Abstract
Human herpesvirus 6A (HHV-6A), Human herpesvirus 6B (HHV-6B), and Human herpesvirus 7 (HHV-7) can persist by establishing a lifelong infection which could have implications on the immunocompetent host. The aim of this work is to contribute with some knowledge about the HHV-6 A/B and HHV-7 infection in healthy individuals. We have carried out a longitudinal study in seropositive healthy individuals for the detection of viral DNA in saliva and plasma samples, and for determining a specific IgG isotype immune response, which enabled the performance of these viruses to be observed over time. Furthermore, an elderly population was transversely studied to provide data of the activity of these viruses in the older population. In the longitudinal study, HHV-6 DNA was occasionally detected and an isotype immune response with a specific IgG1 profile, while in the older group HHV-6 DNA was frequently detected and an isotype immune response with specific IgG1, IgG3, and IgG4. HHV-7 DNA was frequently detected in both groups and isotype patterns of specific IgG1, IgG3, and IgG4. The results of this study highlight that the long-lasting relationship in healthy HHV-6 A/B-infected individuals have the imprint of age groups.
Introduction
Human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) belong to the Roseolovirus genus and the Betaherpesvirinae subfamily. They are genetically related to Cytomegalovirus (CMV) and, just like other human herpesviruses, they persist indefinitely in the infected host (23). Two variants of HHV-6, HHV-6A, and HHV-6B (HHV-6A/B) were initially individualized, but have been recently classified as distinct virus species on the basis of phenotypic and genetic studies (2).
HHV-6A/B and HHV-7 infections are highly prevalent in adult populations and the viruses have a worldwide distribution. Primary infections typically occur in the early childhood and are usually subclinical or associated with more or less severe clinical syndromes. Some of the symptoms associated with these viruses are exanthema subitum, mononucleosis-like disease, encephalitis, or status epileptics, among others (11,20,31,33).
During primoinfection, viral active production occurs and mature virions can be detected in some body compartments, such as the T lymphocytes and salivary glands. Afterward, viruses persist in their host by establishing a lifelong infection. This lifelong infection is characterized by latency, which is maintained primarily in the lymphocytes and salivary glands, from where they can be reactivated (13,26,34). The latency and reactivation stages can be possible due to the fact that HHV-6A/B and HHV-7 develop a wide variety of strategies to modulate host immune responses, facilitating their own spread and persistence (16).
The reactivation stage has been documented extensively in the immunocompromised host, such as AIDS patients or recipients of bone marrow, kidney, or liver transplants, associated with a variety of diseases, such as pneumonitis, encephalitis, or hepatitis (6,9,15,27). In the asymptomatic immunocompetent people, viral activity has also been reported (1,19,22,35). This means that viral reactivation may occur throughout life, which results in a relationship between viruses and hosts over time, and its consequences are difficult to determine.
HHV-6A or HHV-6B have been associated with many clinical manifestations named “associated conditions,” such as multiple sclerosis, heart disease, hypersensitivity, or cancer (4). The relationship between HHV-6A/B activity and some of these diseases is well established, but the true role of HHV-6A/B remains unclear. With regard to the HHV-7, its contribution to some pathological processes in a healthy host, other than the primary infection, is completely unknown. Therefore, despite having made progress in the knowledge of these viruses since its discovery until today, much remains to be done to contribute to a greater understanding of the virus-host relationship.
In previous investigations, we have studied the IgG subclasses and DNA detection of HHV-6A/B and HHV-7 in healthy individuals, in which we have described different patterns of DNA detection for HHV-6A/B and HHV-7 (8). Particularly, a high DNA HHV-7 detection range in plasma samples was described. In addition, other previous results revealed two different immune isotype response patterns in HHV-6A/B infection.
One of them is restrictive to IgG1 in the latent phase of HHV-6A/B infection in healthy children, pregnant women, and transplant patients with stable levels of antibodies, whereas IgG1 and IgG4 were detected in the reactivation stage of HHV-6A/B in bone marrow transplant patients (10). HHV-6A/B IgG4 isotype responses have been demonstrated after measles infection (18). In this way, the specific IgG isotype response to HHV-6A/B and HHV-7 in an infected individual could help to clarify the HHV-6A/B and HHV-7 activity.
