Porcine LFBKαVβ6 cells have been successfully used for diagnostics and propagation of all FMDV serotypes/subtypes. Unfortunately, after initial characterization, these cells showed contamination with bovine viral diarrhea virus (BVDV), a non-cytopathic adventitious agent. Persistent infection with BVDV could interfere with diagnostic tests and, also prevent consideration for other uses, i.e., vaccine production. In this study, we developed a three-prong methodology to completely remove BVDV from LFBKαVβ6 cells. Combined treatment with siRNA against BVDV NS5A, porcine interferon alpha and ribavirin resulted in the elimination of BVDV, as determined by immunohistochemistry analysis, quantitative RT-PCR and RNA sequencing. Importantly, elimination of BVDV from LFBKαVβ6 did not affect FMDV growth and plaque phenotype from different serotypes isolated and propagated in the clean cell line, newly named MGPK αVβ6-C5. Additionally, isolation of FMDV from field oro-pharyngeal samples, was successful at the same sensitivity as in BVDV-contaminated LFBKαVβ6 cells. Our results identified a direct method to efficiently eliminate BVDV from porcine cells without altering FMDV permissiveness, diagnostic value, or potential for use in vaccine production. Furthermore, these cells may provide an improved platform for diagnostics and propagation of other viruses of interest in the veterinary field and the virology community at large.

猪 LFBKαVβ6 细胞已成功用于诊断和传播所有 FMDV 血清型/亚型。不幸的是，在初步表征后，这些细胞显示出被牛病毒性腹泻病毒 (BVDV)，一种非细胞病变的外来因子污染。BVDV 的持续感染可能会干扰诊断测试，还会妨碍考虑其他用途，即疫苗生产。在这项研究中，我们开发了一种三管齐下的方法来从 LFBKαVβ6 细胞中完全去除 BVDV。根据免疫组织化学分析、定量 RT-PCR 和 RNA 测序确定，用针对 BVDV NS5A、猪干扰素 α 和利巴韦林的 siRNA 联合治疗导致 BVDV 的消除。重要的，从 LFBKαVβ6 中消除 BVDV 不影响 FMDV 生长和来自在清洁细胞系中分离和繁殖的不同血清型的斑块表型，新细胞系新命名为 MGPK αVβ6-C5。此外，从野外口咽样本中分离 FMDV，其灵敏度与 BVDV 污染的 LFBKαVβ6 细胞相同。我们的结果确定了一种直接方法，可以在不改变 FMDV 允许性、诊断价值或在疫苗生产中使用的潜力的情况下，有效地从猪细胞中消除 BVDV。此外，这些细胞可以为兽医领域和整个病毒学界中其他感兴趣的病毒的诊断和传播提供改进的平台。以与 BVDV 污染的 LFBKαVβ6 细胞相同的灵敏度获得成功。我们的结果确定了一种直接方法，可以在不改变 FMDV 允许性、诊断价值或在疫苗生产中使用的潜力的情况下，有效地从猪细胞中消除 BVDV。此外，这些细胞可以为兽医领域和整个病毒学界中其他感兴趣的病毒的诊断和传播提供改进的平台。以与 BVDV 污染的 LFBKαVβ6 细胞相同的灵敏度获得成功。我们的结果确定了一种直接方法，可以在不改变 FMDV 允许性、诊断价值或在疫苗生产中使用的潜力的情况下，有效地从猪细胞中消除 BVDV。此外，这些细胞可以为兽医领域和整个病毒学界中其他感兴趣的病毒的诊断和传播提供改进的平台。