Reverse genetics of negative-strand RNA viruses: Closing the circle (original) (raw)
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Advances in Animal and Veterinary Sciences, 2014
Reverse genetics is a cutting-edge tool that has revolutionized molecular virology through which viruses possessing artificial genomes can be rescued from cloned cDNA. This gave the researchers the choice and flexibility to get the modifications in the progeny virions that would be done at the genome level while constructing the cDNA. The idea led to two significant discoveries, with the first that gave an impetus in the area of live attenuated "Differentiation of infected and vaccinated animals" (DIVA) vaccines, and the second that led to a better understanding into the host-virus relationship. The DIVA vaccines developed through the reverse genetics tool have advantages of stable expression of the foreign protein coupled with the fundamental characteristics of the background virus that equates with the wild type. Rescue of DNA viruses and positive sense RNA viruses have been made easy, thanks to the less complicated replication strategies followed by them, but the nonsegmented negative sense RNA viruses needs Ribonucleoprotein (RNP) complex to be provided in vitro to aid in anti-genome complex necessary for their replication. This technology has also played an effective role in identifying the intricacies in viral biology, evolution and replication. This in turn has made a phenomenal progress in identifying the nuances in host-pathogen interactions, thereby establishing new insights in molecular pathogenesis. The complex interplay of viral moieties and the cellular mechanisms that respond to these variations has been simplified to a greater extent with the advent of reverse genetics, and that has changed the way the virulence mechanisms of virus have been addressed so far. All copyrights reserved to Nexus® academic publishers
Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs
Proceedings of the National Academy of Sciences, 1996
We provide the first report, to our knowledge, of a helper-independent system for rescuing a segmented, negative-strand RNA genome virus entirely from cloned cDNAs. Plasmids were constructed containing fulllength cDNA copies of the three Bunyamwera bunyavirus RNA genome segments f lanked by bacteriophage T7 promoter and hepatitis delta virus ribozyme sequences. When cells expressing both bacteriophage T7 RNA polymerase and recombinant Bunyamwera bunyavirus proteins were transfected with these plasmids, full-length antigenome RNAs were transcribed intracellularly, and these in turn were replicated and packaged into infectious bunyavirus particles. The resulting progeny virus contained specific genetic tags characteristic of the parental cDNA clones. Reassortant viruses containing two genome segments of Bunyamwera bunyavirus and one segment of Maguari bunyavirus were also produced following transfection of appropriate plasmids. This accomplishment will allow the full application of recombinant DNA technology to manipulate the bunyavirus genome.
Reverse Genetics of the Negative-Sense Influenza A Virus : Aspects and Applications
2015
Reverse genetics, a technique used to engineer specific mutations into viral genomes, was first performed for DNA viruses, either by transfecting cells with plasmids encoding the viral genome or by heterologous recombination of plasmids bearing viral sequences with the virus genome [1,2]. They were followed by manipulations of positive-sense RNA genomes. Transfection of plasmids, or RNA transcribed from plasmids, containing the poliovirus genome, into susceptible cells led to recovery of infectious poliovirus [3,4]. However, the genomes of negative-sense RNA viruses were less amenable to artificial manipulations in comparison with the DNA and positive-sense RNA viruses. In contrast to positive-sense RNA viruses, the genome of which is also a functional messenger RNA (mRNA), the naked genomic RNA of a negative-sense RNA virus is not able to initiate infection when expressed in or transfected into a permissive cell line. Their genomes are the complement of mRNA and therefore cannot be...
A decade after the generation of a negative-sense RNA virus from cloned cDNA - what have we learned?
The Journal of general virology, 2002
Since the first generation of a negative-sense RNA virus entirely from cloned cDNA in 1994, similar reverse genetics systems have been established for members of most genera of the Rhabdo- and Paramyxoviridae families, as well as for Ebola virus (Filoviridae). The generation of segmented negative-sense RNA viruses was technically more challenging and has lagged behind the recovery of nonsegmented viruses, primarily because of the difficulty of providing more than one genomic RNA segment. A member of the Bunyaviridae family (whose genome is composed of three RNA segments) was first generated from cloned cDNA in 1996, followed in 1999 by the production of influenza virus, which contains eight RNA segments. Thus, reverse genetics, or the de novo synthesis of negative-sense RNA viruses from cloned cDNA, has become a reliable laboratory method that can be used to study this large group of medically and economically important viruses. It provides a powerful tool for dissecting the virus l...
Gene rearrangement attenuates expression and lethality of a nonsegmented negative strand RNA virus
Proceedings of the National Academy of Sciences, 1998
The nonsegmented negative strand RNA viruses comprise hundreds of human, animal, insect, and plant pathogens. Gene expression of these viruses is controlled by the highly conserved order of genes relative to the single transcriptional promoter. We utilized this regulatory mechanism to alter gene expression levels of vesicular stomatitis virus by rearranging the gene order. This report documents that gene expression levels and the viral phenotype can be manipulated in a predictable manner. Translocation of the promoter-proximal nucleocapsid protein gene N, whose product is required stoichiometrically for genome replication, to successive positions down the genome reduced N mRNA and protein expression in a stepwise manner. The reduction in N gene expression resulted in a stepwise decrease in genomic RNA replication. Translocation of the N gene also attenuated the viruses to increasing extents for replication in cultured cells and for lethality in mice, without compromising their ability to elicit protective immunity. Because monopartite negative strand RNA viruses have not been reported to undergo homologous recombination, gene rearrangement should be irreversible and may provide a rational strategy for developing stably attenuated live vaccines against this type of virus.
Vaccines
The current pandemic has highlighted the ever-increasing risk of human to human spread of zoonotic pathogens. A number of medically-relevant zoonotic pathogens are negative-strand RNA viruses (NSVs). NSVs are derived from different virus families. Examples like Ebola are known for causing severe symptoms and high mortality rates. Some, like influenza, are known for their ease of person-to-person transmission and lack of pre-existing immunity, enabling rapid spread across many countries around the globe. Containment of outbreaks of NSVs can be difficult owing to their unpredictability and the absence of effective control measures, such as vaccines and antiviral therapeutics. In addition, there remains a lack of essential knowledge of the host–pathogen response that are induced by NSVs, particularly of the immune responses that provide protection. Vaccines are the most effective method for preventing infectious diseases. In fact, in the event of a pandemic, appropriate vaccine design ...
A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses
Nature Communications, 2021
The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular fulllength viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.