Isolation and Molecular Characterization of Circulating Foot and Mouth Disease Virus in Egypt During 2018-2020

Foot and Mouth Disease (FMD) is a contagious viral disease with high economic losses and primary animal health concerns. FMDV type SAT2 is endemic in Egypt since 2012. This work aimed to characterize the circulating FMDV SAT2 strains genetically in Egypt from 2018 to 2020. A total of 209 vesicular fluids and tongue epithelium were collected from infected cattle and buffaloes in Sharkia, Ismailia, and Dakhlia provinces. All samples were examined by real-time PCR and conventional PCR for FMDV using pan- serotype and serotype-specific primers targeting the VP1 region. Out of 209 samples, 45 infected animals were positive for FMDV SAT2 virus, 29 cattle (21.5%), and 16 buffaloes (13.6%). No FMDV serotype A or O were detected. The highest prevalence of FMDV SAT2 was observed in Sharkia province with a percentage of 10% followed by Ismailia and Dakhlia with a rate of 2.9 and 0.9%, respectively. Three FMDV SAT2 positive samples represented as Sharkia 2018 and Sharkia 2019 and Ismailia 2020 were selected for sequencing and phylogenetic analysis of VP1. Sequencing and phylogenetic analysis of VP1of the three Egyptian strains demonstrated that these strains are closely related to other Egyptian strains in gene bank as Alex 2018 (MK4933346), Ismailia 2018 (MK4933341), and Menofia 2018 (MT199283) with homology ranged from 95.8 to 98.2%. Phylogenetic tree of FMDV SAT2 showed clustering of Sharkia 2018, Sharkia 2019, and Ismailia 2020 with Libya 2012 topotype VII with three amino acid substitutions at the site 24, 28, and 52.

Effective Diagnosis of Foot-And-Mouth Disease Virus (FMDV) Serotypes O and A Based on Optical and Electrochemical Dual-Modal Detection

Foot-and-mouth disease virus (FMDV) is a highly contagious disease that affects cloven-hoofed animals. The traditional diagnostic methods for FMDV have several drawbacks such as cross-reactivity, low sensitivity, and low selectivity. To overcome these drawbacks, we present an optical and electrochemical dual-modal approach for the specific detection of FMDV serotypes O and A by utilizing a magnetic nanoparticle labeling technique with resorufin β-d-glucopyranoside (res-β-glc) and β-glucosidase (β-glc), without the use of typical lateral flow assay or polymerase chain reaction. FMDV serotypes O and A were reacted with pan-FMDV antibodies that recognize all seven FMDV serotypes (O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3). The antigen–antibody complex was then immobilized on magnetic nanoparticles and reacted with β-glc-conjugated FMDV type O or type A antibodies. Subsequently, the addition of res-β-glc resulted in the release of fluorescent resorufin and glucose owing to catalytic hydrolysis by β-glc. The detection limit of fluorescent signals using a fluorescence spectrophotometer was estimated to be log(6.7) and log(5.9) copies/mL for FMDV type O and A, respectively, while that of electrochemical signals using a glucometer was estimated to be log(6.9) and log(6.1) copies/mL for FMDV type O and A, respectively. Compared with a commercially available lateral flow assay diagnostic kit for immunochromatographic detection of FMDV type O and A, this dual-modal detection platform offers approximately four-fold greater sensitivity. This highly sensitive and accurate dual-modal detection method can be used for effective disease diagnosis and treatment, and will find application in the early-stage diagnosis of viral diseases and next-generation diagnostic platforms.

Phage Display Screening of Bovine Antibodies to Foot-and-Mouth Disease Virus and Their Application in a Competitive ELISA for Serodiagnosis

For serodiagnosis of foot-and-mouth disease virus (FMDV), monoclonal antibody (MAb)-based competitive ELISA (cELISA) is commonly used since it allows simple and reproducible detection of antibody response to FMDV. However, the use of mouse-origin MAb as a detection reagent is questionable, as antibody responses to FMDV in mice may differ in epitope structure and preference from those in natural hosts such as cattle and pigs. To take advantage of natural host-derived antibodies, a phage-displayed scFv library was constructed from FMDV-immune cattle and subjected to two separate pannings against inactivated FMDV type O and A. Subsequent ELISA screening revealed high-affinity scFv antibodies specific to a serotype (O or A) as well as those with pan-serotype specificity. When BvO17, an scFv antibody specific to FMDV type O, was tested as a detection reagent in cELISA, it successfully detected FMDV type O antibodies for both serum samples from vaccinated cattle and virus-challenged pigs with even higher sensitivity than a mouse MAb-based commercial FMDV type O antibody detection kit. These results demonstrate the feasibility of using natural host-derived antibodies such as bovine scFv instead of mouse MAb in cELISA for serological detection of antibody response to FMDV in the susceptible animals.

