Salomé Allichon

Pascal Demoly

Luciana Kase Tanno

MD Luciana Kase Tanno

PRE-HOSPITAL AND EMERGENCY CARE OF ANAPHYLACTIC PATIENTS : A CALL FOR BETTER CARE AND COLLABORATION

REMERCIEMENTS

LIST OF TABLES

spécialisées en allergologie en 2017, la quantité et la qualité d'allergologues exerçant sur le territoire français va augmenter et ainsi les ressources de santé pour les patients, dans un monde où l'incidence des maladies allergiques croît [START_REF] Campbell | Fifty years of allergy: 1965-2015: Fifty years of allergy: 1965-2015[END_REF][START_REF] Asher | Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry cross-sectional surveys[END_REF]. En analysant les données de la littérature française, anglo-saxonne et d'Amérique latine à propos de la prise en charge de l'anaphylaxie en pré-hospitalier, nous souhaitons construire des questionnaires facilitant son diagnostic clinique, biologique et thérapeutique, à destination de chaque personne intervenant dans le parcours de soins, du patient à l'allergologue en passant par le médecin généraliste, le pédiatre et/ou l'urgentiste, et ainsi réduire l'incidence de l'anaphylaxie fatale.

BACKGROUND

Anaphylaxis is a severe life-threatening systemic hypersensitivity reaction generally first seen by emergency doctors, general practitioners or pediatricians. The aim of this study is to review the evidence-based data related to the management of anaphylaxis at the pre-hospital and emergency settings in order to possibly propose strategies for prevention and optimization of health care pathways.

METHODS

A systematic review based on five academic databases with qualitative and quantitative analysis of the characteristics of the articles and clinical data was conducted.

RESULTS

8 out of 1278 identified articles were included for analysis, from 3 continents and 3 specialties: allergology, pediatrics and emergency department. Articles insisted on difficulties with diagnosis, [DonnÃAE'à †â€™Ãƒâ€šÃ'©es anonymisÃAE'à †â€™Ãƒâ€šÃ'©es pour especially in pediatric age, and on the delay or under-use of adrenaline. According to the age categories, specific phenotypes at-risk were identified. No studies mentioned the dosage of serum tryptase. Referral to allergist was mentioned in 3 of the 8 documents.

CONCLUSION

Evidence-based data provided highlights of the key issues related to the management of anaphylaxis, such as the difficulties with the diagnosis, lack of collections of samples for tryptase measurement and timely use of adrenaline. More than underlining the need for optimization of heath care pathways for anaphylactic patients, we were able to identify patients at risk to tailor personalized care and suggest questionnaires addressed to health professionals dealing with anaphylaxis in order to improve the quality of care for these patients. KEYWORDS: allergist, anaphylaxis, emergency department, general practitioner, health care pathways, pediatrician, questionnaire, systematic review.

BACKGROUND

In 2019, the World Health Organization International Classification of Diseases 11 (WHO ICD-11) defined anaphylaxis as a severe life-threatening systemic hypersensitivity reaction characterized by being rapid in potentially life-threatening airway, breathing, or circulatory problems and as usually, although not always, associated with skin and mucosal changes (1).

The occurrence of anaphylaxis has increased in recent years. In Europe, it is estimated that about one in 300 people will experience anaphylaxis at some time in their lives [START_REF] Panesar | The epidemiology of anaphylaxis in Europe: a systematic review[END_REF]. Although the number of emergency department visits and hospitalizations associated with anaphylaxis is increasing [START_REF] Turner | Increase in anaphylaxis-related hospitalizations but no increase in fatalities: An analysis of United Kingdom national anaphylaxis data, 1992-2012[END_REF], fatalities remain low [START_REF] Umasunthar | Incidence of fatal food anaphylaxis in people with food allergy: a systematic review and meta-analysis[END_REF] but can be avoidable.

Anaphylaxis requires immediate medical intervention with first line treatment based on intramuscular adrenaline injection and observation during 6 to 12 hours, usually at the emergency department. Even though emergency physicians make the correct diagnosis of anaphylaxis, there is some remain reluctant to administer intramuscular adrenaline [START_REF] Lejeune | Pre-hospital management of paediatric anaphylaxis by French Emergency Medicine physicians: Still to be improved[END_REF]. After an anaphylaxis occurrence, patients should be referred to the allergist to assess the potential cause and to be educated on prevention of recurrences and self-management. Patients must be prescribed one or more epinephrine (adrenaline) auto-injectors (EAI) and educated to use it in the mid-anterolateral thigh, holding it in place for about 3-10 seconds and then to call for medical assistance [START_REF] Muraro | Anaphylaxis: guidelines from the European Academy of Allergy and Clinical Immunology[END_REF]. The limited availability of epinephrine auto-injectors remains a major problem in many countries, as well as their affordability for some patients [START_REF] Cardona | Guía de Actuación en Anafilaxia en Latinoamérica[END_REF][START_REF] Carrillo-Martin | Self-injectable epinephrine: doctors' attitude and patients' adherence in real-life[END_REF][START_REF] Tanno | on behalf of the Joint Allergy Academies*. Action Plan to Ensure Global Availability of Adrenaline Autoinjectors[END_REF].

The most frequent elicitor groups worldwide are food, insect venom and drugs, frequency depending on age, geographical area, and lifestyle [START_REF] Worm | First European data from the network of severe allergic reactions (NORA)[END_REF][START_REF] Tham | Anaphylaxis -Lessons learnt when East meets West[END_REF], explaining why anaphylaxis related to food and insect venom occurs mainly outside the hospital, at home or at school for children.

Anaphylaxis can occur virtually in any setting, therefore, pre-hospital preparedness to manage anaphylaxis is key. Both patients (and their carriers), and Emergency Medical Aid (EMA) health professionals have to be ready to recognize and treat promptly this condition. Education of patients, families, teachers, and formation of health professionals need to insist on urgent and appropriate action.

Most cases of anaphylaxis are first seen by emergency doctors, general practitioners and pediatricians. After referral, diagnosis can be difficult since it is based mostly on the patients' report, worsen by the delay for referral and lack of documentation. Specialized risk stratification is key to avoid new episodes and prevent avoidable deaths. Current health care pathways for anaphylactic patients suffers from the long delays on referral or non referral, increasing the risk for these patients. Delay on referral to allergists can contribute to memory bias from the patients and unprecise clinical history of anaphylaxis episodes. This can contribute to exhaustive investigations, increasing the burden of this condition. Besides, it can lead to false labels of anaphylactic patients as "idiopathic anaphylaxis" in health records.

Anaphylaxis is a public health issue with risk of death that can be avoided with a coordinated health care circuit between first care professionals and the specialist. Patients, families and teachers need to remain up-to-date about how to diagnose, manage and prevent anaphylaxis, especially when potential triggers such as food allergy and medication use rise [START_REF] Anagnostou | Anaphylaxis in Children: Epidemiology, Risk Factors and Management[END_REF].

The aim of this study is to review the evidence-based data related to the management of anaphylaxis at the pre-hospital settings and before referral to the specialist in order to possibly propose strategies for prevention and optimization of health care pathways.

METHODS

Protocol

We performed a systematic review using the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines [START_REF] Page | The PRISMA 2020 statement: an updated guideline for reporting systematic reviews[END_REF]. No ethical approval was required for the performance of this work since it is based on published data.

Patient and Public Involvement

Patients were not involved in the development or conduct of this review.

Data sources and search strategies:

Systematic search of online academic databases PubMed, EMBASE, the Cochrane Library, Lilacs and SciELO was performed from inception to March 23, 2021. The keywords used were broad using database-specific syntaxes and advanced research tools: "anaphylaxis" as MeSH Major Topic AND "care management" as MeSH term, "clinical diagnosis" as MeSH Major Topic AND "anaphylaxis" as MeSH term in PubMed database, using filter "only EMBASE" for EMBASE database, « anaphylaxis » as MeSH term if available in other databases. No restriction of language was done.

Study Selection

We used ZOTERO® software (5.0.96.3) to collect all the files. Duplicates were removed then exclusion by keywords was done (animal model, case report, in vitro, test, urticaria) to finish with manual exclusion on titles not related to the subject. The abstracts of the eligible studies were reviewed focusing on studies about pre-hospital and emergency department anaphylaxis care and excluded in case of non-related topics or if full text was not available. Any doubt was discussed with the last author (LKT).

Data Extraction, Qualitative Analysis

The author extracted the following data from the included studies using two standardized data extraction forms : the first about the characteristics of publication (Table 1) and the second about clinical data (Table 2). Based on these forms, a descriptive analysis was made using basic statistics on Microsoft Excel® sheet.

Support and conflict of interest

No financial support was collected for this study. The first author declares no conflict of interest.

RESULTS

We identified 1278 records from the five databases and report the screening in Figure 1. After removing duplicates, we excluded, based on keywords then manual on title, to have only 145 eligible records. Final exclusion was made on abstracts for non-related topics or if the full text was not available. We included eight full-text papers for analysis [START_REF] Sole | Anaphylaxis in Latin America: a report of the online Latin American survey on anaphylaxis (OLASA)[END_REF][START_REF] Kilger | Acute and preventive management of anaphylaxis in German primary school and kindergarten children[END_REF][START_REF] Misirlioglu | Clinical diagnosis and management of anaphylaxis in infancy[END_REF][START_REF] Chung | Pre-hospital management of anaphylaxis in one Canadian Urban Centre[END_REF][START_REF] Castilano | Pitfalls in anaphylaxis diagnosis and management at a university emergency department[END_REF][START_REF] Aurich | Anaphylaxis in Elderly Patients-Data From the European Anaphylaxis Registry[END_REF][START_REF] Arroabarren | Improving anaphylaxis management in a pediatric emergency department[END_REF][START_REF] Motosue | Increasing Emergency Department Visits for Anaphylaxis, 2005-2014[END_REF].

Descriptive study

From overall 8 articles selected, 3 (37.5%) were published in allergology journals, 2 (25%) in pediatric journal, one in an immunology journal, one in a general science journal and one in a cardiopulmonary emergency journal (12.5% each). Three of the first authors were medical doctors (37.5%), others non specified. First authors were five of allergology (50%), three of pediatrics (37.5%) and one of emergencies (12.5%).

