bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Spodoptera exigua multicapsid nucleopolyhedrovirus ORF17/18, a homologue of Xestia c-nigrum granulovirus ORF129
Authors: IJkel Wilfred F. J. , Roode Els C. , Goldbach Rob W. , Vlak Just M. , Zuidema Douwe 
Published: August 2015

Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) contains a number of genes with a homologue found so far only in a distantly related baculovirus. One of these, SeMNPV ORF17/18 (Se17/18) shares 55% amino acid similarity to ORF129 of Xestia c-nigrum granulovirus (XcGV). Se17/18 was transcribed in cultured S. exigua 301 cells, as a polyadenylated transcript of 1·1 kb. 5′-RACE analysis demonstrated that Se17/18 transcripts started at 134, 131 and 126 nt upstream of the putative translational start codon. These sites overlap with a baculovirus consensus early promoter motif. Se17/18 transcripts were detected by Northern blot analysis and RT–PCR with increasing abundance from 8 h to 24 h post infection (p.i.) and still present until 72 h p.i. A C-terminal GFP-fusion protein of Se17/18 was primarily localized in the cytoplasm of Se301 and Sf21 cells. A chicken polyclonal antiserum was raised that reacted specifically to Se17/18 protein produced in E. coli. However, no immunoreactive protein was detected in SeMNPV-infected Se301 cells and S. exigua larvae, neither in concentrated BV and ODV preparations. These observations and the inability to detect a C-terminal GFP-fusion protein of Se17/18 in Se301 cells using a GFP antibody suggest that Se17/18 protein is present, if at all, in spurious amounts. Based on the low homology of the Se17/18 protein to (methyl) transferases its possible involvement in transcription regulation is discussed.
Interaction of the Cro repressor with the lysis/lysogeny switch of the Lactobacillus casei temperate bacteriophage A2
Authors: Ladero Victor , García Pilar , Alonso Juan C. , Suárez Juan E. 
Published: August 2015

The transcriptional switch region of Lactobacillus casei temperate bacteriophage A2 contains three similar 20 bp operator subsites, O1, O2 and O3, which are interspersed between the divergent promoters P
R and P
L. The Cro protein binds initially to O3, which overlaps the −35 region of P
L, excluding the RNA polymerase (σA-RNAP) from it. This results in the switching off of cI transcription and directs the incoming phage into the lytic cycle. At higher concentrations, Cro also binds to O1 and/or O2, which overlap P
R, probably introducing a bend in the intervening DNA. This interaction induces DNA looping, which provokes the subsequent displacement of σA-RNAP from P
R. Consequently, Cro abolishes the binding of σA-RNAP to the genetic switch of A2 and, presumably, its own synthesis, contributing indirectly to the entry of phage development into its late stages.
Hepatitis A virus polyprotein processing by Escherichia coli proteases
Authors: Pintó Rosa M. , Guix Susana , González-Dankaart Juan F. , Caballero Santiago , Sánchez Gloria , Guo Ke-Jian , Ribes Enric , Bosch Albert 
Published: August 2015

Hepatitis A virus (HAV) encodes a single polyprotein, which is post-translationally processed. This processing represents an essential step in capsid formation. The virus possesses only one protease, 3C, responsible for all cleavages, except for that at the VP1/2A junction region, which is processed by cellular proteases. In this study, data demonstrates that HAV polyprotein processing by Escherichia coli protease(s) leads to the formation of particulate structures. P3 polyprotein processing in E. coli is not dependent on an active 3C protease: the same processing pattern is observed with wild-type 3C or with several 3C mutants. However, this processing pattern is temperature-dependant, since it differs at 37 or 42 °C. The bacterial protease(s) cleave scissile bonds other than those of HAV; this contributes to the low efficiency of particle formation.
Genomic organization of infectious salmon anaemia virus
Authors: Clouthier Sharon C. , Rector Trent , Brown Nathan E. C. , Anderson Eric D. 
Published: August 2015

