scholarly journals Genomic Characterization of Orf Virus Strain D1701-V (Parapoxvirus) and Development of Novel Sites for Multiple Transgene Expression

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 127 ◽  
Author(s):  
Hanns-Joachim Rziha ◽  
Mathias Büttner ◽  
Melanie Müller ◽  
Ferdinand Salomon ◽  
Alena Reguzova ◽  
...  
Keyword(s):  
Viral Infections ◽  
Blot Hybridization ◽  
Insertion Site ◽  
Vero Cells ◽  
Orf Virus ◽  
Marker Genes ◽  
Promoter Strength ◽  
Fluorescent Marker ◽  
Genomic Changes ◽  
Specific Pcr

The Orf virus (ORFV; Parapoxvirus) strain D1701 with an attenuated phenotype and excellent immunogenic capacity is successfully used for the generation of recombinant vaccines against different viral infections. Adaption for growth in Vero cells was accompanied by additional major genomic changes resulting in ORFV strain variant D1701-V. In this study, restriction enzyme mapping, blot hybridization and DNA sequencing of the deleted region s (A, AT and D) in comparison to the predecessor strain D1701-B revealed the loss of 7 open reading frames (ORF008, ORF101, ORF102, ORF114, ORF115, ORF116, ORF117). The suitability of deletion site D for expression of foreign genes is demonstrated using novel synthetic early promoter eP1 and eP2. Comparison of promoter strength showed that the original vegf-e promoter Pv as well as promoter eP2 display an up to 11-fold stronger expression than promoter eP1, irrespective of the insertion site. Successful integration and expression of the fluorescent marker genes is demonstrated by gene- and insertion-site specific PCR assays, fluorescence microscopy and flow cytometry. For the first time ORFV recombinants are generated simultaneously expressing transgenes in two different insertion loci. That allows production of polyvalent vaccines containing several antigens against one or different pathogens in a single vectored ORFV vaccine.

scholarly journals A Novel Inhibitor of the NF-κB Signaling Pathway Encoded by the Parapoxvirus Orf Virus

2010 ◽  
Vol 84 (8) ◽  
pp. 3962-3973 ◽  
Author(s):  
D. G. Diel ◽  
G. Delhon ◽  
S. Luo ◽  
E. F. Flores ◽  
D. L. Rock
Keyword(s):  
Immune Responses ◽  
Signaling Pathway ◽  
Viral Infections ◽  
Nuclear Translocation ◽  
Orf Virus ◽  
Necrosis Factor Alpha ◽  
Iκb Kinase ◽  
Genes Expression ◽  
Tnf Α ◽  
Factor Alpha

ABSTRACT The parapoxvirus orf virus (ORFV) is a pathogen of sheep and goats that has been used as a preventive and therapeutic immunomodulatory agent in several animal species. However, the functions (genes, proteins, and mechanisms of action) evolved by ORFV to modulate and manipulate immune responses are poorly understood. Here, the novel ORFV protein ORFV024 was shown to inhibit activation of the NF-κB signaling pathway, an important modulator of early immune responses against viral infections. Infection of primary ovine cells with an ORFV024 deletion mutant virus resulted in a marked increase in expression of NF-κB-regulated chemokines and other proinflammatory host genes. Expression of ORFV024 in cell cultures significantly decreased lipopolysaccharide (LPS)- and tumor necrosis factor alpha (TNF-α)-induced NF-κB-responsive reporter gene expression. Further, ORFV024 expression decreased TNF-α-induced phosphorylation and nuclear translocation of NF-κB-p65, phosphorylation, and degradation of IκBα, and phosphorylation of IκB kinase (IKK) subunits IKKα and IKKβ, indicating that ORFV024 functions by inhibiting activation of IKKs, the bottleneck for most NF-κB activating stimuli. Although ORFV024 interferes with activation of the NF-κB signaling pathway, its deletion from the OV-IA82 genome had no significant effect on disease severity, progression, and time to resolution in sheep, indicating that ORFV024 is not essential for virus virulence in the natural host. This represents the first description of a NF-κB inhibitor encoded by a parapoxvirus.

