Published online September 1, 2008
PEDIATRICS Vol. 122 No. 3 September 2008, pp. 513-520 (doi:10.1542/peds.2007-2838)
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ARTICLE

Chromosomal Integration of Human Herpesvirus 6 Is the Major Mode of Congenital Human Herpesvirus 6 Infection

Caroline Breese Hall, MDa,b, Mary T. Caserta, MDa, Kenneth Schnabel, MBAa, Lynne M. Shelley, BSa, Andrea S. Marino, BSa, Jennifer A. Carnahan, BSa, Christina Yoo, BSa, Geraldine K. Lofthus, PhDb and Michael P. McDermott, PhDc

Departments of a Pediatrics
b Medicine
c Biostatistics and Computational Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVE. We examined the frequency and characteristics of chromosomally integrated human herpesvirus 6 among congenitally infected children.

METHODS. Infants with and without congenital human herpesvirus 6 infection were prospectively monitored. Cord blood mononuclear cell, peripheral blood mononuclear cell, saliva, urine, and hair follicle samples were examined for human herpesvirus 6 DNA. Human herpesvirus 6 RNA, serum antibody, and chromosomally integrated human herpesvirus 6 levels were also assessed.

RESULTS. Among 85 infants, 43 had congenital infections and 42 had postnatal infections. Most congenital infections (86%) resulted from chromosomally integrated human herpesvirus 6; 6 infants (14%) had transplacental infections. Children with chromosomally integrated human herpesvirus 6 had high viral loads in all sites (mean: 5–6 log10 genomic copies per µg of cellular DNA); among children with transplacental infection or postnatal infection, human herpesvirus 6 DNA was absent in hair samples and inconsistent in other samples, and viral loads were significantly lower. One parent of each child with chromosomally integrated human herpesvirus 6 who had parental hair samples tested had hair containing human herpesvirus 6 DNA. Variant A caused 32% of chromosomally integrated human herpesvirus 6 infections, compared with 2% of postnatal infections. Replicating human herpesvirus 6 was detected only among chromosomally integrated human herpesvirus 6 samples (8% of cord blood mononuclear cells and peripheral blood mononuclear cells). Cord blood human herpesvirus 6 antibody levels were similar among children with chromosomally integrated human herpesvirus 6, transplacental infection, and postnatal infection and between children with maternal and paternal chromosomally integrated human herpesvirus 6 transmission.

CONCLUSIONS. Human herpesvirus 6 congenital infection results primarily from chromosomally integrated virus which is passed through the germ-line. Infants with chromosomally integrated human herpesvirus 6 had high viral loads in all specimens, produced human herpesvirus 6 antibody, and mRNA. The clinical relevance needs study as 1 of 116 newborns may have chromosomally integrated human herpesvirus 6 blood specimens.

Key Words: human herpesvirus 6 • congenital infection • chromosomally integrated infection • transmission

Abbreviations: HHV6—human herpesvirus 6 • CI-HHV6—chromosomally integrated human herpesvirus 6 • TPI—transplacental infection • PNI—postnatal infection • PBMC—peripheral blood mononuclear cell • CBMC—cord blood mononuclear cell • RT—reverse transcription • PCR—polymerase chain reaction • FISH—fluorescence in situ hybridization • CMV—cytomegalovirus

Essentially all children are infected with human herpesvirus 6 (HHV6) within the first 3 years of life, and most have evidence of infection by 12 months of age.1 HHV6 shed in the saliva of asymptomatic contacts is thought to be the major source of transmission to young children.2,3 Congenital HHV6 infection, detected as HHV6 DNA in cord blood, also has been identified as a means of transmission and occurs in ~1% of infants, a rate similar to that of congenital cytomegalovirus (CMV) infection.4–11 In contrast to CMV, however, the HHV6 genome has been shown to be integrated into chromosomes in some individuals, a phenomenon unique to HHV6 among human herpesviruses.12–20 How often chromosomally integrated HHV6 (CI-HHV6) is present in normal individuals is unclear, but it has been estimated to occur in 0.2% to 0.8% of populations in Japan and the United Kingdom.17,18,20 Those individuals were identified primarily through high viral loads (≥1 copy of genomic HHV6 DNA per leukocyte) in their peripheral blood mononuclear cells (PBMCs), which were persistently and markedly higher than levels present in the usual latent infections not associated with CI-HHV6 (1 genomic copy per 104 to 105 leukocytes).17–22 For a few of those individuals, CI-HHV6 was confirmed with fluorescence in situ hybridization (FISH) assays or detection of HHV6 in the hair follicles.14,16–19,23

