Single-round Infectious Particles Research Articles

Single-Round Infectious Particle Antiviral Screening Assays for the Japanese Encephalitis Virus.

Japanese Encephalitis virus (JEV) is a mosquito-borne flavivirus with a positive-sense single-stranded RNA genome that contains a big open reading frame (ORF) flanked by 5′- and 3′- untranslated regions (UTRs). Nearly 30,000 JE cases with 10,000 deaths are still annually reported in East Asia. Although the JEV genotype III vaccine has been licensed, it elicits a lower protection against other genotypes. Moreover, no effective treatment for a JE case is developed. This study constructed a pBR322-based and cytomegaloviruses (CMV) promoter-driven JEV replicon for the production of JEV single-round infectious particles (SRIPs) in a packaging cell line expressing viral structural proteins. Genetic instability of JEV genome cDNA in the pBR322 plasmid was associated with the prokaryotic promoter at 5′ end of the JEV genome that triggers the expression of the structural proteins in E. coli. JEV structural proteins were toxic E. coli, thus the encoding region for structural proteins was replaced by a reporter gene (enhanced green fluorescent protein, EGFP) that was in-frame fused with the first eight amino acids of the C protein at N-terminus and the foot-and-mouth disease virus (FMDV) 2A peptide at C-terminus in a pBR322-based JEV-EGFP replicon. JEV-EGFP SRIPs generated from JEV-EGFP replicon-transfected packaging cells displayed the infectivity with cytopathic effect induction, self-replication of viral genomes, and the expression of EGFP and viral proteins. Moreover, the combination of JEV-EGFP SRIP plus flow cytometry was used to determine the half maximal inhibitory concentration (IC50) values of antiviral agents according to fluorescent intensity and positivity of SRIP-infected packaging cells post treatment. MJ-47, a quinazolinone derivative, significantly inhibited JEV-induced cytopathic effect, reducing the replication and expression of JEV-EGFP replicon in vitro. The IC50 value of 6.28 µM for MJ-47 against JEV was determined by the assay of JEV-EGFP SRIP infection in packaging cells plus flow cytometry that was more sensitive, effective, and efficient compared to the traditional plaque assay. Therefore, the system of JEV-EGFP SRIPs plus flow cytometry was a rapid and reliable platform for screening antiviral agents and evaluating antiviral potency.

Utility of Japanese encephalitis virus subgenomic replicon-based single-round infectious particles as antigens in neutralization tests for Zika virus and three other flaviviruses

The introduction of a foreign virus into an area may cause an outbreak, as with the Zika virus (ZIKV) outbreak in the Americas. Preparedness for handling a viral outbreak involves the development of tests for the serodiagnosis of foreign virus infections. We previously established a gene-based technology to generate some flaviviral antigens useful for functional antibody assays. The technology utilizes a Japanese encephalitis virus subgenomic replicon to generate single-round infectious particles (SRIPs) that possess designed surface antigens. In the present study, we successfully expanded the capacity of SRIPs to four human-pathogenic mosquito-borne flaviviruses that could potentially be introduced from endemic to non-endemic countries: ZIKV, Sepik virus, Wesselsbron virus, and Usutu virus. Flavivirus-crossreactive monoclonal antibodies dose-dependently neutralized these SRIPs. ZIKV-SRIPs also produced antibody-dose-dependent neutralization curves equivalent to those shown by authentic ZIKV particles using sera from a Zika fever patient. The faithful expression of designed surface antigens on SRIPs will allow their use in neutralization tests to diagnose foreign flaviviral infections.

Neutralizing and enhancing antibody responses to five genotypes of dengue virus type 1 (DENV-1) in DENV-1 patients.

