Antiviral prophylaxis may prevent human herpesvirus-6 reactivation in bone marrow transplant recipients.

Human herpesvirus-6 (HHV-6) infects the majority of children under the age of 2 years causing roseola infantum. Following short self-limited disease, the virus enters into a latency phase in peripheral blood lymphocytes (PBL). It has been previously reported that HHV-6 reactivation from latency, in immunocompromised patients undergoing bone marrow transplantation (BMT), could result in febrile illness, pneumonitis, meningitis, and/or encephalitis. In our study, 14 BMT patients received two different antiviral prophylactic therapies: 8 patients received acyclovir, whereas 6 patients received ganciclovir. Clinical manifestations and virus recovery were monitored pre- and post-BMT by polymerase chain reaction tests of cord blood cells cultured with the patients' PBL. No HHV-6 recovery was shown in the 6 patients treated with ganciclovir, whereas 3 of the 8 acyclovir-treated patients experienced virus reactivation 20-21 days post-BMT. One of the 3 patients was asymptomatic but had late engraftment; the second patient had prolonged fever, skin rash, and hemorrhage; the third patient experienced prolonged fever, pneumonitis, marrow rejection, and fatal encephalitis. It is concluded that viral reactivation may be prevented by prophylactic treatment with ganciclovir. Our observation awaits further documentation in prospective randomized trials in high-risk BMT recipients.

Tamplicon-7, a novel T-lymphotropic vector derived from human herpesvirus 7.

We describe the derivation of a novel T-cell-defective virus vector employing the human herpesvirus 7 (HHV-7). The new vector, designated Tamplicon-7, replicates in CD4(+) T cells. The system is composed of a helper virus and defective virus genomes derived by the replication of the input Tamplicon vector. There are two cis-acting functions required for the replication and packaging of the defective virus genomes in the presence of the helper virus: the viral DNA replication origin and the composite cleavage and packaging signal, which directs the cleavage and packaging of defective virus genomes. Viral DNA replication is compatible with the rolling circle mechanism, producing large head-to-tail concatemers of the Tamplicon vector. Thus, in the presence of the helper virus, the replicated vectors are packaged and secreted into the medium. Furthermore, we have shown that the vector can be employed to express a foreign gene, encoding the green fluorescent protein, in the T cells infected with the HHV-7 helper virus. We predict that the Tamplicon-7 vector might be potentially useful for gene therapy of diseases affecting the human CD4(+) T cells, including autoimmune diseases, T-cell lymphomas, and AIDS.

The DNA sequence of the RK strain of human herpesvirus 7.

The complete DNA sequence of human herpesvirus-7 (HHV-7) strain RK was determined following direct cloning of virion DNA fragments into a sequencing vector. The sequence was compared with the previously published complete sequences of HHV-7 strain JI and human herpesvirus-6 (HHV-6) strain U1102. Despite a very close relationship between the two HHV-7 strains, differences are apparent in regions containing tandem reiterations, particularly in the "telomeric" reiterations located near the termini of the large direct repeat at the genome ends, and in a total of 179 additional positions distributed throughout the genome (i.e., about one nucleotide difference per kbp). This extent of divergence implies that the two strains arose from an ancestral virus several thousands of years ago. Differences that affect coding potential do not cluster in particular protein-coding regions, indicating that specific HHV-7 genes have not been measurably subject to unusual evolutionary pressures since divergence. Reassessments of genetic content indicated that the HHV-7 genome contains 84 different genes, whereas the HHV-6 genome contains 85. All HHV-7 genes but 1 have direct HHV-6 counterparts, and all but 2 HHV-6 genes have HHV-7 homologues. Sequence comparisons between HHV-7 and HHV-6 provided evidence that the protein-coding regions of 11 genes are expressed by splicing.

HSV Amplicons in Gene Therapy.

