Foot-and-mouth Disease Virus Serotype Research Articles

Preparation and application of porcine broadly neutralizing monoclonal antibodies in an immunoassay for efficiently detecting neutralizing antibodies against foot-and-mouth disease virus serotype O.

Neutralizing antibodies provide vital protection against foot-and-mouth disease virus (FMDV). The virus neutralization test (VNT) is a gold standard method for the detection of neutralizing antibodies. However, its application is limited due to the requirement for live virus and unsuitability for large-scale serological surveillance. In this study, a porcine broadly neutralizing monoclonal antibody (PO18-10) against FMDV was obtained from the heterologous sequentially vaccinated pig using single-B-cell antibody technology. A competitive enzyme-linked immunosorbent assay (C-ELISA) for detecting neutralizing antibodies against FMDV serotype O was developed using biotinylated PO18-10 as a detector antibody. The sensitivity and specificity of the assay were 100% and 99.55%, respectively, and the positive/negative coincidence rate with VNT was 94%, suggesting that C-ELISA based on natural host-derived monoclonal antibody (mAb) could be a promising tool to detect neutralizing antibodies against FMDV serotype O and evaluate the vaccine efficacy.IMPORTANCEFoot-and-mouth disease virus (FMDV) serotype O is one of the most prevalent serotypes in the world. The neutralizing antibody titers in primo-vaccinated animals are directly related to their level of protection against a virus challenge. The development of a safe, rapid, and accurate method for the detection of the neutralizing antibody is essential for the control and eradication of FMD. In this study, an inter-serotype broadly neutralizing monoclonal antibody PO18-10 was successfully produced using single-B-cell antibody technology from sequentially vaccinated pigs. A competitive ELISA based on this natural host-derived mAb for the detection of neutralizing antibodies against FMDV serotype O was developed and validated. The assay demonstrates high sensitivity, specificity, and coincidence rate with VNT, making it an alternative tool for confirming FMDV infection and evaluating the vaccine efficacy.

Foot-and-mouth disease in Asia.

Foot-and-mouth disease (FMD) is a highly contagious transboundary disease prevalent across the Asian continent, affecting both wild and domestic artiodactyls. The disease is caused by a virus belonging to the Aphthovirus genus of the Picornaviridae family which is categorized into seven serotypes: C, O, A, SAT1, SAT2, SAT3, and Asia1. The virus spreads through direct and indirect contact, including semen, meat, fomites, ingestion, and aerosols. FMD has a severe economic impact due to the high morbidity and mortality, especially in young animals. Prevention of the disease relies on vaccination with the prevalent serotype(s) or the slaughter and destruction of affected animals. This review discusses the prevalence of various FMD virus (FMDV) serotypes across Asia, along with the transmission modes, pathogenesis, immune response, and immune suppression by FMDV. Additionally, the review explores FMD diagnosis, prevention, and control strategies, and highlights future opportunities for research aimed at developing strain-specific viral and bacterial combined vaccines.

A Review of the Utility of Established Cell Lines for Isolation and Propagation of the Southern African Territories Serotypes of Foot-and-Mouth Disease Virus.

Cell culture underpins virus isolation and virus neutralisation tests, which are both gold-standard diagnostic methods for foot-and-mouth disease (FMD). Cell culture is also crucial for the propagation of inactivated foot-and-mouth disease virus (FMDV) vaccines. Both primary cells and cell lines are utilised in FMDV isolation and propagation. Widely used cell lines for FMDV and isolation and propagation include baby hamster kidney cells (BHK-21), swine kidney cells (IB-RS-2), foetal goat tongue (ZZ-R 127), foetal porcine kidney cells (LFBKvB6), bovine kidney cells (BK), human telomerase reverse transcriptase bovine thyroid (hTERT-BTY) and porcine kidney-originating PK-15 or SK 6 cell lines. This review highlights how different receptors and molecules-integrins, heparan sulphate (HS), and the Jumonji C-domain containing Protein 6 (JMJD6)-found on the surface of different cell types contribute to differences experienced with susceptibility and sensitivity of the cells to infection with different serotypes of FMDV. This review specifically focuses on Southern African territory (SAT) serotypes, which are unique to the Southern African context and are often under-investigated in cell line development for FMDV isolation and propagation.

