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

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(14), Jamaica, and the Dominican Republic (13, 17). The rapid geographic expansion of the virus is attributed to movement by viremic birds during local and migratory flight behavior. To date, there is no effective drug treatment against WN virus infection and surveillance and mosquito control measures have not significantly influenced the number of human infections (27). A vaccine against WN virus represents an important approach to the prevention and control of this emerging disease.The ChimeriVax technology has been successfully used to develop a live vaccine against Japanese encephalitis (JE) virus that is now in phase II trials (23). JE virus is a close genetic relative of WN virus (31), a fact that expedited use of this technology to develop multiple WN virus vaccine candidates. The ChimeriVax technology employs the yellow fever (YF) 17D vaccine capsid and nonstructural genes to deliver the envelope genes (prM and E) of other flaviviruses. In the work presented here, the envelope genes of YF 17D were replaced with the corresponding genes of the wild-type WN virus NY99 strain previously described by Lanciotti et al. (19). The resulting YF/WN chimera lacked the mouse neuroinvasive property of WN virus and is less neurovirulent than YF 17D vaccine in both mouse and monkey models. Because WN virus, like other flaviviruses in the genus, is neurotropic for mammals (21, 29), attenuating point mutations were later introduced in the envelope of the YF/WN chimera to further reduce its virulence. Mutation sites were targeted only to regions of the envelope (E) protein gene and were based on previous observations by others (1,3,28,32) pertaining to attenuation phenotypes in related flaviviruses: specifically JE and tick-borne encephalitis viruses. Site-directed mutations in the WN virus E gene of the chimeric prototype vaccine, ChimeriVax-West Nile 01 , (ChimeriVax-WN 01 ) resulted in a significant reduction in virus neurovirulence. Here we discuss a vaccine in a YF vaccine backbone; the WN virus envelope (E) protein mutagenesis rationale; and the assessment of the safety, immunogenicity, efficacy, and genetic stability of these ChimeriVax-WN vaccine candidates in the mouse and macaque models. MATERIALS AND METHODSYF/WN chimeric clones and molecular procedures for virus assembly. Chimeric flaviviruses were constructed with the ChimeriVax two-plasmid technology previously described (9). Briefly, the two-plasmid system provides plasmid stability in Escherichia coli by dividing the cloned YF backbone into two plasmids. This provides smaller plasmids that are more stable to manipulate the YF sequences facilitating replacement of the prM and E genes of the flavivirus target vaccine. The WN virus prM and E genes used were cloned from the WN flamingo isolate 383-99 sequence (GenBank accession no. AF196835; kindly provided by John Roehrig, Centers for Disease Control and Prevention, Fort Collins, Colo.). Virus prME sequence cDNA was obtained by reverse transcription-PCR (RT-PCR) (XL-PCR kit; Applied Biosystems, Foster City, C...

“…ChimeriVax-WN 01 retained the ability to cause lethal encephalitis after i.c. inoculation, a property consistent with that of YF 17D virus (10). We estimated the i.c.…”

Section: Resultsmentioning

confidence: 99%

ChimeriVax-West Nile Virus Live-Attenuated Vaccine: Preclinical Evaluation of Safety, Immunogenicity, and Efficacy

Arroyo

Miller

Catalan

et al. 2004

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150

“…In contrast, direct CNS infection of most YFV strains in mice is lethal (2). This obstacle can be overcome with the use of viscerotropic strains of YFV in hamsters (24,38) or of immunodeficient (e.g., scid) mice with increased sensitivity to YFV by peripheral routes of infection (6). We show in this study that type I and II interferons are crucial in restricting viral replication in the mouse visceral organs, hence adult IFN-␣/␥-R Ϫ/Ϫ mice are uniformly FIG.…”

Section: Discussionmentioning

confidence: 99%

“…The receptor-binding site is thought to be located on dIII. Analyses of neurovirulent variants of YFV-17D, which have acquired E protein reversions to the Asibi sequence, provide support for a role of dIII residues 305, 325, and 380 as well as dI/II hinge region residues 52 and 173 in neurovirulence (6,29,34,35,37). While the location of the dIII residues is consistent with their involvement in receptor binding, the dI/II hinge region changes are speculated to affect fusion based on studies of other flaviviruses (reviewed in reference 27).…”

