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 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.

Simultaneous detection of Dengue virus, Chikungunya virus, Zika virus, Yellow fever virus and West Nile virus

2019 ◽  
Vol 268 ◽  
pp. 53-55 ◽  
Author(s):  
José A. Boga ◽  
Marta E. Alvarez-Arguelles ◽  
Susana Rojo-Alba ◽  
Mercedes Rodríguez ◽  
María de Oña ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Dengue Virus ◽  
Yellow Fever ◽  
Zika Virus ◽  
Chikungunya Virus ◽  
Yellow Fever Virus ◽  
Simultaneous Detection ◽  
Fever Virus ◽  
West Nile ◽  
Virus Zika

Antigenic analysis of West Nile virus strains using monoclonal antibodies

1988 ◽  
Vol 99 (1-2) ◽  
pp. 75-88 ◽  
Author(s):  
Terry G. Besselaar ◽  
N. K. Blackburn
Keyword(s):  
Monoclonal Antibodies ◽  
West Nile Virus ◽  
Antigenic Analysis ◽  
West Nile ◽  
Virus Strains

scholarly journals Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

2009 ◽  
Vol 90 (12) ◽  
pp. 2912-2922 ◽  
Author(s):  
Roy A. Hall ◽  
Si En Tan ◽  
Barbara Selisko ◽  
Rachael Slade ◽  
Jody Hobson-Peters ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
West Nile Virus ◽  
Binding Sites ◽  
Synthetic Peptides ◽  
Structural Model ◽  
Full Length ◽  
Physical Interaction ◽  
Rdrp Activity ◽  
The West ◽  
West Nile

The West Nile virus (WNV) NS5 protein contains a methyltransferase (MTase) domain involved in RNA capping and an RNA-dependent RNA polymerase (RdRp) domain essential for virus replication. Crystal structures of individual WNV MTase and RdRp domains have been solved; however, the structure of full-length NS5 has not been determined. To gain more insight into the structure of NS5 and interactions between the MTase and RdRp domains, we generated a panel of seven monoclonal antibodies (mAbs) to the NS5 protein of WNV (Kunjin strain) and mapped their binding sites using a series of truncated NS5 proteins and synthetic peptides. Binding sites of four mAbs (5D4, 4B6, 5C11 and 6A10) were mapped to residues 354–389 in the fingers subdomain of the RdRp. This is consistent with the ability of these mAbs to inhibit RdRp activity in vitro and suggests that this region represents a potential target for RdRp inhibitors. Using a series of synthetic peptides, we also identified a linear epitope (bound by mAb 5H1) that mapped to a 13 aa stretch surrounding residues 47 and 49 in the MTase domain, a region predicted to interact with the palm subdomain of the RdRp. The failure of one mAb (7G6) to bind both N- and C-terminally truncated NS5 recombinants indicates that the antibody recognizes a conformational epitope that requires the presence of residues in both the MTase and RdRp domains. These data support a structural model of the full-length NS5 molecule that predicts a physical interaction between the MTase and the RdRp domains.

scholarly journals Genetic Diversity in the Collaborative Cross Model Recapitulates Human West Nile Virus Disease Outcomes

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Jessica B. Graham ◽  
Sunil Thomas ◽  
Jessica Swarts ◽  
Aimee A. McMillan ◽  
Martin T. Ferris ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
West Nile Virus ◽  
Virus Infection ◽  
Immune Responses ◽  
West Nile Virus Infection ◽  
Collaborative Cross ◽  
Adaptive Immune Responses ◽  
West Nile ◽  
Adaptive Immune ◽  
Wide Range

ABSTRACT West Nile virus (WNV) is an emerging neuroinvasive flavivirus that now causes significant morbidity and mortality worldwide. The innate and adaptive immune responses to WNV infection have been well studied in C57BL/6J inbred mice, but this model lacks the variations in susceptibility, immunity, and outcome to WNV infection that are observed in humans, thus limiting its usefulness to understand the mechanisms of WNV infection and immunity dynamics. To build a model of WNV infection that captures human infection outcomes, we have used the Collaborative Cross (CC) mouse model. We show that this model, which recapitulates the genetic diversity of the human population, demonstrates diversity in susceptibility and outcomes of WNV infection observed in humans. Using multiple F1 crosses of CC mice, we identified a wide range of susceptibilities to infection, as demonstrated through differences in survival, clinical disease score, viral titer, and innate and adaptive immune responses in both peripheral tissues and the central nervous system. Additionally, we examined the Oas1b alleles in the CC mice and confirmed the previous finding that Oas1b plays a role in susceptibility to WNV; however, even within a given Oas1b allele status, we identified a wide range of strain-specific WNV-associated phenotypes. These results confirmed that the CC model is effective for identifying a repertoire of host genes involved in WNV resistance and susceptibility. The CC effectively models a wide range of WNV clinical, virologic, and immune phenotypes, thus overcoming the limitations of the traditional C57BL/6J model, allowing genetic and mechanistic studies of WNV infection and immunity in differently susceptible populations. IMPORTANCE Mouse models of West Nile virus infection have revealed important details regarding the innate and adaptive immune responses to this emerging viral infection. However, traditional mouse models lack the genetic diversity present in human populations and therefore limit our ability to study various disease outcomes and immunologic mechanisms subsequent to West Nile virus infection. In this study, we used the Collaborative Cross mouse model to more effectively model the wide range of clinical, virologic, and immune phenotypes present upon West Nile virus infection in humans.

