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1 Original article Atypical expression in the detection and quantification of Epstein-Barr virus using real-time PCR Israel Parra-Ortega, Briceida López-Martínez, José Luis Sánchez-Huerta, Armando Vilchis-Ordóñez, and Leticia Barrera Dávila Abstract Background. Epstein-Barr virus (EBV) viral load is useful not only for detection of an active infection but also as a tumor marker for certain malignant forms. In immunocompromised patients it is related to posttransplant lymphoproliferative syndrome (PTLS) with an incidence on the order of 1 to 20%. It is recommended to determine viral load in whole blood and plasma to monitor patients at risk for developing PTLS. Real-time polymerase chain reaction (RT-PCR) is a useful tool for this determination. In these determinations the melting temperature (Tm) plays an important role because changes in Tm suggest that the target sequence has suffered mutations, although the selected regions for detection and quantification of EBV are highly conserved. We undertook this study to describe the findings of the variations identified in the EBV genome and to perform the detection and quantification in samples of pediatric patients using RT-PCR. Methods. Results from 352 pediatric patients were analyzed retrospectively in whom investigation of the EBV was performed in peripheral blood and plasma by RT-PCR. For detection and quantification of EBV, a 166-bp fragment of the genome was amplified using a design of TIB MOLBIOL and the LightCycler equipment with a Tm of 68ºC. Results. Of the 352 patients studied, in 132 (37.5%) presence of EBV was detected and quantified the viral load. In five (3.8%) of the positive patients, a change of the Tm was identified Using electrophoresis running in agarose gel, it was proved that the obtained amplification corresponds to the 166-bp fragment. Conclusion. The specific product and size of the amplified remained unchanged; therefore, we there is a high probability of decrease in the concentration of guanine-cytosine in the target sequence because the Tm showed a decrease in all the reported cases. It is required the sequence of the amplification is required to precisely determine the cause of the decrease in the Tm. Key words: viral load, immunocompromised patients, posttransplant lymphoproliferative syndrome, melting curve, Epstein-Barr virus. Laboratorio Clínico, Hospital Infantil de México Federico Gómez, México, D.F., México Received for publication: Accepted for publication: Introduction Epstein-Barr virus (EBV) was discovered by Burkitt in 1950 after studying a form of cancer affecting the jaw of children and adolescents of equatorial Africa. Subsequently, in 1964, Epstein, Barr and Achong examined biopsies of excised tumors and found a virus similar to the particles of the herpes virus and established that this virus (EBV) was the cause of these tumors

2 Parra OI et al. EBV are large viruses ( nm in diameter) with a nucleocapsid with icosahedral symmetry surrounded by an outer lipid-containing shell. Its genome consists of a double-stranded DNA molecule of kbp and encodes for ~100 proteins. The DNA molecule is flanked on both ends by a variable number of terminal repetitions each ~500 bp in length. 2 EBV is a virus that is widely distributed worldwide, and it is known that >90% of the world population is infected by it. 3 The virus enters a host and causes the infection, generally as a result of oral transmission with someone who is a carrier. 4 When the EBV infection presents during childhood, it is generally not clinically appreciated. However, if it occurs during adolescence or later, it may cause infectious mononucleosis. In immunocompromised individuals, EBV has been related to malignant diseases such as Burkitt s lymphoma, nasopharyngeal carcinoma, non-hodgkin s lymphoma, and posttransplant lymphoproliferative syndrome (PTLS) associated with EBV. 3 During the infection, EBV mainly infects two types of cells epithelial cells and B lymphocytes although under certain circumstances it may also infect T lymphocytes, natural killer cells, smooth muscle cells and astrocytes. 4-6 Initially, EBV fuses with B cells by interaction with glycoprotein gp350/220, predominantly in its membrane with the complement receptor type 2 (CR2/CD21). 7,8 Fusion with the cellular membrane and invasion of the host cell is facilitated by the fusion of a second viral glycoprotein, gp42, to class II MHC molecules and by the addition of three additional viral glycoproteins (gb, gh and gl) B cells infected by the EBV can express four different cycles of the genes and the cycle will depend on the status of the differentiation and localization of the infected B cells. One of these cycles is used to produce infectious viruses; the other three are associated with latent infection and do not produce infectious viruses. 