LMP1 signaling and activation of NF-kappaB in LMP1 transgenic mice.

Transgenic mice expressing Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) under the control of an immunoglobulin heavy-chain promoter and enhancer develop lymphoma at a threefold higher incidence than LMP1-negative mice. In vitro, LMP1 activates numerous signaling pathways including p38, c-Jun N terminal kinase (JNK), phosphatidylinositol 3 kinase (PI3K)/Akt, and NF-kappaB through interactions with tumor necrosis receptor-associated factors (TRAFs). These pathways are frequently activated in EBV-associated malignancies, although their activation cannot be definitively linked to LMP1 expression in vivo. In this study, interactions between LMP1 and TRAFs and the activation of PI3K/Akt, JNK, p38, and NF-kappaB were examined in LMP1 transgenic mice. LMP1 co-immunoprecipitated with TRAFs 1, 2, and 3. Akt, JNK, and p38 were activated in LMP1-positive and -negative splenocytes as well as LMP1-positive and -negative lymphomas. Multiple forms of NF-kappaB were activated in healthy splenocytes from LMP1 transgenic mice, in contrast to healthy splenocytes from LMP1-negative mice. However, in both LMP1-positive and -negative lymphomas, only the oncogenic NF-kappaB c-Rel, was specifically activated. Similarly to EBV-associated malignancies, p53 protein was detected at high levels in the transgenic lymphomas, although mutations were not detected in the p53 gene. These data indicate that NF-kappaB is activated in LMP1-positive healthy splenocytes; however, NF-kappaB c-Rel is specifically activated in both the transgenic lymphomas and in the rare lymphomas that develop in negative mice. The LMP1-mediated activation of NF-kappaB may contribute to the specific activation of c-Rel and lead to the increased development of lymphoma in the LMP1 transgenic mice.

Absence of BRAF mutations in UV-protected mucosal melanomas.

BACKGROUND: Mutations in BRAF have recently been identified in a significant percentage of primary and metastatic cutaneous malignant melanomas. As ultraviolet (UV) exposure may play a role in the development of cutaneous melanoma lesions with BRAF mutations, BRAF mutation frequency in melanomas arising in sites protected from sun exposure may be lower than those from sun-exposed areas. Thus, we determined the BRAF mutation frequency in a panel of 13 mucosal melanomas and compared those data with data from all currently published series of cutaneous melanomas. METHODS: BRAF exon 15 DNA from 13 archival primary mucosal melanomas (eight vulvar, four anorectal, and one laryngeal) was sequenced using intron-based primers. As archival DNA occasionally produces poor-quality template, results were confirmed with a TspRI restriction fragment length polymorphism (RFLP) that distinguishes wild-type BRAF from the common mutant form V599E. A binomial test was used to compare the mutation frequency in the mucosal melanomas with the published mutation frequency in cutaneous melanomas. RESULTS: None of the 13 mucosal melanomas in this series had an exon 15 BRAF mutation, as compared to 54/165 (33%) primary cutaneous melanomas with BRAF mutations in a compilation of all current published studies (p = 0.006). DISCUSSION: These data suggest that UV exposure, plays a role in the genesis of BRAF mutations in cutaneous melanoma, despite the absence of the characteristic C>T or CC>TT mutation signature associated with UV exposure, and suggests mechanisms other than pyrimidine dimer formation are important in UV-induced mutagenesis.

Postnatal development of the vesicular GABA transporter in rat cerebral cortex.

Light and electron microscopic immunocytochemical techniques and Western blotting were used to investigate the postnatal development of the vesicular GABA transporter (VGAT) in the rat somatic sensory cortex. VGAT immunoreactivity was low at birth, it increased gradually through the first and second weeks of life and achieved the adult pattern during the third week. At postnatal day (P)0-P5, VGAT immunoreactivity was associated exclusively to fibers and puncta. Electron microscopic studies performed at P5 showed that all identified synaptic contacts formed by VGAT-positive axonal swellings were of the symmetric type and that a substantial proportion of the boutons appeared not to have formed synapses. From P10 onward, labeled puncta were both scattered in the neuropil and in apposition to unstained cellular profiles; VGAT was also expressed in few GABAergic cell bodies. Western blottings at the same postnatal ages revealed a 55-kDa band whose intensity was weak at P0 (17% of adult), it increased constantly until P15 (P2: 35%; P5: 44%; P10: 68%; P15: 97%), and then leveled off. Overall, the present results show that during neocortical development the expression of VGAT slightly precedes the complete maturation of inhibitory synaptogenesis and suggest that it may contribute to the formation of neocortical GABAergic circuitry.

