Human cancers and viruses: 50 years of Epstein Barr Virus

March marked the 50th anniversary of the discovery of Epstein-Barr Virus (EBV) - an anniversary which the author of this blog almost missed, wouldn’t it have been for an article published Science on March 21st.

Timeline: Animal tumour virus

It was in March of 1964 that Anthony Epstein discovered a Herpesvirus like virus in tumour cells derived from African Burkitt’s lymphoma tissue and thus identified the first human tumour virus. At that time the idea that cancer can be caused by a virus -or any infectious agent- was a matter met with skepticism despite the description of a tumour virus in chicken by Peyton Rous in 1911 (subsequently named Rous Sarcoma Virus or RSV) and the identification of avian leukaemia (myeloblastis) virus. The discovery of Peyton Rous however was preceded by experiments by Vilhelm Ellerman and Oluf Bang in 1908 who published similar results on the viral transmission of avian erythroblastosis. Although met with resistance, these early discoveries were followed by the discovery of the cottontail rabbit papillomavirus in rabbits -the causative agent of transmissible papillomas- and mouse mamary tumour virus (MMTV) in 1933 and 1936 respectively. Further research led to the discovery of the acutely transforming murine retrovirus, a murine polyomavirus (MuPV) as well as the discovery and characterization of a simian Polyomavirus (SV40) in the 1950s and early 1960s. Human Adenovirus’ was shown to induce the formation of tumour in rodents in 1962, thus indicating that human viruses are capable of transforming cells - albeit non-human cells, thus not proving a link between human tumours and viral infections.

Following the discovery of EBV in 1964 it would take another nine years before the link between Burkitt’s lymphoma and EBV was established, although it was shown in 1968 that a EBV-like virus is able to cause T-cell lymphoma in non-human primates (EBV causes B cell lymphoma so this discovery was only a step into the right direction). One of the problems establishing a link between EBV infection and the development of cancer was that the prevalence of EBV within the world population is over 90%. The latent form of EBV can be detected in a small percentage of B-lymphocytes of otherwise healthy individuals and the exposure to EBV can detected by serology. In most cases the infection is asymptomatic and even if the infection causes a disease it is relatively benign disease (infectious mononucleosis) and not cancer. So how can this relatively common and benign virus be the causative agent of a common childhood cancer in Africa, a cancer furthermore mostly confined to Africa and virtually absent in other regions of the world ? The search of additional cancers caused by EBV led to the discovery that almost 100% of nasopharyngeal cancers in Southeast Asia are caused by EBV as well. These observations led to the hypothesis that although EBV may have the ability to transform cells, that presence of EBV itself might not sufficient to cause cancer. At this point it might be worth to take a look at the replication of EBV. 

Following latency EBV can be reactivated and lead to a
persistent infection if not cleared can lead to cancer

Upon infection of the host cell, a short lytic phase during which viral particles are released from the host cell is followed by a latent phase, characterized by only a small viral load. Only a small percentage of infected cells switch from this latent stage to a lytic stage - in other words, it is only in a small number of cases that viral replication is reactivated.

During the latent phase, the genomic DNA of EBV exists in an extrachromosomal state as a closed circular plasmid (“episome”), behaving exactly like host chromosomal DNA. In order to switch to the lytic phase, the first step is the linearization of the genome followed by the expression of early viral proteins, BRLF1 and BZLF1/Zta, the latter only exhibiting low basal levels of expression unless induced by chemical or biological factors.  In general, genes are expressed in three phases, immediate-early, early and late. Immediate-early genes include the transactivators required for the expression of later genes. The EBV genome is amplified during the lytic phase by the viral replication machinery in discrete nuclear replication compartments, promoting an S-phase like arrest of the cell cycle. These replication centres consist of seven viral proteins, BALF5, BBLF4, BSLF1, BBLF2/3, BALF5 (DNA polymerase), BMRF1 (DNA Polymerase processivity factor), BALF2 ( ssDNA binding protein), and BZLF1 (oriLyt binding protein), the assembly being dependent on “later” gene products.

