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PubMed2003

Pathotypical and genotypical characterization of strains of Newcastle disease virus isolated from outbreaks in chicken and goose flocks in some regions of China during 1985-2001.

Twenty-nine strains of Newcastle disease virus (NDV) isolated from outbreaks in chicken and goose flocks in several regions of China during 1985-2001 were characterized pathotypically and genotypically. All except one of these strains were velogenic. For genotyping, phylogenetic tree analysis (nt 47-420), restriction site mapping (nt 334-1682) and residue substitution analysis (residues 4-124) of the F gene were performed using sequences of our isolates and sequences of selected NDV strains from GenBank. The results revealed that most of these newly characterized strains belonged to six novel genetic groups that were designated as VIf, VIg, VIIc, VIId, VIIe and IX. The genotype IX viruses, to which the China challenge strain F48E8 used for vaccine evaluation belonged, were found only in China and still induced sporadic infections in certain areas. Isolates belonging to group VIf and VIg were distinct from previously reported members of genotype VI, with genetic distance from 2.5 to 12.1%. Subgenotype VIIc, VIId and VIIe viruses, which were distributed in clusters in the phylogenetic tree distinct from members of subgenotypes VIIa and VIIb, were responsible for disease outbreaks in chicken and goose flocks and circulated predominantly in southern China in recent years. Finally, cross-protective testing showed that specific-pathogen free (SPF) chickens vaccinated with La Sota vaccines can be fully protected against challenge by strains from genetic groups VIb, VIg, VIId and IX, indicating that the antigenic differences between strains of various genotypes are insufficient to change the cross-protection conferred by the commonly used vaccine.

Amino Acid Sequence↗
PubMed2003

Cell cycle arrest induction by an adenoviral vector expressing HIV-1 Vpr in bovine and feline cells.

An accessory protein, Vpr, of human immunodeficiency virus type 1 (HIV-1) induces the cell cycle G(2)/M arrest in primate cells, but not in rodent cells, suggesting that a species-specific factor might be involved in the phenomenon. To study whether Vpr can cause G(2)/M arrest in non-primate cells, a novel adenoviral vector, Ad-VIG, co-expressing HIV-1 Vpr and green fluorescent protein (GFP) was constructed and infected on cell lines derived from various mammalian species. With its ability to express GFP, Ad-VIG enabled flow cytometric evaluation of transduction efficiency in the infected cells, and Western blot analysis showed successful expression of Vpr in the vector-transduced cells. Upon Ad-VIG infection, human HeLa, African green monkey Vero, feline CRFK, and bovine MDBK cells manifested cell cycle G(2)/M arrest. This is the first study showing that non-primate feline and bovine cells are susceptible to Vpr-induced cell cycle arrest.

Adenoviridae↗
PubMed2004

Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination.

Infectious haematopoietic necrosis virus (IHNV) is a well-studied virus of salmonid fishes. A highly efficacious DNA vaccine has been developed against this virus and studies have demonstrated that this vaccine induces both an early and transient non-specific anti-viral phase as well as long-term specific protection. The mechanisms of the early anti-viral phase are not known, but previous studies noted changes in Mx gene expression, suggesting a role for type I interferon. This study used quantitative real-time reverse transcriptase PCR methodology to compare expression changes over time of a number of cytokine or cytokine-related genes in the spleen of rainbow trout following injection with poly I:C, live IHNV, the IHNV DNA vaccine or a control plasmid encoding the non-antigenic luciferase gene. The target genes included Mx-1, viral haemorrhagic septicaemia virus induced gene 8 (Vig-8), TNF-alpha1, TNF-alpha2, IL-1beta1, IL-8, TGF-beta1 and Hsp70. Poly I:C stimulation induced several genes but the strongest and significant response was observed in the Mx-1 and Vig-8 genes. The live IHN virus induced a significant response in all genes examined except TGF-beta1. The control plasmid construct and the IHNV DNA vaccine marginally induced a number of genes, but the main difference between these two groups was a statistically significant induction of the Mx-1 and Vig-8 genes by the IHNV vaccine only. The gene expression profiles elicited by the live virus and the IHNV DNA vaccine differed in a number of aspects but this study confirms the clear role for a type I interferon-like response in early anti-viral defence.