The glial glutamate transporter GLT-1 is localized both in the vicinity of and at distance from axon terminals in the rat cerebral cortex.

Glutamate transporter-1 (GLT-1) is responsible for the largest proportion of glutamate transport in the brain and the density of GLT-1 molecules inserted in the plasma membrane is highest in regions of high demand. Previous electron microscopic studies in the hippocampus and cerebellum have shown that GLT-1 is concentrated both in the vicinity of and at considerable distance from the synaptic cleft [Chaudry et al., Neuron 15 (1995) 711-721], but little is known about its distribution in the neocortex. We therefore studied the spatial relationships between elements expressing the presynaptic marker synaptophysin and those containing GLT-1 in the rat cerebral cortex using confocal microscopy. Preliminary studies confirmed that GLT-1 positive puncta were exclusively astrocytic processes; moreover, they showed that in most cases GLT-1 positive processes either completely surrounded asymmetric synapses or had no apparent relationship with synapses; occasionally, they were apposed to terminals containing pleomorphic vesicles. In sections double-labeled for GLT-1 and synaptophysin, codistribution analysis revealed that 61.2% of pixels detecting fluorescent emission for GLT-1 immunoreactivity overlapped with pixels detecting synaptophysin. The percentages of GLT-1/synaptophysin codistribution were significantly different from controls. In sections double-labeled for GLT-1 and the vesicular GABA transporter, codistribution analysis revealed that 27% of pixels detecting GLT-1 overlapped with those revealing the vesicular GABA transporter.The remarkable 'synaptic' localization of GLT-1 provides anatomical support for the hypothesis that in the cerebral cortex GLT-1 contributes to shaping fast, point-to-point, excitatory synaptic transmission. Moreover, the considerable fraction of GLT-1 immunoreactivity localized at sites distant from axon terminals supports the notion that glutamate spillout occurs also in the intact brain and suggests that 'extrasynaptic' GLT-1 regulates the diffusion of glutamate escaped from the cleft.

The expression of vesicular glutamate transporters defines two classes of excitatory synapse.

The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.

An acidic motif retains vesicular monoamine transporter 2 on large dense core vesicles.

The release of biogenic amines from large dense core vesicles (LDCVs) depends on localization of the vesicular monoamine transporter VMAT2 to LDCVs. We now find that a cluster of acidic residues including two serines phosphorylated by casein kinase 2 is required for the localization of VMAT2 to LDCVs. Deletion of the acidic cluster promotes the removal of VMAT2 from LDCVs during their maturation. The motif thus acts as a signal for retention on LDCVs. In addition, replacement of the serines by glutamate to mimic phosphorylation promotes the removal of VMAT2 from LDCVs, whereas replacement by alanine to prevent phosphorylation decreases removal. Phosphorylation of the acidic cluster thus appears to reduce the localization of VMAT2 to LDCVs by inactivating a retention mechanism.

Randomised controlled trial of patient education to encourage graded exercise in chronic fatigue syndrome.