Impact of Different Foot and Mouth Disease Vaccine Schemes in Cross-Neutralization Against Heterologous Serotype O Strains in Cattle.

The high antigenic variability of the foot-and-mouth disease virus (FMDV) represents a challenge for developing prophylactic strategies, stressing the need for research into vaccines offering broad protection against a range of virus strains. Here, the heterotypic cross-reaction using different vaccine schemes against serotype O strains was studied, evaluating the impact of revaccination, antigen dose, and incorporation of additional FMDV serotypes. Naïve cattle were immunized with seven distinct FMDV vaccines, receiving three doses of the same formulation at 0, 28, and 56 days post-primary vaccination (dpv). Serum samples were collected up to 70 dpv and tested by a virus-neutralizing test against serotype O strains from a South American lineage and two strains representative of two Asian lineages. Our results showed that vaccines containing the ME-SA topotype O1/Campos strain developed cross-neutralizing responses against the two Asian viruses after the first vaccination. In contrast, significant heterotypic neutralizing antibody titers against the homologous topotype strain were only found after the second vaccination, indicating that the phylogenic relationship may differ from the antigenic profiles for these two viruses. The amount of the O1/Campos strain and the revaccination were essential factors for neutralization against the homologous- and heterologous-type O FMDV viruses. The strain composition of the vaccine was only relevant for cross-neutralization against one of the Asian strains, suggesting potential intra-serotypic divergences for this pattern.

Discovery, recognized antigenic structures, and evolution of cross-serotype broadly neutralizing antibodies from porcine B-cell repertoires against foot-and-mouth disease virus.

It is a great challenge to isolate the broadly neutralizing antibodies (bnAbs) against foot-and-mouth disease virus (FMDV) due to its existence as seven distinct serotypes without cross-protection. Here, by vaccination of pig with FMDV serotypes O and A whole virus antigens, we obtained 10 bnAbs against serotypes O, A and/or Asia1 by dissecting 216 common clonotypes of two serotypes O and A specific porcine B-cell receptor (BCR) gene repertoires containing total 12720 B cell clones, indicating the induction of cross-serotype bnAbs after sequential vaccination with serotypes O and A antigens. The majority of porcine bnAbs (9/10) were derived from terminally differentiated B cells of different clonal lineages, which convergently targeted the conserved "RGDL" motif on structural protein VP1 of FMDV by mimicking receptor recognition to inhibit viral attachment to cells. Cryo-EM complex structures revealed that the other bnAb pOA-2 specifically targets a novel inter-pentamer antigen structure surrounding the viral three-fold axis, with a highly conserved determinant at residue 68 on VP2. This unique binding pattern enabled cross-serotype neutralization by destabilizing the viral particle. The evolutionary analysis of pOA-2 demonstrated its origin from an intermediate B-cell, emphasizing the crucial role of somatic hypermutations (SHMs) in balancing the breadth and potency of neutralization. However, excessive SHMs may deviate from the trajectory of broad neutralization. This study provides a strategy to uncover bnAbs against highly mutable pathogens and the cross-serotype antigenic structures to explore broadly protective FMDV vaccine.

The Current Epizootiological Situation of Three Major Viral Infections Affecting Cattle in Egypt.

One of the major factors hindering efficient livestock production is the presence of high-impact infectious animal diseases, such as foot and mouth disease (FMD), lumpy skin disease (LSD), and bovine ephemeral fever (BEF), which are notable viral infections affecting cattle in Egypt, leading to significant economic losses. FMD is caused by the foot and mouth disease virus (FMDV) of the genus Aphthovirus in the Picornaviridae family. LSD is caused by lumpy skin disease virus (LSDV) of Capripox genus within the Poxviridae family, subfamily Chordopoxvirinae. BEF is caused by bovine ephemeral fever virus (BEFV) of genus Ephemerovirus in the Rhabdoviridae family. FMD is a highly contagious viral infection of domestic and wild cloven-hooved animals and can spread through the wind. On the other hand, LSD and BEF are arthropod-borne viral diseases that mainly affect domestic cattle and water buffalo. Despite government vaccination efforts, these three viral diseases have become widespread in Egypt, with several reported epidemics. Egypt's importation of large numbers of animals from different countries, combined with unregulated animal movements through trading and borders between African countries and Egypt, facilitates the introduction of new FMDV serotypes and lineages not covered by the current vaccination plans. To establish an effective control program, countries need to assess the real epizootic situation of various infectious animal diseases to develop an efficient early warning system. This review provides information about FMD, LSD, and BEF, including their economic impacts, causative viruses, global burden, the situation in Egypt, and the challenges in controlling these diseases.

