A novel real-time PCR method for the determination and. quantification of each cytomegalovirus (CMV) gh-subtype in.

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1 JCM Accepts, published online ahead of print on 23 November 2011 J. Clin. Microbiol. doi: /jcm Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved A novel real-time PCR method for the determination and quantification of each cytomegalovirus (CMV) gh-subtype in clinical samples Kazufumi Ikuta, 1 Ken Ishioka, 1 Yuka Sato, 1 Takashi Imamura, 2 Kimisato Asano, 3 Shin Koyano, 4 Naoki Inoue, 5 and Tatsuo Suzutani 1,* Departments of 1Microbiology, 2 Pediatrics and 3Obstetrics, Fukushima Medical University, School of Medicine, Fukushima, 4Department of Pediatrics, Asahikawa Medical University, School of Medicine, Asahikawa, 5Depertment of Virology I, National Institute of Infectious Diseases, Tokyo, Japan *Corresponding author: Tatsuo Suzutani, M.D., Ph.D. Department of Microbiology, Fukushima Medical University, 1 Hikarigaoka, 16 Fukushima , Japan 17 TEL FAX suzutani@fmu.ac.jp 1

2 19 Running title: Determination and quantification of CMV strains by PCR 20 (Running title: 54 characters, limit: 54 characters include space) 21 Key words: cytomegalovirus, polymorphism, glycoprotein H, real-time PCR, congenital infection To be submitted to: Journal of Clinical Microbiology Downloaded from on May 4, 2018 by guest 2

3 25 ABSTRACT 26 To investigate reinfection in patients with congenital cytomegalovirus 27 (CMV) infection, we established a CMV subtype-specific real-time quantitative PCR method targeting the CMV gh epitope region that can be used for evaluating pathogenic CMV strains in cases of mixed CMV infection. Abstract: 36 words (limit: 50 words) Downloaded from on May 4, 2018 by guest 3

4 34 Cytomegalovirus (CMV) is a causative pathogen of congenital infection 35 that leads to multiple clinical defects, not only at birth but also during the 36 early stages of development (6). Primary infection during pregnancy is a significant risk factor for intrauterine CMV infection. Approximately 30% of pregnant women are CMV seronegative, and 1.1% of them experienced primary infection during pregnancy (10, 13, 14, 18). Compared with primary infection, the chance of CMV reinfection or reactivation is said to be much lower; however, the possibility still exists in neonates born to women who were seropositive prior to pregnancy (1-4, 11, 12, 15, 16). Frequency of reinfection differs greatly according to race, age and infectious disease (7, 15, 17), and reinfection in CMV-seropositive individuals is, therefore, not a rare event. Although there are many CMV strains, immunity against CMV subtype-specific neutralizing epitopes of glycoprotein H (gh) is acquired when CMV-seropositive individuals are infected with a previously unexposed strain (2, 19). Serology by gh offers a clue to understanding 49 immunity against CMV (2, 8). However, the detection of the pathogen itself, 50 not just its antibody, is required for the identification of the pathological 51 strain in cases of CMV infection. We, therefore, established a novel 4

5 52 quantitative PCR method that enables the detailed clarification of CMV 53 reinfection by distinguishing between and quantifying CMV subtypes by gh 54 epitope Seventy-three of 23,757 neonates were diagnosed with congenital CMV infection by the Japanese Congenital Cytomegalovirus Study Group (9). CMV genomic DNA was isolated from urine samples in seven of the 73 cases using a QIAamp DNA mini kit (Qiagen, Valentia, CA) according to the manufacturer s protocol. CMV-specific IgG in blood from neonates was tested by a commercial kit (Enzygnost Anti-CMV IgG, Dade Behring Marburg GmbH, Germany). CMV subtype-specific CMV IgG was tested by ELISA as described previously (8). Quantitative PCR (qpcr) for CMV DNA was carried out with a TaqMan probe as previously reported (5). For CMV subtype-specific quantification and determination, the gh region (amino acid position 27 to 67 52) containing an epitope (position 34 to 43) was targeted for qpcr testing (2, 68 19). Primers to amplify the region were designed based on a consensus 69 sequence between the AD169 and Towne strains, with gh78-97 as the 5

