Pharmacologic profiling reveals lapatinib as a novel antiviral against SARS-CoV-2 in vitro.

The emergence of SARS-CoV-2 virus has resulted in a worldwide pandemic, but effective antiviral therapies are not widely available. To improve treatment options, we conducted a high-throughput screen to uncover compounds that block SARS-CoV-2 infection. A minimally pathogenic human betacoronavirus (OC43) was used to infect physiologically-relevant human pulmonary fibroblasts (MRC5) to facilitate rapid antiviral discovery in a preclinical model. Comprehensive profiling was conducted on more than 600 compounds, with each compound arrayed across 10 dose points. Our screening revealed several FDA-approved agents that can attenuate both OC43 and SARS-CoV-2 viral replication, including lapatinib, doramapimod, and 17-AAG. Importantly, lapatinib inhibited SARS-CoV-2 RNA replication by over 50,000-fold. Further, both lapatinib and doramapimod could be combined with remdesivir to improve antiviral activity in cells. These findings reveal novel therapeutic avenues that could limit SARS-CoV-2 infection.

Evaluating the Antimicrobial Properties of Commercial Hand Sanitizers.

Hand sanitizers have been developed as a convenient means to decontaminate an individual's hands of bacterial pathogens in situations in which soap and water are not available. Yet to our knowledge, no study has compared the antibacterial efficacy of a large collection of hand sanitizers. Using zone of growth inhibition and kill curve assays, we assessed the performance of 46 commercially available hand sanitizers that were obtained from national chain big-box stores, gasoline stations, pharmacies, and boutiques for antibacterial activity toward prototypical Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacterial pathogens. Results revealed substantial variability in the efficacy of many sanitizers evaluated. Formulations following World Health Organization-recommended ingredients (80% ethanol or 75% isopropyl alcohol) or those including benzalkonium chloride as the active principal ingredient displayed excellent antibacterial activity, whereas others exhibited modest or poor activity in the assays performed. Results also revealed that E. coli was generally more susceptible to most sanitizers in comparison to S. aureus and that there was significant strain-to-strain variability in hand sanitizer antimicrobial efficacy regardless of the organism evaluated. Further, tests of a subset of hand sanitizers toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed no direct correlation between antibacterial and antiviral performance, with all ethyl alcohol formulations performing equally well and displaying improved activity in comparison to benzalkonium chloride-containing sanitizer. Taken together, these results indicate that there is likely to be substantial variability in the antimicrobial performance of commercially available hand sanitizers, particularly toward bacterial pathogens, and a need to evaluate the efficacy of sanitizers under development.IMPORTANCE In response to the coronavirus disease 2019 (COVID-19) pandemic, hand hygiene has taken on a prominent role in efforts to mitigate SARS-CoV-2 transmission and infection, which has led to a radical increase in the number and types of hand sanitizers manufactured to meet public demand. To our knowledge, no studies have evaluated or compared the antimicrobial performance of hand sanitizers that are being produced under COVID-19 emergency authorization. Tests of 46 commercially available hand sanitizers purchased from national chain brick-and-mortar stores revealed considerable variability in their antibacterial performance toward two bacterial pathogens of immediate health care concern, S. aureus and E. coli Expanded testing of a subset of hand sanitizers revealed no direct correlation between antibacterial performance of individual sanitizers and their activity toward SARS-CoV-2. These results indicate that as the pandemic subsides, there will be a need to validate the antimicrobial efficacy of sanitizers being produced.

Mechanisms of head stability during gait initiation in young and older women: A neuro-mechanical analysis.

