Specificity analysis of human CD4+ T-cell clones directed against human herpesvirus 6 (HHV-6), HHV-7, and human cytomegalovirus.

In order to clarify antigenic variations among various isolates of human herpesvirus 6 (HHV-6) and cross-reactivity among HHV-6, HHV-7, and human cytomegalovirus (HCMV) in the T-cell immune response, the antigenic specificity of the proliferative response mediated by 232 CD4+ human T-cell clones directed against HHV-6, HHV-7, or HCMV was examined. The results obtained were as follows. (i) Although the majority of T-cell clones directed against HHV-6 proliferated in response to stimulation with all strains of HHV-6 used (U1102, Z29, SF, and HST), 7% (8 of 122) of the T-cell clones showed distinct patterns of proliferative response against strain U1102 (group A) and other strains of HHV-6 (group B). (ii) Of 99 T-cell clones, 71 showed a distinct proliferative response to HHV-6 and HHV-7, whereas 28 proliferated in response to stimulation with both HHV-6 and HHV-7. (iii) A small number of T-cell clones (9 of 232) showed cross-reactivity against HHV-6 and HCMV, and 2 of the 232 clones were reactive with HCMV as well as with HHV-6 and HHV-7. (iv) The specificity of gamma interferon production by T-cell clones following the stimulation with virus antigen was identical to that of their proliferative response. These data thus indicate the presence of antigenic variations among isolates of HHV-6 and also epitopes common to HHV-6 and HHV-7 and to HHV-6, HHV-7, and HCMV which are recognized by CD4+ T cells.     

Identification of the human herpesvirus 6 glycoprotein H and putative large tegument protein genes.

Determination of the nucleotide sequences of two molecular clones of human herpesvirus 6 (HHV-6) (strain GS) and comparison with those of human cytomegalovirus (HCMV) has allowed the identification of the genes for the glycoprotein H (gH) and the putative large tegument protein of HHV-6. Two molecular clones of fragments of HHV-6, the BamHI-G fragment (7,981 bp) of the clone termed pZVB43 and a HindIII fragment (8,717 bp) of the clone termed pZVH14, represent approximately 10% of the HHV-6 genome (16,689). An open reading frame within the BamHI-G fragment was designated the gH gene of HHV-6 because of the extensive sequence similarity of its predicted product (79,549 Da) to the HCMV gH gene product. The predicted product (239,589 Da) of an open reading frame within clone pZVH14 showed homology to the predicted product of the proposed gene of HCMV representing the large tegument protein. Computer analyses indicated a closer relationship of the predicted peptides of these HHV-6 genes to those of HCMV than to those of the other human herpesviruses Epstein-Barr virus, herpes simplex virus type 1, and varicella-zoster virus. The gH gene was more conserved among the human herpesvirus group, while significant sequence similarity of the tegument gene could be found only with that of HCMV. The data reported here with one conserved gene (gH) and a more divergent gene (tegument) support previous reports that HHV-6 and HCMV are more closely related to each other than to the other well-characterized human herpesviruses.     

A human cytomegalovirus function inhibits replication of herpes simplex virus.

Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 micrograms/ml) for 3 or 24 h, conditions known to result in accumulation of HCMV immediate-early and early mRNA, was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5 degrees C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. However, replication of adenovirus, another DNA virus, was not restricted in these cells under the same conditions. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with [3H]thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection. In addition, superinfection with vesicular stomatitis virus ruled out a role for interferon in restriction of HSV replication in this system.     

Efficacy of methylenecyclopropane analogs of nucleosides against herpesvirus replication in vitro