6 70 forward and gh as the reverse primer, respectively (Figure). An 71 amplified PCR product of the gh region from each strain was cloned into the 72 pgem-t Easy Vector (Promega, Madison, WI) and transformed in the E. coli DH-5alfa strain (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The plasmid DNA was then harvested by HiSpeed Plasmid Midi kit (Qiagen) as the standard DNA for the CMV subtype-specific qpcr. Probes to distinguish each strain were designed based on the uniquely sequenced portion of the gh epitope region (position 34 to 39). The qpcr was performed using a StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA). We carried out nested PCR for samples with lower CMV copy numbers. The initial PCR was performed prior to qpcr using primers gh21-38 (5 -CTA CCT CAY CGT CYT CR-3 ) and gh (5 -ATC AAA GAG GAT ACA GGT CTG-3 ) with initialization at 95 C for 5 min, followed by 30 cycles of 95 C for 30 sec, 50 C for 1 min and 72 C for 1 min, and final extension at 72 C for 5 min. 85 PCR products from Case# and #20117 (Table 2) were cloned into 86 a pgem-t Easy Vector (Promega) and CMV subtypes were investigated in 87 each clone by qpcr analysis. 6

7 88 The nucleotide sequences of the CMV gh gene from 7 clinical isolates 89 were determined by fluorescent dye-terminator sequencing. Semi-nested 90 PCR was performed using primers gh21-38 and gh , followed by primers gh69-85 (5 -ACG ATA TGG CGC AGA MG-3 ) and gh PCR products were labeled using a DYEnamic ET terminator Cycle Sequencing kit (GE Healthcare) and read on the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). CMV strain was determined by sequence alignment with data from AD169 and Towne strains (GenBank accession numbers NC_ and FJ , respectively). Sensitivity and specificity of the CMV subtype-specific qpcr were tested using standard plasmids containing the gh region from the AD169 or Towne strain. The AD169 or Towne plasmid DNA was detected from 10 8 to 10 2 copies/reaction with FAM-labeled AD or Vic-labeled To probes, respectively. There were no non-specific detections; i.e., no AD169 or Towne plasmid DNA was detected (at from 10 8 to 10 1 copies/reaction) with the 103 Vic-labeled To or FAM-labeled AD probes, respectively. The sensitivity was 104 increased sufficiently to detect 10 1 copies/reaction by nested PCR. 105 The sensitivity of the CMV subtype-specific qpcr was also tested by 7

8 106 mixing both types of standard plasmid DNA at different ratios to simulate 107 clinical isolates from cases with mixed CMV infection (Table 1). The target 108 plasmid DNA was serially 10-fold diluted from 10 7 to 10 2 copies/reaction, and the other plasmid DNA was mixed in the reaction at 10 7, 10 5 or 10 3 copies/reaction. The signals from the qpcr were then detected with both AD (FAM) and To (Vic) probes as a multiplex qpcr. The results were no longer close to the copy numbers we added nor detectable when the counterpart plasmid DNA exceeded the target 100-fold; for example, it was impossible to detect 10 4 copies/reaction of AD169 plasmid DNA under contamination with 10 7 copies/reaction of Towne plasmid DNA (Table 1A). A level of 10 3 copies/reaction of AD169 plasmid DNA became detectable and closer to the expected copy numbers when the contaminated Towne plasmid DNA was decreased to 10 5 copies/reaction. The results were similar when detecting Towne plasmid DNA by To (Vic) probe under contamination with AD169 plasmid DNA (Table 1B). The relationship between the expected copy 121 numbers (added copy numbers/reaction) and measurements obtained from 122 the qpcr was positively correlated when the copy numbers of the mixed 123 constituents were up to 100-fold that of the plasmid DNA on which the 8

9 124 detection was focused. 125 DNA in urine from seven neonates with congenital CMV infection was 126 provided to evaluate the performance of the method. All seven neonates had IgG antibodies against CMV; three against AD169, three against Towne, and one against both strains (Table 2). Their mothers showed the same CMV IgG antibody patterns as the neonates, indicating the predominant IgG antibodies in the neonates were the maternal antibodies (data not shown). Direct sequencing revealed that four neonates shed AD169-type and three shed Towne-type CMV in their urine, and these results matched the serological results. Results from the CMV subtype-specific qpcr were consistent with the direct sequencing; only AD169-type or Towne-type strain was detected from neonates who had AD169-type or Towne-type strains by direct sequencing from the urine. Case #22383, who had antibodies against both CMV strains, shed only AD169-type CMV strain in his urine. We could not find any cases in which both subtypes where shed in the urine, even 139 though one of the neonates had IgG against both AD169-type and 140 Towne-type strains. We tried to find multiple CMV infections in urine by 141 cloning PCR products. All the clones (308 clones from urine, blood or breast 9

