Comparison of Epstein–Barr virus and Kaposi’s sarcoma-associated herpesvirus viral load in peripheral blood mononuclear cells and oral fluids of HIV-negative individuals aged 3–89 years from Uganda

We previously found that age, sex and malaria were associated with KSHV in individuals from Uganda. In this study, we have evaluated these same factors in relation to EBV in the same specimens. Overall, 74% (oral fluids) and 46% (PBMCs) had detectable EBV. This was significantly higher than observed for KSHV (24% oral fluids and 11% PBMCs). Individuals with EBV in PBMCs were more likely to have KSHV in PBMCs (P = 0.011). The peak age for detection of EBV in oral fluids was 3–5 years while that of KSHV was 6–12 years. In PBMCs, there was a bimodal peak age for detection of EBV (at 3–5 years and 66 + years) while for KSHV there was a single peak at 3–5 years. Individuals with malaria had higher levels of EBV in PBMCs compared to malaria-negative individuals (P = 0.002). In summary, our results show that younger age and malaria are associated with higher levels of EBV and KSHV in PBMCs suggesting malaria impacts immunity to both gamma-herpesviruses. Supplementary Information The online version contains supplementary material available at 10.1186/s13027-023-00516-9.


Introduction
Among the known human herpesviruses, the gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are carcinogenic [1].EBV is causally associated with Burkitt's lymphoma, certain Hodgkin's and non-Hodgkin's lymphomas, nasopharyngeal and gastric carcinomas, while KSHV is the cause of Kaposi's sarcoma, multicentric Castleman's disease (MCD) and primary effusion lymphoma (PEL) [2,3].Of these malignancies, PEL cells are infected with both EBV (~ 80%) and KSHV (100%) [4].In sub-Saharan Africa, both EBV and KSHV infections are very common [5].Infection with both viruses occurs in childhood, with EBV infection occurring earlier than KSHV [6,7].Both EBV and KSHV have been shown to infect and establish latency in several B cell subsets [8,9].However, the interaction between the two viruses has not been extensively studied ex vivo.
Both EBV and KSHV viral load are accurately quantified using real time polymerase chain reaction (PCR) [10][11][12].Detection of both viruses in oral fluids is associated with transmission while detection in blood compartments is associated with disease [13].However, there are differences in the detection of KSHV and EBV in the different blood compartments.Unlike EBV, KSHV is rarely detected in plasma even among individuals with KS [14].On the other hand KSHV can be detected in PBMCs and correlates with KS, suggesting that KSHV is majorly cell associated [15,16].However, only about 50% of KS patients have detectable KSHV in PBMCs and much less in plasma [17].Partily because KS is a tumour of endotherial cells (spindal cells) which don't make part of the PBMCs, and also possibly KSHV infected lymphocytes are mainly in secondary lymphoid organs.KSHV genome copies in PBMC above 10 copies per infected cells represents viral lytic replication, while that below 10 copies per infected cells represents a purely latent infection [17].Therefore measurement of KSHV in PBMCs is very useful to identify individuals at risk of disease.EBV copies in plasma have been linked to disease and have been shown to better predict EBV disease compared to EBV in PBMCs; however, EBV copies in PBMCs are also increased in EBV associated disease [18,19].Therefore EBV in PBMCs could also be very usefull at identifying individuals at risk of disease.
We have previously shown the pattern of KSHV viral load in oral fluids and PBMCs and associated risk factors across a wide age range in HIV-negative individuals from a KSHV endemic area [20].Furthermore, in a Cameroon KS case-control study, we have shown EBV and KSHV viral load interactions in PBMCs and oral fluids [21].However, the Cameroon KS study included only adults some of whom were HIV infected and/or had KS.Here we are showing the pattern of EBV and KSHV viral load in PBMCs and oral fluids and associated risk factors across a wide (3-89 years old) age range in HIV-negative individuals from a KSHV endemic area of Uganda [22].

Study design and population
As reported previously [20], this work was carried out within a rural African cohort, the General Population Cohort (GPC).The GPC is a community-based cohort of about 22,000 people in 25 adjacent villages in southwestern Uganda.It was established in 1989 to investigate the epidemiology of HIV; participants from the GPC have been followed ever since.The seroprevalence of KSHV is > 90% in adults [22].Between July 2017 and November 2017, we nested a cross-sectional study within the GPC enrolling 975 KSHV seropositive (tested previously [13]), HIV-negative individuals aged three to eighty-nine years.Participants were selected randomly after stratification for age and sex.Blood, stool and oral fluids samples were collected from these individuals.Socio-demographic data were collected using standard questionnaires.DNA was extracted from 2 million PBMCs collected and saliva pellets.This DNA was used to quantify both KSHV and EBV.The study was approved by the UVRI-Research and Ethics Committee (REC) (reference number: GC/127/16/09/566), the Uganda National Council for Science and Technology (UNCST) (reference number: HS2123) and LSHTM Ethics Committee (reference number: 11881).Written informed consent was obtained from all adults aged 18 years and above.Parents or guardians consented for children below 18 years, additionally, children aged 8-17 years provided written assent.
Peripheral blood mononuclear cell (PBMC)s and plasma were obtained from the blood for immunological and virological analyses.Study participants were instructed to rinse with 5 mL of Listerine mouthwash, and collect the resulting fluid in a polypropylene tube.Aliquots (of 1 mL each) of oral fluids were spun at 13,000 rcf for 10 min to form oral fluids pellets.Thereafter the supernatant was removed and the oral fluids pellet was stored at − 80 °C.A pellet of two million PBMCs and oral fluids pellets were processed for DNA extraction using a QIAamp blood kit (Qiagen, Valencia, CA), following the manufacturer's instructions.