The RNA genome segment order, nucleotide sequence and the putative encoded proteins were determined for infectious salmon anaemia virus (ISAV). Eight segments of genomic viral RNA between 1·0 and 2·4 kb in length were identified. RNA segments 1–6 each had a predicted single open reading frame encoding the P1, PB1, NP, P2, P3 and HA proteins, respectively. Segment 7 encoded the P4/P5 proteins and segment 8 encoded the P6/P7 proteins. Seven virion proteins with molecular masses between 25 and 72 kDa were found by SDS–PAGE analysis. The 72 and 42 kDa proteins were immunoreactive with ISAV antiserum from Atlantic salmon. The molecular mass of the 72 kDa virion protein suggested that it was the NP protein encoded by segment 3. The amino acid sequence was conserved, sharing 96·6% identity with the NP protein of a Scottish ISAV isolate. Comparison of the amino acid sequences obtained by N-terminal analyses and cDNA nucleotide translation revealed that the 42 and 47 kDa proteins were the HA and P3 proteins encoded by segments 6 and 5, respectively. In addition, analysis provided evidence for their protein synthesis initiation sites. Like the HA protein, the signal sequence and potential glycosylation sites of P3 suggested that it was a surface glycoprotein. The predicted amino acid sequence shared 83·1, 84·0 and 99·6% identity to the predicted P3 protein sequences for ISAV isolates from Norway, Scotland and Maine, respectively. These results establish the specificity, migration, number and nucleotide sequence of the eight RNA segments of the ISAV genome.
Orf virus encodes a functional dUTPase gene
Authors: Cottone R. , Büttner M. , McInnes C. J. , Wood A. R. , Rziha H.-J. 
Published: August 2015

The present study is the first report on the functional activity of a parapoxvirus-encoded dUTPase. The dUTPase gene of the attenuated orf virus (ORFV), strain D1701, was expressed as a bacterial thioredoxin fusion protein.In vitroassays showed that ORFV dUTPase was highly specific for dUTP as substrate. The enzyme was active over a broad pH range (pH 6·0–9·0), with maximal enzymatic activity at pH 7·0 in the presence of Mg2+cations. Kinetic studies of the recombinant ORFV dUTPase revealed an apparentKmof 4·0 μM, which is more similar to that of the mammalian or African swine fever virus enzyme than to theKmof vaccinia virus dUTPase. Enzyme activity was also found with purified ORFV particles, indicating its virion association.
Non Fc receptor-mediated infection of human macrophages by dengue virus serotype 2
Authors: Moreno-Altamirano M. M. Bertha , Sánchez-García F. Javier , Muñoz M. Lourdes 
Published: August 2015

Four human monocyte-derived macrophage membrane proteins, with apparent molecular masses of 27, 45, 67 and 87 kDa, were identified as possible receptors for dengue virus serotype 2 (DEN-2) (Mexican isolate 200787/1983), based on affinity chromatography, immunofluorescence, virus overlay protein-binding assays and Western blotting. Additionally, mouse polyclonal antibodies raised against each of the four proteins were capable of partially inhibitingin vitroDEN-2 infection of monocyte-macrophages, thus supporting the notion of a role for such proteins as DEN-2 receptors. Parallel studies were carried out using the human promonocytic U-937 cell line, both as undifferentiated cells and as monocyte-like phorbol myristate acetate (PMA)-differentiated cells, as target cells. Whereas interaction between DEN-2 and undifferentiated U-937 cells was almost negligible, PMA-differentiated U-937 cells were shown to harbour putative receptors (with molecular masses of 45 and 67 kDa) for DEN-2, similar to those found in human monocyte-derived macrophages. To our knowledge, this is the first report that describes putative receptors for DEN-2 in primary cultures of human macrophages.
Muscovy duck reovirus σC protein is atypically encoded by the smallest genome segment
Authors: Kuntz-Simon Gaëlle , Le Gall-Reculé Ghislaine , de Boisséson Claire , Jestin Véronique 
Published: August 2015

