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Research Project: Intervention Strategies to Support the Global Control and Eradication of Foot-and-Mouth Disease Virus (FMDV)

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Title: An Adventitious agent free clonal cell line that is highly susceptible to foot and mouth disease virus

item LAROCCO, MICHAEL - US Department Of Agriculture (USDA)
item KRUG, PETER - US Department Of Agriculture (USDA)
item AHMED, ZAHEER - Animal And Plant Health Inspection Service (APHIS)
item RODRIGUEZ-CALZADA1, MONICA - Oak Ridge Institute For Science And Education (ORISE)
item Azzinaro, Paul
item Rodriguez, Luis
item De Los Santos, Teresa
item MEDINA, GISSELLE - University Of Kansas

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/21/2021
Publication Date: 6/4/2021
Citation: Larocco, M., Krug, P., Ahmed, Z., Rodriguez-Calzada1, M., Azzinaro, P.A., Rodriguez, L.L., De Los Santos, T.B., Medina, G.N. 2021. An Adventitious agent free clonal cell line that is highly susceptible to foot and mouth disease virus. Journal of Clinical Microbiology.

Interpretive Summary: Foot and mouth disease (FMD) is caused by a highly diverse virus comprising 7 serotypes and dozens of subtypes, most requiring a specific vaccine. FMD vaccines are manufactured by adapting the virus to grow in a hamster-derived cell line (BHK-21) followed by inactivation and formulation with adjuvants. However, during adaptation to the hamster cell line the virus might change and loose its ability to induce protection in vaccinated animals. In addition, some serotypes/ subtypes grow poorly or do not grow at all in BHK-21 cells resulting in difficulties for manufacturing vaccines. An alternate pig cell line was created by introducing proteins used by the virus to infect cattle (called avB6) resulting a cell line (LFBKavb6) that supports growth of all FMDV serotypes and subtypes, and most importantly with need for little or no adaptation. Unfortunately, the LFBKaVb6 cell line was contaminated with another virus from cattle (called BVDV) making it unsuitable for FMD vaccine production. Here we used methodology to clear this virus from the cells without affecting its susceptibility to all FMD viruses. These cells, called MGPKavB6 are now an attractive tool not only for diagnostics but also potentially for vaccine manufacturing.

Technical Abstract: LFBKaVß6 cells of porcine origin have been successfully used for isolation, diagnostics and propagation to high virus titers for all seven FMDV serotypes. These cells have great potential to serve as cell substrates for the production of vaccines, however, presence of adventitious agents may be detrimental for vaccine manufacturing. In this study we discover the presence of the non-cytopathic bovine viral diarrhea virus (BVDV). This contamination increases the awareness about possible interference with FMDV growth in diagnostic assays, as well as potential transmission of pestivirus provided the LFBKaVß cell line is used for FMDV vaccine propagation. In this study, LFBK we developed a three-prong methodology for the complete removal of the spurious BVDV from LFBK aVß6 cells. Combinatorial treatment with siRNA targeting the NS5A protein of BVDV, porcine interferon (IFN) alpha and the polymerase inhibitor ribavirin resulted in the elimination of BVDV as determined by immunohistochemistry analysis and cDNA sequencing. BVDV-free LFBKaVß6 cells were cloned and passaged at least until pass #40 corroborating the absence of genomic BVDV detected by the reverse transcriptase polymerase chain reaction (RT-PCR) and next generation sequencing (NGS). Importantly, elimination of BVDV in LFBKaVß6 did not affect the rate of replication or plaque phenotype of FMDV from different serotypes isolated and propagated in the cell line. In addition, isolation of FMDV from field probang samples was as successful as that previously obtained in BVDV-infected LFBKaVß6 cell line. Our results identified a direct method to efficiently eliminate BVDV from porcine cells without altering FMDV permissiveness and highlight the potential use of these cells not only for FMD diagnostics but also for whole virus vaccine production. Furthermore, these cells provide a platform for testing growth and expansion of other viruses without the risk of contamination with spurious agents usually present in continuous cell lines