Development of novel strategies to control foot-and-mouth disease: Marker vaccines and antivirals

Foot-and-mouth disease (FMD) is economically the most important viral-induced livestock disease worldwide. The disease is highly contagious and FMD virus (FMDV) replicates and spreads extremely rapidly. Outbreaks in previously FMD-free countries, including Taiwan, the United Kingdom, and Uruguay, and the potential use of FMDV by terrorist groups have demonstrated the vulnerability of countries and the need to develop control strategies that can rapidly inhibit or limit disease spread. The current vaccine, an inactivated whole virus preparation, has a number of limitations for use in outbreaks in disease-free countries. We have developed an alternative approach using a genetically engineered FMD subunit vaccine that only contains the portions of the viral genome required for virus capsid assembly and lacks the coding region for most of the viral nonstructural (NS) proteins including the highly immunogenic 3D protein. Thus, animals inoculated with this marker vaccine can readily be differentiated from infected animals using diagnostic assays employing the NS proteins not present in the vaccine and production of this vaccine, which does not contain infectious FMDV, does not require expensive high-containment manufacturing facilities. One inoculation of this subunit vaccine delivered in a replication-defective human adenovirus vector can induce rapid, within 7 days, and relatively long-lasting protection in swine. Similarly cattle inoculated with one dose of this recombinant vector are rapidly protected from direct and contact exposure to virulent virus. Furthermore, cattle given two doses of this vaccine developed high levels of FMDV-specific neutralizing antibodies, but did not develop antibodies against viral NS proteins demonstrating the ability of FMD subunit vaccinated animals to be differentiated from infected animals. To stimulate early protection prior to the vaccine-induced adaptive immune response we inoculated swine with the antiviral agent, type I interferon, and induced complete protection within 1 day. Protection can last for 3-5 days. The combination of the FMD marker vaccine and type I interferon can induce immediate, within 1 day, and long-lasting protection against FMD. Thus, this combination approach successfully addresses a number of concerns of FMD-free countries with the current disease control plan. By rapidly limiting virus replication and spread this strategy may reduce the number of animals that need to be slaughtered during an outbreak.     

Reconstruction of the Transmission History of RNA Virus Outbreaks Using Full Genome Sequences: Foot-and-Mouth Disease Virus in Bulgaria in 2011

Improvements to sequencing protocols and the development of computational phylogenetics have opened up opportunities to study the rapid evolution of RNA viruses in real time. In practical terms, these results can be combined with field data in order to reconstruct spatiotemporal scenarios that describe the origin and transmission pathways of viruses during an epidemic. In the case of notifiable diseases, such as foot-and-mouth disease (FMD), these analyses provide important insights into the epidemiology of field outbreaks that can support disease control programmes. This study reconstructs the origin and transmission history of the FMD outbreaks which occurred during 2011 in Burgas Province, Bulgaria, a country that had been previously FMD-free-without-vaccination since 1996. Nineteen full genome sequences (FGS) of FMD virus (FMDV) were generated and analysed, including eight representative viruses from all of the virus-positive outbreaks of the disease in the country and 11 closely-related contemporary viruses from countries in the region where FMD is endemic (Turkey and Israel). All Bulgarian sequences shared a single putative common ancestor which was closely related to the index case identified in wild boar. The closest relative from outside of Bulgaria was a FMDV collected during 2010 in Bursa (Anatolia, Turkey). Within Bulgaria, two discrete genetic clusters were detected that corresponded to two episodes of outbreaks that occurred during January and March-April 2011. The number of nucleotide substitutions that were present between, and within, these separate clusters provided evidence that undetected FMDV infection had occurred. These conclusions are supported by laboratory data that subsequently identified three additional FMDV-infected livestock premises by serosurveillance, as well as a number of antibody positive wild boar on both sides of the border with Turkish Thrace. This study highlights how FGS analysis can be used as an effective on-the-spot tool to support and help direct epidemiological investigations of field outbreaks.     

Amplification and Characterization of Bull Semen Infected Naturally with Foot-and-mouth Disease Virus Type Asial by RT-PCR

To investigate the security of semen biologically, 15 bull semen samples were collected (of which 5 exhibited clinical signs of Foot-and-mouth disease) and identified by RT-PCR and virus isolation. The results indicated that the semen of the infected bulls were contaminated by Foot-and-mouth disease virus (FMDV), but FMDV was not detected in semen samples from those bulls not showing clinical signs of Foot-and-mouth disease (FMD). This is the first report of the presence of FMDV in bull semen due to natural infection in China. The analysis of the partial sequence of the VP1 gene showed that the virus strain isolated from semen has 97.9% identity with the virus isolated from vesicular liquid of infected bulls showing typical signs of FMD and belonged to the same gene sub-group.     