All studies were original articles (100%) and descriptive (87.5%) except one that was a beforeafter comparative study [START_REF] Arroabarren | Improving anaphylaxis management in a pediatric emergency department[END_REF]. Most of studies were retrospective (75%), two were prospective (25%). Three studies explored populations from Europe (37.5%), two from the USA (25%) and individual documents covered Canada (12.5%), Turkey (12.5%) and Latin America and Portugal (12.5%) populations.

Three studies explored specifically a pediatric population (37.5%), one the elderly population (12.5%). The others had no age limit (50%), including patients from 0.2 to 97 years old.

The number of subjects varied from 60 to 56 212 with a median of 111.5 and the selection of publications covered a total of 58 379 patients.

The definition of anaphylaxis was the World Anaphylaxis Organization's (WAO) [START_REF] Simons | World Allergy Organization Guidelines for the Assessment and Management of Anaphylaxis[END_REF] for one study (12.5%), Sampson and the National Institute of Allergy and Infectious Diseases/Food Allergy and Anaphylaxis Network's one (NIAD/FAAN) [START_REF] Sampson | Second symposium on the definition and management of anaphylaxis: Summary report-Second National Institute of Allergy and Infectious Disease/Food Allergy and Anaphylaxis Network symposium[END_REF] for four studies (50%), and European Academy of Allergy and Clinical Immunology in childhood position paper (EAACI) [START_REF] Muraro | The management of anaphylaxis in childhood: position paper of the European academy of allergology and clinical immunology[END_REF] for two studies (25%). One study (12.5%) used severity degree of anaphylaxis as a definition of anaphylaxis based on Ring and Messmer (Grade ≥ II) [START_REF] Ring | INCIDENCE AND SEVERITY OF ANAPHYLACTOID REACTIONS TO COLLOID VOLUME SUBSTITUTES[END_REF].

The rate of anaphylaxis on emergency admission was 4.8 per 100 000 cases per year for children under fourteen years old in Spain [START_REF] Arroabarren | Improving anaphylaxis management in a pediatric emergency department[END_REF], prevalence calculated at 1.5% in German children [START_REF] Kilger | Acute and preventive management of anaphylaxis in German primary school and kindergarten children[END_REF]. In the USA, the overall rate of anaphylaxis per 100,000 enrollees increased by 101%, from 14.2 in 2005 to 28.6 in 2014 (P < .001) [START_REF] Motosue | Increasing Emergency Department Visits for Anaphylaxis, 2005-2014[END_REF]. Only this last article mentioned a trend with an increase of anaphylaxis in all ages but higher between 5-to 17-years-old.

Clinical data

One study mentioned the ethnic characteristic, of 60% African American [START_REF] Castilano | Pitfalls in anaphylaxis diagnosis and management at a university emergency department[END_REF].

The sex predominance was male in all the three pediatric studies (100%), then female predominance (100%) for the studies with no age limit and the one concerning the elderly (> 65 years old).

Half of the studies (50%) mentioned the location of the episode of anaphylaxis: at home was always the predominant place, but no information was reported regarding trigger per location.

The first main suspected trigger was food in all the pediatric studies (100%), also in three all age studies (75%) and drugs for one (25%). For the study about the elderly, insects were the first cause.

No trigger has been identified or suspected between 11 and 57% in seven studies.

Anaphylaxis was first manifested with cutaneous and respiratory presentation in children and associated with cardiovascular signs in up to 80% of the elderly population (Table 3).

The severity appeared in 50% of the articles with three different classifications [START_REF] Muraro | The management of anaphylaxis in childhood: position paper of the European academy of allergology and clinical immunology[END_REF][START_REF] Ring | INCIDENCE AND SEVERITY OF ANAPHYLACTOID REACTIONS TO COLLOID VOLUME SUBSTITUTES[END_REF][START_REF] Cox | Speaking the same language: The World Allergy Organization Subcutaneous Immunotherapy Systemic Reaction Grading System[END_REF]. Four articles (50%) mentioned fatal or near fatal data, mainly in elderly population. Five studies (62.5%) reported the hospitalization or admission to intensive unit care.

Regarding comorbidities, atopy was mentioned in four studies (50%) (with high rates between 70 and 85% in pediatric studies) and asthma in four studies (50%). Cardiovascular diseases and cardiovascular drugs were mentioned respectively in three (60%) and two (40%) studies covering adult population. Number of episodes of anaphylaxis or previous anaphylaxis was reported in three studies (37.5%).

None of the studies reported tryptase dosage.

First aid management or treatment was mentioned in five studies (62.5%). The use of adrenaline was reported in seven studies (87.5%), antihistamines and steroids in five (62.5%), beta mimetics inhaled treatments in two (25%), and no treatment received in two studies (25%). The use of adrenaline was for pediatric population between 1.2 to 36%, for studies with no age limit between 35 and 54% and 30% for the elderly. Three studies (37.5%) gave information about the prescription of autoinjectors of adrenaline.

Referral to allergist was reported in 3 documents (37.5%). to 3 hours or even longer after the onset of the reaction [START_REF] Sala-Cunill | Biomarkers of anaphylaxis, beyond tryptase[END_REF]. Tryptase is a simple and precious tool for a posteriori diagnosis, especially in case of clinical doubt or repeated reactions with no allergen found. The specialist in allergology sometimes needs to prescribe a dosage to do in case of reaction to remind the emergency physician.

Even though the medical history and tests for suspected allergens by the specialist in allergology are keystones to avoid the recurrence of anaphylaxis, the referral to allergist remained low in the few articles mentioning it. As it is the case for antibiotics with increasing bacterial resistance [START_REF] Staicu | Penicillin Allergy Delabeling: A Multidisciplinary Opportunity[END_REF] a correct labeling and "delabeling" of allergic diseases should expand as it is a systematic question when a doctor collects the medical history of a patient. Anaphylaxis episodes should appear in the medical file of any patient with the confirmed trigger after the allergist evaluation. This evaluation can only be correct if there is a precise history and/or documentation of the episode with a need for collaboration between the doctors managing anaphylaxis at first and the allergist.

Emergency kits can be correctly prescribed if the doctors have an updated formation but it is not enough to ensure that it is correctly used. Education must start from the first reaction, from the doctor to the patient and his entourage. Face-to-face training for patients may improve anaphylaxis knowledge and competence in using autoinjectors [START_REF] Sicherer | Epinephrine for First-aid Management of Anaphylaxis[END_REF].

Limitations :

The study has limitations. Due to the rigorous selection criteria, a limited number of articles were selected which were still heterogeneous. It can also call for more studies in the field to generate comparable data. The comparison of the studies was difficult on several points. Data about anaphylaxis in pre-hospital settings are very scarce and heterogeneous, making comparison between the results of each study difficult. The clinical difficulties for diagnosis of anaphylaxis, missing information on discharge summaries and inaccurate coding are barriers for standardized studies based on codes as ICD. The use of different severity grading system because of no consensus makes the classification of patients in groups that can overlap with others. Data about low-income countries are missing, and other ways of emergency management should be considered as availability of EAI is still a concern [START_REF] Tanno | on behalf of the Joint Allergy Academies*. Action Plan to Ensure Global Availability of Adrenaline Autoinjectors[END_REF].

CONCLUSION

Although the efforts to improve anaphylaxis' management such as the EEACI Task Force in primary care ("Developing a multi-disciplinary approach to the diagnosis and management of allergic disease task force") [START_REF] Ryan | Allergic and hypersensitivity conditions in non-specialist care: Flow-diagrams to support clinical practice[END_REF], there are few outcomes in the past years, with reduced levels of referral to the allergist which implies reduced frequency of diagnosis and risk stratification.

Evidence-based data provided highlights of the key issues related to the management of anaphylaxis, such as the difficulties in the diagnosis, lack of collections of samples for tryptase measures and timely use of adrenaline. More than underlining the need of optimization of heath care pathways for anaphylactic patients, we were able to identify patients at risk to tailor personalized care and suggest questionnaires addressed to health professionals dealing with anaphylaxis in order to improve the quality of care for these patients, which will be validated as the next step of this project. We strongly believe that the data provided here is able to call for public health strategies for better care of anaphylaxis.

LIST OF ABREVIATIONS

CONCLUSION

Le parcours de soins des patients atteints d'anaphylaxie reste à optimiser sur le plan diagnostique, biologique et thérapeutique. À partir des données recueillies et des recommandations, nous avons créé des questionnaires adaptés à chaque échelon du parcours de soins, afin de minimiser le risque de récidive de l'anaphylaxie, parfois létale. MOTS CLES : allergologue, anaphylaxie, questionnaire, médecin généraliste, parcours de soin, pédiatre, revue systématique, urgences

FIGURE 1 :

 1 FIGURE 1: PRISMA FLOWCHART OF THE LITERATURE REVIEW

( 2 )

 2 , mais une cause évitable[START_REF] Pumphrey | Lessons for management of anaphylaxis from a study of fatal reactions: Lessons for management of anaphylaxis[END_REF]. Elle peut se produire dans n'importe quel lieu, l'anaphylaxie induite par les aliments ou les venins d'hyménoptères arrivant la plupart du temps en dehors du système de santé, à domicile ou à l'école pour les enfants. Sa prise en charge a pour recommandation principale l'injection intramusculaire précoce d'adrénaline[START_REF] Cardona | World Allergy Organization Anaphylaxis Guidance 2020[END_REF][START_REF] Tanno | Therapeutic approach of anaphylaxis[END_REF], mais la reconnaissance des symptômes par les patients et les médecins peut parfois être difficile, pouvant ainsi retarder le traitement avec un risque pour la vie du patient. Le dosage de la tryptase sérique est une aide au diagnostic et se réalise par une simple prise de sang idéalement dans les 4 heures suivant le début de la réaction. La plupart des cas d'anaphylaxie sont d'abord pris en charge par les médecins généralistes, pédiatres, urgentistes ou le Service d'Aide Médicale Urgente (SAMU).Cependant leur formation vis-à-vis de l'anaphylaxie est souvent insuffisante malgré les recommandations scientifiques publiées par les sociétés savantes[START_REF] Gfrup | Prise en charge de l'anaphylaxie en médecine d'urgence. Recommandations de la Société française de médecine d'urgence (SFMU) en partenariat avec la Société française d'allergologie (SFA) et le Groupe francophone de réanimation et d'urgences pédiatriques (GFRUP), et le soutien de la Société pédiatrique de pneumologie et d'allergologie (SP2A)[END_REF]. Le parcours de soins en allergologie est pénalisé par une perte d'information lié au biais de mémorisation devant des délais d'attente de plusieurs mois pour une consultation et des patients ou médecins connaissant peu les signes cliniques et les différentes causes à rechercher au moment de l'épisode. Les questionnaires sont des outils diagnostiques ayant démontré leur utilité en médecine. Par exemple, le questionnaire ENDA (European Network for Drug Allergy)(7) est utilisé à travers le monde pour recueillir l'histoire clinique des hypersensibilités médicamenteuses et permet d'orienter le diagnostic et les tests allergologiques indiqués. L'anaphylaxie est une problématique de santé publique souvent négligée dans la formation et le quotidien des médecins généralistes, pédiatres ou urgentistes. C'est pourquoi il existe un réel besoin d'améliorer le diagnostic clinique et biologique avec surtout le dosage de la tryptase, ainsi que le traitement par adrénaline selon les recommandations internationales. Une optimisation du parcours de soins avec orientation vers le spécialiste en allergologie peut permettre d'éviter une récidive d'anaphylaxie, potentiellement mortelle. Avec la création du diplôme d'études

  Data came from different sources: discharge summaries (ICD 9 CM), electronic medical records (ICD 9 and 10 CM), electronic patient care report with Medical Priority Dispatch System MPDS code, standardized questions, questionnaire, European anaphylaxis registry, OLASA (online survey), national administrative claims database. The international classification of diseases (ICD) was used in 2 articles (25%), with 9 and 10 CM versions. The selected documents were published from 2011 to 2019, covering data from 2005 to 2017. The studies covered a mean of 4.4 years, from 4 months to 10 years.

