A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

The Zika virus (ZIKV) has recently attracted major research interest as infection was unexpectedly associated with neurological manifestations in developing foetuses and with Guillain-Barré syndrome in infected adults. Understanding the underlying molecular mechanisms requires reverse genetic systems, which allow manipulation of infectious cDNA clones at will. In the case of flaviviruses, to which ZIKV belongs, several reports have indicated that the construction of full-length cDNA clones is difficult due to toxicity during plasmid amplification in Escherichia coli. Toxicity of flaviviral cDNAs has been linked to the activity of cryptic prokaryotic promoters within the region encoding the structural proteins leading to spurious transcription and expression of toxic viral proteins. Here, we employ an approach based on in silico prediction and mutational silencing of putative promoters to generate full-length cDNA clones of the historical MR766 strain and the contemporary French Polynesian strain H/PF/2013 of ZIKV. While for both strains construction of full-length cDNA clones has failed in the past, we show that our approach generates cDNA clones that are stable on single bacterial plasmids and give rise to infectious viruses with properties similar to those generated by other more complex assembly strategies. Further, we generate luciferase and fluorescent reporter viruses as well as sub-genomic replicons that are fully functional and suitable for various research and drug screening applications. Taken together, this study confirms that in silico prediction and silencing of cryptic prokaryotic promoters is an efficient strategy to generate full-length cDNA clones of flaviviruses and reports novel tools that will facilitate research on ZIKV biology and development of antiviral strategies.

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Tet-Inducible Production of Infectious Zika Virus from the Full-Length cDNA Clones of African- and Asian-Lineage Strains

Viruses ◽

10.3390/v10120700 ◽

2018 ◽

Vol 10 (12) ◽

pp. 700 ◽

Cited By ~ 5

Author(s):

Lizhou Zhang ◽

Wei Ji ◽

Shuang Lyu ◽

Luhua Qiao ◽

Guangxiang Luo

Keyword(s):

Zika Virus ◽

Reverse Genetics ◽

Genetic System ◽

Full Length ◽

Reverse Genetic System ◽

Cdna Clones ◽

Viral Pathogen ◽

Stable Plasmid ◽

Asian Lineage ◽

Delta Virus

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as an important human viral pathogen, causing congenital malformation including microcephaly among infants born to mothers infected with the virus during pregnancy. Phylogenetic analysis suggested that ZIKV can be classified into African and Asian lineages. In this study, we have developed a stable plasmid-based reverse genetic system for robust production of both ZIKV prototype African-lineage MR766 and clinical Asian-lineage FSS13025 strains using a tetracycline (Tet)-controlled gene expression vector. Transcription of the full-length ZIKV RNA is under the control of the Tet-responsive Ptight promoter at the 5′ end and an antigenomic ribozyme of hepatitis delta virus at the 3′ end. The transcription of infectious ZIKV RNA genome was efficiently induced by doxycycline. This novel ZIKV reverse genetics system will be valuable for the study of molecular viral pathogenesis of ZIKV and the development of new vaccines against ZIKV infection.

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Functional Identification of Salt-Stress-Related Genes Using the FOX Hunting System from Ipomoea pes-caprae

International Journal of Molecular Sciences ◽

10.3390/ijms19113446 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3446 ◽

Cited By ~ 6

Author(s):

Mei Zhang ◽

Hui Zhang ◽

Jie-Xuan Zheng ◽

Hui Mo ◽

Kuai-Fei Xia ◽

...

Keyword(s):

Salt Stress ◽

Salt Tolerance ◽

Cdna Library ◽

Molecular Mechanisms ◽

Full Length ◽

Cdna Clones ◽

Functional Screening ◽

Yeast Mutant ◽

Full Length Cdna ◽

Stress Related Genes

Ipomoea pes-caprae is a seashore halophytic plant and is therefore a good model for studying the molecular mechanisms underlying salt and stress tolerance in plant research. Here, we performed Full-length cDNA Over-eXpressor (FOX) gene hunting with a functional screening of a cDNA library using a salt-sensitive yeast mutant strain to isolate the salt-stress-related genes of I. pes-caprae (IpSR genes). The library was screened for genes that complemented the salt defect of yeast mutant AXT3 and could grow in the presence of 75 mM NaCl. We obtained 38 candidate salt-stress-related full-length cDNA clones from the I. pes-caprae cDNA library. The genes are predicted to encode proteins involved in water deficit, reactive oxygen species (ROS) scavenging, cellular vesicle trafficking, metabolic enzymes, and signal transduction factors. When combined with the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, several potential functional salt-tolerance-related genes were emphasized. This approach provides a rapid assay system for the large-scale screening of I. pes-caprae genes involved in the salt stress response and supports the identification of genes responsible for the molecular mechanisms of salt tolerance.

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In silico prediction of a highly immunogenic and conserved epitope against Zika Virus

Informatics in Medicine Unlocked ◽

10.1016/j.imu.2021.100613 ◽

2021 ◽

pp. 100613

Author(s):

Debasish Paul ◽

Imdadul Haque Sharif ◽

Abu Sayem ◽

Hossain Ahmed ◽

Abu Saleh ◽

...

Keyword(s):

Zika Virus ◽

In Silico ◽

In Silico Prediction ◽

Conserved Epitope

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Cloning and characterization of full-length cDNA clones encoding chalcone synthase from the orchid Bromheadia finlaysoniana

Plant Physiology and Biochemistry ◽

10.1016/s0981-9428(98)80013-2 ◽

1998 ◽

Vol 36 (9) ◽

pp. 647-656 ◽

Cited By ~ 18

Author(s):

Chye-Fong Liew ◽

Chong-Jin Goh ◽

Chiang-Shiong Loh ◽

Saw-Hoon Lim

Keyword(s):

Chalcone Synthase ◽

Full Length ◽

Cdna Clones ◽

Full Length Cdna

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Infectious Transcripts from Cloned Genome-Length cDNA of Porcine Reproductive and Respiratory Syndrome Virus

Journal of Virology ◽

10.1128/jvi.72.1.380-387.1998 ◽

1998 ◽

Vol 72 (1) ◽

pp. 380-387 ◽

Cited By ~ 82

Author(s):

J. J. M. Meulenberg ◽

J. N. A. Bos-de Ruijter ◽

R. van de Graaf ◽

G. Wensvoort ◽

R. J. M. Moormann

Keyword(s):

Cdna Clone ◽

De Novo ◽

Infectious Clone ◽

Full Length ◽

Respiratory Syndrome Virus ◽

Cdna Clones ◽

Genome Length ◽

Full Length Cdna ◽

Lung Macrophages ◽

Growth Properties

ABSTRACT The 5′-terminal end of the genomic RNA of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was determined. To construct full-length cDNA clones, the 5′-terminal sequence was ligated to cDNA clones covering the complete genome of LV. When RNA that was transcribed in vitro from these full-length cDNA clones was transfected into BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells, no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. Two nucleotide changes leading to a unique PacI restriction site directly downstream of the ORF7 gene were introduced in the genome-length cDNA clone. The virus recovered from this mutated cDNA clone retained the PacI site, which confirmed the de novo generation of infectious LV from cloned cDNA. These results indicate that the infectious clone of LV enables us to mutagenize the viral genome at specific sites and that it will therefore be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

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