that hsv- <dig> require gene us <dig> to protect itself from apoptosis induced by certain stimuli  <cit> .
finally, the contribution of apoptosis to the pathogenesis of west nile virus  encephalitis has been demonstrated by several studies  <cit> .
CONCLUSIONS
this work is the first step towards elucidating the interactions between oshv- <dig> and its host, the pacific oyster .
it also represents the first study of viral gene expression in oyster families with contrasted susceptibility to virus infection.
in two particular families, contrasted susceptibility was associated with significantly different amounts of virus dna and rna, which suggests that susceptibility of pacific oysters to oshv- <dig> may be affected by genetic variations.
previous studies support the hypothesis of a genetic basis underlying resistance to oshv- <dig> infection in the pacific oyster.
in addition, the observed differences in terms of susceptibility to infection by oshv- <dig> were associated with differences in host gene expression profiles.
finally, the present study is also the first to show positive correlations between viral dna amounts and the expression of selected oyster genes.
these results suggest that the up-regulation of immune related genes may prevent virus replication and open new questions regarding the persistence and/or latency of the virus within the host.
genes of interest could be further evaluated in oyster breeding programs to determine if they confer genetic resistance to oshv- <dig>  such an integrative strategy could be applied to other infectious diseases.

 ostreid herpesvirus type, the causative agent of major economic losses in the pacific oyster industry, is a member of the family malacoherpesviridae from the order herpesvirales. it was in particular identified as the main cause of the massive mortality outbreaks of c. gigas spat recently reported in europe, australia and new zealand. within this context, the control of oshv- infection is considered as a key element to maintain the competitiveness and to increase the sustainability of oyster industry. however, pacific oysters as well as all other marine molluscs have unique features in terms of health management. not only do they live in open marine waters, directly exposed to pathogen infection but they also lack an adaptive immune system. vaccination is therefore not an option to protect them against virus infection. as a consequence, most strategies currently used for other farmed animal species cannot be directly applied to pacific oysters. one of the few applicable and promising approaches to limit the harmful effect of pathogens in oyster production is, selective breeding. in animal populations, susceptibility to virus infection and the modulation of disease progression display inter-individual variability. the study of host factors that control disease susceptibility relies on the multi-approach analysis of individuals demonstrating a differential response to oshv- exposure/infection. this type of data will facilitate the identification of new targets with the potential to be integrated in innovative selection strategies. genes that confer resistance to oshv- may indeed be revealed by combining the “top-down” qtl approach to identify target genomic regions, with “bottom-up” transcript profiling. a qtl analysis for oshv- dna detection and quantification that was previously performed on five f full-sib families highlighted the relevance of further studying their respective genetic controls. oyster gene expression has already been studied, particularly since the oyster genome was sequenced. however, most studies relied on the use of oysters collected in the field which are subjected to multiple stressors and pathogens. in addition, only three studies reported oshv- gene expression while the oshv-1 cycle has never been described at a molecular level. the aim of our study was to determine the genetic basis of differential susceptibility to virus infection among pacific oysters. to overcome the variability of environmental factors, we reproduced the viral infection under controlled laboratory conditions. sixteen pacific oyster families were initially challenged with the virulent variant of oshv- μvar. survival rates were evaluated five days post-injection and used as a measure of their susceptibility to oshv- in a second step, two pacific oyster families with contrasted susceptibility were selected for transcript profiling of virus and five oyster immune related genes.  screening pacific oyster families for oshv- susceptibility two independent experimental infection trials were performed to strengthen the results in terms of survival rates of the bi-parental families of pacific oysters that were challenged. after infection, mortality was monitored daily for a period of 120 hours. control batches systematically displayed 100% survival rates. results showed that susceptibility to oshv- was different among the tested oyster families. survival rates were 2 ±  % for family “a” and 90 ±  % for family “p” 120 hours post infection. based on their survival rates and wilcoxon test, families a and p were selected to represent the most extreme performances in terms of susceptibility to oshv- a typifies the extremely sensitive family and p the less sensitive family with regard to infection by oshv-.figure survival of families of crassostrea gigas oysters at 120 hours post infection during oshv- challenges. two assays were conducted in triplicates for each family. for all families, 100% of survival was reported among negative controls injected with 100 μl of artificial seawater. survival rates of the challenged families and identification of groups of families with significantly distinct performances groups based on the daily monitoring of mortality during 120 hours post infection. lowercase letters design a family group significantly different from