One-Step Reverse-Transcription FRET-PCR for Differential Detection of Five Ebolavirus Species

Ebola is an emerging infectious disease caused by a deadly virus belonging to the family Filoviridae, genus Ebolavirus. Based on their geographical distribution, Ebolavirus has been classified into total five species so far, mainly Zaire, Sudan, Taï Forest, Bundibugyo and Reston. It is important to be able to differentiate the Ebolavirus species as they significantly differ in pathogenicity and more than one species can be present in an area. We have developed a one-step step-down RT-PCR detecting all five Ebolavirus species with high sensitivity (1 copy of Ebolavirus DNA, 10 copies of RNA and 320 copies of RNA spiked in 1 ml whole blood). The primers and FRET-probes we designed enabled us to differentiate five Ebolavirus species by distinct T m (Zaire: flat peaks between 53.0°C and 56.9°C; Sudan: 51.6°C; Reston: flat peaks between 47.5°C and 54.9°C; Tai Forest: 52.8°C; Bundibugyo: dual peaks at 48.9°C and 53.5°C), and by different amplicon sizes (Zaire 255bp, Sudan 211bp, Reston 192bp, Taï Forest 166bp, Bundibugyo 146bp). This one-size-fit-all assay enables the rapid detection and discrimination of the five Ebolavirus species in a single reaction.


Introduction
Ebola Virus Disease (EVD), previously known as Ebola hemorrhagic fever, is an emerging infectious disease caused by a deadly virus of the family Filoviridae, genus Ebolavirus [1,2]. Of the five identified Ebolavirus species, four of them (Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus and Bundibugyo ebolavirus) are known to cause disease in humans, whereas the fifth (Reston ebolavirus) causes disease in non-human primates and swine only [3][4][5][6]. Since human infections were first reported in 1976 on the Ebola River in Democratic Republic of the Congo [7], outbreaks of EVD have occurred into twelve countries (South Sudan, Democratic Republic of Congo, Côte d'Ivoire, Gabon, Uganda, Republic of Congo, Guinea, Philippines, Nigeria, Senegal, Liberia, Mali). There have also been imported cases in Sierra Leone, Spain, and the United States of America [8-10, 3, 11, 12]. By January 25, 2015, a total of 22, 092 cases of Ebola infections have been confirmed, of which 8, 810 cases of EVD have been fatal [12].
While simple or multiplex PCRs have been successfully used to detect Ebolavirus, most of the published assays can only detect a single Ebolavirus species [13,14] or, in the case of those that detect multiple Ebolavirus species, they show reduced sensitivity [15][16][17]. Whereas simple PCR tends to provide high sensitivity but lacks ability to differentiate multiple targets simultaneously [13,14,18], multiplex PCR has the capacity to detect and differentiate multiple targets but tends to have a reduced sensitivity [17,[19][20][21]. The TaqMan RT-PCR assay targeting the nucleoprotein gene of Zaire ebolavirus developed by Huang Y et al. [14] had a wide range in sensitivity, from 10 3 to 10 9 virus copies per reaction. The primers and fluorogenic probe of the one-step qRT-PCR described by Towner JS et al. [13] were specific for Sudan ebolavirus and showed a high sensitivity (10 3 genomic-sense Ebolavirus-RNA copies per ml; one genomicsense RNA per reaction). Panning et al. [19] established a complicated system with 5 primers and 3 probes for Filoviridae with the detection limit from 487 to 4546 RNA copies/ml depending on the species of Ebolavirus. Trombley et al. [20] established a system to detect multiple hemorrhagic fever viruses including Ebolavirus by using 48 TaqMan-based PCR assays with the limit of detection for the assays ranging from 10 to 0.001 plaque-forming units / PCR.
While RT-PCR with high sensitivity and specificity have been widely used to quantify RNA viruses, RNA templates as positive controls were not always available and safe for highly infective pathogens such as SARS virus and highly pathogenic avian influenza viruses. In addition, RNA templates from killed viruses tend to degrade under the influence of RNase and metal ions. In comparison, the VLPs equipped with specific RNA fragments have been widely used as stable, reliable and safe positive controls for RT-PCR [22][23][24].
A generic PCR that can detect and differentiate all Ebolavirus species is needed as increased international movement of people increases the potential of transmission for multiple Ebolavirus species with varying pathogenicity [11,17,[25][26][27][28]. Here we report a highly-sensitive onestep reverse-transcription FRET-PCR capable of rapidly identifying and differentiating all five Ebolavirus species.

