Production of Ebola virus-like particles in Drosophila melanogaster Schneider 2 cells.

In this study, we generated recombinant virus-like particles (VLPs) against family Filoviridae, genus Ebolavirus, species Zaire ebolavirus, strain Makona (EBOV) in Drosophila melanogaster Schneider 2 (S2) cells using the EBOV Makona. S2 cells were cotransfected with four viral plasmids encoding EBOV Makona proteins and protein expression was analyzed by immunoblotting. We confirmed that EBOV Makona proteins were successfully expressed in S2 cells. Additionally, we further examined the formation of intracellular and extracellular VLPs by electron microscopy. eVLPs were produced by sucrose gradient ultracentrifugation of S2 cells transfected with EBOV Makona genes, and production of VLPs was confirmed by immunoblot analysis. Collectively, our findings showed that the S2 cell system could be a promising tool for efficient production of eVLPs.

Implications of and lessons learned from external assurance of eight influenza diagnostics in China.

This study evaluated the ability of laboratories in the Chinese mainland to conduct molecular detection of seasonal A(H1N1), A(H1N1)pdm09, A(H3N2), A(H5N1), A(H7N9), A(H9N2), B(Victoria), and B(Yamagata). Based on a genetically engineered system of virus-like particles (VLPs), the National Center for Clinical Laboratories of China (NCCLs) developed an external quality assessment (EQA) panel. The panel was distributed to 35 laboratories in mainland China to investigate the proficiency of the 16 assays for influenza molecular detection. Using genetic engineering technology, VLPs encapsulating the 37 target genes of 8 influenza viruses were generated. After verification and quantification, 26 influenza virus surrogates with different concentrations were prepared for EQA. Among the 35 participating laboratories, 319 datasets were returned to the NCCLs. Overall, 95.6% (305/319) of datasets correctly reported all 30 samples, while 2.2% (7/319) of datasets with more than one incorrect result were considered as "improvable". A total of 16 misdiagnosed and 18 undiagnosed results were reported. The data analyzed in this study showed good reproducibility in China, but improvements are needed to decrease misdiagnosed and undiagnosed cases, particularly for the A(H9N2) NA gene. Moreover, VLPs are a good alternative specimen type for assay training and proficiency testing purposes.

Diagnostic aptitude of West Nile virus-like particles expressed in insect cells.

West Nile virus is a globally spread zoonotic arbovirus. The laboratory diagnosis of WNV infection relies on virus identification by RT-PCR or on specific antibody detection by serological tests, such as ELISA or virus-neutralization. These methods usually require a preparation of the whole virus as antigen, entailing biosafety issues and therefore requiring BSL-3 facilities. For this reason, recombinant antigenic structures enabling effective antibody recognition comparable to that of the native virions, would be advantageous as diagnostic reagents. WNV virions are enveloped spherical particles made up of 3 structural proteins (C, capsid; M, membrane and E, envelope) enclosing the viral RNA. This study describes the co-expression of these 3 proteins yielding non-infectious virus-like particles (VLPs) and the results of the initial assessment of these VLPs, used instead of the whole virus, that were shown to perform correctly in two different ELISAs for WNV diagnosis.

Complete study demonstrating the absence of rhabdovirus in a distinct Sf9 cell line.