Congenital HHV6 infections have been considered generally to result from reactivated maternal HHV6 infection crossing the placenta. However, CI-HHV6 may be an important and unique mode of transmission of congenital infection. The purpose of this study was first to determine, in a prospectively monitored cohort of children, the occurrence of CI-HHV6 as a mode of congenital infection, identified through the presence of high viral loads in cord blood mononuclear cells (CBMCs) and confirmed through detection of HHV6 in hair follicles. We then aimed to delineate the viral and immunologic features associated with HHV6 infection among congenitally infected infants with CI-HHV6, compared with infants with congenital transplacental infection (TPI) and infants with postnatal infection (PNI). Finally, we sought to determine the presence of CI-HHV6 among the parents of children with congenital HHV6 infection.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Design
From our ongoing studies of children with HHV6 infection,1,4 infants (≥36 weeks of gestation) with and without congenital HHV6 infection, identified through screening of CBMC at birth for the presence of HHV6 DNA, were enrolled from July 2003 through April 15, 2007. For each enrolled infant with congenital infection, an average of 5 children without congenital infection, matched according to gender, race, ethnicity, and date of birth (within 8 weeks), were enrolled and then monitored for the acquisition of primary HHV6 infection. The families were approached for enrollment only after their private physicians approved. Study visits to the Golisano Children's Hospital at the University of Rochester Medical Center were scheduled for 4 to 6, 12 to 15, 24 to 30, and 30 to 36 months of age. Because enrollment was ongoing, the ages and numbers of visits varied among the children. The children were examined and specimens were obtained from all enrolled children for analyses as described below.

Quantitative determination of HHV6 DNA in CBMCs was used to identify among congenitally infected children those with high viral loads characteristic of CI-HHV6 and those with lower viral loads characteristic of TPI.17–22 The groups were confirmed on the basis of the detection or absence of HHV6 DNA in hair follicle samples. In addition, FISH methods were used to demonstrate integration of the HHV6 genome into the chromosomes of a representative sample of congenitally infected infants. The viral and immunologic features were then analyzed among these 2 groups of children with CI-HHV6 and TPI (congenital infection), compared with control children with PNI.

Subject Groups
Children with congenital HHV6 infection were those with HHV6 DNA present in their cord blood. Children with congenital infection resulting from CI-HHV6 were those with high viral loads of HHV6 DNA (≥1 genomic copy per leukocyte, equivalent to 1–2 x 105 HHV6 genomic copies per µg of cellular DNA), which have been used to identify individuals with CI-HHV6,17–20,22 and/or those who had HHV6 DNA detected in hair follicle samples. Children with TPI were those with lower viral loads found in latent infections not associated with CI-HHV6 (1 genomic copy per 104 to 105 leukocytes, equivalent to <1 genomic copy per µg of cellular DNA)21 and no HHV6 DNA in hair follicle samples. Control children were those who had cord blood samples demonstrating negative results for HHV6 DNA, who acquired HHV6 postnatally.

Laboratory Analyses
Specimen Preparation
From 0.5 to 3 mL of whole blood collected in EDTA, mononuclear cells were separated and used for (1) DNA variant-specific polymerase chain reaction (PCR) assays, (2) real-time quantitative PCR assays, (3) reverse transcription (RT)-PCR assays, and (4) FISH analyses. Nucleic acid was purified from 0.05 mL of saliva and 0.2 mL of urine by using RNA isolation kits from Promega (Madison, WI) or Qiagen (Valencia, CA), without DNase treatment. DNA was isolated from 2 or 3 plucked hair follicles by using the Qiagen QIAamp DNA micro kit.

DNA Variant-Specific PCR Assays
Nested PCR assays with virus-specific typing with oligonucleotide probes specific for HHV6 variants A and B were conducted as described previously.1,4 The assays reliably detected ≤10 genomic copies. All experiments were conducted with positive and negative control samples, as well as with β-globin primers to confirm the presence of cellular material and to exclude the presence of inhibitors.

Nested RT-PCR Assays for HHV6
The RT-PCR assays amplified the gp82-105 mRNA of HHV6 and were performed as described previously, except that the probe sequence was modified slightly on the basis of sequence information for HHV6 variant B.24,25 The probe sequence used in these experiments was 5'-GCTCCCGAAAGCGCCATA-3'. The assays for HHV6 detected <10 mRNA copies.

