EP: Prime on page 7 15tlid1225 Grand Round THELANCETID-D-15-01225R1 EMBEDED VIDEO: Confocal microscopy imaging of combined detection of Marseillevirus infection S1473-3099(16)30051-2 A thin section of the lymph node visualised by fluorescence in situ CW hybridisation (Marseillevirus DNA probe in green), immunofluorescence This version saved: 14:14, 26-Jul-16 Embargo: August 4, 2016—23:30 BST (anti-Marseillevirus antibodies in red), and nucleic acid staining with 4’,6-diamidino-2-phenylindole (DAPI)

Marseillevirus in lymphoma: a giant in the lymph node

Sarah Aherfi, Philippe Colson, Gilles Audoly, Claude Nappez, Luc Xerri, Audrey Valensi, Matthieu Million, Hubert Lepidi, Regis Costello, Didier Raoult

The family is a new clade of giant whose original member, marseillevirus, was described in Lancet Infect Dis 2016 2009. These viruses were isolated using Acanthamoeba spp primarily from the environment. Subsequently, a close Published Online relative of marseillevirus was isolated from the faeces of a healthy young man, and others were detected in blood August 4, 2016 samples of blood donors and recipients and in a child with lymph node adenitis. In this Grand Round we describe the http://dx.doi.org/10.1016/ S1473-3099(16)30051-2 detection of marseillevirus by PCR, fluorescence in-situ hybridisation, direct immunofluorescence, and Research Unit on Emerging immunohistochemistry in the lymph node of a 30-year-old woman diagnosed with Hodgkin’s lymphoma, together Infectious and Tropical Diseases with IgG antibodies to marseillevirus. A link with viruses and has been reported for many lymphomas. We (URMITE), CNRS UMR 7278, review the literature describing these associations, the criteria used to consider a causal association, and the IRD 198, Inserm U1095 underlying mechanisms of lymphomagenesis. Our observations suggest that consideration should be given to (S Aherfi PharmD, Prof P Colson PharmD, marseillevirus infections as an additional viral cause or consequence of Hodgkin’s lymphoma, and that this hypothesis G Audoly PhD, C Nappez PhD, should be tested further. A Valensi MRes, M Million MD, Prof H Lepidi MD, Introduction in blood donors and recipients in France and Prof D Raoult MD), Département de Giant viruses emerged during the 21st century following marseillevirus antibodies have been detected in healthy Bio-Pathologie, Oncologie the serendipitous discovery of through the young people in Switzerland.14–16 Moreover, during moléculaire, Hématologie implementation of isolation strategies by co-culture on implementation of marseillevirus serological testing, we et Immunologie des tumeurs , since these viruses replicate within amoebas, identified a strong reactivity to marseillevirus in an (Prof L Xerri MD), and Technological Advances for and inoculation on amoebal cultures was thereafter used 11-month-old boy with lymphadenitis, which was Genomics and Clinics (TAGC), for their study.1,2 Giant viruses are visible at the virion confirmed by FISH and immunohistochemistry analysis Inserm UMR 1090 stage under an optical microscope and contain many of the lymph node, and PCR of blood serum.17 Further (Prof R Costello MD), genes and , in similar numbers to those observed attempts to assess the connection between Aix-Marseille Université, Marseille, France; Méditerranée in small bacteria. Studies done since 2003 by use of marseilleviruses and lymphadenitis in our laboratory led Infection Foundation (IHU), culture isolation and metagenomics have identified that to the discovery of an unexpected case, which we describe Assistance Publique-Hôpitaux giant viruses that infect amoebas are common in water here, of detection of marseillevirus in the lymph node of de Marseille, Marseille, France (S Aherfi, Prof P Colson, and soils worldwide, which suggests human beings are a patient with Hodgkin’s lymphoma. M Million, Prof H Lepidi, 2,3 likely to be exposed. Prof D Raoult); and Institut The presence and potential pathogenic role of these Case presentation Paoli-Calmettes, Marseille, giant viruses in human beings is still poorly understood. A 30-year-old woman living in Marseille was admitted to France (Prof L Xerri) have been increasingly associated with our hospital in May, 2014, for a dry cough and with a 1-year Correspondence to: pneumonia since their discovery in 2003, and two isolates history of lymphadenopathies. She received glucocorticoids­ Prof Didier Raoult, Unité des Rickettsies, Faculté de Médecine, 4–8 were obtained from pneumonia patients. Furthermore, intermittently for asthma. Clinical examination revealed Aix-Marseille Université, Acanthocystis turfacea chlorella 1, of the family bilateral axillar and left subclavicular­ lympha­denitis. PET 13385 Marseille CEDEX 05, France , which is linked to within and CT scanning revealed several hypermetabolic­ [email protected] the proposed order Megavirales, has been found in the lymphadenopathies (cervical, supra­clavicular, axillar, pharynx of people with cognitive disorders.9 The family mediastinal, latero-aortic, coeliac, and in the internal Marseilleviridae is another clade of giant viruses that mammary chain [standardised uptake value: 11·5]), at least infect amoebas, the first member of which, marseillevirus, two splenic nodules, and a diffuse bone marrow was described in 2009.10,11 Marseilleviruses have homogeneous hypermetabolism (figure 1). Laboratory icosahedral capsids with a diameter of around 250 nm parameters revealed T cell (T) and natural killer cell (NK) and have a length of 346–386 kilobase pairs that lymphopenia (0·9 G/L), a slightly raised polynuclear cell encode 444–496 putative proteins.12 Marseilleviruses count (12 G/L), and a high alkaline phosphatase had previously been isolated primarily from the concentration (129 IU/L). Serum electrophoresis environ­ment.10 However, since 2012, they have also been showed no evidence of a monoclonal gammapathy. found in human patients (table 1). Thus, senegalvirus Serological tests were negative for hepatitis B virus, was serendipitously isolated from the stools of a healthy hepatitis C virus, HIV, and cyto­mega­lovirus, and indicated young Senegalese man.13 Subsequently, giant blood previous infection and immunity against Epstein-Barr marseillevirus DNA was found in the metagenome virus. Pathological examination of a left axillar lymph node generated from the blood of a blood donor in Marseille, biopsy sample showed evidence of many Reed-Sternberg France, which was confirmed by assemblingand Hodgkin cells surrounded by an inflammatory approxi­mately 10–13 kilobase pair large contigs, virus granuloma composed of lymphoid elements, macrophages, detection with fluorescence in situ hybridisation (FISH), and eosinophils. Immuno­pheno­typing showed that the immuno­­fluorescence, and serology.14 Additionally, lymphoma cells strongly expressed CD30 but not the CD15, marseillevirus DNA and antibodies have been detected CD45, CD20, CD3, or ALK1 proteins. These results were www.thelancet.com/infection Published online August 4, 2016 http://dx.doi.org/10.1016/S1473-3099(16)30051-2 1 Grand Round

