Analysis of the nucleotide sequence of the coat protein and 3'-untranslated region of two Brazilian Potato virus Y isolates

Two Brazilian Potato virus Y (PVY) isolates were biologically characterized as necrotic (PVY-NBR) and common (PVY-OBR) based upon symptoms on test plants. Additional characterization was performed by sequencing a cDNA corresponding to the 3' terminal region of the viral genome. The sequence consisted of 195 nucleotides (nt) coding part of the nuclear inclusion body b (NIb) gene, 804 nt of the coat protein (CP) gene, and 328 nt (PVY-OBR) or 326 nt (PVY-NBR) of the 3'-untranslated region (UTR). Translation of the sequence resulted in one single open reading frame with part of the NIb and a CP of 267 amino acids. The two isolates shared 95.1% similarity in the CP amino acid sequence. The CP and the 3'-UTR sequence of the Brazilian isolates were compared to those of other PVY isolates previously reported and unrooted phylogenetic trees were constructed. The trees revealed a separation of two distinct clusters, one comprising most of the common strains and the other comprising the necrotic strains. PVY-OBR was clustered in the common group and PVY-NBR in the necrotic one.

Mosaic in Senna occidentalis in southern Brazil induced by a new strain of Soybean mosaic virus

Plants of Senna occidentalis (sin. Cassia occidentalis) with mosaic symptoms were collected near a soybean (Glycine max) field where some plants exhibited symptoms of mosaic and blistering. A preliminary examination of leaf tissue from diseased S. occidentalis by electron microscopy revealed the presence of pinwheel inclusions as well as long flexuous particles, indicating the presence of a potyvirus. Host range, serology, and amino acid sequence from this potyvirus were similar to those from other Brazilian isolates of Soybean mosaic virus (SMV). The 3'- terminal region of the genomic RNA was cloned and a cDNA sequence of 1.9 kb upstream of the poly (A) tract was determined. The sequence contains a single open reading frame and a 3'- non-translated region (NTR) of 259 bp. The nucleotide sequence of the CP gene of SMV-Soc was 98% identical to that of Brazilian isolates SMV-B, SMV-L, and SMV-FT10. The percentage of nucleotide identity of their 3'-NTR's was 91, 98, and 99% in relation to SMV-L, SMV-B, and SMV-FT10, respectively. In contrast to other Brazilian SMV isolates studied, SMV-Soc was able to infect sunflower (Helianthus annuus). Based on these results, the S. occidentalis isolate was identified as a new strain of SMV belonging to the SMV strain, group G5 and was named SMV-Soc. This is the first report of naturaly occurring SMV infecting plants of S. occidentalis in Brazil, adding this weed as a new source of SMV in the field.

Biological and molecular characterization of an isolate of Tobacco streak virus obtained from soybeans in Brazil

A virus was isolated from soybean (Glycine max) plants with symptoms of dwarfing and bud blight in Wenceslau Braz County, Paraná, Brazil. The host range and properties resembled those of Tobacco streak virus (TSV). The purified virus showed three peaks in a frozen sucrose gradient. Antiserum was produced and the virus was serologically related to TSV. Electron microscopy detected 28 nm spherical particles. Coat protein (CP) had a Mr of 29.880 Da. A fragment of 1028 nt was amplified, cloned and sequenced. One open reading frame with 717 nt was identified and associated to the CP. The CP gene shared 83% identity with the sequence of TSV CP from white clover (Trifolium repens) (GenBank CAA25133). This is the first report of the biological and molecular characterization of TSV isolated from soybeans. It is proposed that this isolate be considered a strain of TSV named TSV-BR.

Sequencing and expression analysis of hepcidin mRNA in donkey (Equus asinus) liver

The hypoferremia that is observed during systemic inflammatory processes is mediated by hepcidin, which is a peptide that is mainly synthesized in the livers of several mammalian species. Hepcidin plays a key role in iron metabolism and in the innate immune system. It's up-regulation is particularly useful during acute inflammation, and it restricts the iron availability that is necessary for the growth of pathogenic microorganisms. In this study, the hepcidin mRNA of Equus asinus has been characterized, and the expression of donkey hepcidin in the liver has been determined. The donkey hepcidin sequence has an open reading frame (ORF) of 261 nucleotides, and the deduced corresponding protein sequence has 86 amino acids. The amino acid sequence of donkey hepcidin was most homologous to Equus caballus (98%). The mature donkey hepcidin sequence (25 amino acids) was 100% homologous to the equine mature hepcidin and has eight conserved cysteine residues that are found in all of the investigated hepcidin sequences. The expression profile of donkey hepcidin in the liver was high and was similar to the reference gene expression. The donkey hepcidin sequence was deposited in GenBankTM (HQ902884) and may be useful for additional studies on iron metabolism and the inflammatory process in this species.

Molecular biology and clinical implication of hepatitis C virus

Hepatitis C virus (HCV) was first described in 1989 as the putative viral agent of non-A non-B hepatitis. It is a member of the Flaviviridae family and has been recognized as the major causative agent of chronic liver disease, including chronic active hepatitis, cirrhosis and hepatocellular carcinoma. HCV is a positive RNA virus with a genome containing approximately 9500 nucleotides. It has an open reading frame that encodes a large polyprotein of about 3000 amino acids and is characterized by extensive genetic diversity. HCV has been classified into at least 6 major genotypes with many subtypes and circulates within an infected individual as a number of closely related but distinct variants known as quasispecies. This article reviews aspects of the molecular biology of HCV and their clinical implication.