Quantitative Real-Time PCR Assays
The quantitative PCR assays for the HHV6 U38 gene were performed as described previously,26 except that our forward primer, 5'-TGCTTCTGTAACGTGTCTTGGA-3' (sense), contained 1 less adenine molecule on the 3' end. Only samples with positive HHV6 DNA results in qualitative PCR assays were analyzed with quantitative PCR assays. Samples were purified by using the Wizard SV genomic DNA purification system (Promega) and were assayed in triplicate. Mean results from ≥2 wells were analyzed and reported as genomic copies of HHV6 per µg of cellular DNA (1–2 x 105 HHV6 genomic copies per µg of cellular DNA is equivalent to 1 HHV6 genomic copy per leukocyte).21,27,28

FISH Analyses
Metaphase chromosomal spreads of PBMCs were prepared from Epstein-Barr virus-transformed lymphoblastoid cell lines by using established protocols.29 DNA probes were biotinylated with dATP, and FISH analyses were performed according to published methods.30 FISH signals were observed with fluorescent microscopy and photographed.

Serological Assays
HHV6 antibody levels were determined in indirect immunofluorescence assays by using a clinical HHV6 isolate grown in HSB-2 cells that contained HHV6A and HHV6B genomes.1 Positive results were defined as log2 titers of >3.32 (>1:10 dilution).

Informed Consent
The University of Rochester Research Subjects Review Board approved this study. All families provided informed consent and were compensated for transportation costs and other costs associated with each visit.

Statistical Analyses
Demographic characteristics were compared among the 3 groups of children (those with CI-HHV6, TPI, and PNI) by using {chi}2 tests, Fisher's exact tests, or t tests (for age), as appropriate. Fisher's exact tests were used to compare (1) CI-HHV6 and TPI groups with respect to the proportions of subjects with variant A versus variant B detected in cord blood, (2) CI-HHV6 and PNI groups with respect to the proportions of subjects with variant A detected in samples from ≥1 site at any time after infection, and (3) mothers and fathers with CI-HHV6, detected as HHV6 DNA in hair follicles, with respect to the proportions of samples containing HHV6 variant A. McNemar's test was used to compare paired samples from mothers and fathers of children with CI-HHV6 with respect to the relative frequency of HHV6 DNA detection.

The mean HHV6 antibody titers (logarithmically transformed) in cord blood were compared between CI-HHV6 and PNI groups by using a t test. An analysis of variance model was used to perform pairwise comparisons of the mean HHV6 antibody titers in cord blood among children with maternally derived CI-HHV6, paternally derived CI-HHV6, and PNI. Similar comparisons were performed by using PBMC samples, but a repeated-measures analysis of variance model, with a compound symmetry correlation structure, was used to allow for repeated antibody titer measurements for the same subjects. A similar analysis strategy was used to compare the mean viral loads (logarithmically transformed) in PBMC and saliva samples among the CI-HHV6, TPI, and PNI groups. All reported P values are 2-tailed.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Study Groups
Of 254 children enrolled, 43 had congenital HHV6 infection. Of the 211 children without congenital infection, 42 were control children who acquired HHV6 infection during the study period. None had evidence of acquiring HHV6 infection until ≥1.5 months of age. Among the 43 congenitally infected infants, 37 (86%) were identified as having CI-HHV6 and 6 (14%) as having TPI, according to their quantitative HHV6 DNA levels in CBMCs. The average CBMC viral load among the 43 congenitally infected children was 5.57 log10 genomic copies per µg of cellular DNA (range: 1.90-6.72 log10 genomic copies per µg of cellular DNA). The distribution of the viral loads showed 2 separate groups; 37 children (86%) had a distinctly higher mean viral load (6.06 log10 genomic copies per µg of cellular DNA; range: 5.25-6.72 log10 genomic copies per µg of cellular DNA), and 6 children (14%) had a lower mean load of 2.53 log10 genomic copies per µg of cellular DNA (range: 1.90-3.04 log10 genomic copies per µg of cellular DNA; P < .0001) (Fig 1). The former constituted the group with congenital infection from CI-HHV6 and the latter those with TPI. The characteristics and laboratory findings of the children are presented subsequently according to the 3 groups of children, that is, congenitally infected children with CI-HHV6, congenitally infected children with TPI, and control children.


Figure 1
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  		FIGURE 1 HHV6 viral loads in CBMC, PBMC, hair follicle, urine, and saliva samples from children with CI-HHV6, TPI, and PNI. aAll TPI urines were negative for HHV6 DNA and all except 2 of 100 PNI urines tested were negative.

 
Demographic Characteristics
Congenitally and postnatally infected children were generally similar, although control children, compared with congenitally infected children, were older (not significantly so) and were more likely to have ≥1 sibling (76% vs 51%; P = .02) (Table 1).