Dengue virus (DENV) has four distinct serotypes, DENV-1-4, with four to six genotypes in each serotype. The World Health Organization recommends tetravalent formulations including one genotype of each serotype as safe and effective dengue vaccines. Here, we investigated the impact of genotype on the neutralizing antibody responses to DENV-1 in humans. Convalescent sera collected from patients with primary infection of DENV-1 were examined for neutralizing antibody against single-round infectious particles of the five DENV-1 genotypes (GI-GV). In both GI- and GIV-infected patients, their neutralizing antibody titres against the five genotypes were similar, differing ≤4-fold from the homogenotypic responses. The enhancing activities against the five genotypes were also similar in these sera. Thus, the genotype strains of DENV-1 showed no significant antigenic differences in these patients, suggesting that GI- or GIV-derived vaccine antigens should induce equivalent levels of neutralizing antibodies against all DENV-1 genotypes.

SA14-14-2Rep: A Replicon Vector Derived from the Live-Attenuated Japanese Encephalitis Vaccine SA14-14-2 Virus for Human and Veterinary Medicine

Japanese encephalitis (JE), an acute inflammatory disease of the brain, is caused by infection with Japanese encephalitis virus (JEV). Children are most susceptible to JEV, which is commonly found in Asia. JEV is an enveloped RNA virus that is genetically similar to West Nile, dengue, and yellow fever viruses. No specific antiviral drugs are currently available to treat JE disease, but four types of vaccine are licensed locally to prevent JEV infection. However, the disease continues to be an important global health priority. Of the four vaccines, the most widely used in JE-endemic countries is the live-attenuated vaccine SA14-14-2. In our previous work, we cloned a genomelength cDNA of SA14-14-2 and rescued recombinant viruses entirely from the cloned cDNA (pBac/SA14-14-2). In the present study, we engineered this functional SA14-14-2 cDNA to construct a self-replicating subgenomic replicon (pBac/SA14-14-2Rep) by deleting the coding region for its two viral envelope glycoproteins, prM and E. After transfection of BHK-21 cells with the replicon RNA transcribed in vitro from pBac/SA14-14-2Rep, a combination of confocal microscopic imaging and immunoblotting showed the transient replication of the replicon RNA in the cytoplasm of RNA-transfected cells. Moreover, transfection of the SA14-14-2 replicon RNA into a BHK-21 cell line stably expressing all three JEV structural proteins (C, prM and E) led to the production of singleround infectious particles that packaged the replicon RNA, with a titter of ~105 infectious units per ml. Our SA14-14-2-based replicon represents a valuable tool for studying JEV RNA replication in low-level bio containment facilities and provides a useful platform for developing new vaccine vectors against an array of human and animal pathogens.

Japanese encephalitis virus replicon-based vaccine expressing enterovirus-71 epitope confers dual protection from lethal challenges

BackgroundTo construct safer recombinant flavivirus vaccine, we exploited Japanese encephalitis virus (JEV) replicon-based platform to generate single-round infectious particles (SRIPs) that expressed heterologous neutralizing epitope SP70 derived from enterovirus-71 (EV71). Such pseudo-infectious virus particles, named SRIP-SP70, although are not genuine viable viruses, closely mimic live virus infection to elicit immune responses within one round of viral life cycle.ResultsWe found that, besides gaining of full protection to thwart JEV lethal challenge, female outbred ICR mice, when were immunized with SRIP-SP70 by prime-boost protocol, could not only induce SP70-specific and IgG2a predominant antibodies but also provide their newborns certain degree of protection against EV71 lethal challenge.ConclusionsOur results therefore exemplify that this vaccination strategy could indeed confer an immunized host a dual protective immunity against subsequent lethal challenge from JEV or EV71.

Increased expression of capsid protein in trans enhances production of single-round infectious particles by West Nile virus DNA vaccine candidate.

