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Title: A continuous bovine kidney cell line constitutively expressing bovine alpha v beta 6 integrin has increased susceptibility to foot-and-mouth disease virus

item Larocco, Michael
item Krug, Peter
item KRAMER, ED - Us Deparment Of Homeland Security
item AHMED, ZAHEER - Oak Ridge Institute For Science And Education (ORISE)
item Pacheco Tobin, Juan
item DUQUE, HERNANDO - Animal And Plant Health Inspection Service (APHIS)
item Baxt, Barry
item Rodriguez, Luis

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/14/2013
Publication Date: 3/20/2013
Citation: Larocco, M.A., Krug, P.W., Kramer, E., Ahmed, Z., Pacheco Tobin, J., Duque, H., Baxt, B., Rodriguez, L.L. 2013. A continuous bovine kidney cell line constitutively expressing bovine alpha v beta 6 integrin has increased susceptibility to foot-and-mouth disease virus. Journal of Clinical Microbiology. 51(6):1714-1720. DOI:10.1128/JCM.03370-12.

Interpretive Summary: Foot and mouth disease virus (FMDV) is highly contagious and can cause severe morbidity and mortality in livestock. Effective vaccines do currently exist for FMDV, but they are serotype-specific and do not confer long-term protection. Thus, the rapid detection and serotype determination of a circulating virus during an FMDV outbreak is critical to enable protective intervention to stop the spread of disease. FMDV is normally isolated from tongue tissue or vesicular lesions of infected animals by disrupting and filtering the tissues, then inoculating this filtrate onto susceptible cell culture. Historically, the most susceptible cells to field isolates of FMDV have been primary cells made directly from animal organs such as kidneys or thyroids, but these cells cannot be maintained in cell culture for more than a few days and lose their FMDV susceptibility after cryopreservation. To save resources, many laboratories use continuous cell lines for FMDV detection in clinical isolates but these cell lines are markedly less sensitive to FMDV field isolates, likely due to a lack of FMDV-specific receptors. In order to combine the high FMDV sensitivity of primary cells and the long-term stability of cell lines, we created a bovine kidney cell line that stably expresses a high-affinity FMDV receptor, the integrin alpha v Beta 6. These cells, designated LFBK- alpha v Beta 6, support the replication of all serotypes of FMDV from experimentally infected animals and can detect some FMDV strains with a tenfold higher sensitivity than even primary cells. LFBK- alpha v Beta 6 had excellent performance in a diagnostic setting, detecting field isolates of FMDV both quicker and more efficiently than other cell lines and a primary cell line. LFBK- alpha v Beta 6 cells also were able to detect other vesicular disease viruses, providing a more complete diagnostic result. The use of this cell line in a foreign animal disease diagnostic setting will add to the arsenal of tools for rapid vesicular disease virus detection and will help to reduce the use of animals for the production of primary cell cultures.

Technical Abstract: Foot and mouth disease virus (FMDV) is worldwide problem, limiting the trade of animals and their products from affected countries. The rapid isolation, serotyping and vaccine matching of FMDV from infected animals is critical to enable the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Current virus isolation protocols use baby hamster kidney cells (BHK-21) and other cell lines that are not highly sensitive to some strains of FMDV. Some primary cells have been shown to be more susceptible but are difficult and expensive to make and maintain. Since the alpha v Beta 6 integrin is a principal receptor for FMDV, we produced a novel cell line stably expressing both the alpha v and Beta 6 bovine integrin subunits. This cell line (LFBK- alpha v Beta 6) expressed both transduced genes and showed high susceptibility to FMDV infection for at least 100 cell passages. LFBK- alpha v Beta 6 cells were highly sensitive in detecting all serotypes and subtypes of FMDV including some difficult to grow serotype O viruses, either in samples from experimentally infected animals or diagnostic unknowns. In comparison to other cell types currently used for virus isolation, LFBK- alpha v Beta 6 cells were more effective at detecting FMDV of all serotypes in clinical samples, supporting their use as a more sensitive diagnostic tool for virus isolation.