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At least 55 records · Page 3Linked to original sources
PubMed2004

Responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines.

BACKGROUND: In response to the emergence of severe infection capable of rapid global spread, WHO will issue a pandemic alert. Such alerts are rare; however, on Feb 19, 2003, a pandemic alert was issued in response to human infections caused by an avian H5N1 influenza virus, A/Hong Kong/213/03. H5N1 had been noted once before in human beings in 1997 and killed a third (6/18) of infected people. The 2003 variant seemed to have been transmitted directly from birds to human beings and caused fatal pneumonia in one of two infected individuals. Candidate vaccines were sought, but no avirulent viruses antigenically similar to the pathogen were available, and the isolate killed embryonated chicken eggs. Since traditional strategies of vaccine production were not viable, we sought to produce a candidate reference virus using reverse genetics. METHODS: We removed the polybasic aminoacids that are associated with high virulence from the haemagglutinin cleavage site of A/Hong Kong/213/03 using influenza reverse genetics techniques. A reference vaccine virus was then produced on an A/Puerto Rico/8/34 (PR8) backbone on WHO-approved Vero cells. We assessed this reference virus for pathogenicity in in-vivo and in-vitro assays. FINDINGS: A reference vaccine virus was produced in Good Manufacturing Practice (GMP)-grade facilities in less than 4 weeks from the time of virus isolation. This virus proved to be non-pathogenic in chickens and ferrets and was shown to be stable after multiple passages in embryonated chicken eggs. INTERPRETATION: The ability to produce a candidate reference virus in such a short period of time sets a new standard for rapid response to emerging infectious disease threats and clearly shows the usefulness of reverse genetics for influenza vaccine development. The same technologies and procedures are currently being used to create reference vaccine viruses against the 2004 H5N1 viruses circulating in Asia.

Animals↗
PubMed2003

Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics.

Avian influenza A H5N1 viruses similar to those that infected humans in Hong Kong in 1997 continue to circulate in waterfowl and have reemerged in poultry in the region, raising concerns that these viruses could reappear in humans. The currently licensed trivalent inactivated influenza vaccines contain hemagglutinin (HA) and neuraminidase genes from epidemic strains in a background of internal genes derived from the vaccine donor strain, A/Puerto Rico/8/34 (PR8). Such reassortant candidate vaccine viruses are currently not licensed for the prevention of human infections by H5N1 influenza viruses. A transfectant H5N1/PR8 virus was generated by plasmid-based reverse genetics. The removal of the multibasic amino acid motif in the HA gene associated with high pathogenicity in chickens, and the new genotype of the H5N1/PR8 transfectant virus, attenuated the virus for chickens and mice without altering the antigenicity of the HA. A Formalin-inactivated vaccine prepared from this virus was immunogenic and protected mice from subsequent wild-type H5N1 virus challenge. This is the first successful attempt to develop an H5N1 vaccine seed virus resembling those used in currently licensed influenza A vaccines with properties that make it a promising candidate for further evaluation in humans.

Animals↗
PubMed1992

Reverse genetics of Caenorhabditis elegans.

It is somewhat ironic that animals that are the prime choice for detailed genetic analysis, such as the fruit fly and the nematode, have thus far been largely refractory to reverse genetic analysis. Their detailed genetic map, and small genome size have made them subjects of ambitious genome analysis projects, but there is still no strategy to introduce desired changes into their genomes by homologous recombination. Some alternative approaches have recently become available; this review describes possibilities and unsolved problems for reverse genetics in the nematode Caenorhabditis elegans. The transposon Tc1 could prove to be very useful for the isolation of knock out mutants, and possibly also for introduction of more subtle alterations.

Animals↗
PubMed2001

Reverse genetics system for Uukuniemi virus (Bunyaviridae): RNA polymerase I-catalyzed expression of chimeric viral RNAs.

We describe here the development of a reverse genetics system for the phlebovirus Uukuniemi virus, a member of the Bunyaviridae family, by using RNA polymerase I (pol I)-mediated transcription. Complementary DNAs containing the coding sequence for either chloramphenicol acetyltransferase (CAT) or green fluorescent protein (GFP) (both in antisense orientation) were flanked by the 5'- and 3'-terminal untranslated regions of the Uukuniemi virus sense or complementary RNA derived from the medium-sized (M) RNA segment. This chimeric cDNA (pol I expression cassette) was cloned between the murine pol I promoter and terminator and the plasmid transfected into BHK-21 cells. When such cells were either superinfected with Uukuniemi virus or cotransfected with expression plasmids encoding the L (RNA polymerase), N (nucleoprotein), and NSs (nonstructural protein) viral proteins, strong CAT activity or GFP expression was observed. CAT activity was consistently stronger in cells expressing L plus N than following superinfection. No activity was seen without superinfection, nor was activity detected when either the L or N expression plasmid was omitted. Omitting NSs expression had no effect on CAT activity or GFP expression, indicating that this protein is not needed for viral RNA replication or transcription. CAT activity could be serially passaged to fresh cultures by transferring medium from CAT-expressing cells, indicating that recombinant virus containing the reporter construct had been produced. In summary, we demonstrate that the RNA pol I system, originally developed for influenza virus, which replicates in the nucleus, has strong potential for the development of an efficient reverse genetics system also for Bunyaviridae members, which replicate in the cytoplasm.