West Nile virus (WNV; genus Flavivirus, family Flaviviridae) is an emerging pathogenic arbovirus responsible for outbreaks of encephalitis around the world. Whilst no vaccines are currently available to prevent WNV infection of humans, the use of cDNA copies of flavivirus RNA genomes with large internal deletions within the capsid (C) appears promising. C-deleted vaccines are able to replicate and secrete large amounts of non-infectious immunogenic subviral particles (SVPs) from transfected cells. We have previously generated a WNV DNA vaccine candidate pKUNdC/C where C-deleted WNV cDNA was placed under the control of one copy of the cytomegalovirus (CMV) promoter and the C gene was placed under the control of a second copy of the CMV promoter in the same plasmid DNA. This DNA was shown to generate single-round infectious particles (SRIPs) capable of delivering self-replicating C-deleted RNA producing SVPs to surrounding cells, thus enhancing the vaccine potential. However, the amounts of both SRIPs and SVPs produced from pKUNdC/C DNA were relatively low. In this investigation, we aimed at increasing SRIP production by optimizing trans-C expression via incorporating different forms of C and the use of a more powerful promoter. The construct containing an elongation factor EF1α promoter encoding an extended form of C was demonstrated to produce the highest titres of SRIPs and was immunogenic in mice. Additionally, SRIP and SVP titres were further improved via incorporation of a glycosylation motif in the envelope protein. The optimized DNA yielded ~100-fold greater titres of SRIPs than the original construct, thus providing a promising candidate for further vaccine evaluation.

Incorporation of primary patient-derived glycoproteins into authentic infectious hepatitis C virus particles.

The Japanese fulminant hepatitis-1 (JFH1)-based hepatitis C virus (HCV) infection system has permitted analysis of the complete viral replication cycle in vitro. However, lack of robust infection systems for primary, patient-derived isolates limits systematic functional studies of viral intrahost variation and vaccine development. Therefore, we aimed at developing cell culture models for incorporation of primary patient-derived glycoproteins into infectious HCV particles for in-depth mechanistic studies of envelope gene function. To this end, we first constructed a packaging cell line expressing core, p7, and NS2 based on the highly infectious Jc1 genotype (GT) 2a chimeric genome. We show that this packaging cell line can be transfected with HCV replicons encoding cognate Jc1-derived glycoprotein genes for production of single-round infectious particles by way of trans-complementation. Testing replicons expressing representative envelope protein genes from all major HCV genotypes, we observed that virus production occurred in a genotype- and isolate-dependent fashion. Importantly, primary GT 2 patient-derived glycoproteins were efficiently incorporated into infectious particles. Moreover, replacement of J6 (GT 2a) core, p7, and NS2 with GT 1a-derived H77 proteins allowed production of infectious HCV particles with GT 1 patient-derived glycoproteins. Notably, adaptive mutations known to enhance virus production from GT 1a-2a chimeric genomes further increased virus release. Finally, virus particles with primary patient-derived E1-E2 proteins possessed biophysical properties comparable to Jc1 HCVcc particles, used CD81 for cell entry, were associated with ApoE and could be neutralized by immune sera. This work describes cell culture systems for production of infectious HCV particles with primary envelope protein genes from GT 1 and GT 2-infected patients, thus opening up new opportunities to dissect envelope gene function in an individualized fashion.

Evaluation of single-round infectious, chimeric dengue type 1 virus as an antigen for dengue functional antibody assays

Dengue fever and dengue hemorrhagic fever are endemic throughout tropical and subtropical countries. Four serotypes of dengue viruses (DENV-1 to DENV-4), each with several genotypes including various subclades, are co-distributed in most endemic areas. Infection–neutralizing and -enhancing antibodies are believed to play protective and pathogenic roles, respectively. Measurement of these functional antibodies against a variety of viral strains is thus important for evaluating coverage and safety of dengue vaccine candidates. Although transportation of live virus materials beyond national borders is increasingly limited, this difficulty may be overcome using biotechnology that enables generation of an antibody-assay antigen equivalent to authentic virus based on viral sequence information. A rapid system to produce flavivirus single-round infectious particles (SRIPs) was recently developed using a Japanese encephalitis virus (JEV) subgenomic replicon plasmid. This system allows production of chimeric SRIPs that have surface proteins of other flaviviruses. In the present study, SRIPs of DENV-1 (D1-SRIPs) were evaluated as an antigen for functional antibody assays. Inclusion of the whole mature capsid gene of JEV into the replicon plasmid provided higher D1-SRIP yields than did its exclusion in cases where a DENV-1 surface-protein-expressing plasmid was used for co-transfection of 293T cells with the replicon plasmid. In an assay to measure the balance between neutralizing and enhancing activities, dose (antibody dilution)-dependent activity curves in dengue-immune human sera or mouse monoclonal antibodies obtained using D1-SRIP antigen were equivalent to those obtained using DENV-1 antigen. Similar results were obtained using additional DENV-2 and DENV-3 systems. In a conventional Vero-cell neutralization test, a significant correlation was shown between antibody titers obtained using D1-SRIP and DENV-1 antigens. These results demonstrate the utility of D1-SRIPs as an alternative antigen to authentic DENV-1 in functional antibody assays. SRIP antigens may contribute to dengue vaccine candidate evaluation, understanding of dengue pathogenesis, and development of serodiagnostic systems.

