Metagenomic analysis of nepoviruses: diversity, evolution and identification of a genome region in members of subgroup A that appears to be important for host range.

Data mining and metagenomic analysis of 277 open reading frame sequences of bipartite RNA viruses of the genus Nepovirus, family Secoviridae, were performed, documenting how challenging it can be to unequivocally assign a virus to a particular species, especially those in subgroups A and C, based on some of the currently adopted taxonomic demarcation criteria. This work suggests a possible need for their amendment to accommodate pangenome information. In addition, we revealed a host-dependent structure of arabis mosaic virus (ArMV) populations at a cladistic level and confirmed a phylogeographic structure of grapevine fanleaf virus (GFLV) populations. We also identified new putative recombination events in members of subgroups A, B and C. The evolutionary specificity of some capsid regions of ArMV and GFLV that were described previously and biologically validated as determinants of nematode transmission was circumscribed in silico. Furthermore, a C-terminal segment of the RNA-dependent RNA polymerase of members of subgroup A was predicted to be a putative host range determinant based on statistically supported higher π (substitutions per site) values for GFLV and ArMV isolates infecting Vitis spp. compared with non-Vitis-infecting ArMV isolates. This study illustrates how sequence information obtained via high-throughput sequencing can increase our understanding of mechanisms that modulate virus diversity and evolution and create new opportunities for advancing studies on the biology of economically important plant viruses.

Genetic structure and molecular variability of Grapevine fanleaf virus populations.

To gain insights into the evolutionary mechanisms of Grapevine fanleaf virus (GFLV) from the genus Nepovirus, family Secoviridae, the sequences of the complete coding region of RNA2, including genes 2A(HP), 2B(MP) and 2C(CP), and partial sequence from the RNA1-encoded gene 1E(Pol) of 14 GFLV isolates from three naturally infected California vineyards were characterized. Phylogenetic analyses suggested two to three evolutionarily divergent lineages that did not reflect the vineyard origin of the isolates or an association with rootstock genotype or scion cultivar. Examination of the genetic variability of the California isolates alongside isolates worldwide, for which three RNA1 and 44 RNA2 coding sequences are available, revealed similar patterns of molecular evolution for the different regions within the GFLV genome but distinct selection constraints with the strongest pressure exerted on genes 2C(CP) and 2B(MP), an intermediate level of pressure exerted on gene 1E(Pol), and the weakest pressure exerted on gene 2A(HP). Some of the California isolates resulted from interspecies recombination events between GFLV and Arabis mosaic virus with crossover sites suspected in gene 1E(Pol) and identified in genes 2A(HP) and 2B(MP); and intraspecies recombination events inferred in the four target genes but most frequently observed within gene 2C(CP). This study suggested that purifying selection and recombination are important evolutionary mechanisms in the genetic diversification of GFLV.

Multiple interspecies recombination events within RNA2 of Grapevine fanleaf virus and Arabis mosaic virus.

Sequence alignments and SISCAN analyses inferred multiple interspecies recombination events within RNA2 of strains GHu of Grapevine fanleaf virus (GFLV) and Ta of Arabis mosaic virus (ArMV), two closely related subgroup A nepoviruses in the family Comoviridae. Interspecies recombination events were identified in the 5' untranslated region, the putative homing protein and movement protein genes but not in the coat protein gene and 3' untranslated region. These findings suggest a dynamic relationship between GFLV and ArMV, and a differential selection pressure on RNA2-encoded proteins with constraints in terms of function and co-adaptation that limit interspecies recombination to certain gene segments.

Toward an operational dynamic model for tertiary nitrification by submerged biofiltration.

This work deals with the methodology put in place to fit and validate the parameters of a biofiltration model (BAF) in tertiary nitrification treatment and dynamic conditions. For an average loading rate of 0.65 kg NH4-N/m(3) media/d, different time loading rates are applied inside a filtration-backwash run using a semi-industrial pilot. Comparisons between predicted and observed values on the NH4-N, NO3-N and TSS in treated water and the total head loss deltaP are carried out firstly using default values of BAF parameters. Model predictions overestimate values measured but trends are well reproduced. A sensitivity analysis is carried out and the hierarchy of BAF parameters has been set up classifying them into strong and low influence on the effluent concentrations. Among parameters revealing the strongest influence are those of the filtration module and the mean density of biofilm for the TSS effluent and the total AP, the specific autotrophic growth rate, the maximum biofilm thickness and the reduction coefficient of diffusivity in the biofilm for the NH4-N, NO3-N effluent. Finally, this classification leads to setting a calibration procedure, thanks to specific experimental tests directly measuring some BAF parameters.

Characterization of a naturally occurring recombinant isolate of Grapevine fanleaf virus.

The naturally occurring Grapevine fanleaf virus (GFLV) recombinant isolate A17b was recovered from its grapevine host by sap inoculation and serial passages onto Gomphrena globosa, a pseudo local lesion herbaceous host, and Chenopodium quinoa, a systemic herbaceous host, to characterize some of its biological properties. Sequence analysis of the CP gene, in which a recombinational event was previously detected, demonstrated the genetic stability of recombinant isolate A17b over a 5-year period in its natural host as well as in C. quinoa. Also, recombinant isolate A17b was graft transmissible, as shown by an in vitro heterologous approach, and transmitted by the nematode Xiphinema index as readily as nonrecombinant GFLV isolates. Furthermore, despite a lower pathogenicity on Chenopodium amaranticolor, recombinant isolate A17b had a similar host range and induced similar symptoms in type and severity to nonrecombinant GFLV isolates. Interestingly, the use of infectious chimeric RNA2 transcripts in combination to RNA1 transcripts of GFLV strain F13 suggested no implication of the recombination event in the CP gene of isolate A17b in the reduced pathogenicity on C. amaranticolor. Altogether, recombinant isolate A17b had similar biological properties to GFLV nonrecombinant isolates.

Genetic manipulations of adenovirus type 5 fiber resulting in liver tropism attenuation.

The development of genetically modified adenoviral vectors capable of specifically transducing a given cell population requires the addition and functional presentation of particular tropism determinants within the virus capsid, together with the abrogation of the molecular determinants that dictate their natural tropism in vivo. The human adenovirus serotype 5 (Ad5) first attaches to the cell surface following high-affinity binding of the C-terminal knob of the fiber capsid protein to the coxsackie and adenovirus receptor (CAR). Here we have assessed whether genetic shortening of the fiber shaft (virus BS1), or replacing the Ad5 fiber shaft and knob with their Ad3 counterparts (virus DB6), could cripple this interaction in vitro and in vivo. A 10-fold decrease in the binding of the modified capsids to soluble CAR was evidenced, which correlated with a similar reduction of their ability to transduce CAR-positive cells in vitro. The ability of BS1 to interact with cellular integrins was also impaired, suggesting that the penton base and the short-shafted fiber when embedded in the capsid preclude each other from efficiently interacting with their cognate cell surface receptors (CAR and integrins respectively). BS1 and DB6 intravenous injections in mice further supported a profound impairment of the ability of the capsid-modified viruses to transduce the liver as demonstrated by a 10-fold reduction of intracellular viral DNA and transgene expression. Interestingly enough, the host humoral response was also specifically weakened in BS1- and DB6-inoculated animals. Taken together, these observations indicate that (i) fiber shortening and (ii) pseudo-typing of Ad5-based vectors with the shaft and knob from non-CAR-binding serotypes constitute two promising strategies to successfully attenuate their native tropism in vitro and most importantly in vivo.

Degenerated pIX-IVa2 adenoviral vector sequences lowers reacquisition of the E1 genes during virus amplification in 293 cells.

A critical issue for E1-deleted adenoviral vectors manufactured from 293 cells is the emergence of replication-competent adenovirus (RCA). These contaminants arise through homologous recombination between identical sequences framing the E1 locus displayed by 293 cells, and the vector backbones. Modified recombinogenic sequences (syngen) were thus introduced within the vector backbone, and virus viability and RCA emergence were assessed. Syngen#1 is a synthetic sequence displaying silent point mutations in the pIX and IVa2 coding regions. A side by side comparison of Ad5CMV/p53 (E1-deleted adenovirus expressing the p53 tumor suppressor gene) and AVdeltaE1#1CMV/p53 (with syngen#1 in place of wild-type sequences) demonstrated a normal productivity for the modified construct. The altered sequences did not impair p53-mediated apoptosis in a model tumor cell line. Most importantly, a statistically significant decrease in terms of RCA occurrence could also be demonstrated. Degenerescence of the recombinogenic sequences could be further accentuated by modifying noncoding pIX region (syngen #2), with no effect on virus productivity and stability. We concluded that these vector modifications constitute a feasible strategy to reduce RCA emergence during amplification in 293 cells. This approach could also be applied to decrease reincorporation of the E1 genes during amplification of deltaE1deltaE4 vectors in 293/E4-trans-complementing cells.

RGD inclusion in the hexon monomer provides adenovirus type 5-based vectors with a fiber knob-independent pathway for infection.