1 Virus Samples Case-patient or Country Methods Evidence population 2012 Senegalvirus Stools One healthy young Senegal Metagenomics, amoebal co- Marseillevirus isolated from a human being13 man culture 2013 Giant blood Blood One healthy blood France Metagenomics,5 PCR, FISH, Report suggesting the presence of marseillevirus donor lymphocyte culture, transmission marseilleviruses in the human blood14 electron microscopy, serology 2013 Giant blood Serum 20 blood donors France Serology, PCR Prevalence of 10% of marseillevirus DNA and 15% marseillevirus of antibodies, suggesting frequent human exposure to the virus14 10 2013 Marseillevirus Serum 174 blood donors and France Serology, PCR Higher prevalence in multitransfused patients 22 multitransfused than in blood donors of marseillevirus DNA (9% vs patients with 4%) and IgG antibodies (23% vs 13%) suggesting thalassaemia possible marseillevirus transfusion-transmission15 2013 Lausannevirus Serum 517 young healthy Switzerland Serology Seroprevalence of 1·7–2·5% in a general population, adults confirming human exposure to marseilleviruses16, 15

2013 Marseillevirus Lymph 11-month old boy France PCR, FISH, immunofluorescence, Detection of a marseillevirus in a symptomatic node, with lymphadenitis immunohistochemistry, serology patient and in the lymph node17 serum 2016 Marseillevirus Lymph 30-year-old woman France PCR, FISH, immunofluorescence, Association of a marseillevirus with lymphoma 20 node, with Hodgkin’s immunohistochemistry, serology (present case) plasma lymphoma

FISH=fluorescence in-situ hybridisation.