We have reported previously that purine methylenecyclopropane analogs are potent agents against cytomegaloviruses. In an attempt to extend the activity of these compounds, the 2-amino-6-cyclopropylaminopurine analog, QYL-1064, was selected for further study by modifying the purine 6 substituent. A total of 22 analogs were tested against herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), murine cytomegalovirus (MCMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6) and human herpesvirus type 8 (HHV-8). Ten of the analogs had activity against at least one of the viruses tested. One compound had moderate activity against HSV-1 and six had activity against VZV. All but one compound was active against HCMV with a mean EC50 of 2.1 +/- 0.6 microM, compared with a mean EC50 of 3.9 +/- 0.8 microM for ganciclovir. Of special interest was the fact that eight of the ten compounds were active against both HHV-6A and HHV-6B with mean EC50 values of 6.0 +/- 5.2 mciroM and <2.4 +/- 1.5 microM, respectively. Only two compounds had activity against EBV, whereas all but one compound was active against HHV-8 with a mean EC50 of 3.1 +/- 1.7 microM. These results indicate that members of this series of methylenecyclopropane analogs are highly active against HCMV, HHV-6, and HHV-8 but are less active against HSV, VZV, and EBV.     

The UL97 protein kinase of human cytomegalovirus and homologues in other herpesviruses: impact on virus and host

The human herpesviruses, herpes simplex virus 1 (HSV-1), HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6A (HHV-6A), HHV-6B, HHV-7 and HHV-8, establish persistent infections with possible recurrence during immunosuppression. HCMV replication is inhibited by the nucleoside analogue ganciclovir (GCV), the compound of choice for the treatment of HCMV diseases and preemptive treatment of infections. The viral UL97 protein (pUL97) which shares homologies with protein kinases and bacterial phosphotransferases is able to monophosphorylate GCV. Homologues of pUL97 are found in HSV (UL13), VZV (ORF47), EBV (BGLF4), HHV-6 (U69), HHV-8 (ORF36) as well as in murine CMV (M97) or rat CMV (R97). Several indolocarbazoles have been reported to be specific inhibitors of pUL97. The protein is important for efficient replication of the virus. Autophosphorylation of pUL97 was observed using different experimental systems. Most recently, it has been shown that pUL97 interacts with the DNA polymerase processivity factor pUL44. Indolocarbazole protein kinase inhibitors are promising lead compounds for the development of more specific inhibitors of HCMV.     

Human herpesvirus 6A U27 plays an essential role for the virus propagation

Human herpesvirus 6A (HHV-6A) is a member of the genus Roseolovirus and the subfamily Betaherpesvirinae. It is similar to and human cytomegalovirus (HCMV). HHV-6A encodes a 41 kDa nuclear phosphoprotein, U27, which acts as a processivity factor in the replication of the viral DNA. HHV-6A U27 has 43% amino acid sequence homology with HCMV UL44, which is important for DNA replication. A previous study on HHV-6A U27 revealed that it greatly increases the in vitro DNA synthesis activity of HHV-6A DNA polymerase. However, the role of U27 during the HHV-6A virus replication process remains unclear. In this study, we constructed a U27-deficient HHV-6A mutant (HHV-6ABACU27mut) with a frameshift insertion at the U27 gene using an HHV-6A bacterial artificial chromosome (BAC) system. Viral reconstitution from the mutant BAC DNA was not detected, in contrast to the wild type and the revertant from the U27 mutant. This suggests that U27 plays a critical role in the life cycle of HHV-6A.     

Human herpesvirus 6 is closely related to human cytomegalovirus.

A sequence of 21,858 base pairs from the genome of human herpesvirus 6 (HHV-6) strain U1102 is presented. The sequence has a mean composition of 41% G + C, and the observed frequency of CpG dinucleotides is close to that predicted from this mononucleotide composition. The sequence contains 17 complete open reading frames (ORFs) and part of another at the 5' end of the sequence. The predicted protein products of two of these ORFs have no recognizable homologs in the genomes of other sequenced human herpesviruses (i.e., Epstein-Barr virus [EBV], human cytomegalovirus [HCMV], herpes simplex virus [HSV], and varicella-zoster virus [VZV]). However, the products of nine other ORFs are clearly homologous to a set of genes that is conserved in all other sequenced herpesviruses, including homologs of the alkaline exonuclease, the phosphotransferase, the spliced ORF, and the major capsid protein genes. Measurements of similarity between these homologous sequences showed that HHV-6 is clearly most closely related to HCMV. The degree of relatedness between HHV-6 and HCMV was commensurate with that observed in comparisons between HSV and VZV or EBV and herpesvirus saimiri and significantly greater than its relatedness to EBV, HSV, or VZV. In addition, the gene for the major capsid protein and its 5' neighbor are reoriented with respect to the spliced ORFs in the genomes of both HHV-6 and HCMV relative to the organization observed in EBV, HSV, and VZV. Three ORFs in HHV-6 have recognizable homologs only in the genome of HCMV. Despite differences in gross composition and size, we conclude that the genomes of HHV-6 and HCMV are closely related.     