Evaluation of commercial quadrivalent foot-and-mouth disease vaccines against east African virus strains reveals limited immunogenicity and duration of protection

Foot-and-mouth disease virus (FMDV) causes a contagious disease (FMD) in cloven-hoofed animals. For FMD-endemic countries, vaccination is critical for controlling disease but is rarely monitored, despite substantial funds spent on vaccine purchases. We evaluated antibody responses in cattle to two commercial vaccines each containing antigens of four FMDV serotypes. Sampling was done over 360 days, with serology for each serotype performed using commercially available solid phase competition ELISAs (SPCE) and with virus neutralization tests (VNT) employing regionally relevant test viruses. A primary course of each vaccine was administered to 37 calves, some of which received a second dose after 28 days. Using new production batches of vaccines, all calves received a booster vaccination 180 days post vaccination, while 10 additional naïve calves were also vaccinated using the new batches and followed up for ∼180 days. Simple and general linear models were used to compare antibody responses which varied substantially according to vaccine, dose regime, serotype, and test, but were mostly insufficient to ensure a high likelihood of adequate or sustained probable protection. One of the vaccines administered as a two-dose primary course of vaccination was superior to other options, but even then, data trajectories from VNT responses suggested probable protection of 75 % of calves for 6 months for only one virus serotype. Calves administered with the other vaccine and those given a single primary dose developed low levels of antibodies, offering predicted likely protection lasting less than two months. Individual SPCE results were weakly correlated (r2 = 0.48) to neutralization and associated likelihoods of protection but SPCE and VNT agreed on which vaccine and dose regime performed best. Our findings highlight gaps in immunogenicity of FMD vaccines used in East Africa and reinforce the importance of independent quality control studies to evaluate and improve commercial FMD vaccines and vaccination regimes.

Development and Validation of Serotype-Specific Blocking ELISA for the Detection of Anti-FMDV O/A/Asia1/SAT2 Antibodies.

Foot-and-mouth disease (FMD) is one of the most infectious viral transboundary diseases of livestock, which causes devastating global economic losses. Different enzyme-linked immunosorbent assays (ELISAs) are used for sero-surveillance of the foot-and-mouth disease virus (FMDV). However, more sensitive, accurate, and convenient ELISAs are still required to detect antibodies against FMDV serotypes. The primary goal of this study was to establish serotype-specific monoclonal antibody (mAb)-based blocking ELISAs (mAb-bELISAs) that would provide better performance characteristics or be equivalent in performance characteristics compared with a conventional polyclonal antibody (pAb)-based competitive ELISA (pAb-cELISA). Four mAb-bELISAs were developed using FMDV serotype-specific mAbs for the detection of anti-FMDV/O/A/Asia1/SAT2 antibodies. Using a 50% cut-off, all four mAb-bELISAs exhibited species-independent 99.74%, 98.01%, 96.59%, and 98.55% diagnostic specificity (DSp) and 98.93%, 98.25%, 100%, and 87.50% diagnostic sensitivity (DSe) for FMDV serotypes O, A, Asia1, and SAT2, respectively. In addition, a 100% DSe of serotypes O- and SAT2-specific mAb-bELISAs was observed for porcine sera when the cut-off was 30%. All mAb-bELISAs developed in this study displayed high repeatability/reproducibility without cross-reactivity. Finally, the diagnostic performance of mAb-bELISAs was found to be better than or equivalent to compared with pAb-cELISAs, suggesting that mAb-bELISAs can be used to replace existing pAb-ELISAs for the detection of antibodies against these four FMDV serotypes.

