Immunoglobulin A-induced shift of Epstein-Barr virus tissue tropism.

Increased immunoglobulin A (IgA) antibodies to the Epstein-Barr virus (EBV) appear months to years before the clinical onset of nasopharyngeal carcinoma and define populations at high risk for this EBV-associated epithelial cancer common in south China. In the human HT-29 epithelial cell line, polymeric IgA (pIgA) specific for EBV promoted infection of the otherwise refractory epithelial cells. When bound to pIgA, EBV entered epithelial cells through secretory component-mediated IgA transport but no longer infected B lymphocytes. Such an immune-induced shift in EBV tissue tropism provides a paradigm for endogenous spread of EBV in the immune host that predicts infectious sequelae of epithelium.

Biotin-labeled DNA probes for detection of Epstein-Barr virus by in-situ cytohybridization.

Conventional laboratory diagnosis of EBV-related disease is now performed by one of three methods: serology, lymphocyte transformation assay, or Epstein-Barr nuclear antigen (EBNA) staining of cell preparations. Of these techniques, serology is the most widely used. However, this approach assumes an intact host immune system, which is absent or impaired in some of the more baffling EBV-related syndromes. Detection of infectious virus by the lymphocyte transformation assay is labor-intensive, requires access to human umbilical cord blood lymphocytes, and requires a two-month period of incubation. Although detection of EBNA in tissue imprints is rapid, the anticomplement immunofluorescence assay, when applied to clinical materials, is subject to misinterpretation and requires multiple controls. Because of these difficulties, hybridization analysis of clinical materials for presence of EBV with biotinylated DNA probes promises to have wide-ranging applicability in the clinical microbiology laboratory. These techniques can readily be used in other viral systems and have proved useful for detection of human CMV and HSV DNA and RNA. Extension of the techniques to detection of specific nonviral nucleic acid sequences is the next frontier, limited essentially only by definition of significant target sequences.

Nonurban male homosexuals: epidemiologic, immunologic and virologic characteristics.

Twenty asymptomatic male homosexuals living in North Carolina were evaluated looking at epidemiologic, immunologic and virologic characteristics. In ten subjects selected for inhalant nitrite use a significantly higher frequency of multiple drug abuse and a trend toward greater sexual promiscuity was found in comparison with ten nonnitrite users. None of the 20 subjects had chronic lymphadenopathy. Cytomegalovirus (CMV) was not found in urine, blood or throat washings, but was found in 29% of the subjects' semen specimens--a finding that was significantly linked (P less than .05) to the presence of CMV IgM antibody in serum. There were no abnormal helper lymphocytes: suppressor T lymphocyte ratios (all greater than 1.3) and lymphocyte mitogen stimulations were not different from heterosexual controls in contrast to frequent abnormalities reported in male homosexuals in metropolitan areas. If these immunologic findings are reproducible, they may be important in understanding why the Acquired Immunodeficiency Syndrome has clustered in large cities.

Detection of Epstein-Barr virus genomes in archival tissues by polymerase chain reaction.

We used oligonucleotide primers designed from DNA sequences unique to the long internal direct repeated region of Epstein-Barr virus (EBV) to enzymatically amplify this segment of the EBV genome in formalin-fixed, paraffin-embedded tissues. The products amplified from EBV templates were detected by hybridization with a labeled probe specific for this highly conserved, reiterated region. Epstein-Barr virus-related sequences were detected in the spleen of a patient with infectious mononucleosis, in lung and lymph node specimens from a patient with pulmonary manifestations of infectious mononucleosis, in various tissues from seven immunosuppressed organ transplant recipients with immunoproliferative disorders, and in small biopsy specimens from a patient with nasopharyngeal carcinoma. No viral sequences were detected in 20 histologically normal spleens or 10 lymph nodes. Polymerase chain reaction technology provides an effective means for documenting EBV infection in archival tissues. This approach should facilitate the diagnosis of posttransplant lymphoproliferative disorders and difficult cases of infectious mononucleosis and nasopharyngeal carcinoma.

Epstein-Barr virus transformation of human B lymphocytes despite inhibition of viral polymerase.

Epstein-Barr virus transformed human lymphocytes despite the presence of up to 500 microM acyclovir [9-(2-hydroxyethoxymethyl)guanine], a viral DNA polymerase inhibitor. The transformed cells contained multiple Epstein-Barr virus genome copy numbers. Functional viral DNA polymerase is probably not required for cell transformation and the initial amplification of the viral genome.

