Genetic variation of foot-and-mouth disease virus isolates recovered from persistently infected water buffalo (Bubalus bubalis)

Genetic variation of foot-and-mouth disease virus (FMDV) isolates, serotype O, recovered serially over a 1-year period from persistently infected buffalos was assessed. The persistent state was established experimentally with plaque-purified FMDV, strain O(1)Campos, in five buffalos (Bubalus bubalis). Viral isolates collected from esophageal-pharyngeal (EP) fluids for up to 71 weeks after infection were analyzed at different times by nucleotide sequencing and T(1) RNase oligonucleotide fingerprinting to assess variability in the VP1-coding region and in the complete genome, respectively. Genetic variation increased, although irregularly, with time after infection. The highest values observed for the VP1-coding region and for the whole genome were 2.5% and 1.8%, respectively. High rates of fixation of mutations were observed using both methodologies, reaching values of 0.65 substitutions per nucleotide per year (s/nt/y) and 0.44s/nt/y for nucleotide sequencing and oligonucleotide fingerprinting, respectively, when selected samples recovered at close time periods were analyzed. The data herein indicate that complex mixtures of genotypes may arise during FMDV type O persistent infection in water buffalos, which can act as viral reservoirs and also represent a potential source of viral variants. These results fit within the quasi-species dynamics described for FMDV, in which viral populations are constituted by related, non-identical genomes that evolve independently from each other, and may predominate at a given time.     

Detection of foot-and-mouth disease virus RNA in pharyngeal epithelium biopsy samples obtained from infected cattle: investigation of possible sites of virus replication and persistence

Foot-and-mouth disease (FMD) is a highly contagious viral infection of significant financial importance to the export and trade of agricultural products. The occurrence of persistently infected "carriers" of FMD-virus (FMDV) in ruminant species adds further complications to disease control. There have been significant discrepancies in reports regarding the pathogenesis of FMDV infection in cattle with specific emphasis on the anatomical sites involved in early and persistent virus replication. In this study, collection of small biopsy samples from the dorsal soft palate (DSP) of live animals was used to investigate the level of FMDV RNA present at this site at sequential time points during the infection. Results were compared to measurements of virus excretion in samples of oropharyngeal fluid collected at corresponding time points. Possible sites of virus persistence were investigated through measurements of the levels of FMDV RNA in the DSP as well as mandibular and retropharyngeal lymph nodes beyond 28 days after infection. Results indicated only low levels of FMDV RNA present in samples of pharyngeal epithelia during both early and persistent phases of infection with significantly higher levels of virus detected in pharyngeal excretions. It is concluded that the targeted area for sampling within the DSP does not harbour significant levels of virus replication during acute or persistent FMDV infection in cattle. Furthermore, the DSP and the mandibular and retropharyngeal lymph nodes cannot be concluded to be principal sites for persistence of FMDV.     

Detection of carriers of foot-and-mouth disease virus among vaccinated cattle

To investigate and optimise detection of carriers, we vaccinated 15 calves with an inactivated vaccine based on foot-and-mouth disease virus (FMDV) A Turkey strain and challenged them and two further non-vaccinated calves with the homologous virus four weeks later. To determine transmission to a sensitive animal, we put a sentinel calf among the infected cattle from 60 days post-infection until the end of the experiment at 609 days post-infection. Samples were tested for the presence of FMDV, viral genome, specific IgA antibodies, antibodies against FMDV non-structural (NS) proteins or neutralising antibodies. Virus and viral genome was intermittently isolated from probang samples and the number of isolations decreased over time. During the first 100 days significantly more samples were positive by RT-PCR than by virus isolation (VI), whereas, late after infection more samples were positive by virus isolation. All the inoculated cattle developed high titres of neutralising antibodies that remained high during the entire experiment. An IgA antibody response was intermittently detected in the oropharyngeal fluid of 14 of the 17 calves, while all of them developed detectable levels of antibodies to NS proteins of FMDV in serum, which declined slowly beyond 34 days post-infection. Nevertheless, at 609 days after inoculation, 10 cattle (60%) were still positive by NS ELISA. Of the 17 cattle in our experiment, 16 became carriers. Despite frequent reallocation between a different pair of infected cattle no transmission to the sentinel calf occurred. It remained negative in all assays during the entire experiment. The results of this experiment show that the NS ELISA is currently the most sensitive method to detect carriers in a vaccinated cattle population.     

