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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.

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.

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.

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.

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.

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