RESUMO
Foot-and-mouth disease is a highly contagious disease affecting cloven-hoofed animals, resulting in considerable economic losses. Its causal agent is foot-and-mouth disease virus (FMDV), a picornavirus. Due to its error-prone replication and rapid evolution, the transmission and evolutionary dynamics of FMDV can be studied using genomic epidemiological approaches. To analyze FMDV evolution and identify possible transmission routes in an Argentinean region, field samples that tested positive for FMDV by PCR were obtained from 21 farms located in the Mar Chiquita district. Whole FMDV genome sequences were obtained by PCR amplification in seven fragments and sequencing using the Sanger technique. The genome sequences obtained from these samples were then analyzed using phylogenetic, phylogeographic, and evolutionary approaches. Three local transmission clusters were detected among the sampled viruses. The dataset was analyzed using Bayesian phylodynamic methods with appropriate coalescent and relaxed molecular clock models. The estimated mean viral evolutionary rate was 1.17 × 10- 2 substitutions/site/year. No significant differences in the rate of viral evolution were observed between farms with vaccinated animals and those with unvaccinated animals. The most recent common ancestor of the sampled sequences was dated to approximately one month before the first reported case in the outbreak. Virus transmission started in the south of the district and later dispersed to the west, and finally arrived in the east. Different transmission routes among the studied herds, such as non-replicating vectors and close contact contagion (i.e., aerosols), may be responsible for viral spread.
Assuntos
Vírus da Febre Aftosa , Picornaviridae , Animais , Vírus da Febre Aftosa/genética , Argentina/epidemiologia , Teorema de Bayes , FilogeniaRESUMO
Chimeric virus-like particles are self-assembling structures composed of viral proteins that had been modified to incorporate sequences from different organisms, being able to trigger immune responses against the heterologous sequence. However, the identification of suitable sites for that purpose in the carrier protein is not an easy task. In this work, we describe the generation of rabies chimeric VLPs that expose a major antigenic site of foot-and-mouth disease virus (FMDV) by identifying suitable regions in rabies glycoprotein (RVG), as a proof of concept of a novel heterologous display platform for vaccine applications. To identify adequate sites for insertion of heterologous sequences without altering the correct folding of RVG, we identified regions that were evolutionally non-conserved in Lyssavirus glycoproteins and performed a structural analysis of those regions using a 3D model of RVG trimer that we generated. The heterologous sequence was inserted in three different sites within RVG sequence. In every case, it did not affect the correct folding of the protein and was surface exposed, being recognized by anti-FMDV antibodies in expressing cells as well as in the surface of VLPs. This work sets the base for the development of a heterologous antigen display platform based on rabies VLPs. KEY POINTS: ⢠Adequate regions for foreign epitope display in RVG were found. ⢠G-H loop of FMDV was inserted in three regions of RVG. ⢠The foreign epitope was detected by specific antibodies on fusion proteins. ⢠G-H loop was detected on the surface of chimeric VLPs.
Assuntos
Vírus da Febre Aftosa , Febre Aftosa , Raiva , Vacinas , Animais , Anticorpos Antivirais , Vírus da Febre Aftosa/genética , Glicoproteínas/genéticaRESUMO
The foot-and-mouth disease virus (FMDV) causes a highly infectious disease of all cloven-footed animals. The RNA genome of the virus continuously evolves, leading to the generation of new strains; this necessitates the selection of new vaccine strains to ensure complete protection. Infection with one FMDV serotype does not provide cross-protection against the other FMDV serotypes. Many of the recovered animals may become carriers of the FMDV, but they still remain susceptible to the other serotypes. Coinfection with multiple FMDV serotypes has been reported and studied to understand the virus evolution. Isolation and characterization of all the involved serotypes in the mixed infection case is essential to understand the molecular evolution of the virus. In this study, two cases of coinfection were studied by selective isolation of each of the FMDV serotypes under the cross-serotype-specific immune pressure. It was estimated that the virus present in a minimum of 10-0.92 TCID50 could be isolated from the mixed population containing other serotypes in infective doses of 100.25 TCID50 or less. All involved serotypes present in the mixed infection cases were isolated, without any cross-contamination. Virus characterization revealed that genotype 2 was of serotype A virus from a sample collected in 1995, which was last reported in 1986, indicating a possible subdued prevalence of the genetic group even after vanishing from the field.
