Epstein Barr virus genotypes (EBV1/EBV2) in individuals with infectious mononucleosis in the metropolitan area of Belém, Brazil, between 2005 and 2016

Background: Two types of Epstein Barr virus (EBV1/EBV2) have been shown to infect humans by causing infectious mononucleosis, their genome being very similar, except for regions of the EBNA genes. This study aimed to describe the EBV genotypes in cases of infectious mononucleosis in the metropolitan region of Belém, Brazil, from 2005 to 2016. Methods: A total of 8.295 suspected cases with symptoms/signs of infectious mononucleosis (MI) were investigated by doctors of infectious diseases at the Evandro Chagas Institute Health Care Service from January 2005 to December 2016. In 3.0% (251/8.295) of the cases were positive by enzymatic immunoassay were submitted to PCR for EBNA3C region to detect the types of EBV. Biochemical testing involving aspartate aminotransferase, alanine aminotransferase and gamma-glutamyl transferase were realized. Results: The identification of EBV types by PCR was verified in 30.3% (76/251) of individuals, being 71.1% (54/76) classified as EBV1, 17.1% (13/76) as EBV2 and 11.8% (9/76) as EBV1+EBV2. The number of women infected with EBV1 was higher (61.1% -33/54) than for men (38.9% - 21/54), most were over 14 years old (66.7%-36/54). The main symptoms/clinical signs observed in EBV1 infection were: cervical lymphadenopathy (64.8%-35/54), fever (63%-34/54), headache (20.3%-11/54), arthralgia (20.3%-11/54) and exanthema (18.5%-10/54). In EBV2 infection, it was also detected in all age groups, with the exception of two groups, with an average age of 24 years. The presence of fever in 76.9% (10/13) with an average duration of 18 days and lymphadenopathy in

enzymatic values according to type of EBV was statistically significant (p <0.05) in individuals with EBV1 infection in those over 14 years of age.
Conclusions: A pioneering study that molecularly identified the genotypes of EBV in 30 [1]. EBV was the first oncogenic virus described 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] and the nasopharyngeal carcinoma[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 rates were expressive, even in the younger age groups. These results demonstrated 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 person more susceptible to virus proliferation and may trigger lymphproliferative 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]. Cohen (2000) demonstrated that the difference between sequences encoding EBV nuclear antigens (EBNA2, 3A, 3B and 3C) allows the identification of different genotypes with distinct epidemiological characteristics [12].
According to Young et al. (2000) in the human populations, geographic and ethnic factors may influence the distribution of EBV1 and EBV2, changing the detection rates of these viral genotypes in diseases associated with EBV [13]. Type 1 EBV has been more prevalent in western regions, and type 2 is more often described in sub-Saharan Africa and New Guinea than in other parts of the world [14].
The phenotypic difference between EBV1 and EBV2 is more evident during immortalization of B cells in lymphoblastoid cell lines (LCLs) by EBV1 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 EBV1 [15]. According to Mandell et al. [16] differentiation of genotypes can clarify the different immune responses during viral persistence.
Studies to characterize the circulation of EBV1 and EBV2 types in relation to epidemiological data caused by these agents, such as clinical, demographic (gender, age and origin) and molecular findings in the metropolitan region of Belém, are still lacking.

Laboratory analysis
Serum samples were collected and tested for EBV using an immunoenzymatic assay as well as for biochemical dosages analyses. Peripheral blood mononuclear cells (PBMCs) were separated from the blood sample by Ficoll-Hipaque density gradient centrifugation (Lymphoprep Nycomed Pharma AS, Norway) for use in future PCR tests.

Immunoenzymatic assay (EIA)
EBV screening in samples was carried out using the RIDASCREEN® enzyme immunoassay kit (R-Biopharm, Darmstadt, Germany) which detects IgG and IgM antibodies to viral capsid antigen (VCA), according to manufacturer's guidelines.

Nucleic acid extraction
PBMC DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germantown, MD) according to the manufacturer's protocol.

Identification of the EBV EBNA3C gene
To identify both types of EBV, the primer EBNA-3C was used [17], which due to the primary flanking region can differentiate them by the resulting product: EBV1 153 base pairs (bp) and EBV2 of 246 bp.

Statistical analysis
Results were organized and stored in a database in Microsoft Office Access 2016, Statistical Package for Social Science -SPSS 17.0. A p value less than 0.05 was considered significant [18].

Molecular detection
It was possible to identify by PCR the two types of EBV in 30.3% (76/251) of positive samples by EIA using EBNA-3C primer. The classification of these samples by age and sex showed that the age groups, >15-20 years and >30 years were slightly more affected (37,0%-p= 0.557) in relation of positivity by PCR (Table 1). Regarding gender, no difference was observed in males (30.6% -34/111) compared to females (30.0% -42/140).
Comparing the age of the positive cases with sex, it was found that the majority of cases occurred in males aged >30 (38.1%), however, in females it was higher at age >15-20 years (42.8%).
In the assessment of hepatic function, three age groups were used, as they are considered standard in the analysis of these biochemical parameters. Changes in AST in EBV1 infection were confirmed in 14.8% (8/54) of the cases, with results above the reference values (5-40 U/L) with 19.4% (7/36) age >14 years, and for EBV2 it was 7.7% (1/13), with the only case occurring in the same age group (14.2% -1/7).
For ALT, a value above the reference (2-41 U/L) was also found at age >14 for EBV1 (33. 3%-12/36), showing a significant difference (p-value < 0.05) by the Wilcoxon test, as previously observed for AST at this age.
Regarding EBV2, it was detected in 28.9% (22/76) of the positive EBV cases, with 17.1% (13/76) alone and 11.8% (9/76) co-infected with EBV1 (Table 1). This value was higher than those described by Deng Smatti et al., 2017 [31]. It is important to mention that the positivity observed by EBV2, in the age group > 10-15 was very higher (75%-6/8), when compared with EBV1 (37.5%-3/8). However, these cases were detected in different months of these years. The presence of multiple infections by EBV1 + EBV2 was observed in 11.8% of the positive cases of infectious mononucleosis, where 20.0% (2/10) occurred in children less than five years old (Table 1). This fact emphasize that co-infections are not exclusive to immunocompromised individuals. This association was also verified by Correa  Another finding that stood out in the present investigation was the occurrence of alterations in transaminases (aspartate aminotransferase -AST, alanine aminotransferase -ALT and gamma-glutamyl transferase -GGT), that varied from 19.4-41.7% (Table 2)