Development of standard methods for Zika virus propagation, titration, and purification

Coelho, Sharton Vinícius Antunes; Neris, Rômulo L. S.; Papa, Michelle Premazzi; Schnellrath, Laila C.; Meuren, Lana Monteiro; Tschoeke, Diogo A.; Leomil, Luciana; Verçoza, Brunno Renato Farias; Miranda, Mauro Sayão de; Thompson, Fabiano L.; Poian, Andrea T. Da; Souza, Thiago Moreno L.; Carneiro, Fabiana A.; Damaso, Clarissa R.; Assunção‐Miranda, Iranaia; Arruda, Luciana Barros de

doi:10.1016/j.jviromet.2017.04.011

Cited by 60 publications

(57 citation statements)

References 37 publications

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“…Zika virus strain FSS1302536 was propagated in C6/36 or Vero cells, and viral stocks and experimental samples were titrated on Vero cells by plaque assay (ZIKV) as described previously 99 .…”

Section: Methodsmentioning

confidence: 99%

A global lipid map defines a network essential for Zika virus replication

Leier

Weinstein

Kyle

et al. 2020

Preprint

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26Zika virus (ZIKV), an arbovirus of global concern, remodels intracellular membranes to form 27 replication sites. How ZIKV dysregulates lipid networks to allow this, and consequences for 28 disease, is poorly understood. Here, we performed comprehensive lipidomics to create a lipid 29 network map during ZIKV infection. We found that ZIKV significantly alters host lipid 30 composition, with the most striking changes seen within subclasses of sphingolipids. Ectopic 31 expression of ZIKV NS4B protein resulted in similar changes, demonstrating a role for NS4B in 32 modulating sphingolipid pathways. Disruption of sphingolipid biosynthesis in various cell types, 33including human neural progenitor cells, blocked ZIKV infection. Additionally, the sphingolipid 34 ceramide redistributes to ZIKV replication sites and increasing ceramide levels by multiple 35 pathways sensitizes cells to ZIKV infection. Thus, we identify a sphingolipid metabolic network 36 with a critical role in ZIKV replication and show that ceramide flux is a key mediator of ZIKV 37 infection. Results 112 ZIKV alters the lipid landscape of host cells 113To understand how cellular lipid metabolism is altered by ZIKV infection, we carried out 114 a global lipidomic survey of the model Huh7 human hepatic carcinoma cell line 20 infected for 24 115 or 48 hours with Asian lineage ZIKV strain FSS13025 (Fig. 1a). Lipids extracted from 116 populations of mock and infected cells (n = 5 biological replicates per condition) were analyzed 117 with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (Supplementary Data 118 1 and Supplementary Fig. 1a-f). We identified 340 lipid species spanning the phospholipid, 119 sphingolipid, glycerolipid, and sterol classes for which pairwise comparisons of normalized 120 abundance between mock and infected cells could be made (Supplementary Data 1). Of these, 121 80 species (23.5%) showed significant changes in abundance by 24 hours post-infection (hpi) 122 and 172 species (50.6%) were significantly altered by 48 hpi (P < 0.05, ANOVA or g-test) 123( Supplementary Data 1). Principal component analysis (PCA) of these observations confirmed 124 that infection status (mock or ZIKV) and timepoint (24 or 48 hpi) accounted most of these 125 changes, with changes in lipid composition between mock and infected cells increasing over 126 time (Fig. 1b). 127Next, we examined how ZIKV-induced changes in host lipid composition broke down by 128 subclass and species (Fig. 1c, d). A map of the pairwise correlations of all 340 species at 48 hpi 129 ( Supplementary Fig. 2a) revealed that lipid subclasses largely fell into two groups of species 130 that were either enriched or depleted in abundance ( Supplementary Fig. 2b), suggesting that 131 individual metabolic pathways are up-or down-regulated to create a specific lipid milieu around 132 the events of the viral replication cycle. Supporting this, many of the trends we observed were 133 consistent with earlier reports of functional roles for lipids during flavivirus infection. In ...

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Section: Methodsmentioning

confidence: 99%

A global lipid map defines a network essential for Zika virus replication

Leier

Weinstein

Kyle

et al. 2020

Preprint

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“…The virus was stored at −70 °C for further studies. Detailed protocols to grow the stock virus strains at titers ~10 9 PFU/mL have been described by us previously 13 , 20 .…”

Section: Methodsmentioning

confidence: 99%

Sofosbuvir protects Zika virus-infected mice from mortality, preventing short- and long-term sequelae

Ferreira

Zaverucha-do-Valle

Reis

et al. 2017

Sci Rep

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Zika virus (ZIKV) causes significant public health concerns because of its association with congenital malformations, neurological disorders in adults, and, more recently, death. Considering the necessity to mitigate ZIKV-associated diseases, antiviral interventions are an urgent necessity. Sofosbuvir, a drug in clinical use against hepatitis C virus (HCV), is among the FDA-approved substances endowed with anti-ZIKV activity. In this work, we further investigated the in vivo activity of sofosbuvir against ZIKV. Neonatal Swiss mice were infected with ZIKV (2 × 107 PFU) and treated with sofosbuvir at 20 mg/kg/day, a concentration compatible with pre-clinical development of this drug. We found that sofosbuvir reduced acute levels of ZIKV from 60 to 90% in different anatomical compartments, such as the blood plasma, spleen, kidney, and brain. Early treatment with sofosbuvir doubled the percentage and time of survival of ZIKV-infected animals. Sofosbuvir also prevented the acute neuromotor impairment triggered by ZIKV. In the long-term behavioural analysis of ZIKV-associated sequelae, sofosbuvir prevented loss of hippocampal- and amygdala-dependent memory. Our results indicate that sofosbuvir inhibits ZIKV replication in vivo, which is consistent with the prospective necessity of antiviral drugs to treat ZIKV-infected individuals.

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“…Raw data of supplementary figures (Excel file provided separately). References (45)(46)(47)(48)(49)(50)(51)…”

Section: Supplementary Materialsmentioning

confidence: 99%

Acute and chronic neurological consequences of early-life Zika virus infection in mice

et al. 2018

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Although congenital Zika virus (ZIKV) exposure has been associated with microcephaly and other neurodevelopmental disorders, long-term consequences of perinatal infection are largely unknown. We evaluated short- and long-term neuropathological and behavioral consequences of neonatal ZIKV infection in mice. ZIKV showed brain tropism, causing postnatal-onset microcephaly and several behavioral deficits in adulthood. During the acute phase of infection, mice developed frequent seizures, which were reduced by tumor necrosis factor-α (TNF-α) inhibition. During adulthood, ZIKV replication persisted in neonatally infected mice, and the animals showed increased susceptibility to chemically induced seizures, neurodegeneration, and brain calcifications. Altogether, the results show that neonatal ZIKV infection has long-term neuropathological and behavioral complications in mice and suggest that early inhibition of TNF-α-mediated neuroinflammation might be an effective therapeutic strategy to prevent the development of chronic neurological abnormalities.

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Development of standard methods for Zika virus propagation, titration, and purification

Cited by 60 publications

References 37 publications

A global lipid map defines a network essential for Zika virus replication

A global lipid map defines a network essential for Zika virus replication

Sofosbuvir protects Zika virus-infected mice from mortality, preventing short- and long-term sequelae

Acute and chronic neurological consequences of early-life Zika virus infection in mice

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