2 :

 2 EAACI: European Academy of Allergy and Clinical Immunology EAI: Epinephrine auto-injector GP : General Practitioner ICD : International Classification of Diseases LKT : Dr Luciana Kase Tanno WAO : World Allergy Organization WHO : World Health Organization Table Clinical data of the included publication related to pre-hospital diagnosis and management of anaphylaxis Table 3: Phenotypes of anaphylactic patients according to the age (n : number of papers ; percentages about the mention of the topic by papers out of the papers of the same age category ; F : female / M : male ; CV : cardiovascular ; GI : gastrointestinal ; severity classifications : *Muraro (32) ; **WAO (34) ; ***Ring & Messmer (33) ; ns : not specified)COMMENTAIRECette revue de la littérature à propos de la prise en charge de l'anaphylaxie en pré-hospitalier et aux urgences souligne (i) les difficultés de diagnostic de l'anaphylaxie pour les différentes spécialités impliquées et donc de codage pour des études épidémiologiques standardisées, (ii) les différents phénotypes de patients en fonction de l'âge avec chez les enfants plutôt des garçons atopiques réagissant avec des aliments et chez les personnes âgées des femmes réagissant aux venins d'hyménoptères avec des antécédents cardiovasculaires et symptômes cardiovasculaires au premier plan avec une plus grande sévérité parfois mortelle, (iii) le lieu d'occurrence qui est principalement le domicile, (iv) la sous-utilisation d'adrénaline par les médecins ou les patients disposant de stylo auto-injecteur dans leur trousse d'urgence, (v) l'oubli du dosage de la tryptase sérique pouvant pourtant confirmer une dégranulation mastocytaire lors des situations cliniques douteuses, (vi) l'adressage insuffisant vers le spécialiste en allergologie qui est la pierre angulaire du diagnostic étiologique et de la prévention de la récidive. En se basant sur nos résultats, nous avons créé des questionnaires (en annexe) pour chaque échelon du parcours de soins, patients, généraliste ou pédiatre ou urgentiste, jusqu'à l'allergologue. Leur but est d'améliorer la prise en charge immédiate et à long terme en limitant le risque d'oubli d'informations souvent précieuses pour guider le diagnostic étiologique et ainsi l'éviction de l'allergène coupable afin de réduire l'incidence de l'anaphylaxie et son risque fatal. La place de l'allergologue ne doit pas se trouver seulement à la fin du parcours de soins mais aussi en prévention primaire et secondaire des allergies. Concernant l'anaphylaxie, c'est aux côtés des urgentistes, médecins généralistes, pédiatres et autres spécialités qu'il doit trouver sa place en améliorant les formations initiales et continues, théoriques et pratiques, et aux côtés de la population générale avec des programmes d'éducation de Santé Publique sur les gestes de premiers secours face à l'anaphylaxie et sur le bon usage des stylos auto-injecteurs d'adrénaline. Nous comptons continuer ce projet avec des études de validation des questionnaires auprès des spécialistes en allergologie du CHU de Montpellier et des hôpitaux associés puis au niveau national, francophone et international. INTRODUCTION L'anaphylaxie est une réaction d'hypersensibilité systémique sévère pouvant menacer le pronostic vital à tout âge et caractérisée par une installation rapide de signes cliniques pouvant mettre en danger la vie. Sa prise en charge est réalisée avant tout par les médecins urgentistes, généralistes ou pédiatres. Le but de cette étude est d'analyser les données de la littérature concernant le diagnostic et la prise en charge de l'anaphylaxie en pré-hospitalier et dans les services d'urgences, afin de créer des questionnaires pour optimiser le parcours de soins du patient jusqu'à l'allergologue. METHODES Réalisation d'une revue systématique de la littérature à partir de cinq bases de données avec analyse des données qualitative et quantitative concernant les caractéristiques des publications et les données cliniques. RESULTATS 8 articles sur un total de 1278 identifiés ont été inclus pour l'analyse, provenant de trois continents et émanant de trois spécialités : allergologie, pédiatrie et médecine d'urgence. Les articles soulignaient les difficultés diagnostiques, notamment en pédiatrie, et le traitement recommandé par adrénaline souvent non réalisé ou retardé. Différents phénotypes ont pu être décrits en fonction des âges. Aucune étude ne mentionnait le dosage ou non de la tryptase. L'adressage au spécialiste n'était mentionné que dans 3 articles sur 8.

TABLE 1 : SUMMARY OF CHARACTERISTICS OF THE INCLUDED PUBLICATIONS

 1

TABLE 2 : CLINICAL DATA OF THE INCLUDED PUBLICATION RELATED TO PRE-HOSPITAL DIAGNOSIS AND MANAGEMENT OF ANAPHYLAXIS

 2

TABLE 3 : PHENOTYPES OF ANAPHYLACTIC PATIENTS ACCORDING TO THE AGE

 3 'anaphylaxie est une réaction d'hypersensibilité systémique sévère pouvant menacer le pronostic vital à tout âge et caractérisée par une installation rapide de signes cliniques pouvant mettre en danger la vie (définition de la Classification Internationale des Maladies CIM-11 -n°4A84) (1).C'est une cause rare de mortalité, estimée à moins de 1% du risque de mortalité totale aux USA

	COMMENTAIRE
	ANNEXES

L

50

RESUMÉ

DISCUSSION

This first systematic review aimed to provide an up-to-date overview of anaphylaxis management in pre-hospital settings and emergency care in order to understand the key unmet needs and suggest possible strategies to reduce the risk and prevent avoidable deaths. We were also able to identify phenotypes at risk, who may require special and personalized care.

Limited and heterogeneous data are available in the field and all articles included were published in the last decade, from different countries and involving authors from different specialties, suggesting that anaphylaxis is a recent ubiquitarian topic of interest. Epidemiological data about rate and trend were scarce but confirmed that anaphylaxis is rare but significant and increasing in the last two decades. With the implementation of the new ICD-11 for anaphylaxis, epidemiological studies will be facilitated as their quality for comparability [START_REF] Tanno | Epidemiology of anaphylaxis[END_REF][START_REF] Tanno | Decreasing the undernotification of anaphylaxis deaths in Brazil through the International Classification of Diseases (ICD)-11 revision[END_REF].

Difficulties for clinical recognition of anaphylaxis by patients and health care professionals explained by non-specificity or ambiguity of symptoms, for example with infants (37), delay the pre-hospital management and injection of epinephrine. However, it is the treatment recommended for anaphylaxis but often mis or under used, placing the patient in danger for his life. Although adrenaline has been mentioned as the treatment of choice by the majority of publications, it has been sparkly used in clinical practice as demonstrated by the limited percentage of patients who received this medication. In other words, it means that almost all practicians are aware of the indication of use of adrenaline in cases of anaphylaxis, but this does not reflect in real life practice, probably due to the difficulties with the recognition of anaphylaxis.

The publications covered all ages making an overview of anaphylaxis for all age groups, with distinguished phenotypes at risk (Table 3). Children with anaphylaxis seem to be predominantly boys with atopic diseases, reacting to food allergen at home, with cutaneous symptoms and mild to moderate severity. In adulthood, anaphylaxis seems to be more frequent in female with cardiovascular diseases, due to drugs and/or to food, manifested with cutaneous symptoms. For the elderly population, we also observed female predominance with cardiovascular diseases, with anaphylaxis induced by insect venom, suggesting outdoor activities, and manifested with more severe reactions with cardiovascular symptoms. With identification of phenotypes at risk, the medical examination and investigations can be orientated towards a better personalized medical care.

No article mentioned serum tryptase dosage although it is the only biological test recommended in case of anaphylaxis with its potential help for diagnosis, with levels increased from 15 minutes

LIST OF FIGURES

LIST OF TABLES


Morgan Gaïa

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Eric Pelletier

Patrick Forterre

Chiara Vanni

Antonio Fernandez-Guerra

Olivier Jaillon

Patrick Wincker

Hiroyuki Ogata

Mart Krupovic

Tom O Delmont

Mirusviruses provide a missing link in the evolution of giant viruses Plankton-infecting relatives of herpesviruses clarify the evolutionary trajectory of giant viruses

Keywords: Mirusviricota, mirusviruses, Tara Oceans, plankton, metagenomics, DNA viruses, Giant viruses, eukaryotes, ecology, evolution, major capsid protein, phylogenomics

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The environmental DNA sequencing and bioinformatics legacies of Tara Oceans and anvi'o exposed a putative new phylum dubbed "Mirusviricota" by means of phylogeny-guided genome-Resolved metagenomics. Unexpectedly, the mirusviruses are plankton-infecting relatives of herpesviruses that clarify the evolutionary trajectory of prominent large and giant viruses from another realm.