Epstein-Barr virus and epithelial cells: a possible role for the virus in the development of cervical carcinoma.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with certain B cell lymphomas in humans and also with an epithelial tumour, undifferentiated nasopharyngeal carcinoma. Infection with EBV is widespread and once infected, individuals become life-long virus carriers. Epithelial cells of the pharynx have been implicated as the primary site of EBV persistence. Ectocervical epithelial cell cultures have proved useful for investigating EBV infection in vitro but only recently has EBV been shown to infect the uterine cervix in vivo. The demonstration that EBV replicates in cervical epithelium raises the possibility of venereal transmission, and suggests that the virus may be involved in cervical pathology.

Epstein-Barr virus induction of recombinase-activating genes RAG1 and RAG2.

In experimental B-cell infections, Epstein-Barr virus induced sustained expression of V(D)J recombinase-activating genes RAG1 and RAG2, whose aberrant activity has been implicated in chromosomal translocations in B-cell neoplasms. In cell lines in which RAG1 and RAG2 were detected, virus integrated into cellular DNA rather than assumed the configuration of extrachromosomal episomes. Expression of the Epstein-Barr virus nuclear antigen 1 in transient transfection assays was sufficient to induce both recombinase-activating genes.

A second site for Epstein-Barr virus shedding: the uterine cervix.

Epstein-Barr virus (EBV) infection of the human uterine cervix was detected in 5 out of 28 women by means of culture and cytohybridization analysis of cervical secretions. Cervical samples from 2 of 14 women contained epithelial cells with EBV DNA, and filtered cervical washings from 4 women contained infectious EBV. The discovery of EBV shedding in its cell-free infectious form from the uterine cervix raises the possibility of venereal transmission, neonatal infection, and EBV involvement in cervical pathology.

Acyclovir and Epstein-Barr virus infection.

Acyclovir (ACV) has an ED50 of 0.3 microM against EBV replication in vitro. Based on these and other data we carried out a pilot, double-blind, placebo-controlled trial of ACV for treatment of infectious mononucleosis. Only patients with relatively severe illness requiring hospital management were enrolled. Ten subjects with proven infectious mononucleosis received placebo and 10 ACV. The drug was administered intravenously at 8-hourly intervals in a total daily dosage of 1500 mg/m2 for 5 days. Preliminary analyses of the results indicate that the drug interrupted virus excretion in the oropharynx transiently but had no effect on ability to generate lymphocytic lines from peripheral blood. Symptoms and signs unaffected by ACV were splenomegaly, lymphadenopathy, lethargy, fever and pharyngitis. There was significantly more rapid regain of weight in the ACV-treated group. On the basis of these results we have instituted an out-patient trial of orally administered ACV in patients with less severe illness earlier in its course. We have also begun in-vitro tests of other drugs that might prove to be effective against Epstein-Barr virus infection, and have shown that 9-1 (1,3-dihydroxy-2-propoxymethyl)guanine (BW759) has an ED50 of 0.05 microM.

Interaction of Chlamydia trachomatis with human genital epithelium in culture.

Primary cultures of human endometrial and ectocervical epithelial cells were examined as a new model system to study genital infection by Chlamydia trachomatis. Initial studies demonstrated that these cells were indeed susceptible to chlamydial infection. Inocula, adjusted to produce inclusions in 50 to 80% of equivalent numbers of standard McCoy cells, resulted in infection rates of approximately 15 to 30% for the columnar cells of the endometrium and 5 to 10% for the squamous cells of the ectocervix. Exposure of cultures to DEAE-dextran and centrifugation-assisted inoculation, manipulations reported to enhance infection of HeLa and McCoy cells, did not alter the number of inclusion-positive genital cells. Addition of cycloheximide to the post-inoculation culture medium slightly increased numbers of inclusion-bearing cells while growth of genital cells in hormone-supplemented medium resulted in a variable effect on inclusion development and a significant reduction in the association of radiolabelled organisms with these cells. The basis for the different levels of infection in McCoy versus genital cell cultures was revealed by immunofluorescence analysis of chlamydial association with host cells immediately after inoculation. Chlamydiae failed to adhere to many cells in the genital cell cultures while adherence to McCoy cells was uniform. In addition, the association of radiolabelled C. trachomatis was significantly lower with genital cells than with McCoy cells. Finally, culture conditions were defined which markedly inhibited inclusion development without an immediate loss of chlamydial growth potential. This investigation indicates that primary genital cell cultures are susceptible to chlamydial infection and will be valuable for studies on the nature of C. trachomatis interactions with natural human target cells.

Excretion of the Epstein-Barr virus from the genital tract of men.

