An Improved Reverse Genetics System to Overcome Cell-Type–Dependent Ebola Virus Genome Plasticity

Abstract

Reverse genetics systems represent a key technique for studying replication and pathogenesis of viruses, including Ebola virus (EBOV). During the rescue of recombinant EBOV from Vero cells, a high frequency of mutations was observed throughout the genomes of rescued viruses, including at the RNA editing site of the glycoprotein gene. The influence that such genomic instability could have on downstream uses of rescued virus may be detrimental, and we therefore sought to improve the rescue system. Here we report an improved EBOV rescue system with higher efficiency and genome stability, using a modified full-length EBOV clone in Huh7 cells. Moreover, by evaluating a variety of cells lines, we revealed that EBOV genome instability is cell-type dependent, a fact that has significant implications for the preparation of standard virus stocks. Thus, our improved rescue system will have an impact on both basic and translational research in the filovirus field.

Ebola virus (EBOV), a member of the family Filoviridae and the genus Ebolavirus, causes a severe hemorrhagic fever in humans and in nonhuman primates in Central and Western Africa, with case-fatality rates of 50%–90% [1–3].

Over the past decade, reverse genetics has been used extensively in the filovirus field, and it has become a key technique for studying the molecular biology and pathogenesis of EBOV [4–8]. Such systems have allowed researchers to manipulate the virus genome and generate recombinant viruses from cloned complementary DNA (cDNA), allowing detailed experimental examination of the molecular mechanisms that govern the virus replication cycle and viral protein function [9–11].

The EBOV GP gene encodes at least 3 different glycoproteins, sGP, ssGP, and GP_1,2, and the expression of each of these proteins is controlled by stuttering of the RNA-dependent RNA polymerase (RdRp) at the RNA editing site within the GP gene [12–14]. The editing site comprises a stretch of 7 uridines (7U) in the viral genome that, when left unedited, allows transcription of the sGP mRNA, which is then translated into the nonstructural, secreted GP. Following the stuttering of the RdRp at the editing site, a nontemplate adenosine is inserted into the messenger RNA (mRNA), resulting in a frameshift that produces the transcript for GP_1,2, the spike glycoprotein. Because only approximately 20% of the mRNA transcripts contain the additional adenosine during virus infection, RNA editing serves as a mechanism to regulate GP_1,2 expression. As expected, insertion of a U residue into the editing site within the virus genome results in constitutive expression of GP_1,2 and prevents expression of sGP. Notably, in guinea pigs and nonhuman primates, the EBOV variant containing 8 uridines (8U) at the GP editing site—and therefore expressing only GP_1,2—is rapidly replaced by a variant containing 7U, suggesting that mutations within the RNA editing site may be directly linked to virus replication and pathogenicity [15, 16]. Volchkova et al reported that stable mutations in the EBOV genome appeared predominantly at the RNA editing site of the GP gene during 4–5 passages of the virus in Vero E6 cells, which are commonly used for EBOV amplification and propagation [15]. In addition to the RNA editing site, 3 other mutation sites were identified within the GP and viral protein 24 genes over the course of 5 passages [16]. Indeed, we have also frequently observed mutations in the genome, including at the RNA editing site, of rescued recombinant EBOV (rEBOV) in our own EBOV reverse genetics system using Vero cells. It is likely that the apparent instability of the EBOV genome, and, in particular, the GP RNA editing site, has had an impact on EBOV research, since EBOVs are routinely isolated, passaged, and grown for the preparation of standard virus stocks in Vero or Vero E6 cells.

Here we report an improved EBOV reverse genetics system that results in more efficient generation of rEBOV with greater genomic fidelity. In addition, we demonstrate that the genomic instability of rescued EBOV is cell-type-dependent. Our findings and proposed EBOV reverse genetics system offers stable and accurate EBOV experimental conditions, which will be of significant advantage to both basic and translational filovirus research.