scholarly journals Evolution of Drug Resistance in Experimental Populations of Candida albicans

2000 ◽  
Vol 182 (6) ◽  
pp. 1515-1522 ◽  
Author(s):  
Leah E. Cowen ◽  
Dominique Sanglard ◽  
David Calabrese ◽  
Caroline Sirjusingh ◽  
James B. Anderson ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Marker Genes ◽  
Gene Encoding ◽  
Fluconazole Resistance ◽  
Genomic Changes ◽  
Atp Binding Cassette Transporter ◽  
Experimental Populations ◽  
Major Facilitator ◽  
Different Levels

ABSTRACT Adaptation to inhibitory concentrations of the antifungal agent fluconazole was monitored in replicated experimental populations founded from a single, drug-sensitive cell of the yeast Candida albicans and reared over 330 generations. The concentration of fluconazole was maintained at twice the MIC in six populations; no fluconazole was added to another six populations. All six replicate populations grown with fluconazole adapted to the presence of drug as indicated by an increase in MIC; none of the six populations grown without fluconazole showed any change in MIC. In all populations evolved with drug, increased fluconazole resistance was accompanied by increased resistance to ketoconazole and itraconazole; these populations contained ergosterol in their cell membranes and were amphotericin sensitive. The increase in fluconazole MIC in the six populations evolved with drug followed different trajectories, and these populations achieved different levels of resistance, with distinct overexpression patterns of four genes involved in azole resistance: the ATP-binding cassette transporter genes,CDR1 and CDR2; the gene encoding the target enzyme of the azoles in the ergosterol biosynthetic pathway,ERG11; and the major facilitator gene, MDR1. Selective sweeps in these populations were accompanied by additional genomic changes with no known relationship to drug resistance: loss of heterozygosity in two of the five marker genes assayed and alterations in DNA fingerprints and electrophoretic karyotypes. These results show that chance, in the form of mutations that confer an adaptive advantage, is a determinant in the evolution of azole drug resistance in experimental populations of C. albicans.

scholarly journals A silkworm–baculovirus model for assessing the therapeutic effects of antiviral compounds: characterization and application to the isolation of antivirals from traditional medicines

2008 ◽  
Vol 89 (1) ◽  
pp. 188-194 ◽  
Author(s):  
Yutaka Orihara ◽  
Hiroshi Hamamoto ◽  
Hiroshi Kasuga ◽  
Toru Shimada ◽  
Yasushi Kawaguchi ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Viral Infections ◽  
Antiviral Agents ◽  
Vero Cells ◽  
Infection Model ◽  
Nuclear Magnetic Resonance Analysis ◽  
Therapeutic Effects ◽  
Traditional Medicines ◽  
Baculovirus Infection ◽  
Simplex Virus

Ganciclovir, foscarnet, vidarabine and ribavirin, which are used to treat viral infections in humans, inhibited the proliferation of a baculovirus (Bombyx mori nucleopolyhedrovirus) in BmN4 cells, a cultured silkworm cell line. These antiviral agents inhibited the proliferation of baculovirus in silkworm body fluid and had therapeutic effects. Using the silkworm infection model, the antiviral activity of Kampo medicines was screened and it was found that cinnamon bark, a component of the traditional Japanese medicine Mao-to, had a therapeutic effect. Based on the therapeutic activity, the antiviral substance was purified. Nuclear magnetic resonance analysis of the purified fraction revealed that the antiviral activity was due to cinnzeylanine, which has previously been isolated from Cinnamomum zeylanicum. Cinnzeylanine inhibits the proliferation of herpes simplex virus type 1 in Vero cells. These results suggest that the silkworm–baculovirus infection model is useful for screening antiviral agents that are effective for treating humans infected with DNA viruses.

scholarly journals Diversity of Active Viral Infections within the Sphagnum Microbiome

