Substrate Optimization in Baby Hamster Kidney Cell Culture for Foot and Mouth Disease Virus Vaccine Using the Taguchi Method.

Cell culture is one of the most commonly used techniques in the production of biological products. Many physical and chemical parameters may affect cell growth and proliferation. This study was conducted to investigate the effect of chemical components as supplements using the experimental design method, which aimed at reducing the number of experiments. For this purpose, supplements including chemical components using four levels, with three replications in suspension and batch culture conditions, were examined for 72 hours using the Taguchi experimental design method. From the experiments, it was concluded that the culture media composition had a significant impact on final cell count and pH. High concentrations of different media composition alone were insufficient to ensure higher cell count. According to the results, this insufficiency was associated with an increase of 20% in the number of final cells. In the majority of cultures, the number of final cells at 48 hours increased relative to the number of final cells at 24 hours after culturing the cells.

Evaluation of cytotoxic and antiviral activities of aqueous leaves extracts of different plants against foot and mouth disease virus infection in farming animals.

Cytotoxic and antiviral activity of aqueous leaves extracts of three plants: Azadirachta indica, Moringa oleifera and Morus alba against Foot and Mouth disease virus (FMDV) were determined using MTT assay (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide). Eight different concentrations of each plant were evaluated. Cytotoxic and antiviral activity of each extract was evaluated as cell survival percentage and results were expressed as Means ± S.D. From the tested plant extracts, Azadirachta indica & Moringa oleifera exhibited cytotoxicity at 200 & 100 µ/ml respectively. In case of antiviral assay, Moringa oleifera showed potent antiviral activity (p<0.05) while Azadirachta indica showed significant antiviral activity in the range of 12.5-50 µ/ml & 50-100 µ/ml respectively. In contrast no anti-FMDV activity in the present study was observed with Morus alba, although all the tested concentrations were found to be safe.

Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

Evaluation of antiviral activity of plant extracts against foot and mouth disease virus in vitro.

The aim of this study was to evaluate antiviral activity of chloroformic leaves extracts of three plants: Azadirachta indica, Moringa oleifera and Morus alba against Foot and Mouth disease virus using MTT assay (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide). Antiviral and cytotoxic activity of each extract was evaluated as cell survival percentage and results were expressed as Means ± S.D. The concentrations which resulted in cell survival percentages of greater than 50% are considered to be effective antiviral concentrations. From the tested plant extracts, Moringa oleifera showed potent antiviral activity (p<0.05) while Azadirachta indica showed significant antiviral activity in the range of 1-50µ/ml & 12-100µ/ml respectively. In contrast no antiviral activity was observed by Morus alba as all the tested concentration resulted in significant reduction (p<0.05) in cell survival percentage.

Anti-foot-and-mouth disease virus effects of Chinese herbal kombucha in vivo.

The foot and mouth disease virus (FMDV) is sensitive to acids and can be inactivated by exposure to low pH conditions. Spraying animals at risk of infection with suspensions of acid-forming microorganisms has been identified as a potential strategy for preventing FMD. Kombucha is one of the most strongly acid-forming symbiotic probiotics and could thus be an effective agent with which to implement this strategy. Moreover, certain Chinese herbal extracts are known to have broad-spectrum antiviral effects. Chinese herbal kombucha can be prepared by fermenting Chinese herbal extracts with a kombucha culture. Previous studies demonstrated that Chinese herbal kombucha prepared in this way efficiently inhibits FMDV replication in vitro. To assess the inhibitory effects of Chinese herbal kombucha against FMDV in vitro, swine challenged by intramuscular injection with 1000 SID50 of swine FMDV serotype O strain O/China/99 after treatment with Chinese herbal kombucha were partially protected against infection, as demonstrated by a lack of clinical symptoms and qRT-PCR analysis. In a large scale field trial, spraying cattle in an FMD outbreak zone with kombucha protected against infection. Chinese herbal kombucha may be a useful probiotic agent for managing FMD outbreaks.

Repeated exposure to 5D9, an inhibitor of 3D polymerase, effectively limits the replication of foot-and-mouth disease virus in host cells.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock caused by a highly variable RNA virus (FMDV) that has seven serotypes and more than sixty subtypes. Both prophylactic and post-infection means of controlling the disease outbreak, including universally applicable vaccines and emergency response measures such as therapeutic treatments, are on high demand. In this study, we analyzed the long-term exposure outcome to a previously identified inhibitor of 3D polymerase (FMDV 3Dpol) for controlling FMDV infection and for the selection of resistance mutants. The results showed that no escape mutant viruses were isolated from FMDV A24 Cruzeiro infections in cell culture treated with gradually increasing concentrations of the antiviral compound 5D9 (4-chloro-N'-thieno, [2,3-d]pyrimidin-4-ylbenzenesulfonohydrazide) over ten passages. Biochemical and plaque assays revealed that when 5D9 was used at concentrations within a non-toxic range in cells, it drove the virus to undetectable levels at passage eight to ten. This is in contrast with observations made on parallel control (untreated) passages exhibiting fully viable and stable virus progenies. Collectively, the results demonstrated that under the experimental conditions, treatment with 5D9 does not confer a resistant phenotype and the virus is unable to evade the antiviral effect of the inhibitor. Further efforts using quantitative structure-property relationship (QSPR) based modifications of the 5D9 compound may result in the successful development of an effective in vivo antiviral drug targeting FMDV.

Inactivation of foot-and-mouth disease virus in various bovine tissues used for the production of natural sausage casings.

Bovine intestines, bladders and oesophagus are used for the production of natural casings ("beef casings") as edible sausage containers. Derived from cattle experimentally infected with FMDV (initial dosage 10(4) TCID(50)/mL, strain A Iran 97), these beef casings were treated with sodium chloride (NaCl) or phosphate supplemented salt (P-salt). In addition, different in-vitro experiments using beef casings were done on a small scale with other FMDV strains (A Turkey 06, C-Oberbayern and O(1) Manisa) as "proof of principle". Based on the combined results of the in-vivo and in-vitro experiments, it can be concluded that the storage period of 30 days at 20 °C in NaCl is sufficiently effective to inactivate a possible contamination with FMDV in beef casings and that the usage of P-salt does not clearly enhance the inactivation of FMDV infectivity. Storage of salted beef casings at about 20 °C for 30 days is already part of the Standard Operating Procedures (included in HACCP) of the international casing industry and can therefore be considered as a protective measure for the international trade in natural casings.

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.