all the others. all families were tested by pairs and several groups were identified. comparing two families with contrasted susceptibility to oshv- infection the respective mean survival rates of families a and p were 37% and 95% at hpi and 0% and 90% at hpi. at later time points, family a was entirely dead whereas the survival rate was stable for family p. as indicated previously, the survival rate was 100% for oyster controls.figure kaplan-meier survival curves of two families of crassostrea gigas during infection with oshv- for the third experiment, one tank was used. for the two families, 100% of survival was reported among negative controls injected with 100 μl of artificial seawater. virus dna was initially detected at hpi for family a and hpi for family p. the mean amounts of viral dna increased gradually during the course of infection in family a until all oysters died, whereas it increased up to hpi and decreased until hpi in family p. viral dna amounts were overall lower in family p than in family a. in family a, individual amounts of oshv- dna ranged from to × 10°, to × to × and × to × viral dna copies per ng of total dna at hpi, hpi, hpi and hpi, respectively. in family p, individual amounts of oshv- dna ranged from to × to × to × and to × viral dna copies per ng of total dna at hpi, hpi, hpi and hpi, respectively. no virus dna was detected in oysters injected with artificial seawater.figure virus dna detection curves by real time quantitative pcr in oyster from families a and p after injection oshv-. in both families, a result of dna copies/ng of total dna was reported for negative controls injected with 100 μl of artificial seawater. error bars represent ± standard deviation. detection and expression of oshv- genes in a and p families the level of expression in mantle tissue of oshv- genes was studied in both families by real-time pcr. these particular genes were selected based on literature data and/or their specific function. the full list of oshv- genes is available in segarra et al.. normalized expression levels were calculated for each individual at each time point in both families. two heatmaps were generated. the most striking finding was the existence of different levels of gene expression between both families. all orfs were significantly over-expressed in family a in comparison to family p, particularly at hpi. moreover, initial detection of virus transcripts was performed as early as hpi in family a but did not occur until hpi in family p. in family p, expression levels peaked at and hpi before and decreased at hpi. differences in terms of expression levels were observed between individuals belonging to the same family and collected at identical time points. no viral transcript was detected at hpi in either family.virus transcripts were grouped by the euclidean method to produce a dendrogram comparing the expression levels of all orfs. three clusters were observed for each family. cluster corresponds to high expression levels, regrouped genes in family a and genes in family p. cluster with medium expression levels, comprised genes in family a and genes in family p,. the third and last cluster included three genes in family a and two genes in family p and was characterized by low expression levels. significant differences were observed between the three clusters in family a whereas no difference was detected in family p for these clusters. however, 82% of the analyzed orfs were classified in the same cluster in both families: genes belonged to cluster; genes to cluster and two genes to cluster. in both families, three viral inhibitor of apoptosis genes out of the four ipas that were analysed pertained to cluster.figure heatmap illustrating the level of expression of oshv- genes at different post infection time points in family a and family p. colors represent the fold change. blue color was used to represent the lack of virus detection, whereas red color was employed for highly expressed genes. each row corresponds to one of viral genes and each column represents an individual. the five groups/families of genes were: Ο: putative protein; □: enzyme; ■ ring finger protein; ★: inhibitor apoptosis and ♦: membrane protein. the full list of genes is available in segarra et al.. three clusters were generated for each family: c c and c means of viral gene expression by clusters for each family. different lowercase letters indicate significant differences between clusters. error bars represent ± standard deviation. relative quantification of host gene expression in a and p families in the third experiment, a significant increase or decrease in mrna levels was reported for ifi myd iap and ikb at several times post injection in challenged oysters from both families as compared with negative controls. in family a, ifi gene expression was up-regulated at hpi in infected oysters compared to control oysters. in family p, ifi transcript level was up-regulated from hpi to hpi and hpi comparatively to the control. in family a, the expression level of myd increased significantly at hpi and hpi in infected oysters comparatively to its level of expression in negative control oysters. in family p, myd transcript levels decreased at hpi and subsequently increased at hpi as compared with their level in negative control oysters. however, myd transcript levels were similar in infected and negative control oysters at and hpi. for the iap oyster gene, transcript levels increased significantly at hpi in infected oysters versus controls of family a. in family p, this level was also up-regulated at and hpi in infected oysters versus control oysters. for ikb gene, the number of transcripts increased at hpi in infected oysters in both a and p families.for the gly gene, no significant change was observed between infected and control oysters in family a. in family p, the gly transcript level was down-regulated