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Fluorescence-based assay for phenotypic characterization of human cytomegalovirus polymerase mutations regarding drug susceptibility and viral replicative fitness.

ABSTRACT: One essential prerequisite for genotypic drug susceptibility testing of human cytomegalovirus (HCMV) is the phenotypic characterization of mutations identified in the viral protein kinase gene UL97 and the viral DNA polymerase gene UL54 regarding their quantitative impact on drug susceptibility. We developed a new method for phenotypic characterization of UL54 mutations with regard to polymerase activity, viral replication, and drug susceptibility. To determine the most suitable viral indicator gene, enhanced green fluorescence protein was C-terminally fused to the HCMV early-late protein UL83 (pp65) or the late proteins UL32 (pp150) and UL99 (pp28), resulting in reporter viruses vTB65g, vTB150g, and vTB28g. vTB65g proved to be superior to the other constructs due to its favorable signal-to-noise ratio and was therefore used to establish the optimum conditions for our assay. The UL54 E756K and D413E mutations were introduced into vTB65g by markerless bacterial artificial chromosome mutagenesis, resulting in virus strains vE756Kg and vD413Eg. The drug susceptibility phenotypes of vE756Kg and vD413Eg were comparable to those previously reported. Furthermore, we found a reduced replicative fitness of vE756Kg by measuring fluorescence intensity as well as by conventional virus growth kinetics. Decreased fluorescence signals of vE756Kg- and vD413Eg-infected cells at late times of infection suggested a reduced polymerase activity, which was confirmed by real-time PCR quantification of the newly synthesized viral DNAs. This new fluorescence-based assay is a highly reproducible method for the phenotypic characterization of mutations potentially influencing drug susceptibility, viral replicative fitness, and polymerase activity of HCMV after marker transfer.

SUBMITTER: Chevillotte M

PROVIDER: S-EPMC2737853 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Fluorescence-based assay for phenotypic characterization of human cytomegalovirus polymerase mutations regarding drug susceptibility and viral replicative fitness.

Chevillotte Meike M Schubert Axel A Mertens Thomas T von Einem Jens J

Antimicrobial agents and chemotherapy 20090622 9

One essential prerequisite for genotypic drug susceptibility testing of human cytomegalovirus (HCMV) is the phenotypic characterization of mutations identified in the viral protein kinase gene UL97 and the viral DNA polymerase gene UL54 regarding their quantitative impact on drug susceptibility. We developed a new method for phenotypic characterization of UL54 mutations with regard to polymerase activity, viral replication, and drug susceptibility. To determine the most suitable viral indicator ...[more]

PMID: 19546362

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Project description:BackgroundCongenital cytomegalovirus (CMV) infection is an important cause of hearing loss, and most infants at risk for CMV-associated hearing loss are not identified early in life because of failure to test for the infection. The standard assay for newborn CMV screening is rapid culture performed on saliva specimens obtained at birth, but this assay cannot be automated. Two alternatives--real-time polymerase-chain-reaction (PCR)-based testing of a liquid-saliva or dried-saliva specimen obtained at birth--have been developed.MethodsIn our prospective, multicenter screening study of newborns, we compared real-time PCR assays of liquid-saliva and dried-saliva specimens with rapid culture of saliva specimens obtained at birth.ResultsA total of 177 of 34,989 infants (0.5%; 95% confidence interval [CI], 0.4 to 0.6) were positive for CMV, according to at least one of the three methods. Of 17,662 newborns screened with the use of the liquid-saliva PCR assay, 17,569 were negative for CMV, and the remaining 85 infants (0.5%; 95% CI, 0.4 to 0.6) had positive results on both culture and PCR assay. The sensitivity and specificity of the liquid-saliva PCR assay were 100% (95% CI, 95.8 to 100) and 99.9% (95% CI, 99.9 to 100), respectively, and the positive and negative predictive values were 91.4% (95% CI, 83.8 to 96.2) and 100% (95% CI, 99.9 to 100), respectively. Of 17,327 newborns screened by means of the dried-saliva PCR assay, 74 were positive for CMV, whereas 76 (0.4%; 95% CI, 0.3 to 0.5) were found to be CMV-positive on rapid culture. Sensitivity and specificity of the dried-saliva PCR assay were 97.4% (95% CI, 90.8 to 99.7) and 99.9% (95% CI, 99.9 to 100), respectively. The positive and negative predictive values were 90.2% (95% CI, 81.7 to 95.7) and 99.9% (95% CI, 99.9 to 100), respectively.ConclusionsReal-time PCR assays of both liquid- and dried-saliva specimens showed high sensitivity and specificity for detecting CMV infection and should be considered potential screening tools for CMV in newborns. (Funded by the National Institute on Deafness and Other Communication Disorders.).

Dataset Information

Fluorescence-based assay for phenotypic characterization of human cytomegalovirus polymerase mutations regarding drug susceptibility and viral replicative fitness.

Publications

Fluorescence-based assay for phenotypic characterization of human cytomegalovirus polymerase mutations regarding drug susceptibility and viral replicative fitness.

Similar Datasets

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