Expression of Epstein-Barr virus BMRF-2 and BDLF-3 genes in hairy leukoplakia.

The high level of Epstein-Barr virus (EBV) replication found in hairy leukoplakia (HL) provides a unique opportunity to study EBV expression in the oral epithelium. Screening of a cDNA library from an HL biopsy revealed expression of two genes not previously described in vivo: BMRF-2 and BDLF-3. Sequence analysis of the cDNAs demonstrated several nucleotide changes from the B95-8 sequence. In all six different HL strains studied, only one amino acid change was found in BMRF-2 relative to B95-8 and two amino acid changes were found in the BDLF-3 ORF. mRNA expression of both genes was localized to the lower prickle cell layer of the tongue epithelium. BMRF-2 protein expression was primarily detected in the cell nuclei of the upper prickle cell layer; immunoelectron microscopy revealed that BMRF-2 was associated with the nuclear chromatin. BDLF-3 protein expression was observed in the perinuclear space and cytoplasm of the prickle cells. BDLF-3 has recently been identified as a virion-associated protein, but the functions of BMRF-2 and BDLF-3 have not been elucidated.

Epstein-Barr virus BMRF-2 and BDLF-3 expression in hairy leukoplakia.

Hairy leukoplakia (HL) is a lesion found on the side of the tongue of immunocompromised individuals, including those with human immunodeficiency virus (HIV) infection. The lesion has unique histopathologic features and is characterised by high-level Epstein-Barr virus (EBV) replication, multiple EBV strains, and extensive inter- and intra-strain recombination. Expression of EBV genes spanning the entire viral life cycle from latency-associated genes to late, replicative genes has been detected in the lesion. HL thus provides a unique opportunity to study EBV expression in oral epithelium, and to study expression of novel EBV genes. We therefore constructed a cDNA library from an HL biopsy and detected expression of two genes not previously described in vivo: BMRF-2 and BDLF-3. Sequence analysis of the cDNAs revealed few amino acid changes from the B95-8 sequence. Expression of both genes was localized to the lower prickle cell layer of the tongue epithelium. BMRF-2 protein expression was primarily detected in the cell nuclei of the upper prickle cell layer. BDLF-3 protein expression was observed in the peri-nuclear space and Golgi compartment. The function of these proteins is currently under investigation.

Multicenter study of strains of respiratory syncytial virus.

Two major groups of respiratory syncytial virus (RSV) strains, A and B, have been identified and their patterns of isolation determined in different communities but not simultaneously in multiple communities. In this study, we tested 483 RSV isolates from 14 university laboratories in the United States and Canada for the 1984/1985 and 1985/1986 RSV seasons; 303 (63%) isolates were group A, 114 (24%) were group B, and 66 (14%) could not be grouped. Isolates were subdivided into six subgroups within group A and three within group B; up to six and often four or more different subgroups were isolated in the same laboratory during the same RSV season. The pattern of group and subgroup isolations varied among laboratories during the same year and between years for the same laboratory. These differences suggest that RSV outbreaks are community, possibly regional, but not national phenomena. The ability to identify group and subgroup differences in isolates is a powerful tool for epidemiologic studies of RSV.

The clinical significance of HTLV-I and HTLV-II infection in the AIDS epidemic.

Although early reports of an etiological role of HTLV-I in AIDS are not supported by subsequent epidemiologic and biologic data, all AIDS specialists should be aware of the possible infection of AIDS risk group members with the HTLV viruses. In the United States, HTLV-II and HIV co-exist in high prevalence among IVDU, and coinfected IVDU may progress more quickly to AIDS. The health effects of solitary HTLV-II infection are currently unknown. In Africa and the Caribbean, where HTLV-I is endemic, the epidemic of HIV infection will likewise produce coinfection and possibly faster progression to AIDS. In addition, adult T-cell leukemia/lymphoma and HTLV-associated myelopathy (HAM/TSP) may occur in persons infected with HTLV-I alone. Clinical care of HTLV infected patients is difficult because of societal confusion of these viruses with HIV. The full health effects of both HTLV-I and HTLV-II are not yet known, and even the two known disease outcomes of HTLV-I infection occur in only a few percent of those infected. Without prognostic indicators, and with the long latency from infection to disease, counseling and medical follow-up of patients is difficult. Further research into the clinical effects of HTLV-I and HTLV-II is urgently needed.

Serological confirmation of human T-lymphotropic virus type I infection in healthy blood and plasma donors.

We wished to develop criteria for serological confirmation of human T-lymphotropic virus type I (HTLV-I) infection in healthy donors. Selected serum or plasma samples reactive by HTLV-I enzyme immunosorbent assay or gel-agglutination assays with at least one viral-specific band on Western immunoblot (WIB) were tested in six laboratories by four WIBs and four radioimmunoprecipitation assays (RIPAs) for antibodies to HTLV-I proteins encoded by gag (p19 and p24), env (gp46 and/or gp61), and tax (p40x) genes. One hundred forty-two donor sera were obtained from 38 Japanese, 69 American, and 35 Caribbean blood or plasma donors. Among these samples, WIB assays appeared more sensitive to p24 antibodies, whereas RIPAs were significantly more sensitive to gp61 antibodies. All sera (137) with gp61 antibodies had p24 antibodies. Of the 137 sera positive for p24 and gp61 antibodies, p19 antibodies were detected in 129 sera, and p40x antibodies were detected in 108. In sera with p19 antibodies and antibodies to env- or tax-encoded proteins, p24 antibodies were always present. Antibodies to p40x were not found in the absence of gp61 antibodies. Virological evidence of infection was found in seven American donors by lymphocyte coculture (one HTLV-I, one HTLV-II) or by polymerase chain reaction (three HTLV-I, two HTLV-II). Sera from all seven donors showed p24 and gp46 and/or gp61 antibodies. We suggest that seroreactivity to both p24 and gp46 and/or gp61 by WIB or RIPA or both are suitable criteria to confirm but not to distinguish HTLV-I and HTLV-II infections.

