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Stenfeldt C.,Foreign Animal Disease Research Unit | Stenfeldt C.,Oak Ridge Institute for Science and Education | Pacheco J.M.,Foreign Animal Disease Research Unit | Rodriguez L.L.,Foreign Animal Disease Research Unit | Arzt J.,Foreign Animal Disease Research Unit
PLoS ONE | Year: 2014

A time-course study was performed to elucidate the early events of foot-and-mouth disease virus (FMDV) infection in pigs subsequent to simulated natural, intra-oropharyngeal, inoculation. The earliest detectable event was primary infection in the lingual and paraepiglottic tonsils at 6 hours post inoculation (hpi) characterized by regional localization of viral RNA, viral antigen, and infectious virus. At this time FMDV antigen was localized in cytokeratin-positive epithelial cells and CD172a-expressing leukocytes of the crypt epithelium of the paraepiglottic tonsils. De novo replication of FMDV was first detected in oropharyngeal swab samples at 12 hpi and viremia occurred at 18-24 hpi, approximately 24 hours prior to the appearance of vesicular lesions. From 12 through 78 hpi, microscopic detection of FMDV was consistently localized to cytokeratin-positive cells within morphologically characteristic segments of oropharyngeal tonsil crypt epithelium. During this period, leukocyte populations expressing CD172a, SLA-DQ class II and/or CD8 were found in close proximity to infected epithelial cells, but with little or no co-localization with viral proteins. Similarly, M-cells expressing cytokeratin-18 did not colocalize with FMDV proteins. Intra-epithelial micro-vesicles composed of acantholytic epithelial cells expressing large amounts of structural and non-structural FMDV proteins were present within crypts of the tonsil of the soft palate during peak clinical infection. These findings inculpate the paraepiglottic tonsils as the primary site of FMDV infection in pigs exposed via the gastrointestinal tract. Furthermore, the continuing replication of FMDV in the oropharyngeal tonsils during viremia and peak clinical infection with no concurrent amplification of virus occurring in the lower respiratory tract indicates that these sites are the major source of shedding of FMDV from pigs.


Maree F.F.,Onderstepoort Veterinary Institute | Blignaut B.,Onderstepoort Veterinary Institute | Blignaut B.,University of Pretoria | de Beer T.A.P.,University of Pretoria | And 2 more authors.
Virus Research | Year: 2010

Foot-and-mouth disease virus (FMDV) infects host cells by adhering to the αV subgroup of the integrin family of cellular receptors in a Arg-Gly-Asp (RGD) dependent manner. FMD viruses, propagated in non-host cell cultures are reported to acquire the ability to enter cells via alternative cell surface molecules. Sequencing analysis of SAT1 and SAT2 cell culture-adapted variants showed acquisition of positively charged amino acid residues within surface-exposed loops of the outer capsid structural proteins. The fixation of positively charged residues at position 110-112 in the βF-βG loop of VP1 of SAT1 isolates is thought to correlate with the acquisition of the ability to utilise alternative glycosaminoglycan (GAG) molecules for cell entry. Similarly, two SAT2 viruses that adapted readily to BHK-21 cells accumulated positively charged residues at positions 83 and 85 of the βD-βE loop of VP1. Both regions surround the fivefold axis of the virion. Recombinant viruses containing positively charged residues at position 110 and 112 of VP1 were able to infect CHO-K1 cells (that expresses GAG) and demonstrated increased infectivity in BHK-21 cells. Therefore, recombinant SAT viruses engineered to express substitutions that induce GAG-binding could be exploited in the rational design of vaccine seed stocks with improved growth properties in cell cultures. © 2010 Elsevier B.V.


LaRocco M.,Foreign Animal Disease Research Unit | Krug P.W.,Foreign Animal Disease Research Unit | Kramer E.,McConnell Group Inc. | Ahmed Z.,Foreign Animal Disease Research Unit | And 4 more authors.
Journal of Clinical Microbiology | Year: 2013

Foot-and-mouth disease (FMD) is a worldwide problem limiting the trade of animals and their products from affected countries. The rapid isolation, serotyping, and vaccine matching of FMD virus from disease outbreaks is critical for enabling the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Some primary cells have been shown to be highly susceptible to most strains of FMD virus (FMDV) but are difficult and expensive to prepare and maintain. Since the αVβ 6 integrin is a principal receptor for FMDV, we transduced a bovine kidney cell line to stably express both the αV and β6 bovine integrin subunits. This stable cell line (LFBK-αVβ6) showed β6 expression and enhanced susceptibility to FMDV infection for ≥100 cell passages. LFBK-αVβ6 cells were highly sensitive for detecting all serotypes of FMDV from experimentally infected animals, including the porcinophilic FMDV strain O/TAW/97. In comparison to other cell types that are currently used for virus isolation, LFBK- αVβ6 cells were more effective at detecting FMDV in clinical samples, supporting their use as a more sensitive tool for virus isolation. Copyright © 2013, American Society for Microbiology. All Rights Reserved.