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Joshua M. A. Stough ◽  
Max Kolton ◽  
Joel E. Kostka ◽  
David J. Weston ◽  
Dale A. Pelletier ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Viral Infections ◽  
Model Systems ◽  
Microbial Consortia ◽  
Soil Conditions ◽  
Marker Genes ◽  
Ecological Value ◽  
Metabolic Potential ◽  
Content Type ◽  
Virus Diversity

ABSTRACT Sphagnum-dominated peatlands play an important role in global carbon storage and represent significant sources of economic and ecological value. While recent efforts to describe microbial diversity and metabolic potential of the Sphagnum microbiome have demonstrated the importance of its microbial community, little is known about the viral constituents. We used metatranscriptomics to describe the diversity and activity of viruses infecting microbes within the Sphagnum peat bog. The vegetative portions of six Sphagnum plants were obtained from a peatland in northern Minnesota, and the total RNA was extracted and sequenced. Metatranscriptomes were assembled and contigs were screened for the presence of conserved virus marker genes. Using bacteriophage capsid protein gp23 as a marker for phage diversity, we identified 33 contigs representing undocumented phages that were active in the community at the time of sampling. Similarly, RNA-dependent RNA polymerase and the nucleocytoplasmic large DNA virus (NCLDV) major capsid protein were used as markers for single-stranded RNA (ssRNA) viruses and NCLDV, respectively. In total, 114 contigs were identified as originating from undescribed ssRNA viruses, 22 of which represent nearly complete genomes. An additional 64 contigs were identified as being from NCLDVs. Finally, 7 contigs were identified as putative virophage or polinton-like viruses. We developed co-occurrence networks with these markers in relation to the expression of potential-host housekeeping gene rpb1 to predict virus-host relationships, identifying 13 groups. Together, our approach offers new tools for the identification of virus diversity and interactions in understudied clades and suggests that viruses may play a considerable role in the ecology of the Sphagnum microbiome. IMPORTANCE Sphagnum-dominated peatlands play an important role in maintaining atmospheric carbon dioxide levels by modifying conditions in the surrounding soil to favor the growth of Sphagnum over that of other plant species. This lowers the rate of decomposition and facilitates the accumulation of fixed carbon in the form of partially decomposed biomass. The unique environment produced by Sphagnum enriches for the growth of a diverse microbial consortia that benefit from and support the moss's growth, while also maintaining the hostile soil conditions. While a growing body of research has begun to characterize the microbial groups that colonize Sphagnum, little is currently known about the ecological factors that constrain community structure and define ecosystem function. Top-down population control by viruses is almost completely undescribed. This study provides insight into the significant viral influence on the Sphagnum microbiome and identifies new potential model systems to study virus-host interactions in the peatland ecosystem.

scholarly journals 088 Microprojectile Bombardment-mediated Transformation of Rhododendron `Catawbiense Album' L.

HortScience ◽  
1999 ◽  
Vol 34 (3) ◽  
pp. 456D-456
Author(s):  
Jane E. Knapp ◽  
Mark H. Brand
Keyword(s):  
Microprojectile Bombardment ◽  
Blot Hybridization ◽  
Shoot Proliferation ◽  
Southern Blot Hybridization ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Selective Agent ◽  
Putative Transformants ◽  
High Level

Horticultural improvements in Rhododendron require long periods of time to produce flowering plants by traditional breeding methods. In addition, new trait development by conventional genetics is limited to existing germplasm. Genetic engineering approaches to horticultural improvement offer the possibility for introduction of new traits using foreign DNA from any source. To this end, we have developed a system for the genetic transformation of Rhododendron based on microprojectile bombardment. Leaves from in vitro-grown plantlets of R. `Catawbiense Album' L. were bombarded with the marker genes uidA (GUS) in combination with nptII or hph. Two days post-bombardment, explants were transferred to shoot iniation medium containing either 50 mg/L kanamycin or 2.5 mg/L hygromycin. After 4 weeks, proliferating tissues were transferred to media containing increased levels of selective agent (100 mg/L kanamycin or 5 mg/L hygromycin, respectively). Shoots that regenerated were then excised from necrotic tissues and transferred to shoot proliferation medium containing the high level of selective agent. PCR analysis of putative transformants revealed the presence of the transgenes. Southern blot hybridization confirmed stable transgene integration. Histochemical GUS assays of transformed tissues indicated uniform expression throughout the transgenic plant. With the development of an efficient transformation system, the introduction of genes to confer useful horticultural traits becomes feasible.

