Foot-and-mouth Disease Virus Inactivation Research Articles

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.

Inactivation of foot-and-mouth disease virus in epithelium samples for safe transport and processing in low-containment laboratories

During a foot-and-mouth disease (FMD) outbreak, transport and testing of potentially infectious samples, including epithelium from suspect lesions, presents a biosafety risk, particularly in FMD-free countries. Therefore, treatment to inactivate virus prior to transport is important. Tongue epithelium from cattle infected with FMD virus (FMDV) serotype O (O ALG/3/2014 – Lineage O/ME-SA/Ind-2001d) or A (A IRN/22/2015 – Lineage A/ASIA/G-VII) was incubated in RNAlater, RNA Shield or phosphate-buffered saline (pH 7.4) at room temperature for 2, 6, 24 or 48 h. After incubation, tissues were homogenised and tested by virus titration. Viral RNA in the homogenate was quantified by RT-qPCR, used for sequencing, and transfected into LFBKαVβ6 cells to recover infectious virus.RNAlater reduced A IRN/22/2015 titres by 4 log10 after 24 h, and completely after 48 h incubation. While O ALG/3/2014 was detected by VI after 2, 6 and 24 h, titration yielded no infectious virus, likely as a result of freeze-thawing. RNA Shield was cytotoxic at high concentrations but was effective at inactivating both strains after 24 h. Regardless of reagent or inactivation period, RT-qPCR, VP1 sequencing, and transfection of RNA to recover infectious virus were possible. RNA Shield appears a better choice for FMDV inactivation in tissues, however 24 h incubation is recommended.

Effect of carcase decomposition on the inactivation of foot-and-mouth disease virus under northern Australian conditions.

The control of foot-and-mouth disease virus (FMDV) across northern Australia would likely result in animal carcases that will often be inaccessible for disposal. The aim of this preliminary study was to determine whether the natural pH and/or temperature changes that occur within the skeletal muscle and/or body cavities of a decomposing carcase shot and left in situ in this environment would be sufficient to inactivate FMDV. Study pigs (n = 30), cattle (6), sheep (6) and goats (8) were shot in one of four locations in Queensland. Carcase temperature and pH and ambient temperature were measured every 15-60 min for up to 46 h in two sites per animal: central (thoracic/abdominal cavity) and peripheral (skeletal muscle) or brain. A target pH ≤ 6.0 at any time and/or a target temperature ≥ 43°C for ≥ 7 h or ≥ 49°C for ≥ 1 h were used as proxies for achievement of FMDV inactivation. The target temperature was achieved in only one goat carcase. However, within 16 h of death, the target central and/or peripheral pH was attained in 88-100% of pig, cattle and sheep carcases. Increasing hours since death and death in the late morning/afternoon, relative to the early morning, were positively associated with attaining the target central carcase pH. This preliminary study provided evidence that FMDV inactivation may be achieved in the skeletal muscle and/or body cavities of carcases left under northern Australian conditions, though further work on pH changes in bone marrow are required.

Safe and cost-effective protocol for shipment of samples from Foot-and-Mouth Disease suspected cases for laboratory diagnostic.

An essential step towards the global control and eradication of foot-and-mouth disease (FMD) is the identification of circulating virus strains in endemic regions to implement adequate outbreak control measures. However, due to the high biological risk and the requirement for biological samples to be shipped frozen, the cost of shipping samples becomes one of major obstacles hindering submission of suspected samples to reference laboratories for virus identification. In this study, we report the development of a cost-effective and safe method for shipment of FMD samples. The protocol is based on the inactivation of FMD virus (FMDV) on lateral flow device (LFD, penside test routinely used in the field for rapid immunodetection of FMDV), allowing its subsequent detection and typing by RT-PCR and recovery of live virus upon RNA transfection into permissive cells. After live FMDV collection onto LFD strip and soaking in 0.2% citric acid solution, the virus is totally inactivated. Viral RNA is still detectable by real-time RT-PCR following inactivation, and the virus strain can be characterized by sequencing of the VP1 coding region. In addition, live virus can be rescued by transfecting RNA extract from treated LFD into cells. This protocol should help promoting submission of FMD suspected samples to reference laboratories (by reducing the cost of sample shipping) and thus characterization of FMDV strains circulating in endemic regions.

