Human herpesvirus 6.

The development of techniques for the culture of lymphoid cells and the isolation of viruses that infect these cells led to the discovery of human herpesvirus (HHV) 6 in 1986. At the time, HHV-6 was the first new human herpesvirus to be discovered in roughly a quarter of a century, and its isolation marked the beginning of an era of discovery in herpesvirology, with the identification of HHV-7 and HHV-8 (Kaposi's sarcoma-associated herpesvirus) during the following decade. Like most human herpesviruses, HHV-6 is ubiquitous and capable of establishing a lifelong, latent infection of its host. HHV-6 is particularly efficient at infecting infants and young children, and primary infection with the virus is associated with roseola infantum (exanthem subitum) and, most commonly, an undifferentiated febrile illness. Viral reactivation in the immunocompromised host has been linked to a variety of diseases, including encephalitis, and HHV-6 has been tentatively associated with multiple sclerosis. This article discusses the major properties of HHV-6, its association with human disease, and the pathobiological significance of viral reactivation.

In vitro 1H and 31P NMR spectroscopic evidence of multiple aberrant biochemical pathways in murine trisomy 16 brain development.

Nuclear magnetic resonance (NMR) spectroscopy was used to evaluate cytosolic compounds and membrane phospholipids simultaneously in trisomy 16 (Ts16) and euploid (control) murine brain at fetal day 15 in order to examine the cellular biochemistry that underlies the neurodevelopmental consequences of chromosome triplication in this model of Down syndrome (DS). Proton NMR spectroscopic analysis of brain tissue extracts demonstrated decreased levels of choline and increased levels of myo-inositol (MI) in Ts16 brains compared with control. These data are consistent with the cholinergic deficits and elevated MI levels previously described in Ts16. Compared with euploid brains. Ts16 brains also possess higher levels of creatine, adenosine, and tyrosine. Increased levels of MI and creatine, compounds that are localized to glia, imply abnormalities in the trophic environment of Ts16 brain. Phosphorus NMR spectroscopic analysis of extracts further revealed elevated levels of anionic phospholipid membrane components, such as phosphatidylinositol (PtdIno) and phosphatidylethanolamine, in Ts16 brains. Since these compounds are confined to the inner leaflet of the membrane, the findings suggest that membrane composition is altered specifically in the cytosolic bilayer at this stage. Together our proton and phosphorus NMR spectroscopic results indicate that multiple biochemical pathways are affected in Ts16 brain development. Understanding the effects of these aberrations may elucidate the processes that lead to neural dysfunction and Alzheimer's disease (AD) neuropathology in DS individuals.

Detection of human herpesvirus 6 by reverse transcription-PCR.

The role of human herpesvirus 6 (HHV-6) in disease beyond primary infection remains unclear. We have developed and validated a new reverse transcription-PCR (RT-PCR) assay for HHV-6 that can determine the presence of HHV-6 in clinical specimens and differentiate between latent and replicating virus. Peripheral blood mononuclear cells from 109 children were evaluated for HHV-6 by RT-PCR, DNA PCR, and viral culture. Of these samples, 106 were suitable for analysis. A total of 20 samples were positive for HHV-6 by culture and DNA PCR, of which 19 were positive by RT-PCR (sensitivity, 95%). All 28 samples from children that were negative by viral culture, but positive by DNA PCR, were negative for viral transcripts by our RT-PCR assay. One positive RT-PCR result was observed in 56 samples that were negative by tissue culture and DNA PCR. This indicates a low rate of false-positive results (1.2%) and a specificity of 98.8%. This RT-PCR assay can reliably differentiate between latent and actively replicating HHV-6 and should allow insight into the pathogenesis of this ubiquitous virus.

In vitro proton and phosphorus NMR spectroscopic analysis of murine (C57Bl/6J) brain development.

