Alexander Khromykh

Significance in Replication of the Terminal Nucleotides of the Flavivirus Genome

Publisher: American Society for Microbiology

Date: 10-2003

DOI: 10.1128/JVI.77.19.10623-10629.2003

Abstract: Point mutations that resulted in a substitution of the conserved 3′-penultimate cytidine in genomic RNA or the RNA negative strand of the self- lifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3′-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3′-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of φ6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3′-penultimate cytidines and a 3′-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3′-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.

The in vitro and in vivo antiviral properties of combined monoterpene alcohols against West Nile virus infection

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1016/J.VIROL.2016.04.021

Abstract: West Nile Virus (WNV) is a mosquito-borne flavivirus that can cause neuroinvasive disease in humans and animals for which no therapies are currently available. We studied an established combination of monoterpene alcohols (CMA) derived from Melaleuca alternifolia, against WNV infection. The in vitro results show that CMA exhibits virucidal activity, as well as reduces the viral titres and percentage of infected cells. The antiviral mechanism of action of CMA was studied. We found that CMA did not alter the intracellular pH, neither induced apoptosis, but did induce cell cycle arrest in the G0/G1-phase although that was not the antiviral mechanism. Furthermore, we tested CMA in vivo using IRF 3(-)(/)(-)/7(-/-)mice and it was found that CMA treatment significantly delayed morbidity due to WNV infection, reduced the loss of body weight and reduced the viral titres in brain. These findings suggest that CMA could be a therapeutic agent against WNV infection.

Effect of Wolbachia on replication of West Nile Virus in a mosquito cell line and adult mosqtuitoes

Publisher: American Society for Microbiology

Date: 15-01-2013

DOI: 10.1128/JVI.01837-12

Abstract: Wolbachia as an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown that w Melpop and w Mel strains of Wolbachia inhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g., Plasmodium and filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect of Wolbachia on the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced in Wolbachia -infected Aedes aegypti cells (Aag2). However, the amount of secreted virus was significantly reduced in the presence of Wolbachia . Intrathoracic injections showed that replication of WNV in A. aegypti mosquitoes infected with w Mel strain of Wolbachia was not inhibited, whereas w MelPop strain of Wolbachia significantly reduced the replication of WNV in mosquitoes. Further, when w MelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low in Wolbachia -free mosquitoes and were completely inhibited in the presence of Wolbachia . The results suggest that (i) despite the enhancement of viral genomic RNA replication in the Wolbachia -infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain of Wolbachia , and (iii) replication of the virus in orally fed mosquitoes was completely inhibited in w MelPop strain of Wolbachia .

Identification of residues in West Nile virus premembrane protein that influence viral particle secretion and virulence

Publisher: Microbiology Society

Date: 09-2012

DOI: 10.1099/VIR.0.044453-0

Abstract: The pre-membrane protein (prM) of West Nile virus (WNV) functions as a chaperone for correct folding of the envelope (E) protein, and prevents premature fusion during virus egress. However, little is known about its role in virulence. To investigate this, we compared the amino acid sequences of prM between a highly virulent North American strain (WNV NY99 ) and a weakly virulent Australian subtype (WNV KUN ). Five amino acid differences occur in WNV NY99 compared with WNV KUN (I22V, H43Y, L72S, S105A and A156V). When expressed in mammalian cells, recombinant WNV NY99 prM retained native antigenic structure, and was partially exported to the cell surface. In contrast, WNV KUN prM (in the absence of the E protein) failed to express a conserved conformational epitope and was mostly retained at the pre-Golgi stage. Substitutions in residues 22 (Ile to Val) and 72 (Leu to Ser) restored the antigenic structure and cell surface expression of WNV KUN prM to the same level as that of WNV NY99 , and enhanced the secretion of WNV KUN prME particles when expressed in the presence of E. Introduction of the prM substitutions into a WNV KUN infectious clone (FLSDX) enhanced the secretion of infectious particles in Vero cells, and enhanced virulence in mice. These findings highlight the role of prM in viral particle secretion and virulence, and suggest the involvement of the L72S and I22V substitutions in modulating these activities.

Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Publisher: Elsevier BV

Date: 07-2006

DOI: 10.1016/J.VIROL.2006.03.026

Abstract: Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product formation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation.

Prime‐boost vaccinations using recombinant flavivirus replicon and vaccinia virus vaccines: an ELISPOT analysis

Publisher: Wiley

Date: 03-08-2010

DOI: 10.1038/ICB.2010.99

Abstract: Recombinant Kunjin replicon virus-like particle (VLP), vaccinia virus (rVV) and DNA vaccines were tested in a large series of prime-boost vaccinations using interferon (IFN)γ ELISPOT assays that reflected effector (E), effector memory (EM) and central memory (CM) responses. All vaccine constructs encoded the murine polytope immunogen and responses to four CD8 T-cell epitopes (TYQRTRALV, SYIPSAEKI, YPHFMPTNL and RPQASGVYM) were measured. VLP/rVV out performed (by 14- to 20-fold) DNA/rVV for induction of CM responses, whereas EM responses were only marginally increased. DNA/VLP induced more EM, but not CM responses, than VLP alone, illustrating that DNA priming is not universally beneficial. rVV/VLP gave comparable results to VLP/rVV combinations, although the former induced approximately threefold more E responses, illustrating the utility of poxvirus priming in this setting. Although higher doses of VLP and rVV increased responses after single immunizations, such dose increases provided only marginal benefit in heterologous prime-boost settings. Triple combinations also provided no benefit over two vaccinations. DNA vaccination was associated with broad CM, but not EM responses, and the breadth of EM and E responses was significantly improved by increasing viral vector dose. VLP/rVV, rather than DNA priming, induced T cells with consistently high IFNγ secretion profiles across all ELISPOT measures. Vector-specific CD8 T-cell responses generally correlated well with immunogen-specific responses, although, as expected, single use of each vector reduced the relative levels of vector-specific responses. These experiments illustrate the utility of replicons in heterologous prime-boost vaccinations, and illustrate the ersity of data that can be obtained from ELISPOT analyses.

Determinants of attenuation in the envelope protein of the flavivirus Alfuy

Publisher: Microbiology Society

Date: 10-2011

DOI: 10.1099/VIR.0.034793-0

Abstract: Murray Valley encephalitis virus (MVEV) is a mosquito-borne flavivirus endemic to Australia and Papua New Guinea. Most strains of MVEV cause potentially fatal cases of encephalitis in humans and horses, and have been shown to be highly neuroinvasive in weanling mice. In contrast, the naturally occurring subtype Alfuy virus (ALFV) has never been associated with human disease, nor is it neuroinvasive in weanling mice, even at high doses. To identify viral factors associated with ALFV attenuation, a chimeric infectious clone was constructed containing the structural genes premembrane (prM) and envelope (E) of ALFV swapped into the MVEV genome. The resulting virus (vMVEV/ALFVstr) was no longer neuroinvasive in mice, suggesting that motifs within prM–E of ALFV confer attenuation. To define these motifs further, mutants were constructed by targeting ergent sequences between the MVEV and ALFV E proteins that are known markers of virulence in other encephalitic flaviviruses. MVEV mutants containing a unique ALFV sequence in the flexible hinge region (residues 273–277) or lacking the conserved glycosylation site at position 154 were significantly less neuroinvasive in mice than wild-type MVEV, as determined by delayed time to death or increased LD 50 . Conversely, when the corresponding MVEV sequences were inserted into the vMVEV/ALFVstr chimera, the mutant containing the MVEV hinge sequence was more neuroinvasive than the parental chimera, though not to the same level as wild-type MVEV. These results identify the hinge region and E protein glycosylation as motifs that contribute to the attenuation of ALFV.

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

Publisher: American Society for Microbiology

Date: 24-06-2020

DOI: 10.1128/MSPHERE.00095-20

Abstract: 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.

Zika Virus sfRNA Plays an Essential Role in the Infection of Insects and Mammals

Publisher: MDPI

Date: 07-2020

DOI: 10.3390/PROCEEDINGS2020050112

pDNA-lipoplexes engrafted with flagellin-related peptide induce potent immunity and anti-tumour effects

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1016/J.VACCINE.2011.07.045

Abstract: Complexes of cationic lipids and DNA (lipoplexes) are widely used for non-viral gene delivery and DNA vaccine development, but cationic lipids are toxic and promote non-specific interactions with cells, leading to poor efficacy. Near-neutral lipoplexes, on the other hand, can obviate toxicity, but a convenient means to target them to specific cells such as dendritic cells (DCs) has been lacking. Here, we show that a His-tagged flagellin-derived peptide (denoted 9Flg), previously reported to promote binding of liposomal antigen to TLR5-expressing cells, can be used to target near-neutral pDNA-lipoplexes incorporating the chelator lipid NTA(3)-DTDA (3(nitrilotriacetic acid)-ditetradecylamine) to DCs and other antigen-presenting cells (APCs). Thus, we show that pDNA-lipoplexes engrafted with 9Flg target pDNA to APCs in vitro and in vivo. Following i.v. administration, radiolabelled 9Flg-lipoplexes exhibited increased accumulation in spleen, lung and liver. Vaccination of C57BL/6 mice with 9Flg-lipoplexes containing either pcDNA3.1-SIIN (pSIIN) or a Kunjin virus replicon-based vector (pKUN), each encoding the epitope OVA(257-264) (SIINFEKL), induced Ag-specific T cell priming, and elicited strong cellular immunity as reflected by a marked increase in the number of Ag-responsive IFN-γ-producing CD8(+) T cells. Importantly, compared to i.m. injection of these SIINFEKL-encoding pDNAs in naked form, the i.v. administration of pSIIN or pKUN in 9Flg-lipoplexes to C57BL/6 mice induced a significantly more potent anti-tumour response in the B16-OVA melanoma tumour model. The targeting of near-neutral 9Flg-lipoplexes bearing pDNA encoding tumour antigens to TLR5 on APCs, therefore, is a powerful approach for developing more effective DNA vaccines and immunotherapies.

Subgenomic flaviviral RNAs: What do we know after the first decade of research

Publisher: Elsevier BV

Date: 11-2018

DOI: 10.1016/J.ANTIVIRAL.2018.09.006

Abstract: The common feature of flaviviral infection is the accumulation of abundant virus-derived noncoding RNA, named flaviviral subgenomic RNA (sfRNA) in infected cells. This RNA represents a product of incomplete degradation of viral genomic RNA by the cellular 5'-3' exoribonuclease XRN1 that stalls at the conserved highly structured elements in the 3' untranslated region (UTR). This mechanism of sfRNA generation was discovered a decade ago and since then sfRNA has been a focus of intense research. The ability of flaviviruses to produce sfRNA was shown to be evolutionary conserved in all members of Flavivirus genus. Mutations in the 3'UTR that affect production of sfRNAs and their interactions with host factors showed that sfRNAs are responsible for viral pathogenicity, host adaptation, and emergence of new pathogenic strains. RNA structural elements required for XRN1 stalling have been elucidated and the role of sfRNAs in inhibiting host antiviral responses in arthropod and vertebrate hosts has been demonstrated. Some molecular mechanisms determining these properties of sfRNA have been recently characterized, while other aspects of sfRNA functions remain an open avenue for future research. In this review we summarise the current state of knowledge on the mechanisms of generation and functional roles of sfRNAs in the life cycle of flaviviruses and highlight the gaps in our knowledge to be addressed in the future.

A Novel Bacterium-Free Method for Generation of Flavivirus Infectious DNA by Circular Polymerase Extension Reaction Allows Accurate Recapitulation of Viral Heterogeneity

Publisher: American Society for Microbiology

Date: 15-02-2013

DOI: 10.1128/JVI.03162-12

Abstract: A novel bacterium-free approach for rapid assembly of flavivirus infectious cDNAs using circular polymerase extension reaction was applied to generate infectious cDNA for the virulent New South Wales isolate of the Kunjin strain of West Nile virus (KUNV) that recently emerged in Australia. Recovered virus recapitulated the genetic heterogeneity present in the original isolate. The approach was utilized to generate viral mutants with designed phenotypic properties and to identify E protein glycosylation as one of the virulence determinants.

Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures

Publisher: American Society for Microbiology

Date: 09-1997

DOI: 10.1128/JVI.71.9.6650-6661.1997

Abstract: The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. Comparisons were also made by dual immunolabelling using antibodies to double-stranded RNA (dsRNA), the putative template in the flavivirus replication complex. At 8 h postinfection, the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA located in a perinuclear rim with extensions into the peripheral cytoplasm. By 16 h, at the end of the latent period, all patterns had changed to some discrete perinuclear foci associated with a thick cytoplasmic reticulum. By 24 h, this localization in perinuclear foci was more apparent and some foci were dual labelled with antibodies to dsRNA. In immuno-gold-labelled cryosections of infected cells at 24 h, all antibodies were associated with clusters of induced membrane structures in the perinuclear region. Two important and novel observations were made. First, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 antibodies. Second, adjacent masses of paracrystalline arrays or of convoluted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies. Paired membranes similar in appearance to the rough endoplasmic reticulum were also labelled, but less strongly, with antibodies to the three nonstructural proteins. Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labelled with any of the four antibodies. The collection of induced membranes may represent virus factories in which translation, RNA synthesis, and virus assembly occur.

Generating flavivirus vaccine candidates by modulating interferon sensitivity

Publisher: Informa UK Limited

Date: 09-2009

DOI: 10.1586/ERV.09.73

NS1′ Colocalizes with NS1 and Can Substitute for NS1 in West Nile Virus Replication

Publisher: American Society for Microbiology

Date: 15-08-2013

DOI: 10.1128/JVI.01101-13

Abstract: NS1′ is a C-terminally extended form of the NS1 protein produced only by encephalitic flaviviruses from the Japanese encephalitis virus serogroup. Here we show that West Nile virus (WNV) NS1′ and NS1 localize to the same cellular compartments when expressed from plasmid DNAs and also colocalize to viral RNA replication sites in infected cells. Using complementation analysis with NS1-deleted WNV cDNA, we demonstrated that NS1′ is able to substitute for the crucial function of NS1 in virus replication.

Encapsidation of the Flavivirus Kunjin Replicon RNA by Using a Complementation System Providing Kunjin Virus Structural Proteins in trans

Publisher: American Society for Microbiology

Date: 07-1998

DOI: 10.1128/JVI.72.7.5967-5977.1998

Abstract: Kunjin virus (KUN) replicon RNA was encapsidated by a procedure involving two consecutive electroporations of BHK-21 cells, first with KUN replicon RNA C20DXrep (with prME and most of C deleted) and about 24 h later with a recombinant Semliki Forest virus (SFV) replicon RNA(s) expressing KUN structural proteins. The presence of KUN replicon RNA in encapsidated particles was demonstrated by its lification and expression in newly infected BHK-21 cells, detected by Northern blotting with a KUN-specific probe and by immunofluorescence analysis with anti-NS3 antibodies. No infectious particles were produced when C20DXrep RNA and recombinant SFV RNAs were electroporated simultaneously. When the second electroporation was performed with a single SFV replicon RNA expressing the KUN contiguous prME genes and the KUN C gene together but under control of two separate 26S subgenomic promoters (SFV-prME-C107), a 10-fold-higher titer of infectious particles was achieved than when two different SFV replicon RNAs expressing the KUN C gene (SFV-C107) and prME genes (SFV-prME) separately were used. No SFV replicon RNAs expressing KUN structural proteins were encapsidated in secreted particles. Infectious particles pelleted by ultracentrifugation of the culture fluid from cells sequentially transfected with C20DXrep and SFV-prME-C107 RNAs were neutralized by preincubation with monoclonal antibodies to KUN E protein. Radioimmunoprecipitation analysis with anti-E antibodies of the culture fluid of the doubly transfected cells showed the presence of C, prM/M, and E proteins in the immunoprecipitated particles. Reverse transcription-PCR analysis showed that the immunoprecipitated particles also contained KUN-specific RNA. The encapsidated replicon particles sedimented more slowly than KUN virions in a 5 to 25% sucrose density gradient and were uniformly spherical, with an ∼35-nm diameter, compared with ∼50 nm for KUN virions. The results of this study demonstrate for the first time packaging of flavivirus RNA in trans , and they exclude a role in packaging for virtually all of the structural region. Possible applications of the developed packaging system include the definition of the packaging signal(s) in flavivirus RNA as well as the amino acid motif(s) in the structural proteins involved in RNA encapsidation, virion assembly, and secretion. Furthermore, it could facilitate the development of a noninfectious vaccine delivery system based on encapsidation of a noncytopathic flavivirus replicon expressing heterologous genes.

Translation of the Flavivirus Kunjin NS3 Gene in cis but Not Its RNA Sequence or Secondary Structure Is Essential for Efficient RNA Packaging

Publisher: American Society for Microbiology

Date: 11-2006

DOI: 10.1128/JVI.01559-06

Abstract: Our previous studies using trans -complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76: 10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

Loop de loop: viral RNA evades IFIT1 targeting

Publisher: Elsevier BV

Date: 04-2014

DOI: 10.1016/J.TIM.2014.02.014

Abstract: In a landmark finding published in Science, Hyde et al. have demonstrated that a hairpin RNA structure adjacent to the 5' cap of alphavirus genomic RNA confers the ability of these viruses to evade restriction by the interferon-induced host protein IFIT1.

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein

Publisher: Springer Science and Business Media LLC

Date: 11-2022

DOI: 10.1038/S41380-022-01831-0

Abstract: Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson’s disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson’s disease in COVID-19 infected in iduals, and a potential therapeutic avenue for intervention.

Assessing the potential of unmanned aerial vehicle spraying of aqueous ozone as an outdoor disinfectant for SARS-CoV-2

Publisher: Elsevier BV

Date: 05-2021

DOI: 10.1016/J.ENVRES.2021.110944

Preclinical development of a molecular clamp‐stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2

Publisher: Wiley

Date: 2021

DOI: 10.1002/CTI2.1269

Stable High-Level Expression of Heterologous Genes In Vitro and In Vivo by Noncytopathic DNA-Based Kunjin Virus Replicon Vectors

Publisher: American Society for Microbiology

Date: 05-2000

DOI: 10.1128/JVI.74.9.4394-4403.2000

Abstract: Primary features of the flavivirus Kunjin (KUN) subgenomic replicons include continuous noncytopathic replication in host cell cytoplasm and the ability to be encapsidated into secreted virus-like particles (VLPs). Previously we reported preparation of RNA-based KUN replicon vectors and expression of heterologous genes (HG) in cell culture after RNA transfection or after infection with recombinant KUN VLPs (A. N. Varnavski and A. A. Khromykh, Virology 255:366–375, 1999). In this study we describe the development of the next generation of KUN replicon vectors, which allow synthesis of replicon RNA in vivo from corresponding plasmid DNAs. These DNA-based vectors were able to direct stable expression of β-galactosidase (β-Gal) in several mammalian cell lines, and expression remained high (∼150 pg per cell) throughout cell passaging. The applicability of these vectors in vivo was demonstrated by β-Gal expression in the mouse lung epithelium for at least 8 weeks after intranasal inoculation and induction of anti-β-Gal antibody response after intramuscular inoculation of the β-Gal-encoding KUN replicon DNA. The noncytopathic nature of DNA-based KUN replicon vectors combined with high-level and stability of HG expression in a broad range of host cells should prove them to be useful in a variety of applications in vitro and in vivo.

A Single Amino Acid Substitution in the West Nile Virus Nonstructural Protein NS2A Disables Its Ability To Inhibit Alpha/Beta Interferon Induction and Attenuates Virus Virulence in Mice

Publisher: American Society for Microbiology

Date: 03-2006

DOI: 10.1128/JVI.80.5.2396-2404.2006

Abstract: Alpha/beta interferons (IFN-α/β) are key mediators of the innate immune response against viral infection. The ability of viruses to circumvent IFN-α/β responses plays a crucial role in determining the outcome of infection. In a previous study using subgenomic replicons of the Kunjin subtype of West Nile virus (WNV KUN ), we demonstrated that the nonstructural protein NS2A is a major inhibitor of IFN-β promoter-driven transcription and that a single amino acid substitution in NS2A (Ala30 to Pro [A30P]) dramatically reduced its inhibitory effect (W. J. Liu, H. B. Chen, X. J. Wang, H. Huang, and A. A. Khromykh, J. Virol. 78: 12225-12235). Here we show that incorporation of the A30P mutation into the WNV KUN genome results in a mutant virus which elicits more rapid induction and higher levels of synthesis of IFN-α/β in infected human A549 cells than that detected following wild-type WNV KUN infection. Consequently, replication of the WNV KUN NS2A/A30P mutant virus in these cells known to be high producers of IFN-α/β was abortive. In contrast, both the mutant and the wild-type WNV KUN produced similar-size plaques and replicated with similar efficiency in BHK cells which are known to be deficient in IFN-α/β production. The mutant virus was highly attenuated in neuroinvasiveness and also attenuated in neurovirulence in 3-week-old mice. Surprisingly, the mutant virus was also partially attenuated in IFN-α/βγ receptor knockout mice, suggesting that the A30P mutation may also play a role in more efficient activation of other antiviral pathways in addition to the IFN response. Immunization of wild-type mice with the mutant virus resulted in induction of an antibody response of similar magnitude to that observed in mice immunized with wild-type WNV KUN and gave complete protection against challenge with a lethal dose of the highly virulent New York 99 strain of WNV. The results confirm and extend our previous original findings on the role of the flavivirus NS2A protein in inhibition of a host antiviral response and demonstrate that the targeted disabling of a viral mechanism for evading the IFN response can be applied to the development of live attenuated flavivirus vaccine candidates.

West Nile virus infection and interferon alpha treatment alter the spectrum and the levels of coding and noncoding host RNAs secreted in extracellular vesicles

Publisher: Springer Science and Business Media LLC

Date: 10-06-2019

DOI: 10.1186/S12864-019-5835-6

Virulence and evolution of West Nile Virus, Australia, 1960-2012

Publisher: Centers for Disease Control and Prevention (CDC)

Date: 08-2016

DOI: 10.3201/EID2208.151719

Using a bioaerosol personal sampler in combination with real‐time PCR analysis for rapid detection of airborne viruses

Publisher: Wiley

Date: 18-01-2007

DOI: 10.1111/J.1462-2920.2006.01226.X

Abstract: We have recently developed a new personal s ler and demonstrated its feasibility for detection of viable airborne microorganisms including bacteria, fungi and viruses. To accelerate the time-consuming analytical procedure involving 2-5 days of biological testing, we employed a real-time PCR protocol in conjunction with the personal s ler for collection of airborne viruses. The advantage of this approach is that if the presence of a particular pathogen in the air is detected by the PCR, the remaining collecting liquid can be further analysed by more time-consuming biological methods to estimate the number of airborne infectious/live microorganisms. As s ling of bioaerosols in natural environments is likely to be associated with substantial contamination by a range of microorganisms commonly existing in an ambient air, an investigation of the specificity of detection by targeted PCR analysis is required. Here we present the results of the study on the detection of Influenza virus in the ambient air contaminated with high concentrations of bacteria and fungi using real-time PCR protocol. The combined s ling PCR detection method was found to be fully feasible for the rapid ( approximately 2.5 h) and highly specific (no cross-reactivity) identification of the labile airborne virus in the air containing elevated concentrations of other microorganisms.

A 1958 Isolate of Kedougou Virus (KEDV) from Ndumu, South Africa, Expands the Geographic and Temporal Range of KEDV in Africa

Publisher: MDPI AG

Date: 14-07-2021

DOI: 10.3390/V13071368

Abstract: The mosquito-borne flavivirus, Kedougou virus (KEDV), first isolated in Senegal in 1972, is genetically related to dengue, Zika (ZIKV) and Spondweni viruses (SPOV). Serological surveillance studies in Senegal and isolation of KEDV in the Central African Republic indicate occurrence of KEDV infections in humans, but to date, no disease has been reported. Here, we assembled the coding-complete genome of a 1958 isolate of KEDV from a pool of Aedes circumluteolus mosquitoes collected in Ndumu, KwaZulu-Natal, South Africa. The AR1071 Ndumu KEDV isolate bears 80.51% pairwise nucleotide identity and 93.34% amino acid identity with the prototype DakAar-D1470 strain and was co-isolated with SPOV through intracerebral inoculation of suckling mice and passage on VeroE6 cells. This historical isolate expands the known geographic and temporal range of this relatively unknown flavivirus, aiding future temporal phylogenetic calibration and diagnostic assay refinement.