10 142 milk from Case #22383, and 43 clones from urine from Case #20117) were 143 screened by subtype-specific qpcr and only a single subtype was detected 144 from each individual (data not shown). In agreement with our results, CMV gh subtype (as assessed by antibody assay) was previously reported to match that in the individual s urine (2). In that report, only a single subtype was shed in the urine despite having CMV IgG against both AD169 and Towne subtype. A previous study also reported that the analysis of the genotype of gb, gh, gn, UL144 and UL149 CMV genes revealed no evidence of infection with multiple strains(20). In conclusion our established subtype-specific qpcr is a highly effective method for evaluating multiple CMV infections that escaped neutralization. This study was supported by a Grant-in-Aid for Young Scientists (B) (Ikuta, KAKENHI MO ) from the Ministry of Education, Culture, 157 Sports, Science and Technology, Japan, a Grant for Child Health and 158 Development from the Ministry of Health and Welfare, Japan (Suzutani 159 H20-kodomo-007), and a fellowship from the Kurozumi Medical Foundation 10

11 160 (Ikuta). The authors have no conflicts of interest. 161 We thank Ms. Junko Ito and Mr. Roy Cameron for proofreading this 162 manuscript

12 164 REFERENCES Ahlfors, K., S. A. Ivarsson, and S. Harris Secondary maternal cytomegalovirus infection--a significant cause of congenital disease. Pediatrics 107: Boppana, S. B., L. B. Rivera, K. B. Fowler, M. Mach, and W. J. Britt Intrauterine transmission of cytomegalovirus to infants of women with preconceptional immunity. N Engl J Med 344: Dar, L., S. K. Pati, A. R. Patro, A. K. Deorari, S. Rai, S. Kant, S. Broor, K. B. Fowler, W. J. Britt, and S. B. Boppana Congenital cytomegalovirus infection in a highly seropositive semi-urban population in India. Pediatr Infect Dis J 27: Fowler, K. B., S. Stagno, and R. F. Pass Maternal immunity and prevention of congenital cytomegalovirus infection. Jama 289: Fukushima, E., K. Ishibashi, H. Kaneko, H. Nishimura, N. Inoue, T. Tokumoto, K. Tanabe, K. Ishioka, H. Ogawa, and T. Suzutani Identification of a highly conserved region in the human cytomegalovirus glycoprotein H gene and design of molecular diagnostic methods targeting the region. J Virol Methods 151: Gaytant, M. A., E. A. Steegers, B. A. Semmekrot, H. M. Merkus, and J. M. Galama Congenital cytomegalovirus infection: review of the epidemiology and outcome. Obstet Gynecol Surv 57: Ishibashi, K., T. Tokumoto, H. Shirakawa, K. Hashimoto, N. Kushida, T. Yanagida, K. Shishido, K. Aikawa, O. Yamaguchi, H. Toma, K. Tanabe, and T. Suzutani Strain-specific seroepidemiology and reinfection of cytomegalovirus. Microbes Infect 10: Ishibashi, K., T. Tokumoto, K. Tanabe, H. Shirakawa, K. Hashimoto, N. Kushida, T. Yanagida, N. Inoue, O. Yamaguchi, H. Toma, and T. Suzutani Association of the outcome of renal transplantation with antibody response to cytomegalovirus strain-specific glycoprotein H epitopes. Clin Infect Dis 45: Koyano, S., Inoue, N., Oka, A., Moriuchi, H., Asano, K., Ito, Y., Yamada, H., Yoshikawa, T., Suzutani, T., for the, and J. C. C. S. Group Screening for congenital cytomegalovirus infection using newborn urine samples collected on filter paper: feasibility and outcomes from a multicentre study. BMJ Open in press. 10. Krech, U Complement-fixing antibodies against cytomegalovirus in different parts of the world. Bull World Health Organ 49: Mussi-Pinhata, M. M., A. Y. Yamamoto, R. M. Moura Brito, M. de Lima Isaac, P. F. 12