OBJECTIVE: To assess the efficacy of an educational intervention explaining symptoms to encourage graded exercise in patients with chronic fatigue syndrome. DESIGN: Randomised controlled trial. SETTING: Chronic fatigue clinic and infectious diseases outpatient clinic. SUBJECTS: 148 consecutively referred patients fulfilling Oxford criteria for chronic fatigue syndrome. INTERVENTIONS: Patients randomised to the control group received standardised medical care. Patients randomised to intervention received two individual treatment sessions and two telephone follow up calls, supported by a comprehensive educational pack, describing the role of disrupted physiological regulation in fatigue symptoms and encouraging home based graded exercise. The minimum intervention group had no further treatment, but the telephone intervention group received an additional seven follow up calls and the maximum intervention group an additional seven face to face sessions over four months. MAIN OUTCOME MEASURE: A score of >/=25 or an increase of >/=10 on the SF-36 physical functioning subscale (range 10 to 30) 12 months after randomisation. RESULTS: 21 patients dropped out, mainly from the intervention groups. Intention to treat analysis showed 79 (69%) of patients in the intervention groups achieved a satisfactory outcome in physical functioning compared with two (6%) of controls, who received standardised medical care (P<0.0001). Similar improvements were observed in fatigue, sleep, disability, and mood. No significant differences were found between the three intervention groups. CONCLUSIONS: Treatment incorporating evidence based physiological explanations for symptoms was effective in encouraging self managed graded exercise. This resulted in substantial improvement compared with standardised medical care.

Neuromelanin biosynthesis is driven by excess cytosolic catecholamines not accumulated by synaptic vesicles.

Melanin, the pigment in hair, skin, eyes, and feathers, protects external tissue from damage by UV light. In contrast, neuromelanin (NM) is found in deep brain regions, specifically in loci that degenerate in Parkinson's disease. Although this distribution suggests a role for NM in Parkinson's disease neurodegeneration, the biosynthesis and function of NM have eluded characterization because of lack of an experimental system. We induced NM in rat substantia nigra and PC12 cell cultures by exposure to l-dihydroxyphenylalanine, which is rapidly converted to dopamine (DA) in the cytosol. This pigment was identical to human NM as assessed by paramagnetic resonance and was localized in double membrane autophagic vacuoles identical to NM granules of human substantia nigra. NM synthesis was abolished by adenoviral-mediated overexpression of the synaptic vesicle catecholamine transporter VMAT2, which decreases cytosolic DA by increasing vesicular accumulation of neurotransmitter. The NM is in a stable complex with ferric iron, and NM synthesis was inhibited by the iron chelator desferrioxamine, indicating that cytosolic DA and dihydroxyphenylalanine are oxidized by iron-mediated catalysis to membrane-impermeant quinones and semiquinones. NM synthesis thus results from excess cytosolic catecholamines not accumulated into synaptic vesicles. The permanent accumulation of excess catechols, quinones, and catechol adducts into a membrane-impermeant substance trapped in organelles may provide an antioxidant mechanism for catecholamine neurons. However, NM in organelles associated with secretory pathways may interfere with signaling, as it delays stimulated neurite outgrowth in PC12 cells.

Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.

While the transporters that accumulate classical neurotransmitters in synaptic vesicles have been identified, little is known about how their expression regulates synaptic transmission. We have used adenoviral-mediated transfection to increase expression of the brain vesicular monoamine transporter VMAT2 and presynaptic amperometric recordings to characterize the effects on quantal release. In presynaptic axonal varicosities of ventral midbrain neurons in postnatal culture, VMAT2 overexpression in small synaptic vesicles increased both quantal size and frequency, consistent with the recruitment of synaptic vesicles that do not normally release dopamine. This was confirmed using noncatecholaminergic AtT-20 cells, in which VMAT2 expression induced the quantal release of dopamine. The ability to increase quantal size in vesicles that were already competent for dopamine release was shown in PC12 cells, in which VMAT2 expression increased the quantal size but not the number of release events. These results demonstrate that vesicle transporters limit the rate of transmitter accumulation and can alter synaptic strength through two distinct mechanisms.

Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter.

Previous work has identified two families of proteins that transport classical neurotransmitters into synaptic vesicles, but the protein responsible for vesicular transport of the principal excitatory transmitter glutamate has remained unknown. We demonstrate that a protein that is unrelated to any known neurotransmitter transporters and that was previously suggested to mediate the Na(+)-dependent uptake of inorganic phosphate across the plasma membrane transports glutamate into synaptic vesicles. In addition, we show that this vesicular glutamate transporter, VGLUT1, exhibits a conductance for chloride that is blocked by glutamate.

A phosphorylation site regulates sorting of the vesicular acetylcholine transporter to dense core vesicles.