Crystal structure of the RNA polymerase domain of the West Nile virus non-structural protein 5

Publisher: Elsevier BV

Date: 04-2007

DOI: 10.1074/JBC.M607273200

Expression of mosquito MicroRNA Aae-miR-2940-5p is downregulated in response to west nile virus infection to restrict viral replication

Publisher: American Society for Microbiology

Date: 08-2014

DOI: 10.1128/JVI.00317-14

Abstract: West Nile virus (WNV) is an enveloped virus with a single-stranded positive-sense RNA genome from the Flaviviridae family. WNV is spread by mosquitoes and able to infect humans, causing encephalitis and meningitis that can be fatal it therefore presents a significant risk for human health. In insects, innate response to RNA virus infection mostly relies on RNA interference and JAK/SAT pathways however, some evidence indicates that it can also involve microRNAs (miRNAs). miRNAs are small noncoding RNAs that regulate gene expression at posttranscriptional level and play an important role in a number of processes, including immunity and antiviral response. In this study, we focus on the miRNA-mediated response to WNV in mosquito cells. We demonstrate that in response to WNV infection the expression of a mosquito-specific miRNA, aae-miR-2940, is selectively downregulated in Aedes albopictus cells. This miRNA is known to upregulate the metalloprotease m41 FtsH gene, which we have also shown to be required for efficient WNV replication. Correspondingly, downregulation of aae-miR-2940 reduced the metalloprotease level and restricted WNV replication. Thus, we have identified a novel miRNA-dependent mechanism of antiviral response to WNV in mosquitoes. IMPORTANCE A detailed understanding of vector-pathogen interactions is essential to address the problems posed by vector-borne diseases. Host and viral miRNAs play an important role in regulating expression of viral and host genes involved in endogenous processes, including antiviral response. There has been no evidence to date for the role of mosquito miRNAs in response to flaviviruses. In this study, we show that downregulation of aae-miR-2940 in mosquito cells acts as a potential antiviral mechanism in the mosquito host to inhibit WNV replication by repressing the expression of the metalloprotease m41 FtsH gene, which is required for efficient WNV replication. This is the first identification of an miRNA-dependent antiviral mechanism in mosquitoes, which inhibits replication of WNV. Our findings should facilitate identification of targets in the mosquito genome that can be utilized to suppress vector population and/or limit WNV replication.

Skin-patch delivered subunit vaccine induces broadly neutralising antibodies against SARS-CoV-2 variants of concern

Publisher: Elsevier BV

Date: 08-2022

DOI: 10.1016/J.VACCINE.2022.07.013

Comparison of attenuated and virulent West Nile virus strains in human monocyte-derived dendritic cells as a model of initial human infection

Publisher: Springer Science and Business Media LLC

Date: 22-03-2015

DOI: 10.1186/S12985-015-0279-3

Semliki Forest virus and Kunjin virus RNA replicons elicit comparable cellular immunity but distinct humoral immunity

Publisher: Elsevier BV

Date: 21-07-2005

DOI: 10.1016/J.VACCINE.2005.04.005

Abstract: RNA replicons offer a number of qualities which make them attractive as vaccination vectors. Both alphavirus and flavivirus replicon vaccines have been investigated in preclinical models yet there has been little direct comparison of the two vector systems. To determine whether differences in the biology of the two vectors influence immunogenicity, we compared two prototypic replicon vectors based on Semliki Forest virus (SFV) (alphavirus) and Kunjin virus (KUN) (flavivirus). Both vectors when delivered as naked RNAs elicited comparable CD8+ T cell responses but the SFV vectors elicited greater humoral responses to an encoded cytoplasmic antigen beta-galactosidase. Studies in MHC class II-deficient mice revealed that neither vector could overcome the dependence of CD4+ T cell help in the development of humoral and cellular responses following immunization. These studies indicate that the distinct biology of the two replicon systems may differentially impact the adaptive immune response and this may need to be considered when designing vaccination strategies.

Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity nanobody

Publisher: Springer Science and Business Media LLC

Date: 08-02-2021

DOI: 10.1038/S41598-021-82833-W

Abstract: Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with potential therapeutic applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of Nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent.

Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis

Publisher: American Association for the Advancement of Science (AAAS)

Date: 02-12-2022

DOI: 10.1126/SCIADV.ADD8095

Abstract: All flaviviruses, including Zika virus, produce noncoding subgenomic flaviviral RNA (sfRNA), which plays an important role in viral pathogenesis. However, the exact mechanism of how sfRNA enables viral evasion of antiviral response is not well defined. Here, we show that sfRNA is required for transplacental virus dissemination in pregnant mice and subsequent fetal brain infection. We also show that sfRNA promotes apoptosis of neural progenitor cells in human brain organoids, leading to their disintegration. In infected human placental cells, sfRNA inhibits multiple antiviral pathways and promotes apoptosis, with signal transducer and activator of transcription 1 (STAT1) identified as a key shared factor. We further show that the production of sfRNA leads to reduced phosphorylation and nuclear translocation of STAT1 via a mechanism that involves sfRNA binding to and stabilizing viral protein NS5. Our results suggest the cooperation between viral noncoding RNA and a viral protein as a novel strategy for counteracting antiviral responses.

Noncoding Flavivirus RNA Displays RNA Interference Suppressor Activity in Insect and Mammalian Cells

Publisher: American Society for Microbiology

Date: 15-12-2012

DOI: 10.1128/JVI.01104-12

Abstract: West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae ) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3′-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses.

Kunjin replicon-based simian immunodeficiency virus gag vaccines

Publisher: Elsevier BV

Date: 06-2008

DOI: 10.1016/J.VACCINE.2008.04.001

Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin

Publisher: Elsevier BV

Date: 03-2001

DOI: 10.1016/S0166-0934(00)00270-6

Abstract: The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD-->GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity.

Viral RNA Intermediates as Targets for Detection and Discovery of Novel and Emerging Mosquito-Borne Viruses

Publisher: Public Library of Science (PLoS)

Date: 23-03-2015

DOI: 10.1371/JOURNAL.PNTD.0003629

Last 20 aa of the West Nile virus NS1` protein are responsible for its retention in cells and the formation of unique heat-stable dimers

Publisher: Microbiology Society

Date: 05-2015

DOI: 10.1099/VIR.0.000053

Abstract: West Nile virus (WNV), a mosquito-borne flavivirus, is the major cause of arboviral encephalitis in the USA. As with other members of the Japanese encephalitis virus serogroup, WNV produces an additional non-structural protein, NS1', a C-terminal extended product of NS1 generated as the result of a -1 programmed ribosomal frameshift (PRF). We have previously shown that mutations abolishing the PRF, and consequently NS1', resulted in reduced neuroinvasiveness. However, whether this was caused by the PRF event itself or by the lack of a PRF product, NS1', or a combination of both, remains undetermined. Here, we showed that WNV NS1' formed a unique subpopulation of heat- and low-pH-stable dimers. C-terminal truncations and mutational analysis employing an NS1'-expressing plasmid showed that stability of NS1' dimers was linked to the penultimate 10 aa. To examine the role of NS1' heat-stable dimers in virus replication and pathogenicity, a stop codon mutation was introduced into NS1' to create a WNV producing a truncated version of NS1' lacking the last 20 aa, but not affecting the PRF. NS1' protein produced by this mutant virus was secreted more efficiently than WT NS1', indicating that the sequence of the last 20 aa of NS1' was responsible for its cellular retention. Further analysis of this mutant showed growth kinetics in cells and virulence in weanling mice after peripheral infection similar to the WT WNVKUN, suggesting that full-length NS1' was not essential for virus replication in vitro and for virulence in mice.

Peptide-Based Vaccine against SARS-CoV-2: Peptide Antigen Discovery and Screening of Adjuvant Systems

Publisher: MDPI AG

Date: 13-04-2022

DOI: 10.3390/PHARMACEUTICS14040856

Abstract: The SARS-CoV-2 virus has caused a global crisis, resulting in 0.5 billion infections and over 6 million deaths as of March 2022. Fortunately, infection and hospitalization rates were curbed due to the rollout of DNA and mRNA vaccines. However, the efficacy of these vaccines significantly drops a few months post immunization, from 88% down to 47% in the case of the Pfizer BNT162 vaccine. The emergence of variant strains, especially delta and omicron, have also significantly reduced vaccine efficacy. We propose peptide vaccines as a potential solution to address the inadequacies of the current vaccines. Peptide vaccines can be easily modified to target emerging strains, have greater stability, and do not require cold-chain storage. We screened five peptide fragments (B1–B5) derived from the SARS-CoV-2 spike protein to identify neutralizing B-cell peptide antigens. We then investigated adjuvant systems for efficient stimulation of immune responses against the most promising peptide antigens, including liposomal formulations of polyleucine (L10) and polymethylacrylate (PMA), as well as classical adjuvants (CFA and MF59). Immune efficacy of formulations was evaluated using competitive ELISA, pseudovirion neutralization, and live virus neutralization assays. Unfortunately, peptide conjugation to L10 and PMA dramatically altered the secondary structure, resulting in low antibody neutralization efficacy. Of the peptides tested, only B3 administered with CFA or MF59 was highly immunogenic. Thus, a peptide vaccine relying on B3 may provide an attractive alternative to currently marketed vaccines.

Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay

Publisher: Research Square Platform LLC

Date: 08-07-2021

DOI: 10.21203/RS.3.PEX-1559/V1

Abstract: This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin-converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include: 1) serum heat treatment to prevent non-specific interactions, 2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, 3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and 4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 hours for serum s les this includes the 7.5 hours of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the ‘gold standard’ assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious, time-consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R 2 =0.975, n=25), demonstrating high sensitivity.

Replication and Gene Function in Kunjin Virus

Publisher: Springer Berlin Heidelberg

Date: 2002

DOI: 10.1007/978-3-642-59403-8_16

Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2

Publisher: Cold Spring Harbor Laboratory

Date: 12-06-2023

DOI: 10.1101/2023.06.12.544552

Abstract: Why in iduals with Down Syndrome (DS, trisomy 21) are particularly susceptible to SARS CoV-2 induced neuropathology remains largely unclear. Since the choroid plexus (CP) performs important barrier and immune-interface functions, secretes the cerebrospinal fluid and strongly expresses the ACE2 receptor and the chromosome 21 encoded TMPRSS2 protease, we hypothesized that the CP could play a role in establishing SARS-CoV-2 infection in the brain. To investigate the role of the choroid plexus in SARS-CoV-2 central nervous system infection in DS, we established a new type of brain organoid from DS and isogenic euploid control iPSC that consists of a core of appropriately patterned functional cortical neuronal cell types that is surrounded by a patent and functional choroid plexus (CPCOs). Remarkably, DS-CPCOs not only recapitulated abnormal features of DS cortical development but also revealed defects in ciliogenesis and epithelial cell polarity of the developing choroid plexus. We next demonstrate that the choroid plexus layer facilitates SARS-CoV-2 replication and infection of cortical neuronal cells, and that this is increased in DS-CPCOs. We further show that inhibition of TMPRSS2 and Furin activity inhibits SARS-CoV-2 replication in DS CPCOs to the level observed in euploid organoids. We conclude that CPCOs are a useful model for dissecting the role of the choroid plexus in euploid and DS forebrain development and enables screening for therapeutics that can inhibit SARS-CoV-2 induced neuro-pathogenesis.