Modified from the original planktonic illustration of Noan Le Bescot

Introduction:

Most double-stranded DNA viruses are classified into two major realms: Duplodnaviria and Varidnaviria. Duplodnaviria comprises tailed bacteriophages and related archaeal viruses of the class Caudoviricetes and eukaryotic viruses of the order Herpesvirales. Varidnaviria includes large and giant eukaryotic DNA viruses from the phylum Nucleocytoviricota as well as smaller eukaryotic, bacterial and archaeal viruses with tailless icosahedral capsid 1 . The two realms were established based on the non-homologous sets of virion morphogenesis genes (virion module), including the structurally unrelated major capsid proteins (MCP) with the 'double jelly-roll' and HK97 folds in Varidnaviria and Duplodnaviria, respectively 1 . Both realms are represented across all domains of life, with the respective ancestors thought to date back to the last universal cellular ancestor 2 . Within Duplodnaviria, bacterial and archaeal members of the Caudoviricetes display a continuous range of genome sizes, from ~10 kb to >700 kb, whereas herpesviruses, restricted to animal hosts, are more uniform, with genomes in the range of 100-300 kb. Herpesviruses likely evolved from bacteriophages, but the lack of related viruses outside of the animal kingdom raises questions regarding their exact evolutionary trajectory 3 . Members of the Varidnaviria also display a wide range of genome sizes, from ~10 kb to >2 Mb, but there is a discontinuity in the complexity between large and giant viruses of the Nucleocytoviricota and the rest of varidnaviruses with genomes <50 kb. It has been suggested that Nucleocytoviricota have evolved from a smaller varidnavirus ancestor [4][5][6] , but the complexification entailing acquisition of multiple informational genes (informational module), including those for transcription, such as the multisubunit DNA-dependent RNA polymerase, remains to be fully understood.

The Caudoviricetes and Nucleocytoviricota viruses are prevalent in the sunlit ocean where they play a critical role in regulating the community composition and blooming activity of plankton [7][8][9] . Metagenomic assemblies and genome-resolved metagenomics were used to target these viruses within plankton, complementing culture-dependent virus isolation efforts and leading to our improved appreciation of their genomic diversity 7,8,[10][11][12] . These environmental surveys emphasized the prevalence and functional complexity of DNA viruses in the sunlit ocean and supported the evolutionary demarcation between the virion and informational modules of Varidnaviria and Duplodnaviria realms. Previous efforts to uncover the diversity of large eukaryotic DNA viruses in marine metagenomes largely relied on identification of a nearly full set of Nucleocytoviricota hallmark genes 7,8 . Although this approach ensures high genomic completion and quality of the identified viral genomes, it is by default unsuitable for the discovery of novel marine DNA viruses outside of the Nucleocytoviricota.

Here, we performed a phylogeny-guided genome-resolved metagenomic survey of planktonic DNA viruses containing a DNA-dependent RNA polymerase gene. The survey covers nearly 300 billion metagenomic reads from surface ocean samples of the Tara Oceans expeditions [13][14][15] . We characterized and manually curated >500 Nucleocytoviricota population genomes, expanding the known diversity of the class Megaviricetes and recovering a putative new class. But most notably, our survey led to the discovery of a putative new phylum of large DNA viruses infecting marine eukaryotes, dubbed 'Mirusviricota' (>100 population genomes up to >400 kb in size). These viruses are diverse and prevalent in the sunlit ocean where they likely play a central role in the ecology of abundant planktonic eukaryotes. Members of 'Mirusviricota' contain a virion module characteristic of the realm Duplodnaviria (including a distinct HK97-fold MCP, a possible evolutionary intermediate between the MCPs of Caudoviricetes and Herpesvirales based on protein structure analyses). Yet, these viruses also contain a large fraction of genes more closely related to those of Nucleocytoviricota, including key informational hallmark markers. A nearcomplete mirusvirus genome supports these results. The atypical chimeric attributes of 'Mirusviricota' provide important insights into the evolution of DNA viruses, clarifying the evolution of animal herpesviruses from tailed bacteriophages and giant varidnaviruses from smaller relatives.

Results:

Environmental genomic recovery of a new clade of large DNA viruses DNA-dependent RNA polymerase subunits A (RNApolA) and B (RNApolB) are evolutionarily informative gene markers that are universally conserved in the three cellular domains and occur in most Nucleocytoviricota viruses 5,16 . Here, we performed a comprehensive search for RNApolB genes from the euphotic zone of polar, temperate, and tropical oceans using 798 metagenomes derived from the Tara Oceans expeditions 13 . They correspond to surface waters and deep chlorophyll maximum (DCM) layers from 143 stations covering the Pacific, Atlantic, Indian, Arctic, and Southern Oceans, as well as the Mediterranean and Red Seas, encompassing eight plankton size fractions ranging from 0.8 µm to 2000 µm (Table S1). These 280 billion reads were used as inputs for 11 metagenomic co-assemblies (~12 million contigs longer than 2,500 nucleotides) using geographically related samples, and cellular metagenome-assembled genomes (MAGs) were subsequently characterized 14,15 . We recovered RNApolB genes from these co-assemblies using a broad-spectrum profile hidden Markov model (HMM) and subsequently built a database of more than 2,500 non-redundant protein sequences (similarity <90%; Table S2). Phylogenetic signal for these sequences recapitulated previously observed trends, such as monophyletic groups for Bacteria, Archaea and the three eukaryotic nuclear RNApolB clades 5 , as well as the considerable diversity of Nucleocytoviricota already revealed by means of gene markers 17 and genomeresolved metagenomics 7,8 (Figure S1). The phylogenetic analysis revealed previously undescribed deep-branching lineages with no representatives among known viruses, which we dubbed Mirus (Latin for surprising, strange) once sequential increases in phylogenetic resolution supported their monophyly. With a positioning clearly disconnected from the three domains of life, we hypothesized that the Mirus RNApolB clades may correspond to previously unknown lineages of double-stranded DNA viruses. We performed a two-step phylogeny-guided genome-resolved metagenomic survey of the ~12 million contigs. First, we used the RNApolB genes corresponding to Nucleocytoviricota and Mirus as a compass to identify metagenomic assembly subsets of interest. Then, the sequence composition and differential coverage of contigs across metagenomes were computed independently for each subset, as previously done for the cellular MAGs 15,18 , in order to manually delineate the genomic context of RNApolB genes (see Table S3 and supplemental information for more details). We successfully characterized and curated 587 non-redundant Nucleocytoviricota MAGs (average nucleotide identify <98%) up to 1.45 Mbp in length (average of ~270 Kbp), as well as 111 non-redundant Mirus MAGs up to 438 kbp in length (average of ~200 Kbp). The Mirus MAGs were identified in all the oceanic regions (Table S5), emphasizing their prevalence in the sunlit ocean. This database was supplemented with MAGs from previous metagenomic surveys 7,8 . The final non-redundant genomic resource contained 1,482 marine Nucleocytoviricota MAGs, 224 reference Nucleocytoviricota genomes from culture and cell sorting, and the 111 Mirus MAGs (Table S4). This genomic database, enriched in large and giant marine eukaryotic DNA viruses, has a total volume of 580 Mbp and contains ~0.6 million genes. It was used to map 300 billion metagenomic reads from nine Tara Oceans plankton size fractions (0.2 µm to 2000 µm) 19 . In subsequent sections, we used this database (thereafter called Global Ocean Eukaryotic Viral [GOEV] database) to contextualize the evolutionary history, functional lifestyle and environmental distribution of newly identified Nucleocytoviricota and Mirus MAGs.

Evolutionary links between mirusviruses and the Nucleocytoviricota phylum

The newly assembled Nucleocytoviricota MAGs contain most of the hallmark genes corresponding to the virion and informational modules characteristic of this viral phylum 4,5 (Table S4) and further expand the known diversity of the class Megaviricetes. We also identified one putative new class-level group, 'Proculviricetes' (several MAGs exclusively detected in the Arctic and Southern Oceans) (Figure 1). The Mirus MAGs also contained key genes evolutionarily related to the Nucleocytoviricota informational module, including RNApolA and RNApolB, family B DNA polymerase (DNApolB), and the transcription factor II-S (TFIIS; Figures 1 andS2). For instance, the RNApolA and DNApolB of most Mirus MAGs branched as a sister clade to the order 'Pandoravirales'. Notably, however, the Mirus MAGs were devoid of identifiable homologs of the Nucleocytoviricota virion module, including the hallmark double jelly-roll MCP (Figure 1). Phylogenomic inferences derived from our manually curated database of the four informational gene markers (see Figure 1 and supplemental information) indicate that Mirus represents a new viral clade related to Nucleocytoviricota and organized into seven distinct lineages, M1 to M7 (from the most to least populated), with the latter being represented by a single MAG (Figure 2, Table S4). The Mirus lineages were detected across multiple cellular size fractions. Their distribution suggests they occur as rather large extracellular particles (abundant in the 0.2-3 µm size fraction) and in relation to planktonic eukaryotes (spikes of detection in the larger cellular size fractions), in line with what can be observed for the Nucleocytoviricota viruses (Figures 1 and2). For instance, MAGs from lineages M6 and M4 were enriched in the 3-20 µm and 180-2000 µm size fractions, respectively, in addition to the viral particle size fraction (0.2-3 µm). Collectively, these results suggest that the Mirus MAGs represent viruses of diverse marine eukaryotes with a broad cellular size range (Figure 2A). In addition, mirusviruses were relatively abundant, with a cumulative coverage of 6,479x (i.e., the 111 Mirus MAGs were cumulatively sequenced more than 6,000 times among the considered metagenomes), suggesting that the corresponding viruses infect relatively abundant marine eukaryotes in both temperate and polar waters. Phylogeny-guided host predictions 18 based on co-occurrence patterns using Mirus and eukaryotic MAGs 13 revealed two orders of phytoplankton, Chloropicales (green algae) and Phaeocystales (haptophytes), which showed significant clade-level associations with Mirus subclade M2 (Figure S3 and Table S5). Furthermore, at the individual MAG level, some mirusviruses in other subclades showed a high pairwise association with eukaryotes affiliated to other lineages, such as diatoms and the heterotrophic MAST-4 (Table S5). Together with the large phylogenetic diversity of Mirus MAGs, these results suggest a broad host range of mirusviruses.

