This webpage was produced as an assignment for an undergraduate course at Davidson College
What is Epstein-Barr Virus?
    Epstein-Barr virus (EBV) is a gamma-herpesvirus and is composed of linear DNA about 172 kilobases in length. EBV infects about 95% of the world's adult population between 35 and 40 years of age. Since EBV is a herpesvirus, EBV is dormant in the host forever after primary infection. EBV is responsible for mononucleosis other wise known as ìmonoî. (Baumforth et al., 1999) Mononucleosis is more numerous in countries with high standards of living than in countries with lower standards of living because infection at a younger age leads to a symptom less infections. Thus, in countries where living conditions are crowded and modern methods of sanitation have not been developed, children are exposed to EBV during the first few years of their life, and develop a symptom less infection.  Infection often pass unnoticed, and these individuals then have immunity for the rest of their lives. On the other hand, in countries where the standard of living is high and modern sanitation has been established, exposure to EBV is delayed to young adulthood, ages 15 to 30. Then infection results in symptomatic mononucleosis. (O.R.R. et al., 1981)
How is Epstein-Barr Virus Transmitted?
    EBV is found in the saliva of the host and is mainly transmitted through close personal contact involving the exchange of saliva. EBV has been called ìthe kissing diseaseî since EBV can be transmitted by the exchange of saliva during kissing. EBV can also be transmitted by sharing utensils or drinking containers. Transmission in the air or by blood is very rare. (O.R.R. et al., 1981)
Where is Epstein-Barr Virus Found?
    EBV primarily infects the oropharynx specifically the salivary glands and oral mucosal membrane as well as the nasopharyngeal epithelial tissue. Viral replication takes place in these locations and insures the production of more virus to be passed on to new hosts through saliva. After infection of the oropharynx and nasopharyngeal tissues, EBV infects B-Cells where replication of the virus may also occur, but most importantly EBV can enter a state of latency within the B-Cells allowing it to remain dormant within the host for the remainder of the individual's lifetime. (Baumforth et al., 1999)
What are the Symptoms of EBV?
    An infection by EBV resulting in mononucleosis has no preference for a particular sex nor is there a particular season for EBV. If infected as a child the symptoms are very mild and often unnoticeable but might result in a mild sore throat or a minor case of tonsillitis.  If infected as a adolescent symptoms can be very vague at first. Often an infected individual will complain of fatigue, malaise, headaches, chills, puffy eyes, and loss of appetite.  As the infection progresses symptoms like fever, sore throat, swollen lymph nodes, difficulty in swallowing due to tonsillitis, minor aches and pains, and bleeding gums occur as well as jaundice or a rash in rare cases.  The fever of 101- 105 °F last for about five days but maybe last intermittently for another week. In about 50% of cases the spleen becomes swollen as well. Rarely does an EBV infection result in death.  In cases that this has occurred, air way obstruction due to swollen lymphatic tissue, a ruptured spleen, or inflammation of the heart or tissue surrounding the heart causing a heart attack are the causes of death. In some cases EBV infections last for 6 or more months resulting in a condition called Chronic EBV Syndrome in which the patient's symptoms persist with severity resulting in extreme fatigue, respiratory infections, a severe sore throat, largely swollen lymph nodes, persistent fever and headaches, and depression. (O.R.R. et al., 1981)
How is Epstein-Barr Virus Diagnosed?
    Symptoms of an EBV infection resulting in mononucleosis can mislead diagnosis.  Symptoms may resemble other infections such as the sore throat of a strep infection, the painful stiff neck of meningitis, the abdominal pains of an appendicitis, the rash of measles or even rubella, and even the swollen lymph nodes of certain cancers. The fatigue and loss of appetite resulting from mono may be misinterpreted as infectious hepatitis. Sore throat, high fever, and swollen lymph nodes may be misinterpreted as leukemia. Therefore other methods of detection must be used to detect infection by EBV. (O.R.R. et al., 1981)
