Betaherpesviruses Research Articles

Human Cytomegalovirus-Specific CD4+T-Cell Clones Recognize Cross-Reactive Peptides From the Immediate Early 1 Protein

Human cytomegalovirus (HCMV) is a beta-herpes virus that persists in a latent state in immunocompetent individuals. Both CD4(+) and CD8(+) T lymphocytes have been reported to be present at a high frequency in HCMV-seropositive individuals and are involved in the control of infection. How such frequencies are maintained is not completely understood. We have observed that the canonical HLA-DR8 epitope of the immediate early 1 protein (IE1) contained in the IE1 (156--175) sequence shares homologies with an IE1 sequence contained in part in the previously reported HLA-DR3 epitope, IE1 (91-110). We thus wondered whether such homology in a single protein would translate into recognition of the IE1 homolog sequence by HLA-DR8-restricted CD4(+) cells in addition to the canonical epitope. We found that established HLA-DR8-restricted T cell clones are also able to cross-recognize the IE1 (91--110) peptide, as well as a shorter 14-mer, IE1 (91--104). Moreover, the homolog peptide IE1 (91-110) was able to generate, from a seropositive blood donor, new IE1-specific, HLA-DR8-restricted CD4(+) T cell clones that were also cross-reactive. Those findings may provide clues to the formation and regulation of the T-cell repertoire and memory.

The Crystal Structure of the Herpes Simplex Virus 1 ssDNA-binding Protein Suggests the Structural Basis for Flexible, Cooperative Single-stranded DNA Binding

All organisms including animal viruses use specific proteins to bind single-stranded DNA rapidly in a non-sequence-specific, flexible, and cooperative manner during the DNA replication process. The crystal structure of a 60-residue C-terminal deletion construct of ICP8, the major single-stranded DNA-binding protein from herpes simplex virus-1, was determined at 3.0 A resolution. The structure reveals a novel fold, consisting of a large N-terminal domain (residues 9-1038) and a small C-terminal domain (residues 1049-1129). On the basis of the structure and the nearest neighbor interactions in the crystal, we have presented a model describing the site of single-stranded DNA binding and explaining the basis for cooperative binding. This model agrees with the beaded morphology observed in electron micrographs.

Encephalitis Owing to Human Herpesvirus-6 after Cardiac Transplant

Human herpesvirus-6 (HHV-6), a beta herpesvirus closely related to cytomegalovirus (CMV), infects the majority of the population in childhood. Human herpesvirus-6 can be reactivated in the immunosuppressed patient. After bone marrow and orthotopic liver transplant, it has been linked to various clinical syndromes, including undifferentiated febrile illness, encephalitis, pneumonitis and bone marrow suppression. To date its infectious role after orthotopic heart transplant has not been well documented. We present the case of a 32-year-old cardiac transplant recipient who initially presented 8 weeks after his transplant with high fever and headache. He developed increasing confusion, pulmonary infiltrates and neutropenia. Cytomegalovirus viral loads were negative. Polymerase chain reaction (PCR) of blood and cerebrospinal fluid detected HHV-6 DNA, consistent with HHV-6-related encephalitis, pneumonitis and bone marrow suppression. He was treated with foscarnet with gradual improvement in clinical status. We review the literature on the significance of this virus post cardiac transplant.

Simian cytomegalovirus encodes five rapidly evolving chemokine receptor homologues.

Many herpesviruses, poxviruses and retroviruses encode proteins related to chemokines and chemokine receptors. The first one discovered, US28 of human cytomegalovirus (HCMV), is a 7-transmembrane domain G protein-coupled chemokine receptor able to activate diverse cellular responses, including cell migration and gene expression. A related ORF named US27 is adjacent to US28, but no functions have been defined yet. Recently ORFs 3-7, a cluster of five concatenated ORFs with highest homology to US28 and mammalian chemokine receptors, were sequenced from a prototype "stealth virus", an African green monkey simian CMV (SCMV)-related entity with unusual fungal, bacterial and mammalian gene homologues. Stealth viruses have not yet been independently replicated in tissue culture, and therefore their biological significance remains unclear. ORF3, ORF4, ORF5 and ORF6 are complete ORFs whereas the sequence of ORF7 is incomplete. In the present study, we identified five corresponding ORFs in the genome of a clinical isolate of bonafide simian CMV (SCMV), strain 9610. We found substantial differences between the SCMV and "stealth virus" ORFs, especially for ORF5 where there are 31% non-identities at the amino acid level. Four conserved genes unrelated to chemokines (64K/CAP, DNBI, UL32, and IE2) in SCMV and HCMV had on average 52% identity at the deduced amino acid level, whereas the corresponding values for the SCMV ORFs versus US28 ranged from 21% to 30%, suggesting rapid gene diversification in this cluster. Consistent with this, the amino acid identity for any pairwise comparison among the SCMV ORFs is only 21-52%. The chemokine receptor homologues are estimated to comprise approximately 2-3% of the SCMV genome. HCMV US27 and US28 homologues have also been identified in the chimpanzee CMV genome, whereas mouse and rat CMV lack chemokine receptor homologues. This genomic analysis indicates that SCMV has an unusually high concentration of US28-related chemokine receptor homologues that have arisen by gene duplication and have diverged extensively from their closest relatives in mammals and other beta herpesviruses. The rate of divergence appears to be very rapid compared to other known SCMV genes, suggesting strong positive selection.

