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Ebola virus (EBOV) and Marburg virus (MARV) are enveloped, single-stranded, negative-sense RNA viruses classified into two genera, Ebolavirus and Marburgvirus, in the family Filoviridae, order Mononegavirales[6]. There is a single Marburgvirus species, Lake Victoria marburgvirus (LVMARV), whereas there are five known Ebolavirus species, Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Reston ebolavirus (REBOV), Cote d'Ivoire ebolavirus (CIEBOV) and Bundibugyo (BEBOV) with ZEBOV regarded as the most virulent in humans[23]. The genomic structures of filoviruses are very similar and the genome is approximately 19 kb in length, containing seven genes arranged sequentially in the order nucleoprotein (NP)-viral protein (VP) 35-VP40-glycoprotein-VP30-VP24-RNA polymerase (L)[19].
These viruses are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates with unknown treatments[8]. Since the discovery of Marburg hemorrhagic fever in Germany in 1967, sporadic outbreaks of Marburg and Ebola hemorrhagic fever have been reported from different countries in Central Africa[8]. Incidences have increased in Central Africa since the beginning of the new millennium[3, 4]. Furthermore, two imported cases of Marburg hemorrhagic fever (in the Netherlands and the United States)[2, 20] and one of Ebola hemorrhagic fever (in South Africa)[25] in travelers, have been reported, emphasizing the risk of filovirus infection in non-endemic countries.
Due to its potential for adverse public health impact with mass casualties, EBOV and MARV have been classified as Category-A critical biological agents[17]. These disease-causing hemorrhagic fever RNA viruses have the potential to be used as bioterrorism agents, highlighting the need for their accurate and timely identification. Previous diagnostic methods for EBOV include antigen-capture ELISA testing of serum and blood samples, followed by nested reverse-transcription PCR (RT-PCR) amplification of RNA from the captured virions; however, these methods have limited throughput capacity and need up to a day to acquire the results [7, 22]. Several groups have developed real-time RT-PCR assays that have high sensitivity as well as high throughput capacity[5, 11, 22, 24]. As rapid real-time PCR assays have become routine in diagnostic laboratories, there has been an ever-increasing number of commercially available reagent systems and detection platforms to support these methods.
In China, increasing personal exchange with other counties where sporadic outbreaks have been recorded raises the concerns of potential outbreaks of exotic viruses, however, no reliable diagnostic method has been established for the surveillance of such viruses. In this study, the TaqMan-based, real-time reverse transcription-polymerase chain reaction (RT-PCR) assays targeting the nucleoprotein (NP) genes of ZEBOV and MARV were developed and the sensitivities and specificities were studied. The results indicate that the assays can rapidly detect and identify the gene targets of the genomes of these two filoviruses, which suggests the potential use as a standard detection method in the diagnostics and epidemiological studies of ZEBOV and MARV infections, and may play a role in the development of vaccines and antiviral drugs of these viruses.
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Laboratory procedures for the propagation of EBOV and MARV were performed in the Laboratoire P4 Jean Mérieux in Lyon with requisite containment and safety precautions.
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Virus stock was prepared in the BSL-4 laboratory by infecting Vero cells with a multiplicity of infection (MOI) of 0.01 plaque forming units (pfu)/cell and virus was recovered 5 days post-infection. Supernatants from mock and virus infected cells were transferred into Eppendorf tubes, clarified by centrifugation at 2000 rpm for 5 min and split into fresh tubes. One was treated for RNA extraction and quantification and a second one used for virus titration.
