Analysis of HHV-6 mutations in solid organ transplant recipients at the onset of cytomegalovirus disease and following treatment with intravenous ganciclovir or oral valganciclovir

Journal of Clinical Virology 58 (2013) 279–282

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Analysis of HHV-6 mutations in solid organ transplant recipients at the onset of cytomegalovirus disease and following treatment with intravenous ganciclovir or oral valganciclovir Lotfi Bounaadja, Jocelyne Piret, Nathalie Goyette, Guy Boivin ∗ Infectious Diseases Research Center of the CHU of Québec, and Laval University, Quebec City, Quebec, Canada G1V 4G2

a r t i c l e

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Article history: Received 12 April 2013 Received in revised form 6 June 2013 Accepted 17 June 2013 Keywords: HHV-6 HCMV Co-infections Antivirals Drug resistance mutations

a b s t r a c t Background: Human herpesvirus 6 (HHV-6) and human cytomegalovirus (HCMV) are major opportunistic pathogens in solid organ transplant (SOT) recipients. The use of antivirals for the treatment of HCMV disease can result in the development of drug resistance mutations in HCMV and also potentially in HHV-6. Objectives: The emergence of HHV-6 drug resistance mutations was evaluated in SOT recipients at the onset of HCMV disease and following treatment with ganciclovir (GCV) or valganciclovir (VGCV). Study design: Detection of HHV-6 was performed by real-time PCR from whole blood samples serially obtained from SOT recipients treated for HCMV disease with an induction dose of intravenous GCV or oral VGCV for 21 days followed by VGCV maintenance for 28 days in both arms. Baseline and last positive HHV-6 samples were tested for mutations in the genes encoding the protein kinase (U69) and the DNA polymerase (U38). Results: The rate of HHV-6 viraemia among SOT patients with HCMV disease at baseline was 3.2% (5/155). All isolates belonged to the HHV-6B species. Mutations L213I and Y479H were detected at baseline and at later times in the U69 kinase. Mutation L213I was previously reported as polymorphism whereas the role of mutation Y479H in drug resistance is unknown. Mutations D854E and E855Q found in the DNA polymerase were known as natural variants. Conclusions: The incidence of HHV-6 viraemia in SOT recipients with established HCMV disease before initiation of antiviral therapy was low. Treatment with GCV or VGCV did not induce the emergence of HHV-6 drug resistance mutations. © 2013 Elsevier B.V. All rights reserved.

1. Background Human herpesviruses 6 (HHV-6) A and B are now classified as two genetically distinct species [1]. These viruses along with human cytomegalovirus (HCMV) are common opportunistic pathogens in immunocompromised patients such as solid organ transplant (SOT) recipients [2] and co-infections have been described in this population [3–6]. In the latter setting, clinical manifestations of HHV-6 reactivation or reinfection include fever and/or rash and, less frequently, encephalitis, hepatitis, pneumonitis, bone marrow suppression and graft dysfunction or rejection [7]. The nucleoside analogue ganciclovir (GCV) or its prodrug valganciclovir (VGCV) are generally used for the prevention and treatment of HCMV disease.

Ganciclovir possesses in vitro activity against HHV-6 but its clinical efficacy is only described in case reports [8]. GCV is first phosphorylated by the viral protein kinase encoded by UL97 (HCMV) [9,10] or U69 (HHV-6) [11] gene and then by cellular kinases to be converted into its active triphosphate form which inhibit the viral DNA polymerase encoded by UL54 (HCMV) or U38 (HHV-6) gene [12]. HHV-6 could be exposed to GCV following prolonged prophylaxis/treatment of HCMV disease in immunocompromised patients with the potential development of drug resistance mutations. HCMV resistance to GCV is well-described and resistance mutations emerge generally in the UL97 and more rarely in the UL54 genes [13]. To date, HHV-6 resistance to GCV has not been extensively studied. 2. Objectives

Abbreviations: HHV-6, human herpesvirus 6; HCMV, human cytomegalovirus; SOT, solid organ transplant; GCV, ganciclovir; VGCV, valganciclovir; ND, not determined; NT, not tested. ∗ Corresponding author. Tel.: +1 418 654 2705; fax: +1 418 654 2715. E-mail address: [email protected] (G. Boivin). 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.06.024

The emergence of HHV-6 drug resistance mutations was evaluated in SOT recipients at the onset of HCMV disease and following treatment with intravenous GCV or oral VGCV.