at hpi and hpi in infected oysters compared to controls. then gly transcript levels were up-regulated at hpi in infected oysters versus controls.figure relative expression of three selected genes in family a and family p, determined by real time pcr. myd88: myeloid differentiation factor ikb2: inhibitor of nuclear factor kappab kinase beta, and ifi44: interferon induced protein results at and 144 hours post-infection. expression levels were normalized to ef. a value =  is arbitrarily assigned to controls. *significant difference of gene expression compared to controls by mann–whitney test. relative expression of two selected genes in family a and family p, determined by real time pcr. gly: glypican and iap: inhibitor of apoptosis. results at and 144 hours post-infection. expression levels were normalized to ef. a value =  is arbitrarily assigned to controls. *significant difference of gene expression compared to controls by mann–whitney test. throughout the experiment, the relative expression of these genes did not follow a consistent pattern as they were alternatively up-regulated or down-regulated. this is particularly noticeable in the case of myd ifi and gly. using spearman’s test, a rank correlation was determined between the expression of oyster genes and viral dna amounts. expression levels of myd, ikb, ifi and iap were positively correlated with viral dna amount in family a. in family p, a positive correlation was observed only for ifi and iap.table viral load and correlation with the levels of expression of four selected in family a and family p p value p value in order to compare oyster gene expression levels between families a and p, an rf ratio was calculated. our results show differences concerning the expression level of each gene during experimental challenge in controls in families a and p. nevertheless, at hpi down-regulation of five genes was reported in infected oysters from family a in comparison with family p. at hpi, expression levels were similar in both families. at hpi myd were strongly up-regulated and gly was down-regulated in infected animals belonging to family a compared to family p. finally at hpi, myd and gly were up-regulated in family a as compared with family p.table transcript ratios for each studied gene in families a and p at different time points prior to the onset of mortality discussion the objective of the present study was to compare the susceptibility of pacific oyster families towards experimental infection by oshv- and to further analyze the basis for contrasted susceptibility in two particular families. in this study, the injection protocol was selected in order to obtain a synchronous infection in all animals at the same time. in our experiments, survival at 120 hours post injection was highly variable among the tested families and remained diverse at hpi between families a and p. this result suggests great variability of susceptibility to the viral infection in experimental conditions. sauvage et al. previously suggested that a significant genetic component exists for susceptibility to the viral infection. oshv- dna amounts increased sharply until hpi in family a and to a lesser extent in family p until hpi. the amount of detected oshv- dna decreased at hpi in family p. the peak of viral dna copy number preceded the peak of mortality for both families. these results suggest that the virus replicated better in family a, which was affected by the highest mortality rates. in family p, mortality rates remained low but oshv- was able to replicate. notwithstanding the development of an active infection some individuals belonging to this family appeared to recover from the infection. conversely, verrier et al. showed that resistant cell lines obtained from rhabdovirus-infected fish clones did not support virus production whereas strong virus production took place in a highly susceptible cell line. on the contrary, purcell et al. reported results that are consistent with those observed in the present study, as resistant rainbow trout families showed lower infectious hematopoietic necrosis virus replication, particularly at very early stages of infection. others authors investigated the genetic basis underlying contrasted susceptibility to marek’s disease virus in chicken through experimental trials. heatmap approach was selected to compare the global viral gene expression profiles for the selected oshv- genes in infected pacific oysters from families a and p. although mortality rates remained very low for family p, virus gene transcripts were detected. expression levels were clearly different between family a and family p. cluster studies showed that viral orfs were over-expressed in family a as compared with family p during the course of time. however, most of the analyzed viral genes had a similar expression profile in both families. three different expression profiles were observed in both families: low, moderate and high expression. among the latter, viral iap genes were identified, suggesting that oshv- may actively manipulate host apoptosis in order to multiply itself. interestingly, in oysters from the family demonstrating the highest susceptibility to the viral infection all orfs were up expressed at hpi in comparison to the less susceptible one. as for virus dna detection, expression of gene transcripts confirmed that oshv- was able to replicate in oysters from family p to a lesser extent than in oysters from family a. although virus infection effectively developed in pacific oysters from family p, most animals appeared to recover from the infection. moreover, a decrease in the amount of virus transcripts until they became undetectable at hpi was recorded in family p. this result suggests that the virus may enter a persistence/latency phase as described in vertebrate herpesviruses. however, 70% of oshv- genes encode putative proteins with unknown