Decreased head stability has been reported in older women during locomotor transitions such as the initiation of gait. The aim of the study was to investigate the neuro-mechanical mechanisms underpinning head stabilisation in young and older women during gait initiation. Eleven young (23.1 ±â€¯1.1 yrs) and 12 older (73.9 ±â€¯2.4 yrs) women initiated walking at comfortable speed while focussing on a fixed visual target at eye level. A stereophotogrammetric system was used to assess variability of angular displacement and RMS acceleration of the pelvis, trunk and head, and dynamic stability in the anteroposterior and mediolateral directions. Latency of muscle activation in the sternocleidomastoid, and upper and lower trunk muscles were determined by surface electromyography. Older displayed higher variability of head angular displacement, and a decreased ability to attenuate accelerations from trunk to head, compared to young in the anteroposterior but not mediolateral direction. Moreover, older displayed a delayed onset of sternocleidomastoid activation than young. In conclusion, the age-related decrease in head stability could be attributed to an impaired ability to attenuate accelerations from trunk to head along with delayed onset of neck muscles activation.

Cyclin D mediates tolerance of genome-doubling in cancers with functional p53.

Background: Aneuploidy and chromosomal instability (CIN) are common features of human malignancy that fuel genetic heterogeneity. Although tolerance to tetraploidization, an intermediate state that further exacerbates CIN, is frequently mediated by TP53 dysfunction, we find that some genome-doubled tumours retain wild-type TP53. We sought to understand how tetraploid cells with a functional p53/p21-axis tolerate genome-doubling events. Methods: We performed quantitative proteomics in a diploid/tetraploid pair within a system of multiple independently derived TP53 wild-type tetraploid clones arising spontaneously from a diploid progenitor. We characterized adapted and acute tetraploidization in a variety of flow cytometry and biochemical assays and tested our findings against human tumours through bioinformatics analysis of the TCGA dataset. Results: Cyclin D1 was found to be specifically overexpressed in early but not late passage tetraploid clones, and this overexpression was sufficient to promote tolerance to spontaneous and pharmacologically induced tetraploidy. We provide evidence that this role extends to D-type cyclins and their overexpression confers specific proliferative advantage to tetraploid cells. We demonstrate that tetraploid clones exhibit elevated levels of functional p53 and p21 but override the p53/p21 checkpoint by elevated expression of cyclin D1, via a stoichiometry-dependent and CDK activity-independent mechanism. Tetraploid cells do not exhibit increased sensitivity to abemaciclib, suggesting that cyclin D-overexpressing tumours might not be specifically amenable to treatment with CDK4/6 inhibitors. Conclusions: Our study suggests that D-type cyclin overexpression is an acute event, permissive for rapid adaptation to a genome-doubled state in TP53 wild-type tumours and that its overexpression is dispensable in later stages of tumour progression.

Effect of exercise training on neuromuscular function of elbow flexors and knee extensors of type 2 diabetic patients.

PURPOSE: The effects of exercise training on neuromuscular function of arm and leg muscles in type 2 diabetic patients (T2D) was investigated. METHODS: Eight T2D sedentary male patients (61.0±2.3years) and eight sedentary healthy age matched control subjects (H, 63.9±3.8years) underwent a 16-week supervised combined endurance and resistance exercise program. Before and after training, maximal isometric (MVIC), isokinetic (15, 30, 60, 120, 180, 240°s(-1)) torque and muscle endurance of the elbow flexors (EF) and knee extensors (KE) were assessed. Simultaneously, surface electromyographic signals from biceps brachii (BB) and vastus lateralis (VL) muscles were recorded and muscle fiber conduction velocity (MFCV) estimated. RESULTS: Following training, maximal torque of the KE increased during MVIC and isokinetic contractions at 15 and 30°s(-1) in the T2D (+19.1±2.7% on average; p<0.05) but not in the H group (+7±0.9%; p>0.05). MFCV recorded from the VL during MVIC and during isokinetic contractions at 15 and 30°s(-1) increased (+11.2±1.6% on average; p<0.01), but in the diabetic group only. Muscular endurance was lower in T2D (20.1±0.7s) compared to H (26.9±1.3s), with an associated increase in the MFCV slope after training in the KE muscles only. CONCLUSION: The effect of a combined exercise training on muscle torque appears to be angular velocity-specific in diabetic individuals, with a more pronounced effect on KE muscles and at slow contraction velocities, along with an associated increase in the MFCV. MFCV appears to be a more sensitive marker than torque in detecting the early signs of neuromuscular function reconditioning.