Table 1: Reports of cases confirming the occurrence of marseilleviruses in human beings, by year 25

associated with lymphadenitis19 and for marseillevirus by PCR, which was implemented when we found marseillevirus-like viruses in a paediatric case of 30 lymphadenitis.17 Indeed, the lymph node of the described patient was part of a series of 285 lymph nodes sent to our laboratory for microbial diagnosis between January and August, 2014, of which 281 tested negative for marseillevirus DNA. In particular, this series included 35 ten Hodgkin’s lymphomas and seven non-Hodgkin lymphomas. The three other lymph nodes that were positive by PCR included one mycobacterial infection, one showing inflammatory granulomas, and one showing areas of foamy macrophages in an HIV-infected 40 individual. We obtained from the lymph node of the described patient a 198 base pair long fragment by Sanger sequencing that was 100% identical to the marseillevirus genome (GenBank accession: NC_013756.1). 45 Marseillevirus was also detected by immuno­histo­ chemistry, FISH, and direct immunofluorescence by use of specific mouse polyclonal antibodies, following previously described procedures (figure 2A–H).17 By contrast, no signal generated by FISH or immuno­ 50 fluorescence was observed for 64 other lymph nodes Figure 1: PET and CT scan for the patient reported tested, including 45 from patients with non-Hodgkin Arrows indicate several hypermetabolic cervical, supraclavicular, axillar, mediastinal, latero-aortic, coeliac, and internal mammary chain lymphadenopathies. lymphomas, 14 from patients with Hodgkin’s lymphomas, two from patients with follicular lymphoid consistent with classic nodal Hodgkin’s lymphomas with hyperplasia, and three with healthy histology. Negative mixed cellularity (WHO classification type 3).18 55 and positive controls were Acanthamoeba castellanii The lymph node biopsy sample was also sent for cultures either uninfected or infected with marseillevirus microbiological diagnosis and tested for pathogens strain T19, respectively. Embedding electron microscopy

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A B 1 C

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10 D E DAPI F Fluorescence in-situ hybridisation

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G Immunofluorescence H Combination 20

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Figure 2: Detection of marseillevirus in the pathological lymph node (A, B, C) Immunohistochemical detection of marseillevirus in the patient lymph node by use of a mouse polyclonal anti-marseillevirus antibody at a 1/1000 dilution with haemalum counterstaining. Arrows show the intracellular location of the virus in the macrophage cytoplasm (magnification x100 [A],× 400 [B and C]). (D) Combined detection of marseillevirus infection in a thin section of the lymph node can be shown by FISH (DNA probe 152F1-153R2,12 in green) and immunofluorescence (anti-marseillevirus antibody, in red); nucleic acid was stained50 with 4’,6-diamidino-2-phenylindole (DAPI). The grey box, inset, indicates the region magnified for figures E–H. (E–H) Images at a greater magnification were obtained by DAPI staining (E), FISH (F), immunofluorescence (G), and all combined (H) for a cell from the lymph node section in which marseillevirus was detected. Confocal microscopy settings were in sequential mode between frames to avoid overlapping fluorescence signals between channels. Image averaging methods were used to enhance image quality (see also supplementary movie S1). Electron microscopy images (I,J) of the embedded lymph node show giant virion-resembling structures, which are indicated by arrows (see appendix). See Online for appendix done on a lymph node specimen stripped of paraffin55 to marseillevirus were retrospectively detected on one showed images that might be compatible with giant plasma sample obtained in June, 2014, by ELISA virions (figure 2I,J). Furthermore, IgG antibodies specific (test/cutoff optical density ratio of 1·35), and www.thelancet.com/infection Published online August 4, 2016 http://dx.doi.org/10.1016/S1473-3099(16)30051-2 3 Grand Round

1 A Negative control B Positive control C Case patient

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15 Figure 3: Anti-marseillevirus IgG detection by immunofluorescence in blood serum (A) Negative control (serum from a rabbit not exposed to marseillevirus, at the dilution 1/50), (B) positive control (serum from a rabbit exposed to marseillevirus, at the dilution 1/400), and (C) case patient at magnification × 40 (serum obtained in June, 2014, at the dilution 1/50).

