scholarly journals Construction of a Full-length Infectious Clone of Zika Virus Stably Expressing EGFP Marker in a Eukaryotic Expression System

2020 ◽  
Author(s):  
Jing Gao ◽  
Lingjuan Shi ◽  
Jiayi Chen ◽  
Weizhi Lu ◽  
Jingtai Cai ◽  
...  
Keyword(s):  
Zika Virus ◽  
Reverse Genetics ◽  
Infectious Clone ◽  
Expression System ◽  
Plaque Assay ◽  
Vero Cells ◽  
Full Length ◽  
Egfp Expression ◽  
Wild Type ◽  
Significant Difference

Abstract Background: Zika virus is among the most widely transmitted arboviruses in the world and closely associated with diseases, such as encephalitis, fetal microcephaly, and Guillain–Barré syndrome. The pathogenic mechanism of the virus has not been fully elucidated, and there are no vaccines or specific drugs targeting the virus. To address these issues, the application of reverse genetics is needed for viral reconstruction and reproduction.Methods: Polymerase chain reaction (PCR) was used to merge the full-length Zika virus genome, CMV promoter, intron, EGFP, hepatitis delta virus ribozyme, and SV40 terminator sequence for cloning into a pBAC11 vector through recombination to produce recombinant pBAC-ZIKA-EGFP. The ZIKA–EGFP was rescued by transfection of 293T cells with pBAC-ZIKA-EGFP, and at 7-days post-transfection, the supernatant (P0 generation) was passed through a 0.45-μm membrane and used to infect Vero cells (to produce the P1 generation). Fluorescence-based quantitative PCR, 50% tissue culture infectious dose, and plaque assays were used to measure differences in replication ability and pathogenicity relative to the rescue virus (ZIKA–WT), the sequence of which is consistent with that of the wild-type Zika virus. Additionally, caffeic acid phenethyl ester (CAPE), a nuclear factor kappaB (NF-kB) inhibitor, was used to examine its effect on viral replication.Results: The results showed that ZIKA–EGFP could effectively infect Vero cells, SH-SY5Y cells and C6/36 cells, and cause cytopathic effects on them. ZIKA–EGFP exhibited stable replication and EGFP expression during cell passage for at least six generations, with no significant difference in replication ability relative to the ZIKA–WT. Fluorescent cell foci were observed in the plaque assay while the ZIKA–EGFP was in the absence of phage plaque formation. The inhibition of NF-kB inhibitor on ZIKA-EGFP was observed by fluorescence microscopy, which was consistent with the results of fluorescence quantitative PCR.Conclusions: We constructed an infectious clone of the full-length genome of Zika virus which could replicate with stable EGFP expression in eukaryotic cells during passage. The infectious clone, remaining main characteristics of wild type ZIKA virus could be appied on the studies of reverse genetics, drug screening and gene function of ZIKA virus.

scholarly journals Construction of an infectious clone of Zika virus stably expressing an EGFP marker in a eukaryotic expression system

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Jing Gao ◽  
Jiayi Chen ◽  
Weizhi Lu ◽  
Jintai Cai ◽  
Linjuan Shi ◽  
...  
Keyword(s):  
Zika Virus ◽  
Infectious Clone ◽  
Expression System ◽  
Plaque Assay ◽  
Experimental System ◽  
Genetic System ◽  
Vero Cells ◽  
Eukaryotic Expression ◽  
Reverse Genetic System ◽  
Cmv Promoter

Abstract Background Zika virus is becoming one of the most widely transmitted arboviruses in the world. Development of antiviral inhibitor and vaccine requires an experimental system that allows rapid monitoring of the virus infection. This is achievable with a reverse genetic system. In this study, we constructed an infectious clone for Zika virus that stably expressing EGFP. Methods A PCR-mediated recombination approach was used to assemble the full-length Zika virus genome containing the CMV promoter, intron, EGFP, hepatitis delta virus ribozyme, and SV40 terminator sequence for cloning into the pBAC11 vector to produce recombinant pBAC-ZIKA-EGFP. ZIKA-EGFP virus was rescued by transfection of pBAC-ZIKA-EGFP into 293T cells. The characterization of ZIKA-EGFP virus was determined by qPCR, plaque assay, CCK-8, and Western blot. Results Rescued ZIKA-EGFP virus exhibited stable replication for at least five generations in tissue culture. ZIKA-EGFP can effectively infect C6/36, SH-SY5Y and Vero cells, and cause cytopathic effects on SH-SY5Y and Vero cells. The inhibition of ZIKA-EGFP by NF-κB inhibitor, caffeic acid phenethyl ester was observed by fluorescence microscopy. Conclusion Our results suggested that Zika virus infectious clone with an EGFP marker retained it infectivity as wide-type Zika virus which could be used for drugs screening.

scholarly journals Development of a reverse-genetics system for Avian pneumovirus demonstrates that the small hydrophobic (SH) and attachment (G) genes are not essential for virus viability

