Improved Detection of Emerging Drug - resistant Mutant Cytomegalovirus 1 Subpopulations by Deep Sequencing

17 In immunosuppressed hosts, development of multi-drug resistance complicates the treatment of 18 cytomegalovirus (CMV) infection. Improved genotypic detection of impending drug resistance 19 may follow from recent technical advances. A severely T-cell depleted patient with chronic 20 lymphocytic leukemia developed CMV pneumonia and high plasma viral loads poorly 21 responsive to antiviral therapy. Serial plasma specimens were analyzed for mutant viral 22 populations by conventional and high throughput deep sequencing methods. Uncharacterized 23 mutations were phenotyped for drug resistance using recombinant viruses. Conventional 24 genotyping detected viral UL97 kinase substitution C607Y after ganciclovir treatment, a 25 transient subpopulation of UL54 polymerase L773V first detected 8 weeks after starting 26 foscarnet, and a subpopulation of UL54 codon 981-2 deletion 2 months after adding cidofovir. 27 Deep sequencing of the same serial specimens revealed the same UL54 mutants sooner, along 28 with a more complex evolution of known and newly recognized mutant subpopulations missed 29 by conventional sequencing. UL54 exonuclease substitutions D413N, K513R and C539G were 30 newly shown to confer ganciclovir-cidofovir resistance, while L773V was shown to confer 31 foscarnet resistance and add to the ganciclovir resistance conferred by UL97 C607Y. Increased 32 sequencing depth provided a more timely and detailed diagnosis of mutant viral subpopulations 33 that evolved with changing anti-CMV therapy. 34 35 36 on D ecem er 1, 2017 by gest httpaac.asm .rg/ D ow nladed fom