Herpes simplex virus (HSV) amplicons are defective virus vectors capable of introducing amplified foreign genes into variable types of eukaryotic cells, such as fibroblasts, macrophages, glia, and neurons in different organisms including rodents, monkeys, and human (refs. 1-3; reviewed in ref. 4). The defective viruses follow their nondefective counterparts in the ability to infect mitotic, as well as postmitotic cells. This makes them potentially useful vectors for use in nondividing cells, such as in nerve cells. Available retrovirus vectors employed to date for gene therapy require cell division and therefore cannot be used to target neurons.

Human herpesvirus 7 (HHV-7) DNA: analyses of clones spanning the entire genome.

Human herpesvirus 7 (HHV-7) is a lymphotropic virus which replicates in CD4+ T cells. The majority of DNA sequences of HHV-7, strain RK, were cloned, and the termini of the viral DNA inserts were sequenced. The clones were found to contain sequences homologous to HHV-6 DNA with the average relatedness amounting to 60% in the nucleotide sequence and 53.4% in the corresponding amino acid sequences. Human cytomegalovirus (HCMV) DNA was also found homologous but to a lower extent, while the human alpha and gamma herpesviruses were more distinct. Some genes were found to be better conserved in all herpesviruses (core genes). These findings define the genetic makeup of HHV-7 DNA.

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.

Restriction endonuclease mapping and molecular cloning of the human herpesvirus 6 variant B strain Z29 genome.

Human herpesvirus 6(HHV-6) variants A and B differ in cell tropism, reactivity with monoclonal antibodies, restriction endonuclease profiles, and epidemiology. Nonetheless, comparative nucleotide and amino acid sequences from several genes indicate that the viruses are very highly conserved genetically, The B variant is the major etiologic agent of exanthem subitum and is frequently isolated from children with febrile illness; no disease has been etiologically associated with HHV-6A. One HHV-6A strain has been cloned and sequenced, but similar information and reagents are not available for HHV-6B. We report here the determination of maps of the restriction endonuclease cleavage sites for BamHI, C1aI, HindIII, KpnI, and Sa1I, and the cloning in plasmids and bacteriophages of fragments representing over 95% of the HHV-6B strain Z29 [HHV-6B(Z29)] genome. Hybridization experiments and orientation of several blocks of nucleotide sequence information onto the genomic map indicate that HHV-6A and HHV-6B genomes are colinear.

Coinfection with herpesviruses in young children of HIV-infected women.

Coinfection with herpesviruses in young children born to human immunodeficiency virus (HIV)-infected women was studied with blood samples from children who were 9-12 months and 15-24 months of age. Three groups of children were included: (I) HIV-uninfected, asymptomatic (HIV-); (II) polymerase chain reaction (PCR) and/or culture-positive and asymptomatic or mildly symptomatic (HIV+ asymptomatic); and (III) PCR and/or culture-positive and symptomatic (HIV+ symptomatic). Significantly more of the HIV+ symptomatic patients had cytomegalovirus (CMV) antibody than the HIV patients. In addition, CMV antibody levels were significantly higher in the HIV+ symptomatic patients than in either of the other two groups. Human herpesvirus 7 (HHV-7) antibody titers were significantly different among the three groups of patients; however, no pairwise comparisons were significant. No differences were found for HHV-6 or Epstein-Barr virus (EBV) antibody frequencies or titers. These findings suggest that infection with CMV is a cofactor or an opportunistic infection causing symptomatic HIV infections in young children.

Bone marrow transplant recipients harbor the B variant of human herpesvirus 6.

Lymphotropic herpesviruses have recently been isolated from patients undergoing kidney, liver and bone marrow transplantation (BMT). We have molecularly characterized isolates of HHV-6 from children undergoing allogenic BMT. We show that viruses recovered from the BMT recipients correspond to the B variant of human herpesvirus 6, the etiological agent of exanthem subitum. HHV-6 B strains are known to be associated with infections in the majority of children in infancy or early childhood. Infection in the BMT recipients most likely reflects the reactivation of these viruses during immunosuppressive treatment. Each of the reactivated viruses has its own characteristic heterogeneous (het) sequence. We show that the het sequence is stably unique for each HHV-6 strain. We also show that prolonged viremia can be documented with some of the patients, unlike the characteristic short duration febrile sickness in the exanthem subitum cases of infancy. The mechanism of HHV-6 B reactivation from latency is not known. Furthermore, the full clinical outcome of HHV-6 B viremia in the BMT patients requires further investigation.