METHODS

Cells

African green monkey kidney cells (Vero, Vero E6 and COS7), human embryonic kidney cells (293), and human hepatoma cells (Huh-7; kindly provided by Dr. Matsuura, Osaka university) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), L-glutamine (L-Glu), and penicillin/streptomycin (Pen/St). African green monkey kidney cells (CV-1, BS-C-1, and MA104 clone 1) and human monocytic leukemia cells (THP-1) were maintained in Eagle's minimum essential medium (EMEM) or Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS, L-Glu, and Pen/St. Vero E6 (CRL-1586), COS7 (CRL-1651), CV-1 (CCL-70), BS-C-1 (CCL-26), MA-104 clone 1 (CRL-2378.1), and THP-1 (TIB-202) were obtained from ATCC. BHK-21 expressing T7 RNA polymerase under the control of the chicken β-actin promoter (BHK-T7; kindly provided by Dr. Ito, Gifu university [17]) was maintained in EMEM containing 5% FBS, Pen/St, and 10% triphosphate broth.

Antibodies

To produce rabbit anti-VP40 polyclonal antibodies, a single New Zealand White rabbit was immunized intramuscularly with TiterMax Gold Adjuvant (Sigma-Aldrich) plus 500 µg of purified virus-like particles (VLPs) derived from EBOV VP40–transfected 293 cells. Following the initial inoculation, the rabbit was boosted intramuscularly 2 more times with VLPs plus incomplete Freund's adjuvant (ICFA; Sigma-Aldrich), with approximately 2 weeks between each inoculation. Two weeks later, the rabbit was given a third boost with VLPs plus ICFA; after an additional 2 weeks, rabbit were euthanized and blood specimens were obtained. The collected serum specimen underwent affinity purification using the Nab Protein A Plus Spin Kit (Thermo Scientific). Animal studies were approved by the institutional animal care and use committee and were performed by certified staff in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care.

Construction of EBOV Plasmids

The 3 subgenomic cDNA fragments corresponding to EBOV (strain Mayinga [AF086833]) genome nucleotide positions 1–4727, 4728–11 860, and 10 942–18 959, were subcloned into conventional subcloning plasmids. The T7 RNA polymerase promoter sequence with an additional single guanine (TAATACGACTCACTATAG) and hepatitis delta virus ribozyme followed by T7 terminator were inserted immediately upstream of the leader sequence and immediately downstream of the trailer sequence, respectively. To assemble the full-length genome clone, all subgenomic segments underwent digestion by restriction enzymes and were combined into a plasmid containing a p15A origin and a kanamycin-resistant gene (p15AK-EBOV-HDVRz). An oligonucleotide encoding the hammerhead ribozyme (HamRz [18]) was inserted into the full-length genome plasmid upstream of the 5’ terminal leader region of the EBOV antigenome (p15AK-EBOV-HHRz/HDVRz; Supplementary Figure 2A). The open reading frames (ORFs) encoding NP, VP30, VP35, and L were cloned into expression plasmid pCAGGS, using EcoRI and BglII restriction sites, with a Kozak consensus sequence immediately upstream of the start codon of the ORFs. All plasmid sequences were confirmed by Sanger sequencing before use.

Reverse Genetics

The full-length genome plasmid was cotransfected together with helper plasmids in accordance with established protocols [5]. Briefly, cells were transfected with the 1 µg full-length genome plasmid and helper plasmids 1 µg pCAGGS-NP, 0.5 µg pCAGGS-VP35, 0.3 µg pCAGGS-VP30, 2 µg pCAGGS-L, and 1 µg pCAGGS-T7, using Transit-LT1 (Mirus). Medium was changed to DMEM with 3% FBS 18–24 hours after transfection. To propagate the rescued virus for further experiments, the supernatant was passaged onto newly prepared cells. Virus-infectivity titers (presented as focus-forming units [FFU]) were obtained by counting the number of infected cell foci detected by use of an indirect immunofluorescent antibody assay [19, 20]. EBOV antigen–positive foci were detected with a rabbit polyclonal anti-VP40 antibody and a goat anti-rabbit IgG antibody conjugated to FITC (Sigma).