scholarly journals Development of a Double Antibody Sandwich ELISA for West Nile Virus Detection Using Monoclonal Antibodies against Non-Structural Protein 1

PLoS ONE ◽  
2014 ◽  
Vol 9 (10) ◽  
pp. e108623 ◽  
Author(s):  
Xi-Xia Ding ◽  
Xiao-Feng Li ◽  
Yong-Qiang Deng ◽  
Yong-Hui Guo ◽  
Wei Hao ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
West Nile Virus ◽  
Sandwich Elisa ◽  
Virus Detection ◽  
Structural Protein ◽  
West Nile ◽  
Double Antibody

scholarly journals trans-Packaged West Nile Virus-Like Particles: Infectious Properties In Vitro and in Infected Mosquito Vectors

2004 ◽  
Vol 78 (21) ◽  
pp. 11605-11614 ◽  
Author(s):  
Frank Scholle ◽  
Yvette A. Girard ◽  
Qizu Zhao ◽  
Stephen Higgs ◽  
Peter W. Mason
Keyword(s):  
West Nile Virus ◽  
Cell Lines ◽  
Neutralizing Antibodies ◽  
Fat Body ◽  
Infected Mosquito ◽  
Wild Type Virus ◽  
Coding Region ◽  
West Nile ◽  
Subgenomic Replicon ◽  
Virus Like Particles

ABSTRACT A trans-packaging system for West Nile virus (WNV) subgenomic replicon RNAs (repRNAs), deleted for the structural coding region, was developed. WNV repRNAs were efficiently encapsidated by the WNV C/prM/E structural proteins expressed in trans from replication-competent, noncytopathic Sindbis virus-derived RNAs. Infectious virus-like particles (VLPs) were produced in titers of up to 109 infectious units/ml. WNV VLPs established a single round of infection in a variety of different cell lines without production of progeny virions. The infectious properties of WNV and VLPs were indistinguishable when efficiencies of infection of a number of different cell lines and inhibition of infection by neutralizing antibodies were determined. To investigate the usefulness of VLPs to address biological questions in vivo, Culex pipiens quinquefasciatus mosquitoes were orally and parenterally infected with VLPs, and dissected tissues were analyzed for WNV antigen expression. Antigen-positive cells in midguts of orally infected mosquitoes were detected as early as 2 days postinfection and as late as 8 days. Intrathoracic inoculation of VLPs into mosquitoes demonstrated a dose-dependent pattern of infection of secondary tissues and identified fat body, salivary glands, tracheal cells, and midgut muscle as susceptible WNV VLP infection targets. These results demonstrate that VLPs can serve as a valuable tool for the investigation of tissue tropism during the early stages of infection, where virus spread and the need for biosafety level 3 containment complicate the use of wild-type virus.

scholarly journals A New Clade of Insect-Specific Flaviviruses from Australian Anopheles Mosquitoes Displays Species-Specific Host Restriction

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Agathe M. G. Colmant ◽  
Jody Hobson-Peters ◽  
Helle Bielefeldt-Ohmann ◽  
Andrew F. van den Hurk ◽  
Sonja Hall-Mendelin ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Mosquito Species ◽  
Nucleotide Composition ◽  
Infected Mosquito ◽  
Significant Advance ◽  
West Nile Fever ◽  
Species Determination ◽  
West Nile ◽  
Host Restriction ◽  
Mosquito Genus

ABSTRACT Flaviviruses like dengue, Zika, or West Nile virus infect millions of people each year and are transmitted to humans via infected-mosquito bites. A subset of flaviviruses can only replicate in the mosquito host, and recent studies have shown that some can interfere with pathogenic flaviviruses in mosquitoes and limit the replication and transmission of the latter. The insect-specific flaviviruses (ISFs) reported here form a new Anopheles mosquito-associated clade separate from the Aedes- and Culex-associated ISF clades. The identification of distinct clades for each mosquito genus provides new insights into the evolution and ecology of flaviviruses. One of these viruses was shown to replicate in the midgut of the mosquito host and exhibit the most specialized host restriction reported to date for ISFs. Understanding this unprecedented host restriction in ISFs could help identify the mechanisms involved in the evolution of flaviviruses and their emergence as mosquito-borne pathogens. Flaviviruses are arthropod-borne viruses found worldwide and are responsible for significant human and veterinary diseases, including dengue, Zika, and West Nile fever. Some flaviviruses are insect specific and replicate only in mosquitoes. We report a genetically divergent group of insect-specific flaviviruses from Anopheles mosquitoes that do not replicate in arthropod cell lines or heterologous Anopheles species, exhibiting unprecedented specialization for their host species. Determination of the complete sequences of the RNA genomes of three of these viruses, Karumba virus (KRBV), Haslams Creek virus, and Mac Peak virus (McPV), that are found in high prevalence in some Anopheles mosquito populations and detection of virus-specific proteins, replicative double-stranded RNA, and small interfering RNA responses in the host mosquito species provided strong evidence of a functional replicating virus in the mosquito midgut. Analysis of nucleotide composition in the KRBV and McPV sequences also revealed a pattern consistent with the virus evolving to replicate only in insects. These findings represent a significant advance in our knowledge of mosquito-borne flavivirus ecology, host restriction, and evolution. IMPORTANCE Flaviviruses like dengue, Zika, or West Nile virus infect millions of people each year and are transmitted to humans via infected-mosquito bites. A subset of flaviviruses can only replicate in the mosquito host, and recent studies have shown that some can interfere with pathogenic flaviviruses in mosquitoes and limit the replication and transmission of the latter. The insect-specific flaviviruses (ISFs) reported here form a new Anopheles mosquito-associated clade separate from the Aedes- and Culex-associated ISF clades. The identification of distinct clades for each mosquito genus provides new insights into the evolution and ecology of flaviviruses. One of these viruses was shown to replicate in the midgut of the mosquito host and exhibit the most specialized host restriction reported to date for ISFs. Understanding this unprecedented host restriction in ISFs could help identify the mechanisms involved in the evolution of flaviviruses and their emergence as mosquito-borne pathogens.