HUMAN HERPESVIRUS 6

Background. Human herpesvirus 6 belongs to betaherpesviruses. This is a lymphotropic virus which is widely spread in a population. The most frequent way of virus transmission is by saliva. For this reason the first contact usally occurs early in a childhood period. Clinical manifestation might be expressed as exanthem subitum or roseola infantum. In adults primary infections are a rare event while virus reactivation might be very frequent. Conclusions. Human herpesvirus 6 is believed to be the most neurotropic among all herpesviruses. It even can cause central nervous system disease in an immunocompetent person. The most important role of human herpesvirus 6 is in provoking complications in HIV-infected individuals and in patients after organ transplantations, as it causes immunosupression even more severe. The possible connection of human herpesvirus 6 with multiple sclerosis, chronic fatigue syndrome and some neoplasmas remains to be clarified. Human herpesvirus 6 infection can be most easily diagnosed by serological methods; the virus could be detected by monoclonal antibodies and by the use of the methods of molecular biology.

Latency and reactivation of human cytomegalovirus.

The sequence analysis of herpesviruses suggests they have been evolving with their individual vertebrate hosts for millions of years, and their divergence parallels that of the hosts they infect. Given this time they have been learning to live with their individual hosts, it is not surprising that they have become extremely well adapted to doing so without causing much in the way of obvious disease. A key feature of their strategy for persisting in the host is the ability of all herpesviruses to establish latent infection-a state in which no, or only a very limited set of, viral genes are expressed in cells in which viral DNA persists. The alpha herpesviruses (herpes simplex and varicella zoster virus) establish latency in neuronal cells in sensory ganglia: these are long lived non-dividing cells and the alpha herpesviruses persist in these with expression of only the latency associated transcripts-although the function of these RNA transcripts remains incompletely understood. The principal gamma herpesvirus of humans, Epstein Barr virus (EBV), is latent mainly in B lymphocytes: EBV persistence in B cells is associated with expression of a limited set of viral genes encoding functions necessary for the maintenance of the episomal viral DNA as B cells divide.The mechanism by which the principal beta herpesvirus of humans-human cytomegalovirus (HCMV) persists, is also incompletely understood and the subject of this review. Understanding how HCMV persists has clinical relevance in that its transmission to seronegative recipients might be more easily prevented, and the mechanisms by which it produces disease in the neonate and immunocompromised hosts more easily understood, if we knew more about the cells in which the virus is latent and the way in which it reactivates.

A rapid monitoring system of human herpesviruses reactivation by LightCycler in stem cell transplantation.

To establish a practical monitoring system of human herpesviruses reactivation in patients undergoing stem cell transplantation, we developed a new, very rapid, highly sensitive, and quantitative PCR assay for accurate measurement of human cytomegalovirus (CMV), human herpesvirus 6 (HHV-6) and Epstein-Barr virus (EBV) DNA using LightCycler. The LightCycler system revealed that there was a linear correlation in the wide range of viral template DNA at the indicated number of PCR cycles. Peripheral blood cells were collected from 16 patients undergoing stem cell transplantation. The cut-off level of CMV and HHV-6 was assessed as 10(2) copies/microg and that of EBV as 10(3). High numbers of CMV genomes were detected in 3/13 patients after transplant, and reactivation of HHV-6 was frequently seen, whereas none of the patient showed an elevation of EBV genome copies until the end of the observation period. In the present study, the reactivation of beta herpesviruses is associated with the occurrence of thrombotic microangiopathy (TMA) in two patients undergoing allogeneic BMT. Therefore, it may contribute in clarifying the pathological potential of human herpesviruses using a large number of clinical samples. Our results suggest that this system may be useful for monitoring viral reactivation.