Virus inactivation by salt (NaCl) and phosphate supplemented salt in a 3D collagen matrix model for natural sausage casings

Due to possible presence and spread of contagious animal viruses via natural sausage casings the international trade in these food products is subject to veterinary and public health requirements. In order to manage these restrictions we determined the effect of casing preservation on four highly contagious viruses for livestock: foot-and-mouth-disease virus (FMDV), classical swine fever virus (CSFV), swine vesicular disease virus (SVDV) and African swine fever virus (ASFV). We used an in vitro 3D collagen matrix model in which cells, infected with the four different viruses were embedded in a bovine collagen type I gel matrix and treated with either saturated salt (NaCl) or phosphate supplemented saturated salt at four different temperatures (4, 12, 20 and 25 °C) during a period of 30 days. The results showed that all viruses were faster inactivated at higher temperatures, but that stability of the various viruses at 4 °C differed. Inactivation of FMDV in the 3D collagen matrix model showed a clear temperature and treatment effect on the reduction of FMDV titres. At 4 and 12 °C phosphate supplemented salt showed a very strong FMDV inactivation during the first hour of incubation. Salt (NaCl) only had a minor effect on FMDV inactivation. Phosphate supplemented salt treatment increased the effect temperature had on inactivation of CSFV. In contrast, the salt (NaCl) treatment only increased CSFV inactivation at the higher temperatures (20 °C and 25 °C). Also SVDV inactivation was increased by phosphate supplemented salt, but salt (NaCl) treatment only resulted in a significant decrease of SVDV titre at a few time points. The ASFV results showed that both salt (NaCl) and phosphate supplemented salt were capable to inactivate ASFV within 48 h. In contrast to the other viruses (FMDV, CSFV and SVDV), ASFV was the most stable virus even at higher temperatures. The results obtained in this in vitro model underline the efficacy of a combined treatment using phosphate supplemented salt and storage at 20 °C or higher for a period of 30 days. This treatment may therefore be useful in reducing the animal health risks posed by spread of contagious animal viruses by international trade of natural sausage casings.

Thermal Inactivation of Foot-and-Mouth Disease Virus in Milk Using High-Temperature, Short-Time Pasteurization

Previous studies of laboratory simulation of high temperature, short time pasteurization (HTST) to eliminate foot-and-mouth disease virus (FMDV) in milk have shown that the virus is not completely inactivated at the legal pasteurization minimum (71.7°C/15s) but is inactivated in a flow apparatus at 148°C with holding times of 2 to 3s. It was the intent of this study to determine whether HTST pasteurization conducted in a continuous-flow pasteurizer that simulates commercial operation would enhance FMDV inactivation in milk. Cows were inoculated in the mammary gland with the field strain of FMDV (01/UK). Infected raw whole milk and 2% milk were then pasteurized using an Arm-field pilot-scale, continuous-flow HTST pasteurizer equipped with a plate-and-frame heat exchanger and a holding tube. The milk samples, containing FMDV at levels of up to 104 plaque-forming units/mL, were pasteurized at temperatures ranging from 72 to 95°C at holding times of either 18.6 or 36s. Pasteurization decreased virus infectivity by 4 log10 to undetectable levels in tissue culture. However, residual infectivity was still detectable for selected pasteurized milk samples, as shown by intramuscular and intradermal inoculation of milk into naïve steers. Although HTST pasteurization did not completely inactivate viral infectivity in whole and 2% milk, possibly because a fraction of the virus was protected by the milk fat and the casein proteins, it greatly reduced the risk of natural transmission of FMDV by milk.

