Simple reverse genetics systems for Asian and African Zika viruses

Atieh, Thérèse; Baronti, Cécile; Lamballerie, Xavier de; Nougairède, Antoine

doi:10.1038/srep39384

Cited by 59 publications

(76 citation statements)

References 30 publications

(38 reference statements)

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“…A series of approaches (14,15,24,28,(50)(51)(52) have been applied for the generation of ZIKV reverse genetics platforms. Since direct assembly of the native ZIKV full genome in E. coli had been successful only in a few cases (14,50), most established reverse genetics systems of ZIKV were based on either in vitro assembly of viral cDNA fragments (24,52) or eukaryotic intron-based and HCMV promoter-driven, "infectious DNA" clones (15,28).…”

Section: Discussionmentioning

confidence: 99%

Characterization of cis -Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone

Liu

Huang

et al. 2017

J Virol

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Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5=-SLA promoter and 5=-3= cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses.IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the E. coli host. Moreover, two crucial cis-acting replication elements (5=-SLA and 5=-CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses.

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Section: Discussionmentioning

confidence: 99%

Characterization of cis -Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone

Liu

Huang

et al. 2017

J Virol

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“…Determination of DENV1 titer was done using 50% tissue culture infectious dose -cytopathic effect (TCID50-CPE) as previously described (Atieh et al, 2016;Li et al, 2011). DENV1 (Hawaiian strain) used in this study was a gift from David Perera, University Malaysia Sarawak.…”

Section: Cell Culture and Virusmentioning

confidence: 99%

Revised annotation and characterization of novelAedes albopictusmiRNAs and their potential functions in dengue virus infection

Azlan

Yunus

Azzam

2020

Preprint

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The Asian tiger mosquito, Aedes albopictus (Ae. albopictus), is a highly invasive species that transmit several arboviruses including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV).Although several studies have identified microRNAs (miRNAs) in Ae. albopictus, it is crucial to extend and improve current annotations with the newly improved genome assembly, and the increase number of small RNA-sequencing data. We combined our high-depth sequence data and 26 public datasets to re-annotate Ae. albopictus miRNAs, and found a total of 110 novel mature miRNAs. We discovered that the expression of novel miRNAs was lower than known miRNAs. Furthermore, compared to known miRNAs, novel miRNAs are prone to be expressed in stage-specific manner. Upon DENV infection, a total of 59 novel miRNAs were differentially expressed, and target prediction analysis revealed that miRNA-target genes were involved in lipid metabolism and protein processing in endoplasmic reticulum. Taken together, miRNA annotation profile provided here is the most comprehensive to date, and we believed that this will facilitate future research in understanding virushost interactions particularly on the role of miRNAs. during virus infection, and at diapause condition (Avila-Bonilla et al., 2017;Batz et al., 2017;Liu et al., 2015;Su et al., 2019Su et al., , 2017. These studies suggest the involvement of miRNAs in the regulation of Ae. albopictus development and virus infection.High quality genome coupled with high-depth RNA-sequencing data are important for accurate and comprehensive annotation of coding and non-coding genes. Alignment of small RNA reads to the reference genome is an essential step to annotate miRNAs in a species. Several studies used genome of closely related species as proxy reference to facilitate the miRNA expression analysis (Etebari et al.,

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“…Dengue virus serotype 1 (Hawaiian strain) and Zika virus (Strain H/PF/2013), were propagated in C6/36 cells and titered using BHK-21 cells. Determination of DENV1 titer was done using 50% tissue culture infectious dose -cytopathic effect (TCID50-CPE) as previously described (Li et al, 2011;Atieh et al, 2016). DENV1 used in this study was a gift from Dr. David Perera, University Malaysia Sarawak.…”

Section: Cell Culture and Virusmentioning

confidence: 99%

Identification and characterization of Aedes albopictus long noncoding RNAs provides insights into their roles in development and flavivirus infection

Azlan

Yunus

Azzam

2018

Preprint

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Aedes albopictus (Ae. albopictus) is an important vector of arboviruses such as Dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Long noncoding RNA (lncRNAs) have been identified in other vectors including Aedes aegypti and Anopheles mosquitoes, few of which have been implicated in immunity and viral replication. To identify lncRNAs with potential biological functions in Ae. albopictus, we performed RNA-seq on Ae. albopictus cells infected with DENV and ZIKV, and analyzed them together with public datasets. We identified a total of 23,899 transcripts, 16,089 were intergenic while 3,126 and 4,183 of them were antisense and intronic to annotated genes respectively. Ae. albopictus lncRNAs shared many of the characteristics with their invertebrate and vertebrate counterparts, such as low expression, low GC content, short in length, and low conservation even among closely related species. Compared to protein-coding genes, lncRNAs exhibited higher tendency to be expressed in a stage-specific manner. Besides, expression of lncRNAs and nearest protein-coding genes tended to be correlated, especially for the gene pairs within 1kb from each other.We also discovered that Ae. albopictus lncRNAs have the potential to act as precursors for miRNA and piRNAs, both of which have been implicated in antiviral defense in Aedes mosquito. Upon flavivirus infection, lncRNAs were observed to be differentially expressed, which possibly indicates the involvement of lncRNAs in the host-antiviral defense. Our study provides the first systematic identification of lncRNAs in Ae. albopictus, hence, offering a foundation for future studies of lncRNA functions.

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Simple reverse genetics systems for Asian and African Zika viruses

Cited by 59 publications

References 30 publications

Characterization of cis -Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone

Characterization of cis -Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone

Revised annotation and characterization of novelAedes albopictusmiRNAs and their potential functions in dengue virus infection

Identification and characterization of Aedes albopictus long noncoding RNAs provides insights into their roles in development and flavivirus infection

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