Virus Infection and Interferon (IFN) Enzyme-Linked Immunosorbent Assay (ELISA)

Cells were infected with sequence-confirmed rEBOV at a multiplicity of infection (MOI) of 0.01. Virus was allowed to adsorb for 1 hour, unbound virus was washed away, and a suitable medium for each cell line with 3% FBS was added. At set time points, the supernatants were collected and stored at −80°C for further analysis. To confirm IFN production in infected cells, the supernatants were collected and inactivated by γ-irradiation 5–7 days after infection. The concentration of IFN-α and IFN-γ were measured by the ELISA development kit (MABTECH) according to the manufacturer's instructions.

Minigenome Assay

Cells were transfected with the 0.1 µg p3E5E-minigenome (encoding Renilla Luciferase), 0.02 µg pCAGGS-luc2 (encoding firefly luciferase as a transfection control) [21, 22], and 0.1 µg pCAGGS-NP, 0.05 µg pCAGGS-VP35, 0.03 µg pCAGGS-VP30, 0.2 µg pCAGGS-L, and 0.1 µg pCAGGS-T7, using Transit-LT1. As a negative control, pCAGGS-L was omitted from the mixture of helper plasmids. A total of 24–48 hours after transfection, cells were lysed and reporter activity was analyzed using the Dual-Luciferase reporter assay system (Promega).

Sequence Analysis

Viral RNA was extracted from either supernatants or cells, using the QIAamp viral RNA kit or RNeasy mini kit (Qiagen). Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using Superscript III reverse transcriptase (Life Technologies) and iProof High Fidelity DNA polymerase (Bio-Rad) with virus gene-specific primers. Genome sequences of viruses were analyzed by Sanger sequencing. Transcript fragment lengths were analyzed on an ABI 3730 xl (Applied Biosystems), using capillary electrophoresis with custom FAM-labeled primers in accordance with the manufacturer's recommendations.

To analyze the amounts of EBOV genome containing 7U or 8U stretches at the RNA editing site, we performed rapid transcript quantification analysis (RTQA), as previously described with modifications to facilitate use of gene-specific primers [23]. To quantify U insertions caused by the T7 polymerase, a rescue experiment was performed as described with the omission of pCAGGS-L to avoid viral RNA replication/transcription. Total RNA was extracted from transfected cells at 24 or 48 hours after transfection. To quantify the U insertions that resulted during virus replication, recombinant viruses with either the 7U or 8U genotype or a mixture of 98% 7U and 2% 8U genotypes were passaged repeatedly. At the time of passage, viral genomic RNA was extracted from clarified supernatants. The extracted RNA was treated with RNase-free DNase I (Promega) and then reverse transcribed. The relative amount of cDNAs containing either 7 adenosine (A) or 8 A stretches at the RNA editing site was determined by RTQA.

Statistics

Statistical analysis was performed with the Welch t test. P values of <.05 were considered statistically significant.

Biosafety Statements

All studies with EBOV were performed in the biosafety level 4 facility at the Integrated Research Facility of the Rocky Mountain Laboratories, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Sample inactivation/removal was performed according to standard operating protocols approved by the local institutional biosafety committee.

RESULTS

High Frequency of Genome Mutation in Recombinant EBOV Rescued in Vero Cells

During a series of rEBOV rescue experiments using Vero cells, we observed a high frequency of mutations in the genome of rEBOV (Figure 1). The genomes of 23 clones were fully sequenced, and 78% of those clones possessed ≥1 mutation (Figure 1C). Approximately 60% of the mutations were U insertions in regions of the genome containing U stretches, including the RNA editing site of the GP gene and the translation termination signals. The remaining mutations were both silent and missense nucleotide substitutions that occurred throughout the genome.