Prevalence of IgM and IgG Antibody to HHV-6 and HHV-8 and Results of Plasma PCR to HHV-6 and HHV-7 in a Group of CFS Patients and Healthy Donors

ABSTRACTHuman herpes virus-6 (HHV-6) is a beta herpes virus that was first described in 1986 and which occurs in the form of at least two variants, A and B. Healthy donors in the general population are carriers for mainly the B variant, in whom 90% harbor the DNA of this type in their peripheral blood mononuclear cells (PBMNC). A higher prevalence of this virus has been detected by testing of plasma and PBMNCs by IFA, ELISA and by the nested PCR technique, in addition to direct culture for HHV-6 in certain groups of immunesuppressed patients such as those with multiple sclerosis and HIV. It has also been isolated to a greater degree using these techniques from patients who meet the case definition for the chronic fatigue syndrome (CFS). We determined IgG and IgM antibody titers to HHV-6; IgG to HHV-8 and performed PCR testing for HHV-6 on the plasma of 46 patients with CFS and on 7 healthy donors (HD). We also performed PCR testing for HHV-7 on 15 CFS patients and on 4 HD(s). We found a higher prevalence ...

Association of human herpes virus 6 (HHV-6) with multiple sclerosis: increased IgM response to HHV-6 early antigen and detection of serum HHV-6 DNA.

Viruses have long been suggested to be involved in the etiology of multiple sclerosis (MS). This suggestion is based on (1) epidemiological evidence of childhood exposure to infectious agents and increase in disease exacerbations with viral infection; (2) geographic association of disease susceptibility with evidence of MS clustering; (3) evidence that migration to and from high-risk areas influences the likelihood of developing MS; (4) abnormal immune responses to a variety of viruses; and (5) analogy with animal models and other human diseases in which viruses can cause diseases with long incubation periods, a relapsing-remitting course, and demyelination. Many of these studies involve the demonstration of increased antibody titers to a particular virus, whereas some describe isolation of virus from MS material. However, no virus to date has been definitively associated with this disease. Recently, human herpesvirus 6 (HHV-6), a newly described beta-herpes virus that shares homology with cytomegalovirus (CMV), has been reported to be present in active MS plaques. In order to extend these observations, we have demonstrated increased IgM serum antibody responses to HHV-6 early antigen (p41/38) in patients with relapsing-remitting MS (RRMS), compared with patients with chronic progressive MS (CPMS), patients with other neurologic disease (OND), patients with other autoimmune disease (OID), and normal controls. Given the ubiquitous nature of this virus and the challenging precedent of correlating antiviral antibodies with disease association, these antibody studies have been supported by the detection of HHV-6 DNA from samples of MS serum as a marker of active viral infection.

PCR identification and differentiation of baboon cytomegalovirus from other human and nonhuman primate cytomegalovirus

Background: Differential identification of baboon alpha, beta, and gamma herpesviruses is an essential technology in order to monitor xenozoonotic transmission of baboon viruses to foreign species organ and/or cell recipients. We present polymerase chain reaction techniques that will differentiate known baboon cytomegaloviruses (CMV) from their closely related counterparts found in humans. Methods and Results: Polymerase chain reaction techniques for identification of the known beta herpesviruses commonly present in the baboon are reported. The techniques described also permit the unequivocal differentiation of these virus types from closely related human as well as other nonhuman primate viruses in the family herpesviridae. Methods are based upon sequence analysis of specifically selected genes of baboon CMV. Primer pairs from sequence analyses were selected based upon nonhomologous sequences in gene homologues of human CMV. Unique, baboon species specific amplimers were identified both with ethidium bromide staining and with radiolabeled probe analyses. Conclusions: With the described techniques, it is possible to monitor organ/cell recipients of xenograft transplantation for the establishment of baboon CMV in the foreign human host. Monitoring can be performed throughout the life of the recipient in effort to rule out host susceptibility to baboon CMV, as well as to rule out potential host:donor recombinant viruses formed between the closely related members of herpesvirus family. (Mol Diagn 1996 Sep;1(3):267-273)

Sequence determination and genetic content of an 8.9-kb restriction fragment in the short unique region and the internal inverted repeat of Marek's disease virus type 1 DNA.

The DNA sequence (8.9 kb) covering about 70% of the short unique region (Us) and part of the short inverted repeat of the Marek's disease virus type 1 GA strain was determined. Computer analysis of the sequence showed the presence of nine potential open reading frames (ORFs), consisting of more than 300 nucleotides in the Us region. Of these ORFs, four were found to be homologous to US10 (minor virion protein), US3 (protein kinase), US2, and US6 (gD) in the Us region of alpha-herpesvirus herpes simplex virus type 1. The protein kinase homologue is especially well conserved in alpha-herpesviruses. No counterpart of the nine MDV1 ORFs was found in the beta-herpes virus human cytomegalovirus and gamma-herpesvirus Epstein-Barr virus, suggesting that MDV1 is more similar to the alpha-herpesviruses. The junction of the Us region and the short inverted repeat was also determined by comparison between the sequences of the DNA fragments, including the terminal and internal repeats. Northern blot analysis showed that the Us region within the 8.9 kb sequence was transcriptionally active in MDV1-infected cells.

