scholarly journals A Molecular Determinant of West Nile Virus Secretion and Morphology as a Target for Viral Attenuation

2020 ◽  
Vol 94 (12) ◽  
Author(s):  
Justine Basset ◽  
Julien Burlaud-Gaillard ◽  
Maxence Feher ◽  
Philippe Roingeard ◽  
Félix A. Rey ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rational Design ◽  
Surface Membrane ◽  
Public Health Problem ◽  
M Protein ◽  
Membrane Glycoprotein ◽  
Wild Type ◽  
West Nile ◽  
Viral Attenuation

ABSTRACT West Nile virus (WNV), a member of the Flavivirus genus and currently one of the most common arboviruses worldwide, is associated with severe neurological disease in humans. Its high potential to reemerge and rapidly disseminate makes it a bona fide global public health problem. The surface membrane glycoprotein (M) has been associated with Flavivirus-induced pathogenesis. Here, we identified a key amino acid residue at position 36 of the M protein whose mutation impacts WNV secretion and promotes viral attenuation. We also identified a compensatory site at position M-43 whose mutation stabilizes M-36 substitution both in vitro and in vivo. Moreover, we found that introduction of the two mutations together confers a full attenuation phenotype and protection against wild-type WNV lethal challenge, eliciting potent neutralizing-antibody production in mice. Our study thus establishes the M protein as a new viral target for rational design of attenuated WNV strains. IMPORTANCE West Nile virus (WNV) is a worldwide (re)emerging mosquito-transmitted Flavivirus causing fatal neurological diseases in humans. However, no human vaccine has been yet approved. One of the most effective live-attenuated vaccines was empirically obtained by serial passaging of wild-type yellow fever Flavivirus. However, such an approach is not acceptable nowadays, and the development of a rationally designed vaccine is necessary. Generating molecular infectious clones and mutating specific residues known to be involved in Flavivirus virulence constitute a powerful tool to promote viral attenuation. WNV membrane glycoprotein is thought to carry such essential determinants. Here, we identified two residues of this protein whose substitutions are key to the full and stable attenuation of WNV in vivo, most likely through inhibition of secretion and possible alteration of morphology. Applied to other flaviviruses, this approach should help in designing new vaccines against these viruses, which are an increasing threat to global human health.

scholarly journals Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes

2016 ◽  
Vol 90 (22) ◽  
pp. 10145-10159 ◽  
Author(s):  
G. P. Göertz ◽  
J. J. Fros ◽  
P. Miesen ◽  
C. B. F. Vogels ◽  
M. L. van der Bent ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rna Interference ◽  
Small Rna ◽  
Culex Pipiens ◽  
Biological Function ◽  
Wild Type ◽  
West Nile ◽  
Content Type ◽  
Transmission Rates

ABSTRACT Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5′-3′ exoribonuclease XRN1/Pacman on conserved RNA structures in the 3′ untranslated region (UTR) of the viral genomic RNA. sfRNA production is conserved in insect-specific, mosquito-borne, and tick-borne flaviviruses and flaviviruses with no known vector, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here, we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed growth kinetics similar to those of wild-type WNV in both RNA interference (RNAi)-competent and -compromised mosquito cell lines. Small-RNA deep sequencing of WNV-infected mosquitoes indicated an active small interfering RNA (siRNA)-based antiviral response for both the wild-type and sfRNA1-deficient viruses. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo . Two reproducible small-RNA hot spots within the 3′ UTR/sfRNA of the wild-type virus mapped to RNA stem-loops SL-III and 3′ SL, which stick out of the three-dimensional (3D) sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administered via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production. IMPORTANCE Understanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant noncoding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile virus and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionarily conserved.

scholarly journals Toll-Like Receptor 3 Has a Protective Role against West Nile Virus Infection

2008 ◽  
Vol 82 (21) ◽  
pp. 10349-10358 ◽  
Author(s):  
Stephane Daffis ◽  
Melanie A. Samuel ◽  
Mehul S. Suthar ◽  
Michael Gale ◽  
Michael S. Diamond
Keyword(s):  
West Nile Virus ◽  
Viral Replication ◽  
West Nile Virus Infection ◽  
Protective Role ◽  
Toll Like Receptor ◽  
Wild Type ◽  
West Nile ◽  
Viral Loads