Animals↗
PubMed2006

Vaccinia immune globulin: current policies, preparedness, and product safety and efficacy.

In 1980 the World Health Organization declared that smallpox was eradicated from the world, and routine smallpox vaccination was discontinued. Nevertheless, samples of the smallpox virus (variola virus) were retained for research purposes, not least because of fears that terrorist groups or rogue states might also have kept samples in order to develop a bioweapon. Variola virus represents an effective bioweapon because it is associated with high morbidity and mortality and is highly contagious. Since September 11, 2001, countries around the world have begun to develop policies and preparedness programs to deal with a bioterror attack, including stockpiling of smallpox vaccine. Smallpox vaccine itself may be associated with a number of serious adverse events, which can often be managed with vaccinia immune globulin (VIG). VIG may also be needed as prophylaxis in patients for whom pre-exposure smallpox vaccine is contraindicated (such as those with eczema or pregnant women), although it is currently not licensed in these cases. Two intravenous formulations of VIG (VIGIV Cangene and VIGIV Dynport) have been licensed by the FDA for the management of patients with progressive vaccinia, eczema vaccinatum, severe generalized vaccinia, and extensive body surface involvement or periocular implantation following inadvertent inoculation.

Bioterrorism↗
PubMed2005

Identification and preliminary characterization of vaccinia virus (Dryvax) antigens recognized by vaccinia immune globulin.

Using vaccinia immune globulin (VIG), a high-titer antibody preparation from immunized subjects, we demonstrate that the humoral immune response in humans is directed against numerous antigens in the Dryvax vaccine strain. Western blot and immunoprecipitation analyses revealed highly antigenic proteins associated with both the extracellular enveloped virus and intracellular mature virus forms. The modified vaccinia virus Ankara (MVA), a new generation smallpox vaccine that is attenuated for replication in humans, expresses most, but not all, of the major vaccinia antigens recognized by antibodies in VIG, lacking the highly antigenic protein corresponding to the A-type inclusion body protein. Since new-generation smallpox vaccines such as MVA will require extensive comparison to traditional smallpox vaccines in animal models of immunogenicity and protection, we compared the vaccinia virus antigens recognized by VIG to those recognized by sera from Dryvax and MVA immunized mice. The humoral immune response in immunized mice is qualitatively similar to that in humans.

Animals↗
PubMed2001

Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing.

Virus vectors carrying host-derived sequence inserts induce silencing of the corresponding genes in infected plants. This virus-induced gene silencing (VIGS) is a manifestation of an RNA-mediated defence mechanism that is related to post-transcriptional gene silencing (PTGS) in transgenic plants. Here we describe an infectious cDNA clone of tobacco rattle virus (TRV) that has been modified to facilitate insertion of non-viral sequence and subsequent infection to plants. We show that this vector mediates VIGS of endogenous genes in the absence of virus-induced symptoms. Unlike other RNA virus vectors that have been used previously for VIGS, the TRV construct is able to target host RNAs in the growing points of plants. These features indicate that the TRV vector will have wide application for gene discovery in plants.

Base Sequence↗
PubMed2002

Barley stripe mosaic virus-induced gene silencing in a monocot plant.

RNA silencing of endogenous plant genes can be achieved by virus-mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus-induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive-sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the gammab gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo-bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo-bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild-type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo-bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology-dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA-silencing mechanisms in monocot plant species.

Base Sequence↗
PubMed2005

Inactivation of organellar glutamyl- and seryl-tRNA synthetases leads to developmental arrest of chloroplasts and mitochondria in higher plants.

