The role of HHV-6 and HHV-7 infections in the development of fibromyalgia.

Human herpes virus-6 (HHV-6) and human herpes virus-7 (HHV-7) are immunomodulating viruses potentially affecting the nervous system. We evaluated the influence of HHV-6 and HHV-7 infections on fibromyalgia (FM) clinical course. Forty-three FM patients and 50 control group participants were enrolled. 39.50% (n = 17) FM patients had light A delta and C nerve fiber damage, 27.91% (n = 12) had severe A delta and C nerve fiber damage. 67.44% (n = 29) FM patients had loss of warm sensation in feet, loss of heat pain sensation, and increased cold pain sensation (34.90%, n = 15 in both findings). HHV-6 and HHV-7 genomic sequences in peripheral blood DNA in 23/43 (51.00%) and 34/43 (75.50%) of samples from FM patients and in 3/50 (6.00%) and 26/50 (52.00%) of samples from the control group individuals were detected. Active HHV-6 (plasma viremia) or HHV-7 infection was revealed only in FM patients (4/23, 17.40% and 4/34, 11.80%, respectively). A statistically significant moderate positive correlation was found between A delta and C nerve fiber damage severity and HHV-6 infection (p < 0.01, r = 0.410). 23/43 patients from the FM group and control group participants HHV-6 and 34/45 HHV-7 did have infection markers. A statistically significant moderate positive correlation was found between A delta and C nerve fiber damage severity and HHV-6 infection (p < 0.01, r = 0.410). No difference was found between detection frequency of persistent HHV-6 and HHV-7 infection between FM patients and the control group. Statistically significant correlation was observed between quantitation of changes in QST thermal modalities and HHV-6 infection. There was no correlation between A delta and C nerve fiber damage and HHV-7 infection.

HHV-6A in vitro infection of thyrocytes and T cells alters the expression of miRNA associated to autoimmune thyroiditis.

BACKGROUND: Human herpesviruses have been hypothesized as environmental triggers in the development of autoimmune thyroid diseases (AITD), and in particular active human herpesvirus 6A (HHV-6A) infection was detected in thyrocytes of Hashimoto's thyroiditis (HT) patients, who also show specific anti-viral immune responses. On the other hand, AITD patients display modulation of specific miRNAs in thyroid tissue and blood. We wanted to ascertain whether HHV-6A infection might be correlated to the miRNA dysregulation observed in AITD. METHODS: Human thyroid and T-cell lines were infected in vitro with HHV-6A,-6B or -7, and analysed for miRNAs expression, either by microarray or by specific RT-PCR assays detecting miRNAs associated with AITD in vivo. RESULTS: HHV-6A infection, but not -6B or -7 infections, induced a decrease in miR-155_2 expression and an increase in miR-1238 expression in thyrocytes, as well as an increase in the expression levels of several autoimmunity-associated miRNAs in T lymphocytes, including miR-16_1, miR34a, miR-130a, miR-143_1, miR-202, miR-301b, miR-302c, miR-449b, miR-451_1, and miR-1238_2. CONCLUSIONS: HHV-6A infection modulates miRNAs expression in the cell types involved in the development of AITD. Notably, our in vitro findings correlate with what observed in AITD patients, further supporting the association between HHV-6A infection and AITD development. Moreover, these effects are 6A-specific, emphasizing the differences between the two HHV-6 virus species, and suggesting diverse virus mechanisms of action and therapeutic approaches.

Anti-herpesviral effects of a novel broad range anti-microbial quaternary ammonium silane, K21.

We have created a novel quaternary ammonium silane, K21 through sol-gel chemistry, using an ethoxylated version of an organosilane quaternary ammonium compound and TetraEthyl Ortho Silicate (TEOS) as precursors. Previous studies using the precursor molecule quaternary ammonium compounds (QACs) and a methacryloxy version of K21, primarily designed for use in dental healthcare, have shown inhibited growth properties against several types of gram-positive and gram-negative bacteria including Escherichia coli, Streptococcus mutans, Actinomyces naeslundii and Candida albicans etc. Here we tested the effect of K21 on HSV-1, HHV-6A and HHV-7 in in vitro cell culture infection models. Our results show growth inhibitory effect of K21 on HSV-1, HHV-6A and HHV-7 infection.