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L. Bounaadja et al. / Journal of Clinical Virology 58 (2013) 279–282

Table 1 Primers used for the amplification and sequencing of U69 and U38 genes of both HHV-6 A and B species. Target

Primers

Orientation

Locationa

Use



Sequences 

U69

U69Fd U69Rv

Forward Reverse

5 -GATTAGATTAGGGGAGAGACATCC-3 5 -CAAAAACTGCTGCATTTCCTC-3

104723–104746 106902–106922

First PCR, sequencing

U69

U69Fd GCVKB2b

Forward Reverse

5 -GATTAGATTAGGGGAGAGACATCC-3 5 -CCGAGAACTCGAGCCATAG-3

104723–104746 105588–105606

Nested 1, sequencing

U69

GCVKA1b U69Rvb

Forward Reverse

5 -ATGAAACTGTCGAATGCC-3 5 -GCGTTATATTTTATTAGCATGCAG-3

105320–105337 106841–106864

Nested 2, sequencing

U38

PolBlb PolAb

Forward Reverse

5 -CATGTACAAACGACAAAACC-3 5 -TAGACAGGATCAGGTATAGAGG-3

57475–57494 60573–60594

First PCR, sequencing

U38

PolB2b PolAb

Forward Reverse

5 -CTATAACACAGATTATGCGCC-3 5 -TAGACAGGATCAGGTATAGAGG-3

58777–58797 60573–60594

Nested 1, sequencing

U38

PolB1b PolA2b

Forward Reverse

5 -CGCAAGAAGTCTTTCTCACC-3 5 -GACGGCGTGCATATATATG-3

58091–58110 59534–59552

Nested 2, sequencing

U38

PolBlb PolA3b

Forward Reverse

5 -CATGTACAAACGACAAAACC-3 5 -GGGATAGGTTACAAAGGGGC-3

57475–57494 58882–58901

Nested 3, sequencing

a The location of the primers refers to the nucleotide sequence of HHV-6B strain HST. The specificity of primers for HHV-6 A and B species was confirmed by amplifying HHV-6A strain GS and HHV-6B strain HST prototypes. b Manichanh et al. [25].

3. Study design Clinical specimens were obtained in a randomized open-label multicenter trial (the Victor study) [14,15] in adult SOT recipients with tissue-invasive HCMV disease or syndrome defined according to standard criteria [16]. Patients were randomized to

treatment with intravenous GCV 5 mg/kg twice daily or with oral VGCV 900 mg twice daily (both drugs adjusted for renal function) for an induction period of 21 days, followed by oral VGCV 900 mg once daily for 28 days in both arms. Patients with detectable HCMV viraemia (>200 copies/ml of plasma), previously evaluated for cytomegalovirus resistance mutations to GCV [17], were ret-

Table 2 Detection of HHV-6 DNA in whole blood samples from solid organ transplant recipients and mutations identified in conserved regions of the U69 (codons 122–564) and U38 (codons 1–998) genes. Patients

1

2

3

4

5

Days

HHV-6 DNA load (copies/ml whole blood)

