Inclusion of the Guinea Pig Cytomegalovirus Pentameric Complex in a Live Virus Vaccine Aids Efficacy against Congenital Infection but Is Not Essential for Improving Maternal and Neonatal Outcomes.

The development of a vaccine against congenital human cytomegalovirus (HCMV) infection is a major priority. The pentameric complex (PC) of virion envelope proteins gH, gL, UL128, UL130, and UL131A is a key vaccine target. To determine the importance of immunity to the homologous PC encoded by guinea pig cytomegalovirus (GPCMV) in preventing congenital CMV, PC-intact and PC-deficient live-attenuated vaccines were generated and directly compared for immunogenicity and efficacy against vertical transmission in a vertical transmission model. A virulent PC-intact GPCMV (PC/intact) was modified by galK mutagenesis either to abrogate PC expression (PC/null; containing a frame-shift mutation in GP129, homolog of UL128) or to delete genes encoding three MHC Class I homologs and a protein kinase R (PKR) evasin while retaining the PC (3DX/Δ145). Attenuated vaccines were compared to sham immunization in a two-dose preconception subcutaneous inoculation regimen in GPCMV seronegative Hartley guinea pigs. Vaccines induced transient, low-grade viremia in 5/12 PC/intact-, 2/12 PC/null-, and 1/11 3DX/Δ145-vaccinated animals. Upon completion of the two-dose vaccine series, ELISA titers for the PC/intact group (geometic mean titer (GMT) 13,669) were not significantly different from PC/null (GMT 8127) but were significantly higher than for the 3DX/Δ145 group (GMT 6185; p < 0.01). Dams were challenged with salivary gland-adapted GPCMV in the second trimester. All vaccines conferred protection against maternal viremia. Newborn weights were significantly lower in sham-immunized controls (84.5 ± 2.4 g) compared to PC/intact (96 ± 2.3 g), PC/null (97.6 ± 1.9 g), or 3DX/Δ145 (93 ± 1.7) pups (p < 0.01). Pup mortality in sham-immunized controls was 29/40 (73%) and decreased to 1/44 (2.3%), 2/46 (4.3%), or 4/40 (10%) in PC/intact, PC/null, or 3DX/Δ145 groups, respectively (all p < 0.001 compared to control). Congenital GPCMV transmission occurred in 5/44 (11%), 16/46 (35%), or 29/38 (76%) of pups in PC/intact, PC/null, or 3DX/Δ145 groups, versus 36/40 (90%) in controls. For infected pups, viral loads were lower in pups born to vaccinated dams compared to controls. Sequence analysis demonstrated that infected pups in the vaccine groups had salivary gland-adapted GPCMV and not vaccine strain-specific sequences, indicating that congenital transmission was due to the challenge virus and not vaccine virus. We conclude that inclusion of the PC in a live, attenuated preconception vaccine improves immunogenicity and reduces vertical transmission, but PC-null vaccines are equal to PC-intact vaccines in reducing maternal viremia and protecting against GPCMV-related pup mortality.

Estimating the Risk of Human Herpesvirus 6 and Cytomegalovirus Transmission to Ugandan Infants from Viral Shedding in Saliva by Household Contacts.

Human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) infections are common in early childhood. In a prospective Ugandan birth cohort study, most infants acquired HHV-6 (24/31; 77%) and CMV (20/30; 67%) during follow-up. To assess the transmission risk, we modeled a dose-response relationship between infant HHV-6 and CMV infections and weekly oral viral shedding by mothers and all other ("secondary") children in the home. Oral viral loads that were shed by mothers and secondary children were significantly associated with HHV-6 but not CMV transmission. While secondary children had higher and more frequent HHV-6 shedding than their mothers, they had a lower per-exposure transmission risk, suggesting that transmission to maternal contacts may be more efficient. HHV-6 transmission was relatively inefficient, occurring after <25% of all weekly exposures. Although HHV-6 transmission often occurs following repeated, low dose exposures, we found a non-linear dose-response relationship in which infection risk markedly increases when exposures reached a threshold of > 5 log10 DNA copies/mL. The lack of association between oral CMV shedding and transmission is consistent with breastfeeding being the dominant route of infant infection for that virus. These affirm saliva as the route of HHV-6 transmission and provide benchmarks for developing strategies to reduce the risk of infection and its related morbidity.