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  		TABLE 1 Demographic Features of 85 Enrolled Children With Congenital Infection Acquired Through CI-HHV6 or TPI and Control Children With PNI

 
Detection of HHV6 DNA, Variant, and Replicative State
Among cord blood samples, the HHV6 DNA detected was variant A for 12 infants with CI-HHV6 (32%) and 1 child with TPI (17%; P = .44) (Table 2). Among follow-up PBMC samples from children with CI-HHV6, all contained HHV6 DNA, which was the same variant as detected in their CBMC samples. In comparison, 50% of PBMC samples from children with TPI and 87% from children with PNI contained HHV6 DNA. HHV6 variant B caused all PNI, whereas 21% of CI-HHV6 cases involved HHV6 variant A. Among all samples combined (CBMC, PBMC, saliva, urine, and hair samples), HHV6 variant A was detected for 32% of children with CI-HHV6, compared with 2% of children with PNI (P = .0004). RT-PCR assays for a late gene transcript indicating viral replication were positive in 8% of both CBMC and PBMC samples from children with CI-HHV6 and in none of the samples from the other groups of children.


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  		TABLE 2 HHV6 DNA Variant, Viral State, and IgG Antibody to HHV6 in Cord Blood and Peripheral Blood Samples From Children With Congenital HHV6 Infection Acquired Through CI-HHV6 or TPI and Control Children With PNI

 
Antibody to HHV6
Antibody titers were determined in the cord blood samples, as an indicator of passive maternal antibody levels, and in the subsequently obtained peripheral blood samples to compare the decline and acquisition of HHV6-specific antibody levels among the 3 groups of children (Table 2). Antibody to HHV6 was detected in all cord blood samples obtained from each group. The mean ± SD log2 titers of antibody to HHV6 in the cord blood samples did not differ significantly between children with CI-HHV6 and control infants with PNI (8.27 ± 1.15 vs 8.79 ± 1.39; P = .27).

Because the humoral response to HHV6 infection may be impaired among individuals with CI-HHV6, the levels of passive HHV6-specific antibody in infants with CI-HHV6 whose mothers had CI-HHV6 could differ from those observed among infants with CI-HHV6 whose fathers had CI-HHV6 and those of infants in the PNI group whose parents did not have CI-HHV6. Of the children with CI-HHV6, 18 had mothers with CI-HHV6 and 11 had fathers with CI-HHV6, on the basis of detection of HHV6 DNA in the parents' hair samples. The mean antibody levels present in the cord blood did not differ significantly among children with maternally derived CI-HHV6 (log2 titer: 8.43 ± 1.37), children with paternally derived CI-HHV6 (log2 titer: 8.32 ± 1.10), and children with PNI (log2 titer: 8.79 ± 1.39; P > .30 for each comparison). The mean antibody level in 15 peripheral blood samples from children with maternally derived CI-HHV6 was lower than that in the 47 peripheral blood samples from children with PNI, but this difference was not significant (log2 titer: 5.85 vs 6.60; P > .35). The samples were obtained at a mean age of 13 months from both groups.

Viral Loads of HHV6 DNA in Samples From Congenitally and Postnatally Infected Children
High viral loads of HHV6 DNA were detected in all PBMC, saliva, urine, and hair follicle samples of children with CI-HHV6 (Table 3 and Fig 1). The mean viral loads from each site ranged from 5.13 to 6.07 log10 genomic copies per µg of cellular DNA. The mean and range of viral loads in PBMCs of children with CI-HHV6 were similar to those in their CBMCs. The mean HHV6 loads in PBMCs and saliva of children with CI-HHV6 were significantly greater (P ≤ .0002) than levels detected in the corresponding samples from children with TPI or PNI. Furthermore, the distributions of the viral loads detected in CBMCs and PBMCs from children with CI-HHV6 did not overlap with those detected in the corresponding samples from children with TPI or PNI. HHV6 was detected in all 86 saliva samples from children with CI-HHV6, compared with 45% and 49% of saliva samples from children with TPI and PNI, respectively.


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  		TABLE 3 HHV6 Viral Loads in Samples From Children With Congenital HHV6 Infection From CI-HHV6 or TPI, Compared With Children With PNI

 
FISH Analyses
Lymphoblastoid cell line samples of PBMCs from 5 congenitally infected infants were examined in FISH analyses. Positive FISH signals for HHV6 were detected in 4 cell lines (Fig 2). All were in the CI-HHV6 group, as defined by the presence of HHV6 in high viral loads in CBMCs and in hair follicle samples. The 1 negative specimen was from a congenitally infected child classified as having TPI on the basis of low CBMC viral loads and the absence of HHV6 DNA in hair follicle specimens.