Nucleotide sequence analysis of a 38.5-kilobase-pair region of the genome of human herpesvirus 6 encoding human cytomegalovirus immediate-early gene homologs and transactivating functions.

Human herpesvirus 6 (HHV-6) is prevalent in the human population, with primary infection occurring early in life. Its predominant CD4+ T-lymphocyte tropism, its ability to activate human immunodeficiency virus type 1 (HIV-1) gene expression in vitro, and its upregulation of CD4 expression has led to speculation that HHV-6 may act as a positive cofactor in the progression of HIV infection to AIDS in individuals infected with both viruses. Previous sequencing studies of restricted regions of the 161.5-kbp genome of HHV-6 have demonstrated unequivocally that it is a member of the betaherpesvirus subgroup and have indicated that the HHV-6 genome is generally collinear with the unique long (UL) component of human cytomegalovirus (HCMV). In the work described in this report we have extended these sequencing studies by determining the primary structure of 38.5-kbp of the HHV-6 genome (genomic position 21.0 to 59.5 kbp). Within the sequenced region lie 31 open reading frames, 20 of which are homologous to positional counterparts in HCMV. Of particular significance is the identification of homologs of the HCMV UL36-38 and US22-type genes, which have been shown to encode transactivating proteins. We show that DNA sequences encoding these HHV-6 homologs were able to transactivate HIV-1 long terminal repeat-directed chloramphenicol acetyltransferase expression in cotransfection assays, thus demonstrating functional as well as structural conservation of these betaherpesvirus-specific gene products. Our data therefore confirm the close relationship between HHV-6 and HCMV and identify putative immediate-early regulatory genes of HHV-6 likely to play key roles in lytic replication and possibly also in the interactions between HHV-6 and HIV in dually infected cells.     

Efficacy of Methylenecyclopropane Analogs of Nucleosides Against Herpesvirus Replication In Vitro

Abstract

We have reported previously that purine methylenecyclopropane analogs are potent agents against cytomegaloviruses. In an attempt to extend the activity of these compounds, the 2-amino-6-cyclopropylaminopurine analog, QYL-1064, was selected for further study by modifying the purine 6 substituent. A total of 22 analogs were tested against herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), murine cytomegalovirus (MCMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6) and human herpesvirus type 8 (HHV-8). Ten of the analogs had activity against at least one of the viruses tested. One compound had moderate activity against HSV-1 and six had activity against VZV. All but one compound was active against HCMV with a mean EC₅₀ of 2.1 ± 0.6 µM, compared with a mean EC₅₀ of 3.9 ± 0.8 µM for ganciclovir. Of special interest was the fact that eight of the ten compounds were active against both HHV-6A and HHV-6B with mean EC₅₀ values of 6.0 ± 5.2 µM and <2.4 ± 1.5 µM, respectively. Only two compounds had activity against EBV, whereas all but one compound was active against HHV-8 with a mean EC₅₀ of 3.1 ± 1.7 µM. These results indicate that members of this series of methylenecyclopropane analogs are highly active against HCMV, HHV-6, and HHV-8 but are less active against HSV, VZV, and EBV.     

Identification of the human cytomegalovirus glycoprotein B gene and induction of neutralizing antibodies via its expression in recombinant vaccinia virus.

A human cytomegalovirus (HCMV) glycoprotein gene with homology to glycoprotein B (gB) of herpes simplex virus and Epstein-Barr virus and gpII of varicella zoster virus has been identified by nucleotide sequencing. The gene has been expressed in recombinant vaccinia virus and the gene product recognized by monoclonal antibodies and human immune sera. Rabbits immunized with the recombinant vaccinia virus produced antibodies that immunoprecipitate gB from HCMV-infected cells and neutralize HCMV infectivity in vitro. These data demonstrate a role for this protein in future HCMV vaccines.     