Aminoacyl-tRNA synthetases (ARSs) are key enzymes involved in protein translation, and both cytosolic and organellar forms are present in the genomes of eukaryotes. In this study, we investigated cellular effects of depletion of organellar forms of ARS using virus-induced gene silencing (VIGS) in Nicotiana benthamiana. VIGS of NbERS and NbSRS, which encode organellar GluRS and SerRS, respectively, resulted in a severe leaf-yellowing phenotype. The NbERS and NbSRS genes were ubiquitously expressed in plant tissues, and induced in response to light. Green fluorescent protein (GFP) fusion proteins of the full-length glutamyl-tRNA synthetase (ERS) and seryl-tRNA synthetase (SRS) of Arabidopsis and GFP fusions to the N-terminal extension of these proteins were all dualtargeted to chloroplasts and mitochondria. At the cell level, depletion of NbERS and NbSRS resulted in dramatically reduced numbers of chloroplasts with reduced sizes and chlorophyll content. The numbers and/or physiology of mitochondria were also severely affected. The abnormal chloroplasts lacked most of the thylakoid membranes and appeared to be degenerating, whereas some of them showed doublet morphology, indicating defective chloroplast division. Pulse-field gel electrophoresis analyses demonstrated that chloroplast DNA in subgenomic sizes is the predominant form in the abnormal chloroplasts. Interestingly, despite severe abnormalities in chloroplasts and mitochondria, expression of many nuclear genes encoding chloroplastor mitochondria-targeted proteins, and chlorophyll biosynthesis genes remained unchanged in the ERS and SRS VIGS lines. This is the first report to analyze the effect of ARS disruption on organelle development in plants.

Arabidopsis↗
PubMed2003

Effect of caffeine on target detection and rifle marksmanship.

Thirteen healthy and rifle-trained male military reservists performed shooting sessions on two separate occasions 1 h following the ingestion of placebo or 300 mg of caffeine. Shooting included both friend-foe (FF) and vigilance (VIG) tasks, and were performed in the following order: two FF sequences (4 min each), four VIG sequences (30 min each), and two additional FF sequences. The shooting sessions lasted approximately 2.5 h under outdoor conditions (air temperature range from - 3 to 14 degrees C) and were held 48 h apart in a counter-balanced order. Performance measures during the shooting session included engagement time, friend-foe discrimination, and marksmanship accuracy and precision. Assessments of thermal comfort, tiredness, and debilitating symptoms preceded and followed the shooting session, while a self-assessment on performance was administered post-shooting only. Blood was sampled immediately prior to the beginning of the shooting session and was used to determine plasma caffeine, cortisol, and testosterone levels. Engagement times were faster and certain measures of accuracy and precision were impaired during the later FF and VIG sequences. However, caffeine ingestion had no affect upon any of the marksmanship measures, although it did alleviate cold stress and tiredness. That caffeine ingestion did not affect target detection and rifle marksmanship is a finding that differs from other studies, and is explained by a beneficial arousal caused by the mild level of cold stress experienced by the participants.

Adult↗
PubMed2003

Development of a novel vaccinia-neutralization assay based on reporter-gene expression.

In anticipation of large-scale smallpox vaccination, clinical trials of new vaccine candidates with improved safety profiles, and new vaccinia immune globulin (VIG) products, there is an immediate need to develop new assays to measure vaccinia-specific immune responses. The classical assay to measure vaccinia neutralization, the plaque-reduction neutralization test (PRNT), is slow, labor intensive, and difficult to validate and transfer. Here we describe the development of a novel vaccinia-neutralization assay based on the expression of a reporter gene, beta-galactosidase (beta-Gal). Using a previously constructed vaccinia-beta-Gal recombinant virus, vSC56, we developed a neutralization assay that is rapid, sensitive, and reproducible. The readout is automated. We show that the neutralizing titers, ID(50), for several VIG products measured by our assay were similar to those obtained by PRNTs. A new Food and Drug Administration VIG standard was established for distribution to other laboratories. The new assay will serve as an important tool both for preclinical and clinical trials of new smallpox vaccines and for evaluation of therapeutic agents to treat vaccine-associated adverse reactions.