Figure 1.

High frequency of genomic mutations in virus rescued in Vero cells. Recombinant Ebola virus (EBOV) was rescued in Vero cells from a plasmid containing the full-length EBOV genome complementary DNA (cDNA), and the genome sequence was confirmed by sequencing of extracted viral RNA. A, Schematic diagram demonstrates the positions of genome mutations in full-length cDNA for 23 EBOV clones, which was reverse transcribed from extracted RNA of rEBOV and rEBOV possessing mouse-adapted NP and viral protein 24 (VP24). The size of the arrows indicates the frequency of mutations at that nucleotide position. B, Position and number of mutations observed in 23 clones rescued in Vero cells. C, Type of genomic mutations in rEBOV rescued in Vero cells. We analyzed the full-length sequence of a total of 23 rEBOV clones, either wild type or possessing the mouse-adapted NP and VP24, in Vero cells. The percentage of viruses that possessed no mutations throughout the entire genome is colored in white on the pie chart, and the percentage of viruses that possessed at least 1 mutation is shown in white dots. The bar graph to the right denotes the percentage of viruses that possessed a uridine (U) insertion in a U stretch, the percentage that possessed an amino acid substitution, and the percentage that possessed a silent mutation.

The most common site of mutation was the RNA editing site of the GP gene, where a U insertion increased the stretch of 7U to 8U. To evaluate the efficacy of sGP production in infected cells, we prepared sequence-confirmed rEBOV of a 7U genotype virus (7U-EBOV), an 8U genotype virus (8U-EBOV), and a mixture of approximately half 7U-EBOV and half 8U-EBOV (7/8Uh-EBOV) and compared the concentration of sGP in supernatants and growth kinetics in infected cells (Supplementary Figure 1). The mutation to 8U-EBOV resulted in the lack of expression of the nonstructural sGP protein and increased replication in Vero cells.

Establishment of an Improved EBOV Reverse Genetics System

To improve the rescue efficiency of the EBOV reverse genetics system, the hammerhead ribozyme (HHRz) sequence was inserted upstream of the 5′ terminus (the complementary RNA sense leader region) in the full-length genome plasmid (Supplementary Figure 2A and 2B), since a previous report demonstrated that the HHRz-mediated generation of an authentic 5′ terminus of the viral antigenome improved virus rescue efficiency [18]. Addition of the HHRz sequence to the EBOV minigenome construct did not have a positive impact on reporter gene expression in the minigenome assay in either Vero or Huh7 cells (Figure 2A and 2D). Moreover, virus rescued in Vero cells from full-length genome constructs with (E.HR) or without (E.Ori) the HHRz sequence displayed a similar titer at day 4 after transfection, although at earlier time points the rescue of E.HR did appear slightly more efficient than E.Ori (Figure 2B). In contrast, E.HR showed approximately 1-log higher titer than E.Ori when rescued in Huh7 cells (Figure 2E). To compare the HHRz cleavage efficiency at the 5′ terminus of the transcribed genome in Vero and Huh7 cells, the ratio of cleaved genome RNA after transcription from plasmids was evaluated by RT-PCR (Supplementary Figure 2C and 2D). RNA cleavage in Huh7 appeared slightly more efficient than in Vero cells, perhaps at least partly explaining the increased rescue efficiency.

Figure 2.