Statistical analysis
Statistical analysis was carried out using STATA version 13 (StataCorp, College Station, Texas USA).Graphs were drawn using STATA and GraphPad Prism version 8. Viral load levels were log 10 transformed.First, risk factors associated with viral DNA detection (as a categorical outcome variable) in oral fluids and PBMCs, separately, were obtained using logistic regression modelling.Thereafter, risk factors associated with increasing levels of viral DNA (as a continuous outcome variable) in oral fluids and PBMCs, separately, were determined using linear regression modelling.Chi 2 test, student T-test and one-way ANOVA were used for crude analysis.The false discovery rate (FDR) was used to correct multiple comparisons of antibody data.

Results
Characteristics of the participants included are shown in Table 1.The proportion of individuals with detectable EBV DNA in oral fluids was 74% compared to 24% for KSHV.The median EBV viral load (VL) in oral fluids were 3364 copies/uL while KSHV VL was 401 copies/ uL (Table 1).Prevalence of shedding in oral fluids varied with age: all children aged 3-5 years had EBV in oral fluids whereas adults aged 36-45 years had the lowest proportion (72%).For KSHV, the highest proportion with KSHV DNA was among 6-12-year-olds (30%) whereas adults aged 46-55 years old had the lowest (11%) The patterns of KSHV and EBV shedding with age were similar (Fig. 1A).
Similar to adults from a Cameroon KS case-control study [21], the proportion of individuals with either EBV or KSHV DNA in PBMCs was much lower than the proportion of individuals with either virus in oral fluids (Fig. 1B).
The proportion of individuals with KSHV DNA in oral fluids did not differ between those with and without EBV DNA in oral fluids (Fig. 2A).However, despite the lower prevalence of either virus in PBMCs compared to oral fluids, the proportion of individuals with KSHV DNA in PBMCS was higher among individuals with EBV DNA in PBMCs (14% vs. 9%, P = 0.011) Fig. 2B & Additional file 1: Supplementary Table 1.Both in oral fluids (Fig. 2C) and PBMCs (Fig. 2D), EBV and KSHV DNA levels were positively correlated, although this didn't reach statistical significance.
Age was significantly associated with EBV detection in both oral fluids (P = 0.025) and PBMCs (P = 0.0089).On the other hand, both P. falciparum-infection (detected by malaria rapid diagnostic tests-RDT) and sex were not associated with detection of EBV in oral fluids or PBMCs (Table 2).
Among those with EBV in oral fluids, malaria and sex were not associated with EBV levels.However, among those with EBV in PBMCs, malaria was positively associated with EBV DNA levels.Individuals with malaria had higher levels of EBV DNA in PBMCs compared to individuals without malaria (adjusted regression coefficient 0.43, (0.15-0.71),P = 0.002).Sex was not associated with levels of EBV DNA in PBMCs.Age group was associated with levels of EBV DNA in both oral fluids and PBMCs (Table 3, Fig. 5).

