Chamberland, Roux, Grancher Metchnikoff : Duclaux

Jean-Marc Cavaillon
email: jean-marc.cavaillon@pasteur.fr

Sandra Legout

Charles Chamberland

Émile Roux

Joseph Grancher

The Institut Pasteur was created thanks to worldwide generosity with the aim to welcome and treat rabies patients, to provide a place for scientific research and to offer new teaching programs in microbiology. Louis Pasteur invited his main collaborators, who had accompanied

2

After his main investigations against the spontaneous generation theory, on silk worm disease, fermentation, wine and beer, Pasteur initiated his investigation on infectious diseases and how to prevent them by prior injection of an attenuated microbe. In 1881, at the international medical congress in London, Pasteur used the word "vaccination": "I gave the term vaccination an enlarged meaning, that science I hope will consecrate as an homage to the merit and immense services rendered by one of the greatest men of England, your Jenner. What joy for me to glorify this immortal name on the very soil of the noble and hospitable city of London". But, he could have also paid tribute to Charles-Paul Diday (1813-1894) who had coined the word "vaccination" in 1849, when he reported his amazing works on his anti-syphilitic vaccine. Diday vaccinated 16 patients suffering a genital chancre, by performing an inoculation with the blood from a patient with a tertiary syphilis (Ref. [START_REF] Diday | CP Sur un procédé de vaccination préservatrice de la syphilis constitutionnelle[END_REF]. Of course, Diday was also referring to the work of Edward Jenner who prevented human cow pox thanks to the use of bovine vaccinia. In 1879, Pasteur's first vaccine aimed to protect against avian cholera (a bacteria latter called Pasteurella multicoda). In 1881, Pasteur designed another vaccine against Bacillus anthracis, a major threat in sheep farming. In 1883, he proposed a vaccine against swine erysipelas (Erysipelothrix rhusiopathiae). Working then on rabies, he set up a vaccine which was successfully employed for the first time in humans on the 6 th of July 1885 on a nine-year-old Alsatian shepherd, Joseph Meister. Jean-Baptiste Jupille was the second child to be treated and saved by the vaccine on the 20 th of October 1885. Both would become the door-keepers of the future Institut Pasteur, and Joseph Meister, would eventually kill himself when the Nazis entered in Paris.

The impact of these successes was worldwide. Many doctors and scientists were sent from different countries to learn the technology to prepare such a vaccine against a mostly lethal infection. Anti-rabbies institutes were created all over the world 1 . In 1886, 2,682 people from France and from abroad were treated at rue d'Ulm, with a 1.34% mortality. In 1887, 1,778 people were vaccinated with an improved 1.12% mortality rate (Ref. 2). It was then decided to raise an Institute in Paris which purposes: "the treatment of rabies according to the method developed by M. Pasteur" and "the study of virulent and contagious diseases". In 1887, fundraising for the Institut Pasteur began, with donations from many countries. The major donators were namely: Alexandre III, emperor of Russia, Don Pedro II, emperor of Brazil, The Sultan of Constantinople, and wealthy French people such as the baron Alphonse de Rothschild, or Mrs Boussicaut. But many French people, even very modest ones, also contributed to the funding. The subscription ended to 2 586 680 francs, enough to cover the costs to purchase the land, construct the buildings and to fund three years of operating costs, leaving a grand total of 1 022 894 francs founding money. The piece of land, close to the Montparnasse railway station, which belonged to a family of market gardeners for 150 years was finally chosen. It was considered more appropriate than the one offered by the city of Paris. The architects, M. Eugène Petit and Félicien Brébant refused any payment, and designed a Louis XIII stylish building with large windows and brand-new organized laboratories. The apartment of Louis Pasteur was included within the main building. An out-patient clinic was situated on the ground floor with a large waiting room, a room for bandages, a room for inoculation, a room to gather the administrative archives, and a room for preparing the rabbit spinal cords. In addition to the laboratories within the main building, other rooms were dedicated to photographs, surgery, dissection, and to house incubators. Furthermore, on the first floor, two large rooms were designed for teaching, and a spacious library that was also aimed to welcome official events, was situated facing Pasteur's apartment. Annexes to house workers, storage buildings and animal facilities (for dogs, birds, rabbits, guinea-pigs, horses, and for rabid animals) were built or restored on the same campus.

It was decided that six research units would be housed in the main building under the supervision of six renowned scientists, two of them being medical doctors: Professeur Joseph Granger, MD, Dr. Émile Roux, MD, Dr. Émile Duclaux, Dr.

Charles Chamberland, Dr. Élie Metchnikoff and Dr. Nicolaï Gamaleïa. Most of them had been collaborators of Louis Pasteur, at the École Normale Supérieure, rue d'Ulm, for many years (Table 1), but the two Russian scientists were a more recent encounter and a testimony of Pasteur's love for Russia. Finally, Nicolaï Gamaleïa, although present on the day of the inauguration, and whose name was mentioned during the talks will never join the institute. These five men undeniably participated in the reflection around the design and organization of the institute. In June 1887 the first statutes of the institute, recognizing it of public utility were signed by 16 members including our five musketeers, the first generation of pasteurians. The institute was created not only with the aim to welcome rabies patients and to play a role in national public health, but also to favor scientific research, as well as to offer new teaching programs, particularly within the emerging microbiology field. Another aim was to position the institute within the international scientific community, and a new journal ("Les Annales de l'Institut Pasteur") was launched the year before the opening of the Institute (Ref. 3). Émile Duclaux was nominated as the Editor-in-Chief and financed the journal. In 1887, the other four French partners of the Institute were members of the Editorial Board (Table 2). Very rapidly, the institute extended to different countries: in 1891, Albert Calmette founded the 1 st institute abroad in Saigon (Vietnam) where he studied vaccines against smallpox and rabies. In 1894, Alexandre Yersin was sent to Hong Kong, where he discovered the plague bacillus.

He later then created a laboratory in Nha-Trang (Vietnam).

Inauguration of the Institut Pasteur

In the garden, the bronze statue of Jean-Baptiste Jupille fighting with a rabid dog to protect his friends, made by the French Sculptor Émile-Louis Truffot (1843-1895), witnesses the arrival of many ministers, senators, deputies, ambassadors and representatives of foreign countries, journalists, and many medical and scientific celebrities from both Medical and Scientific Academies. The Institute is decorated with French flags. The ceremony takes places in the big library, which can welcome up to 600 people. The room is decorated with the sculptures of the main donators.

By half-past-noon, the room is already so full that the guest can no longer enter in the library, and gather in the vestibule and on the steps. Even Dr. Roux, is unable to find a place in the library. By one-thirty, Sadi Carnot (1837-murdered in 1894), the He recalled how an official committee was sent from England to enquiry about the rabies vaccine. Initially incredulous, the main reviewer, Sir Victor Alexander Haden Horsley (1857-1916), an accomplished physician, concluded with his committee that Pasteur had indeed discovered a preventive treatment for rabies similar to the cow pox vaccine. He recalled how his colleague, Dr. Alfred Vulpian (1826-1887) had encouraged Pasteur to apply his rabies vaccine to human beings. And finally, he provided statistics to illustrate the amazing success of the rabies vaccine all over the world. The following talk was given by M. Albert Christophle (1830-1904), governor of a French bank who was the treasurer of the project. He was nicknamed "the enthusiastic fairy", for his glorious national and international effort to gather the funds to raise the institute: He also mentioned how, Pasteur, Chamberland and Roux gave up their own royalties on the vaccines (anthrax and rabies) to contribute to the funding of the institute. He ends his talk claiming his admiration and love for Pasteur.

Too much affected, Pasteur asked his son, Jean-Baptiste, to read his talk. He pays tribute to the international generosity, to his Masters who are not here anymore, and to the six collaborators who will join the institute. About research, he made an interesting statement: "To believe that we have found an important scientific fact, to have the fever to announce it, and to constrain days, weeks, sometimes years of fighting oneself, striving to ruin one's own experiences, and proclaiming one's own discovery that when you have exhausted all the contrary assumptions, yes, it is a difficult task." He concludes his talk with a personal thought, illustrating his trauma about the last 1870's war: "I would say that two contrary laws seem today in struggle: a law of blood and death which, imagining every day new means of fighting, obliges the peoples to be always ready for the battle field, and a law of peace, of labor, of salvation, which thinks only of freeing man from the scourges which besiege him. One seeks only violent conquests, the other only the relief of humanity." Warm cheers greet his eloquent speech. Surprisingly, the President of the French Republic, Sadi Carnot, did not say much and only announced that, after the request of Pasteur, he had promoted

A new conceptual organization of research

The personality of Louis Pasteur has undeniably marked the history legacy of his institution. His initial choices weighed heavily on its destiny. During his teaching career he suffered from administrative red tape and too rigid tutelage of the state.

The science of that time was confined to the academic field, to pure theoretical teaching without the experimental complement. Most probably, the creation of a structure like the Institut Pasteur existed for a long time in Pasteur's mind. He wanted to spare his successors the need of "begging" with the public authorities. In a letter of January 12 th , 1886 to the Comte de Laubespin who had just given him a gift of 4000 francs, he mentioned his intent to founder a model establishment in Paris without recourse to the State, with the help of donations and international subscriptions. For Pasteur, it was important to create an attractive place for French and foreign scientists, linked with medicine and similar to those already existing in Germany.

The statutes of the IP reflect its perspectives and wishes. Before being Furthermore, the profits from the sale of vaccines, a point which in the statement of the statutes was not a priority, will gradually steer the Pasteur Institute towards a commercial role.

Beside the statutes, Pasteur also thought about the organization of the staff and entrusted his thoughts to Dr. J. Grancher, in a letter of March 31 st , 1887 "Each head of laboratory will obviously be quite free from his personal work, although obliged however, to certain rules for its expenses. We will need the equivalent of a bursar in a high school. The Assistant Directors will not have to intervene in the works of the head of laboratories, but they will have to adjust and set up the lessons and student work programs in the laboratories, in conjunction with the head of laboratories. We will succeed, I hope, easily to avoid conflicts, by appropriately regulating the attributions and by delimiting the subjects of the studies, either for the young doctors wishing to learn the principles, or for the doctor-professors, scientists, who will want to do personal research and will have distinct and separate investigation rooms." So, if Pasteur's first concern was financial, it is clear that he wanted his successors independence and freedom of work. In an article published in the "Revue Scientifique" of 1874, he wrote "there is no category of science to which we can give the name of applied sciences. There is science and the applications of science, linked together as a fruit to the tree that bore it." Thus, quite a new concept emerged, the Institut Pasteur would be a private institute, depending as little as possible on the State financial support, and instead based on donations, and incomes from its own applied research, and providing a teaching activity, independent from the University but offering new areas of courses with practical works. The very first course on microbiology, established by Dr. Emile Roux, started in 1889, and soon became a must for physicians and scientists from all over the world.