Diagnostic and prophylactic potential of a stabilized foot-and-mouth disease serotype Asia1 virus like particles designed through a structure guided approach

Intact capsids of foot-and-mouth disease virus (FMDV) play a vital role in eliciting a protective immune response. Any change in the physico-chemical environment of the capsids results in dissociation and poor immunogenicity. Structural bioinfomatics studies have been carried out to predict the amino acids at the interpentameric region that resulted in the identification of mutant virus-like particles(VLPs) of FMDV serotype Asia1/IND/63/1972. The insect cell expressed VLPs were evaluated for their stability by sandwich ELISA. Among 10 mutants, S93H showed maximum retention of antigenicity at different temperatures, indicating its higher thermal stability as revealed by the in-silico analysis and retained the antigenic sites of the virus demonstrated by Sandwich ELISA. The concordant results of the liquid phase blocking ELISA for estimation of antibody titre of known sera with stable mutant VLP as antigen in place of virus antigen demonstrate its diagnostic potential. The stable mutant VLP elicited a robust immune response with 85.6 % protection in guinea pigs against virus challenge. The stabilized VLP based antigen requires minimum biosafety and cold storage for production and transit besides, complying with differentiation of infected from vaccinated animals. It can effectively replace the conventional virus handling during antigen production for prophylactic and diagnostic use.

Protein characterization of an Indonesian isolate of foot and mouth disease virus inactivated with formaldehyde and binary ethylenimine.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-footed animals. It is a major threat to livestock production worldwide, causing significant economic losses. Inactivation of FMD virus (FMDV) is crucial for vaccine development and control of outbreaks. However, traditional inactivation methods can sometimes damage the viral protein, affecting vaccine efficacy. Therefore, finding new inactivating agents that effectively inactivate the virus while preserving the integrity of its proteins is an important research area. This study investigated the optimal materials (0.04% formaldehyde, 0.001 M binary ethylenimine [BEI], or a combination) for inactivating and preserving the specific molecular weight of Serotype O FMDV protein. This study used serotype O FMDV isolated from several areas of East Java. The virus was inoculated into baby hamster kidney-21 cells, and the titer was calculated using the TCID50 Assay. The virus was inactivated using 0.04% formaldehyde, 0.001 M BEI, or a combination of 0.04% formaldehyde and 0.001 M BEI. Inactive viral proteins were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. Serotype O FMDV can be inactivated using 0.04% formaldehyde while preserving specific FMDV proteins, specifically VP0 and VP3 with a molecular weight (MW) of 36 kDa and VP3 with a MW of 24 kDa. Serotype O FMDV can be inactivated by 0.001 M BEI while preserving specific FMDV proteins, specifically VP0 with a MW of 35 kDa, VP3 with a MW of 28 kDa, and VP1 with a MW of 23 kDa. FMDV serotype O can be inactivated using a combination of 0.04% formaldehyde and 0.001 M BEI while preserving specific FMDV proteins, specifically VP0 and VP3 with a MW of 36 kDa and VP3 with a MW of 24 kDa. This study found that 0.04% formaldehyde, alone or in combination with 0.001 M BEI, was effective for inactivating and preserving the specific molecular weight of Serotype O FMDV protein. The limitation of this study was the inactivations of the virus have not yet been tested for their potency on experimental animals. Further research is warranted to investigate the inactivation kinetics of these materials, including their potency on experimental animals. Additionally, a comparison of the inactivation rates between 0.04% formaldehyde alone and the combination with BEI would help to determine the optimal inactivation agent for future applications.

Development of recombinant proteins for vaccine candidates against serotypes O and A of Foot-and-Mouth Disease virus in Bangladesh.

Frequent vaccine failure leading to recurrent outbreaks of Foot-and-Mouth Disease (FMD) in livestock populations necessitates the development of a customizable vaccine platform comprising potential antigenic determinants of circulating lineages of FMD viruses. Artificially designed, chimaeric protein-based recombinant vaccines are novel approaches to combat the phylogenetically diverse FMD Virus (FMDV) strains. Among seven recognized serotypes, only serotypes O and A are dominantly circulating in Bangladesh and neighbouring countries of Asia, where transboundary transmission, recurrent outbreaks and emergence of novel lineages of FMDV are highly prevalent. The objective of this study was to develop multi-epitope recombinant proteins, procuring immunogenicity against circulating diverse genotypes of FMDV serotypes O and A. Two chimaeric proteins, named B1 (41.0 kDa) and B3 (39.3 kDa), have been designed to incorporate potential B-cell and T-cell epitopes selected from multiple FMDV strains, including previously reported and newly emerged sub-lineages. After expression, characterization and immunization of guinea pigs with a considerable antigen load of B1 and B3 followed by serological assays revealed the significant protective immunogenicity, developed from the higher (100 µg) doses of both antigens, against most of the currently prevalent serotype O and A strains of FMDV. The efficient expression, antigenic stability, and multivalent immunogenic potency of the chimaeric proteins strongly indicate their credibility as novel vaccine candidates for existing serotypes O and A of FMDV in Bangladesh and surrounding territories.