immunofluorescence, which showed strong reactivity20 which is partly attributed to improved diagnostic tests and (figure 3). This serology was done with serum samples the emergence of AIDS-associated non-Hodgkin lympho­ obtained from rabbits and mice exposed to marseillevirus mas. Following this period, the prevalence of non-Hodgkin and adsorbed on A castellanii, serum samples from lymphoma has plateaued. animals not exposed to marseillevirus, and human being serum samples that had been found seronegative and 25 Associations of infectious agents with lymphomas and seropositive for marseillevirus in previously published mechanisms underlying a causal link experiments.15 Although aetiologies of Hodgkin’s lymphoma and From July 2014, the patient underwent a standard non-Hodgkin lymphoma have not been comprehensively chemotherapy regimen for Hodgkin’s lymphomas characterised, considerable evidence pointing to the consisting of two courses of bleomycin, etoposide, 30 association of several pathogens with lymphomagenesis doxorubicin, cyclophosphamide, vincristine, procarbazine, has been presented. The possible infectious nature of and prednisolone. Complete remission was achieved, and Hodgkin’s lymphomas was suspected from the 1960s on chemotherapy was scaled back to four additional courses the basis of histological characteristics (inflammatory of a combination of doxorubicin, bleomycin, vincristine, infiltrate surrounding the malignant cells), bimodal age and dacarbazine. 35 distribution (third decade and sixth/seventh decade), geographical incidence variations, and social-class risk Review and discussion factors of this disease.24,25 The contribution of the Classification and epidemiology of lymphomas Epstein-Barr virus to the development of some Hodgkin’s Hodgkin’s lymphomas are characterised by large, mono­ lymphoma was then established, and their association nucleated Hodgkin and multinucleated Reed-Sternberg 40 with other infectious agents was described. In a study cells, which originate from germinal B cells but most involving 7414 patients with Hodgkin’s lymphoma and frequently express non-B-cell genes.20 The disease has been 29 240 controls, the risk of Hodgkin’s lymphoma was classified in two main entities, classic Hodgkin’s lympho­ significantly increased by 11% in the event of any mas and nodular lymphocyte-predominant Hodgkin’s previous infection history.25 As for Hodgkin’s lymphomas, lymphomas.18 The estimated incidence of Hodgkin’s 45 the aetiologies and pathophysiology of non-Hodgkin lymphomas is 2 ·7–2· 8 cases per 100 000 per year in the lymphomas remain incompletely understood. Risk USA and the UK and has been largely stable over the past factors that were associated with development of these two decades.20,21 The incidence is greater in men than in cancer subtypes include genetic susceptibility, infectious women, and peaks in young adults—in whom Hodgkin’s agents, and blood transfusions.26–31 The parallel between lymphoma is one of the most common cancers—and 50 an increase in erythrocyte blood transfusions and people older than 60 years. Other lymphomas include a lymphomas27–31 raises the question of an unknown, blood heterogeneous group of lympho­proliferative disorders with transmitted agent associated with lymphoma. Against distinct histological, immuno­phenotypic, genetic, bio­ this backdrop, the marseillevirus, which has been found logical, epidemiological, and clinical features.22 These in the blood of asymptomatic blood donors, might be a non-Hodgkin lymphomas account for approximately 5·1% 55 candidate.14,32 of all cancer cases. Their incidence has increased in many Three main mechanisms underlying the association high-income countries between the 1950s and the 1990s,23 of pathogens with lymphomas have been described.26

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First, lymphotropic viruses can induce the direct 1 herpes virus 8,36 Merkel cell polyomavirus,37 hepatitis B transformation of lymphocytes, leading to clonal virus,38 and hepatitis C virus,39 and bacteria such as H expansion. This process is related to viral genome pylori,40 C jejuni,41 Chlamydia psittaci,42 Borrelia persistence in a latent form, in infected cells, whether burgdorferi,43 and Coxiella burnetii44 have been identified or not they are integrated into the host DNA, and can 5 in association with lymphomas (table 2). involve viral oncogenic proteins. Second, agents such as Helicobacter pylori, Campylobacter jejuni, or hepatitis Criteria for causal link between infectious agents and C virus could act via prolonged antigenic stimulation. lymphomas Thus, given particular host predispositions,­ which Causal relationships between infectious agents and probably include immune, genetic, and epigenetic 10 cancer were first described a century ago, although this determinants, chronic stimulation of the immune association was subsequently overlooked for several system induced by an infectious agent might lead to decades.49,50 Infectious agents are considered to cause 16% the selection of abnormal B cell clones and, ultimately, of malignancies worldwide, 7% of malignancies occurring to lymphoma. Finally, immuno­suppression caused by in high-income countries, and 23% of those occurring in HIV can favour the carcinogenesis­ process induced by 15 low-income and middle-income countries.45 In terms of other viruses. This outcome implies that the natural other malignant neoplasms, it is difficult to define the mechanisms­ of immune surveillance of cancer cells causative principles for infectious agents in lymphomas, are overcome. Overall, viruses such as Epstein-Barr particularly in cases of rare associations or, conversely, in virus,33 HIV,34 human T lymphotropic virus,35 human cases of a high prevalence of the infectious agent in the 20