The musketeers

Pasteur gathered personalities from different origins, backgrounds and disciplines, whose scientific styles have combined their specificities in a beneficial and complementary way. Pasteur had offered to Duclaux, Chamberland, Roux and Grancher, his main collaborators who were working with him at the École Nationale Supérieure, the possibility to head a research laboratory or a medical entity. All had been closely associated with the works of their Master who had acquired so much fame. The connection with Metchnikoff was far more recent, since it followed a visit that the great Russian Scientist had made in 1887 when he was looking for a place where to work, after he had left the Bacteriology Institute that he had chaired in Odessa. After having met Robert Koch in Berlin, Metchnikoff made the decision to join the new institute. Together, with the putative Gamaleïa, the Russian connection was a great chance for the other members. Both were speaking Russian, German, French, and English while the French partners did not master foreign languages. Toussaint (1847-1890) had prepared an efficient vaccine using an antiseptic treatment with carbolic acid. In fact, Chamberland did use a vaccine that had been chemically treated, but using potassium dichromate. The need to prepare a huge quantity of doses was the pressure leading to create a spin-off vaccine, in addition to the research laboratory. Chamberland was in charge of the vaccine preparation establishing a kind of industrial production unit in a private laboratory, 14 rue

Vauquelin, with the participation of four assistant chemists. Then the number of doses went from 164 000 in 1881, to 700 000 in 1882, with a further increase of 900 00 in 1885 (Ref. 12). But in 1882, a crisis occurred after reports of vaccination accidents. Indeed, the quality, the reproducibility, standardization and efficacy of the vaccine was not easily achieved, particularly when the preparations had to be sent abroad, in distant countries.

When the Institute opened, Pasteur entrusted to him the direction of the practical service, that of preparation of vaccines, supported in this task by his brother-in-law Charles Rebour. In this laboratory, he studies the "reports of microbia with hygiene".

In fact, Chamberland, in addition to having solid knowledge in physics, had extensive knowledge in chemistry. He published extensively on antiseptic substances to determine the most effective methods for disinfecting objects and premises. He studied the microbicidal action of chlorine in the gaseous state or in solution of chloride of lime, but also the destructive power of essential oils and hydrogen peroxide. With his collaborator C. Jouan, he carried out studies on Pasteurella.

In 1885 he was elected as deputy from Jura and contributed to laws in public health and hygiene. In addition to his duties as head of department, he was also a member of the first editorial board of the Annals of the Institut Pasteur and his laboratory was the first to be functional. He was accompanied by his second wife, Olga, who was a gifted artist but who also became his laboratory assistant, publishing some works herself on anthrax vaccine and on germfree tadpoles. Olga was also very helpful to host and in organizing the stays of the foreign trainees. From 1886 to 1892, Gamaleïa shared his life between Odessa and Paris.

Despite their long collaborative years, the relationship between Metchnikoff and Gamaleïa began to deteriorate. Although, Duclaux reiterated the invitation to join the institute, Gamaleïa pursued his career in Russia, and never joined the Institute where his venue was scheduled at the very beginning. In 1888, he published a quite fascinating work (Ref. [START_REF] Schwartz | The Institut Pasteur: 120 years of research in microbiology[END_REF]. For the first time he showed that bacteria don't need to be alive to induce fever. Furthermore, he showed that filtered alcoholic extracts of spleen from pyretic sheep induced fever within 30 minutes when injected in rabbits.

5 th

 5 President of the French Republic, arrives in a carriage in front of the steps, welcomed by the Republican Guard which plays the Marseillaise. Mr. Pasteur, after having walked down the steps to meet the President, takes place in an armchair just close to that of the President. The first talk is given by M. Bertrand, General Secretary of the French Academy of Sciences. He recalled a statement by Émile Verdet, a French physicist who worked at École Normale Supérieure, told him a long time ago: "Pasteur, does not know the limits of science. I fear for his sterile efforts! He loves insoluble problems." He concluded: "The problems which, for half a century have tormented your mind without rest, are no longer insoluble nowadays. It is to thank you in the name of science, to rejoice in the name of humanity, to glorify all of us together in the name of France, that we are together today." Then, Prof. Grancher, the medical doctor who supported and assisted Pasteur in his first rabies vaccine injection in humans gave a long and detailed talk about the rabies vaccination and addressed the different discoveries of Pasteur in the field of infectious diseases. He paid tribute to Pasteur's collaborators, Roux and Chamberland who contributed to his famous investigations on anthrax and rabies.

  Grancher and Duclaux as officer of the Legion of Honor, and Chantemesse, a medical doctor who worked with Pasteur, as a knight of the Legion of Honor. Brébant, the architect was made officer in the order of Academic Palms by Edouard Lockroy, the Minister of Public Instruction (of note the other architect, Eugène Petit, had passed away before the inauguration). Then, Pasteur invited the President to visit the Institute (REF. 4, 5).

  adopted by the Council of State, they went through very tentative stages of elaboration. Pasteur had a clear vision of what a research laboratory should be, but less about what an institute should be. Under the influence of friends who held high office in the administrative (especially the State Councilor Tisserand, the Ministry of Agriculture), he charged his collaborator Joseph Grancher to negotiate with the government, a recognition of Institut Pasteur as a "public utility" institution. The decree and the statutes were accepted on June 4 th , 1887. The objectives of the Institute remain those of the Limited Company. In a letter to the Minister of Public Education (Liard, December 22, 1888), he seems to want to use the contribution of the state only in case of budget deficit. The first financial statements mention the contribution of the State as a regular income finally required for a balanced budget.

  Amazingly, a common tragedy links Metchnikoff, Roux and Duclaux. All three had lost their first wifes: Metchnikoff lost Ludmilla Fédorovna in 1873 because of tuberculosis (Ref. 6). Likewise, in 1878, Roux lost Rose A. Shedlock because of tuberculosis (Ref. 7). Both died in Madeira, a place commonly visited with the hope to offer better air and sunny days to tubercular patients (A famous scientist, Paul Langherans also died in Madeira in 1888, due to a kidney complication of his tuberculosis). In 1880, Duclaux lost Mathilde Briot, because of puerperal fever, following the birth of their third son (Ref. 8). Most probably these misfortunes have favored the relationship and friendship between them. a. Émile Duclaux Émile Duclaux is the first of the five musketeers to have joined Louis Pasteur.After his aggregation of Sciences at École Nationale Supérieure, he joined his laboratory in 1862 at the time the fight against the spontaneous generation was going on (ending to the demonstration of micro-organisms within the environment and the air). He participated to the demonstration on wine making by Pasteur, who had moved in laboratory in Arbois (Table3). Soon he was nominated as professor of chemistry at the University of Clermont-Ferrand (1867), and then as professor of physics in Lyon (1873). Back to Paris in 1878, he taught physics and meteorology, and ran a course of biological chemistry at the Sorbonne (Ref. 9). After the opening of Institut Pasteur, Duclaux transferred his course to the institute as well as the laboratory of physiology-chemistry he headed at Ecole des Hautes études. He was entirely associated with Pasteur's work, even if he was rarely co-author in the publications of his Mentor. He only co-signed two papers with Pasteur, when Roux and Chamberland had co-signed together some 60 papers. He covered a broad range of subjects. His first works were on alcoholic fermentation, which was the topic of his thesis. He worked on the influence of air and temperatures on the birth and the life of silk worms. He studied grape phylloxera, addressing its geographical spread and the means to prevent it. He investigated molecular balance and capillary tension of liquids. From 1877 to 1883, in response to the request of the Ministry of Agriculture he investigated the preparation of Cantal cheese and on its diseases. He then investigated gastric, pancreatic and intestinal digestion. He also made some personal investigations on the production of enzymes by Aspergillus and Penicilium, establishing that the production of enzymes is related to added nutrients, known as enzymatic and adaptation observed in bacteria, yeasts, and molds. It was a most important contribution, which was poorly recognized due to its lost within his huge treatise on microbiology (Ref. 9). From May to July 1879, he personally contributed to an important paper of his Mentor on the demonstration of germs in the etiology of some infectious diseases (Ref. 10). He repeatedly provided samples from his own boils! From those, Pasteur identified the presence of Staphylococcus aureus, which he also found in bone samples of a 12 year old girl suffering osteomyelitis (Ref. 10). Due to Pasteur old age and poor health, Duclaux was the master piece, in establishing the Institute, and its running. As mentioned previously, he created the "Annales de l'Institut Pasteur (Journal de Microbiologie)", assuming the operating cost from his own money and all the editing work. In addition of being the official channel for the work carried out in Pasteur's house, there were also numerous reports on articles published elsewhere. After his death, Institut Pasteur, under Roux's proposals, bought the journal to his heirs and ensured them a comfortable life annuity (Ref. 11). Duclaux was also playing the role of scientific facilitator, in addition to his own research activities and teaching duties at the Agronomic Institute and at the Sciences University. He organized a cafeteria/restaurant "The golden microbe", a truely scientific and social place. Duclaux would have loved to operate a brewing school and to sell beer yeast. As it was against Pasteur's view, he eventually gave in. Two years after its opening,an extension was built to meet the need to make antituberculosis vaccine. But it was only in 1896, that Duclaux favored the purchase of land just on the other side of the street, thanks to the generous donation of Mrs Lebaudy. On May 1899, a new land was bought, extending the campus until rue de Vaugirard. Duclaux played a key role in the establishment of a hospital specifically designed to host patients with infectious diseases, and a new research building, both inaugurated in 1900. Thus, Duclaux finalized his dream to open a new research building devoted to biological chemistry Duclaux logically succeeded Pasteur, after his death on September 28 th , 1895, and remained the director until his own death, on May 2 nd , 1904. In 1888 he became member of the Sciences Academy, and in 1894, member of the National Academy of Medicine. He was made "Commander" of the Legion of Honor in 1895. Co-founder of the "French League for the Defense of Human and Citizen's Rights", he was the vicepresident and took an active part during the Dreyfus affair. Duclaux wrote few books including one on his beloved master published in 1896: "Pasteur the story of a mind". b. Charles Chamberland Chamberland is the handyman, the inventor and the hygienist in our group of five. In 1875, Charles Chamberland joined the laboratory of physiological chemistry of Louis Pasteur as an assistant one year after receiving his aggregation from the École Normale Supérieure. He was commissioned to repeat an experiment carried out by Henry Charlton Bastian (1837-1915), professor of pathological anatomy at London's University College Medical School, a supporter of the theory of spontaneous generation, and to demonstrate that the experimental approach and the conclusions were false. Pasteur associated him with his publication on the "Theory of germs and its applications to medicine and surgery" in which they show that each disease corresponds to a particular germ (1878). He was a valuable and recognized collaborator for Pasteur, since he was associated with the works on the etiology of anthrax, on anthrax vaccination, vaccination against swine erysipelas, and anti-rabies vaccination. From 1878 to 1888, he co-signed on 85 papers with Pasteur. Their collaboration soon turned into friendship, and Pasteur even agreed to be his wedding witness in 1889. Charles Chamberland established the rules of the sterilization of culture media, rules which he developed in his thesis of sciences (1879). As a very ingenious and inventive person, he improved the sterilization methods and the laboratory equipment, the Chevallier-Appert water bath with pressure gauge. Furthermore, he has built a disinfection oven, an autoclave, a tool that has become indispensable in laboratories and in industry, not to mention the invention of porous walls made of porcelain clay in the hollow cylindrical shape (candle filters) aimed to retain the particles suspended in the liquids. Chamberland has played a major role in the preparation of the Pouilly-Le-Fort experiment, which allowed the most successful and convincing demonstration of the anthrax vaccination in front of a general public. The nature of the vaccine against anthrax was never divulged by Pasteur and Chamberland. Pasteur was favoring attenuated vaccine by exposure of bacteria to air, while, in Toulouse, his rival, Henry

