RNA viruses and human inflammatory heart diseases: from viral metagenomics to animal model – HeartVir

Over the past decade, metagenomic techniques, which are based on massive sequencing of whole genomes without prior knowledge on the initial sequence or the targeted agent, have set themselves as a powerful tool in the discovery of new viruses in the medical field. As part of this project, we use a large-scale metagenomic approach to search for and to characterize viruses associated with idiopathic myopericarditis. Once identified, the presence of the(se) virus(es) is confirmed in the initial sample and its molecular and serological prevalence is studied through a comprehensive epidemiological study on a cohort of patients with idiopathic myopericarditis over the past three years. A collective effort is also focused on the isolation of the virus based on recent work published in the field in order to produce sufficient virions to generate an animal model to better understand the physiopathology of the infection.

The first phase of this project has consisted into the development, validation and publication of a robust protocol for purifying viral particles that remain infectious (which is a requirement for subsequent tests of isolation and cell culture production) while limiting contamination by nucleic acids of the host for the sequencing step (Temmam S. et al., 2016). This methodological development has been applied to a pericardial fluid sample and allows to discover a new gemycircularvirus in a young patient (14 years) with relapsing pericarditis (Halary S. et al., 2016). This virus is being characterized and a molecular and serological prevalence study of this virus in blood of patients suffering (myo)-pericardites and controls is underway. We are also performing cultivation attempts on several supports in order to produce sufficient infectious particles to analyze the physiopathology of this viral infection in an animal model.
Depending on the sample type and preservation conditions, a high proportion of contaminating human sequences was observed in several metagenomes. We thus have developed an approach based on subtractive hybridization and capture of human nucleic acids with a biotin/streptavidin complex directly from the extracted. This protocol, which will be submitted soon for publication, has been successfully applied to blood samples of patient with acute myocarditis. A bioinformatics approach based on similarity networks applied to viruses (Halary S. et al., 2016) allowed us to detect sequences related to new retrovirus and/or retrotransposons whose role is being currently investigated. Finally, we used a protocol adapted to mammalian tissues (Temmam S. et al., 2016) to treat heart valves of 12 patients with sterile endocarditis and sequencing results are being analyzed.

From a scientific perspective, this project provides crucial information on the causes of myopericarditis and an extensive knowledge of the epidemiology and pathophysiology of the infection, knowledge that in turn will open new therapeutic perspectives. From a technical point of view, this project is based on the latest innovations for obtaining purified viral nucleic acids (free from host contaminants), high-throughput sequencing and sequence analyzes. The protocol developed in this project can also be more generally applied to other pathologies in the search for novel RNA/DNA viral candidate (particularly in the context of emerging viral diseases). Finally, many socio-economic benefits may result from this project. As acute myopericarditis is a potentially fatal disease and frequently evolving into recurrent or chronic form, its economic cost to the scale of the society is significant. Through a better understanding of both etiological agents responsible for myopericarditis and physiopathology of the infection, the results of this project will help the medical teams to determine the best support possible and the most appropriate treatment strategy for a given patient. This will result in an improved prognosis and thus a reduction in medical costs.

1. Temmam S., Monteil-Bouchard S., Robert C., Pascalis H., Blanc-Tailleur C., Jardot P., Charrel R., Raoult D., and Desnues C. (2015) Host-associated metagenomics: a guide to generate infectious RNA viromes. PLoS One 2;10(10):e0139810
This article proposes a methodological guide to generate infectious RNA viromes.

2. Temmam S., Davoust B., Chaber A.L., Lignereux Y., Michelle C., Monteil-Bouchard S., Raoult D. and Desnues C. (2016) Screening for viral pathogens in African simian bushmeat seized at a French airport. Transb. Emerging Dis. doi: 10.1111/tbed.12481.
This article proposes a rigorous protocol to detect viral pathogens from mammalian tissues. This protocol is used for biopsies and cardiac valves.

3. Halary S., Temmam S. and Desnues C. (2016) Viral metagenomics: are we missing the giants? Current Op. Microb. 31:34-43. doi: 10.1016/j.mib
This article proposes an approach based on similarity networks to detect divergent viruses in metagenomic datasets.

4. Halary S., Duraisamy R., Fancello L., Monteil-Bouchard S., Jardot P., Biagini P., Gouriet F., Raoult D. and Desnues C. (2016) Novel ssDNA circular viruses in pericardial fluid of patient with recurrent pericarditis. Emerg. Infect. Dis. 22(10):1839-41
This article reports the discovery of a new gemycircularviruse associated with pericarditis in a young patient.

The HearVir project aims at a better understanding and thus an improved diagnostic of idiopathic inflammatory heart diseases by using a combination of high-throughput sequencing, culturing, and epidemiology. Among inflammatory heart diseases, myocarditis and pericarditis are characterized by an inflammation of the myocardium and the pericardial sac, respectively. Clinical manifestations of myopericarditis are variable from acute to chronic, asymptomatic to very severe and even life-threatening forms. Among known etiological infectious agents, viruses (in mono or co-infections) largely prevail over other causes of myocarditis. However, more than 50 % of the cases still remain idiopathic i.e., diagnostic tests fail in determining the etiological cause of disease. Although current methods are good for detecting known viruses, they are very ineffective for discovering novel ones. Diagnosing novel viral infections is difficult mainly due to our inability to culture viruses on cell lines in the laboratory, the small size and the low nucleic acid content of viruses, and the lack of a conserved genetic element among viral genomes that could be used for PCR-based analyses.

Since the past 10 years, metagenomics, the sequenced-based analysis of the whole collection of genomes directly isolated from a sample, has emerged has a powerful tool for discovering completely new viruses or viruses that escaped current diagnostic screening tests. In this project, we propose to use a blind metagenomic approach for a complete description of the RNA viruses associated with idiopathic myopericarditis. Once identified and confirmed in the original sample, this project aims to link the presence of this (these) virus(es) to the pathology throughout an exhaustive epidemiological study performed on a 3-year cohort of patients with myopericarditis. In this project, we also want to bring some collective efforts on culture by using the latest developments of the domain in order to both produce antibodies that can retrospectively be used for epidemiology and to generate an animal model for studying the pathophysiology of the viral infection. The HeartVir project reunites together and toward a common objective, a strong and complementary team composed of scientific experts in the field of characterization of RNA viruses from complex samples, viral metagenomics, physiopathology and animal models of viral infections, associated with among the best physicians in cardiology and infectious diseases.