Epstein-Barr Virus (EBV) or Human Herpesvirus 4- An Overview

Epstein-Barr virus (EBV) is a member of the herpesvirus family and one of the most common human viruses. It causes infectious mononucleosis and is associated with various cancers and autoimmune diseases.

Structure of EBV

The structure of EBV is similar to other herpesviruses. It consists of a double-stranded DNA genome surrounded by an icosahedral protein capsid containing 162 capsomers. A protein tegument is present between the capsid and the envelope embedded with glycoproteins that play a part in cell tropism, host range, and cell recognition. The mature virions are approximately 120 to 180 nm in diameter.

The following diagram shows a simplified structure of EBV:

Subtypes of EBV

There are currently two recognized subtypes of EBV; Type 1 and Type 2, also referred to as Type A and Type B, respectively. These subtypes differ from one another at the EBV nuclear antigen loci (EBNA). EBNA are proteins expressed by the virus during latency that help the virus evade the immune system and maintain its episomal state in the infected cells.

Type 1 EBV is more prevalent and more efficient in transforming B cells than Type 2 EBV. Type 2 EBV is more common in Africa and is associated with Burkitt`s lymphoma, a type of cancer that affects B cells.

The following table summarizes some of the differences between Type 1 and Type 2 EBV:

The genomic structure of the EBV virus consists of a linear, double-stranded DNA of approximately 172 Kbp genome size, which encodes for more than 85 genes. The genome consists of uniquely tandem repeats of DNA fragments, whose number varies among different EBV isolates. Most proteins encoded by the virus are involved in the virus metabolism, replication cycles, and building the structural compartments like nucleocapsid, tegument proteins, and the envelope.

The EBV genome also consists of several latent genes that remain untranslated during the lytic phase and several RNA genes that are expressed during latency. During latency, the EBV DNA usually persists as multiple circular episomes inside the infected cells. However, it can also integrate with the chromosomal DNA and persist as integrated DNA.

The EBV genome can be divided into two regions: the unique region (UR) and the terminal repeat region (TR). The UR contains most of the viral genes and is flanked by two TRs at both ends. The TR contains multiple copies of a 533 bp sequence that serves as a site for viral DNA replication and recombination. The UR can be further subdivided into four regions: BamHI W, BamHI C, BamHI A rightward transcript (BART), and BamHI A leftward transcript (BALF).

The BamHI W region contains genes that encode for EBV nuclear antigens (EBNAs) that are essential for viral latency and immortalization of B cells. The BamHI C region contains genes that encode for latent membrane proteins (LMPs) that are involved in B cell activation and transformation. The BART region contains genes that encode for non-coding RNAs (ncRNAs) that regulate viral gene expression and host cell functions. The BALF region contains genes that encode for lytic proteins that are involved in viral replication and release.

The EBV genome also contains several internal repeat regions that are dispersed throughout the UR. These include internal repeat 1 (IR1), internal repeat 2 (IR2), internal repeat 3 (IR3), internal repeat 4 (IR4), family of repeats (FR), and dyad symmetry element (DS). These repeat regions contain regulatory elements, such as promoters, enhancers, and oriP (origin of plasmid replication), that control viral gene expression and replication.

The EBV genome is highly variable among different strains and isolates. The main sources of variation are the number and arrangement of the TRs and the IRs, as well as point mutations and deletions in the UR. These variations can affect the viral tropism, pathogenicity, and immune evasion strategies.

The following table summarizes the main features of the EBV genome structure:

Epstein-Barr Virus (EBV) is a common human herpesvirus that infects more than 90% of the world`s population . EBV infection can cause various diseases, ranging from asymptomatic or mild infections to severe complications such as infectious mononucleosis (IM), lymphoproliferative disorders, and autoimmune diseases .

The epidemiology and prevalence of EBV infection vary depending on several factors, such as age, geographic location, socioeconomic status, and immune status of the host . The following table summarizes some of the key aspects of EBV epidemiology and prevalence:

The primary route of transmission for the EBV virus is through saliva containing the infected cells. The virus can spread through activities like:

• Kissing
• Sharing drinks and food
• Using the same cups, eating utensils, or toothbrushes
• Having contact with toys that children have drooled on

EBV can also spread through other body fluids like blood and semen during sexual contact, blood transfusions, and organ transplantations . Although blood banks undergo screening procedures for infectious pathogens, the risk of transmission of untested pathogens, such as EBV, still remains of concern.

