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

ABSTRACT 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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Envelope Protein Glycosylation Mediates Zika Virus Pathogenesis

Journal of Virology ◽

10.1128/jvi.00113-19 ◽

2019 ◽

Vol 93 (12) ◽

Cited By ~ 29

Author(s):

Derek L. Carbaugh ◽

Ralph S. Baric ◽

Helen M. Lazear

Keyword(s):

Zika Virus ◽

Envelope Protein ◽

Glycosylation Site ◽

Viral Envelope ◽

Protein Glycosylation ◽

Guillain Barre Syndrome ◽

Viral Loads ◽

E Protein ◽

Guillain Barré Syndrome ◽

Guillain Barré

ABSTRACT Zika virus (ZIKV) is an emerging mosquito-borne flavivirus. Recent ZIKV outbreaks have produced serious human disease, including neurodevelopmental malformations (congenital Zika syndrome) and Guillain-Barré syndrome. These outcomes were not associated with ZIKV infection prior to 2013, raising the possibility that viral genetic changes could contribute to new clinical manifestations. All contemporary ZIKV isolates encode an N-linked glycosylation site in the envelope (E) protein (N154), but this glycosylation site is absent in many historical ZIKV isolates. Here, we investigated the role of E protein glycosylation in ZIKV pathogenesis using two contemporary Asian-lineage strains (H/PF/2013 and PRVABC59) and the historical African-lineage strain (MR766). We found that glycosylated viruses were highly pathogenic in Ifnar1−/− mice. In contrast, nonglycosylated viruses were attenuated, producing lower viral loads in the serum and brain when inoculated subcutaneously but remaining neurovirulent when inoculated intracranially. These results suggest that E glycosylation is advantageous in the periphery but not within the brain. Accordingly, we found that glycosylation facilitated infection of cells expressing the lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) or DC-SIGN-related (DC-SIGNR), suggesting that inefficient infection of lectin-expressing leukocytes could contribute to the attenuation of nonglycosylated ZIKV in mice. IMPORTANCE It is unclear why the ability of Zika virus (ZIKV) to cause serious disease, including Guillain-Barré syndrome and birth defects, was not recognized until recent outbreaks. One contributing factor could be genetic differences between contemporary ZIKV strains and historical ZIKV strains. All isolates from recent outbreaks encode a viral envelope protein that is glycosylated, whereas many historical ZIKV strains lack this glycosylation. We generated nonglycosylated ZIKV mutants from contemporary and historical strains and evaluated their virulence in mice. We found that nonglycosylated viruses were attenuated and produced lower viral loads in serum and brains. Our studies suggest that envelope protein glycosylation contributes to ZIKV pathogenesis, possibly by facilitating attachment to and infection of lectin-expressing leukocytes.

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Memantine treatment reduces the incidence of flaccid paralysis in a zika virus mouse model of temporary paralysis with similarities to Guillain-Barré syndrome

Antiviral Chemistry and Chemotherapy ◽

10.1177/2040206620950143 ◽

2020 ◽

Vol 28 ◽

pp. 204020662095014

Author(s):

Venkatraman Siddharthan ◽

Hong Wang ◽

Alexandre LR de Oliveira ◽

Xin Dai ◽

John D Morrey

Keyword(s):

Mouse Model ◽

Receptor Antagonist ◽

Zika Virus ◽

Clinical Evidence ◽

Flaccid Paralysis ◽

Guillain Barre Syndrome ◽

Aspartate Receptor ◽

Memantine Treatment ◽

Guillain Barré Syndrome ◽

Guillain Barré

Clinical evidence suggests that Zika virus contributes to Guillain-Barré syndrome that causes temporary paralysis. We utilized a recently described Zika virus mouse model of temporary flaccid paralysis to address the hypothesis that treatment with an N-methyl-D-aspartate receptor antagonist, memantine, can reduce the incidence of paralysis. Aged interferon alpha/beta-receptor knockout mice were used because of their sublethal susceptibility to Zika virus infection. Fifteen to twenty-five percent of mice infected with a Puerto Rico strain of Zika virus develop acute flaccid paralysis beginning at days 8–9 and peaked at days 10–12. Mice recover from paralysis within a week of onset. In two independent studies, twice daily oral administration of memantine at 60 mg/kg/day on days 4 through 9 after viral challenge significantly reduced the incidence of paralysis. No efficacy was observed with treatments from days 9 through 12. Memantine treatment in cell culture or mice did not affect viral titers. These data indicate that early treatment of memantine before onset of paralysis is efficacious, but treatments beyond the onset of paralysis were not efficacious. The effect of this N-methyl-D-aspartate receptor antagonist on the incidence of Zika virus-induced paralysis may provide guidance for investigations on the mechanism of paralysis.

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Faculty Opinions recommendation of Guillain-Barré Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726197809.793542616 ◽

2018 ◽

Author(s):

Gavin Screaton

Keyword(s):

Virus Infection ◽

Zika Virus ◽

Case Control Study ◽

French Polynesia ◽

Case Control ◽

Guillain Barre Syndrome ◽

Zika Virus Infection ◽

Guillain Barré Syndrome ◽

Guillain Barré ◽

Control Study

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Zika Virus and the Guillain–Barré Syndrome — Case Series from Seven Countries

New England Journal of Medicine ◽

10.1056/nejmc1609015 ◽

2016 ◽

Vol 375 (16) ◽

pp. 1598-1601 ◽

Cited By ~ 194

Author(s):

Thais dos Santos ◽

Angel Rodriguez ◽

Maria Almiron ◽

Antonio Sanhueza ◽

Pilar Ramon ◽

...