Discussion
This study presents the following observations: (1) both in oral fluids and PBMCs, EBV is detected more frequently and in higher quantities compared to KSHV, as shown previously [21]; (2) both viruses are more likely to be detected in children's oral fluids and in PBMCs than in other age groups; (3) Individuals with KSHV in PBMCs are more likely to have EBV in PBMCs as well; (4) Infection with asymptomatic P. falciparum malaria is associated with higher EBV viral load in PBMCs; (5) in addition to IgG antibody levels to the KSHV K8.1, IgG antibody levels to KSHV ORF65 and K10.5 are higher in individuals with detectable KSHV in PBMCs and oral fluids while IgG antibodies to the KSHV ORF38 and ORF25 are higher in individuals with detectable KSHV in oral fluids only.
EBV is ubiquitous in all human populations with over 90% of adults infected [30] whereas KSHV is limited to specific areas or high-risk populations, most notably in sub-Saharan Africa [31].The findings suggest that EBV is more easily transmitted than KSHV.The mechanism leading to the difference in transmissibility between the two viruses is not documented.This study and previous studies showing that EBV DNA is more frequently detected in oral fluids and at higher levels than KSHV contributes to our understanding of the differences in transmission patterns between the two viruses.The difference cannot be solely explained by cell tropism because both KSHV and EBV infect several types of cells, some of which overlap.EBV infects B lymphocytes, epithelial cells, T lymphocytes, NK cells, monocytes, smooth muscle cells and follicular dendritic cells using CD21, HLA-II, integrins and EphA2 for attachment, internalization and entry [32].KSHV infects endothelial cells, fibroblasts, monocytes, epithelial cells, B lymphocytes, macrophages and dendritic cells using HSPGs, DC-SIGN, EphAs and integrins for attachment and entry [33].
EBV and KSHV are more likely to be detected in children compared to adults.This might be attributed to a more recent infection with the viruses.Viral control may have not been well established in children and could be developed over time as individuals age.Furthermore, the high burden of malaria infection in children could be driving viral reactivation of KSHV and EBV.We observed that individuals with detectable KSHV in PBMCs are more likely to have detectable EBV as well, we speculate that systemic factors affecting viral immune control including immunosuppression, Th2 skew, immune regulation, and immune cell dysfunction could affect the control of both viruses.
Epidemiology studies have linked EBV and P. falciparum to Burkitt's lymphoma [34].Both EBV and P. falciparum upregulate AID expression while AID expression has been shown to contribute to c-MYC translocation and mutation [35][36][37].c-MYC translocation is a hallmark of Burkitt lymphoma development [36].Additionally, chronic exposure to P. falciparum has been shown to reactivate EBV, increasing the number of latently infected B cells with EBV [38].Possibly P. falciparum impairs T cell immunity to EBV through immune suppression   The strength of this study was the large sample size (over 800 individuals analysed) and the inclusion of males and females across the life course (3-89 years).Furthermore, all individuals analysed were HIV uninfected, so the impact of HIV on viral reactivation is not a concern.Although HIV has been shown to dramatically reactivate both viruses, in endemic regions, transmission of both viruses occurs in childhood before HIV acquisition for most individuals.The major weakness of this study is the cross-sectional design of the study.

Conclusion
EBV is more frequently detected and at higher levels, both in PBMCs and oral fluids than is KSHV.Viral detection of both KSHV and EBV is more frequent in children compared to adults.This might in part be explained by the burden of P. falciparum infection in children and the recent viral infection.The mechanism through which P. falciparum affects both KSHV and EBV warrants further investigation.• thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year

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Fig. 2
Fig. 2 Relationship between KSHV and EBV viral load in oral fluids or PBMCs.A proportion of individuals with KSHV in saliva among those with or without EBV in saliva; B proportion of with KSHV in PBMCs among those with or without EBV in PBMCs; C correlation between EBV and KSHV DNA levels in saliva; D correlation between EBV and KSHV DNA levels in PBMCs; EBV and KSHV Viral load quantified using qPCR.Graphs were drawn in GraphPad Prism version 8 (A, B) and STATA version 13 (C, D).P values and correlation coefficient obtained in STATA version 13.Chi 2 test used in A, and logistics regression adjusting for age and sex in B

Fig. 3
Fig. 3 IgG responses to KSHV and EBV proteins.A proportion of individuals with a seropositive response to KSHV/EBV proteins.B proportion of individuals with a seropositive response to KSHV proteins by age group.C mean IgG median fluorescent intensities-MFI to KSHV/EBV proteins.D mean IgG MFI to KSHV proteins by age group.Error bars represent standard deviations (C, D) or 95% confidence intervals (A, B).P value obtained using chi 2 test (B) or one-way ANOVA (D) in STATA version 13.Graphs were drawn using GraphPad Prism version 8

Fig. 5
Fig. 5 EBV viral copies in oral fluids (A) and PBMCs (B) by malaria parasitaemia status.P values obtained from a student T test.Malaria parasitaemia determined using Rapid diagnostic tests (RDT).EBV quantified in oral fluids and PBMCs using qPCR

Table 1
Population characteristics and infection status a Asymptomatic malaria by rapid diagnostic test (RDT).Viral load detected using qPCR

Table 2
Factors associated with EBV detection in oral fluids or PBMCs*Adjusted for age group, sex and malaria parasitaemia.a Asymptomatic malaria by rapid diagnostic test (RDT).Logistic regression modelling done using STATA version 13.Viral load detected using qPCR

Table 3
Factors associated with EBV viral load among individuals with detectable EBV in oral fluids or PBMCs*Adjusted for age group, sex and malaria parasitaemia.a Asymptomatic malaria by rapid diagnostic test (RDT).Logistic regression modelling done using STATA version 13.Viral load detected using qPCR