It is generally considered that host cell factors contribute to the absence or control of viral replication independent of cellular factors during the latent phase, suggesting that factors such as immunodeficiency contribute to the establishment of EBV induced malignancy. While this is the case in patients diagnosed with Infectious Mononucleosis, this is also the case in patients diagnosed with EBV positive nasopharyngeal cancer and Burkitt’s lymphoma. The difference might be an underlying immunodeficiency that predispose to EBV associated malignancy, in particular those affecting cytotoxic T-lymphocytes and Natural Killer (NK) cells - such as but not limited to HIV infection or  "X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia" (XMEN) disease, the latter a genetic disease, the former leading to the depletion of cytotoxic T-lymphocytes. The role of the immunogenic status of the host in the control of viral malignancy is further highlighted in the development of HIV associated Kaposi Sarcoma, a cancer caused by another Herpesvirus, Human Herpesvirus 8 (HHV 8 or KSHV). Similar to EBV, the infection of HIV predisposes humans to this form of skin cancer and KSHV positive lymphoma by switching a latent infection to a lytic infection with the release of viral particles concomitant with a depletion of cytotoxic T-lymphocytes.
In the case of EBV however, a late protein expressed during the lytic phase of the replication cycle, BPLF1, has been shown to play a role in the immune evasion by targeting the antiviral Toll like pathway and the activation of NF-κB. In the absence of BPLF1, EBV activates NF-κB via TLR-2, TLR-3, and TLR-9 activation. BPLF1 is also cleaved by Caspase-1, thus localizing to the nucleus where it increases the accumulation of Cullin-RING-Ligase substrates and thus increasing viral replication via an unknown mechanism.

Timeline: Human tumour viruses

The role of viruses and infectious agents in the development of human cancer were found to be more common than originally thought. The 1980s saw the discovery of the first human retrovirus to be linked to cancer (HTLV-1), the role of high risk human Papillomavirus in cervical cancer and the identification of viruses causing hepatocellular cancer (HBV/HCV). In parallel, the first anticancer vaccines were developed and approved (Hepatitis B and HPV), followed by vaccination programs. More recent discoveries include Merkel Cell Polyomavirus -the causative agent of Merkel Cell Carcinoma- and the discovery that EBV might also induce about 10% of stomach cancers as well as lupus erythematosis. The discovery of endogenous retrovirus in animals and humans lead to the hypothesis that endogenous human retroviruses such as XMRV are the causative agent of prostate cancer, a link which however was dismissed (indeed XMRV owes its existence is due to a contamination of cell lines). 

Not only lead these discoveries to vaccines and improved diagnosis of cancer, but also to a better understanding of cellular processes such as the DNA damage response, the induction of cell death and its role in the development of cancer.

Oncogenic viruses such as EBV, KSHV, HBV, or HPV have been shown to interact with a variety of cellular proteins, thus modulating apoptotic pathways. The most famous is surely the interaction of HPV E6 mediated inactivation of p53 – also targeted by EBV EBNA1 in Hodgkin’s lymphoma. Other examples include HTLV-1 Tax and HBZ or KSHV LANA1. Targets not only include p53, but also the Retinoblastoma (Rb) protein, signaling proteins such as Akt, mTOR, or PI3-Kinase, the Interferon receptor or the Interferon Regulatory Factor to name a few.

Common to all tumour virus is the extended period of time of latency and the relative rare incidence of cancer. Interest in this field was certainly renewed by the appearance of HIV in the 1980s, whose long-term consequences often include rare cancers.

Further reading

Moore PS, & Chang Y (2010). Why do viruses cause cancer? Highlights of the first century of human tumour virology. Nature reviews. Cancer, 10 (12), 878-89 PMID: 21102637 

Butel, J., & Fan, H. (2012). The diversity of human cancer viruses Current Opinion in Virology, 2 (4), 449-452 DOI: 10.1016/j.coviro.2012.07.002

Hammerschmidt, W., & Sugden, B. (2013). Replication of Epstein-Barr Viral DNA Cold Spring Harbor Perspectives in Biology, 5 (1) DOI: 10.1101/cshperspect.a013029 

Murata T, Sato Y, & Kimura H (2014). Modes of infection and oncogenesis by the Epstein-Barr virus. Reviews in medical virology PMID: 24578255 

Daikoku T, Kudoh A, Fujita M, Sugaya Y, Isomura H, Shirata N, & Tsurumi T (2005). Architecture of replication compartments formed during Epstein-Barr virus lytic replication. Journal of virology, 79 (6), 3409-18 PMID: 15731235 