Keyword(s):

Zika Virus ◽

Case Series ◽

Guillain Barre Syndrome ◽

Guillain Barré Syndrome ◽

Guillain Barré

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Zika Virus Infection as a Cause of Congenital Brain Abnormalities and Guillain–Barré Syndrome: Systematic Review

PLoS Medicine ◽

10.1371/journal.pmed.1002203 ◽

2017 ◽

Vol 14 (1) ◽

pp. e1002203 ◽

Cited By ~ 219

Author(s):

Fabienne Krauer ◽

Maurane Riesen ◽

Ludovic Reveiz ◽

Olufemi T. Oladapo ◽

Ruth Martínez-Vega ◽

...

Keyword(s):

Systematic Review ◽

Virus Infection ◽

Zika Virus ◽

Guillain Barre Syndrome ◽

Brain Abnormalities ◽

Zika Virus Infection ◽

Guillain Barré Syndrome ◽

Guillain Barré

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Zika Virus–Associated Neurological Disease in the Adult: Guillain–Barré Syndrome, Encephalitis, and Myelitis

Seminars in Reproductive Medicine ◽

10.1055/s-0036-1592066 ◽

2016 ◽

Vol 34 (05) ◽

pp. 273-279 ◽

Cited By ~ 43

Author(s):

Laura Muñoz ◽

Paula Barreras ◽

Carlos Pardo

Keyword(s):

Zika Virus ◽

Neurological Disease ◽

Guillain Barre Syndrome ◽

Guillain Barré Syndrome ◽

Guillain Barré

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The re-emergence of Zika in Brazil in 2020: a case of Guillain Barré Syndrome during the low season for arboviral infections

Journal of Travel Medicine ◽

10.1093/jtm/taaa165 ◽

2020 ◽

Vol 27 (7) ◽

Cited By ~ 2

Author(s):

Kevan M Akrami ◽

Betania Mara Freitas de Nogueira ◽

Mateus Santana do Rosário ◽

Laise de Moraes ◽

Marli Tenório Cordeiro ◽

...

Keyword(s):

Zika Virus ◽

Guillain Barre Syndrome ◽

Guillain Barré Syndrome ◽

Guillain Barré

Zika virus cases in Brazil have diminished since emergence in 2015. We report Guillain Barré Syndrome caused by Zika and possible Chikungunya co-infection during an expected low arboviral season. This case highlights the importance of clinical vigilance for Zika in those with neurological syndromes outside typical arboviral season.

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Zika virus infection as a cause of congenital brain abnormalities and Guillain-Barré syndrome: From systematic review to living systematic review

F1000Research ◽

10.12688/f1000research.13704.1 ◽

2018 ◽

Vol 7 ◽

pp. 196 ◽

Cited By ~ 14

Author(s):

Michel Jacques Counotte ◽

Dianne Egli-Gany ◽

Maurane Riesen ◽

Million Abraha ◽

Teegwendé Valérie Porgo ◽

...

Keyword(s):

Systematic Review ◽

Zika Virus ◽

Congenital Abnormalities ◽

Causal Link ◽

The Body ◽

Guillain Barre Syndrome ◽

Sufficient Evidence ◽

Zikv Infection ◽

Guillain Barré Syndrome ◽

Guillain Barré

Background. The Zika virus (ZIKV) outbreak in the Americas has caused international concern due to neurological sequelae linked to the infection, such as microcephaly and Guillain-Barré syndrome (GBS). The World Health Organization stated that there is “sufficient evidence to conclude that Zika virus is a cause of congenital abnormalities and is a trigger of GBS”. This conclusion was based on a systematic review of the evidence published until 30.05.2016. Since then, the body of evidence has grown substantially, leading to this update of that systematic review with new evidence published from 30.05.2016 – 18.01.2017, update 1. Methods. We review evidence on the causal link between ZIKV infection and adverse congenital outcomes and the causal link between ZIKV infection and GBS or immune-mediated thrombocytopaenia purpura. We also describe the transition of the review into a living systematic review, a review that is continually updated. Results. Between 30.05.2016 and 18.01.2017, we identified 2413 publications, of which 101 publications were included. The evidence added in this update confirms the conclusion of a causal association between ZIKV and adverse congenital outcomes. New findings expand the evidence base in the dimensions of biological plausibility, strength of association, animal experiments and specificity. For GBS, the body of evidence has grown during the search period for update 1, but only for dimensions that were already populated in the previous version. There is still a limited understanding of the biological pathways that potentially cause the occurrence of autoimmune disease following ZIKV infection. Conclusions. This systematic review confirms previous conclusions that ZIKV is a cause of congenital abnormalities, including microcephaly, and is a trigger of GBS. The transition to living systematic review techniques and methodology provides a proof of concept for the use of these methods to synthesise evidence about an emerging pathogen such as ZIKV.

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