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PubMed2004

From lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza.

Over the past eight years, cases of human infection with highly pathogenic avian influenza viruses have raised international concern that we could be on the brink of a global influenza pandemic. Many of these human infections have proved fatal and if the viruses had been able to transmit efficiently from person to person, the effects would have been devastating. How can we arm ourselves against this pandemic threat when these viruses are too dangerous to use in conventional vaccine production? Recent technological developments (reverse genetics) have allowed us to manipulate the influenza virus genome so that we can construct safe, high-yielding vaccine strains. However, the transition of reverse-genetic technologies from the research laboratory to the manufacturing environment has presented new challenges for vaccine manufacturers as well as veterinary and public health authorities.

Disease Outbreaks↗
PubMed2005

TILLING moves beyond functional genomics into crop improvement.

Transgenic methods have been successfully applied to trait improvement in a number of crops. However, reverse genetics studies by transgenic means are not practical in many commercially important crops, hampering investigations into gene function and the development of novel and improved cultivars. A nontransgenic method for reverse genetics called Targeting Induced Local Lesions IN Genomes (TILLING) has been developed as a method for inducing and identifying novel genetic variation, and has been demonstrated in the model plant, Arabidopsis thaliana. Recently, TILLING has been extended to the improvement of crop plants and shows great promise as a general method for both functional genomics and modulation of key traits in diverse crops.

Agriculture↗
PubMed2000

Target selected insertional mutagenesis on chromosome IV of Arabidopsis using the En-I transposon system.

Reverse genetics using insertional mutagenesis is an efficient experimental strategy for assessing gene functions. The maize Enhancer-Inhibitor (En-I) transposable element system was used to develop an effective reverse genetics strategy in Arabidopsis based on transposons. To generate insertion mutations in a specific chromosomal region we developed a strategy for local transposition mutagenesis. A small population of 960 plants, containing independent I transpositions was used to study local mutagenesis on chromosome IV of Arabidopsis. A total of 15 genes, located on chromosome IV, were tested for I insertions and included genes identified by the European ESSA I sequencing programme. These genes were of particular interest since homologies to other genes and gene families were identified, but their exact functions were unknown. Somatic insertions were identified for all genes tested in a few specific plants. Analysis of these progeny plants over several generations revealed that the ability to generate somatic insertions in the target gene were heritable. These genotypes that show high levels of somatic insertions can be used to identify germinal insertions in the progeny.

Arabidopsis↗
PubMed1999

Plant tagnology.

Transposable elements have been used as an effective mutagen and as a tool to clone tagged genes. Insertion of a transposable element into a gene can lead to loss- or gain-of-function, changes in expression pattern, or can have no effect on gene function at all, depending on whether the insertion took place in coding or non-coding regions of the gene. Cloning transposable elements from different plant species has made them available as a tool for the isolation of tagged genes using homologous or heterologous tagging strategies. Based on these transposons, new elements have been engineered bearing reporter genes that can be used for expression analysis of the tagged gene, or resistance genes that can be used to select for knockout insertions. While many genes have been cloned using transposon tagging following traditional forward genetics strategies, gene cloning has ceased to be the rate-limiting step in the process of determining sequence-function relations in several important plant model species. Large-scale insertion mutagenesis and identification of insertion sites following a reverse genetics strategy appears to be the best method for unravelling the biological role of the thousands of genes with unknown functions identified by genome or expressed sequence tag (EST) sequencing projects. Here we review the progress in forward tagging technologies and discuss reverse genetics strategies and their applications in different model species.

Journal Article↗
PubMed2006

Mutations conferring zanamivir resistance in human influenza virus N2 neuraminidases compromise virus fitness and are not stably maintained in vitro.

