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Measles remains an important problem in Africa, where human immunodeficiency virus type 1 (HIV-1) infection is prevalent. To identify the consequences of coinfection, Zambian children hospitalized with measles were studied at entry, discharge, and 1 month after discharge. All children had low lymphocyte and eosinophil counts at entry and high leukocyte and monocyte counts during recovery. The death of cultured lymphocytes was more prolonged for HIV-positive children. CD38 and Fas were increased on CD4+ and CD8+ lymphocytes, and CD28 was decreased on CD8+ lymphocytes in all children. Abnormalities in CD4+ and CD8+ lymphocyte percentages and CD28 expression associated with HIV infection were preserved during measles. Therefore, the patterns of changes in leukocyte counts were similar, with little evidence that measles exacerbated HIV-associated lymphocyte abnormalities. Copyright © 2005 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Background: Plasma thrombin generation has been used to characterize trauma-induced coagulopathy, but description of whole blood thrombin generation is lacking. This study aimed to evaluate plasma and whole blood thrombin generation in healthy volunteers and trauma patients. We hypothesized that (1) plasma and whole blood thrombin generation are distinct, (2) whole blood thrombin generation is more pronounced in trauma patients than in healthy volunteers, and (3) thrombin generation correlates with clinical coagulation assays., Methods: Blood was collected from healthy volunteers and trauma patients at a single, level-1 trauma center. Whole blood thrombin generation was assessed with a prototype point-of-care whole blood thrombin generation device, and plasma thrombin generation was measured with a calibrated automated thrombogram analogue. Plasma and whole blood thrombin generation were compared and correlated with international normalized ratio and thrombelastography., Results: Overall, 10 healthy volunteers (average age 30, 50% men) were included and 58 trauma patients (average age 34, 76% men, 55% blunt mechanism, and with a median new injury severity score of 17) were included. Plasma and whole blood thrombin generation differed with more robust thrombin generation in plasma. Trauma patients had a significantly increased whole blood thrombin generation compared with healthy volunteers]. Plasma thrombin generation correlated with international normalized ratio, whereas whole blood thrombin generation did not correlate with thrombelastography., Conclusion: Plasma and whole blood thrombin generation are distinct, highlighting the need to perform standardized assays to better understand their correlation and to assess how whole blood thrombin generation confers differential outcomes in trauma., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: We investigated associations between maternal characteristics, access to care, and obstetrical complications including near miss status on admission or during hospitalization on perinatal outcomes among Indonesian singletons., Methods: We prospectively collected data on inborn singletons at two hospitals in East Java. Data included socio-demographics, reproductive, obstetric and neonatal variables. Reduced multivariable models were constructed. Outcomes of interest included low and very low birthweight (LBW/VLBW), asphyxia and death., Results: Referral from a care facility was associated with a reduced risk of LBW and VLBW [AOR = 0.28, 95% CI = 0.11-0.69, AOR = 0.18, 95% CI = 0.04-0.75, respectively], stillbirth [AOR = 0.41, 95% CI = 0.18-0.95], and neonatal death [AOR = 0.2, 95% CI = 0.05-0.81]. Mothers age <20 years increased the risk of VLBW [AOR = 6.39, 95% CI = 1.82-22.35] and neonatal death [AOR = 4.10, 95% CI = 1.29-13.02]. Malpresentation on admission increased the risk of asphyxia [AOR = 4.65, 95% CI = 2.23-9.70], stillbirth [AOR = 3.96, 95% CI = 1.41-11.15], and perinatal death [AOR = 3.89 95% CI = 1.42-10.64], as did poor prenatal care (PNC) [AOR = 11.67, 95%CI = 2.71-16.62]. Near-miss on admission increased the risk of neonatal [AOR = 11.67, 95% CI = 2.08-65.65] and perinatal death [AOR = 13.08 95% CI = 3.77-45.37]., Conclusions: Mothers in labor should be encouraged to seek care early and taught to identify early danger signs. Adequate PNC significantly reduced perinatal deaths. Improved hospital management of malpresentation may significantly reduce perinatal morbidity and mortality. The importance of hospital-based prospective studies helps evaluate specific areas of need in training of obstetrical care providers.