Animals↗
PubMed2001

Reverse genetics system for the avian coronavirus infectious bronchitis virus.

Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.

Animals↗
PubMed2025

Establishment of reverse genetics systems for Colorado tick fever virus.

The Colorado tick fever virus (CTFV), which has 12-segmented double-stranded RNA genomes, is a pathogenic arbovirus that causes severe diseases in humans. However, little progress has been made in the analysis of replication mechanisms and pathogenicity. This virological constraint is due to the absence of a reverse genetics system for CTFV; therefore, we aimed to establish the system. Initially, the efficacy of CTFV replication was investigated in various cell lines. CTFV was found to grow in many cell types derived from different hosts and organs. Subsequently, BHK-T7 cells stably expressing T7 RNA polymerase were transfected with plasmids encoding each of the 12 CTFV gene segments, expression plasmids encoding all CTFV proteins, and a vaccinia virus RNA-capping enzyme. Following transfection, the cells were co-cultured with Vero or HeLa cells. Using this system, we rescued monoreassortants and recombinant viruses harboring peptide-tagged viral proteins. Furthermore, an improved system using Expi293F cells expressing T7 RNA polymerase was established, which enabled the generation of recombinant reporter CTFVs. In conclusion, these reverse genetics systems for CTFV will greatly contribute to the understanding of viral replication mechanisms, pathogenesis, and transmission, ultimately facilitating the development of rational treatments and candidate vaccines.

Animals↗
PubMed2005

Reverse genetics of negative-stranded RNA viruses: a global perspective.

The advent of reverse genetics technology has revolutionized the field of RNA viruses. It is now possible to manipulate even negative-stranded RNA viruses at will, and evaluate the effects of these changes on the biology and pathogenesis of these viruses. The fundamental insights gleaned from the reverse genetics-based studies over the last several years have provided a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. The recombinant viruses have been exploited also as vectors for devising targeted therapies for non-viral diseases such as malignancies, and in gene therapy for inherited disorders. This review provides a brief summary of the stumbling blocks and the successes in the development of the technology for the negative-stranded RNA viruses. The many and varied applications of the recombinant vectors are also outlined.

Animals↗
PubMed1995

Reverse genetics system for generation of an influenza A virus mutant containing a deletion of the carboxyl-terminal residue of M2 protein.

We established a reverse genetics system for the M gene of influenza A virus, using amantadine resistance as a selection criterion. Transfection of an artificial M ribonucleoprotein complex of A/Puerto Rico/8/34 (H1N1), a naturally occurring amantadine-resistant virus, and superinfection with amantadine-sensitive A/equine/Miami/1/63 (H3N8), followed by cultivation in the presence of the drug, led to the generation of a transfectant virus with the A/Puerto Rico/8/34 (H1N1) M gene. With this system, we attempted to generate a virus containing a deletion in an M-gene product (M2 protein). Viruses lacking the carboxyl-terminal Glu of M2, but not those lacking 5 or 10 carboxyl-terminal residues, were rescued in the presence of amantadine. These findings indicate that carboxyl-terminal residues of the M2 protein play an important role in influenza virus replication. The M-gene-based reverse genetics system will allow the study of different M-gene mutations to achieve a balance between attenuation and virus replication, thus facilitating the production of live vaccine strains.

Amantadine↗
PubMed1998

Forward and reverse genetic approaches to synaptogenesis.

Genetic approaches at the neuromuscular synapse are leading investigation into the mechanisms of synaptogenesis. The marriage of classical (forward) and reverse genetic techniques allows the isolation and analysis of novel proteins involved in synaptic maturation and the functional in vivo characterization of previously identified synaptic proteins. Of particular interest are recent advances using mouse reverse genetics and Drosophila forward genetics.

Animals↗
PubMed2006

Reassortment and modification of hemagglutinin cleavage motif of avian/WSN influenza viruses generated by reverse genetics that correlate with attenuation.