Human herpesvirus-7 infection of the CNS with acute myelitis in an adult bone marrow recipient.

The beta-herpesviruses, human herpesviruses-6 and -7 (HHV-6 and HHV-7), are closely related and have very similar biological behaviour. While HHV-6 is associated with encephalitis in immunosuppressed adults, HHV-7 is not recognised as a cause of neurological disease in such patients. This report describes the identification of a reactivated HHV-7 infection in the cerebrospinal fluid of an adult who presented with an acute myelitis 11 months after unrelated donor bone marrow transplant.

Human herpesvirus 6 and human herpesvirus 7: emerging pathogens in transplant patients.

Human herpesvirus 6 (HHV-6) and HHV-7 are two recently identified beta-herpesviruses, genetically related to human cytomegalovirus (CMV). Infection with both viruses is common worldwide with rates of seropositivity in adults over 90%. Infection with both viruses usually occurs in early childhood. In this age group HHV-6 is a cause of febrile illness including exanthem subitum, and likewise, primary HHV-7 infection has been associated with febrile illness. Similar to the other human herpesviruses, in particular CMV, the viruses have the potential for enhanced pathogenicity in the immunocompromised host. Active infection with both viruses is common following bone marrow or solid organ transplantation, most likely through reactivation of recipient's virus or re-infection considering their high prevalence in the population. Both viruses can be detected by PCR in the peripheral blood of healthy individuals and although the significance of blood-borne transmission is not clear, a preliminary study suggested that it was not significant for HHV-6. However, there is growing evidence that these viruses may be medically important in the post-transplant period. In bone marrow transplant patients HHV-6 has been associated with a range of clinical disease including encephalitis, interstitial pneumonitis, early and late graft failure and bone marrow suppression. There is also growing evidence for potential interactions among the beta-herpesviruses in liver and renal transplant patients. HHV-6 infection has been associated with an increased risk of developing CMV disease and opportunistic infections and HHV-7 infection has also been linked to an increased risk of CMV disease.

Reactivation of human herpesvirus 6 and 7 in pregnant women.

To elucidate the roles of human herpesvirus (HHV)-6 and -7 in pregnant women, peripheral blood samples and genital tract secretions were collected serially from pregnant women, and both serological testing and polymerase chain reaction (PCR) were carried out to detect viral DNA in the secretions. HHV-6 or HHV-7 Immunoglobulin(Ig)M antibodies were not detected in 432 plasma samples collected from pregnant women and cord blood, but IgG antibodies against both viruses were detected in all plasma samples. Significant increases in HHV-6 and HHV-7 IgG antibodies were observed in two (1.6%) and three (2.4%) pregnant women respectively of a total of 123 cases. HHV-6 DNA was detected in the genital tract in three (3.7%) of 82 pregnant women at the first trimester, and in 10 (12.2%) of the same women in the third trimester. The detection rate in the third trimester was significantly higher than that in the first trimester (P = 0.043). Although HHV-7 DNA was detected in the genital tract of two (2.7%) and seven (9.6%) pregnant women of a total of 73 during the first and third trimesters respectively, there was no statistical difference in the detection rate of the viral DNA between the trimesters. Because a significant increase in HHV-6 IgG antibodies was detected in only two pregnant women, it was not possible to carry out statistical analysis to determine the relationship between HHV-6 infection and associated clinical features. Although there was a significant increase in HHV-7 antibody titers in three pregnant women, a positive correlation between the virus infection and the clinical features was not demonstrated. There was no statistical association between virus shedding in the genital tract and the clinical features examined in this study.

Inverse relationship between human herpesvirus-6 and -7 detection after allogeneic and autologous stem cell transplantation.