Vesicular transport proteins package classical neurotransmitters for regulated exocytotic release, and localize to at least two distinct types of secretory vesicles. In PC12 cells, the vesicular acetylcholine transporter (VAChT) localizes preferentially to synaptic-like microvesicles (SLMVs), whereas the closely related vesicular monoamine transporters (VMATs) localize preferentially to large dense core vesicles (LDCVs). VAChT and the VMATs contain COOH-terminal, cytoplasmic dileucine motifs required for internalization from the plasma membrane. We now show that VAChT undergoes regulated phosphorylation by protein kinase C on a serine (Ser-480) five residues upstream of the dileucine motif. Replacement of Ser-480 by glutamate, to mimic the phosphorylation event, increases the localization of VAChT to LDCVs. Conversely, the VMATs contain two glutamates upstream of their dileucine-like motif, and replacement of these residues by alanine conversely reduces sorting to LDCVs. The results provide some of the first information about sequences involved in sorting to LDCVs. Since the location of the transporters determines which vesicles store classical neurotransmitters, a change in VAChT trafficking due to phosphorylation may also influence the mode of transmitter release.

Evidence for early hematopoietic progenitor cell involvement in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) represents a subtype of acute myeloid leukemia with characteristic morphologic, molecular, and immunophenotypic features. Previous immunophenotypic analyses have shown that leukemic cells in APL typically express the myeloid markers CD33 and CD13 but lack expression of the early hematopoietic progenitor cell antigens CD34 and HLA-DR. We analyzed selected immunophenotypic features of APL by flow cytometry and showed that 7 (41%) of 17 cases contained significant subsets of CD34+ leukemic cells: CD34+ myeloid cells predominated in 2 APL cases. By using a fluorescence-activated cell sorter-fluorescence in situ hybridization approach, we confirmed that the CD34+ cells harbored the t(15;17) translocation characteristic of APL. By using the same experimental approach, CD34+ populations were stratified into primitive CD34+ CD38- and committed CD34+ CD38+ progenitor cell subpopulations; cells in both subsets contained the t(15;17) translocation. The knowledge that APL may be partly or largely CD34+ is important for proper diagnosis. Furthermore, identification of the t(15;17) translocation in CD34+ CD38- blasts indicates that, in at least some cases, the leukemogenic mutation in APL occurs within primitive hematopoietic progenitor cells.

Signature amino acid changes in latent membrane protein 1 distinguish Epstein-Barr virus strains.

Sequence variations in the Epstein-Barr virus (EBV) latent membrane protein 1 gene have been described in numerous EBV-associated tumors with some of these variations, most notably a 30-base pair deletion in the cytoplasmic carboxyl-terminal domain, suggested as associated with an increase in tumorigenicity. In this study, EBV DNA sequence was determined from 92 tissue specimens or cell lines, including nasopharyngeal carcinoma, oral hairy leukoplakia, post-transplant lymphoma, post-transplant without pathology, mononucleosis, Burkitt's lymphoma, parotid tumor, and normal from distinct geographical regions. The amino- and carboxyl-terminal sequences and, in some cases, the full-length sequences of latent membrane protein 1 were determined. Characteristic sequence patterns distinguished strains, with the carboxyl-terminal sequence being the most informative in distinguishing among the strains. Phylogenetic relationships between strains were determined, as were signature amino acid changes that discriminate between them. A correlation between strain and disease or strain and geographic location was not detected. The sequence variation and signature sequences identified at least seven distinct strains, as well as hybrid strains that apparently result from recombination.

Membrane trafficking of neurotransmitter transporters in the regulation of synaptic transmission.

Many psychoactive drugs influence the transport of neurotransmitters across biological membranes, suggesting that the physiological regulation of neurotransmitter transport might contribute to normal and perhaps abnormal behaviour. Over the past few years, molecular characterization of the neurotransmitter transporters has enabled investigation of their subcellular location and regulation. The analysis of location suggests that membrane trafficking has an important role in the normal function of these proteins. One of the major regulatory mechanisms also involves changes in localization that might contribute to synaptic plasticity. This article discusses recent work on the membrane trafficking of neurotransmitter transporters and its role in regulating their activity.

