scholarly journals Structural gene (prME) chimeras of St Louis encephalitis virus and West Nile virus exhibit altered in vitro cytopathic and growth phenotypes

2012 ◽  
Vol 93 (1) ◽  
pp. 39-49 ◽  
Author(s):  
Payal D. Maharaj ◽  
Michael Anishchenko ◽  
Stanley A. Langevin ◽  
Ying Fang ◽  
William K. Reisen ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Structural Gene ◽  
Growth Patterns ◽  
Encephalitis Virus ◽  
Mosquito Vectors ◽  
West Nile ◽  
Genetic Elements ◽  
Wide Range ◽  
Chimeric Viruses

Despite utilizing the same avian hosts and mosquito vectors, St Louis encephalitis virus (SLEV) and West Nile virus (WNV) display dissimilar vector-infectivity and vertebrate-pathogenic phenotypes. SLEV exhibits a low oral infection threshold for Culex mosquito vectors and is avirulent in avian hosts, producing low-magnitude viraemias. In contrast, WNV is less orally infective to mosquitoes and elicits high-magnitude viraemias in a wide range of avian species. In order to identify the genetic determinants of these different phenotypes and to assess the utility of mosquito and vertebrate cell lines for recapitulating in vivo differences observed between these viruses, reciprocal WNV and SLEV pre-membrane and envelope protein (prME) chimeric viruses were generated and growth of these mutant viruses was characterized in mammalian (Vero), avian (duck) and mosquito [Aedes (C6/36) and Culex (CT)] cells. In both vertebrate lines, WNV grew to 100-fold higher titres than SLEV, and growth and cytopathogenicity phenotypes, determined by chimeric phenotypes, were modulated by genetic elements outside the prME gene region. Both chimeras exhibited distinctive growth patterns from those of SLEV in C6/36 cells, indicating the role of both structural and non-structural gene regions for growth in this cell line. In contrast, growth of chimeric viruses was indistinguishable from that of virus containing homologous prME genes in CT cells, indicating that structural genetic elements could specifically dictate growth differences of these viruses in relevant vectors. These data provide genetic insight into divergent enzootic maintenance strategies that could also be useful for the assessment of emergence mechanisms of closely related flaviviruses.

scholarly journals Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence

2016 ◽  
Vol 90 (9) ◽  
pp. 4757-4770 ◽  
Author(s):  
Alexander J. McAuley ◽  
Maricela Torres ◽  
Jessica A. Plante ◽  
Claire Y.-H. Huang ◽  
Dennis A. Bente ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Receptor Binding ◽  
Encephalitis Virus ◽  
Target Cells ◽  
West Nile ◽  
E Protein ◽  
Domain Iii ◽  
Chimeric Viruses

ABSTRACTFlaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of human infections annually. The envelope (E) protein of flaviviruses comprises three structural domains, of which domain III (EIII) represents a discrete subunit. The EIII gene sequence typically encodes epitopes recognized by virus-specific, potently neutralizing antibodies, and EIII is believed to play a major role in receptor binding. In order to assess potential interactions between EIII and the remainder of the E protein and to assess the effects of EIII sequence substitutions on the antigenicity, growth, and virulence of a representative flavivirus, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from nine flaviviruses with various levels of genetic diversity from WNV were substituted. Of the constructs tested, chimeras containing EIIIs from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimerasin vitroandin vivorevealed differences in growth and virulence between the viruses, within vivopathogenesis often not being correlated within vitrogrowth. Taken together, the data demonstrate that substitutions of EIII can allow the generation of viable chimeric viruses with significantly altered antigenicity and virulence.IMPORTANCEThe envelope (E) glycoprotein is the major protein present on the surface of flavivirus virions and is responsible for mediating virus binding and entry into target cells. Several viable West Nile virus (WNV) variants with chimeric E proteins in which the putative receptor-binding domain (EIII) sequences of other mosquito-borne flaviviruses were substituted in place of the WNV EIII were recovered, although the substitution of several more divergent EIII sequences was not tolerated. The differences in virulence and tissue tropism observed with the chimeric viruses indicate a significant role for this sequence in determining the pathogenesis of the virus within the mammalian host. Our studies demonstrate that these chimeras are viable and suggest that such recombinant viruses may be useful for investigation of domain-specific antibody responses and the more extensive definition of the contributions of EIII to the tropism and pathogenesis of WNV or other flaviviruses.

