Inactivation of Foot-and-Mouth Disease Virus with Ethylenimine

Foot-and-mouth disease virus (FMDV) was inactivated by ethylenimine (EI) at three concentrations and two temperatures. Comparison of inactivation kinetics and the antigenic and immunogenic potency of EI and N-acetylethylenimine (AEI)-inactivated FMDV indicates that EI has nearly optimal characteristics as an inactivant for FMDV vaccine preparation. Although AEI-inactivated FMDV has proved to be a potent specific immunogen, an equivalent percentage of EI inactivated FMDV at substantially faster rates and produced an equally potent immunogen. In addition, EI inactivated FMDV at rates that were essentially linear throughout the loss of nearly all measurable infectivity.     

Inactivation of Foot-and-Mouth Disease Virus by Citric Acid and Sodium Carbonate with Deicers

Three out of five outbreaks of foot-and-mouth disease (FMD) since 2010 in the Republic of Korea have occurred in the winter. At the freezing temperatures, it was impossible to spray disinfectant on the surfaces of vehicles, roads, and farm premises because the disinfectant would be frozen shortly after discharge and the surfaces of the roads or machines would become slippery in cold weather. In this study, we added chemical deicers (ethylene glycol, propylene glycol, sodium chloride, calcium chloride, ethyl alcohol, and commercial windshield washer fluid) to keep disinfectants (0.2% citric acid and 4% sodium carbonate) from freezing, and we tested their virucidal efficacies under simulated cold temperatures in a tube. The 0.2% citric acid could reduce the virus titer 4 logs at −20°C with all the deicers. On the other hand, 4% sodium carbonate showed little virucidal activity at −20°C within 30 min, although it resisted being frozen with the function of the deicers. In conclusion, for the winter season, we may recommend the use of citric acid (>0.2%) diluted in 30% ethyl alcohol or 25% sodium chloride solvent, depending on its purpose.     

Thermal Inactivation of Foot-and-Mouth Disease Viruses in Suspension

The heat resistance of foot-and-mouth disease virus (FMDV) strains isolated from outbreaks in Thailand was investigated in phosphate-buffered saline (PBS) at 50, 60, 70, 80, 90, and 100°C. The first-order kinetic model fitted most of the observed linear inactivation curves. The ranges of decimal-reduction time (D value) of FMDV strains at 50, 60, 70, 80, 90, and 100°C were 732 to 1,275 s, 16.37 to 42.00 s, 6.06 to 10.87 s, 2.84 to 5.99 s, 1.65 to 3.18 s, and 1.90 to 2.94 s, respectively. The heat resistances of FMDV strains at lower temperature (50°C) were not serotype specific. The effective inactivating temperature is approximately 60°C. Heat resistances of FMDV strains at 90 and 100°C were not statistically different (P > 0.05), while the FMDV serotype O (OPN) appeared to be the most heat resistant at 60 to 80°C. The other observed inactivation curves were linear with shoulder or tailing (biphasic curves). The shoulder effect was mostly observed at 90 and 100°C, while the tailing effect was mostly observed at 50 to 80°C. The adjusted D values in the case of shoulder and tailing effects did not affect the overall estimated heat resistance of these FMDV strains, so even unadjusted D values of deviant inactivation curves were legitimate. The z values of FMDV serotypes O, A, and Asia 1 were 21.78 to 23.26, 20.75 to 22.79, and 19.87°C, respectively. The z values of FMDV strains studied were not statistically significantly different (P > 0.05). The results of this study indicated that the heat resistance in PBS of FMDV strains from Thailand was much less than had been reported for foreign epidemic FMDV strains.     