Quantitative Risk Assessment of FMD Virus Transmission via Water

Foot-and-mouth disease (FMD) is a viral disease of domesticated and wild cloven-hoofed animals. FMD virus is known to spread by direct contact between infected and susceptible animals, by animal products such as meat and milk, by the airborne route, and mechanical transfer on people, wild animals, birds, and by vehicles. During the outbreak of 2001 in the Netherlands, milk from dairy cattle was illegally discharged into the sewerage as a consequence of transport prohibition. This may lead to contaminated discharges of biologically treated and raw sewage in surface water that is given to cattle to drink. The objective of the present study was to assess the probability of infecting dairy cows that were drinking FMD virus contaminated surface water due to illegal discharges of contaminated milk. So, the following data were collected from literature: FMD virus inactivation in aqueous environments, FMD virus concentrations in milk, dilution in sewage water, virus removal by sewage treatment, dilution in surface water, water consumption of cows, size of a herd in a meadow, and dose-response data for ingested FMD virus by cattle. In the case of 1.6 x 10(2) FMD virus per milliliter in milk and discharge of treated sewage in surface water, the probability of infecting a herd of cows was estimated to be 3.3 x 10(-7) to 8.5 x 10(-5), dependent on dilution in the receiving surface water. In the case of discharge of raw sewage, all probabilities of infection were 100 times higher. In the case of little dilution in small rivers, the high level of 8.5 x 10(-3) is reached. For 10(4) times higher FMD virus concentrations in milk, the probabilities of infecting a herd of cows are high in the case of discharge of treated sewage (3.3 x 10(-3) to 5.7 x 10(-1)) and very high in the case of discharge of raw sewage (0.28-1.0). It can be concluded that illegal and uncontrolled discharges of contaminated milk into the sewerage system may lead to high risks to other cattle farms at 6-50 km distance of the location of discharge within one day. This clearly underlines current measures that prohibit such discharges, and also asks for strict control. This risk assessment clearly demonstrated the potential significance of FMD virus transmission via water, and the results will be useful on an international scale, and could also serve as a basis for other FMD risk-assessment models.

Immunogenicity of namogram to milligram quantities of inactivated foot-and-mouth disease virus. I. Relative virus-neutralizing potency of guinea pig sera.

Quantitative antigen dose-neutralizing antibody response curves were established in guinea pigs for purified foot-and-mouth disease virus (FMDV), type A, strain 119, inactivated for 48 hr with N-acetylethyleneimine (AEI). Inactivation of FMDV by 0.05% AEI at 25 C occurred without virus degradation and followed first-order kinetics over a 10(8)-fold decrease in plaque-forming units (PFU) extrapolating to 10(-5) PFU/ml at 48 hr. The AEI-treated virus was administered in doses ranging from 10 ng to 2.62 mg, alone or emulsified in oil adjuvant. Sigmoidal dose-response curves were obtained with 160 ng as the minimum effective dose. The maximum effective dose was 163 mug and 2.62 mg or more at 6 and 28 through 84 days postinoculation, respectively. Oil adjuvant had little effect at 6 days postinoculation, but its use markedly increased the amount of neutralizing antibody obtained at the later testing periods.

Immunogenicity of Nanogram to Milligram Quantities of Inactivated Foot-and-Mouth Disease Virus. I. Relative Virus-neutralizing Potency of Guinea Pig Sera

Quantitative antigen dose-neutralizing antibody response curves were established in guinea pigs for purified foot-and-mouth disease virus (FMDV), type A, strain 119, inactivated for 48 hr with N-acetylethyleneimine (AEI). Inactivation of FMDV by 0.05% AEI at 25 C occurred without virus degradation and followed first-order kinetics over a 10(8)-fold decrease in plaque-forming units (PFU) extrapolating to 10(-5) PFU/ml at 48 hr. The AEI-treated virus was administered in doses ranging from 10 ng to 2.62 mg, alone or emulsified in oil adjuvant. Sigmoidal dose-response curves were obtained with 160 ng as the minimum effective dose. The maximum effective dose was 163 mug and 2.62 mg or more at 6 and 28 through 84 days postinoculation, respectively. Oil adjuvant had little effect at 6 days postinoculation, but its use markedly increased the amount of neutralizing antibody obtained at the later testing periods.

The inactivation of foot-and-mouth disease virus at ionic-strength dependent isoelectric points.

Abstract Foot-and-mouth disease virus (FMDV) isoelectric point (pI) values, determined by cellulose acetate electrophoresis, were linear with respect to the square root of the ionic strength. This effect for proteins is attributed to the preferential binding of anions. The Longsworth and Jacobsen equation, pI=pI0−(AΓ/2)/(1+BΓ/2), was evaluated for FMDV; Γ/2 is the ionic strength, pI0 is the isoionic point 7.6, and constants A and B are 21.5 and 4.2, respectively. With pI values of 3.25−7.5 estimated from this equation, it was found that FMDV was always inactivated at its pl, while at 2 pH units above its pI there was no loss of infectivity. Storing virus at conditions greater than 2 pH units above isoelectric points at alkaline pH also resulted in inactivation. Thus, virus inactivation or stability can be predetermined over a range of hydrogen ion concentrations and ionic strengths. Inactivation of FMDV below pH 7.0 in isotonic solution or degradation at pH 7.5 in hypotonic solution (Γ/2 0.002) which previously appeared to occur by two independent mechanisms are both the result of isoelectric inactivation.