Gene EBNA3C: Type of Infection by EBV (EBV1 and EBV2) Correlation With Clinical and Biochemical Parameters (AST, ALT and GGT) in Individuals With Infectious Mononucleosis in the Metropolitan Area of Belém, Pará, 2005-2016

Background: Two types of EBV (EBV1 and 2) have been shown to infect humans. This study aimed to detect the types of EBV that cause infectious mononucleosis and correlate these viral types with biochemical parameters (AST, ALT and GGT) in the metropolitan region of Belém from 2005 to 2016. Methods: A total of 76 cases of infectious mononucleosis were processed at the Instituto Evandro Chagas, Ananindeua, Brazil. PCR was used to analyze the EBNA3C region for the recognition of EBV types. Biochemical testing (AST, ALT and GGT) was performed by the COBAS INTEGRA clinical biochemistry PLUS 400 / ROCHE automatic analyzer. The data were evaluated using the Statistical Package for Social Science - SPSS 17.0 and GraphPadPrism 7.0 for Windows (GraphPad software, San Diego, CA, USA). Results: EBV1 infection was observed in 71.1% (54/76) of individuals, among whom those > 14 years constituted 66.7% (36/54); the average age was 23 years, and the number of women infected was higher (61.1% 33/54) than that of men 38.9%21/54). The symptoms/clinical signs observed in infection by EBV1 were cervical lymphadenopathy in 64.8% (35/54), fever in 63% (34/54), headache and arthralgia in 20.3% (11/54), and exanthema in 18.5% (10/54). Infection by EBV2 was observed in only 17.1% (13/76) of cases. Coinfection by EBV1 and EBV2, most frequently showing symptoms of fever and cervical lymphadenopathy, occurred in 66.7% (6/9) and 55.6% (5/9) of individuals. Conclusion: EBV1 was predominant in 71% of clinical cases of infectious mononucleosis. The correlation of biochemical parameters in infection by EBV1, EBV2, and coinfection by EBV1/2 revealed a statistically significant difference in mean changes of EBV1

subfamily Gammaherpesvirinae, genus Lymphocryptovirus and species Human gammaherpesvirus 4 1. EBV was the first oncogenic virus found to infect humans 2,3. The hexagonal nucleocapsid viral particles were formed with linear, double stranded, enveloped DNA, with a diameter of 180 to 200 nm 4.
Although symptomatic infections with these viruses occur in benign form, EBV has been implicated in the genesis of a variety of lymphoproliferative disorders and severe epithelial neoplasms, such as African Burkitt's lymphoma 5, carcinoma and nasopharyngeal 6.
In Brazil, several studies have recorded the high frequency of antibodies in the studied populations. Studies conducted by Monteiro et al. (1998) found that at least 70% of the serum samples analyzed in the city of Belém, state of Pará, contain IgG antibodies to EBV, at the outpatient clinic level ranging from 53.8% to 95.6%, or in the community (81.1% to 100%) 7.
Positive indexes were expressive, even in the youngest lowest age groups. These results suggest that active and recent infection (infectious mononucleosis) was detected in 10.6% (25/234) of children and adolescents in northern Brazil 8.
Data from Young and Murray (2003) demonstrated that EBV is present in approximately 90% of individuals and is controlled by the immune system, mainly by cellular immunity, which may make the subject more susceptible to virus proliferation and may trigger lymphoproliferative disorders 9,10.
It is increasingly important to identify the epidemiological characteristics associated with the risk of EBV infection in populations to reduce the clinical conditions associated with possible morbidity and mortality 11.
The difference between the sequences encoding EBV nuclear antigens (EBNA2, 3A, 3B, and 3C) allows for the identification of different genotypes with distinct epidemiological characteristics 12.
According to Young et al. (2000), human and geographic assemblages can influence the distribution of EBV genotypes 1 and 2, which change with rates of detection of these viral genotypes in diseases associated with EBV 13. Type 1 EBV has a higher incidence in western regions, and type 2 is found more frequently in sub-Saharan Africa and New Guinea than in other parts of the world 14.
The phenotypic difference between EBV1 and 2 is more evident during immortalization of B cells in EBV1 lymphoblastoid cell lines (LCLs) compared to EBV2. This fact reinforces the biological and functional difference between the two viral types, where B cell immortalization in vitro was shown to be more effective by type 1 EBV 15. According to Mandell et al. (2001), differentiation of genotypes can clarify the different immune responses during viral persistence 16.
The metropolitan area of Belém still lacks studies to characterize the different types of circulating EBV (EBV1 and EBV2) associated with clinical and demographic characteristics (gender, age and origin) and molecular findings that describe the epidemiology of the infection caused by these viral agents due to its characteristics of viral persistence that can induce chronic infections and reactivations in human populations with genetic competence to possible oncogenic events.
The objective of this study was to detect the types of Epstein-Barr virus (EBV1 and EBV2) EBV1 infection was observed in 71.1% (54/76); the most frequent age group was >14 years old, with 66.7% (36/54) of the individuals. Since the age range was 23 years, the number of women was 61.1% (33/54), which was higher than in men, with 38.9% (21/54).
In the assessment of hepatic function, alterations to AST in EBV1 infection were confirmed in 14.8% (8/54) of cases with results above the reference values (5-40 U/L) and 7.7% (1/13) for EBV2. It is worth mentioning that the reference values are the same in the three age groups (Fig 2). When analyzing the mean values by age group, the range of >14 years was outside the normality limits with a higher frequency of lterations, thus presenting a statistically significant difference (p-value < 0.05). Values above the reference values (2-41 U/L) were also observed for ALT (Fig 3). The mean value was outside the limits of normality in the age groups of 6 to 14 years and >14 years, presenting a significant difference (p-value < 0.05) by the Wilcoxon test.

Fig 3. Evaluation of AST of individuals with EBV 1, EBV 2 and EBV 1 and 2 infection.
For the evaluation of GGT, some altered results were observed, but no changes were found in the age group of 2 to 5 years, given the reference values: (5 -55 U/L). Changes in the age group from 6 to 14 years were all above normal, given the same reference range of the previous age group, and the greatest number of alterations was observed in the range of >14 years, above or below, given reference values: (12 -43 U/L) (Fig 4). The mean value was outside the limits of normality in the age group of > 14 years, and the mean values presented a significant difference (p-value <0.05) by the Wilcoxon test.      Evaluation of GGT activity in individuals with EBV 1, EBV 2 and EBV 1 and 2 infection.