Inactivation of foot-and-mouth disease virus in epithelium samples for safe transport and processing in low-containment laboratories

Abstract

During a foot-and-mouth disease (FMD) outbreak, transport and testing of potentially infectious samples, including epithelium from suspect lesions, presents a biosafety risk, particularly in FMD-free countries. Therefore, treatment to inactivate virus prior to transport is important. Tongue epithelium from cattle infected with FMD virus (FMDV) serotype O (O ALG/3/2014 - Lineage O/ME-SA/Ind-2001d) or A (A IRN/22/2015 - Lineage A/ASIA/G-VII) was incubated in RNAlater, RNA Shield or phosphate-buffered saline (pH 7.4) at room temperature for 2, 6, 24 or 48 h. After incubation, tissues were homogenised and tested by virus titration. Viral RNA in the homogenate was quantified by RT-qPCR, used for sequencing, and transfected into LFBK_αVβ6 cells to recover infectious virus. RNAlater reduced A IRN/22/2015 titres by 4 log₁₀ after 24 h, and completely after 48 h incubation. While O ALG/3/2014 was detected by VI after 2, 6 and 24 h, titration yielded no infectious virus, likely as a result of freeze-thawing. RNA Shield was cytotoxic at high concentrations but was effective at inactivating both strains after 24 h. Regardless of reagent or inactivation period, RT-qPCR, VP1 sequencing, and transfection of RNA to recover infectious virus were possible. RNA Shield appears a better choice for FMDV inactivation in tissues, however 24 h incubation is recommended.

Keywords: Foot-and-mouth disease virus; Inactivation; Sample transport; Tissue samples.