Validation of binary ethyleneimine (BEI) used as an inactivant for foot and mouth disease tissue culture vaccine

Spatial trend of Foot and Mouth Disease virus (FMDV) serotypes in cattle and buffaloes, Pakistan

Foot and mouth disease (FMD) virus: Quantification of whole virus particles during the vaccine manufacturing process by size exclusion chromatography

Animal products such as raw salted cowhide are thought to have the potential to transmit the foot and mouth disease (FMD) virus from the infected zone. Indonesia imports raw salted cowhide from Malaysia, so it has the potential to transmit FMD to Indonesia which enters through Tanjung Priok Port. This study aimed to investigate the presence of the FMD virus in raw salted cowhide from Malaysia. The number of samples was collected from each container of raw salted cowhide imported through Tanjung Priok Port during August–December 2022. A total of 21 samples were obtained from 21 bulk containers containing raw salted cowhide. Real time q Polymerase Chain Reaction (RT-qPCR) was used to investigate foot and mouth disease virus in samples. The RT-qPCR screening test on 21 samples reported that salted raw cowhide was free from the FMD virus. Continuous monitoring and surveillance protocols for salted rawhide imported from non-free countries need to be carried out at other points of entry.

Enzyme labelled immunosorbent assays (ELISA) have been developed to detect and quantify foot and mouth disease (FMD) virus using flexible plastic microtitre plates. The methods were successful for the specific detection of FMD virus and were 50 to 100 times more sensitive than the complement fixation test. The application of the ELISA techniques to FMD virus typing and subtyping, and to the assay of antigen concentration during manufacture of vaccines is discussed.

Foot and mouth disease (FMD) is a major Transboundary Animal Disease (TAD) which causes major economics losses to the developing countries. In Pakistan the disease is considered endemic and outbreaks are still being reported. Rapid diagnosis of the disease and Isolation of FMD virus is important to confirm viral subtyping and allow for the development of effective vaccines against the specific subtypes. Carrier animals are the major source of current outbreaks of FMD in Pakistan. Current study was planned and conducted for isolation of FMD virus from persistently infected animals by using LFBK cell line and comparison of LFBK and LFBK αVβ6 for isolation of FMDV isolates from recent FMD outbreaks. A total of 120 serum samples were collected from persistently infected riverine buffaloes and examined for the presence of FMD virus Non-Structural Proteins by using NSP-ELISA. Of 120 sera samples 23 animals were found positive for NSP’s. The Oro-pharyngeal fluids (OP) were collected from NSP-ELISA positive animals. The OP fluids samples were treated with Tri-cholo-Tri-flouro-Ethane (TTE) and inoculated onto LFBK cell line. Out of 23 OP fluid samples 11 exhibited CPE’s. A total of six (06) FMD viruses were confirmed by rRT-PCR and characterized by Indirect Sandwich ELISA as type O, Asia-1and A. The FMD virus isolates were acquired from FAO-UN project on FMD in Pakistan. All isolates were inoculated on both of the cell lines and observed for the development of CPEs. We found that the newly modified LFBK αVβ6 cell line exhibited CPEs more rapidly after 18-20 hours, while LFBK cell line CPEs developed after 24 to 48 hours. TCID50 calculated on LFBK αVβ6 was higher for the all the serotypes tested than LFBK cell line. Percentage of CPEs in LFBK αVβ6 per plate resulted higher than over LFBK cell line.

Interferon is an important cytokine that plays a critical role in the initial host defense against viral infection. Recombinant human adenoviruses expressing human interferon-α (Ad-HIFNα) or pig interferon-β fused with interleukin-18 (Ad-PIFNβ-IL18) were constructed and used to induce an early protective response against foot and mouth disease (FMD). To analyze the antiviral effect, bovine thyroid and porcine kidney IBRS-2 cells and ICR mice were treated with Ad-HIFNα, Ad-PIFNβ-IL18, and cocktail of Ad-HIFNα and Ad-PIFNβ-IL18. The survival rate of suckling mice was monitored after foot and mouth disease virus (FMDV) challenge following intra-peritoneal (IP) administration of appropriate adenovirus. Indirect antigen ELISA was performed to evaluate inhibition of FMDV replication following challenge with the FMDV O, A, or Asia 1 serotypes in vitro. These recombinant adenoviruses reduced the replication of FMDV in susceptible cells, thereby decreasing the fatality in mice, suggesting that they can be a useful control method for the early protection against FMD infection in livestock after field trial.