Expression of human cytomegalovirus immediate early antigens is responsible for a novel chromatin structure of infected human embryonic lung cells

Whereas human embryonic lung (HEL) cells displayed chromatin fibers composed of a repeat of conventional nucleosomes of 15 nm in diameter, human cytomegalovirus (HCMV) infection induced transient appearance of a novel chromatin structure composed of a repeat of large ellipsoids of 45-65 nm X 15-30 nm with linkers of 50-60 nm long and 6-7 nm thick. Essentially the same change in chromatin structure could be induced when uninfected HEL cell nuclei were incubated in vitro with a 0.4 M NaCl nuclear extract from HCMV-infected HEL cells expressing immediate early antigens (IEA's) or with a similar nuclear extract from NIH/3T3 cells constitutively expressing HCMV IEA's. The latter cell line was established by transformation of the mouse cells with a plasmid carrying the HCMV major immediate early and immediate early 2 genes. These results together with those of control experiments suggest that the expression of IEA's is directly or indirectly responsible for the appearance of the novel chromatin structure in HCMV-infected HEL cells.     

Amino acid changes within conserved region III of the herpes simplex virus and human cytomegalovirus DNA polymerases confer resistance to 4-oxo-dihydroquinolines,a novel class of herpesvirus antiviral agents

The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.     

Selective killing of subacute sclerosing panencephalitis virus-infected cells by liposomes containing fragment A of diphtheria toxin

A more than 99% reduction in infectivity of cultures infected with subacute sclerosing panencephalitis (SSPE) virus was obtained by treating the cultures with liposomes containing fragment A of diphtheria toxin. In the mixed cultures of human embryonic lung (HEL) cells and SSPE virus-infected HEL cells (SSPE cells), SSPE virus in SSPE cells invaded the surrounding HEL cells successively, forming syncytial giant cells to destroy the whole cultures. By adding liposomes containing fragment A of diphtheria toxin to the mixed cultures, we were successful in killing the SSPE cells selectively and as the result of elimination of SSPE cells from the cultures, the cultures appeared to be cured by uninfected HEL cell growth.     

Human cytomegalovirus tegument protein pp71 (ppUL82) enhances the infectivity of viral DNA and accelerates the infectious cycle.

Three tegument proteins of human cytomegalovirus (HCMV), ppUL82 (pp71), pUL69, and ppUL83 (pp65), were examined for the ability to stimulate the production of infectious virus from human diploid fibroblasts transfected with viral DNA. Although viral DNA alone had a low intrinsic infectivity of 3 to 8 plaques/microg of viral DNA, cotransfection of a plasmid expressing pp71 increased the infectivity of HCMV DNA 30- to 80-fold. The increase in infectivity produced by pp71 was reflected in an increased number of nuclei observed to express high levels of the major immediate-early proteins IE1 and IE2. Cotransfection of viral DNA with plasmids directing expression of IE1 and IE2 also resulted in extensive IE1 and IE2 expression in the transfected cells; however, the infectivity of viral DNA was only marginally increased. pp71 also facilitated late gene expression, virus transmission to adjacent cells, and plaque formation. In contrast, expression of pUL69 reduced the pp71- and IE1/IE2-mediated enhancement of HCMV DNA infectivity and also failed to produce any increase in the number of cells expressing IE1 and IE2 over that seen with viral DNA alone. Expression of pp65 did not alter the infectivity of HCMV DNA, nor did it modify the effects of pp71 or pUL69. These results imply that pp71 plays a critical role in the initiation of infection apart from its function as a transactivator of IE1 and IE2.     

Identification and expression of a human cytomegalovirus glycoprotein with homology to the Epstein-Barr virus BXLF2 product, varicella-zoster virus gpIII, and herpes simplex virus type 1 glycoprotein H.