Detection of human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) DNA in endocervical samples from a positive and negative HPV woman of Córdoba, Argentina.

AIMS: The purpose of the present study was to elucidate the presence of human herpesvirus 6A (HHV-6A), HHV-6B and HHV-7 in samples of the uterine cervix through detection of viral DNA. We analysed normal tissues, samples with low-grade squamous intraepithelial lesions (LSILs) and high-grade squamous intraepithelial lesions (HSILs). We correlated the presence of HHV-6 and HHV-7 with the finding of human papillomavirus (HPV) in mucosal samples. METHODS: Cervical samples were examined and grouped as follows: group 1 (n=29), normal cytology; group 2 (n=61), samples with LSIL; group 3 (n=35), samples with HSIL. Molecular biology examinations were performed in all samples to detect HHV-6, HHV-7 and HPV DNA and to typify HHV-6 species. RESULTS: Group 1: normal cytology and HPV (-): HHV-6: 6.8% (2/29), HHV-7: 79.3% (23/29); group 2: LSIL and HPV (-): HHV-6: 93.1% (27/29), HHV-7: 96.5% (28/29); LSIL and HPV (+): HHV-6: 0% (0/32), HHV-7: 90.6% (29/32); group 3: HSIL and HPV (-): HHV-6: 20% (2/10), HHV-7: 70% (7/10); HSIL HPV (+): HHV-6: 12% (3/25), HHV-7: 68% (17/25). HHV-6A DNA was not detected in any samples. CONCLUSIONS: (1) Both HHV-6 and HHV-7 infect the mucosal cells of the cervix with higher prevalence of HHV-7. (2) The higher prevalence of HHV-6 in LSIL HPV (-) samples compared with those with normal cytology indicates that it constitutes a possible risk factor for atypia production. (3) The presence of HHV-7 in all samples questions its role in the production of atypia. (4) The finding of HHV-6 and HHV-7 suggests that the cervical mucosa is a possible transmission pathway for these viruses.

Comparative Analysis of Roseoloviruses in Humans, Pigs, Mice, and Other Species.

Viruses of the genus Roseolovirus belong to the subfamily Betaherpesvirinae, family Herpesviridae. Roseoloviruses have been studied in humans, mice and pigs, but they are likely also present in other species. This is the first comparative analysis of roseoloviruses in humans and animals. The human roseoloviruses human herpesvirus 6A (HHV-6A), 6B (HHV-6B), and 7 (HHV-7) are relatively well characterized. In contrast, little is known about the murine roseolovirus (MRV), also known as murine thymic virus (MTV) or murine thymic lymphotrophic virus (MTLV), and the porcine roseolovirus (PRV), initially incorrectly named porcine cytomegalovirus (PCMV). Human roseoloviruses have gained attention because they can cause severe diseases including encephalitis in immunocompromised transplant and AIDS patients and febrile seizures in infants. They have been linked to a number of neurological diseases in the immunocompetent including multiple sclerosis (MS) and Alzheimer's. However, to prove the causality in the latter disease associations is challenging due to the high prevalence of these viruses in the human population. PCMV/PRV has attracted attention because it may be transmitted and pose a risk in xenotransplantation, e.g., the transplantation of pig organs into humans. Most importantly, all roseoloviruses are immunosuppressive, the humoral and cellular immune responses against these viruses are not well studied and vaccines as well as effective antivirals are not available.

Multiple transmissions of chromosomally integrated human herpesvirus-6 in one family.

