Packaging the replicon RNA of the Far-Eastern subtype of tick-borne encephalitis virus into single-round infectious particles: development of a heterologous gene delivery system

Yoshii, Kentarou; Hayasaka, Daisuke; Goto, Akiko; Kawakami, Kazue; Kariwa, Hiroaki; Takashima, Ikuo

doi:10.1016/j.vaccine.2005.03.004

Cited by 25 publications

(30 citation statements)

References 49 publications

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“…As shown in Fig. 2B, sequential transfection with the Oshima-REP-luc2A replicon and the pcTBECME plasmid resulted in the production of VLPs, and luciferase expression was confirmed in the cells that were infected with VLPs that contained the Oshima-REP-luc2A replicon (luc-VLPs).In previous studies, the envelope glycoproteins of the VLPs exhibited the same antigenicities as those of the authentic virion (Yoshii et al, 2005;Yoshii et al, 2008). To apply the VLPs that express reporter genes to neutralization testing, we examined whether infection with VLPs was neutralized by virus-specific neutralizing antibodies.…”

mentioning

confidence: 56%

“…The pcTBECME plasmid, which contains the TBEV virus C-prM-E genes (Yoshii et al, 2005), was used for the expression of TBEV structural proteins to be used for packaging the replicon RNA.…”

Section: Gfp Was Detected In the Bhk Cells By Fluorescence Microscopymentioning

confidence: 99%

“…To prepare the VLPs, 293T cells were first transfected with Oshima-REP-GFP2A or Oshima-REP-luc2A, followed 8 h later by a second transfection with the pcTBECME plasmid, as described previously (Yoshii et al, 2005). Following the second transfection, the culture supernatant that contained the VLPs was harvested and used to infect naïve cells.…”

Section: Gfp Was Detected In the Bhk Cells By Fluorescence Microscopymentioning

confidence: 99%

“…The levels of luciferase activity in the transfected cells increased significantly as the levels of replicon RNA increased (Fig. 1C).The pcTBECME plasmid, which contains the TBEV virus C-prM-E genes (Yoshii et al, 2005), was used for the expression of TBEV structural proteins to be used for packaging the replicon RNA.To prepare the VLPs, 293T cells were first transfected with Oshima-REP-GFP2A or Oshima-REP-luc2A, followed 8 h later by a second transfection with the pcTBECME plasmid, as described previously (Yoshii et al, 2005). Following the second transfection, the culture supernatant that contained the VLPs was harvested and used to infect naïve cells.…”

mentioning

confidence: 91%

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Establishment of a neutralization test involving reporter gene-expressing virus-like particles of tick-borne encephalitis virus

Yoshii

Ikawa

Chiba

et al. 2009

Journal of Virological Methods

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SummaryPreviously, a system for packaging tick-borne encephalitis virus (TBEV) subgenomic replicon RNAs into single-round infectious virus-like particles (VLPs) was developed. In the present study, VLPs were applied to measuring the levels of neutralizing antibodies against TBEV as an alternative to performing neutralization tests with live virus. As markers of VLP infection, the genes for GFP and luciferase were inserted into the TBEV replicon, which was then packaged into VLPs. The reporter genes were expressed in cells that were infected with the VLPs, and this infection was inhibited by neutralizing antibodies to TBEV. Serum samples from wild rodents were used to evaluate the neutralization test using VLPs. All the sera that were positive in the conventional neutralization test were also found to be positive in the neutralization test using VLPs, and there were highly significant correlations between the neutralization titres obtained using the native virus and those using VLPs.These results indicate that VLPs that express reporter genes represent a useful and safe alternative to conventional neutralization testing using live virus.Keywords: tick-borne encephalitis, virus-like particles, and neutralization test 3 TextTick-borne encephalitis virus (TBEV), which belongs to the family Flaviviridae genus Flavivirus, causes fatal encephalitis in humans with serious sequelae (Dumpis et al., 1999). TBE occurs widely across Europe, Russia, and Far-Eastern Asia, including Japan (Blaskovic et al., 1967; Korenberg and Kovalevskii, 1999; Lindgren and Gustafson, 2001;Ormaasen et al., 2001;Roggendorf et al., 1981), and more than 10,000 cases of the disease are reported annually. TBEV can be divided into three subtypes: 1) the Far-Eastern subtype, known as Russian spring summer encephalitis (RSSE) virus; 2) the European subtype, known as Central European encephalitis (CEE) virus; and 3) the Siberian subtype (Bakhvalova et al., 2000;Ecker et al., 1999). TBEV is transmitted by tick bites and is maintained in the zoonotic transmission cycle between ticks and wild vertebrate hosts, with humans acting as accidental hosts. The major tick vector of the European subtype is Ixodes ricinus, whereas I. persulcatus is the major tick vector for the other two viral subtypes (Ecker et al., 1999;Gaunt et al., 2001;Hayasaka et al., 2001;Lundkvist et al., 2001).Effective diagnosis of TBE infection relies on the detection of specific antibodies. However, the presence of cross-reactive antigenic structures among flaviviruses makes it difficult to differentiate between TBEV and other flavivirus infections/vaccinations using IgG-ELISA and the HI test (Holzmann et al., 1996). For cases that involve contact with other flaviviruses or vaccinations, a neutralizing test, which is the most specific serological test, is required to confirm the diagnosis of TBE infection. However, since TBEV is classified as a biosafety level 3 or 4 virus, a high-level biocontainment facility is required to handle the live virus required for neutralization testing. V...