Hypervariable region 5 (HVR5) is a hydrophilic, serotypically nonconserved loop of the hexon monomer which extrudes from the adenovirus (Ad) capsid. We have replaced the HVR5 sequence of Ad5 with that of heterologous peptides and studied their effects on virus viability and peptide accessibility. A poliovirus model epitope was first inserted in a series of nine "isogenic" viruses that differed in their flanking spacers. Whereas virus productivity was not profoundly altered by any of these modifications, immunoprecipitation experiments under nondenaturing conditions demonstrated that epitope recognition by its cognate monoclonal antibody (C3 MAb) was strongly linker dependent and correlated perfectly with the ability of C3 MAb to inhibit transgene delivery and expression. An alphav-specific ligand (DCRGDCF) was then inserted in a suitable linker context to investigate whether hexon-modified capsids would enhance the transduction of cells displaying limiting amounts of the virus attachment receptors. Interestingly, although hexon has never been implicated in Ad entry, the modified virus significantly increased the transduction of human vascular smooth muscle cells in vitro. Competition experiments with 293 cells saturated with recombinant knob further indicated that the hexon-modified virus could use an additional, knob-independent pathway for entry. We concluded that genetic engineering of the Ad5 hexon monomer constitutes a novel and feasible approach to equip the virus with additional targeting ligands.

Therapeutic benefit of ciliary neurotrophic factor in progressive motor neuronopathy depends on the route of delivery.

Ciliary neurotrophic factor (CNTF) has demonstrated therapeutic effects in several mouse mutants with motoneuronal degeneration. However, the poor bioavailability and toxic side effects of recombinant CNTF protein have complicated its use in patients with amyotrophic lateral sclerosis. CNTF gene transfer strategies were developed but faced the question of whether CNTF should be delivered to motoneuron cell bodies or to their axons or muscle targets. To address this issue, we have used an adenoviral vector (AdCNTF) coding for a secretable form of CNTF and compared different routes of its administration in the mouse mutant progressive motor neuronopathy (pmn). Intramuscular, intravenous, and intracerebroventricular injections of AdCNTF or the control vector AdlacZ resulted in transgene expression in skeletal muscle fibers, hepatocytes, and ependymal cells, respectively, as determined by histochemistry and reverse transcription-polymerase chain reaction. AdCNTF intramuscularly treated and intravenously treated pmn mice showed a 25% increase in mean life span and a reduced degeneration of phrenic myelinated nerve fibers, which correlated with elevated CNTF serum bioactivities. In contrast, intracerebroventricular AdCNTF administration did not affect the mean life span or motor axonal degeneration of pmn mice. The differential efficacy of peripheral and central CNTF vector administrations might be of interest for future studies in human motor neuron diseases.

Adenovirus-mediated transfer of the neurotrophin-3 gene into skeletal muscle of pmn mice: therapeutic effects and mechanisms of action.

Several neurotrophic factors (CNTF, BDNF, IGF-1) have been suggested for the treatment of motor neuron diseases. In ALS patients, however, the repeated subcutaneous injection of these factors as recombinant proteins is complicated by their toxicity or poor bioavailability. We have constructed an adenovirus vector coding for neurotrophin-3 (AdNT-3) allowing for stable and/or targeted delivery of NT-3 to motoneurons. The intramuscular administration of this vector was tested in the mouse mutant pmn (progressive motor neuronopathy). AdNT-3-treated pmn mice showed prolonged lifespan, improved neuromuscular function, reduced motor axonal degeneration and efficient reinnervation of muscle fibres. NT-3 protein and also adenovirus vectors, when injected into muscle, can be transported by motoneurons via retrograde axonal transport to their cell bodies in the spinal cord. Using ELISA and RT-PCR analyses in muscle, spinal cord and serum of AdNT-3-treated pmn mice, we have investigated the contribution of these processes to the observed therapeutic effects. Our results suggest that most if not all therapeutic benefit was due to the continuous systemic liberation of adenoviral NT-3. Therefore, viral gene therapy vectors auch as adenoviruses, AAVs, lentiviruses and new types of gene transfer not based on viral vectors that allow for efficient in vivo liberation of neurotrophic factors have potential for the future treatment of human motor neuron diseases.

Phenotypic alteration of astrocytes induced by ciliary neurotrophic factor in the intact adult brain, As revealed by adenovirus-mediated gene transfer.

