Neutralizing antibody assay for human herpesvirus-6

Antibody to human herpesvirus-6 (HHV-6) was measured in cord blood mononuclear cell cultures by a neutralization (NT) test, in which the presence or absence of characteristic large cell formation in cells infected with HHV-6 was used as an indicator for neutralization of the virus. The NT test could measure antibodies during and just after the appearance of the skin rash in patients with exanthem subitum. The levels of antibodies measured by the NT test was generally higher than that by an indirect immunofluorescence assay.     

Activation of human herpesvirus-6 in children with acute measles.

Virological and serological studies were carried out prospectively to evaluate the possible activation of human herpesvirus-6 (HHV-6) in 50 infants and children with acute measles by isolation of HHV-6 from peripheral blood and by determining neutralizing antibodies to the virus. All but 5 patients (90%) were seropositive to HHV-6 in the acute stage of measles and 18 (40%) had a significant increase in HHV-6 antibody titers thereafter, whereas only 2 of 27 patients who were initially seropositive to Epstein-Barr virus (EBV) viral capsid antigen (VCA) had a significant rise in antibody titers to EBV VCA. Among 18 patients with a significant increase in HHV-6 titers, the virus was isolated from the peripheral blood mononuclear cells of three patients in the early convalescent stage of measles. These results indicate that activation of HHV-6 may occur frequently a few weeks after primary infection with the measles virus.     

Detection of human herpesvirus 6 DNAs in samples from several body sites of patients with exanthem subitum and their mothers by polymerase chain reaction assay

Polymerase chain reaction amplification was used to detect human herpesvirus 6 (HHV-6) DNAs in peripheral blood mononuclear cells (MNCs), plasma, saliva, stool, and urine from three patients with exanthem subitum and in peripheral blood MNCs, plasma, and saliva from their mothers. HHV-6 DNAs were detected in MNCs during and after the disease and were found in plasma only in the acute phase. The virus DNAs were also detected in saliva after recovery from the illness and were found persistently or intermittently in stool but not in urine samples after the onset of the disease. In contrast, one of the three mothers excreted HHV-6 DNAs persistently in saliva. None of the mothers had the virus DNAs in peripheral blood MNCs and plasma nor a significant increase in antibody titers to HHV-6 after possible exposure from their children. These findings suggest systemic replication of HHV-6 during the acute phase in patients with exanthem subitum and persistent infection of the virus in several organs after recovery from the disease. © 1995 Wiley-Liss, Inc.     

Reactivation of human herpesvirus 6 by infection of human herpesvirus 7

We have attempted to reactivate human herpesvirus 6 (HHV-6) by infection with HHV-7 using childhood exanthem subitum patients in vitro. Peripheral blood mononuclear cells (PBMCs) were collected from children who had a history of exanthem subitum(ES) by HHV-6 and were infected by human herpesvirus 7 (HHV-7) in vitro. The antigen positive rate to HHV-6 started to increase 7 days after the infection and reached a maximum by Day 15 using an immunofluorescence antibody test. The copy number of HHV-6 DNA also increased in the samples in 10 days after infection in vitro. No antigen or increase in DNA was detected in PBMCs, that were mock-infected or infected with supernatant of stock virus after ultracentrifugation, suggesting that an infection by HHV-7 is necessary to reactivate HHV-6. In the paired sera samples during the acute and the convalescent phases of ES, seven to ten bands, that were specific for HHV-6, were recognized in samples from the acute phase, and at least 5 dominant polypeptides were found more intensively after HHV-7 infection.     

Detection of human herpesvirus 6 DNA in throat swabs by polymerase chain reaction

The detection of human herpesvirus 6 (HHV-6) DNA was carried out in throat swabs of adults and children by the polymerase chain reaction, and isolation of virus was also attempted from peripheral mononuclear cells. Although virus was isolated only from peripheral blood mononuclear cells of infants with exanthem subitum, HHV-6 DNA was detected in one of 30 healthy adults (3%), two of nine adults (22%) with common cold, two of 10 infants (20%) with exanthem subitum, four of 39 febrile children (10%) with antibody to HHV-6 (including three of 10 infants aged under 1 year old, and one of 29 children aged over 1 year old). However, HHV-6 DNA was not detected in samples from healthy neonates.     