Virus-like particle-based multipathogen vaccine of FMD and SVA elicits balanced and broad protective efficacy in mice and pigs

Inactivated vaccines lack the capability to serologically differentiate between infected and vaccinated animals, thereby impeding the effective eradication of pathogen. Conversely, vaccines based on virus-like particles (VLPs) emulate natural viruses in both size and antigenic structure, presenting a promising alternative to overcome these limitations. As the complexity of swine infectious diseases increases, the increase of vaccine types and doses may intensify the stress response. This exacerbation can lead to diminished productivity, failure of immunization, and elevated costs. Given the critical dynamics of co-infection and the clinically indistinguishable symptoms associated with foot-and-mouth disease virus (FMDV) and senecavirus A (SVA), there is a dire need for an efficacious intervention. To address these challenges, we developed a combined vaccine composed of three distinct VLPs, specifically designed to target SVA and FMDV serotypes O and A. Our research demonstrates that this trivalent VLP vaccine induces antigen-specific and robust serum antibody responses, comparable to those produced by the respective monovalent vaccines. Moreover, the immune sera from the combined VLP vaccine strongly neutralized FMDV type A and O, and SVA, with neutralization titers comparable to those of the individual vaccines, indicating a high level of immunogenic compatibility among the three VLP components. Importantly, the combined VLPs vaccines-immunized sera conferred efficient protection against single or mixed infections with FMDV type A and O, and SVA viruses in pigs. In contrast, individual vaccines could only protect pigs against homologous virus infections and not against heterologous challenges. This study presents a novel combined vaccines candidate against FMD and SVA, and provides new insights for the development of combination vaccines for other viral swine diseases.

Foot-and-mouth disease-associated myocarditis is age dependent in suckling calves

Myocarditis is considered a fatal form of foot-and-mouth disease (FMD) in suckling calves. In the present study, a total of 17 calves under 4 months of age and suspected clinically for FMD were examined for clinical lesions, respiratory rate, heart rate, and heart rhythm. Lesion samples, saliva, nasal swabs, and whole blood were collected from suspected calves and subjected to Sandwich ELISA and reverse transcription multiplex polymerase chain reaction (RT-mPCR) for detection and serotyping of FMD virus (FMDV). The samples were found to be positive for FMDV serotype “O”. Myocarditis was suspected in 6 calves based on tachypnoea, tachycardia, and gallop rhythm. Serum aspartate aminotransferase (AST), creatinine kinase myocardial band (CK-MB) and lactate dehydrogenase (LDH), and cardiac troponins (cTnI) were measured. Mean serum AST, cTn-I and LDH were significantly higher (P < 0.001) in < 2 months old FMD-infected calves showing clinical signs suggestive of myocarditis (264.833 ± 4.16; 11.650 ± 0.34 and 1213.33 ± 29.06) than those without myocarditis (< 2 months old: 110.00 ± 0.00, 0.06 ± 0.00, 1050.00 ± 0.00; > 2 months < 4 months: 83.00 ± 3.00, 0.05 ± 0.02, 1159.00 ± 27.63) and healthy control groups (< 2 months old: 67.50 ± 3.10, 0.047 ± 0.01, 1120.00 ± 31.62; > 2 months < 4 months: 72.83 ± 2.09, 0.47 ± 0.00, 1160.00 ± 18.44). However, mean serum CK-MB did not differ significantly amongst the groups. Four calves under 2 months old died and a necropsy revealed the presence of a pathognomic gross lesion of the myocardial form of FMD known as “tigroid heart”. Histopathology confirmed myocarditis. This study also reports the relevance of clinical and histopathological findings and biochemical markers in diagnosing FMD-related myocarditis in suckling calves.

