Epstein-Barr Virus: More maladies than merely mono
Part 4 of “The human herpesviruses: much more than you wanted to know”
0. Actionable advice
Unfortunately there’s not much you can do about EBV. Most people are already infected, and if you’re not, the measures you’d need to take to avoid infection would be quite strict. Plus, the severity of primary infection increases with age, so trying to avoid infection and failing would be worse than not trying at all.
For a virus most people have never heard of, Epstein-Barr virus (EBV) has a surprisingly big impact on human health. EBV infects the vast majority of the human population, and causes serious effects in a small (though not insignificant) fraction. Those who have heard of EBV mostly associate it with mononucleosis, which is certainly an unpleasant disease. However, this is far from the worst that EBV can do.
First, EBV infections are responsible for approximately 1 in 70 of all cancer cases, accounting for about 265,000 cases per year globally (1, 2). With the decline in HPV prevalence due to vaccination, EBV is now the virus that’s most likely to give you cancer.
Second, EBV is likely responsible for a large number of autoimmune diseases, including Sjögren’s syndrome, rheumatoid arthritis (RA), lupus, and multiple sclerosis (MS) (3–5). Interestingly, recent research has also claimed that EBV reactivation is responsible for “long COVID” symptoms, although I’m not fully convinced about this.
Overall, EBV is a strong contender for the worst human herpesvirus. Every year, it causes the loss of 4.6 million disability-adjusted life years due to cancers alone (1), and probably another ~2 million to autoimmune diseases like multiple sclerosis.
(The only other contender is cytomegalovirus, which will be the topic of my next post.)
To put this in context, EBV is about 20% as bad as malaria (which causes the loss of 33.4 million DALYs each year). Malaria is widely considered a huge threat to public health, and although EBV isn’t quite as bad, it’s still pretty terrible.
2. Virology of EBV
EBV, also known as human herpesvirus 4, is one of the two gammaherpesviruses known to infect humans (the other being Kaposi’s sarcoma virus). The overall viral life cycle is similar to what I described previously for HSV, but there are some important differences, primarily regarding viral tropism (i.e., which cells get infected) and latency.
Unlike the alphaherpesviruses, EBV does not establish latency in neurons. Instead, the reservoir is memory B cells, which (like neurons) are a long-lived cell type. EBV can also undergo latency in epithelial cells. EBV has a rather different strategy for latency than the alphaherpesviruses. Rather than simply remaining dormant, EBV extensively alters the behavior of the host cell in order to benefit the virus (6).
EBV undergoes three distinct stages of latency, with varying levels of viral gene expression. The initial stage of latency established after a cell is infected is latency III. In this stage, EBV forces the infected cell to divide and proliferate, producing more infected progeny. This activity is also what gives rise to the EBV-associated cancers, although in cancers the overall gene expression pattern is often different from latency III and closer to the other latency stages.
Some of the infected cells in latency III will then switch to latency II, driving them to enter a resting state and differentiate into memory B cells, which can persist for years. Latency I is the most dormant stage of EBV, where very few viral genes are expressed, and takes place in these memory cells.
In all stages of latency, the viral genome is present as a circular piece of DNA (an episome) that replicates along with the host cell. This replication is mediated by the viral protein EBNA1, which recruits the host DNA polymerase to the viral origin of replication in order to copy the viral genome. This is crucial, since otherwise the viral genome would be lost when the host cell divides. (The alphaherpesviruses don’t have to worry about this since they establish latency in neurons, which don’t divide.)
In addition to its function in viral replication, EBNA1 has a repetitive region that blocks it from being recognized by the immune system (7). Along with other immune-evading tactics, this means that EBV-infected cells cannot be cleared by the host.
3. Epidemiology of EBV
EBV infects almost every human; the seroprevalence is estimated at above 90% in developed countries, and close to 100% globally. A study in England showed that 55% of the population was already infected by age 11, and by age 24 this had increased to 93% (8). The average age at infection was younger in ethnic minorities.
Most primary EBV infections in children are asymptomatic, but in adolescents and adults, mononucleosis symptoms are frequent, occurring at a frequency of roughly 75% (9). EBV is mainly transmitted by contact with saliva, which gives mononucleosis the nickname “the kissing disease”. Infected individuals shed EBV in saliva throughout their lives.