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Viruses were titrated by plaque assay on Vero cells. Briefly, six-well plates containing Vero cells were incubated for 1 h at 37 ℃ in a 5 % CO2 incubator with 1 mL of serial 1:10 dilutions of virus. Cells were washed twice with Dulbeco's minimum essential medium (DMEM) without fetal calf serum (FCS) and covered with 2 mL of 1.6 % carboxymethylcellulose in DMEM containing 5 % FCS. After incubation for 4-5 days at 37 ℃ in 5 % CO2, the medium was removed, and the cells were washed with PBS and fixed with 10 % buffered-PBS formalin. To detect viral antigen, cells were incubated with virus-specific rabbit sera, followed by addition of HRP-conjugated anti-rabbit goat IgG antibody (SIGMA). Plaques were counted and the titer expressed as pfu/mL after DAB (3, 3'-iaminobenzidine, tetrahydrochloride) incubation (SIGMA).
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Viral RNA was extracted from 140 μL of supernatant from virus-infected Vero cells using the RNA extraction kit (Qiagen Inc., USA) following the manufacturer's instructions. The extracts were resuspended in 60 μL of AVE Buffer, aliquoted and stored at -70 ℃ before RT-PCR amplification was carried out.
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The ZEBOV and MARV in vitro RNA transcripts were synthesized using the T7 RiboMAXTM expression large-scale RNA production system (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, following RT-PCR amplification of the partial NP gene (primers are listed in Table 1), the products underwent phenol: chloroform extraction and ethanol precipitation[18], and the resulting DNA pellets were resuspended in 30 μL of Tris-EDTA buffer. The in vitro transcription reactions were carried out for 30 min at 37 ℃ with 2 μg DNA. Two units of RQ1 RNase-free DNase (Promega) were then added and incubation was continued for a further 60 min. The preparations were incubated for 15 min at 70 ℃ to inactivate the DNase. The transcripts were extracted using an RNeasy mini kit (Qiagen, Hilden, Germany), and resuspended in 50 μL of DEPC-treated water. The concentration of RNA transcript was determined by measuring the optical density (OD) at 260 nm. RNA transcripts were stored at -70 ℃ for further use.
Table 1. Primers and probes used in the study.
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The primers and probes, which were used in these 2 assays for targeting the NP genes of ZEBOV and MARV, are listed in Table 1. The conserved fragments of the NP genes were chosen after alignment using ClustalW[15] and the primers and probes were designed using the Primer Premier software package version 5.0 (Fig. 1).
Figure 1. Alignment of ZEBOV and MARV NP gene sequences and TaqMan RT-PCR primer and probe sequences used in this study. Dots indicate the positions identical to ZEBOV strain, Mayinga (NC 002549) or MARV strain Musoke (NC_001608) sequences. The numbers indicate the respective nucleotide positions in the Mayinga and Musoke strain genome sequences. Genbank accession numbers of the nucleotide sequences used in this study are NC 002549 (Mayinga), AY354458 (Kikwit), AF272001, AF499101, AY142960, EU224440, AF054908, J04337, Y09358, L11365 and NC 001608 (Musoke), AY358025 (Ozolin), DQ447656 (Angola), EF446131 (Ravn), EF446132 (Ci67), Z29337 (Popp).
Quantitative RT-PCR assays were performed using the DNA Engine Opticon 1 system and a Roche LightCycle 2.0 Instrument. The one-step RT-PCR system (QIAGEN one-step RT-PCR Kit, Qiagen, Hilden, Germany) was used for uninterrupted thermal cycling. A master mix reaction was prepared and dispensed in 22-μL aliquots into accompanying thin-walled microAmp optical tubes. Then 1-3 μL of RNA transcript or RNA extract from stock virus or infected cell supernatants was added to each tube. The final reaction mixture contained 400 nmol/L of each primer and 100 nmol/L of the probe. Prior to amplification the RNA was reverse transcribed at 50 ℃ for 30 min. This was followed by one cycle of denaturation at 94 ℃ for 5 min. Next, PCR amplification was carried out for 45 cycles at 94 ℃ for 15 s and 60 ℃ for 1 min. The fluorescence was read at the end of this second step allowing a continuous monitoring of the amount of RNA. Amplification products were later confirmed by sequencing.