Prolonged measles virus shedding in human immunodeficiency virus-infected children, detected by reverse transcriptase-polymerase chain reaction.

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.

Isolation of mononuclear cells from tonsillar tissue.

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.

Reverse transcription-PCR detection of Mycobacterium leprae in clinical specimens.

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 effect of Edmonston-Zagreb and Schwarz measles vaccines on immune response in infants.

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.

Changes within T cell receptor V beta subsets in infants following measles vaccination.

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.

In situ detection of lytic Epstein-Barr virus infection: expression of the NotI early gene and viral interleukin-10 late gene in clinical specimens.

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.

The lytic origin of herpesvirus papio is highly homologous to Epstein-Barr virus ori-Lyt: evolutionary conservation of transcriptional activation and replication signals.

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 of herpesvirus papio diverges significantly from the type A and type B EBNA-2 proteins of Epstein-Barr virus but retains an efficient transactivation domain with a conserved hydrophobic motif.

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.