Temporal and spatial spread of Lettuce mosaic virus in lettuce crops in central Spain: factors involved in Lettuce mosaic virus epidemics

Lettuce mosaic virus (LMV) is transmitted by aphid vectors in a nonpersistent manner as well as by seeds. The virus causes severe disease outbreaks in commercial lettuce crops in several regions of Spain. The temporal and spatial patterns of spread of LMV were studied in autumn 2002 in the central region of Spain. Symptomatic lettuce (var. Cazorla) plant samples were collected weekly, first at the seedling stage from the greenhouse nursery and later outdoors after transplantation. The exact position of symptomatic plants sampled in the field was recorded and then material was tested by enzyme‐linked immunosorbent assay to assess virus infection. Cumulative spatial data for infected plants at different growth stages were analysed using spatial analysis by distance indices. For temporal analysis, the monomolecular, Gompertz, logistic and exponential models were evaluated for goodness of fit to the entire set of disease progress data obtained. The results indicated that the disease progress curve of LMV epidemics in the selected area is best described by a Gompertz model and that the epidemic follows a polycyclic disease progression. Our data suggest that secondary cycle of spread occurs when noncolonising aphid species land on the primary infected plants (probably coming from infected seed) and move to adjacent plants before leaving the crop. The role of weeds growing close to lettuce fields as potential inoculum sources of virus and the aphid species most likely involved in the transmission of LMV were also identified.     

Cattle transport network predicts endemic and epidemic foot-and-mouth disease risk on farms in Turkey

The structure of contact networks affects the likelihood of disease spread at the population scale and the risk of infection at any given node. Though this has been well characterized for both theoretical and empirical networks for the spread of epidemics on completely susceptible networks, the long-term impact of network structure on risk of infection with an endemic pathogen, where nodes can be infected more than once, has been less well characterized. Here, we analyze detailed records of the transportation of cattle among farms in Turkey to characterize the global and local attributes of the directed—weighted shipments network between 2007-2012. We then study the correlations between network properties and the likelihood of infection with, or exposure to, foot-and-mouth disease (FMD) over the same time period using recorded outbreaks. The shipments network shows a complex combination of features (local and global) that have not been previously reported in other networks of shipments; i.e. small-worldness, scale-freeness, modular structure, among others. We find that nodes that were either infected or at high risk of infection with FMD (within one link from an infected farm) had disproportionately higher degree, were more central (eigenvector centrality and coreness), and were more likely to be net recipients of shipments compared to those that were always more than 2 links away from an infected farm. High in-degree (i.e. many shipments received) was the best univariate predictor of infection. Low in-coreness (i.e. peripheral nodes) was the best univariate predictor of nodes always more than 2 links away from an infected farm. These results are robust across the three different serotypes of FMD observed in Turkey and during periods of low-endemic prevalence and high-prevalence outbreaks.     

用口蹄疫病毒非结构蛋白区分注苗动物和自然感染动物研究进展

口蹄疫是由口蹄疫病毒引起的偶蹄类动物烈性传染病,疫苗接种是防治口蹄疫暴发的主要措施之一,而要控制口蹄疫流行,首先要将病毒感染动物从疫苗接种群体中区分开来。以口蹄疫病毒非结构蛋白（NSP）为抗原,检测动物体内的NSP抗体是一种很好的区分感染动物和疫苗免疫动物的诊断方法,许多实验室开展了相关研究,并取得了一定的成绩。     

Development of a Foot-and-Mouth Disease Virus Serotype A Empty Capsid Subunit Vaccine Using Silkworm (Bombyx mori) Pupae

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that inflicts severe economic losses in the livestock industry. In 2009, FMDV serotype A caused outbreaks of FMD in cattle in China. Although an inactivated virus vaccine has proven effective to control FMD, its use may lead to new disease outbreaks due to a possible incomplete inactivation of the virus during the manufacturing process. Here, we expressed the P1-2A and the 3C coding regions of a serotype A FMDV field isolate in silkworm pupae (Bombyx mori) and evaluated the immunogenicity of the expression products. Four of five cattle vaccinated with these proteins developed high titers of FMDV-specific antibody and were completely protected against virulent homologous virus challenge with 10,000 50% bovine infectious doses (BID(50)). Furthermore, the 50% bovine protective dose (PD(50)) test was performed to assess the bovine potency of the empty capsid subunit vaccine and was shown to achieve 4.33 PD(50) per dose. These data provide evidence that silkworm pupae can be used to express immunogenic FMDV proteins. This strategy might be used to develop a new generation of empty capsid subunit vaccines against a variety of diseases.     

