Ebola Virus RNA Research Articles

Emergence of Divergent Zaire Ebola Virus Strains in Democratic Republic of the Congo in 2007 and 2008

Zaire ebolavirus was responsible for 2 outbreaks in Democratic Republic of the Congo (DRC), in 1976 and 1995. The virus reemerged in DRC 12 years later, causing 2 successive outbreaks in the Luebo region, Kasai Occidental province, in 2007 and 2008. Viruses of each outbreak were isolated and the full-length genomes were characterized. Phylogenetic analysis was then undertaken to characterize the relationships with previously described viruses. The 2 Luebo viruses are nearly identical but are not related to lineage A viruses known in DRC or to descendants of the lineage B viruses encountered in the Gabon-Republic of the Congo area, with which they do, however, share a common ancestor. Our findings strongly suggest that the Luebo 2007 outbreak did not result from viral spread from previously identified foci but from an independent viral emergence. The previously identified epidemiological link with migratory bat species known to carry Zaire ebolavirus RNA support the hypothesis of viral spillover from this widely dispersed reservoir. The high level of similarity between the Luebo2007 and Luebo2008 viruses suggests that local wildlife populations (most likely bats) became infected and allowed local viral persistence and reemergence from year to year.

Cross-platform evaluation of commercial real-time reverse transcription PCR master mix kits using a quantitative 5′nuclease assay for Ebola virus

Selection of optimal reaction master mix reagents is essential to obtain the best performance with diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Every year the number of commercially available master mix kits increases, so it is prudent to periodically evaluate kits on the market. In this study we evaluated five commercial real-time RT-PCR master mix kits, the RealMasterMix RT-PCR ROX kit, the AgPath-ID One-Step RT-PCR kit, the SuperScript III Platinum One-step Quantitative RT-PCR system, the QuantiTect Probe RT-PCR kit, and the LightCycler RNA HybProbe amplification kit, using a 5′nuclease assay for Ebola virus. The kits were evaluated using the manufacturer’s recommended conditions, as well as conditions which have been used with the Ebola virus assay during outbreaks. When evaluated for use in Ebola virus RNA detection, the AgPath-ID kit resulted in the greatest sensitivity in comparison to the other four kits. The efficacy of the AgPath-ID kit was instrument-independent in the five real-time PCR platforms tested. This study demonstrated that Ebola virus RNA detection was not equivalent among the master mix reagents studied and, thus, that this variable can affect real-time RT-PCR assay sensitivity. Furthermore, this study rates the master mix reagents for their suitability, providing diagnostic laboratories the option to select from these kits to suit their specific laboratory needs for real-time RT-PCR.

Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study

SummaryBackgroundWe previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV) RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful for screening prospective countermeasures, they are frequently not useful for prediction of efficacy in the more stringent non-human primate models. We therefore assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever.MethodsA combination of modified siRNAs targeting the ZEBOV L polymerase (EK-1 mod), viral protein (VP) 24 (VP24-1160 mod), and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given these pooled anti-ZEBOV siRNAs (2 mg/kg per dose, bolus intravenous infusion) after 30 min, and on days 1, 3, and 5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after 30 min, and on days 1, 2, 3, 4, 5, and 6 after challenge with ZEBOV.FindingsTwo (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs were protected from lethal ZEBOV infection, whereas all macaques given seven postexposure treatments were protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that might have been related to viral infection.InterpretationThis complete postexposure protection against ZEBOV in non-human primates provides a model for the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an effective postexposure treatment strategy for people infected with Ebola virus, and suggest that this strategy might also be useful for treatment of other emerging viral infections.FundingDefense Threat Reduction Agency.

Are we any closer to combating Ebola infections?

Are we any closer to combating Ebola infections?

Analysis of human peripheral blood samples from fatal and nonfatal cases of Ebola (Sudan) hemorrhagic fever: cellular responses, virus load, and nitric oxide levels.

Peripheral blood samples obtained from patients during an outbreak of Ebola virus (Sudan species) disease in Uganda in 2000 were used to phenotype peripheral blood mononuclear cells (PBMC), quantitate gene expression, measure antigenemia, and determine nitric oxide levels. It was determined that as the severity of disease increased in infected patients, there was a corresponding increase in antigenemia and leukopenia. Blood smears revealed thrombocytopenia, a left shift in neutrophils (in some cases degenerating), and atypical lymphocytes. Infected patients who died had reduced numbers of T cells, CD8(+) T cells, and activated (HLA-DR(+)) CD8(+) T cells, while the opposite was noted for patients who survived the disease. Expression levels of cytokines, Fas antigen, and Fas ligand (TaqMan quantitation) in PBMC from infected patients were not significantly different from those in uninfected patients (treated in the same isolation wards), nor was there a significant increase in expression compared to healthy volunteers (United States). This unresponsive state of PBMC from infected patients despite high levels of circulating antigen and virus replication suggests that some form of immunosuppression had developed. Ebola virus RNA levels (virus load) in PBMC specimens were found to be much higher in infected patients who died than patients who survived the disease. Similarly, blood levels of nitric oxide were much higher in fatal cases (increasing with disease severity), and extremely elevated levels (>/=150 microM) would have negatively affected vascular tone and contributed to virus-induced shock.

Diagnosis of Ebola haemorrhagic fever by RT-PCR in an epidemic setting

This study reports the first field evaluation of a new diagnostic technique for Ebola virus disease with sensitivity and specificity. Ebola virus causes rare but fulminating outbreaks in Equatorial Africa. Rapid differentiation from other infections is critical for timely implementation of public health measures. Patients usually die before developing antibodies, necessitating rapid virus detection. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed, implemented and evaluated at Centre International de Recherches Médicales de Franceville (CIRMF) in Gabon, to detect Ebola viral RNA in peripheral blood mononuclear cells (PBMC). Twenty-six laboratory-confirmed patients during and 5 after the acute phase of Ebola haemorrhagic fever, 15 healthy controls and 20 febrile patients not infected with Ebola virus were studied. RT-PCR results were compared with ELISA antigen capture, and Ebola specific IgM and IgG antibody detection. Ebola virus RNA was amplified from 26/26 specimens from the acute phase, 3/5 during recovery, 0/20 febrile patients and 1/15 negative controls. Sensitivity of RT-PCR in identifying acute infection and early convalescence compared with antigen or IgM detection was 100% and 91% respectively, and specificity compared with antigen detection and IgM assay combined was 97%. Antigen capture detected only 83% of those identified by PCR, and IgM only 67%. Ebola virus RNA was detected in all 13 fatalities, only 5 of whom had IgM and none IgG. RT-PCR detected Ebola RNA in PBMC one to three weeks after disappearance of symptoms when antigen was undetectable. RT-PCR was the most sensitive method and able to detect virus from early acute disease throughout early recovery.