A putative novel rhabdovirus (SfRV) was previously identified in a Spodoptera frugiperda cell line (Sf9 cells [ATCC CRL-1711 lot 58078522]) by next generation sequencing and extensive bioinformatic analysis. We performed an extensive analysis of our Sf9 cell bank (ATCC CRL-1711 lot 5814 [Sf9L5814]) to determine whether this virus was already present in cells obtained from ATCC in 1987. Inverse PCR of DNA isolated from Sf9 L5814 cellular DNA revealed integration of SfRV sequences in the cellular genome. RT-PCR of total RNA showed a deletion of 320 nucleotides in the SfRV RNA that includes the transcriptional motifs for genes X and L. Concentrated cell culture supernatant was analyzed by sucrose density gradient centrifugation and revealed a single band at a density of 1.14 g/ml. This fraction was further analysed by electron microscopy and showed amorphous and particulate debris that did not resemble a rhabdovirus in morphology or size. SDS-PAGE analysis confirmed that the protein composition did not contain the typical five rhabdovirus structural proteins and LC-MS/MS analysis revealed primarily of exosomal marker proteins, the SfRV N protein, and truncated forms of SfRV N, P, and G proteins. The SfRV L gene fragment RNA sequence was recovered from the supernatant after ultracentrifugation of the 1.14 g/ml fraction treated with diethyl ether suggesting that the SfRV L gene fragment sequence is not associated with a diethyl ether resistant nucleocapsid. Interestingly, the 1.14 g/ml fraction was able to transfer baculovirus DNA into Sf9L5814 cells, consistent with the presence of functional exosomes. Our results demonstrate the absence of viral particles in ATCC CRL-1711 lot 5814 Sf9 cells in contrast to a previous study that suggested the presence of infectious rhabdoviral particles in Sf9 cells from a different lot. This study highlights how cell lines with different lineages may present different virosomes and therefore no general conclusions can be drawn across Sf9 cells from different laboratories.

Generation of porcine reproductive and respiratory syndrome (PRRS) virus-like-particles (VLPs) with different protein composition.

The causative agent of Porcine Reproductive and Respiratory Syndrome (PRRS) is an enveloped ssRNA (+) virus belonging to the Arteriviridae family. Gp5 and M proteins form disulfide-linked heterodimers that constitute the major components of PRRSV envelope. Gp2, Gp3, Gp4 and E are the minor structural proteins, being the first three incorporated as multimeric complexes in the virus surface. The disease has become one of the most important causes of economic losses in the swine industry. Despite efforts to design an effective vaccine, the available ones allow only partial protection. In the last years, VLPs have become good vaccine alternatives because of safety issues and their potential to activate both branches of the immunological response. The characteristics of recombinant baculoviruses as heterologous expression system have been exploited for the production of VLPs of a wide variety of viruses. In this work, two multiple baculovirus expression vectors (BEVs) with PRRS virus envelope proteins were engineered in order to generate PRRS VLPs: on the one hand, Gp5 and M cDNAs were cloned to generate the pBAC-Gp5M vector; on the other hand, Gp2, Gp3, Gp4 and E cDNAs have been cloned to generate the pBAC-Gp234E vector. The corresponding recombinant baculoviruses BAC-Gp5M and BAC-Gp234E were employed to produce two types of VLPs: basic Gp5M VLPs, by the simultaneous expression of Gp5 and M proteins; and complete VLPs, by the co-expression of the six PRRS proteins after co-infection. The characterization of VLPs by Western blot confirmed the presence of the recombinant proteins using the available specific antibodies (Abs). The analysis by Electron microscopy showed that the two types of VLPs were indistinguishable between them, being similar in shape and size to the native PRRS virus. This system represents a potential alternative for vaccine development and a useful tool to study the implication of specific PRRS proteins in the response against the virus.

Synthesis of empty african horse sickness virus particles.

As a means to develop African horse sickness (AHS) vaccines that are safe and DIVA compliant, we investigated the synthesis of empty African horse sickness virus (AHSV) particles. The emphasis of this study was on the assembly of the major viral core (VP3 and VP7) and outer capsid proteins (VP2 and VP5) into architecturally complex, heteromultimeric nanosized particles. The production of fully assembled core-like particles (CLPs) was accomplished in vivo by baculovirus-mediated co-synthesis of VP3 and VP7. The two different outer capsid proteins were capable of associating independently of each other with preformed cores to yield partial virus-like particles (VLPs). Complete VLPs were synthesized, albeit with a low yield. Crystalline formation of AHSV VP7 trimers is thought to impede high-level CLP production. Consequently, we engineered and co-synthesized VP3 with a more hydrophilic mutant VP7, resulting in an increase in the turnover of CLPs.

Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells.