On the contrary, it is often noted that elderly individuals are more vulnerable to infectious diseases. If so, potentially viral activity of HHV-6A/B or HHV-7 could be modified by old age, as it was demonstrated for other human herpesviruses such as CMV or EBV (30). Aging is a complex process affecting a wide variety of physiological functions, including the development and maintenance of the peripheral immune system (25). It is widely accepted that the aging process attenuates the host ability to mount a robust or effective immune response. Many of these changes are exacerbated by or even caused by chronic T cell stimulation by persistent antigen, more particularly described in CMV infections (14,32). Nevertheless, there are no reports of HHV-6A/B and HHV-7 activity in elderly individuals.
One of the aims of this research performed in healthy individuals was to study the long-term relationship between the host and HHV-6A/B and HHV-7 infection. In this way, two studies were conducted. One of them was a longitudinal study carried out in seropositive healthy individuals analyzed during 18 months. During this period, the individuals were sequentially screened for HHV-6A/B and HHV-7 IgG subclasses and DNA detection patterns in saliva and plasma samples. The other one, the transversal study, focused on elderly individuals. Therefore, the aim of this study is contribute to understand the long-lasting relationship in healthy HHV-6A/B- and HHV-7-infected individuals and age-related patterns of DNA detection and IgG subclasses.
Materials and Methods
Population study and samples
Two studies were carried out: a longitudinal and a transversal one. In both cases, all individuals were HHV-6A/B and HHV-7 seropositive by indirect immunofluorescence assays and were studied for HHV-6A/B and HHV-7 IgG subclasses and DNA detection patterns in saliva and plasma samples. The longitudinal study was conducted in each of the 10 individuals (5 men and 5 women; average: 30 years; between 23 and 42 years), who were studied over 18 months. Each individual's saliva, serum, and plasma samples were taken every 3 months. The transverse study included 18 healthy older adults (8 men and 10 women; average: 79 years; between 68 and 93 years).
No individuals had medical records of systemic disease, neither infectious nor noninfectious, or autoimmune syndromes. Whole saliva, serum, and plasma samples were collected from each individual. Saliva (1 mL) was obtained without any stimulus by spitting the collected saliva into sterile containers, and peripheral blood (5 mL) was collected into either an EDTA tube or a plain tube for the isolation of plasma or serum, respectively. When blood samples were taken and when the plasma separation was performed, reasonable precautions were taken so that the blood would not suffer lysis. All samples were stored at −20°C until analyzed.
Participants were informed of the nature and purpose of the serological investigations. Voluntary informed consent for participation in the studies was obtained from each individual in accordance with the ethical principles enumerated in the Declaration of Helsinki and the additional requirements of local and national authorities (National Normative 5330/97, National Administration of Food, Medicine and Technology).
DNA extraction
DNA was extracted from a 0.5 mL sample of plasma or saliva using a phenol–chloroform protocol. In brief, each sample was resuspended in 0.5 mL extraction buffer [10 mM Tris–HCl (pH 8.0), 5 mM EDTA, and 0.1% SDS] and vortexed. After 10 min at room temperature, 1 mL phenol–chloroform was added. After 30 min at 56°C, the mixture was centrifuged at 12,000 g for 30 min and the aqueous phase was separated. After overnight incubation of the samples with absolute ethanol at −20°C, samples were pelleted by centrifugation at 12,000 g for 30 min. Ethanol was discarded, and the precipitated DNA was air-dried and resuspended in 50 mL double-distilled water. All DNA extracts were stored at −20°C.