Isolation, Genetic and Antigenic Characterization of Circulating Foot and Mouth Disease Virus

Foot and Mouth Disease (FMD) is a highly contagious viral illness that predominantly affects cloven-hoofed animals. To identify and profile the predominant FMDV serotypes circulating in Baluchistan bovine and buffalo populations. Twenty tissue samples were collected and analyzed using sandwich indirect ELISA and RT-PCR to identify the presence and genetic characteristics of FMD virus. We identified 'O' serotype of FMD virus in all specimens. RT-PCR analysis yielded an impressive 90% positivity rate, confirming its diagnostic efficacy. While observations of LFBK cell line lead to the isolation of 18 distinct viruses with distinct Cytopathic Effects, two samples lacked detectable CPEs. Lower CT values, those observed in sample PK- 16 with CT of 15, indicated higher initial concentration of the target nucleic acid, indicating a potential for higher viral burden.The endemic prevalence of 'O' serotype in Balochistan necessitated targeted serotype-specific interventions, strengthened surveillance and strategic vaccination campaigns.

Potent small molecule inhibitors against the 3C protease of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is one of the most devastating diseases of livestock which can cause significant economic losses, especially when introduced to FMD-free countries. FMD virus (FMDV) belongs to the family Picornaviridae and is antigenically heterogeneous with seven established serotypes. The prevailing preventive and control strategies are limited to restriction of animal movement and elimination of infected or exposed animals, which can be potentially combined with vaccination. However, FMD vaccination has limitations including delayed protection and lack of cross-protection against different serotypes. Recently, antiviral drug use for FMD outbreaks has increasingly been recognized as a potential tool to augment the existing early response strategies, but limited research has been reported on potential antiviral compounds for FMDV. FMDV 3C protease (3Cpro) cleaves the viral-encoded polyprotein into mature and functional proteins during viral replication. The essential role of viral 3Cpro in viral replication and the high conservation of 3Cpro among different FMDV serotypes make it an excellent target for antiviral drug development. We have previously reported multiple series of inhibitors against picornavirus 3Cpro or 3C-like proteases (3CLpros) encoded by coronaviruses or caliciviruses. In this study, we conducted structure-activity relationship studies for our in-house focused compound library containing 3Cpro or 3CLpro inhibitors against FMDV 3Cpro using enzyme and cell-based assays. Herein, we report the discovery of aldehyde and α-ketoamide inhibitors of FMDV 3Cpro with high potency. These data inform future preclinical studies that are related to the advancement of these compounds further along the drug development pathway.IMPORTANCEFood-and-mouth disease (FMD) virus (FMDV) causes devastating disease in cloven-hoofed animals with a significant economic impact. Emergency response to FMD outbreaks to limit FMD spread is critical, and the use of antivirals may overcome the limitations of existing control measures by providing immediate protection for susceptible animals. FMDV encodes 3C protease (3Cpro), which is essential for virus replication and an attractive target for antiviral drug discovery. Here, we report a structure-activity relationship study on multiple series of protease inhibitors and identified potent inhibitors of FMDV 3Cpro. Our results suggest that these compounds have the potential for further development as FMD antivirals.

Porcine interferon-α linked to the porcine IgG-Fc induces prolonged and broad-spectrum antiviral effects against foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is an economically important disease, and the FMD virus (FMDV) can spread rapidly in susceptible animals. FMD is usually controlled through vaccination. However, commercial FMD vaccines are only effective 4–7 days after vaccination. Furthermore, FMDV comprises seven serotypes and various topotypes, and these aspects should be considered when selecting a vaccine. Antiviral agents could provide rapid and broad protection against FMDV. Therefore, this study aimed to develop a fusion protein of consensus porcine interferon-α and Fc portion of porcine antibody IgG (poIFN-α-Fc) using a baculovirus expression system to develop a novel antiviral agent against FMDV. We measured the antiviral effects of the poIFN-α-Fc protein against FMDV and the enhanced duration in vitro and in vivo. The broad-spectrum antiviral effects were tested against seven FMDV serotypes, porcine reproductive and respiratory syndrome virus (PRRSV), and bovine enterovirus (BEV). Furthermore, the early protective effects and neutralizing antibody levels were tested by co-injecting poIFN-α-Fc and an FMD-inactivated vaccine into mice or pigs. Sustained antiviral effects in pig sera and mice were observed, and pigs injected with a combination of the poIFN-α-Fc and an inactivated FMD vaccine were protected against FMDV in a dose-dependent manner at 2- and 4-days post-vaccination. In addition, combined with the inactivated FMD vaccine, poIFN-α-Fc increased the neutralizing antibody levels in mice. Therefore, poIFN-α-Fc is a potential broad-spectrum antiviral and adjuvant candidate that can be used with inactivated FMD vaccines to protect pigs against FMDV.