Foot-and-mouth disease virus (O/UKG/2001) is poorly transmitted between sheep by the airborne route

Foot-and-mouth disease virus (FMDV) can be spread by the airborne route and therefore atmospheric dispersion models have been developed to predict where the virus might spread during a disease outbreak. Airborne transmission between sheep of the FMDV strain involved in the outbreak in Europe in 2001 (O/UKG/2001) was studied experimentally. Recipient animals were exposed to two donor sheep excreting virus for 2, 4, 6, 8 or 24 h. Although FMDV was detected in air samples collected during challenge, none of the recipient sheep became infected. These data suggest that O/UKG/2001 is not efficiently transmitted by the airborne route between sheep.     

Quantitative analysis of foot-and-mouth disease virus RNA duration in tissues of experimentally infected pigs

Quantitative analysis of the duration of foot-and-mouth disease virus (FMDV) RNA in tissues was carried out in pigs experimentally infected with FMDV O UKG 34/2001 and O SKR 1/2000. The results showed that the viral RNA was still detectable in cervical lymph nodes, mandibular lymph nodes and tonsils collected from both inoculated and contact pigs at 28 days post infection. There was no detectable viral RNA in the soft palate or pharynx, which are thought to be tissue sites for viral persistence in cattle. Further study is needed to clarify whether this difference has significance in terms of viral clearance in pigs.     

口蹄疫病毒感染细胞的研究进展

口蹄疫是引起偶蹄动物的急性发热性水泡性疾病,具有高度接触传染性,对发病国家和地区经济有破坏性作用和不良的政治影响。口蹄疫病原体为小RNA病毒科的口蹄疫病毒,是一类单股正链RNA病毒,该病毒有多种血清型及其亚型,相互之间无交叉保护力或保护力极其有限。目前,口蹄疫病毒感染的分子机理还不是很清楚。口蹄疫病毒感染细胞的过程主要包括病毒与细胞的吸附、病毒穿透细胞壁进入细胞、病毒粒子的脱衣壳、病毒RNA的翻译转录、病毒基因组的复制以及病毒粒子的成熟过程,最后是成熟的病毒粒子衣壳包装成为完整病毒。文章就口蹄疫病毒感染细胞的过程做一概述。     

Foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) is an aphthovirus of the family Picornaviridae and the etiological agent of the economically most important animal disease. As a typical picornavirus, FMD virions are nonenveloped particles of icosahedral symmetry and its genome is a single stranded RNA of about 8500 nucleotides and of positive polarity. FMDV RNA is infectious and it replicates via a complementary, minus strand RNA. FMDV RNA replication is error-prone so that viral populations consist of mutant spectra (quasispecies) rather than a defined genomic sequence. Therefore FMDV in nature is genetically and antigenically diverse. This poses important challenges for the diagnosis, prevention and control of FMD. A deeper understanding of FMDV population complexity and evolution has suggested requirements for a new generation of anti-FMD vaccines. This is relevant to the current debate on the adequacy of non-vaccination versus vaccination policies for the control of FMD.

Résumé

Le virus de la fièvre aphteuse est un aphtovirus de la famille des Picornaviridae et l'agent de la maladie animale la plus importante sur le plan économique. En tant que picornavirus typique, le virus de la fièvre aphteuse est nu, sous forme d'icosaèdre et son génome comprend un acide ribonucléique monobrin avec environ 8500 nucléotides et une polarité positive. L'acide ribonucléique de ce virus est infectieux et il se réplique par l'intermédiaire d'un brin d'ARN moins, complémentaire. La réplication de l'acide nucléique de ce virus conduit à des erreurs, de telle sorte que les populations virales comprennent un ensemble de mutants (quasi espèce) plutôt qu'une séquence génomique bien définie. Par suite, le virus de la fièvre aphteuse est génétiquement et antigéniquement varié. Ceci entraîne des difficultés importantes pour le diagnostic, la prévention et la maîtrise de la fièvre aphteuse. Une connaissance plus approfondie de la complexité et de l'évolution de la population de ce virus a conduit à des besoins pour une nouvelle génération de vaccines aphteux. Ceci est lié au débat actuel sur le choix d'une politique de vaccination ou de non-vaccination dans la lutte contre la fièvre aphteuse.     