HIV-1 virus-like particles (VLPs) have great potential as new-generation vaccines. The novel CAP-T cell line is used for the first time to produce Gag-GFP HIV-1 VLPs by means of polyethylenimine (PEI)-mediated transient transfection. CAP-T cells are adapted to grow to high cell densities in serum-free medium, and are able to express complex recombinant proteins with human post-translational modifications. Furthermore, this cell line is easily transfected with PEI, which offers the flexibility to rapidly generate and screen a number of candidates in preclinical studies. Transient transfection optimization of CAP-T cells has been performed systematically in this work. It is determined that for optimal production, cells need to be growing at mid-exponential phase, Protein Expression Medium (PEM) medium has to be added post-transfection, and cells can be transfected by independent addition of DNA and PEI with no prior complexation. A Box-Behnken experimental design is used to optimize cell density at time of transfection, DNA/cell and PEI/cell ratios. The optimal conditions determined are transfection at a density of 3.3E + 06 cells/mL with 0.5 pg of DNA/cell and 3 pg of PEI/cell. Using the optimized protocol, 6 × 10(10) VLP/mL are obtained, demonstrating that CAP-T is a highly efficient cell line for the production of HIV-1 VLPs and potentially other complex viral-based biotherapeutics.

Production of Brazilian human norovirus VLPs and comparison of purification methods.

Noroviruses (NVs) are responsible for most cases of human nonbacterial gastroenteritis worldwide. Some parameters for the purification of NV virus-like particles (VLPs) such as ease of production and yield were studied for future development of vaccines and diagnostic tools. In this study, VLPs were produced by the expression of the VP1 and VP2 gene cassette of the Brazilian NV isolate, and two purification methods were compared: cesium chloride (CsCl) gradient centrifugation and ion-exchange chromatography (IEC). IEC produced more and purer VLPs of NV compared to CsCl gradient centrifugation.

Production of Brazilian human norovirus VLPs and comparison of purification methods

Abstract Noroviruses (NVs) are responsible for most cases of human nonbacterial gastroenteritis worldwide. Some parameters for the purification of NV virus-like particles (VLPs) such as ease of production and yield were studied for future development of vaccines and diagnostic tools. In this study, VLPs were produced by the expression of the VP1 and VP2 gene cassette of the Brazilian NV isolate, and two purification methods were compared: cesium chloride (CsCl) gradient centrifugation and ion-exchange chromatography (IEC). IEC produced more and purer VLPs of NV compared to CsCl gradient centrifugation.

The application of virus-like particles as vaccines and biological vehicles.

Virus-like particles (VLPs) can be spontaneously self-assembled by viral structural proteins under appropriate conditions in vitro while excluding the genetic material and potential replication probability. In addition, VLPs possess several features including can be rapidly produced in large quantities through existing expression systems, highly resembling native viruses in terms of conformation and appearance, and displaying repeated cluster of epitopes. Their capsids can be modified via genetic insertion or chemical conjugation which facilitating the multivalent display of a homologous or heterogeneous epitope antigen. Therefore, VLPs are considered as a safe and effective candidate of prophylactic and therapeutic vaccines. VLPs, with a diameter of approximately 20 to 150 nm, also have the characteristics of nanometer materials, such as large surface area, surface-accessible amino acids with reactive moieties (e.g., lysine and glutamic acid residues), inerratic spatial structure, and good biocompatibility. Therefore, assembled VLPs have great potential as a delivery system for specifically carrying a variety of materials. This review summarized recent researches on VLP development as vaccines and biological vehicles, which demonstrated the advantages and potential of VLPs in disease control and prevention and diagnosis. Then, the prospect of VLP biology application in the future is discussed as well.

A protocol for the gentle purification of virus-like particles produced in plants.

The purpose of the protocol is to extract and purify virus-like particles (VLPs) that have been produced in plants. More specifically, this method is well suited to the purification of chimaeric and genetically modified VLPs that do not have native surface properties. This will be the case for VLPs used in antigen display experiments. Such particles are often more fragile than their wild-type infectious virus counterparts, and as such can be damaged or lost during procedures that involve pelleting or precipitating the particles. The method presented here is based on ultracentrifugation and density gradients, with no pelleting or precipitation step. It makes virtually no assumptions about the yield of recombinant VLPs or their properties, which means that this protocol is ideally suited to screening new constructs which are expected to lead to the formation of VLPs. This protocol will allow the researcher to determine whether the construct does indeed form VLPs, and if it does, will reduce the likelihood of those particles being lost or damaged during the purification process. Because of its non-specific nature, this protocol may also be suited to the purification of viruses of unknown nature from leaf material where an infection is suspected.

