ABSTRACT Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct.

As infecções causadas por dengue representam uma das mais prevalentes doenças transmitidas por artrópodes mundialmente, causando um amplo espectro de manifestações clínicas. A tecnologia de clones infecciosos é uma importante ferramenta para o entendimento da biologia do vírus da dengue (DENV). Clones de cDNA infeccioso têm sido construídos para muitos vírus de RNA com cadeia positiva e são ferramentas valiosas para o estudo dos mecanismos moleculares envolvidos na replicação do genoma viral, montagem da partícula viral, patogênese viral, e desenvolvimento de vacinas. No presente trabalho, nós descrevemos o desenvolvimento de um clone infeccioso baseado num isolado primário brasileiro do vírus Dengue sorotipo 3 (DENV3), genótipo III. Usando uma estratégia de dois plasmídeos, o genoma viral foi dividido em duas partes e os fragmentos gerados foram clonados separadamente num vetor shuttlelevedura-bactéria. Os plasmídeos foram construídos pela técnica de recombinação homóloga em levedura e a transcrição do genoma completo foi realizada a partir ligação in vitro das duas partes do genoma. O transcrito de DENV3 se mostrou infeccioso quando transfectado em células BHK-21 e a identidade do clone infeccioso foi confirmada por caracterização in vitro. A cinética de crescimento do DENV3 gerado neste sistema foi indistinguível do vírus parental. Este sistema representa uma poderosa ferramenta que ajudará na elucidação de aspectos moleculares da biologia do DENV bem como no estudo de mutações associadas com patogênese do DENV.

Dengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis.

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.

O replicon de RNA derivado do genoma de Flavivirus é uma ferramenta valiosa para o estudo de replicação viral independente da montagem e da maturação do virion, além de possuir um grande potencial para expressão de genes heterólogos. Neste estudo nós descrevemos a construção de replicons subgenômicos do vírus da febre amarela utilizando a técnica de recombinação homóloga em levedura. O plasmídeo contendo o replicon do vírus febre amarela cepa 17D (pBSC-repYFV-17D), caracterizado anteriormente, foi manipulado para a expressão heteróloga dos genes repórteres green fluorescent protein (repYFV-17D-GFP) e firelly luciferase (repYFV-17D-Luc). Ambos os replicons foram construídos por recombinação homóloga entre o vetor pBSC-repYFV-17D linearizado e o produto de PCR contendo 25 nucleotídeos terminais homólogos incorporados aos oligonucleotídeos iniciadores. A organização genômica dos construídos é semelhante ao repYFV-17D, com inserção de um gene repórter entre os restantes 63 nucleotídeos N-terminais da proteína do capsídeo e 72 nucleotídeos C-terminais da proteína E. Os replicons repYFV-17D-GFP e repYFV-17D-Luc mostraram uma eficiente replicação e expressão dos genes repórteres. A técnica de recombinação homóloga em levedura usada neste estudo demonstrou ser aplicável à manipulação do genoma do vírus da febre amarela para a construção de replicons subgenômicos.

RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potencial for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSC-repYFV-17D), previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP) and firefly luciferase (repYFV-17D-Luc) reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons.

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.

RESUMO A fim de estabelecer um parâmetro para a avaliação da viabilidade após o descongelamento, os resultados do cultivo de embriões frescos de camundongos, no meio PBS Modificado sem a adição de C02, foram comparados aos resultados obtidos após o cultivo no meio de Whitten Modificado em atmosfera com 5% de C02. Foram coletadas 1458 mórulas provenientes de fêmeas Mus musculus da cepa Suíço Albina CF 1, previamente superovuladas com 6-8UI de eCG e 6-8UI de HCG, com intervalo de 48 horas. Destas, 1051 foram cultivadas em PBS Modificado acrescido de 20% de SFB, a 37°C e 95% de umidade. As restantes 407 foram cultivadas no meio de Whitten Modificado, acrescido de 3% de BSA, a 37°C, 95% de umidade e 5% de C02. As avaliações foram realizadas com 24h de cultivo, sendo considerados os embriões que atingiram os estágios de blastocisto inicial (Bi), blastocisto (Bl), blastocisto expandido (Bx), blastocisto em eclosão (Bh) ou blastocisto eclodido (Be), e após 48h de cultivo os que atingiram pelo menos o estágio de blastocisto expandido. Os resultados obtidos após 24h (87,9 e 89,4%) e após 48h de cultivo (95,1 e 93,8%) em PBS e Whitten respectivamente, não apresentaram diferenças estatisticamente significativas (p < 0,05).

SUMMARY The development of fresh mouse embryos in Modified PBS without adding C02 was compared to the development in Modified Whitten supplemented with 5% de C02 in order to establish a parameter to assess the viability after thawing. One thousand and four hundred and fifty eight morulae were collected from Strain CF1 Swiss Albino Mus musculus females which had been previously superovulated with 6-8UI of PMSG and 6-8UI of HCG 48h apart. One thousand and fifty one morulae were cultured in Modified PBS + 20% FCS, at 37°C and 95% of humidity. The others 407 were cultured in Modified Whitten's medium + 3% BSA, at 37 °C, 95% of humidity and 5% of C02. At the evaluation, performed after 24h of culture, were considered developed the embryos that reached early blastocyst, expanded blastocyst, hatching blastocyst and hatched blastocyts stages. After 48h of culture were considered developed the embryos that reached at least the expanded blastocyst stage. The results obtained after 24h (87.9 and 89.4%) and 48h of culture (95.2 and 93.8%) in PBS and Whitten, respectively, were similar (p < 0,05).