Since March 1997 two strains of foot and mouth disease (FMD) virus have found their way into Taiwan, causing severe outbreaks in pigs and in Chinese yellow cattle. Outbreaks occurred in March 1997 were caused by a pig-adapted virus strain (O/Taiwan/97) which did not infect other species of cloven-hoofed animals by natural route. The epidemic spread over the whole region of Taiwan within two months and the aftermath was 6,147 pig farms infected and 3,850,746 pigs destroyed. In June 1999, the second strain of FMD virus (O/Taiwan/99) was isolated from the Chinese yellow cattle in the Kinmen Prefecture and in the western part of Taiwan. By the end of 1999, Chinese yellow cattle were the only species infected and those infected cattle did not develop pathological lesions. Seroconversions of serum neutralization antibody and on non-structural protein (NSP) antibodies were the best indicators for infection in non-vaccinated herds. The infected animals, however, excreted infectious levels of virus to infect new hosts. Based on the detection of the specific antibody to FMD virus, and virus isolation from oesophageal-pharyngeal (OP) fluid samples, ten herds of Chinese yellow cattle located in Kinmen and Taiwan were declared to have been infected. During the period of January to March 2000, however, five outbreaks caused by FMD virus similar to the O/Taiwan/99 virus occurred in four prefectures of Taiwan. The infected species included goats, Chinese yellow cattle and dairy cattle. Those outbreaks have caused high mortality in goat kids under two weeks old and also developed typical clinical signs of infection in dairy cattle.

It was reported that the sera of convalescent animals contain antibodies to foot and mouth disease (FMD) virus (FMDV) 2C, highly conserved nonstructural protein (NSP), whereas the sera of vaccinated animals do not. But ELISA methods using this protein were not reported and developed until recently. In this study, NSP 2C peptides were synthesized within the amino acid sequence of the conserved 2C nonstructural region of FMDV according to the sequences from Genbank database and used for identifying antigenic determinants. One of the synthesized thirteen peptides gave strong positive reactivity with most of the sera from 13 FMD infected farms, but not with sera from vaccinated and non-infected animals. Moreover, with the sera collected through serial bleedings from four cattle and five goats infected with FMDV O/SKR/2000 experimentally, positive results were obtained in two species after 10 days post infection (DPI). Therefore, we tried to develop and evaluate this ELISA based on 2C peptides. In comparison with the commercial NSP ELISA, the 2C peptide based ELISA method showed good specificity and sensitivity. These results demonstrate that the synthetic 2C peptide ELISA can be a complementary marker to differentiate FMDV-infected from vaccinated on a herd basis.

Foot and mouth disease (FMD) virus was reported as an outbreak in Indonesia in April 2022 and belonged to serotype O/ME-SA/Ind-2001e is spread in the country. This study aimed to detect the causative agent based on clinical symptoms in cattle that have the vesicle in the mouth and hooves. A total of 25 samples were collected during August 2022 from Lamongan and Surabaya, Indonesia. FMD was identified in 58% (7/12) using reverse transcription polymerase chain reaction (RT-PCR), respectively. The samples were performed using universal primers with 328 bp length for primary diagnosis of FMD. These findings indicate that the spread of FMD viruses is highly contagious, so rapid and accurate diagnosis is needed as an effort to control and monitor FMD viruses.

Milk is seen as a chief source of protein and other biologically available nutrients for human beings. Pakistan, the fourth largest milk-producing country, is badly affected by the contagious transboundary apthoviral disease of ungulate animals; the foot and mouth disease (FMD) virus. FMD is endemic in Pakistan and has caused significant economic loss to the dairy industry in the form of a profound decrease in milk production and increased morbidity and deaths of dairy animals. Inclusively, the case fatality ratio of FMD was 15.11%. Of the seven FMDV serotypes, (O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3), three serotypes (O, A, and Asia-1) are endemic in Pakistan. Rapid and highly sensitive diagnostic tools are required for efficient control of this disease. Presently, FMD in the laboratory is diagnosed via ELISA and molecular approaches, i.e., RT-PCR. Serotype-specific RT-PCR analysis not only confirms ELISA serotyping results but can also be used for the screening of ELISA negative samples. Genotypically, FMDV serotype O has a topotype (Middle East–South Asia (ME–SA) and lineage PanAsia-2) that is reported frequently from different areas of Pakistan. Confirmed cases of serotype A and Asia-1 are also reported. The information gathered can be used for understanding the molecular epidemiology of FMD in Pakistan. Further studies on the molecular dynamics of FMD could be useful for ensuring the timely diagnosis of this deadly pathogen, which would ultimately be beneficial for the mass vaccination programs of FMD in Pakistan.

