Development of a droplet digital PCR assay for quantification of the proviral load of bovine leukemia virus.

Droplet digital PCR (ddPCR) is a highly sensitive tool developed for the detection and quantification of short-sequence variants-a tool that offers unparalleled precision enabling measurement of smaller-fold changes. We describe here the use of ddPCR for the detection of Bovine leukemia virus (BLV) DNA provirus. Serum samples and whole blood from experimentally infected sheep and naturally infected cattle were analyzed through ddPCR to detect the BLV gp51 gene, and then compared with serologic and molecular tests. The ddPCR assay was significantly more accurate and sensitive than AGID, ELISA, nested PCR, and quantitative PCR. The limit of detection of ddPCR was 3.3 copies/µL, detecting positive experimentally infected sheep beginning at 6 d post-infection. The ddPCR methodology offers a promising tool for evaluating the BLV proviral load, particularly for the detection of low viral loads.

Metagenomic analysis of the microbiota from the crop of an invasive snail reveals a rich reservoir of novel genes.

The shortage of petroleum reserves and the increase in CO(2) emissions have raised global concerns and highlighted the importance of adopting sustainable energy sources. Second-generation ethanol made from lignocellulosic materials is considered to be one of the most promising fuels for vehicles. The giant snail Achatina fulica is an agricultural pest whose biotechnological potential has been largely untested. Here, the composition of the microbial population within the crop of this invasive land snail, as well as key genes involved in various biochemical pathways, have been explored for the first time. In a high-throughput approach, 318 Mbp of 454-Titanium shotgun metagenomic sequencing data were obtained. The predominant bacterial phylum found was Proteobacteria, followed by Bacteroidetes and Firmicutes. Viruses, Fungi, and Archaea were present to lesser extents. The functional analysis reveals a variety of microbial genes that could assist the host in the degradation of recalcitrant lignocellulose, detoxification of xenobiotics, and synthesis of essential amino acids and vitamins, contributing to the adaptability and wide-ranging diet of this snail. More than 2,700 genes encoding glycoside hydrolase (GH) domains and carbohydrate-binding modules were detected. When we compared GH profiles, we found an abundance of sequences coding for oligosaccharide-degrading enzymes (36%), very similar to those from wallabies and giant pandas, as well as many novel cellulase and hemicellulase coding sequences, which points to this model as a remarkable potential source of enzymes for the biofuel industry. Furthermore, this work is a major step toward the understanding of the unique genetic profile of the land snail holobiont.

Molecular analysis of Brazilian infectious bronchitis field isolates by reverse transcription-polymerase chain reaction, restriction fragment length polymorphism, and partial sequencing of the N gene.

Molecular analysis of 15 Brazilian infectious bronchitis virus (IBV) isolates, obtained from clinical outbreaks of the disease in chickens (broilers or layers) in the state of Minas Gerais (Brazil) between 1972 and 1989, is reported. Using the N protein gene as target, IBVs were analyzed by reverse transcription-polymerase chain reaction/restriction fragment length polymorphism (RT-PCR/RFLP) with the restriction enzymes AvaII, HphI, Sau96I, and Tsp509I and cDNA sequencing. Results obtained from those isolates were compared to 19 sequences available in GenBank. N gene RFLP profiles, cDNA sequences, and predicted amino acid composition were used for the construction of dendrograms. Brazilian isolates were grouped into one distinct group. Identity of predicted N protein amino acid composition varied from 45% (between isolates G and 208) up to 99% (PM 1 and PM2), and, when compared to the other IBVs, the amino acid identity was from 42% (Q3/88 and G) up to 97% (D41 and PM1). The great genetic diversity was shown to occur before the official use of vaccination in Brazil and has remained thereafter.

Análise funcional de genes regulados por glicocorticóides na reversão fenotípica tumoral-normal de células STI de glioma de rato / Functional analysis of glucocorticoid-regulated genes in the tumor to normal phenotypic reversion of ST1 rat glioma cells

As linhagens celulares ST1 e P7 (ARMELIN E ARMELIN, 1983), isoladas no laboratório a partir da linhagem C6 de glioma de rato, constituem modelos interessantes para o estudo de ação de glicocorticóides por mostrarem, respectivamente, hiperesponsividade e resistência ao tratamento com o hormônio quando observados morfologia, tempo de dobramento, densidade de saturação, crescimento em suspensão de agarose e tumorigenicidade em camundos "nude". Em trabalhos anteriores, foram isoladas de bibliotecas de cDNA de células ST1, através de hibridização diferencial, seqüências correspondentes a metalotioneína 1, metalotioneína 2, glicoproteína "alfa"-1 ácida e pelo menos um transcrito de um retrovírus endógeno

Acute and latent infection in mice with a virulent strain of Aujeszky's disease virus

Acute and latent infections with the Brazilian LA031 strain of Aujeszky's disease virus (ADV) were established in mice. Ultraviolet irradiated ADV administered subcutaneously was a successful way to establish latent infection. The presence of ADV was detected by PCR. Two sets of 22-mer primers were synthesized and used to amplify gG glycoprotein gene sequences in acute and latent infected trigeminal nerve ganglia. The specificity of the amplification was verified by dot-blot hybridization.

Acute and latent infection in mice with a virulent strain of Aujeszkys disease virus

Acute and latent infections with the Brazilian LA031 strain of Aujeszkys disease virus (ADV) were established in mice. Ultraviolet irradiated ADV administered subcutaneously was a successful way to establish latent infection. The presence of ADV was detected by PCR. Two sets of 22-mer primers were synthesized and used to amplify gG glycoprotein gene sequences in acute and latent infected trigeminal nerve ganglia. The specificity of the amplification was verified by dot-blot hybridization.

Infecções dos tipos aguda e latente foram obtidas em camundongos infectados com a amostra brasileira LA031 do vírus da doença de Aujeszky (ADV). A administração subcutânea da amostra de ADV irradiada com luz ultravioleta foi indispensável para se estabelecer a infecção latente. A presença do ADV em animais foi detectada por PCR. Oligonucleotídeos de 22 mer foram sintetizados e usados para amplificar sequências do gene da glicoproteína gG no gânglio do nervo trigêmio em camundongos com infecção aguda e latente. A especificidade da amplificação foi verificada por hibridização do tipo dot-blot.