Estrogen-dependent in vitro transcription from the vitellogenin promoter in liver nuclear extracts.

One approach to analyzing the molecular mechanisms of gene expression in vivo is to reconstitute these events in cell-free systems in vitro. Although there is some evidence for tissue-specific transcription in vitro, transcriptionally active extracts that mimic a steroid hormone-dependent enhancement of transcription have not been described. In the study reported here, nuclear extracts of liver from the frog Xenopus laevis were capable of estrogen-dependent induction of a homologous vitellogenin promoter that contained the estrogen-responsive element.

Evaluation of peanut genotypes for resistance to Tomato spotted wilt virus by mechanical and thrips inoculation

The objective of this work was to evaluate the reactions of three peanut breeding lines (IC-10, IC-34, and ICGV 86388) to Tomato spotted wilt virus (TSWV) by mechanical and thrips inoculation, under greenhouse conditions, and compare them to the reactions of cultivars SunOleic, Georgia Green, and the breeding line C11-2-39. TSWV infection by mechanical inoculation was visually assessed using an index ranging from 0 (no symptoms) to 4 (apical death). Enzyme-linked immunosorbent assay was used to confirm TSWV infection from both mechanical and thrips inoculations. IC-10, IC-34, ICGV 86388, and C11-2-39 were more resistant than the cultivars SunOleic and Georgia Green based on mechanical inoculation. Upon thrips inoculation only IC-34 and ICGV-86388 were infected by TSWV, as demonstrated by reverse transcription polymerase chain reaction (RT-PCR), although no symptoms of infection were observed. The peanut breeding lines IC-10, IC-34, and ICGV 86388 show higher level of resistance to TSWV than cultivar Georgia Green considered a standard for TSWV resistance.

Recovery of a persistent Canine distemper virus expressing the enhanced green fluorescent protein from cloned cDNA.

Wild-type A75/17-Canine distemper virus (CDV) is a highly virulent strain, which induces a persistent infection in the central nervous system (CNS) with demyelinating disease. Wild-type A75/17-CDV, which is unable to replicate in cell lines to detectable levels, was adapted to grow in Vero cells and was designated A75/17-V. Sequence comparison between the two genomes revealed seven nucleotide differences located in the phosphoprotein (P), the matrix (M) and the large (L) genes. The P gene is polycistronic and encodes two auxiliary proteins, V and C, besides the P protein. The mutations resulted in amino acid changes in the P and V, but not in the C protein, as well as in the M and L proteins. Here, a rescue system was developed for the A75/17-V strain, which was shown to be attenuated in vivo, but retains a persistent infection phenotype in Vero cells. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein (eGFP) was inserted at the 3' proximal position in the A75/17-V cDNA clone. Reverse genetics technology will allow us to characterize the genetic determinants of A75/17-V CDV persistent infection in cell culture.

Molecular interactions involved in the transactivation of the human T-cell leukemia virus type 1 promoter mediated by Tax and CREB-2 (ATF-4).

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.

Stability of Citrus tristeza virus protective isolates in field conditions

The objective of this work was to monitor the maintenance of Citrus tristeza virus (CTV) protective isolates stability in selected clones of 'Pêra' sweet orange (Citrus sinensis), preimmunized or naturally infected by the virus, after successive clonal propagations. The work was carried out in field conditions in the north of Paraná State, Brazil. Coat protein gene (CPG) analysis of 33 isolates collected from 16 clones of 'Pêra' sweet orange was performed using single strand conformational polymorphism (SSCP). Initially, the isolates were characterized by symptoms of stem pitting observed in clones. Then viral genome was extracted and used as template for the amplification of CPG by reverse transcription polimerase chain reaction (RTPCR). RTPCR products electrophoretic profiles were analyzed using the Jaccard coefficient and the UPGMA method. The majority of the clones had weak to moderate stem pitting symptoms and its CTV isolates showed alterations in the SSCP profiles. However, the stability of the protective complex has been maintained, except for isolates from two analised clones. Low genetic variability was observed within the isolates during the studied years.

The histone-interacting domain of nuclear factor I activates simian virus 40 DNA replication in vivo.

Efficient initiation of SV40 DNA replication requires transcription factors that bind auxiliary sequences flanking the minimally required origin. To evaluate the possibility that transcription factors may activate SV40 replication by acting on the chromatin structure of the origin, we used an in vivo replication system in which we targeted GAL4 fusion proteins to the minimally required origin. We found that the proline-rich transcriptional activation domain of nuclear factor I (NF-I), which has been previously shown to interact with histone H3, specifically activates replication. Evaluation of a series of deletion and point mutants of NF-I indicates that the H3-binding domain and the replication activity coincide perfectly. Assays with other transcription factors, such as Sp1, confirmed the correlation between the interaction with H3 and the activation of replication. These findings imply that transcription factors such as NF-I can activate SV40 replication via direct interaction with chromatin components, thereby contributing to the relief of nucleosomal repression at the SV40 origin.

Reovirus infection activates JNK and the JNK-dependent transcription factor c-Jun.