PCR positive for

Mutations identified in

U69

U38

U69

U38

Antiviral therapy 0 3 7

1.60 × 103 ND 1.39 × 103

+ ND +

+ ND +

L213I ND L213I

D854E, E855Q ND D854E, E855Q

Antiviral therapy 0 3 7 10 14 17 21

7.53 × 103 1.04 × 104 1.36 × 104 8.54 × 103 7.32 × 103 3.42 × 103 7.75 × 103

+ + + + + + +

+ + + + + + +

L213I NT NT NT NT NT L213I

D854E, E855Q NT NT NT NT NT D854E, E855Q

1.04 × 104 ND 0.61 × 103 1.92 × 103

+ + + ND

+ + + +

Y479H NT NT ND

D854E, E855Q NT NT NT

2.56 × 103 3.10 × 103

ND +

+ +

ND Y479H

NT D854E, E855Q

Antiviral therapy 0 3 7 10

6.61 × 105 1.11 × 105 1.32 × 104 1.18 × 104

+ + + +

+ + + +

L213I NT NT L213I

D854E, E855Q NT NT D854E, E855Q

Antiviral therapy 0 3 7 10 14 17

3.25 × 103 2.61 × 103 1.13 × 103 1.07 × 103 1.23 × 103 0.59 × 103

+ + + + + ND

+ + + ND + +

L213I NT NT NT L213I ND

D854E, E855Q NT NT ND D854E, E855Q D854E, E855Q

Antiviral therapy 0 3 7 10 Follow-up 90 180

HHV-6 DNA loads are the mean of two amplification reactions. The limit of detection of this assay using external plasmid standards is 1 and 10 copies for HHV-6 A and B20 , respectively. ND, not detected; NT, not tested.

L. Bounaadja et al. / Journal of Clinical Virology 58 (2013) 279–282

rospectively tested for the emergence of HHV-6 drug resistance mutations. Blood samples were typically obtained on day 0 prior to initiation of antiviral therapy and then on days 3, 7, 10, 14, 17, 28, 35, 42 and 49 over the course of treatment. Follow-up samples were collected on days 90 and 180 and at time of relapsing HCMV disease. Viral DNA load was determined in whole blood specimens (to achieve higher sensitivity [18]) collected prior to initiation of antiviral therapy (day 0) and in samples serially obtained from HHV-6-positive patients by a real-time PCR assay on a LightCycler instrument using primers designed to amplify the U65-U66 genes of both A and B species as previously described [19,20]. Conserved regions of the U69 and U38 genes were amplified by a first PCR for all HHV-6-positive samples. Nested PCR assays were then performed for both genes using the same primers for both HHV-6 A and B (Table 1). PCR procedures were similar for the two target genes and for the first and nested PCRs. The PCR mix consisted of 5× GC buffer (10 ␮l), DMSO (2.5 ␮l), dNTPs (200 ␮M each), primers (0.5 ␮M each), Phusion® High-Fidelity DNA Polymerase (1 Unit) and extracted DNA or PCR product (1 ␮l). After a first cycle of 30 s at 98 ◦ C, 35 cycles were run as follows: 10 s at 98 ◦ C, 30 s at 60 ◦ C and 1 min/kb at 72 ◦ C with a final elongation step of 10 min at 72 ◦ C. DNA sequencing was performed on PCR products obtained on day 0 and on the last HHV-6 PCR-positive day then the U69 and U38 gene sequences were compared for each patient. Nucleotide sequences were translated to amino acid sequences and aligned with those published for HHV-6A strain U1102 and HHV-6B strain HST (Genebank accession number NC 001664 and AB021506, respectively) to discriminate both species. 4. Results Among the 275 SOT recipients monitored for HCMV drug resistance mutations [17], whole blood samples obtained on day 0 from 155 patients (56%) were available in sufficient amount for the HHV-6 sub-study. Among tested patients, the frequency of HHV6 viraemia was found to be 3.2% (5/155) at baseline. All positive samples consisted of the HHV-6B species. For these 5 patients, HHV-6 persisted over the course of testing which varied from 7 to 180 days (Table 2). Whole blood specimens can contain latent or lytic replicative virus as well as chromosomally integrated HHV-6 (ciHHV-6) which results in abnormally high viral DNA levels (>5.5log10 copies/ml) [21]. Therefore, the discrimination of an active infection from a latent state and ciHHV-6 may rely on the viral DNA load fluctuation [22]. A decrease of HHV-6 DNA levels of at least 1 log over the course of antiviral therapy has been associated with a lytic infection. Thus, HHV-6 DNA load in patients 1, 3 and 4 suggests the detection of a lytic infection whereas that of patients 2 and 5 possibly correlates to the detection of latent viruses. The presence of HHV-6 in the clinical specimens from these 5 patients was further confirmed by U69 and U38 gene amplifications (Table 2). All mutations identified in the U69 and U38 genes in comparison with prototype sequences were present on day 0 and on the last tested day. In the U69 kinase, the Y479H and L213I mutations were identified in the isolates of patient 3 and of all the other patients, respectively. All HHV-6 isolates also had mutations D854E and E855Q in the U38 DNA polymerase. 5. Discussion The rate of HHV-6 viraemia in SOT recipients at the onset of HCMV disease and receiving no antiviral treatment was low (3.2%). All isolates belonged to HHV-6B species. HHV-6 reactivation was