In vivo gene delivery and expression by bacteriophage lambda vectors.

AIMS: Bacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo. METHODS AND RESULTS: Mice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo. Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer. CONCLUSIONS: Unmodified lambda phage particles are capable of transducing mammalian cells in vivo, and may be taken up -- at least in part -- by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo, and suggest new approaches that may enhance the efficiency of this process.

Efficient gene transfer into human monocyte-derived macrophages using defective lentiviral vectors.

Gene therapy is a promising approach for the treatment of neurological disorders. However, current approaches to gene transfer in the central nervous system (CNS) are limited by the lack of effective, but non-invasive methods to deliver transgenes across the blood-brain barrier (BBB). In an effort to begin to explore the use of migratory monocytes as vehicles for delivery of therapeutic and antiviral genes into the CNS, we have utilized three HIV-based transfer vectors encoding cis-acting elements but lacking either structural genes (gag/pol and env), most accessory genes (vif, vpr and nef) and/or rev. These defective lentiviral vectors (DLV) encode the green fluorescent protein (GFP), display potent antiviral activity in CD4+ lymphocytes and can be mobilized by wild-type HIV-1 DLV were generated by transient transfection of 293T cells. Vector titers ranged from 4.2-6.6 x 10(6) infectious units (IU)/ml prior to concentration (by ultracentrifugation) and were equal to or higher than 1 x 10(9) IU/ml after concentration. Primary human monocyte-derived macrophages (MDM) were exposed to DLV resulting in efficiencies of transduction ranging from 14 to 26%. GFP expression in transduced MDM remained stable for more than 8 weeks without apparent cytopathic effect. Given the previously reported antiviral activities of these DLV and their lack of cytopathic effects on primary MDM, it may be possible to use these vectors to inhibit HIV-1 replication within the CNS.

Epitope mapping of human herpesvirus-7 gp65 using monoclonal antibodies.

Human herpesvirus (HHV)-7 encodes a unique 65-kDa heparin-binding glycoprotein, designated gp65. This molecule is thought to play a role in virus attachment and entry. To obtain reagents to map the structure and function of HHV-7 gp65, we produced monoclonal antibodies to this molecule. Ten monoclonal antibodies reacting with gp65 on ELISA were subdivided in four groups on the basis of their isotype and differential reactivity with (i) native versus denatured forms of gp65, and (ii) mature (virion-associated) versus immature (cell-associated) forms of the molecule. We were able to map the binding epitopes for eight of these ten antibodies, and these were found to cluster to one site on gp65 (amino acids 239-278); within this region, the antibodies reacted with at least three distinct domains (244-251, 255-262, 263-278). The reasons for the apparent immunodominance of this region are uncertain. Taken together, this panel of antibodies constitutes an extensive and well-characterized set of HHV-7 specific antibodies that may have utility for future analyses of the structure/function of gp65, and for studies on the virus life cycle.

Traditional tooth bud gouging in a Ugandan family: a report involving three sisters.

It has been reported in the literature that some rural populations of Sub-Saharan and Eastern Africa and other isolated areas around the world, practise gouging or enucleation of primary tooth buds to cure childhood illnesses. The unerupted primary canine tooth bud is believed to be the cause of febrile illness, diarrhoea, and vomiting; prevalent in infants in these areas of the world. Tooth bud gouging has implications for the developing dentition, and is a potential risk to the health and life of the child. Reported prevalence ranges from 22%-90%. From the information in this case report, the practise of tooth bud gouging is no longer confined to rural areas and may well be performed by communities that have emigrated to the UK. The three sisters described, now resident in the UK, suffered tooth gouging in a city clinic in Uganda. The damage caused to the primary and permanent dentition is described and treatment planning and options are suggested for each case to restore structure and function. Appropriate provision of healthcare and education could avoid the potential long-term damage to the health of the child and their developing dentition by the practise of tooth bud gouging, whether it occurs in the developing or developed world.