Chromosomally integrated human herpesvirus-6 (ciHHV-6) can be transmitted from parent to child or via allogeneic hematopoietic cell transplantation (HCT). We report a case of ciHHV-6 transmitted via syngeneic HCT, and vertically across 3 generations. ciHHV-6 was transmitted from a parent to the patient and her identical twin, and from the patient to her son. The patient underwent syngeneic HCT as rescue from chemotherapy-induced aplasia during which ciHHV-6 was re-transmitted to her, this time from her identical twin. This is the first report, to our knowledge, of a patient acquiring ciHHV-6 once via germline from a parent and again via syngeneic HCT from an identical twin.

Possible chromosomal and germline integration of human herpesvirus 7.

Human herpesvirus 7 (HHV-7) is a betaherpesvirus, and is phylogenetically related to both HHV-6A and HHV-6B. The presence of telomeric repeat sequences at both ends of its genome should make it equally likely to integrate into the human telomere as HHV-6. However, numerous studies have failed to detect germline integration of HHV-7, suggesting an important difference between the HHV-6A/-6B and HHV-7 genomes. In search of possible germline integrated HHV-7, we developed a sensitive and quantitative real-time PCR assay and discovered that primers designed against some parts of the HHV-7 genome can frequently miss HHV-7 positive clinical samples even though they work efficiently in cell-culture-derived HHV-7 positive materials. Using a primer pair against the U90 ORF of HHV-7, we identified a possible case of germline integration of HHV-7 with one copy of viral genome per cell in both peripheral blood cells and hair follicles. Chromosomal integration of HHV-7 in these individuals was confirmed by fluorescence in situ hybridization analysis. Germline integration of HHV-7 was further confirmed by detection of ~2.6 copies of HHV-7 in the hair follicles of one of the parents. Our results shed light on the complex nature of the HHV-7 genome in human-derived materials in comparison to cell-culture-derived materials and show the need for stringent criteria in the selection of primers for epidemiological HHV-7 studies.

Transmission of chromosomally integrated human herpesvirus 6 via cord blood transplantation.

Chromosomally integrated human herpesvirus 6 (ciHHV-6) can be transmitted via allogeneic hematopoietic cell transplantation. To date, only a few cases have been reported. Here, we report a case identified as transmission of ciHHV-6 via cord blood transplantation. Distinguishing transmission of ciHHV-6 from HHV-6 reactivation in cases with high titer of HHV-6 DNA load after transplantation is important to prevent unnecessary exposure to antiviral drugs that could be toxic.

Efficient identification of inherited chromosomally integrated human herpesvirus 6 using specimen pooling.

BACKGROUND: Human herpesvirus 6 (HHV-6) has a unique ability to integrate into chromosomal telomeres. Vertical transmission via germ cell integration results in offspring with inherited chromosomally integrated (ci)HHV-6 in all nucleated cells, affecting ∼1% of the population. OBJECTIVES: Inherited ciHHV-6 may be a direct or indirect mediator of human disease, but efficient identification of affected individuals is a fundamental roadblock to larger studies exploring the clinical importance of this condition. STUDY DESIGN: A group testing strategy was designed to efficiently identify individuals with inherited ciHHV-6. DNA was extracted from 2496 cellular samples from hematopoietic cell transplant (HCT) donor-recipient pairs. Pools of 12 samples were screened for HHV-6 DNA with quantitative (q)PCR. Individual samples from high positive pools were tested with qPCR, and high positive individual samples were tested for inherited ciHHV-6 using droplet digital (dd)PCR to determine HHV-6 DNA copies/cellular genome. RESULTS: Thirty-one pools had high positive HHV-6 DNA detection with >10(3) HHV-6 DNA copies/µg. Each pool had one sample with >10(4) copies/µg HHV-6 DNA. Inherited ciHHV-6 was confirmed by ddPCR in every high positive sample (>10(3) HHV-6 DNA copies/µg), yielding a prevalence of 1.5% in HCT recipients and 0.96% in donors. We performed 580 qPCR tests to screen 2496 samples for inherited ciHHV-6, a 77% reduction in testing. CONCLUSIONS: Inherited ciHHV-6 can be efficiently identified by specimen pooling coupled with modern molecular techniques. This algorithm can be used to facilitate cost-effective identification of patients with inherited ciHHV-6, thereby removing a major hurdle for large-scale study of its clinical impact.

