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  1. Simple reverse genetics systems for Asian and African Zika viruses
  2. Reverse Genetics of RNA Viruses: Applications and Perspectives :: Book :: ChemistryViews
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Includes numerous examples of cutting- edge applications of reverse genetics within each of the RNA viral groups. Written by a team of international experts, including some of the leading researchers in the field. Free Access. Summary PDF Request permissions. Tools Get online access For authors. Email or Customer ID.

Forgot password? Old Password. Rescue of other full-length RNA viruses soon followed, and included vesicular stomatitis virus VSV [ 39 , 72 ], measles virus MV [ 58 ], and many others [ 5 , 44 ]. Notwithstanding the early success in the rescue of individual influenza virus segments from cDNAs, the successful construction and rescue of minigenomes of a majority of the SNS RNA viruses 1 , 6 , 16 , 24 , 26 , 27 , 37 , 38 , 56 ], and the continuous improvement in rescue technology, so far, only three of SNS viruses have been recovered from cDNA constructs.

Simple reverse genetics systems for Asian and African Zika viruses

The first report in this category of viruses was the rescue in of the bunyamwera virus [ 7 ]. In , the eight-genome-segmented influenza A was rescued by Neumann et al. Fodor et al. Hoffman et al. The vRNA and mRNA was generated simultaneously from these constructs, thereby reducing the total number of plasmids required for rescue to just eight instead of The six segmented Thogoto virus was rescued in by using a combination of T7 and Pol I driven systems for protein expression and vRNA transcription, respectively [ 69 ].

The Pol I system, initiated by Hobom and colleagues, [ 75 ] have several advantage over the T7 polymerase system: The Pol I enzyme is expressed in the nucleolus of all eukaryotic cells and therefore does not need to be provided in trans. Transcripts generated by the Pol I constructs have precise viral ends, i. Also, the Pol I system does not have the potential inherent disadvantage of vaccinia virus-mediated recombination. The Pol I system is ideally suited for viruses like the influenza virus or the bornavirus that transcribe in the nucleus.

8 Replication of positive stranded RNA virus

However, the majority of RNA viruses replicate in the cytoplasm, and in this case, it is necessary for the plasmids to enter the nucleus to undergo transcription and then the transcripts need to exit to the cytoplasm to complete the downstream processes. Nevertheless, the location of the enzyme does not appear to be a factor since Flick et al.


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  • Reverse genetics of RNA viruses : applications and perspectives!

Reverse genetics-based studies have had a dramatic effect in expanding our knowledge of the molecular biology and pathogenesis of RNA viruses and has put to rest many issues that were impossible to address by conventional virological or biochemical procedures. Reverse genetic analysis has been particularly successfully applied to the identification and characterization of the cis -acting elements of RNA viruses.

A selected group of some of the more revealing studies is reviewed below in more detail.

Experimental evidence for extended promoters in the paramyxoviruses was first reported in a SeV study [ 65 ] and subsequently confirmed for paramyxovirus simian virus 5 SV5 [ 41 ] and MV [ 70 ]. The impact of the experimental design and procedures used in the characterization of cis -acting sequences was underscored from the results of the MV investigation: mutational analysis of the 18 nucleotide region of MV AGP internal replication control element clearly demonstrated that mutating combinations of some nucleotides had a profound effect on replication efficiency although when individually substituted, they appeared not to be required.

Results of mutational analysis in the same study showed further, that the nucleotides at only some of the positions of the internal element were essential for functionality. However, the use of an in vivo nucleotide selection procedure revealed that with continued replication, a preference for the conservation of wild type nucleotides across the entire element was clearly evident.

These results indicated that all the nucleotides in this region were required for replication, although to varying degrees; the essential nucleotides may be involved in sequence specific binding with precise regions of the homologous polymerase while the preferred nucleotides may be contributing to the energetics of binding [ 73 ].

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A replication model described in this study [ 70 ] proposes that to initiate and maintain optimal replication efficiency, the polymerase complex proteins need to make multiple contacts with the terminal and internal nucleotides of the promoter; some of these are essential and while others are preferred.

Precise spacing of the sequence elements with reference to each other may be needed for correct positioning of the polymerase to initiate replication; the need for precise spacing between the cis -acting sequences was indicated from experimental data in SeV [ 65 ] and SV5 [ 41 ] studies, and from our unpublished data, which showed that nucleotide deletions in the region intervening the terminal and internal elements were not tolerated.