A transforming fragment within the direct repeat region of human herpesvirus type 6 that transactivates HIV-1.

HHV-6 infection has been associated with several malignancies including non-Hodgkin's lymphoma and Hodgkin's disease by the presence of high antibody titer and/or the presence of HHV-6 DNA. To understand their oncogenic potential, SalI restriction fragments from HHV-6 strain U1102 were transfected into NIH3T3 cells to assess transforming ability. A 3.9-kbp SalI-L DNA fragment spanning the junction of the direct repeat left (DRL) and unique long segment (UL) regions of HHV-6 induced foci of morphologically altered cells. The SalI-L transformed NIH3T3 focal lines induced tumors in nude mice within 2 weeks. The retention of HHV-6 specific DNA observed in SalI-L transformed cells and their tumor-derived lines suggest a possible maintenance function. Since both HHV-6 infection as well as transforming fragments from other DNA viruses have been shown to transactivate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR), SalI-L was examined for transactivation activity. SalI-L up-regulated HIV-1 LTR CAT 10-15 fold in both monkey CV-1 and human T Jurkat cells. The further study of the SalI-L transforming fragment exhibiting transactivation of HIV-1 LTR will elucidate whether these two activities are encoded by a single gene and will aid in the understanding of the interaction between HHV-6 and HIV-1 as it relates to progression of AIDS and/or AIDS-related malignancies.

Minireview: the herpes simplex virus amplicon--a versatile defective virus vector.

The Herpes simplex virus (HSV) amplicon was designed in our laboratory, as a defective virus vector, capable of shuttle delivery of DNA sequences and genes from prokaryotic to eukaryotic cells, tissues or organs. The HSV vector was termed "amplicon" to delineate the fact that it carried the cloned sequences of interest, within amplified concatemeric defective genomes, packaged in HSV virions. Employing the replication functions of their helper virus, the amplicons are wide tropic vectors, capable of entry, replication, and gene expression in varied types of cells, both in vitro, and in vivo. In this brief review, the amplicons will be revisited, beginning with studies of the naturally occurring defective genomes, where many of the virologic aspects of the system were established. Properties of the amplicon system and its components, will be enlightened. The cis acting functions required for amplicon propagation will be briefly described. This will follow with examples of our studies of prokaryotic, viral and eukaryotic DNA sequences, which were amplified and expressed within HSV defective genomes. Studies designed to examine the involvement of helper viruses will be described, including our analyses of host shutoff mutant helper viruses. Finally, recent studies of other laboratories will be reviewed, with emphasis on what appears to be the use of amplicons as neurotropic vectors, towards potential gene therapy.

Seroepidemiology of human herpesvirus 7 in healthy children and adults in Japan.

The isolation of human herpesvirus 7 (HHV-7) from saliva and blood, and the prevalence of antibodies to the virus in healthy individuals were investigated in Japan. By cocultivating samples with phytohemagglutinin-P-stimulated cord blood mononuclear cells, HHV-7 was isolated from the saliva of 1 of 20 children and from 4 of 38 adults but not from their blood. The isolates were confirmed as closely related to RK strain of HHV-7, but not to U1102 (human herpesvirus 6, HHV-6 type A) or Z29 (HHV-6 type B) strains by restriction cleavage patterns of the DNA. The virus antibody of 330 healthy children and adults was measured with an indirect immunofluorescence assay, using one of our isolates (FG7-6). The positivity rate of antibody was 40% in the first 2 months of life, declined during the first 6 months, then gradually increased and was 45% at 1-4 years of age. It reached the highest level (60%) at 11-13 years of age and was maintained until the end of the third decade, then decreased thereafter. Additionally, no simultaneous rise in the antibody titers was observed in 7 virologically confirmed exanthem subitum patients.

Human herpesvirus 7 is a constitutive inhabitant of adult human saliva.

