Coxsackievirus-B4 Infection Can Induce the Expression of Human Endogenous Retrovirus W in Primary Cells

Figures

• 

  Figure 1. Cultures of primary human pancreatic ductal cells infected with Coxsackievirus B4 (CV-B4). (a) Pancreatic primary cells were obtained from five brain-dead donors. (b) Cultured cells infected with CV-B4 E2 or CV-B4 JVB (multiplicity of infection (MOI): 0.1) and observed under an inverted microscope (magnification ×100) after 48 h of incubation. (c) Viability of the cultured cells assessed using Orangu®. Optical density values are normalized using the viability value of uninfected cells (mock = 100%). (d) The level of infectious viral particles in cultured supernatants harvested 6 and 48 h post-inoculation at MOI 0.1 determined by titration on HEp-2 cells. Results are expressed as TCID50/mL. Means ± SD of log-transformed values of five independent experiments are shown.

• 

  Figure 2. Human Endogenous Retrovirus W Envelope (HERV-W ENV) mRNA expression in primary human pancreatic ductal cells infected by Coxsackievirus B4 (CV-B4). Pancreatic primary cells obtained from five brain-dead donors were infected with CV-B4 E2 (multiplicity of infection (MOI) 0.01, 0.1 or 1). Total RNA was extracted from the cells with TriReagent, and the level of intracellular HERV-W ENV mRNA in each culture was assessed using RT-qPCR normalized with the level of beta-actin mRNA.Each result is the mean of two or three determinations; furthermore, each RT-PCR datum is the mean of two technical duplicates. The relative expression of HERV-W ENV mRNA in CV-B4 E2 infected cultures compared with that in mock-infected cultures was calculated using the 2−ΔΔCt method. (a) The individual relative expression of HERV-W ENV for each culture is shown. ND: not done due to an insufficient number of cells. (b) The maximum individual relative expression of HERV-W ENV for each culture is shown. The dotted line indicates a relative expression of 2.

• 

  Figure 3. Levels of Human Endogenous Retrovirus W Envelope (HERV-W ENV) protein in primary pancreatic ductal cells infected by Coxsackievirus B4 (CV-B4). The levels of intracellular HERV-ENV protein in ductal cells obtained from two brain-dead donors were evaluated using capillary automated Western blot (Simple Western technology, ProteinSimple®). The cultures were inoculated with CV-B4 E2 at multiplicity of infection 0.1 and 1. Total protein lysates were analyzed on a 66-440kDa separation matrix, and HERV-W Env was detected using GN_mAb_Env01 antibody. (a) Electrophoregrams provided the migration profile of the target, and the intensity of the specific signal was precisely quantified using the area under curve (AUC) determination. (b) Signal quantification of the main peak corresponding to the monomeric form of HERV-W Env not carrying post-translational modifications (black arrow) was presented in a digital Western blot. (c) Results are expressed as the relative quantity of ENV in infected cultures compared with the non-specific background signal in mock-infected cultures. Results are presented as the mean ± SD of two independent experiments. The dotted line indicates the non-specific…

• 

  Figure 4. Relative expression of Human Endogenous Retrovirus W Envelope (HERV-W ENV) and enteroviral RNA in peripheral blood mononuclear cells (PBMCs) after inoculation with coxsackievirus B4 (CV-B4). PBMCs were isolated from buffy coats of six healthy donors and inoculated with CV-B4 E2 at multiplicity of infection 1 or with CV-B4 E2 mixed with human immune serum (dilution 1/1000), followed by incubation for 4 h. Thereafter, the cell layers were rinsed three times and incubated overnight. The cell layers were then washed once to eliminate non-adherent cells, and adherent cells were recovered to determine the level of HERV-W ENV mRNA and enteroviral RNA using RT-qPCR after normalization with beta-actin mRNA expression. For each condition, the result, which is the mean of two determinations, is the relative expression of the marker compared with the control calculated using the 2–∆∆Ct method. Relative expression of HERV-W ENV mRNA in PBMCs from (a) donors 1, 3, 5, and 6 (mean ± SD), (b) donor 2, and (c) donor 4. (d) Individual representation of relative expression of enteroviral RNA in PBMCs of donors 1–6.

• 

  Figure 5. Infection of human primary macrophage cultures by Coxsackievirus B4 (CV-B4). Peripheral blood mononuclear cells from 13 healthy donors were isolated as previously described and cultured for 24 h. Thereafter, floating cells were removed to obtain a monocyte-enriched culture that was treated with macrophage-colony stimulating factor. Seven days later, the cultures were infected with CV-B4 E2 or CV-B4 JVB (multiplicity of infection: 1); (a) 24 and 48 h post-infection, the cellular viability was measured using the Orangu® viability assay, and results are expressed as the percentage of viability compared with controls (Mean ± SD, n = 13). (b) The supernatants are harvested 6 and 48 h post-infection to evaluate the level of infectious particles by titration, and the results are expressed as TCID50/mL; mean ± SD of log-transformed values are shown (n = 13). The dotted line indicates the threshold of the viral titration method (log 101.5 TCID50/mL).

• 

  Figure 6. Individual representation of Human Endogenous Retrovirus W Envelope (HERV-W ENV) mRNA expression in macrophages infected by Coxsackievirus B4 (CV-B4). Macrophages from 13 healthy donors were prepared as previously described in the legend of Figure 5. Total RNA was extracted from the cells 6, 24, and 48 h post infection with TriReagent, and the level of intracellular HERV-W ENV mRNA in each culture was assessed in duplicate using RT-qPCR normalized with beta-actin mRNA expression. Each result is the mean of two or three determinations; furthermore, each RT-PCR datum is the mean of two technical duplicates. The relative expression of HERV-W ENV mRNA in CV-B4 E2-infected cultures compared with that in mock-infected cultures is calculated using the 2-ΔΔCt method. (a) The individual relative expression of HERV-W ENV for each culture is shown. ND: not done due to an insufficient number of cells. (b) The maximum individual relative expression of HERV-W ENV for each culture is shown. The dashed line represents a relative expression of 2.