BACKGROUND: Viruses resistant to zanamivir have been generated in vitro, but no resistant virus has yet been isolated from a zanamivir-treated immunocompetent patient. In contrast most resistant viruses isolated from oseltamivir-treated patients correspond to those selected in vitro. However, despite mutations being in conserved residues in the neuraminidase (NA) they do not confer resistance in all NA subtypes. OBJECTIVES AND METHODS: We have used reverse genetics and the recombinant baculovirus expression system for investigating reasons for the lack of isolation of zanamivir-resistant H3N2 viruses and for further exploring subtype-specific oseltamivir resistance. RESULTS: H3N2 viruses generated by reverse genetics with H274Y, R292K E119V and E119D mutations were rescued. Those with E119G, E119A or R152K mutations could only be rescued in the presence of exogenous NA and after passage in the absence of exogenous NA only isolates that had reverted to the wild-type NA or, surprisingly, E119G/A to E119V NA were isolated. Mutations conferring zanamivir resistance significantly affected enzyme activity, virus replication or NA thermal stability. E119V viruses were stable and grew to similar titres as wild-type virus, consistent with their isolation from oseltamivir-treated patients. Mutations conferring oseltamivir resistance in N1 (H274Y) and B (R152K) NAs also conferred resistance in recombinant G70C N9 NA expressed in insect cells. CONCLUSIONS: These data suggest that zanamivir-resistant H3N2 viruses may not readily arise in vivo due to their poor viability. The G70C N9 NA may also provide a useful model for understanding the structural basis of subtype-specific drug resistance.

Animals↗
PubMed2006

Identification and characterization of Arabidopsis thaliana genes involved in xylem secondary cell walls.

The xylem of higher plants offers support to aerial portions of the plant body and serves as conduit for the translocation of water and nutrients. Terminal differentiation of xylem cells typically involves deposition of thick secondary cell walls. This is a dynamic cellular process accompanied by enhanced rates of cellulose deposition and the induction of synthesis of specific secondary-wall matrix polysaccharides and lignin. The secondary cell wall is essential for the function of conductive and supportive xylem tissues. Recently, significant progress has been made in identifying the genes responsible for xylem secondary cell wall formation. However, our present knowledge is still insufficient to account for the molecular processes by which this complex system operates. To acquire further information about xylem secondary cell walls, we initially focused our research effort on a set of genes specifically implicated in secondary cell wall formation, as well as on loss-of-function mutants. Results from two microarray screens identified several key candidate genes responsible for secondary cell wall formation. Reverse genetic analyses led to the identification of a glycine-rich protein involved in maintaining the stable structure of protoxylem, which is essential for the transport of water and nutrients. A combination of expression analyses and reverse genetics allows us to systematically identify new genes required for the development of physical properties of the xylem secondary wall.

Arabidopsis↗
PubMed2005

Making waves in cancer research: new models in the zebrafish.

The zebrafish (Danio rerio) has proven to be a powerful vertebrate model system for the genetic analysis of developmental pathways and is only beginning to be exploited as a model for human disease and clinical research. The attributes that have led to the emergence of the zebrafish as a preeminent embryological model, including its capacity for forward and reverse genetic analyses, provides a unique opportunity to uncover novel insights into the molecular genetics of cancer. Some of the advantages of the zebrafish animal model system include fecundity, with each female capable of laying 200-300 eggs per week, external fertilization that permits manipulation of embryos ex utero, and rapid development of optically clear embryos, which allows the direct observation of developing internal organs and tissues in vivo. The zebrafish is amenable to transgenic and both forward and reverse genetic strategies that can be used to identify or generate zebrafish models of different types of cancer and may also present significant advantages for the discovery of tumor suppressor genes that promote tumorigenesis when mutationally inactivated. Importantly, the transparency and accessibility of the zebrafish embryo allows the unprecedented direct analysis of pathologic processes in vivo, including neoplastic cell transformation and tumorigenic progression. Ultimately, high-throughput modifier screens based on zebrafish cancer models can lead to the identification of chemicals or genes involved in the suppression or prevention of the malignant phenotype. The identification of small molecules or gene products through such screens will serve as ideal entry points for novel drug development for the treatment of cancer. This review focuses on the current technology that takes advantage of the zebrafish model system to further our understanding of the genetic basis of cancer and its treatment.