Xenotransplantation and porcine cytomegalovirus.

Porcine microorganisms may be transmitted to the human recipient when xenotransplantation with pig cells, tissues, and organs will be performed. Most of such microorganisms can be eliminated from the donor pig by specified or designated pathogen-free production of the animals. As human cytomegalovirus causes severe transplant rejection in allotransplantation, considerable concern is warranted on the potential pathogenicity of porcine cytomegalovirus (PCMV) in the setting of xenotransplantation. On the other hand, despite having a similar name, PCMV is different from HCMV. The impact of PCMV infection on pigs is known; however, the influence of PCMV on the human transplant recipient is unclear. However, first transplantations of pig organs infected with PCMV into non-human primates were associated with a significant reduction of the survival time of the transplants. Sensitive detection methods and strategies for elimination of PCMV from donor herds are required.

Quantitative polymerase chain reaction for detection of human herpesvirus-7 infection in umbilical cord blood donors.

OBJECTIVE: Umbilical cord blood (UCB) has been a reasonable alternative to granulocyte colony-stimulating factor-mobilized peripheral blood or bone marrow, as a source of hematopoietic stem cells with a lower risk of graft-versus-host disease. In immunocompromised hosts after transplantation, the risk of viral infection in adults, especially with beta-herpesviruses such as human herpesvirus-7 (HHV-7), may be increased. This virus in immunocompromised patients can be reactivated from latency and converted to an active phase. Therefore, light-upon-extension real-time polymerase chain reaction (PCR) was developed to assess the prevalence and load of HHV-7 in the plasma and buffy coat of donors. METHODS: About 825 UCB samples under standard protocol from donors were collected. Then, DNA from plasma and buffy coat was extracted and quantitative real-time PCR was performed with light-upon-extension primers. RESULTS: Overall, HHV-7 was detected in 3.64% (30/825) of UCB donors. HHV-7 DNA was detected in 26 (3.2%) buffy coat samples (latent infection), and only 4 (0.48%) of them were positive for HHV-7 DNA in plasma samples (active infection); the mean HHV-7 viral load was 1.31 × 10(1) copies/mL in latent infection, and 1.94 × 10(5) copies/mL in active infection. CONCLUSIONS: We suggest that real-time PCR in plasma and buffy coat could be a useful method to detect active and latent HHV-7 infection in UCB donors and determine its role in subsequent transmission events.

Molecular and biological characterization of a new isolate of guinea pig cytomegalovirus.

Development of a vaccine against congenital infection with human cytomegalovirus is complicated by the issue of re-infection, with subsequent vertical transmission, in women with pre-conception immunity to the virus. The study of experimental therapeutic prevention of re-infection would ideally be undertaken in a small animal model, such as the guinea pig cytomegalovirus (GPCMV) model, prior to human clinical trials. However, the ability to model re-infection in the GPCMV model has been limited by availability of only one strain of virus, the 22122 strain, isolated in 1957. In this report, we describe the isolation of a new GPCMV strain, the CIDMTR strain. This strain demonstrated morphological characteristics of a typical Herpesvirinae by electron microscopy. Illumina and PacBio sequencing demonstrated a genome of 232,778 nt. Novel open reading frames ORFs not found in reference strain 22122 included an additional MHC Class I homolog near the right genome terminus. The CIDMTR strain was capable of dissemination in immune compromised guinea pigs, and was found to be capable of congenital transmission in GPCMV-immune dams previously infected with salivary gland­adapted strain 22122 virus. The availability of a new GPCMV strain should facilitate study of re-infection in this small animal model.