To identify immune factors present during the acute rash phase of measles and associations with outcome and human immunodeficiency virus type 1 (HIV-1) coinfection, we measured the plasma levels of 22 cytokines and chemokines in Zambian children hospitalized with measles (n = 148) and control children (n = 44). Children with measles had higher levels of innate cytokines tumor necrosis factor (TNF) α, interleukin 1β (IL-1β), interleukin 18, and interleukin 6; chemokines CCL2, CCL4, CCL11, CCL22, CXCL8, and CXCL10; and T-cell cytokines interferon γ, and interleukin 2, 10, and 17. Children who died in the hospital had higher levels of TNF-α, IL-1β, interleukin 12p70; CCL2, CCL4, CCL13, CCL17, CXCL8, CXCL10; and interleukin 2 and interferon γ than children who survived, and lower levels of interleukin 4. Children coinfected with HIV-1 had higher levels of TNF-α and IL-1β than HIV-uninfected children with measles, and lower levels of interleukin 4 and 5. Therefore, acute measles was characterized by activation of macrophages and T cells producing type 1, but not type 2, cytokines, which was more pronounced in fatal disease., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

This Indonesian study evaluates associations between near-miss status/death with maternal demographic, health care characteristics, and obstetrical complications, comparing results using retrospective and prospective data. The main outcome measures were obstetric conditions and socio-economic factors to predict near-miss/death. We abstracted all obstetric admissions (1,358 retrospective and 1,240 prospective) from two district hospitals in East Java, Indonesia between 4/1/2009 and 5/15/2010. Prospective data added socio-economic status, access to care and referral patterns. Reduced logistic models were constructed, and multivariate analyses used to assess association of risk variables to outcome. Using multivariate analysis, variables associated with risk of near-miss/death include postpartum hemorrhage (retrospective AOR 5.41, 95 % CI 2.64-11.08; prospective AOR 10.45, 95 % CI 5.59-19.52) and severe preeclampsia/eclampsia (retrospective AOR 1.94, 95 % CI 1.05-3.57; prospective AOR 3.26, 95 % CI 1.79-5.94). Associations with near-miss/death were seen for antepartum hemorrhage in retrospective data (AOR 9.34, 95 % CI 4.34-20.13), and prospectively for poverty (AOR 2.17, 95 % CI 1.33-3.54) and delivering outside the hospital (AOR 2.04, 95 % CI 1.08-3.82). Postpartum hemorrhage and severe preeclampsia/eclampsia are leading causes of near-miss/death in Indonesia. Poverty and delivery outside the hospital are significant risk factors. Prompt recognition of complications, timely referrals, standardized care protocols, prompt hospital triage, and structured provider education may reduce obstetric mortality and morbidity. Retrospective data were reliable, but prospective data provided valuable information about barriers to care and referral patterns.

Background: Endemic transmission of measles continues in many countries that have a high human immunodeficiency virus (HIV) burden. The effects that HIV infection has on immune responses to measles and to measles vaccine can impact measles elimination efforts. Assays to measure antibody include the enzyme immunoassay (EIA), which measures immunoglobulin G (IgG) to all measles virus (MV) proteins, and the plaque reduction neutralization (PRN) assay, which measures antibody to the hemagglutinin and correlates with protection. Antibody avidity may affect neutralizing capacity., Methods: HIV-infected and HIV-uninfected Zambian children were studied after measles vaccination (n=44) or MV infection (n=57). Laboratory or wild-type MV strains were used to infect Vero or Vero/signaling lymphocyte-activation molecule (SLAM) cells in PRN assays. IgG to MV was measured by EIA, and avidity was determined by ammonium thiocyanate dissociation., Results: HIV infection impaired EIA IgG responses after vaccination and measles but not PRN responses measured using laboratory-adapted MV. Avidity was lower among HIV-infected children 3 months after vaccination and 1 and 3 months after measles. Neutralization of wild-type MV infection of Vero/SLAM cells correlated with IgG avidity., Conclusion: Lower antibody quality and quantity in HIV-infected children after measles vaccination raise challenges for assuring the long-term protection of these children. Antibody quality in children receiving antiretroviral therapy requires assessment.

