Herpesviruses Slide Set

Epstein-Barr Virus

 

PROPERTIES

member of the herpesvirus family
ds DNA enveloped virus
nucleocapsid 100 nm in diameter, with 162 capsomers
membrane is derived by budding of immature particles through cell membrane and is required for infectivity.
genome is a linear ds DNA molecule with 172 kbp
the viral genome does not normally integrate into the cellular DNA but forms circular episomes which reside in the nucleus.
the genome is large enough to code for 100 - 200 proteins but only a few have been identified.

The proteins characterized thus far fall into the following groups :

A._Latent_proteins

1. EB viral nuclear antigen complex (EBNA) - EBNA is now known to be a complex of at least 6 proteins ie. EBNA 1-6. Their functions have yet to be understood but EBNA 1 is thought to be essential for the maintenance of the episomal state of EBV in infected cells and binds to the origin of replication. EBNA 1 is expressed in all known virus carrying cells, but its expression may be lost when a lytic cycle ensues. EBNA 2 has two phenotypes, A and B, strain A, which is found mainly in Caucasians, is more efficient in immortalizing B lymphocytes than strain B, which is mainly found in Africa. However, neither strains have any specific disease association.

2. Latent membrane protein (LMP) - LMP is thought to be a membrane associated protein which is found in the virus particle as well as the infected cells. The structure of LMP resembles a growth factor receptor which may play a role in the immortalization process. The transfection of LMP into NIH 3T3 cells causes a tumourigenic phenotype to be expressed.

3. Terminal protein - this new protein family was described recently but its function is unknown. The terminal proteins are only expressed when the viral genome is circularized as its reading frame spans the terminal repeat sequences.

4. Lymphocyte-detected membrane antigen (LYDMA) - LYDMA is the name given to viral coded proteins in expressed cells which are recognized by EBV specific Tc cells. LMP and EBNA-2 have been shown to act in such a capacity as antigenic targets for Tc cells.

B._Lytic_cycle_proteins

1. Membrane antigen (MA) - MA consists of at least 3 glycoproteins, gp 350, gp 250 and gp85, which are present on the viral envelope and the cell membranes of infected cells. These molecules mediate the attachment of the virus to the cell and are antibodies against MA are virus neutralizing.

2. Early antigen complex (EA) - EA is a complex which is only expressed in infected cells undergoing the lytic cycle. The functions of the proteins of the EA complex are as yet unknown.

3. Viral capsid antigen complex (VCA) - the VCA complex comprises of the structural proteins which make up the viral capsid and are synthesized late in the lytic cycle.

Man is the natural host for EBV though some primates eg. tamarins, gibbon, owl monkey and squirrel monkey may be infected experimentally. In humans the only cell types known to be infected are (i) squamous epithelial cells, and (ii) B lymphocytes. These are the only cells which express the receptor for EBV ie. CR2, the receptor for the C3d component of complement. The outcome of EBV infection in these cell types are different. In squamous epithelial cells, a lytic infection occurs with the production of progeny and cell death. In B cells, immortalization takes place, with a minority of cells undergoing the lytic cycle. Until recently, it has not been possible to grow EBV in squamous epithelial cells.

EBV_Immortalization

Human B lymphocytes are immortalized when infected by EBV in vitro as well as vivo. The resting B cells undergo blast transformation with an increase in HLA-DR expression, nuclear size, and cytoplasmic volume and also the expression of certain cellular genes eg. CD23. CD23 is also thought to play a role in the immortalization process. After 36 hours, DNA synthesis is initiated and cell division takes place. Immunoglobulin is detected in 1 to 2% of B cells and is secreted into the culture medium. These early changes seen in B-cells after infection is similar to those seen after activation by B-cell mitogens. However, the polyclonal activation of B-cells is totally independent of T- cells and the immortalization is a permanent event.

All cells that are immortalized express CD34 (cluster designation 23) and EBNA. LMP becomes detectable after EBNA at 48 hours postinfection. This viral gene expression (EBNA 1-6, LMP) is seen in the majority of cells in a lymphoblastoid cell line and is termed "latent gene expression". A minority of cells in a lymphoblastoid cell line at any one time enters the lytic cycle with the resultant production of viral progeny and cell death. Different cell lines have varying degrees of permissiveness for viral replication, ranging from <1% in cell lines derived from umbilical cord cell lines to 15% in the B95-8 cell line derived from tamarin. Most cell lines can be induced by some agents to produce more virus eg. TPA, anti-IgM Ab. These agents induce the maturation of the B-cells.
 

