| 1. |
- Andersson, Cecilia, et al.
(författare)
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In situ rolling circle amplification detection of Crimean Congo hemorrhagic fever virus (CCHFV) complementary and viral RNA
- 2012
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Ingår i: Virology. - : Elsevier. - 0042-6822 .- 1096-0341. ; 426:2, s. 87-92
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Tidskriftsartikel (refereegranskat)abstract
- Crimean Congo hemorrhagic fever virus (CCHFV) is a human pathogen that causes a severe disease with high fatality rate for which there is currently no specific treatment. Knowledge regarding its replication cycle is also highly limited. In this study we developed an in situ technique for studying the different stages during the replication of CCHFV. By integrating reverse transcription, padlock probes, and rolling circle amplification, we were able to detect and differentiate between viral RNA (vRNA) and complementary RNA (cRNA) molecules, and to detect viral protein within the same cell. These data demonstrate that CCHFV nucleocapsid protein (NP) is detectable already at 6 hours post infection in vRNA- and cRNA-positive cells. Confocal microscopy showed that cRNA is enriched and co-localized to a large extent with NP in the perinuclear area, while vRNA has a more random distribution in the cytoplasm with only some co-localize with NP. However, vRNA and cRNA did not appear to co-localize directly.
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| 2. |
- Jaaskelainen, Anne J., et al.
(författare)
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Development and Evaluation of a Real-Time RT-qPCR for Detection of Crimean-Congo Hemorrhagic Fever Virus Representing Different Genotypes
- 2014
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Ingår i: Vector Borne and Zoonotic Diseases. - : Mary Ann Liebert. - 1530-3667 .- 1557-7759. ; 14:12, s. 870-872
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Tidskriftsartikel (refereegranskat)abstract
- Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease caused by a nairovirus belonging to family Bunyaviridae. The CCHF virus (CCHFV) can be transmitted to humans by Hyalomma ticks as well as by direct contact with infected body fluids or tissues from viremic livestock or humans. Our aim was to set up a fast RT-qPCR for detection of the different CCHFV genotypes in clinical samples, including an inactivation step to make the sample handling possible in lower biosafety levels (BSL) than BSL-4. This method was evaluated against commercial reference assays and international External Quality Assessment (EQA) samples. The analytical limit of detection for the developed CCHFV-S RT-qPCR was 11 CCHFV genomes per reaction. After exclusion of four dubious samples, we studied 38 CCHFV-positive samples (using reference tests) of which 38 were found positive by CCHFV-S RT-qPCR, suggesting a sensitivity of 100%. CCHFV-S RT q-PCR detected all eight different CCHFV strains representing five different CCHFV genotypes. In conclusion, the CCHFV-S RT-qPCR described in this study was evaluated using various sources of CCHFV samples and shown to be an accurate tool to detect human CCHFV infection caused by different genotypes of the virus.
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| 3. |
- Ke, Rongqin, et al.
(författare)
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Colorimetric Nucleic Acid Testing Assay for RNA Virus Detection Based on Circle-to-Circle Amplification of Padlock Probes
- 2011
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 49:12, s. 4279-4285
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Tidskriftsartikel (refereegranskat)abstract
- We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 103 viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing different viral strain variants could be detected. We analyzed 34 CCHF patient samples, and all patients were correctly diagnosed when the results were compared to those of the current real-time PCR method. This is the first time that highly specific padlock probes have been applied to detection of a highly variable target sequence typical of RNA viruses.
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| 4. |
- Mousavi-Jazi, Mehrdad, et al.
(författare)
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Healthy individuals immune response to the Bulgarian Crimean-Congo hemorrhagic fever virus vaccine
- 2012
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Ingår i: Vaccine. - : Elsevier. - 0264-410X .- 1873-2518. ; 30:44, s. 6225-6229
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Tidskriftsartikel (refereegranskat)abstract
- Crimean-Congo hemorrhagic fever virus (CCHFV) poses a great threat to public health due to its high mortality and transmission rate and wide geographical distribution. There is currently no specific antiviral therapy for CCHF. This study provides the first in-depth analysis of the cellular and humoral immune response in healthy individuals following injection of inactivated Bulgarian vaccine, the only CCHFV vaccine available at present. Vaccinated individuals developed robust, anti-CCHFV-specific T-cell activity as measured by IFN-gamma ELISpot assay. The frequency of IFN-gamma secreting T-cells was 10-fold higher in individuals after vaccination with four doses than after one single dose. High levels of CCHFV antibodies were observed following the first dose, but repeated doses were required to achieve antibodies with neutralizing activity against CCHFV. However, the neutralizing activity in these groups was low.