Animals↗
PubMed2006

Invertebrate studies and their ongoing contributions to neuroscience.

Invertebrates have been deployed very successfully in experimental studies of the nervous system and neuromuscular junctions. Many important discoveries on axonal conduction, synaptic transmission, integrative neurobiology and behaviour have been made by investigations of these remarkable animals. Their advantages as model organisms for investigations of nervous systems include (a) the large diameter of neurons, glia and muscle cells of some invertebrates, thereby facilitating microelectrode recordings; (b) simple nervous systems with few neurons, enhancing the tractability of neuronal circuitry; and (c) well-defined behaviours, which lend themselves to physiological and genetic dissection. Genetic model organisms such as Drosophila melanogaster and Caenorhabditis elegans have provided powerful genetic approaches to central questions concerning nervous system development, learning and memory and the cellular and molecular basis of behaviour. The process of attributing function to particular gene products has been greatly accelerated in recent years with access to entire genome sequences and the application of reverse genetic (e.g. RNA interference, RNAi) and other post-genome technologies (e.g. microarrays). Studies of many other invertebrates, notably the honeybee (Apis mellifera), a nudibranch mollusc (Aplysia californica), locusts, lobsters, crabs, annelids and jellyfish have all assisted in the development of major concepts in neuroscience. The future is equally bright with ease of access to genome-wide reverse genetic technologies, and the development of optical recordings using voltage and intracellular calcium sensors genetically targeted to selected individual and groups of neurons.

Animals↗
PubMed2006

EMS mutagenesis of Arabidopsis.

A powerful approach for determining the biological functions of genes in an organism is to produce mutants with altered phenotypes and physiological responses. Various approaches for mutagenesis involving chemical, irradiation, and insertional methods have been developed; each has advantages and disadvantages for the study of gene function. In this post-genomic era, the use of reverse genetic approaches to understanding the role of genes in growth and development has become widespread. With development of new techniques such as targeting induced local lesions in genomes (TILLING), ethyl methanesulfonate (EMS) mutagenesis can be used for both forward and reverse genetic studies. Generation of diverse mutant alleles in the same gene provides critical tools to understand the role of these genes in the function of the organism. Here we describe the general method of EMS mutagenesis for the molecular genetic model plant Arabidopsis thaliana.

Arabidopsis↗
PubMed2003

Genetic systems of West Nile virus and their potential applications.

Prevention and treatment of West Nile virus (WNV) and other flavivirus infections have become a global public health priority. This fact is highlighted by the recent epidemic of WNV in the US. Since the first outbreak of WNV in the US in 1999, the virus has caused over 4000 known human cases and at least 263 human deaths. The epidemic of WNV in the US in 2002 represents the largest meningoencephalitis outbreak in the western hemisphere, and the largest WNV outbreak ever reported. Reverse genetic systems of WNV are extremely useful tools for studying many aspects of the virus, including viral replication, pathogenesis, antiviral therapy and vaccine development. In this review the current status of the reverse genetic systems of WNV and their potential applications are discussed.

Antiviral Agents↗
PubMed1984

Prostaglandins and mutagenesis: prevention and/or reversibility of genetic damage induced by benzo (a) pyrene in the bone marrow cells of mice by prostaglandin E1.

Prostaglandin E1 and thromboxane A2 (PGE1 and TXA2) have been proposed to bind to DNA, regulate gene action and prevent mutagenesis. Benzo (a) pyrene (BP) is a known mutagen and tumor promotor. BP-induced damage to the bone marrow cells of mice was prevented and/or reversed by PGE1 and by colchicine, an agent which may enhance PGE1 synthesis and TXA2 synthesis or action. PGF2 alpha did not have any action. Imidazole, a selective TXA2 synthesis inhibitor, enhanced the mutagenic action of benzo (a) pyrene. These results lend support to the concept that an altered PG system may have a role in the pathogenesis of mutagenesis and carcinogenesis.

Alprostadil↗
PubMed1975

Induction by RNA of inositol independence in Neurospora crassa.