We report for the first time in vitro proton and phosphorus NMR spectroscopic analyses of murine brain development from fetal to adult stages. Chloroform-methanol extracts from C57B16/J mouse brain, at ages ranging from 15 days in utero (F15) to adult, permitted the simultaneous investigation of both cytosolic and membrane phospholipid compartments. The protein content of murine brain was determined and used for quantitation of individual metabolite levels. Proton NMR spectroscopy revealed that NAA, considered a neuronal marker, is undetectable at F15. Glutamate, GABA and creatine, however, are present at this time. All four compounds reach maximum levels at 21 days postnatal (P21). Choline and alanine levels are at their peak in fetal brain and progressively fall as the brain develops. Phosphorus NMR spectroscopy shows that phosphatidylcholine, phosphatidylinositol, sphingomyelin, and phosphatidylserine increase steadily from F15 to P21.

Primary human herpesvirus 7 infection: a comparison of human herpesvirus 7 and human herpesvirus 6 infections in children.

OBJECTIVE: To define the clinical and virologic characteristics of primary human herpesvirus 7 (HHV-7) infection and to compare these characteristics with those of primary human herpesvirus 6 (HHV-6) infection. STUDY DESIGN: A prospective convenience sample study of 496 children < or =3 years old. HHV-7 and HHV-6 infections were identified by viral isolation. Polymerase chain reaction and serology for HHV-7 and HHV-6 were performed. Clinical and laboratory characteristics of patients were obtained from medical records and follow-up interviews. RESULTS: Children with primary HHV-7 infection (n = 8) were identified and compared with children with primary HHV-6 infection (n = 29) detected during the same time period. All children were febrile (mean temperature 39.8 degrees C) with no difference in the degree of fever, frequency of rash, or gastrointestinal complications between the groups. The median age of children with primary HHV-7 infection was 26 months, significantly older than that of children with primary HHV-6 infection (median, 9 months). Children with primary HHV-7 infection were also more likely than those with primary HHV-6 infection to have seizures associated with the illness (P = .004). CONCLUSION: Primary infection with HHV-7 can cause a highly febrile illness in childhood, complicated by seizures. The serologic diagnosis of primary HHV-6 and HHV-7 infections may be confounded by cross-reacting antibodies.

Blood-brain barrier integrity in Alzheimer's disease patients and elderly control subjects.

A defective blood-brain barrier (BBB) has been postulated to be present in Alzheimer's disease (AD), which would allow circulating beta-amyloid peptide to enter the brain. The authors tested this hypothesis by studying BBB function in 14 individuals with probable AD and 9 elderly control subjects. A computed tomographic method was used to measure blood-to-brain transport (K1), tissue-to-blood efflux (k2), tissue plasma space (Vp), and tissue extracellular space (Ve) of meglumine iothalamate. Repeated-measures analysis of variance indicated no significant differences between the groups for any of the measures. The authors conclude that there is no generalized abnormality of the blood-brain barrier in AD.

Persistence of human herpesvirus 6 according to site and variant: possible greater neurotropism of variant A.

Little is known of the persistence and pathogenicity of human herpesvirus 6 (HHV-6) after primary infection, including the role of strain variant. Over 2 to 5 years, 2,716 children and 149 families were studied. Peripheral blood mononuclear cell (PBMC), saliva, and cerebrospinal fluid (CSF) specimens were examined for HHV-6 DNA and variant. Ninety-nine percent of isolates causing primary infection were HHV-6 variant B (HHV-6B), which predominated in 95%-98% of the variants persisting in PBMC and saliva specimens from children and adults. Of 668 CSF samples, 13% contained HHV-6 DNA; of 77 children examined after primary infection, 61% had HHV-6 DNA detected only in their CSF and 39% had HHV-6 DNA in both CSF and PBMCs. HHV-6 variant A (HHV-6A) was detected significantly (P = .0001) more frequently in CSF than in PBMCs or saliva. In children for whom HHV-6 was identified in both CSF and PBMCs, PBMCs contained only HHV-6B, while CSF contained HHV-6A or HHV-6B, not both. Thus, in patients with dual infection, only HHV-6A persisted in CSF, which suggests that HHV-6A has greater neurotropism. Findings for adults indicate that dual infection occurs; variant persistence is similar to that for children. The frequency of HHV-6A infection increased little with age, thereby indicating that HHV-6A infection remains uncommon into adulthood. This study suggests that HHV-6 variants have different immunobiologic courses and neurotropism.