ABSTRACT Protection against West Nile virus (WNV) infection requires rapid viral sensing and the generation of an interferon (IFN) response. Mice lacking IFN regulatory factor 3 (IRF-3) show increased vulnerability to WNV infection with enhanced viral replication and blunted IFN-stimulated gene (ISG) responses. IRF-3 functions downstream of several viral sensors, including Toll-like receptor 3 (TLR3), RIG-I, and MDA5. Cell culture studies suggest that host recognizes WNV in part, through the cytoplasmic helicase RIG-I and to a lesser extent, MDA5, both of which activate ISG expression through IRF-3. However, the role of TLR3 in vivo in recognizing viral RNA and activating antiviral defense pathways has remained controversial. We show here that an absence of TLR3 enhances WNV mortality in mice and increases viral burden in the brain. Compared to congenic wild-type controls, TLR3−/− mice showed relatively modest changes in peripheral viral loads. Consistent with this, little difference in multistep viral growth kinetics or IFN-α/β induction was observed between wild-type and TLR3−/− fibroblasts, macrophages, and dendritic cells. In contrast, a deficiency of TLR3 was associated with enhanced viral replication in primary cortical neuron cultures and greater WNV infection in central nervous system neurons after intracranial inoculation. Taken together, our data suggest that TLR3 serves a protective role against WNV in part, by restricting replication in neurons.

scholarly journals The recently identified flavivirus Bamaga virus is transmitted horizontally by Culex mosquitoes and interferes with West Nile virus replication in vitro and transmission in vivo

2018 ◽  
Vol 12 (10) ◽  
pp. e0006886 ◽  
Author(s):  
Agathe M. G. Colmant ◽  
Sonja Hall-Mendelin ◽  
Scott A. Ritchie ◽  
Helle Bielefeldt-Ohmann ◽  
Jessica J. Harrison ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Virus Replication ◽  
West Nile ◽  
Culex Mosquitoes

scholarly journals Human Monoclonal Fab Antibodies Against West Nile Virus and its Neutralizing Activity Analyzed in Vitro and in Vivo

2009 ◽  
Vol 01 (01) ◽  
pp. 036-042 ◽  
Author(s):  
Tao Duan ◽  
Monique Ferguson ◽  
Lintian Yuan ◽  
Fangling Xu ◽  
Guangyu Li
Keyword(s):  
West Nile Virus ◽  
West Nile ◽  
Neutralizing Activity

Genetic impairment of AMPKα2 signaling does not reduce muscle glucose uptake during treadmill exercise in mice

2009 ◽  
Vol 297 (4) ◽  
pp. E924-E934 ◽  
Author(s):  
Stine J. Maarbjerg ◽  
Sebastian B. Jørgensen ◽  
Adam J. Rose ◽  
Jacob Jeppesen ◽  
Thomas E. Jensen ◽  
...  
Keyword(s):  
Glucose Uptake ◽  
Glucose Transporter ◽  
Treadmill Exercise ◽  
Surface Membrane ◽  
Absolute Intensity ◽  
Wild Type ◽  
Running Speed ◽  
Mouse Muscle ◽  
Glucose Clearance

Some studies suggest that the 5′-AMP-activated protein kinase (AMPK) is important in regulating muscle glucose uptake in response to intense electrically stimulated contractions. However, it is unknown whether AMPK regulates muscle glucose uptake during in vivo exercise. We studied this in male and female mice overexpressing kinase-dead AMPKα2 (AMPK-KD) in skeletal and heart muscles. Wild-type and AMPK-KD mice were exercised at the same absolute intensity and the same relative intensity (30 and 70% of individual maximal running speed) to correct for reduced exercise capacity of the AMPK-KD mouse. Muscle glucose clearance was measured using 2-deoxy-[3H]glucose as tracer. In wild-type mice, glucose clearance was increased at 30 and 70% of maximal running speed by 40 and 350% in the quadriceps muscle and by 120 and 380% in gastrocnemius muscle, respectively. Glucose clearance was not lower in AMPK-KD muscles compared with wild-type regardless of whether animals were exercised at the same relative or the same absolute intensity. In agreement, surface membrane content of the glucose transporter GLUT4 was increased similarly in AMPK-KD and wild-type muscle in response to running. We also measured signaling of alternative exercise-sensitive pathways that might be compensatorily increased in AMPK-KD muscles. However, increases in phosphorylation of CaMKII, Trisk95, p38 MAPK, and ERK1/2 were not higher in AMPK-KD than in WT muscle. Collectively, these findings suggest that AMPKα2 signaling is not essential in regulating glucose uptake in mouse skeletal muscle during treadmill exercise and that other mechanisms play a central role.

scholarly journals Mutations in West Nile virus nonstructural proteins that facilitate replicon persistence in vitro attenuate virus replication in vitro and in vivo

Virology ◽  
2007 ◽  
Vol 364 (1) ◽  
pp. 184-195 ◽  
Author(s):  
Shannan L. Rossi ◽  
Rafik Fayzulin ◽  
Nathan Dewsbury ◽  
Nigel Bourne ◽  
Peter W. Mason
Keyword(s):  
West Nile Virus ◽  
Virus Replication ◽  
Nonstructural Proteins ◽  
West Nile

scholarly journals Point mutations in the West Nile virus (Flaviviridae; Flavivirus) RNA-dependent RNA polymerase alter viral fitness in a host-dependent manner in vitro and in vivo

Virology ◽  
2012 ◽  
Vol 427 (1) ◽  
pp. 18-24 ◽  
Author(s):  
Greta A. Van Slyke ◽  
Alexander T. Ciota ◽  
Graham G. Willsey ◽  
Joachim Jaeger ◽  
Pei-Yong Shi ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rna Polymerase ◽  
Point Mutations ◽  
Viral Fitness ◽  
Dependent Manner ◽  
The West ◽  
Rna Dependent Rna Polymerase ◽  
West Nile

scholarly journals Interferon Regulatory Factor IRF-7 Induces the Antiviral Alpha Interferon Response and Protects against Lethal West Nile Virus Infection

2008 ◽  
Vol 82 (17) ◽  
pp. 8465-8475 ◽  
Author(s):  
Stephane Daffis ◽  
Melanie A. Samuel ◽  
Mehul S. Suthar ◽  
Brian C. Keller ◽  
Michael Gale ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Cortical Neurons ◽  
Interferon Regulatory Factor ◽  
Interferon Response ◽  
Type I ◽  
Type I Ifn ◽  
Regulatory Factor ◽  
West Nile

ABSTRACT Type I interferon (IFN-α/β) comprises a family of immunomodulatory cytokines that are critical for controlling viral infections. In cell culture, many RNA viruses trigger IFN responses through the binding of RNA recognition molecules (RIG-I, MDA5, and TLR-3) and induction of interferon regulatory factor IRF-3-dependent gene transcription. Recent studies with West Nile virus (WNV) have shown that type I IFN is essential for restricting infection and that a deficiency of IRF-3 results in enhanced lethality. However, IRF-3 was not required for optimal systemic IFN production in vivo or in vitro in macrophages. To begin to define the transcriptional factors that regulate type I IFN after WNV infection, we evaluated IFN induction and virus control in IRF-7−/− mice. Compared to congenic wild-type mice, IRF-7−/− mice showed increased lethality after WNV infection and developed early and elevated WNV burdens in both peripheral and central nervous system tissues. As a correlate, a deficiency of IRF-7 blunted the systemic type I IFN response in mice. Consistent with this, IFN-α gene expression and protein production were reduced and viral titers were increased in IRF-7−/− primary macrophages, fibroblasts, dendritic cells, and cortical neurons. In contrast, in these cells the IFN-β response remained largely intact. Our data suggest that the early protective IFN-α response against WNV occurs through an IRF-7-dependent transcriptional signal.

scholarly journals Early Production of Type I Interferon during West Nile Virus Infection: Role for Lymphoid Tissues in IRF3-Independent Interferon Production

2007 ◽  
Vol 81 (17) ◽  
pp. 9100-9108 ◽  
Author(s):  
Nigel Bourne ◽  
Frank Scholle ◽  
Maria Carlan Silva ◽  
Shannan L. Rossi ◽  
Nathan Dewsbury ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Type I Interferon ◽  
High Titer ◽  
West Nile Virus Infection ◽  
Lymphoid Tissues ◽  
Genome Replication ◽  
Type I ◽  
West Nile

ABSTRACT Infection of cells with flaviviruses in vitro is reduced by pretreatment with small amounts of type I interferon (IFN-α/β). Similarly, pretreatment of animals with IFN and experiments using mice defective in IFN signaling have indicated a role for IFN in controlling flavivirus disease in vivo. These data, along with findings that flavivirus-infected cells block IFN signaling, suggest that flavivirus infection can trigger an IFN response. To investigate IFN gene induction by the very first cells infected during in vivo infection with the flavivirus West Nile virus (WNV), we infected mice with high-titer preparations of WNV virus-like particles (VLPs), which initiate viral genome replication in cells but fail to spread. These studies demonstrated a brisk production of IFN in vivo, with peak levels of over 1,000 units/ml detected in sera between 8 and 24 h after inoculation by either the intraperitoneal or footpad route. The IFN response was dependent on genome replication, and WNV genomes and WNV antigen-positive cells were readily detected in the popliteal lymph nodes (pLN) of VLP-inoculated mice. High levels of IFN mRNA transcripts and functional IFN were also produced in VLP-inoculated IFN regulatory factor 3 null (IRF3−/−) mice, indicating that IFN production was independent of the IRF3 pathways to IFN gene transcription, consistent with the IFN type produced (predominantly α).

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