Assuntos
Coinfecção , Vírus da Febre Aftosa , Febre Aftosa , Animais , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/isolamento & purificação , Filogenia , SorogrupoRESUMO
In this study, we compiled 84-year worth (1934-2017) of genomic and epidemiological data of foot-and-mouth disease virus (FMDV), and performed comprehensive analyses to determine its early origin and transmission route. We found that recombination is a key feature of FMDV, and that the genomic regions coding for structural and non-structural proteins have markedly different evolutionary histories, and evolve at different rates. Despite all of these differences, analyses of both structural and non-structural protein coding regions consistently suggested that the most recent common ancestor of FMDV could be dated back to the Middle Age, ~ 200 to 300 years earlier than previously thought. The ancestors of the Euro-Asiatic and SAT strains could be dated back to the mid-seventeenth century, and to the mid-fifteenth to mid-sixteenth century, respectively. Our results implicated Mediterranean counties as an early geographical origin of FMDV before spreading to Europe and subsequently to Asia and South America.
Assuntos
Vírus da Febre Aftosa/genética , Animais , Ásia , Europa (Continente) , Evolução Molecular , Febre Aftosa/virologia , Genômica , Epidemiologia Molecular/métodos , Fases de Leitura Aberta/genética , Filogenia , América do Sul , Proteínas não Estruturais Virais/genéticaRESUMO
Cocal virus (COCV) is one of the causative agents of vesicular stomatitis, presenting clinical signs indistinguishable from those caused by foot-and-mouth disease virus (FMDV). Therefore, the differentiation of these two viruses via laboratory diagnosis is essential. The objective of this study was to develop and validate a real-time quantitative PCR (RT-qPCR) protocol for the diagnosis of COCV directly from epithelial samples. The method developed had 97% accuracy at 3950 pfu and a repeatability error of 1.29%. RT-qPCR was able to distinguish COCV from other viruses that cause vesicular diseases, an important factor because seroneutralization may produce cross-reactivity between COCV and vesicular stomatitis Alagoas virus (VSAV). No epithelial sample originating from vesicular disease outbreaks between 2014 and 2018 in Brazil was positive for COCV.
Assuntos
Estomatite Vesicular/diagnóstico , Estomatite Vesicular/virologia , Vesiculovirus/genética , Animais , Brasil , Vírus de DNA/genética , Febre Aftosa/diagnóstico , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
Deep sequencing of viral genomes is a powerful tool to study RNA virus complexity. However, the analysis of next-generation sequencing data might be challenging for researchers who have never approached the study of viral quasispecies by this methodology. In this work we present a suitable and affordable guide to explore the sub-consensus variability and to reconstruct viral quasispecies from Illumina sequencing data. The guide includes a complete analysis pipeline along with user-friendly descriptions of software and file formats. In addition, we assessed the feasibility of the workflow proposed by analyzing a set of foot-and-mouth disease viruses (FMDV) with different degrees of variability. This guide introduces the analysis of quasispecies of FMDV and other viruses through this kind of approach.
Assuntos
Vírus da Febre Aftosa/genética , Haplótipos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Quase-Espécies , Animais , Vírus da Febre Aftosa/classificação , Genes ViraisRESUMO
This study investigates the historical temporal trend and geographical distribution of the foot-and-mouth disease virus (FMDv) serotype C in South America; discussing the findings within the context of the actions and strategies carried out for the elimination of foot-and-mouth disease (FMD). This is the first time that such a comprehensive historical compilation has been carried out in the Region; hence, the study is intended as a reference and source of evidence about the presence/absence of FMDv serotype C in South America. Data on the occurrence of FMD were sourced from the Weekly Epidemiological Reports submitted by the countries to Pan American Foot-and-Mouth Disease Center (PANAFTOSA-PAHO/WHO) since 1972, and complemented with other sources of information from the 1968-1971 period. The temporal distribution was examined with local weighted regression (LOESS) to identify two temporal trends, that is, "smoothed" and "over-adjusted", utilising the time-series with the total number of cases per year, at Regional level. Thereafter the outbreaks were aggregated by decades and mapped by the first subnational administrative level. As a result, two major peaks of occurrence were identified, one in the 70s, with up to 1,193 outbreaks, and another in the 80s, with 380. Overall, the investigations show a clear regressive trend in the occurrence of serotype C, with a reduction in the number of outbreaks over-time, and with the subsequent reduction of affected locations. This study illustrates the contrast between the very limited presence over the last 20 years - with only one event in 2004 - and the epidemic situation in the 1970s and 1980s, and suggests that serotype C of FMDv is no longer present in the Region.
