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

2001 ◽  
Vol 75 (22) ◽  
pp. 10912-10922 ◽  
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.

scholarly journals Neuroadapted Yellow Fever Virus 17D: Determinants in the Envelope Protein Govern Neuroinvasiveness for SCID Mice

2003 ◽  
Vol 77 (22) ◽  
pp. 12232-12242 ◽  
Author(s):  
Michael Nickells ◽  
Thomas J. Chambers
Keyword(s):  
Mouse Model ◽  
Yellow Fever ◽  
Scid Mouse ◽  
Yellow Fever Virus ◽  
Scid Mice ◽  
Survival Times ◽  
E Protein ◽  
Average Survival ◽  
Scid Mouse Model ◽  
Domain Iii

ABSTRACT A molecular clone of mouse-neuroadapted yellow fever 17D virus (SPYF-MN) was used to identify critical determinants of viral neuroinvasiveness in a SCID mouse model. Virus derived from this clone differs from nonneuroinvasive YF5.2iv virus at 29 nucleotide positions, encoding 13 predicted amino acid substitutions and 2 substitutions in the 3′ untranslated region (UTR). The virulence determinants of SPYF-MN for SCID mice were identified by constructing and characterizing intratypic viruses in which the E protein of SPYF-MN was expressed in the YF5.2iv background (SPYF-E) or the E protein of YF5.2iv was expressed in the SPYF-MN background (YF5.2-E). SPYF-E caused lethal encephalitis in young adult SCID mice after intraperitoneal inoculation, with average survival times and tissue virus burdens resembling those of mice inoculated with the parental SPYF-MN virus. To define which domains of the E protein are involved in neuroinvasiveness, two viruses were tested in which the amino acid substitutions in domains I-II and III were segregated. This revealed that substitutions in domain III (residues 305, 326, and 380) were critical for the neuroinvasive phenotype, based on average survival times and tissue burdens of infectious virus. Comparison of growth properties of the various intratypic viruses in cell culture indicated that no inherent defects in replication efficiency were likely to account for the biological differences observed in these experiments. These findings demonstrate that the E protein is a critical factor for yellow fever virus neuropathogenesis in the SCID mouse model and that the neuroinvasive properties depend principally on functions contributed by domain III of this protein. To assess whether critical determinants for neuroinvasion of normal ICR mice by SPYF virus were also in the E protein, sequences of viruses recovered from brains of ICR mice succumbing to encephalitis with the parental SPYF virus were derived. No differences were found in the E protein; however, two substitutions were present in the 3′ UTR compared to that of SPYF-MN, one of which is predicted to alter RNA secondary structure in this region. These findings suggest that the 3′ UTR may also affect neuroinvasiveness of SPYF virus in the mouse model.

Attenuation of aerosolized yellow fever virus after passage in cell culture.

1966 ◽  
Vol 30 (3) ◽  
pp. 615-623
Author(s):  
H J Hearn ◽  
W A Chappell ◽  
P Demchak ◽  
J W Kominik
Keyword(s):  
Cell Culture ◽  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Fever Virus

Location of a neutralization determinant in the E protein of yellow fever virus (17D vaccine strain)

Virology ◽  
1987 ◽  
Vol 161 (2) ◽  
pp. 474-478 ◽  
Author(s):  
M. Lobigs ◽  
L. Dalgarno ◽  
J.J. Schlesinger ◽  
R.C. Weir
Keyword(s):  
Yellow Fever ◽  
Vaccine Strain ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
E Protein

scholarly journals Production of Pseudoinfectious Yellow Fever Virus with a Two-Component Genome

2007 ◽  
Vol 81 (21) ◽  
pp. 11737-11748 ◽  
Author(s):  
Alexandr V. Shustov ◽  
Peter W. Mason ◽  
Ilya Frolov
Keyword(s):  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
Structural Proteins ◽  
Multiplicity Of Infection ◽  
Nonstructural Proteins ◽  
Replication Complex ◽  
Packaging Process ◽  
E Protein ◽  
Two Component