Amplification and characterization of bull semen infected naturally with foot-and-mouth disease virus type Asia1 by RT-PCR

To investigate the security of semen biologically, 15 bull semen samples were collected (of which 5 exhibited clinical signs of Foot-and-mouth disease) and identified by RT-PCR and virus isolation. The results indicated that the semen of the infected bulls were contaminated by Foot-and-mouth disease virus (FMDV), but FMDV was not detected in semen samples from those bulls not showing clinical signs of Foot-and-mouth disease (FMD). This is the first report of the presence of FMDV in bull semen due to natural infection in China. The analysis of the partial sequence of the VP1 gene showed that the virus strain isolated from semen has 97.9% identity with the virus isolated from vesicular liquid of infected bulls showing typical signs of FMD and belonged to the same gene sub-group. Foundation items: State Science and Technology Support Program (2006DAD06A03) and Hi-tech Research and Development Program of China 863 (2006AA10A204).     

Serosurveillance for foot-and-mouth disease in Mongolian gazelles (Procapra gutturosa) and livestock on the Eastern Steppe of Mongolia

Foot-and-mouth disease (FMD) is a highly contagious, viral disease that affects most ruminant and porcine species, and periodic outbreaks on Mongolia's Eastern Steppe affect Mongolian gazelles (Procapra gutturosa) and livestock. During 2005-08, we collected sera from 36 and 57 calf and adult gazelles, respectively, and from adult domestic animals sympatric with the gazelles, including 138 sheep (Ovis aries), 140 goats (Capra aegagrus hircus), 139 Bactrian camels (Camelus bactrianus), and 138 cattle (Bos taurus). Our goal was to determine whether the prevalence of the antibody to foot-and-mouth disease virus (FMDV) in gazelles declined relative to previous estimates in the absence of FMD outbreaks. Overall, 2.0% (95% CI 0.7-3.3%, n=555) of the four livestock species were antibody-positive for nonstructural proteins of FMDV (FMDV-NS), whereas 30.3% (95% CI 26.5-34.1%, n=555) had antibodies for structural proteins (i.e., vaccination-derived antibodies). Seven of 57 free-ranging gazelle calves (7.5%, 95%CI 1.6-12.4%) were FMDV-NS positive. None of 36 adult gazelles sampled in 2008 were antibody-positive for exposure to FMDV, indicating a significant decline (χ(2)=18.99; P<0.001; df=1) in antibody prevalence among gazelles from the same area during a livestock outbreak in 2001. The episodic nature of FMD outbreaks on the Eastern Steppe, Mongolia, with evidence of FMDV exposure in gazelles only during or following concurrent outbreaks in livestock, suggests that FMDV may spill over into the gazelle population during livestock outbreaks and that successful control of FMD on the Eastern Steppe requires a focus on control in livestock populations through vaccination.     

Antibody estimation by indirect complement fixation test for foot-and-mouth disease in cattle.

Antibody against foot-and-mouth disease (FMD) virus was measured by the indirect complement fixation (ICF) test. For this test serum samples were collected from cattle experimentally infected with FMD virus of O, A and Asia 1 types, as well as cattle infected in the field. Two types of antigen were used. One was antigen derived from infected lingual epithelial culture prepared by Frenkel's method with each type of the virus. The other was antigen derived from the lingual epithelium of cattle infected by virus inoculation. ICF antibody began to be dectected about 4 5 days after inoculation. It reached a maximum titer 10 14 days after inoculation, remaining at this titer for about a week or two, and then decreased gradually. It was, however, detectable even 63 days after inoculation. The rise and fall of ICF antibody was parallel with that of neutralizing antibody, although that antibody was always lower in titer than this. ICF antibody was detected type-specifically from cattle infected experimentally and naturally. These results indicated that the ICF test was available for the routine serological diagnosis and epizootiological investigation and research.     

Typing foot-and-mouth disease virus by fluorescent antibody technique.

Typing of foot-and-mouth disease (FMD) virus was performed by the direct fluorescent antibody (FA) technique. Type-specific FA was prepared from the following two sorts of procedures: (1) FA against live virus (FA-live) was prepared from hyperimmune serum taken from guinea pigs having received live FMD virus. Then it was adsorbed with concentrated heterotype antigen. (2) FA against inactivated virus (FA-Inact) was prepared from antiserum taken from guinea pigs immunized with purified FMD virus inactivated with acetylethyleneimine. Seventeen strains of FMD virus (seven strains of type A, seven strains of type O, and three strains of thpe C) were used. Type-specific FMD virus antigen was detected distinctly from the monolayer of BHK cells infected with each type of virus and fixed in acetone, in spite of negative results obtained from the cells fixed in methyl alcohol. All the 17 strains were typed successfully by the implementation of these two FA methods.     