Although muscovy duck reovirus (DRV) shares properties with the reovirus isolated from chicken, commonly named avian reovirus (ARV), the two virus species are antigenically different. Similar to the DRV σB-encoded gene (1201 bp long) previously identified, the three other double-stranded RNA small genome segments of DRV have been cloned and sequenced. They were 1325, 1191 and 1124 bp long, respectively, and contained conserved terminal sequences common to ARVs. They coded for single expression products, except the smallest (S4), which contained two overlapping open reading frames (ORF1 and ORF2). BLAST analyses revealed that the proteins encoded by the 1325 and 1191 bp genes shared high identity levels with ARV σA and σNS, respectively, and to a lesser extent with other orthoreovirus counterparts. No homology was found for the S4 ORF1-encoded p10 protein. The 29·4 kDa product encoded by S4 ORF2 appeared to be 25% identical to ARV S1 ORF3-encoded σC, a cell-attachment oligomer inducing type-specific neutralizing antibodies. Introduction of large gaps in the N-terminal part of the DRV protein was necessary to improve DRV and ARV σC amino acid sequence alignments. However, a leucine zipper motif was conserved and secondary structure analyses predicted a three-stranded α-helical coiled-coil feature at this amino portion. Thus, despite extensive sequence divergence, DRV σC was suggested to be structurally and probably functionally related to ARV σC. This work provides evidence for the diversity of the polycistronic S class genes of reoviruses isolated from birds and raises the question of the relative classification of DRV in the Orthoreovirus genus.
Nucleotide sequences of segments 1, 3 and 4 of the genome of Bombyx mori cypovirus 1 encoding putative capsid proteins VP1, VP3 and VP4, respectively
Authors: Hagiwara Kyoji , Rao Shujing , Scott Simon W. , Carner Gerald R. 
Published: August 2015

The complete nucleotide sequences of genomic segments S1, S3 and S4 from Bombyx mori cypovirus 1 (BmCPV-1) have been determined. The segments consisted of 4190, 3846 and 3262 nucleotides encoding putative proteins of 1333, 1239 and 1058 amino acids with molecular masses of approximately 148, 140 and 120 kDa (p148, p140 and p120, respectively). All segments possess a single open reading frame. Homology searches showed that all three proteins have homologies to proteins of Rice ragged stunt virus, a member of the genus Oryzavirus within the family Reoviridae. Partial homologies of p140 to structural proteins in other viruses were also found. The predicted molecular masses and the homologies with structural proteins in other viruses lead us to suggest that S1, S3 and S4 encode the capsid proteins VP1, VP3, and VP4, respectively, of BmCPV-1.
Quasispecies in the 5′ untranslated genomic region of bovine viral diarrhoea virus from a single individual
Authors: Jones Leandro Roberto , Zandomeni Rubén , Weber E. Laura 
Published: August 2015

The variability of the 5′ untranslated genomic region (5′UTR) of bovine viral diarrhoea virus (BVDV) RNA obtained from a single individual was analysed. Lung, kidney and spleen tissues from a naturally infected foetus were used as the source of viral RNA. A fragment of 288 bases of the internal ribosome entry site from the BVDV 5′UTR was amplified by RT–PCR using a proofreading DNA polymerase. PCR products were cloned into pGem and, subsequently, transformed intoEscherichia coli. The single-strand conformational polymorphisms of 158 lung-derived clones were analysed; a total of 11 banding patterns was observed. DNAs corresponding to all patterns were sequenced. Of the randomly selected clones, 11 and 10 clones derived from the kidney and spleen, respectively, were also sequenced. All sequences presented differences ranging from 1 to 6 nt substitutions. Analysis of the secondary structure of the variant sequences and comparisons to variant nucleotide sites from the 5′UTR of several BVDV isolates showed that the observed changes were almost free of randomness. Clustering and phylogenetic analyses suggested the existence of low-kinetic variants. BVDV quasispecies may be involved in establishing persistent infections by means of eluding maternal antibodies. The methods described here may be adapted easily both to analyse large numbers of samples from other genomic regions and for the study of BVDV quasispecies evolution in other systems.
Cloning of complete genome sets of six dsRNA viruses using an improved cloning method for large dsRNA genes
Authors: Potgieter A. C. , Steele A. D. , van Dijk A. A. 
Published: August 2015