Production of single-round infectious chimeric flaviviruses with DNA-based Japanese encephalitis virus replicon

A method for rapid production of single-round infectious particles (SRIPs) of flavivirus would be useful for viral mutagenesis studies. Here, we established a DNA-based production system for SRIPs of flavivirus. We constructed a Japanese encephalitis virus (JEV) subgenomic replicon plasmid, which lacked the C-prM-E (capsid-pre-membrane-envelope) coding region, under the control of the cytomegalovirus promoter. When the JEV replicon plasmid was transiently co-transfected with a JEV C-prM-E expression plasmid into 293T cells, SRIPs were produced, indicating successful trans-complementation with JEV structural proteins. Equivalent production levels were observed when C and prM-E proteins were provided separately. Furthermore, dengue types 1-4, West Nile, yellow fever or tick-borne encephalitis virus prM-E proteins could be utilized for production of chimaeric flavivirus SRIPs, although the production was less efficient for dengue and yellow fever viruses. These results indicated that our plasmid-based system is suitable for investigating the life cycles of flaviviruses, diagnostic applications and development of safer vaccine candidates.

Natural selection of adaptive mutations in non-structural genes increases trans-encapsidation of hepatitis C virus replicons lacking envelope protein genes

A trans-packaging system for hepatitis C virus (HCV) replicons lacking envelope glycoproteins was developed. The replicons were efficiently encapsidated into infectious particles after expression in trans of homologous HCV envelope proteins under the control of an adenoviral vector. Interestingly, expression in trans of core or core, p7 and NS2 with envelope proteins did not enhance trans-encapsidation. Expression of heterologous envelope proteins, in the presence or absence of heterologous core, p7 and NS2, did not rescue single-round infectious particle production. To increase the titre of homologous, single-round infectious particles in our system, successive cycles of trans-encapsidation and infection were performed. Four cycles resulted in a 100-fold increase in the yield of particles. Sequence analysis revealed a total of 16 potential adaptive mutations in two independent experiments. Except for a core mutation in one experiment, all the mutations were located in non-structural regions mainly in NS5A (four in domain III and two near the junction with the NS5B gene). Reverse genetics studies suggested that D2437A and S2443T adaptive mutations, which are located at the NS5A-B cleavage site did not affect viral replication, but enhanced the single-round infectious particles assembly only in trans-encapsidation model. In conclusion, our trans-encapsidation system enables the production of HCV single-round infectious particles. This system is adaptable and can positively select variants. The adapted variants promote trans-encapsidation and should constitute a valuable tool in the development of replicon-based HCV vaccines.

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

Previously, 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.

Sub-genomic replicon and virus-like particles of Omsk hemorrhagic fever virus

Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis serocomplex of flaviviruses, and causes hemorrhagic disease in humans. To investigate the molecular mechanisms involved in OHFV pathogenesis, we constructed several subgenomic OHFV replicons containing large deletions in the structural region. Replicon RNA was introduced into BHK cells by transfection and the production of viral proteins was monitored by IFA. GFP and luciferase genes were inserted into the OHFV replicon, and these reporter genes were expressed in cells harboring replicating replicon RNA. OHFV replicons were packaged into single-round infectious virus-like particles (VLPs) by sequential transfection with replicon RNA and a plasmid expressing the viral structural proteins. Reporter genes were expressed in cells infected with VLPs, and the infection was inhibited by neutralizing antibodies. These replicon and VLP systems will be useful tools for investigating the molecular mechanism of OHFV pathogenicity.