scholarly journals Identification of Novel Small-Molecule Inhibitors of West Nile Virus Infection

2007 ◽  
Vol 81 (21) ◽  
pp. 11992-12004 ◽  
Author(s):  
Amine O. Noueiry ◽  
Paul D. Olivo ◽  
Urszula Slomczynska ◽  
Yi Zhou ◽  
Ben Buscher ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Yellow Fever ◽  
High Throughput Screening ◽  
Yellow Fever Virus ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chemical Library ◽  
Entry Site ◽  
West Nile ◽  
Subgenomic Replicon

ABSTRACT West Nile virus (WNV) has spread throughout the United States and Canada and now annually causes a clinical spectrum of human disease ranging from a self-limiting acute febrile illness to acute flaccid paralysis and lethal encephalitis. No therapy or vaccine is currently approved for use in humans. Using high-throughput screening assays that included a luciferase expressing WNV subgenomic replicon and an NS1 capture enzyme-linked immunosorbent assay, we evaluated a chemical library of over 80,000 compounds for their capacity to inhibit WNV replication. We identified 10 compounds with strong inhibitory activity against genetically diverse WNV and Kunjin virus isolates. Many of the inhibitory compounds belonged to a chemical family of secondary sulfonamides and have not been described previously to inhibit WNV or other related or unrelated viruses. Several of these compounds inhibited WNV infection in the submicromolar range, had selectivity indices of greater than 10, and inhibited replication of other flaviviruses, including dengue and yellow fever viruses. One of the most promising compounds, AP30451, specifically blocked translation of a yellow fever virus replicon but not a Sindbis virus replicon or an internal ribosome entry site containing mRNA. Overall, these compounds comprise a novel class of promising inhibitors for therapy against WNV and other flavivirus infections in humans.

scholarly journals Structural basis for Zika envelope domain III recognition by a germline version of a recurrent neutralizing antibody

2020 ◽  
Vol 117 (18) ◽  
pp. 9865-9875
Author(s):  
Shannon R. Esswein ◽  
Harry B. Gristick ◽  
Andrea Jurado ◽  
Avery Peace ◽  
Jennifer R. Keeffe ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Envelope Protein ◽  
Yellow Fever Virus ◽  
Cross Reactivity ◽  
Affinity Maturation ◽  
Protein Domain ◽  
Structural Basis ◽  
Maturation Process ◽  
West Nile ◽  
Domain Iii

Recent epidemics demonstrate the global threat of Zika virus (ZIKV), a flavivirus transmitted by mosquitoes. Although infection is usually asymptomatic or mild, newborns of infected mothers can display severe symptoms, including neurodevelopmental abnormalities and microcephaly. Given the large-scale spread, symptom severity, and lack of treatment or prophylaxis, a safe and effective ZIKV vaccine is urgently needed. However, vaccine design is complicated by concern that elicited antibodies (Abs) may cross-react with other flaviviruses that share a similar envelope protein, such as dengue virus, West Nile virus, and yellow fever virus. This cross-reactivity may worsen symptoms of a subsequent infection through Ab-dependent enhancement. To better understand the neutralizing Ab response and risk of Ab-dependent enhancement, further information on germline Ab binding to ZIKV and the maturation process that gives rise to potently neutralizing Abs is needed. Here we use binding and structural studies to compare mature and inferred-germline Ab binding to envelope protein domain III of ZIKV and other flaviviruses. We show that affinity maturation of the light-chain variable domain is important for strong binding of the recurrent VH3-23/VK1-5 neutralizing Abs to ZIKV envelope protein domain III, and identify interacting residues that contribute to weak, cross-reactive binding to West Nile virus. These findings provide insight into the affinity maturation process and potential cross-reactivity of VH3-23/VK1-5 neutralizing Abs, informing precautions for protein-based vaccines designed to elicit germline versions of neutralizing Abs.

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