Comparison of specific serological assays for diagnosing human herpesvirus 6 infection after liver transplantation.

Cross-reactivity between human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) antibodies and the reliability of specific serological assays were analyzed for 12 patients with concurrent HHV-6 and HHV-7 antibody responses after transplantation with a liver from a living relative by using an immunofluorescence assay (IFA). A neutralizing antibody titer assay (NT) and an immunoblot assay (IB) designed to detect immunoglobulin M (IgM) antibody to the HHV-6 immunodominant 101-kDa protein were compared in the diagnosis of an active HHV-6 infection. A total of 9 of 12 patients demonstrated concurrent HHV-6 and HHV-7 antibody responses, including increased IgG titers and/or the presence of IgM by IFA, and were thus analyzed for cross-reactive antibody to heterologous virus. The average percentages of residual antibody to HHV-6 and HHV-7 after absorption with HHV-6 antigen were 32.6% (range, 6 to 50%) and 55.6% (range, 35 to 100%), respectively. All 12 patients were subsequently analyzed for HHV-6 antibody by using IB and NT. IB detected IgM antibody to the 101-kDa protein in 75% (9 of 12) of the recipients. A significant rise in the NT antibody titer was detected in the same nine samples. However, HHV-6 DNA was detected by PCR in only five of nine plasma samples collected from recipients with a specific serologic response against HHV-6.

An immunoblot assay for detection of immunoglobulin M antibody to human herpesvirus 6.

We identified the human herpesvirus 6 (HHV-6)-dominant immunoglobulin M (IgM)-reactive virion protein as being the same 101-kDa protein (101K) previously identified as the major IgG immunoreactive protein and a specific serologic marker of HHV-6 infection. An immunoblot assay (IB) to detect HHV-6-specific IgM antibodies against the 101K protein in human serum samples was developed. The assay was validated by using acute- and convalescent-phase serum collected from children under 2 years of age in which we previously detected IgG seroconversion to the HHV-6 101K protein. Of 32 serum pairs which previously demonstrated IgG seroconversion to the 101K protein, 29 had IgM reactivity to the same protein in the acute-phase sample and the remaining 3 had reactivity in the convalescent-phase sample. We also detected HHV-6 IgM activity in sera collected from individuals > or =4 years of age who were also IgM seropositive to measles or rubella. Results of cross-adsorption studies using measles virus-, rubella virus-, and HHV-6-infected cells as the adsorbing antigen indicated no cross-reactivity between measles or rubella IgM and HHV-6 IgM in human serum samples. The IgM IB detected HHV-6-specific IgM antibody to the 101K protein in 78% (63 of 81) of tested acute-phase serum collected from young children with an undifferentiated rash illness by using a single serum dilution.

Human herpesviruses 6 and 7 in chronic fatigue syndrome: a case-control study.

We conducted this study to determine whether infection with human herpesvirus (HHV) 6A, HHV-6B, or HHV-7 differed between patients with chronic fatigue syndrome and control subjects. We recruited 26 patients and 52 nonfatigued matched control subjects from Atlanta. Serum samples were tested by enzyme immunoassay for seroreactivity to HHV-6, and all were seropositive. Lymphocyte specimens were cocultivated with cord blood lymphocytes and assayed for HHV-6 and HHV-7; neither virus was isolated. Finally, lymphocytes were tested by use of 3 polymerase chain reaction methods for HHV-6A, HHV-6B, and HHV-7 DNA. HHV-6A or HHV-6B DNA was detected in 17 (22.4%) of 76 samples, and there were no significant differences (by matched analyses) between patients (3 [11.5%] of 26) and control subjects (14 [28%] of 50). HHV-7 DNA was detected in 14 subjects, and although control subjects (12 [24%]) were more likely than patients (2 [7.7%]) to be positive, the difference was not statistically significant. We found no evidence that active or latent infection with HHV-6A, HHV-6B, HHV-7, or any combination these 3 HHVs is associated with chronic fatigue syndrome.

Comparison of serologic assays and PCR for diagnosis of human herpesvirus 8 infection.