Preparation of Ebolavirus RNA transcripts
Since it is not allowed to use viable Ebolavirus for research in China and none of five Ebolavirus species was available to us, transcripts made from the plasmids described above and virus-likeparticles (VLPs) containing Ebolavirus RNA fragment were used as positive controls. The transcription reaction was run with MEGAscript Kit (Ambion by life technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. After incubation at 37°C for 4h, the transcribed products were treated with TURBO DNase (Ambion by life technologies, California, USA), followed by precipitation with lithium chloride. Finally, the yield of RNA transcripts was determined with the Quant-iT RiboGreen RNA Kit (Molecular Probes, Eugene, OR, USA) for preparation of the RNA standards (10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 copies /10μl).

Construction and expression of virus-like particles (VLPs) containing Ebolavirus NP RNA
In addition to the positive controls of plasmids and transcripts described above, we prepared also the VLPs containing the Ebolavirus NP RNA fragment to further verify the specificity and reverse-transcription efficiency of the established PCR in this study. The MS2 VLPs containing the NP RNA fragments of each of these five Ebolavirus species were prepared as the method described by Drosten et al. [29]. The primers (forward primer: 5'-ATGAATTCTCCTGCTCA ACTTCCTGTCG-3'; reverse primer: 5'-GCAAGCTTGTTAGTAGATGCCGGAGTTT-3') of MS2 phage (ATCC 15591-B1) assembly protein gene, coat protein gene and the fragment of Eblolavirus RNA NP gene was synthesized according to their sequences from GenBank data (Gene Accession#: KC24280) and were used to amplify their cDNAs by RT-PCR. Expression vector pTrc99a (Pharmacia) and their cDNA fragments were ligated with T4 DNA ligase after digestion with restriction enzyme. The prokaryotic expression was obtained by transformation of recombinant plasmids into E. coli JM109.The obtained VLPs were purified and tested by quantitative analysis and RT-PCR.

Inoculation of transcribed Ebolavirus RNA and VLPs in human blood
Ten μl serially diluted transcribed RNAs and VLPs (10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 copies/10 μl) were thoroughly mixed with 50 μl human whole blood (collected in EDTA) and 150 μl 1× PBS for RNA extraction (High Pure RNA Isolation Kit, Roche, Mannheim, Germany). After vortexing for 15s with Lysis/-Binding Buffer and Red Cell Lysis Buffer (Roche Diagnostics GmbH, Mannheim, Germany), the blood samples were transferred to the High Pure filter tube and RNA eluted with 60 μl elution buffer which was stored at -80°C until thawed at room temperature for RT-PCR.

One-step FRET RT-PCR for Ebolavirus
Primers and probes. The full genome sequences of five Ebolavirus species (two representing GenBank Accession # for Zaire ebolavirus NC_002549, KC24280; Sudan ebolavirus NC_006432, KC54539; Reston ebolavirus NC_004161, JX477166; Taï Forest ebolavirus NC_014372, FJ217162; Bundibugyo ebolavirus NC_014373, KC545396), and those two monophyletic genus (Marburgvirus KC545388, NC_001608; Cuevavirus JF828358, NC_016144) were obtained from GenBank (Fig 1). Based on the conserved and variable regions determined by Clustal Multiple Alignment, we identified highly conserved regions in 1-146 bp of the genome which were common to all five Ebolavirus species and used for the forward primer, 6-FAM probe and LCRed 640 probe (Fig 1, Table 1). The regions between 146 bp and 255 bp were highly conserved for individual Ebolavirus species but were highly variable among the different species and were thus used to design five reverse primers to specifically amplify only a single Ebolavirus species (Fig 1, Table 1). The primers and probes demonstrated high level of mismatches with Cuevavirus (35-40 mismatches) and Marburgvirus (no corresponding fragment).
Specificity of the one-step RT FRET-PCR. The BLASTN with the organism options of including and excluding Ebolavirus (taxid: 186536) was performed on each of the primers and probes to verify the specificity of the designed oligonucleotides we designed. Specificity of the one-step RT FRET-PCR was also evaluated by amplifying each of five plasmids containing portions of each Ebolavirus species (Zaire ebolavirus, Sudan ebolavirus, Reston, ebolavirus Taï Forest ebolavirus and Bundibugyo ebolavirus), and a mixture of the five plasmids (Fig 3). In addition, the following oligonucleotide polymers were synthesized (GenScript, Nanjing, China) to further test the sensitivity of the established PCR: 1) the first 100 bp of the PCR amplicon for EVD Zaire (contains forward primer and two probes); 2) the complimentary sequences of the first 100 bp of the PCR amplicon for EVD Zaire; 3) the 33-250 bp of the the PCR amplicon for EVD Zaire (contains two probes and five downstream primers).
The specificity of the established PCR in this work was further tested on the nucleic acids of other pathogens causing hemorrhagic fever or similar clinical signs as Ebolavirus (Bunyavirus, Japanese encephalitis virus, Cuevavirus, Dengue virus, Yellow fever virus, Hanta virus, forest encephalitis virus, Lassa fever virus, Marburg virus,; Nucleic acids of these viruses were provided by the National Institute for Food and Drug Control, China Food and Drug Administration). The PCR products from the reactions with the plasmids of the individual Ebolavirus species and the mixture containing them all, were verified by electrophoresis through a 4%  (Fig 4). A high concentration (4%) of BIOWEST Gel (gel strength750, gelling range around 36-39°C, and melting range around 87-89°C) ensures efficient differentiation of the small sizes of PCR fragments in this study.