Figure 2
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  		FIGURE 2 Representative FISH analysis of metaphase chromosomes from a child with high HHV6 DNA levels in cord blood (>5 log10 genomic copies per µg of cellular DNA) and HHV6 DNA detected in his hair follicles. The arrow shows symmetrical signals of HHV6 DNA on both chromatids of a single chromosome.

 
HHV6 DNA in Hair Follicle Samples From Parents
HHV6 DNA was detected in hair follicle specimens from 1 parent of all children with CI-HHV6 with available parental samples (Table 4). In 1 family with 2 enrolled children, HHV6 DNA was detected in the hair follicle samples of the mother and both children.


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  		TABLE 4 Detection of HHV6 DNA in Hair Follicles of Parents of Children With Congenital HHV6 Infection From CI-HHV6 or TPI and Children With PNI

 
Among the 56 parental hair follicle samples examined, similar proportions of the maternal and paternal hair samples contained HHV6 DNA (60% and 42%, respectively; P = .56). The mean viral load in the hair follicle samples of the mothers was lower than that in paternal samples but not significantly (P = .12). Variant A was detected in 44% of maternal hair samples and in 18% of paternal hair samples (P = .23). The variant identified among the hair samples of family members was the same as that identified in their child in all cases.

HHV6 DNA was not detected in hair samples obtained from the parents of children with PNI. Four parents of TPI children, 2 fathers and 2 mothers, had samples tested, and both mothers were positive. Although these mothers had CI-HHV6, the child's infection was transplacental as the children's hair follicle samples did not contain HHV6 DNA and the viral loads in their CBMC and PBMC samples were low and not consistently present. Hair samples also were obtained from 21 parents of an additional group of control children who were being monitored for acquisition of PNI but who did not become infected during the study period and thus could not be included in the PNI group. None of those parental or infant hair samples contained HHV6 DNA.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study's prospective evaluation of infants since birth has produced novel observations concerning congenital HHV6 infection. Congenital HHV6 infection can occur through both transplacental transmission and the unique mechanism of CI-HHV6. Furthermore, CI-HHV6 caused the great majority (86%) of congenital HHV6 infections. Among congenitally infected infants, CI-HHV6 can be identified as the mechanism of transmission on the basis of high viral loads in the cord and all samples and detection of HHV6 DNA in hair follicle specimens. This was confirmed among 5 congenitally infected infants (12%) through FISH analysis. In addition, CI-HHV6 congenital infection most likely occurred through germ-line transmission, as demonstrated by the detection of HHV6 DNA in hair follicle samples from 1 parent in families with children with CI-HHV6 but in none of the hair follicle samples from parents of children with PNI. Although HHV6 may become chromosomally integrated through other mechanisms, these findings indicate that spontaneous integration occurring during gestation or after birth was unlikely to be the mechanism of CI-HHV6 infection in these children.12,31

Because all mothers of infants with congenital HHV6 infection have had previous HHV6 infection, the assumption has been that congenital HHV6 infection, like congenital CMV infection, results primarily from transplacental passage of maternal reactivated infection or reinfection with HHV6. The 2 viruses result in congenital infection at notably similar rates of ~1%,8,9 and CMV is also a common but not universal infection. However, important differences between HHV6 and CMV congenital infections exist, as noted by Pass.32 Congenital CMV infection results from maternal infection, which may be a primary infection, reinfection, or reactivation of latent virus.32,33 For congenital infection to occur after this maternal infection, transplacental transmission of actively replicating virus would be critical. However, we have infrequently been able to document active replication of HHV6 in maternal samples of PBMCs and other sites or in cord or neonatal blood samples of congenitally infected infants.4,34 This would support our current findings that only a small proportion of congenital HHV6 infections result from reactivation or reinfection in the mother.

The importance of CI-HHV6 congenital infection depends in part on the biological ramifications of CI-HHV6. Little is known, however, about the viral state of CI-HHV6 and whether individuals with CI-HHV6 produce a normal immune response or demonstrate immune tolerance to subsequent infection with HHV6.

The humoral immune response has been variable in the few individuals reported with CI-HHV6.13,17,22,23 Among 4 reports examining 3 to 7 patients with CI-HHV6, most but not all had detectable HHV6 antibody, but the antibody in some patients was not neutralizing.13,17,22,23,35All of our children with congenital infection had antibody to HHV6 in their cord blood, which suggests that all of their mothers produced a humoral response to HHV6. The children with CI-HHV6 had lower mean levels of HHV6 titers in their cord and peripheral blood samples, compared with the mean levels detected in children with TPI or in the control children with PNI, but the differences were not statistically significant. Furthermore, the kinetics of the HHV6 antibody titers seemed similar for children with and without CI-HHV6. Both showed the expected decline of passive antibody levels, followed by a titer increase in samples obtained after a few months of age. Most infants with CI-HHV6, therefore, seem able to mount a humoral immune response to HHV6 from infection acquired after birth or possibly to the integrated genome's production of viral particles. The finding that the mean HHV6 antibody titer in the cord blood of infants whose mothers had CI-HHV6 was similar to that of infants with paternally acquired CI-HHV6 and to that of children without congenital infection further suggests that individuals with CI-HHV6 can produce a specific humoral response to HHV6.