Detection by polymerase chain reaction amplification of human herpesvirus 6 DNA in peripheral blood of patients with exanthem subitum.

The polymerase chain reaction was used to detect human herpesvirus 6 (HHV-6) DNA in peripheral blood mononuclear cells from patients with exanthem subitum. Amplified products were detected by agarose gel electrophoresis and dot blot hybridization with a cloned DNA probe. No cross-hybridization with DNAs of five other human herpesviruses was observed in this system, and all HHV-6 strains gave positive reactions when the primer pairs were used. Immunoglobulin M antibody appeared about 5 days after the onset of disease, reaching a maximum after about 2 to 3 weeks and then decreasing to less than 1:10 1 month after the onset of disease in almost all patients. Mononuclear cells from seven patients with exanthem subitum all gave positive reactions in this system, and viral DNA was found in samples even during the convalescent phase of the disease. We conclude that this test, using polymerase chain reaction amplification, which takes only 1 to 2 days, is useful for the diagnosis of HHV-6 infection.     

Primary human herpesvirus-6 infection is frequently overlooked as a cause of febrile fits in young children

Sera from 248 children aged 30–179 weeks were tested retrospectively for human herpesvirus-6 (HHV-6) IgG antibody avidity. Twenty-five children presented with febrile fits, in one case with a rash, exanthem subitum resulting from primary HHV-6 infection was diagnosed at the time, but in the others HHV-6 was not considered in the differential diagnosis even though two patients had a rash. In fact, five of the 25 patients experienced primary infection as shown by low avidity HHV-6 antibody. Although nine other of these patients were seronegative in the acute phase of the illness, and unfortunately convalescent sera were not available to confirm primary HHV-6 infection, seronegativity was clearly a risk factor for febrile fits (P = 0.03, odds ratio 3.14 for all children; P = 0.002, odds ratio 7.20 for children aged 70–179 weeks), and primary HHV-6 infection was a very likely diagnosis. The remaining 11 children had high avidity antibody, so HHV-6 was excluded as a cause of their febrile fits. HHV-6 may frequently be a cause of febrile fits, often without the typical rash of exanthem subitum, and this diagnosis is often overlooked by clinicians.     

Phenotypic and genetic polymorphisms among human herpesvirus-6 isolates from North American infants.

Fifteen human herpesvirus-6 (HHV-6) isolates from North American infants with primary infection manifest as febrile or roseola (exanthem subitum) like illnesses were characterized phenotypically on the basis of their in vitro growth in continuous T-cell lines and primary human mononuclear cells and by their reactivity with monoclonal antibodies. All isolates replicated efficiently in primary human cord blood mononuclear cells, but five distinct patterns of viral replication in human cells lines were observed. Two of the HHV-6 isolates from infants were found to replicate in HSB-2 cells, a property associated with so-called group A viruses, which had previously been isolated only from adults. These same isolates also reacted with a panel of A-specific monoclonal antibodies. Genomic characterization of viral isolates using well-characterized restriction site polymorphisms indicated that these two isolates contained a mixture of both A- and B-type genomes, in different proportions. These data suggest that not all HHV-6 isolates can be categorized into one of two broad groups and that such segregation of HHV-6 isolates may in fact be misleading.     

Isolation of HHV-6-related virus from children affected by infectious syndrome

Summary Peripheral blood cultures from children showing symptoms of generic infectious disease have been examined for human herpesvirus-6 (HHV-6). Two HHV-6-related isolates have been obtained: one from a child for whom a clinical diagnosis of exanthema subitum has been made when a typical rash appeared, and one from a child in whom the infectious symptomatology has maintained the characters of nonspecific syndrome. All the syndrome diagnosed as exanthema subitum resulted as being associated with fresh infection by HHV-6-related virus.     