Circulation of foot-and-mouth disease serotypes, risk factors, and their effect on hematological and biochemical profiles among cattle and buffalo in Quetta, Balochistan, Pakistan.

Foot-and-mouth disease (FMD) is an infectious disease of cloven-hoofed animals, including buffalo, cattle, sheep, goats, and pigs, causing major economic losses to the local farmers and, overall, to the national economy of the country. This study aimed to detect FMDV serotypes in year-round FMD outbreaks, hematological and biochemical changes, and oxidative stress in FMDV-infected cattle and buffaloes in the district of Quetta, Balochistan, Pakistan, and the socioeconomic impact of FMD outbreaks on farmers. We conducted a cross-sectional study in the district of Quetta, Balochistan, Pakistan, where FMD virus (FMDV) serotypes were detected by enzyme-linked immunosorbent assay (ELISA). Hematological, biochemical, and oxidative analyses were performed by analyzing the blood of FMDV-infected and non-infected animals. Information on the associated risk factors was obtained through a structured questionnaire by interviewing farmers in each FMD-affected farm. Thirty-four out of 38 farms (89%, 95% confidence interval [CI]: 75%-97%) were positive for FMD by ELISA. Higher FMD infection was detected in farms with a herd size of <50 animals (50%, 17/34), followed by >100 animals (32%, 11/34) and 51-100 animals (18%, 6/34). Fifty-seven percent (114/200, 95% CI: 50%-64%) of animals were positive for FMD. Of these, 61% (69/114) were cattle and 39% (45/114) were buffalo. FMD positivity was higher in females (86%, 98/114) than in males (14%, 16/114) and higher in animals older than 2 years of age (52%, 59/114). On average, farmers lose U.S. dollars 3000 annually due to FMD outbreaks. Animals infected with FMDV had significantly (p ≤ 0.05) white blood cell counts and significantly (p ≤ 0.05) lower hemoglobin and total protein concentrations in buffalo and cattle, whereas infected cattle showed significantly (p ≤ 0.05) lower albumin levels. Globulin levels were lower in buffaloes infected. Alanine aminotransferase levels were lower in infected cattle (p ≤ 0.05). Creatinine levels were higher in infected buffalo (p ≤ 0.05). Urea and phosphorus concentrations were higher in FMDV-infected cattle and buffalo (p ≤ 0.05). Calcium levels were lower in infected cattle and buffalo (p ≤ 0.05). Catalase enzyme activity in infected cattle and buffaloes was significantly lower (p < 0.05). Lipid peroxidation was significantly higher in FMDV-infected cattle and buffalo (p ≤ 0.05). This study confirmed serotype O circulation among cattle and buffalo in year-long FMD outbreaks in the Quetta District of Balochistan. Blood analysis identified a parameter deviated from the normal level due to FMDV infection. In addition, the outbreak of FMD has a significant negative economic impact on livestock farmers.

Evaluation of specific chicken IgY antibody value developing diagnostic capture antibody ELISA kit against Foot and Mouth disease.

