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 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 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 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 Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes

1998 ◽  
Vol 72 (11) ◽  
pp. 8756-8764 ◽  
Author(s):  
Elke Mühlberger ◽  
Beate Lötfering ◽  
Hans-Dieter Klenk ◽  
Stephan Becker
Keyword(s):  
Expression System ◽  
Genetic System ◽  
Marburg Virus ◽  
Reverse Genetic System ◽  
Expression Ratio ◽  
Nucleocapsid Proteins ◽  
Replication System ◽  
P Proteins ◽  
Transcriptional Start Sites ◽  
Cat Gene

ABSTRACT This paper describes the first reconstituted replication system established for a member of the Filoviridae, Marburg virus (MBGV). MBGV minigenomes containing the leader and trailer regions of the MBGV genome and the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsidated and could be passaged. Unlike most other members of the orderMononegavirales, filoviruses possess four proteins presumed to be components of the nucleocapsid (NP, VP35, VP30, and L). To determine the protein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 expression system. Northern blot analysis of viral RNA revealed that three nucleocapsid proteins (NP, VP35, and L) were essential and sufficient for transcription as well as replication and encapsidation. These data indicate that VP35, rather than VP30, is the functional homologue of rhabdo- and paramyxovirus P proteins. The reconstituted replication system was profoundly affected by the NP-to-VP35 expression ratio. To investigate whether CAT gene expression was achieved entirely by mRNA or in part by full-length plus-strand minigenomes, a copy-back minireplicon containing the CAT gene but lacking MBGV-specific transcriptional start sites was employed in the artificial replication system. This construct was replicated without accompanying CAT activity. It was concluded that the CAT activity reflected MBGV-specific transcription and not replication.

scholarly journals Characterization of Rose rosette virus and development of reverse genetic system for studying virus accumulation and movement in whole plants

2019 ◽  
Author(s):  
Jeanmarie Verchot ◽  
Venura Herath ◽  
Cesar D. Urrutia ◽  
Mathieu Gayral ◽  
Kelsey Lyle ◽  
...  
Keyword(s):  
Infectious Clone ◽  
Systemic Infection ◽  
Genetic System ◽  
Open Reading Frame ◽  
Reverse Genetic System ◽  
Reverse Genetic ◽  
Glycoprotein Precursor ◽  
Reading Frame ◽  
Negative Strand ◽  
Rose Rosette

ABSTRACTRose rosette virus (RRV) is an Emaravirus, a negative-sense RNA virus with a 7-segmented genome that is enclosed by a double membrane. While the genome sequences of many emaraviruses are reported, there is negligible information concerning virus replication and movement in host plants. Computational methods determined that RNA1 encoded the RNA dependent RNA polymerase (RdRp), RNA2 encoded glycoprotein precursor, and the RNA3 encoded the nucleocapsid (N), all share significant homologies with similar proteins of the Orthobunyavirus family. The RRV terminal UTR sequences are complementary and share significant identity with the UTR sequences of Bunyamwera virus. We report a minireplicon system and a full length infectious clone of RRV, which are the first for any emaravirus species. The photoreversible fluorescent iLOV protein was used to replace the RNA5 open reading frame (R5-iLOV). We demonstrate that agro-infiltration of Nicotiana benthamiana leaves to deliver RNA1, RNA3, and R5-iLOV cDNAs led to iLOV expression. A mutation was introduced into the RdRp active site and iLOV expression was eliminated. Delivery of four segments or seven segments of the RRV infectious clone produced systemic infection in N. benthamiana and rose plants. iLOV was also fused to the glycoprotein precursor (R2-iLOV). Using confocal microscopy, the R2-iLOV was seen in spherical bodies along membrane strands inside N. benthamiana epidermal cells. This new technology will enable future research to functionally characterize the RRV proteins, to study the virus-host interactions governing local and systemic infection, and examine the subcellular functions of the Gc.IMPORTANCERRV has emerged as a severe threat to cultivated roses, causing millions of dollars in losses to commercial producers. The majority of the viral gene products have not been researched or characterized until now. We constructed a minireplicon system and an infectious clone of the seven-segmented RRV genome that is contained in a binary vector and delivered by Agrobacterium. This technology has been slow to develop for viruses with negative-strand RNA genomes. It has been especially tricky for plant viruses with multicomponent negative-strand RNA genomes. We report the first reverse genetic system for a member of the genus Emaravirus, Rose rosette virus (RRV). We introduced the iLOV fluorescent protein as a fusion to the Gc protein and as a replacement for the open reading frame in genome segment 5. This game-changing reverse genetic system creates new opportunities for studying negative-strand RNA viruses in plants.

scholarly journals Development of a Reverse Genetic System for Studying Rose Rosette Virus in Whole Plants

2020 ◽  
Vol 33 (10) ◽  
pp. 1209-1221 ◽  
Author(s):  
Jeanmarie Verchot ◽  
Venura Herath ◽  
Cesar D. Urrutia ◽  
Mathieu Gayral ◽  
Kelsey Lyle ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Rna Virus ◽  
Infectious Clone ◽  
Systemic Infection ◽  
Genetic System ◽  
Polymerase Activity ◽  
Full Length ◽  
Reverse Genetic System ◽  
Reading Frame ◽  
Rose Rosette