Located on the inside of the throat, the paired palatine tonsils form part of the first major barrier protecting the digestive and respiratory tracts from potentially invading microorganisms. The tonsils have a surface of stratified squamous epithelium that extends into deep and branched crypts lined by reticulated epithelium, which in parts may only be one cell thick. Organized in the sub-epithelial space are B cell rich lymphoid follicles. T cells are mostly located in the extra-follicular spaces with a very high CD4:CD8 T cell ratio. In addition to the T and B cell subsets, six phenotypes of dendritic cells (DC) have been identified in the tonsils: Langerhans cells in the squamous epithelium, germinal center DC, and follicular DC in the germinal center, and another three DC phenotypes that are located in the extra-follicular area (interdigitating DC, plasmacytoid DC, and lympho-epithelial symbiosis-DC). Here, we describe the isolation of tonsil mononuclear cells from fresh human tonsil., (Copyright 2009 by John Wiley & Sons, Inc.)

Background: Measles remains a significant cause of vaccine-preventable mortality in sub-Saharan Africa, yet few studies have investigated risk factors for measles mortality in regions of high human immunodeficiency virus type 1 (HIV-1) prevalence., Methods: Between January 1998 and July 2003, children with clinically diagnosed measles who were hospitalized at the University Teaching Hospital in Lusaka, Zambia, were enrolled in an observational study. Demographic and clinical information was recorded at enrollment and at discharge or death. Measles was confirmed by detection of antimeasles virus immunoglobulin M antibodies, and HIV-1 infection was confirmed by detection of HIV-1 RNA., Results: Of 1474 enrolled children, 1227 (83%) had confirmed measles and known HIV-1 infection status. Almost one-third of the HIV-1-infected children with measles were <9 months of age, the age of routine measles vaccination, compared with one-fourth of the uninfected children (P = .07). Death occurred during hospitalization in 23 (12.2%) of the HIV-1-infected children and 45 (4.3%) of the HIV-1-uninfected children (p < .001) with measles. After adjusting for age, sex, and measles vaccination status, HIV-1 infection (odds ratio, 2.5; 95% confidence interval, 1.4-4.6), < or =8 years of maternal education (odds ratio, 2.4; 95% confidence interval, 1.2-4.8), and the presence of a desquamating rash (odds ratio, 2.2, 95% confidence interval, 1.3-3.6) were significant predictors of mortality due to measles., Conclusions: In a region of high HIV-1 prevalence, coinfection with HIV-1 more than doubled the odds of death in hospitalized children with measles. Increased mortality among HIV-1-infected children is further evidence that greater efforts are necessary to reduce transmission of the measles virus in regions of high HIV-1 prevalence.

Background: Achieving the level of population immunity required for measles elimination may be difficult in regions of high human immunodeficiency virus type 1 (HIV-1) prevalence, because HIV-1-infected children may be less likely to respond to or maintain protective antibody levels after vaccination., Methods: We conducted a prospective study of the immunogenicity of standard-titer measles vaccine administered at 9 months of age to HIV-1-infected and uninfected children in Lusaka, Zambia., Results: From May 2000 to November 2002, 696 children aged 2-8 months were enrolled. Within 6 months of vaccination, 88% of 50 HIV-1-infected children developed antibody levels of >or=120 mIU/mL, compared with 94% of 98 HIV-seronegative children and 94% of 211 HIV-seropositive but uninfected children (P=.3). By 27 months after vaccination, however, only half of the 18 HIV-1-infected children who survived and returned for follow-up maintained measles antibody levels >or=120 mIU/mL, compared with 89% of 71 uninfected children (P=.001) and in contrast with 92% of 12 HIV-1-infected children revaccinated during a supplemental measles immunization activity., Conclusions: Although HIV-1-infected children showed good primary antibody responses to measles vaccine, their rapid waning of antibody suggests that measles vaccination campaigns may need to be repeated more frequently in areas of high HIV-1 prevalence.

Immunizations are of particular importance for human immunodeficiency virus type 1(HIV-1)-infected children as they are at increased risk of severe disease and death from several vaccine-preventable diseases. Outside the United States, however, research on the impact of the HIV-1 epidemic on childhood immunization coverage is sparse. We conducted a nested case-control study in hospitalized children with measles to assess whether HIV-1 infection was a risk factor for incomplete immunization with diphtheria-tetanus-pertussis vaccine (DTP) and oral polio vaccine (OPV). Of 473 children, whose immunization status was determined from the immunization record or maternal recall, 23% were incompletely immunized and 19% were HIV-1 infected. After adjusting for age, sex, and measles vaccination status, HIV-1 infection was significantly associated with incomplete immunization with DTP and OPV (adjusted OR 1.9; 95% CI 1.1, 3.3). In a subset of children for whom information on maternal education was available, less than 7 years of school education was a risk factor for incomplete immunization (adjusted OR 3.7; 95% CI 1.8. 7.5). Children from homes with more than three children were twice as likely to be incompletely immunized as those from homes with one to three children. Our findings suggest that HIV-1-infected children are at increased risk of vaccine-preventable diseases not only because of impaired immune responses but because of lower rates of vaccine coverage.

Age-related changes in lymphocyte subsets in HIV-uninfected Zambian children are described. The total lymphocyte count and numbers of CD4+ and CD8+ T-lymphocytes declined with increasing age, while the percentage of CD4+ and CD8+ T-lymphocytes changed little during childhood. Girls between the ages of 12 and 71 months had a higher percentage of CD4+ T-lymphocytes and a higher CD4:CD8 ratio than did boys of a similar age.

We have developed a colorimetric microtitre plate hybridization assay in order to simplify detection of Mycobacterium leprae in clinical specimens. This system detects the products amplified by a sensitive RT-PCR assay targeting a species-specific sequence of the bacterial 16S rRNA. The assay detected as few as 10 bacilli isolated from infected nude mouse lymph nodes or human skin biopsies. Sensitivity for diagnosis of clinical specimens was assessed for 58 tissue biopsies from untreated leprosy patients. The assay detected M. leprae RT-PCR products in 100% of biopsies from patients with multibacillary disease and 80% of biopsies from patients with paucibacillary disease, for an overall sensitivity of 91.3%. The test was highly specific as no RT-PCR products were amplified from skin biopsies of normal individuals or patients with skin diseases other than leprosy. The colorimetric assay is faster, more sensitive, and simplifies detection of RT-PCR products compared to Southern blot analysis. It may be useful for diagnosis of difficult cases of leprosy, and, since RNA is rapidly degraded after cell death, it may be appropriate for assessing response to therapy and for distinguishing relapse from reaction.

Measles virus infects thymic epithelia, induces a transient lymphopenia, and impairs cell-mediated immunity, but thymic function during measles has not been well characterized. Thirty Zambian children hospitalized with measles were studied at entry, hospital discharge, and at 1-month follow-up and compared to 17 healthy children. During hospitalization, percentages of naïve (CD62L+, CD45RA+) CD4+ and CD8+ T lymphocytes decreased (P = 0.01 for both), and activated (HLA-DR+, CD25+, or CD69+) CD4+ and CD8+ T lymphocytes increased (P = 0.02 and 0.03, respectively). T-cell receptor rearrangement excision circles (TRECs) in measles patients were increased in CD8+ T cells at entry compared to levels at hospital discharge (P = 0.02) and follow-up (P = 0.04). In CD4+ T cells, the increase in TRECS occurred later but was more sustained. At discharge, TRECs in CD4+ T cells (P = 0.05) and circulating levels of interleukin-7 (P = 0.007) were increased compared to control values and remained elevated for 1 month, similar to observations in two measles virus-infected rhesus monkeys. These findings suggest that a decrease in thymic output is not the cause of the lymphopenia and depressed cellular immunity associated with measles.

To determine the effect of measles virus infection on cytokine production in children from sub-Saharan Africa, temporal changes in cytokine production in vivo were analyzed and the T cell sources of type 1 and type 2 cytokines were identified in Zambian children with measles. The immune response during measles involved early type 1 responses, with production of interferon-gamma by CD8(+) T cells and of interleukin (IL)-2 by CD4(+) T cells. Subsequently, more-prolonged increases were observed in the type 2 cytokines IL-4 and IL-13, both produced by CD4(+) T cells. IL-5 was regulated differently from IL-4 and IL-13: levels were low compared with levels in control children and were reflected in lower eosinophil counts during measles. Immunoglobulin E was lower in children with measles, despite high levels of IL-4 and IL-13. Plasma levels of IL-10 were elevated for weeks, potentially contributing to impaired cellular immunity and depressed hypersensitivity responses following measles.

Measles is associated with immunosuppression and increased susceptibility to secondary infections and is a particular problem in developing countries. Lymphocyte changes accompanying immune activation and regulation of the immune response may contribute to immunosuppression. To evaluate lymphocyte changes during measles, children (n = 274) hospitalized with measles in Lusaka, Zambia, were evaluated at entry, discharge, and 1-month follow-up and compared to healthy Zambian children (n = 98). Lymphopenia was present on hospital admission and reflected decreased CD4 and CD8 T cells but resolved quickly. Lymphopenia was most marked in girls, in those with temperatures of >38.5 degrees C, and in malnourished children. CD4/CD8 ratios were decreased at all time points and were lower in boys than in girls at discharge and follow-up. Spontaneous death occurred in cultured lymphocytes, and the proportions of freshly isolated cells undergoing apoptosis, based on annexin V and propidium iodide staining, were increased. Surface Fas was increased on both CD4 and CD8 T cells compared to controls, and expression was greater on CD4 T cells and was inversely correlated with lymphocyte viability in culture at study entry. Mitogen stimulation of lymphocytes improved viability, but inhibitors of Fas, tumor necrosis factor (TNF)-related apoptosis-inducing ligand, and TNF did not. Plasma levels of beta(2) microglobulin and soluble Fas, Fas ligand, CD8, CD4, and TNF receptor were increased, and soluble CD8 was higher in boys than in girls. The multiple effects of measles on lymphocytes from Zambian children include decreased numbers in circulation, increased activation, and increased susceptibility to cell death, with substantive differences in the magnitude of these changes between boys and girls.

Measles in persons coinfected with human immunodeficiency virus (HIV) has been reported to be unusual in its presentation and frequently fatal. To determine the effect of HIV coinfection on the clinical features and outcome of measles, a prospective study of hospitalized children with measles was conducted between January 1998 and October 2000 in Lusaka, Zambia. One-sixth (17%) of 546 children hospitalized with laboratory-confirmed measles were coinfected with HIV. One-third of the HIV-infected children hospitalized with confirmed measles were <9 months old, compared with 23% of HIV-uninfected children (P=.03). Few differences in clinical manifestations, complications, or mortality were found between HIV-infected and HIV-uninfected children with measles. HIV-infected children constitute a significant proportion of children hospitalized with measles in countries with high HIV prevalence and are more likely to be younger than the age for routine measles immunization.

To determine the effect of measles virus coinfection on plasma human immunodeficiency virus (HIV) RNA levels, a prospective study of hospitalized children with measles was conducted between January 1998 and October 2000 in Lusaka, Zambia. Plasma HIV RNA levels were measured during acute measles and 1 month after hospital discharge. The median plasma HIV RNA level in 33 children with measles who were followed longitudinally was 5339 copies/mL at study entry, 60,121 copies/mL at hospital discharge, and 387,148 copies/mL at 1-month follow-up. The median plasma HIV RNA level in children without acute illness was 228,454 copies/mL. Plasma levels of immune activation markers were elevated during the period of reduced plasma HIV RNA. Plasma levels of several potential HIV suppressive factors also were elevated during acute measles. HIV replication is transiently suppressed during acute measles at a time of intense immune activation.

This unit describes a protocol for the derivation of lymphoid cell populations from tonsillar tissue. The procedure can also be applied with little or no modification to spleen and lymph node tissue. Human tonsils are the most readily available lymphoid organs and are often used as a source of large numbers of cells characteristic of local lymphoid tissue. Mononuclear cells are isolated by mechanical disruption of tonsil tissue followed by Ficoll-Hypaque gradient centrifugation. Cells may then be separated into subpopulations according to general protocols used for peripheral blood lymphocytes.

A reverse transcriptase-polymerase chain reaction assay was used to detect measles virus RNA in peripheral blood mononuclear cells, urine, and nasopharyngeal specimens from Zambian children during hospitalization and approximately 1-2 months after discharge. Of 47 children, 29 (61.7%) had prolonged measles virus shedding, as defined by detection of measles virus RNA in > or =1 specimen obtained 30-61 days after rash onset. Ten (90.9%) of 11 human immunodeficiency virus (HIV)-infected children had prolonged measles virus shedding, compared with 19 (52.8%) of 36 HIV-uninfected children (P=.02). Prolonged measles virus shedding did not correlate with levels of measles virus-specific antibody. HIV-infected children with measles may have a prolonged infectious period that potentially enhances measles virus transmission and hinders measles control.