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.

Neurotrophins prevent HIV Tat-induced neuronal apoptosis via a nuclear factor-kappaB (NF-kappaB)-dependent mechanism.

HIV-1 associated dementia is thought to be caused by neuronal damage and death in response to the production of soluble neurotoxic factors by virally infected mononuclear phagocytes. These neurotoxins include HIV-1 Tat. The ability of neurotrophins to promote cell survival prompted us to examine whether neurotrophins might also be capable of opposing the pro-apoptotic effects of Tat. Here, we show that Tat-induced neuronal apoptosis in primary cultures of rat cerebellar granule cells and in neuronally differentiated human SK-N-MC cells is profoundly inhibited by brain-derived neurotrophic factor, nerve growth factor and activity-dependent neurotrophic factor nonamer peptide. These neurotrophins activated the transcription factor NF-kappaB, and inhibition of NF-kappaB activation using a super-repressor IkappaB-alpha mutant was found to block the survival-promoting activity of the neurotrophins. Reporter gene assays and immunoblot experiments revealed that the neurotrophins also up-regulated the expression of Bcl-2, at both the transcriptional and protein levels. Overexpression of the super-repressor IkappaB-alpha mutant prevented this induction of Bcl-2 expression. Moreover, overexpression of either Bcl-2, alone, or the RelA subunit of NF-kappaB, alone, protected neurons from Tat-induced apoptosis. These findings suggest that the activation of NF-kappaB by neurotrophic factors may promote survival of neurons exposed to Tat, via regulation of anti-apoptotic genes including Bcl-2.

Activation of glycogen synthase kinase 3 beta (GSK-3beta) by platelet activating factor mediates migration and cell death in cerebellar granule neurons.

Children with vertically acquired HIV-1 can present with a rapidly progressive encephalopathy and neuronal apoptosis in the first 12-18 months of life. Furthermore, abnormal prenatal platelet activating factor (PAF) signalling may result in lissencephaly, a disorder of neuronal migration. PAF, produced from human immunodeficiency virus type 1 (HIV-1) -infected brain-resident macrophages, induces neuronal apoptosis in cultured cerebellar granule neurons (CGNs) in part by activating glycogen synthase kinase 3 beta (GSK-3beta). However, PAF can also inhibit migration of CGNs that are dispersed and allowed to reaggregate. Therefore, we investigated the biological effects following activation of GSK-3beta by PAF, and whether these effects were dependent on the culture conditions of the CGNs. We show here that activation of neuronal GSK-3beta by PAF is receptor-specific, with similar kinetics of activation in both monolayer cultures of CGNs that have ceased to migrate and reaggregate cultures of CGNs that are actively migrating. However, PAF receptor activation in reaggregated CGNs inhibits neuronal migration and induces approximately half the level of neuronal apoptosis compared with PAF-treated CGN cultures that have ceased to migrate. PAF-mediated inhibition of neuronal migration in reaggregated CGNs or induction of apoptosis in CGNs that have ceased to migrate can be reversed by either PAF receptor antagonists, or the GSK-3beta inhibitors lithium or valproic acid, in a dose-dependent manner. Abnormal PAF signalling that results in GSK-3beta overactivation may represent a common mechanism for pathological defects in neuronal migration in the prenatal period and neuronal apoptosis in the postnatal period.

Adenovirus type 7 penton purification of soluble pentamers from Escherichia coli and development of an integrin-dependent gene delivery system.

