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.     

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.     

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.     

Surface-Active Agents for Isolation of the Core Component of Avian Myeloblastosis Virus

Sixty-one surface-active agents were evaluated in a procedure designed to assess their ability to remove the envelope from the core component of avian myeloblastosis virus (AMV). The procedure consisted of centrifugation of intact AMV through a series of sucrose gradients each containing an upper layer of agent at one of eight concentrations between 0.01 and 10%. The effectiveness of an agent in producing AMV cores was indicated by (i) the appearance of light-scattering bands in the region of core buoyant density in gradient tubes; (ii) the range of surfactant concentration over which these bands appeared; and (iii) an electron microscopy assessment by the negative-staining technique of the relative proportion of core to non-core material in each of these bands. Six nonionic surfactants were selected by this screening method for comparison in regard to recovery of core protein and endogenous ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase activity, as well as further morphologic evaluation by electron microscopy. The nonionic surfactants of the polyoxyethylene alcohol class (particularly, Sterox SL) were most effective. Nonionic surfactants of the polyoxyethylene alkylphenol class (particularly, Nonidet P-40) were also effective. Sterox SL and Nonidet P-40 each gave a more than fivefold increase in specific activity of endogenous RNA-dependent DNA polymerase, and each gave a low recovery of core protein. Sterox SL did not interfere to the extent that Nonidet P-40 did in procedures which involved spectrophotometric assay at 260 nm. The use of Sterox SL resulted in the least envelope contamination of core preparations by electron microscopy examination, the most recovery of protein and endogenous RNA-dependent DNA polymerase activity, and a core buoyant density in sucrose of 1.27 g/ml.     

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-MouthDisease 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。     

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.     

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。     

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。     

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

Viroporins are a family of low-molecular-weight hydrophobic transmembrane proteins that are encoded by various animal viruses. Viroporins form transmembrane pores in host cells via oligomerization, thereby destroying cellular homeostasis and inducing cytopathy for virus replication and virion release. Among the Picornaviridae family of viruses, the 2B protein encoded by enteroviruses is well understood, whereas the viroporin activity of the 2B protein encoded by the foot-and-mouth disease virus (FMDV) has not yet been described. An analysis of the FMDV 2B protein domains by computer-aided programs conducted in this study revealed that this protein may contain two transmembrane regions. Further biochemical, biophysical and functional studies revealed that the protein possesses a number of features typical of a viroporin when it is overexpressed in bacterial and mammalian cells as well as in FMDV-infected cells. The protein was found to be mainly localized in the endoplasmic reticulum (ER), with both the N- and C-terminal domains stretched into the cytosol. It exhibited cytotoxicity in Escherichia coli, which attenuated 2B protein expression. The release of virions from cells infected with FMDV was inhibited by amantadine, a viroporin inhibitor. The 2B protein monomers interacted with each other to form both intracellular and extracellular oligomers. The Ca²⁺ concentration in the cells increased, and the integrity of the cytoplasmic membrane was disrupted in cells that expressed the 2B protein. Moreover, the 2B protein induced intense autophagy in host cells. All of the results of this study demonstrate that the FMDV 2B protein has properties that are also found in other viroporins and may be involved in the infection mechanism of FMDV.     

Serotype-Specific Transmission and Waning Immunity of Endemic Foot-and-Mouth Disease Virus in Cameroon