Genetic relationships between bovine herpesvirus 4 and the gammaherpesviruses epstein-Barr virus and herpesvirus saimiri

The overall arrangement of genes in the unique central part of the bovine herpesvirus type 4 (BHV-4) genome has been deduced by analysis of short DNA sequences. Twenty-three genes conserved in at least one of the completely sequenced herpesviruses have been identified and localized. All of these genes encoded amino acid sequences with higher similarity to proteins of the gammaherpesviruses Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS) than to the homologous products of the alphaherpesviruses varicella-zoster virus and herpes simplex virus type 1 or the betaherpesvirus human cytomegalovirus. The genome organization of BHV-4 had also an overall collinearity with that of the gammaherpesviruses EBV and HVS. Furthermore, the BHV-4 genes content and arrangement were more similar to those of HVS than to those of EBV, suggesting that BHV-4 and HVS are evolutionarily more closely related to each other than either are to EBV. BHV-4 DNA sequences were generally deficient in CpG dinucleotide. This CpG deficiency is characteristic of gammaherpesvirus genomes and suggests that the BHV-4 latent genome is extensively methylated. Despite several biological features similar to those of beta herpesviruses, BHV-4 displays the molecular characteristics of the representative members of the gammaherpesvirinae subfamily.

Characterization of the DNA polymerase gene of human herpesvirus 6

The construction of a recombinant bacteriophage lambda library containing overlapping clones covering 155 kbp of the 161-kbp genome of the Ugandan U1102 isolate of human herpesvirus 6 (HHV-6) is described. The use of degenerate-primer polymerase chain reaction allowed the isolation of a DNA probe for the DNA polymerase gene of HHV-6, which was subsequently used to isolate and position the pol gene on the physical map of the viral genome. A 4.4-kbp EcoRI DNA restriction fragment containing the pol gene was isolated and sequenced. The open reading frames flanking the pol gene code for the HHV-6 glycoprotein B gene and the human cytomegalovirus UL53 homolog. This arrangement is different from that seen in the alpha and gamma herpesvirus families, lending further support to the notion that HHV-6 is a member of the beta herpesvirus group.

Physical structure and molecular cloning of equine cytomegalovirus DNA

Restriction endonuclease (RE) mapping studies and molecular hybridization analyses were conducted to determine the molecular structure of the genome of equine cytomegalovirus (ECMV). The ECMV genome is a linear, double-stranded DNA with a molecular size of 126 ± 0.6 MDa (189 kbp). A library of cloned BamHI, EcoRI, and HindIII fragments of the viral genome was used to construct RE maps. Individual 32P-labeled cloned DNA fragments were hybridized to Southern blots of viral genomic DNA digested to completion with BamHI, EcoRI, HindIII, or SalI. These analyses revealed that the ECMV genome consists of a 97-MDa unique long region which is bracketed by repeated sequences. At one terminus of the genome, a 21.3-MDa segment of repeated sequences with no apparent unique sequences was identified. At the other terminus, a 6-MDa unique region bracketed by 2.4-MDa repeat segments was identified. No submolar RE fragments were identified upon digestion of the ECMV genome with BamHI, EcoRI, HindIII, Sall, or other REs, including BclI, BglII, NruI, and XbaI. The genome possesses only two termini as judged by λ exonuclease digestion and by T4 DNA polymerase end-labeling of the intact DNA followed by digestion with BamHI, EcoRI, HindIII, SalI, BclI, BglII, NruI, or XbaI. In addition, Southern blot analysis of DNA extracted from ECMV-infected rabbit kidney cells revealed that only one viral DNA fragment within the intracellular viral DNA pool contains fused genomic termini. Taken together, these observations indicate that the ECMV genome does not isomerize and suggest that the genome of ECMV may be unique among those of the herpesviruses and especially those of the beta herpesviruses (cytomegaloviruses) since it contains regions of extensive internal homology yet does not undergo isomerization. Lastly, the relatively small size of the viral genome indicates an evolutionary diversification among the cytomegaloviruses.

Detailed characterization of an apparently unspliced beta herpes simplex virus type 1 gene mapping in the interior of another.

We precisely localized the coding region and determined the nucleotide sequence of a 1.2-kilobase beta herpes simplex virus type 1 mRNA which underlies the 3' region of the 5.2-kilobase beta mRNA mapping in HindIII fragment K. This mRNA, which lacks readily detectable splices, has its own promoter by the criteria of identification of putative herpes simplex virus type 1 control sequences and in vitro transcription by a Manley polymerase system.