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.     

Enzyme-linked immunosorbent assay for detection of IgG antibody to human herpesvirus 6

A lysate of human herpesvirus 6 (HHV-6)-infected cord blood mononuclear cells was used as antigen for enzyme-linked immunosorbent assay for the detection of IgG antibody to HHV-6. Antibody responses after exanthem subitum were well correlated with clinical recovery from the disease and the level of antibody activities was well correlated with indirect immunofluorescence assay and the neutralization test. Seroconversion to other human herpesvirus, including cytomegalovirus, was not observed in infants with exanthem subitum. All of the infants had by the age of 1 month antibodies to HHV-6, which decreased with age to the lowest level at the age of 3 to 6 months and then increased and reached the maximum level by 1 to 2 years of age. After 3 years of age, the prevalence was almost stable.     

Atypical clinical features of a human herpesvirus-6 infection in a neonate

A case of neonatal human herpesvirus 6 (HHV-6) B infection is presented. Although HHV-6 B was isolated from peripheral blood at the onset of the illness, a significant increase in viral antibody titers was not observed. The patient had a slight fever with generalized maculopapular skin rash and an increased number of atypical lymphocytes, which is quite different from the typical clinical features of exanthem subitum.     

Establishment of titration system for human herpesvirus 6 and evaluation of neutralizing antibody response to the virus.

The susceptibilities of seven T-cell lines to human herpesvirus 6 (HHV-6) infection were examined. MT-4 cells were the most susceptible of these lines to infection with this virus. Therefore, chemically adhered MT-4 cell monolayers were used for infectious HHV-6 assay by indirect immunofluorescent-antibody (IFA) staining. When cell monolayers were fixed 30 to 45 h postinfection, the foci stained with IFA were easy to count and a linear relationship was observed between the number of foci and the virus concentration. MT-4 cell monolayers were also used for a focus reduction neutralizing-antibody test. In this test, sera from patients in the convalescent stage of exanthem subitum all showed significant neutralizing activity (1:80 to 1:320), whereas sera from patients in the acute stage of disease showed no detectable neutralizing activity. The titers of neutralizing antibody correlated well with the levels of anti-HHV-6 antibodies detected by IFA.     

Human Herpesvirus 6 and Febrile Convulsions

Human herpesvirus 6 (HHV-6) is a ubiquitous virus that causes the childhood febrile illness exanthem subitum. Primary infection usually occurs in the first few years of life and the virus is considered to be one of the most common causes of acute febrile illness in childhood. HHV-6 can infect the central nervous system, causing meningitis or encephalitis after an acute attack of exanthem subitum. Febrile convulsions are common in early childhood, affecting 2--3% of children between 6 months and 5 years of age. Primary HHV-6 infection has been reported to be complicated by febrile convulsions in 8--50% of cases. An association between HHV-6 and febrile convulsions was established on the basis of: the similarity between the age groups in which febrile convulsions and HHV-6 infection occur, the high incidence of febrile convulsions after HHV-6 infection and the neurotropic properties of the virus. The exact role of HHV-6 in the pathogenesis of febrile convulsions remains unclear.     

Latent human herpesvirus-6 DNA is sparsely distributed in peripheral blood lymphocytes of healthy adults and patients with lymphocytic disorders