Comparison of rescue efficiency with additional ribozyme sequence in Vero cells (A–C) or Huh7 cells (D–F). A and D, The cleavability of the 5’ terminus of the genome with or without the hammerhead ribozyme (HHRz) sequence was evaluated by a minigenome assay. Cells were transfected with minigenome plasmid (Renilla luciferase), with or without the HHRz sequence, together with helper plasmids. Twenty-four hours after transfection, cells were lysed and tested for luciferase activity. Results were standardized to firefly luciferase activity and are expressed as relative luciferase units (RLU) ± SD (n = 3). **P < .01. B and E, Comparison of the growth kinetics of recombinant Ebola virus (rEBOV) in transfected cells. Cells were transfected with either p15AK-EBOV-HDVRz or p15AK- EBOV-HHRz/HDVRz along with helper plasmids. Twenty-four hours after transfection, medium was replaced, and supernatants were collected at the indicated time points until day 7. Virus titers were determined by the focus-forming unit assay (n = 3; *P < .05). C and F, To evaluate the mutation frequency of rEBOV rescued in Vero or Huh7 cells, 10 clones of E.HR were rescued in each cell line, and the entire genome was subsequently sequenced. The number of clones possessing any mutation is depicted in the bar graph on the left. The detected mutations are depicted by type in the bar graph on the right. Abbreviation: U, uridine.

Next, we compared the genome stability during virus generation and propagation in Vero or Huh7 cells. Ten virus clones of E.HR were rescued in Vero or Huh7 cells, and viral RNA was extracted from each rescued virus. The extracted RNA was amplified by RT-PCR, and the entire viral genome was sequenced (Figure 2C and 2F). Although mutations (including U insertions, and silent and missense nucleotide substitutions) were detected in viruses rescued in both Vero and Huh7 cells, the frequency and proportion of mutated viruses rescued in each of these cell types varied dramatically. Mutations were detected in 8 of 10 viruses rescued in Vero cells, whereas only 3 of 10 viruses rescued on Huh7 cells contained a mutation.

In addition, we compared the rescue efficiency of rEBOV in 5 cell lines routinely used for the rescue of RNA viruses and known to support EBOV replication: Vero, Huh7, BHK-T7, COS7, and 293 cells. Huh7 cells showed the highest rescue efficiency among the cell lines tested (Figure 3), but lower transfection and polymerase activity were observed in Huh7 cells (Supplementary Figure 3). On the other hand, 293 cells showed high transfection efficiency, and BHK-T7 cells exhibited the highest polymerase activity; however, rEBOV was produced slowly in these cells. We also evaluated IFN-γ and IFN-α production in infected cells because contamination of IFNs in inocula may affect further experiments. No detectable level of IFNs was detected in either Huh7 or Vero cells infected with EBOV (data not shown).

Figure 3.

Comparison of rescue efficiency in 5 cell lines. A, Vero, Huh7, BHK-T7, COS7, and 293 cells were transfected with p15AK-EBOV-HHRz/HDVRz along with helper plasmids. Supernatants of transfected cells were collected at the indicated time points, and virus titers were determined by the focus-forming unit (FFU) assay (n = 3). B, Each cell line was inoculated with recombinant Ebola virus (rEBOV) at a multiplicity of infection of 0.01, and supernatants of infected cells were collected at the indicated time points. Virus titers were determined by the FFU assay (n = 3). Huh7 cells supported the highest levels of EBOV replication, demonstrated by the virus titer, which reached approximately 10⁷ FFU/mL by day 4 after infection. In Vero and BHK-T7 cells, rEBOV had lower replication rates. rEBOV showed rapid growth in COS7 and 293 cells, as well as in Huh7 cells.

Viral RNA Replication by the Viral RdRp Is a Critical Driver of Cell-Type–Dependent Genome Plasticity

To elucidate the mechanisms of EBOV genomic RNA instability in Vero cells, we focused on the RNA editing site of the GP gene. We first examined whether the T7 RNA polymerase was responsible for adding the A residues at the RNA editing site during the initial transcription of the antigenomic sense of full-length EBOV RNA, since Volchkov et al reported GP mRNA editing by the T7 RNA polymerase [12]. Approximately 1%–2% of virus RNA genomes that were initially transcribed by the T7 RNA polymerase from the full-length plasmid possessed the 8U sequence at the RNA editing site in both Vero and Huh7 cells (Figure 4A).