Epstein-Barr virus (EBV) DNA was detected by polymerase chain reaction in urethral discharges from 10 (48%) of 21 men attending a clinic for sexually transmitted diseases. Five of the 10 subjects were concurrently excreting virus from their oropharynx. These findings indicate that EBV is shed from more than one mucosal site in seropositive adult males. Involvement of the genital tract raises the possibility of sexual transmission as well as EBV-induced genital tract pathology.

Detection of cell-free Epstein-Barr virus DNA in serum during acute infectious mononucleosis.

Infectious Epstein-Barr virus (EBV) is shed from the oropharynx of infected hosts intermittently throughout life, but in the peripheral circulation the viral genome characteristically maintains itself in a noninfectious, cell-associated form. Sera from 125 persons with heterophil-positive acute infectious mononucleosis or EBV-associated nasopharyngeal carcinoma or who were healthy virus carriers were examined for evidence of cell-free viral DNA. EBV DNA suggesting viremia was detected in 11 (27%) of 41 infectious mononucleosis patients by polymerase chain reaction analysis but infrequently in healthy seropositive carriers and patients with nasopharyngeal carcinoma. In serial samples examined from 2 patients, serum EBV DNA was detected over a 3-day interval. Viral DNA was found in concert with one serologic marker of acute infection, EBV-specific polymeric IgA, that could affect patterns of viral spread and clinical symptomatology.

Evasion of host immune responses by tumours and viruses.

Viruses and tumours use various mechanisms to avoid immune surveillance. Oncogenic viruses have achieved a balance with the immune system through evolutionary time to ensure long-term persistence. Mutations that promote escape mechanisms favouring tumour growth to the detriment of host survival through reproductive age offer no selective advantage and will not generally be maintained in the viral genome that persists in nature. Conventional (non-oncogenic) and tumour viruses interact with various immune mediators and T cells in different ways. Oncogenic viruses cannot operate solely in the context of a lytic cycle, though this may be characteristic of the initial phase of infection that is limited by the acute immune response. Some oncogenic viruses interact with normal cellular growth control and signalling mechanisms. Synthesis of key viral proteins may be tightly controlled in replicating cells that are subject to T cell surveillance, such as basal epithelia, while productive infection occurs in non-proliferating progeny that are lost under normal physiological conditions, such as desquamating epithelia. Tumorigenesis may be an aberrant consequence of the molecular mechanisms needed to maintain this pattern of viral growth regulation in the context of the cell cycle. Vaccines designed to limit the acute phase of infection with cell-free oncogenic viruses should be as effective as those for conventional viruses.

Spontaneous loss of viral episomes accompanying Epstein-Barr virus reactivation in a Burkitt's lymphoma cell line.

Life-long viral persistence is a hallmark of human herpesvirus infection. In the Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell line, Mutu, spontaneous loss of all viral episomes accompanied productive viral DNA replication. The molecular configuration of intracellular EBV DNA evolved from monoclonal episomes in cells retaining the original tumor phenotype to predominantly replicating linear DNA and, subsequently, only integrated forms in BL cells that had acquired the lymphoblastoid cell phenotype. Transient appearance of deleted, rearranged WZhet EBV DNA capable of disrupting viral latency, along with the integration of viral DNA into human chromosomes, indicates a genetic instability in the host cell which, if duplicated in vivo, may affect configuration and persistence of the viral genome in expanding malignant cell clones.

Detection of Epstein-Barr virus genome in ocular tissues.

BACKGROUND: Epstein-Barr virus (EBV) is a ubiquitous mucosal pathogen with a propensity for lifelong, asymptomatic persistence. Because of reported association between EBV and ocular inflammatory disorders, we tested ocular tissues from normal eyes for presence of the EBV genome. METHODS: Ten freshly harvested cadaveric human eyes were dissected into limbal cornea, central cornea, aqueous humor, iris, vitreous humor, and optic nerve. Total cellular DNA preparations were screened for DNA sequences specific to EBV's large internal repeat region. After Southern transfer, polymerase chain reaction products were detected by a 32P-labeled oligonucleotide probe specific to amplified sequences internal to the polymerase chain reaction primers. RESULTS: Seven of ten eyes from deceased donors yielded a polymerase chain reaction product, indicating presence of EBV genome. In all, 12 (20%) of 60 cadaveric ocular samples contained EBV DNA. Only the optic nerve was consistently negative for EBV DNA. CONCLUSIONS: Detection of EBV DNA in cadaveric ocular tissues indicates a broad anatomic distribution of this persistent mucosal pathogen. The frequency with which EBV was found at apparently normal ocular sites raises the possibility for viral involvement in disease states, but emphasizes the need for specific criteria to implicate EBV in ocular pathology.