Cloning full-length large (>3 kb) dsRNA genome segments from small amounts of dsRNA has thus far remained problematic. Here, a single-primer amplification sequence-independent dsRNA cloning procedure was perfected for large genes and tailored for routine use to clone complete genome sets or individual genes. Nine complete viral genome sets were amplified by PCR, namely those of two human rotaviruses, two African horsesickness viruses (AHSV), two equine encephalosis viruses (EEV), one bluetongue virus (BTV), one reovirus and bacteriophage Φ12. Of these amplified genomes, six complete genome sets were cloned for viruses with genes ranging in size from 0·8 to 6·8 kb. Rotavirus dsRNA was extracted directly from stool samples. Co-expressed EEV VP3 and VP7 assembled into core-like particles that have typical orbivirus capsomeres. This work presents the first EEV sequence data and establishes that EEV genes have the same conserved termini (5′ GUU and UAC 3′) and coding assignment as AHSV and BTV. To clone complete genome sets, one-tube reactions were developed for oligo-ligation, cDNA synthesis and PCR amplification. The method is simple and efficient compared to other methods. Complete genomes can be cloned from as little as 1 ng dsRNA and a considerably reduced number of PCR cycles (22–30 cycles compared to 30–35 of other methods). This progress with cloning large dsRNA genes is important for recombinant vaccine development and determination of the role of terminal sequences for replication and gene expression.
Felis domesticus papillomavirus, isolated from a skin lesion, is related to canine oral papillomavirus and contains a 1·3 kb non-coding region between the E2 and L2 open reading frames
Authors: Terai Masanori , Burk Robert D. 
Published: August 2015

We have characterized the complete genome (8300 bp) of an isolate of Felis domesticus papillomavirus (FdPV) from a domestic cat with cutaneous papillomatosis. A BLAST homology search using the nucleotide sequence of the L1 open reading frame demonstrated that the FdPV genome was most closely related to canine oral papillomavirus (COPV). A 384 bp non-coding region (NCR) was found between the end of L1 and the beginning of E6, and a 1·3 kbp NCR was located between the end of E2 and the beginning of L2. Phylogenetic analysis placed FdPV in the E3 clade with COPV. Both viruses contain the atypical second NCR, which has no homology with sequences in existing databases.
Nuclease activities and cell death processes associated with the development of surface cultures of Streptomyces antibioticus ETH 7451
Authors: Fernández Marisol , Sánchez Jesús 
Published: August 2015

The presence and significance of developmentally regulated nucleases in Streptomyces antibioticus ETH 7451 has been studied in relation to the lytic processes occurring during differentiation. The cell-death processes have been followed in surface cultures by a propidium iodide viability assay. This has allowed the visualization of dead (membrane-damaged, red fluorescent) and live (membrane-intact, green fluorescent) mycelium during development, and has facilitated the analysis of the role of nucleases in these processes. A parallel activity-gel analysis showed the appearance of 20–22 kDa, 34 kDa and 44 kDa nucleases, the latter appearing only when aerial mycelium is formed. The appearance of these nucleases shows a remarkable correlation with the death process of the mycelium during differentiation and with chromosomal DNA degradation. The 20–22 kDa enzymes are possibly related to the lytic phenomena taking place in the vegetative substrate mycelium before the emergence of the reproductive aerial mycelium, whereas the function of the 44 kDa nuclease seems to be related to the sporulation step. The 20–22 kDa nucleases require Ca2+ for activity and are inhibited by Zn2+. The nucleases are loosely bound to the cell wall from where they can be liberated by simple washing. Conceivably, these enzymes work together and co-ordinate to achieve an efficient hydrolysis of DNA from dying cells. The results show that the biochemical reactions related with the lytic DNA degradation during the programmed cell death are notably conserved in Streptomyces. Some of the features of the process and the biochemical characteristics of the enzymes involved are analogous to those taking place during the DNA fragmentation processes in eukaryotic apoptotic cells.
A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components
The GenBank accession number for the sequence reported in this paper is AF120104.
Authors: Roos Stefan , Jonsson Hans 
Published: August 2015

A gene from Lactobacillus reuteri 1063 encoding a cell-surface protein, designated Mub, that adheres to mucus components in vitro has been cloned and sequenced. The deduced amino acid sequence of Mub (358 kDa) shows the presence of 14 approximately 200 aa repeats and features typical for other cell-surface proteins of Gram-positive bacteria. Fusion proteins consisting of different repeats of Mub and the maltose-binding protein (MBP) were produced. These proteins adhered to pig mucus components, with molecular masses ranging from <0·1 to >2 MDa, to pig gastric mucin and to hen intestinal mucus. The binding of Mub to mucus components occurred in the pH range 3–7·4, with maximum binding at pH 4–5 and could be partly inhibited by the glycoprotein fetuin. Affinity-purified antibodies against recombinant Mub were used in immunofluorescence microscopy to demonstrate the presence of Mub on the cell surface of strain 1063. By using the antibodies in a Western blot analysis, Mub could also be detected in the growth medium. The results implicate Mub as a cell-surface protein that is involved in Lactobacillus interactions with mucin and in colonization of the digestive tract.
Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of a recA gene fragment
Authors: Waleron Małgorzata , Waleron Krzysztof , Podhajska Anna J , Łojkowska Ewa 
Published: August 2015

