Draft Genome Sequences of Eight Crimean-Congo Hemorrhagic Fever Virus Strains

Koehler, Jeff W; Delp, Korey L.; Kearney, Brian; Conrad, Turner; Schoepp, Randal J.; Garrison, Aura R.; Altamura, Louis A.; Rossi, Cynthia A.; Minogue, Timothy D.

doi:10.1128/genomea.00240-17

Cited by 3 publications

(5 citation statements)

References 9 publications

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“…The resultant metagenomic diagnosis of Capnocytophaga canimorsus on a plasma specimen positive for Gram-negative rods beats the blood culture positive result by several hours and had no effect on treatment as the clinicians had already covered for infections associated with sepsis and dog bites. Perhaps a major takeaway from the story is how Food and Drug Administration (FDA) sanctioning of databases (in this case, a manufacturer's matrix-assisted laser desorption/ionization-time of flight [MALDI-TOF] database) affects the utility of less biased techniques [40]. A similar lack of actionability is seen in several of the other cases that have been reported to date [1,[41][42][43][44][45].…”

Section: Species Transmission Epidemiologymentioning

confidence: 99%

The challenge of diagnostic metagenomics

Greninger

2018

Expert Review of Molecular Diagnostics

116

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Diagnostic metagenomics and its associated trail of publications are spreading across the world. Multiple clinical labs in the United States, Europe, and Asia have gone to considerable lengths to optimize and validate a range of protocols for agnostically detecting viral, bacterial, fungal, and eukaryotic parasite nucleic acid across a range of patient specimens to aid in diagnosis for particularly recalcitrant cases. Others see a role for diagnostic metagenomics as a frontline diagnostic to replace other microbiological testing. Areas covered: There are considerable barriers to adoption for diagnostic metagenomics, including analytical sensitivity, interpretation, actionability, turnaround time, antimicrobial susceptibility, clinical utility, laboratory workflow, trial comparators, cost, and reimbursement. Expert commentary: Metagenomics is unlikely to become 'one test to rule them all' any time soon, not least because it is not indicative of historical infection like some of the highest volume tests in the clinical virology lab, viral serologies. The high cost and low marginal utility compared to 'standard of care' diagnostics have forced metagenomics to be mostly used for last-ditch cases. However, waiting for such patients to declare themselves as being diagnostically challenging in turn likely lessens the diagnostic yield and actionability of the information. Significant reductions in the cost of metagenomic sequencing are required for it to move up in the diagnostic pipeline. This review covers these associated obstacles of metagenomics, arguing for a parsimonious role in last-ditch diagnostics and awaiting the answer of many outstanding questions regarding its adoption.

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Section: Species Transmission Epidemiologymentioning

confidence: 99%

The challenge of diagnostic metagenomics

Greninger

2018

Expert Review of Molecular Diagnostics

116

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“…Since the development of the Garrison assay, 20 we ( Table 2) and others 15 identified decreased assay performance including nondetection of several CCHFV strains (JD-206, Drosdov, and DAK8194). To address this problem, we conducted deep sequencing of multiple 27 The S segment consensus sequences for these viruses were aligned to identify mismatches within the assay target region (Figure 1). Multiple nucleotide variants identified in the primer and probe region for each strain sequenced ( Figure 1) could have negatively impacted primer/probe binding.…”

Section: Resultsmentioning

confidence: 99%

“…For assay primer/probe design optimization, sequences from the UCC virus strains used in this study were sequenced and analyzed. 27 While S segments for some of these viruses have previously been sequenced 28 and there have been additional GenBank submissions by Lofts, Hodgson, and Smith, the specific viruses used in this study were sequenced to 1) characterize the virus stocks being used, 2) characterize strains not previously sequenced, and 3) meet the FDA-ARGOS reference genome standards (Bio-Project #PRJNA231221 27 The S segments from these sequenced viruses were aligned, and the assay target region was isolated for variant analysis and assay redesign. In addition, existing CCHFV S segment sequences from GenBank that covered the assay target region were aligned with the CLC Genomics Workbench (Supplemental Figure 1).…”

Section: Methodsmentioning

confidence: 99%

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

Koehler

Delp

Hall

et al. 2018

The American Journal of Tropical Medicine and Hygiene

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Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus 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.

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“…We attempted to amplify CCHFV segments L, M, and S from tick pool 159A using segment-specific primers modified for Nextera-based sequencing ( Table 2 ) ( 21 , 22 ); however, only the M segment amplified. In brief, we used 5 µL of extracted nucleic acid from tick homogenate to reverse transcribe the genome and amplify the cDNA of the M segment using the SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity DNA Polymerase (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…We analyzed sequencing reads with the CLC Genomics Workbench (QIAGEN), filtered and trimmed them for quality, and assembled the complete M segment de novo. We generated the final consensus sequence by remapping the trimmed reads to the de novo consensus sequence as previously described ( 22 ).…”

Section: Methodsmentioning

confidence: 99%

Crimean-Congo Hemorrhagic Fever Virus, Mongolia, 2013–2014

Voorhees¹,

Padilla²,

Jamsransuren³

et al. 2018

Emerg. Infect. Dis.

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During 2013–2014, we collected 1,926 serum samples from humans and 4,583 ticks (Hyalomma asiaticum or Dermacentor nuttalli) in select regions of Mongolia to determine the risk for Crimean-Congo hemorrhagic fever virus (CCHFV) infection among humans in this country. Testing of human serum samples by ELISA demonstrated an overall CCHFV antibody prevalence of 1.4%; Bayankhongor Province had the highest prevalence, 2.63%. We pooled and analyzed tick specimens by real-time reverse transcription PCR; 1 CCHFV-positive H. asiaticum tick pool from Ömnögovi was identified. In phylogenetic analyses, the virus’s partial small segment clustered with CCHFV isolates from Central Asia, and the complete medium segment grouped with CCHFV isolates from Africa, Asia, and the Middle East. This study confirms CCHFV endemicity in Mongolia and provides information on risk for CCHFV infection. Further research is needed to better define the risk for CCHFV disease to improve risk mitigation, diagnostics, and surveillance.

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Draft Genome Sequences of Eight Crimean-Congo Hemorrhagic Fever Virus Strains

Cited by 3 publications

References 9 publications

The challenge of diagnostic metagenomics

The challenge of diagnostic metagenomics

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

Crimean-Congo Hemorrhagic Fever Virus, Mongolia, 2013–2014

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