Interestingly, EBV is so good at evading the immune system that an existing EBV infection does not always provide immunity against a subsequent infection. This can only really be confirmed (via genotyping) when the second infection occurs with a different strain of EBV. However, this superinfection can happen when the two strains only have minor genetic differences, and are antigenically very similar (10). Therefore, this is not a phenomenon of antibodies failing to protect against EBV variants, but rather antibodies failing to protect against EBV at all.
The symptoms resulting from primary EBV infection are known as mononucleosis (or more rarely, as “glandular fever”). The name mononucleosis is due to the large proliferation of mononuclear cells in the blood, which are EBV-infected B cells. It is this proliferation of cells which causes the symptoms, rather than viral infection itself. As such, mononucleosis has a relatively long incubation period (typically 5–6 weeks from infection to symptom onset), during which infected individuals are highly contagious, since the viral load is higher than at any other point.
Mononucleosis symptoms, which typically last 1–2 weeks, include fever, sore throat, an enlarged spleen and lymph nodes (due to proliferating cells), and debilitating fatigue. The fatigue is particularly noteworthy, since it can persist for long after the initial infection. The overall rates of chronic fatigue at 6, 12, and 24 months after mononucleosis in adolescence were estimated as 15%, 7%, and 4%, respectively (11). The rates were higher in females than in males. EBV has also been proposed as a cause of “chronic fatigue syndrome” but there’s not a lot of high-quality evidence for this, largely because close to the entire population is infected, yet chronic fatigue is rare.
5. EBV and cancer
EBV causes cells to proliferate, and this can lead to cancer. On the level of molecular biology, many EBV proteins inhibit apoptosis, and others activate several different signaling pathways to stimulate cell division (12). EBV is a powerful carcinogen, but alone it is not sufficient to drive cancer, except in immunosuppressed individuals.Usually other mutations are necessary. However, the process of B cell activation naturally leads to a high rate of mutation, which sometimes causes cancer.
The main sites of EBV infection are epithelial tissues and immune cells, and thus these are also the main tissues of origin for EBV-positive cancers. Infection of immune cells leads to a wide variety of lymphoproliferative diseases, including Burkitt’s lymphoma, Hodgkin’s lymphoma, and several forms of other B and T cell lymphomas. Infection of other cells can lead to nasopharyngeal carcinoma, gastric carcinoma, and leiomyosarcoma. In total, EBV is estimated to cause approximately 1.5% of all human cancers, leading to an annual loss of 4.6 million disability-adjusted life years (1).
Although EBV causes cancers all over the world, the rates of particular cancers vary by population (12). Some of these are easily explained: for example, the high rate of Burkitt’s lymphoma in children in sub-Saharan Africa is explained by co-infection with EBV and malaria, resulting in an extremely high rate of B cell activation. However, other observations, such as the fact that EBV-positive nasopharyngeal carcinoma is 50-fold more frequent in southeast Asia than elsewhere, are difficult to explain. This is not likely related to any variation in EBV, and may result from human genetic variation, but the cause has not been identified.
6. EBV and autoimmune disease
For cancer, EBV has been caught red-handed, with its genome inside tumor cells. Indeed, measurement of EBV DNA in the blood can provide a way to detect EBV-associated cancers.
But cancer isn’t the only nasty long-term effect of EBV infection. In addition, there is a considerable amount of evidence linking EBV and autoimmune disease (3). Rheumatoid arthritis, lupus, and Sjogren’s syndrome are all characterized by antibodies targeting the patient’s own tissue (known as auto-antibodies). The molecular mimicry between EBV and human proteins can lead to the production of auto-antibodies in response to EBV infection (5).
However, causation is difficult to prove here, largely due to the fact that EBV is also widespread in people without autoimmune disease. For example, 99.5% of people with lupus were EBV-positive, but so were 95% of a control group (5). What’s likely going on is that the random process of antibody generation makes a small number people susceptible to autoimmune disease in response to EBV infection.
The evidence is probably best for EBV causing multiple sclerosis (MS), which involves an immune attack on the myelin protecting neurons. The rate of MS is about 15-fold lower in EBV-negative individuals compared to EBV-positive ones (4). This risk is increased in those with a history of symptomatic mononucleosis (rather than asymptomatic primary infection). Furthermore, MS in young people, when it occurs, always occurs subsequent to EBV infection (13). However, as with cancer, EBV alone is not sufficient to cause MS.