The HHV6 antibody in individuals with CI-HHV6, however, may not be protective. Two mothers of infants with transplacental infection had hair samples tested and both had CI-HHV6. This suggests that mothers with CI-HHV6 may produce congenitally infected infants through germ-line passage of CI-HHV6 or TPI, or possibly both mechanisms. Furthermore, the transplacental infection may be the result of replicating maternal CI-HHV6.

The viral state of CI-HHV6 is potentially integral to the immune response and to the consequences of CI-HHV6 congenital infection. Information on the viral state, however, has been limited and equivocal. CI-HHV6 has been shown to be nonreplicating in vitro, and replication has not been detected in a limited number of patients with lymphoproliferative diseases or in family members of individuals with CI-HHV6.12,13,15,17,36 HHV6 mRNA was detected in 8% of both cord blood and PBMC samples from our children with CI-HHV6 but in none of the samples from children with TPI or PNI. Detection of HHV6 mRNA in cord blood could indicate replication of CI-HHV6 or transcription of all or part of the viral genome during human gene transcription. However, detection of mRNA in PBMCs also could result from reinfection with a new HHV6 strain. Previously we demonstrated mRNA in PBMC samples from a small proportion (3.3%) of normal children years after initial infection, which indicates that reinfection or reactivation of latent virus can occur in immunocompetent children.34

The rate of CI-HHV6 transmission did not seem to be affected by the parental source, because the proportions of infants with CI-HHV6 with maternal versus paternal inheritance were not significantly different and their viral loads were similar. However, variant A was detected significantly more often in blood samples from children with CI-HHV6 than in samples from children with PNI. This may indicate that HHV6 variant A has a greater propensity than HHV6 variant B to become chromosomally integrated at specific chromosomal sites. All of our patients with CI-HHV6 who had samples evaluated in FISH analyses demonstrated symmetrical integration of HHV6 at the ends of both chromatids of a single chromosome. Very few other individuals or families reported as having CI-HHV6 have been analyzed with FISH studies, and only 2 children, both with neonatal problems, were examined during infancy.14,17–19 The signal of HHV6 integration in these children also came from only 1 homolog of 1 chromosome.19 In the few chromosomes identified with CI-HHV6, the ends of the telomeres have been the predominately identified sites.12,13,16–19,23 This theoretically could be of clinical importance, because the ends of telomeres seem to be integral to the maintenance of normal immune function.37

The potential immunologic, biological, and long-term manifestations associated with CI-HHV6 are unknown. Our findings demonstrate that transcription of viral genes, either from the integrated genome or from lack of protection against reinfection, can occur in children with CI-HHV6. Persistent viral gene activity in multiple sites, including the central nervous system, might enhance the risk of subsequent developmental deficits, as occurs with congenital CMV infection.8,10,11,32 This deserves further study. If 86% of the 1% of infants born with congenital HHV6 infection have CI-HHV6, as suggested by this study, then 1 of every 116 live-born infants may have CI-HHV6, which may result in an appreciable, but currently unrecognized, health burden.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the National Institute of Child Health and Human Development (grant RO1 HD 44430-01), the National Center for Research Resources, National Institutes of Health (General Clinical Research Center grant 5 MO1 RR00044), and the HHV6 Foundation.


   FOOTNOTES
 
Accepted Dec 18, 2007.

Address correspondence to Caroline Breese Hall, MD, Department of Pediatrics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Box 689, Rochester, NY 14642. E-mail: caroline_hall{at}urmc.rochester.edu

The authors have indicated they have no financial relationships relevant to this article to disclose.


What's Known on This Subject

We showed previously that HHV6 causes congenital infection in 1% of newborns, similar to the closely related cytomegalovirus, and it is assumed that congenital infection occurs through transplacental transmission of virus during maternal reinfection or reactivation of HHV6.

 

What This Study Adds

Eighty-six percent of HHV6 congenital infections resulted from chromosomally integrated HHV6, a unique mode of viral congenital infection. Infants with chromosomally integrated HHV6 had distinctive findings, suggesting the currently unknown biological, immunologic, and viral state of the chromosomally integrated virus.