Tsurumi, T., Fujita, M., & Kudoh, A. (2005). Latent and lytic Epstein-Barr virus replication strategies Reviews in Medical Virology, 15 (1), 3-15 DOI: 10.1002/rmv.441

van Gent M, Braem SG, de Jong A, Delagic N, Peeters JG, Boer IG, Moynagh PN, Kremmer E, Wiertz EJ, Ovaa H, Griffin BD, & Ressing ME (2014). Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling. PLoS pathogens, 10 (2) PMID: 24586164 

Rickinson, A., Long, H., Palendira, U., Münz, C., & Hislop, A. (2014). Cellular immune controls over Epstein–Barr virus infection: new lessons from the clinic and the laboratory Trends in Immunology DOI: 10.1016/j.it.2014.01.003

Gastaldello S, Chen X, Callegari S, & Masucci MG (2013). Caspase-1 promotes Epstein-Barr virus replication by targeting the large tegument protein deneddylase to the nucleus of productively infected cells. PLoS pathogens, 9 (10) PMID: 24130483 

van Gent M, Braem SG, de Jong A, Delagic N, Peeters JG, Boer IG, Moynagh PN, Kremmer E, Wiertz EJ, Ovaa H, Griffin BD, & Ressing ME (2014). Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling. PLoS pathogens, 10 (2) PMID: 24586164

Li FY, Chaigne-Delalande B, Su H, Uzel G, Matthews H, & Lenardo MJ (2014). XMEN disease: a new primary immunodeficiency affecting Mg2+ regulation of immunity against Epstein-Barr virus. Blood PMID: 24550228 

Arnaud F, Varela M, Spencer TE, & Palmarini M (2008). Coevolution of endogenous betaretroviruses of sheep and their host. Cellular and molecular life sciences : CMLS, 65 (21), 3422-32 PMID: 18818869 

Hohn O, Krause H, Barbarotto P, Niederstadt L, Beimforde N, Denner J, Miller K, Kurth R, & Bannert N (2009). Lack of evidence for xenotropic murine leukemia virus-related virus(XMRV) in German prostate cancer patients. Retrovirology, 6 PMID: 19835577 

Bhardwaj N, & Coffin JM (2014). Endogenous retroviruses and human cancer: is there anything to the rumors? Cell host & microbe, 15 (3), 255-9 PMID: 24629332 

White, E. (1998). Regulation of Apoptosis by Adenovirus E1A and E1B Oncogenes Seminars in Virology, 8 (6), 505-513 DOI: 10.1006/smvy.1998.0155 

Allison AB, Kevin Keel M, Philips JE, Cartoceti AN, Munk BA, Nemeth NM, Welsh TI, Thomas JM, Crum JM, Lichtenwalner AB, Fadly AM, Zavala G, Holmes EC, & Brown JD (2014). Avian oncogenesis induced by lymphoproliferative disease virus: a neglected or emerging retroviral pathogen? Virology, 450-451, 2-12 PMID: 24503062 

Fuentes-González, A., Contreras-Paredes, A., Manzo-Merino, J., & Lizano, M. (2013). The modulation of apoptosis by oncogenic viruses Virology Journal, 10 (1) DOI: 10.1186/1743-422X-10-182

Encephalitis lethargica and Influenza: a case of mistaken identity with a twist

Following the Influenza epidemic in 1918, physicians observed an increase in cases of Encephalitis lethargica  (EL), the acute (often lethal) phase followed by post encephalitic parkinsonism (PEP), the latter affecting patients for several decades.

Based on the observation that these patients might have been infected with the 1918 Influenza virus, the conclusion was reached that a long-term consequence of Influenza might be the development of a neurological disease. This notion was supported by experimental studies in mice inoculated with neurovirulent variants of Influenza virus strains A/NWS/33 and A/WSN/33  - both strains which are highly lab adapted, but still used in Influenza virus research - as well as circumstantial evidence suggested that patients in 1918 died not only of pulmonary edema but also of neurogenic congestive heart failure. Furthermore one of the variants of the 1918 Influenza virus contains single change in the amino acid sequence that changes its binding properties to receptors, thus making the virus -potentially- neurovirulent

Other evidence dating back to the 1918/1919 epidemic suggested that the neuropathology of EL during and after the epidemic was unique. Arguments against a link between the Influenza virus epidemic and the occurrence of EL were already presented in the 1920s, mainly pointing out that the increase in EL preceded the epidemic by at least two years. This assumes however that the epidemic truly started in 1918 - as I pointed out earlier there is some evidence that local outbreaks of Influenza occurred in 1915 in both Great Britain and France.