7. EBV and other rare diseases
EBV has also been implicated in several neurological diseases, including “Alice in Wonderland syndrome” (14), viral encephalitis (15), and cerebellar ataxia (16). Given the high prevalence of EBV, it’s possible that many rare diseases only occur in EBV-positive people, and we just don’t notice because the control group is too small.
Since “long COVID” is in the news recently, I should just mention that EBV reactivation has been proposed as a cause. One widely publicized study found a rate of EBV reactivation in 20 of 30 long COVID patients (mainly suffering from fatigue, >90 days post SARS-CoV-2 positive test) compared with 2 of 20 people who had COVID but recovered (17). Still, I think the evidence is far from conclusive. This is just a correlation, and it’s also possible that the causation goes the other way (immune cell activation during long COVID causing EBV reactivation) or that it’s completely unrelated. Still, since EBV is known to cause fatigue, it’s not completely implausible.
For more about long COVID, I suggest this FAQ which I helped author, or the Astral Codex Ten writeup.
8. EBV in biotechnology
Besides causing diseases, EBV is also useful in biotechnology. The earliest use of EBV was to induce proliferation in cultured B cells, allowing the establishment of cell lines. More recently, EBV-based episomal vectors have been used to express genes in human cells. These episomal vectors contain the EBV replication origin, as well as a DNA sequence encoding EBNA1. The EBNA1 protein recruits host DNA polymerase to the replication origin, meaning that the plasmid replicates every time the cell does. This can be used for stable expression of genes in human cells, without integrating the genes into the genome. If EBNA1 expression is shut off, then the plasmid will be lost, leaving the cell’s genome unchanged. One application of this is for reprogramming skin fibroblasts to induced pluripotent stem cells (iPSCs) (18). The iPSCs that I use in my research were generated by this method.
EBV can sometimes cause nuisances for research as well. Most human genomes are sequenced from saliva, where EBV DNA is present alongside human DNA. To avoid misidentification of EBV sequences as human, a “decoy genome” of EBV is included in the human reference genome. I’ve had my genome sequenced, and there were definitely some EBV sequences present in the sample.
9. What is to be done?
EBV is clearly a large burden to human health, mainly due to the cancers that it causes. Therefore, developing an effective vaccine against EBV would be highly beneficial. However, this would also be extremely challenging, given that anti-EBV antibodies are mostly ineffective at preventing infection. The human immune system may simply be outmatched.
Interestingly, to my knowledge EBV is the only curable human herpesvirus. There was a case study of an EBV-positive patient who became EBV-negative after receiving a bone marrow transplant from her EBV-negative brother (19). During the transplant process (which was performed to treat leukemia), all the patient’s immune cells were killed by cyclophosphamide chemotherapy, in combination with radiation delivered to the bone marrow. The patient also received acyclovir to treat a recurrence of HSV. After this treatment, the patient remained EBV-negative until being re-infected by a new strain of EBV from her husband approximately 4 years later. This case study shows that it is possible in principle to cure EBV. However, given the side effects of wiping out all the immune cells in the body, the cure is worse than the disease. Hopefully, this can lead to more targeted cures in the future. Still, it would be important to prevent re-infection afterwards. As of now, this can only be done by avoiding contact with EBV-positive people, who are the vast majority of humans.
One final note about why EBV preventions and cures are important: although natural EBV is not a huge threat, if a more oncogenic variant of EBV arose, either randomly or through malicious action, the results could be extremely bad. (Just how bad is left as an exercise for the reader.) So even though this seems like an intractable challenge, I still think it deserves attention.
1. G. Khan, C. Fitzmaurice, M. Naghavi, L. A. Ahmed, Global and regional incidence, mortality and disability-adjusted life-years for Epstein-Barr virus-attributable malignancies, 1990–2017. BMJ Open. 10, e037505 (2020).
2. H. Sung, J. Ferlay, R. L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, F. Bray, Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA A Cancer J Clin. 71, 209–249 (2021).
3. G. Houen, N. H. Trier, Epstein-Barr Virus and Systemic Autoimmune Diseases. Front. Immunol.11, 587380 (2021).
4. A. Ascherio, K. L. Munger, Epstein–Barr Virus Infection and Multiple Sclerosis: A Review. J Neuroimmune Pharmacol. 5, 271–277 (2010).
5. É. Toussirot, J. Roudier, Epstein–Barr virus in autoimmune diseases. Best Practice & Research Clinical Rheumatology. 22, 883–896 (2008).
