Rapid detection and quantification of RNA of Crimean-Congo Hemorrhagic Fever Virus by Real-Time Reversed Transcription-PCR

Bladh Linda (1), Mousavi-Jazi Mehrdad (1) ,Lundkvist Åke (1,2), Nilsson Mikael (1)

1. Kunskapscentrum för Mikrobiologisk Beredskap, Smittskyddsinstitutet
2. Mikrobiologiskt och Tumörbiologiskt Centrum, Karolinska Institutet

Viral hemorrhagic fevers (VHFs) refer to a group of diseases that are caused by several different families of viruses. The symptoms vary by the type of VHF, but initial signs and symptoms often include high fever, fatigue, myalgia and exhaustion. Patients with severe VHF often show signs of bleeding under the skin, in internal organs, or from body orifices like the mouth, eyes, or ears. Some viruses that cause hemorrhagic fever can spread from one person to another, once an initial person has become infected, e.g. Ebola, Marburg, Lassa and Crimean-Congo hemorrhagic fever viruses.

The ability to rapidly recognize VHF viruses is critical to limit further spread of the disease. A rapid, sensitive and specific laboratory diagnostic test is therefore necessary to confirm outbreaks and to distinguish VHF from other diseases that may present with similar initial clinical symptoms.

PCR has been successfully applied in the diagnosis of VHFs. However, most of the PCRs are relatively time-consuming as you have to run the PCR assay by assay for different pathogens and also include a post-PCR step with agarose gel analysis of the PCR product. In some instances it is also necessary to have a second round of nested amplification which may increase the risk of false positive results.

We have developed a method for detection of CCHF virus by real-time PCR with ABI PRISM 7900 sequence detection system and TaqMan chemistry. The method is based on sequence specific primers and a probe specific to the nucleocapsid gene of CCHF.

The method was tested and evaluated on clinical samples from Iran. Fifteen samples were analysed and the results were in agreement with previous results from the established method of reversed transcription PCR for CCHF.

We are currently developing real-time PCRs for detection of Marburg, Ebola and Lassa viruses. The aim is to establish sensitive and specific real-time PCR assays for all known viral hemorrhagic fever viruses.

Keynote speaker - Andersson S et al.: Phylogeny and Distribution of Vector-Borne Pathogens: What to Expect from Genomics?

Keynote speaker - Barbour A et al.: Interrupting transmission of Lyme borreliosis by targeting a reservoir for vaccination: a longitudinal study of a field site in North America

Keynote speaker - Broman T et al.: Campylobacter jejuni and wild birds

Keynote speaker - Broman T et al.: Natural reservoirs and vectors of Francisella tularensis in Sweden

Keynote speaker - Fouchier R: Influenza virus zoonoses

Keynote speaker - Fouchier R: A Novel Corona Virus Causing Severe Acute Respiratory Syndrome

Keynote speaker - Lundström J: Intercontinental dispersal and local adaptation of a mosquito-borne bird virus

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Bladh L et al.: Rapid detection and quantification of RNA of Crimean-Congo Hemorrhagic Fever Virus by Real-Time Reversed Transcription-PCR

Dahlgren D et al.: Survival of Campylobacter jejuni within Acanthamoeba polyphaga; a possible transmission route.

Ehrenborg C et al.: Genetic diversity over short geographic distances and no host specificity among Bartonella grahamii infecting woodland rodents of central Sweden

Ekerfelt C et al.: Involvement of cytolytic immune cells in human Lyme borreliosis - indication of intracellular persistance of the Borrelia spirochete?

Ekstrom J-O et al.: Characterization of the Ljungan virus

Haglund M: Characterization of human TBEV-strains from Sweden and a short review of the phylogenetic relationships within TBEV and Louping Ill.

Jarefors S et al.: Suppressed response to Borrelia-antigen in patients co-exposed to Borrelia burgdorferi and Anaplasma phagocytophila

Johansson M et al.: Development of molecular diagnostics for Orthopoxvirus

Mirazimi A et al.: Phatogenesis and the role of innate immunity in emerging patogen (Crimean Congo Heamorraghic fever virus)

Nejedla P et al.: A Six-year study of the presence of Borrelia burgdorferi s.l. in Ixodes ricinus ticks in the town park of Brno, Czech Republic.

Olsson G et al.: Hot zones in time and space on human hantavirus infections in northern sweden

Palo T et al.: Changing climate and emerging infectious diseases

Persson T et al.: Biological mosquito control in Sweden and risk assessment for non-target wetland insects

Schäfer M et al.: Biological diversity versus risk for mosquito nuisance and disease transmission in constructed wetlands

Shinikar S et al.: Genetic analysis of Crimean-Congo Hemorrhagic Fever virus in Iran

Skarphedinsson S et al.: Detection by PCR of Anaplasma in Danish Roe deer

Vostal K et al.: A Five-year Study of the Presence of Borrelia and Antibodies to Borrelia in Small Rodents.

Widhe M et al.: Borrelia specific IFN-γ and IL-4 secretion in CSF and blood during the course of Human Lyme Borreliosis: relation to clinical outcome

You E et al.: Host Immune Mechanisms in Recurrent Lyme Erythema Infection with Focus on the Cytokines Interleukin-4, Interferon-gamma and Interleukin-10

Zakovska A et al.: Spirochaetes and pathogenic Borrelia burgdorferi s.l. in mosquitoes (larvae and adults) in the Czech Republic