What is the relation between Influenza virus and neuropathological abnormalities, namely death of neuronal cells? As I mentioned, mice were infected with neurovirulent strains of A/NWS/33 and A/WSN/33 and the brain tissue was subsequently analyzed for abnormalities. Following nasal inoculation of susceptible mice with the WSN virus, cytotoxic T- Lymphocytes are activated as well as Microglia and neuronal cells of the olfactory bulb are infected. As part of the antiviral response, cytotoxic CD8+ T- Lymphocytes produce cytokines and release cytolytic proteins such Perforin, Granzyme B, and FasL. FasL -or Fas ligand- can bind to the Fas receptor and induces the external/Caspase-3 dependent pathway of apoptosis, in other words leading to cell death, whilst

Perforin/Granzyme B inserts into the cell membrane and forms pores - similar to a bag filled with water which slowly leaks if a tiny hole is present. 

Infection of neuronal cells (as well of T- Lymphocytes) with Influenza A induces the expression of both the Fas ligand and the Fas receptor, thus inducing the clearance of infected cells in an autocrine manner. As mentioned in a different post, the 1918 virus also expressed a PB1-F2 protein with a high capacity for apoptosis. Taken together this explains the why the experimental neurovirulent strain of WSN induces cell death of (mouse) neurons. The infection of Microglia  - which are antigen presenting cells and thus part of the immune system- with neurovirulent WSN leads not only to the activation of macrophages, but also to the secretion of neurotrophin (NT), Annexin V and basal fibroblast growth factor (bFGF) into the surrounding tissue, thus protecting non infected neuronal cells. Activated macrophages on the other hand, secrete tumor necrosis factor (TNF)-α, reactive oxygen Intermediates (ROI) and Nitric Oxyde (NO), all of them leading to the death of infected cells and -potentially- to inflammation. 

The importance of the neuroprotective effect was also highlighted by studies in mice using the neurovirulent strain R404BP virus which is a experimental variant of the neurovirulent A/H1N1/WSN expres-sing the matrix and neuraminidase genes of the neurovirulent WSN strain (H1N1) and other genes of the non-neurovirulent A/Aichi/2/68 strain (H3N2). This strain is only fatal if injected directly into the CNS of mice but not upon nasal infection of the olfactory neuroepithelium, suggesting that the viral infection is cleared after the initial infection.

If Influenza would have been the causative agent of Encephalitis lethargica traces of the Influenza genome or viral particles should be detectable in brain specimens from individuals who died from Encephalitis lethargica. In order to detect remains of viral particles or whole viral particles, brain specimens from patients who died either of modern EL or PEP were analyzed by both Transmission electron microscopy and immunohistochemistry.

Indeed 27nm virus like particles (VLP) were detected in the cytoplasm and in nuclei of neurons in samples of confirmed classic Encephalitis lethargica patients and both larger (50 nm) and smaller particles (27nm) intranuclear particles in modern EL cases as well as in a PEP case. Immunohistochemistry analysis however revealed that these particles derived from two Enteroviruses, Poliovirus and Coxsackievirus B4. Both viruses are known to cause viral meningitis. These results were confirmed by real time PCR and by comparing cell cultures infected with Poliovirus and Coxsackievirus B4. The VLP observed in the patient samples therefore most likely represent viral factories.