Assuntos
Búfalos , Doenças dos Bovinos , Vírus da Febre Aftosa/fisiologia , Febre Aftosa , Doenças das Cabras , Doenças dos Ovinos , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/transmissão , Doenças dos Bovinos/virologia , Febre Aftosa/diagnóstico , Febre Aftosa/prevenção & controle , Febre Aftosa/transmissão , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , Doenças das Cabras/diagnóstico , Doenças das Cabras/prevenção & controle , Doenças das Cabras/transmissão , Doenças das Cabras/virologia , Cabras , Sorogrupo , Ovinos , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/prevenção & controle , Doenças dos Ovinos/transmissão , Doenças dos Ovinos/virologia , América do Sul , Análise Espaço-TemporalRESUMO
Foot-and-mouth-disease (FMD) is a highly contagious disease of domestic animals which can result in substantial economic losses, caused by the FMD virus (FMDV). The aim of this study was to develop and standardize a novel reverse transcriptase droplet digital PCR (RT-ddPCR) assay for the quantification of FMDV RNA. This assay was based upon an OIE-recognized real-time RT-PCR that detects the 3D-encoding region of FMDV. The limit of detection at 101.4 TCID50/mL and 26.5 copies was determined using FMDV-A24-Cruzeiro-virus and a plasmid containing the 3D-FMDV sequences, respectively. FMDV O, A and C serotypes and 11 species of non-FMDV were used to confirm the sensitivity and specificity of the assay. The RT-ddPCR was standardized using 60 bovine samples (representing negative and positive samples of epithelium and/or oesophageal-pharyngeal [OP] fluid) from animals suspected of vesicular diseases and previously tested by RT-qPCR. The RT-ddPCR showed robustness, sensitivity, specificity and accuracy, with similar results to the RT-qPCR. Moreover, the new RT-ddPCR diagnostic tool allowed the absolute quantification of FMDV RNA from epithelium and OP-fluid samples, as well as having the advantages of direct quantification by endpoint, eliminating the need for a calibration standard curve required in quantitative real-time RT-PCR.
Assuntos
Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/virologia , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Carga Viral/métodos , Animais , Bovinos , Doenças dos Bovinos/virologia , Vírus da Febre Aftosa/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Sensibilidade e Especificidade , Sorogrupo , Carga Viral/normasRESUMO
A foot-and-mouth disease virus (FMDV) DNA-launched reporter replicon containing a luciferase gene was used to assess the impact of non-structural (NS) protein 3A on viral replication. Independent deletions within the N-terminal region (amino acid [aa] residues 6 to 24) and the central hydrophobic region (HR, aa 59 to 76) of FMDV NS protein 3A were engineered, and luciferase activity in lysates of control and mutated replicon-transfected cells was measured. Triple alanine replacements of the N-terminal triplet Arg 18- His 19 -Glu 20 and a single alanine substitution of the highly charged Glu 20 residue both resulted in a 70-80% reduction in luciferase activity when compared with wild-type controls. Alanine substitution of the 17 aa present in the central HR, on the other hand, resulted in complete inhibition of luciferase activity and in the accumulation of the mutated 3A within the cell nucleus according to immunofluorescence analysis. Our results suggest that both the aa sequence around the putatively exposed hydrophilic E20 residue at the N-terminus of the protein and the hydrophobic tract located between aa 59 and 76 are of major relevance for maintaining the functionality of the 3A protein and preventing its mislocalization into the cell nucleus.
Assuntos
Vírus da Febre Aftosa/genética , Replicon , Proteínas não Estruturais Virais/química , Replicação Viral/genética , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Linhagem Celular , Núcleo Celular/virologia , Cricetinae , Replicação do DNA , Febre Aftosa/virologia , Vírus da Febre Aftosa/química , Vírus da Febre Aftosa/fisiologia , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Luciferases , Mutação , Domínios Proteicos , Deleção de Sequência , Proteínas não Estruturais Virais/genéticaRESUMO
In 2010 serotype O foot-and-mouth disease virus of the Mya98 lineage/SEA topotype spread into most East Asian countries. During 2010-2011 it was responsible for major outbreaks in the Republic of Korea where a monovalent O/Manisa vaccine (belonging to the ME-SA topotype) was applied to help control the outbreaks. Subsequently, all susceptible animals were vaccinated every 6â¯months with a vaccine containing the O/Manisa antigen. Despite vaccination, the disease re-occurred in 2014 and afterwards almost annually. This study focuses on the in vivo efficacy in pigs of a high quality monovalent commercial O1/Campos vaccine against heterologous challenge with a representative 2015 isolate from the Jincheon Province of the Republic of Korea. Initially, viral characterizations and r1 determinations were performed on six viruses recovered in that region during 2014-2015, centering on their relationship with the well characterized and widely available O1/Campos vaccine strain. Genetic and antigenic analysis indicated a close similarity among 2014-2015 Korean isolates and with the previous 2010 virus, with distinct differences with the O1/Campos strain. Virus neutralisation tests using O1/Campos cattle and pig post vaccination sera and recent Korean outbreak viruses predicted acceptable cross-protection after a single vaccination, as indicated by r1 values, and in pigs, by expectancy of protection. In agreement with the in vitro estimates, in vivo challenge with a selected field isolate indicated that O1/Campos primo vaccinated pigs were protected, resulting in a PD50 value of nearly 10. The results indicated that good quality oil vaccines containing the O1/Campos strain can successfully be used against isolates belonging to the O Mya98/SEA topotype.