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reported in 40–50% of SOT recipients between 2 and 6 weeks after transplantation and is mostly due to HHV-6B [7]. A rate of HHV-6 detection of 9.4% (28/298) on day 0 was reported in the same cohort of SOT recipients than the one we investigated [22]. The lower frequency of HHV-6 detection in our study could be explained by the use of a different real-time PCR assay. HHV-6 DNA load decreased by at least 1 log over the course of antiviral therapy in 3 HHV-6positive patients suggesting a lytic infection whereas the constant viral DNA load in samples from the other 2 patients is possibly indicative of a latent infection. The emergence of drug-resistant HHV-6 strains in the clinic has been described only in a few case reports [23–25]. In our study, all HHV-6-positive samples had mutations in the U69 and U38 genes compared to the prototype strain on day 0 and on the last tested day. The mutation L213I, located in the nucleotide-binding site of the U69 kinase, was previously attributed to natural polymorphism by using a recombinant baculovirus system [23,26]. The mutation Y479H is located close to the sub-domain XI of the U69 kinase. Several mutations in this sub-domain were associated with HHV-6 resistance to GCV when evaluated by a recombinant baculovirus antiviral susceptibility assay [26,27]. No homology exists between the corresponding amino acids of HHV-6 and HCMV kinases (Y479 in U69 versus G623 in UL97). The role of the Y479H mutation in HHV-6 resistance to GCV should thus be investigated by using a recombinant baculovirus system [26] or recombinant phenotyping [28,29]. Mutations D854E and E855Q in the U38 DNA polymerase correspond probably to natural polymorphisms, as these amino acids are present in the prototype HHV-6A strain U1102. Thus, no emergence of putative drug resistance mutations was observed in the U69 and U38 genes of HHV-6 following 49 days of GCV or VGCV therapy. Additional studies are needed to evaluate the emergence of HHV-6 mutations during extended antiviral prophylaxis. Funding G.B. is the holder of the Canada research chair on emerging viruses and antiviral resistance. Competing interests None declared. Ethical approval This study was approved by research ethics committees of all participating centres. References [1] Adams MJ, Carstens EB. Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2012). Arch Virol 2012;157:1411–22. [2] Mendez JC, Dockrell DH, Espy MJ, Smith TF, Wilson JA, Harmsen WS, et al. Human beta-herpesvirus interactions in solid organ transplant recipients. J Infect Dis 2001;183:179–84. [3] DesJardin JA, Gibbons L, Cho E, Supran SE, Falagas ME, Werner BG, et al. Human herpesvirus 6 reactivation is associated with cytomegalovirus infection and syndromes in kidney transplant recipients at risk for primary cytomegalovirus infection. J Infect Dis 1998;178:1783–6. [4] DesJardin JA, Cho E, Supran S, Gibbons L, Werner BG, Snydman DR. Association of human herpesvirus 6 reactivation with severe cytomegalovirusassociated disease in orthotopic liver transplant recipients. Clin Infect Dis 2001;33:1358–62. [5] Razonable RR, Rivero A, Brown RA, Hart GD, Espy MJ, van Cruijsen H, et al. Detection of simultaneous beta-herpesvirus infections in clinical syndromes due to defined cytomegalovirus infection. Clin Transplant 2003;17:114–20. [6] Harma M, Hockerstedt K, Lyytikainen O, Lautenschlager I. HHV-6 and HHV-7 antigenemia related to CMV infection after liver transplantation. J Med Virol 2006;78:800–5. [7] De Bolle L, Naesens L, De Clercq E. Update on human herpesvirus 6 biology, clinical features, and therapy. Clin Microbiol Rev 2005;18:217–45.

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