The effect of purified wild-type RNA (allo-RNA) on genetic reversion of inositol-requiring mutant 89601 of Neurospora crassa is described. The mutant (inos minus) strain, on treatment with the wild-type RNA preparation, was found to revert to wild type (inos+) in significant numbers. RNA from the mutant (iso-RNA) and allo-RNA digested by RNase were ineffective in causing genetic reversion at the inositol locus. The allo-RNA-induced revertants were stable and showed a Mendelian transmission of the inos+ character.

Inositol↗
PubMed2000

Reversal of fortune for Drosophila geneticists?

Although Drosophila is a wonderful model organism, there is one molecular arena where it lags far behind its yeast and mouse model counterparts. Reverse genetics, whereby a piece of DNA is integrated into a target gene such that the gene is disrupted or replaced, is not easy in Drosophila. As Engels explains in his provocative Perspective, this may be set to change with the description of a new method for reverse genetics in Drosophila (Rong and Golic). This new technique should ensure that Drosophila remains the darling of geneticists for many years to come.

Animals↗
PubMed1993

MASH genes and the logic of neural crest cell lineage diversification.

Avian embryos have traditionally been the system of choice for studying neural crest development, but the combination of reverse genetics and clonal culture should allow new insights to be gained from mammalian systems as well. We describe one of the first examples where a targeted mutation in a developmental control gene, isolated on the basis of its homology to Drosophila neural determination genes, causes a highly selective phenotype affecting the early development of a subset of neural crest derivatives. Detailed analysis of the cellular phenotype and expression pattern of the gene, called MASH-1, have led to novel insights into the genetic logic that controls neural crest development. Most important, some features of the phenotype appear inconsistent with the predictions of current models of neural crest lineage diversification. These unexpected results have forced a reevaluation of our thinking about some aspects of neural crest development, and illustrate the power of the reverse genetic approach to reveal unanticipated features of complex biological systems as well as to suggest new directions for future study.

Animals↗
PubMed2005

Generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection.

A reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) has been described in which a full-length cDNA, corresponding to the IBV (Beaudette-CK) genome, was inserted into the vaccinia virus genome following in vitro assembly of three contiguous cDNAs [Casais, R., Thiel, V., Siddell, S.G., Cavanagh, D., Britton, P., 2001. Reverse genetics system for the avian coronavirus infectious bronchitis virus. J. Virol. 75, 12359-12369]. The method has subsequently been used to generate a recombinant IBV expressing a chimaeric S gene [Casais, R., Dove, B., Cavanagh, D., Britton, P., 2003. Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism. J. Virol. 77, 9084-9089]. Use of vaccinia virus as a vector for the full-length cDNA of the IBV genome has the advantage that modifications can be made to the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. We describe the use of homologous recombination as a method for modifying the Beaudette full-length cDNA, within the vaccinia virus genome, without the requirement for in vitro assembly of the IBV cDNA. To demonstrate the feasibility of the method we exchanged the ectodomain of the Beaudette spike gene for the corresponding region from IBV M41 and generated two recombinant infectious bronchitis viruses (rIBVs) expressing the chimaeric S protein, validating the method as an alternative way for generating rIBVs.

Animals↗
PubMed2006

Rescue of the prototypic Arenavirus LCMV entirely from plasmid.

We document a helper-independent reverse genetics system for rescuing infectious arenaviruses from cloned cDNAs. We constructed plasmids containing full-length cDNAs of the antigenomic (ag) L and S segments of the Armstrong (ARM) strain of the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) flanked at their 5'- and 3'-termini by the T7 RNA polymerase (T7RP) promoter and ribozyme sequences, respectively. These plasmids directed intracellular synthesis of viral L and S ag RNA species in cells expressing plasmid-supplied T7RP. Co-expression of plasmid-supplied LCMV trans-acting factors, nucleoprotein (NP) and polymerase (L), resulted in replication and expression of L and S ag and genome RNA species, and generation of LCMV infectious progeny termed rT7/LCMV. The recombinant rT7/LCMV was unequivocally identified based on a genetic tag introduced in the recombinant S segment. In addition, rT7/LCMV exhibited growth and biological properties predicted for an ARM-like LCMV. To our knowledge, this is the first documented Arenavirus rescue, as well as of an ambisense negative strand (NS) RNA virus, entirely from cloned cDNAs. Our results extend the use of reverse genetic approaches for DNA-mediated virus rescue to all known virus families with NS RNA genome.