Prevalence in cases Odds ratio Lines of evidence in human beings Epstein-Barr virus Hodgkin’s lymphoma Europe, USA: 20–50% 4 if the patient has a High antibody titres; China: 50% past history25 of cell-free Epstein-Barr virus DNA in plasma or serum samples; Peru: 94% infectious detection of monoclonal Epstein-Barr virus in Reed-Sternberg cells; mononucleosis expression of Epstein-Barr virus latent membrane proteins; detection of RNA transcripts in tumour cells Burkitt’s lymphoma Sub-Saharan Africa Sub-Saharan Africa: High antibody titres; (endemic): 100% >20 cell-free Epstein-Barr virus DNA in plasma or serum samples; Europe, USA: 20% 30 Epstein-Barr virus detection in tumour cells Nasal T/NK lymphomas 100% ·· High antibody titres; cell-free Epstein-Barr virus DNA in plasma or serum samples; Epstein-Barr virus detection in tumour cells Post-transplant lymphoma 77% ·· Cell-free Epstein-Barr virus DNA in plasma or serum samples; high Epstein-Barr virus DNA load is predictive of lymphoma 35 development AIDS-associated lymphoma 30–100% depending on the ·· ·· type of lymphoma HIV Hodgkin’s lymphoma ·· 5–30 ·· Systemic non-Hodgkin lymphoma ·· ·· 40 ·· Primary CNS lymphoma 100% ·· ·· Primary effusion lymphoma ·· ·· ·· Hepatitis B virus Non-Hodgkin lymphoma 10% but great 2·6 ·· heterogeneity by region 45 Hepatitis C virus B-cell lymphomas ·· Europe, USA, Japan, Anti-tumour response with antiviral therapy South Korea, Australia, Canada, Egypt: 2–2·4 Borrelia burgdorferi ·· ·· 50 ·· Cutaneous marginal zone ·· ·· The organism is shown to be present in lesioned skin of patients lymphoma with primary cutaneous B-cell lymphoma Coxiella burnetii ·· ·· ·· Diffuse large-B-cell lymphoma; ·· ·· ·· Follicular lymphoma 55 (Table 2 continues on next page)

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1 Prevalence in cases Odds ratio Lines of evidence in humans (Continued from previous page) Human T lymphotropic virus 1 Adult T cell lymphoma/leukaemia 100% ·· 5 ·· Human herpes virus 8 Primary effusion lymphomas 100% in AIDS-associated ·· Detection of human herpes virus 8 DNA in tumour malignancies Diffuse large-B-cell lymphomas ·· ·· ··

Multicentric Castleman’s ·· ·· 10 Detection of human herpes virus 8 DNA in tumour disease-associated plasmablastic non-Hodgkin lymphomas Merkel cell polyomavirus Diffuse large-B-cell lymphoma ·· ·· Epidemiological data based on serological findings Campylobacter jejuni ·· ·· ·· 15 Immuno-proliferative small ·· ·· Molecular detection in lymphoma biopsies and intestinal disease immunohistochemistry; lymphoma response after antimicrobial treatment Helicobacter pylori ·· ·· ·· Mucosa-associated lymphoid 86% but varies according 2·8 Epidemiological data; anti-tumour response with antimicrobial tissue lymphoma to geographical areas 20 therapy Primary gastric non-Hodgkin 6·3 Acquisition of gastric lymphoid tissue is shown in response to local lymphomas infection Chlamydia psittaci ·· ·· ·· Ocular adnexal marginal zone Up to 23% but great ·· Detection of bacterial DNA in tumour biopsy specimens lymphoma variations according to geographical areas 25

