Epstein-Barr Virus Type 2 Infects T Cells in Healthy Kenyan Children

Abstract

Background: The 2 strains of Epstein-Barr virus (EBV), EBV type 1 (EBV-1) and EBV-2, differ in latency genes, suggesting that they use distinct mechanisms to establish latency. We previously reported that EBV-2 infects T cells in vitro. In this study, we tested the possibility that EBV-2 infects T cells in vivo.

Methods: Purified T-cell fractions isolated from children positive for EBV-1 or EBV-2 and their mothers were examined for the presence of EBV and for EBV type.

Results: We detected EBV-2 in all T-cell samples obtained from EBV-2-infected children at 12 months of age, with some children retaining EBV-2-positive T cells through 24 months of age, suggesting that EBV-2 persists in T cells. We were unable to detect EBV-2 in T-cell samples from mothers but could detect EBV-2 in samples of their breast milk and saliva.

Conclusions: These data suggest that EBV-2 uses T cells as an additional latency reservoir but that, over time, the frequency of infected T cells may drop below detectable levels. Alternatively, EBV-2 may establish a prolonged transient infection in the T-cell compartment. Collectively, these novel findings demonstrate that EBV-2 infects T cells in vivo and suggest EBV-2 may use the T-cell compartment to establish latency.

Keywords: Burkitt lymphoma; Epstein-Barr virus; T lymphocytes; cellular tropism.

Figure1.

Prevalence of Epstein-Barr virus (EBV) types in the Kisumu, Kenya, infant population and EBV types detectable in saliva and breast milk specimens from the infants’ mothers. A, Blood specimens were collected from infant donors at 6 weeks, 10 weeks, 14 weeks, 18 weeks, 6 months, 9 months, and 12 months of age. DNA was isolated from blood specimens and used to perform quantitative polymerase chain reaction (qPCR) analysis to detect BALF5 in the EBV genome (and thereby identify EBV-positive samples (84 collected at age 6 weeks, 62 collected at 10 weeks, 37 collected at 14 weeks, 23 collected at 18 weeks, 28 collected at 6 months, 28 collected at 9 months, and 32 collected at 12 months). DNA samples that were positive for EBV genome were further analyzed to determine the EBV type present in each sample, via a multiplex reverse-transcription PCR for EBNA3c that distinguishes EBV type 1 (EBV-1) and EBV-2; for 3 children, we determined the EBV type for samples collected ≥1 time point. DNA samples that were positive for EBV-1 or EBV-2 through the first year of life were used to determine the type-specific EBV prevalence in this population. Infants were considered coinfected if both EBV-1 and EBV-2 were detected at any time point tested. Prevalence data for samples in which the EBV type could be determined are shown in bar graph (22 of 35 were positive for EBV-1, 8 of 35 were positive for EBV-2, and 5 of 35 were positive for both types). B, Saliva and breast milk specimens were collected from infants’ mothers at postpartum week 6, and DNA was isolated. Multiplex quantitative PCR analysis of BALF5 in the EBV genome was performed to determine the log number of EBV copies per milliliter of saliva and breast milk. Additionally, multiplex reverse-transcription PCR analysis to detect EBNA3c was performed to identify the EBV type present in each sample. Abbreviation: ND, not detected.

Figure 2.

Epstein-Barr virus type 2 (EBV-2) infects T cells in vivo. Peripheral blood mononuclear cells (PBMCs) were collected from infant donors at 1, 2, and 24 months of age. Non–T-cell and T-cell fractions were isolated from the total PBMC population via magnetic columns, and DNA was subsequently isolated. Multiplex quantitative polymerase chain reaction (PCR) analysis of BALF5 in the EBV genome was performed to determine the log EBV copy number per 1 × 10⁶ cells for each cellular fraction collected at ages 12 months (A) and 24 months (B). Additionally, a multiplex reverse-transcription PCR to detect EBNA3c was performed to identify the EBV type infecting each cell fraction. Post sort purity analysis for CD3 and CD19 was performed on all non–T-cell and T-cell fractions. Representative flow plots for infants 2 and 8 are shown under the corresponding bar graphs. Abbreviation: ND, not detected.

Figure 3.

Epstein-Barr virus type 2 (EBV-2) is not detected in the T-cell compartment of adults. Peripheral blood mononuclear cells (PBMCs) were collected from mothers at the time of enrollment into the study. Non–T-cell and T-cell fractions were isolated from the total PBMC population via magnetic columns, and DNA was subsequently isolated. Multiplex quantitative polymerase chain reaction (PCR) analysis of BALF5 in the EBV genome was performed to determine the log number of EBV copies per 1 × 10⁶ cells for each cellular fraction. Additionally, a multiplex reverse-transcription PCR analysis to detect EBNA3c was performed to identify the EBV type infecting each cell fraction. Postsorting purity analysis for CD3 and CD19 was performed on all non–T-cell and T-cell fractions. Representative flow plots for infants 2 and 8 are shown under the corresponding bar graphs. Abbreviation: ND, not detected.