The work of this team concentrates on the analysis of new giant viruses in the environment and in foodstuffs, from ecology to genetic study and their potential involvement in human pathologies. At the border of this work, we will work on research and analyses of new viral agents in immune compromised individuals. The analysis will be done according to 4 research orientations' of the team to knowing research of the giant viruses and the small intracellular bacteria of protozoa in the environment, the genetics of these giant viruses and these bacteria, their potential role in human pathology and finally the search and the analysis for emergent viruses in human pathology for immunocompromised patients.
The principal axis of the team will continue to be researching new environmental agents, primarily bacteria and viruses associated with the protozoa. First of all, the continuation of research for these new agents will be done by widening the environments and the supports tested to isolate them. Until now we researched these agents quasi exclusively in water by using amoebas such as Acanthamoeba as culture system. This approach was very effective since it allowed to isolate many new viral and bacterial agents [Pagnier I, et al. Isolation and identification of amoeba-resisting bacteria from water in human environment by using an Acanthamoeba polyphaga co-culture procedure. 2008. Environ Microbiol. 10:1135-1144; La Scola B et al. The virophage as a unique parasite of the giant mimivirus. 2008. Nature. 455:100-104 ; Pagnier I et al. 2009. Emerging Mycobacteria spp. in cooling towers. Emerg Infect Dis. 15:121-122; Evstigneeva A et al. 2009. Amoeba co-culture of soil specimens recovered 33 different bacteria, including four new species and Streptococcus pneumoniae. Microbiology. 155:657-664; Boyer M et al, 2009. Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms. Proc Natl Acad Sci U S A. 106:21848-53; La Scola B et al, 2010. A burden of new giant viruses from the environment and their tentative characterization by MALDI-TOF mass-spectrometry. Intervirology. 53:344-53.].
The new environmental specimens tested will be the various human environments, particularly grounds and foodstuffs, plants and animals. We will test also the human samples often colonised by amoebas, particularly the lenses or liquids from lenses coming from patients with keratitis. As regards the culture supports, we will add to the amoebas of the gender Acanthamoeba the other protozoa to be used as support of culture with these new agents, for example ciliates and flagellates, but also algae. Beside the protozoa which we started to cultivate in the laboratory after having ordered them in collections of micro-organisms cultures, we will use also protozoa which we already isolated. In order to improve our techniques of isolation which require long handling, we are currently preparing high flow farming systems by agar co-inoculation. Indeed, the current procedure using a liquid medium with various antibiotics is effective but tiresome because it requires to carry out many staining to visualize growth of a microbial agent. Simultaneously with a better samples preparation by successive filtrations, we will inoculate our samples in agar on nonconfluent amoeba layers. That will make it possible to to visualize the presence of pathogenic agents for the amoebas by the simple macroscopic examination of the agar according to a method comparable with that we had developed to study the pathogenic capacity of various strains of P. aeruginosa (Fenner L et al, Are clinical isolates of Pseudomonas aeruginosa more virulent than hospital environmental isolates in amebal co-culture test ? Crit Care Med. 2006 34:823-8). This system of inoculation on Acanthamoeba is at the end of its development and is ready to be used, for the other protozoa it remains to be developed. This part of the inoculation work will be carried out only by our team and will be coordinated by our post-doctorant I. Pagnier (planned for a MCF position). She will frame with B La Scola a team of 3 PhD students. The first one will manage the development of the culture on the protozoa other than the amoebas gender Acanthamoeba and the characterisation preliminaries of the micro-organisms inoculated within this framework. A second one will manage the high flow inoculation on the amoebas Acanthamoeba and also the preliminary characterisation of the micro-organisms isolated within this framework. Originating from Tunisia, this PhD student will realise its PhD in co-supervision with our doctoral school and will study moreover various environmental samples from his country.
After the isolation of these new agents we will study their genetics. After the first characterization, the virus and bacteria strains promissing to be representative of completely new species (electronic microscopy, sequence of one or two conserved genes, direct MALDI-TOF MS) will be analysed by complete genome sequencing. Philippe Colson who is finishing a bioinformatic analysis of the viral genomes post-doctoral training course in the E. Koonin laboratory in Bethesda in the NIH, will be the person in charge of this field. He will continue his work in collaboration with the E Koonin team and with the bio-informatics team of the URMITE. Bernard La Scola will continue to manage the genomic analysis of the new original bacterial agents which we will discover and sequence. For the most original agents, we will go further in the study of their ecology and their way of life, in particular by studying their mode of multiplication in the amoebas by analysis with electronic and confocal microscopy. For the same agents we will analyse the most original processes of multiplication by microarrays in order to study genes up-regulated in the amoeba as well as in the micro-organism during the infection and the various steps of the multiplication cycle. Another PhD student under the direction of P. Colson will be directly in charge of this work.
With respect to the human infections, the research of the implication of the previous and new giant viruses as well as the intracellular bacteria of amoebas will be done by 2 different approaches. A first approach will be based on the realisation of high flow sero-epidemiologic studies by ELISA technique using the various species giant viruses as antigens. The URMITE laboratory has significant collections of serums classified by syndromes (pneumonias, meningoencephalitis, endocarditis, gastroenteritis, eruptive fever, adenopathies…) which will be tested in a serial way. The second approach will be targeted, aiming at detecting by PCR (using systems of starters based on studies on genomes) the ADN as of the these viruses in clinical samples (essentially for pneumonias and diarrhoeas or directed by preceding sero-epidemiologic studies). At last, the positive clinical specimens will be inoculated in co-cultures with protozoa. Hubert Lepidi will be in charge of the anatomo-pathology with immunohistochimy analyses on biopsy samples of positive patients. As for gastroenteritis, we will collect stool specimens in the Federation of microbiology in the hospital of Timone in Marseilles, which receives more than 20 stools of diarrheal patients per day and on our collaborations overseas, particularly with our partner IRD which will be able to give us access to stools of patients of Senegal. A preliminary work on a stools sample of a Senegalese patient allowed the isolation of the first human giant virus.
