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. 2015 Oct 1;212 Suppl 2(Suppl 2):S129-37.
doi: 10.1093/infdis/jiu681. Epub 2015 Mar 24.

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

Yoshimi Tsuda  1 , Thomas Hoenen  2 , Logan Banadyga  3 , Carla Weisend  3 , Stacy M Ricklefs  4 , Stephen F Porcella  4 , Hideki Ebihara  3
Affiliations

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

Yoshimi Tsuda et al. J Infect Dis. .

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.

Keywords: Ebola virus; RNA editing site; mutation; reverse genetics system.

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Figures

Figure 1.
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.
Figure 2.
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.
Figure 3.
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.
Figure 4.
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.
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