The presence of infected cells in the uterine cervix and semen is also suggestive of transmission through the sexual route.

EBV can be spread by using objects, such as a toothbrush or drinking glass, that an infected person recently used. The virus probably survives on an object at least as long as the object remains moist.

The first time you get infected with EBV (primary EBV infection) you can spread the virus for weeks and even before you have symptoms. Once the virus is in your body, it stays there in a latent (inactive) state. If the virus reactivates, you can potentially spread EBV to others no matter how much time has passed since the initial infection.

The replication process of EBV can be divided into two phases: the lytic phase and the latent phase. The lytic phase is when the virus actively produces new viral particles and infects new cells. The latent phase is when the virus remains dormant in the infected cells and does not produce new viral particles.

Lytic phase

The lytic phase of EBV replication involves the following steps:

1. Attachment/Adsorption. After the entry of the virus through any of the transmission routes, the B cells and epithelial cells act as host cells for the virus. The virus attaches to receptors like CD21 and integrin proteins on the host cells by interaction of the receptors with the viral glycoproteins.
2. Penetration. The EBV virus penetrates into the host cells through the process of fusion. The viral protein gp42 interacts with the HLA class II molecule of the B cell, while the EBV gH/gL envelope protein interacts with the αvβ6/8 integrins of the epithelial cells that induce the fusion with the host cell and allow the entry of the viral particle.
3. Uncoating. Through the action of host lysosomal enzymes, the capsid gets separated from the viral genome, and the viral DNA is released into the cytoplasm that enters the nucleus.
4. Biosynthesis. The linear dsDNA of the EBV gets converted to circular DNA and is replicated through the rolling circle mechanism. During the viral latency, latent genes are transcribed, and the circular DNA can persist for decades before entering the lytic phase. In the lytic phase, the intermediate-early, early, and late mRNA are synthesized that leave the nucleus and get translated to proteins in the free ribosomes or ribosomes on the endoplasmic reticulum.
5. Assembly. The capsid proteins enter the nucleus and form the nucleocapsid with the viral genome, and bud off the nuclear membrane with a single membrane envelope.
6. Maturation. The maturation of the virus takes place in the endoplasmic reticulum and Golgi bodies and gets released back to the cytoplasm.
7. Release. The viruses are released from the infected host cells through lysis of the host cell membrane or exocytosis.

Latent phase

The latent phase of EBV replication involves the following steps:

1. Integration or episomal persistence. The circular DNA of EBV can either integrate into the host chromosomal DNA or persist as episomes in the nucleus of infected cells.
2. Latent gene expression. Depending on different latency programs, different sets of latent genes are expressed in infected cells that regulate various cellular functions such as proliferation, differentiation, apoptosis, immune evasion, etc.
3. Reactivation. Under certain stimuli such as stress, immunosuppression, or inflammation, some latently infected cells can switch to lytic replication and produce new viral particles that can infect new cells or be shed in saliva or other body fluids.

The pathogenesis of EBV infection can be divided into three phases: primary infection and lytic replication, latency, and reactivation.

Primary Infection and Lytic Replication

The primary infection usually begins in the tonsillar region, with the virus mainly affecting the B cells and epithelial cells. The virus attaches to CD21 receptors on the B cells through the viral gp350, followed by interaction of viral gp42 with HLA class II molecules of the B cells that induce fusion with the host membrane. In epithelial cells, EBV BMRF-2 protein interacts with the β1 integrins, followed by the interaction of EBV gH/gL envelope protein with αvβ6/8 integrins initiating their fusion .

The viral replication, transcription, and translation produce a range of gene products where the early products are involved in many functions, including replication, metabolism, and suppression of antigen processing, while the late gene products are involved in the production of structural proteins and products involved in immune evasion.

The infected B cells are triggered to differentiate into memory B cells and released into peripheral circulation resulting in high virus levels in the blood. Although the number of infected B cells decreases with time after the onset of symptoms from primary infection, they are never completely eliminated .