Animals↗
PubMed1995

Electroporation of influenza virus ribonucleoprotein complexes for rescue of the nucleoprotein and matrix genes.

Reverse genetics has been successfully used for the generation of recombinant influenza virus with altered biological properties. The standard method is based on DEAE-dextran transfection of in vitro reconstituted influenza virus ribonucleoprotein complex (RNP) into helper virus infected cells with subsequent selection of the recombinant viruses. Here we report the utilization of electroporation for reverse genetics of influenza virus as an improvement over the standard method. In a neuraminidase (NA) gene rescue system, we were able to demonstrate that electroporation of in vitro reconstituted NA RNP of influenza A/WSN/33 (H1N1) virus into WSN/HK virus infected cells allows the rescue of the transfectant WSN virus. The titer of transfectant virus obtained using electroporation is comparable to that generated using the DEAE-dextran transfection method. More significantly, the ratio of transfectant virus to helper virus is as much as 20-fold greater than that achieved using the DEAE-dextran system. We have also used electroporation to generate recombinant influenza virus carrying cDNA-derived matrix (M) gene or nucleoprotein (NP) gene of the WSN virus by using the temperature-sensitive (ts) mutants ts51 and ts56 as helper viruses. In the case of electroporation of M gene RNP, 88% of the viruses isolated after selection at 39 degrees C were transfectants. In contrast, the majority of viruses obtained using the DEAE-dextran transfection method were revertants of the helper virus. The NP-gene transfectant was only generated by the electroporation method. Our results suggest that electroporation of influenza virus RNP may be a useful method for generation of recombinant influenza viruses, especially in a system in which a ts mutant is used as helper virus.

Animals↗
PubMed2026

Comparative dynamics of Japanese encephalitis virus adaptation in porcine macrophages and insect cells.

BACKGROUND: Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne Orthoflavivirus that circulates primarily in birds and pigs. Previous observations of vector-free transmission between pigs indicates the possibility of single-host cycling in swine. Therefore, the aim of this work was to investigate the evolutionary pressure of single host cycling using a relevant primary cell culture model. METHODS: To investigate whether such single-host cycles affect viral infectivity, fitness and genomic adaptations, two strains and a reverse genetic cDNA-derived clone of JEV were serially passaged 12 times in primary porcine monocyte-derived macrophages (MDMs), in Aedes albopictus-derived C6/36 cells, and alternately between both cell types. Next-generation sequencing analysis was used to identify selected single nucleotide variants (SNVs) and haplotypes. Phenotype-to-genotype connections were confirmed using reverse genetics. RESULTS: For all viruses, serial passaging in MDMs - but not in C6/36 cells - led to a rapid increase in relative infectivity toward MDMs, accompanied by reduced plaque sizes in porcine endothelial cells. In contrast to C6/36 cells, MDM imposed a strong selective pressure, rapidly favoring selection of many SNVs and viral haplotypes. In addition, we identified a dominant selection of mutants with glutamic acid to lysine substitutions at positions 49 or 138 in the E protein, which explained the small plaque phenotype and caused viral sensitivity to heparin-mediated inhibition of attachment, indicating enhanced virus binding to glycosaminoglycans (GAG). The E138K mutant also explained the increased relative infectivity for MDM. CONCLUSION: This work demonstrates a high evolutionary pressure on JEV in MDM causing rapid selections of minor haplotypes. Furthermore, the efficient selection of E49K and E138K SNV, which were responsible for the phenotype, are likely caused by a selective pressure for GAG binding, observed in vitro with other mammalian cells.

Animals↗