The following aspect of the project relating to the emergent viruses will be interested in the plants viruses and will be coordinated by P. Colson. A recent metagenomic work showed that the majority of the RNA viruses present in the human digestive tract were viruses of the pepper (pepper mild mottle virus (PMMoV)) found infectious with very high titles. Other studies showed that the administration by oral way of plants virus induced an immune response in mice, and some plant viruses could be cultivated on human cellular lines and be detected in bodies of mouse after inoculation by oral or systemic route. Moreover, the possible implication of plants viruses in human diseases was discussed several decades ago. Indeed, for the viruses of the tobacco mosaic, which are like PMMoV extremely resistant viruses like Tobamovirus, respiratory samples of patients with medical history of pneumopathy and lung cancer could be recovered. In the first work, we detected by PCR of PMMoV in more half of the foodstuffs containing pepper which we tested. We showed that PMMoV was infectious by inoculation with sensitive plants; moreover, we confirmed that this virus can be present in the human digestive tract since we detected it in the stools of 7% of the adult patients [Colson P et al. Pepper mild mottle virus, a plant virus associated with specific immune responses, fever, abdominal pains, and pruritus in humans. Plos One 2010, 5:e100410]. In parallel, we observed among patients carrying PMMoV in stools a specific antibody response directed against the virus, and a case controls study found that the presence of PMMoV was significantly associated with fever, abdominal pains and a pruritus. All these data suggest the interest to revalue the relation and the pathogenic role of the plant viruses of plants in human and animal. We will continue our work on Tobamovirus by analysing the prevalence and the persistence of these viruses in various samples, tissues and organs coming from patients, or animals after experimental inoculation. We will study in particular with Y. Berland and V. Moal an active file of kidney transplanted patients. An interregional PHRC currently aims at determining the incidence of the Hepatitis E Virus (VHE) and PMMoV in the stools of kidney or liver transplanted patients, at determining the clinical impact of these viruses, the factors of risk associated with their presence and to characterise the viruses identified by sequencing, culture and electronic microscopy. We will also study the specific immune responses to viral proteins. In parallel, we will set up farming systems of Tobamovirus on sensitive plants and cellular cultures of human or animal lines, and will study the interactions between these viruses and the human or animal cells.
The last part of this work will more generally relate to research and analysis of emergent viruses in human pathology in immunocompromised patients. This part of work will be coordinated for microbiology research by P. Colson and on the clinical research by Y. Berland and V. Moal. The clinical department held by Y. Berland includes a significant population of immunocompromised patients, primarily renal transplanted patients under immunosuppressive treatment. These patients frequently develop infectious pathologies (unexplained hepatitis, fevers, pneumonias, diarrhoeas) without detected etiologic agent. Within the framework of our research, those patients will be sampled and samples will be analyzed by viral metagenomic according to a procedure close to those already used in recent studies (Nakamura S et al, 2009, Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach. 4:e4219). P. Colson and V. Moal will continue in parallel their work on hepatitis E virus, emergent virus whose transmission in the tropical and subtropical countries is done primarily by water and who is suspected to be an agent of swine zoonoses in the industrialised countries. Its epidemiology in Europe, where hepatitis E was recently regarded as an imported disease, remains to be clarified. Moreover VHE was identified since 2008 like an emergent agent of chronic hepatitis and cirrhosis among the patients receiving a kidney and liver transplant and patients infected by the VIH. We before described the existence of a porcine reservoir of the VHE in France [Kaba M et al. Frequent transmission of hepatitis E virus among piglets in farms in Southern France. J Med Virol. 2009;81:1750-9] and identified a food source of transmission for some of the cases diagnosed at the Timone hospital (Colson P et al. Pig liver sausage as a source of hepatitis E virus transmission to humans. J Infect Dis, 2010; in press). We also described the first case of hepatitis E transmitted by transfusion in France [Colson P et al., Transfusion-associated hepatitis E, France. Emerg Infect Dis. 2007;13(4):648-9] and described chronic cases of infections with VHE among transplanted kidney patients [Gérolami R et al., Hepatitis E virus as an emerging cause of chronic liver disease in organ transplant recipients. J Hepatol.2009;50:6 22-4] and among seropositive VIH patients (Colson P et al., Acute and chronic hepatitis E in patients infected with human immunodeficiency virus. J Viral Hepatitis 2010, in press). Finally we described the first case of cirrhosis complicating chronic hepatitis E in a patient receiving a kidney transplant [Gerolami R et al., Chronic hepatitis E with cirrhosis in a kidney-transplant recipient. N Engl J Med 2008;358:859-60]. We will continue the study of the sources and modes of transmission of the hepatitis E virus in the general population and that of the immunocompromised patients, by testing in particular various food environmental sources, as well as the characterization of the viruses and the immune answers during chronic infections. Currently is carried out a clinical research (AORC) the purpose of which is to determine if there is a peripheral signature specific of chronic hepatitis E among kidney transplanted patients with the study of the RNA messengers transcribed to the peripheral blood level.