Background: Foot and mouth disease (FMD) is ubiquitous worldwide but endemic in many countries of Africa, Asia, South America, and the Middle East. Many reasons contribute to the incidence of viral diseases even in vaccinated animals. These reasons include low antigenic payload, low PD50, improper formulation, unstable vaccine containing antigen, and genetically different from field strain. Among these, the most important one is the low antigenic load per dose of the vaccine. Vaccine failure is mainly due to the direct use of virus suspension in the vaccine without the concentration of viral antigen. Another reason to concentrate the antigen is small volume storage in the vaccine bank. These issues are mostly concerned with developing countries like Pakistan which lack antigen concentration technology. The concentration of the virus is a major milestone to be achieved for the production of an effective vaccine as well as for the diagnostic tool.Methods: Different techniques including precipitation with polyethylene glycol, ammonium sulfate, methanol, and filtration through an ultra-filter membrane were used for the concentration of viral suspension. Antigen quantification in terms of µg/ml was determined through size exclusion chromatography by using Sephacryl S-300 as a stationary phase.Results: Percentage recovery of FMDV calculated through analysis of chromatograms found 77.80%, 59.75%, 32.50%, and 13.83% for polyethylene glycol, ammonium sulfate, ultra-filtration, and methanol treated samples respectively.Conclusion: Classical polyethylene glycol precipitation showed a maximum percentage recovery of foot and mouth disease virus as compared to other concentration methods.Keywords: Foot and mouth disease virus (FMDV); Concentration Methods; Polyethylene Glycol (PEG); Size Exclusion Chromatography (SEC); Sephacryl S-300

Antiviral activity of merimepodib against foot and mouth disease virus in vitro and in vivo

Foot and mouth disease virus (FMDV) is the cause of foot and mouth disease (FMD) outbreaks in livestock worldwide, which affects domestic and international trade, resulting in significant economic losses and social consequences. For efficient monitoring and prevention of FMD outbreaks, the need for improved strategies to control FMDV and achieve FMD-free status with various control measures including vaccination can be established. In vaccinology, major advances and discoveries in vaccination variations including DNA and protein subunit vaccines proved to be more economical and sustainable. To develop a safe vaccine for animals, possible antigenic genes or antigens need to be identified and characterised. The FMDV is a single-stranded RNA virus consisting of a capsid precursor polypeptide, P1, which encodes for four structural proteins (VP4-1), leading to antigenic variation and VP1 potentially carrying the key epitope for vaccine development. This study aims to identify and characterise the capsid polypeptide, P1 and capsid protein, VP1 of the Malaysian FMDV serotype O and serotype A isolates. The nucleotide and protein sequences were identified based on the FMD outbreaks in Malaysia and the antigenicity of the P1 and VP1 was predicted by Kolaskar and Tongaonkar's semi-empirical method. Subsequently, the P1 and VP1 genes were inserted into pET-28a, respectively, and used for protein expression analysis. The P1 and VP1 were predicted to be antigenic via in silico analysis and successfully expressed and characterised through in vitro analysis. Hence, this study can be exploited as a tool to design a new novel vaccine for vaccine development against FMD in bovines.

Foot and mouth disease (FMD) is a highly contagious viral disease of clovenhooved animals that has devastating outcomes.Apart from vaccination, there is no antiviral therapy available to treat the on-going FMD.The current trial was carried out to investigate the antiviral activity of Withania somnifera (W.somnifera) and Curcuma longa (C.longa) plants traditionally used against Foot and mouth disease virus (FMDV).Tissue culture technique along with 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to evaluate the antiviral properties of two medicinal plants using the FMDV.The ethanol and ethyl acetate extracts of W. somnifera and C. longa were used to determine in-vitro antiviral activity against the FMDV serotype-O.The maximum non-toxic dose (MNTD) of ethanolic and ethyl acetate extract of W. somnifera roots and C. longa were 0.625 mg/ml, 0.312 mg/ml, 5 mg/ml, and 0.625 mg/ml, respectively.For antiviral activity test, 10 tissue culture infective dose 50 (TCID-50) of FMDV type-O was incubated with MNTD of each of the plant extracts for one hour prior to inoculation on cells.The preliminary antiviral extracts were tested for 3 days, and the cytopathic effects were assessed by MTT dye uptake assay.The percentage protection of baby hamster kidney-21 (BHK-21) cells against FMDV by using MNTD of ethanolic and ethyl acetate extracts of W. somnifera and C. longa were found 77.4,76.3, 55.4, and 67.1%, respectively.Results indicated that the extract of W. somnifera possesses more antiviral effect as compared to C. longa, although the ethanolic extracts of both plants showed highest activity as compared to their ethyl acetate extracts.As these two medicinal plants possess good antiviral activities in-vitro against the FMDV Serotype O, therefore, further research is needed to be carried out by performing in-vivo trials, so that their antiviral properties may be assessed, and their use may be recommended in the field.

The carrier state in foot and mouth disease—an immunological review