Sharma R.K.,Allahabad University | Rai D.K.,Allahabad University | Rai D.K.,Foreign Animal Disease Research Unit | Sharma B.,Allahabad University
Cellular and Molecular Biology | Year: 2012

The farmers in general get exposed to different chemicals including pesticides. Many of these compounds are capable of inducing mutations in DNA and lead to several diseases including cancer. Carbofuran is a broad spectrum pesticide and frequently used in agricultural practices in India. In this study we intended to evaluate DNA damage infl icted by pesticide exposure in human blood lymphocytes under in vitro condition. The lymphocytes were exposed to varying concentrations of carbofuran (0-50μM) and analyzed by means of the micronucleus (MN) test. The results obtained showed signifi cant increase in MN frequency after exposure to 5, 10, 25 and 50μM of carbofuran as compared to the control group. The frequencies of MN were observed to be in concentration dependent manner. As we further increase the concentration of carbofuran, we observed signifi cant decrease in the mean percentage of binucleated cells (70-49%) and increase in the number of micronu-clei formed per 1000 binucleated cells. Simultaneously, we also observed reduction in Cytokinesis-Block Proliferation index (CBPI) with increase in the carbofuran concentrations. The results indicate that this pesticide may exhibit genotoxic effect at higher concentrations. This study emphasizes the need to reinforce the good practices campaigns in order to enlighten those who work with pesticides and also to make them aware about the importance of using protective measures. © 2012.


Zhu J.J.,Foreign Animal Disease Research Unit | Arzt J.,Foreign Animal Disease Research Unit | Puckette M.C.,Foreign Animal Disease Research Unit | Smoliga G.R.,Foreign Animal Disease Research Unit | And 2 more authors.
PLoS ONE | Year: 2013

Foot-and-mouth disease virus (FMDV) targets specific tissues for primary infection, secondary high-titer replication (e.g. foot and mouth where it causes typical vesicular lesions) and long-term persistence at some primary replication sites. Although integrin αVβ6 receptor has been identified as primary FMDV receptors in animals, their tissue distribution alone fails to explain these highly selective tropism-driven events. Thus, other molecular mechanisms must play roles in determining this tissue specificity. We hypothesized that differences in certain biological activities due to differential gene expression determine FMDV tropism and applied whole genome gene expression profiling to identify genes differentially expressed between FMDV-targeted and non-targeted tissues in terms of supporting primary infection, secondary replication including vesicular lesions, and persistence. Using statistical and bioinformatic tools to analyze the differential gene expression, we identified mechanisms that could explain FMDV tissue tropism based on its association with differential expression of integrin αVβ6 heterodimeric receptor (FMDV receptor), fibronectin (ligand of the receptor), IL-1 cytokines, death receptors and the ligands, and multiple genes in the biological pathways involved in extracellular matrix turnover and interferon signaling found in this study. Our results together with reported findings indicate that differences in (1) FMDV receptor availability and accessibility, (2) type I interferon-inducible immune response, and (3) ability to clear virus infected cells via death receptor signaling play roles in determining FMDV tissue tropism and the additional increase of high extracellular matrix turnover induced by FMDV infection, likely via triggering the signaling of highly expressed IL-1 cytokines, play a key role in the pathogenesis of vesicular lesions.


Maree F.F.,Onderstepoort Veterinary Institute | Blignaut B.,Onderstepoort Veterinary Institute | Blignaut B.,University of Pretoria | Aschenbrenner L.,Foreign Animal Disease Research Unit | And 2 more authors.
Virus Research | Year: 2011

The three SAT serotype viruses, endemic in Africa, are well known for their difficulty to adapt to cell culture. The viral mechanism involved in foot-and-mouth disease virus (FMDV) tissue tropism and cell-entry is not well understood. A recombinant, small plaque-forming virus (vSAT1tc), derived from a tissue culture-adapted SAT1 virus (SAR/9/81tc), revealed four amino acid substitutions (VP3 Asp192→Tyr; VP3 Ser217→Ile; VP1 Ala69→Gly and VP1 Asn110→Lys) in the capsid, compared to the SAR/9/81wt isolate collected from infected impala epithelium. One substitution added a positively charged lysine residue to the short βF-βG loop of VP1. Furthermore, vSAT1tc displayed a high affinity for CHO-K1 cells possibly via interaction with negatively charged sulphated polysaccharides while SAT1 impala strain relied strongly on α Vβ6 integrin receptors for cell entry. The cell culture adaptation and small plaque phenotype of vSAT1tc was accompanied by differences in particle aggregation and significant differences in acid stability. Based on limited cross neutralization data, the antigenic features seem to be unchanged. Thus, acquisition of positively charged residues in the virion may be beneficial for adaptation of SAT type field strains to cell culture. © 2010 Elsevier B.V.