A practitioner's guide to human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7).

Human herpesvirus-6 (HHV-6) and HHV-7 are newly recognized ubiquitous human viruses first discovered in patients with AIDS or lymphoproliferative disorders. Much more information is available about the clinical characteristics of infection with HHV-6 than HHV-7. Primary infection with HHV-6 occurs in early childhood and is most commonly manifested as an undifferentiated highly febrile illness, with seizures noted to be the most common complication. A subset of children develop the classic manifestations of roseola infantum or exanthem subitum. Other neurologic diseases in adults such as encephalitis and multiple sclerosis also have been linked to HHV-6; however, the role of HHV-6 in these clinical entities has not been fully elucidated. Although HHV-6 and HIV are both tropic for CD4+ lymphocytes and interact in vitro, there is no evidence at present that HHV-6 plays a role in HIV disease. HHV-7 is similar to HHV-6 in genetic organization and structure. Little is known of the clinical characteristics of infection with HHV-7 or its ability to cause disease in children or reactivation in adults.

Neuroinvasion and persistence of human herpesvirus 6 in children.

Human herpesvirus 6 (HHV-6) causes a febrile illness in children and has been implicated as a cause of encephalitis and recurrent seizures. Paired samples of cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) from 487 children were evaluated by nested polymerase chain reaction (PCR) for evidence of current or past infection with HHV-6. PBMC were also cultured for isolation of HHV-6. These data were correlated with the patients' clinical information. HHV-6 DNA was detected in 72 (14.8%) of 487 CSF samples. HHV-6 persistence was documented in 142 children by PCR detection of HHV-6 DNA in PBMC or CSF (or both) in the absence of primary HHV-6 infection; the central nervous system was the only site of HHV-6 DNA persistence in 28.9%. HHV-6 DNA can be detected in the CSF of children during and after primary infection, and the central nervous system may be the sole site of persistence.

Development of the GABAergic phenotype in murine spinal cord-dorsal root ganglion cultures.

Murine spinal cord and dorsal root ganglion GABAergic neurons, derived from 12-day-old fetuses, were examined autoradiographically, biochemically and immunocytochemically in vitro to determine the timecourse of appearance and maturation of this phenotype and the extent and mode of its innervation of target neurons. Specific 3H-GABA uptake into spinal cord neurons was the first property to develop and was present at the earliest time studied, one day in vitro. Immunocytochemical localization of glutamic acid decarboxylase (GAD) revealed positively stained neurons beginning at four days. At five days in vitro, electron microscopic immunocytochemistry revealed GAD-immunoreactive (GAD-IMR) boutons investing neuronal perikarya as well as neuronal processes. By one week in vitro, GAD-IMR neurons constituted 27% of the total population and GAD-IMR boutons could be seen contacting every cell with a neuronal morphology. The mode of investment of target neurons by GAD-IMR boutons was not circumscribed to either soma or dendrites but usually involved the entire neuronal perimeter and did not change with time in culture. Three morphologically distinct types of GAD-IMR neurons were evident: a small, bipolar type; a medium-sized multipolar neuron which was the most common and a large, multipolar type, resembling a motoneuron. A small population (8%) of dorsal root ganglion neurons was found to contain GAD both biochemically and immunocytochemically but was never invested by GAD-IMR boutons. GAD activity in vitro paralleled in vivo levels with maximal activity being reached at four weeks in vitro and 10 days postnatally in the intact mouse spinal cord. Murine spinal cord GABAergic neurons are a morphologically diverse and abundant neuronal population with extensive, precocious innervation of all other neuronal phenotypes in vitro suggesting that GABA has a widespread influence over other developing neuronal systems in the murine spinal cord.

