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Background: Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus family Bunyaviridae, which are enveloped viruses containing tripartite, negative polarity, single-stranded RNA. CCHF is characterized by high case mortality, occurring in Asia, Africa, the Middle East and Europe. Currently, there are no specific treatments or licensed vaccines available for CCHFV. Recently, two research groups have found adult mice with defective interferon responses allowed to lethal CCHFV infection. These mouse models could provide invaluable information for further studies. Efforts to develop a vaccine against CCHFV are being made. To determine the efficacy of vaccine candidates it is important to conduct serological studies that can accurately measure levels of protective antibodies. In the present study, a pseudo-plaque reduction neutralization test (PPRNT) based on enzyme-catalyzed color development of infected cells probed with anti-CCHFV antibodies was used to measure neutralization antibody of CCHFV., Methods: Sixty-nine human serum samples (20 acute and 49 convalescent) were tested. The presence of CCHFV antibodies was determined and confirmed by a commercial ELISA kit. CCHFV RNA was determined by RT-PCR. All the samples were analyzed by PPRNT and fluorescent focus reduction neutralization test (FFRNT) to measure of CCHFV-neutralizing antibodies., Results: Pseudo-plaque reduction neutralization test showed a high sensitivity (98%), specificity (100%) and agreement (96,6%) in qualitative comparison with those of the FFRNT. There was a high correlation between the titers obtained in PPRNT and FFRNT (R2 = 0.92). The inter- and intra-assay variation of PPRNT revealed good reproducibility and positive cut-off of PPRNT was defined as 1:4 by the geometric mean titers for the individual samples distributed., Conclusion: The pseudo-plaque reduction neutralization test described in this study is a fast, reproducible and sensitive method for the measurement of CCHF neutralizing antibodies. This novel assay could serve as useful tools for CCHF research in epidemiology, vaccine development and other studies of immunity. It also provides an alternative to PRNT when viruses with no or poor CPE in cell culture.

A pseudo-plaque assay was developed for detection and quantitation of Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06. Enzyme-catalyzed color development of infected cells probed with anti-Crimean-Congo hemorrhagic fever virus antibodies was used for determining the titer of Crimean-Congo hemorrhagic fever Turkey-Kelkit06 and for its detection in samples from persons infected with the Crimean-Congo hemorrhagic fever virus. The pseudo-plaque assay accuracy was confirmed by comparing pseudo-plaque assay titers with fluorescent immunofocus assay and focus formation assay titers using three stocks of virus. No significant difference in virus titers of Crimean-Congo hemorrhagic fever Turkey-Kelkit06 among the three methods was observed. The pseudo-plaque assay is more sensitive than the fluorescent immunofocus assay for detecting the virus in primary isolates of Crimean-Congo hemorrhagic fever virus collected from humans, but no difference in sensitivity between the two methods was observed in the cell-adapted strain of Crimean-Congo hemorrhagic fever Turkey-Kelkit06. The pseudo-plaque assay is suitable for titration of Crimean-Congo hemorrhagic fever Turkey-Kelkit06, which does not develop plaques, suggesting it may also be suitable for the detection of other viruses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis which is common in Africa, Asia, Eastern Europe, and the Balkan Peninsula. CCHF has been reported in Turkey with high frequency since 2002. The aim of the present study was to investigate the genetic diversity and genetic relationship between CCHF virus (CCHFV) isolates derived from infected patients over a 2-year period (2009 and 2010) in several provinces of Turkey. Serum samples (n = 48) were selected from CCHFV RNA positive patients and subjected to sequence analysis of the gene regions encoding the S (48 samples) and M (14 samples) segments. The nucleotide sequence alignments showed that the nucleic acid relatedness of CCHFV isolates ranged from 95.7% to 100% and from 93.7% to 100% for S and M segments, respectively. Phylogenetic analysis of both segment sequences revealed that CCHFV isolates circulating in Turkey belonged to the European lineage I and were closely related to the viruses found in the Eastern European-Russian and Balkan Peninsula. The M gene segment-based phylogenetic analysis suggested that 2/14 CCHFV isolates (KYSR3159/09 and YZGT714/10) had additional genetic variations. The results of the present study confirmed that the CCHFV isolates present in Turkey associated with human disease had high genetic homology in S segment, but some variability in the M segment of the RNA., (Copyright © 2012 Wiley Periodicals, Inc.)

The goal of this study was to investigate the molecular epidemiology of Crimean-Congo hemorrhagic fever virus (CCHFV) in Turkey. The study was performed on a total of 48 confirmed human CCHF cases from 2006 to 2008. The majority of the CCHF viral strains in Turkey were found to belong to the European lineage. Local CCHF viral strains are grouped into two main clusters, which can be further divided into two sub-groups. We also identified an AP92-like virus causing clinical disease in Corum (a mid-Anatolian province). Phylogenetic analysis revealed that the most recent CCHFV infections were caused by intrinsic (or native) CCHF viral strains, which we identified as the local topotype. Comparison of deduced amino acid sequences of S-segment RNAs indicated that the local topotype was derived from viruses of previous years, most likely by a low rate recombination. No genetic differences, based on S- and M-segment RNA sequences, were found between human and tick viral isolates. This data suggest that replication of CCHFV in the tick vector, whether Rhiphicephalus spp. or Hyalomma spp., has no effect on the viral genomic structure., (Copyright 2010 Elsevier B.V. All rights reserved.)

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of a tick-borne disease with high mortality rates in humans. The distribution of CCHFV includes over 30 countries in Asia, the Middle East, southeastern Europe, and Africa. It was first recognized in Turkey in 2002, with an increasing number of cases reported between 2002 and 2009. Recent analysis of complete genome sequences of CCHFV isolates has revealed that the genomic plasticity of the virus is surprisingly high for an arthropod-borne virus. We have determined the complete nucleotide and deduced amino acid sequences of strain CCHFV Turkey-Kelkit06 isolated from the blood of a patient in an endemic region of Turkey in 2006. The complete sequence length of the CCHFV Turkey-Kelkit06 strain is 19,186 nt, consisting of a 1673 nt S segment, a 5364 nt M segment, and a 12,149 nt L segment. Based on the analysis of S, M, and L segments, CCHFV Turkey-Kelkit06 clustered in Group V, which represents the Europe/Turkey geographic lineage. Although glycoproteins encoded by the M gene are the most variable part of the CCHFV Turkey-Kelkit06 strain, some functional domains of the glycoproteins are well conserved. Here, we report the complete sequence and genome organization of the CCHFV Turkey-Kelkit06 strain and its phylogenetic relationship to other strains of CCHFV. Collecting data on viral sequences among isolates from CCHF epidemics may provide valuable information regarding the molecular basis of the epidemic potential of the virus., ((c) 2009 Elsevier B.V. All rights reserved.)