    Since EBV can produce certain antigens known as heterophil antigens, a Paul-Bunnell test or heterophil antibody test is often used to detect an EBV infection. Since heterophil antibodies have the ability to clot sheep or horse red blood cells, a small amount of the patient's blood is added to some sheep or horse blood to see if clotting occurs. Testing for EBV in this manner and obtaining a titer of 1:10 means the patient has mono. Often diagnosis using this test is misleading since younger children do not produce large enough amounts of heterophil antibodies to obtain the necessary titer, but they still might have the infection. Therefore other test are used when the Paul-Bunnell test is not positive in patients suspected of having an EBV infection. Also other infections can produce heterophil antibodies as well and mislead diagnosis. Thus other methods such as obtaining a serotype profile are used. (O.R.R. et al., 1981)
    Here a measurement of the different antibodies produced during infection against the different antigens that EBV displays during its infection is obtained. Other than heterophil antigens, EBV produces early antigens (EAs) 2-4 weeks into infection and viral capsid antigens (VCAs) 4-6 weeks into infection both of which are produced during the acute phase of infection. During the latent phase of infection, Epstein-Barr virus nuclear antigens (EBNA) are produced. A measurement of antibody response to these antigens allows for an identification of current infection, recent infection, or past infection. During acute EBV infection IgG and IgM antibodies to EAs and VCAs are elevated and no anti-EBNA antibodies are present which represents a current infection. A recent infection is characterized by small amounts of IgM antibodies to EAs, large amounts of IgG antibodies to VCAs, and no anti-EBNA antibodies. For a past infection moderate IgG and anti-EBNA antibodies are present which continue at moderation for life. (Baumforth et al., 1999)
    Enzyme-linked immunosorbent assay test (ELISA) or immunofluorescence can be used to detect EBV antibodies and thus an EBV infection. Also a Southern blot analysis of DNA accompanied with polymerase chain reaction (PCR) of saliva and throat washings can be used to detect EBV. (Ikuta et al., 1999)
What does Epstein-Barr Virus Do?
    After infecting the oropharynx tissues such as the salivary glands, EBV then infects and transforms B-Cells into B-Cells with unlimited growth potential. Cytotoxic T-Cells and Natural Killer Cells are then activated in defense against EBV and regulate the elimination of infected B-Cells. In extreme cases when long lasting infection results, agammaglobulinemia, the lack of gamma globulin, occurs due to T-Cells severely suppressing the B-Cells. In such cases Chronic EBV Syndrome is observed, and symptoms from EBV last for up to six months due to the severity of B-Cell suppression. At this time other infections can occur due to the suppression of the immune system which can complicate recovery. The occurring infections are treated and eventually the patient recovers. On the other hand, if the T-Cell response is not strong enough, unrestricted growth of B-Cell lymphocytes occurs resulting in a lymphoma. Normally an EBV infection occurs, and the patient observes the usual symptoms such as fever and swollen lymphatic tissue that result from the immune system fighting the proliferation of infected B-Cells until full recovery is accomplished. (O.R.R. et al., 1981)
How does Epstein-Barr Virus Operate?
    EBV suppresses the immune system through several mechanisms and spreads throughout the host. EBV infects B-Cells by binding to the CD21 receptor on the B-Cell surface resulting in internalization of the virus. Either EBV causes the B-Cell to enter a lytic phase or to progress towards a latent phase depending on which proteins are synthesized during infection. (Baumforth et al., 1999)
    During the lytic phase, new viruses are synthesized and eventually the B-Cell lyses releasing the viruses which then enter other non infected B-Cells. Also during the lytic phase, the proteins BZLF1 and BRLF1, viral transactivator proteins, are expressed on the B-Cells, and BCRF1 and BHRF1, lytic genes, are expressed as well. (Baumforth et al., 1999)