Kunjin virus replicons: an RNA-based, non-cytopathic viral vector system for protein production, vaccine and gene therapy applications

Publisher: Informa UK Limited

Date: 26-01-2006

DOI: 10.1517/14712598.6.2.135

Abstract: The application of viral vectors for gene expression and delivery is rapidly evolving, with several entering clinical trials. However, a number of issues, including safety, gene expression levels, cell selectivity and antivector immunity, are driving the search for new vector systems. A number of replicon-based vectors derived from positive-strand RNA viruses have recently been developed, and this paper reviews the current knowledge on the first flavivirus replicon system, which is based on the Australian flavivirus Kunjin (KUN). Like most replicon systems, KUN replicons can be delivered as DNA, RNA or virus-like particles, they replicate their RNA in the cytoplasm and direct prolonged high-level gene expression. However, unlike most alphavirus replicon systems, KUN replicons are non-cytopathic, with transfected cells able to ide, allowing the establishment of cell lines stably expressing replicon RNA and heterologous genes. As vaccine vectors KUN replicons can induce potent, long-lived, protective, immunogen-specific CD8+ T cell immunity, a feature potentially related to extended production of antigen and double-stranded RNA-induced 'danger signals'. The identification of KUN replicon mutants that induce increased levels of IFN-alpha/beta has also spawned investigation of KUN replicons for use in cancer gene therapy. The unique characteristics of KUN replicons may thus make them suitable for specific protein production, vaccine and gene therapy applications.

A paucigranulocytic asthma host environment promotes the emergence of virulent influenza viral variants

Publisher: eLife Sciences Publications, Ltd

Date: 16-02-2021

DOI: 10.7554/ELIFE.61803

Abstract: Influenza virus has a high mutation rate, such that within one host different viral variants can emerge. Evidence suggests that influenza virus variants are more prevalent in pregnant and/or obese in iduals due to their impaired interferon response. We have recently shown that the non-allergic, paucigranulocytic subtype of asthma is associated with impaired type I interferon production. Here, we seek to address if this is associated with an increased emergence of influenza virus variants. Compared to controls, mice with paucigranulocytic asthma had increased disease severity and an increased emergence of influenza virus variants. Specifically, PB1 mutations exclusively detected in asthmatic mice were associated with increased polymerase activity. Furthermore, asthmatic host-derived virus led to increased disease severity in wild-type mice. Taken together, these data suggest that at least a subset of patients with asthma may be more susceptible to severe influenza and may be a possible source of new influenza virus variants.

Characterization of virulent West Nile virus Kunjin strain, Australia, 2011

Publisher: Centers for Disease Control and Prevention (CDC)

Date: 05-2012

DOI: 10.3201/EID1805.111720

De Novo Generation and Characterization of New Zika Virus Isolate Using Sequence Data from a Microcephaly Case

Publisher: American Society for Microbiology

Date: 28-06-2017

DOI: 10.1128/MSPHEREDIRECT.00190-17

Abstract: The major complications of an ongoing Zika virus outbreak in the Americas and Asia are congenital defects caused by the virus’s ability to cross the placenta and infect the fetal brain. The ability to generate molecular tools to analyze viral isolates from the current outbreak is essential for furthering our understanding of how these viruses cause congenital defects. The majority of existing viral isolates and infectious cDNA clones generated from them have undergone various numbers of passages in cell culture and/or suckling mice, which is likely to result in the accumulation of adaptive mutations that may affect viral properties. The approach described herein allows rapid generation of new, fully functional Zika virus isolates directly from deep sequencing data from virus-infected tissues without the need for prior virus passaging and for the generation and propagation of full-length cDNA clones. The approach should be applicable to other medically important flaviviruses and perhaps other positive-strand RNA viruses.

Reporter Flaviviruses as Tools to Demonstrate Homologous and Heterologous Superinfection Exclusion

Publisher: MDPI AG

Date: 08-07-2022

DOI: 10.3390/V14071501

Abstract: Binjari virus (BinJV) is a lineage II or dual-host affiliated insect-specific flavivirus previously demonstrated as replication-deficient in vertebrate cells. Previous studies have shown that BinJV is tolerant to exchanging its structural proteins (prM-E) with pathogenic flaviviruses, making it a safe backbone for flavivirus vaccines. Here, we report generation by circular polymerase extension reaction of BinJV expressing zsGreen or mCherry fluorescent protein. Recovered BinJV reporter viruses grew to high titres (107−8 FFU/mL) in Aedes albopictus C6/36 cells assayed using immunoplaque assays (iPA). We also demonstrate that BinJV reporters could be semi-quantified live in vitro using a fluorescence microplate reader with an observed linear correlation between quantified fluorescence of BinJV reporter virus-infected C6/36 cells and iPA-quantitated virus titres. The utility of the BinJV reporter viruses was then examined in homologous and heterologous superinfection exclusion assays. We demonstrate that primary infection of C6/36 cells with BinJVzsGreen completely inhibits a secondary infection with homologous BinJVmCherry or heterologous ZIKVmCherry using fluorescence microscopy and virus quantitation by iPA. Finally, BinJVzsGreen infections were examined in vivo by microinjection of Aedes aegypti with BinJVzsGreen. At seven days post-infection, a strong fluorescence in the vicinity of salivary glands was detected in frozen sections. This is the first report on the construction of reporter viruses for lineage II insect-specific flaviviruses and establishes a tractable system for exploring flavivirus superinfection exclusion in vitro and in vivo.

A Kunjin replicon vector encoding granulocyte macrophage colony-stimulating factor for intra-tumoral gene therapy

Publisher: Springer Science and Business Media LLC

Date: 18-12-2008

DOI: 10.1038/GT.2008.169

Abstract: We have recently developed a non-cytopathic RNA replicon-based viral vector system based on the flavivirus Kunjin. Here, we illustrate the utility of the Kunjin replicon system for gene therapy. Intra-tumoral injections of Kunjin replicon virus-like particles encoding granulocyte colony-stimulating factor were able to cure >50% of established subcutaneous CT26 colon carcinoma and B16-OVA melanomas. Regression of CT26 tumours correlated with the induction of anti-cancer CD8 T cells, and treatment of subcutaneous CT26 tumours also resulted in the regression of CT26 lung metastases. Only a few immune-based strategies are able to cure these aggressive tumours once they are of a reasonable size, illustrating the potential of this vector system for intra-tumoral gene therapy applications.

Dermal Delivery of a SARS-CoV-2 Subunit Vaccine Induces Immunogenicity against Variants of Concern

Publisher: MDPI AG

Date: 08-04-2022

DOI: 10.3390/VACCINES10040578

Abstract: The ongoing coronavirus disease 2019 (COVID-19) pandemic continues to disrupt essential health services in 90 percent of countries today. The spike (S) protein found on the surface of the causative agent, the SARS-CoV-2 virus, has been the prime target for current vaccine research since antibodies directed against the S protein were found to neutralize the virus. However, as new variants emerge, mutations within the spike protein have given rise to potential immune evasion of the response generated by the current generation of SARS-CoV-2 vaccines. In this study, a modified, HexaPro S protein subunit vaccine, delivered using a needle-free high-density microarray patch (HD-MAP), was investigated for its immunogenicity and virus-neutralizing abilities. Mice given two doses of the vaccine candidate generated potent antibody responses capable of neutralizing the parental SARS-CoV-2 virus as well as the variants of concern, Alpha and Delta. These results demonstrate that this alternative vaccination strategy has the potential to mitigate the effect of emerging viral variants.

The Naturally Attenuated Kunjin Strain of West Nile Virus Shows Enhanced Sensitivity to the Host Type I Interferon Response

Publisher: American Society for Microbiology

Date: 06-2011

DOI: 10.1128/JVI.00232-11

Abstract: The host determinants that contribute to attenuation of the naturally occurring nonpathogenic strain of West Nile virus (WNV), the Kunjin strain (WNV KUN ), remain unknown. Here, we show that compared to a highly pathogenic North American strain, WNV KUN exhibited an enhanced sensitivity to the antiviral effects of type I interferon. Our studies establish that the virulence of WNV KUN can be restored in cells and mice deficient in specific interferon regulatory factors (IRFs) or the common type I interferon receptor. Thus, WNV KUN is attenuated primarily through its enhanced restriction by type I interferon- and IRF-3-dependent mechanisms.

The distinguishing NS5-M114V mutation in American Zika virus isolates has negligible impacts on virus replication and transmission potential

Publisher: Public Library of Science (PLoS)

Date: 10-05-2022

DOI: 10.1371/JOURNAL.PNTD.0010426

Abstract: During 2015–2016, outbreaks of Zika virus (ZIKV) occurred in Southeast Asia and the Americas. Most ZIKV infections in humans are asymptomatic, while clinical manifestation is usually a self-limiting febrile disease with maculopapular rash. However, ZIKV is capable of inducing a range of severe neurological complications collectively described as congenital Zika syndrome (CZS). Notably, the scale and magnitude of outbreaks in Southeast Asia were significantly smaller compared to those in the Americas. Sequence comparison between epidemic-associated ZIKV strains from Southeast Asia with those from the Americas revealed a methionine to valine substitution at residue position 114 of the NS5 protein (NS5-M114V) in all the American isolates. Using an American isolate of ZIKV (Natal), we investigated the impact of NS5-M114V mutation on virus replication in cells, virulence in interferon (IFN) α/β receptor knockout ( Ifnar -/- ) mice, as well as replication and transmission potential in Aedes aegypti mosquitoes. We demonstrated that NS5-M114V mutation had insignificant effect on ZIKV replication efficiency in cells, its ability to degrade STAT2, and virulence in vivo , albeit viremia was slightly prolonged in mice. Furthermore, NS5-M114V mutation decreased mosquito infection and dissemination rates but had no effect on virus secretion into the saliva. Taken together, our findings support the notion that NS5-M114V mutation is unlikely to be a major determinant for virus replication and transmission potential.

Statement in Support of: “Virology under the Microscope—a Call for Rational Discourse”

Publisher: American Society for Microbiology

Date: 27-06-2023

DOI: 10.1128/MBIO.00815-23

A vaccinia-based single vector construct multi-pathogen vaccine protects against both Zika and chikungunya viruses

Publisher: Springer Science and Business Media LLC

Date: 26-03-2018

DOI: 10.1038/S41467-018-03662-6

Abstract: Zika and chikungunya viruses have caused major epidemics and are transmitted by Aedes aegypti and/or Aedes albopictu s mosquitoes. The “Sementis Copenhagen Vector” (SCV) system is a recently developed vaccinia-based, multiplication-defective, vaccine vector technology that allows manufacture in modified CHO cells. Herein we describe a single-vector construct SCV vaccine that encodes the structural polyprotein cassettes of both Zika and chikungunya viruses from different loci. A single vaccination of mice induces neutralizing antibodies to both viruses in wild-type and IFNAR −/− mice and protects against (i) chikungunya virus viremia and arthritis in wild-type mice, (ii) Zika virus viremia and fetal lacental infection in female IFNAR −/− mice, and (iii) Zika virus viremia and testes infection and pathology in male IFNAR −/− mice. To our knowledge this represents the first single-vector construct, multi-pathogen vaccine encoding large polyproteins, and offers both simplified manufacturing and formulation, and reduced “shot burden” for these often co-circulating arboviruses.

A Highly Structured, Nuclease-Resistant, Noncoding RNA Produced by Flaviviruses Is Required for Pathogenicity

Publisher: Elsevier BV

Date: 12-2008

DOI: 10.1016/J.CHOM.2008.10.007

Abstract: Viral noncoding RNAs have been shown to play an important role in virus-host interplay to facilitate virus replication. We report that members of the genus Flavivirus, a large group of medically important encephalitic RNA viruses, produce a unique and highly structured noncoding RNA of 0.3-0.5 kb derived from the 3' untranslated region of the viral genome. Using West Nile virus as a model, we show that this subgenomic RNA is a product of incomplete degradation of viral genomic RNA by cellular ribonucleases. Highly conserved RNA structures located at the beginning of the 3' untranslated region render this RNA resistant to nucleases, and the resulting subgenomic RNA product is essential for virus-induced cytopathicity and pathogenicity. Thus, flaviviruses evolved a unique strategy to generate a noncoding RNA product that allows them to kill the host more efficiently.

cis - and trans -Acting Elements in Flavivirus RNA Replication

Publisher: American Society for Microbiology

Date: 04-2000

DOI: 10.1128/JVI.74.7.3253-3263.2000

Abstract: Most of the seven flavivirus nonstructural proteins (NS1 to NS5) encoded in the distal two-thirds of the RNA positive-sense genome are believed to be essential components of RNA replication complexes. To explore the functional relationships of these components in RNA replication, we used trans -complementation analysis of full-length infectious RNAs of Kunjin (KUN) virus with a range of lethal in-frame deletions in the nonstructural coding region, using as helper a repBHK cell line stably producing functional replication complexes from KUN replicon RNA. Recently we showed that replication of KUN RNAs with large carboxy-terminal deletions including the entire RNA polymerase region in the NS5 gene, representing 34 to 75% of the NS5 coding content, could be complemented after transfection into repBHK cells. In this study we have demonstrated that KUN RNAs with deletions of 84 to 97% of the NS1 gene, or of 13 to 63% of the NS3 gene including the entire helicase region, were also complemented in repBHK cells with variable efficiencies. In contrast, KUN RNAs with deletions in any of the other four nonstructural genes NS2A, NS2B, NS4A, and NS4B were not complemented. We have also demonstrated successful trans complementation of KUN RNAs containing either combined double deletions in the NS1 and NS5 genes or triple deletions in the NS1, NS3, and NS5 genes comprising as much as 38% of the entire nonstructural coding content. Based on these and our previous complementation results, we have generated a map of cis - and trans -acting elements in RNA replication for the nonstructural coding region of the flavivirus genome. These results are discussed in the context of our model on formation and composition of the flavivirus replication complex, and we suggest molecular mechanisms by which functions of some defective components of the replication complex can be complemented by their wild-type counterparts expressed from another (helper) RNA molecule.