Detection of HHV-6A/B and HHV-7 DNA
HHV-6A/B and HHV-7 DNA were detected in saliva and plasma samples by nested PCR using primers that target the major capsid protein gene for HHV-6A/B and U10, an α gene for HHV-7 (7,28). The first set of primers for the detection of HHV-6A/B included EX1 (forward primer): 5′-AAGATGGCCACCCCCTCGATGATGCCGCAGT-3′, and EX2 (reverse primer): 5′-CACTTATGTGGTGGCGTTGCCGGCCGAGAACGG-3′. The first set of primers for the detection of HHV-7 included HV7 (forward primer): 5′-TATCCCAGCTGTTTTCATATATAGTAAC-3′, and HV8 (reverse primer): 5′-GCCTTGCGGTAGCACTAGATTTTTTG-3′. For each reaction, a total of 50 mL contained 10 × PCR buffer, 400 mM dNTPs, 0.2 mM primers, 0.6 mL MgCl2 (50 mM), and 1 U Taq DNA polymerase (Invitrogen).
For HHV-6, a total of 40 cycles included denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min. For HHV-7, a total of 40 cycles included denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min.
The second PCR primer set for HHV-6A/B included IN1 (forward primer), 5′-CACATCGCCGGACAGGATGCTTCGGAGTA-3′, and IN2 (reverse primer), 5′-GTGTTGTGAGCCATGGGGAAGAAGGTGGC-3′. For HHV-7, the second PCR primer set included HV7N1 (forward primer): 5′-ACCAATTCAGTTTTCATCCAG-3′, and HV8N2 reverse primer): 5′-TTGAAGAGGAGAATTCTGTAC-3′. Reactions were performed in a total volume of 50 mL that contained 10× PCR buffer, 200 mM dNTPs, 0.2 mM primers, 0.6 mL MgCl2 (50 mM), and 1U Taq DNA polymerase (Invitrogen).
For HHV-6, a total of 40 cycles included denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 2 min. For HHV-7, a total of 40 cycles included denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min. HHV-6A/B- or HHV-7-positive control was processed in parallel in each reaction.
To exclude the possibility of contamination during the PCR, two negative controls were amplified in each experiment. One of them consisted of negative plasma and the other one included all the reagents, except the sample DNA. Filter tips were used, and reagent mix for each step in the nested PCR reaction was done in separate rooms. The sensitivities of the sets of primers for both HHV-6A/B and HHV-7 PCR amplifications were checked by a limiting dilution and proved to be 10–100 DNA copies/mL.
HHV-6A and HHV-6B typification
HHV-6A and HHV-6B were detected by specific primers previously described (5). For HHV-6A, O15 (5′-CGGTGTCACACAGCATGAACTCTC-3′) and O16 (5′-ACTCGGAATGAGGTCAACTTCT-3′) primers were used in a total of 50 mL contained 10 × PCR buffer, 400 mM dNTPs, 0.2 mM primers, 3 μL MgCl2 (50 mM), and 1 U Taq DNA polymerase (Invitrogen). A total of 35 cycles, including denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min were done. For HHV-6B: O10 (5′-GATCCGACGCCTACAAACAC-3′) and O17 (5′-GCAAAACCAAGAATTGTCCAG-3′) primers were used. HHV-6B PCR conditions were identical to HHV-6A, but an annealing temperature of 63°C was used. Expected PCR products, 631 pb for HHV-6A and 727 pb for HHV-6B, were detected in 1% agarose gel stained with ethidium bromide.
Serological assays
Samples were analyzed for the presence of HHV-6A/B and HHV-7 IgG antibodies using indirect immunofluorescence assays. Slides were prepared using Molt-3 lymphoblastoid cells infected with the HHV-6 variant, B strain Z29, and SupT-1 cells were infected with the SB HHV-7 viral strain [kindly provided by the Centers for Disease Control and Prevention (CDC), Atlanta, GA]. In parallel, slides of uninfected cells were prepared as negative controls.
For IgG detection, samples were diluted geometrically in PBS from 1:10. Positive and negative serum control samples (also provided by the CDC) were included in each run. For the IgG isotype test, sera were diluted 1:20, and the reaction was stained using FITC-conjugated mouse monoclonal antibodies against human IgG1, IgG2, IgG3, and IgG4 (Sigma, St Louis, MO). A fluorescein-conjugated goat anti-rat mouse IgG antibody was diluted 1:100. Optimal dilutions of monoclonal antibodies used were titrated against reference sera. The dependent variable for analysis of the data was transformed into a base 2 logarithms. The geometrical mean titer (GMT) of the antibodies was calculated and the results are expressed as antilogarithms.
Statistical analysis
The Shapiro–Wilk's test was used to compare the frequency distribution of antibody titers with a normal distribution (p = 0.004). Inferences were made using nonparametric tests. The Spearman's correlation coefficient and the Wilcoxon test were performed. The p-values less than 0.05 were considered statistically significant.
Results
Longitudinal study
The results of the monitoring of 10 individuals over 18 months for HHV-6A/B and HHV-7 are shown in Tables 1 and 2, respectively. Both viruses developed an independent activity in the same individual. With respect to the results for HHV-6A/B, only a single individual presented DNA in plasma in two out of five plasma samples (individual number 5, 2nd and 5th plasma samples). DNA in saliva was detected in the individuals 1 (3rd sample) and 4 (1st, 2nd, 3rd, 5th, and 6th samples). All HHV-6A/B PCR-positive samples were typified as HHV-6B.
Results of HHV-6A/B Longitudinal Study
n.d., not determinate; S, saliva sample; P, plasma sample.
Results of HHV−7 Longitudinal Study
n.d., not determinate; S, saliva sample; P, plasma sample.
Ten individuals showed specific IgG1 in all samples, and they were specific IgG 2 negative. Some individuals showed positivity for specific IgG3 or IgG4 along the study. With respect to the results for HHV-7, most of the samples of saliva and plasma obtained from the 10 individuals during the study showed the presence of DNA. The 10 individuals showed specific IgG 1 in all samples and were specific IgG 2 and 4 negative. With the exception of the individuals 6 and 10, the rest showed positivity for specific IgG3 at least in one of the samples taken during the monitoring.
Transversal study
As shown in Table 3, HHV-6A/B DNA was detected in elderly individuals in 72.2% of the 18 plasma samples tested, but in none of the saliva samples of the same patients. All HHV-6 PCR positive samples were typified as HHV-6B. In contrast, HHV-7 DNA was detected in 94.4% of the plasma samples and in 90.9% of the saliva samples. We established statistically significant difference in reference to the detection of HHV-7 DNA in saliva samples in relationship to HHV-6A/B DNA (p < 0.0001 χ2 Test of Pearson).
Transversal Study: Detection of HHV-6B and HHV-7 DNA in Saliva and Plasma Samples
Table 4 lists the concentration and the isotype response of HHV-6A/B and HHV-7 antibodies detected. There was no correlation between the antibody titers determined for HHV-6A/B and HHV-7 (Spearman's correlation coefficient 0.22; p = 0.25). No significant difference was observed for each of the GMTs according to the individual's age or sex (Wilcoxon test). Specific HHV-6A/B and HHV-7 IgG1 were detected in 100% of the 18 individuals, which all resulted in specific IgG2 negative. Specific HHV-6A/B IgG3 and IgG4 were detected in 70.5% and 58.8% of sera samples, respectively. Specific HHV-7 IgG3 and IgG4 were detected in 58.8% and 29.4% of sera samples, respectively. No significant difference was observed in reference to the isotype profile.
Transversal Study: Concentration and IgG Isotype Profile of HHV-6A/B and HV-7 Antibodies in Sera Samples
GMT, geometrical mean titer.
Discussion
Comparison of the data from a longitudinal and a cross-sectional study can provide insight into the biology of long-life infections such as HHV-6A/B and HHV-7. In this way, the results of the longitudinal study showed that only an individual presented HHV-6B DNA in plasma in two out of five samples. On the contrary, HHV-7 DNA was detected in all the studied individuals. This difference between the proportions, in which these viruses were detected, was observed in saliva samples too. These results could indicate that there is an increased activity of the HHV-7 with respect to the HHV-6B in the studied population. It is worth noting that in a previous study carried out by our working group, HHV-7 DNA was detected in 90% of the plasma samples of healthy blood donor in Cordoba, Argentina (8).
Another observation we did in this longitudinal study is that a high proportion of healthy individuals with detectable HHV-7 DNA in plasma is maintained over time; at least during the period in which those individuals in this study were tested. This result suggests that the HHV-7 could have a long period of active replication in an immunocompetent individual, suggesting that the HHV-7 has a chronic infection model rather than a latency-reactivation one. In the present study, we used a nested PCR for HHV-6A/B and HHV-7 DNA detection, which has been used in other works (7,27).
