Establishing a molecular toolbox of lineage-specific real-time RT-PCR assays for the characterization of foot-and-mouth disease viruses circulating in Asia.

Foot-and-mouth disease (FMD) is endemic in many Asian countries, with outbreaks occurring regularly due to viruses from serotypes O, A, and Asia1 that co-circulate in the region. The ability to rapidly characterize new virus occurrences provides critical information to understand the epidemiology and risks associated with field outbreaks, and helps in the selection of appropriate vaccines to control the disease. FMD lineage-specific characterization is usually determined through sequencing; however, this capacity is not always readily available. In this study, we provide a panel of real-time RT-PCR (rRT-PCR) assays to allow differentiation of the FMD virus (FMDV) lineages known to have been co-circulating in Asia during 2020. This panel included five new rRT-PCR assays designed to detect lineages O/ME-SA/PanAsia-PanAsia-2, O/ME-SA/Ind-2001, O/SEA/Mya-98, O/CATHAY, and A/ASIA/Sea-97, along with three published rRT-PCR assays for A/ASIA/Iran-05, A/ASIA/G-VII, and Asia1 serotypes. Samples of known FMD lineage (n = 85) were tested in parallel with all eight lineage-specific assays and an established 3D pan-FMD rRT-PCR assay, and comparative limit of detection (LOD) experiments were conducted for the five newly developed assays. All samples (85/85) were assigned to the correct serotype, and the correct lineage was assigned for 70 out of 85 samples where amplification only occurred with the homologous assay. For 13 out of 85 of the samples, there was amplification in two assays; however, the correct lineage could be designated based on the strongest Ct values for 12 out of 13 samples. An incorrect lineage was assigned for 3 out of 85 samples. The amplification efficiencies for the five new rRT-PCR assays ranged between 79.7 and 100.5%, with nucleic acid dilution experiments demonstrating broadly equivalent limits of detection when compared to the 3D pan-FMD rRT-PCR assay. These new tests, together with other published lineage-specific rRT-PCR assays, constitute a panel of assays (or molecular toolbox) that can be selected for use in FMD endemic countries (individually or a subset of the assays depending on region/lineages known to be circulating) for rapid characterization of the FMDV lineages circulating in Asia at a relatively low cost. This molecular toolbox will enhance the ability of national laboratories in endemic settings to accurately characterize circulating FMDV strains and facilitate prompt implementation of control strategies, and may be particularly useful in settings where it is difficult to access sequencing capability.

Genotyping of foot-and-mouth disease viruses collected in Sudan between 2009 and 2018.

Foot-and-mouth disease (FMD) is widely distributed in Sudan where outbreaks occur on an annual basis especially during the winter months (December-February). This study aimed to increase our understanding of the epidemiological patterns of FMD in Sudan and connections to neighbouring countries by characterizing the genetic sequences of FMD viruses (FMDV) collected from samples collected in 10 Sudanese states over a 10-year period (between 2009 and 2018). FMDV was detected in 91 of the 265 samples using an antigen-detection ELISA. Three serotypes were detected: O (46.2%), A (34.0%), and SAT 2 (19.8%). Fifty-two of these samples were submitted for sequence analyses, generating sequences that were characterized as belonging to O/EA-3 (n = 17), A/AFRICA/G-IV (n = 23) and SAT 2/VII/Alx-12 (n = 12) viral lineages. Phylogenetic analyses provided evidence that FMDV lineages were maintained within Sudan, and also highlighted epidemiological connections to FMD outbreaks reported in neighbouring countries in East and North Africa (such as Ethiopia and Egypt). This study motivates continued FMD surveillance in Sudan to monitor the circulating viral lineages and broader initiatives to improve our understanding of the epidemiological risks in the region.

Elimination of Non-cytopathic Bovine Viral Diarrhea Virus From the LFBK-αvß6 Cell Line.

