Fig 1.
SDS-PAGE and western blotting of the LCDV cellular receptor-27.8kDa protein.
Lane 1: Molecular mass marker; Lane 2, 3: SDS-PAGE of FG and HINAE cell membrane protein, stained with coomassie bule; Lane 4, 5: SDS-PAGE of FG and HINAE cell cytoplasm protein, stained with coomassie bule; Lane 6, 7: reaction with anti-27.8R MAbs showed only one 27.8 kDa in FG and HINAE cell membrane protein; Lane 8, 9: reaction with anti-27.8R MAbs showed no band in FG and HINAE cell cytoplasm protein; Lane 10, 11: anti-WSSV MAb 1D5 instead of anti-27.8R MAbs served as negative controls.
Fig 2.
Co-localization of LCDV and 27.8R in FG and HINAE cell surface.
FG cells (A) and HINAE cells (B) were exposed to LCDV at 22°C for 2 h and stained with mouse anti-27.8R MAbs and rabbit anti-LCDV serum for detection of 27.8R (green) and LCDV (red) simultaneously. The merged yellow signals (arrows) indicated the co-localization of LCDV and 27.8R protein on cell surface. Cell nuclei were counterstained in blue by DAPI. Scale bar = 20 μm. (a) and (b) were the higher magnification view of the co-localized area in FG and HINAE cells, respectively, scale bar = 5 μm.
Fig 3.
Dose response of LCDV-induced 27.8R expression in FG and HIANE cells.
FG and HINAE Cells were infected with LCDV at a MOI of 0.003, 0.03, 0.3 and 3.0 respectively. The cells without LCDV infection served as control. The cells were sampled at 48 h post infection. Error bars represented standard deviation (SD). Data represented the absorbance value at 405 nm (mean ± SD; n = 3) and were compared by Student’s t test. The asterisk represented the statistical significance (p < 0.05) as compared with the control.
Fig 4.
Dynamics of LCDV copies in FG and HINAE cells post LCDV infection investigated by qPCR.
(A) Standard curve of LCDV MCP qPCR assays. The X-axis showed the positive control plasmid copy number in Log 10 value, and the Y-axis indicated the corresponding cycle threshold (Ct) value. R2: coefficient of determination. (B) Changes of LCDV copies in FG and HINAE cells post LCDV infection. 0 h represented un-infected cells. Error bars represented SD. Data represented the number of LCDV copies per microgram of total DNA in the cell samples (mean ± SD; n = 3).
Fig 5.
Dynamic expression of 27.8R in FG (A) and HINAE (B) cells post LCDV infection detected by sandwich ELISA.
The cells were infected with LCDV at a MOI of 3.0 and sampled at different time points post infection. Error bars represented SD. Data represented the absorbance value at 405 nm (mean ± SD; n = 3) and were compared by Student’s t test. Un-infected cells (0 h) represented the negative control. The asterisk represented the statistical significance (p < 0.05) as compared with the negative control.
Fig 6.
Blocking effect of anti-27.8R MAbs on 27.8R expression.
The cells were pre-incubated with different concentration of anti-27.8R MAbs. Experimental groups were challenged by LCDV at a MOI of 3.0, and the cells without LCDV infection served as control groups. The cells were sampled at 48 h post infection. Error bars represented SD. Data represented the absorbance value at 405 nm (mean ± SD; n = 3) and were compared by Student’s t test. The asterisk represented the statistical significance (p < 0.05) as compared with the control group.
Fig 7.
Inhibition of anti-27.8R MAbs on LCDV replication.
At 48 h post infection, the quantity of LCDV loads in FG (A) and HINAE (B) cells pre-incubated with different concentration of anti-27.8R MAbs was showed. Error bars represented SD. Data represented the number of LCDV copies per microgram of total DNA in the cell samples (mean ± SD; n = 3) and were compared by One-way ANOVO. Means with different letters were significantly different (p < 0.05).
Fig 8.
LCDV infection inhibition assays using anti-27.8R MAbs showing the cytopathic effect (CPE) in FG cells at 120 h p.i. (a-f) and HINAE cells at 168 h p.i. (A-F).
Black arrows: dying cells with typical cell detachment and rounding; (*) CPE. (a) FG cell cultures without LCDV infection. (b) ++++: Pre-incubation with anti-WSSV MAb 1D5. (c−f) FG cells pre-incubated with 0.04, 0.4, 4 and 40 μg of anti-27.8R MAbs, respectively. (c) ++++, (d) ++, (e)++ (f) +. Scale bars = 100 μm. (A) HINAE cell cultures without LCDV infection. (B) ++++: Pre-incubation with anti-WSSV MAb 1D5. (C−F) HINAE cells pre-incubated with 0.04, 0.4, 4 and 40 μg of anti-27.8R MAbs, respectively. (C) ++++, (D) +++, (E) ++, (F) +. Scale bars = 200 μm.
Table 1.
In vitro LCDV infection inhibition assays using anti-27.8R MAbs.