function, which makes the study of virus interactions and of the viral cycle particularly difficult. other studies would be necessary to further unravel the development of the cycle of oshv- including functional genomics analysis. our data also shows high variability of virus gene expression in both oyster families. this could be explained by the involvement of more than one oshv- genotype in the infectious process. however, partial sequencing and genotyping of six viral microsatellite loci indicated the presence of a unique oshv- genotype, in both families after the onset of viral infection. the observed variability may therefore be interpreted as the presence in each oyster family of individuals with different degrees of susceptibility to the same oshv- genotype. these results confirm a genetic basis for susceptibility to oshv- infection in the pacific oyster. moreover, they suggest that oysters belonging to family p, the less susceptible one, could be able to circumvent infection by oshv- after experimental inoculation of the virus challenge. in this family, a phase of active virus replication took place, as demonstrated by the initial increase of viral dna and rna. ensuing this phase, virus dna and rna amounts decreased rapidly suggesting that the host immune defense had been activated and that it might be effective to limit virus multiplication. to explore this hypothesis, the expression of immune related genes was also monitored by real time pcr. the expression levels of five host genes, interferon induced protein, glypican, inhibitor of nuclear factor kappab kinase beta, inhibitor of apoptosis ) were thus analyzed in families a and p. in our study, transcript ratios shown small differences in control oysters suggesting a family effect on the gene expression. nevertheless, this ratio was highly different in infected oysters, especially at and hpi for myd it was previously shown that the expression of three of these genes in c. gigas haemocytes is modulated after a contact with oshv-. in the case of ikb decreased abundance of its transcripts was associated with a certain level of resistance to summer mortality. finally, an inhibitor of apoptosis was selected for this study due to the fact that it could play a pivotal role in the pacific oyster immune defense. myd transcripts were up-regulated at hpi in infected pacific oysters from both families, although up-regulation was higher in family a. in this family, the expression level of myd was positively correlated with viral dna amounts in family a whereas no significant correlation was detected in family p. myd is a universal adapter for the tlr/il-1r family. it has been described in many species including humans, fish, drosophila, and molluscs. several studies demonstrated that myd88-deficient mice were susceptible to hsv- infection. the signaling pathway via myd is initiated to activate nuclear factor-kappa b, c-jun nh terminal kinase and mitogen-activated proteins kinase. ikb transcripts showed significant up-regulation only at hpi in both families and expression levels were almost identical in both families. nevertheless, the positive correlation of the expression level of ikb and viral dna amounts was only observed in family a. ikb is a member of nf-kappab signal pathway and plays an important role in regulating the innate immune response of invertebrates. zhang et al. described ikb mrna up-regulation in hemocytes at different time-points after contact with vibrio alginolyticus in pearl oyster. over-expression of both myd and ikb genes suggests a crucial role of the nf-kappab signal pathway in virus recognition and cell activation. some viruses, such as the african swine fever virus, hiv or ebv have evolved strategies to interfere with nf-kb activation in order to evade the immune response. however, viruses can also activate nf-kb to block apoptosis and prolong survival of the host cell in order to gain time for replication and increase the production of viral progeny. although myd is reported as a key element in the activation of immunity, over-expression of this gene appeared higher in pacific oysters belonging to family a, which is more susceptible to oshv- infection. in this context, the over expression of myd could be interpreted more as a marker of infectious processes and viral replication than that of an effective antiviral response. gagnaire et al. previously suggested that gene over-expression in pacific oysters could lead to tissue injury and thus result in higher mortality rates. defense mechanisms may play a key role in pathogenesis as they induce cell and tissue damages. here, ifi was highly up-regulated in both families and continued to increase in family p at hpi. the detection of increasing levels of ifi transcripts in oysters from this family between hpi and hpi was thus concomitant with the detection of decreasing amounts of oshv- dna and rna by real-time pcr. nevertheless, the analysis of data collected throughout the entire experiment reveals an overall positive correlation between the expression of this gene and viral dna amounts in both families. this result suggests that ifi may be a key element for effective antiviral defense against infection by oshv- infection as it provides pacific oysters with the ability to circumvent the virus proliferation. ifi gene is a member of the ifns inducible gene family and may function as a mediator of antiviral activity against hepatitis c or d virus infections through interferons. however, the role of ifi in antiviral immune response remains unclear. the glypican transcript level decreased significantly hpi in family p whereas no change was observed in family a as shown by the rf value. glypicans belong to a family of heparan sulfate proteoglycans that are linked to the cell surface by a glycosylphosphatidylinositol anchor. heparan sulfate proteoglycans may be used by different