ABSTRACT Application of genetically modified, deficient-in-replication flaviviruses that are incapable of developing productive, spreading infection is a promising means of designing safe and effective vaccines. Here we describe a two-component genome yellow fever virus (YFV) replication system in which each of the genomes encodes complete sets of nonstructural proteins that form the replication complex but expresses either only capsid or prM/E instead of the entire structural polyprotein. Upon delivery to the same cell, these genomes produce together all of the viral structural proteins, and cells release a combination of virions with both types of genomes packaged into separate particles. In tissue culture, this modified YFV can be further passaged at an escalating scale by using a high multiplicity of infection (MOI). However, at a low MOI, only one of the genomes is delivered into the cells, and infection cannot spread. The replicating prM/E-encoding genome produces extracellular E protein in the form of secreted subviral particles that are known to be an effective immunogen. The presented strategy of developing viruses defective in replication might be applied to other flaviviruses, and these two-component genome viruses can be useful for diagnostic or vaccine applications, including the delivery and expression of heterologous genes. In addition, the achieved separation of the capsid-coding sequence and the cyclization signal in the YFV genome provides a new means for studying the mechanism of the flavivirus packaging process.

scholarly journals Vaccine and Wild-Type Strains of Yellow Fever Virus Engage Distinct Entry Mechanisms and Differentially Stimulate Antiviral Immune Responses

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Maria Dolores Fernandez-Garcia ◽  
Laurent Meertens ◽  
Maxime Chazal ◽  
Mohamed Lamine Hafirassou ◽  
Ophélie Dejarnac ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Virus Entry ◽  
Fever Virus ◽  
Host Cells ◽  
Target Cells ◽  
Wild Type ◽  
E Protein ◽  
Virulence Attenuation ◽  
Molecular Determinants

ABSTRACTThe live attenuated yellow fever virus (YFV) vaccine 17D stands as a “gold standard” for a successful vaccine. 17D was developed empirically by passaging the wild-type Asibi strain in mouse and chicken embryo tissues. Despite its immense success, the molecular determinants for virulence attenuation and immunogenicity of the 17D vaccine are poorly understood. 17D evolved several mutations in its genome, most of which lie within the envelope (E) protein. Given the major role played by the YFV E protein during virus entry, it has been hypothesized that the residues that diverge between the Asibi and 17D E proteins may be key determinants of attenuation. In this study, we define the process of YFV entry into target cells and investigate its implication in the activation of the antiviral cytokine response. We found that Asibi infects host cells exclusively via the classical clathrin-mediated endocytosis, while 17D exploits a clathrin-independent pathway for infectious entry. We demonstrate that the mutations in the 17D E protein acquired during the attenuation process are sufficient to explain the differential entry of Asibi versus 17D. Interestingly, we show that 17D binds to and infects host cells more efficiently than Asibi, which culminates in increased delivery of viral RNA into the cytosol and robust activation of the cytokine-mediated antiviral response. Overall, our study reveals that 17D vaccine and Asibi enter target cells through distinct mechanisms and highlights a link between 17D attenuation, virus entry, and immune activation.IMPORTANCEThe yellow fever virus (YFV) vaccine 17D is one of the safest and most effective live virus vaccines ever developed. The molecular determinants for virulence attenuation and immunogenicity of 17D are poorly understood. 17D was generated by serially passaging the virulent Asibi strain in vertebrate tissues. Here we examined the entry mechanisms engaged by YFV Asibi and the 17D vaccine. We found the two viruses use different entry pathways. We show that the mutations differentiating the Asibi envelope (E) protein from the 17D E protein, which arose during attenuation, are key determinants for the use of these distinct entry routes. Finally, we demonstrate that 17D binds and enters host cells more efficiently than Asibi. This results in a higher uptake of viral RNA into the cytoplasm and consequently a greater cytokine-mediated antiviral response. Overall, our data provide new insights into the biology of YFV infection and the mechanisms of viral attenuation.

scholarly journals Efficacy of 2′-C-methylcytidine against yellow fever virus in cell culture and in a hamster model