The LFBK-αvß6 cell line is highly sensitive for the isolation of foot-and-mouth disease virus (FMDV) and porcinophilic vesicular viruses. However, LFBK-αvß6 cells are contaminated with a non-cytopathic bovine viral diarrhea virus (BVDV), which complicates handling procedures in areas where other cell lines are maintained, as well downstream use of viral isolates. In this study, we used an aromatic cationic compound (DB772) to treat LFBK-αvß6 cells using an approach that has been previously used to eliminate persistent BVDV from fetal fibroblast cell lines. After three cell passages with 4 µM DB772, BVDV could no longer be detected in unclarified cell suspensions using a pan-pestivirus real-time RT-PCR assay, and remained undetectable after treatment was stopped (nine passages) for an additional 28 passages. The analytical sensitivity of the DB772-treated LFBK-αvß6 cultures (renamed WRL-LFBK-αvß6) to titrations of FMDV and other vesicular virus isolates was comparable to untreated LFBK-αvß6 cells. These new BVDV-free cells can be handled without the risk of cross-contaminating other cells lines or reagents, and used for routine diagnostics, in vivo studies and/or preparation of new vaccine strains.

Evaluation of Cell Lines for the Isolation of Foot-and-Mouth Disease Virus and Other Viruses Causing Vesicular Disease.

The most sensitive cell culture system for the isolation of foot-and-mouth disease virus (FMDV) is primary bovine thyroid (BTY) cells. However, BTY cells are seldom used because of the challenges associated with sourcing thyroids from FMDV-negative calves (particularly in FMD endemic countries), and the costs and time required to regularly prepare batches of cells. Two continuous cell lines, a fetal goat tongue cell line (ZZ-R 127) and a fetal porcine kidney cell line (LFBK-αVß6), have been shown to be highly sensitive to FMDV. Here, we assessed the sensitivity of ZZ-R 127 and LFBK-αVß6 cells relative to primary BTY cells by titrating a range of FMDV original samples and isolates. Both the ZZ-R 127 and LFBK-αVß6 cells were susceptible to FMDV for >100 passages, and there were no significant differences in sensitivity relative to primary BTY cells. Notably, the LFBK-αVß6 cell line was highly sensitive to the O/CATHAY porcine-adapted FMDV strain. These results support the use of ZZ-R 127 and LFBK-αVß6 as sensitive alternatives to BTY cells for the isolation of FMDV, and highlight the use of LFBK-αVß6 cells as an additional tool for the isolation of porcinophilic viruses.

Defining the relative performance of isothermal assays that can be used for rapid and sensitive detection of foot-and-mouth disease virus.

This study describes the first multiway comparison of portable isothermal assays for the detection of foot-and-mouth disease virus (FMDV), benchmarked against real-time reverse transcription RT-PCR (rRT-PCR). The selected isothermal chemistries included reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA). The analytical sensitivity of RT-LAMP was comparable to rRT-PCR (101 RNA copies), while RT-RPA was one log10 less sensitive (102 RNA copies). Diagnostic performance was evaluated using a panel of 35 samples from FMDV-positive cattle and eight samples from cattle infected with other vesicular viruses. Assay concordance for RT-LAMP and RT-RPA was 86-98% and 67-77%, respectively, when compared to rRT-PCR, with discordant samples consistently having high rRT-PCR cycle threshold values (no false-positives were detected for any assay). In addition, a hierarchy of sample preparation methods, from robotic extraction to simple dilution of samples, for epithelial suspensions, serum and oesophageal-pharyngeal (OP) fluid were evaluated. Results obtained for RT-LAMP confirmed that FMDV RNA can be detected in the absence of RNA extraction. However, simple sample preparation methods were less encouraging for RT-RPA, with accurate results only obtained when using RNA extraction. Although the evaluation of assay performance is specific to the conditions tested in this study, the compatibility of RT-LAMP chemistry with multiple sample types, both in the presence and absence of nucleic acid extraction, provides advantages over alternative isothermal chemistries and alternative pen-side diagnostics such as antigen-detection lateral-flow devices. These characteristics of RT-LAMP enable the assay to be performed over a large diagnostic detection window, providing a realistic means to rapidly confirm positive FMD cases close to the point of sampling.