Mirusviruses are functionally divergent from Nucleocytoviricota clades

We used AGNOSTOS 20 to characterize 29,414 non-singleton gene clusters from the GOEV database (Table S6). Clustering of Nucleocytoviricota and Mirus genomes based on the occurrence of these gene clusters emphasized the complex functional makeup of the Megaviricetes and Pokkesviricetes classes, with some clades (e.g., the Imitervirales and Algavirales) split into multiple groups (Figure 3). In contrast, the Mirus MAGs clustered together, with the lineages being organized into distinct groups that largely recapitulated phylogenomic signals. Overall, the distribution of gene clusters, which is agnostic of any functional inferences, indicates that Mirus MAGs share genomic traits sufficiently distinct from those occurring in the known Nucleocytoviricota lineages. This comparative genomic analysis strongly supports the monophyly of the Mirus MAGs, corroborating the inferences from the phylogenomic analysis of the informational module. Among the most widespread gene clusters within the viral genomic database, thirtyfive gene clusters occurred in more than 50% of the 111 Mirus MAGs, representing some of the core genes for this newly identified clade (Table S6). Critically, most of these clusters appear to be unique to this clade and 24 lacked any functional annotation (Figure 3A). Among the 11 Mirus core genes with a functional annotation, five were detected in <10% of the known Nucleocytoviricota lineages. They correspond to trypsin and M16-family peptidase (proteases), viral rhodopsin (ion-conducting pathway), TATA-binding protein (transcription), and a histone (DNA stability) (Figure 3B). Phylogenetic trees of histones and viral rhodopsins show several monophyletic clades of Mirus sequences, suggesting that they are relatively distant from the corresponding proteins of both Nucleocytoviricota and marine planktonic eukaryotes (Figure 4). Notably, heliorhodopsins encoded by Micromonas populations (prominent green algae widespread in the sunlit ocean) might have been acquired from mirusviruses through horizontal gene transfer. In contrast to Mirus, 20 out of the 28 gene clusters shared by more than 50% of the Nucleocytoviricota genomes within the GOEV database are functionally annotated. Analysis of these core gene clusters showed that they are highly depleted if not entirely missing in Mirus: RNA pol5, RNA pol10, ribonuclease 3 and the SWIB protein (transcription), ResIII helicase and D5 NTPase (DNA replication), and deoxyribonucleoside kinase (nucleotide metabolism) (Figure 3B). Overall, comparative genomics and the functional inferences strongly suggest that Mirus viruses display a unique lifestyle within plankton with a specific functional gene complement -as compared to the known Nucleocytoviricota lineages -that remains largely enigmatic due to the predominance of core genes for which functional predictions are challenging.

Mirus is a putative phylum ('Mirusviricota') within the realm Duplodnaviria

Given that major virion morphogenesis proteins are typically conserved across members of a particular virus lineage, we focused on the core genes defined in the comparative genomic analysis for identification of the 'missing' virion module of Mirus. We compared HMMs of the Mirus core proteins to a comprehensive HMM database of viral and cellular proteins. This analysis provided no hits for either the double jelly-roll MCP or the FtsK-like genome packaging ATPase, two virion module markers characteristic of the realm Varidnaviria. Instead, this comparison provided a remote hit to a HK97-fold MCP encoded by a Caudoviricetes bacteriophage. The corresponding function occurs in 82% of the Mirus MAGs, mostly in single copy. The hit covers 68% of the template MCP (268/351 aa; YP_004306755), but encompasses only an N-terminal fragment of the considerably longer (>800 aa) query Mirus HMM. To validate the result of the profile-profile comparison and to understand the discrepancy between the phage and Mirus protein sizes, we predicted the 3D structure of the corresponding Mirus protein using AlphaFold2 21,22 and RoseTTAFold 23 . Both programs produced similar models with a readily recognizable HK97-fold domain (Figure 5), corresponding to the N-terminal region recognized in the profile-profile comparisons. Notably, in herpesvirus MCPs, the HK97-fold domain, referred to as the 'floor' domain, responsible for capsid shell formation, is embellished with a 'tower' domain which projects away from the surface of the assembled capsid 24 . The 'tower' domain is an insertion within the A-subdomain of the core HK97-fold 24,25 . Remarkably, the Mirus protein contained an insertion within the A-subdomain, equivalent to the 'tower' domain of herpesvirus MCPs (Figure 5). Such 'tower' domains have not been thus far described for any member of the Caudoviricetes, including the so-called jumbo phages (i.e., phages with very large genome 26 ), explaining the limited coverage of the hit in profile-profile comparisons 18 . The size of the Mirus MCP 'tower' suggests that this protein could represent an evolutionary intermediate between Caudoviricetes (no tower) and Herpesvirales (larger tower) MCPs. Protein divergences prevented any resolutive phylogenetic analyses to compare the HK97-fold MCP of Herpesvirales, Caudoviricetes and Mirus. Yet, phylogenetic inferences of the Mirus MCP alone perfectly recapitulates the seven clades initially identified based on the informational module (Figure 2, panel C), indicative of coevolution of the two functional modules. Consistent with the identification of the HK97-fold MCP, further profile-profile comparisons uncovered all other components of the Duplodnaviria-specific virion module, including the terminase (ATPase-nuclease, key component of the DNA packaging machine), portal protein and the capsid maturation protease (Pfam PF04586, known as assemblin in herpesviruses). The presence of the entire virion module genes in mirusviruses strongly suggests that they are bona fide large DNA viruses capable of forming virus particles similar to those of viruses in the realm Duplodnaviria.

Analysis of the gene content of Nucleocytoviricota, Herpesvirales and mirusviruses indicates that DNApolB is a rare hallmark core gene shared by the three groups of eukaryote-infecting DNA viruses. Phylogenetic inferences of this marker using not only the viral genomic database but also a wide range of eukaryotic and additional viral lineages 27 supported the evolutionary distance of mirusviruses, being distinct from all other known clades of double-stranded DNA viruses (Figure 6). The Mirus DNApolB was positioned in the vicinity of Herpesviridae, Megaviricetes and the Zeta group of eukaryotes (the closest Nucleocytoviricota relative being 'Pandoravirales'), distant from both Pokkesviricetes and Caudoviricetes.

The chimeric nature of 'Mirusviricota' links two major realms of DNA viruses

On the one hand, mirusviruses belong to the realm Duplodnaviria based on their virion module (Figures 2 and5). On the other hand, hallmark informational markers RNApolA, RNApolB and TFIIS (Figure S2), that are all missing in known Herpesvirales lineages, display high sequence similarities to the corresponding proteins encoded by members of the phylum Nucleocytoviricota. Such atypical distribution of the 'Mirusviricota' attributes places this virus group in between the two major realms of DNA viruses, Duplodnaviria (virion module) and Varidnaviria (informational module and other core functions). To further validate the genomic content of mirusviruses and to exclude the possibility of artificial chimerism, we created an HMM for the newly identified 'Mirusviricota' MCP and used it as bait to search for complete genomes in additional databases. First, we only found two 'Mirusviricota' MCPs in a comprehensive viral genomic resource from the <0.2 µm size fraction of the surface oceans (GOV2 10 ), suggesting that virions in this clade are usually >0.2 µm in size. We subsequently screened the 'Mirusviricota' MCP in a database containing hundreds of metagenomic assemblies from the 0.2-3 µm size fraction of the surface oceans 28 . We found a contiguous 'Mirusviricota' genome in the Mediterranean Sea with a length of 431.5 kb, just 6 kb shorter than the longest 'Mirusviricota' MAG. This near-complete contiguous genome contains all marker genes of 'Mirusviricota' and is affiliated to the clade M2 based on both the information module and MCP phylogenies (see Figure 2, panels B and C).

We compared the 355 genes found in the near-complete contiguous genome to two comprehensive genomic databases corresponding to the realms Duplodnaviria (748,546 genes and 57,295 HMMs) and Varidnaviria (269,523 genes and 16,689 HMMs) 29 and found 86 significant hits (HMM searches, e-value <e-6). Only six of them had better matches within the Duplodnaviria database (hits to phages and jumbo phages) and included the terminase protein (best match for a Vibriophage). The remaining 80 genes had better matches within the Varidnaviria (hits to Nucleocytoviricota only) and occurred relatively homogeneously across the genome (Figure 7). These include the RNApolA, RNApolB, DNApolB, topoisomerase II, TATAbinding protein, histone, multiple heliorhodopsins, Ras-related GTPases, cell surface receptor, ubiquitin, and trypsin. While the evolutionary trajectories of the corresponding genes remain uncertain, the shared gene content emphasizes a strong functional connectivity between mirusviruses and the large and giant DNA viruses within the realm Varidnaviria. The near-complete contiguous genome also included two unusually long genes. The first one, 34.6 kb in length, is longer than any gene in the Duplodnaviria and Nucleocytoviricota genomic databases and might represent a record holder in terms of length for a viral gene. NCBI blast provided a weak hit for a YadA-like family protein (cell attachment) used by pathogenic bacteria to invade eukaryotic cells. The second one is 25.1 kb in length and NCBI blast provided a weak hit for a Hyalin Repeat domain also involved in bacterial cell adhesion. Looking at gene lengths more broadly, we also found genes >20 Kbp in Nucleocytoviricota (up to 30.7 kb) and Caudoviricetes (up to 25,7 kb) but not in Herpesvirales (longest gene of 13.9 kb only). Thus, mirusviruses contain unusually long genes, a trait shared with certain members of the Nucleocytoviricota and Caudoviricetes, which may represent common strategies for invading planktonic cells.

Overall, the near-complete contiguous 'Mirusviricota' genome perfectly recapitulated the chimeric attributes of this putative phylum as initially observed based on >100 manually curated MAGs. Sequence similarities between proteins indicated that key functionalities within and beyond the informational module connect 'Mirusviricota' to Nucleocytoviricota. Since 'Mirusviricota' belongs to the Duplodnaviria based on the presence of this realm-specific virion module, we demonstrated that the genomic content of this putative new phylum fills a critical evolutionary gap between the two major realms of double-stranded DNA viruses.