Latency

The EBV genome mostly persists in the B cells as episomes or as integrated DNA and may also exist in the epithelial cells. During the latent phase, replication occurs through the host DNA polymerase, and limited EBNA (EBV nuclear antigen) and LMP (latent membrane proteins) are expressed. The latency phase occurs as three different latency programs .

Through transcription, the latent EBV gene can multiply in memory cells, induce B cell differentiation, activate the naïve B cells, or even restrict the expression of all genes. During type I latency, only EBNA1 is expressed, as observed in Burkitt’s lymphoma. CD8 T cells are produced in response to specific EBV antigens but not against EBNA1, which helps them evade the host immune response. During type II latency, EBNA1 and LMP1/2A are expressed as seen in nasopharyngeal carcinoma and Hodgkin’s lymphoma. All the latency gene products are produced during the type III latency observed during acute infectious mononucleosis complication where the infected B cells become immortalized, and a large number variety of antibodies are produced against it.

Reactivation

The exact cause for the viral reactivation is not yet clear however chronic psychological stress and the weakened immune system of the host have been observed to induce the reactivation. The latently infected cells further infect new B cells and epithelial cells that aid in the viral shedding and transmission. The proportion of EBV-infected cells that are in the lytic or latent phase at any given time is, however, not known.

Epstein-Barr virus (EBV) infection can cause a wide range of clinical manifestations, depending on the age of the host, the immune status, and the type of latency program. Some of the common and rare clinical manifestations and complications of EBV infection are:

• Infectious mononucleosis (IM): This is the most common manifestation of EBV infection, especially in adolescents and young adults. It is characterized by fever, sore throat, lymphadenopathy, splenomegaly, and atypical lymphocytosis. IM usually resolves within 4 to 6 weeks, but some patients may experience prolonged fatigue and malaise for months. IM can also cause complications such as hepatitis, splenic rupture, hemolytic anemia, thrombocytopenia, and neurological disorders (such as encephalitis, meningitis, Guillain-Barré syndrome, and Bell`s palsy) .

• Lymphoproliferative disorders: EBV is associated with several types of lymphomas and leukemias that arise from the uncontrolled proliferation of infected B cells or T cells. These include Burkitt lymphoma, Hodgkin lymphoma, diffuse large B cell lymphoma, post-transplant lymphoproliferative disorder (PTLD), chronic active EBV infection (CAEBV), and T cell or NK cell lymphomas . The risk of developing these malignancies depends on several factors such as genetic susceptibility, immunosuppression, co-infection with other viruses (such as HIV or malaria), and environmental exposure.

• Epithelial malignancies: EBV is also implicated in the development of some epithelial cancers that originate from the infected epithelial cells of the nasopharynx, stomach, or salivary glands. These include nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and salivary gland carcinoma . The pathogenesis of these cancers involves the expression of latent EBV genes that induce cellular proliferation, invasion, angiogenesis, and immune evasion. The incidence of these cancers varies geographically and is influenced by genetic factors, dietary habits, and co-infection with other bacteria (such as Helicobacter pylori).

• Autoimmune diseases: EBV infection has been suggested to play a role in the etiology of some autoimmune diseases that are characterized by chronic inflammation and tissue damage. These include multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren syndrome (SS), and autoimmune thyroiditis . The proposed mechanisms by which EBV may trigger autoimmunity include molecular mimicry, bystander activation, epitope spreading, polyclonal activation, and loss of tolerance.

Diagnosing EBV infection can be challenging because the symptoms are similar to other illnesses. However, there are some tests that can help confirm the diagnosis and rule out other possible causes. These tests include:

• Peripheral blood smear: This test involves examining a drop of blood under a microscope to look for atypical lymphocytes, also known as Downey cells. These are activated CD8 T cells that are produced in response to the EBV-infected B cells. The presence of atypical lymphocytes is suggestive of infectious mononucleosis caused by EBV .
• Heterophile antibody test: This test detects antibodies that react with antigens from other species, such as sheep or horse red blood cells. These antibodies are produced by some people with EBV infection and are called heterophile antibodies. This test is considered the best initial diagnostic test for EBV-related infectious mononucleosis . However, it may not be reliable in the first week of symptoms due to low antibody levels, and it may also give false negative results in some cases . Additionally, it may not differentiate between EBV and other causes of heterophile antibodies, such as cytomegalovirus (CMV) or toxoplasmosis.
• EBV-specific antibody test: This test measures the levels of antibodies that are specific to EBV antigens, such as viral capsid antigen (VCA), early antigen (EA), and Epstein-Barr nuclear antigen (EBNA). These antibodies can help determine the stage and type of EBV infection. For example, VCA IgM and IgG antibodies indicate acute infection, while EBNA IgG antibodies indicate past infection . This test has higher sensitivity and specificity than the heterophile antibody test, but it is also more expensive and time-consuming.
• Viral detection and quantification: This test involves detecting and measuring the amount of EBV DNA or RNA in blood or tissue samples using molecular techniques, such as polymerase chain reaction (PCR) or in situ hybridization (ISH). This test can help diagnose active or latent EBV infection, monitor viral load, and assess the risk of complications or malignancies associated with EBV . However, this test is not widely available and may not be necessary for most cases of EBV infection.

There is no specific treatment for the EBV virus infection. However, symptomatic management of infectious mononucleosis can be done by the use of antipyretics and analgesics to reduce fever and pain during the acute phases. Oral rehydration and a nutritional diet can be especially important for febrile patients.

Proper bed rest can also help in the recovery of the patients. However, strenuous physical activities should be avoided as they may increase the risk of splenic rupture. Patients with enlarged spleen should be advised to wear an abdominal binder and avoid contact sports for at least four weeks after the onset of symptoms.

In some cases, corticosteroids may be prescribed to reduce the inflammation of the throat, tonsils, or spleen. Corticosteroids can also help in the management of hemolytic anemia, thrombocytopenia, or neurological complications caused by EBV infection. However, corticosteroids have many side effects and should be used with caution and under medical supervision.

Antiviral drugs like acyclovir and valacyclovir can also be used in the treatment of EBV infection. These drugs inhibit the viral DNA polymerase and prevent the replication of the virus. However, antiviral drugs have limited efficacy in reducing the duration or severity of symptoms of infectious mononucleosis. They may be more useful in preventing or treating EBV-associated complications like oral hairy leukoplakia, lymphoproliferative disorders, or nasopharyngeal carcinoma.

In rare cases, patients with severe or life-threatening complications of EBV infection may require hospitalization and intensive care. These complications may include airway obstruction, respiratory failure, hepatic failure, renal failure, disseminated intravascular coagulation, or hemophagocytic lymphohistiocytosis. In such cases, supportive measures like oxygen therapy, mechanical ventilation, fluid resuscitation, blood transfusion, dialysis, or plasmapheresis may be needed.

The treatment of EBV-associated cancers depends on the type and stage of the malignancy. Chemotherapy, radiotherapy, immunotherapy, or surgery may be used alone or in combination to treat these cancers. Some targeted therapies like rituximab (a monoclonal antibody against CD20 antigen on B cells) or pembrolizumab (a checkpoint inhibitor that blocks PD-1 receptor on T cells) have shown promising results in treating EBV-associated lymphomas and nasopharyngeal carcinoma.

The treatment of chronic active Epstein-Barr virus infection (CAEBV) is challenging as there is no definitive cure for this condition. CAEBV is characterized by persistent or recurrent symptoms of EBV infection and high viral load in the blood or tissues for more than six months. CAEBV can cause severe organ damage and increased risk of malignancy. The treatment options for CAEBV include antiviral drugs, immunosuppressive drugs, immunomodulatory drugs, stem cell transplantation, or gene therapy. However, these treatments are experimental and have variable outcomes.

Epstein-Barr virus (EBV) is a highly prevalent and contagious virus that can cause various diseases and complications in humans. Although most people are infected with EBV during their childhood, some may experience severe symptoms or develop chronic conditions later in life. Therefore, it is important to prevent and control the spread of EBV infection as much as possible.