Development of EV71 virus-like particle purification processes.

Enterovirus 71 (EV71) causes the outbreaks of hand-foot-and-mouth disease and results in deaths of hundreds of young children. EV71 virus-like particles (VLPs) are empty capsids consisting of viral structural proteins and can elicit potent immune responses, thus holding promise as an EV71 vaccine candidate. However, an efficient, scalable production and purification scheme is missing. For mass production of EV71 VLPs, this study aimed to develop a production and chromatography-based purification process. We first demonstrated the successful EV71 VLPs production in the stirred-tank bioreactor in which High Five™ cells were infected with a recombinant baculovirus co-expressing EV71 structural polyprotein P1 and protease 3CD. The culture supernatant containing the VLPs was subjected to tangential flow filtration (TFF) for concentration/diafiltration, which enabled the removal of >80% of proteins while recovering >80% of VLPs. The concentrated VLPs were next subjected to hydroxyapatite chromatography (HAC) in which the VLPs were mainly found in the flow through. After another TFF concentration/diafiltration, the VLPs were purified by size-exclusion chromatography (SEC) and concentrated/diafiltered by a final TFF. The integrated process yielded an overall VLPs recovery of ≈ 36% and a purity of ≈ 83%, which was better or comparable to the recovery and purity for the purification of live EV71 virus particles. This process thus may move the EV71 VLPs vaccine one step closer to the clinical applications.

Production methods for viral particles.

Viral particles and virus-like particles (VLPs) or capsids are becoming important vehicles and templates in bio-imaging, drug delivery and materials sciences. Viral particles are prepared by infecting the host organism but VLPs are obtained from cells that express a capsid protein. Some VLPs are disassembled and then re-assembled to incorporate a material of interest. Cell-free systems, which are amenable to manipulating the viral assembly process, are also available for producing viral particles. Regardless of the production system employed, the particles are functionalized by genetic and/or chemical engineering. Here, we review various methods for producing and functionalizing viral particles and VLPs, and we discuss the merits of each system.

Development of a versatile and stable internal control system for RT-qPCR assays.

RT-qPCR, an established method for the detection of RNA viruses, requires internal RNA controls for the correct interpretation of PCR results. Robust and versatile RT-PCR controls can be achieved for example by packaging RNA into a virus-derived protein shell. In this study a MS2-based internal control system was developed, that allows stable and universal packing of different RNAs into non-infectious, non-lytic MS2-based viral like particles (VLPs). Two competitive internal controls for a hantavirus assay and a Crimean-Congo Hemorrhagic Fever Virus (CCHFV) assay were cloned for the expression of VLPs. The expression of VLPs containing the RNA of interest could be induced with arabinose in Escherichia coli. The VLPs proved to be temperature resistant and could be frozen and thawed several times without degradation. Distinction of IC RNA from the target RNA was facilitated by a clear shift in the melting temperature or by specific hybridization signals. Furthermore, target and IC PCR amplification could be easily distinguished by their size in gel-electrophoretic analyses. Limits of detection were determined, demonstrating that the application of the IC did not reduce the sensitivity of the target RT-qPCR reactions. The system can be adapted to nearly any required sequence, resulting in a highly flexible method with broad range applications.

Characterization of human bocavirus-like particles generated by recombinant baculoviruses.