An open reading frame with the characteristics of a glycoprotein-coding sequence was identified by nucleotide sequencing of human cytomegalovirus (HCMV) genomic DNA. The predicted amino acid sequence was homologous with glycoprotein H of herpes simplex virus type 1 and the homologous protein of Epstein-Barr virus (BXLF2 gene product) and varicella-zoster virus (gpIII). Recombinant vaccinia viruses that expressed this gene were constructed. A glycoprotein of approximately 86 kilodaltons was immunoprecipitated from cells infected with the recombinant viruses and from HCMV-infected cells with a monoclonal antibody that efficiently neutralized HCMV infectivity. In HCMV-infected MRC5 cells, this glycoprotein was present on nuclear and cytoplasmic membranes, but in recombinant vaccinia virus-infected cells it accumulated predominantly on the nuclear membrane.     

ACYCLIC/CARBOCYCLIC GUANOSINE ANALOGUES AS ANTI-HERPESVIRUS AGENTS

Several guanosine analogues, i.e. acyclovir (and its oral prodrug valaciclovir), penciclovir (in its oral prodrug form, famciclovir) and ganciclovir, are widely used for the treatment of herpesvirus [i.e. herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2),varicella-zoster virus (VZV) and/or human cytomegalovirus (HCMV)] infections. In recent years, several new guanosine analogues have been developed, including the 3-membered cyclopropylmethyl and-methenyl derivatives (A-5021 and synguanol) and the 6-membered D-and L-cyclohexenyl derivatives. The activity of the acyclic/carbocyclic guanosine analogues has been determined against a wide spectrum of viruses, including the HSV-1, HSV-2, VZV, HCMV, and also human herpesviruses type 6 (HHV-6), type 7 (HHV-7) and type 8 (HHV-8), and hepatitis B virus (HBV). The new guanosine analogues (i.e. A-5021 and D- and L-cyclohexenyl G) were found to be particularly active against those viruses (HSV-1, HSV-2, VZV) that encode for a specific thymidine kinase (TK), suggesting that their antiviral activity (at least partially) depends on phosphorylation by the virus-induced TK. Marked antiviral activity was also noted with A-5021 against HHV-6 and with D- and L-cyclohexenyl G against HCMV and HBV. The antiviral activity of the acyclic/carbocyclic nucleoside analogues could be markedly potentiated by mycophenolic acid, a potent inhibitor of inosine 5′-monophosphate (IMP) dehydrogenase. The new carbocyclic guanosine analogues (i.e. A-5021 and D- andL-cyclohexenyl G) hold great promise, not only as antiviral agents for the treatment of herpesvirus infections, but also an antitumor agents for the combined gene therapy/chemotherapy of cancer, provided that (part of) the tumor cells have been transduced by the viral (HSV-1, VZV) TK gene.     

Acyclic/carbocyclic guanosine analogues as anti-herpesvirus agents

Several guanosine analogues, i.e. acyclovir (and its oral prodrug valaciclovir), penciclovir (in its oral prodrug form, famciclovir) and ganciclovir, are widely used for the treatment of herpesvirus [i.e. herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2), varicella-zoster virus (VZV) and/or human cytomegalovirus (HCMV)] infections. In recent years, several new guanosine analogues have been developed, including the 3-membered cyclopropylmethyl and -methenyl derivatives (A-5021 and synguanol) and the 6-membered D- and L-cyclohexenyl derivatives. The activity of the acyclic/carbocyclic guanosine analogues has been determined against a wide spectrum of viruses, including the HSV-1, HSV-2, VZV, HCMV, and also human herpesviruses type 6 (HHV-6), type 7 (HHV-7) and type 8 (HHV-8), and hepatitis B virus (HBV). The new guanosine analogues (i.e. A-5021 and D- and L-cyclohexenyl G) were found to be particularly active against those viruses (HSV-1, HSV-2, VZV) that encode for a specific thymidine kinase (TK), suggesting that their antiviral activity (at least partially) depends on phosphorylation by the virus-induced TK. Marked antiviral activity was also noted with A-5021 against HHV-6 and with D- and L-cyclohexenyl G against HCMV and HBV. The antiviral activity of the acyclic/carbocyclic nucleoside analogues could be markedly potentiated by mycophenolic acid, a potent inhibitor of inosine 5'-monophosphate (IMP) dehydrogenase. The new carbocyclic guanosine analogues (i.e. A-5021 and D- and L-cyclohexenyl G) hold great promise, not only as antiviral agents for the treatment of herpesvirus infections, but also an antitumor agents for the combined gene therapy/chemotherapy of cancer, provided that (part of) the tumor cells have been transduced by the viral (HSV-1, VZV) TK gene.     