A broadly protective antibody that targets the flavivirus NS1 protein

Publisher: American Association for the Advancement of Science (AAAS)

Date: 08-01-2021

DOI: 10.1126/SCIENCE.ABB9425

Abstract: Flaviviruses are a group of RNA viruses that include the human pathogens dengue virus, Zika virus, and West Nile virus. The envelope protein (E) on the virus surface has been the target of vaccine development, but problems have arisen with antibodies against E, leading to enhanced infection. Now, Modhiran et al. and Biering et al. describe two different antibodies that bind to the flavivirus NS1 protein and prevent it from disrupting epithelial cells, which is associated with severe disease. Both antibodies cross-react with multiple flavivirus NS1 proteins. The antibodies reduce viremia and increase survival in mouse models of flavivirus disease. Both papers include structures of NS1 bound to an antibody, which give insight into the protective mechanism. Science , this issue p. 190 , p. 194

RNA Structures Required for Production of Subgenomic Flavivirus RNA

Publisher: American Society for Microbiology

Date: 11-2010

DOI: 10.1128/JVI.01159-10

Abstract: Flaviviruses are a group of single-stranded, positive-sense RNA viruses causing ∼100 million infections per year. We have recently shown that flaviviruses produce a unique, small, noncoding RNA (∼0.5 kb) derived from the 3′ untranslated region (UTR) of the genomic RNA (gRNA), which is required for flavivirus-induced cytopathicity and pathogenicity (G. P. Pijlman et al., Cell Host Microbe, 4: 579-591, 2008). This RNA (subgenomic flavivirus RNA [sfRNA]) is a product of incomplete degradation of gRNA presumably by the cellular 5′-3′ exoribonuclease XRN1, which stalls on the rigid secondary structure stem-loop II (SL-II) located at the beginning of the 3′ UTR. Mutations or deletions of various secondary structures in the 3′ UTR resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3). Here, we investigated in detail the importance of West Nile virus Kunjin (WNV KUN ) 3′ UTR secondary structures as well as tertiary interactions for sfRNA formation. We show that secondary structures SL-IV and dumbbell 1 (DB1) downstream of SL-II are able to prevent further degradation of gRNA when the SL-II structure is deleted, leading to production of sfRNA2 and sfRNA3, respectively. We also show that a number of pseudoknot (PK) interactions, in particular PK1 stabilizing SL-II and PK3 stabilizing DB1, are required for protection of gRNA from nuclease degradation and production of sfRNA. Our results show that PK interactions play a vital role in the production of nuclease-resistant sfRNA, which is essential for viral cytopathicity in cells and pathogenicity in mice.

An Optimized High-Throughput Immuno-Plaque Assay for SARS-CoV-2

Publisher: Frontiers Media SA

Date: 12-02-2021

DOI: 10.3389/FMICB.2021.625136

Abstract: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 and is capable of human-to-human transmission and rapid global spread. The rapid emergence and global spread of SARS-CoV-2 has encouraged the establishment of a rapid, sensitive, and reliable viral detection and quantification methodology. Here, we present an alternative assay, termed immuno-plaque assay (iPA), which utilizes a combination of plaque assay and immunofluorescence techniques. We have extensively optimized the conditions for SARS-CoV-2 infection and demonstrated the great flexibility of iPA detection using several antibodies and dual-probing with two distinct epitope-specific antibodies. In addition, we showed that iPA could be utilized for ultra-high-throughput viral titration and neutralization assay within 24 h and is amenable to a 384-well format. These advantages will significantly accelerate SARS-CoV-2 research outcomes during this pandemic period.

Regulated cleavages at the West Nile virus NS4A-2K-NS4B junctions play a major role in rearranging cytoplasmic membranes and Golgi trafficking of the NS4A protein

Publisher: American Society for Microbiology

Date: 05-2006

DOI: 10.1128/JVI.80.9.4623-4632.2006

Abstract: A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS2B-3-4A, as well as that of in idual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the in idual NS4A and NS4B proteins. In idual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.

RNA binding properties of core protein of the flavivirus Kunjin

Publisher: Springer Science and Business Media LLC

Date: 03-1996

DOI: 10.1007/BF01718326

Viral determinants in the NS3 helicase and 2K peptide that promote West Nile virus resistance to antiviral action of 2′,5′-oligoadenylate synthetase 1b

Publisher: Elsevier BV

Date: 03-2010

DOI: 10.1016/J.VIROL.2009.12.036

Abstract: The interferon-inducible 2',5'-oligoadenylate synthetase 1b (Oas1b) protein inhibits West Nile virus (WNV) infection by preventing viral RNA (vRNA) accumulation in infected cells. Serial passage of WNV in Oas1b-expressing mouse cells selected a virus variant with improved growth capacity. Two major amino acid substitutions were identified in this Oas1b-resistant WNV variant: NS3-S365G in the ATPase/helicase domain of NS3 and 2K-V9M in the C-terminal segment of NS4A. To assess their effect on antiviral activity of Oas1b, the NS3 and 2K mutations were engineered into an infectious WNV cDNA clone. The NS3 mutation alters requirement of ATP for ATPase activity and attenuates Oas1b-mediated suppression of vRNA accumulation. However, growth of NS3-mutant virus remains impaired in Oas1b-expressing cells. Only the 2K-V9M mutation efficiently rescued viral growth by promoting vRNA replication. Thus, WNV resistance to Oas1b antiviral action could be attributed to the 2K-V9M substitution with a potential role of NS3-S365G through rescue of vRNA accumulation.

Internal Ribosome Entry Site-Based Attenuation of a Flavivirus Candidate Vaccine and Evaluation of the Effect of Beta Interferon Coexpression on Vaccine Properties

Publisher: American Society for Microbiology

Date: 15-02-2014

DOI: 10.1128/JVI.03051-13

Abstract: Infectious clone technologies allow the rational design of live attenuated viral vaccines with the possibility of vaccine-driven coexpression of immunomodulatory molecules for additional vaccine safety and efficacy. The latter could lead to novel strategies for vaccine protection against infectious diseases where traditional approaches have failed. Here we show for the flavivirus Murray Valley encephalitis virus (MVEV) that incorporation of the internal ribosome entry site (IRES) of Encephalomyocarditis virus between the capsid and prM genes strongly attenuated virulence and that the resulting bicistronic virus was both genetically stable and potently immunogenic. Furthermore, the novel bicistronic genome organization facilitated the generation of a recombinant virus carrying an beta interferon (IFN-β) gene. Given the importance of IFNs in limiting virus dissemination and in efficient induction of memory B and T cell antiviral immunity, we hypothesized that coexpression of the cytokine with the live vaccine might further increase virulence attenuation without loss of immunogenicity. We found that bicistronic mouse IFN-β coexpressing MVEV yielded high virus and IFN titers in cultured cells that do not respond to the coexpressed IFN. However, in IFN response-sufficient cell cultures and mice, the virus produced a self-limiting infection. Nevertheless, the attenuated virus triggered robust innate and adaptive immune responses evidenced by the induced expression of Mx proteins (used as a sensitive biomarker for measuring the type I IFN response) and the generation of neutralizing antibodies, respectively. IMPORTANCE The family Flaviviridae includes a number of important human pathogens, such as Dengue virus , Yellow fever virus , Japanese encephalitis virus , West Nile virus , and Hepatitis C virus . Flaviviruses infect large numbers of in iduals on all continents. For ex le, as many as 100 million people are infected annually with Dengue virus , and 150 million people suffer a chronic infection with Hepatitis C virus . However, protective vaccines against dengue and hepatitis C are still missing, and improved vaccines against other flaviviral diseases are needed. The present study investigated the effects of a redesigned flaviviral genome and the coexpression of an antiviral protein (interferon) on virus replication, pathogenicity, and immunogenicity. Our findings may aid in the rational design of a new class of well-tolerated and safe vaccines.

Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay

Publisher: MDPI AG

Date: 16-12-2021

DOI: 10.3390/VACCINES9121493

Abstract: This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for serum s les this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the ‘gold standard’ assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R2 = 0.975, n = 25), demonstrating high sensitivity.

Role of nonstructural protein NS2A in flavivirus assembly

Publisher: American Society for Microbiology

Date: 15-05-2008

DOI: 10.1128/JVI.00002-08

Abstract: Flavivirus nonstructural (NS) proteins are involved in RNA replication and modulation of the host antiviral response however, evidence is mounting that some NS proteins also have essential roles in virus assembly. Kunjin virus (KUN) NS2A is a small, hydrophobic, transmembrane protein that is part of the replication complex and inhibits interferon induction. Previously, we have shown that an isoleucine (I)-to-asparagine (N) substitution at position 59 of the NS2A protein blocked the production of secreted virus particles in cells electroporated with viral RNA carrying this mutation. We now show that prolonged incubation of mutant KUN NS2A-I59N replicon RNA, in an inducible BHK-derived packaging cell line (expressing KUN structural proteins C, prM, and E), generated escape mutants that rescued the secretion of infectious virus-like particles. Sequencing identified three groups of revertants that included (i) reversions to wild-type, hydrophobic Ile, (ii) pseudorevertants to more hydrophobic residues (Ser, Thr, and Tyr) at codon 59, and (iii) pseudorevertants retaining Asn at NS2A codon 59 but containing a compensatory mutation (Thr-to-Pro) at NS2A codon 149. Engineering hydrophobic residues at NS2A position 59 or the compensatory T149P mutation into NS2A-I59N replicon RNA restored the assembly of secreted virus-like particles in packaging cells. T149P mutation also rescued virus production when introduced into the full-length KUN RNA containing an NS2A-I59N mutation. Immunofluorescence and electron microscopy analyses of NS2A-I59N replicon-expressing cells showed a distinct lack of virus-induced membranes normally present in cells expressing wild-type replicon RNA. The compensatory mutation NS2A-T149P restored the induction of membrane structures to a level similar to those observed during wild-type replication. The results further confirm the role of NS2A in virus assembly, demonstrate the importance of hydrophobic residues at codon 59 in this process, implicate the involvement of NS2A in the biogenesis of virus-induced membranes, and suggest a vital role for the virus-induced membranes in virus assembly.

The West Nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced MxA protein

Publisher: Elsevier BV

Date: 2014

DOI: 10.1016/J.VIROL.2013.10.005

Abstract: Flaviviruses have evolved means to evade host innate immune responses. Recent evidence suggests this is due to prevention of interferon production and signaling in flavivirus-infected cells. Here we show that the interferon-induced MxA protein can sequester the West Nile virus strain Kunjin virus (WNVKUN) capsid protein in cytoplasmic tubular structures in an expression-replication system. This sequestering resulted in reduced titers of secreted WNVKUN particles. We show by electron microscopy, tomography and 3D modeling that these cytoplasmic tubular structures form organized bundles. Additionally we show that recombinant ER-targeted MxA can restrict production of infectious WNVKUN under conditions of virus infection. Our results indicate a co-ordinated and compartmentalized WNVKUN assembly process may prevent recognition of viral components by MxA, particularly the capsid protein. This recognition can be exploited if MxA is targeted to intracellular sites of WNVKUN assembly. This results in further understanding of the mechanisms of flavivirus evasion from the immune system.