Discussion

Our phylogeny-guided genome-resolved metagenomic survey of plankton in the surface of five oceans and two seas exposed a major clade of large eukaryotic DNA viruses, with genomes up to >400 Kbp in length, that are diverse and prevalent in the sunlit ocean. This clade, dubbed 'Mirusviricota', corresponds to a putative new phylum within the realm Duplodnaviria that until now included the bacteria-and archaea-infecting Caudoviricetes and animal-infecting Herpesvirales. The 'Mirusviricota' phylum is organized into at least seven sub-clades that might correspond to distinct families. Although both mirusviruses and Herpesvirales include the eukaryote-infecting duplodnaviruses, they display very different genomic features. Most notably, mirusviruses substantially deviates from all other previously characterized groups of DNA viruses, with the virion morphogenesis module (a basis for highest-rank viral taxonomy) affiliated to the realm Duplodnaviria and the informational module closely related to that of large and giant viruses within the realm Varidnaviria. These chimeric attributes were recapitulated in a near-complete contiguous genome of 431.5 Kbp and could explain why previous environmental surveys did not identify this putative phylum despite its considerable metagenomic signal. 'Mirusviricota' has a substantial functional overlap with large and giant eukaryotic varidnaviruses that goes well beyond the informational module. The discovery of 'Mirusviricota' is a reminder that we have not yet grasped the full ecological and evolutionary complexity of even the most abundant double-stranded DNA viruses in key ecosystems such as the surface of our oceans and seas.

To our knowledge, 'Mirusviricota' is the first identified eukaryotic virus clade within the Duplodnaviria realm that is widespread within plankton. Biogeographic signal computed by means of genome-wide metagenomic read recruitments indicates that mirusviruses are prevalent and relatively abundant in various regions of the sunlit oceans, from pole to pole. In addition, distribution patterns across planktonic cellular size fractions mirrored those observed for the Nucleocytoviricota lineages, strongly suggesting that mirusviruses actively infect eukaryotic plankton, possibly with a broad distribution of the cellular sizes. Viral-host predictions using the eukaryotic environmental genomic data characterized from Tara Oceans 15 linked some mirusviruses to several eukaryotic lineages including key phototrophs (e.g., haptophytes) that now await experimental validations. In addition, the strong signal for 'Mirusviricota' in the 0.2-3 µm size fraction contrasts with a lack of signal for its major capsid protein in a comprehensive viral genomic resource from the <0.2 µm size fraction 10 . This suggests that the virion of 'Mirusviricota', which remains to be visualized, is larger than 0.2 µm. Indeed, the virions of alloherpesviruses, which infect fish and have substantially smaller genomes compared to mirusviruses, are up to 0.2 µm in diameter 30 . In terms of functions, comparative genomics indicates that 'Mirusviricota' viruses have a rather complex lifestyle (wide array of functions), and encode phylogenetically distinct branches of H3 histones (proteins involved in chromatin formation within the eukaryotic cells 31 ) and heliorhodopsins (viral rhodopsins, light-gated channels already identified in Nucleocytoviricota lineages, for which functional role remains unclear 32,33 ). Analysis of the biogeography and functional gene repertoire suggest that mirusviruses influence the ecology of key marine eukaryotes, using a distinct lifestyle that resembles, to some extent, that of marine varidnaviruses. For instance, phylogenetic analysis suggests that a core Micromonas heliorhodopsin has originated from a mirusvirus, implicating them as important players in the evolution of planktonic eukaryotes by means of gene flow. Concurrently, the close sequence similarity between mirusvirus and Micromonas heliorhodopsins indicates that green algae might serve as hosts for some of the 'Mirusviricota' lineages. As of now, the prevalence and diversification of 'Mirusviricota' indicates that we just started exploring the full extent of the genomic diversity of this virus group within plankton, and possibly beyond. Moving forward, the genomics of 'Mirusviricota' could help solve many hypotheses that orbit around the large and giant eukaryotic DNA viruses, from their potential roles in eukaryogenesis 5,[34][35][36][37][38] , to their regulation of ecologically critical marine eukaryotes 39,40 .

Viruses of the Herpesvirales and Nucleocytoviricota are descendants of two ancient virus lineages, Duplodnaviria and Varidnaviria, respectively, with the corresponding ancestors antedating the last universal cellular ancestor (LUCA) 1,2 . Nevertheless, the exact evolutionary trajectories and the identity of the respective most recent common ancestors of these prominent eukaryote-infecting double-stranded DNA viral clades remain elusive, in part due to the lack of known intermediate states.

Particularly puzzling is the evolutionary trench between relatively simple varidnaviruses (those infecting Bacteria and Archaea, as well as virophages and Adenoviridae), with minimalistic gene repertoires for virion formation and genome replication, and complex eukaryotic varidnaviruses (the Nucleocytoviricota) with large and giant genomes encoding multiple functions, including nearly complete genome replication and transcription machineries. Similarly enigmatic is the gap between the ubiquitous Caudoviricetes, some of which rival Nucleocytoviricota in terms of functional complexity and richness of their gene repertoires, and Herpesvirales, which are restricted to animal hosts and uniformly lack the transcription machinery and practice nuclear replication. The chimeric attributes of 'Mirusviricota' might now help to clarify both of these long-standing questions. It is possible that genome complexity observed in the contemporary 'Mirusviricota' lineages has been attained and inherited from a common ancestor with large Caudoviricetes, an ancient virus lineage which is believed to have diversified at the time of LUCA. Indeed, most of the proteins involved in genome replication and nucleotide metabolism conserved in mirusviruses are also widespread in Caudoviricetes. The closer sequences similarity of these proteins to homologs in Nucleocytoviricota could then suggest that the genes were transferred between ancestors of the two groups after the divergence of 'Mirusviricota' and Caudoviricetes. Such transfer of informational module from a mirusvirus to the ancestor of Nucleocytoviricota would explain a sudden transition from small varidnaviruses to complex Nucleocytoviricota. It is unlikely that the complexity of the Nucleocytoviricota genomes has evolved through gradual accretion of informational genes in a particular lineage of varidnaviruses. The described scenario provides a logical explanation to the emergence of Nucleocytoviricota, without invoking unknown and unknowable extinct virus lineages. Beside the core functions responsible for virion formation and information processing, another important component of both Nucleocytoviricota and 'Mirusviricota' genomes, specifically related to eukaryote-specific aspects of virus-host interactions, has likely evolved more recently though horizontal gene transfers or convergent gene acquisition from different sources, facilitated by shared hosts and ecosystems. Finally, the identification of 'Mirusviricota' extends the presence of duplodnaviruses beyond animals to eukaryotic plankton hosts, strongly suggesting their ancient association with eukaryotes. The presence of the tower domain in both 'Mirusviricota' and Herpesvirales MCPs is suggestive of their common ancestry, rather than independent evolution from distinct Caudoviricetes clades. With the shorter size of the tower domain and considering later emergence of animals compared to unicellular eukaryotes, 'Mirusviricota' viruses might more closely resemble the ancestral state of eukaryotic duplodnaviruses. If so, animal herpesviruses would have evolved by reductive evolution, in particular, by losing the transcription machinery and switching to nuclear replication 1,2 .

Given the relatively early stage of viral metagenomics, it is more than likely that other intermediate states between 'Mirusviricota' and Herpesvirales on one hand, and 'Mirusviricota' and large and giant viruses (the Nucleocytoviricota) on the other hand also exist. Their discovery, coupled with phylogenomic analyses and genomewide 3D structure comparisons will be instrumental in further refinement of our understanding of the evolutionary trajectories of prominent eukaryotic DNA viruses within the realms Duplodnaviria and Varidnaviria.

Material & methods:

considers the sequence composition, differential coverage, GC-content, and taxonomic signal of each contig, and displayed the eight hallmark genes as individual layers as well 149 additional orthologous groups often found in reference NCLDVs 5 as a single extra layer for guidance. During binning, no restriction was applied in term of number of giant virus core gene markers present, as long as the signal suggested the occurrence of a putative NCLDV MAG. Note that while some metagenomic blocks contained a limited number of NCLDV MAGs, others contained dozens. Finally, we individually refined all the NCLDV MAGs >50kbp in length as outlined in Delmont and Eren 53 , and renamed contigs they contained according to their MAG ID.

A non-redundant genomic database of marine NCLDVs. We incorporated into our database marine NCLDV MAGs characterized using automatic binning by Schulz et al. 7 (n=743) and Moniruzzaman et al. 8 (n=444), in part using Tara Oceans metagenomes. We also incorporated 235 reference NCLDV genomes mostly characterized by means of cultivation but also cell sorting within plankton 54 . We determined the average nucleotide identity (ANI) of each pair of NCLDV MAGs using the dnadiff tool from the MUMmer package 55 v4.0b2. MAGs were considered redundant when their ANI was >98% (minimum alignment of >25% of the smaller MAG in each comparison). Manually curated MAGs were selected to represent a group of redundant MAGs. For groups lacking manually curated MAGs, the longest MAG was selected. This analysis provided a non-redundant genomic database of 1,593 marine MAGs plus 224 reference genomes. We created a single CONTIGs database for this set of NCLDV genomes using anvi'o. Prodigal 44 was used to identify genes.

Curation of hallmark genes. The amino-acid sequence datasets for RNApolA, RNApolB, DNApolB, and TFIIS were manually curated through BLASTp alignments (BLAST 56 v2.10.1) and phylogenetic reconstructions, as previously described for eukaryotic hallmark genes 41 . Briefly, multiple sequences for a single hallmark gene within the same MAG were inspected based on their position in a corresponding single-protein phylogenetic tree performed with the same protocol as described above ("Diversity of DNA-dependent RNA polymerase B subunit genes" section). The genome's multiple sequences were then aligned with BLASTp to their closest reference sequence, and to each other. In case of important overlap with >95% identity (likely corresponding to a recent duplication event), only the longest sequence was conserved; in case of clear split, the sequences were fused and accordingly labeled for further inspection. Finally, RNApolA and RNApolB sequences shorter than 200 aa were also removed, as DNApolB sequences shorter than 100 aa, and TFIIS sequences shorter than 25 aa. This step created a set of curated hallmark genes.

Alignments, trimming, and single-protein phylogenetic analyses. For each of the four curated hallmark genes, the sequences were aligned with MAFFT 50 v7.464 and the FFT-NS-i algorithm with default parameters. Sites with more than 50% gaps were trimmed using Goalign v0.3.5 (https://www.github.com/evolbioinfo/goalign). IQ-TREE 52 v1.6.2 was used for the phylogenetic reconstructions, with the ModelFinder 51 Plus option to determine the best fitting model according to BIC. Supports were computed from 1,000 replicates for the Shimodaira-Hasegawa (SH)like approximation likelihood ratio (aLRT) 57 and ultrafast bootstrap approximation (UFBoot 58 ). As per IQ-TREE manual, supports were deemed good when SH-like aLRT >= 80% and UFBoot >= 95%. Anvi'o v7.1 was used to visualize and root the phylogenetic trees.

