Sequence Optimized Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever virus (CCHFV; family Bunyaviridae, genus Nairovirus) infection of humans can result in a disease spectrum ranging from a nonspecific febrile illness to hemorrhagic fever manifestations with a mortality rate of 5%–30%.1 The relatively low rate of disease in seropositive populations has spurred research into potential host susceptibility factors,2–5 although the availability of appropriate supportive care may provide a more direct correlation with clinical outcomes. CCHFV is predominantly transmitted by ixodid ticks of the genus Hyalomma, and CCHFV has a wide geographic distribution with endemic foci in Eastern Europe, sub-Saharan and southern Africa, the Middle East, and Asia.6,7 Handling of tick-infested livestock and proximity to vegetated areas with high tick burdens are significant risk factors for CCHFV infection. In addition, nosocomial exposure to CCHFV-infected individuals in low resource facilities can result in severe disease among healthcare workers.1,7 CCHFV is an enveloped virus with a trisegmented, negative-sense RNA genome that encodes an RNA-dependent RNA polymerase (L), two major structural glycoproteins (GN and GC), and a nucleoprotein (N) on the L, M, and S genome segments, respectively. CCHFV has the largest genome of any bunyavirus at 19.1 kb total with 12.1, 5.4, and 1.6 kb in the three genome segments, respectively. To date, the National Institute of Allergy and Infectious Diseases (NIAID) Virus Pathogen Database and Analysis Resource (ViPR) lists complete genome sequences available for 54 (L), 75 (M), and 102 (S) genome segments of CCHFV strains.8 Pairwise alignments of these sequences indicate that their mean sequence identities are 89.4% (L), 80.0% (M), and 88.1% (S). Currently, there are no CCHFV vaccines or therapeutics approved for human use by the United States Food and Drug Administration, although immunoglobulin therapy and ribavirin have been used abroad with mixed results.9 In the absence of approved countermeasures, effective diagnostics remain an invaluable means to identify and control CCHFV outbreaks. A variety of assay platforms for CCHFV can detect viral nucleic acids to include low density macroarrays,10 high density resequencing arrays,11 padlock probes with colorimetric readout,12 loop-mediated isothermal amplification (LAMP),13 and polymerase chain reaction.14–18 Real-time reverse-transcription polymerase chain reaction (RT-PCR) remains the gold standard for quantitative, sensitive, and specific detection of CCHFV; however, these assays have sensitivity issues due to the genetic diversity of different CCHFV strains.19 Previously, Garrison et al.20 developed a TaqMan MGB real-time RT-PCR assay (Garrison assay) capable of detecting eighteen strains of CCHFV. Subsequent testing of this assay identified several additional strains which were undetectable by this assay.15 We suspected whether the inherent diversity of CCHFV genome contributed to inefficient primer/probe hybridization. To improve the assay performance, we sequenced these isolates and designed a set of degenerate primers and probes to take into account CCHFV diversity in the assay target region. This optimization increased assay sensitivity compared with the original Garrison assay and to a commonly used assay developed by Atkinson et al.15,21–26