Human bocavirus (HBoV) is a nonenveloped, single-stranded DNA virus, classified recently into the genus Bocavirus in the family Parvoviridae. A recombinant baculovirus expression system was used to express the major capsid protein VP2 of HBoV1, HBoV2, HBoV3 and HBoV4 in insect cells. A large amount of the 61-kDa VP2 capsid protein (p61) of HBoVs was generated and efficiently released into the supernatant. The capsid protein was self-assembled into 22-nm-dia. virus-like particles (VLPs) with a buoyant density of 1.30g/cm(3). The morphology of HBoVs-LPs was similar to that of the native HBoV particles, and immunogenic studies demonstrated the cross-reactivity among HBoV1, HBoV2, HBoV3 and HBoV4. When VP1 and VP2 protein of HBoV1 were co-expressed in insect cells, both proteins were detected in the same fraction after CsCl gradient centrifugation, suggesting that the VP1 protein is a minor structural protein of HBoVs. We developed an ELISA using purified VLPs as the antigen and used it to detect antibodies against HBoV1, HBoV2, HBoV3 and HBoV4. A high prevalence of antibodies against HBoVs was found in a general population of healthy Japanese, indicating that HBoVs have spread throughout Japan.

Suitability and perspectives on using recombinant insect cells for the production of virus-like particles.

Virus-like particles (VLPs) can be produced in recombinant protein production systems by expressing viral surface proteins that spontaneously assemble into particulate structures similar to authentic viral or subviral particles. VLPs serve as excellent platforms for the development of safe and effective vaccines and diagnostic antigens. Among various recombinant protein production systems, the baculovirus-insect cell system has been used extensively for the production of a wide variety of VLPs. This system is already employed for the manufacture of a licensed human papillomavirus-like particle vaccine. However, the baculovirus-insect cell system has several inherent limitations including contamination of VLPs with progeny baculovirus particles. Stably transformed insect cells have emerged as attractive alternatives to the baculovirus-insect cell system. Different types of VLPs, with or without an envelope and composed of either single or multiple structural proteins, have been produced in stably transformed insect cells. VLPs produced by stably transformed insect cells have successfully elicited immune responses in vivo. In some cases, the yield of VLPs attained with recombinant insect cells was comparable to, or higher than, that obtained by baculovirus-infected insect cells. Recombinant insect cells offer a promising approach to the development and production of VLPs.

Diagnostic potential and antigenic properties of recombinant tick-borne encephalitis virus subviral particles expressed in mammalian cells from Semliki Forest virus replicons.

The precursor membrane envelope (prME) proteins of all three tick-borne encephalitis virus (TBEV) subtypes were produced based on expression from Semliki Forest virus (SFV) replicons transcribed from recombinant plasmids. Vero E6 cells transfected by these plasmids showed specific reactivities in immunofluorescence and immunoblot assays by monoclonal antibodies against European and Far-Eastern subtype strains of TBEV, indicating proper folding of the expressed glycoproteins. The prME glycoproteins were secreted into the cell culture supernatant, forming TBEV subviral particles of 20 to 30 nm in diameter. IgM µ-capture and IgG monoclonal antibody (MAb)-capture enzyme immunoassays (EIAs) were developed based on prME Karelia-94 (Siberian subtype) particles. Altogether, 140 human serum samples were tested using these assays, and the results were compared to those obtained with a commercial IgM EIA, an in-house µ-capture IgM assay based on baculovirus-expressed antigen, a commercial IgG EIA, and a hemagglutination inhibition test. Compared to reference enzyme-linked immunosorbent assays (ELISAs), the sensitivities of the generated µ-capture IgM SFV-prME and IgG MAb-capture SFV-prME EIAs were 97.4 to 100% and 98.7%, respectively, and the specificities of the two assays were 100%. IgM and IgG immunofluorescence assays (IFAs) were created based on Vero E6 cells transfected with the recombinant plasmid carrying the TBEV Karelia-94 prME glycoproteins. The IgM IFA was 100% concordant with the µ-capture IgM bac-prME ELISA. The IgG IFA sensitivity and specificity were 98.7% and 100%, respectively, compared to those of the commercial ELISA. In conclusion, the tests developed based on SFV replicon-driven expression of TBEV glycoproteins provide safe and robust alternatives for conducting TBEV serology.

Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells.

