Alternatively we have used both purified proteins, synthetic peptides and recombinant proteins. GP125 has been used purified from P3HR1 line with which it has obtained 95% sensitivity and specificity of 100%.

In regard to the use of synthetic peptides, was applied to p62, whose sequence corresponds to a region of EBNA1, to develop a test to differentiate IgM and IgG responses.

The test however suffered from a lack of sensitivity when compared with benchmarks, in fact its sensitivity is very similar to tests that measure AH.

Very recently, trials have used synthetic peptides VAC, p18 or a fraction thereof of 56 amino acids, which have the immunodominant regions of the capsid antigen of these tests have only limited information but very satisfying in terms of correlation between serology and clinic patients.

There are ELISA tests that detect IgG antibodies to EBNA (using the recombinant p72 and p58). and IgG, IgM and IgA against EA, EA employing recombinant (p54 and p138) with values of sensitivity and specificity of 94 and 100% respectively which enable the diagnosis of MI when used together as a profile.

On the other hand, has developed a compact ELISA used as antigen a mixture of VCA, EA and EBNA, controlled with monoclonal antibodies directed against p58, GP125, and p72 IgM detection with this assay shows a sensitivity of 100% and a specificity of 89%.

With these techniques in the course of primary infection occurring anti-VCA responses, both IgM and IgG isotype, in a virtually simultaneous. In most cases reach the peak in the time symptoms appear, or a few days later, so in most cases it is possible to detect IgG seroconversion. Between 2 and 3 months after onset of illness, the IgM response falls to undetectable levels, while IgG remains at good levels throughout the life of the individual.

A significant proportion of patients (about 85%) developed IgG against early antigen diffuse component (EA-D), whose peak was reached a few days after onset, and usually disappears within a few months. Antibodies to EBNA appear weeks or even several months after the onset of the disease.

Thus, the profile of antibodies against primary infection by EBV is characterized by the presence of IgG and IgM responses against the capsid antigen in the absence of anti-EBNA. This profile is presented in more than 90% of cases of IM caused by EBV.

In a few patients displayed abnormal patterns with early onset of anti-EBNA and others is not possible to detect the IgM response. In cases where no typical profile obtained is very useful the tests for the characterization of specific IgG avidity.

In recent years methods have been developed for IgG and IgM anti-VCA and IgG anti-EBNA, based on the use of recombinant proteins. When compared the IFIs and conventional techniques using recombinant proteins for detection of IgM anti-VCA some outlying result is obtained because the cell lines used in IFI express not only AC but some other antigens against which also IgM response occurs.

The fluorescence assay for anti-EBNA with recombinant antigen has the advantage of using the technique of IFI, easier development of IFAC. A novel alternative is to apply a test of IFAC on a cell line that expressed both EBNA and VCA and EA. The method offers the advantage of allowing a single determination on a single well, to establish whether it is an acute infection, past or absent depending on the pattern of fluorescence obtained.

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Specific serological response

EBV has a complex antigen. Are three classes of antigenic systems: the latent phase antigens, antigens early replicative and late antigens. Not all are important for diagnosis. Among the former, there are the EBV nuclear antigens (EBNA1 and 2). Despite being the first to receive the antibody response to EBNA-1, which occurs in all infected individuals, is a late marker of infection. Among the early antigens (EA) and depending on your location there are two types: diffuse (EA-D in the nucleus and cytoplasm) and restricted (EA-R, only in cytoplasm).

The response of anti-EA is not universal and indicates that the cell has entered a lytic cycle and producer. Finally, among the late ones, the antigen of the virus capsid (VCA) is expressed equally abundantly in the lytic and productive infection, and induces a response of the IgM isotype in the primary disease that lasts 2-3 months, and IgG isotype , which remains detectable for life. 

Techniques and antigens used in indirect diagnosis.