Avian influenza associated with H9N2 and H5N1 subtypes of avian influenza viruses (AIVs) has raised great concerns in China. To study this problem, reverse genetics has been employed. Three reassortants, rgH9N2, rgH5N1 and rgH5N2, were prepared and compared. Their hemagglutinin (HA) and neuraminidase (NA) genes originated from Chinese AIV isolates of H9N2 or H5N1 subtype, while the rest of their genes were derived from A/WSN/33(H1N1) virus (WSN). In the H5 HA reassortants, the multibasic cleavage site was converted to a monobasic one. The results demonstrated that the reassortants did not produce CPE on MDCK cells in the absence of trypsin, showed egg-adaptation phenotype and stability of HA and NA during consecutive egg passages, and were not lethal to chickens and mice. However, the rgH5N1 reassortant exhibited a residual virulence in terms of lethality to chick embryos and pathogenesis in chickens. It can be concluded that (i) the genetic modification of H5 HA attenuated the H5 reassortants, (ii) the presence of internal WSN proteins contributed to the attenuated properties of the reassortants independently on H5 HA, and (iii) also the overall genome composition contributed to virulence differences. This report provides further contribution of reverse genetics to the knowledge of virulence of influenza viruses.

Animals↗
PubMed2003

Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis.

Chemical mutagenesis has been the workhorse of traditional genetics, but it has not been possible to determine underlying rates or distributions of mutations from phenotypic screens. However, reverse-genetic screens can be used to provide an unbiased ascertainment of mutation statistics. Here we report a comprehensive analysis of approximately 1900 ethyl methanesulfonate (EMS)-induced mutations in 192 Arabidopsis thaliana target genes from a large-scale TILLING reverse-genetic project, about two orders of magnitude larger than previous such efforts. From this large data set, we are able to draw strong inferences about the occurrence and randomness of chemically induced mutations. We provide evidence that we have detected the large majority of mutations in the regions screened and confirm the robustness of the high-throughput TILLING method; therefore, any deviations from randomness can be attributed to selectional or mutational biases. Overall, we detect twice as many heterozygotes as homozygotes, as expected; however, for mutations that are predicted to truncate an encoded protein, we detect a ratio of 3.6:1, indicating selection against homozygous deleterious mutations. As expected for alkylation of guanine by EMS, >99% of mutations are G/C-to-A/T transitions. A nearest-neighbor bias around the mutated base pair suggests that mismatch repair counteracts alkylation damage.

Arabidopsis↗
PubMed2027

Molecular Cloning and Reverse Genetics.

This chapter describes a detailed molecular biology protocol for introducing specific point mutations into the chikungunya virus (CHIKV) genome using a reverse genetics strategy. The method utilizes an overlapping PCR-based approach to generate a mutated DNA fragment, which is then cloned into a pre-engineered CHIKV infectious clone plasmid. The protocol covers all major steps, from the initial PCRs to create the mutated insert to its digestion and ligation into the vector. It also includes procedures for bacterial transformation, colony screening via PCR and Sanger sequencing to confirm the mutation, and plasmid purification via miniprep. The document is structured with a clear introduction, a list of all required reagents and equipment, and a step-by-step methods section.

Cloning, Molecular↗
PubMed1992

Tracking disease genes by reverse genetics.

Increasingly, human genes are being identified by the "reverse genetics", or "positional cloning" approach. This molecular genetic strategy is particularly useful in mental illness, for which no readily detectable functional alterations are present to indicate candidate genes. The positional cloning procedure is briefly described. Significant examples of successful positional cloning are presented, including the fragile-X mental retardation syndrome gene. The study of gene expression may be complicated by genetic and non-genetic variability. Genomic imprinting may play a role in several mental illnesses, and may provide an explanation for the unusual inheritance pattern in fragile-X syndrome, for the phenotypic differences observed between Angelman and Prader-Willi syndromes, and for the juvenile onset form of Huntington disease. DNA instability may explain disease anticipation in fragile-X syndrome and myotonic dystrophy. Finally, the prospects of improvements in positional cloning methods for tracking genes responsible for mental illness are briefly discussed.

Chromosome Mapping↗
PubMedDate not supplied

Paramyxovirus replication and pathogenesis. Reverse genetics transforms understanding.

A recent breakthrough in the field of nonsegmented negative strand RNA viruses (Mononegavirales), including paramyxoviruses, is the establishment of a system to recover an infectious virus entirely from complementary DNA and hence allow reverse genetics. Mutations can now be introduced into viral genomes at will and the resulting phenotypes studied as long as the introduced mutations are not lethal. This technology is being successfully applied to answer outstanding questions regarding the roles of viral components in replication and their contribution to pathogenicity, which are difficult to address using conventional virology. For instance, how the paramyxovirus accessory proteins V and C contribute to actual viral replication and pathogenesis has remained unanswered since their first description more than 20 years ago. Using Sendai virus, which causes fatal pneumonia in mice, it has been shown that the V protein is completely dispensable for viral replication in cell cultures but encodes a luxury function required for pathogenesis in vivo. The Sendai virus C proteins were also defined to be nonessential gene products which greatly contributed to replication both in vitro and in vivo. It is also now possible to design live vaccines by introducing predetermined or plausible attenuating mutations. In addition, the use of paramyxoviruses to express foreign genes has also become feasible. Paramyxovirus reverse genetics is thus renovating our understanding of viral replication and pathogenesis and will further mark an era in recombinant technology for disease prevention and gene therapy.