scholarly journals Virucidal Activity of Gold Nanoparticles Synthesized by Green Chemistry Using Garlic Extract

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1111 ◽  
Author(s):  
Mayra A. Meléndez-Villanueva ◽  
Karla Morán-Santibañez ◽  
Juan J. Martínez-Sanmiguel ◽  
Raúl Rangel-López ◽  
Marco A. Garza-Navarro ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Antiviral Activity ◽  
Measles Virus ◽  
Viral Infections ◽  
Vero Cells ◽  
Garlic Extract ◽  
World Health ◽  
Viral Particles ◽  
Virucidal Effect ◽  
Health Organization

Measles virus (MeV) is a paramyxovirus that infects humans, principally children. Despite the existence of an effective and safe vaccine, the number of cases of measles has increased due to lack of vaccination coverage. The World Health Organization (WHO) reports that the number of cases worldwide multiplied fourfold between January and March 2019, to 112,000. Today, there is no treatment available for MeV. In recent years, it has been demonstrated that natural extracts (herbal or algal) with antiviral activity can also work as reducing agents that, in combination with nanotechnology, offer an innovative option to counteract viral infections. Here, we synthetized and evaluated the antiviral activity of gold nanoparticles using garlic extract (Allium sativa) as a reducing agent (AuNPs-As). These nanoparticles actively inhibited MeV replication in Vero cells at a 50% effective concentration (EC50) of 8.829 µg/mL, and the selectivity index (SI) obtained was 16.05. AuNPs-As likely inhibit viral infection by blocking viral particles directly, showing a potent virucidal effect. Gold nanoparticles may be useful as a promising strategy for treating and controlling the infection of MeV and other related enveloped viruses.

Pandemic strains of O3:K6Vibrio parahaemolyticusin the aquatic environment of Bangladesh

2004 ◽  
Vol 50 (10) ◽  
pp. 827-834 ◽  
Author(s):  
M Sirajul Islam ◽  
Rizwana Tasmin ◽  
Sirajul Islam Khan ◽  
Habibul Bari Mahmud Bakht ◽  
Zahid Hayat Mahmood ◽  
...  
Keyword(s):  
Aquatic Environment ◽  
Vibrio Parahaemolyticus ◽  
Blot Hybridization ◽  
Virulence Gene ◽  
Gene Probe ◽  
Chromosomal Dna ◽  
Specific Pcr ◽  
Pandemic Strains ◽  
Positive Results ◽  
Tdh Gene

A total of 1500 environmental strains of Vibrio parahaemolyticus, isolated from the aquatic environment of Bangladesh, were screened for the presence of a major V. parahaemolyticus virulence factor, the thermostable direct haemolysin (tdh) gene, by the colony blot hybridization method using a digoxigenin-labeled tdh gene probe. Of 1500 strains, 5 carried the tdh sequence, which was further confirmed by PCR using primers specific for the tdh gene. Examination by PCR confirmed that the 5 strains were V. parahamolyticus and lacked the thermostable direct haemolysin-related haemolysin (trh) gene, the alternative major virulence gene known to be absent in pandemic strains. All 5 strains gave positive Kanagawa phenomenon reaction with characteristic β-haemolysis on Wagatsuma agar medium. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated, in all 5 strains, the presence of 2 tdh genes common to strains positive for Kanagawa phenomenon. However, the 5 strains were found to belong to 3 different serotypes (O3:K29, O4:K37, and O3:K6). The 2 with pandemic serotype O3:K6 gave positive results in group-specific PCR and ORF8 PCR assays, characteristics unique to the pandemic clone. Clonal variations among the 5 isolates were analyzed by comparing RAPD and ribotyping patterns. Results showed different patterns for the 3 serotypes, but the pattern was identical among the O3:K6 strains. This is the first report on the isolation of pandemic O3:K6 strains of V. parahaemolyticus from the aquatic environment of Bangladesh.Key words: pandemic strains, Vibrio parahaemolyticus, aquatic environment.

