Comparative Evaluation of the Foot-and-Mouth Disease Virus Permissive LF-BK αVβ6 Cell Line for Senecavirus A Research

Abstract

Senecavirus A (SVA) is a member of the family Picornaviridae and enzootic in domestic swine. SVA can induce vesicular lesions that are clinically indistinguishable from Foot-and-mouth disease, a major cause of global trade barriers and agricultural productivity losses worldwide. The LF-BK α_Vβ₆ cell line is a porcine-derived cell line transformed to stably express an α_Vβ₆ bovine integrin and primarily used for enhanced propagation of Foot-and-mouth disease virus (FMDV). Due to the high biosecurity requirements for working with FMDV, SVA has been considered as a surrogate virus to test and evaluate new technologies and countermeasures. Herein we conducted a series of comparative evaluation in vitro studies between SVA and FMDV using the LF-BK α_Vβ₆ cell line. These include utilization of LF-BK α_Vβ₆ cells for field virus isolation, production of high virus titers, and evaluating serological reactivity and virus susceptibility to porcine type I interferons. These four methodologies utilizing LF-BK α_Vβ₆ cells were applicable to research with SVA and results support the current use of SVA as a surrogate for FMDV.

Keywords: FMDV; LF-BK αVβ6; Senecavirus A; cell line; interferon; isolation; swine.

Figure 1

Supernatant from an IB-RS-2 flask inoculated with a submitted field sample cultured in three porcine-derived cell lines, LF-BK α_Vβ₆, PK-15, or IB-RS-2. (A) PEG precipitated supernatant was applied to discontinuous sucrose gradients ranging in density from 20% to 65% and demonstrated diffuse materials at all densities. (B) Western blotting of harvested gradient layers demonstrated strong SVA antigen in 20% and 35% fractions from LF-BK α_Vβ₆ cultured samples and weak antigen in the 20% fraction for PK-15 and IB-RS-2 cultured samples. (C) The 20% and 35% fractions from the LF-BK α_Vβ₆ gradient were examined by transmission electron microscopy using negative staining, and viral particles were observed. Both samples contained particles of the expected size for SVA. (D) The 20% fraction also contained structures resembling empty SVA procapsids.

Figure 2

(A) Plaque-purified SVA produced 3 to 7 mm plaques on LF-BK α_Vβ₆ cells after 48 h of incubation and (B) demonstrated strong reactivity to polyclonal anti-SVA VP1 antibody.

Figure 3

(A) Cesium chloride gradient of PEG precipitated supernatant from SVA-LP8 inoculated LF-BK α_Vβ₆ cells produced a discrete white band just above the 1.42 g/cm³ density. (B) The band harvested from the gradient demonstrated bands of the expected size for individual viral proteins and capsid subunits when run on a denaturing gel and stained. Polyclonal anti-SVA VP1 antibody was reactive with individual VP1 as well as VP1 containing capsid subunits.

Figure 4

SVA titers, in log₁₀ TCID₅₀/mL, obtained from LF-BK α_Vβ₆ (blue), PK-15 (green), and BHK-21 (orange), and IB-RS-2 (dark blue) cell lines following infection with ≈2.5 log₁₀ TCID₅₀/mL of SVA. Titer was determined by the presentation of CPE at 24, 48, and 72 h post inoculation on LF-BK α_Vβ₆ cells and averaged together, the standard deviation is represented by error bars.

Figure 5

Mean virus neutralizing antibody titers (log₁₀) against SVA, blue, and FMDV, orange. No statistically significant differences in SVA titers between unvaccinated and FMD vaccinated swine are observed unlike neutralizing titers against FMD which increased in response to FMD vaccination. * represents p < 0.001 using a Student’s paired t-test.