Human herpesvirus-6 (HHV-6) can be regularly isolated from peripheral blood mononuclear cells (PBMC) of children suffering from exanthema subitum, but only rarely from PBMC of adults. Although the high prevalence of HHV-6 infection in early childhood seems to result from cell-free infectious virus shedded in saliva of healthy adults, latent HHV-6 infection is supposed to occur in lymphocytes. Therefore, we performed polymerase chain reaction (PCR) with DNA from PBMC of 44 healthy adults, 31 HIV-seropositive individuals and 33 patients with leukaemia or lymphoproliferative disorders. As positive control served PBMC from 11 children with exanthema subitum and as negative control PBMC from 20 newborns. Whereas HHV-6-specific sequences were detected in PBMC from all children with exanthema subitum and never in PBMC from newborns, they were found in PBMC of 9% of healthy adults and HIV-seropositive individuals and in 16% of the patients with lymphoproliferative disorders. Apparently detection of HHV-6 DNA in PBMC was neither limited by low sensitivity of the HHV-6 PCR assay, which detected less than ten copies of cloned HHV-6 DNA, nor by a low rate of latently infected individuals, but was limited by the number of lymphocytes subjected to PCR. It is supposed that the presence of latent HHV-6 DNA in lymphocytes is common, but that infected lymphocytes are rare (1 infected cell in 10⁵ lymphocytes).Although the results do not support a definite propagation of HHV-6 in HIV-seropositive individuals or in patients with leukaemia or lymphoproliferative disorders, they cannot exclude HHV-6 acting as co-factor in HIV infection or in lymphoproliferative disorders.     

Molecular epidemiological studies of human herpesvirus 6 in families

The mode of transmission of human herpesvirus 6 (HHV-6) was investigated by molecular epidemiological techniques in four families. HHV-6 was isolated from the peripheral blood of seven infants with exanthem subitum, including siblings, and from the saliva of two mothers. These isolates were examined for genetic relatedness by means of restriction enzymes analysis of purified DNA. While the profiles of the digestion patterns differed among families, they were very similar in a particular family. These results may suggest that HHV-6 is transmitted primarily from mother to child. © 1994 Wiley-Liss, Inc.     

Distribution of virus in blood components during the viremia of measles

Summary Measles virus was isolated from four cases of human measles 8 hours to 29 hours after the onset of exanthem when the washed leucocyte fraction of blood was employed as inoculum, but isolation was not obtained from the RBC or plasma components even with larger inocula. In one case, virus was isolated from all fractions of blood drawn at the onset of rash, but in significantly higher quantity from the WBC component.Dedicated to ProfessorJohn F. Enders on the occasion of his 70th birthday.This study was supported in part by grant E1101 from the National Institute of Allergy and Infectious Diseases.This work was done in 1957 to 1959 when the author was Research Associate, Vincent Memorial Laboratories, Massachusetts General Hospital.     

Genomic polymorphism, growth properties, and immunologic variations in human herpesvirus-6 isolates.

Fifteen human herpesvirus-6 (HHV-6) isolates from normal donors and patients with AIDS, systemic lupus erythematosis, chronic fatigue syndrome, collagen-vascular disease, leukopenia, bone marrow transplants, Exanthem subitum (roseola), and atypical polyclonal lymphoproliferation were studied for their tropism to fresh human cord blood mononuclear cells, growth in continuous T cell lines, reactivity to monoclonal antibodies, and by restriction enzyme banding patterns. All isolates replicated efficiently in human cord blood mononuclear cells, but mitogen stimulation of the cells prior to infection was required. The ability to infect continuous T-cell lines varied with the isolates. Isolates similar to GS prototype infected HSB2 and Sup T1 cells and did not infect Molt-3 cells, whereas isolates similar to Z-29 infected Molt-3 cells but not HSB2 and Sup T1 cells. Some of the monoclonal antibodies directed against the HHV-6 (GS) isolate showed reactivity with all isolates tested, but others only reacted with HHV-6 isolates similar to the GS isolate and not with those similar to Z-29 isolate. Restriction enzyme analysis using EcoRI, BamHI, and HindIII revealed that HHV-6 isolates from roseola, bone marrow transplant, leukopenia, and an HIV-1-positive AIDS patient from Zaire (Z-29) were closely related but distinct from GS type HHV-6 isolates. Based on the above findings, we propose that, like herpes simplex virus types 1 and 2, the 15 HHV-6 isolates analyzed can be divided into group A (GS type) and group B (Z-29 type).