  photography to microscopic preparation (creation of the microphotography laboratory). In a paper published in 1887, he detailed all means to apply photography to the study of microbes (Ref.[START_REF] Roux | La photographie appliquée à l'étude des microbes[END_REF]. He was also a hygienist. He contributed to disseminate the Pasteurian principles and led the "Higher Council of Public Hygiene"

  These works led Gamaleïa to write a book on bacterial poisons (1892). Gamaleïa served as director of the Odessa Bacteriological Institute from 1896 to 1908. To fight plague in Odessa, he contributed to a campaign of de-infectation and rat control. In 1910 (till 1913), he published and edited the journal "Hygiene and Sanitation" which welcomed eugenic publications, including his own editorials. He also advocated his eugenic views in different lectures delivered in St Petersburg or Tartu (Ref. 25). In 1912, he was director of an institute in St Petersburg before moving to Moscow in 1930 to take over the Central Institute of Epidemiology and Bacteriology. He was corresponding member of the USSR Academy of Sciences (1939), honorary academician (1940), and academician of the Academy of Medical Sciences of the USSR (1945). Nowadays, the National Research Center for Epidemiology and Microbiology is named after this famous Russian scientist. Conclusions When Pasteur gathered his five musketeers, he contributed to the foundation of an original place where scientists and physicians could cooperate, illustrating the statement of Charles Richet (Nobel Prize, 1913) who was among the 16 founding members: "To oppose the physician to the physiologist, and the scientist to the clinician, means that one has failed to understand physiology and medicine". Pasteur invited esteemed colleagues who later had become friends. And for most of them, have been working and supporting his research over a long period of time. He welcomed two Russian scientists, illustrating Pasteur's support for their country. His institution coined a new model favoring the independence of researchers, teaching, applied sciences and its putative incomes, and the offer of an international window within its global network. In 1889, twenty-seven people were listed in the staff of the institute; nowadays around 2500 people are working on the Parisian campus which remains a leading place within the French and international landscape of biological research (Ref. 29).

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .

 1234567 Figure 1. On the French banknote of 5 francs (1966), the portrait of Louis Pasteur was shown in front of his historical building and associated with the sculpture of Jean-Baptiste Jupille fighting a rabid dog (by Émile-Louis Truffot) Figure 2. Portrait of Émile Duclaux by Ernest Bordes (circa 1904) Figure 3. Portrait of Charles Chamberland (circa 1900) Figure 4. Portrait of Émile Roux while teaching by Albert Edelfelt (1895) Figure 5. Portrait of Joseph Grancher Figure 6. Portrait of Elie Metchnikoff by William Laparra (1911) Figure 7. Portrait of Nikolaï Gamaleïa

  Émile Roux started his medical studies in Clermont-Ferrand and became the assistant of Émile Duclaux who was in charge of the chemistry lectures at the University of Science in Clermont-Ferrand. In 1877, Roux moved to Paris and pursued his medical studies at the military school of Medicine (Hôpital du Val de , "the strangling angel of children", as it was called which was killing thousands of young children and had a great impact. The Parisian newspaper 'Le petit journal' published a picture of Emile Roux saving a child on its front page (24 September 1894). The French daily newspaper 'Le Figaro' also echoed this success and launched for a call for donations. The newspaper gathered more than a million francs, which were more than welcomed by the research institute and to buy new horses (Ref. 14). In recognition of the discovery of the protective immunity provided by serotherapy, Emil von Behring was awarded, the first Nobel prize in medicine or physiology in 1901. In his Nobel lecture, Berhing paid tribute to his colleagues Löffler and Roux. Between 1901 and 1932 Emile Roux received 115 nominations for the Nobel Prize, but they were in vain. French medical scientists headed by Emile Roux

	In 1872, Grâce), but left before defending his thesis (maybe because of his difficulties with the
	military authority or because of his love affair with Rose A. Shedlock, herself a
	medical student). Meeting by chance Duclaux, he was asked by his mentor to assist
	him for the lectures he was giving on fermentation at La Sorbonne. In 1878, Pasteur
	asked Duclaux if he knew a medical doctor who could assist him in his studies on
	infectious diseases (Ref. 8); and Duclaux introduced Roux to Pasteur. In 1883, Roux
	became the deputy-director of Pasteur's laboratory at the École Normale Supérieure,
	Behring (1854-1917) and Kitasato Shibasaburō (1853-1931) showed that sera
	obtained against the toxin could protect. Then, Roux introduced the use of horses to
	. In 1904, when prepare the immune sera. A great step forward was taken when Roux at the Pasteur
	Duclaux died, he became Deputy Director of the Institute. No doubt he could have Institute began to use horses for immunization on a large scale to obtain serum
	had a great scientific and administrative influence for the Pasteur Institute, but against diphtheria toxin, a method that Behring immediately adopted. In 1894 with
	disease killed him prematurely at the age of 57 due to lung cancer.
	c. Émile Roux

rue d'Ulm. Since 1879, Roux had contributed to Pasteur's investigations on anthrax, then on rabbies. Between 1879 and 1884, Roux would become co-author of 74 papers with Pasteur. In 1883, he was sent to Egypt with Edmond Nocard (1850-1903), Isidore Straus (1845-1896) and Louis Thuillier (1856-1883) to study an epidemic of cholera. There, Thuillier died because of the cholera. During his investigation, Roux conceived that the vibrio of cholera discovered by Robert Koch gains its toxicity through the production of a soluble poison, a "ptomaïne". This concept was key when he undertook his investigation on diphtheria in 1888, together with Alexandre Yersin (1863-1943) (Ref. 13). In 1884, Friedrich Löffler (1852-1915), a collaborator of Robert Koch, isolated and cultured the bacterium identified by Edwin Klebs (1834-1913) an assistant of Rudolf Virchow. Soon after they were convinced that the disease was due to a toxin (Ref. 14). The validation of the hypothesis came from the work of Roux and Yersin. Between 1888 and 1890, they published three reports illustrating that the injected filtrate of cultures of Löffler's bacillus into guinea pigs, rabbits and pigeons would kill them (Ref. 15). In 1890, in Berlin, Emil Adolf von Louis Martin (1864-1946), his colleague of Institut Pasteur, and Dr. Auguste Chaillou (1866-1915), from Necker Hospital, he published the treatment of 300 children with a 50% decreased in mortality (from 50% to 25%). These results were achieved on a devastating disease

  The institute became thanks to its links with the Kindia station in Guinea, one of the very first place to investigate on chimpanzees. Furthermore, Metchnikoff wrote two philosophy books on optimism. A sense of life he had acquired later on after two attempts to kill himself, and many other similar ideas when he had to fight for the cellular immunity against the German school who was supporting the humoral immunity. The wise Nobel committee awarded both leaders, Paul Ehrlich (1854-1915) and Metchnikoff in 1908. Admirably, Metchnikoff also favored research on humoral immunity in his own laboratory, and Jules Bordet (1870-1961) initiated works on alexin, a humoral activity, named "complement" by Ehrlich himself. These investigations of Jules Bordet were awarded with the Nobel prize in 1919. In 1914, Paul h visited the Institut Pasteur. In the Berliner Klinische Wochenschrift on March 16 th , 1914, a few months before the war between France and Germany, Roux and Metchnikoff paid tribute to the work of Paul Ehrlich, illustrating the reciprocal consideration between the French and German scientists In 1890, Metchnikoff joined the editorial board of the Annales de l'Institut Pasteur. Since the beginning of the course organized by Roux, he gave lectures on inflammation. Those gathered in a book published in 1892 were translated in English the following year. Wen, in 1904, Roux became Director of the institute, he nominated Metchnikoff as a deputy-director. But in May 1909, Metchnikoff sent a letter of resignation from this position to the President of the Administrative Council,arguing he had too many collaborators to supervise. It appears that his request was not taken into consideration since he remained deputy-director until his death, which happened in Louis Pasteur's apartment on July 15 th , 1916. For his 70 th birthday, Metchnikoff as its director. Fluent in French, the links he had established with Pasteur were so good that Pasteur sent him to London to follow the works of the British investigative committee on the rabies vaccine. He contributed to studies on the different parameters that affected the vaccine preparation. He realized that Russian rabbits were smaller than the French ones, that their spinal cords dried faster, that the protection was dependent of the amounts used for vaccination, and that, in agreement with Roux observation, there was a seasonal influence on the preparation of the rabies vaccine (Ref.[START_REF] Gamaleia | Sur les vaccinations préventives de la rage[END_REF]. He mentioned that there was a need to acquire immunity before the rabies virus reaches the central nervous system. Like Grancher did, Gamaleïa tested on himself the vaccine to demonstrate its safety.Then, he similarly investigated the parameters that influenced the preparation of the anthrax vaccine. He reported experiments performed on more than 300 sheep, and a few dogs, rabbits, and rats (Ref.[START_REF] Gamalieia | Etude sur la vaccination charbonneuse[END_REF]. He reported that certain preparations could kill the sheep, and established that the fever induced by the vaccine was a prerequisite for the vaccine efficacy. Why such expensive efforts to better define the vaccine. Well, during the summer 1887, it happened that an anthrax vaccination organized by the bacteriology station of Odessa ended to the death of 80% of the vaccinated animals, i.e. 3549 sheeps, for a cost of more than 40 000 Ref. 28). Gamaleïa showed that this bacterium was lethal to chickens, pigeons and also guinea pigs. But Gamaleïa failed to reproduce his results with Parisian pigeons. On April 4 th , 1889 Pasteur sent him a letter requesting him to be able to reproduce his results. Pasteur precised, that despite the mention of his name in the Annales de L'Institut Pasteur and in the Official Journal of the Republic, the administrative Council of the institute would not yet validate his venue.