Foot-and-Mouth Disease Virus Modulates Cellular Vimentin for Virus Survival

Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Aphthovirus within the Picornaviridae family. During infection with FMDV, several host cell membrane rearrangements occur to form sites of viral replication. FMDV protein 2C is part of the replication complex and thought to have multiple roles during virus replication. To better understand the role of 2C in the process of virus replication, we have been using a yeast two-hybrid approach to identify host proteins that interact with 2C. We recently reported that cellular Beclin1 is a natural ligand of 2C and that it is involved in the autophagy pathway, which was shown to be important for FMDV replication. Here, we report that cellular vimentin is also a specific host binding partner for 2C. The 2C-vimentin interaction was further confirmed by coimmunoprecipitation and immunofluorescence staining to occur in FMDV-infected cells. It was shown that upon infection a vimentin structure forms around 2C and that this structure is later resolved or disappears. Interestingly, overexpression of vimentin had no effect on virus replication; however, overexpression of a truncated dominant-negative form of vimentin resulted in a significant decrease in viral yield. Acrylamide, which causes disruption of vimentin filaments, also inhibited viral yield. Alanine scanning mutagenesis was used to map the specific amino acid residues in 2C critical for vimentin binding. Using reverse genetics, we identified 2C residues that are necessary for virus growth, suggesting that the interaction between FMDV 2C and cellular vimentin is essential for virus replication.     

口蹄疫病毒诱导宿主细胞凋亡的研究

本文报道了口蹄疫病毒（Foot-and-Mouth Disease Virus,FMDV）在体外诱导PK－15细胞凋亡的研究结果,采用Hoechst33258荧光探针、DNA凝胶电泳、脱氧核糖核酸转移酶介导的制品末端标记（TUNEL）技术均检测到了典型的细胞凋亡,结果显示：使用感染性滴度为4.8lgTCID50/mL的口蹄疫病毒感染PK－15细胞,在培养32h后,荧光探针检测呈现典型的凋亡细胞核固缩和梅花状碎裂核,并伴随有凋亡小体出现,调亡率约为20％;DNA凝胶电泳显示ladder梯带;末端标记检测到强绿色荧光标记物结合于凋亡细胞核上。研究结果提示：口蹄疫病毒可以在体外诱导宿主细胞凋亡,细胞凋亡是其致细胞病变死亡的重要途径之一。     

口蹄疫病毒诱导宿主细胞凋亡的研究

本文报道了口蹄疫病毒（Foot-and-Mouth Disease Virus,FMDV）在体外诱导PK-15细胞凋亡的研究结果。采用Hoechst33258荧光探针、DNA凝胶电泳、脱氧核糖核酸转移酶介导的缺口末端标记（TUNEL）技术均检测到了典型的细胞凋亡。结果显示使用感染性滴度为4.8lgTCID50/mL的口蹄疫病毒感染PK-15细胞,在培养32?h后荧光探针检测呈现典型的凋亡细胞核固缩和梅花状碎裂核,并伴随有凋亡小体出现,凋亡率约为20%;DNA凝胶电泳显示ladder梯带;末端标记检测到强绿色荧光标记物结合于凋亡细胞核上。研究结果提示口蹄疫病毒可以在体外诱导宿主细胞凋亡,细胞凋亡是其致细胞病变死亡的重要途径之一。     

Passive Hemagglutination-Inhibition Test for Typing Foot-and-Mouth Disease Virus

In addition to currently used serological tests for the occurrence of foot-and-mouth disease virus (FMDV), a specific "passive" hemagglutination-inhibition (HAI) test has been developed as a supplement. Serial twofold dilutions of antiserum (0.05 ml) were mixed with 0.05 ml of a constant concentration of FMDV. After incubating for 30 min at 37 C, agglutinating antibodies were determined by adding 0.1 ml of 2.5% virus-sensitized erythrocytes. The minimum concentration of antiserum required to agglutinate the erythrocytes defined the inhibition in the HAI test. Similar tests using different concentrations of virus to inhibit antibodies were carried out in parallel fashion. The relationship between the logarithm of the HAI titer and the concentration of inhibiting virus was nearly first order (P > 0.25). The slope was used as a measure of the relative specificities of the antigen-antibody interaction and was independent of concentration. The HAI test was type-, subtype-, strain-, and variant-specific with the viral antigens used. In particular, typing was performed directly on bovine antisera.     