Retrospective analysis of an outbreak of B virus infection in a colony of DeBrazza's monkeys (Cercopithecus neglectus)

In 1981, an outbreak of herpetic disease developed in a colony of DeBrazza's monkeys (Cercopithecus neglectus). In seven of eight infected animals, clinical signs of infection included vesicular and ulcerative lesions on the lips, tongue, and/or palate. Histologic examination of lesions revealed intranuclear inclusion bodies, and electron microscopy revealed nucleocapsids and virions with typical herpesvirus morphology. Although a virus was isolated that appeared similar to monkey B virus, techniques available at the time did not allow precise identification of the virus. Analysis of serum from one surviving monkey collected 12 years after the outbreak revealed a pattern of reactivity characteristic of B virus-positive serum on the basis of results of ELISA and western immunoblot analysis. Polymerase chain reaction analysis of archived paraffin-embedded tissue specimens and molecular analysis of the one viral isolate obtained from a DeBrazza's monkey indicated that the virus responsible for the outbreak was a new genotype of B virus. Testing of sera from lion-tailed macaques (Macaca silenus) housed in an adjacent cage at the same zoo indicated that these animals harbored this virus and, thus, were the likely source of the virus that infected the DeBrazza's monkeys. This study documents usefulness of archiving samples from disease outbreaks for later analysis. In addition, this incident underscores the importance of considering herpes B virus infection when outbreaks of disease having characteristics of herpetic infections develop in nonhuman primates kept at institutions that also house macaques.     

A method to detect major serotypes of foot-and-mouth disease virus

Nucleic acid sequence-based amplification (NASBA) is an isothermal technique that allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A rapid and specific NASBA technique was developed, allowing the detection of foot-and-mouth disease virus genetic material in a range of sample material, including preserved skin biopsy material from infected animals, vaccines prepared from denatured cell-free material, and cell-free antigen-based detection kits. A single pair of DNA oligonucleotide primers was able to amplify examples of all major FMD virus subtypes. The amplified viral RNA was detected by electrochemiluminescence. The method was at least as sensitive as existing cell-free antigen detection methods.     

Bayesian analysis of experimental epidemics of foot-and-mouth disease

We investigate the transmission dynamics of a certain type of foot-and-mouth disease (FMD) virus under experimental conditions. Previous analyses of experimental data from FMD outbreaks in non-homogeneously mixing populations of sheep have suggested a decline in viraemic level through serial passage of the virus, but these do not take into account possible variation in the length of the chain of viral transmission for each animal, which is implicit in the non-observed transmission process. We consider a susceptible-exposed-infectious-removed non-Markovian compartmental model for partially observed epidemic processes, and we employ powerful methodology (Markov chain Monte Carlo) for statistical inference, to address epidemiological issues under a Bayesian framework that accounts for all available information and associated uncertainty in a coherent approach. The analysis allows us to investigate the posterior distribution of the hidden transmission history of the epidemic, and thus to determine the effect of the length of the infection chain on the recorded viraemic levels, based on the posterior distribution of a p-value. Parameter estimates of the epidemiological characteristics of the disease are also obtained. The results reveal a possible decline in viraemia in one of the two experimental outbreaks. Our model also suggests that individual infectivity is related to the level of viraemia.     

Foot-and-mouth disease virus: biology and prospects for disease control

Foot-and-mouth disease virus (FMDV) is the causative agent of a disease that constitutes one of the main animal health concerns, as evidenced by the devastating outbreaks that occurred in different areas of the world over the last few years. In this review, we summarise important features of FMDV, aspects of its interactions with cells and hosts as well as current and new strategies for FMD control by vaccination.     

Evaluation of Infectivity,Virulence and Transmission of FDMV Field Strains of Serotypes O and A Isolated In 2010 from Outbreaks in the Republic of Korea

Since the early 2000s outbreaks of foot-and-mouth disease (FMD) have been described in several previously FMD-free Asian nations, including the Republic of Korea (South Korea). One outbreak with FMD virus (FDMV) serotype A and two with serotype O occurred in South Korea in 2010/2011. The causative viruses belonged to lineages that had been spreading in South East Asia, far East and East Asia since 2009 and presented a great threat to the countries in that region. Most FMDV strains infect ruminants and pigs, as it happened during the outbreaks of FMDV serotype O in South Korea. Contrastingly, the strain of serotype A affected only ruminants. Based upon these findings, the intention of the work described in the current report was to characterize and compare the infectivity, virulence and transmission of both strains under laboratory conditions in cattle and pigs, by direct inoculation and contact exposure. As expected, FMDV serotype O was highly virulent in both cattle and swine by contact exposure and direct inoculation. Surprisingly, FMDV serotype A was highly virulent in swine, but was less infectious in cattle by contact exposure to infected swine or cattle. Interestingly, similar quantities of aerosolized FMDV RNA were detected during experiments with viruses of serotypes O and A. Specific virus-host interaction of A/SKR/2010 could affect the transmission of this strain to cattle, and this may explain in part the limited spread of the serotype A epizootic.     