4 EBV causes a latent infection with sporadic reactivations because the greater part of the population of infected human B lymphocytes maintain a latent state of replication and viral genetic expression; few enter a productive phase of the virus unless the individual is immunocompromised. When this happens, the virus colonizes epithelial cells of the nasopharynx where it establishes its lytic cycle of replication, causing a localized inflammatory response that produces a pharyngeal exudate. In this manner the virus is carried lymphatically towards the local lymph nodes, which causes a viremia that develops local and generalized lymphadenopathy as well as splenomegaly. The pathogenic mechanisms by which EBV is capable of inducing the appearance of tumors is related to the codified proteins by some of the genes expressed in the latent infection. These products induce different effects on the cell. EBVinfected B cells can express a well-defined group of latent genes including six nuclear antigens (EB- NAs) and three latent membrane proteins (LMP1, LMP2A, LMP2B). Of these latency proteins, LMP1 is considered the strongest oncoprotein and is essential for the immortalization of B cells as well as sharing functional properties with members of the superfamily of receptors of tumor necrosis factor, particularly CD40. 4,12 The disease associated with greatest frequency to EBV in children who have undergone transplants is PTLS, which is the neoplastic proliferation of B cells in immunocompromised individuals. The incidence of PTLS is found on the order of 1-20%. PTLS is an entity that has a broad spectrum of clinical presentation ranging from localized to systemic forms including B-cell lymphoma. Among the risk factors for the development of PTLS are the combination of donor seropositive/recipient seronegative, having received anti-lymphocyte globulin for induction of immunosuppression and treatment with OKT-3 for early rejection. 13,14 Clinical laboratories have increased the use of viral load as a tool for the diagnosis and monitoring of virus-related illnesses. EBV viral load not only serves to detect an active infec- 414 Bol Med Hosp Infant Mex

3 Atypical expression in the detection and quantification of Epstein-Barr virus using real-time PCR tion, but also serves as a tumor marker for some malignancies. Some publications recommend determination of the total EBV viral load in the blood and peripheral blood plasma in order to provide follow-up for PTLS patients. However, there are no cutoff values after which development of PTLS can be predicted. Also, there is no information regarding the possibility of developing EBV disease when an active infection is detected because not all patients who suffer an active infection develop PTLS. 14,15 According to these determinations, the melting temperature (Tm) plays an important role because it serves to identify the product of the polymerase chain reaction. Tm changes in this sequence suggest that the target has mutated even though the selected genes for the detection of EBV are highly preserved. The aim of this investigation is to describe the findings of the variations identified in the EBV genome when performing detection and quantification in pediatric patient specimens using RT-PCR technology. For EBV detection and quantification, a 166-bp fragment of the genome was amplified using a design from TIBMOL Biol and LightCycler equipment. Also, as an internal positive control a set of primers and a probe that amplifies a 278-bp fragment was used. The reaction mixture was brought to a final volume of 20 ml using 5 ml of DNA extracted from each of the samples. The reaction mixtures were subjected to a cycle of sample denaturation and activation of the enzyme at a temperature of 95 C for 10 min, followed by 50 cycles of amplification for 5 sec at 95 C, 10 sec at 60 C, 15 sec at 72 C. Then a melting curve is used to identify the PCR product derived from the DNA of EBV, with a program of 1 cycle for 20 sec at 95 C, 20 sec at 40 C and 10 sec at 85 C with an increase in temperature of 0.2 C/sec and in continuous fluorescence acquisition mode. The amplification was designed to have a specific melting temperature (Tm) of 68 C. In all cases, the manufacturer s instructions were followed. Subjects and Methods A retrospective analysis was done of the results of 352 pediatric patients. EBV detection and quantification in peripheral blood and plasma by means of RT-PCR was analyzed. Three ml of EDTA anticoagulated peripheral blood was used. An aliquot of 400 ml of whole blood was separated. The remaining sample was centrifuged at 3000 x g for 10 min and an aliquot of 400 ml of plasma was taken. For DNA extraction, a Pure Great Compac computer with a set of reagents (Magna Pure Compact Nucleic Acid Isolation Kit I, Roche Molecular Diagnostics) was used. Cycles for DNA extraction from 400 ml of whole blood with an eluted volume of 200 ml and a total nucleic acid cycle from 400 ml of plasma with an eluted volume of 100 ml were used. Results Of the 352 patients studied, we detected the presence of EBV in 132 (37.5%) patients and the viral load was quantified. Amplification curves are shown in Figure 1, including the six points of the quantification curve ( ) of five samples, of which three had positive results, two negative results and the negative control. In five patients, equivalent to 3.8% of positive patients, a change in Tm was identified: a patient with a Tm of 57.4 C (Figure 2a), two patients with 61.5 C (Figure 2b), one with 62 C and another with 62.5 C. Quantification of EBV in these patients with decreased Tm was performed twice and at different times, observing the same behavior, confirming that they are findings of the EBV and not the analytical process. 415