Adenoviral gene therapy vectors suffer from the disadvantages of toxicity and immunogenicity associated with the expression of adenoviral genes from the vector backbone. We report here an alternative strategy for gene delivery that utilizes a single component of the adenoviral type 7 capsid, the penton base (Ad7PB). The Ad7PB gene was sequenced and its amino-acid composition was deduced from its nucleotide sequence. The penton was expressed in Escherichia coli as a soluble C-terminal fusion with glutathione S-transferase (GST-Ad7PB) and was purified by single-step affinity chromatography. Both GST-Ad7PB and cleaved (GST-free) Ad7PB retained the ability to fold into pentamers as observed by electron microscopy. GST-Ad7PB was able to bind a synthetic peptide (FK20) derived from the Ad type 7 fiber and retard DNA through a polylysine chain present at the C-terminus of this linker peptide. GST-Ad7PB was an effective cell transfecting agent when assayed on 293 cells. Transfection was not dependent upon the presence of lysosomotropic agents indicating efficient endosome escape capability. Excess of an RGD-containing peptide derived from Ad7PB was able to inhibit transfection indicating specific integrin-mediated uptake of the GST-Ad7PB-FK20-DNA complexes. We propose that Ad7 pentons can be developed into integrin-specific gene delivery agents.

Identification and analysis of a novel heparin-binding glycoprotein encoded by human herpesvirus 7.

Human herpesvirus 6 (HHV-6) and HHV-7 are closely related betaherpesviruses that encode a number of genes with no known counterparts in other herpesviruses. The product of one such gene is the HHV-6 glycoprotein gp82-105, which is a major virion component and a target for neutralizing antibodies. A 1.7-kb cDNA clone from HHV-7 was identified which contains a large open reading frame capable of encoding a predicted primary translational product of 468 amino acids (54 kDa) with 13 cysteine residues and 9 potential N-linked glycosylation sites. This putative protein, which we have termed gp65, was homologous to HHV-6 gp105 (30% identity) and contained a single potential membrane-spanning domain located near its amino terminus. Comparison of the cDNA sequence with that of the viral genome revealed that the gene encoding gp65 contains eight exons, spanning almost 6 kb of the viral genome at the right (3') end of the HHV-7 genome. Northern (RNA) blot analysis with poly(A)(+) RNA from HHV-7-infected cells revealed that the cDNA insert hybridized to a single major RNA species of 1.7 kb. Antiserum raised against a purified, recombinant form of gp65 recognized a protein of roughly 65 kDa in sucrose density gradient-purified HHV-7 preparations; treatment with PNGase F reduced this glycoprotein to a putative precursor of approximately 50 kDa. Gp65-specific antiserum also neutralized the infectivity of HHV-7, while matched preimmune serum did not do so. Finally, analysis of the biochemical properties of recombinant gp65 revealed a specific interaction with heparin and heparan sulfate proteoglycans and not with closely related molecules such as N-acetylheparin and de-N-sulfated heparin. At least two domains of the protein were found to contribute to heparin binding. Taken together, these findings suggest that HHV-7 gp65 may contribute to viral attachment to cell surface proteoglycans.

Pathogenesis and treatment of HIV-1 infection: recent developments (Y2K update).

Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of acquired immunodeficiency syndrome (AIDS). The pathogenesis of HIV-1-induced disease is complex and characterized by the interplay of both viral and host factors, which together determine the outcome of infection. An improved understanding of the pathogenic mechanisms of AIDS, combined with recent insights into the dynamics of viral infection may provide powerful new opportunities for therapeutic intervention against this virus.

Neuronal fractalkine expression in HIV-1 encephalitis: roles for macrophage recruitment and neuroprotection in the central nervous system.

HIV-1 infection of the brain results in chronic inflammation, contributing to the neuropathogenesis of HIV-1 associated neurologic disease. HIV-1-infected mononuclear phagocytes (MP) present in inflammatory infiltrates produce neurotoxins that mediate inflammation, dysfunction, and neuronal apoptosis. Neurologic disease is correlated with the relative number of MP in and around inflammatory infiltrates and not viral burden. It is unclear whether these cells also play a neuroprotective role. We show that the chemokine, fractalkine (FKN), is markedly up-regulated in neurons and neuropil in brain tissue from pediatric patients with HIV-1 encephalitis (HIVE) compared with those without HIVE, or that were HIV-1 seronegative. FKN receptors are expressed on both neurons and microglia in patients with HIVE. These receptors are localized to cytoplasmic structures which are characterized by a vesicular appearance in neurons which may be in cell-to-cell contact with MPs. FKN colocalizes with glutamate in these neurons. Similar findings are observed in brain tissue from an adult patient with HIVE. FKN is able to potently induce the migration of primary human monocytes across an endothelial cell/primary human fetal astrocyte trans-well bilayer, and is neuroprotective to cultured neurons when coadministered with either the HIV-1 neurotoxin platelet activating factor (PAF) or the regulatory HIV-1 gene product Tat. Thus focal inflammation in brain tissue with HIVE may up-regulate neuronal FKN levels, which in turn may be a neuroimmune modulator recruiting peripheral macrophages into the brain, and in a paracrine fashion protecting glutamatergic neurons.