The presence of HHV-6A/B and HHV-7 DNA in plasma is used as a marker of active infection. Nevertheless, Achour et al. checked that HHV-6A/B DNA detection in plasma does not necessarily reflect viremia because this HHV-6A/B DNA may have originated from lysis of circulating infected cells in the blood (3).
Approximately 1% of the human population worldwide harbors inherited chromosomally integrated HHV-6A and HHV-6B (24). Individuals with HHV-6A/B chromosomal integration but no active infection could have a positive reaction in qualitative or quantitative PCR done in plasma, serum, or blood samples (12). In this way, it should be clarified that there is still no reliable method to detect active infection or reactivation of HHV-6A/B. However, that measuring of the viral load could have been more informative about the state of replication of these viruses.
Current serological assays do not permit discrimination of HHV-6A and HHV-6B. As per our serologic results, they showed two individuals who have seroconversion to HHV-6. However, nonspecific DNA in saliva or plasma was detected, suggesting that seroconversion could be due to antigenic exposure but not to endogen reactivation. Also, an individual showed seroconversion to HHV-7, but in this case, specific DNA was detected in saliva or plasma samples.
In relationship to specific IgG subclasses profile observed in the longitudinal study, we found no correlation between any specific pattern and the presence of HHV-6A/B DNA. Different was the case of HHV-7, where five out of 10 individuals were shown to be positive for HHV-7-specific
In the transverse study, we observed a 72.2% proportion of HHV-6B DNA detection in plasma samples of older adults. On the contrary, it is striking that HHV-6 DNA was not detected in the saliva samples. We did not find an explanation of why the HHV-6 DNA was not detected in the saliva of any elderly person who had the virus in the plasma. It could be interesting to investigate if in older the HHV-6B activity could be compartmentalized in lymphocytes more than in saliva.
The presence of HHV-6B in plasma could reflect a reactivation associated to aging. In this way, this was demonstrated for other human herpesviruses, such as HSV and CMV (29,30). In view of our results, we can hypothesize that the same applies to the HHV-6B. The activity of HHV-6B in old age could deserve more attention. In the case of HHV-7, as well as in the longitudinal study, a high range of DNA detection in plasma was observed. In this way, it could be that the HHV-7 is more active in the general population than in the elderly, but this should be more investigated.
In the specific IgG subclasses profile observed in the elderly, particularly striking is the appearance of a high percentage of individuals with IgG3 specific for HHV-6A/B, which could be due to active infection. In older individuals, the reduction of cell-mediated immunity by aging can lead to herpesviruses reactivation, and it could be interesting to investigate if there is any relationship between IgG3 and the reduction of cell-immunity. Also, it is important to draw attention to the 58.8% and 29.4% of individuals who present specific IgG4 to HHV-6A/B and HHV-7, respectively. The correlation between HHV-6A/B reactivation and the presence of specific IgG4 was described in a previous study (18).
Viral DNA presence in the plasma of the healthy population opens new questions regarding its interpretation in specific clinical situations or its eventual viral transmission by blood; for example, in transfusions or in the donation of organs.
Recently HHV-6A and HHV-6B were classified as different viruses. Getting to a better understanding of these two viruses is necessary to classify HHV-6A/B since much of the existing information in the bibliography does not distinguish between these two variants.
One limitation of this work was the low number of individuals included in both longitudinal and transversal studies. Thus, some suggestions derived from our results could require studies that include a large number of individuals. Despite this, preliminary result shows a clear difference between DNA detection and the IgG subclass patterns of HHV-6A/B and HHV-7 in healthy individuals. Our results seem to be important since no previous longitudinal studies related to the behavior of these viruses and which focus the attention on the HHV-6A/B activity in elderly subjects were done.
Footnotes
Acknowledgment
This work was supported by grants from SeCyT, Universidad Nacional de La Rioja, La Rioja, Argentina.
Author Disclosure Statement
No competing financial interests exist.