Figure 4.

Genetic instability of the RNA editing site during virus generation or propagation. A, Vero or Huh7 cells were transfected with p15AK-EBOV-HHRz/HDVRz along with helper plasmids. At 24 and 48 hours after transfection, cells were lysed, and viral RNA was extracted. B, Sequence-confirmed clones of 7-uridine Ebola virus (7U-EBOV), 8U-EBOV, or a mix of viruses containing 98% 7U-EBOV and 2% 8U-EBOV (7/8U-EBOV) were used to inoculate Vero or Huh7 cells at a multiplicity of infection (MOI) of 0.01. Viruses were diluted 1:500 with fresh medium and blind passaged 5 times, and viral RNA was extracted from supernatant at each passage. C, Several African green monkey kidney cell lines (Vero, Vero E6, Vero-IRF3 [Vero cells transiently transfected with IRF3 expression plasmid], COS7, CV-1, BS-C-1, and MA104 clone 1 cells) were infected with 7/8U-EBOV at a MOI of 0.01. Viruses were blind passaged 3 times, and viral RNA was extracted at each passage. In all cases, extracted RNA was treated with DNase I and transcribed by reverse transcriptase. A region of the genome composing the RNA editing site was amplified from complementary DNA by polymerase chain reaction analysis and subjected to genotyping by rapid transcript quantification analysis.

To demonstrate the contribution of the RdRp to EBOV genomic instability, we prepared inocula consisting of clones of 7U-EBOV or 8U-EBOV, or a mixture of the 7U and 8U viruses at a ratio of 98:2 (7/8U-EBOV) that mimicked the RNA populations produced upon initial transcription by T7 RNA polymerase in the transfected cells. Following 5 serial passages of the viruses, the sequences at the RNA editing site were analyzed for each passage. In Vero cells infected with 7U-EBOV, approximately 40% of viral RNA after passage 3 possessed the 8U sequence at the RNA editing site, and the population of 8U sequence remained around 40% during further passage (Figure 4B). The appearance of an 8U-EBOV population was even more evident in cells infected with the 7/8U-EBOV inoculum, such that the proportion of the population that was 8U-EBOV increased from 2% to 88% after only 2 passages. In contrast, the 8U sequence was not detected in Huh7 cells infected with 7U-EBOV, even after 5 passages, and when infected with 7/8U-EBOV the population of 8U-EBOV decreased to almost 0 after only 2 passages in Huh7 cells. In addition, 17% of 8U-EBOV reverted to 7U-EBOV after 5 passages in Huh7 cells. We also analyzed the whole genome sequence of 7U-EBOV passaged in Vero and Huh7 cells. No mutations were detected during 3 passages in Huh7 cells, whereas in Vero cells all 3 clones were mutated to 8U-EBOV, and 1 possessed an additional mutation in the transcription termination signal of the L gene (data not shown).

To investigate whether the instability of the genome sequence of passaged virus was specific to Vero cells, we compared virus sequences obtained from passaging EBOV in 7 African green monkey kidney cell lines. Vero, Vero E6, COS7, CV-1, BS-C-1, MA104, and Vero-IRF3 (Vero cells transiently transfected with an IRF3 expression plasmid) were infected with 7/8U-EBOV and passaged 3 times in each cell line. After only 2 passages, the ratio of 8U-EBOV increased by >80% in most of the cell lines analyzed (Figure 4C). Conversely, in COS7 cells, approximately 70% of viral RNA still possessed the 7U sequence after 3 passages. Therefore, EBOV genome instability does not appear to be specific to Vero E6 cells and may rather be a cell-type–dependent phenomenon.