Chimeric viruses between Rocio and West Nile: the role for Rocio prM-E proteins in virulence and inhibition of interferon-alpha/beta signaling

Publisher: Springer Science and Business Media LLC

Date: 20-03-2017

DOI: 10.1038/SREP44642

Abstract: Mosquito-transmitted flavivirus Rocio (ROCV) was responsible for an outbreak of encephalitis in the Ribeira Valley, located in the south coast of Sao Paulo State, Brazil, in 1975–1976. ROCV also causes fatal encephalitis in adult mice. Seroprevalence studies in humans, horses and water buffaloes in different regions of Brazil have suggested that ROCV is still circulating in the country, indicating the risk of re-emergence of this virus. West Nile virus (WNV) is also a mosquito-transmitted encephalitic flavivirus, however, WNV strains circulating in Australia have not been associated with outbreaks of disease in humans and exhibit low virulence in adult mice. To identify viral determinants of ROCV virulence, we have generated reciprocal chimeric viruses between ROCV and the Australian strain of WNV by swapping structural prM and E genes. Chimeric WNV containing ROCV prM-E genes replicated more efficiently than WNV or chimeric ROCV containing WNV prM-E genes in mammalian cells, was as virulent as ROCV in adult mice, and inhibited type I IFN signaling as efficiently as ROCV. The results show that ROCV prM and E proteins are major virulence determinants and identify unexpected function of these proteins in inhibition of type I interferon response.

Ancestral SARS-CoV-2, but not Omicron, replicates less efficiently in primary pediatric nasal epithelial cells

Publisher: Public Library of Science (PLoS)

Date: 08-2022

DOI: 10.1371/JOURNAL.PBIO.3001728

Abstract: Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air–liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.

Infectious DNAs derived from insect-specific flavivirus genomes enable identification of pre- and post-entry host restrictions in vertebrate cells

Publisher: Springer Science and Business Media LLC

Date: 07-06-2017

DOI: 10.1038/S41598-017-03120-1

Abstract: Flaviviruses such as West Nile virus (WNV), dengue virus and Zika virus are mosquito-borne pathogens that cause significant human diseases. A novel group of insect-specific flaviviruses (ISFs), which only replicate in mosquitoes, have also been identified. However, little is known about the mechanisms of ISF host restriction. We report the generation of infectious cDNA from two Australian ISFs, Parramatta River virus (PaRV) and Palm Creek virus (PCV). Using circular polymerase extension cloning (CPEC) with a modified OpIE2 insect promoter, infectious cDNA was generated and transfected directly into mosquito cells to produce infectious virus indistinguishable from wild-type virus. When infectious PaRV cDNA under transcriptional control of a mammalian promoter was used to transfect mouse embryo fibroblasts, the virus failed to initiate replication even when cell entry steps were by-passed and the type I interferon response was lacking. We also used CPEC to generate viable chimeric viruses between PCV and WNV. Analysis of these hybrid viruses revealed that ISFs are also restricted from replication in vertebrate cells at the point of entry. The approaches described here to generate infectious ISF DNAs and chimeric viruses provide unique tools to further dissect the mechanisms of their host restriction.

Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis

Publisher: Cold Spring Harbor Laboratory

Date: 19-05-2021

DOI: 10.1101/2021.05.18.444753

Abstract: Zika virus (ZIKV) is a re-emerging pathogenic flavivirus, which causes microcephaly in infants and poses a continuing threat to public health. ZIKV, like all other flaviviruses, produces highly abundant noncoding RNA known as subgenomic flaviviral RNA (sfRNA). Herein we utilized wild-type and mutant ZIKV defective in production of sfRNA to elucidate for the first time how production of sfRNA affects all aspects of ZIKV pathogenesis. We found that in mouse pregnancy model of infection sfRNA is required for trans-placental dissemination of ZIKV and subsequent infection of fetal brain. Using human brain organoids, we showed that sfRNA promotes apoptosis of neural progenitor cells leading to profound cytopathicity and disintegration of organoids. We also found by transcriptome profiling and gene network analysis that in infected human placental cells sfRNA inhibits multiple antiviral pathways and promotes apoptosis with STAT1 identified as a key shared factor linking these two interconnected sfRNA activities. We further showed for the first time that sfRNA inhibits phosphorylation and nuclear translocation of STAT1 by a novel mechanism which involves binding to and stabilizing viral protein NS5. This allows accumulation of NS5 at the levels required for efficient inhibition of STAT1 phosphorylation. Thus, we elucidated the molecular mechanism by which ZIKV sfRNA exerts its functions in vertebrate hosts and discovered a co-operation between viral noncoding RNA and a viral protein as a novel strategy employed by viruses to counteract antiviral responses.

A recombinant platform for flavivirus vaccines and diagnostics using chimeras of a new insect-specific virus.

Publisher: American Association for the Advancement of Science (AAAS)

Date: 11-12-2019

DOI: 10.1126/SCITRANSLMED.AAX7888

Abstract: Flaviviruses such as dengue, yellow fever, Zika, West Nile, and Japanese encephalitis virus present substantial global health burdens. New vaccines are being sought to address safety and manufacturing issues associated with current live attenuated vaccines. Here, we describe a new insect-specific flavivirus, Binjari virus, which was found to be remarkably tolerant for exchange of its structural protein genes (prME) with those of the aforementioned pathogenic vertebrate-infecting flaviviruses (VIFs). Chimeric BinJ/VIF-prME viruses remained replication defective in vertebrate cells but replicated with high efficiency in mosquito cells. Cryo-electron microscopy and monoclonal antibody binding studies illustrated that the chimeric BinJ/VIF-prME virus particles were structurally and immunologically similar to their parental VIFs. Pilot manufacturing in C6/36 cells suggests that high yields can be reached up to 10

DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus

Publisher: Proceedings of the National Academy of Sciences

Date: 13-08-2003

DOI: 10.1073/PNAS.1834270100

Abstract: A plasmid DNA directing transcription of the infectious full-length RNA genome of Kunjin (KUN) virus in vivo from a mammalian expression promoter was used to vaccinate mice intramuscularly. The KUN viral cDNA encoded in the plasmid contained the mutation in the NS1 protein (Pro-250 to Leu) previously shown to attenuate KUN virus in weanling mice. KUN virus was isolated from the blood of immunized mice 3-4 days after DNA inoculation, demonstrating that infectious RNA was being transcribed in vivo however, no symptoms of virus-induced disease were observed. By 19 days postimmunization, neutralizing antibody was detected in the serum of immunized animals. On challenge with lethal doses of the virulent New York strain of West Nile (WN) or wild-type KUN virus intracerebrally or intraperitoneally, mice immunized with as little as 0.1-1 μg of KUN plasmid DNA were solidly protected against disease. This finding correlated with neutralization data in vitro showing that serum from KUN DNA-immunized mice neutralized KUN and WN viruses with similar efficiencies. The results demonstrate that delivery of an attenuated but replicating KUN virus via a plasmid DNA vector may provide an effective vaccination strategy against virulent strains of WN virus.

West Nile virus NS2A protein facilitates virus-induced apoptosis independently of interferon response

Publisher: Microbiology Society

Date: 02-2013

DOI: 10.1099/VIR.0.047076-0

Abstract: The flavivirus NS2A protein is a small, multifunctional protein, involved in replication, virion formation and regulation of the innate immune response. Using the Kunjin strain of West Nile virus (WNV KUN ) we previously demonstrated that a single amino acid change from alanine to proline at position 30 of the NS2A protein (A30P) reduced viral cytopathicity in cells and virulence in mice. To further investigate functions of the NS2A protein we have substituted alanine at position 30 with different amino acids (A30 mutants) in a WNV KUN infectious clone. The virulence of mutant viruses in wild-type (WT) and IRF3/IRF7 double-knockout mice was influenced by the amino acid change and ranged from high to low in the order of WT A30L A30E A30P/A30G. Moreover, infection of beta interferon (IFN-β)-deficient Vero cells with A30P virus showed less pronounced chromosomal DNA degradation and lower percentage of cells with positive TUNEL labelling than in WT virus infection, indicating a role for the WT NS2A protein in IFN-independent apoptotic cell death.

West Nile virus-induced cytoplasmic membrane structures provide partial protection against the interferon-induced antiviral MxA protein

Publisher: Microbiology Society

Date: 11-2007

DOI: 10.1099/VIR.0.83125-0

Abstract: The human MxA protein is a type I and III interferon (IFN)-induced protein with proven antiviral activity against RNA viruses. In this study, we investigated the effect of MxA expression on the replication of West Nile Virus strain Kunjin (WNV KUN ). Pretreatment of A549 cells with IFN- α lead to increased expression of MxA, which contributed to inhibition of WNV KUN replication and secretion. However, in Vero cells stably expressing the MxA protein, WNV KUN replication, maturation and secretion was not inhibited. Biochemical and subcellular localization studies of WNV KUN proteins and MxA suggest that the MxA activity was not compromised by a flavivirus-encoded antagonist. Instead, we show that characteristic membranous structures induced during WNV KUN replication provide partial protection from MxA, possibly by ‘hiding’ WNV KUN replication components. This distinct compartmentalization of viral replication and components of the cellular antiviral response may be an evolutionary mechanism by which flaviviruses can hide from host surveillance.

Monoclonal Antibodies Specific for SARS-CoV-2 Spike Protein Suitable for Multiple Applications for Current Variants of Concern

Publisher: MDPI AG

Date: 31-12-2022

DOI: 10.3390/V15010139

Abstract: The global coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spawned an ongoing demand for new research reagents and interventions. Herein we describe a panel of monoclonal antibodies raised against SARS-CoV-2. One antibody showed excellent utility for immunohistochemistry, clearly staining infected cells in formalin-fixed and paraffin embedded lungs and brains of mice infected with the original and the omicron variants of SARS-CoV-2. We demonstrate the reactivity to multiple variants of concern using ELISAs and describe the use of the antibodies in indirect immunofluorescence assays, Western blots, and rapid antigen tests. Finally, we illustrate the ability of two antibodies to reduce significantly viral tissue titers in K18-hACE2 transgenic mice infected with the original and an omicron isolate of SARS-CoV-2.

Structural analysis of 3’UTRs in insect flaviviruses reveals novel determinant of sfRNA biogenesis and provides new insights into flavivirus evolution

Publisher: Cold Spring Harbor Laboratory

Date: 24-06-2021

DOI: 10.1101/2021.06.23.449515

Abstract: Insect-specific flaviviruses (ISFs) circulate in nature due to vertical transmission in mosquitoes and do not infect vertebrates. ISFs include two distinct lineages – classical ISFs (cISFs) that evolved independently and dual host associated ISFs (dISFs) that are proposed to erge from mosquito-borne flaviviruses (MBFs). Compared to pathogenic flaviviruses, ISFs are relatively poorly studied, and their molecular biology remains largely unexplored. In this study we focused on the characterisation of ISF 3’UTRs and their ability to produce subgenomic flaviviral RNAs – noncoding viral RNAs that are known as important determinants of transmission and replication of pathogenetic flaviviruses. We demonstrated that cISFs and dISFs produce sfRNAs by employing a highly conserved mechanism of resistance to degradation by the cellular 5’-3’ exoribonuclease XRN1. We determined the secondary structures of complete 3’UTRs and experimentally identified structured RNA elements that resist degradation by XRN1 (xrRNAs) in ergent representatives of cISF and dISF clades. We discovered a novel class of xrRNAs in dISFs and identified structurally ergent xrRNA in Anopheles -associated cISFs. Phylogenetic analyses based on sequences and secondary structures of xrRNAs and complete 3’UTRs reveal that xrRNAs of cISFs and MBFs/dISFs evolved from a common xrRNA ancestor similar to the xrRNA of Anopheles -associated cISFs. Additionally, we found that duplications of xrRNAs occurred independently in ISF and MBF clades. Using ISF mutants deficient in the production of sfRNAs, we found that in idual sfRNAs of ISFs have redundant functions. We conclude that duplicated xrRNAs were selected in the evolution of flaviviruses to ensure that sfRNA is produced if one of the xrRNAs lose XRN1 resistance due to mutations or misfolding.