Data are from several sources.38,39,42,44–48

Table 2: Infectious agents associated with lymphomas

30 general population. This difficulty can be attributed to the NK lymphomas; in these associations the alteration of latency between infection and tumour development and growth regulation in B cells and induction of B-cell the existence of multiple cofactors. Additionally, some transformation have been shown.46,54 Human herpes infectious agents might not be directly oncogenic, but virus 836,55 also recognised as carcinogenic, is linked to could modify the phenotype of a pre-existing tumour 35 primary effusion lymphomas and related to diffuse leading to progression. Several causality criteria have large-B-cell lymphomas. Human T lymphotropic virus 1 been proposed for infectious agents since Henle-Koch’s is associated with adult T-cell leukaemia/lymphoma.56 hypotheses were put forward—notably Bradford and Merkel cell polyomavirus seropositivity was found to be Hill’s criteria—and causative principles have evolved in significantly higher in patients with diffuse large-B-cell line with technological improvements and introduction 40 lymphomas than in controls,57 and an increased risk of of new tests.51–53 These criteria rely mostly on non-Hodgkin lymphomas has been observed after epidemio­logical data, including those inferred from Merkel cell polyomavirus infection.58 Hepatitis B virus serology, results from various molecular-based qualitative and hepatitis C virus might double the overall risk of and quantitative approaches, and evidence of oncogenicity developing a non-Hodgkin lymphoma.39,59 H pylori is in animal models and culture cell experiments (table 3). 45 associated with gastric mucosa-associated lymphoid Molecular criteria include detection of the infectious tissue lymphoma in up to 92% of cases.60–62 C jejuni has agent in tumours and high bacterial or viral loads in been associated with immunoproliferative small tumours, or other body sites. A single criterion is not intestinal disease, a variant of extranodal marginal zone sufficient to consider the infectious agent as being causal, lymphoma.63,64 C psittaci might be associated with ocular but not all criteria are necessary to establish the causal 50 adnexal marginal zone lymphoma, and regression of this link. These criteria have been variously fulfilled in cases lymphoma has been observed post-eradication of this in which infectious agents were associated with bacteria by antibiotic therapy, although the association is lymphomas. still under debate.42,65 B burgdorferi has been associated with some instances of skin marginal zone lymphoma in Infectious agents associated with lymphomas 55 Europe, but this association was not found in another Epstein-Barr virus has been associated with Burkitt’s, case series.43,66,67 In 2015, Melenotte and colleagues44 first Hodgkin’s, HIV-associated, post-transplant, and T and described an association between C burnetii and B-cell