DISCUSSION

Recently, the genome plasticity of EBOV in Vero cells was demonstrated by several studies [15, 16], and such plasticity may influence several aspects of filovirus research, since Vero or Vero-derived cells have been used as the standard cell lines for propagation of EBOV stocks. Mutations arising during virus propagations may alter the phenotype of the virus from the original wild-type virus. One of the potential measures to prevent the evolution of quasispecies in virus stocks is the use of reverse genetics, in which rescued recombinant virus is derived from a single full-length clone with a defined genome sequence. However, in our current reverse genetics system, unwanted mutations were detected in the genome of rEBOV clones with a frequency as high as 80% (Figure 1C). Although it is possible that some of these mutations were induced during the RT-PCR step before sequencing, duplicate sequencing of extracted RNA produced identical results, suggesting that RT-PCR was not the cause of these mutations and implying, instead, that the mutations were induced during virus rescue. Importantly, approximately 40% of the mutations resulted in amino acid substitutions or U insertions in the RNA editing site of the GP gene, possibly altering the phenotype of EBOV. The EBOV GP gene encodes the nonstructural sGP as well as structural GP_1,2 through transcriptional editing at the RNA editing site on sGP/GP_1,2 mRNA [12, 13]. The insertion of a U at the RNA editing site of the GP gene in the viral genome results in a lack of sGP expression due to a loss of sGP as a primary transcript in the GP mRNA. Notably, sGP constitutes approximately 75% of expressed glycoprotein during infection and may play an important role in pathogenesis [14, 15, 24]. The absence of sGP expression following GP RNA editing may, therefore, have significant impacts on studies of virus replication and pathogenesis that use recombinant viruses.

To elucidate mechanisms of EBOV genomic RNA instability, especially at the RNA editing site, we analyzed populations of viral RNA possessing 7U and 8U sequences. Interestingly, the 8U-EBOV population increased to >90% after only 3 passages of 7/8U-EBOV in Vero cells, but decreased to almost 0% after 2 passages in Huh7 cells (Figure 4B). In addition, the population of 8U-EBOV increased during passages of 7/8U-EBOV in Vero cells, as well as in many other African green monkey kidney cell lines (Figure 4C). Notably, the amount of 8U-EBOV did not increase in COS7 cells, suggesting that these cells may not possess the cellular factors that accelerate EBOV genome instability or, alternatively, that there are factors that increase genome fidelity.

The current full-length genome plasmid possesses a HDVRz sequence at the 3′ end of the EBOV genome to provide an authentic 3′ end. Previously, it was reported that the addition of the HHRz sequence to the 5′ end of the virus genome enhanced the cleavage activity and improved the rescue efficiency of other negative-sense RNA virus reverse genetics systems [18]. In the EBOV reverse genetics system, the addition of the HHRz sequence enhanced rescue efficiency in Huh7 cells but had little effect in Vero cells (Figure 2). The activity of ribozyme sequences may depend not only on the cleaved sequence but also on the cell type. Moreover, Huh7 was the most potent of the 5 cell lines tested for both generation and propagation of virus. RNA polymerase activity and transfection efficiency were similar among all cell lines tested, although the rescue efficacy was different in each cell line. For example, 293 cells were highly transfectable and readily supported EBOV replication, but rEBOV was produced slowly in these cells, and only a low amount of virus was detected 5 days after transfection. These results suggest that a cell line's susceptibility to transfection and/or virus replication does not necessarily correlate with the efficiency of the EBOV reverse genetics system. We also evaluated IFN production in infected cells but were unable to detect any IFN produced in Huh7 cells. Accordingly, it has been reported that Huh7 cells have a limited ability to produce and respond to IFNs [25], implying that the effects of IFN contamination in virus inoculum may be minimal. Thus, we demonstrated that Huh7 cells, which showed high EBOV growth and genome stability but also low IFN production against EBOV infection, are an alternate cell line for rEBOV generation and propagation.

In conclusion, we have developed an improved EBOV reverse genetics system in which rEBOV was generated with high efficiency and genomic fidelity. Our improved system will be useful for studying the molecular mechanisms of EBOV replication and pathogenicity, as well as for the development of vaccines.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.