scholarly journals Polyandrous Mexican Fruit Flies: Second Male Paternity and Biological Attributes of Transgenic Strains

Insects ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 5
Author(s):  
Betzabé Verónica-Murrieta ◽  
José Salvador Meza ◽  
Martha Lucía Baena ◽  
Gerardo Alvarado-Castillo ◽  
Diana Pérez-Staples
Keyword(s):  
Sterile Insect Technique ◽  
Mass Rearing ◽  
Larval Survival ◽  
Anastrepha Ludens ◽  
Marker Genes ◽  
Fluorescent Marker ◽  
Significant Difference ◽  
Lower Ability ◽  
Biological Attributes ◽  
Transgenic Strains

Anastrepha ludens (Diptera: Tephritidae), is a damaging agricultural pest. Currently, the Sterile Insect Technique (SIT) is used as part of its control. The SIT consists of the mass-rearing, sterilization, and release of insects in target areas. Sterile males mate with wild females, and prevent them from laying fertile eggs. However, even if females mate with sterile males, they can then remate with a second male. If this second male is wild, then this could reduce the efficiency of the SIT by producing viable offspring. The amount of progeny produced by second males (P2 values) for A. ludens is unknown. Here, we evaluated the biological attributes, mating competitiveness, and the proportion of male paternity gained by the second male, using strains that carry fluorescent marker genes and can be potentially used to develop transgenic sexing strains. Furthermore, the transgenic strains were irradiated, to test their ability to induce sterility in females. We found that the 443-G strain had significantly higher larval survival than the 419-R strain. No significant difference was found between the two strains in their mating probability with wild females. We found P2 values between 67 and 74% for the 419-R and the 443-G strain, respectively. Second male sperm precedence only decreased slightly after 12 days, suggesting that sperm from the first and second male is not mixing with time, but rather the second male’s sperm prevails. Furthermore, sterile 443-G males induced significantly higher sterility in females than sterile males from the 419-R strain. The apparent lower ability of the 443-G strain to inhibit female remating should be further investigated. Knowledge of the pre and postcopulatory performance of transgenic strains will help in understanding their potential for control.

scholarly journals Evaluation of a Herbicide-resistant Trait Conferred by the Bar Gene Driven by Four Distinct Promoters in Transgenic Blueberry Plants

2008 ◽  
Vol 133 (4) ◽  
pp. 605-611 ◽  
Author(s):  
Guo-Qing Song ◽  
Kenneth C. Sink ◽  
Peter W. Callow ◽  
Rebecca Baughan ◽  
James F. Hancock
Keyword(s):  
Transgenic Plants ◽  
Herbicide Resistance ◽  
Single Copy ◽  
Vaccinium Corymbosum ◽  
Cauliflower Mosaic Virus ◽  
Leaf Damage ◽  
Marker Genes ◽  
Promoter Strength ◽  
Herbicide Resistant ◽  
Selectable Marker Genes

