The Incubation Period of Primary Epstein-Barr Virus Infection: Viral Dynamics and Immunologic Events

<div><p>Epstein-Barr virus (EBV) is a human herpesvirus that causes acute infectious mononucleosis and is associated with cancer and autoimmune disease. While many studies have been performed examining acute disease in adults following primary infection, little is known about the virological and immunological events during EBV’s lengthy 6 week incubation period owing to the challenge of collecting samples from this stage of infection. We conducted a prospective study in college students with special emphasis on frequent screening to capture blood and oral wash samples during the incubation period. Here we describe the viral dissemination and immune response in the 6 weeks prior to onset of acute infectious mononucleosis symptoms. While virus is presumed to be present in the oral cavity from time of transmission, we did not detect viral genomes in the oral wash until one week before symptom onset, at which time viral genomes were present in high copy numbers, suggesting loss of initial viral replication control. In contrast, using a sensitive nested PCR method, we detected viral genomes at low levels in blood about 3 weeks before symptoms. However, high levels of EBV in the blood were only observed close to symptom onset–coincident with or just after increased viral detection in the oral cavity. These data imply that B cells are the major reservoir of virus in the oral cavity prior to infectious mononucleosis. The early presence of viral genomes in the blood, even at low levels, correlated with a striking decrease in the number of circulating plasmacytoid dendritic cells well before symptom onset, which remained depressed throughout convalescence. On the other hand, natural killer cells expanded only after symptom onset. Likewise, CD4+ Foxp3+ regulatory T cells decreased two fold, but only after symptom onset. We observed no substantial virus specific CD8 T cell expansion during the incubation period, although polyclonal CD8 activation was detected in concert with viral genomes increasing in the blood and oral cavity, possibly due to a systemic type I interferon response. This study provides the first description of events during the incubation period of natural EBV infection in humans and definitive data upon which to formulate theories of viral control and disease pathogenesis.</p></div

Plasmacytoid DC declined in the circulation during the incubation period and remained depressed through convalescence.

<p>(A) Representative flow cytometry plots of pDC frequencies amongst non-lymphoid cells (CD3, CD56, CD14, CD20 negative) from samples collected at multiple timepoints for one subject (5524). (B) The percentage of pDC from 5524 over time. (C) Frequencies of pDC over time are shown for all subjects. (D) Frequencies of conventional DC (cDC) (CD11c⁺, HLA-DR⁺ cells) are shown over time for all subjects. (E) Numbers of pDC per mL of whole blood are shown for all subjects. (F) shows the percentage of pDC in samples where viral genomes were detected in the blood by nested PCR (Blood lo) or qPCR (Blood hi). Statistical analysis was performed using a one-way ANOVA with multiple test comparison. Light pink symbols indicate a significant difference (p<0.05) compared to pre-infection; darker pink symbols (p<0.001); red symbols (p<0.0001). Gray symbols indicate no statistical difference.</p

CD4⁺ Foxp3⁺ T cells transiently decline in the circulation at symptom onset during AIM.

<p>(A) Frequency of Foxp3⁺CD25⁺ cells amongst total CD4⁺ T cells data plotted over time for a representative individual (subject 7112). (B) Normalized frequency of Foxp3⁺ CD4 T cells over time in all subjects. Foxp3⁺ frequencies were normalized to each subject’s pre-infection baseline due to substantial variation in this population between individuals. (C) Number of CD4+ T cells per mL of whole blood over time. Statistics were performed using a one-way ANOVA with multiple test comparison. Gray values are not statistically different. Red value p<0.0001 compared to pre.</p

Viral genome detection during the incubation period.

<p>Quantitative viral load was determined by qPCR using DNA from oral wash cell pellets (A) or blood (B). Data are expressed as Log₁₀ viral genome copies/mL of sample. The dashed gray line represents the limit of detection. (C) and (D) show the time to the first positive measurement for each subject for viral genomes detected in the blood (D) by non-quantitative nested PCR (filled squares) or qPCR (filled inverted triangles), or in the oral cavity (C) by nested or qPCR (same results were obtained with both assays) (open circles). The theoretical presence of virus shown in (C) is the estimated time period in which study participants were initially exposed to oral virus. (E) In sequential samples from the incubation period, subjects were scored for which compartment viral genomes were first detected in: blood by nested PCR (blood (lo)), blood by qPCR (blood (hi)), oral, or a simultaneous positive in both compartments. (F) Shows an inset comparing blood and oral cavity for the time period close to symptom onset. The results for twenty-six subjects who had a sample collected within the first two weeks of symptom onset are shown.</p

CD8 T cell activation occurred during the incubation period, although not an EBV specific response.