EPIDEMIOLOGY

In developed countries, 2 peaks of infection are seen : the first in very young preschool children aged 1 - 6 and the second in adolescents and young adults aged 14 - 20. 80 - 90% of the adult population have been infected by the virus. In developing countries, infection occurs at a much earlier age so that 90% of children over the age of 2 are seropositive.

Once infected, a lifelong carrier state develops whereby a low grade infection is kept in check by the immune defenses. Low grade virus replication and shedding can be demonstrated in the epithelial cells of the pharynx of all seropositive individuals. Furthermore a few EBV-immortalized B-cells can be demonstrated in the circulation which are continually cleared by immune surveillance mechanisms. Whether these B-cells are true latent EBV infected B-cells or cells which become infected as they pass through Waldeyer's ring in the pharynx is uncertain. EBV is associated with several diseases state where it may act as the direct agent or as one of several cofactors. These diseases include :-

1. Infectious Mononucleosis
2. Burkitt's lymphoma
3. Nasopharyngeal carcinoma
4. Lymphoproliferative disease and lymphoma in the immunosuppressed.
5. X-linked lymphoproliferative syndrome
6. Chronic infectious mononucleosis
7. Oral leukoplakia in AIDS patients
8. Chronic interstitial pneumonitis in AIDS patients.

Since virus shedding can be demonstrated in the oropharynx of most seropositive individuals, it is thought that transmission occurs mainly by the oral route. Thus IM has been dubbed the "kissing disease". More recently, virus replication has been detected in vivo in the uterine cervix, thus raising the possibility of sexual route for the transmission of IM (Similar to CMV). IM has also been acquired by individuals receiving large amounts of blood.
 

Infectious Mononucleosis

Infectious mononucleosis is an acute self-limiting lymphoproliferative disease resulting from primary infection with EBV. Primary EBV infection is usually subclinical in childhood but where the infection is delayed until adolescents and young adulthood (15 - 25), there is a 50% chance that the person infected will develop IM. EB virus enters the body through the mouth and establishes a productive infection in the pharyngeal epithelial cells. B-cells become infected and are disseminated throughout the body via the bloodstream. During acute primary infection, IgA, IgM and IgG to VCA as well as IgG to EA(D) and MA are induced. Antibodies to EBNA, a latent phase protein, are not usually detectable in the serum until the convalescent period. Heterophile antibodies appear early in IM, but their relationship to the virus is unclear. A variety of autoantibodies are also found in IM, which includes cold agglutinins, rheumatoid factor, anti-nuclear antibodies and antibodies to smooth muscle. These antibodies are thought to be due to the polyclonal activation of B-cells. They are usually transient and harmless. The atypical lymphocytes characteristically seen in IM are CD8 cytotoxic lymphocytes that react against EBV-infected B cells in a non-MHC restricted manner. These Tc cells probably play an important role in recovery from the virus infection.

Clinical Features - The incubation period is not known as the source of infection is rarely recognized. Characteristically IM begins with a sore throat and swelling of the neck, accompanied by fever, malaise, sweating, chills, headaches, stiff neck, anorexia and vague abdominal discomfort. The sore throat occurs in 80 - 90% of patients and is usually mild in nature and clears in 7 - 14 days. Occasionally, gross tonsillar enlargement may lead to pharyngeal obstruction. Secondary infection of the pharynx with B-haemolytic streptococcus occurs in 25% of cases. Less commonly, patients may present with jaundice, cough, myalgia or symptoms of one of the neurological complications of IM. Lymphadenopathy is present in the majority of cases, the cervical lymph nodes are most commonly involved but generalized lymphadenopathy may occur which may last for several weeks.