Animals↗
PubMed2003

Virus-induced gene silencing of jasmonate-induced direct defences, nicotine and trypsin proteinase-inhibitors in Nicotiana attenuata.

Research into the molecular basis of plant-insect interactions is hampered by the inability to alter the expression of individual genes in plants growing under natural conditions. The ability of virus-induced gene silencing (VIGS) to silence the expression of two jasmonate-induced genes known to mediate the expression of two potent direct defences (nicotine and proteinase inhibitors) that are produced in different tissues (roots and shoots, respectively) in Nicotiana attenuata is documented here. Fragments of consensus sequences of N. attenuata's putrescine N-methyltransferase (PMT) and trypsin inhibitor (TI) genes were cloned in sense, anti-sense and inverted repeat orientations into the Tobacco Rattle Virus (TRV) to trigger post-transcriptional gene silencing by Agrobacterium-mediated inoculation in plants previously elicited with methyl jasmonate (MeJA) or left as controls. MeJA treatment elicited 2.4- and 9.8-fold increases in the concentrations of nicotine and proteinase inhibitors, respectively, and inoculation with constructs containing appropriate genes inhibited these MeJA-induced increases and halved constitutive accumulations, regardless of the orientation of the gene fragment. Root PMT transcript levels were significantly elevated in MeJA-treated plants 10 h after elicitation, but not in plants inoculated with the appropriate TRV constructs 9 d prior to MeJA treatment, demonstrating that VIGS was responsible for the inhibition of these potent direct defences. While additional research is required to minimize the effects on plant growth and the risks of using such constructs in natural settings, it is concluded that VIGS has a potential to manipulate the expression of genes important for ecological interactions.

Base Sequence↗
PubMed2006

CDNA-AFLP combined with functional analysis reveals novel genes involved in the hypersensitive response.

To identify genes required for the hypersensitive response (HR), we performed expression profiling of tomato plants mounting a synchronized HR, followed by functional analysis of differentially expressed genes. By cDNA-AFLP analysis, the expression profile of tomato plants containing both the Cf-4 resistance gene against Cladosporium fulvum and the matching Avr4 avirulence gene of this fungus was compared with that of control plants. About 1% of the transcript-derived fragments (442 out of 50,000) were derived from a differentially expressed gene. Based on their sequence and expression, 192 fragments, referred to as Avr4-responsive tomato (ART) fragments, were selected for VIGS (virus-induced gene silencing) in Cf-4-transgenic Nicotiana benthamiana. Inoculated plants were analyzed for compromised HR by agroinfiltration of either the C. fulvum Avr4 gene or the Inf1 gene of Phytophthora infestans, which invokes a HR in wild-type N. benthamiana. VIGS using 15 of the ART fragments resulted in a compromised HR, whereas VIGS with fragments of ART genes encoding HSP90, a nuclear GTPase, an L19 ribosomal protein, and most interestingly, a nucleotide binding-leucine rich repeat (NB-LRR)-type protein severely suppressed the HR induced both by Avr4 and Inf1. Requirement of an NB-LRR protein (designated NRC1, for NB-LRR protein required for HR-associated cell death 1) for Cf resistance protein function as well as Inf1-mediated HR suggests a convergence of signaling pathways and supports the recent observation that NB-LRR proteins play a role in signal transduction cascades downstream of resistance proteins.

Algal Proteins↗
PubMed2005

An investigation of the therapeutic value of vaccinia-immune IgG in a mouse pneumonia model.