viruses including herpesviruses as cellular receptors. binding of a viral protein to heparan sulfate is the first step in a cascade of interactions between viruses and cells that is required for viral entry into the cells and the initiation of infection. cho cells treated with heparinases that prevent heparan sulfate biosynthesis have reduced capacity to bind hsv- and are at least partially resistant to hsv- infection. therefore, our results suggests that reduced glypican levels in family p may obstruct or hinder to some extent oshv- entry in host cells and contribute to this family decreased susceptibility to viral infection. iap cellular transcripts were respectively up-regulated in families a and p between hpi and hpi and between hpi and hpi. our study showed a positive correlation between the level of iap transcripts and the amounts of viral dna in both families. iap proteins are conserved throughout animal evolution and can block apoptosis. apoptosis is one of the major mechanisms of anti-viral response. over-expression of iap could be a reaction to the apoptotic process induced by oshv- infection. nevertheless, some pathogens enter a cell and inhibit apoptosis to increase their life span. all the members of the gamma-herpesvirus family encode genes that are able to inhibit apoptosis including, one or two bcl- homologues. moreover, studies have shown that hsv- require gene us to protect itself from apoptosis induced by certain stimuli. finally, the contribution of apoptosis to the pathogenesis of west nile virus encephalitis has been demonstrated by several studies.  this work is the first step towards elucidating the interactions between oshv- and its host, the pacific oyster. it also represents the first study of viral gene expression in oyster families with contrasted susceptibility to virus infection. in two particular families, contrasted susceptibility was associated with significantly different amounts of virus dna and rna, which suggests that susceptibility of pacific oysters to oshv- may be affected by genetic variations. previous studies support the hypothesis of a genetic basis underlying resistance to oshv- infection in the pacific oyster. in addition, the observed differences in terms of susceptibility to infection by oshv- were associated with differences in host gene expression profiles. finally, the present study is also the first to show positive correlations between viral dna amounts and the expression of selected oyster genes. these results suggest that the up-regulation of immune related genes may prevent virus replication and open new questions regarding the persistence and/or latency of the virus within the host. genes of interest could be further evaluated in oyster breeding programs to determine if they confer genetic resistance to oshv- such an integrative strategy could be applied to other infectious diseases. 
however, few studies have been performed to understand and follow viral gene expression, as it has been done in vertebrate herpesviruses. massive mortality outbreaks affecting pacific oyster spat in various countries have been associated with the detection of a herpesvirus called ostreid herpesvirus type. initial results provided information on the virus cycle of oshv- based on the kinetics of viral dna replication and transcription and host defense mechanisms against the virus. in this work, experimental infection trials of c. gigas spat with oshv- were conducted in order to test the susceptibility of several bi-parental oyster families to this virus and to analyze host-pathogen interactions using in vivo transcriptomic approaches. the divergent response of these oyster families in terms of mortality confirmed that susceptibility to oshv- infection has a significant genetic component. two families with contrasted survival rates were selected. moreover, the present study showed a positive correlation between viral dna amounts and the level of expression of selected oyster genes. in the two selected families, the detected amounts of viral dna and rna were significantly different. a total of viral genes and five host genes were monitored by real-time pcr. this result suggests that pacific oysters are genetically diverse in terms of their susceptibility to oshv- infection.
 massive mortality outbreaks affecting pacific oyster spat in various countries have been associated with the detection of a herpesvirus called ostreid herpesvirus type. however, few studies have been performed to understand and follow viral gene expression, as it has been done in vertebrate herpesviruses. in this work, experimental infection trials of c. gigas spat with oshv- were conducted in order to test the susceptibility of several bi-parental oyster families to this virus and to analyze host-pathogen interactions using in vivo transcriptomic approaches.  the divergent response of these oyster families in terms of mortality confirmed that susceptibility to oshv- infection has a significant genetic component. two families with contrasted survival rates were selected. a total of viral genes and five host genes were monitored by real-time pcr. initial results provided information on the virus cycle of oshv- based on the kinetics of viral dna replication and transcription and host defense mechanisms against the virus.  in the two selected families, the detected amounts of viral dna and rna were significantly different. this result suggests that pacific oysters are genetically diverse in terms of their susceptibility to oshv- infection. this contrasted susceptibility was associated with dissimilar host gene expression profiles. moreover, the present study showed a positive correlation between viral dna amounts and the level of expression of selected oyster genes. keywords interactionsostreid herpesvirus 1gene expressionsusceptibilitycrassostrea gigasissue-copyright-statement© the author 2014 