6. W. Amon, P. J. Farrell, Reactivation of Epstein-Barr virus from latency. Rev. Med. Virol.15, 149–156 (2005).
7. J. Levitskaya, M. Coram, V. Levitsky, S. Imreh, P. M. Steigerwald-Mullen, G. Klein, M. G. Kurilla, M. G. Masucci, Inhibition of antigen processing by the internal repeat region of the Epstein–Barr virus nuclear antigen-1. Nature. 375, 685–688 (1995).
8. J. R. Winter, G. S. Taylor, O. G. Thomas, C. Jackson, J. E. A. Lewis, H. R. Stagg, Predictors of Epstein-Barr virus serostatus in young people in England. BMC Infect Dis. 19, 1007 (2019).
9. O. A. Odumade, K. A. Hogquist, H. H. Balfour, Progress and Problems in Understanding and Managing Primary Epstein-Barr Virus Infections. Clin Microbiol Rev. 24, 193–209 (2011).
10. D. M. Walling, A. L. Brown, W. Etienne, W. A. Keitel, P. D. Ling, Multiple Epstein-Barr Virus Infections in Healthy Individuals. J Virol. 77, 6546–6550 (2003).
11. B. Z. Katz, Y. Shiraishi, C. J. Mears, H. J. Binns, R. Taylor, Chronic Fatigue Syndrome After Infectious Mononucleosis in Adolescents. PEDIATRICS. 124, 189–193 (2009).
12. P. J. Farrell, Epstein–Barr Virus and Cancer. Annual Review of Pathology: Mechanisms of Disease, 27 (2018).
13. L. I. Levin, K. L. Munger, E. J. O’Reilly, K. I. Falk, A. Ascherio, Primary infection with the epstein-barr virus and risk of multiple sclerosis. Ann Neurol.67, 824–830 (2010).
14. M. Cinbis, S. Aysun, Alice in Wonderland syndrome as an initial manifestation of Epstein-Barr virus infection. British Journal of Ophthalmology. 76, 316–316 (1992).
15. M. T. Koning, T. Brik, R. Hagenbeek, I. van den Wijngaard, A case of fulminant Epstein-Barr virus encephalitis in an immune-competent adult. J. Neurovirol.25, 422–425 (2019).
16. K. Ali, C. Lawthom, Epstein-Barr virus-associated cerebellar ataxia. Case Reports. 2013, bcr2013009171–bcr2013009171 (2013).
17. J. E. Gold, R. A. Okyay, W. E. Licht, D. J. Hurley, Investigation of Long COVID Prevalence and Its Relationship to Epstein-Barr Virus Reactivation. Pathogens. 10, 763 (2021).
18. A. M. Drozd, M. P. Walczak, S. Piaskowski, E. Stoczynska-Fidelus, P. Rieske, D. P. Grzela, Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system. Stem Cell Res Ther. 6, 122 (2015).
19. J. W. Gratama, M. A. Oosterveer, F. E. Zwaan, J. Lepoutre, G. Klein, I. Ernberg, Eradication of Epstein-Barr virus by allogeneic bone marrow transplantation: implications for sites of viral latency. Proceedings of the National Academy of Sciences. 85, 8693–8696 (1988).
The “bubble boy” David Vetter died of Burkitt’s lymphoma caused by EBV acquired from a bone marrow transplant from his sister.
Very interesting, thank you!
Thanks for the deep and detailed analysis.
EBV seems like arguably the most successful virus in history: it infects 90% of one of the most viable species (lifespan X population) and persists for life.
A few questions if you don't mind:
1. Are you on Twitter?
2. I don't have access to paper 17. Did the reactivation occur in B cells or epithelial cells? Did they reveal the specific factors or signaling pathways for reactivation? It makes sense for EBV to reactivate when the immune system is stressed, but I want to learn more about the specific triggers.
3. To replicate cell lines, why can't you manufacture EBNA1 with mRNA technology and avoid the plasmid altogether?
4. You mention, "the rate of MS is about 15-fold lower in EBV-negative individuals compared to EBV-positive ones." What's the lower bound (roughly) for meaningfulness? For instance, would 5x be considered meaningful? 2x?
5. Why are anti-EBV antibodies mostly ineffective at preventing infection? Don't we have a clear understanding of EBV antigens?