	After
	having initiated his career as an embryologist, Metchnikoff made his main discovery
	on phagocytosis in 1882 while he was in Messina. The contribution of macrophages
	and microphages (the name given to the neutrophils) during infectious diseases has
	been his main conductive thread, and most of his trainees at Institut Pasteur
	contributed to this research (Ref. 22-24). But he also identified new macrophages
	such as the alveolar macrophages with Nicolaï Tchistovitch (1860-1926) and
	microglial cells, then called neuronophages, with Félix Mesnil (1868-1938) and
	Michel Weinberg (1868-1940). But Metchnikoff had also addressed all the processes
	associated with the function of the phagocytes. With Arthur Hugenschmidt (1862-
	1929), and Georges Gabritchevsky (1860-1907) he studied chemotactism. The later
	coined the word pinocytosis. With Sir Marc Armand Ruffer (1859-1917), he studied
	efferocytosis. Of note, after his stay in Metchnikoff's laboratory, Ruffer would be the
	most fervent advocate of the phagocytosis process in UK. With Constantin Levaditi (1874 -1953) they observed netosis. But the work of Metchnikoff, once at Institut Pasteur covered far wider field of investigation. Metchnikoff can be considered as the father of the microbiota, which he studied in humans and various animal species. He also promoted the use of probiotics and coined the word "gerontology". He demonstrated the link between certain bacterial metabolites and the occurrence of atheromatous plaque associated with ageing. With Eugène Wollman (1883-1943) and Michel Cohendy (1873-1940), they obtained germ-free flies and germ-free chicks, respectively. Finally, with Roux, he worked on syphilis, introducing investigations on chimpanzees. Their work ended to the proposal of calomel, a mercuric treatment that was proposed by the US army to its soldiers during world war reputation, and your work and that of your students have greatly contributed to its glory". f. The missing sixth: Nikolaï Gamaleïa Nikolaï Gamaleïa (1859-1949), a Russian physician and microbiologist was present among the 600 people gathered in the library during the inauguration of Institut Pasteur, and his name was officially mentioned to lead the unit entitled "Comparative microbiology" (microbie comparée). However, he will never join the institute. This episode in the history of the creation of Institut Pasteur is rather opaque and the few elements that we address will perhaps offer a begin to explain I. Roux said: "The Institut Pasteur owes you much, you brought the prestige of your this intriguing absence.
	On February 1886, the Odessa Medical Society had sent him to learn the
	new anti-rabies treatment developed by Pasteur (Ref. 25). He spent four months in
	Paris, before contributing to the birth of the first Russian bacteriology station in June
	1886, with

rubles. The owner asked Metchnikoff and Gamaleïa to reimburse half the price and went to trial. Of course, the lay press echoed this disaster. No doubt that a major mistake had been made, particularly, the large-scale use of a vaccine not beforehand tested.

Another difficulty arose in Gamaleïa's scientific life. In 1888, studying the sanitary status of the bird market, he identified a new pathology of fowl cholera, he called "choleric gastroenteritis of birds". He reported that this new pathogen was close to that of the avian cholera, but it induced a disease without fever. Gamaleïa isolated and identified the bacteria, he called "Vibrio metchnikovi" to pay tribute to his Mentor (

Table 1 .

 1 First encounter of Louis Pasteur with his five associates

	Name	Degree	Year	Age *	Place
	Emile DUCLAUX (1840-1904)	Agrégation	1862	48	Laboratory of L. Pasteur, Ecole Normale. Works as a laboratory associate
	Charles CHAMBERLAND Agrégation (1851-1908)	1875	39	Laboratory of L. Pasteur, Ecole Normale. Works as a laboratory associate
	Emile ROUX (1853-1933)	MD	1878	35	Contact rue D'Ulm thanks to Duclaux. Works as a laboratory associate
	Joseph GRANCHER (1843-1907)	MD	1884	45	International Congress of Medicine, Copenhagen. Called in 1885 to perform the very first human rabies vaccine
	Elie METCHNIKOFF (1845-1916)	Doctor Sci.	1887	43	Rue d'Ulm after a visit to L. Pasteur while looking for a job

* Age at the opening of Institut Pasteur

Acknowledgements. The authors thank the phototèque of Institut Pasteur for providing all the pictures but one. For Figure 5, we thank BIU Santé, Paris for their kind contribution. The authors thank Orhan Rasid, Emma Patey and Michael Connor for their collective efforts in English editing.

where he was entrusted with the direction of the newly created physiological chemistry laboratory.


Stanislas Pol
email: stanislas.pol@aphp.fr

PhD, DrSc Sylvie Lagaye
email: sylvie.lagaye@inserm.fr

Stanislas Pr

MD Pol

The remarkable history of the hepatitis C virus

The infection with the hepatitis C virus (HCV) is an example of the translational research success. The reciprocal interactions between clinicians and scientists have allowed in 30 years the initiation of empirical treatments by interferon, the discovery of the virus, the development of serological and virological tools for diagnosis but also for prognosis (the non-invasive biochemical or morphological fibrosis tests, the predictors of the specific immune response including genetic IL28B polymorphisms). Finally, well-tolerated and effective treatments with oral antivirals inhibiting HCV non-structural viral proteins involved in viral replication have been marketed this last decade, allowing the cure of all infected subjects. HCV chronic infection, which is a public health issue, is a hepatic disease which may lead to a cirrhosis and an hepatocellular carcinoma (HCC) but also a systemic disease with extra-hepatic manifestations either associated with a cryoglobulinemic vasculitis or chronic inflammation. The HCV infection is the only chronic viral infection which may be cured: the so-called sustained virologic response, defined by undetectable HCV RNA 12 weeks after the end of the treatment, significantly reduces the risk of morbidity and mortality associated with hepatic and extra-hepatic manifestations which are mainly reversible.

The history of HCV ends with the pangenotypic efficacy of the multiple combinations, easy to use for 8 to 12 weeks with one to three pills per day and little problems of tolerance. This explains the short 30 years from the virus discovery to the viral hepatitis elimination policy proposed by the World Health Organization (WHO) in 2016. The hepatitis C virus (HCV) infection is an example of the translational research success. The reciprocal interactions between clinicians and scientists have allowed in 30 years the initiation of empirical treatments by interferon, the discovery of the virus, the development of serological (ELISA, RIA) and virological (reverse transcriptase polymerase chain reaction or RT-PCR enabling the qualitative and quantitative detection of viral RNA, genotyping, resistance analysis such as basic virology) diagnostic tests with an increasing sensitivity and specificity. In parallel, non-invasive biochemical or morphological fibrosis tests, predictors of the specific immune response including genetic (IL28B) have been developed and finally, radically effective treatments with HCV-specific oral antivirals inhibiting non-structural viral proteins involved in viral replication have been marketed this last decade, allowing the cure of all infected subjects. The elimination agenda of the World Health Organization (WHO) illustrates these major advances but must not hide the global challenges of tomorrow. If the diagnosis, efficacy and tolerability of treatment are no longer an issue, less than 1% of the 71 million infected individuals worldwide has been treated and the majority of patients are unaware of their infection: the next challenges are therefore mainly to improve the screening and access to treatment for this frequent infection which represents a public health although that is the only viral chronic infection that can be cured.

Epidemiology

Between 1990 and 2013, the viral hepatitis rised from the tenth to seventh place worldwide as a cause of death today, higher than the mortality from infection with HIV (HIV), malaria and tuberculosis [START_REF] Stanaway | The global burden of viral hepatitis from 1990 to 2013: findings from the Global Burden of Disease Study 2013[END_REF]: The viral hepatitis appear as the leading cause of infectious mortality in the world despite the HCV prevalence decreased from approximately 170 million chronic carriers worldwide in 1999 [START_REF] Cohen | The scientific challenge of hepatitis C[END_REF] to 71 million in 2017 [START_REF] Observatory | Global prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study[END_REF]4]. This reduction in prevalence is linked to reducing the risk of nosocomial infection, improving access to an efficient antiviral treatment (still a minority), but also to the mortality of infected subjects. We now consider that 1% of the world's population is infected with HCV with over one million seven hundred and fifty thousand new infections in 2015 mainly related to the parenteral risk, intravenous drug use in the northern countries but also the lack of hemovigilance in countries of intermediate or low economy [START_REF] Observatory | Global prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study[END_REF]4]; 2.3 million people are co-infected with HIV and HCV.