The most preferred method for the detection of foot-and-mouth disease (FMD) viral antigen and identification of viral serotype is the enzyme-linked immunosorbent assay (ELISA). Diagnostic tests with high sensitivity are necessary both to distinguish infected vaccinated animals and execute disease control programs for the identification of the carrier animals. The current strategies for the detection of FMD virus are mainly based on the capture antibody (sandwich) ELISA test. The usage of laying pullets as an animal bioreactor for the production of specific egg yolk antibodies (IgY) has increased in recent years due to its high yield, affinity, low price, and quick production turnover. The present study aimed to produce a concentrated and purified IgY polyclonal antibody to design a capture antibody ELISA kit against the FMD virus (FMDV) serotype A. At first, laying hens were immunized with inactivated FMDV serotype virus, and then, on days 14, 21, and 28 following vaccination, the eggs and sera were collected. Afterward, the IgY polyclonal antibodies were extracted and purified from the chicken egg yolk using a polyethylene glycol 6000-ethanol precipitation procedure. Extracts were filtered, purified by ion exchange chromatography, and dialyzed. The purified IgY concentration, estimated by Bradford assay, confirmed its presence by SDS-PAGE and Western blot and also its specific immune reaction by Ouchterlony double immunodiffusion and Dot blot tests. Moreover, for achieving the optimum concentration of antigen/antibody (sera) in sandwich ELISA, a checkerboardtitrationtest was set up based on indirect ELISA results. Eventually, 119 previously confirmed samples (including 80 positive and 39 negative) by both real-time polymerase chain reaction (quantitative PCR, qPCR) and a commercial ELISA kit were used for evaluation of the sensitivity and accuracy of our developed Capture antibody ELISA kit. In this manner, the sensitivity and specificity of our designed kit were 100% and 98%, respectively. Accordingly, the present developed capture ELISA kit based on IgY had high sensitivity and specificity for FMD virus detection and it could be used in the future for both commercial detecting and serotyping applications.

Evaluation of a Vaccine Candidate Designed for Broad-Spectrum Protection against Type A Foot-and-Mouth Disease in Asia.

Foot-and-mouth disease (FMD) vaccines are currently the most powerful protective and preventive measures used to control FMD. In this study, the chimeric vaccine strain containing antigenic epitopes from the FMD virus serotype A, which belongs to the ASIA topotype, was produced and evaluated. The chimeric vaccine strains contain sea-97/G1 (VP4, VP2, VP3) and A22 Iraq (VP1) or G-VII (VP1) for use in FMD vaccines in Asia. The 50% protective dose was determined in mice. Vaccinated mice were challenged with three different type A viruses (Sea-97/G1, Sea-97/G2, G-VII clade) seven days post-vaccination (dpv), and mice that received the vaccine candidates were protected against the three viruses. The protective capability of one of the vaccine candidates was evaluated in pigs. Vaccinated pigs were challenged with three different type A viruses (Sea-97/G1, Sea-97/G2, G-VII clade) at 28 dpv, and pigs that received the vaccine candidate were protected against the three viruses. The results showed that this vaccine candidate, which was designed to provide protection against FMD in Asia, efficiently protected pigs against virus challenge and thus has potential as a broad-spectrum vaccine for various epidemic FMD viruses.

Analysis of Coinfection Pathogens From Foot‐and‐Mouth Disease Virus Persistently Infected Cattle Using Oxford Nanopore Sequencing

The persistent infection caused by foot‐and‐mouth disease virus (FMDV) still lacks a reliable explanation, as its etiology and maintenance are intricate and potentially involve concurrent infections with multiple pathogens. In this study, we utilized the nanopore platform for direct sequencing of clinical samples obtained from cattle persistently infected with FMDV serotype O and investigated the distribution characteristics of coinfecting pathogens in their pharyngeal region. Briefly, we exploited Oxford Nanopore sequencing technology to generate high‐quality and sufficient sequence data for the comprehensive characterization of microbial genomes. Furthermore, we performed sequence comparison, alignment, and phylogenetic tree construction. Our findings revealed a total of 23 viruses in FMDV carrier bovine, with FMDV, bovine orthopneumovirus, and Choristoneura fumiferana granulovirus emerging as the top three identified pathogens. The analysis unexpectedly revealed the presence of porcine circovirus type 2 and pepper mild mottle virus among the viral genes detected in the bovine FMDV carrier. Compared to noncarrier, carrier bovine of FMDV exhibited a greater diversity and abundance of mycoplasma types as well as reads counts. Therefore, we propose that the establishment and perpetuation of persistent FMDV infection may be attributed to the simultaneous presence of other viral agents and mycoplasmas. These findings highlight the significance of investigating multipathogen coinfection in elucidating the etiology of persistent FMD virus infection.