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

Weger-Lucarelli, James; Duggal, Nisha K.; Bullard-Feibelman, Kristen M.; Veselinović, Milena; Romo, Hannah; Nguyen, Chilinh; Rückert, Claudia; Brault, Aaron C.; Bowen, Richard A.; Stenglein, Mark D.; Geiss, Brian J.; Ebel, Gregory D.

doi:10.1128/jvi.01765-16

Cited by 101 publications

(116 citation statements)

References 24 publications

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“…We set out to examine if N-linked glycosylation within the VNDT motif plays any role in ZIKV pathogenicity in an immunocompromised-mouse model. Although infectious clones of MR766 viruses (25,28,29), as well as several other ZIKV isolates (26,27,47), have been reported, the role of VNDT or glycosylation in this region in pathogenicity has not been explored. A recent study using ectopic expression and pseudoparticle formation with a WNV replicon showed that N-glycosylation of ZIKV E protein is necessary for efficient particle assembly and infectivity (48).…”

Section: Discussionmentioning

confidence: 99%

“…In fact, a recent study has shown that a single amino acid substitution (A188V) in the viral NS1 protein is critical for NS1 antigenemia, which in turn facilitates ZIKV acquisition by mosquitoes from viremic animals (24). With the availability of infectious clones of ZIKV (25)(26)(27)(28)(29)47) from historical and contemporary isolates, it will be possible to further examine the specific changes in the viral genome that are responsible for viral transmission, replication, virulence, and tissue tropism.…”

Section: Discussionmentioning

confidence: 99%

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Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion

Annamalai

Pattnaik

Sahoo

et al. 2017

J Virol

111

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Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion

Annamalai

Pattnaik

Sahoo

et al. 2017

J Virol

111

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“…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). In the present study, we successfully utilized the group II intron P.li.LSUI2 to stabilize the cDNA clone of ZIKV in a commonly used E. coli host strain.…”

Section: Discussionmentioning

confidence: 99%

“…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%

“…To bypass this obstacle, different approaches have been tested. Early reverse genetics systems of flaviviruses were usually based on the in vitro ligation of separately cloned viral 5=-half and 3=-half cDNA fragments (16,22), and similar strategies are still being utilized (23)(24)(25). For the generation of a full-length flavivirus infectious clone, the screening of different E. coli strains is usually needed (17), and the optimization of the assembly sequence of viral cDNA fragments is often required (18).…”

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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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Leveraging insect viruses and genetic manipulation for sustainable agricultural pest control

Sun,

Fu,

et al. 2023

Pest Management Science

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Insect viruses have long been recognized for their potential as biological control agents in managing agricultural pests. Despite their promise, challenges such as host specificity, virulence, and limited viral resources have hindered their widespread application. The need for efficient discovery and utilization of insect viruses is paramount for sustainable agricultural practices. With the introduction of high‐throughput sequencing (HTS) technologies, the discovery and identification of new insect viruses have been significantly accelerated, enriching the available resources for pest management. Furthermore, advancements in reverse genetics tools have enabled the development of multifunctional viral expression vectors, enhancing the specificity and efficacy of insect viruses against targeted pests. This review provides a comprehensive overview of the methodologies for insect virus identification using HTS and the potential of genetically modified insect viruses in pest control. Genetically engineered insect viruses offer a promising avenue for targeted and efficient pest management. While HTS technologies have revolutionized the discovery of insect viruses, challenges remain. Addressing these challenges and harnessing the potential of these technologies will be pivotal in shaping sustainable agricultural management strategies in the future.This article is protected by copyright. All rights reserved.

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

Cited by 101 publications

References 24 publications

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion

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

Leveraging insect viruses and genetic manipulation for sustainable agricultural pest control

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