ABSTRACT
The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.
Subject(s)
Animals , Chick Embryo , Homologous Recombination , Infectious bursal disease virus/genetics , Reverse Genetics/methods , Brazil , Cells, Cultured , Fibroblasts/virology , Genetic Vectors , Genomic Instability , Infectious bursal disease virus/isolation & purification , Infectious bursal disease virus/physiology , Saccharomyces cerevisiae/genetics , Transfection , Virus Cultivation , Virus ReplicationABSTRACT
The Atlantic Rainforest does not have a uniform physiognomy, its relief determines different environmental conditions that define the composition of its flora and fauna. Within this ecosystem, bromeliads that form tanks with their leaves hold water reservoirs throughout the year, maintaining complex food chains, based mainly on autotrophic and heterotrophic bacteria. Some works concluded that the water held by tank bromeliads concentrate the microbial diversity of their ecosystem. To investigate the bacterial diversity and the potential biotechnology of these ecosystems, tank bromeliads of the Neoregelia cruenta species from the Atlantic Rainforest in Brazil were used as models for this research. Bacteria isolated from these models were tested for production of bioactive compounds. DGGE of the water held by tank bromeliads was performed in different seasons, locations and sun exposure to verify whether these environmental factors affect bacterial communities. The DGGE bands profile showed no grouping of bacterial community by the environmental factors tested. Most of the isolates demonstrated promising activities in the tests performed. Collectively, these results suggest that tank bromeliads of the N. cruenta species provide important habitats for a diverse microbial community, suggesting that each tank forms a distinct micro-habitat. These tanks can be considered excellent sources for the search for new enzymes and/or new bioactive composites of microbial origin.
Subject(s)
Heterotrophic Bacteria , Bromeliaceae , Phytochemicals , Microbiota , Autotrophic ProcessesABSTRACT
This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.
Subject(s)
Biodiversity , Eukaryotic Cells/cytology , DNA, Bacterial , Environmental Microbiology , Elapidae/microbiology , In Vitro Techniques , Polymerase Chain Reaction/methods , Soil Microbiology , Methods , Guidelines as Topic , SoilABSTRACT
This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.
ABSTRACT
1. Twenty-eight male albino rats were evaluted for audiogenic seizure sensitivity by systematic observation and the recording of behavior by ethological methods. The animals were subjected to high-intensity acoustic stimulation and their behavior was evaluated by reference to an audiogenic severity index (SI). Animals were classified as susceptible (S) or resistant (R) depending on the SI value. R. animals were: 1) subjected to chemical lesion of the substantia nigra compacta with 6-hydroxydopamine (60HDA), followed by SI quantitation, contralateral electolytic lesion osf the substantia nigra reticulata and new SIevaluation (N = 10) received 0,9% saline followed by SI evaluation, contralateral sham (mechanical) lesion and new SI calculation; 3) another group (N = 10) was subjected to unilateral electrolytical lesion of the substantia nigra reticulata and SI evaluation. 2. Effects of asymmetry were observed after chemical or electrolytic lesions, but these alterations correlated only with increased audiogenic sensitivity in rats with electrolytic lesions in the substantia nigra reticulada. No bhevioral changes were observed in any of the cocntrols. The amplhetamine-induced rotational behavior presented a definite left pattern (ipsilateral to the 60HDA lesion) for the animals with bilateral lesions, with an asymmetry index of 98%, whereas the sham-lesioned controls showed a 60% asymmetry index which was not significant. 3. The relationship between asymmetry and ssimultaneous audiogenic sensitivity may correspond to changes in the basal ganglia possibly in the hypersensitive postsynaotic portions of the substantia nigra reticulata efferents