13 de Carvalho e Oliveira, S. Boppana, and W. J. Britt Birth prevalence and natural history of congenital cytomegalovirus infection in a highly seroimmune population. Clin Infect Dis 49: Nagamori, T., S. Koyano, N. Inoue, H. Yamada, M. Oshima, T. Minematsu, and K. Fujieda Single cytomegalovirus strain associated with fetal loss and then congenital infection of a subsequent child born to the same mother. J Clin Virol 49: Nishimura, N., H. Kimura, Y. Yabuta, N. Tanaka, Y. Ito, K. Ishikawa, C. Suzuki, and T. Morishima Prevalence of maternal cytomegalovirus (CMV) antibody and detection of CMV DNA in amniotic fluid. Microbiol Immunol 43: Rosenthal, S. L., L. R. Stanberry, F. M. Biro, M. Slaoui, M. Francotte, M. Koutsoukos, M. Hayes, and D. I. Bernstein Seroprevalence of herpes simplex virus types 1 and 2 and cytomegalovirus in adolescents. Clin Infect Dis 24: Ross, S. A., N. Arora, Z. Novak, K. B. Fowler, W. J. Britt, and S. B. Boppana Cytomegalovirus reinfections in healthy seroimmune women. J Infect Dis 201: Ross, S. A., K. B. Fowler, G. Ashrith, S. Stagno, W. J. Britt, R. F. Pass, and S. B. Boppana Hearing loss in children with congenital cytomegalovirus infection born to mothers with preexisting immunity. J Pediatr 148: Ross, S. A., Z. Novak, G. Ashrith, L. B. Rivera, W. J. Britt, S. Hedges, J. R. Schwebke, and A. S. Boppana Association between genital tract cytomegalovirus infection and bacterial vaginosis. J Infect Dis 192: Stagno, S., and R. J. Whitley Herpesvirus infections of pregnancy. Part I: Cytomegalovirus and Epstein-Barr virus infections. N Engl J Med 313: Urban, M., W. Britt, and M. Mach The dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific. J Virol 66: Yan, H., S. Koyano, Y. Inami, Y. Yamamoto, T. Suzutani, M. Mizuguchi, H. Ushijima, I. Kurane, and N. Inoue Genetic variations in the gb, UL144 and UL149 genes of human cytomegalovirus strains collected from congenitally and postnatally infected Japanese children. Arch Virol 153:

14 232 FIGURE LEGEND Figure Sequence of HCMV gh region. A. Sequence of gh in CMV were compared between 19 isolates. Twelve sequences were from GenBank, each accession number is: 1 NC_001347, 2 AB , 3 AB , 4 AB , 5 AB , 6 AB , 7 AB , 8 U , 9 FJ , 10 AB , 11 AB and 12 AB B. Primers and Probes for CMV subtype-specific real-time PCR. R: A or G, M: A or C, Y: C or T, S: G or C, D: G, A or T. Downloaded from on May 4, 2018 by guest 14

15 A. Mixed Towne copies Expected AD169 copies (±0.07)x (±0.01)x (±0.09)x (±0.01)x (±0.02)x (±0.15)x (±0.01)x (±0.01)x (±0.04)x undet. 1.13(±0.09)x (±0.07)x undet. 0.65(±0.13)x (±0.12)x undet. undet. 1.33(±0.21)x10 2 B. Expected Mixed AD169 copies Towne copies (±0.01)x (±0.04)x (±0.01)x (±0.01)x (±0.18)x (±0.21)x (±0.61)x (±0.24)x (±0.12)x undet. 0.58(±0.04)x (±0.05)x undet. undet. 0.71(±0.08)x undet. undet. 1.27(±0.31)x10 2 Table 1. Sensitivity of HCMV strain-specific real-time quantitative PCR.

16 CMV DNA in urine CMV IgG antibody ID Copies per ul CMV gh CMV AD To sequencing CMV AD To x x10 4 < AD x x10 2 < AD x x10 2 < AD x x10 4 < AD x10 1 < * To x10 4 < 1.4x10 4 To x10 5 < 1.1x10 5 To *: positive by nested PCR Table 2. Determination of CMV strain in neonates with congenital CMV infection.

17 Figure. A. AD169 1 CGC GGA CGC CGC ATC CGA AGC GCT GGA CCC TCA CGC ATT TCA CCT ACT ACT CAA CAC CTA CGG GAG ACC CAT CCG CTT CC 30615(gH) 2... A A G G (gH) 3... A G (gH) 4... A G G. H2-8(gH) 5... A G H3-26(gH) 6... A G G. T136(gH) 7... A G Pt(gH) 8... A G T A G A G A G A A G Towne 9... A.. A...AT C A. A.. G G H A.. A....T C A. A.. G G G. T A.. A....T C A. A.. G G G. T A.. A....T C A. A.. G G G A.. G.T C G. A.. G G.. G A.. A....T C A. A.. G G A.. A....T C A. A.. G G B. Primers gh 78-97: CGC RGA MGC CRY ATC CGA AS gh : GSA ADC GGA TGG GTC TCC CGT Probes AD: FAM- CTG GAC CCT CAC GCG T -MGB To: Vic- CTG GAC AAA GCG TTT CA -MGB

c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP

c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP Supplementary Figure 1 Establishment of the gain- and loss-of-function experiments and cell survival assays. a Relative expression of mature mir-484 30 20 10 0 **** **** NCP mir- 484P NCP mir- 484P b Relative