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non-Hodgkin lymphomas. Finally, people with HIV 1 Present case infection have a relative risk ratio varying from 23 to 353 of developing lymphoma in the absence of antiretroviral Clinical experiments therapy, compared with the general population, with an Epidemiology, serology incidence of Hodgkin’s lymphomas that is five to 30 times 5 Greater prevalence of the infectious agent in lymphoma patients than in controls ND higher, a greatly increased risk of diffuse large-B-cell Exposure to the infectious agent precedes lymphoma development ND lymphomas, primary lymphomas of the CNS, and Elevated or abnormal titres of antibodies to the infectious agent Yes Burkitt’s lymphoma, which are AIDS-defining events Molecular biology (table 2).68–70 Detection of DNA/RNA of the infectious agent in tumour Yes 10 High infectious agent DNA/RNA load in tumour ND Marseillevirus Higher infectious agent DNA/RNA load in lymphoma patients than in controls ND We have reported here the presence of a giant Detection of microbial or viral transcripts in tumour by fluorescence in-situ hybridisation ND marseillevirus in the lymph node of the described Metagenomics patient, which has been linked to an association with Detection of infectious agent sequences in the metagenome generated from a ND lymphoma. This is an important finding, since it is based 15 tumour specimen on convergent results obtained with three different Immunohistochemistry techniques (PCR, FISH, and immunodetection). These Detection of microbial or viral antigens in tumour Yes techniques showed signals similar to those of positive Phenotyping/genotyping controls and co-localised with 4', 6-diamidino-2- Bacterial or viral monoclonality in malignant cells ND phenylindole (DAPI) staining under confocal microscopy. 20 Culture We noted no reactivity when these techniques were used Isolation of the infectious agent from the lymphoma patient No on several negative controls. Moreover, we observed a Therapy strong seropositivity to marseillevirus. Lymphoma regression with specific antimicrobial therapy or cytotoxic T cells ND The link between marseillevirus and Hodgkin’s In-vitro experiments lymphoma cannot be established on the basis of this 25 Culture cells single study. The observed association between Ability to immortalise cell lines ND marseillevirus and lymphomas might be a rare Culture cells occurrence. Of the Hodgkin’s lymph nodes tested during Ability to modify migration and invasion phenotypes of cells ND this study for the presence of marseillevirus DNA, one of Animal model ten patients were tested by PCR and one of 14 tested by 30 Carcinogenicity in animal models ND FISH were positive. That notwithstanding, several causality criteria are met (table 3). Our observation ND=not determined. suggests that marseillevirus or close relatives could be Table 3: Evidence from technical approaches for determining role of infectious agents in lymphoma 51–53 emerging pathogens involved in lymphadenitis, as previously reported in a child patient.17 Additionally, their 35 detection in peripheral blood from asymptomatic people was associated with an increased risk of diffuse with high prevalence rates, ranging from 4% to 10%, large-B-cell lymphomas (odds ratio, 1·18; 95% confidence raises the question as to whether some individuals could interval 1·09–1·27), marginal zone lymphoma become chronic carriers (table 1). Moreover, marseillevirus (1·22, 1·03–1·45), lymphoplasmacytic lymphoma (1·59, DNA has been found in multitransfused patients, at a 40 1·32–1·92), T cell non-Hodgkin lymphoma (1·30, higher prevalence (9%) than those observed for blood 1·10–1·54), and Hodgkin’s lymphoma (1·28, 1·04–1·57). donors (4%), and a similar association has been observed Apart from the marginal zone lymphoma, however, this with marseillevirus antibodies (23% in multitransfused increased risk was observed only during the shortest patients vs 13% in blood donors).14,15 Taken together, these latency periods. No significant association had been findings support the hypothesis of possible transmission 45 found between transfusion and non-Hodgkin of marseillevirus via blood transfusion.32 This finding is lymphomas in earlier studies.71,72 Nevertheless, some of noteworthy because previous studies have indicated an these data incite us to consider the possible involvement increased risk of lymphoma development in transfusion of novel blood-borne viruses, including marseillevirus, in recipients, although this association remains lympho­magenesis.31 The understanding of the contro­versial.27,28,71,72 Particularly, Hjalgrim and others29 50 connection between infections and lymphomas are likely showed that blood transfusions were associated with a to be incomplete, since new associations have been significant increase in the risk of cancers, and reported a reported as for the case of C burnetii.44 standardised incidence ratio of 7·2 for non-Hodgkin The presence and persistence in the blood of lymphoma and 9·6 for Hodgkin’s lymphoma, during the marseillevirus could explain viral dissemination and first 6 months after transfusion. In another study30 in the 55 lymph node attainment. Marseillevirus might enter USA involving 77 488 patients with haematological monocytes or lymphocytes, then disseminate from the malignancies and 154 509 controls, blood transfusion peripheral blood and migrate to the lymph nodes through www.thelancet.com/infection Published online August 4, 2016 http://dx.doi.org/10.1016/S1473-3099(16)30051-2 7 Grand Round