There are major regional disparities in this prevalence since the most exposed areas are Egypt or Mongolia with 15% of the historically infected population. In these countries, the contamination was mainly nosocomial due to the absence of hemovigilance in Mongolia or due to the systematic treatment of schistosomiasis without single-use equipment in Egypt [START_REF] Thursz | HCV transmission in industrialized countries and resourceconstrained areas[END_REF]. Other regions such as West Africa or Central Africa are heavily infected with approximately 5% to 8% of the contaminated population, not only because of nosocomial transmission but also because of certain "folkloric" practices (scarification, excision, cupping in Japan or barber in Sicily). In northern countries, the prevalence of Hepatitis C is less than 1% with a steady decrease in prevalence and incidence over the past 20 years: as an example, in France, the prevalence among insured persons decreased from 1.2% in 1996 to 0.8% in 2011 and probably 0.47% in 2017 and this decline is related to the high rate of the screening and antiviral treatment and to the pro-active policy of harm reduction especially among intravenous drug users (IVDU).

The overall mortality attributable to viral hepatitis in 2015 is approximately 720 000 deaths from cirrhosis and 470,000 deaths from hepatocellular carcinoma (HCC) with an increase of 22% since 2000 [4]. Although the hepatitis B virus infection mortality is almost twice as high, despite a vaccine that has been available for more than 30 years, about 400 000 people die each year because of their chronic hepatitis C infection, mainly because of cirrhosis (about 2/3) but also because of hepatocellular carcinoma (1/3) [4].

The history of the hepatitis C virus

The history of HCV is unique in the history of microbiology because its discovery was late, in 1988 occurring after the first empiric therapeutic trials but later developments were extremely fast since in [START_REF] Arazi | In Vivo Dynamical Interactions between CD4 Tregs, CD8 Tregs and CD4+ CD25-Cells in Mice[END_REF] This policy, promoted by the WHO and initiated by at least 12 countries in the world, is not strictly speaking a policy of elimination since it is expected a reduction of new infections by 30% in 2020 and 90% in 2030 and a reduction in hepatitis-related mortality of 10 and 65%, respectively [4].

In 1988, Michael Houghton's team isolated complementary DNA from the blood of a person infected with a "non-A non-B" virus, allowing the isolation of viral RNA and the rapid development of serological diagnostic tests. Elisa tests (EIA) and radio-immunoassays (RIA, now abandoned) were developed in parallel [START_REF] Choo | Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome[END_REF]; their sensitivity and specificity are now excellent. The presence of anti-HCV antibodies testified to the past exposure to HCV but did not determine the active nature of the infection, suspected by the presence of hypertransaminasemia but absent in a quarter of chronically infected patients. The only limit of the serology is the seroconversion time of about 4 to 10 weeks after contamination compared to HCV viremia, which is detectable by RT-PCR within 4 days post-infection.

The detection of HCV RNA by RT-PCR (and in particular of the negative strand) allows itself to assert the active nature of the infection. The detection thresholds have been lowered over the past 20 years and are now around 1.2 log, i.e 12 IU/mL. HCV RNA is detectable in the liver [START_REF] Mcguinness | Intrahepatic hepatitis C RNA levels do not correlate with degree of liver injury in patients with chronic hepatitis C[END_REF] or in the peripheral blood mononuclear cells and, of course, in the serum where it is classically sought to affirm the active infection. The isolation of the HCV RNA allowed the sequencing of the virus and the definition of different genotypes (numbered from 1 to 7) and subtypes (a, b, c ...) [START_REF] Zein | Clinical significance of hepatitis C virus genotypes[END_REF]. Quantitative tests of viremia have been developed, allowing, for a given patient, the quantification and genotypic identification, at first, conditioning the therapeutic choices and the duration of treatment in the interferon era and enabling communities to trace contaminations and migrations.

Prior to identification of HCV RNA [START_REF] Choo | Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome[END_REF], standard Interferon therapies for non-A non-B hepatitis began in the late 80s (Fig. 1) [START_REF] Bisceglie | Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial[END_REF]. It had been shown that 3 to 5 million Interferon units, 5 days a week or every 2 days for 24, 48 or 72 weeks allowed a viral eradication or sustained virological response (RVS) defined by undetectable HCV-RNA within 24 weeks after the end of treatment and corresponding to virological cure [START_REF] Bisceglie | Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial[END_REF]. In parallel to the standard virological tests, a serological identification of the HCV capsid antigen has been developed; this test, which has a comparable sensitivity to HCV-RNA quantification, has not experience the expected developments but could be a less expensive and as effective test as the viral load quantification.

The development of the serological tests allowed on the one hand the identification of the subjects having met the HCV (presence of anti-HCV antibodies) and the detection of the HCV RNA by RT-PCR in the serum the identification of the subjects having an active infection.

Thus a patient with anti-HCV antibodies and undetectable HCV RNA is a patient exposed but cured of his infection. Serological tests allowed for a mass screening including blood donors.

After the identification of hepatitis B virus markers (anti-HBc or HBs antigen) leading to the exclusion of blood donation, the identification of anti-HCV antibodies reduced the risk of "transfusion" contamination by HCV related to blood products (blood, immunoglobulins, anti-hemophilic factors, fresh frozen plasma ...). Before this screening, the risks were approximately 5% to 10% per transfused blood pellet; they are today about 1 to 700,000 to 1 million, corresponding to occult infections despite the viral genomic diagnosis in blood bank.

The HCV identification thus had a major individual and collective effect, transforming the diagnosis and prevention of nosocomial or community-based risk, particularly among drugusing patients.

The major scientific steps

HCV life cycle

HCV is a small, enveloped, positive single-stranded RNA virus belonging to the Flaviviridae family, genus Hepacivirus. The enveloped particles have an icosahedral diameter of 56 to 65 nm [START_REF] Kaito | Hepatitis C virus particle detected by immunoelectron microscopic study[END_REF], while the viral core is around 45 nm [START_REF] Gastaminza | Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture[END_REF]. It is important to understand the key stages of the HCV viral cycle in order to understand the mode of action of different treatments even if the molecular mechanisms underlying this cycle are not completely understood and remain extremely complex. We have therefore selected important steps in this cycle and their protein actors, in order to introduce therapeutic objectives and the particular relationship of HCV with the liver. The first step in the HCV replication cycle is its entry into the cell, through the interaction of its E1 and E2 surface glycoproteins with the baso-lateral hepatocyte membrane, in contact with the blood stream. HCV can be associated with lipoproteins in the serum, thus escaping neutralizing antibodies [START_REF] Dubuisson | Virology and Cell Biology of the Hepatitis C Virus Life Cycle: an update[END_REF]. As many other viruses, the initiation of HCV entry seems to utilize for attachment glycosaminoglycans (GAGs) [START_REF] Belouzard | Hepatitis C virus entry into the hepatocyte[END_REF][START_REF] Brazzoli | CD81 is a central regulator of cellular events required for hepatitis C virus infection of human hepatocytes[END_REF]. The HCV particle interacting with lipoprotein, it is more likely that the apolipoprotein E is responsible for the interaction with GAGs [START_REF] Pileri | Binding of Hepatitis C Virus to CD81[END_REF]. Many membrane molecules seem to be the target of the pathogen and allow its entry, including ubiquitous CD81 (tetraspanin family protein -TSPAN28) [START_REF] Brazzoli | CD81 is a central regulator of cellular events required for hepatitis C virus infection of human hepatocytes[END_REF][START_REF] Pileri | Binding of Hepatitis C Virus to CD81[END_REF], the LDL receptor, claudin-1 (Cldn1) or occludin (OCLN) [START_REF] Evans | Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry[END_REF][START_REF] Harris | CD81 and claudin-1 coreceptor association: role in hepatitis C virus entry[END_REF][START_REF] Ploss | Human occludin is a hepatitis C virus entry factor required for infection of mouse cells[END_REF]. In vivo, its entry into the cell is done in several steps: the envelope glycoprotein E2 interacts with a co-receptor that is scavenger receptor B1 (SR-B1), and with CD81 [START_REF] Catanese | Role of scavenger receptor class B type I in hepatitis C virus entry: kinetics and molecular determinants[END_REF]. The interaction between CD81 and the E2 glycoprotein appears to be essential for initiating the adsorption [START_REF] Dubuisson | Virology and Cell Biology of the Hepatitis C Virus Life Cycle: an update[END_REF], then the receptor complex with attached virion is moving to the tight junction, where an interaction with proteins claudin-1 and occluding set up. Finally, other cellular factors such as Epidermal Growth Factor (EGF) receptor [START_REF] Lupberger | EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy[END_REF] and the Niemann-Pick C1-like 1 (NPC1L1) cholesterol uptake receptor [START_REF] Sainz | Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor[END_REF] are likely involved in HCV entry. Subsequently, the virus is internalized in clathrin vesicles and fused with early endosomes [START_REF] Coller | RNA interference and single particle tracking analysis of hepatitis C virus endocytosis[END_REF][START_REF] Blanchard | Hepatitis C virus-like particle morphogenesis[END_REF][START_REF] Blanchard | Hepatitis C virus entry depends on clathrin-mediated endocytosis[END_REF][START_REF] Meertens | Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles[END_REF]. The acidification of the vacuole allows the membrane fusion of the virus, the virus capsid is then released and destroyed while the viral RNA is released in the cytosol. Once in the cystosol, the viral RNA is used for both processes the replication and the polyprotein translation. The RNA translation into polyprotein occurs in endoplasmic reticulum (ER) and is initiated by binding of the 5'UTR IRES to the ribosome [START_REF] Lindenbach | Complete replication of hepatitis C virus in cell culture[END_REF]. The primary translation product is ~3000 amino acid long polyprotein precursor which contains structural and non-structural proteins of HCV. Then, the polyprotein is cleaved by host and viral proteases into three structural proteins (Core protein, envelop proteins E1 and E2) as well as seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) of the viral replication machinery [START_REF] Dubuisson | Virology and Cell Biology of the Hepatitis C Virus Life Cycle: an update[END_REF] and in addition a Frameshift protein (F protein) or Alternate reading frame protein (ARFP). The functions of ARFP in the viral life cycle remain to be elucidated [START_REF] Xu | Hepatitis C virus F protein is a short-lived protein associated with the endoplasmic reticulum[END_REF][START_REF] Komurian-Pradel | Antigenic relevance of F protein in chronic hepatitis C virus infection[END_REF] and could modulate the dendritic cells function and stimulate the T cell responses [START_REF] Samrat | Alternate Reading Frame Protein (F Protein) of Hepatitis C Virus: Paradoxical Effects of Activation and Apoptosis on Human Dendritic Cells Lead to Stimulation of T Cells[END_REF][START_REF] Arazi | In Vivo Dynamical Interactions between CD4 Tregs, CD8 Tregs and CD4+ CD25-Cells in Mice[END_REF].