Unfortunately, there is no vaccine available for EBV infection yet. However, some preventive measures that can be adopted include:

• Avoiding close contact with people who have active EBV infection or symptoms of infectious mononucleosis (IM), such as fever, sore throat, swollen lymph nodes, and fatigue. This includes avoiding activities like kissing, sharing foods, drinks, personal belongings like toothbrushes, eating utensils, etc. with people infected with EBV.
• Practicing good personal hygiene habits, such as washing hands frequently with soap and water, especially before eating or touching the mouth or nose. This can help reduce the transmission of the virus through saliva or droplets.
• Keeping the immune system healthy and strong by eating a balanced diet, getting enough sleep, exercising regularly, and managing stress. This can help the body fight off the virus and prevent complications.
• Seeking medical attention if experiencing symptoms of EBV infection or IM, such as fever, sore throat, swollen lymph nodes, and fatigue. A doctor can diagnose the infection by performing blood tests and prescribe appropriate treatment options. Antiviral drugs, such as acyclovir or valacyclovir, may help reduce the severity and duration of symptoms. Corticosteroids may help reduce inflammation and swelling in severe cases. Symptomatic treatment, such as antipyretics and analgesics, may help relieve fever and pain.
• Avoiding strenuous physical activities or contact sports until fully recovered from EBV infection or IM. This can prevent the risk of rupturing the spleen, which can be enlarged due to the infection. The spleen is an organ that helps filter the blood and fight infections. A ruptured spleen can cause internal bleeding and shock, which can be life-threatening.
• Following proper screening and testing procedures for blood donation or transfusion. This can reduce the risk of transmitting EBV through blood products. Blood banks usually test for antibodies against EBV to determine the donor`s status. However, some people may have low levels of antibodies or be in the window period between infection and antibody production. Therefore, additional tests, such as PCR or EBERs, may be needed to detect the presence of the virus in the blood.

By following these preventive measures, one can reduce the chances of getting infected with EBV or spreading it to others. However, since EBV is so common and widespread in the population, it may not be possible to avoid it completely. Therefore, it is also important to monitor one`s health and seek medical help if needed.

Future Prospects of Epstein-Barr Virus (EBV) Vaccine

The development of a safe and effective vaccine for EBV has been a long-standing goal of many researchers and public health experts. EBV is a major cause of infectious mononucleosis and is associated with several cancers and autoimmune diseases. A vaccine that can prevent EBV infection or reduce its severity and complications could have a significant impact on global health.

However, the development of an EBV vaccine faces many challenges, such as the complexity of the virus, the diversity of its strains and antigens, the lack of animal models that fully mimic human infection, and the ethical issues of testing a vaccine in healthy young adults who are at risk of primary infection.

Despite these challenges, several vaccine candidates have been proposed and tested in preclinical and clinical studies over the years. Most of these candidates target the viral glycoprotein gp350, which is involved in the attachment and entry of the virus into B cells and epithelial cells. However, none of these candidates have shown sufficient efficacy or safety to warrant further development.

Recently, new approaches and technologies have emerged that may offer new opportunities for EBV vaccine development. For example:

• Moderna has initiated a phase I clinical trial of an mRNA vaccine for EBV that targets four glycoproteins on the virus surface: gp350, gH/gL, gB, and gp42. This vaccine aims to elicit broad and robust immune responses against multiple viral antigens that are involved in cell tropism, host range, and cell recognition. The trial will enroll healthy adults aged 18 to 30 years who are either EBV seronegative or seropositive and will evaluate the safety and immunogenicity of different doses of the vaccine.
• NIH has launched an early-stage clinical trial to evaluate an investigational preventive vaccine for EBV that also targets gp350. The vaccine uses a recombinant protein subunit adjuvanted with Matrix-M™, a saponin-based adjuvant developed by Novavax. The trial will enroll 40 healthy volunteers aged 18 to 29 years who are either EBV seronegative or seropositive and will assess the safety and immune response of the vaccine.
• Several researchers have proposed novel vaccine design approaches based on polymer-based nanoparticles, effective adjuvants, and antibody-guided vaccine design that may enhance the immunogenicity and efficacy of EBV vaccines. These approaches aim to overcome some of the limitations of conventional vaccines, such as low antigen stability, poor antigen presentation, weak neutralizing antibody responses, and suboptimal T cell responses.

In conclusion, there is a strong rationale and urgency for developing an EBV vaccine that can prevent or reduce the burden of EBV-associated diseases. Although there is no approved vaccine for EBV yet, recent advances in vaccine technology and immunology may offer new avenues for achieving this goal. Further research and collaboration among scientists, clinicians, industry partners, and regulatory agencies are needed to overcome the remaining challenges and bring an EBV vaccine closer to reality.

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