 


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hall CB, Long CE, Schnabel KC, et al. Human herpesvirus-6 infection in children: a prospective study of complications and reactivation. N Engl J Med. 1994;331 (7):432 –438[Abstract/Free Full Text]
  2. Mukai T, Yamamoto T, Kondo T, et al. Molecular epidemiological studies of human herpesvirus 6 in families. J Med Virol. 1994;42 (3):224 –227[Web of Science][Medline]
  3. De Bolle L, Naesens L, De Clercq E. Update on human herpesvirus 6 biology, clinical features, and therapy. Clin Microbiol Rev. 2005;18 (1):217 –245[Abstract/Free Full Text]
  4. Hall CB, Caserta MT, Schnabel KC, et al. Congenital infections with human herpesviruses 6 and 7. J Pediatr. 2004;145 (4):472 –477[CrossRef][Web of Science][Medline]
  5. Adams O, Krempe C, Kogler G, Wernet P, Scheid A. Congenital infections with human herpesvirus 6. J Infect Dis. 1998;178 (2):544 –546[Web of Science][Medline]
  6. Dahl H, Fjaertoft G, Norsted T, Wang FZ, Mousavi-Jazi M, Linde A. Reactivation of human herpesvirus 6 during pregnancy. J Infect Dis. 1999;180 (6):2035 –2038[CrossRef][Web of Science][Medline]
  7. Daibata M, Miyoshi I. Presence of human herpesvirus 6 DNA in cord blood cells. J Infect Dis. 1999;179 (4):1046 –1047[Web of Science][Medline]
  8. Dollard SC, Grosse SD, Ross DS. New estimates of the prevalence of neurological and sensory sequelae and mortality associated with congenital cytomegalovirus infection. Rev Med Virol. 2007;17 (5):355 –363[CrossRef][Web of Science][Medline]
  9. Schleiss MR. Vertically transmitted herpesvirus infections. Herpes. 2003;10 (1):4 –11[Medline]
  10. Fowler KB, Stagno S, Pass RF, Britt WJ, Boll TJ, Alford CA. The outcome of congential cytomegalovirus infection in relation to maternal antibody status. N Engl J Med. 1992;326 (10):663 –667[Abstract]
  11. Boppana SB, Pass RF, Britt WJ. Virus-specific antibody responses in mothers and their newborn infants with asymptomatic congenital cytomegalovirs infections. J Infect Dis. 1993;167 (1):72 –77[Web of Science][Medline]
  12. Torelli G, Barozzi P, Marasca R, et al. Targeted integration of human herpesvirus 6 in the p arm of chromosome 17 of human peripheral blood mononuclear cells in vivo. J Med Virol. 1995;46 (3):178 –188[Web of Science][Medline]
  13. Luppi M, Marasca R, Barozzi P, et al. Three cases of human herpesvirus-6 latent infection: integration of viral genome in peripheral blood mononuclear cell DNA. J Med Virol. 1993;40 (1):44 –52[Web of Science][Medline]
  14. Daibata M, Taguchi T, Sawada T, Taguchi H, Miyoshi I. Chromosomal transmission of human herpesvirus 6 DNA in acute lymphoblastic leukaemia. Lancet. 1998;352 (9127):543 –544[CrossRef][Medline]
  15. Daibata M, Taguchi T, Taguchi H, Miyoshi I. Integration of human herpesvirus 6 in a Burkitt's lymphoma cell line. Br J Haematol. 1998;102 (5):1307 –1313[CrossRef][Web of Science][Medline]
  16. Morris C, Luppi M, McDonald M, Barozzi P, Torelli G. Fine mapping of an apparently targeted latent human herpesvirus type 6 integration site in chromosome band 17p13.3. J Med Virol. 1999;58 (1):69 –75[CrossRef][Web of Science][Medline]
  17. Tanaka-Taya K, Sashihara J, Kurahashi H, et al. Human herpesvirus 6 (HHV-6) is transmitted from parent to child in an integrated form and characterization of cases with chromosomally integrated HHV-6 DNA. J Med Virol. 2004;73 (3):465 –473[CrossRef][Web of Science][Medline]
  18. Clark DA, Nacheva EP, Leong HN, et al. Transmission of integrated human herpesvirus 6 through stem cell transplantation: implications for laboratory diagnosis. J Infect Dis. 2006;193 (7):912 –916[CrossRef][Web of Science][Medline]
  19. Ward KN, Leong HN, Nacheva EP, et al. Human herpesvirus 6 chromosomal integration in immunocompetent patients results in high levels of viral DNA in blood, sera, and hair follicles. J Clin Microbiol. 2006;44 (4):1571 –1574[Abstract/Free Full Text]
  20. Leong HN, Tuke PW, Tedder RS, et al. The prevalence of chromosomally integrated human herpesvirus 6 genomes in the blood of UK blood donors. J Med Virol. 2007;79 (1):45 –51[CrossRef][Web of Science][Medline]
  21. Clark DA, Ait-Khaled M, Wheeler AC, et al. Quantification of human herpesvirus 6 in immunocompetent persons and post-mortem tissues from AIDS patients by PCR. J Gen Virol. 1996;77 (9):2271 –2275[Abstract/Free Full Text]
  22. Ward KN, Thiruchelvam AD, Couto-Parada X. Unexpected occasional persistence of high levels of HHV-6 DNA in sera: detection of variants A and B. J Med Virol. 2005;76 (4):563 –570[CrossRef][Web of Science][Medline]
  23. Daibata M, Taguchi T, Nemoto Y, Taguchi H, Miyoshi I. Inheritance of chromosomally integrated human herpesvirus 6 DNA. Blood. 1999;94 (5):1545 –1549[Abstract/Free Full Text]
  24. Norton RA, Caserta MT, Hall CB, Schnabel K, Hocknell P, Dewhurst S. Detection of human herpesvirus 6 by reverse transcription-PCR. J Clin Microbiol. 1999;37 (11):3672 –3675[Abstract/Free Full Text]
  25. Dominguez G, Dambaugh TR, Stamey FR, Dewhurst S, Inoue N, Pellett PE. Human herpesvirus 6B genome sequence: coding content and comparison with human herpesvirus 6A. J Virol. 1999;73 (10):8040 –8052[Abstract/Free Full Text]
  26. Zhen Z, Bradel-Tretheway B, Sumagin S, Bidlack JM, Dewhurst S. The human herpesvirus 6 G protein-coupled receptor homolog U51 positively regulates virus replication and enhances cell-cell fusion in vitro. J Virol. 2005;79 (18):11914 –11924[Abstract/Free Full Text]
  27. Qiagen. Nucleic acids and proteins: mammalian cells and tissues. Available at: www1.qiagen.com/resources/info/nucleic_acids_and_proteins_mammalian_cells_and_tissues.aspx? Accessed November 19, 2007
  28. Jefferies WM, Turner JC, Lobo M, Gwaltney JM Jr. Low plasma levels of adrenocorticotropic hormone in patients with acute influenza. Clin Infect Dis. 1998;26 (3):708 –710[Web of Science][Medline]
  29. Omori F, Messner HA, Ye C, et al. Nontargeted stable integration of recombinant adeno-associated virus into human leukemia and lymphoma cell lines as evaluated by fluorescence in situ hybridization. Hum Gene Ther. 1999;10 (4):537 –543[CrossRef][Web of Science][Medline]
  30. Heng HH, Tsui LC. Modes of DAPI banding and simultaneous in situ hybridization. Chromosoma. 1993;102 (5):325 –332[CrossRef][Web of Science][Medline]
  31. Luppi M, Barozzi P, Bosco R, et al. Human herpesvirus 6 latency characterized by high viral load: chromosomal integration in many, but not all, cells. J Infect Dis. 2006;194 (7):1020 –1021[CrossRef][Web of Science][Medline]
  32. Pass RF. HHV6 and HHV7: persistence and vertical transmission. J Pediatr. 2004;145 (4):432 –434[CrossRef][Web of Science][Medline]
  33. Pass RF, Fowler KB, Boppana SB, Britt WJ, Stagno S. Congenital cytomegalovirus infection following first trimester maternal infection: symptoms at birth and outcome. J Clin Virol. 2006;35 (2):216 –220[CrossRef][Web of Science][Medline]
  34. Caserta MT, McDermott MP, Dewhurst S, et al. Human herpesvirus 6 (HHV6) DNA persistence and reactivation in healthy children. J Pediatr. 2004;145 (4):478 –484[CrossRef][Web of Science][Medline]
  35. Luppi M, Barozzi P, Marasca R, Torelli G. Integration of human herpesvirus-6 (HHV-6) genome in chromosome 17 in two lymphoma patients. Leukemia. 1994;8 (suppl 1):S41 –S45[Web of Science][Medline]
  36. Bandobashi K, Daibata M, Kamioka M, et al. Human herpesvirus 6 (HHV-6)-positive Burkitt's lymphoma: establishment of a novel cell line infected with HHV-6. Blood. 1997;90 (3):1200 –1207[Abstract/Free Full Text]
  37. Artandi SE. Telomeres, telomerase, and human disease. N Engl J Med. 2006;355 (12):1195 –1197[Free Full Text]
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