The viral RNA will be replicated by the protein NS5B, the RNA-dependent RNA polymerase (RdRp) containing the GDD motif in its active site [START_REF] Xu | Synthesis of a novel hepatitis C virus protein by ribosomal frameshift[END_REF]. For HCV RNA replication, the polarized positive HCV RNA genome synthesizes a negative strand HCV RNA by the NS5B RNA-dependent RNA polymerase. The newly synthesized negative strand of HCV RNA may act as a template to synthesize the positive strand of viral RNA [START_REF] Paul | Hepatitis C Virus Replication and Assembly: Living on the Fat of the Land[END_REF][START_REF] Ashfaq | An overview of HCV molecular biology, replication and immune responses[END_REF][START_REF] Moradpour | Replication of hepatitis C virus[END_REF].

After an accumulation of structural proteins and the viral RNA in the cytosol, the morphogenesis of virions can start. HCV needs a liver-specific microRNA named miR122 to replicate properly. The latter recruits proteins in 5' viral RNAs, thus preventing their degradation by intracellular exonucleases [START_REF] Jopling | Modulation of Hepatitis C Virus RNA Abundance by a Liver-Specific MicroRNA[END_REF][START_REF] Shimakami | Stabilization of hepatitis C virus RNA by an Ago2-miR-122 complex[END_REF].

The life cycle of HCV seems to be related to that of lipoproteins: beyond its association with them to escape the immune system in the blood and facilitate its entry into hepatocytes, they are also necessary for its morphogenesis. The Core protein, forming the viral capsid, will bind to intracellular lipid droplets to initiate the virion morphogenesis. The replication of the virus completely changes the distribution of intracellular lipid droplets: they are physiologically distributed equitably throughout the cytosol of the hepatocytes but are found concentrated in the perinuclear domain during the replication of the virus.

In addition, apolipoproteins A1, B, C1, C3 and E are found on the surface of the envelope of viruses. Only Apo E seems strictly necessary for the viability of virions. Finally, if one blocks certain proteins necessary for the genesis of VLDL, such as MPT (microsomal triglyceride transfer protein), the virus can no longer replicate. The NS5A protein plays an important role in the assembly of the virus, in the lipid protein C-droplet stage. The lipid-capsid combination will surround the freshly replicated RNA and then bind with the other structural proteins of the glycoprotein envelope (E1 and E2) derived from the endoplasmic reticulum [START_REF] Boulant | Hepatitis C Virus Core Protein Induces Lipid Droplet Redistribution in a Microtubuleand Dynein-Dependent Manner[END_REF][START_REF] Bartenschlager | Assembly of infectious hepatitis C virus particles[END_REF][START_REF] Hueging | Apolipoprotein E Codetermines Tissue Tropism of Hepatitis C Virus and Is Crucial for Viral Cell-to-Cell Transmission by Contributing to a Postenvelopment Step of Assembly[END_REF][START_REF] Huang | Hepatitis C Virus Production by Human Hepatocytes Dependent on Assembly and Secretion of Very Low-Density Lipoproteins[END_REF][START_REF] Masaki | Interaction of Hepatitis C Virus Nonstructural Protein 5A with Core Protein Is Critical for the Production of Infectious Virus Particles[END_REF]. After entering the cell, its replication and translation of its proteins, as well as the assembly of its various components, the virion is ready to be exocyted and to infect new cells [START_REF] Scheel | Understanding the hepatitis C virus life cycle paves the way for high effective therapies[END_REF].

Cloning and sequencing of HCV, pseudo-particles: the understanding of HCV replication cycle

The HCV genome was identified in 1989 by cloning it from infected chimpanzee, while in humans the amounts were too low for detection [START_REF] Choo | Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome[END_REF]. The first complete full-length HCV cDNA clone was constructed from the HCV strain H77 (genotype 1a). The HCV RNA transcribed from this clone, then followed by several full-length HCV RNAs was found to be infectious after intrahepatic injection in a chimpanzee. The HCV viremia was detected at week 1 and increased from 1 × 10 2 genomes/mL to 1 × 10 6 genomes/mL at week 8 [START_REF] Kolykhalov | Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA[END_REF][START_REF] Yanagi | Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee[END_REF].

Since these HCV clones were found to replicate inefficiently in vitro, this limitation was resolved by R. Bartenschlager et al when subgenomic HCV replicon, cloned from the HCV genome was constructed after transfection into the Huh7 cells [START_REF] Lohmann | Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line[END_REF]. Several studies demonstrated that the virus and host factors were important for the HCV replication in cells; some mutations in the wide-type (wt) consensus sequence efficiently contributed to the replication and the adaptation to the host cells but these replicons with adaptive mutations could replicate with a high efficiency, they were not able to produce infectious particles in vitro. Finally, a selectable HCV replicon was constructed containing the full-length HCV cDNA of the genotype 2a infectious clone JFH-1 (the only HCV strain reported to induce fulminant HCV-related hepatitis) and was shown to produced infectious particles in vitro and in vivo [START_REF] Blight | Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication[END_REF]. The most efficient construct is the genotype 2a/2a clone which consists of J6CF and JFH-1 derived sequence [START_REF] Blight | Efficient initiation of HCV RNA replication in cell culture[END_REF]. The HCV replicon is remarkably valuable for studying HCV replication and for testing new antiviral drugs. The HCV subgenomic replicons containing reporter genes (luciferase, secreted alkaline phosphatase and chloramphenicol transferase) facilitated the study of the HCV infection. This high-throughput screening assay allowed the visualization and tracking of the HCV replication complex in living host cells without affecting HCV replication [START_REF] Wakita | Production of infectious hepatitis C virus in tissue culture from a cloned viral genome[END_REF][START_REF] Pietschmann | Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras[END_REF].

HCV pseudotyped particles were constructed with chimeric genes expressing HCV (genotype 1a) envelope E1 and E2 proteins (HCVpp) and the transmembrane and cytoplasmic tail of vesicular stomatitis virus G protein. These pseudotyped particles allowed a detailed study of the role of HCV receptors in the early steps of HCV infection (adsorption, and viral entry) [START_REF] Murray | Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells[END_REF][START_REF] Bartosch | Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor[END_REF] and for testing new antiviral drugs [START_REF] Gottwein | Development and characterization of hepatitis C virus genotype 1-7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs[END_REF]. All these major discoveries have been previously detailed [START_REF] Lindenbach | Complete replication of hepatitis C virus in cell culture[END_REF][START_REF] Morozov | Hepatitis C virus: Morphogenesis, infection and therapy[END_REF].

Non-invasive tests of fibrosis improving screening and individualizing therapies:

Given their limited efficacy and their poor tolerance, interferon-based therapies were restricted to patients with significant fibrosis. The evaluation of hepatic lesions was only performed by liver biopsy. This has been revolutionized by the development of non-invasive fibrosis tests evaluating both the necro-inflammation activity (A) and the fibrosis stage according to different scoring system [START_REF] Bedossa | An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group[END_REF]. At the same time blood tests (Fibrotest, Fibrometer, Hepascore, FIB-4 or APRI) [START_REF] Poynard | Overview of the diagnostic value of biochemical markers of liver fibrosis (FibroTest, HCV FibroSure) and necrosis (ActiTest) in patients with chronic hepatitis C[END_REF][START_REF] Oberti | Noninvasive diagnosis of hepatic fibrosis or cirrhosis[END_REF] or morphological tests including pulse elastometry [START_REF] Castéra | Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C[END_REF][START_REF] Cassinotto | Non-invasive assessment of liver fibrosis with impulse elastography: comparison of Supersonic Shear Imaging with ARFI and FibroScan(R)[END_REF] which measures a shearing force corresponding to the liver stiffness makes it possible. These tools make it possible to evaluate F fibrosis on a conventional Metavir scale of 0 to 4, where F0 and F1 correspond to a null or minimal fibrosis as opposed to medium F2 fibrosis, extensive F3 or cirrhotic F4 which justify not only a therapeutic treatment but also a follow-up of the patients because of the risks of hepatocellular carcinoma.

The polymorphism of IL28B

Well-established on-treatment and baseline predictors of sustained virological response (SVR) to pegylated interferon and ribavirin (PEG-IFN/RBV) in patients with chronic hepatitis C virus (HCV) genotype 1 infection include rapid virological response (RVR; undetectable HCV RNA at week 4), low baseline viral load (<600 000 IU/mL), non-black race, and absence of severe fibrosis or insulin resistance [START_REF] Hoofnagle | Peginterferon and ribavirin for chronic hepatitis C[END_REF][START_REF] Mchutchison | Peginterferon alfa-2b or alfa-2a with ribavirin for treatment of hepatitis C infection[END_REF]. For some years, low serum levels of the 10k Da interferon gamma-induced protein (IP-10) have also been associated with a better PEG-IFN/RBV response [START_REF] Romero | Interferon (IFN)-gamma-inducible protein-10: association with histological results, viral kinetics, and outcome during treatment with pegylated IFN-alpha 2a and ribavirin for chronic hepatitis C virus infection[END_REF].

Candidate genes have long been targeted to explain the differences in host antiviral response, and it is now well established that host genetics plays a role in the response to IFN-based therapy in HCV infection [START_REF] Thomas | Genetic variation in IL28B and spontaneous clearance of hepatitis C virus[END_REF]. Five GWAS investigations described several single nucleotide polymorphisms (SNPs) in the IL28B gene region on chromosome 19 as being highly predictive of spontaneous clearance of acute hepatitis C infection [START_REF] Thomas | Genetic variation in IL28B and spontaneous clearance of hepatitis C virus[END_REF], response to PEG-IFN α/RBV therapy in the general population as well as in human immunodeficiency virus (HIV) co-infected individuals and liver transplant recipients in whom both donor and recipient IL28B haplotypes contribute to the probability of treatment response [START_REF] Ge | Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance[END_REF][START_REF] Mccarthy | Replicated association between an IL28B gene variant and a sustained response to pegylated interferon and ribavirin[END_REF].