Human herpesvirus-6 (HHV-6) and -7 were analyzed in 25 and 18 patients with allogeneic (allo) and autologous (auto) stem cell transplantation (SCT), respectively, by weekly examination of viral DNA in peripheral mononuclear cells using semiquantitative PCR and serologic tests up to 12 weeks after SCT. HHV-6 DNA was detected in 29.6% and 27.9% of samples after allo- and auto-SCT, respectively. The proportions of HHV-6-DNA-positive samples increased in week 3 and 4 after allo-SCT, and in week 1 to 3 after auto-SCT. The frequency of HHV-7 DNA detection, however, was higher after auto-SCT (24.7%) than allo-SCT (12.8%) (P 10(2) copies of HHV-6 DNA (/10(5) cells) on two consecutive occasions were allo-SCT recipients and three showed clinical episodes. Conversely, three of five patients with continuous reactivation of HHV-7 were auto-SCT recipients. Thus, the frequencies of HHV-6 and -7 DNA detection showed an inverse relationship comparing allo- and auto-SCT, suggesting a different mechanism may regulate HHV-6 and -7 reactivation.

Propagation and characterization of human herpesvirus-7 (HHV-7) isolates in a continuous T-lymphoblastoid cell line (SupT1).

After initial culture of HHV-7 in PHA-stimulated human cord blood mononuclear cells (HCBMC), six HHV-7 isolates were propagated successfully in an immature continuous T-lymphoblastoid cell line SupT1. All six isolates infected efficiently the SupT1 cells, and the infected cells became grossly enlarged and multinucleated 7-21 days post-infection. Various stages of HHV-7 morphogenesis were detected. Cell-free supernatants from HHV-7-infected SupT1 cells were infectious to HCBMC as well as to SupT1 cells. The HHV-7-infected SupT1 and HCBMC cell lysates contained more infectious virus than the centrifuged cell culture fluid supernates from the same culture. The HHV-7 isolates H7-2, H7-3, JHC, and JB, concentrated 500 times, had average infectivity titers of 10(3.0) TCID50/ml while strains H7-4 and KHR titered approximately 1-2 logs higher. When all six HHV-7 isolates were propagated in SupT1 and culture fluid supernatants were examined 14-21 days post-infection by negative stain electron microscopy they contained an average of 1.9 x 10(9) virus particles/liter. IFA and ELISA, using HHV-7/SupT1 cell lysate as an antigen, seem to correlate well in detecting high and low HHV-7 antibody in sera from chronic fatigue patients and healthy donors as controls. HHV-7 from SupT1 cell culture was free of HHV-6 and other human herpesviruses as tested by PCR, and the HHV-7 PCR signal was still strong when the viral preparation was diluted to 4.82 x 10(2) genome copies. Since HCBMC are expensive to obtain and available in only small amounts, it is difficult to obtain large quantities of HHV-7 antigen. On the other hand, the SupT1 cell is an excellent source to produce consistently sufficient quantities of HHV-7 for purification studies, development of immunodiagnostics, in vivo infectivity studies, evaluation of antiviral drugs, and molecular biological studies.

SupT-1: a cell system suitable for an efficient propagation of both HHV-7 and HHV-6 variants A and B.

HHV-7 growth on Sup-T1, an immature T-cell line, was studied using different HHV-7 isolates obtained in our laboratory. Titration of viral yields showed that all the virus isolates propagate on this cell line more efficiently than in cord blood lymphocytes, the cells usually recommended for HHV-7 growth. The permissivity of Sup-T1 to HHV-6, whose ability to replicate in these cells was still unknown, was also investigated using two virus isolates representative of variants A and B respectively. Both isolates were able to propagate on Sup-T1 and viral titres were similar to those obtained in cord blood lymphocytes. As the efficient propagation of both HHV-7 and HHV-6 isolates in Sup-T1 cultures, these cells may replace more time consuming and expensive cord blood lymphocyte preparations for the propagation of both the viruses.

Biologic properties of human herpesvirus 7 strain SB.