Rose rosette virus (RRV) is a negative-sense RNA virus with a seven-segmented genome that is enclosed by a double membrane. We constructed an unconventional minireplicon system encoding the antigenomic (ag)RNA1 (encoding the viral RNA-dependent RNA polymerase [RdRp]), agRNA3 (encoding the nucleocapsid protein [N]), and a modified agRNA5 containing the coding sequence for the iLOV protein in place of the P5 open reading frame (R5-iLOV). iLOV expression from the R5-iLOV template was amplified by activities of the RdRp and N proteins in Nicotiana benthamiana leaves. A mutation was introduced into the RdRp catalytic domain and iLOV expression was eliminated, indicating RNA1-encoded polymerase activity drives iLOV expression from the R5-iLOV template. Fluorescence from the replicon was highest at 3 days postinoculation (dpi) and declined at 7 and 13 dpi. Addition of the tomato bushy stunt virus (TBSV) P19 silencing-suppressor protein prolonged expression until 7 dpi. A full-length infectious clone system was constructed of seven binary plasmids encoding each of the seven genome segments. Agro-delivery of constructs encoding RRV RNAs 1 through 4 or RNAs 1 through 7 to N. benthamiana plants produced systemic infection. Finally, agro-delivery of the full-length RRV infectious clone including all segments produced systemic infection within 60 dpi. This advance opens new opportunities for studying RRV infection biology.

scholarly journals Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis

2006 ◽  
Vol 80 (21) ◽  
pp. 10900-10906 ◽  
Author(s):  
Fernando Almazán ◽  
Marta L. DeDiego ◽  
Carmen Galán ◽  
David Escors ◽  
Enrique Álvarez ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Cdna Clone ◽  
Rna Synthesis ◽  
Infectious Clone ◽  
Genetic System ◽  
Infectious Cdna Clone ◽  
Reverse Genetic System ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Processing Enzymes

ABSTRACT The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2′-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

scholarly journals Reverse genetic system, genetically stable reporter viruses and packaged subgenomic replicon based on a Brazilian Zika virus isolate

2017 ◽  
Vol 98 (11) ◽  
pp. 2712-2724 ◽  
Author(s):  
Margit Mutso ◽  
Sirle Saul ◽  
Kai Rausalu ◽  
Olga Susova ◽  
Eva Žusinaite ◽  
...  
Keyword(s):  
Zika Virus ◽  
Virus Isolate ◽  
Genetic System ◽  
Reverse Genetic System ◽  
Reverse Genetic ◽  
Subgenomic Replicon

scholarly journals Zika Virus Replication Is Substantially Inhibited by Novel Favipiravir and Interferon Alpha Combination Regimens

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Camilly P. Pires de Mello ◽  
Xun Tao ◽  
Tae Hwan Kim ◽  
Jürgen B. Bulitta ◽  
Jaime L. Rodriquez ◽  
...  
Keyword(s):  
Interferon Alpha ◽  
Zika Virus ◽  
Time Course ◽  
Plaque Assay ◽  
Vero Cells ◽  
Health Concern ◽  
Combination Regimen ◽  
Viral Inhibition ◽  
Dosage Regimens ◽  
Viral Burden

ABSTRACT Zika virus (ZIKV) is a major public health concern due to its overwhelming spread into the Americas. Currently, there are neither licensed vaccines nor antiviral therapies available for the treatment of ZIKV. We aimed to identify and rationally optimize effective therapeutic regimens for ZIKV by evaluating the antiviral potentials of the approved broad-spectrum antiviral agents favipiravir (FAV), interferon alpha (IFN), and ribavirin (RBV) as single agents and in combinations. For these studies, Vero cells were infected with ZIKV in the presence of increasing concentrations of FAV, IFN, or/and RBV for 4 days. Supernatants were harvested daily, and the viral burden was quantified by a plaque assay on Vero cells. The time course of the viral burden during treatment in vitro was characterized by a novel translational, mechanism-based model, which was subsequently used to rationally optimize combination dosage regimens. The combination regimen of FAV plus IFN provided the greatest extent of viral inhibition without cytotoxicity, reducing the viral burden by 4.4 log10 PFU/ml at concentrations of 250 μM FAV and 100 IU/ml IFN. Importantly, these concentrations are achievable in humans. The translational, mechanism-based model yielded unbiased and reasonably precise curve fits. Simulations with the model predicted that clinically relevant regimens of FAV plus IFN would markedly reduce viral burdens in humans, resulting in at least a 10,000-fold reduction in the amount of the virus during the first 4 days of treatment. These findings highlight the substantial promise of rationally optimized combination dosage regimens of FAV plus IFN, which should be further investigated to combat ZIKV.

scholarly journals Study on the recovery of CTN-1V5 strain rabies virus with reverse genetics system

2014 ◽  
Vol 3 (3) ◽  
pp. 90
Author(s):  
Yunpeng Wang ◽  
Shouchun Cao ◽  
Leitai Shi ◽  
Jia Li ◽  
Guanmu Dong
Keyword(s):  
Rna Polymerase Ii ◽  
Rabies Virus ◽  
Reverse Genetics ◽  
Hammerhead Ribozyme ◽  
Vero Cells ◽  
Virus Genome ◽  
Eukaryotic Expression ◽  
Full Length ◽  
Direct Immunofluorescence ◽  
Cmv Promoter

Use molecular cloning techniques. The CTN-1V5 strains of rabies virus genome is divided into six fragments in order to cloned into Eukaryotic expression vector PVAX 1.0(+). Recombinant full length genomic cDNA was flanked by a hammerhead ribozyme and hepatitis delta virus ribozyme. four helper plasmids encoding the nucleoprotein, phosphoprotein,Glycoprotein and the large protein were constructed and co-transfected with a plasmid containing the full-length CTN viral genome into Vero cells. Recombinant CTN-1V5 virus was successfully recovered from the cloned cDNA under the control of a CMV promoter driven by RNA polymerase II. The recovered CTN-1V5 virus was identified by direct Immunofluorescence technique with anti-nucleoprotein monoclonal-antibody. A group of mice was challenged with the recombinant-strain by intracerebral inoculation, resulting in 100% morbidity.and the titer of the recombinant CTN-1V5 virus was 5.0 log LD50/mL.

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