Resolving hallmark genes occurring multiple times. We manually inspected all the duplicated sequences (hallmark genes detected multiple times in the same genome) that remained after the curation step, in the context of the individual phylogenetic trees (see previous section). First, duplicates were treated as putative contaminations based on major individual (i.e. not conserved within a clade) incongruences with the position of the corresponding genome in the other singleprotein trees. The putative contaminants were easily identified and removed. Second, we identified hallmark gene paralogs encapsulating entire clades and/or subclades (Fig S2 ), suggesting that the duplication event occurred before the diversification of the concerned viral clades. This is notably the case for the majority of Imitervirales, which have two paralogs of the RNApolB. These paralogs were conserved for single-protein trees, but only the paralog clades with the shortest branch were conserved for congruence inspection and concatenation. Finally, we also detected a small clade of Algavirales viruses containing a homolog of TFIIS branching distantly from the ordinary TFIIS type, suggesting a gene acquisition. These sequences were not included in subsequent analyses. This step created a set of curated and duplicate-free hallmark genes.

Identification and modeling of the Mirus major capsid protein.

The putative major capsid protein of Mirus as well as the other morphogenetic module proteins were identified using HHsearch against the publicly available Pfam v35, PDB70, and UniProt/Swiss-Prot viral protein databases 59,60 . The candidate MCP was then modeled using the AlphaFold2 21,22 and RoseTTAFold 23 . The resulting 3D models were then compared to the major capsid protein structures of phage HK97 and human cytomegalovirus and visualized using ChimeraX 61 .

Supermatrix phylogenetic analysis of NCLDV genomes. Concatenations of the four aligned and trimmed curated and duplicated-free hallmark genes (methods as described above) were performed in order to increase the resolution of the phylogenetic tree. Genomes only containing TFIIS out of the four hallmark genes were excluded. For the remaining MAGs and reference genomes, missing sequences were replaced with gaps. Ambiguous genomes, determined based on the presence of major and isolated (i.e. not a clade pattern) incongruences within single and concatenated proteins trees, as well as on frequent long branches and unstable positions in taxon sampling inferences, were removed. The concatenated phylogenetic trees were reconstructed using IQ-TREE 52 v1.6.2 with the best fitting model according to the BIC from the ModelFinder 51 Plus option. The resulting tree was then used as a guide tree for a phylogenetic reconstruction based on the site-specific frequency PMSF mixture model 62 (LG+C30+F+R10). For the concatenated trees, supports were computed from 1,000 replicates for the Shimodaira-Hasegawa (SH)-like approximation likelihood ratio (aLRT) 57 and ultrafast bootstrap approximation (UFBoot 58 ). As per IQ-TREE manual, supports were deemed good when SH-like aLRT >= 80% and UFBoot >= 95%. Anvi'o v7.1 was used to visualize and root the phylogenetic trees.

Taxonomic inference of NCLDV MAGs. We determined the taxonomy of NCLDV MAGs based on the phylogenetic analysis results, using guidance from the reference genomes as well as previous taxonomical inferences by Schulz et al. , Moniruzzaman et al. 8 and Aylward et al. 16 .

Biogeography of NCLDV genomes. We performed a mapping of all metagenomes to calculate the mean coverage and detection of the marine NCLDV genomic database. Briefly, we used BWA v0.7.15 (minimum identity of 90%) and a FASTA file containing the 1,593 MAGs and 224 reference genomes to recruit short reads from all 937 metagenomes. We considered MAGs were detected in a given filter when >25% of their length was covered by reads to minimize non-specific read recruitments 63 . The number of recruited reads below this cut-off was set to 0 before determining vertical coverage and percent of recruited reads.

Viral-host predictions. Ecological network analysis was performed using a relative abundance matrix of 'Mirusviricota' MAGs in the pico-size fractions (0.22-3 µm) and relative abundances of eukaryotic genomes in the following five size fractions: 0.8-5 µm, 3-20 µm, 20-180 µm, 180-2,000 µm, and 0.8-2000 µm. To create the input files for network inference, we combined the Mirus matrix with each of the eukaryotic matrices (corresponding to different size fractions), and only the samples represented by both virus and eukaryotes were placed in new files. Relative abundances in the newly generated matrices were normalized using centered logratio (clr) transformation to Mirus and eukaryotes separately. Only MAGs observed in at least three samples were considered. Ecological network was built using FlashWeave 64 v0.15.0 sensitive model with Julia v1.3.1. A threshold to determine the statistical significance was set to alpha = 0.01. To compare the performance of FlashWeave to naive correlations, we calculated the spearman rank correlation coefficient using the package Scipy v1.3.1. To build a global Mirus-eukaryote interactome, we pooled associations from the five size fractions by keeping the best positive or negative associations of each genome pair (i.e., the edges with the highest absolute weights). We used a phylogeny-guided filtering approach, Taxon Interaction Mapper (TIM) 18 , to predict the host using the global virus-eukaryote interactome. All the virus-eukaryote associations were mapped on the 'Mirusviricota' informational module phylogenetic tree to calculate the significance of the enrichment of specific associations using TIM. TIM provided a list of nodes in the viral tree and associated NCBI taxonomies (order, class, and phylum) of eukaryotes that show significant enrichment in the leaves under the nodes. commitment to transparency and openness. Tara Oceans (which includes the Tara Oceans and Tara Oceans Polar Circle expeditions) would not exist without the leadership of the Tara Oceans Foundation and the continuous support of 23 institutes (https://oceans.taraexpeditions.org/). Some of the computations were performed using the platine, titane and curie HPC machine provided through GENCI grants (t2011076389, t2012076389, t2013036389, t2014036389, t2015036389 and t2016036389). This study was in part supported by Japan Society for the Promotion of Science The maximum-likelihood phylogenetic tree is based on 2,728 RNApolB sequences more than 800 amino acids in length with similarity <90% (gray color) identified from 11 large marine metagenomic co-assemblies. This analysis also includes 262 reference RNApolB sequences (red color in the first layer) corresponding to known archaeal, bacterial, eukaryotic and giant virus lineages for perspective. The second layer shows the number of RNApolB sequences from the 11 metagenomic co-assemblies that match to the selected amino acid sequence with identity >90%. Finally, RNApolB new lineages are displayed in green.

Supplemental figures:

Figure 1 :

 1 Figure 1: Evolutionary relationship between Nucleocytoviricota and mirusviruses. The figure displays a maximum-likelihood phylogenetic tree built from the GOEV database (1,722 genomes) based on a concatenation of manually curated RNApolA, RNApolB, DNApolB and TFIIS genes (3,715 amino acid positions) using the PMSF mixture model and rooted between the Pokkesviricetes and the rest. The tree was decorated with layers of complementary information and visualized with anvi'o.

Figure 2 :

 2 Figure 2: Genomic statistics and evolution of mirusviruses. Panel A displays genomic and environmental statistics for the seven 'Mirusviricota' clades. Panel B displays a maximum-likelihood phylogenetic tree built from the 'Mirusviricota' MAGs based on a concatenation of four hallmark informational genes (RNApolA, RNApolB, DNApolB, TFIIS; 3,715 amino acid positions) using the LG+F+R7 model. Panel C displays a maximum-likelihood phylogenetic tree built from the 'Mirusviricota' MAGs based on the Major Capsid Protein (701 amino acid positions) using the LG+R6 model. Both trees were rooted between clade M6 and other clades. Values at nodes represent branch supports (out of 100) calculated by the SH-like aLTR (1,000 replicates; left score) and ultrafast bootstrap approximations (1,000 replicates; right score).

Figure 3 :

 3 Figure 3: Functional clustering of abundant and widespread marine viruses within mirusviruses and Nucleocytoviricota. In panel A, the inner tree is a clustering of 'Mirusviricota' and Nucleocytoviricota genomes >100kbp in length based on the occurrence of all the non-singleton gene clusters (Euclidean distance), rooted with the Chordopoxvirinae subfamily of Poxviridae genomes. Layers of information display the main taxonomy of Nucleocytoviricota as well as the occurrence of 60 gene clusters detected in at least 50% of 'Mirusviricota' or Nucleocytoviricota. The 60 gene clusters are clustered based on their occurrence (absence/presence) across the genomes. Panel B displays the occurrence of gene clusters of known functions detected in at least 50% of 'Mirusviricota' or Nucleocytoviricota genomes.

Figure 4 :

 4 Figure 4: The phylogeny of histones and heliorhodopsins. Left panel displays a maximumlikelihood phylogenetic of histones occurring in MAGs of eukaryotes, Nucleocytoviricota and mirusviruses. Mainly eukaryotic clades distant from H2-H3-H4 were excluded in order to focus on the more restrained viral signal. Right panel displays a maximum-likelihood phylogenetic tree of heliorhodopsins occurring in eukaryotes, Nucleocytoviricota and mirusviruses.

Figure 5 :

 5 Figure 5: 3D structure of the major capsid protein (MCP). The figure displays MCP 3D structures for Escherichia phage HK97 (Caudoviricetes), a reference genome for the mirusviruses (estimated using Alphafold), and the human cytomegalovirus (Herpesvirales).

Figure 6 :

 6 Figure 6: Phylogeny of the DNApolB hallmark genes. The figure displays a maximum-likelihood phylogenetic tree of DNA-polymerase B-family sequences (1,080 sites, 2,213 sequences) from the database described herein, Duplodnaviria and Baculoviridae sequences from the NCBI viral genomic database, and eukaryotic and viral sequences from Kazlauskas et al.27 . Eukaryotic Epsilon-type and related sequences were used as outgroup.

Figure 7 :

 7 Figure 7: A near-complete genome for 'Mirusviricota'. The figure displays the length of 355 genes found in a near complete genome of 'Mirusviricota' (clade M2), along with their link to two viral domains (gene versus HMM signal). The figure also highlights hallmark genes for the informational and particle modules of the virus.

  (JSPS) KAKENHI (18H02279, 22H00384), Research Unit for Development of Global Sustainability, Kyoto University Research Coordination Alliance, and the International Collaborative Research Program of the Institute for Chemical Research, Kyoto University (2022-26, 2021-29, 2020-28). M.K. was supported by grants from the l'Agence Nationale de la Recherche (ANR-20-CE20-0009-02 and ANR-21-CE11-0001-01). Part of computational work was performed at the SuperComputer System, Institute for Chemical Research, Kyoto University. This article is contribution number XXX of Tara Oceans.