The growth characteristics of human herpesvirus 7 strain SB (HHV-7 (SB)) were studied in human umbilical cord blood lymphocyte (CBL) cultures. The virus has approximately a 4-day growth cycle, as measured by immunofluorescence analysis, quantitation of the relative viral DNA concentration, and examination of infected cells by electron microscopy on consecutive days post-infection. By systematically varying the culture media components, improved culturing conditions were established. Activated lymphocytes were required for virus growth. HHV-7(SB) grew best in phytohemagglutinin-stimulated CBL cultured in media containing 0.01 mg/ml hydrocortisone. Addition of recombinant human interleukin 2 (IL-2) at concentrations exceeding 1-10 U/ml inhibited virus growth in most CBL cultures. Addition of exogenous IL-2 to the culture media had no effect on viral DNA production. However, the percentage of virus antigen-positive cells was highest when 0.1-1 U/ml was added to the media. Differences in the ability of individual CBL cultures to replicate HHV-7(SB) was not explained by differing CD4+ cell concentrations. However, individual cultures varied in the level of endogenous IL-2 production, which may contribute to the virus growth variability in CBL. HHV-7(SB) grew in the CD4-positive T-cell line SupT1, but not in a variety of other lymphocyte, fibroblast, or epithelial cell lines. Nine compounds were tested for antiviral activity against HHV-7 in vitro. Phosphonoformic acid inhibited virus growth with a 50% effective concentration of 4.8 microM. Ganciclovir (200 microM) and phosphonoacetic acid (100 microM) inhibited more than 90% of virus production. None of the compounds were cytotoxic at concentrations which inhibited the virus. A generalized increase in host cell protein synthesis was also observed in virus-infected cells similar to that seen in CBL infected with human herpesvirus 6.

Growth of human herpesvirus 6 in HEPG2 cells.

HepG2 cells, a well differentiated liver cell line, were shown to be permissive for both human herpesvirus 6 (HHV-6) A and B strains by three independent methods of analysis: detection of viral antigens, viral DNA sequences and infectious virus. HepG2 cell infection with HHV-6 resulted in functional damage as shown by the increased release in the culture medium of some hepatocyte markers. Cells surviving the acute infection were serially passaged without showing cytopathic effect, but, some months later, HHV-6 DNA was still present in the cells and virus induction with a phorbol ester was successful. A possible pathogenetic role of HHV-6 in liver diseases is discussed. Experiments of HepG2 infection with human herpesvirus 7 (HHV-7) were also carried out. The lack of an efficient virus replication suggested a difficulty for HHV-7 to infect hepatic cells.

In vitro activation of human herpesviruses 6 and 7 from latency.

Human herpesviruses 6 and 7 (HHV-6 and HHV-7) are prevalent lymphotropic viruses that infect more than 80% of children at infancy or during early childhood. Infection ranges from asymptomatic to severe disease. HHV-6B causes exanthem subitum. The virus can be recovered from peripheral blood mononuclear cells during the acute phase of exanthem subitum, but the host remains latently infected throughout life. In immunocompromised patients undergoing kidney, liver, or bone marrow transplantation latent HHV-6B is reactivated, at times causing severe or fatal disease. Here, we describe the establishment of an in vitro system for reactivation of HHV-6B and HHV-7 from latency. HHV-7 is reactivated from latently infected peripheral blood mononuclear cells by T-cell activation. HHV-6B could not be reactivated under similar conditions; however, the latent HHV-6B could be recovered after the cells were infected with HHV-7. Once reactivated, the HHV-6B genomes became prominent and the HHV-7 disappeared. We conclude that HHV-7 can provide a transacting function(s) mediating HHV-6 reactivating from latency. Understanding the activation process is critical for the development of treatments to control the activation of latent viruses so as to avoid these sometimes life threatening infections in transplant recipients.

Biological and molecular characteristics of human herpesvirus 7: in vitro growth optimization and development of a syncytia inhibition test.

Two isolates of human herpesvirus 7 (HHV-7) were recovered from phytohemagglutinin-activated peripheral blood mononuclear cells of a patient with chronic fatigue syndrome and of a healthy blood donor. A genetic polymorphism between the two isolates was detected by Southern blot analysis using a novel HHV-7 genomic clone (pVL8) as a probe. We developed optimized conditions for the in vitro propagation of HHV-7 by using enriched populations of activated CD4+ T lymphocytes derived from normal peripheral blood, resulting in the production of high-titered extracellular virus (> 10(6) cell culture infectious doses/ml). Bona fide syncytia formation was documented both in normal CD4+ T lymphocytes and in the Sup-T1 CD4+ T-cell line following infection with high-titered HHV-7. To identify neutralizing antibodies to HHV-7, a syncytia-inhibition test was developed. Variable titers of syncytia-neutralizing antibodies were detected in all the human sera tested, thus confirming the high prevalence of HHV-7 in the human population.