Thermal Inactivation of Newcastle Disease Virus

The rate of destruction of hemagglutinins and infectivity of Newcastle disease virus was determined over a temperature range of 37.8 to 60 C. From the calculated values of deltaH and deltaS, it was concluded that inactivation of the hemagglutinating activity and viral infectivity was due to protein denaturation.     

Inhibition of Host Cell Ribosomal Ribonucleic Acid Methylation by Foot-and-Mouth Disease Virus

A study of protein and ribonucleic acid (RNA) synthesis in cells infected by foot-and-mouth disease virus has indicated possible mechanisms of viral control over host cell metabolism. Foot-and-mouth disease virus infection of baby hamster kidney cells resulted in 50% inhibition of host cell protein synthesis at 180 min postinfection. A viral-induced interference with host cell RNA methylation was observed to be more rapidly inhibited than protein synthesis. To determine the nature of methylation inhibition, the kinetics of several host cell methylated RNA species were examined subsequent to virus infection. Data from sucrose zonal centrifugation and methylated albumin kieselguhr chromatography showed that methylation of nuclear RNA was inhibited 50% at 60 min postinfection. Inhibition of nuclear ribosomal RNA precursors and formation of nascent ribosomes correlated with inhibition kinetics of nuclear RNA methylation. It is suggested that the viral interference with the host nuclear RNA methylation is directly responsible for the observed loss of nascent ribosome formation. Moreover, early in the infectious cycle, methylation inhibition of host cell RNA could, in part, account for the cessation of host protein synthesis.     

口蹄疫病毒RT-LAMP检测方法的建立

利用逆转录环介导等温核酸扩增技术(RT-LAMP),建立了口蹄疫病毒快速检测方法,同时评价了该方法的灵敏性和特异性。结果表明,根据口蹄疫病毒多聚蛋白基因保守区段设计的LAMP引物能够在65℃下,1 h内实现目标核酸区段的大量扩增,检测结果可直接用肉眼判断。该检测体系具有极高的特异性,只能检测到目标病毒,与其他类似病毒如猪水泡病病毒、猪瘟病毒、猪细小病毒等无交叉反应,可检测到10-5稀释度的目标病毒核酸量,比普通RT-PCR的灵敏性高100倍,比荧光PCR高10倍。     

Biotechnological Approach to a new Foot-and-Mouth Disease Virus Vaccine

Abstract

The 2A region of the foot-and-mouth disease virus (FMDV) encodes a short sequence that mediates self-processing by a novel translational effect. Translation elongation arrest leads to release of the nascent polypeptide and re-initiation at the next in-frame codon. In this way discrete translation products are derived from a single open read-size of 2A peptides compared to internal promoters or IRES sequences makes them ideal candidates for use in size-restricted viral and nonviral vectors. Additionally, the diversity of the 2A sequence minimizes the chances for homologous recombination which is an important consideration when using retroviral or lentiviral systems. One outstanding question is the effect of the 2A “tag” attached to the C-terminus of the upstream protein. This may interfere with function, or more importantly may present a new epitope that could be subject to immunological surveillance. However, the attachment of extra amino acids is a routine method for labelling transgene products while leaving their function intact (e.g. tags such as the His tag and Myc tag). To our knowledge, the 2A tag does not impair activity and expression - proteins that require authentic termini, or are N-/C- terminally modified, can be introduced as the first or final polyprotein domain, respectively. In any event, strategies have now been devised that allow removal of the 2A linker (see François et al., 2004; Fang et al., 2005). The “unwanted” tag may however stick – antibodies directed against 2A can be used to detect the gene cloned upstream (Ryan and Drew, 1994; de Felipe et al., 2003, 2006). Lastly, the presence of a proline residue at the N-terminus of the downstream protein, as a relict of the 2A self-cleaving process, does not normally interfere with function – it does, however, confer high protein stability (Varshavsky, 1992).