An interaction between the methyltransferase and RNA dependent RNA polymerase domains of the West Nile virus NS5 protein

Publisher: Microbiology Society

Date: 09-2013

DOI: 10.1099/VIR.0.054395-0

Abstract: The flavivirus nonstructural protein 5 (NS5) is a large protein that is structurally conserved among members of the genus, making it an attractive target for antiviral drug development. The protein contains a methyltransferase (MTase) domain and an RNA dependent RNA polymerase (POL) domain. Previous studies with dengue viruses have identified a genetic interaction between residues 46–49 in the αA3-motif in the MTase and residue 512 in POL. These genetic interactions are consistent with structural modelling of these domains in West Nile virus (WNV) NS5 that predict close proximity of these regions of the two domains, and potentially a functional interaction mediated via the αA3-motif. To demonstrate an interaction between the MTase and POL domains of the WNV NS5 protein, we co-expressed affinity-tagged recombinant MTase and POL proteins in human embryonic kidney cells with simian virus 40 large T antigen (HEK293T cells) and performed pulldown assays using an antibody to the flag tag on POL. Western blot analysis with an anti-MTase mAb revealed that the MTase protein was specifically co-immunoprecipitated with POL, providing the first evidence of a specific interaction between these domains. To further assess the role of the αA3 helix in this interaction, selected residues in this motif were mutated in the recombinant MTase and the effect on POL interaction determined by the pulldown assay. These mutations were also introduced into a WNV infectious clone (FLSDX) and the replication properties of these mutant viruses assessed. While none of the αA3 mutations had a significant effect on the MTase–POL association in pulldown assays, suggesting that these residues were not specific to the interaction, an E46L mutation completely abolished virus viability indicating a critical requirement of this residue in replication. Failure to generate compensatory mutations in POL to rescue replication, even after several passages of the transfection supernatant in Vero cells, precluded further conclusion of the role of this residue in the context of MTase–POL interactions.

Kunjin Virus Replicon-Based Vaccines Expressing Ebola Virus Glycoprotein GP Protect the Guinea Pig Against Lethal Ebola Virus Infection

Publisher: Oxford University Press (OUP)

Date: 11-2011

DOI: 10.1093/INFDIS/JIR347

West Nile Virus Core Protein

Publisher: Elsevier BV

Date: 07-2004

DOI: 10.1016/J.STR.2004.04.024

Functional non-coding RNAs derived from the flavivirus 3' untranslated region

Publisher: Elsevier BV

Date: 08-2015

DOI: 10.1016/J.VIRUSRES.2015.01.026

Abstract: Flaviviruses are single-stranded positive sense RNA enveloped viruses. The flavivirus genus includes important human pathogens such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), and Murray Valley encephalitis virus (MVEV). In addition to the viral proteins and viral genomic RNA, flaviviruses produce at least two functional non-coding RNAs derived from the 3' untranslated region (3'UTR), the subgenomic flavivirus RNA (sfRNA) and a putative WNV miRNA (KUN-miR-1). In this review we summarize published data from studies with WNV, YFV, DENV, JEV, and MVEV on sfRNA production following incomplete degradation of the viral genomic RNA by the cellular 5'-3' exoribonuclease 1 (XRN1), RNA structural elements involved in stalling XRN1 to generate sfRNA, and functions of sfRNA in modulating cellular mRNA decay and RNAi pathways as well as in modulating anti-viral type I interferon response. In addition, we also summarize data on the mechanisms of biogenesis of 3'UTR-derived KUN-miR-1 and its function in WNV replication in mosquito host, along with recent findings on a discovery of a second potential flaviviral miRNA vsRNA5, derived from the 3'UTR of DENV. This review thus summarizes the known mechanisms of generation and the functions of flaviviral 3'UTR-derived non-coding RNAs.

Single-round infectious particles enhance immunogenicity of a DNA vaccine against West Nile virus

Publisher: Springer Science and Business Media LLC

Date: 20-04-2008

DOI: 10.1038/NBT1400

Abstract: DNA vaccines encoding replication-defective viruses are safer than inactivated or live attenuated viruses but may fail to stimulate an immune response sufficient for effective vaccination. We augment the protective capacity of a capsid-deleted flavivirus DNA vaccine by co-expressing the capsid protein from a separate promoter. In transfected cells, the capsid-deleted RNA transcript is replicated and translated to produce secreted virus-like particles lacking the nucleocapsid. This RNA is also packaged with the help of co-expressed capsid protein to form secreted single-round infectious particles (SRIPs) that deliver the RNA into neighboring cells. In SRIP-infected cells, the RNA is replicated again and produces additional virus-like particles, but in the absence of capsid RNA no SRIPs are formed and no further spread occurs. Compared with an otherwise identical construct that does not encode capsid, our vaccine offers better protection to mice after lethal West Nile virus infection. It also elicits virus-neutralizing antibodies in horses. This approach may enable vaccination against pathogenic flaviviruses other than West Nile virus.

Analysis of Adaptive Mutations in Kunjin Virus Replicon RNA Reveals a Novel Role for the Flavivirus Nonstructural Protein NS2A in Inhibition of Beta Interferon Promoter-Driven Transcription

Publisher: American Society for Microbiology

Date: 15-11-2004

DOI: 10.1128/JVI.78.22.12225-12235.2004

Abstract: The establishment of persistent noncytopathic replication by replicon RNAs of a number of positive-strand RNA viruses usually leads to generation of adaptive mutations in nonstructural genes. Some of these adaptive mutations (e.g., in hepatitis C virus) increase the ability of RNA replication to resist the antiviral action of alpha/beta interferon (IFN-α/β) others (e.g., in Sindbis virus) may also lead to more efficient IFN production. Using puromycin-selectable Kunjin virus (KUN) replicon RNA, we identified two adaptive mutations in the NS2A gene (producing Ala30-to-Pro and Asn101-to-Asp mutations in the gene product for simplicity, these will be referred to hereafter as Ala30-to-Pro and Asn101-to-Asp mutations) that, when introduced in idually or together into the original wild-type (wt) replicon RNA, resulted in ∼15- to 50-fold more efficient establishment of persistent replication in hamster (BHK21) and human (HEK293 and HEp-2) cell lines. Transfection with a reporter plasmid carrying the luciferase gene under the control of the IFN-β promoter resulted in ∼6- to 7-fold-higher luciferase expression in HEp-2 cells stably expressing KUN replicon RNA with an Ala30-to-Pro mutation in the NS2A gene compared to that observed in HEp-2 cells stably expressing KUN replicon RNA with the wt NS2A gene. Moreover, cotransfection of plasmids expressing in idual wt or Ala30-to-Pro-mutated NS2A genes with the IFN-β promoter reporter plasmid, followed by infection with Semliki Forest virus to activate IFN-β promoter-driven transcription, showed ∼7-fold inhibition of luciferase expression by the wt but not by the Ala30-to-Pro-mutated NS2A protein. The results show for the first time a role for the flavivirus nonstructural protein NS2A in inhibition of IFN-β promoter-driven transcription and identify a single-amino-acid mutation in NS2A that dramatically reduces this inhibitory activity. The findings determine a new function for NS2A in virus-host interactions, extend the range of KUN replicon vectors for noncytopathic gene expression, and identify NS2A as a new target for attenuation in the development of live flavivirus vaccines.

Structural analysis of 3'UTRs in insect flaviviruses reveals novel determinants of sfRNA biogenesis and provides new insights into flavivirus evolution

Publisher: Springer Science and Business Media LLC

Date: 11-03-2022

DOI: 10.1038/S41467-022-28977-3

Abstract: Subgenomic flaviviral RNAs (sfRNAs) are virus-derived noncoding RNAs produced by pathogenic mosquito-borne flaviviruses (MBF) to counteract the host antiviral response. To date, the ability of non-pathogenic flaviviruses to produce and utilise sfRNAs remains largely unexplored, and it is unclear what role XRN1 resistance plays in flavivirus evolution and host adaptation. Herein the production of sfRNAs by several insect-specific flaviviruses (ISFs) that replicate exclusively in mosquitoes is shown, and the secondary structures of their complete 3’UTRs are determined. The xrRNAs responsible for the biogenesis of ISF sfRNAs are also identified, and the role of these sfRNAs in virus replication is demonstrated. We demonstrate that 3’UTRs of all classical ISFs, except Anopheles spp -asscoaited viruses, and of the dual-host associated ISF Binjari virus contain duplicated xrRNAs. We also reveal novel structural elements in the 3’UTRs of dual host-associated and Anopheles -associated classical ISFs. Structure-based phylogenetic analysis demonstrates that xrRNAs identified in Anopheles spp -associated ISF are likely ancestral to xrRNAs of ISFs and MBFs. In addition, our data provide evidence that duplicated xrRNAs are selected in the evolution of flaviviruses to provide functional redundancy, which preserves the production of sfRNAs if one of the structures is disabled by mutations or misfolding.

Virulence determinants between New York 99 and Kunjin strains of West Nile virus

Publisher: Elsevier BV

Date: 05-2011

DOI: 10.1016/J.VIROL.2011.03.008

NS1′ of Flaviviruses in the Japanese Encephalitis Virus Serogroup Is a Product of Ribosomal Frameshifting and Plays a Role in Viral Neuroinvasiveness

Publisher: American Society for Microbiology

Date: 02-2010

DOI: 10.1128/JVI.01979-09

Abstract: Flavivirus NS1 is a nonstructural protein involved in virus replication and regulation of the innate immune response. Interestingly, a larger NS1-related protein, NS1′, is often detected during infection with the members of the Japanese encephalitis virus serogroup of flaviviruses. However, how NS1′ is made and what role it performs in the viral life cycle have not been determined. Here we provide experimental evidence that NS1′ is the product of a −1 ribosomal frameshift event that occurs at a conserved slippery heptanucleotide motif located near the beginning of the NS2A gene and is stimulated by a downstream RNA pseudoknot structure. Using site-directed mutagenesis of these sequence elements in an infectious clone of the Kunjin subtype of West Nile virus, we demonstrate that NS1′ plays a role in viral neuroinvasiveness.

Cholesterol manipulation by West Nile virus perturbs the cellular immune response

Publisher: Elsevier BV

Date: 10-2007

DOI: 10.1016/J.CHOM.2007.09.003

Abstract: Complex membrane structures induced by West Nile virus (WNV), an enveloped RNA virus, are required for efficient viral replication. How these membranes are induced and how they facilitate the viral life cycle are unknown. We show that WNV modulates host cell cholesterol homeostasis by upregulating cholesterol biosynthesis and redistributing cholesterol to viral replication membranes. Manipulating cholesterol levels and altering concentrations of cellular geranylgeranylated proteins had a deleterious effect on virus replication. Depletion of the key cholesterol-synthesizing enzyme 3-hydroxy-methyglutaryl-CoA reductase drastically h ered virus replication. Significantly, virus-induced redistribution of cellular cholesterol downregulated the interferon-stimulated Jak-STAT antiviral signaling response to infection. This defect could be partially restored by exogenous addition of cholesterol, which increased the ability of infected cells to respond to interferon. We propose that, by manipulating cellular cholesterol, WNV utilizes the cellular response to cholesterol deficiency and dependence of antiviral signaling pathways on cholesterol-rich microdomains to facilitate viral replication and survival.

Subgenomic replicons of the flavivirus Kunjin: construction and applications

Publisher: American Society for Microbiology

Date: 02-1997

DOI: 10.1128/JVI.71.2.1497-1505.1997

Abstract: Several Kunjin virus (KUN) subgenomic replicons containing large deletions in the structural region (C-prM-E) and in the 3' untranslated region (3'UTR) of the genome have been constructed. Replicon RNA deltaME with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the C gene to nucleotide 2403 near the carboxy terminus of the E gene, inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (from nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated efficiently in electroporated BHK21 cells. A further deletion from C20rep of 53 nucleotides, reducing the coding sequence in core protein to two codons (C2rep RNA), resulted in abolishment of RNA replication. Replicon deltaME/76 with a deletion of 76 nucleotides in the 3'UTR of deltaME RNA (nucleotides 10423 to 10498) replicated efficiently, whereas replicon deltaME/352 with a larger deletion of 352 nucleotides (nucleotides 10423 to 10774), including two conserved sequences RCS3 and CS3, was significantly inhibited in RNA replication. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive strand and the negative strand of KUN RNA, we inserted a chlor henicol acetyltransferase (CAT) gene into the 3'UTR of deltaME/76 RNA under control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA in both plus (deltaME/76CAT[+])- and minus (deltaME/76CAT[-])-sense orientations. Although insertion of the IRES-CAT cassette in the plus-sense orientation resulted in a significant (10- to 20-fold) reduction of RNA replication compared to that of the parental deltaME/76 RNA, CAT expression was readily detected in electroporated BHK cells. No CAT expression was detected after electroporation of RNA containing the IRES-CAT cassette inserted in the minus-sense orientation despite its apparently more efficient replication (similar to that of deltaME/76 RNA) this result indicated that KUN negative-strand RNA was probably not released from its template after synthesis. Replacement of the CAT gene in the deltaME/76CAT(+) RNA with the neomycin gene (Neo) enabled selection and recovery of a BHK cell culture in which the majority of cells were continuously expressing the replicon RNA for 41 days (nine passages) without apparent cytopathic effect. The constructed KUN replicons should provide valuable tools to study flavivirus RNA replication as well as providing possible vectors for a long-lasting and noncytopathic RNA virus expression system.

Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs

Publisher: Public Library of Science (PLoS)

Date: 06-01-2022

DOI: 10.1371/JOURNAL.PPAT.1010202

Abstract: The exogenous small interfering RNA (exo-siRNA) pathway is a key antiviral mechanism in the Aedes aegypti mosquito, a widely distributed vector of human-pathogenic arboviruses. This pathway is induced by virus-derived double-stranded RNAs (dsRNA) that are cleaved by the ribonuclease Dicer 2 (Dcr2) into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are used by the effector protein Argonaute 2 within the RNA-induced silencing complex to cleave target viral RNA. Dcr2 contains several domains crucial for its activities, including helicase and RNase III domains. In Drosophila melanogaster Dcr2, the helicase domain has been associated with binding to dsRNA with blunt-ended termini and a processive siRNA production mechanism, while the platform-PAZ domains bind dsRNA with 3’ overhangs and subsequent distributive siRNA production. Here we analyzed the contributions of the helicase and RNase III domains in Ae . aegypti Dcr2 to antiviral activity and to the exo-siRNA pathway. Conserved amino acids in the helicase and RNase III domains were identified to investigate Dcr2 antiviral activity in an Ae . aegypti -derived Dcr2 knockout cell line by reporter assays and infection with mosquito-borne Semliki Forest virus ( Togaviridae , Alphavirus ). Functionally relevant amino acids were found to be conserved in haplotype Dcr2 sequences from field-derived Ae . aegypti across different continents. The helicase and RNase III domains were critical for silencing activity and 21 nt vsiRNA production, with RNase III domain activity alone determined to be insufficient for antiviral activity. Analysis of 21 nt vsiRNA sequences (produced by functional Dcr2) to assess the distribution and phasing along the viral genome revealed erse yet highly consistent vsiRNA pools, with predominantly short or long sequence overlaps including 19 nt overlaps (the latter representing most likely true Dcr2 cleavage products). Combined with the importance of the Dcr2 helicase domain, this suggests that the majority of 21 nt vsiRNAs originate by processive cleavage. This study sheds new light on Ae . aegypti Dcr2 functions and properties in this important arbovirus vector species.

Nucleic Acid-Based Infectious and Pseudo-Infectious Flavivirus Vaccines

Publisher: Springer Basel

Date: 29-09-2010

DOI: 10.1007/978-3-0346-0277-8_13

The I22V and L72S substitutions in West Nile virus prM protein promote enhanced prM/E heterodimerisation and nucleocapsid incorporation

Publisher: Springer Science and Business Media LLC

Date: 07-05-2015

DOI: 10.1186/S12985-015-0303-7

Helicase domain of west nile virus NS3 protein plays a role in inhibition of type I interferon signalling

Publisher: MDPI AG

Date: 02-11-2017

DOI: 10.3390/V9110326

Crystal Structure and Activity of Kunjin Virus NS3 Helicase; Protease and Helicase Domain Assembly in the Full Length NS3 Protein

Publisher: Elsevier BV

Date: 09-2007

DOI: 10.1016/J.JMB.2007.06.055

Abstract: Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-triphosphatase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication. In addition, NS3 assists the initiation of replication by unwinding the RNA secondary structure in the 3' non-translated region (NTR). We report here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase domain (residues 186-619 NS3:186-619) from Kunjin virus, an Australian variant of the West Nile virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/recognition. The NS3:186-619 construct occurs as a dimer in solution and in the crystals. We show that NS3:186-619 displays both ATPase and RTPase activities, that it can unwind a double-stranded RNA substrate, being however inactive on a double-stranded DNA substrate. Analysis of different constructs shows that full length NS3 displays increased helicase activity, suggesting that the protease domain plays an assisting role in the RNA unwinding process. The structural interaction between the helicase and protease domain has been assessed using small angle X-ray scattering on full length NS3, disclosing that the protease and helicase domains build a rather elongated molecular assembly differing from that observed in the NS3 protein from hepatitis C virus.

Systematic analysis of viral genes responsible for differential virulence between American and Australian West Nile virus strains

Publisher: Microbiology Society

Date: 06-2015

DOI: 10.1099/VIR.0.000069

Abstract: A variant Australian West Nile virus (WNV) strain, WNVNSW2011, emerged in 2011 causing an unprecedented outbreak of encephalitis in horses in south-eastern Australia. However, no human cases associated with this strain have yet been reported. Studies using mouse models for WNV pathogenesis showed that WNVNSW2011 was less virulent than the human-pathogenic American strain of WNV, New York 99 (WNVNY99). To identify viral genes and mutations responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, we constructed chimeric viruses with substitution of large genomic regions coding for the structural genes, non-structural genes and untranslated regions, as well as seven in idual non-structural gene chimeras, using a modified circular polymerase extension cloning method. Our results showed that the complete non-structural region of WNVNSW2011, when substituted with that of WNVNY99, significantly enhanced viral replication and the ability to suppress type I IFN response in cells, resulting in higher virulence in mice. Analysis of the in idual non-structural gene chimeras showed a predominant contribution of WNVNY99 NS3 to increased virus replication and evasion of IFN response in cells, and to virulence in mice. Other WNVNY99 non-structural proteins (NS2A, NS4B and NS5) were shown to contribute to the modulation of IFN response. Thus a combination of non-structural proteins, likely NS2A, NS3, NS4B and NS5, is primarily responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, and accumulative mutations within these proteins would likely be required for the Australian WNVNSW2011 strain to become significantly more virulent.

Multiple Immune Factors Are Involved in Controlling Acute and Chronic Chikungunya Virus Infection

Publisher: Public Library of Science (PLoS)

Date: 04-12-2014

DOI: 10.1371/JOURNAL.PNTD.0003354

West Nile Virus Genome with Glycosylated Envelope Protein and Deletion of Alpha Helices 1, 2, and 4 in the Capsid Protein Is Noninfectious and Efficiently Secretes Subviral Particles

Publisher: American Society for Microbiology

Date: 12-2013

DOI: 10.1128/JVI.01552-13

Abstract: Flavivirus genomes with deletions in the capsid (C) gene are attractive vaccine candidates, as they secrete highly immunogenic subviral particles (SVPs) without generating infectious virus. Here, we report that cytomegalovirus promoter-driven cDNA of West Nile virus Kunjin (KUNV) containing a glycosylation motif in the envelope (E) gene and a combined deletion of alpha helices 1, 2, and 4 in C produces significantly more SVPs than KUNV cDNAs with nonglycosylated E and various other deletions in C.

A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) encodes a divergent class 1b XRN1-resistant RNA element

Publisher: Cold Spring Harbor Laboratory

Date: 06-07-2023

DOI: 10.1101/2023.07.06.547906

Abstract: Flavivirids are small, enveloped, positive-sense RNA viruses from the Flaviviridae family with genomes between ∼9-13kb. Metatranscriptomic analyses of metazoan organisms have revealed a ersity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. To date, however, no flavivirus-like or flavivirid has been identified in hibians. To remedy this, we investigated the virome of the European common frog ( Rana temporaria ) in the United Kingdom, utilising high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid ∼11.2kb in length. The virus encodes a single open reading frame of 3456 amino acids and 5’ and 3’ untranslated regions (UTRs) of 227 and 666nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus , respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently ergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but ergent RaTV, 95.64-97.39% pairwise nucleotide identity, were also obtained from the Poole and Deal s les, indicating that RaTV is widespread in UK frog s les. Bioinformatic analyses of putative secondary structures in the 3′-UTR of RaTV indicated a potential exoribonuclease-resistant RNA (xrRNA) structure identified in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro XRN1 digestion assay showing that RaTV likely forms a ergent but functionally homologous XRN1-resistant xrRNA.

Essential Role of Cyclization Sequences in Flavivirus RNA Replication

Publisher: American Society for Microbiology

Date: 15-07-2001

DOI: 10.1128/JVI.75.14.6719-6728.2001

Abstract: A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5′- and 3′-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5′ genomic region (first ∼160 nucleotides) and the 3′ untranslated region (last ∼115 nucleotides) for a range of mosquito-borne Flavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5′ or 3′) in the replicon RNA of the mosquito-borne Kunjin virus were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5′ or only the 3′ cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for lification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5′- and 3′-terminal regions of three tick-borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito-borne species. They base-paired with Δ G values of approximately −20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5′ and 3′ complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirus species.

Efficient trans -Complementation of the Flavivirus Kunjin NS5 Protein but Not of the NS1 Protein Requires Its Coexpression with Other Components of the Viral Replicase

Publisher: American Society for Microbiology

Date: 12-1999

DOI: 10.1128/JVI.73.12.10272-10280.1999

Abstract: Successful trans -complementation of the defective Kunjin virus (KUN) RNA FLd GDD with a deletion of the RNA polymerase motif GDD in the NS5 gene by using a BHK cell line, repBHK, that continuously produced a functionally active KUN replication complex (RC) from replicon RNA was recently reported (A. A. Khromykh, M. T. Kenney, and E. G. Westaway, J. Virol. 72:7270–7279, 1998). In order to identify whether this complementation of FLd GDD RNA was provided by the wild-type NS5 protein alone or with the help of other nonstructural (NS) proteins also expressed in repBHK cells, we generated BHK cell lines stably producing the in idual NS5 protein (SRns5BHK) or the NS1-NS5 polyprotein (SRns1-5BHK) by using a heterologous expression vector based on a modified noncytopathic Sindbis replicon. Western blot analysis with anti-NS5 antibodies showed that the level of production of NS5 was significantly higher in SRns5BHK cells than in SRns1-5BHK cells. Despite the higher level of expressed NS5, trans -complementation of FLd GDD RNA was much less efficient in SRns5BHK cells than in SRns1-5BHK cells and produced at least 100-fold less of the secreted complemented virus. In contrast, efficient complementation of KUN RNA with lethal cysteine-to-alanine mutations in the NS1 gene was achieved both in BHK cells producing the in idual KUN NS1 protein from the Sindbis replicon vector and in repBHK cells, with both cell lines expressing similar amounts of NS1 protein. These results clearly demonstrate that flavivirus NS5 coexpressed with other components of the viral replicase possesses much higher functional ( trans -complementing) activity than in idually expressed NS5 and that efficient trans -complementation of mutated flavivirus NS1 and NS5 proteins occurs by different mechanisms. The results are interpreted and discussed in relation to our proposed model of formation of the flavivirus RC largely based on previous ultrastructural and biochemical analyses of KUN replication.

Vaccine Development Against West Nile Virus

Publisher: Springer New York

Date: 2009

DOI: 10.1007/978-0-387-79840-0_20

Programmed Ribosomal Frameshift Alters Expression of West Nile Virus Genes and Facilitates Virus Replication in Birds and Mosquitoes

Publisher: Public Library of Science (PLoS)

Date: 06-11-2014

DOI: 10.1371/JOURNAL.PPAT.1004447