Development of viral infectious clones and their applications based on yeast and bacterial artificial chromosome platforms

Abstract

Infectious Clones represent a foundational technique in the field of reverse genetics, allowing for the construction and manipulation of full-length viral genomes. The main methods currently used for constructing viral infectious clones include Transformation-associated recombination (TAR), which is based on Yeast Artificial Chromosome (YAC) and Bacterial Artificial Chromosome (BAC). The YAC and BAC systems are powerful tools that enable the clones and manipulation of large DNA fragments, making them well-suited for the construction of full-length viral genomes. These methods have been successfully applied to construct infectious clones for a wide range of viruses, including coronaviruses, herpesviruses, flaviviruses and baculoviruses. The rescued recombinant viruses from these infectious clones have been widely used in various research areas, such as vaccine development, antiviral drug screening, pathogenesis and virulence studies, gene therapy and vector design. However, as different viruses possess unique biological characteristics, the challenge remains in how to rapidly obtain infectious clones for future research. In summary, this review introduced the development and applications of infectious clones, with a focus on the YAC, BAC and combined YAC-BAC technologies. We emphasize the importance of these platforms in various research areas and aim to provide deeper insights that can advance the platform and broaden its application horizons.

Keywords: Application; Bacterial artificial chromosome (BAC); Reverse genetics; Virus Infectious Clones; Yeast artificial chromosome (YAC).

Fig. 1

Overview of the strategies and processes involved in constructing viral infectious clones using Yeast Artificial Chromosome (YAC), Bacterial Artificial Chromosome (BAC), and YAC-BAC combined systems. a The full-length viral genome is amplified from clinical isolated viruses or sequence databases by PCR or chemical synthesis. Then the genome is divided into overlapping fragments and ligated with a promoter for later experiments. b YAC based systems. Overlapping DNA fragments were delivered into S.cerevisiae yeast along with a linearized YAC/TAR vector. Homologous recombination in the yeast cells assembles the DNA fragments to generate the YAC vector containing the viral full-length cDNA. The infectious clones are then produced in vitro by plasmid linearization and run-off T7 RNA polymerase-based transcription. Virus rescue is initiated by transfection of RNA into susceptible cells, followed by virus production and amplification. c BAC based systems. Restriction endonucleases were used to insert the DNA fragments into a BAC vector, and ligation to generate the viral full-length cDNA. The recombinant constructs are transformed into E. Coli for selective cultivation, and positive transformants are amplified and transfected into host cells for virus rescue. d YAC-BAC systems. Overlapping DNA fragments are delivered into yeast cells along with a linearized YAC vector, and all DNA fragments are assembled by homologous recombination to generate the YAC plasmid containing the viral full-length cDNA. The YAC plasmid is then transformed into E. Coli to produce a BAC plasmid, which is subsequently used to rescue recombinant viruses by transfecting it into susceptible cells

Fig. 2

Overview of recombinant viruses generated by infectious cloning and their applications in biomedical research, such as vaccine development, antiviral drug discovery, pathogenesis and virulence studies, gene therapy and vector design. a Examples for recombinant virus models in virology research, including replicons, oncolytic viruses, reporter gene viruses, and viral vectors. b The application of infectious cloning in vaccine development. Recombinant attenuated viruses constructed by infectious cloning can protect the challenge of wild type viruses in animal models. For example, in a mouse model, those immunized with a recombinant attenuated virus survived from wild-type virus challenge, while the control mice immunized with PBS did not. Survival curve and pathological results indicate that the recombinant virus could serve as a candidate attenuated live vaccine. c Recombinant viruses can facilitate antiviral drugs discovery. By culturing recombinant virus with candidate antiviral drugs, researchers can evaluate the efficacy of these drugs. The use of recombinant virus with specific advantages (e.g., reporter genes, attenuation), allows for the statistically determination of the half-maximum inhibitory concentration of the drugs. d The recombinant viruses for viral pathogenesis and virulence. By comparing the recombinant virus with specific mutation to their wild-type viruses, researchers can evaluate differences in growth characteristics and virulence in vitro and in vivo. e Recombinant viruses for gene therapy and vector design. The virus recombinant virus can be used in vivo, allowing for the analysis of various tissue samples and detection of therapeutic effects

Fig. 3

Overview of the types of oncolytic viruses and their mechanisms in targeting and destroying cancer cells. Oncolytic viruses that are commonly used in anti-tumor research including poliovirus, herpesvirus, poxvirus, adenovirus, measles virus, and coxsackievirus. There are mainly two aspects of oncolytic viruses for anti-tumor effect. One is direct oncolytic effect. Oncolytic viruses specifically replicate in tumor cells, it causes lysis of the tumor cells. The other is activation of antitumor immunity, immunogenic cell death induced by oncolytic virus exposure leads to the release of various molecules and recruitment of immune cells such as macrophages and dendritic cells, inducing the body to produce an immune response