Results and Discussion
Our one-step RT FRET-PCR was capable of detecting a single DNA copy and 10 RNA copies of all five Ebolavirus species (Figs 3 and 5). When whole blood spiked with transcribed Ebolavirus RNA and VLPs were tested, the detection limit was 100 copies of RNA per PCR reaction (Fig 5), equivalent to 320 copies of Ebolavirus in 1 ml whole blood. Triplicates were performed for each assay. The BLASTN results for each of the 8 oligonucleotides we used in the one-step RT FRET-PCR showed they were specific for the Ebolavirus species and did not cross-react with partial amplicons or other viruses in the family Filoviridae. No PCR amplification was observed with nucleic acids of eight types of pathogens which induce hemorrhagic fever or similar clinical signs as Ebolavirus.
Due to the different levels of mismatches between the probes we designed and the PCR amplicons we generated from the different Ebolavirus species (Fig 1), melting curve analysis of the PCRs products showed unique and distinct T m for each of the five Ebolavirus species (Fig 2): Zaire: flat dual peaks between 53.0°C and 56.9°C; Sudan: 51.6°C; Reston: flat dual peaks between 47.5°C and 54.9°C; Taï Forest: 52.8°C; Bundibugyo: dual peaks at 48.9°C and 53.5°C. The temperatures and shapes of the melting curves were consistent even with changes in the concentrations of Ebolavirus used in the reaction (Fig 2). Furthermore, the specific reverse primers we designed gave amplicons of different sizes for each of the five Ebolavirus species (Zaire ebolavirus 255bp; Sudan ebolavirus 211bp; Reston ebolavirus 192bp; Taï Forest ebolavirus 166bp; Bundibugyo ebolavirus 146bp) (Fig 4). The detection sensitivity, melting temperatures and amplicon sizes of each of these five Ebolavirus species were confirmed when the established RT-PCR was applied on Ebolavirus NP RNA-containing VLPs (Fig 6).
In the one-step RT-PCR established in this study, the combination of a single set of forward primer and probes with five different reverse primers gave very high sensitivity and differential ability. While the system detects all five Ebolavirus with highly sensitivity (1 copy of DNA, 10 copies of RNA, or 320 copies of RNA / 1 ml whole blood), the unique T m distribution enables the convenient differentiation of the five Ebolavirus species (Fig 2). In laboratories that do not have real-time PCR machines and melting curve analysis, our primers can be used in standard PCRs and the five Ebolavirus species differentiated by the specific size of the amplicon identified for each species in gel electrophoresis (Fig 4). The different concentrations of transcribed Zaire ebolavirus RNA (10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 /10μl) were detected with the one-step reverse transcription FRET-PCR described in this study, and the detection limitation was 10 copies per reaction system. B: Serial concentrations of transcribed Zaire ebolavirus RNAs (10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 /10μl) were spiked into human whole blood and were extracted with RNA purification kit, followed by the one-step reverse transcription FRET-PCR established in this study. The detection limitation was 100 RNA copies per reaction, equivalent to 320 copies of Zaire Ebolavirus in 1 ml whole blood. Triplicates (A) or duplicates (B) were performed for each concentration of tested Ebolavirus RNA.  While RNA templates as positive controls were not always available and safe for highly infective pathogens such as SARS virus and highly pathogenic avian influenza viruses, the VLPs equipped with specific RNA fragments have been widely used as stable, reliable and safe positive controls for RT-PCR [22][23][24]. The VLPs containing Ebolavirus NP RNA established in this work are adopted as standards to evaluate and certify Ebolavirus-related commercial diagnostic kits by National Institute for Food and Drug Control, Food and Drug Administration of China.
While simple PCR tends to provide high sensitivity but lacks ability to differentiate multiple targets simultaneously, multiplex PCR has the capacity to detect and differentiate multiple targets but tends to have a reduced sensitivity. Our study has established a novel highly-sensitive one-step RT FRET-PCR with one set of upstream primer /probes and five downstream primers that can rapidly identify and differentiate all five Ebolavirus species.