Whether substitution of the essential nucleotides in a promoter element disrupts a required structure in the RNA template or destabilizes a preferred nucleotide specific interaction between the template and the polymerase is not clear. The proposed structure consists of a six base-pair rod structure in the distal element and two stem—loop structures of two intra-strand short-range base pairs; the latter support an exposed tetranucleotide loop within each branch of the proximal element in an overall oblique organization.

A complete set of single substitutions and double complementary mutations showed that the number of invariant nucleotides in this structure was few and retention of structural integrity of the region was paramount. Interestingly, the invariant nucleotides are located within the tetranucleotide loop structure which is likely to facilitate its interaction with the polymerase complex proteins.

Reverse Genetics of RNA Viruses: Applications and Perspectives :: Book :: ChemistryViews

The corkscrew conformation, verified in recent [ 15 ] and previous studies, is more complex than the two other promoter models predicted previously for this virus, the panhandle and the fork structure [ reviewed in 44 ]. Most of the viruses in the Bunyaviridae are also likely to have a corkscrew conformation based on computer generated secondary structure predictions of the promoter regions of these viruses [ 25 ], but this needs experimental evaluation.

The successful generation of recombinant viruses and the ability to manipulate their genomes at will has made it possible to ask questions pertaining to the molecular biology and disease pathogenesis of RNA viruses and as a result, our understanding of these aspects of the RNA viruses continues to grow. The ability of the recombinant viruses to stably express foreign genetic material has also resulted in the conception of novel therapeutic [ 4 ] and disease prevention paradigms [ 5 , 11 ], and the potential of these viruses in such applications is being tapped at an ever increasing rate.


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The paramyxo- and rhabdoviruses are particularly suited for the stable insertion of foreign genetic information for several reasons: the encapsidated genomes of these viruses make elimination of the inserted sequence by homologous recombination a very unlikely event; genome integrity is likely to be further enforced by the requirement by a majority of the paramyxoviruses for the rule of six : deletions that disrupt this requirement would result in non-viable genomes.

The genomes of these viruses are able to accept and stably express up to 5 kb of introduced genetic material so that making bivalent or even multivalent vaccines would be a reality. Moreover, introduction of extra transcription units is achieved with relative ease by the introduction of additional gene stop and start signals. Finally, the levels of expression of the inserted gene can be controlled by careful selection of the point of insertion [ 71 ].

Most importantly, recombinant viruses widely used as vectors, like the measles vaccine virus, already have a long safety record as live attenuated vaccines; other viruses such as SeV and the VSV vaccine virus which are considered non-pathogenic to humans are also widely used as vectors. Importantly, the life cycle of these viruses is confined to the cytoplasm, and therefore such vectors carry no accompanying risk of unwanted chromosomal integration. Segmented viruses have also been used as vectors of foreign genetic material [reviewed in 44 ]. The simple approach of introducing additional genome segments in these viruses is not successful however, because segments which are not essential for virus survival are rapidly lost by re-assortment or become non-functional through mutation.

Creative approaches have been used to circumvent this problem: the segmented genomes have been successfully used as vectors by making fusion genes mediated through insertion of internal ribosomal entry sites IRES or by the insertion of the 17aa long protease 2A sequence of foot and mouth disease virus between the viral gene and the foreign genetic material. Alternatively, selected regions of foreign genetic material such as the T cell specific epitope s have been incorporated into the stalk region of NA, or the antigenic sites of the influenza virus HA genes.

A variety of approaches has been undertaken to use recombinant viruses to generate live attenuated vaccines. For example, attenuating point mutations corresponding to host range, cold adaptation, temperature sensitive phenotypes or others have been identified and some have been inserted into vaccine or wild type backgrounds to generate vaccine candidates [ 11 , 50 , 51 ]. Mutations which alter enzyme cleavage specificity, e. A class of live attenuated recombinant virus involves the alteration or elimination of the non-structural interferon antagonist genes.