scholarly journals Antigenic Characterization of New Lineage II Insect-Specific Flaviviruses in Australian Mosquitoes and Identification of Host Restriction Factors

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Jessica J. Harrison ◽  
Jody Hobson-Peters ◽  
Agathe M. G. Colmant ◽  
Joanna Koh ◽  
Natalee D. Newton ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
West Nile Virus ◽  
Cell Lines ◽  
Yellow Fever Virus ◽  
West Nile ◽  
Host Restriction ◽  
Antiviral Responses ◽  
Vertebrate Cell ◽  
Wide Range ◽  
Chimeric Viruses

ABSTRACT We describe two new insect-specific flaviviruses (ISFs) isolated from mosquitoes in Australia, Binjari virus (BinJV) and Hidden Valley virus (HVV), that grow efficiently in mosquito cells but fail to replicate in a range of vertebrate cell lines. Phylogenetic analysis revealed that BinJV and HVV were closely related (90% amino acid sequence identity) and clustered with lineage II (dual-host affiliated) ISFs, including the Lammi and Nounané viruses. Using a panel of monoclonal antibodies prepared to BinJV viral proteins, we confirmed a close relationship between HVV and BinJV and revealed that they were antigenically quite divergent from other lineage II ISFs. We also constructed chimeric viruses between BinJV and the vertebrate-infecting West Nile virus (WNV) by swapping the structural genes (prM and E) to produce BinJ/WNVKUN-prME and WNVKUN/BinJV-prME. This allowed us to assess the role of different regions of the BinJV genome in vertebrate host restriction and revealed that while BinJV structural proteins facilitated entry to vertebrate cells, the process was inefficient. In contrast, the BinJV replicative components in wild-type BinJV and BinJ/WNVKUN-prME failed to initiate replication in a wide range of vertebrate cell lines at 37°C, including cells lacking components of the innate immune response. However, trace levels of replication of BinJ/WNVKUN-prME could be detected in some cultures of mouse embryo fibroblasts (MEFs) deficient in antiviral responses (IFNAR−/− MEFs or RNase L−/− MEFs) incubated at 34°C after inoculation. This suggests that BinJV replication in vertebrate cells is temperature sensitive and restricted at multiple stages of cellular infection, including inefficient cell entry and susceptibility to antiviral responses. IMPORTANCE The globally important flavivirus pathogens West Nile virus, Zika virus, dengue viruses, and yellow fever virus can infect mosquito vectors and be transmitted to humans and other vertebrate species in which they cause significant levels of disease and mortality. However, the subgroup of closely related flaviviruses, known as lineage II insect-specific flaviviruses (Lin II ISFs), only infect mosquitoes and cannot replicate in cells of vertebrate origin. Our data are the first to uncover the mechanisms that restrict the growth of Lin II ISFs in vertebrate cells and provides new insights into the evolution of these viruses and the mechanisms associated with host switching that may allow new mosquito-borne viral diseases to emerge. The new reagents generated in this study, including the first Lin II ISF-reactive monoclonal antibodies and Lin II ISF mutants and chimeric viruses, also provide new tools and approaches to enable further research advances in this field.

scholarly journals In Vitro Resistance Selection and In Vivo Efficacy of Morpholino Oligomers against West Nile Virus

2007 ◽  
Vol 51 (7) ◽  
pp. 2470-2482 ◽  
Author(s):  
Tia S. Deas ◽  
Corey J. Bennett ◽  
Susan A. Jones ◽  
Mark Tilgner ◽  
Ping Ren ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Binding Site ◽  
Viral Genome ◽  
Infectious Clone ◽  
Encephalitis Virus ◽  
West Nile ◽  
In Vivo Efficacy ◽  
Conserved Sequence