Splenic enlargement and tenderness occurs in 50 - 60% of patients, and hepatomegaly occurs in 15 - 25% of patients. Clinically apparent jaundice appears in 5 - 10% of cases. Fevers of 38 - 40oc are commonly present during the first 2 weeks of IM. Pharyngitis and palatal petechiae occur during the first week of illness which may be accompanied by a gray-white membrane. Periorbital oedema may also be present. 2 forms of rashes occur in IM, a morbilliform eruption which lasts 24 - 48 hrs and a maculopapular rash which occurs following treatment with ampicillin in virtually 100% of patients treated. Signs of rare complications such as meningitis and encephalitis may be present. IM is rare in children and when it occurs, the disease is usually mild. In the elderly, the onset is often insidious and occasionally bizarre. The disease can be serious with hepatic, renal and CNS involvement. IM during pregnancy is not associated with any deleterious effects on the fetus. However, rare cases of fatal lymphoproliferative disease in pregnant women who develop IM has been reported. IM usually lasts for several weeks. Recovered patients occasionally complain of recurrent attacks of fatigue typical of the "persistent viral fatigue syndrome". Some patients suffer relapses of IM like illnesses within one year following the initial attack, with a positive heterophil antibody test. The exact nature of these relapses are unclear at present.

Immunocompromised patients - Where primary EBV infection occurs in immunocompromised patients, particularly after organ transplantation, it may result in abnormal disease with GI symptoms and/or signs of renal graft rejection and failure. Some of these primary infections go on to develop into lymphoproliferative disease and lymphoma.

Complications - IM is usually a benign and self-limiting disease. Certain morbid complications may arise which account for 30 deaths per annum in the US. These complications include :-

1. CNS - CNS complications include meningitis, encephalitis and the Guillain-Barre syndrome. These complications may precede, accompany or postdate IM by several weeks. Recovery is usual. Although CNS disorders may occur as the sole manifestation of an EBV infection, most cases arise during the course of IM.

2. Hepatic - almost all IM patients have deranged LFTs, overt jaundice is uncommon (5-10%), and complete recovery is the rule. More severe cases include massive hepatic cirrhosis and Reye's syndrome.

3. Splenic rupture - this is a very rare complication of IM and when it occurs, urgent surgery is required.

4. Pharyngeal and tracheal obstruction - these may occur due to massive enlargement and oedema of the tonsils and adenoids. A short course of steroids is indicated

5. Immunological complications - these include haemolytic and aplastic anaemia, thrombocytopenia, hypogammaglobulinaemia, agranulocytosis and haemophagocytosis. EBV induced autoantibodies may be involved in these cases.

6. Chronic IM - rarely, chronic cases of IM occurs where symptoms persists for years and death may result from lymphamatous disease. In these cases, there is the persistence of the acute profile of IM, with elevated titres of IgG against VCA and EA-D antigens. There is an absence of antibodies against EBNA 1 and a positive monospot test. High titres of virus can be isolated from throat washings.
 

Diagnosis - the laboratory diagnosis of IM is usually made by the heterophil antibody test, which had been reported to be 98% sensitive for IM. However, false negatives occur commonly in patients under the age of 5 years and anti-VCA IgM testing should be carried out in those instances.

Treatment - the management of IM is mainly supportive. The sore throat may be extremely painful and regular analgesics are needed. Some physicians regularly treat cases of IM with corticosteroids but others prefer to reserve them for severe cases.
   

BURKITT'S_LYMPHOMA

Burkitt's lymphoma (BL) occurs endemically in parts of Africa (where it is the commonest childhood tumour) and Papua New Guinea and is restricted to areas with holoendemic malaria. BL also occurs sporadically throughout the world. However, whereas almost 100% of African BL is associated with EBV, only 12% of cases of sporadic BL are associated with EBV. The evidence that EBV is involved in the pathogenesis of BL is as follows ;-

1. Multiple copies of EBV genome can be demonstrated in the monoclonal tumour cells of BL. EBV MA and EBNA-1 antigens can be detected from all tumour cells and furthermore, infectious EBV particles can be recovered from tumour cell lines.

2. All patients with BL have antibodies to EBV antigens. Furthermore, BL patients are shown to have much higher levels of such antibodies than comparable seropositive subjects. Elevated levels of anti-VCA may be used for screening purposes for BL as there is a 30 fold increase in risk of developing BL in those children with an anti-VCA titre of two doubling dilutions or higher than the general population.