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mentioning

confidence: 56%

“…The pcTBECME plasmid, which contains the TBEV virus C-prM-E genes (Yoshii et al, 2005), was used for the expression of TBEV structural proteins to be used for packaging the replicon RNA.…”

Section: Gfp Was Detected In the Bhk Cells By Fluorescence Microscopymentioning

confidence: 99%

Section: Gfp Was Detected In the Bhk Cells By Fluorescence Microscopymentioning

confidence: 99%

mentioning

confidence: 91%

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Establishment of a neutralization test involving reporter gene-expressing virus-like particles of tick-borne encephalitis virus

Yoshii

Ikawa

Chiba

et al. 2009

Journal of Virological Methods

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“…The RNAs were synthesized as described previously (Yoshii et al, 2005). Briefly, the plasmids were prepared for run-off transcription by digestion with XbaI restriction endonuclease, and the resulting template DNAs were in vitro transcribed using the mMESSAGE mMACHINE T7 Kit (Ambion) in a 20-μl reaction mixture that contained an additional 1 μl of 20 mM GTP solution.…”

Section: Rna Synthesis-transfectionmentioning

confidence: 99%

Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

et al. 2011

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Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis serocomplex of flaviviruses, and causes hemorrhagic disease in humans. In this study, an infectious cDNA of OHFV was constructed to investigate the molecular mechanisms involved in OHFV pathogenesis for the first time. Our cDNA clone was capable of producing infectious virus which is genetically identical to the parental Guriev strain, and the recombinant virus showed similar biological properties to the parental virus including growth kinetics and virulence characteristics. While characterizing the cDNAs, fortuitous mutations at NS2A position 46 and NS5 position 836 were found to affect viral production. By using a viral replicon expressing luciferase, it was shown that both of the mutations produced a defect in RNA replication and that the NS5 mutation induced a temperature-sensitive phenotype, indicating the importance of these residues in RNA replication.This infectious cDNA will be a useful tool to study the replication and pathogenesis of OHFV.

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Nucleic Acid-Based Infectious and Pseudo-Infectious Flavivirus Vaccines

Roby

Hall

Khromykh

2010

Replicating Vaccines

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The genus Flavivirus contains a number of important pathogens of humans including yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), and West Nile virus (WNV). Despite causing significant morbidity and mortality worldwide, commercially available vaccines only exist for YFV (live-attenuated), TBEV, and JEV (inactivated). Flavivirus vaccine research has been driven by the need for cheap, safe, thermally stable, and efficacious preparations amenable to use in developing nations. The creation of infectious cDNA clones of various flaviviruses has led to the development of genetically engineered, nucleic acid-delivered, attenuated live vaccine candidates. These provide effective immunity from a single immunisation, however share the same safety concerns as traditional live-attenuated vaccines. The generation of large internal deletions in the capsid gene of flavivirus genomes creates a vaccine that secretes large amounts of immunogenic prM/E particles from self-replicating RNA but does not form a spreading infection. Packaging of these capsid-deleted RNAs into virus-like particles (VLPs) using a cell line that produces capsid gene from another expression vector creates a pseudoinfectious vaccine that elicits a highly efficient immune response from a single dose and is safer than infectious virus. However, production of these VLPs is cumbersome and the resulting product is heat labile. Providing the capsid gene in trans from another promoter but within the same plasmid DNA as the capsid-deleted viral genome creates a DNA vaccine capable of producing VLPs in vivo. Uptake of this plasmid DNA results in the generation of self-replicating, capsid-deleted RNA and the capsid protein in the same cell, leading to production of secreted single-round infectious particles (SRIPs). These SRIPs then deliver capsid-deleted RNA to adjacent cells where it replicates to produce more prM/E particles. As functional

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Packaging the replicon RNA of the Far-Eastern subtype of tick-borne encephalitis virus into single-round infectious particles: development of a heterologous gene delivery system

Cited by 25 publications

References 49 publications

Establishment of a neutralization test involving reporter gene-expressing virus-like particles of tick-borne encephalitis virus

Establishment of a neutralization test involving reporter gene-expressing virus-like particles of tick-borne encephalitis virus

Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

Nucleic Acid-Based Infectious and Pseudo-Infectious Flavivirus Vaccines

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