Flavivirus DNA vaccine with a kick

Immunization with single-round infectious particles enhances potency of a DNA vaccine against West Nile virus.

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

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.

Construction and application of chimeric virus-like particles of tick-borne encephalitis virus and mosquito-borne Japanese encephalitis virus

We have previously reported a system for packaging tick-borne encephalitis (TBE) virus subgenomic replicon RNAs into single-round infectious virus-like particles (VLPs) by using in trans expression of viral C/prM/E structural proteins. In this study, the trans-packaging system was applied to the generation of chimeric VLPs with mosquito-borne Japanese encephalitis (JE) virus. Although trans-expression of TBE virus C and JE virus prM/E proteins resulted in the secretion of VLPs, the expression of JE virus C/prM/E proteins did not lead to the secretion of VLPs, suggesting that homologous interaction between C and non-structural proteins or the genomic RNA is important for efficient assembly of infectious particles. Neutralization testing showed that the antigenic characteristics of the VLPs were similar to those of the native virus. Furthermore, the infectivities of the TBE virus- and JE virus-enveloped VLPs for the ISE6 tick cell line and C6/36 mosquito cell line were investigated. The VLPs were able to enter only those cells that were derived from the natural vectors for the respective viruses. TBE virus replicon RNA packaged in VLPs produced TBE virus non-structural proteins in tick cells, but could neither replicate nor produce viral proteins in mosquito cells. These findings indicate the importance of specific cellular factors for virus entry and replication during flavivirus infection of arthropods. These results demonstrate that chimeric VLPs are useful tools for the study of viral genome packaging and cellular factors involved in vector specificity, with the additional safety aspect that these chimeric VLPs can be used instead of full-length chimeric viruses.

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

The sub-genomic replicon of tick-borne encephalitis (TBE) virus (Far-Eastern subtype) was packaged into infectious particles by providing the viral structural proteins in trans. Sequential transfection of TBE replicon RNA and a plasmid that expressed the structural proteins led to the secretion of infectious particles that contained TBE replicon RNA. The secreted particles had single-round infectivity, which was inhibited by TBE virus-neutralizing antibody. The physical structure of the particles was almost identical to that of infectious virions, and the packaged replicon RNA showed no recombination with the mRNAs of the viral structural proteins. Furthermore, heterologous genes were successfully delivered and expressed by packaging TBE replicon RNA with inserted GFP and Neo genes. This replicon packaging system may be a useful tool for the molecular study of the TBE virus genome packaging mechanism, and for the development of vaccine delivery systems.

Cleavage of protein prM is necessary for infection of BHK-21 cells by tick-borne encephalitis virus.

Flavivirus particles are synthesized in an immature form containing heterodimers of the proteins prM and E. Shortly before release from the cell, prM is cleaved by the host protease furin to yield mature virions. In this study, the furin-mediated cleavage of the tick-borne encephalitis (TBE) virus protein prM was prevented by specific mutagenesis of the cleavage site. This resulted in the production of immature TBE virions, which were shown to be completely non-infectious in BHK-21 cells. This finding contrasted with previous studies in which immature flavivirus particles produced by other techniques were shown to have considerable residual infectivity. The structural integrity of the mutant virus particles was confirmed by the characterization of physical and antigenic properties. Most importantly, infectivity could be restored by the addition of trypsin, which presumably cleaved protein prM at one of the monobasic sites retained in the mutated sequence. In the presence of trypsin, the mutant could be passaged repeatedly in BHK-21 cells, but if the protease was removed, the activated particles could initiate only a single round of infection, which again generated non-infectious virus progeny. These observations provide evidence that the infectivity of flaviviruses depends on the endoproteolytic cleavage of protein prM, which probably has a regulatory function rather than a direct role in virus entry. Moreover, the results illustrate that mutation of the furin cleavage site is a convenient way to produce single-round infectious flavivirus particles, which may be useful in vaccine and vector development.