| Autor: |
Koehler JW; Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland., Delp KL; Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland., Hall AT; Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland., Olschner SP; Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland., Kearney BJ; Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland., Garrison AR; Virology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland., Altamura LA; Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland., Rossi CA; Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland., Minogue TD; Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland. |
| Abstrakt: |
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus within the family Bunyaviridae. Infection can result in general myalgia, fever, and headache with some patients developing hemorrhagic fever with mortality rates ranging from 5% to 30%. CCHFV has a wide geographic range that includes Africa, Asia, the Middle East, and Europe with nucleotide sequence variation approaching 20% across the three negative-sense RNA genome segments. While phylogenetic clustering generally aligns with geographic origin of individual strains, distribution can be wide due to tick/CCHFV dispersion via migrating birds. This sequence diversity negatively impacts existing molecular diagnostic assays, leading to false negative diagnostic results. Here, we updated a previously developed CCHFV real-time reverse transcription polymerase chain reaction (RT-PCR) assay to include strains not detected using that original assay. Deep sequencing of eight different CCHFV strains, including three that were not detectable using the original assay, identified sequence variants within this assay target region. New primers and probe based on the sequencing results and newly deposited sequences in GenBank greatly improved assay sensitivity and inclusivity with the exception of the genetically diverse strain AP92. For example, we observed a four log improvement in IbAr10200 detection with a new limit of detection of 256 PFU/mL. Subsequent comparison of this assay to another commonly used CCHFV real-time RT-PCR assay targeting a different region of the viral genome showed improved detection, and both assays could be used to mitigate CCHFV diversity for diagnostics. Overall, this work demonstrated the importance of continued viral sequencing efforts for robust diagnostic assay development. |