What are the Function of the Lytic Genes?

         BCRF1: makes a homologue similar to interleukin-10 (IL-10). IL-10 inhibits TH1 T-Cells; Since TH1 T-Cells are inhibited, the secretion of interferon-gamma, a macrophage activating cytokine, by the TH1 T-Cells does not occur. Thus EBV suppresses the immune system by inhibiting the activation of macrophages. (Baumforth et al., 1999)
         BHRF1: makes a protein like BCL-2 which can protect the cell during the lytic phase from apoptosis. (Baumforth et al., 1999)

    In the lytic phase, the BARF1 protein is secreted from EBV infected B-Cells during lytic replication and binds to colony-stimulating factor-1 (CSF-1), a cytokine that induces macrophage activation. Thus, by secreting BARF1, EBV suppresses macrophage activation. (Strockbine et al., 1998). Nonetheless at the end of the lytic phase, a downregulation of latent genes occurs and the cell undergoes death releasing newly replicated viruses. (Baumforth et al., 1999)
    During the latent phase, EBV causes B-Cells to become lymphoblastoid cells, B-Cells with accelerated growth. Replication of the virus does not occur, and the B-Cell becomes a reservoir for EBV. In the latent phase, certain genes are expressed resulting in proteins which signify this phase: six nuclear proteins called Epstein-Barr virus nuclear antigens EBNA1, EBNA2, EBNA3, EBNA3B, EBNA3C, and EBNA leader protein (EBNA-LP), the latent proteins: LMP1, LMP2A, LMP2B, and the Epstein-Barr early RNAs: EBER1 and EBER2. (Baumforth et al., 1999)

What are the Functions of the Latent Proteins?
        EBNA1: EBNA1 is a DNA binding protein that controls the replication of the virus within infected cells. EBAN1 is required for maintaining latency. (Baumforth et al., 1999)
        EBNA2: this protein results in B-Cell transformation by upregulating the genes that code for CD23, CD21, LMP1, and LMP2. (Baumforth et al., 1999)
        EBNA3A, EBNA3B, and EBNA3C: Little is known about these three proteins except that EBNA3A and EBNA3C are necessary for EBV transformation but EBNA3B is not. EBNA3C transactivates EBNA2 regulated genes but blocks the EBNA2 transactivation of LMP1 and LMP2. (Baumforth et al., 1999)
        EBNA-LP: EBAN-LP along with EBNA2 are the first EBV proteins to be expressed after infection of the B-Cell. They act together to activate cyclin D2 synthesis which allows the B-Cell to enter the G1 phase of the cell cycle. (Baumforth et al., 1999)
        LMP1: LMP1 is a protein similar to ion channels and G protein receptors. When LMP1 genes are expressed in latent cells a dramatic change in the phenotype of the B-Cell results. LMP1 is involved in processes that ultimately result in increased expression of cell adhesive molecules resulting in cell clumping, expression of CD23, CD39, CD40, and CD44, and decreased expression of CD10. LMP1 can also protect the B-Cell from apoptosis by inducing BCL-2, MCL-1 and A20, anti-apoptotic proteins. LMP1 also induces the production of interleukin-6 and interleukin-10. LMP1 accomplishes its function by signaling through four different pathways: the nuclear factor kB (NF-kB) pathway, the c-Jun N terminal kinase ((JNK)-AP-1) pathway, the p38/MAPK (mitogen activated protein kinase) pathway, and the Janus kinase ((JAK)-STAT (signal transducers and activators of transcription)) pathway. (Baumforth et al., 1999)
        LMP2A and LMP2B: LMP2A obstructs signaling pathways that are trigger by the binding of the B-Cell antigen to the B-Cell receptor complex. LMP2A proteins compete with SRC proteins and SYK proteins to block the kinases that are activated by those proteins. Blockage prevents the transition of the B-Cell into the lytic phase maintaining EBV latency. Little is known about LMP2B, but what is suspected is that this protein interacts with LMP2A and ultimately restores the SRC and SYK kinases allowing B-Cell receptor signal transduction. (Baumforth et al., 1999)
        EBER1 and EBER2: EBER1 and EBER2 are small polyadenylated RNAs and are the most abundant EBV RNAs in the latency infected cell; thus they are used as markers for detection of latent EBV infections. But, the main function of EBER1 and EBER2 is to complex with the interferon inducible protein kinase, PKR, which causes the inhibition of the antiviral and antiproliferation effects of interferons, thus counteracting PKRs effects allowing for the proliferation of the B-Cell. (Baumforth et al., 1999)

    However there is a small number of lymphoblastoid cells that go from that latent stage back into the lytic stage, and this is mediated by BZLF1 and BRLF1. (Baumforth et al., 1999)

    EBV can also infect Natural Killer Cells (NK Cells). The receptor for EBV on NK Cells is not know, but EBV can enter NK Cells without being phagocytosed which was determined from a study conducted in which NK Cells were treated with cytochalasin B, a phagocytosis inhibitor, and the EBV genome was still found inside the NK Cells. The rest of the study determined that EBV induced apoptosis of NK cells and that this was Fas/FasL apoptosis determined from the observation that Fas/FasL expression increased on NK Cells when treated with EBV. Thus EBV is able to exert its immunosuppressive effects on NK Cells as well. (Larochelle et al., 1998)
    Also when EBV interacts with NK Cells, the production of interleukin-8 (IL-8) results which is a chemokine that attracts neutrophils. This interaction also causes neutrophils to secrete Macrophage Inflammatory Protein-1alpha (MAP-1alpha) which attracts B-Cells to the site of infection. This result is beneficial to EBV because it ultimately allows more B-Cells to locate at an area of infection giving EBV more B-Cells to infect. This further allows the spread of infection. (McColl et al., 1997)

 How Does the Immune System React to EBV?

    Cytotoxic CD8 T-Cells specific for a certain EBV antigen find B-Cells bearing their antigen and kill them; NK Cells can also kill EBV infected B-Cells. During the lytic stage, CD8 T-Cells specific for BZLF1 and BRLF1 proteins find EBV infected B-Cells bearing these proteins and kill them. During the latent stage, CD8 T-Cells for latent proteins regulate the amount of latent protein expressing EBV infected B-Cells also. (Steven et al., 1997)
    Constant exposure to IL-10, as seen in patients with chronic EBV infections, results in anti-IL-10 antibodies being made. These antibodies are hypothesized to be produced by CD5 B-Cells which are known to be the source of autoantibodies such as ones against IL-10, and CD5 B-Cells have been shown to increase by three fold during an EBV infection. Thus the immune system fights back with anti-IL-10 antibodies to unsuppress macrophage activation. (Tanner et al., 1997)
    Infected B-Cells can be caused to undergo CD95-CD95L-mediated apoptosis (Fas/FasL apoptosis) by CD4 T-Cells. Some TH1 CD4 T-Cells bear CD95L and can bind to EBV infected B-Cells bearing the CD95 receptor ultimately resulting in apoptosis. This normal mechanism of  regulation of B-Cell growth is observed during EBV infection. (Wilson et al., 1998)
Are There Different Strains of EBV?
        There are two strains of EBV, EBV-1 and EBV-2, which differ in the regions that code for the EBNAs and EBERs. EBV-2 is less effective at causing B-Cell growth and proliferation than EBV-1. This is mainly a result of differences in the coding regions of the gene for EBNA2. (Baumforth et al., 1999)
What is the Treatment for EBV?
    There is no treatment for an EBV infection resulting in mononucleosis except to treat the symptoms. An infection by EBV can last from a few days to months. The exact duration is not know. Rest, limited activity, and plenty of fluids is the usual treatment. Strenuous exercise is avoided so as not to rupture or damage the swollen spleen. Aspirin or tylenol is given for headaches. Salt gargles are recommended for sore throats. The infection seems to go away on its own over time. (O.R.R. et al., 1981)
    There are no antibiotics available to treat EBV. Antibiotics are only given to treat secondary infections during mono. If a patient comes down with a streptococcal throat infection, the individual is treated with penicillin or erythromycin. However in some cases when penicillin is administered to a patient, a rash develops which is misinterpreted as an allergic response to penicillin. Therefore penicillin is not administered to patients with mono because the patient may mistakenly be told not to take penicillin in the future. If the spleen is ruptured surgery should follow. Lastly, corticosteriods such as prednisone are often given to lesson symptoms such as a swollen heart or tissues surrounding the heart and breathing obstruction. (O.R.R. et al., 1981)
 Are There Vaccines for EBV?
    There is no current vaccine for EBV but one is being studied that contains the protein EBNA3 which will hopefully result in activation of the immune system resulting in antibodies against EBV but without the symptoms of a usual EBV infection. (O.R.R. et al., 1981)
How is EBV Involved in cancers?
    EBV has been known to be involved with several cancers. Burkittís lymphoma is characterized by chromosomal translocations involving chromosome 8 and chromosome 14, 2, or 22 which results in the deregulation of MYC protein expression resulting in increased B-Cell proliferation. The presence of EBV in B-Cells with Burkittís lymphoma has been correlated with a break in chromosome 8 involving the MYC locus that is different than the break in chromosome 8 that is found in B-Cells with Burkittís lymphoma that do not contain EBV. Thus each type of Burkittís lymphoma results in a different method of deregulation of the MYC protein but with the same result, increased B-Cell proliferation. (Baumforth et al., 1999)
    EBV has also been linked to nasopharyngeal carcinoma since EBV has been found in undifferentiated nasopharyngeal carcinomas. PCR has revealed that LMP2A and LMP2B genes are expressed in these carcinomas, and other latent transcripts have been found running through the BAMHI A region of the EBV genome in the opposite direction of the conventional lytic cycle MRNAs transcribed over this region. Thus EBV may be the cause of nasopharyngeal carcinomas. (Baumforth et al., 1999)
    Similar BAMHI A transcripts have also been found in Hodgkinís lymphoma, and thus EBV has been linked to Hodgkinís as well. (Baumforth et al., 1999)