The foot-and-mouth disease virus (FMDV) structural protein precursor, P1-2A, is cleaved by the virus-encoded 3C protease (3C(pro)) into the capsid proteins VP0, VP1 and VP3 (and 2A). In some systems, it is difficult to produce large amounts of these processed capsid proteins since 3C(pro) can be toxic for cells. The expression level of 3C(pro) activity has now been reduced relative to the P1-2A, and the effect on the yield of processed capsid proteins and their assembly into empty capsid particles within mammalian cells has been determined. Using a vaccinia-virus-based transient expression system, P1-2A (from serotypes O and A) and 3C(pro) were expressed from monocistronic cDNA cassettes as P1-2A-3C, or from dicistronic cassettes with the 3C(pro) expression dependent on a mutant FMDV internal ribosome entry site (IRES) (designated P1-2A-mIRES-3C). The effects of using a mutant 3C(pro) with reduced catalytic activity or using two different mutant IRES elements (the wt GNRA tetraloop sequence GCGA converted, in the cDNA, to GAGA or GTTA) were analysed. For both serotypes, the P1-2A-mIRES-3C construct containing the inefficient GTTA mutant IRES produced the highest amount of processed capsid proteins. These products self-assembled to form FMDV empty capsid particles, which have a related, but distinct, morphology (as determined by electron microscopy and reconstruction) from that determined previously by X-ray crystallography. The assembled empty capsids bind, in a divalent cation-dependent manner, to the RGD-dependent integrin αvß6, a cellular receptor for FMDV, and are recognized appropriately in serotype-specific antigen ELISAs.

Development and validation of a quantitation assay for fluorescently tagged HIV-1 virus-like particles.

Upon expression, the Gag polyprotein of HIV-1 assembles spontaneously in the vicinity of the plasma membrane giving rise to enveloped virus-like particles (VLPs). These particulate immunogens offer great promise as HIV-1 vaccines. Robust VLP production and purification processes are required to generate VLPs of sufficient quality and quantity for both pre-clinical and clinical evaluation. The availability of simple, fast and reliable quantitation tools is critical to develop, optimize and monitor such processes. Traditionally, enzyme-linked immunosorbent assays (ELISA) are used to quantify p24 antigen concentrations, which reflect tightly virus particle concentrations. However, this quantitation technique is not only time-consuming, laborious and costly but it is also prone to methodological variability. As an alternative, the development and validation of a fluorescence-based quantitation assay for Gag VLPs is presented here. A Gag polyprotein fused to the enhanced green fluorescent protein was used for generation of fluorescent VLPs. A purified standard reference Gag-GFP VLP material was prepared and characterized in house. The method was validated according to ICH guidelines. The validation characteristics evaluated included accuracy, precision, specificity, linearity, range and limit of detection. The method showed to be specific for Gag-GFP. The fluorescence signal correlated well with p24 concentrations measured using a reference p24 ELISA assay. The method showed little variability compared to ELISA and was linear over a 3-log range. The limit of detection was ~10 ng of p24/mL. Finally, fluorescence-based titers were in good agreement with those obtained using transmission electron microscopy and nanoparticle tracking analysis. This simple, rapid and cost-effective quantitation assay should facilitate development and optimization of bioprocessing strategies for Gag-based VLPs.

In vivo self-assembly of TMV-like particles in yeast and bacteria for nanotechnological applications.

Heterologous expression of tobacco mosaic virus coat protein and in vivo assembly of rod-shaped TMV-like particles encapsidating viral or host RNA were compared between Escherichia coli and Schizosaccharomyces pombe. TMV-like particles were produced in both hosts, irrespective of whether the TMV origin of assembly was present. The additional plasmid providing an OAS-containing RNA was able to alter the length distribution of the TMV-like particles. Plant and yeast-expressed CP behaved similarly upon isoelectric focusing, whereas CP expressed in bacteria migrated differently. After purification by buoyant density centrifugation, the encapsidated nucleic acids were determined to be of host origin as well as of viral origin. OAS-containing mRNA was packaged preferentially in yeast to some extent (8%). In consequence, the majority of TMV-like particles showed the same length distribution similar to those in the absence of OAS-containing mRNA, likely due to host RNA being primarily encapsidated. Notwithstanding this limitation for tailoring particle sizes, the heterologous expression system provides a new avenue to deliver versatile nucleoprotein scaffolds for a diversity of nanotechnological applications, without the need for an infectious virus. The results are discussed with reference to the competition of translation and packaging as well as to the selective decay of TMV RNA.