Genotypic characterization, based on the analysis of restriction fragment length polymorphism of the recA gene fragment PCR product (recA PCR-RFLP), was performed on members of the former Erwinia genus. PCR primers deduced from published recA gene sequences of Erwinia carotovora allowed the amplification of an approximately 730 bp DNA fragment from each of the 19 Erwinia species tested. Amplified recA fragments were compared using RFLP analysis with four endonucleases (AluI, HinfI, TasI and Tru1I), allowing the detection of characteristic patterns of RFLP products for most of the Erwinia species. Between one and three specific RFLP groups were identified among most of the species tested (Erwinia amylovora, Erwinia ananas, Erwinia cacticida, Erwinia cypripedii, Erwinia herbicola, Erwinia mallotivora, Erwinia milletiae, Erwinia nigrifluens, Erwinia persicina, Erwinia psidii, Erwinia quercina, Erwinia rhapontici, Erwinia rubrifaciens, Erwinia salicis, Erwinia stewartii, Erwinia tracheiphila, Erwinia uredovora, Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. betavasculorum, Erwinia carotovora subsp. odorifera and Erwinia carotovora subsp. wasabiae). However, in two cases, Erwinia chrysanthemi and Erwinia carotovora subsp. carotovora, 15 and 18 specific RFLP groups were detected, respectively. The variability of genetic patterns within these bacteria could be explained in terms of their geographic origin and/or wide host-range. The results indicated that PCR-RFLP analysis of the recA gene fragment is a useful tool for identification of species and subspecies belonging to the former Erwinia genus, as well as for differentiation of strains within E. carotovora subsp. carotovora and E. chrysanthemi.
Leaf traits drive differences in biomass partitioning among major plant functional types
Authors: Duursma Remko A. , Falster Daniel S. 
Published: August 2015

SummaryThe partitioning of biomass into leaves and stems is one of the most uncertain and influential components of global vegetation models (GVMs). Although GVMs typically assume that the major woody plant functional types (PFTs) differ in biomass partitioning, empirical studies have not been able to justify these differences. Here we test for differences between PFTs in partitioning of biomass between leaves and stems.We use the recently published Biomass And Allometry Database (BAAD), a large database including observations for individual plants. The database covers the global climate space, allowing us to test for direct climate effects in addition to PFT.The leaf mass fraction (LMF, leaf / total aboveground biomass) varied strongly between PFTs (as defined by deciduous vs. evergreen and gymnosperm vs. angiosperm). We found that LMF, once corrected for plant height, was proportional to leaf mass per area across PFTs. As a result, the PFTs did not differ in the amount of leaf area supported per unit above ground biomass. We found only weak and inconsistent effects of climate on biomass partitioning.Combined, these results uncover fundamental rules in how plants are constructed and allow for systematic benchmarking of biomass partitioning routines in GVMs.
Systems Biology Approach to Model the Life Cycle ofTrypanosoma cruzi
Authors: Carrea Alejandra , Diambra Luis 
Published: August 2015

AbstractDue to recent advances in reprogramming cell phenotypes, many efforts have been dedicated to developing reverse engineering procedures for the identification of gene regulatory networks that emulate dynamical properties associated with the cell fates of a given biological system. In this work, we propose a systems biology approach for the reconstruction of the gene regulatory network underlying the dynamics of theTrypanosoma cruzi’s life cycle. By means of an optimisation procedure, we embedded the steady state maintenance, and the known phenotypic transitions between these steady states in response to environmental cues, into the dynamics of a gene network model. In the resulting network architecture we identified a small subnetwork, formed by seven interconnected nodes, that controls the parasite’s life cycle. The present approach could be useful for better understanding other single cell organisms with multiple developmental stages.AbbreviationsGRNgene regulatory networkSVDsingular value decompositionTStrans-sialidase