1 patient with Hodgkin’s lymphoma, opens a new field of Search strategy and selection criteria research into previously unknown infectious agents in We identified data for this Grand Round through searches of human beings by use of promising new techniques to PubMed with the following terms: lymphoma AND infections study the human virome, particularly metagenomics. OR “infectious agents associated lymphoma” OR “Hodgkin’s 5 The use of these techniques could allow the disease” AND epidemiology; lymphoma* (infectio* OR virus* characterisation­ of lymphomas associated with OR bacteria) (causal*[TIAB] OR cause[TIAB] OR epidemics of novel infectious agents. Finally, this etiolog*[TIAB]); and the following MeSH terms: “Hodgkin finding highlights the need to continue further Disease/cytology”[MeSH] OR “Hodgkin Disease/ systematic investigation of marseillevirus to determine diagnosis”[MeSH] OR “Hodgkin Disease/epidemiology”[Mesh] 10 the association with lymphomas. Additionally, wider OR “Hodgkin Disease/etiology”[MeSH] OR “Hodgkin Disease/ investigations into the role of infectious agents as genetics”[MeSH] OR “Hodgkin Disease/history”[MeSH] OR cofactors in lymphomas need to be addressed because “Hodgkin Disease/microbiology”[MeSH] OR “Hodgkin the epidemiology is not yet fully understood. Disease/mortality”[MeSH] OR “Hodgkin Disease/ Contributors physiology”[MeSH]). References from relevant articles were 15 SA, PC, RC, MM and DR wrote the manuscript. GA and AV did the also searched. The search was limited to articles published in FISH analyses. CN and SA did the serological analyses. HL did immunohistochemistry on the lymphoma biopsy sample. LX English between Jan 1, 1960, and March 10, 2016. participated in the discussion. RC provided the clinical information. DR designed and supervised the study. Declaration of interests lymphatic vessels. Thus, marseillevirus has been shown 20 We declare no competing interests. Funding sources had no role in the to enter into Jurkat cells, an immortalised line of human design and conduct of the study, collection, management, analysis, and T lymphocyte cells, which supports the possibility of a interpretation of the data, nor the preparation, review or approval of the manuscript. viral lymphotropism.14 Moreover, in the patient described in the case study and in a previous report of a child with Acknowledgments 17 We thank Jean-Pierre Baudoin for acquiring the electron microscopy adenitis, the immunohistochemistry showed the 25 images. presence of marseillevirus in macrophages. Similarly, it References has been reported that mimivirus, the first discovered 1 La Scola B, Audic S, Robert C, et al. A giant virus in amoebae. giant virus of Acanthamoeba spp and a relative of Science 2003; 299: 2033. marseillevirus, is capable of entering mouse and human 2 Sharma V, Colson P, Pontarotti P, Raoult D. Mimivirus inaugurated in the 21st century the beginning of a reclassification of viruses. macrophages, which was shown to occur through 30 Curr Opin Microbiol 2016; 31: 16–24. 73 phagocytosis. Additionally, this entry process was 3 Aherfi S, Colson P, La Scola B, Raoult D. Giant viruses of amoebas: similar to that observed for mimivirus into Acanthamoeba an update. Front Microbiol 2016; 7: 349. spp itself. The size of these giant viruses is probably a key 4 Bousbia S, Papazian L, Saux P, et al. Serologic prevalence of -associated microorganisms in intensive care unit determinant that enables their ingestion by phagocytic pneumonia patients. PLoS One 2013; 8: e58111. cells, independently of any specific receptor, which might 35 5 Raoult D, Renesto P, Brouqui P. Laboratory infection of a subsequently result in a broad range of potential hosts.2,73 technician by mimivirus. Ann Intern Med 2006; 144: 702–03. 6 La Scola B, Marrie TJ, Auffray JP, Raoult D. Mimivirus in Finally, the large gene content of marseillevirus consists pneumonia patients. Emerg Infect Dis 2005; 11: 449–52. of about 450 genes, which is more than five times higher 7 Saadi H, Pagnier I, Colson P, et al. First isolation of Mimivirus in a than in Epstein-Barr virus.12 Additionally, the patient with pneumonia. Clin Infect Dis 2013; 57: e127–34. marseillevirus genome has been described as a mosaic of 40 8 Saadi H, Reteno DG, Colson P, et al. Shan virus: a new mimivirus isolated from the stool of a Tunisian patient with pneumonia. genes inferred to originate from , archaea, Intervirology 2013; 56: 424–29. bacteria, and other viruses.10 Moreover, more than half of 9 Yolken RH, Jones-Brando L, Dunigan DD, et al. Chlorovirus ATCV-1 the genes encode what are known as hypothetical proteins, is part of the human oropharyngeal virome and is associated with changes in cognitive functions in humans and mice. whose function is unknown, and most of them have no Proc Natl Acad Sci USA 2014; 111: 16106–11. available homologues in sequence databases outside the 45 10 Boyer M, Yutin N, Pagnier I, et al. Giant Marseillevirus highlights the members of Marseilleviridae. Therefore, marseillevirus role of amoebae as a melting pot in emergence of chimeric microorganisms. Proc Natl Acad Sci USA 2009; 106: 21848–53. has a strong potential of interacting with the human 11 Colson P, Pagnier I, Yoosuf N, et al. “Marseilleviridae”, a new family immune system and genes, which might include of giant viruses infecting amoebae. Arch Virol 2013; 158: 915–20. oncogenes and tumour suppressor genes. 12 Aherfi S, La Scola B, Pagnier I, Raoult D, Colson P. The expanding 50 family Marseilleviridae. 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