To characterize specific serological, considered the reference methods were the techniques of immunofluorescence (IFAT) for IgG and IgM anti-VCA, and IgG anti-EA, and anticomplement (IFAC) for anti-EBNA. Indirect methods are developed on slides containing cells almost exclusively express antigens of interest.

In the case of AC cell line is most often used P3HR1 for IgG anti-EA line is used to optimize chemically induced Raji antigen expression by inhibiting the production of virions .. To avoid interference resulting from the presence of rheumatoid factor, measurements of anti-VCA IgM should be performed after removal of IgG in the sample, for which the most commonly used procedure is the use of an anti-human IgG antiserum.

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In general, the best procedure for laboratory diagnosis of viral infections is isolation of the virus or any of its components or products. While acknowledging cell types naturally susceptible to infection by EBV in vitro is reduced to B cells, which are processed in a technologically complex and slow process, so that isolation is an imp procedure for most laboratories diagnostics.

The identification of either virus antigens has proved an adequate approximation. Finally, the detection of HBV DNA by the chain reaction of polymerase has shown a good performance comparative serological studies for the diagnosis of mononucleosis in the acute phase.

INDIRECT DIAGNOSIS

Given the drawbacks of direct diagnosis, EBV infections are made fundamentally by serology.

Heterophil antibody

Since long before the description of the virus and its relationship with infectious mononucleosis, applied the Paul-Bunnell test for heterophil antibody identification (AH). These are IgM antibodies directed against antigens present on the surface of erythrocytes of different species and occurring in more than 80% of adult cases and less frequently (less than 50%) in children. However, and despite not correspond to a specific response against the virus, they are fairly specific for the disease caused by the virus.

Currently different approaches are used to detect HA, from the classical agglutination of erythrocytes from cows, sheep or horses, after differential absorption with guinea pig kidney extract until ELISA or agglutination of latex particles sensitized with membrane antigens of bovine erythrocytes, which offer greater ease in interpreting results. All have similar performance. There are some studies indicating that agglutination methods are more sensitive than immunoassays with a specificity equivalent.

The incubation period of about a month. After a prodromal period, characterized by chills, sweating, fever and malaise, the disease occurs, which in its most typical form includes the triad of sore throat, fever and lymphadenopathy.

They also appear frequently hepatosplenomegaly and rash. Most cases remits spontaneously within 3 to 4 weeks, although fatigue can last a little longer.

There are some important neurological complications (meningoencephalitis and Guillain Barre syndrome), laryngeal obstruction or rupture of the spleen. In most cases the recovery is complete by symptomatic treatment.

Since the virus immortalizes and transforms cells in culture, has always suspected the involvement of this agent in Endemic Burkitt’s lymphoma. This lymphoma is endemic in some regions of Africa has in its cells virions and viral DNA sequences. It was thought that act by activating the EBV oncogene c-myc, the presence of a gene in the virus itself.

Recently it is suspected that the role played by this virus is that of cofactor as in sporadic LB sequences are not viruses. It appears that infection with Plasmodium, which causes a disturbance important role “caretaker” of T cells may be the main factor for the development of this tumor in these endemic areas.

The characteristic serological response in patients with nasopharyngeal carcinoma Undifferentiated also been associated with this virus. LBE Unlike the cells in this tumor, also endemic in the Far East are of epithelial lineage. Again we think that there is also another factor in the development of this tumor. Phorbol esters, a widely used medicinal herb may be the cofactor.

There is increasing evidence that immunosuppression or impaired function of T cells promotes the occurrence of lymphoproliferative disease in EBV-infected patients. Hairy leukoplakia in HIV-positive patients is one example.

Chronic Fatigue Syndrome (CFS) with chronic fatigue, fever, muscle weakness and inability to concentrate has tried to link with this virus. The only evidence of this association is at a certain elevation of antibody titers against the virus. Also described this syndrome in association with other viruses and the importance that the patient gave his disease. Therefore this association is unclear.

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EBNA1 is required for that automantenga DNA in lymphocytes that are activated and allows the virus to remain LPM2 limiting latent gene expression in the membrane. This minimal expression of the repertoire of viral proteins allows the virus to minimize the number of targets for the immune system.

Another defense mechanism is BCRF1 gene expression that produces a protein, EBV IL-10, similar in 80% of interleukin 10 (IL-10). Thus, the virus inhibits the production of interferon and synthesis of IL-1 and IL-12, thus altering the differentiation of B lymphocytes. The third mechanism that can be used for persisting EBV is its ability to decrease the production and expression of HLA I and also of “carrier proteins” in the infected cells also appears that EBNA1 can inhibit the mechanism of processing of viral antigens. This will achieve a reduction in antigen expression on cell membranes and protection against the destructive power of T8 lymphocytes.

Epidemiology

It is a widely distributed virus, estimated that about 90% of adults have been infected. It is thought that 70% of the population is infected before age 30. The seroepidemiology has shown that in underdeveloped countries asymptomatic infection in early life is more often while on the contrary, in countries with better standard of living many individuals escape from primary infection until adolescence . When primary infection is delayed virus tends to cause symptoms.

When infection occurs cell begins production of viral proteins. These proteins include early antigen (EA), the capsid (VCA) and membrane glycoproteins (MA).

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Viral Pathogenesis and Persistence

The virus is transmitted by infected saliva and reaches the oropharynx epithelial cells where it replicates in production of virions and cell lysis. B cells are infected as they pass through the oropharynx or the epithelium of the postnasal space. The virus uses the cell to contact one of its envelope protein, gp350, which binds to the cellular receptor CD21 (the same that has for the C3d of complement).

Most of the antibodies produced during infection are directed against this protein and have existed since it attempts to develop a vaccine against this infection. In the acute phase only a small number of B cells with viral replication allows expression of all viral antigens, virion formation and ultimately cell lysis. Most of them express only a limited number of genes and do not allow at this time viral replication (latent infection).

Sometime during this latent phase among these cells can enter the activity and allow a full replicative cycle. Acutely infected cells are controlled by NK and T cells that proliferate in large numbers, this increase is responsible for cell enlargement of lymph nodes, spleen and liver that can be seen in the acute phase of infection . In the convalescence phase and latency is the latter the most important mechanism for monitoring and control.

The Epstein-Barr virus (EBV) is a human herpes virus type 4, lymphotropic, providing for cells whose latent infection. It has been shown that this virus is mainly responsible for Infect mononucleosis (IM), a disease of adolescence and childhood.

It can also produce certain forms of cancer, including undifferentiated nasopharyngeal carcinoma (CNI), Endemic Burkitt’s Lymphoma (LBE) or B cell lymphomas in patients with acquired or congenital immunodeficiencies and there is great controversy over the role of this virus as a cause of chronic illness, in particular in regard to the Chronic Fatigue Syndrome (CFS).

Structure

Like every family is a large virus, encapsulated with a double-stranded DNA. It is about 150 nm.Its capsid is icosahedral with 162 capsomers and all the virus is enveloped by a cover with glycoproteins.

The space between the deck and the capsid, tegument, is full of proteins and viral enzymes. It is sensitive to acids, solvents, detergents, and drying. The genome consists of double-stranded linear DNA of different sizes. It has two sections, one long (UL) and one short (UC), each flanked by two groups of direct repeats of DNA (LR) as, unlike other types of herpes virus replicates with groups of indirect shows a single isomeric configuration.

Replication

The molecular processes of replication of herpes virus are regulated by viral and cellular factors. Schematically say that the viral protein synthesis takes place in three phases: 1) immediate early protein synthesis necessary for the primary synthesis of nucleic acids and other viral proteins, 2) synthesis of specific proteins and viral genome and 3 ) synthesis of late structural proteins and 4) the viral and cellular factors determine whether the virus causes a lytic infection, persistent or latent.

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