Prevalence of respiratory syncytial virus subgroups over six consecutive outbreaks: 1981-1987.

Indirect immunofluorescence with strain-specific monoclonal antibodies was used to determine the phenotype of respiratory syncytial virus (RSV) isolates obtained from infants hospitalized in greater Boston over six successive outbreaks from 1981 to 1987. Of 981 isolates, 591 (60%) were classified as subgroup A and 383 (39%) as subgroup B. The prevalence of subgroups varied both between and within yearly outbreaks. In 1983-84 and 1984-85, both subgroups circulated concurrently and in almost equal proportions; in 1981-82, 1982-83, and 1985-86 subgroup A was dominant, accounting for 93% of all RSV isolates; and in 1986-87 subgroup B accounted for 89% of all RSV isolates. In some outbreaks both geographic and temporal clustering of subgroups occurred. No major differences in age, gender, or frequency of nosocomially acquired RSV between infants infected with either subgroup were seen, either overall or between or within yearly outbreaks. An expanded panel of monoclonal antibodies revealed further heterogeneity among subgroup A isolates. Comparison of these results with similar studies from other geographic locations indicated that the pattern of RSV subgroup prevalence is a localized phenomenon.

Evaluation of a commercially available direct immunofluorescent staining reagent for the detection of respiratory syncytial virus in respiratory secretions.

A commercially-available direct immunofluorescence (IF) reagent (Imagen; Boots-Celltech, Slough, Berkshire, United Kingdom) was similar in sensitivity and specificity to the conventional indirect IF test for the detection of respiratory syncytial virus in respiratory secretions. Both IF tests were more sensitive than culture, particularly for specimens transported from outside the institution.

Comparison of washed nasopharyngeal cells and whole nasal secretions for detection of respiratory syncytial virus antigens by enzyme-linked immunosorbent assay.

We compared washed nasal epithelial cells with unfractionated nasal secretions as sources of respiratory syncytial virus (RSV) antigens in an indirect enzyme-linked immunosorbent assay (ELISA). Of 28 infants positive for RSV by virus isolation or direct immunofluorescence or both, 27 (96%) were positive by ELISA with whole nasal secretions, whereas only 19 (68%) were positive by ELISA with the matching washed-cell fractions. Furthermore, the ELISA absorbances obtained with nasal secretions were significantly greater than those seen with washed-cell fractions, indicating that whole nasal secretions contain relatively greater amounts of RSV antigens as measured by ELISA.

Enzyme-linked immunosorbent assay for detection of respiratory syncytial virus infection: application to clinical samples.

An enzyme-linked immunosorbent assay (ELISA) for respiratory syncytial virus antigens was applied to the rapid diagnosis of acute infections in children and was compared with viral culture and immunofluorescence tests. The ELISA test employed commercially available reagents and was run on a day-to-day basis as specimens were received in the laboratory. Sensitivity and specificity by ELISA were 82 and 95%, respectively, compared with culture. In the same specimens, the sensitivity and specificity by immunofluorescence were 86 and 96%, respectively. Nasopharyngeal aspirates were proven to be a better source of viral antigen than were nasopharyngeal swabs. ELISA-positive samples remained positive even when left unrefrigerated for a week or mailed to the laboratory in plastic containers. Respiratory syncytial virus ELISA, like culture, became negative as the disease progressed and showed no superiority over culture for diagnosis late in the illness.

Isolation and transformation of primary mesenchymal cells of the chick embryo.

Pure primary mesenchymal cells from definitive streak stage chick embryos have been prepared free of epiblast and hypoblast cells. These cells have the potential in culture to differentiate into erythroid cells, beating heart muscle tissue, chondrocytes and epithelial cells. Transformation in vitro of pure primary mesenchymal cells by avian erythroblastosis virus (wt-AEV) and a temperature-sensitive mutant (ts34-AEV) gave rise to rapidly growing cells which remained largely undifferentiated, could be cloned in semi-solid medium and could be maintained for up to 3 months in culture. The majority of mesenchymal cells transformed by wt-AEV (MAE cells) are benzidine-negative. Gel electrophoresis of radioactively labeled cell proteins, immunoprecipitated with specific antisera against chicken hemoglobin, showed that MAE cell clones synthesize the alpha D, pi (or pi') and some unidentified "globin" polypeptide chains. Treatment of MAE cell clones with 1.0 mM n-butyrate stops cell proliferation reversibly and causes an increased synthesis of alpha D and pi (or pi') globin polypeptide chains. In certain clones of mesenchymal cells transformed by a temperature-sensitive mutant of the virus, ts34-AEV (MAE-ts34 cells), benzidine-positive cells can be induced by a shift from 37 degrees to 41 degrees C. The ability of the clone to undergo an increase in benzidine-positivity by temperature shift is decreased with the age of the clone. Different clones show a variable proportion of cells which are positive by immunofluorescence for both globin and chicken-specific histone H5. The alpha A and alpha D globin chains are synthesized in MAE-ts34 clones, but the ratios and quantities of these chains vary for different clones. Temperature shift made little difference in the types and quantities of globin chains synthesized; the increase in benzidine positivity is probably due to an increase in heme biosynthesis.