 

References

1. Baumforth K.R., Young L.S., Flavell K.J., et al. The Epstein-Barr virus and its association with human cancers. Journal of Molecular Pathology 1999; 52:307-322.
2. Ikuta K., Satoh Y., Hoshikawa Y., Siarjenji T. Detection of Epstein-Barr virus in salivas and throat washings in healthy adults and children. Microbes and Infection 1999; 2:115-120.
3. Larochelle B., Flamand L., Gourde P., et al. Epstein-Barr Virus Infects and Induces Apoptosis in Human Neutrophils. Blood 1998; 92:291-299.
4. McColl S.R., Roberge C.J., Larachelle B., et al. EBV Induces the Prodution and Release of IL-8 and Macrophage Inflammatory Protein-1alpha in Human Neutrophils. The Journal of Immunology 1997; 159:6164-6168.
5. Office of Research Reporting (O.R.R.), Public Response National Institute of Allergy and Infectious Diseases, National Institute of Health. Mononucleosis. Bethesda: National Institute of Health; 1981. p1-6.
6. Steven N.M., Annels N.E., Kumar A., et al. Immediate Early and Early Lytic Cycle Proteins Are Frequent Targets of the Epstein-Barr Virus-induced Cytotoxic T Cell Response. J. Exp. Med. 1997; 185:1605-1618.
7. Strockbine L.D., Cohen J. I., Farrah T., et al. The Epstein-Barr Virus BARF1 Gene Encodes a Novel, Soluble Colony-Stimulatin Factor-1 Receptor. Journal of Virology 1998; 72:4015-4021.
8. Tanner J.E., Diaz-Miltoma F., Rooney C.M., et al. Anti-Interleukin-10 Antibodies in Patients with Chronic Active Epstein-Barr Virus Infection.  The Journal of Infectious Disease 1997; 176:1454-14.
9. Wilson A.D., Redchenko I., Williams N.A., Morgan A.J. CD4+ T cells inhibit growth or Epstein-Barr virus-transformed B Cells through Cd95-CD95 ligand-mediated apoptosis. International Immunology 1998; 10:1149-1157.
 


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