Figure S1 :

 S1 Figure S1: Evolutionary diversity of the DNA-dependent RNA polymerase B in the sunlit ocean.The maximum-likelihood phylogenetic tree is based on 2,728 RNApolB sequences more than 800 amino acids in length with similarity <90% (gray color) identified from 11 large marine metagenomic co-assemblies. This analysis also includes 262 reference RNApolB sequences (red color in the first layer) corresponding to known archaeal, bacterial, eukaryotic and giant virus lineages for perspective. The second layer shows the number of RNApolB sequences from the 11 metagenomic co-assemblies that match to the selected amino acid sequence with identity >90%. Finally, RNApolB new lineages are displayed in green.

Figure S2 :

 S2 Figure S2: Single-protein phylogenies of the four informational hallmark genes in the GOEV database. Maximum-likelihood phylogenetic trees of the DNA-dependent RNA polymerase subunits A and B, the DNA polymerase B family, and Transcription Factor II-S.In each tree, the branches corresponding to mirusviruses are colored in red. The layers provide additional information, from innermost to outermost: the source (red for the MAGs in our survey, black for references, uncolored for MAGs from other surveys), the presence/absence of hallmark genes in the corresponding genomes, and their taxonomy.

Table S6 :

 S6 Occurrence of gene clusters with remote homologies for the GOEV database. Statistics are included.

Table S7 :

 S7 Functional annotations for genes found in the GOEV database. Statistics are included.

Acknowledgments:

Our survey was made possible by two scientific endeavors: the sampling and sequencing efforts by the Tara Oceans Project, and the bioinformatics and visualization capabilities afforded by anvi'o. We are indebted to all who contributed to these efforts, as well as other open-source bioinformatics tools for their

Tara Oceans metagenomes. We analyzed a total of 937 Tara Oceans metagenomes available at the EBI under project PRJEB402 (https://www.ebi.ac.uk/ena/browser/view/PRJEB402). Table S1 reports general information (including the number of reads and environmental metadata) for each metagenome.

Constrained automatic binning with CONCOCT. The 798 metagenomes corresponding to size fractions ranging from 0.8 µm to 2 mm were previously organized into 11 'metagenomic sets' based upon their geographic coordinates 14,41 . Those 0.28 trillion reads were used as inputs for 11 metagenomic co-assemblies using MEGAHIT 42 v1.1.1, and the contig header names were simplified in the resulting assembly outputs using anvi'o 43 v6.1. Co-assemblies yielded 78 million contigs longer than 1,000 nucleotides for a total volume of 150.7 Gbp. Constrained automatic binning was performed on each co-assembly output, focusing only on the 11.9 million contigs longer than 2,500 nucleotides. Briefly, (1) anvi'o profiled contigs using Prodigal 44 v2.6.3 with default parameters to identify an initial set of genes, (2) we mapped short reads from the metagenomic set to the contig using BWA v0.7.15 45 (minimum identity of 95%) and stored the recruited reads as BAM files using samtools 46 , (3) anvi'o profiled each BAM file to estimate the coverage and detection statistics of each contig, and combined mapping profiles into a merged profile database for each metagenomic set. We then clustered contigs with the automatic binning algorithm CONCOCT 47 by constraining the number of clusters per metagenomic set to a number ranging from 50 to 400 depending on the set (total of 2,550 metagenomic blocks from ~12 million contigs).

Diversity of DNA-dependent RNA polymerase B subunit genes. We used HMMER 48 v3.1b2 to detect genes matching to the DNA-dependent RNA polymerase B subunit (RNApolB) among all 2,550 metagenomic blocks based on a single HMM model. We used CD-HIT 49 to create a non-redundant database of RNApolB genes at the amino acid level with sequence similarity <90% (longest hit was selected for each cluster). Short sequences were excluded. Finally, we included reference RNApolB amino acid sequences from Bacteria, Archaea, Eukarya and giant viruses 5 : The sequences were aligned with MAFFT 50 v7.464 and the FFT-NS-i algorithm with default parameters, and trimmed at >50% gaps with Goalign v0.3.5 (https://www.github.com/evolbioinfo/goalign). We performed a phylogenetic reconstruction using the best fitting model according to the Bayesian Information Criterion (BIC) from the ModelFinder 51 Plus option with IQ-TREE 52 v1.6.2. We visualized and rooted the phylogeny using anvi'o. This tree allowed us to identify new RNApolB clades.

Phylogeny-guided genome-resolved metagenomics. Each metagenomic block containing at least one of the RNApolB genes of interest (see previous section) was manually binned using the anvi'o interactive interface to specifically search for NCLDV MAGs. First, we used HMMER 48 v3.1b2 to identify eight hallmark genes (eight distinct HMM runs within anvi'o) as well 149 additional orthologous groups often found in reference NCLDVs 5 (a single HMM run within anvi'o). The interface Cosmopolitan score. Using metagenomes from the Station subset 1 (n=757; excludes the 0.8-2000 µm size fraction lacking in the first leg of the Tara Oceans expeditions), NCLDV MAGs and reference genomes were assigned a "cosmopolitan score" based on their detection across 119 stations, as previously computed for eukaryotic MAGs 39 .

AGNOSTOS functional aggregation inference. AGNOSTOS partitioned protein coding genes from the marine NCLDV genomic database in groups connected by remote homologies, and categorized those groups as members of the known or unknown coding sequence space based on the workflow described in Vanni et al. 2020 20 . AGNOSTOS produces groups of genes with low functional entropy as shown in Vanni et al. 2020 20 and Delmont et al. 2020 41 allowing us to provide functional annotation (Pfam domain architectures) for some of the gene clusters using remote homology methods.

Functional inferences of NCLDV genomes. Genes from the marine NCLDV genomic database were BLASTP against Virus-Host DB 65 , RefSeq 66 , UniRef90 67 , NCVOGs 68 , and NCBI nr database using Diamond 69 v2.0.6 with a cut-off E-value 1 × 10 -5 . A recently published GVOG database 16 was also used in annotation using hmmer 48 v3.2.1 search with E-value 1 × 10 -3 as a significant threshold. In addition, KEGG Orthology (KO) and functional categories were assigned with the Eggnog-Mapper 70 v2.1.5. Finally, tRNAscan-SE 71 v2.0.7 predicted 7,734 tRNAs.

Realm assignation of genes. Two in-house HMM databases were created as follows. First, all coding sequences (CDS) labeled as "Nucleocytoviricota" were removed from the Varidnaviria CDS dataset (N= 53,776) in the Virus-Host Database (VHDB 29 , May 2022). To this dataset, Tara Ocean NCLDV MAGs (all were manually curated) and 235 reference NCLDV genomes were integrated. The final Nucleocytoviricota protein database contained 269,523 CDS. Similarly, we replaced all Herpesvirales CDS in the VHDB Duplodnaviria CDS dataset with Herpesvirales protein sequences downloaded from NCBI in April 2022. Additionally, a marine Caudovirales database including jumbo phage environmental genomes 12,72 was integrated into the Duplodnaviria proteins. The final Duplodnaviria protein database contained 748,546 proteins. Proteins in the two databases were independently clustered at 30% sequence identity (-c 0.4 --cov-mode 5), using Linclust in MMseqs 73 v13-45111. Gene clusters with fewer than 3 genes were removed, and the remaining gene clusters were aligned using 50 v 7.487. HMM files (N= 16,689 and 57,259 for Varidnaviria and Duplodnaviria, respectively) were created using the hmmhuild in HMMER3 74 v3.2.1. All proteins in the near-complete 'Mirusviricota' genome were searched against the two custom HMM databases using the hmmsearch with a cut-off E-value 1 × 10 -6 .

Statistical analyses.

A "greater" Fisher's exact test was employed to identify KO functions as well as gene clusters with remote homologies that are differentially occurring between the 111 'Mirusviricota' MAGs on one side, and all other NCLDVs Mirusviruses provide a missing link in the evolution of giant viruses in the database on the other side. P-values were corrected using the Benjamini-Hochberg procedure in R, and values <0.05 were considered significant.

Naming of Mirus and Procul. The latin adjective "Mirus" (surprising, strange) was selected to describe the putative new Duplodnaviria phylum: the 'Mirusviricota'. The latin adverb "Procul" (away, at distance, far off) was selected to describe the putative new class of NCLDVs discovered from the Arctic and Southern Oceans: the 'Proculviricetes'.

Data availability. Data our study generated has been made publicly available at https://doi.org/10.6084/m9.figshare.20284713.v1. This link provides access to (1) the RNApolB genes reconstructed from the Tara Oceans assemblies (along with references), (2) individual FASTA files for the 1,593 non-redundant marine NCLDV and Mirus MAGs (including the 697 manually curated MAGs from our survey) and 224 reference NCLDV genomes contained in the GOEV database, (3) the GOEV anvi'o CONTIGS database, (4) genes and proteins found in the GOEV database, (5) manually curated hallmark genes and corresponding phylogenies, (6) HMMs for hallmark genes, (7) the supplemental tables, (8) and the supplemental information document.

Contributions:

Tom O. Delmont conducted the study. Morgan Gaïa, Lingjie Meng, Mart Krupovic, Chiara Vanni, Eric Pelletier and Tom O. Delmont performed the primary data analysis. Tom O. Delmont completed the genome-resolved metagenomic analysis. Morgan Gaïa and Tom O. Delmont curated the marker genes and identified the biological duplicates. Morgan Gaïa performed phylogenetic and phylogenomic analyses. Lingjie Meng performed functional analyses and virus-host predictions. Hiroyuki Ogata supervised Lingjie Meng. Chiara Vanni produced gene cluster with remote homologies. Mart Krupovic discovered the major capsid protein of 'Mirusviricota' and other key genes of the virion module, linking the two realms. Eric Pelletier performed comparative genomic and biogeographic analyses. All the authors contributed to interpreting the data and writing the manuscript.

Conflict of interest:

Authors declare having no conflicts of interest.

Supplemental tables:

Table S1: Description of 937 Tara Oceans metagenomes.

Table S2: DNA-dependent RNA polymerase subunit B genes characterized from the sunlit ocean.

Table S3: Metabins containing DNA-dependent RNA polymerase subunit B genes of interest and used for genome-resolved metagenomics (see supplemental information for more details).

Table S4: Genomic and environmental statistics for the GOEV database.

Table S5:

Biogeographic signal for the GOEV database.