口蹄疫病毒3C蛋白酶的结构及功能研究进展

口蹄疫病毒能引起牛、羊等偶蹄动物发生高度接触性的传染病口蹄疲,该病常常影响着全球畜牧业的发展.FMDV是小RNA病毒科口蹄疫病毒属的成员,口蹄疫病毒为单股正链RNA病毒,病毒基因组全长约8.5 kb,基因组分为5'非编码区、3'非编码区和一个开放阅读框(ORF).基因组的中部是一大的开放阅读框,编码一多聚蛋白,多聚蛋白在翻译的同时,经二级裂解后,形成3种病毒结构蛋白(VP0,VP3和VP1)和8种非结构蛋白(L,2A,2B,2C,3A,3B,3C和3D).其中3C全长639 bp,编码213个氨基酸.3C蛋白酶是小RNA病毒的共同裂解酶,在多聚蛋白成熟过程中起着极为重要的作用,且在抗病毒药物打靶方面具有一定研究价值.因此,对3C蛋白酶的结构及功能研究进展进行综述很有必要.     

Performance of a foot-and-mouth disease virus reverse transcription-polymerase chain reaction with amplification controls between three real-time instruments.

The foot-and-mouth disease virus (FMDV) is a member of the picornavirus family, possessing an 8-kb single-stranded RNA genome of positive polarity. It is highly contagious among several livestock species and can lead to severe economic consequences, as evidenced by the UK outbreak in 2001. The usage of real-time polymerase chain reaction has facilitated rapid detection of FMDV. Several real-time PCR instruments are available with various capabilities, such as portability and high sample volume analysis. Primers and a dual-labeled TaqMan probe were optimized to detect a single, highly conserved 88-bp segment of the FMDV 3D (RNA polymerase) gene. To increase the confidence of the RT-PCR result, a positive amplification control was synthesized to detect potential false-positive results due to contamination if a wild-type virus is used as positive control. In addition, a preventative measure against false-negative results was developed in which endogenous beta actin mRNA is coamplified by RT-PCR. Assay performance was compared on the LightCycler1.2 (Roche), the SmartCyclerII (Cepheid), and the SDS 7900HT (ABI). These assays successfully identified the FMDV genome and beta actin mRNA from several sources of infected nasal and oral swabs, as well as probang samples.     

Analysis of synonymous codon usage in foot-and-mouth disease virus

In this study, we calculate the relative synonymous codon usage (RSCU) values and codon usage bias (CUB) values to carry out a comparative analysis of codon usage pattern for open reading frames (ORFs) among 85 samples which belong to all seven serotypes of foot-and-mouth disease virus (FMDV). Although the degree of CUB for ORFs is a relatively slight, there is a significant variation for CUB among different serotypes, which is mainly determined by codon usage pattern depending on RSCU. By comparison with RSCU values for all samples, although RSCU values fail to show the relationship of specific-lineage serotype, there are two main genetic populations existing in FMDV, namely (i) serotypes Asia 1, A, C &O; (ii) serotypes SAT 1, 2 & 3. This interesting characteristic may be formed by the mechanism of RNA virus recombination. The analysis of quantitative & qualitative evaluation based on CUB indicates interesting characteristic of codon usage, which suggests that more FMDV genome diversity may exist in specific-lineage serotypes rather than exist randomly. Furthermore, the relationship between amino acids and codon usage pattern indicates that mutation pressure rather than translational selection in nature is the important determinant of the codon usage bias observed. Our work might give some sight into some characteristics of FMDV ORF and some evolutionary information of this virus.     

