EBV is a ubiquitous herpesvirus that is the causative agent of acute infectious mononucleosis and is associated with nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin and non-Hodgkin lymphomas, other lymphoproliferative disorders in immunodeficient individuals, and gastric carcinoma.
Properties of the Virus
The EBV DNA genome contains about 172 kbp, has a G + C content of 59%, and encodes about 100 genes. There are two major strains of EBV, types A and B.
A. Biology of Epstein-Barr Virus
The major target cell for EBV is the B lymphocyte. When human B lymphocytes are infected with EBV, continuous cell lines can be established, indicating that cells have been immortalized by the virus. Very few of the immortalized cells produce infectious virus. Laboratory studies of EBV are hampered by the lack of a fully permissive cell system able to propagate the virus.
EBV initiates infection of B cells by binding to the viral receptor, which is the receptor for the C3d component of complement (CR2 or CD21). EBV directly enters a latent state in the lymphocyte without undergoing a period of complete viral replication. The hallmarks of latency are viral persistence, restricted virus expression, and the potential for reactivation and lytic replication.
The efficiency of B-cell immortalization by EBV is quite high. When virus binds to the cell surface, cells are activated to enter the cell cycle. Subsequently, a limited repertoire of EBV genes is expressed, and the cells are able to proliferate indefinitely. The linear EBV genome forms a circle and is amplified during the cell cycle S phase; the majority of viral DNA in the immortalized cells exists as circular episomes.
EBV-immortalized B lymphocytes express differentiated functions, such as secretion of immunoglobulin. B-cell activation products (eg, CD23) are also expressed. Several pat terns of latent viral gene expression are recognized based on the spectrum of proteins and transcripts expressed. These include EBV nuclear antigens (EBNA1, 2, 3A-3C, LP), latent membrane proteins (LMP1, 2), and small untranslated RNAs (EBERs).
At any given time, very few cells (< 10%) in an immortalized population release virus particles. Latency can be disrupted and the EBV genome activated to replicate in a cell by a variety of stimuli, including chemical-inducing agents or cross-linking cell surface immunoglobulin.
EBV can replicate in vivo in epithelial cells of the oropharynx, parotid gland, and uterine cervix; it is found in epithelial cells of some nasopharyngeal carcinomas. Although epithelial cells in vivo contain an EBV receptor, the receptor is lost from cultured cells.
EBV is associated with a number of lymphoproliferative disorders. Viral gene expression in these cells is limited and varies from only EBNA1 to the full complement of proteins found in latently infected B cells.
B. Viral Antigens
EBV antigens are divided into three classes based on the phase of the viral life cycle in which they are expressed: (1) Latent phase antigens are synthesized by latently infected cells. These include the EBNAs and the LMPs. Their expression reveals that an EBV genome is present. Only EBNA1, needed to maintain the viral DNA episomes, is invariably expressed; expression of the other latent phase antigens may be regulated in different cells. LMP1 mimics an activated growth factor receptor. (2) Early antigens are nonstructural proteins whose synthesis is not dependent on viral DNA replication. The expression of early antigens indicates the onset of productive viral replication. (3) Late antigens are the structural components of the viral capsid (viral capsid anti gen) and viral envelope (glycoproteins). They are produced abundantly in cells undergoing productive viral infection.
C. Experimental Animal Infections
EBV is highly species specific for humans. However, cotton top tamarins inoculated with EBV frequently develop fatal malignant lymphomas.
Pathogenesis and Pathology
A. Primary Infection
EBV is commonly transmitted by infected saliva and initiates infection in the oropharynx. Viral replication occurs in epithelial cells (or surface B lymphocytes) of the pharynx and salivary glands. Many people shed low levels of virus for weeks to months after infection. Infected B cells spread the infection from the oropharynx throughout the body. In nor mal individuals, most virus-infected cells are eliminated, but small numbers of latently infected lymphocytes persist for the lifetime of the host (one in 105–106 B cells).
Primary infections in children are usually subclinical, but if they occur in young adults, acute infectious mononucleosis often develops. Mononucleosis is a polyclonal stimulation of lymphocytes. EBV-infected B cells synthesize immunoglobulin. Autoantibodies are typical of the disease, with heterophil antibody that reacts with antigens on sheep erythrocytes detectable in acute cases.
B. Reactivation from Latency
Reactivations of EBV latent infections can occur, as evidenced by increased levels of virus in saliva and of DNA in blood cells. These are usually clinically silent. Immunosuppression is known to reactivate infection, sometimes with serious consequences.
Clinical Findings
Most primary infections in children are asymptomatic. In adolescents and young adults, the classic syndrome associated with primary infection is infectious mononucleosis (∼50% of infections). EBV is also associated with several types of cancer.
A. Infectious Mononucleosis
After an incubation period of 30–50 days, symptoms of head ache, fever, malaise, fatigue, and sore throat occur. Enlarged lymph nodes and spleen are characteristic. Some patients develop signs of hepatitis.
The typical illness is self-limited and lasts for 2–4 weeks. During the disease, there is an increase in the number of circulating white blood cells, with a predominance of lymphocytes. Many of these are large, atypical T lymphocytes. Low-grade fever and malaise may persist for weeks to months after acute illness. Complications are rare in normal hosts.
B. Cancer
EBV is associated with Burkitt lymphoma, nasopharyngeal carcinoma, Hodgkin and non-Hodgkin lymphomas, and gastric carcinoma. EBV-associated posttransplant lymphoproliferative disorders are a complication for immunodeficient patients. Sera from patients with Burkitt lymphoma or nasopharyngeal carcinoma contain elevated levels of antibody to virus-specific antigens, and the tumor tissues contain EBV DNA and express a limited number of viral genes.
Burkitt lymphoma is a B cell lymphoma commonly presenting as a tumor of the jaw in African children and young adults. Most African tumors (>90%) contain EBV DNA and express EBNA1 antigen. In other parts of the world, only about 20% of Burkitt lymphomas contain EBV DNA. It is speculated that EBV may be involved at an early stage in Burkitt lymphoma by immortalizing B cells. Malaria, a recognized cofactor, may foster enlargement of the pool of EBV-infected cells. Finally, there are characteristic chromosome translocations that involve immunoglobulin genes and result in deregulation of expression of the c-myc proto-oncogene.
Nasopharyngeal carcinoma is a cancer of epithelial cells and is common in males of Chinese and southeastern Asian origin. EBV DNA is regularly found in nasopharyngeal carcinoma cells, and patients have high levels of antibody to EBV. EBNA1 and LMP1 are expressed. Genetic and environmental factors are believed to be important in the development of nasopharyngeal carcinoma.
Immunodeficient patients are susceptible to EBV induced lymphoproliferative diseases that may be fatal. From 1% to 10% of transplant patients develop an EBV-associated lymphoproliferative disorder, often when experiencing a primary infection. Aggressive monoclonal B-cell lymphomas may subsequently develop.
AIDS patients are susceptible to EBV-associated lymphomas and oral hairy leukoplakia, a wart-like growth that develops on the tongue; it is an epithelial focus of EBV replication. Virtually all central nervous system non-Hodgkin lymphomas are associated with EBV, but fewer than 50% of systemic lymphomas are EBV positive. In addition, EBV is associated with classic Hodgkin disease, with the viral genome detected in the malignant Reed-Sternberg cells in up to 50% of cases.
Immunity
EBV infections elicit an intense immune response consisting of antibodies against many virus-specific proteins, a number of cell-mediated responses, and secretion of lymphokines. Cell-mediated immunity and cytotoxic T cells are important in limiting primary infections and controlling chronic infections.
Serologic testing to determine the pattern of specific antibodies to different classes of EBV antigens is the usual means of ascertaining a patient’s status with regard to EBV infection.
Laboratory Diagnosis
A. Molecular Detection
PCR assays for EBV viral DNA can detect virus in blood, body fluids, and tissues. Quantitative PCR methods can determine viral load and are used to monitor for early development of post-transplant lymphoproliferative disorder (PTLD) in transplant patients. Testing of plasma will detect circulating viremia (often associated with PTLD progression), while whole blood can detect EBV integrated into WBC genomes or latent infections. Nucleic acid hybridization can detect EBV in patient tissues. EBER RNAs are abundantly expressed in both latently infected and lytically infected cells and provide a useful diagnostic target for detection of EBV-infected cells by hybridization. Viral antigens can be demonstrated directly in lymphoid tissues and in nasopharyngeal carcinomas. During the acute phase of infection, about 1% of circulating lymphocytes will contain EBV markers; after recovery from infection, about one in 1 million B lymphocytes will carry the virus.