2004 ◽  
Vol 85 (11) ◽  
pp. 3219-3227 ◽  
Author(s):  
Clive J. Naylor ◽  
Paul A. Brown ◽  
Nicole Edworthy ◽  
Roger Ling ◽  
Richard C. Jones ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Vero Cells ◽  
Full Length ◽  
Wild Type Virus ◽  
Wild Type ◽  
T7 Promoter ◽  
Restriction Endonuclease Site ◽  
Avian Pneumovirus ◽  
Full Length Genome ◽  
Small Hydrophobic

Avian pneumovirus (APV) is a member of the genus Metapneumovirus of the subfamily Pneumovirinae. This study describes the development of a reverse-genetics system for APV. A minigenome system was used to optimize the expression of the nucleoprotein, phosphoprotein, M2 and large polymerase proteins when transfected into Vero cells under the control of the bacteriophage T7 promoter. Subsequently, cDNA was transcribed from the virion RNA to make a full-length antigenome, which was also cloned under the control of the T7 promoter. Transfection of the full-length genome plasmid, together with the plasmids expressing the functional proteins in the transcription and replication complex, generated APV in the transfected cells. The recombinant virus was passaged and was identified by cytopathic effect (CPE) that was typical of APV, the presence of a unique restriction-endonuclease site in the cDNA copy of the genome and immunofluorescence staining with anti-APV antibodies. Replacement of the full-length wild-type antigenome with one lacking the small hydrophobic (SH) protein and the attachment (G) genes generated a virus that grew more slowly and produced atypical CPE with syncytia much larger than those seen with wild-type virus.

scholarly journals An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 547 ◽  
Author(s):  
Silvia Márquez-Jurado ◽  
Aitor Nogales ◽  
Ginés Ávila-Pérez ◽  
Francisco Iborra ◽  
Luis Martínez-Sobrido ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Zika Virus ◽  
Rna Synthesis ◽  
Infectious Clone ◽  
Vero Cells ◽  
Viral Rna ◽  
Single Amino Acid ◽  
Reverse Genetic System ◽  
Infectious Clones

The recent outbreaks of Zika virus (ZIKV), its association with Guillain–Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies.

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

Viruses ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 368 ◽  
Author(s):  
Maximilian Münster ◽  
Anna Płaszczyca ◽  
Mirko Cortese ◽  
Christopher Neufeldt ◽  
Sarah Goellner ◽  
...  
Keyword(s):  
Zika Virus ◽  
In Silico ◽  
Reverse Genetics ◽  
Molecular Mechanisms ◽  
Full Length ◽  
Cdna Clones ◽  
In Silico Prediction ◽  
Fluorescent Reporter ◽  
Full Length Cdna ◽  
Spurious Transcription

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.

scholarly journals Volume and surface changes in vero cell and its nucleus after infection with measles virus: a stereological study

2011 ◽  
Vol 2 (1) ◽  
pp. 18
Author(s):  
Ali Noorafshan ◽  
Mohammad Motamedifar ◽  
Saied Karbalay-Doust
Keyword(s):  
Vero Cell ◽  
Post Infection ◽  
Measles Virus ◽  
Plaque Assay ◽  
Vero Cells ◽  
Uninfected Control ◽  
Mean Cell Volume ◽  
Significant Difference ◽  
Infected Cells ◽  
The Mean

Measles virus has no or indistinctive cytopathic effects (CPE) in cell couture system. Employment of some detecting methods like plaque assay or stereologic experiments, as a method of detecting of viral infection in the cells would be applicable. The aim of this study was investigating the early changes in quantitative parameters of measles virus infected Vero cells. Stereological methods using invariator, were applied for the first time to estimate cell and nucleus volume and cell surface of the infected Vero cell line with the measles virus.This method can be applied on other cultured cells.Vero cells grown in tissue culture plates for 48 hours at 36˚C were infected with 100TCID50 of AiK strain of measles virus. Volume and surface of the infected Vero cells were studied at 4, 9 and 25 hours post infection along with uninfected control cells. The mean cell volume and surface of the cells infected with measles virus, increased ~87% and ~50%, respectively, 4 hours post-infection, as compared with the uninfected control. The nuclei did not show any differences. The mean parameters of infected cells in other time intervals showed no significant difference comparing with the control cells. Although there are other specific methods, stereology may be used as an integrated protocol to detect cytophatic changes of the measles virus infected cells early in the permissive cell culture system.

scholarly journals Leu-to-Phe substitution at prM146 decreases the growth ability of Zika virus and partially reduces its pathogenicity in mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takuya Inagaki ◽  
Satoshi Taniguchi ◽  
Yasuhiro Kawai ◽  
Takahiro Maeki ◽  
Eri Nakayama ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zika Virus ◽  
Reverse Genetics ◽  
Cultured Cells ◽  
Congenital Infection ◽  
Febrile Illness ◽  
Vero Cells ◽  
Lower Growth ◽  
Asian Strain ◽  
Growth Ability