A reverse transcription (RT)-PCR assay targeting the 16S rRNA of Mycobacterium leprae was developed to detect the organism in clinical specimens. A 171-bp fragment was amplified when M. leprae RNA was used as a template but not when a panel of RNAs from 28 potentially cross-reacting mycobacterial species, seven genera related to Mycobacterium, and three organisms normally found among skin or nose flora were tested. As few as 10 organisms isolated from infected tissue could be detected, confirming the sensitivity of the assay. When the test was applied to clinical specimens, M. leprae was detected in 82% of skin biopsy specimens obtained from untreated leprosy patients, while skin biopsy specimens from healthy volunteers and patients with other dermatological disorders were negative. The sensitivity of the RT-PCR was higher than that of slit skin smear staining for acid-fast bacilli or acid-fast staining of fixed biopsy specimens since 53% of acid-fast bacillus-negative biopsy specimens were RT-PCR positive. Because 16S rRNA is rapidly degraded upon cell death, the assay may detect only viable organisms and may prove to be useful in assessing the efficacy of chemotherapy.

The effects of measles immunization on immune responses in infants and the roles of vaccine strain and age of immunization are not known. Eighty-eight children were immunized at 6 or 9 months of age with the Edmonston-Zagreb (EZ) or Schwarz (SW6, SW9) strain of measles vaccine. Children were studied before and 2 weeks and 3 months after immunization. Seroconversion was similar, but geometric mean neutralizing titers at 3 months differed by vaccine group: SW9, 1367 mIU/mL; SW6, 982; and EZ, 303 (P = .003). Mitogen-induced lymphoproliferation was decreased at 2 weeks in the SW9 group and at 3 months in all groups and was negatively correlated with measles antibody level at 3 months (r = -.387, P = .003). CD8 T cells, soluble CD8, neopterin, and beta2-microglobulin were increased at 2 weeks in the SW9 group, and soluble CD8 and beta2-microglobulin remained elevated at 3 months. Therefore, measles immunization resulted in suppression of lymphoproliferation, which was most evident in infants with the highest antibody responses and most immune activation.

Measles produces immune suppression which contributes to an increased susceptibility to other infections. Recently, high titered measles vaccines have been linked to increased long-term mortality among some female recipients. Because the mechanisms by which wild-type or attenuated live-vaccine strains of measles virus alter subsequent immune responses are not fully understood, this prompted an examination of the changes within the peripheral blood T cell receptor V beta repertoire following measles immunization. Twenty-four 6- and 9-month-old infants were studied at 2 weeks and 3 months following immunization by semiquantitative reverse transcription-polymerase chain reaction. There was a significant increase in V beta 2 expression (P less than 0.05), and a decrease in the V beta 4 subset (P less than 0.03) 2 weeks following vaccination with subsequent return to baselines at 3 months in vaccine recipients who seroconverted. These data suggest that measles virus may affect immune responses in part by altering the T cell receptor repertoire.

Riboprobes that detect two genes expressed only during productive infection were developed to characterize the clinical spectrum of Epstein-Barr virus (EBV) lytic infection and identify diseases that may be responsive to antiviral drug therapy. The NotI antisense probe hybridizes to tandem repeats in the abundant early lytic cycle BHLF1 mRNA. Transcripts were detected in lytically infected cell lines, AIDS-associated oral hairy leukoplakia, bone marrow of a patient with virus-associated hemophagocytic syndrome, and spleen of an AIDS patient but not in EBV-positive primary central nervous system lymphomas or in circulating EBV-infected B cells from a patient with acute infectious mononucleosis. The viral (v) interleukin-10 (IL-10) probe hybridizes to the unique 5' end of the late lytic cycle BCRF1 mRNA, which encodes a protein homologous to the human cytokine IL-10. The vIL-10 probe detected transcripts in lytically infected cell lines and within the differentiated layers of oral hairy leukoplakia.