3. EBV is able to transform human B-lymphocytes in vitro.

4. EBV can cause tumours in subhuman primates.

Lymphoid cell lines can be grown from BL biopsy materials but they differ from the cell lines derived from in vitro transformation in that BL cell lines are monoclonal in nature whereas in vitro derived cell lines are polyclonal. Also BL derived cell lines have a greater malignant potential. BL cells show a reciprocal translocation between the long arm of chromosome 8 and chromosomes 14, 2 or 22. These translocations result in the c-myc oncogene being transferred to the Immunoglobulin heavy chain gene region (14), the light kappa (2) or lambda regions (14). These regions are thought to be transcriptionally active and results in the deregulation of the c-myc gene. It is uncertain whether EBV actually causes these translocations or that these translocations have already occurred by the time of infection and thus further malignant transformation by EBV. The latter is more likely.

Cofactors - although EBV is ubiquitous, EBV associated BL occurs almost exclusively in parts of the world where malaria is holoendemic. In areas where malaria had been successfully eradicated, the incidence of BL has declined dramatically. Also BL is rare in children with the sickle cell trait which is known to protect against malaria. The actual mechanism through which malaria act as a cofactor is uncertain. There is evidence that chronic infection by malaria leads to a decrease in cellular immunity and thus immunosurveillance. Another possibility is that malaria leads to a sizable increase in the pool of B cells that can be infected.

Clinical Features - BL usually occurs in children aged 3 - 13 years with a peak incidence at 6 - 7 yrs. In areas where BL is endemic ie. some parts of Africa and New Guinea, the incidence is around 15 per 100000 children aged 5 - 10 years and is the commonest childhood malignancy. BL is more common in boys than girls and arises extranodally typically in the area of the jaw. The tumour is usually found to be multifocal at presentation, the other sites involved being the bone marrow, GI tract, ovaries and breast. BL is usually a highly malignant tumour with death supervening within 4 months of onset in untreated cases, although self-cures have occasionally been reported. The tumour is, however highly sensitive to chemotherapy.

Diagnosis - In endemic areas, BL can often be diagnosed from the typical clinical features. Histology of biopsy specimens should reveal a poorly differentiated lymphocytic lymphoma. The tumour can be stained with antibodies to lambda light chains which should reveal a monoclonal tumour of B-cell origin. In over 90% of cases, the cells express IgM at the cell surface.

Treatment - BL is very sensitive to chemotherapy, one dose of cyclophosphamide often being sufficient to cause complete remission of the tumour mass. The prognosis is good after a full course of treatment if given early.

Prevention - Prevention can theoretically be achieved by the eradication of malaria, or by vaccination against EBV. Where the eradication of malaria have been achieved eg. in parts of New Guinea, the incidence of BL had dropped dramatically.
   

NASOPHARYNGEAL_CARCINOMA

NPC is a malignant tumour of the squamous epithelium of the nasopharynx. It is very prevalent in S. China, where it is the commonest tumour in men and the second commonest in women. The tumour is rare in most parts of the world, though pockets occur in N. and C. Africa, Malaysia, Alaska, and Iceland. The most undifferentiated form of the tumour is always associated with EBV whereas the rarer, more differentiated forms are not consistently so. The evidence that EBV is involved in the pathogenesis of NPC is as follows :-

1. Multiple copies of the EBV genome can be detected in the malignant cells of 100% of undifferentiated NPC. All the malignant cells express EBNA-1 and some have been reported to express LMP. Furthermore, infectious EBV particles can be recovered from NPC cell lines.

2. 100% of sera from undifferentiated NPC patients have high-titre antibodies to EB-viral antigens. As in BL, antibodies against VCA are at a 10 times geometric mean titre. IgG and IgA levels against EA (D) and VCA rise as the disease progress and may be used for screening and monitoring purposes. VCA and EA (D) IgA are also uniquely found in the saliva of NPC patients.
 