Animals↗
PubMed2005

The role of reverse genetics in the development of vaccines against respiratory viruses.

Despite their significance, the only available vaccines against respiratory viruses are those for the prevention of influenza. Attempts have been made to produce vaccines against other respiratory viruses using traditional techniques, but have met with little success. Reverse genetics, although still a relatively new tool for the manipulation of negative-strand RNA viruses, has great potential for the preparation of vaccines against many of the common respiratory viruses. In the preparation of live vaccines, reverse genetics systems allow the direct modification of the specific regions in the genomes of negative-stranded RNA viruses concerned with attenuation; the ultimate goal is the introduction of site-specific mutations through a cDNA intermediate in order to develop strains with the requisite attenuation, antigenic and growth properties needed in a vaccine. These techniques can also be used to disarm potentially highly pathogenic viruses, such as emerging H5N1 avian influenza viruses, in order to facilitate large-scale preparation of viruses for use in inactivated vaccines under conditions of manufacturing safety. Before these vaccines become available, residual issues concerned with intellectual property rights to the technology and its application will need to be resolved.

Animals↗
PubMed2004

Influenza vaccines generated by reverse genetics.

Influenza viruses cause annual epidemics and occasional pandemics of acute respiratory disease. Vaccination is the primary means to prevent and control the disease. However, influenza viruses undergo continual antigenic variation, which requires the annual reformulation of trivalent influenza vaccines, making influenza unique among pathogens for which vaccines have been developed. The segmented nature of the influenza virus genome allows for the traditional reassortment between two viruses in a coinfected cell. This technique has long been used to generate strains for the preparation of either inactivated or live attenuated influenza vaccines. Recent advancements in reverse genetics techniques now make it possible to generate influenza viruses entirely from cloned plasmid DNA by cotransfection of appropriate cells with 8 or 12 plasmids encoding the influenza virion sense RNA and/or mRNA. Once regulatory issues have been addressed, this technology will enable the routine and rapid generation of strains for either inactivated or live attenuated influenza vaccine. In addition, the technology offers the potential for new vaccine strategies based on the generation of genetically engineered donors attenuated through directed mutation of one or more internal genes. Reverse genetics techniques are also proving to be important for the development of pandemic influenza vaccines, because the technology provides a means to modify genes to remove virulence determinants found in highly pathogenic avian strains. The future of influenza prevention and control lies in the application of this powerful technology for the generation of safe and more effective influenza vaccines.

Animals↗
PubMed1987

A "reverse genetic" approach to autosomal dominant polycystic kidney disease.

Biochemical, anatomical, pathophysiological and clinical studies of autosomal dominant "adult-type" polycystic kidney disease have cast little light on the underlying biochemical defect which causes the disease. The advent of recombinant DNA technology permits a novel approach to its pathophysiology. In this approach, termed "reverse genetics", the mutation which produces the disease is first localised by genetic linkage. This is followed by the identification and cloning of the "disease gene" itself, and the characterisation of its mutations. The recent assignment of the polycystic kidney disease mutation to the short arm of chromosome 16 is thus the first step in a reverse genetic approach to an understanding of the molecular pathology of this disorder.

Genes, Dominant↗
PubMed1993

Co-chairman's remarks: reverse genetics: directed modification of DNA for functional analysis.

The classic paradigm for identifying the genetic basis of a particular organism's properties proceeds from the phenotype to the gene, and thence to the molecular structures of the corresponding DNA, RNA and protein. 'Positional cloning' of disease genes and the molecular characterization of the responsible mutations (inappropriately referred to as reverse genetics, initially) exemplifies this approach. Now, the ability to clone, modify and test the biological activities of DNA segments provides a new approach, referred to as 'reverse genetics'. This paradigm begins with a segment of DNA whose molecular structure is known, and proceeds to explore the gene's contribution to the organism's phenotype; thus, the experimental path is from the gene as a nucleotide sequence to the corresponding phenotypic characteristic. Such a strategy follows from the ability to modify these sequences in highly directed and nearly unlimited ways, and to assess the phenotypic relevance of such alterations either in vitro, in cultured cells, or even in whole organisms. This approach permits the full panoply of molecular techniques to be used for creating uniquely altered structures and obviates the reliance on chance events as the source of mutations. As a consequence, the range of questions that can be studied is greatly expanded, and the information that is obtained is all the richer.

Animals↗