AbstractZika virus (ZIKV) is a mosquito-borne flavivirus that causes febrile illness. The recent spread of ZIKV from Asia to the Americas via the Pacific region has revealed unprecedented features of ZIKV, including transplacental congenital infection causing microcephaly. Amino acid changes have been hypothesized to underlie the spread and novel features of American ZIKV strains; however, the relationship between genetic changes and the epidemic remains controversial. A comparison of the characteristics of a Southeast Asian strain (NIID123) and an American strain (PRVABC59) revealed that the latter had a higher replication ability in cultured cells and higher virulence in mice. In this study, we aimed to identify the genetic region of ZIKV responsible for these different characteristics using reverse genetics. A chimeric NIID123 strain in which the E protein was replaced with that of PRVABC59 showed a lower growth ability than the recombinant wild-type strain. Adaptation of the chimeric NIID123 to Vero cells induced a Phe-to-Leu amino acid substitution at position 146 of the prM protein; PRVABC59 also has Leu at this position. Leu at this position was found to be responsible for the viral replication ability and partially, for the pathogenicity in mouse testes.

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

Viruses ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 700 ◽  
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.

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

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Zhong-Yu Liu ◽  
Jiu-Yang Yu ◽  
Xing-Yao Huang ◽  
Hang Fan ◽  
Xiao-Feng Li ◽  
...  
Keyword(s):  
Zika Virus ◽  
Reverse Genetics ◽  
Infectious Clone ◽  
Neurological Complications ◽  
Cdna Clones ◽  
Content Type ◽  
Infectious Clones ◽  
Global Concern ◽  
Rna Elements ◽  
Self Splicing

ABSTRACT 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.

scholarly journals Chloroquine Inhibits Dengue Virus Type 2 Replication in Vero Cells but Not in C6/36 Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Kleber Juvenal Silva Farias ◽  
Paula Renata Lima Machado ◽  
Benedito Antônio Lopes da Fonseca
Keyword(s):  
Dengue Virus ◽  
Virus Type ◽  
Mammalian Cells ◽  
Plaque Assay ◽  
Antiviral Drug ◽  
Vero Cells ◽  
Significant Difference ◽  
Dengue Virus Type 2

Dengue viruses are the most important arthropod-borne viruses in terms of morbidity and mortality in the world. Since there is no dengue vaccine available for human use, we have set out to investigate the use of chloroquine as an antiviral drug against dengue. Chloroquine, an amine acidotropic drug known to affect intracellular exocytic pathways by increasing endosomal pH, was used in the in vitro treatment of Vero and C6/36 cells infected with dengue virus type 2 (DENV-2). Real-time RT-PCR and plaque assays were used to quantify the DENV-2 load in infected Vero and C6/36 cells after chloroquine treatment. Our results showed that a dose of 50 μg/ml of chloroquine was not toxic to the cells and induced a statistically significant inhibition of virus production in infected Vero cells when compared to untreated cells. In C6/36 cells, chloroquine does not induce a statistically significant difference in viral replication when compared to untreated cells, showing that this virus uses an unlikely pathway of penetration in these cells, and results were also confirmed by the plaque assay (PFU). These data suggest that the inhibition of virus infection induced by chloroquine is due to interference with acidic vesicles in mammalian cells.

scholarly journals Functional dissection of Ctr4 and Ctr5 amino-terminal regions reveals motifs with redundant roles in copper transport

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 209-222 ◽  
Author(s):  
Jude Beaudoin ◽  
Julie Laliberté ◽  
Simon Labbé
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Expression System ◽  
Copper Transport ◽  
Full Length ◽  
Proper Function ◽  
Copper Uptake ◽  
Wild Type ◽  
Amino Terminal ◽  
Redundant Functions

Copper uptake in the fission yeast Schizosaccharomyces pombe is carried out by a heteromeric complex formed by two proteins, Ctr4 and Ctr5. In this study, a stable expression system using integrative plasmids was developed to investigate the respective roles of Ctr4 and Ctr5 in copper transport. It was shown that expression of full-length Ctr4 or truncated Ctr4 containing residues 106–289 was required for localization of Ctr5 to the plasma membrane. Likewise, when the full-length Ctr5 or truncated Ctr5 from residues 44–173 was co-expressed with Ctr4, this protein was visualized at the periphery of the cell. To determine the importance of the Mets motifs (consisting of five methionines arranged as Met-X2-Met-X-Met, where X is any amino acid) of Ctr4 and Ctr5 in the heteroprotein complex, we co-expressed Ctr5 lacking the Mets motif and Cys-X-Met-X-Met sequence with wild-type Ctr4 or its mutant derivatives. Conversely, Ctr4 lacking the Mets motif and Met122 was expressed with wild-type Ctr5 or its mutant derivatives. These experiments revealed that the five Mets motifs of Ctr4 and the Ctr4 residue Met122 have equally important roles in copper assimilation. Furthermore, the two partially overlapping Mets motifs and the Cys-X-Met-X-Met sequence in Ctr5 have redundant functions in copper transport, with the latter sequence making a greater contribution than the former. Together, the data reveal that co-expression of both Ctr4 and Ctr5 is necessary for the proper function and localization of the heteroprotein complex to the plasma membrane. Once on the cell surface, the N-terminal regions of Ctr4 and Ctr5 can function independently to transport copper; however, the greatest efficiency is achieved when both N termini are present.

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