<p>(A), Time to first response for three distinct immune parameters is shown: CD38 upregulation on total CD8⁺ T cells (filled squares), an increased CD8 to CD4 T cell ratio (open diamonds), or the presence of EBV tetramer binding CD8⁺ T cells above background (0.4%) (filled circles). (B) Frequency of CD8⁺ T cells expressing CD38 over time. (C) Ratio of CD8⁺ to CD4⁺ T cells over time. Statistics were performed using a one-way ANOVA with multiple test comparison. Pink symbols indicate a significant difference (p<0.05) compared to pre-infection. Red symbols indicate a significant difference (p<0.0001) compared to pre-infection. Gray symbols indicate no statistical difference.</p

Gene expression signatures during the incubation period showed distinct kinetic patterns.

<p>43 EBV infection signature genes were measured in total PBMC RNA. Functional categorization of the genes is shown at left. Fold change (FC) in expression was calculated compared to pre-infection samples. Heirarchical clustering (above) showed three distinct groupings: no signature (red), type I IFN (blue), and type II IFN/cell cycle (green). Subject numbers and sample collection date are indicated at top. The presence of viral genomes by nested (lo) or qPCR (hi) is noted below, along with the range of sample days (relative to symptom onset) that each signature was observed in.</p

NKG2A⁺ NK cells were expanded during AIM, but not during the incubation period.

<p>(A) Percentage and number of NK cells that are CD56^bright (immature) decreases during the first 50 days after symptom onset. (B) The percentage and number of NK cells that are CD56^dim NKG2A⁺ KIR^- increases, and remains elevated. Statistics were performed using a one-way ANOVA with multiple test comparison. Pink symbols indicate a significant difference (p<0.05) compared to pre-infection. Red symbols indicate a significant difference (p<0.0001) compared to pre-infection. Gray symbols indicate no statistical difference.</p

Primary EBV Infection Induces an Expression Profile Distinct from Other Viruses but Similar to Hemophagocytic Syndromes

<div><p>Epstein-Barr Virus (EBV) causes infectious mononucleosis and establishes lifelong infection associated with cancer and autoimmune disease. To better understand immunity to EBV, we performed a prospective study of natural infection in healthy humans. Transcriptome analysis defined a striking and reproducible expression profile during acute infection but no lasting gene changes were apparent during latent infection. Comparing the EBV response profile to multiple other acute viral infections, including influenza A (influenza), respiratory syncytial virus (RSV), human rhinovirus (HRV), attenuated yellow fever virus (YFV), and Dengue fever virus (DENV), revealed similarity only to DENV. The signature shared by EBV and DENV was also present in patients with hemophagocytic syndromes, suggesting these two viruses cause uncontrolled inflammatory responses. Interestingly, while EBV induced a strong type I interferon response, a subset of interferon induced genes, including <i>MX1, HERC5</i>, and <i>OAS1</i>, were not upregulated, suggesting a mechanism by which viral antagonism of immunity results in a profound inflammatory response. These data provide an important first description of the response to a natural herpesvirus infection in humans.</p></div

Type II IRG gene expression correlates with CD8 lymphocytosis in primary EBV infection.

<p>Data show the correlation between IRG gene expression and CD8 lymphocytosis in 42 subjects at timepoints within the first two weeks after symptom onset during primary EBV infection. The number of CD8 T cells per mL of peripheral blood was determined by flow cytometry (note: average at baseline was 0.25×10⁶). Superarray analysis by qPCR was performed to determine average fold changes for groups of Type I IRGs and Type II IRGs. Pearson correlation coefficients (r-values) and p-values are shown.</p

A distinct gene expression profile is apparent during acute EBV infection, but not latent infection.

<p>(A) Microarray analysis was performed on pre-infection, acute, and latent timepoints for the 10 subjects with primary EBV infection (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085422#pone.0085422.s005" target="_blank">Table S1</a>). 464 genes were shown to be significantly changed during the primary response to EBV at a fold change of ≥2 and a p-value ≤0.05. No genes were significantly changed during the latent phase of infection using the same criteria. (B) Ingenuity Pathway Analysis of the 464 acute genes revealed 14 pathways that were enriched amongst the genes that changed during primary EBV. These had a significant p-value (the negative log is shown) following evaluation with the Benjamini-Hochberg multiple tests correction. (C) A heatmap representation of the highest (≥3 fold) gene changes during the acute and latent stages of EBV infection.</p