Human herpesvirus 6A infection and immunopathogenesis in humanized Rag2⁻/⁻ γc⁻/⁻ mice.

Although serious human diseases have been correlated with human herpesvirus 6A (HHV-6A) and HHV-6B, the lack of animal models has prevented studies which would more definitively link these viral infections to disease. HHV-6A and HHV-6B have recently been classified as two distinct viruses, and in this study we focused specifically on developing an in vivo model for HHV-6A. Here we show that Rag2⁻/⁻γc⁻/⁻ mice humanized with cord blood-derived human hematopoietic stem cells produce human T cells that express the major HHV-6A receptor, CD46. Both cell-associated and cell-free viral transmission of HHV-6A into the peritoneal cavity resulted in detectable viral DNA in at least one of the samples (blood, bone marrow, etc.) analyzed from nearly all engrafted mice. Organs and cells positive for HHV-6A DNA were the plasma and cellular blood fractions, bone marrow, lymph node, and thymic samples; control mice had undetectable viral DNA. We also noted viral pathogenic effects on certain T cell populations. Specific thymocyte populations, including CD3⁻ CD4⁺ CD8⁻ and CD3⁺ CD4⁻ cells, were significantly modified in humanized mice infected by cell-associated transmission. In addition, we detected significantly increased proportions of CD4⁺ CD8⁺ cells in the blood of animals infected by cell-free transmission. These findings provide additional evidence that HHV-6A may play a role in human immunodeficiencies. These results indicate that humanized mice can be used to study HHV-6A in vivo infection and replication as well as aspects of viral pathogenesis.

Persistent human herpesvirus-6 infection in patients with an inherited form of the virus.

Human herpesvirus-6 (HHV-6)A and 6B are ubiquitous betaherpesviruses viruses with lymphotropic and neurotropic potential. As reported earlier, these viruses establish latency by integration into the telomeres of host chromosomes. Chromosomally integrated HHV-6 (CIHHV-6) can be transmitted vertically from parent to child. Some CIHHV-6 patients are suffering from neurological symptoms, while others remain asymptomatic. Four patients with CIHHV-6 and CNS dysfunction were treated with valganciclovir or foscarnet. HHV-6 replication was detected by reverse transcriptase polymerase chain reaction amplification of a late envelope glycoprotein. In this study we also compared the inherited and persistent HHV-6 viruses by DNA sequencing. The prevalence of CIHHV-6 in this cohort of adult patients from the USA suffering from a wide range of neurological symptoms including long-term fatigue were found significantly greater than the reported 0.8% in the general population. Long-term antiviral therapy inhibited HHV-6 replication as documented by loss of viral mRNA production. Sequence comparison of the mRNA and the inherited viral genome revealed that the transcript is produced by an exogenous virus. In conclusion, the data presented here document that some individuals with CIHHV-6 are infected persistently with exogenous HHV-6 strains that lead to a wide range of neurological symptoms; the proposed name for this condition is inherited herpesvirus 6 syndrome or IHS.

Skin recruitment of monomyeloid precursors involves human herpesvirus-6 reactivation in drug allergy.