2010 ◽  
Vol 86 (3) ◽  
pp. 261-267 ◽  
Author(s):  
Justin G. Julander ◽  
Ashok K. Jha ◽  
Jung-Ae Choi ◽  
Kie-Hoon Jung ◽  
Donald F. Smee ◽  
...  
Keyword(s):  
Cell Culture ◽  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
Hamster Model

scholarly journals E Protein Domain III Determinants of Yellow Fever Virus 17D Vaccine Strain Enhance Binding to Glycosaminoglycans, Impede Virus Spread, and Attenuate Virulence

2008 ◽  
Vol 82 (12) ◽  
pp. 6024-6033 ◽  
Author(s):  
Eva Lee ◽  
Mario Lobigs
Keyword(s):  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
Protein Domain ◽  
Type I ◽  
Wild Type ◽  
E Protein ◽  
Virulence Attenuation ◽  
Domain Iii

ABSTRACT The yellow fever virus (YFV) 17D strain is one of the most effective live vaccines for human use, but the in vivo mechanisms for virulence attenuation of the vaccine and the corresponding molecular determinants remain elusive. The vaccine differs phenotypically from wild-type YFV by the loss of viscerotropism, despite replicative fitness in cell culture, and genetically by 20 amino acid changes predominantly located in the envelope (E) protein. We show that three residues in E protein domain III inhibit spread of 17D in extraneural tissues and attenuate virulence in type I/II interferon-deficient mice. One of these residues (Arg380) is a dominant glycosaminoglycan-binding determinant, which mainly accounts for more rapid in vivo clearance of 17D from the bloodstream in comparison to 17D-derived variants with wild-type-like E protein. While other mutations will account for loss of neurotropism and phenotypic stability, the described impact of E protein domain III changes on virus dissemination and virulence is the first rational explanation for the safety of the 17D vaccine in humans.

scholarly journals 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

2004 ◽  
Vol 78 (14) ◽  
pp. 7418-7426 ◽  
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.

scholarly journals Neuroblastoma Cell-Adapted Yellow Fever 17D Virus: Characterization of a Viral Variant Associated with Persistent Infection and Decreased Virus Spread

2002 ◽  
Vol 76 (12) ◽  
pp. 6172-6184 ◽  
Author(s):  
Leonsia A. Vlaycheva ◽  
Thomas J. Chambers
Keyword(s):  
Cell Culture ◽  
Cell Lines ◽  
Yellow Fever ◽  
Persistent Infection ◽  
Yellow Fever Virus ◽  
Neuroblastoma Cell ◽  
Nucleotide Sequence Analysis ◽  
Plaque Size ◽  
Surface Binding ◽  
E Protein

ABSTRACT Serial passage of yellow fever 17D virus (YF5.2iv, derived from an infectious molecular clone) on mouse neuroblastoma (NB41A3) cells established a persistent noncytopathic infection associated with a variant virus. This virus (NB15a) was dramatically reduced in plaque formation and exhibited impaired replication kinetics on all cell lines examined compared to the parental virus. Nucleotide sequence analysis of NB15a revealed a substitution in domain III of the envelope (E) protein at residue 360, where an aspartic acid residue was replaced by glycine. Single mutations were also found within the NS2A and NS3 proteins. Engineering of YF5.2iv virus to contain the E360 substitution yielded a virus (G360 mutant) whose plaque size and growth efficiency in cell culture resembled those of NB15a. Compared with YF5.2iv, both NB15a and G360 were markedly restricted for spread through Vero cell monolayers and mildly restricted in C6/36 cells. On NB41A3 cells, spread of the viruses was similar, but all three were generally inefficient compared with spread in other cell lines. Compared to YF5.2iv virus, NB15a was uniformly impaired in its ability to penetrate different cell lines, but a difference in cell surface binding was detected only on NB41A3 cells, where NB15a appeared less efficient. Despite its small plaque size, impaired growth, and decreased penetration efficiency, NB15a did not differ from YF5.2iv in mouse neurovirulence testing, based on mortality rates and average survival times after intracerebral inoculation of young adult mice. The data indicate that persistence of yellow fever virus in NB41A3 cells is associated with a mutation in the receptor binding domain of the E protein that impairs the virus entry process in cell culture. However, the phenotypic changes which occur in the virus as a result of the persistent infection in vitro do not correlate with attenuation during pathogenesis in the mouse central nervous system.

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