Abstract. Dengue is currently regarded as a major public health problem worldwide. In a hyperendemic region during an outbreak, we detected the co-circulation of all Dengue virus (DENV) serotypes including two different genotypes of DENV-3 and DENV-4, and concurrent infections with up to three serotypes were identified in symptomatic patients. A total of 49 acute phase plasma samples from patients clinically suspected of dengue were collected during the 4 weeks of May 2013. DENV-1-4 was detected by reverse transcriptase semi-nested polymerase chain reaction in 33 samples (67.3%), of which 26 DNA fragments were sequenced. Twenty samples (76.9%) were identified with a single DENV serotype and six (23.1%) with more than one serotype. DENV-3 was the predominant serotype of the outbreak. On the basis of phylogenetic analyses, DENV-1 isolates belong to genotype V, DENV-2 to American-Asian genotype, DENV-3 to genotypes I and III, and DENV-4 to genotypes I and II.Dengue is currently regarded globally as the most important mosquito-borne viral disease as it is a major public health problem worldwide.1 The disease is caused by four antigenically and genetically distinct viruses designated dengue virus 1-4 (DENV-1-4), which are subdivided into distinct genotypes. 2,3DENVs are transmitted by mosquito vectors, primarily Aedes aegypti.4 DENV infection is often unapparent but can lead to a wide range of clinical manifestations from mild disease to severe dengue cases with bleeding, plasma leakage, and shock. 5In dengue-endemic countries, the co-circulation of multiple DENV serotypes in the same area has been described with concurrent infections. 1,6 The first reported case of concurrent infection with two serotypes (DENV-1 and DENV-4) occurred in Puerto Rico in 1982. 4 Since this time, dual infections have been reported in locals in New Caledonia, Taiwan, China, Singapore, and India. In 1999, an infection with three serotypes (DENV-1/DENV-3/DENV-4) was detected in two patients from Indonesia and one from Mexico. 4,6 In this study, we report, for the first time in Brazil, the cocirculation and coinfection of DENV-1-4 during a severe outbreak in an endemic area of southeast Brazil. Our data raise new questions about biological and public health aspects related to dengue occurrence in Brazil.The study was performed in Contagem, Minas Gerais, which borders the city of Belo Horizonte (state capital), and it has an area of 195,268 km 2 with approximately 603,442 inhabitants. In 2013, Contagem underwent a dengue epidemic with the highest number of notified cases that has been ever recorded (23,436) and three deaths. In May 2013, a total of 49 acute phase plasma samples were collected during 4 weeks from clinically suspected dengue patients admitted to Geraldo Pinto Vieira Hospital in Contagem. Until this month, 22,808 (97.3%) cases had been reported. This research was approved by the Ethical Committee of UFMG. The residential addresses of DENV-positive patients are plotted on the map of Contagem shown in Figure 1.Viral RNA was extrac...
Yeast cells need to respond to a variety of stresses found in such different conditions as gastrointestinal tract after probiotic ingestion or fermentation vat during ethanol production. In the present study, H+ neutralisation capacity, membrane fatty acid composition, H+-ATPase activity, and cytosolic Ca2+ concentration were evaluated in yeast cells used for probiotic (Saccharomyces boulardii) and laboratory (Saccharomyces cerevisiae W303) purposes, as well as in some W303 mutant strains for ENA1 gene and S. cerevisiae BY4741. Results show that the H+ internal concentration of yeast is regulated by several systems, including the plasma membrane H+-ATPase, and that Ena1p has an important but undefined role in the cellular response to acid. Membrane fatty acid composition of S. cerevisiae W303 strain was affected by exposure to acidic pH, but the presence of 86 mM NaCl prevented this effect, whereas membrane fatty acid composition of S. boulardii was unaffected by acidic pH. We also demonstrated that the acid stress response is dependent on calcium metabolism and blocked by FK 506.
BackgroundL-ascorbic acid (L-AA) is naturally synthesized in plants from D-glucose by 10 steps pathway. The pathway branch to synthesize L-galactose, the key intermediate for L-ascorbic acid biosynthesis, has been recently elucidated. Budding yeast produces an 5-carbon ascorbic acid analogue Dehydro-D-arabinono 1,4-lactone (D-DAL), which is synthesized from D-arabinose. Yeast is able to synthesize L-ascorbic acid only if it is cultivated in the presence of one of its precursors: L-galactose, L-galactono 1,4-lactone, or L-gulono 1,4-lactone extracted from plants or animals. To avoid feeding the yeast culture with this “L” enantiomer, we engineered Kluyveromyces lactis with L-galactose biosynthesis pathway genes: GDP-mannose 3,5-epimerase (GME), GDP-L-galactose phosphorylase (VTC2) and L-galactose-1-phosphate phosphatase (VTC4) isolated from Arabidopsis thaliana.ResultsPlasmids were constructed and modified such that the cloned plant genes were targeted to the K. lactis LAC4 Locus by homologous recombination and that the expression was associated to the growth on D-galactose or lactose. Upon K. lactis transformation, GME was under the control of the native LAC4 promoter whereas VTC2 and VTC4 were expressed from the S. cerevisiae promoters GPD1 and ADH1 respectively. The expression in K. lactis, of the L-galactose biosynthesis genes was determined by Reverse Transcriptase-PCR and western blotting. The recombinant yeasts were capable to produce about 30 mg.L-1 of L-ascorbic acid in 48 hours of cultivation when cultured on rich medium with 2% (w/v) D-galactose. We also evaluated the L-AA production culturing recombinant recombinant strains in cheese whey, a waste product during cheese production, as an alternative source of lactose.ConclusionsThis work is the first attempt to engineer K. lactis cells for L-ascorbic acid biosynthesis by a fermentation process without any trace of “L” isomers precursors in the culture medium. We have engineered K. lactis strains capable of converting lactose and D-galactose into L-galactose, by the integration of the genes from the A. thaliana L-galactose pathway. L-galactose is a rare sugar, which is one of the main precursors for L-AA production.
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