Transient developmental expression of monoamine transporters in the rodent forebrain.

Neurons in first-order sensory thalamic nuclei have been shown to express functional plasma membrane serotonin (SERT) and vesicular monoamine (VMAT2) transporters during early postnatal development. In the present study, we provide an extensive description of the spatial and the temporal patterns of VMAT2 and SERT expression, during early embryonic development and postnatal life, by using in situ hybridization and immunocytochemistry. VMAT2 and SERT genes are transiently expressed in a wide population of non-monoaminergic neurons in the central and peripheral nervous system with a large overlap in the temporal and spatial pattern of expression of both genes. A selective pattern of expression of both genes was observed in the thalamus with expression limited to the dorsal thalamus and more particularly to primary sensory relay nuclei that convey point to point projection maps. Transient expression of the transporters was also observed in sensory cranial nerves, in the hippocampus, cerebral cortex, septum, and amygdala. VMAT2 and SERT gene expression was not necessarily linked, as some neural populations expressed only VMAT2, while others only contained SERT. Since VMAT2 serves to transport catecholamines besides serotonin, we examined the developmental expression of the plasma membrane dopamine and norepinephrine transporters but found no transient expression of these genes. Despite minor temporal disparities, VMAT2 and SERT extinguished almost simultaneously during the second and third weeks of post-natal life. These expressions did not seem to be dependent on peripheral neural inputs, since monocular enucleations and infraorbital nerve cuts effected on the day of birth, did not modify the period of transporter expression or of extinction.

The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons.

A transporter thought to mediate accumulation of GABA into synaptic vesicles has recently been cloned (McIntire et al., 1997). This vesicular GABA transporter (VGAT), the first vesicular amino acid transporter to be molecularly identified, differs in structure from previously cloned vesicular neurotransmitter transporters and defines a novel gene family. Here we use antibodies specific for N- and C-terminal epitopes of VGAT to localize the protein in the rat CNS. VGAT is highly concentrated in the nerve endings of GABAergic neurons in the brain and spinal cord but also in glycinergic nerve endings. In contrast, hippocampal mossy fiber boutons, which although glutamatergic are known to contain GABA, lack VGAT immunoreactivity. Post-embedding immunogold quantification shows that the protein specifically associates with synaptic vesicles. Triple labeling for VGAT, GABA, and glycine in the lateral oliva superior revealed a higher expression of VGAT in nerve endings rich in GABA, with or without glycine, than in others rich in glycine only. Although the great majority of nerve terminals containing GABA or glycine are immunopositive for VGAT, subpopulations of nerve endings rich in GABA or glycine appear to lack the protein. Additional vesicular transporters or alternative modes of release may therefore contribute to the inhibitory neurotransmission mediated by these two amino acids.

Novel bcl-2 breakpoints in patients with follicular lymphoma.

Using genomic DNA from patients with follicular lymphoma, we performed polymerase chain reaction (PCR) amplifications to detect t(14;18) translocations. Unexpectedly large products of approximately 1 kilobase (kb) were detected by gel electrophoresis in 2 of 50 positive cases. In these 2 cases, sequence analyses showed novel breakpoints in the 3' untranslated region of bcl-2, approximately 800 bp downstream of the major breakpoint region (mbr). The breakpoints in IgH occurred in JH4 in one patient and JH5 in the other. Sequences just upstream of the new bcl-2 breakpoints suggest a mechanism of translocation that may include minisatellite core-mediated recombination. In one of our two patients with novel bcl-2 breakpoints, the approximately 1 kb product obtained using conventional mbr primers was detectable only when a nested PCR was performed. These findings have important implications for diagnosis and minimal residual disease detection in t(14;18)-positive lymphomas.

Expression of the Epstein-Barr virus latent membrane protein 1 induces B cell lymphoma in transgenic mice.