Cofactors - NPC is a genetically restricted tumour, being most common in the Southern Chinese, with intermediate frequency in some Negro and Mongoloid races and rare in Caucasians. Studies have shown that first-generation immigrants from S. China retain the high incidence of the disease, with the later generations showing a decline in incidence. This suggests that environmental as well as genetic factors are involved. NPC is especially associated with certain HLA haplotypes eg.HLA A2. More genetic linkage studies demonstrated the presence of NPC susceptibility genes near the MHC genes. Environmental factors are thought to play a role, in particularly the consumption of salted fish and foods containing nitrosamines.

Clinical Features - NPC occurs at a rate of 98 per 100,000 of the population of Southern China and is more common in men than women. The EBV associated undifferentiated type arises mainly in younger patients whereas the more differentiated types occur in older patients and constitute the bulk of the sporadic cases. The tumour most commonly arise in the posterior wall of the nasopharynx in the fossa of Rosenmuller, where it often remains silent and metastasizes to the local lymph nodes. The most common presentation of NPC is bilateral enlargement of the glands in the neck. The primary tumour may be very small and difficult to locate. Less frequently, the patient may present with the symptoms of invasion by the primary tumour eg. nasal obstruction, postnasal discharge, epistaxis, partial deafness and cranial nerve palsies. If untreated, the disease is rapidly fatal due to the development of laryngeal and pharyngeal obstruction.

Diagnosis - the diagnosis of NPC is usually made on histological examination of biopsy material. 3 types of NPC are recognized on histological appearance :-

1. a well differentiated SCC
2. a non-keratinizing carcinoma
3. an undifferentiated carcinoma

Serum antibodies to EBV antigens can be used to confirm the diagnosis and monitor the progress of the disease. Recent studies have demonstrated the value of testing for persistent high levels of serum IgA to VCA in screening for early lesions of the disease. It is hoped that early lesions can be diagnosed in this way and treated, and then monitored.

Treatment - NPC is difficult to treat surgically because of the early metastasis to regional lymph nodes. The tumour is resistant to chemotherapy and radiotherapy is the treatment of choice. However, because the tumour usually presents late, the prognosis is poor with a 5 year survival rate of 20%. It should in theory be possible to prevent the development of NPC with the use of an EBV vaccine at an early age.
 

EBV_Infection in the Immunocompromised

After primary infection, EBV maintains a steady low grade latent infection in the body. This latent infection is normally kept in check by the cellular defenses which, if impaired later in life, may lead to the reactivation of the virus to cause clinical disease. In a few cases, lymphoproliferative lesions and lymphoma may develop. These lesions tend to be extranodal and in unusual sites such as the GI tract or the CNS.

1. X-linked Lymphoproliferative Syndrome

Many families are known where the male members are unable to mount an effective cell-mediated immune response to EBV infection. The defective gene is located on the X chromosome. This condition accounts for half of the fatal cases of IM, the other half being sporadic with an equal age distribution. Clinically the affected male members are healthy until primary EBV infection occurs. The course of the disease can be either (i) fulminating and rapidly fatal, or (ii) progress on to a chronic phase which may culminate in a fatal B-cell lymphoproliferative disease, variously described as immunoblastic lymphoma, plasmacytoma and American Burkitt's lymphoma. These tumours often occur in the CNS or GI tract.

Many abnormal laboratory findings have been reported in X-LPS, including defects in NK cell activity and a defect in EBV specific T cell cytotoxicity. The pattern of antibodies to EBV antigens is also abnormal, with high titres of anti-VCA and EA antibodies and low EBNA antibodies. This pattern is identical to that seen in many immunosuppressed states and probably reflects increased viral replication leading to raised VCA and EA with decreased killing of infected cells resulting in low anti-EBNA titres. Female carriers show a milder derangement of antibody pattern but with normal EBV specific T-cell killing.

2. Transplant Recipients

Post Transplant Lymphoproliferative Disorder (PTLD) is thought to be a lymphoproliferation of EBV infected B-cells arising in the setting of over immunosuppression. The patients at risk are those who encounter EBV as a primary infection during the post-transplant course. The proliferation may be seen anywhere lymphoid tissue presides, although in lung transplant recipients, presentation in the allograft is relatively common. Histopathological manifestation appears as nodular sheets of atypical lymphoid cell which are not dissimilar to Non-Hodgkins lymphomas. Some cases are similar to lymphomatoid granulomatosis or T-cell rich B-cell lymphomas with a large subset of reactive T-cells. Reduction in immunosuppression often results in regression of PTLD.