Aware of the factors that influence expression levels it is important to empirically design any co-expression cassette to ensure the polyprotein is the most suitable arrangement in respect to desired function. As a form of control of protein biogenesis, 2A sequences are much more wide-spread than was first suspected. To appease different and opposing sensibilities, 2A variants that are not found in mammalian viruses can be used just as effectively for the production of multiple protein products. Although a relative new-kid-on-the-block in terms of co-expression studies, 2A can safely be considered an “established” player. It is clear that assorted 2A-derived proteins with diverse and distinct localized functions may be stably expressed in several different cell types demonstrating the applicability of this technology in biomedicine and biotechnology. The biotechnological applications of 2A are continually updated on www.st-andrews.ac.uk/ryanlab/Index.htm We envisage that 2A technology will become one of the predominant strategies for multigene delivery in the coming years.     

Differentiation of Virus Types in Foot-and-Mouth Disease (FMD)

An improved complement-fixation technique in foot-and-mouth disease is described in details. In the development of the technique consideration has been given to the established presence of an antigen-excessphenomenon as well as to the linear relationship between amounts of immune serum used and complement fixed. The increased accuracy of the technique has made it possible more precise and rapid to detect even small serological differences.     

Foot-and-Mouth Disease Virus: Stability of Neutralizing Antibody After Freeze-drying and Air-drying

It is possible to stabilize foot-and-mouth disease antiserum by either freeze-drying in bottles or air-drying on paper for periods of 7 months.     

Membrane Topology and Cellular Dynamics of Foot-and-Mouth Disease Virus 3A Protein

Foot-and-mouth disease virus non-structural protein 3A plays important roles in virus replication, virulence and host-range; nevertheless little is known on the interactions that this protein can establish with different cell components. In this work, we have performed in vivo dynamic studies from cells transiently expressing the green fluorescent protein (GFP) fused to the complete 3A (GFP3A) and versions including different 3A mutations. The results revealed the presence of a mobile fraction of GFP3A, which was found increased in most of the mutants analyzed, and the location of 3A in a continuous compartment in the cytoplasm. A dual behavior was also observed for GFP3A upon cell fractionation, being the protein equally recovered from the cytosolic and membrane fractions, a ratio that was also observed when the insoluble fraction was further fractioned, even in the presence of detergent. Similar results were observed in the fractionation of GFP3ABBB, a 3A protein precursor required for initiating RNA replication. A nonintegral membrane protein topology of FMDV 3A was supported by the lack of glycosylation of versions of 3A in which each of the protein termini was fused to a glycosylation acceptor tag, as well as by their accessibility to degradation by proteases. According to this model 3A would interact with membranes through its central hydrophobic region exposing its N- and C- termini to the cytosol, where interactions between viral and cellular proteins required for virus replication are expected to occur.     

口蹄疫病毒单克隆抗体的制备及应用研究进展

口蹄疫是严重影响全球政治经济的烈性动物传染病,快速诊断及有效防治对口蹄疫的防控具有重要意义。单克隆抗体具有高特异性、均质、活性单一等优点,在生物医学领域中有广泛用途。目前,国内外学者制备了多种抗口蹄疫病毒的单克隆抗体,并应用于口蹄疫病毒抗原定型、疫苗量化、抗体水平监测、自然感染与疫苗免疫动物的鉴别诊断,以及口蹄疫病毒抗原表位分析等方面。我们简要综述口蹄疫病毒单克隆抗体的制备及应用进展。     

Some Surface-Active Agents and Their Virucidal Effect on Foot-and-Mouth Disease Virus

Selected cationic and anionic surface-active compounds were tested to determine their virucidal effect on the foot-and-mouth disease virus, type O, strain M11, propagated in primary calf kidney cells. The chemical inactivation of the virus was tested with 0.5, 1.0, 2.0, and 5.0% concentrations of the selected compounds. Virus controls with pH adjusted to cover the expected range of the mixtures of the chemicals and virus were also tested. The absence of virus from the mixtures of chemical and virus after reaction at 28 C for 2 hr was assayed by inoculating suckling mice with the mixtures. One cationic compound, alkyl methyl isoquinilinium chloride, showed considerable antiviral activity due largely to pH effect. The use of the surface-active agents investigated in this study, in the presence of organic material, would not be recommended as virucides.