mentioning

confidence: 99%

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

Lee

Lobigs

2008

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.The live, attenuated yellow fever virus (YFV) strain 17D vaccine has been used safely and effectively in over 500 million individuals over the past 70 years. This excellent safety record also underpins its potential application as a platform for chimeric vaccines against other medically important flaviviruses (dengue, Japanese encephalitis, and West Nile viruses), which are currently in clinical trials (reviewed in reference 13). Despite knowledge of the complete genome sequences of 17D (33) and its virulent parent (10), the Asibi strain isolated in Africa in 1927, the in vivo mechanism(s) and molecular determinants for virulence attenuation of the YFV vaccine are still not completely resolved. YFV strain 17D was the first human vaccine derived from repeated passage in tissue culture (reviewed in reference 39). More than 230 passages in embryonic mouse tissue (18 passages), chicken embryo tissue (50 passages), and chicken embryo tissue from which the head and spinal chord had been removed (152 passages) separate the 17D from the Asibi strain. The adaptation of 17D to growth in mouse and chicken embryonal tissues culture has resulted in loss of viscerotropism, a property which accounts for the major disease manifestations of yellow fever in primates: high viremia, hepatitis, renal dysfunction, hemorrhagic fever, and circulatory shock (reviewed in reference 28); it also resulted in reduced neurotropism and loss of tropism for mosquitoes, the obligatory vector in the natural transmission cycle of YFV. The complex passage history of YFV strain 17D has given rise to as many as 68 nucleotide mutations and 32 amino acid changes in the viral RNA genome (10). However, when the Asibi genome is compared to the consensus sequence of different 17D vaccine strains derived from it, the number of differences potentially contributing to the attenuated virulence phenotype can be reduced to 20 amino acids and 4 nucleotides in the 3Ј untranslated region...

“…The RGD motif is a known participant in the interaction of a number of ligands with cell receptors from the integrin superfamily (8). Residues of the RGD motif, as well as RGDassociated regions, have previously been shown to influence the activity of a number of enveloped and nonenveloped viruses (4,10,11,13,15,17,21,27,36,42,44,47,49,50,52,56). Moreover, as evidence of the importance of this region, a 10-mer DENV-2 synthetic peptide sequence that overlapped the flavivirus Mut-5-like region (i.e., flavivirus amino acid residues at the Mut-5 positions) inhibited the binding of a DENV-2 domain III envelope protein to mosquito but not mammalian cells, indicating that this region has more specificity for insect cells (16).…”

Section: Discussionmentioning

confidence: 99%

Genetic Determinants of Sindbis Virus Mosquito Infection Are Associated with a Highly Conserved Alphavirus and Flavivirus Envelope Sequence

Pierro

Powers

Olson

2008

Wild-type Sindbis virus (SINV) strain MRE16 efficiently infects Aedes aegypti midgut epithelial cells (MEC), but laboratory-derived neurovirulent SINV strain TE/52J infects MEC poorly. SINV determinants for MEC infection have been localized to the E2 glycoprotein. The E2 amino acid sequences of MRE16 and TE/52J differ at 60 residue sites. To identify the genetic determinants of MEC infection of MRE16, the TE/52J virus genome was altered to contain either domain chimeras or more focused nucleotide substitutions of MRE16. The growth patterns of derived viruses in cell culture were determined, as were the midgut infection rates (MIR) in A. aegypti mosquitoes. The results showed that substitutions of MRE16 E2 aa 95 to 96 and 116 to 119 into the TE/52J virus increased MIR both independently and in combination with each other. In addition, a unique PPF/.GDS amino acid motif was located between these two sites that was found to be a highly conserved sequence among alphaviruses and flaviviruses but not other arboviruses.The majority of medically important mosquito-borne alphaviruses (family Togaviridae) and flaviviruses (family Flaviviridae) are vectored by two Culicinae mosquito genera, Aedes and Culex. The mosquito Aedes aegypti is a major vector in the epidemic disease cycles of the flaviviruses dengue virus (DENV) and yellow fever virus, as well as being a competent vector for a number of alphaviruses, including the prototypic alphavirus Sindbis virus (SINV). There are three major genotypes of SINV, the Paleoarctic-Ethiopian (P/E), which is found in Europe and Africa, the Oriental-Australian (O/A), which is found in Asia and Australasia, and the Southwest genotype, which is found in the southwestern region of Western Australia (28,34,40,41). SINV strain MRE16 is an O/A genotype isolated between 1966 and 1969 from a pool of Culex tritaeniorhynchus mosquitoes in Malaysia (20a). A full-length infectious clone (IC) of MRE16 has been generated, and it is currently the model O/A genotype being investigated in laboratory mosquito infection assays (24,25,29,31,43). MRE16 was previously found to have a midgut infection rate (MIR) in A. aegypti mosquitoes of 100% at 7 days postinfection (dpi) (24). In contrast, recombinant SINV strain TE/5Ј2J (P/E genotype), a double subgenomic SINV constructed from a chimeric mouse neurovirulent variant of SINV AR339 called TE12, infects less than 15% of A. aegypti mosquito midguts when analyzed similarly (31).The MRE16 E2 gene has been implicated as having genetic determinants of midgut infection (31). Sequence comparison between the SINV TE/5Ј2J and MRE16 E2 genes identified approximately 316 nucleotide (nt) (24.9%) and 60 amino acid (aa) residue (14.2%) differences (43). In this work, SINV strain TE/5Ј2J was mutated to generate viruses containing MRE16 sequences within the E2 gene (as either chimeras or point mutations) and the viruses were evaluated for the ability to productively infect the MEC of A. aegypti mosquitoes. The data demonstrated that TE/5Ј2J viruses having the specific MRE1...