B. Isolation of Virus
EBV can be isolated from saliva, peripheral blood, or lymphoid tissue by immortalization of normal human lymphocytes, usually obtained from umbilical cord blood. This assay is laborious and time consuming (6–8 weeks), requires specialized facilities, and is seldom performed. It is also possible to culture “spontaneously transformed” B lymphocytes from virus-infected patients. Any recovered immortalizing agent is confirmed as EBV by detection of EBV DNA or virus-specific antigens in the immortalized lymphocytes.
C. Serology
Common serologic procedures for detection of EBV antibodies include enzyme-linked immunosorbent assays, immunoblot assays, and indirect immunofluorescence tests using EBV-positive lymphoid cells.
The typical pattern of antibody responses to EBV-specific antigens after a primary infection is shown in Figure 1. Early in acute disease, a transient rise in IgM antibodies to viral capsid antigen (VCA) occurs, replaced within weeks by IgG antibodies to this antigen, which persist for life. Slightly later, antibodies to the early antigen (EA) develop that persist for several months. Several weeks after acute infection, antibodies to EBNA and the membrane antigen arise and persist throughout life.
Fig1. Typical pattern of antibody formation to Epstein-Barr virus (EBV)-specific antigens after a primary infection. Individuals with recent infection have immunoglobulin M (IgM) and IgG antibodies to the viral capsid antigen (VCA IgM, VCA IgG); only the IgG antibodies persist for years. Transient heterophil antibodies develop that can agglutinate sheep cells. Antibodies to early antigens (EA) develop in many patients and persist for several months. Several weeks after acute infection, antibodies to EBV nuclear antigens (EBNA) and membrane antigen appear and persist for life. (Reprinted from Gulley ML, Tang W: Laboratory assays for Epstein-Barr virus-related disease. J Mol Diagn 2008;10:279–292 with permission from the American Society for Investigative Pathology and the Association for Molecular Pathology.)
The less-specific heterophil agglutination test may be used to diagnose acute EBV infections. In the course of infectious mononucleosis, most patients develop transient heterophil antibodies that agglutinate sheep cells. Commercially available spot tests are convenient.
Serologic tests for EBV antibodies require some interpretation. The presence of antibody of the IgM type to the viral capsid antigen is indicative of current infection. Antibody of the IgG type to the viral capsid antigen is a marker of past infection and indicates immunity. Early antigen antibodies are generally evidence of current viral infection, although such antibodies are often found in patients with Burkitt lymphoma or nasopharyngeal carcinoma. Antibodies to the EBNA antigens reveal past infection with EBV, although detection of a rise in anti-EBNA antibody titer suggests a primary infection. Not all persons develop antibody to EBNA.
Epidemiology
EBV is common in all parts of the world, with more than 90% of adults being seropositive. It is transmitted primarily by contact with oropharyngeal secretions. In developing areas, infections occur early in life; more than 90% of children are infected by age 6 years. These infections in early childhood usually occur without any recognizable disease. The inapparent infections result in permanent immunity to infectious mononucleosis. In industrialized nations, more than 50% of EBV infections are delayed until late adolescence and young adulthood. In almost half of cases, the infection is manifested by infectious mononucleosis. There are an estimated 100,000 cases of infectious mononucleosis annually in the United States.
Prevention, Treatment, and Control
There is no EBV vaccine available.
Acyclovir reduces EBV shedding from the oropharynx during the period of drug administration, but it does not affect the number of EBV-immortalized B cells. Acyclovir has no effect on the symptoms of mononucleosis and is of no proved benefit in the treatment of EBV-associated lymphomas in immunocompromised patients.
Adoptive transfer of EBV-reactive T cells shows promise as a treatment for EBV-related lymphoproliferative disease.
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