Synthesis of the ciliary neurotrophic factor (CNTF) and its specific receptor (CNTFRalpha) is widespread in the intact CNS, but potential biological roles for this system remain elusive. Contradictory results have been obtained concerning a possible effect on the morphological and biochemical phenotype of astrocytes. To reassess this question, we have taken advantage of adenovirus-mediated gene transfer into the rat brain to obtain the local release of CNTF. Stereotaxic administration of CNTF recombinant adenovirus vectors into the striatum led to phenotypic changes in astrocytes located in regions that were related axonally to striatal neurons at the injection site. Astrocytes appeared hypertrophied and displayed an increase in both GFAP and CNTF immunoreactivity. This response was observed up to 5 weeks after injection, the longest time studied. It was not observed after the administration of a control vector. The methodology used in the present study, allowing us to analyze the effect of the factor in areas remote from the injection site, has provided conclusive evidence that CNTF affects the astroglial phenotype in the intact CNS. The characteristics of these effects may explain why contradictory results have been obtained previously, because this signaling system seems to have a low efficiency and therefore requires a high local concentration of the factor close to the target cells. One might speculate as to the involvement of a CNTF astroglio-astroglial signaling system in the organized response of a population of astrocytes to changes in CNS homeostasis detected locally, even by a single cell.

Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses.

We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity. Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression. A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice. The immune response to the virally encoded beta-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver. Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CpG dinucleotides. However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter may be critical to maintain transgene expression from these attenuated adenovirus vectors.

Gene therapy of murine motor neuron disease using adenoviral vectors for neurotrophic factors.

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy cause progressive paralysis, often leading to premature death. Neurotrophic factors have been suggested as therapeutic agents for motor neuron diseases, but their clinical use as injected recombinant protein was limited by toxicity and/or poor bioavailability. We demonstrate here that adenovirus-mediated gene transfer of neurotrophin-3 (NT-3) can produce substantial therapeutic effects in the mouse mutant pmn (progressive motor neuronopathy). After intramuscular injection of the NT-3 adenoviral vector, pmn mice showed a 50% increase in life span, reduced loss of motor axons and improved neuromuscular function as assessed by electromyography. These results were further improved by coinjecting an adenoviral vector coding for ciliary neurotrophic factor. Therefore, adenovirus-mediated gene transfer of neurotrophic factors offers new prospects for the treatment of motor neuron diseases.

Recombinational construction in Escherichia coli of infectious adenoviral genomes.

A two-step gene replacement procedure was developed that generates infectious adenoviral genomes through homologous recombination in Escherichia coli. As a prerequisite, a human adenovirus serotype 5 (Ad5)-derived genome was first introduced as a PacI restriction fragment into an incP-derived replicon which, in contrast to ColE1-derivatives (e.g., pBR322 or pUC plasmids), is functional in a polA mutant of E. coli. Any modification can be introduced at will following two consecutive homologous recombinations between the incP/Ad5 replicon and the ColE1 plasmid. The overall procedure requires only the in vitro engineering of the ColE1-derivative by flanking the desired modification with small stretches of identical sequences. In the first step, a cointegrate between the tetracycline-resistant incP/Ad5 replicon and the kanamycin-resistant ColE1-derivative is selected by growing the polA host in the presence of both antibiotics. Resolution of this cointegrate is further selected in sucrose growth conditions due to the loss of a conditional suicide marker (the sacB gene of Bacillus subtilis) present in the ColE1 plasmid, leading to unmodified and modified incP/Ad5 replicons that can be differentiated upon restriction analysis. Consecutive rounds of this two-step cloning procedure allowed the introduction of multiple independent modifications within the virus genome, with no requirement for an intermediate virus. The potential of this procedure is demonstrated by the recovery of several E1E3E4-deleted adenoviruses following transfection of the corresponding E. coli-derived genomes in IGRP2 cells.

Restoration of hexosaminidase A activity in human Tay-Sachs fibroblasts via adenoviral vector-mediated gene transfer.

Tay-Sachs disease (TSD) is a lysosomal storage disease due to hexosaminidase A deficiency caused by mutations in the gene for alpha-chain (Hex alpha). A human Hex alpha cDNA was subcloned into the adenoviral plasmid pAdRSV. Hex alpha. Replication-deficient adenovirus was generated by homologous recombination in 293 cells. Human fibroblasts from a patient suffering from TSD were infected with the recombinant adenovirus. TSD fibroblasts expressing the recombinant alpha-chain had an enzyme activity on the natural substrate ranging from 40 to 84% of the normal. The corrected cells secreted up to 25 times more Hex alpha than control fibroblasts. The Hex alpha encoded by the adenovirus was shown to be correctly transported into the lysosomes and to normalize the impaired degradation of GM2 ganglioside in TSD fibroblasts.

Rapid regulation of a cyclic AMP-specific phosphodiesterase (PDE IV) by forskolin and isoproterenol in LRM55 astroglial cells.

Elevation of intracellular cyclic AMP (cAMP) levels by incubation of intact LRM55 astroglial cells with 0.1 mM forskolin or 0.1 microM isoproterenol (IPR) caused a rapid increase in soluble cAMP phospho-diesterase (PDE) activity. Activation did not require de novo protein synthesis and reached a maximum of > or = 100% increase over basal PDE activity after 15 min of treatment. The increase in activity was recovered in a single peak (peak 3) following DEAE chromatography; the other two peaks separated by this procedure showed no change. Peak 3 had all the characteristics of PDE IV: it was sensitive to rolipram, was insensitive to CI-930 and cyclic GMP (cGMP), had a high affinity for cAMP (K(m) approximately equal to 4 microM), and had a very low affinity for cGMP (K(m) > 100 microM). Forskolin treatment resulted in an increase of the Vmax of peak 3 without affecting its K(m). In vitro treatment of peak 3 with the catalytic subunit of protein kinase A increased activity, whereas treatment with alkaline phosphatase decreased activity. The rapid activation of this specific PDE in response to forskolin and IPR represents a novel regulation of PDE IV by a mechanism that seems to involve its phosphorylation by a cAMP-dependent protein kinase.

Direct intracerebral nerve growth factor gene transfer using a recombinant adenovirus: effect on basal forebrain cholinergic neurons during aging.

Gene therapy in the nervous system offers an attractive strategy for the administration of therapeutic factors in a potentially region-specific, sustained, and well-tolerated manner. We tested the trophic effect of a recombinant adenovirus encoding nerve growth factor (AdNGF) in vivo on basal forebrain cholinergic neurons of aged rats, a neuronal population affected during normal and pathological aging. Three weeks after unilateral injection of the recombinant adenovirus into the nucleus basalis magnocellularis, a significant increase in the somal areas of cholinergic neurons ipsilateral to the injection was observed. No increase was detected in animals receiving a recombinant adenovirus carrying the Escherichia coli Lac Z reporter gene. Injected animals did not lose weight, an adverse effect usually described after intracerebroventricular infusion of NGF, and no tissue loss or massive local inflammatory response was observed around injection sites. Thus, a single intracerebral injection of AdNGF produces trophic effects similar to those resulting from chronic intracerebroventricular high levels of NGF. These findings indicate that recombinant adenoviruses encoding growth factors are potentially powerful tools for improving neuronal deficits associated with degenerative processes.

Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit.

Transgene expression after the administration of recombinant adenovirus with E1 deleted is constantly transient. It is admitted that E1A-substituting activities of cellular or viral origin allow viral antigen synthesis and trigger cytotoxic lymphocyte-mediated clearance of the recipient cells. Our approach to solving this problem relies on the additional deletion of the E4 region from the vector backbone as this region upregulates viral gene expression at both transcriptional and posttranscriptional levels. As a prerequisite to the construction of E1 E4 doubly defective adenoviruses, we investigated the possibility of transcomplementing both functions within a single cell. In particular, the distal ORF6+ORF7 segment from the E4 locus of adenovirus type 5 was cloned under the control of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Following transfection into 293 cells, clone IGRP2 was retained and characterized as it can rescue the growth defect of all E1+ E4- adenoviral deletants tested. DNA and RNA analysis experiments verified that the mouse mammary tumor virus promoter drives the expression of the ORF6+ORF7 unit and permits its bona fide alternative splicing, generating ORF6/7 mRNA in addition to the ORF6-expressing primary transcript. Importantly, IGRP2 cells sustain cell confluence for a period longer than that of 293 parental cells and allow the plaque purification of E1- or E4- defective viruses. The dual expression of E1 and E4 regulatory genes within IGRP2 cells is demonstrated by the construction, plaque purification, and helper-free propagation of recombinant lacZ-encoding doubly defective adenoviruses harboring different E4 deletions. In addition, the emergence, if any, of replicative particles during viral propagation in this novel packaging cell line will be drastically impaired as only a limited segment of E4 has been integrated.

Transplantation to the rat brain of human neural progenitors that were genetically modified using adenoviruses.

Transplantations for neurological disorders are limited by the supply of human fetal tissue. To generate larger numbers of cells of appropriate phenotype, we investigated whether human neural progenitors expanded in vitro could be modified with recombinant adenoviruses. Strong expression of beta-galactosidase was obtained in vitro. Two or three weeks after transplantation of engineered cells to the rat brain, we observed a small percentage of surviving neuroblasts strongly expressing beta-galactosidase in four out of 13 rats. Thus human precursor cells that have been genetically modified using adenoviruses are a promising tool for ex vivo gene therapy of neurodegenerative diseases.