A variety of assays for the diagnosis human herpesvirus 8 (HHV-8) infection have been reported. We compared several such assays with a panel of 88 specimens from human immunodeficiency virus (HIV)-infected patients with Kaposi's sarcoma (KS) (current-KS patients; n = 30), HIV-infected patients who later developed KS (later-KS patients; n = 13), HIV-infected patients without KS (no-KS patients; n = 25), and healthy blood donors (n = 20). PCR assays were also performed with purified peripheral blood mononuclear cells (PBMCs) to confirm positive serologic test results. The order of sensitivity of the serologic assays (most to least) in detecting HHV-8 infection in current-KS patients was the mouse monoclonal antibody-enhanced immunofluorescence assay (MIFA) for lytic antigen (97%), the orfK8.1 peptide enzyme immunoassay (EIA) (87%), the orf65 peptide EIA (87%), MIFA for latent antigen (83%), the Advanced Biotechnologies, Inc., EIA (80%), and the orf65 immunoblot assay (80%). Combination of the results of the two peptide EIAs (combined peptide EIAs) increased the sensitivity to 93%. For detection of infection in later-KS patients, the MIFA for lytic antigen (100%), the orfK8.1 peptide EIA (85%), and combined peptide EIAs (92%) were the most sensitive. Smaller percentages of no-KS patients were found to be positive (16 to 56%). Most positive specimens from the current-KS and later-KS groups were positive by multiple assays, while positive specimens from the no-KS group tended to be positive only by a single assay. PCR with PBMCs for portions of the HHV-8 orf65 and gB genes were positive for less than half of current-KS and later-KS patients and even fewer of the no-KS patients. The concordance between serologic assays was high. We propose screening by the combined peptide EIAs. For specimens that test weakly positive, we recommend that MIFA for lytic antigen be done. A positive result with a titer of >/=1:40 would be called HHV-8 positive. A negative or low titer would be called HHV-8 negative. If a population has a high percentage of persons who test positive by the combined peptide EIAs, then a MIFA could be performed with the negative specimens to determine if any positive specimens are being missed. Alternatively, if a population has a low percentage that test positive, then a MIFA could be performed with a subset of the negative specimens for the same reason. As described above, only a titer of >/=1:40 would be considered HHV-8 positive.

Glycoprotein B of human herpesvirus 8 is a component of the virion in a cleaved form composed of amino- and carboxyl-terminal fragments.

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) is the only known human member of the Rhadinovirus genus of the gammaherpesvirus subfamily. Antibodies against peptides representing portions of the amino- and carboxyl-termini of HHV-8 gB were produced and used to detect gB expression in Vero cells transfected with the gB gene, in the HHV-8-harboring cell line, BCBL-1, and in purified virions. Expression of gB was detected in approximately 3% of uninduced BCBL-1 cells, while up to 30% of the cells expressed gB after 12-O-tetradecanoylphorbol-13-acetate (TPA) induction of virus replication. Indirect immunofluorescence assays and confocal microscopy showed that gB was distributed throughout the cytoplasm of BCBL-1 cells and transfected Vero cells. Immunoblot analyses of virion preparations revealed the presence of full-length as well as two smaller than full-length gB-derived species corresponding to the amino- and carboxy-terminal portions of gB, respectively. Biochemical analysis of the gB carbohydrate moieties using glycosylation inhibitors revealed that gB contained N-linked oligosaccharides of the high-mannose type, characteristic of precursor carbohydrate chains added in the endoplasmic reticulum.

Antiviral screening assays for human herpesviruses 6 and 7.

Human herpesviruses 6 and 7 are ubiquitous herpesviruses that normally infect their hosts early in life. There are two variant groups of human herpesvirus 6 (HHV-6): variants A (HHV-6A) and B (HHV-6B). Variant A has not been unambiguously associated with a specific disease but may contribute to disease in immunocompromised patients; variant B is the major etiologic agent of roseola (roseola infantum or exanthem subitum) and other febrile illnesses of young children, and has been associated with disease in immunocompromised patients. HHV-6B is frequently present in plaque regions in the brains of multiple sclerosis patients, although an etiologic association has not been proven. Human herpesvirus 7 (HHV-7) has been associated with some cases of roseola. The clinical spectrum of these viruses remains to be completely defined. Braun et al. (1) recently described three clinical scenarios that might warrant the use of antivirals to treat HHV-6 infections: (1) transplant recipients with idiopathic pneumonitis (2), multiple sclerosis patients, and (3) patients with HHV-6-associated encephalitis. For HHV-7, cases of neurologic involvement during primary infection might warrant investigation (2).

Human herpesvirus 7.

Human herpesvirus 7, reported in 1990 is a lymphotropic member of the betaherpesvirus subfamily of herpesviruses. The virus is highly seroprevalent, primary infection usually occurs during childhood, and it has been associated with cases of exanthem subitum, pityriasis rosea, neurological manifestations and transplant complications. The latter two may warrant antiviral intervention, in vitro studies have shown that HHV-7 is susceptible to several nucleoside phosphonate compounds. In vitro, the virus has approximately a 5 day growth cycle in cultured lymphocytes; in vivo, latency is established in peripheral blood T-cells and a persistent infection is established in salivary gland tissue from which infectious virus is constitutively shed in saliva. The HHV-7 genome is approximately 145 kb and encodes at least 84 different proteins. Studies characterising HHV-7 gene products and the required interactions between viral and cellular genes necessary for virus replication, persistence and latency are in their infancy. HHV-7 infection has a variety of effects on host cells including upregulation of interleukin 15 and down-modulation of the cell surface molecule CD4; the latter serves as the cellular membrane receptor for HHV-7. Since HIV also infects T-cells via the CD4 molecule, the interactions of these viruses within T-cells during the course of AIDS are important areas of investigation.

Induction of human herpesvirus-8 DNA replication and transcription by butyrate and TPA in BCBL-1 cells.

Human herpesvirus-8 (HHV-8) is a gammaherpesvirus that is present primarily in a state of low level persistence in primary effusion lymphoma cell lines. Using BCBL-1 cells that harbour HHV-8 but lack Epstein-Barr virus, we demonstrate that sodium butyrate is much more effective than the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) at inducing high levels of class II and III virus transcription and viral DNA replication, but also initiates apoptosis. Apoptosis occurs prior to assembly of virions when high concentrations of butyrate (1-3 mM) are used, whereas reduction of butyrate concentration to 0.3 mM decreases the rate of apoptosis and results in production and secretion of enveloped virions that are visualized at high number by electron microscopy in approximately 20% of BCBL-1 cells. Butyrate induces much higher levels of multiple class II and class III transcripts than does TPA, including v-MIPI, v-IL-6, v-Bcl-2, vGPCR and ORF26. A decrease in concentration of butyrate from 3 to 0.3 mM delays the peak induction of these genes, but peak levels remain higher than peak levels in response to TPA. These studies indicate that the massive apoptosis induced by 3 mM butyrate could be diminished and delayed by reduction of butyrate concentration to 0.3 mM, thereby allowing expression of high levels of lytic-associated genes and production of high yields of HHV-8 virions.

The effect of human herpesvirus-6 (HHV-6) on cultured human neural cells: oligodendrocytes and microglia.

Human herpesvirus-6 (HHV-6) is a betaherpesvirus that has been frequently associated with pediatric encephalitis. In 1995 Challoner et al reported that HHV-6 variant B (HHV-6B) was linked to multiple sclerosis (MS) due to the presence of viral DNA and antigen in the oligodendrocytes surrounding MS plaques. These findings led us to examine HHV-6B's in vitro tropism for primary neural cells. HIV-6B mediated cell-to-cell fusion in cultured adult oligodendroglia. Infection of oligodendrocytes was further confirmed by transmission electron microscopy (EM), which showed the presence of intracellular HHV-6 particles, and by PCR for HHV-6 DNA. However, the release of infectious virus was low or undetectable in multiple experiments. Microglia were also susceptible to infection by HHV-6B, as demonstrated by an antigen capture assay. We did not detect infection of a differentiated neuronal cell line (NT2D). Our findings suggest that HHV-6B infection of oligodendrocytes and/or microglia could potentially play a role in neuropathogenesis.

Mapping and serodiagnostic application of a dominant epitope within the human herpesvirus 8 ORF 65-encoded protein.

A dominant epitope within the human herpesvirus 8 (HHV8) ORF 65-encoded protein was mapped to an 8-amino-acid (aa) sequence (RKPPSGKK [aa 162 to 169]) by an amino acid replacement method. Using a 14-aa peptide (P4) encompassing this epitope as the antigen, we developed an enzyme immunoassay for HHV8 antibodies. The presence of P4 antibodies in a panel of 61 human serum specimens was highly correlated with biopsy-confirmed Kaposi's sarcoma. The homologous Epstein-Barr virus peptide derived from BFBR3-encoded protein did not interfere with the assay, suggesting that P4 is specific for HHV8.

Biologic properties of human herpesvirus 7 strain SB.

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

Seroconversion to human herpesvirus 6 and human herpesvirus 7 among Brazilian children with clinical diagnoses of measles or rubella.

We collected acute-phase and convalescent-phase serum samples from Brazilian patients who presented with exanthem of unknown origin and evaluated these samples by means of an immunoblot assay for seroconversion to human herpesvirus 6 (HIV-6) or human herpesvirus 7 (HIV-7). Measles or rubella had been clinically diagnosed in all these patients, but their sera were negative for antibodies to both measles virus and rubella virus. Twenty percent of the patients clearly seroconverted to HHV-6 after manifestation of the exanthem, and 8% seroconverted to HHV-7. All seroconversions to HHV-6 occurred in children aged < or = 5 years; a 41% frequency of seroconversion to HHV-6 was noted among children between 3 months and 23 months of age, whereas seroconversions to HHV-7 were detected during infancy and through adulthood. Our data indicate that primary infections due to HHV-6 or HHV-7 can be misdiagnosed as measles or rubella.

Evaluation of immunoassays for detection of antibodies to human herpesvirus 7.

An enzyme immunoassay (EIA), an immunoblot assay (IB), and an indirect immunofluorescence assay were developed for detection of human herpesvirus 7 (HHV-7) antibodies in human serum. Cross-absorption studies with EIA or IFA using HHV-7 and human herpesvirus 6 (HHV-6) antigens indicated that most human sera contain cross-reactive HHV-6 and HHV-7 antibodies and that the degree of cross-reactivity varies between individual serum specimens. Inhibition of homologous antibody activity by absorption with heterologous virus ranged from 0 to 57% by EIA. However, for every sample tested, absorption with homologous virus removed more activity than did heterologous virus. An 89-kDa protein was identified as an HHV-7-specific serologic marker by IB. Activity to this protein was not removed by absorption with HHV-6 antigen. Of the three assays, the EIA was the most sensitive (94%), while the IB was the most specific (94%). Approximately 80% of specimens collected from German adults and children older than 2 years were positive for HHV-7 antibodies by these assays.

Physical and genetic maps of the human herpesvirus 7 strain SB genome.

Human herpesvirus 7 (HHV-7) is a close relative of human herpesvirus 6A (HHV-6A) and human herpesvirus 6B (HHV-6B) based on limited biologic and genetic data. In this work we describe physical and genetic maps for HHV-7 strain SB [HHV-7(SB)], which was obtained from the saliva of a healthy adult. The HHV-7(SB) genome length is approximately 144 kb by clamped homogeneous electric field gel electrophoresis and approximately 135 kb by summation of restriction endonuclease fragments. We constructed plasmid clones and PCR amplimers that span the HHV-7 genome, except for the genomic termini, and determined the maps of the restriction endonuclease cleavage sites for BamHI, PstI, and SacI. The HHV-7(SB) genome is composed of a single unique region of approximately 122 kb bounded at each end by a 6 kb direct repeat. Homologs to thirty-five herpesvirus genes were identified. The highest similarity was with the HHV-6 genes, with an average amino acid identity of 50%, followed by the human cytomegalovirus counterpart. The genomic and genetic maps indicated that the HHV-7 and HHV-6 genomes are colinear. There was no sequence variation in a segment of the gene encoding the DNA polymerase-associated factor homolog among six HHV-7 isolates, while the corresponding segment of the HHV-6A and HHV-6B counterparts differed by 4.6%. These data support previous observations that the closest genetic relatives of HHV-7 are betaherpesviruses.

Intragenomic linear amplification of human herpesvirus 6B oriLyt suggests acquisition of oriLyt by transposition.

We identified some passage lineages of human herpesvirus 6 variant B (HHV-6B) strain Z29 that contain as many as 12 tandem copies of a genomic segment that corresponds almost precisely to a previously identified minimal efficient origin of lytic replication (oriLyt). Analysis of nucleotide sequences in the vicinity of the amplified segment suggests that the amplification occurred as a two-step process, with the first step being a rare sequence duplication mediated through directly repeated sequences located near the termini of the amplified segment and the second step occurring via homologous recombination through the duplicated sequence. These results demonstrate that oriLyt has been amplified in some virus stocks and indicate that (i) origin amplification confers a growth advantage on the virus in cell culture and (ii) laboratory-passaged HHV-6B genomes can accommodate additional nucleotide sequences and thus may be useful gene transfer vectors. The structures of the amplified segment and its adjacent sequences together suggest that HHV-6B or a progenitor virus acquired oriLyt by transposition from an unknown source.

A strongly immunoreactive virion protein of human herpesvirus 6 variant B strain Z29: identification and characterization of the gene and mapping of a variant-specific monoclonal antibody reactive epitope.

We previously identified a 101-kDa apparent molecular mass polypeptide (101K) as the major immunoreactive virion protein of human herpesvirus 6 variant B strain Z29 [HHV-6B(Z29)] and found that the human immune response to this protein is HHV-6-specific (Yamamoto, M., Black, J. B., Stewart, J. A., Lopez, C., and Pellett, P. E., 1990, J. Clin. Microbiol. 28, 1957-1962). We report here the identification and characterization of the gene encoding 101K. We found 81% amino acid identity between an HHV-6B(Z29) open reading frame (ORF) and its homolog in HHV-6A strain U1102 [HHV-6A(U1102)]. The product of this gene was identified as 101K on the basis of both the reactivity of a 101K-specific monoclonal antibody (MAb C3108-103) with a bacterially expressed portion of the gene and the reactivity of polyclonal rabbit antibodies raised against the bacterially expressed protein with 101K expressed by HHV-6B(Z29)-infected cells. MAb C3108-103 reacted with eight of eight variant B isolates and none of six variant A isolates, indicating that it is a variant-specific MAb. The MAb reactivity was mapped to an eight-amino-acid segment of 101K. HHV-6A(U1102) differs from HHV-6B(Z29) by two amino acids in this region; substitution mapping with synthetic oligopeptides mapped the variant B specificity to Asp723, this explaining the failure of the MAb to react with variant A proteins. A set of transcripts appropriately sized for expression of 101K was identified and precisely mapped. The transcripts originated down-stream from either of two TATA boxes located 139 bp apart in the region 5' to the 101K ORF, with one 5'-species being much more abundant. Two independent polyadenylation sites were identified; the canonical polyadenylation signal located 3' to the 101K ORF was used much more frequently than was the atypical polyadenylation signal located within the 101K ORF. These results suggest a complex regulatory mechanism for this gene.

Frequent isolation of human herpesvirus 7 from saliva.

We obtained isolates of human herpesvirus 7 (HHV-7) from 6 of 8 healthy adults by culturing saliva with human umbilical cord blood lymphocytes. These isolates were identified as HHV-7 on the basis of comparisons of restriction endonuclease fragment profiles and hybridization with HHV-7 strain RK DNA. The isolates could be differentiated from HHV-7 strain RK and from each other by their restriction endonuclease fragment profiles. We confirm the finding of frequent isolation of HHV-7 from saliva of healthy adults and report the first dual isolation of human herpesvirus 6 (HHV-6) and HHV-7 from a single saliva specimen. We also describe an in situ hybridization assay that can distinguish between HHV-6 and HHV-7.

Effects of temperature, potassium concentration, and sugar on human spermatozoa motility: a cell preservation model from reproductive medicine.

Two experiments were performed in vitro with human sperm from a panel of 11 donors. In both experiments, washed sperm from each donor were divided into four equivolume samples, three of which were resuspended in various formulations of Tyrode's solution and the fourth was resuspended in its own seminal plasma. Each of the four samples was then stored at 37 or 3 degrees C for fixed intervals of 0, 2, 6, 12, and 24 h. Motility was assessed at 37 degrees C for all samples at the end of each interval. The results indicate that sperm survival in vitro at 3 degrees C was significantly enhanced by 20 mM K+ in Tyrode's solution relative to Tyrode's with less K+. A metabolizable sugar such as glucose was essential to maintaining sperm viability in K(+)-free media. The addition of raffinose to media containing glucose improved motility of sperm stored at 3 degrees C for 6 h.

Induction of host cell protein synthesis by human herpesvirus 6.

We observed an increase in host cell protein synthesis in human cord blood lymphocytes (CBL) infected with human herpesvirus 6 relative to uninfected cultures. The magnitude of this effect could not be explained by a smaller decrease in cell number in the infected cultures. The induction of host cell protein synthesis by HHV-6 does not appear to be mediated by a stable soluble factor present in the infected cell culture supernatant. When CBL were infected with virus that had been exposed to ultraviolet irradiation (UV) for various intervals, we found that the level of increase in cell number, host protein synthesis, viral DNA and viral antigen was inversely proportional to the length of time of virus exposure to UV. No increase in cell number or host cell protein synthesis was seen in CBL infected in the presence of 50 micrograms/ml phosphonoacetic acid, an inhibitor of HHV-6 DNA replication. These results indicate that components of input virions do not induce the increased protein synthesis and that the induction is dependent on viral DNA replication.