We report the frequent isolation of human herpesvirus 7 from the saliva of healthy adults. Virus isolates recovered from different individuals exhibited minimal restriction enzyme polymorphism, which was mostly confined to heterogeneous (het) sequences in the genome. DNAs of isolates recovered from the same individual over a period of several months showed the same characteristic het fragments, indicating the stability of the het sequences upon virus replication and shedding in vivo. In contrast to the results of previous reports, human herpesvirus 6, the causative agent of roseola infantum, could not be isolated from the saliva specimens, raising questions regarding oral transmission of human herpesvirus 6 and human herpesvirus 7 to young children.

HHV-6 and HHV-7 as exogenous agents in human lymphocytes.

Human herpesviruses 6 and 7 (HHV-6 and HHV-7), the newly recognized lymphotropic members of the herpesvirus family, were isolated from peripheral blood mononuclear cells (PBMC) of healthy individuals. HHV-7 was recovered from PBMC after exposing the cells to conditions leading to T cell activation. No virus could be recovered from quiescent non-activated cultures, suggesting that the virus resided latently in these cells and that it could be induced from latency by T cell activation. HHV-6 could not be recovered from PBMC using similar approaches. However, it could be repeatedly reactivated from PBMC after infection of the cells with HHV-7. Upon further passaging in culture, HHV-6 took over the population and virus stocks appeared to be clear of the original HHV-7 helper virus. The results of this study suggest that genomes of the lymphotropic herpesviruses are resident in PBMC of healthy adults. HHV-7 can supply the functions required for its reactivation from latency. It can also act as a helper virus for the reactivation of HHV-6 from latency. Following the initial reactivation step, HHV-6 can replicate autonomously in the cells. These results may be relevant to the findings that HHV-6 infection is induced in immunosuppressed patients and in patients undergoing organ transplantation. Furthermore, these studies may exemplify situations whereby propagation of known agent(s) in vitro results in the activation of human pathogens resident latently in the cells.

Human herpesvirus 7: antigenic properties and prevalence in children and adults.

The recent isolation of human herpesvirus 7 (HHV-7) from activated CD4+ T lymphocytes of a healthy individual raises questions regarding the prevalence of this virus in humans and its immunological relationship to previously characterized human herpesviruses. We report that HHV-7 is a ubiquitous virus which is immunologically distinct from the highly prevalent T-lymphotropic HHV-6. Thus, (i) only two of six monoclonal antibodies to HHV-6 cross-reacted with HHV-7-infected cells, (ii) Western immunoblot analyses of viral proteins revealed different patterns for HHV-6- and HHV-7-infected cells, (iii) tests of sequential serum samples from children revealed seroconversion to HHV-6 without concomitant seroconversion to HHV-7, and (iv) in some instances HHV-7 infection occurred in the presence of high titers of HHV-6 antibodies, suggesting the lack of apparent protection of children seropositive for HHV-6 against subsequent infection with HHV-7. On the basis of the analyses of sera from children and adults it can be concluded that HHV-7 is a prevalent human herpesvirus which, like other human herpesviruses, infects during childhood. The age of infection appears to be somewhat later than the very early age documented for HHV-6.

Differentiation between two distinct classes of viruses now classified as human herpesvirus 6.

Human herpesvirus 6 (HHV-6) causes exanthem subitum (ES, roseola infantum), a childhood disease characterized by high fever and skin rash. We have analyzed restriction enzyme cleavage patterns of the DNAs of ES virus isolates from Japan and the United States. The patterns of all the ES viral DNAs were highly conserved, except for variable sequences within the terminal repeat sequences. They resembled closely the restriction enzyme patterns of the Z29 strain of HHV-6 but were distinct from those of the U1102 strain. That all ES isolates were closely related whereas the U1102 patterns were very different suggests that the U1102 strain represents a distinct virus. Moreover, the ES isolates all resembled the Z29 strain and not the U1102 strain with respect to reactivity with HHV-6 monoclonal antibodies. These findings provide evidence for the existence of two distinct classes of viruses previously classified as HHV-6. Whereas the Z29-like viruses are involved in ES infections, the association of the U1102-like viruses with human disease has yet to be determined.