Such altered viruses are attenuated because they have reduced, or no capacity to counteract host interferon responses [ 14 , 19 , 63 , 67 ]. Rearrangement of genes also results in attenuation [ 71 ]. Replacing the promoter region s of a virus with that of a closely related virus has the same effect. This was demonstrated by us using MV and CDV minigenome chimeras our unpublished data , and the validity of this approach for vaccine purposes was reported by Chapman et al. Recombinant viruses carrying the glycoprotein gene of a related or an unrelated virus instead of its own have been used for vaccine purposes [ 5 , 11 , 14 , 59 ].

For example, in one study, virus binding was redirected by creating MV H gene-anti CD38 fusion gene, a myeloma cell marker, to create a recombinant measles virus with altered tropism, with the ultimate aim of targeted destruction of the malignant cells [ 55 ]. Pseudo-type viruses have been used as substitutes for investigations involving highly pathogenic viruses which otherwise would require bio-containment: for example, a G gene deletion mutant of VSV carrying Ebola virus glycoprotein was used to probe the receptor usage of this virus [ 62 ].

Similarly, a VSV pseudo-type carrying Hantaan or Seoul virus envelope protein was used as an effective replacement for the complete virus to develop a safe neutralization assay for Hantaan virus [ 46 ].

Selected cytokine genes have been inserted in VSV and other viral vectors to modulate immune responses [ 59 ]. Recombinant paramyxoviruses are likely to be valuable for gene therapy because these vectors have several obvious advantages over the conventional retroviral and adenoviral vectors used for this purpose. For example, SeV vectors have been used to express foreign genes efficiently in non-dividing cells such as neurons [ 32 ]. Gene transfer to murine lung epithelium mediated by a replication competent SeV vector appeared also to be effective [ 74 ].

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The exploration of recombinant viruses for therapy and prophylaxis has just begun, and innovative and novel applications continue to grow and show exciting promise for the future. The above discourse underscores the undisputed importance of reverse genetics in improving our understanding of RNA viruses at the molecular level.

However, in many instances, a cautionary note needs to be introduced in the interpretation of data generated by this approach since the results may depend on the systems used. The following are some examples. Tissue culture based studies may produce information that may not reflect that which results from interaction of the virus and its human or animal host. A case in point is the studies investigating the role of non-structural proteins such as the C and V, encoded by the P cistron of MV and SeV.

In tissue culture based studies, these proteins appear to be dispensable since recombinant viruses lacking these genes were able to replicate and produce plaque sizes and virus titer comparable to those obtained from wild type viruses. However, growth of the deletion mutants was highly restricted in both the upper and lower airways of chimpanzees indicating the role of these non-structural proteins in pathogenesis [reviewed in 5 , 42 , 59 ].

Evaluation of the role of the cis -acting elements by mutational analysis should also be viewed with a caveat because it is conceivable that compensatory changes may be introduced elsewhere to restore the effects of the designed mutations. Experimental design and procedures used may also alter the interpretation of data [ 70 ].

Minigenome based data of the sequence characteristics or the viral proteins may need to be validated in full length recombinant viruses because the interactions of the different viral proteins with one another and with the host milieu are likely to play a role in the outcome. Importantly, majority of the evaluations currently undertaken are based on tissue culture adapted laboratory strains such as Edmonston B measles virus, and the results obtained may not apply to the circulating wild-type viruses.

Knowledge of RNA viruses has grown exponentially since the availability of reverse genetics technology, and has provided insights into the biology and pathogenesis of RNA viruses which were previously impossible to address. However, it also has raised many questions and produced conflicting data, some of which may relate to the differences in the viruses or experimental systems used, or may be due to factors that we have failed to fathom yet. Many questions still remain to be answered. Reverse Genetics of RNA Viruses provides a comprehensive account of the very latest developments in reverse genetics of RNA viruses through a wide range of applications within each of the core virus groups including; positive sense, negative sense and double stranded RNA viruses.

Written by a team of international experts in the field, it provides a unique insight into how the field has developed, what problems are being addressed now and where applications may lead in the future. It will prove invaluable to bioscience, medical and veterinary students, those starting research in this area as well as other researchers and teachers needing to update their knowledge of this fast-moving field. Dr Anne Bridgen , previously of The University of Ulster is a molecular virologist with extensive research and teaching experience. Dr Bridgen knows the field and its main players well and has both the knowledge and experience to bring individual expert contributions together around the common theme of reverse genetics.

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