Diagnosis Based on 1D Gene Analysis of the Foot-And-Mouth Disease Virus

A foot and mouth disease (FMD) is an acute, febrile and high contagious viral disease in many cloven hoofed domestic animals and more than 70 wild ones, resulting in severe finantial loss throughout the world. Choosing the correct seeds for foot-and-mouth disease (FMD) is an important issue in protecting this disease, and it is urgently necessary to establish a plan for vaccination in areas affected by FMD and to protect new foot-and-mouth disease. The genetic diversity and antigenic diversity of foot-and-mouth disease virus (FMDV) make eradication through vaccination difficult. Variable antigenic types exist in different geographic areas, and research projects on existing antigenic types are necessary to select vaccine in such an environment. From this, in this paper, oligonucleotide primers for detection and Selecting Serotype of FMDV were newly designed and synthesized. In addition, the nucleotide sequence of the 1D gene was aligned, the mutation region was determined, and the homology and phylogenetic relationship of the nucleotide sequence were analyzed. The antigenicity of the FMDV type O strains in the Democratic People's Republic of Korea and the correspondence between them were examined. The neutralization reaction was used to examine antigenicity between FMDV to select waxy seeds. To prevent FMD through this primer design and experimental method, the nucleic acid of FMDV was amplified by RT-PCR. Then, the nucleotide sequence of the 1D gene corresponding to the virus VP1 protein was analyzed and compared to select a seed vaccine strain.

Development of Monoclonal Antibody Specific to Foot-and-Mouth Disease Virus Type A for Serodiagnosis

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease affecting cloven-hoofed livestock worldwide. FMD virus (FMDV) type A is one of the most common causes of FMD outbreaks among the seven FMDV serotypes, and its serological diagnosis is therefore important to confirm FMDV type A infection and to determine FMD vaccine efficacy. Here, we generated monoclonal antibodies (mAbs) specific to FMDV type A via hybridoma systems using an inactivated FMDV type A (A22/Iraq/1964) and found 4 monoclones (#29, #106, #108, and #109) with high binding reactivity to FMDV type A among 594 primary clones. In particular, the #106 mAb had a higher binding reactivity to the inactivated FMDV type A than the other mAbs and a commercial mAb. Moreover, the #106 mAb showed no cross-reactivity to inactivated FMDV type South African territories 1, 2, and 3, and low reactivity to inactivated FMDV type O (O1 Manisa). Importantly, the solid-phase competitive ELISA (SPCE) using horseradish peroxidase (HRP)-conjugated #106 mAb detected FMDV type A-specific Abs in sera from FMD type A-vaccinated cattle more effectively than a commercial SPCE. These results suggest that the newly developed FMDV type A-specific mAb might be useful for diagnostic approaches for detecting Abs against FMDV type A.

A gold nanoparticle strip for simultaneously evaluating FMDV immunized antibody level and discriminating FMDV vaccinated animals from infected animals

A gold nanoparticle strip was developed for rapidly evaluating FMDV type O antibody level and simultaneously discriminating FMDV vaccinated animals from infected animals.

A rapid immunochromatographic strip for neutralizing antibodies detection of foot and mouth disease virus serotype O

Based on the BSA-Pep antigen, a test strip was developed to evaluate the neutralizing antibody of serum samples from swine vaccinated with FMDV type O vaccine rapidly. The BSA-Pep used as a detector was labeled with colloidal gold.

Molecular Characterization of Foot-and-Mouth Disease Virus Type O from Wild Pig in Bangladesh

Foot-and-mouth disease (FMD) is a disease of all non-avian livestock animals which costs direct and indirect economic burden of 6.5-21 billion USD per year worldwide in endemic countries. In Bangladesh, FMD is endemic and mainly caused by foot-and-mouth disease virus (FMDV) serotypes O, A and Asia1. Among FMD susceptible animals cattle, sheep and goat were reported to be more susceptible to this virus, whereas, pig was reported as amplifier of the virus. To date there is no epidemiological data in Bangladesh defining the circulation of FMDV in pig population. This investigation first reports the circulation of FMDV in wild pigs of Bangladesh, its molecular characterization and genotyping. To pursue this, tissue sample from ruptured vesicles of mammary gland was collected from wild pig suspected to be infected with FMD followed by RT-PCR amplification, sequencing and phylogenetic study of the VP1 gene, which is the most variable region of FMDV genome. The virus was identified as FMDV serotype O and phylogenetically clustered together with India 2001 (Ind2001) lineage under middle-east, south Asia (ME-SA) topotype. Within the clade of Ind2001 lineage, FMDV from pig formed a sub-clade with 2013 sequences of cattle which indicates the phylogenetic relatedness of the virus from pig with circulatory virus in cattle population of Bangladesh in 2013. Pair-wise local alignment of the FMDV type O VP1 sequence of pig with other local cattle FMDV type O VP1 sequences of 2012 and 2013 supported the phylogenetic affiliation of the pig FMDV with the circulatory virus in cattle population of Bangladesh in 2013, whereas nucleotide sequences of cattle FMDV VP1 of 2012 were found to be 7-8% divergent compared to pig FMDV VP1. Phylogenetic and pair-wise alignment data conclusively revealed that (i) homologous circulation of the FMDV type O (Ind2001 lineage) occurs in both animal traits; and (ii) FMDV type O VP1 sequence of pig origin is distantly related to 2012 local FMDV type O VP1 sequences of cattle origin, but closely related to 2013 local cattle FMDV type O VP1.Bangladesh J Microbiol, Volume 31, Number 1-2,June-Dec 2014, pp 41-45