It is well known that there is an increased incidence of malignancy following organ transplantation and maintenance immunosuppression. Malignant lymphoma accounts for 20% of such malignancies. Lymphomas have been reported to develop in 1 - 13% of patients following renal transplantation, 7.3% of cardiac transplants. Post transplant lymphomas have also been reported in bone marrow and heart/lung transplant recipients. The tumours tend to occur in the CNS, the GI tract, or in the transplanted organ. Most can be shown to carry the EBV genome and the cells express EBNA. High titres of antibodies to EA and VCA are present, which suggests increased virus replication. In around 50% of those developing EBV-associated lymphoma, there is evidence of recent primary infection.

It has been shown that the immunosuppressive therapy following transplantation reduces the CMI to EBV. Indeed, cessation of immunosuppressive had been accompanied by the regression of the lymphomas in some cases. Lymphomas in the immunocompromized are usually of the B cell type and classified as non-Hodgkin’s large cell lymphoma. They are usually extranodal, in the brain, gut and the transplanted organ and arise in 1-10% of transplant recipients. Around 80% of these tumours carry EBV genome and express EBNA. The large cell lymphomas express the same antigens as the lymphoblastoid cell lines, including the 2 viral oncogenes and cd 23. The majority of these tumours are monoclonal in nature.

AIDS

EBV-associated tumours occur frequently in patients with AIDS. Lymphomas are the second commonest malignancy in AIDS patients. However, not all lymphomas in AIDS patients are associated with EBV. The EBV associated tumours are (i) primary lymphoma of the brain and, (ii) Burkitt's lymphoma (50% of these tumours are similar to the African type and associated with EBV, whilst the other 50% are EBV genome negative). Recently, oral hairy leukoplakia has been described in HIV- seropositive individuals, forming multiple characteristic lesions on the lateral side of the tongue. DNA hybridization studies have shown active EBV replication in these lesions. In infants with AIDS, a lymphocytic infiltration of the lungs termed chronic interstitial pneumonitis in which the lymphocytes carry the EBV genome. However, it is thought that the syndrome is probably due to the direct action of HIV rather than EBV.
   

Laboratory_Diagnosis of EBV Infections

1. Infectious Mononucleosis - the classical finding in IM is the presence of atypical mononuclear cells in the blood. Lymphocytes would account for more than 50% of leucocytes present and of these, 20% are atypical. Atypical lymphocytes may also be seen in infections by CMV, hepatitis, influenza B, and rubella, but they are most prominent in IM. Abnormal LFTs are also present in the majority of patients. The diagnosis of IM may be suspected on clinical grounds and the findings of atypical lymphocytes, However, a firm diagnosis can only be made on serological testing. 2 types of serological tests are generally used for the diagnosis of IM.

The Heterophil antibody test is commonly called the Paul-Bunnel test, or the Monospot test (if done on slides). This test detects an antibody which causes agglutination of red blood cells from another species. False negative heterophil Ab test results occur and are more common in children under 14 years, especially under the age of 5 years. A possibility is that priming exposure to the unknown "heterophil antigen" has not taken place in very young children and thus no secondary response will arise on polyclonal stimulation. False positive heterophil Ab results are fewer in number than false negative results. Positive heterophil Ab results may last more than 6 months after the onset of IM and can occur in asymptomatic primary infection. This may be responsible for some of the "false" positive results.

IgM to VCA by indirect immunofluorescence is the best serological test available for the diagnosis of IM. However, this test is time consuming and results may vary between different laboratories. Also false positive results in the IgM may result from the cross linking between EBV specific IgG and anti-IgM conjugate by rheumatoid factor. If rheumatoid factor is present in the serum, it should be absorbed with staphylococcal protein A before testing. For these reasons, most laboratories rely mainly on the heterophil antibody test. A pre- illness specimen is rarely available to demonstrate a rise in IgG antibodies to VCA. High levels of VCA IgG are not diagnostic of acute infection. VCA IgM is generally used in the diagnosis of acute infection and IgG as an immune status screen.