Maree F.F.,Onderstepoort Veterinary Institute | Maree F.F.,University of Pretoria | Blignaut B.,Onderstepoort Veterinary Institute | Blignaut B.,University of Pretoria | And 3 more authors.
PLoS ONE | Year: 2013

Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces. © 2013 Maree et al.


Krug P.W.,Foreign Animal Disease Research Unit | Larson C.R.,Foreign Animal Disease Research Unit | Larson C.R.,Oak Ridge Institute for Science and Education | Eslami A.C.,Foreign Animal Disease Research Unit | And 2 more authors.
Veterinary Microbiology | Year: 2012

Transboundary animal disease viruses such as foot-and-mouth disease virus (FMDV) and African swine fever virus (ASFV) are highly contagious and cause severe morbidity and mortality in livestock. Proper disinfection during an outbreak can help prevent virus spread and will shorten the time for contaminated agriculture facilities to return to food production. Wood surfaces are prevalent at these locations, but there is no standardized method for porous surface disinfection; commercial disinfectants are only certified for use on hard, nonporous surfaces. To model porous surface disinfection in the laboratory, FMDV and ASFV stocks were dried on wood coupons and exposed to citric acid or sodium hypochlorite. We found that 2% citric acid was effective at inactivating both viruses dried on a wood surface by 30. min at 22 °C While 2000. ppm sodium hypochlorite was capable of inactivating ASFV on wood under these conditions, this chemical did not meet the 4-log disinfection threshold for FMDV. Taken together, our data supports the use of chemical disinfectants containing at least 2% citric acid for porous surface disinfection of FMDV and ASFV. © 2011.


Rai D.K.,Foreign Animal Disease Research Unit | Rieder E.,Foreign Animal Disease Research Unit
International Journal of Molecular Sciences | Year: 2012

Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ∼43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3Dpol). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3Dpol (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to Aphthovirus RdRps. © 2012 by the authors; licensee MDPI, Basel, Switzerland.


Lawrence P.,Foreign Animal Disease Research Unit | Larocco M.,Foreign Animal Disease Research Unit | Baxt B.,Foreign Animal Disease Research Unit | Rieder E.,Foreign Animal Disease Research Unit
Virology Journal | Year: 2013

Background: Foot-and-mouth disease virus (FMDV) initiates infection via recognition of one of at least four cell-surface integrin molecules αvβ1, αvβ3, αvβ6, or αvβ8 by a highly conserved Arg-Gly-Asp (RGD) amino acid sequence motif located in the G-H loop of VP1. Within the animal host, the αvβ 6 interaction is believed to be the most relevant. Sub-neutralizing levels of soluble secreted αvβ6 (ssαvβ6) was used as a selective pressure during passages in vitro to explore the plasticity of that interaction. Results: Genetically stable soluble integrin resistant (SIR) FMDV mutants derived from A24 Cruzeiro were selected after just 3 passages in cell culture in the presence of sub-neutralizing levels of ssαvβ6. SIR mutants were characterized by: replication on selective cell lines, plaque morphology, relative sensitivity to ssαvβ6 neutralization, relative ability to utilize αvβ 6 for infection, as well as sequence and structural changes. All SIR mutants maintained an affinity for αvβ6. Some developed the ability to attach to cells expressing heparan sulfate (HS) proteoglycan, while others appear to have developed affinity for a still unknown third receptor. Two classes of SIR mutants were selected that were highly or moderately resistant to neutralization by ssαvβ 6. Highly resistant mutants displayed a G145D substitution (RGD to RDD), while moderately resistant viruses exhibited a L150P/R substitution at the conserved RGD + 4 position. VP1 G-H loop homology models for the A-type SIR mutants illustrated potential structural changes within the integrin-binding motif by these 2 groups of mutations. Treatment of O1 Campos with ssαvβ6 resulted in 3 SIR mutants with a positively charged VP3 mutation allowing for HS binding. Conclusions: These findings illustrate how FMDV particles rapidly gain resistance to soluble receptor prophylactic measures in vitro. Two different serotypes developed distinct capsid mutations to circumvent the presence of sub-neutralizing levels of the soluble cognate receptor, all of which resulted in a modified receptor tropism that expanded the cell types susceptible to FMDV. The identification of some of these adaptive mutations in known FMDV isolates suggests these findings have implications beyond the cell culture system explored in these studies. © 2013 Lawrence et al.; licensee BioMed Central Ltd.

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