Four chimeric bialaphos resistance (bar) genes driven by different promoters were evaluated for production of herbicide-resistant ‘Legacy’ blueberry plants (73.4% Vaccinium corymbosum L. and 25% Vaccinium darrowi Camp) through Agrobacterium tumefaciens (Smith & Towns.) Conn.-mediated transformation. When the bars were used as selectable marker genes, different promoters yielded different transformation frequencies. Three chimeric bar genes with the promoter nopaline synthase (nos), cauliflower mosaic virus (CaMV) 35S, or CaMV 34S yielded transgenic plants, whereas a synthetic (Aocs)3AmasPmas promoter did not lead to successful regeneration of transgenic plants. In addition, herbicide resistance in bar-expressing plants was influenced by the promoter strength. Under controlled environmental conditions, 3-month-old plants from six single-copy transgenic events with 35S∷bar or nos∷bar, as well as those nontransgenic plants, were sprayed with herbicide glufosinate ammonium (GS) at five levels (0, 750, 1500, 3000, and 6000 mg·L−1). Evaluations on leaf damage 2 weeks after spraying indicated that all transgenic plants exhibited much higher herbicide resistance than nontransgenic plants. Additionally, the transgenic plants with the 35S∷bar showed a higher herbicide resistance than those with the nos∷bar. After application of 6000 mg·L−1 GS, over 90% of the leaves from plants with the 35S∷bar and 19.5% to 51.5% of the leaves from plants with the nos∷bar showed no symptom of herbicide damage, whereas only 5% of leaves from the nontransgenic had no damage. One-year-old, field-grown plants from four transgenic events with the nos∷bar were evaluated for herbicide resistance after spraying with 750 mg·L−1 GS. Transgenic plants survived with variations in the level of foliar damage; in contrast, all nontransgenic plants died. This study is the first investigation of different promoters for engineering transgenic blueberry plants.

scholarly journals Diversity of active viral infections within the Sphagnum microbiome

2018 ◽  
Author(s):  
Joshua M.A. Stough ◽  
Max Kolton ◽  
Joel E. Kostka ◽  
David J. Weston ◽  
Dale A. Pelletier ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Viral Infections ◽  
Housekeeping Gene ◽  
Model Systems ◽  
Microbial Consortia ◽  
Soil Conditions ◽  
Marker Genes ◽  
Ecological Value ◽  
Metabolic Potential ◽  
Virus Diversity

AbstractSphagnum-dominated peatlands play an important role in global carbon storage and represent significant sources of economic and ecological value. While recent efforts to describe microbial diversity and metabolic potential of the Sphagnum microbiome have demonstrated the importance of its microbial community, little is known about the viral constituents. We used metatranscriptomics to describe the diversity and activity of viruses infecting microbes within the Sphagnum peat bog. The vegetative portions of 6 Sphagnum plants were obtained from a peatland in northern Minnesota and total RNA extracted and sequenced. Metatranscriptomes were assembled and contigs screened for the presence of conserved virus marker genes. Using bacteriophage capsid protein, gp23, as a marker for phage diversity, we identified 33 contigs representing undocumented phage s that were active in the community at the time of sampling. Similarly, RNA-dependent RNA polymerase and the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) major capsid protein were used as markers for ssRNA viruses and NCLDV, respectively. In total 114 contigs were identified as originating from undescribed ssRNA viruses, 22 of which represent near-complete genomes. An additional 64 contigs were identified as being from NCLDVs. Finally, 7 contigs were identified as putative virophage or polinto-like viruses. We developed co-occurrence networks with these markers in relation to the expression of potential-host housekeeping gene rpb1 to predict virus-host relationships, identifying 13 groups. Together, our approach offers new tools for the identification of virus diversity and interactions in understudied clades, and suggest viruses may play a considerable role in the ecology of the Sphagnum microbiome.SignificanceSphagnum-dominated peatlands play an important role in maintaining atmospheric carbon dioxide levels by modifying conditions in the surrounding soil to favor its own growth over other plant species. This slows rates of decomposition and facilitates the accumulation of fixed carbon in the form of partially decomposed biomass. The unique environment produced by Sphagnum enriches for the growth of a diverse microbial consortia that benefit from and support the moss’s growth, while also maintaining the hostile soil conditions. While a growing body of research has begun to characterize the microbial groups that colonize Sphagnum, little is currently known about the ecological factors that constrain community structure and define ecosystem function. Top-down population control by viruses is almost completely undescribed. This study provides insight into the significant viral influence on the Sphagnum microbiome, and identifying new potential model systems to study virus-host interactions in the peatland ecosystem.

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