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Evaluation of Signature Erosion in Ebola Virus Due to Genomic Drift and Its Impact on the Performance of Diagnostic Assays.

Sozhamannan S, Holland MY, Hall AT, Negrón DA, Ivancich M, Koehler JW, Minogue TD, Campbell CE, Berger WJ, Christopher GW, Goodwin BG, Smith MA - Viruses (2015)

Bottom Line: Genome sequence analyses of the 2014 Ebola Virus (EBOV) isolates revealed a potential problem with the diagnostic assays currently in use; i.e., drifting genomic profiles of the virus may affect the sensitivity or even produce false-negative results.We evaluated signature erosion in ebolavirus molecular assays using an in silico approach and found frequent potential false-negative and false-positive results.The new signatures identified in this study may be evaluated with real-time reverse transcription PCR (rRT-PCR) assay development and validation.

View Article: PubMed Central - PubMed

Affiliation: Critical Reagents Program, Medical Countermeasure Systems Annex, 110 Thomas Johnson Drive, Frederick, MD 21702, USA. shanmuga.sozhamannan.ctr@mail.mil.

ABSTRACT
Genome sequence analyses of the 2014 Ebola Virus (EBOV) isolates revealed a potential problem with the diagnostic assays currently in use; i.e., drifting genomic profiles of the virus may affect the sensitivity or even produce false-negative results. We evaluated signature erosion in ebolavirus molecular assays using an in silico approach and found frequent potential false-negative and false-positive results. We further empirically evaluated many EBOV assays, under real time PCR conditions using EBOV Kikwit (1995) and Makona (2014) RNA templates. These results revealed differences in performance between assays but were comparable between the old and new EBOV templates. Using a whole genome approach and a novel algorithm, termed BioVelocity, we identified new signatures that are unique to each of EBOV, Sudan virus (SUDV), and Reston virus (RESTV). Interestingly, many of the current assay signatures do not fall within these regions, indicating a potential drawback in the past assay design strategies. The new signatures identified in this study may be evaluated with real-time reverse transcription PCR (rRT-PCR) assay development and validation. In addition, we discuss regulatory implications and timely availability to impact a rapidly evolving outbreak using existing but perhaps less than optimal assays versus redesign these assays for addressing genomic changes.

No MeSH data available.


Related in: MedlinePlus

Various ebolavirus-specific Conserved Sequence Domains (CSD) vs. Signature Sequence Domains (SSD) identified using BioVelocity. The bars are color coded according to the k-mer size.
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viruses-07-02763-f004: Various ebolavirus-specific Conserved Sequence Domains (CSD) vs. Signature Sequence Domains (SSD) identified using BioVelocity. The bars are color coded according to the k-mer size.

Mentions: Using this approach we discovered ten unique signatures, large enough to be used to develop PCR assays that are present and unique to EBOV at 100% conservation. The results of this analysis, i.e., number of features as a function of k-mer size are presented in Figure 4. The sequences of the best candidate BioVelocity (BV) signatures and their lengths are presented in Table S4 and their respective locations on the reference genome in Figure 1. In order to validate that these signatures are unique to EBOV, we used BLAST analysis to query these regions in all Ebolavirus sequences (Table S5) and a summary of the results is presented in the form of a heat map (Figure 5). None of the ten signatures are present at 100% identity in any of the other species. The identities varied from 46%–88% depending on the signature, target genes, and species, indicating that these are potentially useful EBOV specific PCR signatures. Interestingly, none of the current PCR assay targets tested by PSET overlapped with the BioVelocity signatures except two (EBOV_9 and EBOV_10) which partially overlapped the BV signatures in this region (Table S5, Figure 1).


Evaluation of Signature Erosion in Ebola Virus Due to Genomic Drift and Its Impact on the Performance of Diagnostic Assays.

Sozhamannan S, Holland MY, Hall AT, Negrón DA, Ivancich M, Koehler JW, Minogue TD, Campbell CE, Berger WJ, Christopher GW, Goodwin BG, Smith MA - Viruses (2015)

Various ebolavirus-specific Conserved Sequence Domains (CSD) vs. Signature Sequence Domains (SSD) identified using BioVelocity. The bars are color coded according to the k-mer size.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488730&req=5

viruses-07-02763-f004: Various ebolavirus-specific Conserved Sequence Domains (CSD) vs. Signature Sequence Domains (SSD) identified using BioVelocity. The bars are color coded according to the k-mer size.
Mentions: Using this approach we discovered ten unique signatures, large enough to be used to develop PCR assays that are present and unique to EBOV at 100% conservation. The results of this analysis, i.e., number of features as a function of k-mer size are presented in Figure 4. The sequences of the best candidate BioVelocity (BV) signatures and their lengths are presented in Table S4 and their respective locations on the reference genome in Figure 1. In order to validate that these signatures are unique to EBOV, we used BLAST analysis to query these regions in all Ebolavirus sequences (Table S5) and a summary of the results is presented in the form of a heat map (Figure 5). None of the ten signatures are present at 100% identity in any of the other species. The identities varied from 46%–88% depending on the signature, target genes, and species, indicating that these are potentially useful EBOV specific PCR signatures. Interestingly, none of the current PCR assay targets tested by PSET overlapped with the BioVelocity signatures except two (EBOV_9 and EBOV_10) which partially overlapped the BV signatures in this region (Table S5, Figure 1).

Bottom Line: Genome sequence analyses of the 2014 Ebola Virus (EBOV) isolates revealed a potential problem with the diagnostic assays currently in use; i.e., drifting genomic profiles of the virus may affect the sensitivity or even produce false-negative results.We evaluated signature erosion in ebolavirus molecular assays using an in silico approach and found frequent potential false-negative and false-positive results.The new signatures identified in this study may be evaluated with real-time reverse transcription PCR (rRT-PCR) assay development and validation.

View Article: PubMed Central - PubMed

Affiliation: Critical Reagents Program, Medical Countermeasure Systems Annex, 110 Thomas Johnson Drive, Frederick, MD 21702, USA. shanmuga.sozhamannan.ctr@mail.mil.

ABSTRACT
Genome sequence analyses of the 2014 Ebola Virus (EBOV) isolates revealed a potential problem with the diagnostic assays currently in use; i.e., drifting genomic profiles of the virus may affect the sensitivity or even produce false-negative results. We evaluated signature erosion in ebolavirus molecular assays using an in silico approach and found frequent potential false-negative and false-positive results. We further empirically evaluated many EBOV assays, under real time PCR conditions using EBOV Kikwit (1995) and Makona (2014) RNA templates. These results revealed differences in performance between assays but were comparable between the old and new EBOV templates. Using a whole genome approach and a novel algorithm, termed BioVelocity, we identified new signatures that are unique to each of EBOV, Sudan virus (SUDV), and Reston virus (RESTV). Interestingly, many of the current assay signatures do not fall within these regions, indicating a potential drawback in the past assay design strategies. The new signatures identified in this study may be evaluated with real-time reverse transcription PCR (rRT-PCR) assay development and validation. In addition, we discuss regulatory implications and timely availability to impact a rapidly evolving outbreak using existing but perhaps less than optimal assays versus redesign these assays for addressing genomic changes.

No MeSH data available.


Related in: MedlinePlus