Functional interplay between nuclear factor-kappaB and c-Jun integrated by coactivator p300 determines the survival of nerve growth factor-dependent PC12 cells.

Nerve growth factor (NGF) activates the transcription factors nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) in sympathetic neurons. Whereas NGF-inducible NF-kappaB is required for the survival of neurons, c-Jun has the ability to promote neuronal death. In this report, we have examined the effect of NGF withdrawal on c-Jun and NF-kappaB transcription factors in PC12 cells differentiated to a neuronal phenotype. We show that the withdrawal of NGF from these cultures results in de novo synthesis of c-Jun, increase in AP-1 activity, and down-regulation of NF-kappaB activity. To investigate how the signal transduction pathways activating c-Jun and NF-kappaB are differentially regulated by NGF, we performed transcriptional analyses. Expression of ReIA (NF-kappaB) suppressed the c-Jun-dependent transcription of c-jun, and this effect was reversed by overexpression of the coactivator p300. RelA's effects on c-Jun transcription were mediated by competitive binding of the carboxy-terminal region of RelA to the CH1 domain of p300, which also binds to c-Jun; deletion of this region abrogated the ability of RelA to inhibit c-Jun activity. Furthermore, the inhibition of endogenous NF-kappaB in NGF-maintained neuronal PC12 cells led to the induction of c-Jun synthesis and a marked increase in cell death. Together, these studies demonstrate a functional interaction between NF-kappaB and c-Jun and suggest a novel mechanism of NF-kappaB-mediated neuroprotection.

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.

Human herpesvirus 6B genome sequence: coding content and comparison with human herpesvirus 6A.

Human herpesvirus 6 variants A and B (HHV-6A and HHV-6B) are closely related viruses that can be readily distinguished by comparison of restriction endonuclease profiles and nucleotide sequences. The viruses are similar with respect to genomic and genetic organization, and their genomes cross-hybridize extensively, but they differ in biological and epidemiologic features. Differences include infectivity of T-cell lines, patterns of reactivity with monoclonal antibodies, and disease associations. Here we report the complete genome sequence of HHV-6B strain Z29 [HHV-6B(Z29)], describe its genetic content, and present an analysis of the relationships between HHV-6A and HHV-6B. As sequenced, the HHV-6B(Z29) genome is 162,114 bp long and is composed of a 144,528-bp unique segment (U) bracketed by 8,793-bp direct repeats (DR). The genomic sequence allows prediction of a total of 119 unique open reading frames (ORFs), 9 of which are present only in HHV-6B. Splicing is predicted in 11 genes, resulting in the 119 ORFs composing 97 unique genes. The overall nucleotide sequence identity between HHV-6A and HHV-6B is 90%. The most divergent regions are DR and the right end of U, spanning ORFs U86 to U100. These regions have 85 and 72% nucleotide sequence identity, respectively. The amino acid sequences of 13 of the 17 ORFs at the right end of U differ by more than 10%, with the notable exception of U94, the adeno-associated virus type 2 rep homolog, which differs by only 2.4%. This region also includes putative cis-acting sequences that are likely to be involved in transcriptional regulation of the major immediate-early locus. The catalog of variant-specific genetic differences resulting from our comparison of the genome sequences adds support to previous data indicating that HHV-6A and HHV-6B are distinct herpesvirus species.