Virological Surveillance of Aedes aegypti Vectors Identifies All Four Dengue Serotypes in a Hyperendemic Region.

Dengue virus (DENV) 1-4 is the etiological agent of dengue, the most important viral infection transmitted by Aedes spp mosquitoes to humans. Our goal was to identify the circulating DENV in Aedes aegypti collected in an area of Brazil where all four DENV serotypes had already been detected in humans, understand the epidemiology better, and to test the vector as a virological surveillance tool. Twenty-eight larvae pools and 174 females of Aedes aegypti were screened by reverse transcriptase quantitative polymerase chain reaction and semi-nested PCR assays. PCR products were sequenced, and phylogenetic analyses were performed. Nine larvae pools (32.1%) were positive for DENV, four (44.4%) with DENV-3, and five (55.6%) with more than one serotype. Fifteen females (8.6%) were positive for any DENV serotype. 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. We demonstrate for the first time the co-circulation of all four DENV serotypes in larvae pools and adult Aedes aegypti in a hyperendemic area. This scenario represents a challenge for disease control and reinforces the importance of virological surveillance in the vector as a tool for predicting circulating DENV serotypes in humans.

First report of infectious spleen and kidney necrosis virus in Nile tilapia in Brazil.

In June 2020, an atypical fatal outbreak in a Brazilian Nile tilapia farm was investigated. Twenty-three animals were collected and different tissues were used for bacterial isolation, histopathological and electron microscopic examination and viral detection using molecular methods. A large number of megalocytes were observed in the histopathological analysis of several tissues. Icosahedral virions, with a diameter of approximately 160 nm, were visualized inside the megalocytes through transmission electron microscopy of the spleen tissue. The virions were confirmed to be infectious spleen and kidney necrosis virus (ISKNV) through PCR and sequencing analyses of the fish samples. Phylogenetic analysis indicated that the virus belongs to the Clade 1 of ISKNV. This viral pathogen is associated with high mortality in the early stages of cultured Nile tilapia in the United States, Thailand and Ghana; however, until now, there have been no reports from ISKNV affecting cultured fish in Brazil. Additionally, in 14 out of 23 sampled fish, Streptococcus agalactiae, Edwardsiella tarda or Aeromonas hydrophila infections were also detected. This is the first report of fatal ISKNV infections in the Brazilian Nile tilapia fish farms and represents a new challenge to the aquaculture sector in the country.

Effects of temperature changes in the transcriptional profile of the emerging fish pathogen Francisella noatunensis subsp. orientalis.

One of the major challenges in Nile tilapia (Oreochromis niloticus L.) farming is the occurrence of bacterial infections, and the Francisella noatunensis subsp. orientalis (FNO) is an important pathogen that has emerged in last decades. Francisellosis outbreaks have been reported in the literature as occurring seasonally when water temperature is below 24 °C. The aim of this study was to quantify the median lethal doses (LD50) of FNO in experimental challenges at 28 °C and 22 °C, and to investigate the impact of temperature changes in whole genome expression using microarray technology. The LD50 for Nile tilapia at 28 °C was ∼105.7, whereas at 22 °C, the LD50 was ∼102.2, showing that the decrease in temperature enhanced disease outcome. Out of 1917 genes screened, a total of 31 and 19 genes were down- and up-regulated at 22 °C, respectively. These genes were grouped by orthology into functional categories of: amino acid, inorganic ion, and carbohydrate transport and metabolism; transcription; and posttranslational modification, protein turnover, and chaperones. Expression of genes related to metabolism, oxidative stress, and thermal shock were regulated by temperature changes, reflecting an ability of FNO to adapt to the environment. Expression of virulence genes usually required for the Francisella genus was not changed between tested temperatures, including that of genes located on the Francisella Pathogenicity Island.

Multiple bluetongue virus serotypes causing death in Brazilian dwarf brocket deer (Mazama nana) in Brazil, 2015-2016.

Bela Vista Biological Sanctuary (RBV) is a protected area of Itaipu Binacional, a hydroelectric power company located on the border of Brazil and Paraguay. A captive population of Brazilian dwarf brocket deer (Mazama nana, Cervidae, Artiodactyla) is maintained for conservation purposes. Despite the reproductive success of the animals, outbreaks of a fatal hemorrhagic disease have been registered over the years, compromising conservation efforts. In order to identify the etiological agents of these hemorrhagic diseases, 32 captive Brazilian dwarf brockets were sampled to investigate bluetongue virus (BTV), epizootic hemorrhagic disease (EHD), and adenovirus hemorrhagic disease (AHD), in 2015. Only one deer (1/32; 3.12%) was seropositive for BTV. After this survey, five animals died in the early autumn of 2015 and 2016, again presenting clinical signs of hemorrhagic disease. Using RT-qPCR, RT-PCR and DNA sequencing, five BTV serotypes (3, 14, 18, 19, and 22) were identified in blood and tissues collected during necropsies. These BTV serotypes had not been previously described or isolated in Brazil, either in wild or domestic ruminants. Additionally, differential diagnosis was performed for EHD and AHD, but all samples were negative for both diseases. The multiple distinct BTV serotypes identified in these outbreaks resulted in a high lethality (100%) of Brazilian dwarf brockets and indicated that various BTV serotypes are circulating in the area.

Identification of bluetongue virus serotypes 1, 4, and 17 co-infections in sheep flocks during outbreaks in Brazil.

Bluetongue (BT) is a vector-borne viral disease caused by the Bluetongue virus (BTV), an Orbivirus from the Reoviridae family, affecting domestic and wild ruminants. BTV circulation in Brazil was first reported in 1978, and several serological surveys indicate that the virus is widespread, although with varied prevalence. In 2014, BT outbreaks affected sheep flocks in Rio Grande do Sul state, causing significant mortality (18.4%; 91/495) in BTV-infected sheep. In total, seven farms were monitored, and one or two sheep from each farm that died due to clinical signs of BT were necropsied. Apathy, pyrexia, anorexia, tachycardia, respiratory, and digestive disorders were noted. Additionally, an abortion was recorded in one of the monitored farms. The main gross lesions observed were pulmonary edema, anterior-ventral pulmonary consolidation, muscular necrosis in the esophagus and in the ventral serratus muscle, and hemorrhagic lesions in the heart. The blood and tissue samples were tested for BTV RNA detection by RT-qPCR targeting the segment 10. Positive samples were used for viral isolation. The isolated BTVs were typed by conventional RT-PCR targeting the segment 2 of the 26 BTV serotypes, followed by sequencing analysis. BTV-1, BTV-4 and BTV-17 were identified in the analyzed samples. Double or triple BTV co-infections with these serotypes were detected. We report the occurrence of BT outbreaks related to BTV-1, BTV-4 and BTV-17 infections and co-infections causing clinical signs in sheep flocks in Southern Brazil, with significant mortality and lethality rates.

Genome Sequence of Bluetongue virus Serotype 17 Isolated in Brazil in 2014.

The complete genome sequence of Bluetongue virus (BTV) serotype 17 strain 17/BRA/2014/73, isolated from a sheep in Brazil in 2014, is reported here. All segments clustered with western topotype strains and indicated reassortment events with other BTV from the Americas. The strain 17/BRA/2014/73 represents a novel reference strain for BTV-17 from South America.

Metabolic engineering of Kluyveromyces lactis for L-ascorbic acid (vitamin C) biosynthesis.

BACKGROUND: L-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. RESULTS: Plasmids 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. CONCLUSIONS: This 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.