Vaccinia-immune globulin (VIG) was used to treat severe complications of smallpox vaccination, but its use was controversial because it resolved disease in only some clinical cases. VIG is a pool of hyperimmune sera collected from individuals with a high neutralizing titre against the intracellular mature form (IMV) of vaccinia virus (VACV), but activity against the extracellular enveloped form (EEV) was often not considered. Here, the efficacy of anti-VACV antibodies (Abs) in protecting mice from intranasal infection with the VACV strain Western Reserve (WR) was evaluated. Mice were immunized passively with hyperimmune rabbit Abs (IgG) generated against inactivated IMV or produced following infection by VACV; subsequently, animals were challenged with VACV WR. The results demonstrated that: (i) good protection requires Abs to EEV in addition to IMV; (ii) Abs were effective when given before or up to 4 days after infection; and (iii) protection of mice from VACV WR correlated with a reduction of virus replication in lungs, but not in brain. In agreement with studies conducted before smallpox was eradicated and recent studies using EEV antigens for immunization, this study reiterates the importance of anti-EEV Abs in protecting against orthopoxvirus infection and illustrates the need to evaluate both anti-IMV and anti-EEV neutralizing Abs in VIG.

Animals↗
PubMed2005

Involvement of PPS3 phosphorylated by elicitor-responsive mitogen-activated protein kinases in the regulation of plant cell death.

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant innate immunity. Overexpression of StMEK1(DD), a constitutively active MAPK kinase that activates salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), provokes hypersensitive response-like cell death in Nicotiana benthamiana. Here we purified a 51-kD MAPK, which was activated in potato (Solanum tuberosum) tubers treated with hyphal wall elicitor of a plant pathogen, and isolated the cDNA designated StMPK1. The deduced amino acid sequence of the StMPK1 showed strong similarity to stress-responsive MAPKs, such as tobacco (Nicotiana tabacum) SIPK and Arabidopsis (Arabidopsis thaliana) AtMPK6. To investigate the downstream signaling of StMPK1, we identified several proteins phosphorylated by StMPK1 (PPSs) using an in vitro expression cloning method. To dissect the biological function of PPSs in the plant defense, we employed virus-induced gene silencing (VIGS) in N. benthamiana. VIGS of NbPPS3 significantly delayed cell death induced by the transient expression of StMEK1(DD) and treatment with hyphal wall elicitor. Furthermore, the mobility shift of NbPPS3 on SDS-polyacrylamide gel was induced by transient expression of StMEK1(DD). The mobility shift of NbPPS3 induced by StMEK1(DD) was not compromised by VIGS of WIPK or SIPK alone, but drastically reduced by the silencing of both WIPK and SIPK. This work strongly supports the idea that PPS3 is a physiological substrate of StMPK1 and is involved in cell death activated by a MAPK cascade.

Amino Acid Sequence↗
PubMed2006

Silencing of a germin-like gene in Nicotiana attenuata improves performance of native herbivores.

Germins and germin-like proteins (GLPs) are known to function in pathogen resistance, but their involvement in defense against insect herbivores is poorly understood. In the native tobacco Nicotiana attenuata, attack from the specialist herbivore Manduca sexta or elicitation by adding larval oral secretions (OS) to wounds up-regulates transcripts of a GLP. To understand the function of this gene, which occurs as a single copy, we cloned the full-length NaGLP and silenced its expression in N. attenuata by expressing a 250-bp fragment in an antisense orientation with an Agrobacterium-based transformation system and by virus-induced gene silencing (VIGS). Homozygous lines harboring a single insert and VIGS plants had significantly reduced constitutive (measured in roots) and elicited NaGLP transcript levels (in leaves). Silencing NaGLP improved M. sexta larval performance and Tupiocoris notatus preference, two native herbivores of N. attenuata. Silencing NaGLP also attenuated the OS-induced hydrogen peroxide (H(2)O(2)), diterpene glycosides, and trypsin proteinase inhibitor responses, which may explain the observed susceptibility of antisense or VIGS plants to herbivore attack and increased nicotine contents, but did not influence the OS-elicited jasmonate and salicylate bursts, or the release of the volatile organic compounds (limonene, cis-alpha-bergamotene, and germacrene-A) that function as an indirect defense. This suggests that NaGLP is involved in H(2)O(2) production and might also be related to ethylene production and/or perception, which in turn influences the defense responses of N. attenuata via H(2)O(2) and ethylene-signaling pathways.

Acetates↗
PubMed2005

Virus-induced gene silencing in tomato fruit.

Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants. Here we report that either by syringe-infiltrating the tobacco rattle virus (TRV)-vector into the surface, stem or carpopodium of a tomato fruit attached to the plant or by vacuum-infiltrating into a tomato fruit detached from the plant, TRV can efficiently spread and replicate in the tomato fruit. Although VIGS can be performed in tomato fruit by all of the means mentioned above, the most effective method is to inject the TRV-vector into the carpopodium of young fruit attached to the plant about 10 days after pollination. Several reporter genes related to ethylene responses and fruit ripening, including LeCTR1 and LeEILs genes, were also successfully silenced by this method during fruit development. In addition, we found that the silencing of the LeEIN2 gene results in the suppression of tomato fruit ripening. The results of our study indicate that the application of VIGS techniques by the described methods can be successfully applied to tomato fruit and is a valuable tool for studying functions of the relevant genes during fruit developing.

Fruit↗
PubMed2006

Prohibitin is involved in mitochondrial biogenesis in plants.

Prohibitin, which consists of two subunits PHB1 and PHB2, plays a role in cell-cycle progression, senescence, apoptosis, and maintenance of mitochondrial function in mammals and yeast. In this study, we examined the role of prohibitins in plants by using virus-induced gene silencing (VIGS) of two prohibitin subunit genes of Nicotiana benthamiana, designated NbPHB1 and NbPHB2. NbPHB1 and NbPHB2 were targeted to the mitochondria, and their gene expression was suppressed during senescence. VIGS of NbPHB2 caused severe growth inhibition, leaf yellowing and symptoms of cell death, whereas VIGS of NbPHB1 resulted in a milder phenotype. At the cellular level, depletion of these subunits affected mitochondria by severely reducing their number and/or mass, and by causing morphological and physiological abnormalities. Suppression of prohibitin function resulted in a 10- to 20-fold higher production of reactive oxygen species and induced premature leaf senescence. Finally, disruption of prohibitin function rendered the plants more susceptible to various oxidative stress-inducing reagents, including H(2)O(2), paraquat, antimycin A and salicylic acid. These results suggest that prohibitins play a crucial role in mitochondrial biogenesis and protection against stress and senescence in plant cells.

DNA, Complementary↗
PubMed1994

Vigabatrin and lamotrigine in refractory epilepsy.

Epilepsy arises from an imbalance of inhibitory and excitatory influences in the brain. Vigabatrin (VIG) decreases the breakdown of the inhibitory neurotransmitter gamma-aminobutyric acid, whereas lamotrigine (LTG) reduces presynaptic excitatory amino acid release. 22 patients with refractory epilepsy, treated with an anticonvulsant regimen containing VIG, entered a balanced, double blind, placebo controlled, crossover trial of additional LTG. Treatment periods of 12 weeks (25 mg, 50 mg, 100 mg LTG twice daily for four weeks at each dose, and matched placebo) were followed by wash out intervals of four weeks. 14 of the 20 patients completing the study improved, resulting in a significant fall in seizure days and numbers. Analysis of seizure type confirmed a beneficial effect on partial and secondary generalised tonic-clonic seizures. At the highest LTG dose (200 mg daily) there was a median fall of 37% in seizure count with nine (45%) patients reporting > 50% reduction. Three of these patients were seizure free during this month of treatment. Side effects were minimal throughout the study. Concentrations of other antiepileptic drugs, including those of carbamazepine 10,11-epoxide, were not modified by LTG. This study suggests a substantial efficacy for a regimen containing VIG and LTG. Combinations of drugs with complementary modes of action may provide a rational pharmacological approach to the management of refractory epilepsy.

Anticonvulsants↗