Direct contact transmission of three different foot-and-mouth disease virus strains in swine demonstrates important strain-specific differences

A novel direct contact transmission model for the study of foot-and-mouth disease virus (FMDV) infection of swine was utilized to investigate transmission characteristics of three FMDV strains belonging to serotypes A, O and Asia1. Each strain demonstrated distinct transmission characteristics and required different exposure times to achieve successful contact transmission. While a 4h exposure was sufficient for strain A24 Cruzeiro (A24Cru), both O1 Manisa and Asia1 Shamir transmission required 18h or more. Viral excretion levels from donors (for all three strains) and virus present in room air (for A24Cru and O1 Manisa) were evaluated and associated with clinical signs and observed transmission pattern. Although all directly inoculated donor animals showed acute FMD, A24Cru had the highest levels of viral shedding in saliva and nasal swabs followed by O1 Manisa and Asia1 Shamir. Virus levels in room air were higher and were detected longer for A24Cru than for O1 Manisa. These results provide direct evidence for important strain-specific variation in transmission characteristics and emphasize the need for thorough evaluation of different FMDV viral strains using a well defined contact transmission methodology. This information is critical for vaccine and biotherapeutic efficacy testing, pathogenesis and disease modeling of FMDV transmission.     

Genome analysis and development of infectious cDNA clone of a virulence-attenuated strain of foot-and-mouth disease virus type Asia 1 from China

The RNA genome sequence of the rabbit passage-attenuated strain of foot-and-mouth disease virus (FMDV) Asia 1, ZB/CHA/58(att), was determined to be 8165 nt in length excluding the poly(C) tract in the 5′ UTR and the poly(A) tail at the 3′ end. ZB/CHA/58(att) was most similar to the vaccine strain Asia 1/YNBS/58 in genome sequence and there were no deletions or insertions within the deduced polyprotein between ZB/CHA/58(att) and YNBS/58, but there were a total of 25 substitutions at the amino acid level and an extra 19-nt stretch in the 5′ UTR was found in ZB/CHA/58(att). An infectious full-length cDNA clone of ZB/CHA/58(att) was developed. Infectious virus could be recovered in BHK-21 cells transfected with the synthetic viral RNA transcribed in vitro. The plaque morphology, growth kinetics and antigenic profile of the infectious clone-derived virus (termed tZB) were indistinguishable from those induced by the parental virus. Furthermore, the virulence properties of ZB/CHA/58(att) and tZB were found to be highly similar in the mouse model. The availability of genome sequence information and infectious cDNA clone of the FMDV ZB/CHA/58(att) lays a new ground for further investigation of FMDV virulence determinants and development of new potent vaccine to FMD.     

光生物素标记的口蹄疫病毒cDNA探针的制备及其对口蹄疫病毒RNA检测的试验研究

本文首次报道了利用pF1034质粒制备FMDV O型特异性探针,并通过PCR反应扩增FMDO_1K株病毒基因组的第2962位与3071位之间共110bp序列,制备了能检测O型、A型和亚洲I型FMDV RNA的群(组)特异性探针。用硝酸纤维素膜斑点杂交试验表明,二者均能检测出10pg水平的O_9K毒株的纯RNA;但前者只与O型FMDV RNA杂交,与A型及亚洲I型FMDV RNA无交叉杂交现象;而后者则能与O型、A型和亚洲I型的FMDV RNA发生杂交反应。对照试验显示:此两种探针与SVDV ssRNA、BTV dsRNA、EHDV dsRNA、DHV ssRNA、PRV DNA、乳鼠组织细胞RNA、BHK_(21)克隆13细胞RNA及DNA等均不出现交叉杂交现象,但与IBR DNA有假阳性杂交反应。     

Use of in situ hybridization for the detection of foot-and-mouth disease virus in cell culture

Biotinylated complementary DNA (cDNA) and RNA probes were prepared from a specific and highly conserved section of the foot-and-mouth disease virus (FMDV) genome coding for the RNA-dependent RNA polymerase. Hybridization was conducted on FMDV-infected, bovine enterovirus (BEV)-infected, and noninfected swine kidney cell cultures. The detection system utilized the enzyme system streptavidin-alkaline phosphatase, the substrate phosphate, and the chromogen nitroblue tetrazolium. Intense cytoplasmic granular staining was present at 2 and 4 hr postinfection (hpi), with less staining observed at 24 hpi. The staining was specific for FMDV, as indicated by a lack of staining of noninfected cells and BEV-infected cells. With the RNA probe, positive cells were detected up to the highest viral dilution assayed, which was approximately 96 TCID50. The cDNA probe was slightly less sensitive, detecting positive cells at 10-fold lower dilutions. This technique could prove useful in the diagnosis of foot-and-mouth disease in animals or in the detection of FMDV in biologics submitted for importation.     