Viral infection often perturbs host cell signaling pathways including those involving mitogen-activated protein kinases (MAPKs). We now show that reovirus infection results in the selective activation of c-Jun N-terminal kinase (JNK). Reovirus-induced JNK activation is associated with an increase in the phosphorylation of the JNK-dependent transcription factor c-Jun. Reovirus serotype 3 prototype strains Abney (T3A) and Dearing (T3D) induce significantly more JNK activation and c-Jun phosphorylation than does the serotype 1 prototypic strain Lang (T1L). T3D and T3A also induce more apoptosis in infected cells than T1L, and there was a significant correlation between the ability of these viruses to phosphorylate c-Jun and induce apoptosis. However, reovirus-induced apoptosis, but not reovirus-induced c-Jun phosphorylation, is inhibited by blocking TRAIL/receptor binding, suggesting that apoptosis and c-Jun phosphorylation involve parallel rather than identical pathways. Strain-specific differences in JNK activation are determined by the reovirus S1 and M2 gene segments, which encode viral outer capsid proteins (sigma1 and mu1c) involved in receptor binding and host cell membrane penetration. These same gene segments also determine differences in the capacity of reovirus strains to induce apoptosis, and again a significant correlation between the capacity of T1L x T3D reassortant reoviruses to both activate JNK and phosphorylate c-Jun and to induce apoptosis was shown. The extracellular signal-related kinase (ERK) is also activated in a strain-specific manner following reovirus infection. Unlike JNK activation, ERK activation could not be mapped to specific reovirus gene segments, suggesting that ERK activation and JNK activation are triggered by different events during virus-host cell interaction.

Role of mutations in the phosphoprotein on RNA polymerase activity of canine distemper virus following cell culture adaptation

Abstract: The canine distemper virus A75/17 wild-type strain, which is unable to replicate in cell lines, was adapted to growth in Vero cells. Sequence comparison between the A75/17 and the Vero cell-adapted A75/17-V virus revealed 7 amino acid differences between the 2 viruses. Three of these were located in the matrix protein, three in the phosphoprotein also changing the V protein but not the C protein and one in the large protein. The phosphoprotein and the large protein constituted the viral RNA polymerase whose activity was studied by transfection experiments using a reverse genetic system with a plasmid encoding a minireplicon and expression plasmids encoding the nucleocapsid protein and the viral RNA polymerase subunits. Surprinsingly, the enzyme of A75/17 CDV was significantly more active in cell lines compared to the polymerase of A75/17-V CDV. The decrease in overall enzyme activity was found to be due to both decreased replication and transcription activity. This polymerase attenuation was confirmed in CHO cells infection stably expressing the dog SLAM receptor mainly found in dog's lymphoid organs and allowing both virus strains to enter these cells at the same efficiency. A75/17-V CDV replicated more slowly in CHODogSLAM cells than A75/17 CDV and syncytium formation was significantly decreased compared to A75/17 infected CHODogSLAM cells.. Cell culture adaptation lead to an attenuated virus strain both in vitro and in vivo with decreased polymerase activity and syncytium forming capability showing an important role of the polymerase in determining the phenoytpe of the virus. In addition, this reduced phenotype of A75/17-V CDV was shown to be due to the P mutations in the P protein only, showing an important function of the polycistronic P gene in the adaptation process. The role of the matrix protein was found not to have any effect on polymerase activity, however its participation in the adaptation process still needs to be elucidated. The accessory proteins V and C were shown to act on polymerase activity, but their functions in virus pathogenicity and in inhibiting the interferon system have not been studied in this thesis. The V proteins have an activating effect on the polymerase of both the A75/17 and the A75/17-V CDV strains. Although the C protein amino acid sequence was not changed during adaptation of wild-type canine distemper virus in Vero cells, the C protein was demonstrated to have opposite effects on polymerase activity of both virus strains suggesting a different interaction of the C protein with the proteins forming the polymerase complex, which could modulate polymeras activity. These effects were demonstrated by transfection experiments and studying recombinant viruses not expressing the C protein. Thus, the abrogation of the C protein decrease the activity of the wild-type polymerase. In contrast, the polymerase activity of the Vero cell- adapted virus is enhanced in the absence of the C protein and this has also been demonstrated with a recombinant virus, which grew faster in the first 48 hours of infection. Future studies will focus on the generation of recombinant wild-type viruses, which should be very helpful in understanding the molecular mechanisms underlying the adaptation process and the loss of pathogenicity.

Localization and fine structure of a vaccinia virus gene encoding an envelope antigen.

The major antigen on the envelope of extracellular vaccinia virus particles is a polypeptide with an apparent molecular weight of 37,000 (p37K; G. Hiller and K. Weber, J. Virol. 55:651-659, 1985). The gene encoding p37K was mapped in the vaccinia virus genome by hybrid selection of RNA followed by in vitro translation. p37K was then identified among the in vitro translation products by immunoprecipitation with a monoclonal antibody. The gene is located close to the right-hand end of the HindIII F fragment. The corresponding region of the DNA was sequenced, and an open reading frame encoding a polypeptide of 41,748 daltons was observed. The 5' end of the mRNA, as defined by nuclease S1 analysis, maps within only a few nucleotides of the translation initiation codon. Examination of the DNA sequence around the putative initiation site of transcription revealed a characteristic sequence, TAAATG, which includes the ATG translation initiation codon and which is conserved in all but one late gene so far analyzed. It is therefore likely that this sequence is an important regulatory signal for late gene expression in vaccinia virus.

RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

Transgenic passionfruit expressing RNA derived from Cowpea aphid-borne mosaic virus is resistant to passionfruit woodiness disease

Sixteen transgenic yellow passionfruit (Passiflora spp.) plants (R0) were obtained which express a non-translatable transgenic RNA corresponding to the 3' region of the NIb gene and the 5' region of the CP gene, derived from the genome of a Brazilian isolate of Cowpea aphid-borne mosaic virus (CABMV). The transgenic plants were propagated by stem cuttings and challenged by sap inoculation with isolates CABMV-MG1 and CABMV-PE1. One transgenic plant (TE5-10) was resistant to the isolate CABMV-MG1, but susceptible to CABMV-PE1. The remaining transgenic plants developed systemic symptoms, equal to non-transformed plants, when inoculated with either isolate. The absence of virus in TE5-10 plants was confirmed by indirect ELISA. Transcription analysis of the transgene demonstrated that the TE5-10 plant did not accumulate transgenic mRNA, even before inoculation. After inoculation, viral RNA was only detected in plants inoculated with CABMV-PE1. These results confirm that the transgenic plant TE5-10 is resistant to isolate CABMV-MG1, and suggest that the resistance mechanism is post-transcriptional gene silencing, which is already activated in the transgenic plants before virus inoculation.

Detection of rabies virus nucleoprotein-RNA in several organs outside the Central Nervous System in naturally-infected vampire bats

Rabies is a neurological disease, but the rabies virus spread to several organs outside the central nervous system (CNS). The rabies virus antigen or RNA has been identified from the salivary glands, the lungs, the kidneys, the heart and the liver. This work aimed to identify the presence of the rabies virus in non-neuronal organs from naturally-infected vampire bats and to study the rabies virus in the salivary glands of healthy vampire bats. Out of the five bats that were positive for rabies in the CNS, by fluorescent antibody test (FAT), viral isolation in N2A cells and reverse transcription - polymerase chain reaction (RT-PCR), 100% (5/5) were positive for rabies in samples of the tongue and the heart, 80% (4/5) in the kidneys, 40% (2/5) in samples of the salivary glands and the lungs, and 20% (1/5) in the liver by RT-PCR test. All the nine bats that were negative for rabies in the CNS, by FAT, viral isolation and RT-PCR were negative for rabies in the salivary glands by RT-PCR test. Possible consequences for rabies epidemiology and pathogenesis are discussed in this work.

Diagnosis and clinic-pathological findings of influenza virus infection in Brazilian pigs

Influenza A virus (IAV) is a respiratory pathogen of pigs and is associated with the porcine respiratory disease complex (PRDC), along with other respiratory infectious agents. The aim of this study was to diagnose and to perform a clinic-pathological characterization of influenza virus infection in Brazilian pigs. Lung samples from 86 pigs in 37 farrow-to-finish and two farrow-to-feeder operations located in the States of Minas Gerais, São Paulo, Paraná, Rio Grande do Sul, Santa Catarina, and Mato Grosso were studied. Virus detection was performed by virus isolation and quantitative real time reverse-transcription PCR (qRT-PCR). Pathologic examination and immunohistochemistry (IHC) were performed in 60 lung formalin-fixed paraffin-embedded tissue fragments. Affected animals showed coughing, sneezing, nasal discharge, hyperthermia, inactivity, apathy, anorexia, weight loss and growth delay, which lasted for five to 10 days. Influenza virus was isolated from 31 (36.0%) lung samples and 36 (41.9%) were positive for qRT-PCR. Thirty-eight (63.3%) lung samples were positive by IHC and the most frequent microscopic lesion observed was inflammatory infiltrate in the alveoli, bronchiole, or bronchi wall or lumen (76.7%). These results indicate that influenza virus is circulating and causing disease in pigs in several Brazilian states.

Genomic characterization of Brazilian hepatitis C virus genotypes 1a and 1b

Parts of 5' non-coding (5' NC) and of E1 envelope regions of the hepatitis C virus (HCV) genome were amplified from sera of 26 Brazilian anti-HCV antibody-positive patients using the reverse transcription-polymerase chain reaction (RT-PCR). Fourteen samples were PCR positive with primers from the 5' NC region and 8 of them were also positive with primers from the E1 region. A genomic segment of 176 bp from the E1 region of 7 isolates was directly sequenced from PCR products. The sequences were compared with those of HCV strains isolated in other countries and the Brazilian isolates were classified by phylogenetic analysis into genotypes 1a and 1b. This could have a clinical importance since it has been shown that individuals infected with type 1 viruses are less likely to respond to treatment with interferon than individuals infected with types 2 and 3 viruses. Two quasispecies isolated from the same patient with an interval of 13 months differed by two base substitutions (1.1%). The sequence of another isolate presented a three-nucleotide deletion at codon 329

The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV) is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22). Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.