The latent membrane protein 1 (LMP1) of the Epstein-Barr virus has transforming properties in rodent fibroblasts and is expressed in most of the cancers associated with Epstein-Barr virus (EBV) infection including posttransplant lymphomas, Hodgkin's disease, nasopharyngeal carcinoma, and AIDS-related lymphomas. In this study, three lineages of LMP1 transgenic mice were established with LMP1 expressed under the control of the Ig heavy chain promoter and enhancer. Lymphoma developed in all three lineages, and the incidence of lymphoma increased significantly with age with lymphomas developing in 42% of transgenic mice over 18 months. The expression of LMP1 was detected at high levels in the lymphoma tissues but only at trace levels in normal lymphoid tissues. Gene rearrangement of the Ig heavy chain indicated monoclonality or oligoclonality in all lymphomas, some of the lymphoid hyperplastic spleens, and some histologically normal spleens. These data reveal that LMP1, without the expression of other EBV genes, is oncogenic in vivo and indicate that LMP1 is a major contributing factor to the development of EBV-associated lymphomas.

High-efficiency expression and characterization of the synaptic-vesicle monoamine transporter from baculovirus-infected insect cells.

The full-length cDNA for the rat synaptic-vesicle monoamine transporter (VMAT2) containing a C-terminal polyhistidine epitope has been engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) insect cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed at levels of 7.8x10(6) transporters per cell, as assessed by [3H]dihydrotetrabenazine binding. A 1l culture of infected cells produced approx. 15 nmol (900 microg) of transporter. rVMAT2 expressed in the Sf9 cells bound [3H]dihydrotetrabenazine with a KD of 31.2 nM and a Bmax of 19.9 pmol/mg. Two polypeptides of 55 and 63 kDa were identified using the photolabel, 7-azido-8-[125I]iodoketanserin ([125I]AZIK). Photoaffinity labelling of rVMAT2 by 1 nM [125I]AZIK was protectable by 10 microM tetrabenazine and 10 microM 7-aminoketanserin. Digitonin-solubilized VMAT2 was purified to greater than 95% homogeneity using immobilized Ni2+-affinity chromatography, followed by lectin (Concanavalin A) chromatography. The purified transporter migrates as a single broad band with a molecular mass of approx. 63kDa, as analyzed by SDS/PAGE. The purified transporter retained the ability to bind ligands ([125I]AZIK and [3H]dihydrotetrabenazine). The purified VMAT2 bound [3H]dihydrotetrabenazine with a KD of 86.2 nM. As is the case with the monoamine transporter from bovine chromaffin granule membranes, purified VMAT2 is covalently modified by dicyclohexylcarbodi-imide (DCCD) and is specifically labelled by [14C]DCCD. This labelling is inhibited by tetrabenazine and ketanserin. These data indicate that VMAT2 can be overexpressed using the baculovirus expression system and purified.

Epstein-Barr virus strain variation in nasopharyngeal carcinoma from the endemic and non-endemic regions of China.

Nasopharyngeal carcinoma (NPC) occurs with a striking geographic incidence and is endemic in parts of southern China, where it is the major cause of cancer death. Epstein-Barr virus (EBV) is detected in all cells of the majority of NPC cases regardless of geographic origin. A small subset of EBV genes is expressed in NPC, including the latent membrane protein (LMP-1). LMP-1 is essential for transformation of B lymphocytes and is considered to be the EBV oncogene. This analysis of the DNA sequence variation within the LMP-1 gene reveals a consensus sequence for a strain, denoted China1, which predominates in East Asia where NPC is endemic. The China1 strain is characterized by nucleotide changes at 13 loci in the amino terminal portion of the LMP-1 gene when compared with the B95-8 prototype, including a point mutation resulting in the loss of an Xho1 restriction site. This strain was present in 9 of 15 NPC biopsy specimens from the endemic region and in 7 of 13 from northern China, where NPC is non-endemic. A second strain, China2, was detected in 4 of 15 endemic isolates and in 2 of 13 non-endemic isolates; this strain was characterized by a cluster of 5 nucleotide changes in the amino terminal portion of LMP-1 in addition to those seen in China1. It was also marked by distinct changes in the carboxy terminal region of LMP-1 including the retention of amino acids 343-352. All China1 isolates were EBV type 1, whereas the China2 isolates did not correlate with EBV type. Phylogenetic relationships between these 2 strains were determined, as were signature amino acid alterations that discriminate between them.