The susceptibilities of fish clones and cell lines to VHSV infection are highly correlated.

<p>(A) Kaplan-Meier estimation of survival function for every fish clone. This estimation is calculated from the 9 independent waterborne infections with VHSV corresponding to 5166 infected fish (including data from (49)). (B) Relative risks relative to the B57 clone (R = 1). Relative risks are estimated from the same dataset as above using the “Survival kit” software. The program also computes a chi² test between each clone pair. Letters above each column design a clone significantly different from all the others. All paired tests indicated statistically significant differences between clones. (C) Correlation between risks of fish clones and viral production 96 hours post infection. Linear regression: R = 0.99. (D) Correlation between risks of fish clones and N viral gene expression 4 hours post infection in cell lines. Linear regression: R = 0.85.</p

Early IFN- dependent and independent viral inhibition in B57 cells.

<p>(A): The expression of shIFNφ1 transcript in A22 and B57 cells after VHSV infection was measured by qPCR 4, 8 and 24 hours post infection by VHSV or in mock infected cells (ctrl). mRNA copy numbers were normalized on ß-actin expression (measured ratio of shIFNφ1 mRNA/actin mRNA). The mean of three experiments is shown. (B): Expression of N viral gene in cells after VHSV 07-71 infection at different time. Expression of gene is measured by qPCR after 4, 8 and 24 hours of infection by VHSV 07-71 or without viral infection (ctrl). Gene expression is evaluated relative to ß-actin expression (measured ratio of VHSV N mRNA/actin mRNA). The mean of three experiments is shown. (C): Viral genomic RNA (strand+ plus strand−) was quantified using qPCR. The virus replication was assessed by the ratio of virus genome at 6 h versus 2 h post-inoculation. (D): The mRNA encoding the viral N protein was quantified in parallel and the ratio N mRNA 6 hours post inoculation/N mRNA 2 hours post inoculation is represented.</p

qPCR primers sequences.

<p>qPCR primers sequences.</p

Fibroblastic cell lines from double haploid fish clones show different susceptibilities to VHSV infection.

<p>(A). Fibroblast-like cells from the B57 line. Bar 50 µm. (B): Monolayer destruction 3 days post infection with different MOI of VHSV. Cells were incubated 3 days with the virus inoculum, then fixed and colored with crystal violet. (C) Quantification of CPE after VHSV infection (MOI 1): cells were infected as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033935#s2" target="_blank">Materials and Methods</a>, colored with DAPI 3 days post infection, and nuclei counted using the ImageJ software. Three independent infections were performed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033935#s3" target="_blank">Results</a> are shown as ratios of cell counts in infected wells to cell counts before infection. This ratio may be >1 when cell growth occurs after infection in the absence of cytopathic effect.</p

The resistant phenotype of B57 cells results from combined early IFNφ1 and intrinsic immunity.

<p>(A): Expression of the functional IFNφ1 (shIFNφ1) in cell lines. Gene expression was measured by qPCR 4 hours post infection by VHSV (MOI 1) or in mock infected cells (ctrl). shIFNφ1 transcript copy numbers were normalized on the ß-actin expression (measured ratio of mRNA of interest/ß-actin mRNA). The mean of three experiments is shown. (B): Expression of IFN φ1, MxI and Mx3 interferon induced genes and viral N mRNA in B57 and A22 cell lines. Expression was measured by qPCR 4 hours post infection or in control cells. Higher amount of template was used, allowing detection of the basic expression levels of the different genes. Primers used are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033935#pone-0033935-t001" target="_blank">Table 1</a>.</p

Different resistant phenotypes of cell lines A2 and B57.

<p>(A): viral production in cells infected by VHSV at MOI 4. Two independent infections were performed. Viral titer was estimated by plaque assay on EPC cells in duplicates. (B): CPE after VHSV infection at MOI 1 and MOI 4. Cells were fixed 3 days post infection and colored with crystal violet.</p