ABSTRACT We characterize in vitro resistance to and demonstrate the in vivo efficacy of two antisense phosphorodiamidate morpholino oligomers (PMOs) against West Nile virus (WNV). Both PMOs were conjugated with an Arg-rich peptide. One peptide-conjugated PMO (PPMO) binds to the 5′ terminus of the viral genome (5′-end PPMO); the other targets an essential 3′ RNA element required for genome cyclization (3′ conserved sequence I [3′ CSI] PPMO). The 3′ CSI PPMO displayed a broad spectrum of antiflavivirus activity, suppressing WNV, Japanese encephalitis virus, and St. Louis encephalitis virus, as demonstrated by reductions in viral titers of 3 to 5 logs in cell cultures, likely due to the absolute conservation of the 3′ CSI PPMO-targeted sequences among these viruses. The selection and sequencing of PPMO-resistant WNV showed that the 5′-end-PPMO-resistant viruses contained two to three mismatches within the PPMO-binding site whereas the 3′ CSI PPMO-resistant viruses accumulated mutations outside the PPMO-targeted region. The mutagenesis of a WNV infectious clone demonstrated that the mismatches within the PPMO-binding site were responsible for the 5′-end PPMO resistance. In contrast, a U insertion or a G deletion located within the 3′-terminal stem-loop of the viral genome was the determinant of the 3′ CSI PPMO resistance. In a mouse model, both the 5′-end and 3′ CSI PPMOs (administered at 100 or 200 μg/day) partially protected mice from WNV disease, with minimal to no PPMO-mediated toxicity. A higher treatment dose (300 μg/day) caused toxicity. Unconjugated PMOs (3 mg/day) showed neither efficacy nor toxicity, suggesting the importance of the peptide conjugate for efficacy. The results suggest that a modification of the peptide conjugate composition to reduce its toxicity yet maintain its ability to effectively deliver PMO into cells may improve PMO-mediated therapy.

scholarly journals Selective constraint and adaptive potential of West Nile virus within and among naturally infected avian hosts and mosquito vectors

2018 ◽  
Vol 4 (1) ◽  
Author(s):  
Chase W Nelson ◽  
Samuel D Sibley ◽  
Sergios-Orestis Kolokotronis ◽  
Gabriel L Hamer ◽  
Christina M Newman ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Selective Constraint ◽  
Mosquito Vectors ◽  
Adaptive Potential ◽  
West Nile

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

Surveillance on antibodies against West Nile virus, Usutu virus, tick-borne encephalitis virus and Tribeč virus in wild birds in Drienovská wetland, Slovakia

Biologia ◽  
2019 ◽  
Vol 74 (7) ◽  
pp. 813-820 ◽  
Author(s):  
Tomáš Csank ◽  
Ľuboš Korytár ◽  
Terézia Pošiváková ◽  
Tamás Bakonyi ◽  
Juraj Pistl ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Wild Birds ◽  
Encephalitis Virus ◽  
West Nile ◽  
Usutu Virus ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

scholarly journals Extinction of West Nile Virus by Favipiravir through Lethal Mutagenesis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Estela Escribano-Romero ◽  
Nereida Jiménez de Oya ◽  
Esteban Domingo ◽  
Juan Carlos Saiz
Keyword(s):  
West Nile Virus ◽  
Mutation Frequency ◽  
Ebola Virus ◽  
Rna Viruses ◽  
Lethal Dose ◽  
Antiviral Agent ◽  
West Nile ◽  
Lethal Mutagenesis ◽  
Mutant Spectrum

ABSTRACT Favipiravir is an antiviral agent effective against several RNA viruses. The drug has been shown to protect mice against experimental infection with a lethal dose of West Nile virus (WNV), a mosquito-borne flavivirus responsible for outbreaks of meningitis and encephalitis for which no antiviral therapy has been licensed; however, the mechanism of action of the drug is still not well understood. Here, we describe the potent in vitro antiviral activity of favipiravir against WNV, showing that it decreases virus-specific infectivity and drives the virus to extinction. Two passages of WNV in the presence of 1 mM favipiravir—a concentration that is more than 10-fold lower than its 50% cytotoxic concentration (CC50)—resulted in a significant increase in mutation frequency in the mutant spectrum and in a bias toward A→G and G→A transitions relative to the population passaged in the absence of the drug. These data, together with the fact that the drug is already licensed in Japan against influenza virus and in a clinical trial against Ebola virus, point to favipiravir as a promising antiviral agent to fight medically relevant flaviviral infections, such as that caused by WNV.

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