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Abstract
The integrins αvβ1, αvβ3, αvβ6, and αvβ8 are known to be the natural receptors of foot-and-mouth disease virus (FMDV). Among them, integrin αvβ6 is considered a major receptor for FMDV. We performed protein expression of full-length bovine integrins αv, β3, and β6 and confirmed the high efficiency of bovine α vβ6 as the FMDV receptor in FMDV non-permissive SW 480 cells. Next, we established the black goat kidney (BGK) cell line, stably expressing bovine integrin β6 (BGK-β6-4). We observed that BGK-β6-4 cells had significantly enhanced sensitivity to FMDV compared with that of BGK cells (P<0.05). In addition, BGK-β6-4 cells had equal or higher sensitivity to several serotypes of FMDV compared with that of other FMDV permissive cell lines, such as BHK-21 and IBRS-2. In conclusion, we established a promising novel goat cell line, BGK-β6-4, which can be used to isolate or culture FMDV. Furthermore, the BGK-β6-4 cell line may serve as a promising tool for studying integrin α vβ6 receptor functions.
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Figure 1.
Detection of bovine integrins αvβ3 and αvβ6 heterodimer expression in SW480 cells using indirect immunofluorescence assay (IFA). SW-480 cells were co-transfected with vectors expressing bovine αv and β6 (a) or β3 (b) subunits. Protein expression was detected by IFA after 48 h of transfection using an anti-human β6 Mab (2076Z) (a) or an anti-human integrin αvβ3 Mab (LM609) (b). Untransfected cells treated with the same primary and secondary antibodies were used as negative controls (c and d).
Figure 2.
Comparison of viral infection efficiency in the cells expressing bovine αvβ3 and αvβ6 integrins. SW480 cells were co-transfected with vectors expressing bovine αv and β6 or β3 subunits. Twenty-four hours after transfection, the cells were infected with 104.5 TCID50 of FMDV O/SKR/2002, Asia 1/M0G/05, or A22/IRQ 24/64 viral suspensions. The supernatant was collected at 8 days after the infections, and viral RNA was extracted. FMDV quantification was performed using real-time RT PCR. An unpaired t-test was performed for statistical analysis by GraphPad InStat software (∗P< 0.05, ∗∗P< 0.01, ∗∗∗P < 0.001). Error bars indicate standard deviations from the mean.
Figure 3.
Comparative virus titration in BGK-β6 stable cell lines. Four stable BGK cell lines, expressing bovine integrin β6 (BGK-β6-1, BGK-β6-2, BGK-β6-3, and BGK-β6-4), in addition to BGK and IBRS-2 cell lines were used for titrating FMDV O/SKR/2002 or C3 Resende. Virus titration was measured 72 h post FMDV infection. TCID50 was calculated by Reed and Muench method (14). An unpaired t-test was performed for statistical analysis by GraphPad InStat software (∗P< 0.05, ∗∗P< 0.01). Error bars indicate standard deviations from the mean.
Figure 4.
Comparative titration of various strains of FMDV in BGK-β6-4, BGK, IBRS-2, and BHK-21 cell lines. BGK-ß6-4, BGK, IBRS-2, and BHK-21 cell lines were used for titrating FMDV A22/IRQ 24/64, A/Pocheon/SKR/2010, Asia1/MOG/05, SAT2/ZIM 5/81, and SAT3/ZIM 4/81. Virus titration was measured 72 h post FMDV infection. TCIID50 was calculated by Reed and Muench method (14). An unpaired t-test was performed for statistical analysis by GraphPad InStat software (∗P< 0.05, ∗∗P< 0.01). Error bars indicate standard deviations from the mean.
Table 1.
Passage history of FMDV strains used in this study
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