Foot-and-mouth disease virus (FMDV) causes morbidity and mortality in a range of animals and threatens local economies by acting as a barrier to international trade. The outbreak in the United Kingdom in 2001 that cost billions to control highlighted the risk that the pathogen poses to agriculture. In response, several mathematical models have been developed to parameterize and predict both transmission dynamics and optimal disease control. However, a lack of understanding of the multi-strain etiology prevents characterization of multi-strain dynamics. Here, we use data from FMDV serology in an endemic setting to probe strain-specific transmission and immunodynamics. Five serotypes of FMDV affect cattle in the Far North Region of Cameroon. We fit both catalytic and reverse catalytic models to serological data to estimate the force of infection and the rate of waning immunity, and to detect periods of sustained transmission. For serotypes SAT2, SAT3, and type A, a model assuming life-long immunity fit better. For serotypes SAT1 and type O, the better-fit model suggests that immunity may wane over time. Our analysis further indicates that type O has the greatest force of infection and the longest duration of immunity. Estimates for the force of infection were time-varying and indicated that serotypes SAT1 and O displayed endemic dynamics, serotype A displayed epidemic dynamics, and SAT2 and SAT3 did not sustain local chains of transmission. Since these results were obtained from the same population at the same time, they highlight important differences in transmission specific to each serotype. They also show that immunity wanes at rates specific to each serotype, which influences patterns of local persistence. Overall, this work shows that viral serotypes can differ significantly in their epidemiological and immunological characteristics. Patterns and processes that drive transmission in endemic settings must consider complex viral dynamics for accurate representation and interpretation.     

Effect of Foot-and-Mouth Disease Virus Infection on the Frequency,Phenotype and Function of Circulating Dendritic Cells in Cattle

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that causes one of the most devastating diseases in cloven-hoofed animals. Disease symptoms develop within 2 to 3 days of exposure and include fever and vesicular lesions on the tongue and hooves. Dendritic cells (DC) play an essential role in protective immune responses against pathogens. Therefore, investigating their role during FMDV infection would lead to a better understanding of host-pathogen interactions. In this study, following infection of cattle with FMDV, we investigated the frequency and function of conventional (cDC) and plasmacytoid DC (pDC) in blood by using multi-color flow cytometry. We show that the frequency of cDC and pDC increased following FMDV infection and peaked 3 to 4 days post-infection. During peak viremia, the cattle became lymphopenic, the expression of MHC class II molecules on cDC and pDC was dramatically down-regulated, the processing of exogenous antigen by cDC and pDC was impaired, and there was an increase in IL-10 production by DC and monocytes. Notably, after clearance of FMDV from the blood, MHC class II expression returned to pre-infection levels. Altogether, our study demonstrates that in cattle, FMDV inhibits the function of DC, thereby retarding the initiation of adaptive immune responses, potentially enhancing virus shedding during the acute phase of infection.     

重组鸡痘病毒和DNA疫苗对口蹄疫病毒的豚鼠免疫保护

将构建的携带FMDV衣壳蛋白P1-2A和蛋白酶3C编码基因的重组鸡痘病毒活载体疫苗vUTAL3CP1以及编码FMDV P1-2A基因和猪IL-18基因的重组DNA疫苗pVIRIL18P1,分别以单独和混合的方式给豚鼠进行2次免疫,然后测定FMDV特异性结合抗体、中和抗体和T淋巴细胞增殖反应,并用250 ID50的FMDV进行攻击,观察其保护效果.结果表明这2种基因工程疫苗均能诱导豚鼠产生特异性的体液免疫及细胞免疫应答.其中以vUTAL3CP1两次免疫组的效果最好,其诱导的抗体水平已接近于常规灭活疫苗,而细胞免疫水平则比后者高得多.攻击保护结果表明该组完全保护率可达3/4,而另外两组也具有一定保护效果.上述研究结果为进一步进行大动物免疫攻毒试验,并最终筛选出最佳疫苗和免疫程序奠定了基础.     

Complement-Fixation Analysis of Four Subtypes of Foot-and-Mouth Disease Virus Type A

Complement-fixation patterns were established for four subtypes of foot-and-mouth disease virus by block assays against homologous and heterologous antiserum. Inhibition of fixation by excess antigen was observed in most homologous systems but rarely in the heterologous systems. The heterologous antibody titers were, in all instances, considerably lower than those for the homologous systems. Although relatively high dilutions of antiserum may be desirable for subtyping, higher concentrations of antibody should be used for determining serological types.