Testing for EBNA antibodies may be of use in the window period. Anti-EBNA-1 antibodies do not usually arise until convalescence. Anti-EBNA-2 antibodies arise earlier in the illness and fall to low or undetectable levels during convalescence. The absence of EBNA- antibodies should not be regarded as diagnostic for IM as they are often undetectable in chronic IM, and conversely, they may be present soon after the onset of IM. Another possible confirmatory test is EBV-IgG avidity. The elution principle (avidity-index) is generally used for VCA-IgG.

2. Chronic IM - signs and symptoms of chronic IM range from fever, Pharyngitis, malaise, myalgia, and lymphadenopathy to potentially life-threatening problems such as anemia, thrombocytopenia, hupoglobulinaemia, and pneumonitis. Onset of chronic IM follows acute IM and may be due to impaired cell-mediated response to the virus. To meet the criteria for a diagnosis of chronic IM, three conditions need to be satisfied; (1) the symptoms should have persisted for at least 12 months (2) onset of persistent symptoms should have been preceded by a proven case of IM; (3) there should be evidence of active EBV infection . Highly elevated VCA and EA antibodies are often observed (>1024), EBNA-2 antibodies are frequently higher than those of EBNA-1 (the reverse of the situation is found in normal seropositive individuals), a positive heterophil antibody result may be seen, and more rarely, anti-VCA IgM may be detected. Similar antibody profiles have been observed in patients suffering from post-viral fatigue syndrome. It may be that some PVFS patients were, in fact suffering from chronic IM.

3. Burkitt’s Lymphoma - histology of biopsy specimens should reveal a poorly differentiated lymphocytic lymphoma. The tumour can be stained with antibodies to lambda light chains which should reveal a monoclonal tumour of B-cell origin. In over 90% of cases, the cells express IgM at the cell surface. The presence of EBV in tumour cells can be demonstrated by hybridization or the detection of EBNA-1. However, both these methods are technically demanding and therefore a diagnosis of BL is usually made on clinical and histological grounds. Children with BL have highly elevated titre of antibodies to EBV which may decrease following treatment and remission. Therefore, the determination of antibody levels may have a role in the monitoring of treatment. Although EBV serology might be of value in the early diagnosis of BL, such monitoring is not feasible on financial and practical terms.

4. Nasopharyngeal Carcinoma - the diagnosis of NPC is usually made on histological examination of biopsy material. However, the presence of EBV DNA and EBNA-1 can be readily demonstrated. Serum antibodies to EBV antigens can be used to confirm the diagnosis and monitor the progress of the disease, especially serum IgA to EA, VCA and ENA-1.. Recent studies have demonstrated the value of testing for persistent high levels of serum IgA to VCA in screening for early lesions of the disease and also for monitoring treatment.

5. X-linked Lymphoproliferative syndrome - the diagnosis of XLPS is much the same as for IM. In addition, EBNA-positive infiltrating lymphocytes can often be detected in post mortem or biopsy material. Serological studies of XLPS families commonly reveal a carrier state in healthy female relatives as evidenced by elevated EA and/or VCA titres. In such cases, genetic counseling may be given.

6. Post transplant lymphomproliferative disease - it is possible to detect EBV-DNA and EBNA in most of these lesions. The demonstration of EBNA positive cells is probably the most suitable method. Unlike BL and NPC, other latent antigens, ie. all the EBNAs and LMPA are expressed. The majority of EBV-associated post transplant lymphoproliferative lesions appear to occur following primary EBV infection. Serological diagnosis of primary infection is usually made retrospectively on sera taken for other purposes, as these patients rarely exhibit symptoms of IM. It may be possible to demonstrate a seroconversion. Patients who were seropositive prior to the transplant may have an antibody profile suggestive of reactivation.
   

VACCINE_DEVELOPMENT

A vaccine against EBV which prevents primary EBV infection should be able to control both BL and NPC. Such a vaccine must be given early in life. Such a vaccine would also be useful in seronegative organ transplant recipients and those developing severe IM, such as the male offspring of X-linked proliferative syndrome carriers. The antigen chosen for vaccine development is the MA antigen gp 340/220 as antibodies against this antigen are virus neutralizing. Inoculation of cotton top tamarins by purified gp 340/220 was able to protect the animals by subsequent virus challenge.
 

Herpesviruses Slide Set