Human herpesvirus-6 infection in children. A prospective study of complications and reactivation.

BACKGROUND: Infection with human herpesvirus-6 (HHV-6) is nearly universal in infancy or early childhood. However, the course of this infection, its complications, and its potential for persistence or reactivation remain unclear. METHODS: We studied infants and children under the age of three years who presented to our emergency department with acute illnesses. Infants and young children without acute illness were studied as controls. HHV-6 infection was identified by blood-mononuclear-cell culture, serologic testing, and the polymerase chain reaction (PCR). RESULTS: No primary HHV-6 infection was found among 582 infants and young children with acute nonfebrile illnesses or among 352 controls without acute illness. Of 1653 infants and young children with acute febrile illnesses, 160 (9.7 percent) had primary HHV-6 infection, as documented by viremia and seroconversion. They ranged in age from 2 weeks to 25 months; 23 percent were under the age of 6 months. HHV-6 infections accounted for 20 percent of 365 visits to the emergency department for febrile illnesses among children 6 to 12 months old. Of the 160 infants and young children with acute HHV-6 infections, 21 (13 percent) were hospitalized, and 21 had seizures. Often the seizures appeared late and were prolonged or recurrent. HHV-6 infections accounted for one third of all febrile seizures in children up to the age of two years. In follow-up studies over a period of one to two years, the HHV-6 genome persisted in blood mononuclear cells after primary infection in 37 of 56 children (66 percent). Reactivation, sometimes with febrile illnesses, was suggested by subsequent increases in antibody titers in 16 percent (30 of 187) and by PCR in 6 percent (17 of 278). No recurrent viremia was detected. Of 41 healthy newborns studied, 12 (29 percent) had the HHV-6 genome in their blood mononuclear cells; nevertheless, 6 of these newborns subsequently had primary HHV-6 infections. CONCLUSIONS: In infants and young children HHV-6 infection is a major cause of visits to the emergency department, febrile seizures, and hospitalizations. Perinatal transmission may occur, with possible asymptomatic, transient, or persistent neonatal infection.

Neuropeptide Y immunoreactive neurons in murine trisomy 16 cortical cultures. Plasticity of expression and differentiation.

Neuropeptide Y (NPY)-containing neurons are depleted in the cortices of individuals with Alzheimer disease (AD), yet spared in the striatum of patients with Huntington chorea. It is unknown whether this neuronal phenotype is inherently susceptible to the neurodegenerative processes that are a hallmark of AD. To study this question, the murine trisomy 16 model of Down syndrome and Alzheimer disease was investigated. Since trisomic fetuses die in utero, studies were carried out on primary cultures of dissociated cortical neurons. These were prepared from 15-d gestational trisomy 16 fetuses and their littermate euploid controls, and examined by immunocytochemical staining for neuropeptide Y at 7 and 12 d in vitro. Trisomy 16 neurons were also grown on euploid glial carpets, whereas euploid neurons were grown on trisomic glia. The results demonstrate a significant increase in the number of NPY neurons and a stunting in the dendritic arbor of these neurons in trisomic vs euploid cortex. Both of these parameters could be normalized by direct contact with euploid glia. When euploid cortex was plated on trisomic glia, the number of NPY neurons and their morphology were altered so that they began to resemble trisomic NPY cortical neurons. These results indicate a dysregulation of NPY neuronal expression and differentiation in trisomy 16 cortex that are modifiable by interaction with euploid glia and imply an abnormal trophic (glial) environment in trisomic cortex.

Human herpesvirus-6.

Human herpesvirus-6 (HHV-6), a mature T-cell lymphotropic virus, is a newly recognized member of the herpes virus family and shares limited homology with human cytomegalovirus. HHV-6 has been identified as the etiologic agent of the childhood illness exanthem subitum (roseola infantum). Widespread acquisition of this virus has been demonstrated to occur early in childhood, with fever as the most consistent manifestation of primary infection. Reactivation of latent infection has been postulated and may be associated with lymphoproliferative disorders or other diseases of immune function.

