Dataset Information

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

ABSTRACT: Zika virus (ZIKV; family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available that lack significant attenuation, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated infection and transmission rates in Aedes aegypti mosquitoes comparable to those of the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis. IMPORTANCE:ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barré syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with in vitro and in vivo characteristics similar to those of the primary isolate, providing a critical tool to study ZIKV infection and disease.

SUBMITTER: Weger-Lucarelli J

PROVIDER: S-EPMC5165210 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

Weger-Lucarelli James J Duggal Nisha K NK Bullard-Feibelman Kristen K Veselinovic Milena M Romo Hannah H Nguyen Chilinh C Rückert Claudia C Brault Aaron C AC Bowen Richard A RA Stenglein Mark M Geiss Brian J BJ Ebel Gregory D GD

Journal of virology 20161216 1

Zika virus (ZIKV; family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available that lack significant attenuation, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system bas ...[more]

PMID: 27795432

Similar Datasets

Project description: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.

Project description:An infectious nonpathogenic molecular clone (19-2-6A) of equine infectious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fragment amplified by PCR techniques from a pathogenic variant (EIAV(PV)) of the cell culture-adapted strain of EIAV (EIAV(PR)). This substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the S1 (encoding the second exon of tat), S2, and S3 (encoding the second exon of rev) open reading frames, the complete env gene (including the first exon of rev), and the 3' long terminal repeat (LTR). Modified 19-2-6A molecular clones were designated EIAV(PV3.3), and infection of a single pony (678) with viruses derived from a mixture of five of these molecular clones induced clinical signs of acute equine infectious anemia (EIA) at 23 days postinfection (dpi). As a consequence of this initial study, a single molecular clone, EIAV(PV3.3#3) (redesignated EIAV(UK)), was selected for further study and inoculated into two ponies (613 and 614) and two horses (700 and 764). Pony 614 and the two horses developed febrile responses by 12 dpi, which was accompanied by a 48 to 64% reduction in platelet number, whereas pony 613 did not develop fever (40.6 degrees C) until 76 dpi. EIAV could be isolated from the plasma of these animals by 5 to 7 dpi, and all became seropositive for antibodies to this virus by 21 dpi. Analysis of the complete nucleotide sequence demonstrated that the 3.3-kbp 3' fragment of EIAV(UK) differed from the consensus sequence of EIAV(PV) by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAV(PV) consensus sequence was observed in the hypervariable region of the LTR. However, EIAV(UK) was found to contain an unusual 68-bp nucleotide insertion/duplication in a normally conserved region of the LTR sequence. These results demonstrate that substitution of a 3.3-kbp fragment from the EIAV(PV) strain into the infectious nonpathogenic molecular clone 19-2-6A leads to the production of progeny virus particles with the ability to induce clinical signs of EIA. Therefore, EIAV(UK), which is the first pathogenic, cell culture-adapted molecular clone of EIAV to be described, should be of value in identifying viral determinants of pathogenicity.

Dataset Information

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

Publications

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets