Infection of SCID mice with Montana Myotis leukoencephalitis virus as a model for flavivirus encephalitis

We have established a convenient animal model for flavivirus encephalitis using Montana Myotis leukoencephalitis virus (MMLV), a bat flavivirus. This virus has the same genomic organization, and contains the same conserved motifs in genes that encode potential antiviral targets, as flaviviruses that cause disease in man (N. Charlier et al., accompanying paper), and has a similar particle size (approximately 40 nm). MMLV replicates well in Vero cells and appears to be equally as sensitive as yellow fever virus and dengue fever virus to a selection of experimental antiviral agents. Cells infected with MMLV show dilation of the endoplasmic reticulum, a characteristic of flavivirus infection. Intraperitoneal, intranasal or direct intracerebral inoculation of SCID mice with MMLV resulted in encephalitis ultimately leading to death, whereas immunocompetent mice were refractory to either intranasal or intraperitoneal infection with MMLV. Viral RNA and/or antigens were detected in the brain and serum of MMLV-infected SCID mice, but not in any other organ examined: MMLV was detected in the olfactory lobes, the cerebral cortex, the limbic structures, the midbrain, cerebellum and medulla oblongata. Infection was confined to neurons. Treatment with the interferon-α/β inducer poly(I)·poly(C) protected SCID mice against MMLV-induced morbidity and mortality, and this protection correlated with a reduction in infectious virus titre and viral RNA load. This validates the MMLV model for use in antiviral drug studies. The MMLV SCID model may, therefore, be attractive for the study of chemoprophylactic or chemotherapeutic strategies against flavivirus infections causing encephalitis.

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Exchanging the Yellow Fever Virus Envelope Proteins with Modoc Virus prM and E Proteins Results in a Chimeric Virus That Is Neuroinvasive in SCID Mice

Journal of Virology ◽

10.1128/jvi.78.14.7418-7426.2004 ◽

2004 ◽

Vol 78 (14) ◽

pp. 7418-7426 ◽

Cited By ~ 16

Author(s):

Nathalie Charlier ◽

Richard Molenkamp ◽

Pieter Leyssen ◽

Jan Paeshuyse ◽

Christian Drosten ◽

...

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Vero Cells ◽

Envelope Proteins ◽

Scid Mice ◽

Fever Virus ◽

Chimeric Virus ◽

E Proteins ◽

Virus Envelope

ABSTRACT A chimeric flavivirus infectious cDNA was constructed by exchanging the premembrane (prM) and envelope (E) genes of the yellow fever virus vaccine strain 17D (YF17D) with the corresponding genes of Modoc virus (MOD). This latter virus belongs to the cluster of the “not-known vector” flaviviruses and is, unlike YF17D, neuroinvasive in SCID mice. Replication of in vitro-transcribed RNA from this chimeric flavivirus was shown by [3H]uridine labeling and RNA analysis. Expression of the MOD prM and E proteins was monitored by radioimmunoprecipitation and revealed that the MOD proteins were correctly and efficiently produced from the chimeric precursor protein. The MOD E protein was shown to be N-linked glycosylated, whereas prM, as predicted from the genome sequence, did not contain N-linked carbohydrates. In Vero cells, the chimeric virus replicated with a similar efficiency as the parental viruses, although it formed smaller plaques than YF17D and MOD. In SCID mice that had been infected intraperitoneally with the chimeric virus, the viral load increased steadily until death. The MOD/YF virus, like MOD from which it had acquired the prM and E structural proteins, but unlike YF, proved neuroinvasive in SCID mice. Animals developed neurological symptoms about 15 days after inoculation and died shortly thereafter. The distribution of MOD/YF RNA in the brain of infected mice was similar to that observed in MOD-infected mice. The observations provide compelling evidence that the determinants of neuroinvasiveness of flaviviruses are entirely located in the envelope proteins prM and E.

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Immature particles and capsid-free viral RNA produced by Yellow fever virus-infected cells stimulate plasmacytoid dendritic cells to secrete interferons

Scientific Reports ◽

10.1038/s41598-018-29235-7 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 9

Author(s):

Laura Sinigaglia ◽

Ségolène Gracias ◽

Elodie Décembre ◽

Matthieu Fritz ◽

Daniela Bruni ◽

...

Keyword(s):

Dendritic Cells ◽

Yellow Fever ◽

Yellow Fever Virus ◽

Plasmacytoid Dendritic Cells ◽

Fever Virus ◽

Viral Rna ◽

Infected Cells

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Adaptation of yellow fever virus 17D to Vero cells is associated with mutations in structural and non-structural protein genes

Virus Research ◽

10.1016/j.virusres.2013.04.003 ◽

2013 ◽

Vol 176 (1-2) ◽

pp. 280-284 ◽

Cited By ~ 8

Author(s):

David W.C. Beasley ◽

Merribeth Morin ◽

Ashley R. Lamb ◽

Edward Hayman ◽

Douglas M. Watts ◽

...

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Vero Cells ◽

Fever Virus ◽

Structural Protein

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FDA Approved Drugs Efavirenz, Tipranavir, and Dasabuvir Inhibit Replication of Multiple Flaviviruses In Vitro

Proceedings ◽

10.3390/proceedings2020050006 ◽

2020 ◽

Vol 50 (1) ◽

pp. 6

Author(s):

Michal Stefanik ◽

Fortunatus C Ezebuo ◽

Jan Haviernik ◽

Ikemefuna C. Uzochukwu ◽

Martina Fojtikova ◽

...

Keyword(s):

Yellow Fever Virus ◽

Antiviral Drug ◽

Antiviral Effect ◽

Vero Cells ◽

Significant Inhibition ◽

Structural Protein ◽

Inhibition Effect ◽

Tick Borne Encephalitis Virus ◽

Approved Drugs

Arthropod-borne flaviviruses such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Zika virus (ZIKV), Dengue virus (DENV), and yellow fever virus (YFV) cause several serious life-threatening syndromes (encephalitis, miscarriages, paralysis, etc.). No effective antiviral therapy against these viruses has been approved yet. We selected, via in silico modeling, 12 U.S. Food and Drug Administration (FDA)-approved antiviral drugs (paritaprevir, dolutegravir, raltegravir, efavirenz, elvitegravir, tipranavir, saquinavir, dasabuvir, delavirdine, maraviroc, trifluridine, and tauroursodeoxycholic acid) for their interaction with ZIKV proteins (NS3 helicase and protease, non-structural protein 5 (NS5) RNA-dependent RNA polymerase, and methyltransferase). Only three of them were active against ZIKV, namely, dasabuvir (ABT-333), efavirenz, and tipranavir. These compounds inhibit virus replication of ZIKV (MR-766 and Paraiba_01) in Vero cells; therefore, we tested these compounds against other medically important flaviviruses WNV (13-104 and Eg101) and TBEV (Hypr). Dasabuvir was originally developed as an antiviral drug against hepatitis C virus (HCV); tipranavir and efavirenz are used for treating human immunodeficiency virus (HIV) infection. The antiviral effects of efavirenz, tipranavir, and dasabuvir were tested for ZIKV in HUH-7, astrocytes (HBCA), and UKF-NB-4 cells, where we also identified a significant inhibition effect of these compounds. For Vero cells, efavirenz inhibited all investigated viruses with EC50 ranging from 9.70 to 29.26 µM; the tipranavir inhibition effect was from 16.19 (WNV 13-104) to 27.47 µM (TBEV), while the strongest and most robust antiviral effect was demonstrated in the case of dasabuvir (EC50 values ranging from 9.09 (TBEV) to 10.85 µM (WNV 13-104)). These results warrant further research of these drugs, either individually or in combination, as possible pan-flavivirus inhibitors.

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Purification of yellow fever virus produced in Vero cells for inactivated vaccine manufacture

Vaccine ◽

10.1016/j.vaccine.2019.04.077 ◽

2019 ◽

Vol 37 (24) ◽

pp. 3214-3220 ◽

Cited By ~ 9

Author(s):

Tânia P. Pato ◽

Marta C.O. Souza ◽

Diogo A. Mattos ◽

Elena Caride ◽

Davis F. Ferreira ◽

...

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Vero Cells ◽

Fever Virus ◽

Inactivated Vaccine

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Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

Journal of Virology ◽

10.1128/jvi.03077-15 ◽

2016 ◽

Vol 90 (6) ◽

pp. 3212-3228 ◽

Cited By ~ 17

Author(s):

Leonia Bozzacco ◽

Zhigang Yi ◽

Ursula Andreo ◽

Claire R. Conklin ◽

Melody M. H. Li ◽

...

Keyword(s):

Protein Folding ◽

Yellow Fever ◽

Virus Replication ◽

Yellow Fever Virus ◽

Rna Replication ◽

Fever Virus ◽

Viral Rna ◽

Wild Type ◽

Polyprotein Processing ◽

Wide Range

ABSTRACTDNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication.IMPORTANCEFlaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development.

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Establishment of an antiviral assay system and identification of severe fever with thrombocytopenia syndrome virus inhibitors

Antiviral Chemistry and Chemotherapy ◽

10.1177/2040206617740303 ◽

2017 ◽

Vol 25 (3) ◽

pp. 83-89 ◽

Cited By ~ 10

Author(s):

Masanori Baba ◽

Masaaki Toyama ◽

Norikazu Sakakibara ◽

Mika Okamoto ◽

Naomichi Arima ◽

...

Keyword(s):

Antiviral Agents ◽

Vero Cells ◽

Assay System ◽

Viral Rna ◽

University Hospital ◽

Selective Inhibitors ◽

Reverse Transcription Pcr ◽

The Family ◽

Severe Fever

Aims Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease. SFTS is epidemic in Asia, and its fatality rate is around 30% in Japan. The causative virus severe fever with thrombocytopenia syndrome virus (SFTSV) is a phlebovirus of the family Phenuiviridae (the order Bunyavirales). Although effective treatments are required, there are no antiviral agents currently approved for clinical use. Ribavirin and favipiravir were examined for their anti-SFTSV activity and found to be selective inhibitors of SFTSV replication in vitro. However, their activity was not sufficient. Therefore, it is mandatory to identify novel compounds active against SFTSV. To this end, we have established a safe and rapid assay system for screening selective inhibitors of SFTSV. Methods The virus was isolated from SFTS patients treated in Kagoshima University Hospital. Vero cells were infected with SFTSV and incubated in the presence of various concentrations of test compounds. After three days, the cells were examined for their intracellular viral RNA levels by real-time reverse transcription-PCR without extracting viral RNA. The cytotoxicity of test compounds was determined by a tetrazolium dye method. Results Among the test compounds, the antimalarial agent amodiaquine was identified as a selective inhibitor of SFTSV replication. Its 50% effective concentration (EC50) and cytotoxic concentration (CC50) were 19.1 ± 5.1 and >100 µM, respectively. The EC50 value of amodiaquine was comparable to those of ribavirin and favipiravir. Conclusion Amodiaquine is considered to be a promising lead of novel anti-SFTSV agents, and evaluating the anti-SFTSV activity of its derivatives is in progress.

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Favipiravir Inhibits Mayaro Virus Infection in Mice

Viruses ◽

10.3390/v13112213 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2213

Author(s):

Michèle Bengue ◽

Ai-rada Pintong ◽

Florian Liegeois ◽

Antoine Nougairède ◽

Rodolphe Hamel ◽

...