Herpesvirus papio (HVP) is a B-lymphotropic baboon virus with an estimated 40% homology to Epstein-Barr virus (EBV). We have cloned and sequenced ori-Lyt of herpesvirus papio and found a striking degree of nucleotide homology (89%) with ori-Lyt of EBV. Transcriptional elements form an integral part of EBV ori-Lyt. The promoter and enhancer domains of EBV ori-Lyt are conserved in herpesvirus papio. The EBV ori-Lyt promoter contains four binding sites for the EBV lytic cycle transactivator Zta, and the enhancer includes one Zta and two Rta response elements. All five of the Zta response elements and one of the Rta motifs are conserved in HVP ori-Lyt, and the HVP DS-L leftward promoter and the enhancer were activated in transient transfection assays by the EBV Zta and Rta transactivators. The EBV ori-Lyt enhancer contains a palindromic sequence, GGTCAGCTGACC, centered on a PvuII restriction site. This sequence, with a single base change, is also present in the HVP ori-Lyt enhancer. DNase I footprinting demonstrated that the PvuII sequence was bound by a protein present in a Raji nuclear extract. Mobility shift and competition assays using oligonucleotide probes identified this sequence as a binding site for the cellular transcription factor MLTF. Mutagenesis of the binding site indicated that MLTF contributes significantly to the constitutive activity of the ori-Lyt enhancer. The high degree of conservation of cis-acting signal sequences in HVP ori-Lyt was further emphasized by the finding that an HVP ori-Lyt-containing plasmid was replicated in Vero cells by a set of cotransfected EBV replication genes. The central domain of EBV ori-Lyt contains two related AT-rich palindromes, one of which is partially duplicated in the HVP sequence. The AT-rich palindromes are functionally important cis-acting motifs. Deletion of these palindromes severely diminished replication of an ori-Lyt target plasmid.

EBNA-2 contributes to the establishment of Epstein-Barr virus (EBV) latency in B cells and to the resultant alterations in B-cell growth pattern by up-regulating expression from specific viral and cellular promoters. We have taken a comparative approach toward characterizing functional domains within EBNA-2. To this end, we have cloned and sequenced the EBNA-2 gene from the closely related baboon virus herpesvirus papio (HVP). All human EBV isolates have either a type A or type B EBNA-2 gene. However, the HVP EBNA-2 gene falls into neither the type A category nor the type B category, suggesting that the separation into these two subtypes may have been a recent evolutionary event. Comparison of the predicted amino acid sequences indicates 37% amino acid identity with EBV type A EBNA-2 and 35% amino acid identity with type B EBNA-2. To define the domains of EBNA-2 required for transcriptional activation, the DNA binding domain of the GAL4 protein was fused to overlapping segments of EBV EBNA-2. This approach identified a 40-amino-acid (40-aa) EBNA-2 activation domain located between aa 437 and 477. Transactivation ability was completely lost when the amino-terminal boundary of this domain was moved to aa 441, indicating that the motif at aa 437 to 440, Pro-Ile-Leu-Phe, contains residues critical for function. The aa 437 boundary identified in these experiments coincides precisely with a block of conserved sequences in HVP EBNA-2, and the comparable carboxy-terminal region of HVP EBNA-2 also functioned as a strong transcriptional activation domain when fused to the Gal4(1-147) protein. The EBV and HVP EBNA-2 activation domains share a mixed proline-rich, negatively charged character with a striking conservation of positionally equivalent hydrophobic residues. The importance of the individual amino acids making up the Pro-Ile-Leu-Phe motif was examined by mutagenesis. Any alteration of these residues was found to reduce transactivation efficiency, with changes at the Pro-437 and Phe-440 positions producing the most deleterious effects. Activation of the EBV latency C promoter by EBNA-2 was shown to be dependent on the presence of the carboxy-terminal activation domain. However, this requirement was generic, rather than specific, since the EBNA-2 activation domain could be replaced with those from the herpes simplex virus (HSV) VP16 protein or the EBV Rta protein. Potential karyophilic signals within EBNA-2 were examined by introducing oligonucleotides encoding positively charged amino acid groupings that might serve in this capacity into a cytoplasmic test protein, HSV delta IE175, and by examining the intracellular localization of the resulting proteins. This assay identified a strong nuclear localization signal between EBV amino acids (aa) 478 to 485, which was conserved in HVP, and a weaker noncanonical signal between EBV aa 341 to 355, which was not conserved in HVP.