ABSTRACT
Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.
Subject(s)
Dengue Virus/genetics , Reverse Genetics , RNA, Viral/genetics , Virus Replication/genetics , Escherichia coli/genetics , Genetic Vectors/genetics , PlasmidsABSTRACT
A busca por produtos de qualidade exige do produtor mudanças no sistema de produção de suíno que priorizem, em particular, o bem-estar do animal. As mudanças são necessárias para atender à demanda da sociedade e ampliar os mercados internos e externos. O bem-estar na espécie suína pode ser avaliado por meio das respostas comportamentais, fisiológicas, ligadas à sanidade e à produção. Em função do exposto, o objetivo deste trabalho é revisar a literatura em relação aos critérios científicos utilizados para indicar o bem-estar da espécie suína nos sistemas de produção.
In order to obtain good quality products, the swine production system should prioritize the animals welfare. Changes are necessary to meet society's demand and expand domestic and foreign markets. The swine's welfare can be assessed by behavioral and physiological traits related to health and production. Thus, our goal is to review in the literature what are the scientific criteria that are used to indicate the swine welfare in the production systems.