Mutations in SRY and WT1 genes required for gonadal development are not responsible for XY partial gonadal dysgenesis

The WT1 transcription factor regulates SRY expression during the initial steps of the sex determination process in humans, activating a gene cascade leading to testis differentiation. In addition to causing Wilms' tumor, mutations in WT1 are often responsible for urogenital defects in men, while SRY mutations are mainly related to 46,XY pure gonadal dysgenesis. In order to evaluate their role in abnormal testicular organogenesis, we screened for SRY and WT1 gene mutations in 10 children with XY partial gonadal dysgenesis, 2 of whom with a history of Wilms' tumor. The open reading frame and 360 bp of the 5' flanking sequence of the SRY gene, and the ten exons and intron boundaries of the WT1 gene were amplified by PCR of genomic DNA. Single-strand conformation polymorphism was initially used for WT1 mutation screening. Since shifts in fragment migration were only observed for intron/exon 4, the ten WT1 exons from all patients were sequenced manually. No mutations were detected in the SRY 5' untranslated region or within SRY open-reading frame sequences. WT1 sequencing revealed one missense mutation (D396N) in the ninth exon of a patient who also had Wilms' tumor. In addition, two silent point mutations were found in the first exon including one described here for the first time. Some non-coding sequence variations were detected, representing one new (IVS4+85A>G) and two already described (-7ATG T>G, IVS9-49 T>C) single nucleotide polymorphisms. Therefore, mutations in two major genes required for gonadal development, SRY and WT1, are not responsible for XY partial gonadal dysgenesis.

Molecular and cellular pathogenesis of autosomal recessive polycystic kidney disease

Autosomal recessive polycystic kidney disease (ARPKD) is an inherited disease characterized by a malformation complex which includes cystically dilated tubules in the kidneys and ductal plate malformation in the liver. The disorder is observed primarily in infancy and childhood, being responsible for significant pediatric morbidity and mortality. All typical forms of ARPKD are caused by mutations in a single gene, PKHD1 (polycystic kidney and hepatic disease 1). This gene has a minimum of 86 exons, assembled into multiple differentially spliced transcripts and has its highest level of expression in kidney, pancreas and liver. Mutational analyses revealed that all patients with both mutations associated with truncation of the longest open reading frame-encoded protein displayed the severe phenotype. This product, polyductin, is a 4,074-amino acid protein expressed in the cytoplasm, plasma membrane and primary apical cilia, a structure that has been implicated in the pathogenesis of different polycystic kidney diseases. In fact, cholangiocytes isolated from an ARPKD rat model develop shorter and dysmorphic cilia, suggesting polyductin to be important for normal ciliary morphology. Polyductin seems also to participate in tubule morphogenesis and cell mitotic orientation along the tubular axis. The recent advances in the understanding of in vitro and animal models of polycystic kidney diseases have shed light on the molecular and cellular mechanisms of cyst formation and progression, allowing the initiation of therapeutic strategy designing and promising perspectives for ARPKD patients. It is notable that vasopressin V2 receptor antagonists can inhibit/halt the renal cystic disease progression in an orthologous rat model of human ARPKD.

Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%), an extended strand (17.13%), a ß turn (5.61%), and a random coil (43.30%). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.

Expression of a hantavirus N protein and its efficacy as antigen in immune assays

Hantavirus cardiopulmonary syndrome (HCPS) has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric β-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.

Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667 - a multifunctional KRAB/C2H2 zinc finger protein

Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C2H2 motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.

Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α

A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

Inovação aberta na gestão pública: análise do plano de ação brasileiro para a Open Government Partnership

Este estudo objetivou analisar o Plano de Ação brasileiro para o Governo Aberto, baseado na teoria da inovação aberta na gestão pública. Utilizou-se de uma pesquisa documental, com vistas a aprofundar o conhecimento do fenômeno em questão. O documento foi escolhido intencionalmente, por ser exemplo basilar das políticas públicas relacionadas à inovação aberta brasileira. Os resultados mostram que os compromissos firmados pelo governo brasileiro estão consoantes com o processo de inovação aberta pública. As ações previstas no Plano estão especificamente relacionadas a transparência, abertura de dados e preparação do corpo estatal para o processo aberto de inovação.

Mid-point for open-ended income category and the effect of equivalence scales on the income-health relationship

To estimate the mid-point of an open-ended income category and to assess the impact of two equivalence scales on income-health associations. Data were obtained from the 2010 Brazilian Oral Health Survey ( Pesquisa Nacional de Saúde Bucal – SBBrasil 2010). Income was converted from categorical to two continuous variables ( per capita and equivalized) for each mid-point. The median mid-point was R$ 14,523.50 and the mean, R$ 24,507.10. When per capita income was applied, 53% of the population were below the poverty line, compared with 15% with equivalized income. The magnitude of income-health associations was similar for continuous income, but categorized equivalized income tended to decrease the strength of association.