Keyword(s):

Antiviral Treatment ◽

Antiviral Drug ◽

Viral Rna ◽

In Vivo Model ◽

Blood Levels ◽

Viral Particles ◽

Mayaro Virus ◽

Immunocompetent Mice

Mayaro virus (MAYV) is an emergent alphavirus that causes MAYV fever. It is often associated with debilitating symptoms, particularly arthralgia and myalgia. MAYV infection is becoming a considerable health issue that, unfortunately, lacks a specific antiviral treatment. Favipiravir, a broad-spectrum antiviral drug, has recently been shown to exert anti-MAYV activity in vitro. In the present study, the potential of Favipiravir to inhibit MAYV replication in an in vivo model was evaluated. Immunocompetent mice were orally administrated 300 mg/kg/dose of Favipiravir at pre-, concurrent-, or post-MAYV infection. The results showed a significant reduction in infectious viral particles and viral RNA transcripts in the tissues and blood of the pre- and concurrently treated infected mice. A significant reduction in the presence of both viral RNA transcript and infectious viral particles in the tissue and blood of pre- and concurrently treated infected mice was observed. By contrast, Favipiravir treatment post-MAYV infection did not result in a reduction in viral replication. Interestingly, Favipiravir strongly decreased the blood levels of the liver disease markers aspartate- and alanine aminotransferase in the pre- and concurrently treated MAYV-infected mice. Taken together, these results suggest that Favipiravir is a potent antiviral drug when administered in a timely manner.

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Neuroadapted Yellow Fever Virus 17D: Genetic and Biological Characterization of a Highly Mouse-Neurovirulent Virus and Its Infectious Molecular Clone

Journal of Virology ◽

10.1128/jvi.75.22.10912-10922.2001 ◽

2001 ◽

Vol 75 (22) ◽

pp. 10912-10922 ◽

Cited By ~ 32

Author(s):

Thomas J. Chambers ◽

Michael Nickells

Keyword(s):

Cell Culture ◽

Survival Time ◽

Yellow Fever ◽

Yellow Fever Virus ◽

Scid Mice ◽

Fever Virus ◽

Nucleotide Substitutions ◽

Average Survival Time ◽

E Protein ◽

Average Survival

ABSTRACT A neuroadapted strain of yellow fever virus (YFV) 17D derived from a multiply mouse brain-passaged virus (Porterfield YF17D) was additionally passaged in SCID and normal mice. The virulence properties of this virus (SPYF) could be distinguished from nonneuroadapted virus (YF5.2iv, 17D infectious clone) by decreased average survival time in SCID mice after peripheral inoculation, decreased average survival time in normal adult mice after intracerebral inoculation, and occurrence of neuroinvasiveness in normal mice. SPYF exhibited more efficient growth in peripheral tissues of SCID mice than YF5.2iv, resulting in a more rapid accumulation of virus burden, but with low-titer viremia, at the time of fatal encephalitis. In cell culture, SPYF was less efficient in replication than YF5.2iv in all cell lines tested. The complete nucleotide sequence of SPYF revealed 29 nucleotide substitutions relative to YF5.2iv, and these were distributed throughout the genome. There were a total of 13 predicted amino acid substitutions, some of which correspond to known differences among the Asibi, French viscerotropic virus, French neurotropic vaccine, and YF17D vaccine strains. The envelope (E) protein contained five substitutions, within all three functional domains. Substitutions were also present in regions encoding the NS1, NS2A, NS4A, and NS5 proteins and in the 3′ untranslated region (UTR). Construction of YFV harboring all of the identified coding nucleotide substitutions and those in the 3′ UTR yielded a virus whose cell culture and pathogenic properties, particularly neurovirulence and neuroinvasiveness for SCID mice, generally resembled those of the original SPYF isolate. These findings implicate the E protein and possibly other regions of the genome as virulence determinants during pathogenesis of neuroadapted YF17D virus in mice. The determinants affect replication efficiency in both neural and extraneural tissues of the mouse and confer some limited host-range differences in cultured cells of nonmurine origin.

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