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| 5. |
- Papa, Anna, et al.
(författare)
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Crimean-Congo Hemorrhagic Fever Virus, Greece
- 2014
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Ingår i: Emerging Infectious Diseases. - : U.S. National Center for Infectious Diseases. - 1080-6040 .- 1080-6059. ; 20:2, s. 288-290
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Tidskriftsartikel (refereegranskat)abstract
- Seroprevalence of Crimean-Congo hemorrhagic fever virus (CCHFV) is high in some regions of Greece, but only 1 case of disease has been reported. We used 4 methods to test 118 serum samples that were positive for CCHFV IgG by commercial ELISA and confirmed the positive results. A nonpathogenic or low-pathogenicity strain may be circulating.
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| 6. |
- Rosenstierne, Maiken W., et al.
(författare)
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The Microbial Detection Array for Detection of Emerging Viruses in Clinical Samples - A Useful Panmicrobial Diagnostic Tool
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 9:6, s. e0100813-
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Tidskriftsartikel (refereegranskat)abstract
- Emerging viruses are usually endemic to tropical and sub-tropical regions of the world, but increased global travel, climate change and changes in lifestyle are believed to contribute to the spread of these viruses into new regions. Many of these viruses cause similar disease symptoms as other emerging viruses or common infections, making these unexpected pathogens difficult to diagnose. Broad-spectrum pathogen detection microarrays containing probes for all sequenced viruses and bacteria can provide rapid identification of viruses, guiding decisions about treatment and appropriate case management. We report a modified Whole Transcriptome Amplification (WTA) method that increases unbiased amplification, particular of RNA viruses. Using this modified WTA method, we tested the specificity and sensitivity of the Lawrence Livermore Microbial Detection Array (LLMDA) against a wide range of emerging viruses present in both non-clinical and clinical samples using two different microarray data analysis methods.
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| 7. |
- Wang, Yi, et al.
(författare)
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Structure of Crimean-Congo Hemorrhagic Fever Virus Nucleoprotein: Superhelical Homo-Oligomers and the Role of Caspase-3 Cleavage
- 2012
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Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 86:22, s. 12294-12303
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Tidskriftsartikel (refereegranskat)abstract
- Crimean-Congo hemorrhagic fever, a severe hemorrhagic disease found throughout Africa, Europe, and Asia, is caused by the tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is a negative-sense single-stranded RNA (ssRNA) virus belonging to the Nairovirus genus of the Bunyaviridae family. Its genome of three single-stranded RNA segments is encapsidated by the nucleocapsid protein (CCHFV N) to form the ribonucleoprotein complex. This ribonucleoprotein complex is required during replication and transcription of the viral genomic RNA. Here, we present the crystal structures of the CCHFV N in two distinct forms, an oligomeric form comprised of double antiparallel superhelices and a monomeric form. The head-to-tail interaction of the stalk region of one CCHFV N subunit with the base of the globular body of the adjacent subunit stabilizes the helical organization of the oligomeric form of CCHFV N. It also masks the conserved caspase-3 cleavage site present at the tip of the stalk region from host cell caspase-3 interaction and cleavage. By incubation with primer-length ssRNAs, we also obtained the crystal structure of CCHFV N in its monomeric form, which is similar to a recently published structure. The conformational change of CCHFV N upon deoligomerization results in the exposure of the caspase-3 cleavage site and subjects CCHFV N to caspase-3 cleavage. Mutations of this cleavage site inhibit cleavage by caspase-3 and result in enhanced viral polymerase activity. Thus, cleavage of CCHFV N by host cell caspase-3 appears to be crucial for controlling viral RNA synthesis and represents an important host defense mechanism against CCHFV infection.
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