Establishment and functional characterization of human herpesvirus 6-specific CD4+ human T-cell clones.

In order to clarify the protective immune responses against a newly identified herpesvirus, human herpesvirus 6 (HHV-6), we established HHV-6-specific human T-cell clones and examined their functional properties. Five CD3+CD4+CD8- T-cell clones, which proliferated in response to stimulation with two different strains of HHV-6 in the presence of autologous antigen-presenting cells but not with herpes simplex virus type 1 or human cytomegalovirus, were established from peripheral blood lymphocytes of a healthy individual. The proliferative response of all T-cell clones to HHV-6 antigen was inhibited by addition of anti-HLA-DR monoclonal antibody, indicating that these clones were human leukocyte antigen (HLA) class II DR restricted. Of the five clones, two lysed HHV-6-infected autologous lymphoblasts, but not HHV-6-infected allogeneic cells or natural killer-sensitive K562 cells (group 1); one showed cytotoxicity against HHV-6-infected autologous lymphoblasts as well as HHV-6-infected allogeneic cells and K562 cells (group 2); and the remaining two showed no cytotoxic activity (group 3). The cytotoxic activity of group 1 was inhibited by addition of anti-HLA-DR monoclonal antibody to the culture, whereas this monoclonal antibody had no effect on the cytotoxicity of group 2 and did not induce the cytotoxicity of group 3. Perforin, which is one of the mediators of cytotoxicity, was abundantly expressed in group 1 and 2 clones. Moreover, all groups of clones produced gamma interferon after culture with antigen-presenting cells followed by HHV-6 antigen stimulation. These results suggest that HHV-6-specific CD4+ T cells have heterogeneous functions.     

Detection of varicella-zoster virus DNA by field-inversion gel electrophoresis

A new method for detection of varicella-zoster virus (VZV) DNA using field-inversion gel electrophoresis (FIGE) was devised. VZV-genomic DNA could be differentiated from the host cell DNA of human embryonic lung (HEL) fibroblasts infected with VZV under electrophoretic conditions allowing resolution of linear and double-stranded DNAs in the 49-230 kilobase pairs (Kb) range. The detection of VZV-genomic DNA from infected HEL cells was successful regardless of whether the VZV was a laboratory strain, live vaccine strain, or fresh isolate. Under the same electrophoretic conditions, DNA of VZV-infected HEL cells could be clearly differentiated from DNA obtained from HEL cells infected with herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), or human cytomegalovirus (HCMV). Furthermore, VZV genomic DNA could be detected from as small a sample as 1.9 x 10(4) VZV-infected HEL cells. Finally, we could detect VZV genomic DNA from 10 samples of vesicle tissue (blister lids, each about 1-4 mm2) and one sample of vesicle fluid (about 5 microliters) obtained from patients diagnosed as having herpes-zoster. The results of this study indicate that FIGE is a simple and promising method for the detection of VZV from clinical materials as well as infected in vitro cultured cells.     

Application of a new medium supplement for propagation and storage of human cytomegalovirus

A new medium supplement, NU-SERUM, was evaluated for cultivation of human embryonic lung fibroblasts (HEL) and for propagation and storage of human cytomegalovirus (HCMV). NU-SERUM was comparable to fetal bovine serum (FBS) in promoting rapid growth of HEL if they were seeded at a sufficient density. HCMV replicated quite satisfactorily in HEL cultured with media supplemented with NU-SERUM as well as FBS. Inactivation of HCMV at 37 C occurred similarly when the medium contained FBS or NU-SERUM. However, at -70 C, HCMV was less stable in NU-SERUM-containing medium than in FBS-containing medium. Sorbitol added to the NU-SERUM-containing medium improved the unstableness of HCMV at -70 C, and HCMV was storable with such medium. Thus, NU-SERUM is useful as an alternative to FBS not only for growth of HEL but also for propagation and storage of HCMV.