A Reverse Genetics Platform That Spans the Zika Virus Family Tree

Widman, Douglas G.; Young, Ellen; Yount, Boyd; Plante, Kenneth S.; Gallichotte, Emily N.; Carbaugh, Derek L.; Peck, Kayla M.; Plante, Jessica A.; Swanstrom, Jesica; Heise, Mark T.; Lazear, Helen M.; Baric, Ralph S.

doi:10.1128/mbio.02014-16

Cited by 60 publications

(77 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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%

“…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%

See 1 more Smart Citation

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

Annamalai

Pattnaik

Sahoo

et al. 2017

J Virol

111

View full text Add to dashboard Cite

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.

show abstract

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

View full text Add to dashboard Cite

show abstract

“…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%

“…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%

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

View full text Add to dashboard Cite

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.

show abstract

“…Although ZIKV was first identified more than 70 years ago (1), its ability to cause congenital infection and fetal malformations was discovered only after the explosive 2015 epidemic in South America, where more than 1.5 million people were infected (2). Some have speculated that genetic evolution of ZIKV may have contributed to some of the new clinical manifestations identified in the recent outbreaks ( 3, 4 ). Indeed, the emergence of a mutation in the prM gene (S139N) in post-epidemic strains was associated with greater infectivity and injury of neuroprogenitor cells (5).…”

Section: Introductionmentioning

confidence: 99%

Zika virus–related neurotropic flaviviruses infect human placental explants and cause fetal demise in mice

et al. 2018

View full text Add to dashboard Cite

Although Zika virus (ZIKV) infection in pregnant women can cause placental damage, intrauterine growth restriction, microcephaly, and fetal demise, these disease manifestations only became apparent in the context of a large epidemic in the Americas. We hypothesized that ZIKV is not unique among arboviruses in its ability to cause congenital infection. To evaluate this, we tested the capacity of four emerging arboviruses (West Nile virus, WNV; Powassan virus, POWV; chikungunya virus, CHIKV; and Mayaro virus, MAYV) from related (flavivirus) and unrelated (alphavirus) genera to infect the placenta and fetus in immunocompetent, wild-type mice. Whereas all four viruses caused placental infection, only infection with the neurotropic flaviviruses (WNV and POWV) resulted in fetal demise. WNV and POWV also replicated efficiently in second-trimester human maternal (decidua) and fetal (chorionic villi and fetal membrane) explants whereas CHIKV and MAYV replicated less efficiently. In mice, RNA in situ hybridization and histopathological analysis revealed that WNV infected the placenta and fetal central nervous system, causing injury to the developing brain. In comparison, CHIKV and MAYV did not cause substantive placental or fetal damage despite evidence of vertical transmission. Based on the susceptibility of human maternal and fetal tissue explants and pathogenesis experiments in immunocompetent mice, other emerging neurotropic flaviviruses may share with ZIKV the capacity for transplacental transmission, and subsequent infection and injury to the developing fetus.

show abstract

A Reverse Genetics Platform That Spans the Zika Virus Family Tree

Cited by 60 publications

References 54 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

Zika virus–related neurotropic flaviviruses infect human placental explants and cause fetal demise in mice

Contact Info

Product

Resources

About