Increased number of somatostatin-immunoreactive neurons in primary cultures of trisomy 16 mouse neocortex.

The gene encoding for pre-prosomatostatin is located on chromosome 16 of the mouse. To determine the effect of an extra copy of this gene on somatostatin expression in neurons, primary disaggregated cultures of neocortex prepared from 15 days gestational Trisomy 16 mice and their littermate euploid controls were subjected to immunocytochemical staining for somatostatin, neuropeptide Y and glutamic acid decarboxylase. The results demonstrate a selective and significant increase in the number of somatostatin-immunoreactive neurons.

Electron microscopy of glutamate decarboxylase (GAD) immunoreactivity in the inner plexiform layer of the rhesus monkey retina.

With indirect immunofluorescence, glutamate decarboxylase (GAD), the GABA synthesizing enzyme, was localized to cell bodies in the inner half of the inner nuclear layer and a few in the outer tier of the ganglion cell layer in the rhesus monkey retina. In the inner plexiform layer there were three strongly GAD-immunoreactive laminae separated by two less immunoreactive laminae. Electron microscopy demonstrated that the GAD was contained in amacrine cells and these GAD-immunoreactive amacrines were primarily pre- and postsynaptic to biopolar cell axon terminals. The GAD-containing processes possessed small synaptic vesicles and formed synapses that could be characterized as symmetrical. Large, dense-cored vesicles were often found in the cell bodies and synaptic processes of the GAD-immunoreactive amacrine cells. As the vast majority of the synaptic input and output of the GAD-containing amacrine cells was to and from bipolar cells and the strongest GAD-immunoreactivity correlated with the endings of bipolar cells that connect with a single cone, the functional effects of GABA in the primate retina are likely to be found in the responses of single cone pathways in the inner plexiform layer.

Morphological and histochemical characterization of three types of dopamine-containing neurons in primary cultures of mouse and rat spinal cord.

Although the anatomical localization and distribution of the neurotransmitter, dopamine, has been extensively studied in the vertebrate central nervous system, the cell bodies of neurons which synthesize and store this transmitter were not thought to be present in the spinal cord. Using the formaldehyde-glutaraldehyde (Faglu) method for the fluorescent visualization of catecholamines, and immunohistochemistry with antisera to the catecholamine synthetic enzymes, we have found in primary cultures of mouse and rat spinal cord three morphologically distinct types of intrinsic spinal cord neurons that contain a catecholamine and the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).

An epinephrine-containing pathway in avian spinal cord: development and localization.

We have studied the uptake mechanism, biochemistry and autoradiographic localization of a descending epinephrine-containing pathway in the chick spinal cord. This epinephrine (E) projection has a developmental timetable (appears at 14 days in ovo) that is different from those of the serotonin (5-HT) and norepinephrine (NE) projections which appear at 8 and 12 days respectively. E possesses its own uptake mechanism with different pharmacological specificities from those of the NE and 5-HT uptake mechanisms. Phenylethanolamine-N-methyltransferase (PNMT), the enzyme that converts NE to E, is present in the cord at 14 days in ovo which is the same time that the uptake mechanism is detectable. Transection of the spinal cord at upper thoracic levels almost completely eliminates the uptake mechanism and PNMT activity below the transection, indicating a supraspinal origin of this pathway. E can first be detected fluorimetrically at 12 days in ovo but at this age E appears not to be of supraspinal origin since transmission at 5 days in ovo does not deplete the spinal cord of E. However, transection of the spinal cord at 3 days post-hatching does markedly reduce the E content by 12 days. Autoradiographic analysis after uptake with [3H]E shows a circumscribed localization of the uptake of E to the neuropil of the preganglionic sympathetic nucleus (nucleus of Terni). These observations demonstrate the presence of a separate descending epinephrine-containing projection in the avian spinal cord which terminates predominantly on preganglionic sympathetic neurons. This pathway may be the major central autonomic pathway in the avian spinal cord.