Patterned entry and egress by Epstein-Barr virus in polarized CR2-positive epithelial cells.

In polarized epithelium direction of viral entry and release correlates with proclivity of a virus to establish local versus systemic infection. The Epstein-Barr virus (EBV), whose principal tissue reservoir is B lymphocytes, also has disease manifestations in epithelium, suggesting intertissue spread potentially influenced by epithelial cell polarity. We stably transfected the B lymphocyte EBV receptor (CR2/CD21) into Madin-Darby canine kidney (MDCK) epithelial cells used extensively to study effects of cell polarity on infection by both DNA and RNA viruses. CR2/CD21 was detected on both apical and basolateral surfaces of polarized MDCK cells, with predominant expression basolaterally. However, infectivity was up to four-fold greater apically, suggesting that endogenous cell surface molecules, sorted asymmetrically onto polarized plasma membranes, may be involved in EBV entry into MDCK cells. EBV gp350/220, a replicative cycle glycoprotein added to the virus envelope on egress through the cell membrane, was immunolocalized by confocal microscopy to basolateral cell surfaces only. Apical entry of EBV with subsequent basolateral release of newly replicated virus favors systemic infection by viral dissemination to underlying lymphocytic aggregations. Under conditions of long-term culture, latent EBV was not stably maintained in these cells, suggesting that the epithelial phase of acute EBV infection may be transient.

Epstein-Barr virus receptors on human pharyngeal epithelia.

The apparently strict tropism of Epstein-Barr virus (EBV) for B lymphocytes has been attributed to the existence of a B-lineage-specific surface molecule, the C3d receptor, which also functions as a receptor for EBV. Two monoclonal antibodies against different determinants on the EBV/C3d receptor of B cells were shown to react with pharyngeal epithelia in a cell differentiation-dependent manner. These findings, which raise the possibility of direct virus entry into a naturally exposed epithelium, strengthen the evidence in favour of an epithelial reservoir of EBV infection in vivo and identify a means whereby the virus/epithelium interactions leading to nasopharyngeal carcinoma might be initiated.

A transformation-incompetent, nuclear antigen 2-deleted Epstein-Barr virus associated with replicative infection.

Epstein-Barr virus (EBV) obtained directly from the oropharynx was used to detect viral DNA deleted for the EBV nuclear antigen 2 (EBNA2)-encoding gene that is essential for lymphocyte transformation. By polymerase chain reaction analysis, the deletion was found in virus from 5 of 33 healthy adult donors and 11 of 12 patients with concurrent human immunodeficiency virus infection. Lymphoblastoid cell lines that produce standard transforming EBV also harbored EBNA2-deleted virus in cells permissive of EBV replication. In vitro infectivity studies indicated that the DNA is packaged and transmissible, with biologic properties similar to those of a laboratory mutant, P3HR-1, which also lacks the EBNA2 gene. These findings, obtained from productively infected cell systems, provide evidence for the existence in nature of a transformation-incompetent EBV variant that may facilitate EBV persistence and the emergence of reactivation diseases.

Epithelial cell polarization is a determinant in the infectious outcome of immunoglobulin A-mediated entry by Epstein-Barr virus.

Diseases of the nasopharyngeal epithelium due to Epstein-Barr virus (EBV) infection typically occur in chronic virus carriers with preexisting virus-specific antibodies. In vitro studies have shown that EBV-specific immunoglobulin A (IgA) promotes infection of human epithelial cells, otherwise refractory to EBV, via the polymeric immunoglobulin receptor (pIgR). To determine if EBV similarly exploits IgA transport mechanisms in vivo, we examined the fate of IgA-EBV complexes in the blood of mice, where pIgR-mediated transcytosis of IgA immune complexes through hepatocytes eliminates exogenous antigens from the circulation. By PCR analysis we showed hepatobiliary transport of IgA-EBV in viremic mice, but without detectable hepatocellular infection by immunostaining. Because efficient transport of EBV immune complexes might avert an infectious outcome, we modulated the transcytotic pathway in polarized Madin-Darby canine kidney (MDCK) cells transfected with pIgR to determine the effect on viral antigen expression. Like hepatocytes in vivo, MDCK cells in polarized monolayers translocated IgA-EBV from the basal cell face into apical medium without evidence for infection. However, when exposed to IgA-EBV as unpolarized single-cell suspensions, MDCK cells expressed EBV immediate-early and early antigens. These results suggest that pIgR-mediated transcytosis of pIgA-EBV through epithelium facilitates endogenous spread of EBV in long-term virus carriers, with infection being confined to cells with altered polarity from prior cytopathology.