The GWAS data demonstrated an association between variations at the IL28B gene locus and outcomes in HCV infection, but did not identify a causal variant responsible for these effects: the specific immunologic mechanisms involved in HCV clearance associated with IL28B genotype remain elusive and a functional link between IL28B genotype and liver cytokine expression has not been established [START_REF] Morrow | Comparative ability of IL-12 and IL-28B to regulate Treg populations and enhance adaptive cellular immunity[END_REF]. IL28B encodes for IFN-λ3 which belongs to the family of type III IFNs; type III INFs effect their antiviral activity by activating the JAK-STAT pathway which leads to the induction of IFN stimulated genes (ISGs) from interferon stimulated response elements (ISREs) in the nucleus [START_REF] Morrow | Comparative ability of IL-12 and IL-28B to regulate Treg populations and enhance adaptive cellular immunity[END_REF]. Thus, it is involved in the T-cell adaptive immune response [START_REF] Freeman | The presence of an intrahepatic cytotoxic T lymphocyte response is associated with low viral load in patients with chronic hepatitis C virus infection[END_REF] and IL28B has been associated with increased CD8 + cytotoxic T cell responses. Interestingly, it has been demonstrated that in non-responders, some interferon-stimulated genes were upregulated before treatment. In addition, minor alleles of IL28B polymorphisms (i.e. rs8099917 G and rs12979860 T) have been associated with reduced IL28B expression in peripheral blood mononuclear cells [START_REF] Freeman | The presence of an intrahepatic cytotoxic T lymphocyte response is associated with low viral load in patients with chronic hepatitis C virus infection[END_REF]. Thus, IL28B genotypes may play a role in viral containment, and recent results suggest that IL28B polymorphisms associated with poor HCV clearance may actually be protective against hepatic necro-inflammation and fibrosis progression, particularly in patients with HCV genotypes other than 1 [START_REF] Bochud | ANRS HC EP 26 Genoscan Study Group. IL28B alleles associated with poor hepatitis C virus (HCV) clearance protect against inflammation and fibrosis in patients infected with non-1 HCV genotypes[END_REF].

A chronic infection with liver and extra-hepatic consequences

The natural history of viral infection C is characterized by hepatotropism and lymphotropism of the virus (Fig. 2). Hepatotropism accounts for the risks of chronic hepatitis (there is no risk of fulminant hepatitis outside the only strain of genotype 2 at the origin of the first replicon) and of cirrhosis and hepatocellular carcinoma like all chronic hepatitis [START_REF] Trinchet | Complications and competing risks of death in compensated viral cirrhosis (ANRS CO12 CirVir prospective cohort)[END_REF]. Lymphotropism is characterized by HCV replication within B cells and explains the detection of cryoglobulinemia in about half of infected patients. This cryoglobulin is predominantly of type II associating a monoclonal IgM contingent and a polyclonal IgG contingent, and more rarely a type III cryoglobulinemia. It is a protein complex associating the virus with antiviral antibodies and rheumatoid factor that will be deposited in the walls of small and mediumcaliber vessels causing cryoglobulinemic vasculitis responsible for cutaneous involvement (purpura, necrotizing vasculitis), rheumatologic (polyarthritis of the small joints), renal (membranoproliferative glomerulonephritis) and neurological manifestations with frequent peripheral neuropathies and rarely central attacks [START_REF] Cacoub | Impact of sustained virological response on the extrahepatic manifestations of chronic hepatitis C: a meta-analysis[END_REF][START_REF] Negro | Extrahepatic morbidity and mortality of chronic hepatitis C[END_REF]. At most, the B lymphocyte infection can lead to a clonal selection responsible for lymphoma predominantly Non-Hodgkin B-cell lymphoma (splenic villous lymphoma, but sometimes more diffuse lymphomas) [START_REF] Pozzato | Hepatitis C virus and non-Hodgkin's lymphomas: meta-analysis of epidemiology data and therapy options[END_REF][START_REF] Pol | Extra-hepatic cancers and HCV[END_REF].

The chronic infection that occurs in three quarters of infected subjects is also responsible for chronic inflammation that will lead to extra-hepatic manifestations associating neurocognitive disorders, insulin resistance with a risk 1.5 times higher of diabetes, a risk two to three times higher of cardio-, cerebro-or reno-vascular diseases and an increased risk of extra-hepatic cancers [START_REF] Cacoub | Impact of sustained virological response on the extrahepatic manifestations of chronic hepatitis C: a meta-analysis[END_REF][START_REF] Negro | Extrahepatic morbidity and mortality of chronic hepatitis C[END_REF][START_REF] Pozzato | Hepatitis C virus and non-Hodgkin's lymphomas: meta-analysis of epidemiology data and therapy options[END_REF][START_REF] Pol | Extra-hepatic cancers and HCV[END_REF][START_REF] Lee | Chronic hepatitis C virus infection increases mortality from hepatic and extrahepatic diseases: a communitybased long-term prospective study[END_REF][START_REF] Hsu | Association between antiviral treatment and extrahepatic outcomes in patients with hepatitis C virus infection[END_REF]. These liver and extra-hepatic events account for a ten-fold higher mortality in patients with antibodies to HCV with detectable viral C RNA compared to those without detectable HCV RNA or in patients who have never met HCV [START_REF] Lee | Chronic hepatitis C virus infection increases mortality from hepatic and extrahepatic diseases: a communitybased long-term prospective study[END_REF][START_REF] Hsu | Association between antiviral treatment and extrahepatic outcomes in patients with hepatitis C virus infection[END_REF]. Extrahepatic mortality is twice as frequent in patients with active infection as in patients without active viral infection C or without prior HCV exposure. When HCV infection is confirmed, it is important to evaluate the clinical consequences of hepatic and extra-hepatic infections. The evaluation of hepatic lesions was previously performed only on the basis of a liver biopsy and has been revolutionized by the development of non-invasive fibrosis tests (see above).

Patients with "significant" fibrosis (extensive F3 or cirrhotic F4) or even intermediate (F2) but with hepatic co-morbidities justify not only a priority therapeutic management and therefore a virological cure, a hygiene and dietary education to reduce chronic alcohol consumption, overweight or metabolic syndrome (liver co-morbidities) but also a follow-up because of the risk of occurrence of hepatocellular carcinoma, admittedly reduced but not zero [START_REF] Trinchet | Complications and competing risks of death in compensated viral cirrhosis (ANRS CO12 CirVir prospective cohort)[END_REF].

Thus, HCV infection is not only a hepatic infection but a systemic disease [START_REF] Cacoub | Impact of sustained virological response on the extrahepatic manifestations of chronic hepatitis C: a meta-analysis[END_REF][START_REF] Negro | Extrahepatic morbidity and mortality of chronic hepatitis C[END_REF][START_REF] Pozzato | Hepatitis C virus and non-Hodgkin's lymphomas: meta-analysis of epidemiology data and therapy options[END_REF][START_REF] Pol | Extra-hepatic cancers and HCV[END_REF][START_REF] Lee | Chronic hepatitis C virus infection increases mortality from hepatic and extrahepatic diseases: a communitybased long-term prospective study[END_REF][START_REF] Hsu | Association between antiviral treatment and extrahepatic outcomes in patients with hepatitis C virus infection[END_REF] whose consequences are less related to a direct toxicity of the virus than to immuno-mediated mechanisms: chronic hepatitis is mainly related to a destruction of hepatocytes by specific cytotoxic T lymphocytes recognizing the viral antigens expressed on the surface of the cells.

In immunocompetent and immunocompromised patients with little, or no intrahepatic damage, including inflammation, high levels of the HCV replication have been reported [START_REF] Spengler | Immunopathogenesis in hepatitis C virus cirrhosis[END_REF].

In about 30% of HCV liver transplanted patients, despite the high levels of HCV replication, a recurrent hepatitis is developed one year after transplantation. However, high levels of an intrahepatic HCV replication are usually tolerated by the host immune system. A lymphomononuclear infiltrate represented mainly by CD8 + T cells is expected to play a major role in the viral containment, though other subsets, such as CD4 + T and natural killer (NK) cells, and regulatory T cells (Treg) are considered [START_REF] Koziel | Intrahepatic cytotoxic T lymphocytes specific for hepatitis C virus in persons with chronic hepatitis[END_REF]. The intrahepatic CD4 + and CD8 + T cells can recognize HCV structural and nonstructural antigens [START_REF] Schirren | Liverderived hepatitis C virus (HCV)-specific CD4(+) T cells recognize multiple HCV epitopes and produce interferon gamma[END_REF]. However, why in most patients the immune response cannot resolve the infection, remains obscure. In fact, cytotoxic CD8 + T cell-mediated killing could be blunt by a predominant Treg response [START_REF] Alatrakchi | Regulatory T cells and viral liver disease[END_REF].

The only chronic viral infection that is virologically cured

The biology of HCV is simple with a positive-polarity HCV-RNA that will be translated into a polyprotein that will be split by a protease into different structural and non-structural proteins. The NS3/4 protease, the NS5B polymerase are like the key enzymes in viral replication as well as the NS5A protein or replication complex which participates not only in the replication of the viral RNA but also in the assembly of the viral particles. The specific inhibition of these proteins from the years 2005 allowed a control of the viral multiplication [START_REF] Buhler | New targets for antiviral therapy of chronic hepatitis C[END_REF]. Viral replication and viral organogenesis are exclusively cytoplasmic, which facilitates the targeting of antiviral drugs. In contrast to HIV or hepatitis B virus (HBV), there is no reservoir, no pro-viral DNA, no micro-chromosome (HBV cccDNA) and no genomic integration. This explains that one can obtain with the treatments a virologic cure, the so-called SVR, defined by undetectable HCV-RNA at 12 weeks after the infection or after the end of the treatment corresponds to a true virological cure. HCV RNA undetectability in the serum is accompanied by undetectability in peripheral blood mononuclear cells and in hepatocytes testifying to the complete and lasting nature of virologic cure [START_REF] Fontaine | Recovery from chronic hepatitis C in long-term responders to ribavirin plus interferon alfa[END_REF]. This cure is confirmed by organ transplantation of infected subjects who have benefited from effective treatment, unlike HBV for example, transplantation (derogatory) of these organs, and despite deep immunosuppression, does not lead to any infection [START_REF] Parlati | Evidence of HCV recovery after therapy of hepatitis C virus infection by direct acting antivirals[END_REF].

