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In JoVE (1)
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Articles by Carlos Maluquer de Motes in JoVE
JoVE Immunology and Infection
माइक्रोबियल स्रोत विशिष्ट मानव और पशु वायरस का उपयोग ट्रैकिंग के लिए लागत प्रभावी विधि
अध्ययन मल / मूत्र या पानी में नाइट्रेट मानव / शूकरीय / गोजातीय डीएनए वायरस, एडिनोवायरस और polyomaviruses, MST उपकरण के रूप में प्रस्तावित की विशिष्ट मात्रा का ठहराव के लिए qPCR का उपयोग करके संदूषण संदूषण के स्रोत की पहचान के लिए एक लागत प्रभावी विधि का वर्णन करता है.
Other articles by Carlos Maluquer de Motes on PubMed
Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination
In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.
Identification of Human and Animal Adenoviruses and Polyomaviruses for Determination of Sources of Fecal Contamination in the Environment
The Adenoviridae and Polyomaviridae families comprise a wide diversity of viruses which may be excreted for long periods in feces or urine. In this study, a preliminary analysis of the prevalence in the environment and the potential usefulness as source-tracking tools of human and animal adenoviruses and polyomaviruses has been developed. Molecular assays based on PCR specifically targeting human adenoviruses (HAdV), porcine adenoviruses (PAdV), bovine adenoviruses (BAdV), and bovine polyomaviruses (BPyV) were applied to environmental samples including urban sewage, slaughterhouse, and river water samples. PAdV and BPyV were detected in a very high percentage of samples potentially affected by either porcine or bovine fecal contamination, respectively. However, BAdV were detected in only one sample, showing a lower prevalence than BPyV in the wastewater samples analyzed. The 22 slaughterhouse samples with fecal contamination of animal origin showed negative results for the presence of HAdV. The river water samples analyzed were positive for the presence of both human and animal adenoviruses and polyomaviruses, indicating the existence of diverse sources of contamination. The identities of the viruses detected were confirmed by analyses of the amplified sequences. All BPyV isolates showed a 97% similarity in nucleotide sequences. This is the first time that PAdV5, BAdV6, and BPyV have been reported to occur in environmental samples. Human and porcine adenoviruses and human and bovine polyomaviruses are proposed as tools for evaluating the presence of viral contamination and for tracking the origin of fecal/urine contamination in environmental samples.
Excretion of BSE and Scrapie Prions in Stools from Murine Models
Faeces from infected animals have been suggested as a potential source of contamination and transmission of prion diseases in the environment. This work describes the development of a procedure for the detection of PrP(res) in stools which is based on a detergent-based extraction and immunoprecipitation (IP). The procedure was evaluated by analyzing TSE-spiked sheep and mice faeces, and proved to be specific for PrP(res) with sensitivities of 5-10 microg of infected brain tissue. In order to analyze the shedding of prions, we studied stools from orally inoculated mice over 4-days post-inoculation and also stools from terminally sick scrapie-infected mice. PrP(res) was only detected in stools shortly after the oral ingestion of TSE agents. The procedure described could be a useful tool for studying the excretion of prions and for evaluating potential environmental contamination by prions.
Development of a Quantitative PCR Assay for the Quantitation of Bovine Polyomavirus As a Microbial Source-tracking Tool
Adenoviruses and polyomaviruses are two distinct DNA viral families that are excreted in high concentrations and distributed in human and animal populations. Targeting specific virus included in these families has proved to be a promising and useful tool for tracing specifically sources of environmental contamination. In this study, a quantitative PCR assay that is specific for bovine polyomaviruses was developed and used to determine the excretion level and concentration of bovine polyomaviruses in urine and environmental samples, including urban sewage, slaughterhouse sewage, and river water. A set of primers and a TaqMan probe were designed to target a 77-bp region of the bovine polyomavirus VP1 gene, and the conditions of the reaction were optimized. A detection limit was established at 1-10 genome copies per test tube. The assay was specific and produced negative results when samples containing human or porcine fecal contamination were analyzed. This is, to our knowledge, the first description of bovine polyomaviruses excreted in bovine urine samples (mean values of 10(4) GC/l). Bovine polyomaviruses were also detected and quantified in slaughterhouse wastewater and river waters, which shows the spread of these viruses in many environmental samples containing contamination of bovine origin. The procedure described in this paper provides a quantitative source-tracking tool for the analysis of bovine polyomaviruses as indicators of the presence of bovine contamination in environmental samples.
Isolation of a Novel Monkey Adenovirus Reveals a New Phylogenetic Clade in the Evolutionary History of Simian Adenoviruses
Adenoviruses of primates include human (HAdV) and simian (SAdV) isolates classified into 8 species (Human Adenovirus A to G, and Simian Adenovirus A). In this study, a novel adenovirus was isolated from a colony of cynomolgus macaques (Macaca fascicularis) and subcultured in VERO cells. Its complete genome was purified and a region encompassing the hexon gene, the protease gene, the DNA binding protein (DBP) and the 100 kDa protein was amplified by PCR and sequenced by primer walking. Sequence analysis of these four genes showed that the new isolate had 80% identity to other primate adenoviruses and lacked recombination events. The study of the evolutionary relationships of this new monkey AdV based on the combined sequences of the four genes supported a close relationship to SAdV-3 and SAdV-6, lineages isolated from Rhesus monkeys. The clade formed by these three types is separated from the remaining clades and establishes a novel branch that is related to species HAdV-A, F and G. However, the genetic distance corresponding to the newly isolated monkey AdV considerably differs from these as to belong to a new, not yet established species. Results presented here widen our knowledge on SAdV and represents an important contribution to the understanding of the evolutionary history of primate adenoviruses.
Inhibition of Apoptosis and NF-κB Activation by Vaccinia Protein N1 Occur Via Distinct Binding Surfaces and Make Different Contributions to Virulence
Vaccinia virus (VACV) protein N1 is an intracellular virulence factor and belongs to a family of VACV B-cell lymphoma (Bcl)-2-like proteins whose members inhibit apoptosis or activation of pro-inflammatory transcription factors, such as interferon (IFN) regulatory factor-3 (IRF-3) and nuclear factor-κB (NF-κB). Unusually, N1 inhibits both apoptosis and NF-κB activation. To understand how N1 exerts these different functions, we have mutated residues in the Bcl-2-like surface groove and at the interface used to form N1 homodimers. Mutagenesis of the surface groove abolished only the N1 anti-apoptotic activity and protein crystallography showed these mutants differed from wild-type N1 only at the site of mutation. Conversely, mutagenesis of the dimer interface converted N1 to a monomer and affected only inhibition of NF-κB activation. Collectively, these data show that N1 inhibits pro-inflammatory and pro-apoptotic signalling using independent surfaces of the protein. To determine the relative contribution of each activity to virus virulence, mutant N1 alleles were introduced into a VACV strain lacking N1 and the virulence of these viruses was analysed after intradermal and intranasal inoculation in mice. In both models, VACV containing a mutant N1 unable to inhibit apoptosis had similar virulence to wild-type virus, whereas VACV containing a mutant N1 impaired for NF-κB inhibition induced an attenuated infection similar to that of the N1-deleted virus. This indicates that anti-apoptotic activity of N1 does not drive virulence in these in vivo models, and highlights the importance of pro-inflammatory signalling in the immune response against viral infections.