口蹄疫病毒3C蛋白酶结构与功能及应用研究进展

口蹄疫病毒(FMDV)3C蛋白酶是FMDV基因组编码中具有酶学活性的病毒产物之一,在FMDV编码蛋白的成熟和子代病毒在宿主细胞体内大量扩增中发挥着重要作用。3C蛋白酶能剪切多聚蛋白,降解特定的蛋白质,是宿主细胞中重要的毒力因子。3C蛋白酶能调控蛋白的转录和翻译,使宿主细胞内的干扰素等多种抗病毒基因低水平表达,使FMDV逃避宿主的天然免疫。论文主要综述了FMDV 3C蛋白酶的结构、生物学功能,并介绍了其在研制新型疫苗中的应用,以期为今后FMDV 3C蛋白酶的研究、新型疫苗的研发提供参考。     

Transmission of a pestivirus infection in a population of Pyrenean chamois

Outbreaks of a previously unrecorded disease have recently affected Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations across the mountain range. A pestivirus was hypothesized to be the cause of this emerging disease and this type of virus can cross the species barrier and be transmitted to or from wildlife. Using an epidemiological survey conducted from 1995 to 2004 at Orlu, France, we characterized the virus and analyzed its transmission. A phylogenetic analysis of viral sequences and virus neutralization tests showed that the virus belonged to the newly described border disease virus-4 group. The increase of seroprevalence with age indicated that infection can occur at any age and resulted in lifelong immunity. Overall, 70.3% of 323 samples were positive for anti-p80 antibodies and 10.2% of 167 samples showed viremia, as demonstrated by either positive ELISA antigen test or RT-PCR. Infection has thus been widespread in this population since 1995, whereas no mass mortality or clinical signs have been observed. Incidence and seroprevalence varied seasonally and according to number of individuals aged less than 2 years old in the population, so viral transmission was dependent on host population age structure. We propose that the virus is now endemic in this population and is likely detrimental for reproduction and juveniles. Further investigation is needed to estimate the impact of pestivirus on host population dynamics and the risk of cross-transmission to farm animals.     

Phylodynamic reconstruction of O CATHAY topotype foot-and-mouth disease virus epidemics in the Philippines

Reconstructing the evolutionary history, demographic signal and dispersal processes from viral genome sequences contributes to our understanding of the epidemiological dynamics underlying epizootic events. In this study, a Bayesian phylogenetic framework was used to explore the phylodynamics and spatio-temporal dispersion of the O CATHAY topotype of foot-and-mouth disease virus (FMDV) that caused epidemics in the Philippines between 1994 and 2005. Sequences of the FMDV genome encoding the VP1 showed that the O CATHAY FMD epizootic in the Philippines resulted from a single introduction and was characterised by three main transmission hubs in Rizal, Bulacan and Manila Provinces. From a wider regional perspective, phylogenetic reconstruction of all available O CATHAY VP1 nucleotide sequences identified three distinct sub-lineages associated with country-based clusters originating in Hong Kong Special Administrative Region (SAR), the Philippines and Taiwan. The root of this phylogenetic tree was located in Hong Kong SAR, representing the most likely source for the introduction of this lineage into the Philippines and Taiwan. The reconstructed O CATHAY phylodynamics revealed three chronologically distinct evolutionary phases, culminating in a reduction in viral diversity over the final 10 years. The analysis suggests that viruses from the O CATHAY topotype have been continually maintained within swine industries close to Hong Kong SAR, following the extinction of virus lineages from the Philippines and the reduced number of FMD cases in Taiwan.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0090-y) contains supplementary material, which is available to authorized users.