BACKGROUND: In drug-induced hypersensitivity syndrome (DIHS), latent human herpesvirus (HHV)-6 is frequently reactivated in association with flaring of symptoms such as fever and hepatitis. We recently demonstrated an emergence of monomyeloid precursors expressing HHV-6 antigen in the circulation during this clinical course. METHODS: To clarify the mechanism of HHV-6 reactivation, we immunologically investigated peripheral blood mononuclear cells (PBMCs), skin-infiltrating cells, and lymphocytes expanded from skin lesions of patients with DIHS. RESULTS: The circulating monomyeloid precursors in the patients with DIHS were mostly CD11b(+) CD13(+) CD14(-) CD16(high) and showed substantial expression of skin-associated molecules, such as CCR4. CD13(+) CD14(-) cells were also found in the DIHS skin lesions, suggesting skin recruitment of this cell population. We detected high levels of high-mobility group box (HMGB)-1 in blood and skin lesions in the active phase of patients with DIHS and showed that recombinant HMGB-1 had functional chemoattractant activity for monocytes/monomyeloid precursors in vitro. HHV-6 infection of the skin-resident CD4(+) T cells was confirmed by the presence of its genome and antigen. This infection was likely to be mediated by monomyeloid precursors recruited to the skin, because normal CD4(+) T cells gained HHV-6 antigen after in vitro coculture with highly virus-loaded monomyeloid precursors from the patients. CONCLUSIONS: Our results suggest that monomyeloid precursors harboring HHV-6 are navigated by HMGB-1 released from damaged skin and probably cause HHV-6 transmission to skin-infiltrating CD4(+) T cells, which is an indispensable event for HHV-6 replication. These findings implicate the skin as a cryptic and primary site for initiating HHV-6 reactivation.

[Acute human herpesvirus 6 (HHV-6) infections: when and how to treat?]. / Infections aiguës à herpèsvirus humain 6 (HHV-6) : quand et comment traiter ?

The pathogenicity of human herpesvirus 6 (HHV-6) still raises numerous questions. Acute HHV-6 infections correspond to primary infections, reactivations or exogenous reinfections. The expression of related clinical symptoms is highly variable but may be extremely severe, particularly among immunocompromised patients. The prototypic severe disease associated with these infections is limbic encephalitis occurring in stem cell transplant recipients. The diagnosis of acute HHV-6 infections relies on the quantitation of viral load in whole blood by means of quantitative PCR. The demonstration of HHV-6 causative role in the genesis of clinical symptoms requires additional investigations such as the search for HHV-6 DNA in cerebrospinal fluid in case of encephalitis. The chromosomal integration of HHV-6 DNA is a rare event among HHV-6-infected subjects but may alter the interpretation of virological results. Therapy with ganciclovir, foscarnet or cidofovir has not yet clear indications but, at the current stage of knowledge, only concerns the treatment of highly symptomatic infections. The usefulness of prophylactic or pre-emptive antiviral chemotherapy has not yet been convincingly demonstrated. Treatment efficacy must be checked through a clinical and virological follow up, based in part on quantitative PCR approaches. Controlled studies are urgently needed with the goal of evaluating the cost-effectiveness of HHV-6 follow up and therapy in different clinical situations.

The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro.

Previous research has suggested that human herpesvirus-6 (HHV-6) may integrate into host cell chromosomes and be vertically transmitted in the germ line, but the evidence--primarily fluorescence in situ hybridization (FISH)--is indirect. We sought, first, to definitively test these two hypotheses. Peripheral blood mononuclear cells (PBMCs) were isolated from families in which several members, including at least one parent and child, had unusually high copy numbers of HHV-6 DNA per milliliter of blood. FISH confirmed that HHV-6 DNA colocalized with telomeric regions of one allele on chromosomes 17p13.3, 18q23, and 22q13.3, and that the integration site was identical among members of the same family. Integration of the HHV-6 genome into TTAGGG telomere repeats was confirmed by additional methods and sequencing of the integration site. Partial sequencing of the viral genome identified the same integrated HHV-6A strain within members of families, confirming vertical transmission of the viral genome. We next asked whether HHV-6A infection of naïve cell lines could lead to integration. Following infection of naïve Jjhan and HEK-293 cell lines by HHV-6, the virus integrated into telomeres. Reactivation of integrated HHV-6A virus from individuals' PBMCs as well as cell lines was successfully accomplished by compounds known to induce latent herpesvirus replication. Finally, no circular episomal forms were detected even by PCR. Taken together, the data suggest that HHV-6 is unique among human herpesviruses: it specifically and efficiently integrates into telomeres of chromosomes during latency rather than forming episomes, and the integrated viral genome is capable of producing virions.