4 Parra OI et al. Also, an electrophoretic shift in agarose gel was done, and it was found that the amplified fragment obtained corresponds to the expected size of the EBV (166 bp) and for the internal positive control a fragment of 278 bp, which is useful to demonstrate that there are no PCR inhibitors in the reaction mixture (Figure 3). The specific product is obtained with the electrophoretic shift, and the amplified size does not show variations. According to previous results, we may conclude that there is a high probability of decrease in the concentration of guaninecytosine in the target sequence because the Tm Este experienced documento a decrease es elaborado of 10.6 C por Medigraphic in one case, 6.5 C in another two cases, and 6 C and 5.5 C in two more cases, with respect to the expected Tm. However, it is necessary to sequence the amplifications to precisely determine precisely the cause of decreased Tm. This finding raises several questions that we hope to clarify in the future: Amplification curves Fluorescence (640) MP 10 6 MN Cycles Figure 1. Amplification curves. Quantification curve with concentrations ranging from 10 6 to NC, negative control; PS, positive samples; NS, negative samples. -(d/dt) Fluorescence (640) Melting peaks Melting peaks a 57.4 C b 61.5 C (d/dt) Fluorescence (640) Temperature o C Temperature o C Figure 2. Curves showing the specific melting temperature (Tm). (a) Standard 10 3 quantification curves with a Tm of 68 C of a patient with a Tm of 57.4 C and negative control (NC). (b) Standard 10 3 quantification curves with a Tm of 68 C, two patients with a Tm of 61.5 C and negative control (NC). 416 Bol Med Hosp Infant Mex

5 Atypical expression in the detection and quantification of Epstein-Barr virus using real-time PCR MP EST ) Is the change of Tm caused by a decrease in the concentration of guanine-cytosine in the target sequence? ) Does this change have any effect on EBV pathogenesis? 3) Does it have any clinical impact in patients infected with EBV? 100 Figure 3. Electrophoretic shift in agarose gel. MP, molecular weight marker 100 bp; EST. Amplified from the point on the curve Amplified from the five samples with lower Tm. Correspondence to: M. en C. José Luis Sánchez Huerta Laboratorio Clínico Hospital Infantil de México Federico Gómez México, D.F., México jlshuerta@yahoo.com.mx References 1. Kieff E, Rickinson AB. Epstein-Barr virus and its replication. PA: Lippincott Williams & Wilkins; p Rickinson AB, Kieff E. Epstein-Barr virus and its replication. PA: Lippincott Williams & Wilkins; p Young KA, Herbert AP, Barlow PN, Holers VM, Hannan JP. Molecular basis of the interaction between complement receptor type 2 (CR2/CD21) and Epstein-Barr virus glycoprotein gp350. J Virol 2008;82: Thorley-Lawson DA. Epstein-Barr virus: exploiting the immune system. Nat Rev Immunol 2001;1: Rickinson AB, Kieff E. Epstein-Barr virus and its replication. PA: Lippincott Williams & Wilkins; 2001; pp Menet A, Speth C, Larcher C, Prodinger WM, Schwendinger MG, Chan P, et al. Epstein-Barr virus infection of human astrocyte cell lines. J Virol 1999;73: Fingeroth JD, Weis JJ, Tedder TF, Strominger JL, Biro PA, Fearon DT. Epstein-Barr virus receptor of human B lymphocytes is the C3d receptor CR2. Proc Natl Acad Sci USA 1984;81: Frade R, Barel M, Ehlin-Henriksson B, Klein G. gp140, the C3d receptor of human B lymphocytes, is also the Epstein-Barr virus receptor. Proc Natl Acad Sci USA 1985;82: Haan KM, Lee SK, Longnecker R. Different functional domains in the cytoplasmic tail of glycoprotein B are involved in Epstein-Barr virus-induced membrane fusion. Virology 2001;290: Haddad RS, Hutt-Fletcher LM. Depletion of glycoprotein gp85 from virosomes made with Epstein-Barr virus proteins abolishes their ability to fuse with virus receptor-bearing cells. J Virol 1989;63: Hutt-Fletcher LM. Epstein-Barr virus entry. J Virol 2007;81: Terrin L, Dal Col J, Rampazzo E, Zancai P, Pedrotti M, Ammirabile G, et al. Latent membrane protein 1 of Epstein-Barr virus activates the htert promoter and enhances telomerase activity in B lymphocytes. J Virol 2008;82: Kogan DL, Burroughs M, Emre S, Fishbein T, Moscona A, Ramson C, et al. Prospective longitudinal analysis of quantitative Epstein-Barr virus polymerase chain reaction in pediatric liver transplant recipients. Transplantation 1999;67: Green M, Cacciarelli TV, Mazariegos GV, Sigurdsson L, Qu L, Rowe DT, et al. Serial measurement of Epstein-Barr viral load in peripheral blood in pediatric liver transplant recipients during treatment for posttransplant lymphoproliferative disease. Transplantation 1998;66: Patel S, Zuckerman M, Smith M. Real-time quantitative PCR of Epstein-Barr virus BZLF1 DNA using the LightCycler. J Virol Methods 2003;109: