Construction of an infectious cDNA clone of Enterovirus 71: Insights into the factors ensuring experimental success

Lazouskaya, Natallia V.; Palombo, Enzo A.; Poh, Chit Laa; Barton, Peter

doi:10.1016/j.jviromet.2013.12.005

Cited by 8 publications

(4 citation statements)

References 40 publications

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“…In previous studies, infectious clones were successfully developed for a number of enteroviruses with different methods. EV71 or CA16 cDNA clones were established by assembling segmented cDNA fragments of the viral genome into a DNA vector via specific cleavage sites [15, 23, 24], and CA6 or Echo25 cDNA clones were constructed by utilizing In-Fusion Cloning to assemble the full length of viral genome and vector, which was difficult to operate. [16, 19].…”

Section: Discussionmentioning

confidence: 99%

Construction and characterization of an infectious cDNA clone of coxsackievirus A 10

Liu

Dan

Zhao

et al. 2019

Virol J

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Background Coxsackievirus A10 (CA10) constitutes one of the four major pathogens causing hand, foot and mouth disease in infants. Infectious clones are of great importance for studying viral gene functions and pathogenic mechanism. However, there is no report on the construction of CA10 infectious clones. Methods The whole genome of CA10 derived from a clinical isolate was amplified into two fragments and ligated into a linearized plasmid vector in one step by In-Fusion Cloning. The obtained CA10 cDNA clones and plasmids encoding T7 RNA polymerase were co-transfected into 293 T cells to rescue CA10 virus. The rescued virus was identified by SDS-PAGE, Western blotting and transmission electron microscopic. One-day-old ICR mice were intracerebrally inoculated with the CA10 virus and clinical symptoms were observed. Multiple tissues of moribund mice were harvested for analysis of pathogenic changes and viral distribution by using H&E staining, real-time PCR and immunohistochemical staining. Results CA10 viruses were rescued from the constructed cDNA clone and reached a maximum titer of 10 8.125 TCID 50 /mL after one generation in RD cells. The virus exhibited similar physical and chemical properties to those of the parental virus. It also showed high virulence and the ability to induce death of neonatal ICR mice. Severe necrotizing myositis, intestinal villus interstitial edema and severe alveolar shrinkage were observed in infected mice. The viral antigen and the maximum amount of viral RNA were detected in limb skeletal muscles, which suggested that the limb skeletal muscles were the most likely site of viral replication. Conclusion Infectious clones of CA10 were successfully constructed for the first time, which will facilitate the establishment of standardized neonatal mouse models infected with CA10 for the evaluation of vaccines and antiviral drugs, as well as preservation and sharing of model strains.

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Section: Discussionmentioning

confidence: 99%

Construction and characterization of an infectious cDNA clone of coxsackievirus A 10

Liu

Dan

Zhao

et al. 2019

Virol J

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“…Multiple studies suggest that transfection of in vitro synthesized infectious RNA with precise 3’ ends showed no difference in virus titers when compared with infectious RNA with additional non-viral nucleotides at the 3’ end [ 45 , 53 ]. However, our data demonstrated that the presence of a self-cleavage HDV ribozyme sequence at the 3’ end significantly increased overall virus yield of the DNA-launched infectious clone after transfection.…”

Section: Discussionmentioning

confidence: 99%

Enterovirus A71 DNA-Launched Infectious Clone as a Robust Reverse Genetic Tool

et al. 2016

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Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot and mouth disease, and is occasionally associated with neurological complications and death in children. Reverse genetics is widely used in the field of virology for functional study of viral genes. For EV-A71, such tools are limited to clones that are transcriptionally controlled by T7/SP6 bacteriophage promoter. This is often time-consuming and expensive. Here, we describe the development of infectious plasmid DNA-based EV-A71 clones, for which EV-A71 genome expression is under transcriptional control by the CMV-intermediate early promoter and SV40 transcriptional-termination signal. Transfection of this EV-A71 infectious DNA produces good virus yield similar to in vitro-transcribed EV-A71 infectious RNA, 6.4 and 5.8 log10PFU/ml, respectively. Infectious plasmid with enhanced green fluorescence protein and Nano luciferase reporter genes also produced good virus titers, with 4.3 and 5.0 log10 PFU/ml, respectively. Another infectious plasmid with both CMV and T7 promoters was also developed for easy manipulation of in vitro transcription or direct plasmid transfection. Transfection with either dual-promoter infectious plasmid DNA or infectious RNA derived from this dual-promoter clone produced infectious viral particles. Incorporation of hepatitis delta virus ribozyme, which yields precise 3’ ends of the DNA-launched EV-A71 genomic transcripts, increased infectious viral production. In contrast, the incorporation of hammerhead ribozyme in the DNA-launched EV-A71 resulted in lower virus yield, but improved the virus titers for T7 promoter-derived infectious RNA. This study describes rapid and robust reverse genetic tools for EV-A71.

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“…Barring a gross genetic defect, the sampled RNA genomes found in vivo are functional in vitro . Studies of Enterovirus 71 have shown that cloning from half genomes and construction of recombinant genomes versus cloning from full-length PCR amplicons led to less functional clones from the same virus stock which again suggests that laboratory-induced recombination is detrimental to virus integrity [25]. Although viruses such as HIV-1 can often generate recombinants in vivo , these likely emerge only after in vivo selection for replication competent viruses and that in the absence of this selection in vitro, constructed chimeras will more likely produce defective genomes.…”

Section: Discussionmentioning

confidence: 99%

Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

et al. 2014

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The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that could be universally applied to the study of human viral pathogens.

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Construction of an infectious cDNA clone of Enterovirus 71: Insights into the factors ensuring experimental success

Cited by 8 publications

References 40 publications

Construction and characterization of an infectious cDNA clone of coxsackievirus A 10

Construction and characterization of an infectious cDNA clone of coxsackievirus A 10

Enterovirus A71 DNA-Launched Infectious Clone as a Robust Reverse Genetic Tool

Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

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