3A蛋白104–115位氨基酸缺失口蹄疫A型标记病毒的构建

【目的】构建一株含3A非结构蛋白104–115位氨基酸缺失的口蹄疫A型标记病毒,分析其生物学特性和发展标记疫苗的潜力。【方法】采用融合PCR技术,在当前流行毒株A/Sea-97/CHA/2014全长感染性克隆p QAHN中引入3A104–115位氨基酸的缺失,构建全长重组质粒。全长质粒经NotI线化后转染表达T7RNA聚合酶的稳定细胞系,拯救标记病毒。RT-PCR、序列分析、间接免疫荧光和Western blotting鉴定标记病毒。噬斑表型和一步生长曲线分析标记病毒的生物学特性,并用实验室开发的针对3A优势表位(AEKNPLE)的阻断ELISA方法分析其区分亲本和标记病毒感染的动物。【结果】成功拯救到一株含3A 104–115位氨基酸缺失的口蹄疫A型标记病毒,3A表位的缺失没有影响标记病毒的噬斑表型和一步生长曲线。3A单抗阻断ELISA可以明显区分标记病毒和亲本病毒感染的动物。【结论】本研究构建的3A蛋白104–115位氨基酸缺失的标记病毒可以作为发展口蹄疫鉴别诊断疫苗的候选毒株,用于我国未来口蹄疫A型的有效防控。     

型内嵌合口蹄疫病毒全长感染性cDNA克隆的构建

【目的】近年来,O型口蹄疫的不断暴发严重危害了我国畜牧业的发展,其病原——O型口蹄疫病毒已演化出3种谱系:中国型猪毒系、泛亚系和缅甸98系。其中中国型猪毒系病毒高度嗜猪,对养猪业危害最大。目前应用的疫苗已不能有效保护中国型猪毒系变异株的流行,这给我国猪口蹄疫的防控带来了极大的困难。为了进一步发展免疫原性好、抗原谱广的猪O型口蹄疫疫苗候选株,本研究以O/HN/93现用疫苗毒株的感染性克隆为骨架,用流行的新猪毒系病毒的部分VP3和VP1基因(主要是替换VP1蛋白上的B-C环和G-H环)替换疫苗毒株的相应部分,构建了嵌合的FMDV全长cDNA克隆。【方法】线化的嵌合全长质粒和表达T7 RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞,体内转录拯救嵌合病毒。【结果】嵌合全长质粒转染BHK-21细胞36h后,出现明显的FMDV致细胞病变效应。对收获的病毒分别用RT-PCR、间接免疫荧光、电子显微镜观察结果证实成功拯救到嵌合的FMDV。拯救的病毒乳鼠致病性试验结果表明该拯救病毒对乳鼠的致病力减弱。该嵌合病毒的成功拯救为研制口蹄疫新型疫苗等奠定了基础。     

Development and Utilization of VHH Antibodies Derived from Camelus Dromedarius Against Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) is an acute, highly contagious, and economically devastating viral disease of domestic and wildlife species. For effective implementation of FMD control program, there is an imperative need for developing a rapid, sensitive, and specific diagnostics which help in the identification of serotypes involved in the outbreaks. The humoral immune response of the Camelidae is unique since in these animals 75% of circulating antibodies are constituted by heavy-chain antibodies and 25% are conventional immunoglobulin with two identical heavy chains. In the present study, we developed and characterized FMD virus-specific single-domain heavy-chain antibodies (VHHs) against inactivated whole-virus antigens of FMDV serotypes O (INDR2/1975), A (IND40/2000), and Asia 1 (IND63/1972) vaccine strains. After six rounds of panning and enrichment, these VHHs were stably expressed in Escherichia coli cells. The VHHs directed against outer capsid proteins of FMD virus were successfully utilized as the capture antibody in liquid-phase blocking ELISA (LPBE) thus replacing rabbit coating antibodies. Our study demonstrated the utility of FMD virus-specific VHHs as potential candidates in FMD research and diagnostic application.