Clinico-biological benefits of virological cure and reversibility of manifestations

Virologic cure is usually accompanied by clinical improvement or even clinical cure of liver and extra-hepatic manifestations [START_REF] Negro | Extrahepatic morbidity and mortality of chronic hepatitis C[END_REF][START_REF] Hsu | Association between antiviral treatment and extrahepatic outcomes in patients with hepatitis C virus infection[END_REF][START_REF] Aasld-Idsa | Recommendations for testing, managing, and treating hepatitis C[END_REF][START_REF] Liver | EASL Recommendations on Treatment of Hepatitis C[END_REF][START_REF] Pol | Reversibility of hepatitis C virus-related cirrhosis[END_REF][START_REF] Mallet | Brief communication: the relationship of regression of cirrhosis to outcome in chronic hepatitis C[END_REF][START_REF] Hermine | Regression of splenic lymphoma with lymphocytes after treatment of hepatitis C virus infection[END_REF][START_REF] Mahale | The effect of sustained virological response on the risk of extrahepatic manifestations of hepatitis C virus infection[END_REF][START_REF] Cacoub | Prognostic value of viral eradication for major adverse cardiovascular events in hepatitis C cirrhotic patients[END_REF]. This explains the expected reduction in mortality from hepatic and extra-hepatic impacts of chronic infection. As an example in the prospective CIRVIR cirrhotic compensated cohort of ANRS-INSERM, we observed a reduction in the risk of the HCC occurrence at 3 and 5 years in cured patients (13.6 versus 1.9 and 21), 8 versus 3.3% incidence of HCC) [START_REF] Nahon | Incidence of Hepatocellular Carcinoma after Direct Antiviral Therapy for HCV in Patients with Cirrhosis Included in Surveillance Programs[END_REF]; in cured patients, HCC is observed only in cases of hepatic comorbidity (metabolic syndrome, overweight, diabetes, alcohol abuse). In addition to the reduced risk of HCC observed in cirrhotic and non-cirrhotic patients, a virological healing also reduces liver and overall mortality in patients [START_REF] Hsu | Association between antiviral treatment and extrahepatic outcomes in patients with hepatitis C virus infection[END_REF][START_REF] Carrat | Clinical outcomes in patients with chronic hepatitis C following direct-acting antiviral therapy: a prospective cohort study[END_REF]. The reduction in hepatic mortality is at least in part, related to the ability to remodel fibrosis at all stages of the disease, including the possibility of cirrhosis reversibility that contributes to this reduction in mortality [START_REF] Pol | Reversibility of hepatitis C virus-related cirrhosis[END_REF][START_REF] Mallet | Brief communication: the relationship of regression of cirrhosis to outcome in chronic hepatitis C[END_REF]. Alongside the significant reduction in liver risk (HCC and decompensation) in cirrhotic patients, there is a similar reduction in the risk of bacterial infections and a reduction in vascular risks (myocardial infarction, stroke or peripheral arterial disease) with a reduction from 9.1 to 2.3% at 3 years and 12.3 versus 3.5% at 5 years in cirrhotic patients cured compared with non-cured patients [START_REF] Nahon | Eradication of Hepatitis C Virus Infection in Patients With Cirrhosis Reduces Risk of Liver and Non-Liver Complications[END_REF]. An extensive fibrosis or cirrhosis is associated with an increased risk of carotid arteriosclerosis, and oral antiviral therapies allow rapid reduction of carotid intimal thickness [START_REF] Petta | Cardiovascular diseases and HCV infection: a simple association or more?[END_REF].

Antiviral treatments

For more than 20 years Interferon for its antiviral and immune-stimulatory properties has been used as the main treatment for chronic infection with HCV [START_REF] Bisceglie | Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial[END_REF][START_REF] Hoofnagle | Peginterferon and ribavirin for chronic hepatitis C[END_REF][START_REF] Mchutchison | Peginterferon alfa-2b or alfa-2a with ribavirin for treatment of hepatitis C infection[END_REF].

Pegylation after 1997 resulted in weekly subcutaneous rather than tri-weekly injections, and the addition of Ribavirin, a nucleoside analogue, in the early 1990s significantly increased the treatment efficacy [START_REF] Manns | Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial[END_REF]. Their limitations were mainly poor clinical tolerance (flu-like syndrome, acuity of dysimmunitary conditions, neurocognitive disorders aggravated by Ribavirin) and biological (myelosuppression with neutropenia and thrombocytopenia for Interferon, haemolytic anemia for ribavirin). The SVR rate increased from about 6% to at most 50% with 48-week treatments for the most common genotypes 1 and 4 (24 weeks for genotypes 2 and 3 with a PVR rate of about 75%) [START_REF] Hoofnagle | Peginterferon and ribavirin for chronic hepatitis C[END_REF]. A large number of factors limited therapeutic efficacy, extensive fibrosis, overweight, genotype 1, HIV-associated infection or insulin resistance (see above). This limited efficiency and this difficult tolerance of interferon-based treatments explain that availability of direct oral antivirals, specific inhibitors of viral proteins, has been a real therapeutic revolution. The first protease inhibitors, Telaprevir and Boceprevir, used from 2011 to 2014, were combined with standard treatment with pegylated interferon and ribavirin: they allowed a halving of the treatment duration of genotypes 1 and 4 (24 weeks) and cured about three quarter of the patients [START_REF] Hézode | Triple therapy in treatment-experienced patients with HCV-cirrhosis in a multicentre cohort of the French Early Access Programme (ANRS CO20-CUPIC) -NCT01514890[END_REF] but the safe issues remained. Since 2014, the combination of 2 to 3 antivirals has been used to cure almost all patients [START_REF] Buhler | New targets for antiviral therapy of chronic hepatitis C[END_REF][START_REF] Aasld-Idsa | Recommendations for testing, managing, and treating hepatitis C[END_REF][START_REF] Liver | EASL Recommendations on Treatment of Hepatitis C[END_REF][START_REF] Zeuzem | Glecaprevirpibrentasvir for 8 or 12 weeks in HCV genotype 1 or 3 infection[END_REF][START_REF] Jacobson | Efficacy of 8 weeks of sofosbuvir, velpatasvir, and voxilaprevir in patients with chronic HCV infection: 2 phase 3 randomized trials[END_REF][START_REF] Kwo | High SVR rates upon 8-or 12-week treatment with glecaprevir and pibrentasvir in patients with chronic HCV genotype 1-6 infection without cirrhosis[END_REF][START_REF] Puoti | High SVR12 with 8week and 12-week glecaprevir/pibrentasvir therapy: an integrated analysis of HCV genotype 1-6 patients without cirrhosis[END_REF][100][101]; the duration of the pangenotypic treatments available since 2017 are 8 to 12 weeks with one to three capsules per day (Fig. 1). The efficacy of these pangenotypic treatments (RVP> 97%) completely removed the factors of poor response to treatment that can be given in all clinical situations [START_REF] Zeuzem | Glecaprevirpibrentasvir for 8 or 12 weeks in HCV genotype 1 or 3 infection[END_REF][START_REF] Jacobson | Efficacy of 8 weeks of sofosbuvir, velpatasvir, and voxilaprevir in patients with chronic HCV infection: 2 phase 3 randomized trials[END_REF][START_REF] Kwo | High SVR rates upon 8-or 12-week treatment with glecaprevir and pibrentasvir in patients with chronic HCV genotype 1-6 infection without cirrhosis[END_REF][START_REF] Puoti | High SVR12 with 8week and 12-week glecaprevir/pibrentasvir therapy: an integrated analysis of HCV genotype 1-6 patients without cirrhosis[END_REF][100].

Therapeutic recommendations are today to treat all patients infected with HCV by prioritizing those with advanced liver diseases (fibrosis) or extra-hepatic (vasculitis), risks of rapid progression of fibrosis (liver comorbidities or transplants) or risks of community diffusion [START_REF] Aasld-Idsa | Recommendations for testing, managing, and treating hepatitis C[END_REF][START_REF] Liver | EASL Recommendations on Treatment of Hepatitis C[END_REF]. The benefit of these treatments is not only individual but also collective by reducing the risk of infection and infection/re-infection in risk communities such as men who have sex with men (MSM) [101] or drug addicts [102,103].

The only limits that can be avoided today are the drug interactions, the therapeutic observance and the rare side effects associated with these treatments [104].

In summary, the history of HCV ends with this great pangenotypic efficacy of multiple combinations, easy to use 8 to 12 weeks with one to three pill-s per day and little problem of tolerance. The virological cure allows a clinical benefit with at worst a stabilization and at best a reversibility of the clinico-biological manifestations. This explains the short 30 years from the discovery of the virus to a policy of elimination proposed by the WHO in 2016 [4].

HCV Elimination

With the antiviral efficacy and good tolerance of pangenotypic drugs, the elimination (and not eradication in the absence of an effective vaccine) is feasible provided that the screening and access to care policies are improved. It is considered that only 1% of the infected population worldwide has been treated and cured [105], this is, of course, linked to the policy lack of screening and access to care but also the drugs price.

  years from HCV isolation of the virus, the reciprocal interactions between the research and the clinical field have led to the development of reliable serological and virological diagnostic tests, the development of effective treatments allowing to hope for the cure of all the patients and the efficacy of HCV elimination policies elimination programs [4].

Fig. 1

 1 Fig. 1 Summary of the hepatitis C virus history and the antiviral treatments against the Hepatitis C virus infection. IFN = Interferon; RBV = Ribavirin; the protease inhibitors are in red (TVR = Telaprevir; BOC = Boceprevir; SMV = Simeprevir; PTV/r = Paritaprevir boosted by ritonavir; GZR = Grazoprevir; G = Glecaprevir; VOX = Voxilaprevir); the polymerase inhibitors NS5B are in yellow (SOF = Sofosbuvir; DSV = Dasabuvir) and the replication complex NS5A inhibitors are in white (LDV = Ledipasvir; DCV = Daclatasvir; EBR = Elabsvir; VEL = Velpatasvir; P = Pibentrasvir).

Fig. 2

 2 Fig. 2 The natural history of HCV infection combining hepatic and extra-hepatic manifestations.

prisons, addiction centers, maternity wards, migrant care centers, psychiatric services. The provision of easy-to-use and low-cost treatment should make this elimination possible.

The elimination as defined by WHO is feasible in European countries, and even planned for 2025 in France. It is expected in Egypt but seems difficult in the US because of the epidemic HCV related to intravenous drug use but it seems impossible in sub-Saharan Africa.