Does it mean that the 1918 Influenza virus and modern Influenza viruses do not infect the neuronal system? No it doesn’t. But as I pointed out the infection of neuronal cells does not only have apoptotic effects but also induces neuroprotection in mice. Other Influenza viruses that show a high degree of neuropathogenesis are those that are classified as highly pathogenic avian influenza viruses, including A/H7N1, A/H5N1, and A/Whistling Swan/Shimane/499/83 (H5N3). As in the case of the most recent human pandemic Influenza virus, A/H1N1/2009, these exhibit both α2,3- and α2,6-linked sialic acid receptor binding properties (i.e. they bind both to human and avian cells as well to cells located in both the upper and lower human respiratory tract) and exhibit neurological symptoms. Experimental infection of the human neuroblastoma cell lines SK-N-SH and SH-SY5Y the human GBM847 glioblastoma patient isolate with A/CA/7/2009 (an isolate of pandemic A/H1N1/2009) showed that those are permissive for A/CA/7/2009. Hypothetically it is possible that neurovirulent Influenza viruses might infect neurons and then -over time- use the neuronal network to to migrate to the brain, thus leading to neurodegenerative diseases, such as Parkinson. So far however no link has been proven.

What about Encephalitis lethargica then? After all it might be an autoimmune disease and not caused (directly) by pathogens.

However, as the controversy surrounding Encephalitis lethargica illustrates is that seemingly benign viruses such as Enteroviruses harbor serious side effects and emphasizes the importance for a careful analysis of epidemiological data. 

Further reading:

Maurizi CP (2010). Influenza caused epidemic encephalitis (encephalitis lethargica): the circumstantial evidence and a challenge to the nonbelievers. Medical hypotheses, 74 (5), 798-801 PMID: 20060230

Foley PB (2009). Encephalitis lethargica and the influenza virus. II. The influenza pandemic of 1918/19 and encephalitis lethargica: epidemiology and symptoms. Journal of neural transmission (Vienna, Austria : 1996), 116 (10), 1295-308 PMID: 19707848

Fujimoto I, Takizawa T, Ohba Y, & Nakanishi Y (1998). Co-expression of Fas and Fas-ligand on the surface of influenza virus-infected cells. Cell death and differentiation, 5 (5), 426-31 PMID: 10200492 

Ward AC (1996). Neurovirulence of influenza A virus. Journal of neurovirology, 2 (3), 139-51 PMID: 8799206

Nichols JE, Niles JA, & Roberts NJ Jr (2001). Human lymphocyte apoptosis after exposure to influenza A virus. Journal of virology, 75 (13), 5921-9 PMID: 11390593

Gomes, C., Ferreira, R., George, J., Sanches, R., Rodrigues, D., Gonçalves, N., & Cunha, R. (2013). Activation of microglial cells triggers a release of brain-derived neurotrophic factor (BDNF) inducing their proliferation in an adenosine A2A receptor-dependent manner: A2A receptor blockade prevents BDNF release and proliferation of microglia Journal of Neuroinflammation, 10 (1) DOI: 10.1186/1742-2094-10-16

Chaves AJ, Busquets N, Valle R, Rivas R, Vergara-Alert J, Dolz R, Ramis A, Darji A, & Majó N (2011). Neuropathogenesis of a highly pathogenic avian influenza virus (H7N1) in experimentally infected chickens. Veterinary research, 42 PMID: 21982125

Mori I, Goshima F, Imai Y, Kohsaka S, Sugiyama T, Yoshida T, Yokochi T, Nishiyama Y, & Kimura Y (2002). Olfactory receptor neurons prevent dissemination of neurovirulent influenza A virus into the brain by undergoing virus-induced apoptosis. The Journal of general virology, 83 (Pt 9), 2109-16 PMID: 12185263

Lo KC, Geddes JF, Daniels RS, & Oxford JS (2003). Lack of detection of influenza genes in archived formalin-fixed, paraffin wax-embedded brain samples of encephalitis lethargica patients from 1916 to 1920. Virchows Archiv : an international journal of pathology, 442 (6), 591-6 PMID: 12695912

McCall S, Henry JM, Reid AH, & Taubenberger JK (2001). Influenza RNA not detected in archival brain tissues from acute encephalitis lethargica cases or in postencephalitic Parkinson cases. Journal of neuropathology and experimental neurology, 60 (7), 696-704 PMID: 11444798

Dale RC, Church AJ, Surtees RA, Lees AJ, Adcock JE, Harding B, Neville BG, & Giovannoni G (2004). Encephalitis lethargica syndrome: 20 new cases and evidence of basal ganglia autoimmunity. Brain : a journal of neurology, 127 (Pt 1), 21-33 PMID: 14570817