Klenkia terrae resistant to DNA extraction in germ-free mice stools illustrates the extraction pitfall faced by metagenomics.

Over the past decade, metagenomics has become the preferred method for exploring complex microbiota such as human gut microbiota. However, several bias affecting the results of microbiota composition, such as those due to DNA extraction, have been reported. These bias have been confirmed with the development of culturomics technique. In the present study, we report the contamination of a gnotobiotic mice unit with a bacterium first detected by gram staining. Scanning electron microscopy and transmission electron microscopy permitted to detect a bacterium with a thick cell wall. However, in parallel, the first attempt to identify and culture this bacterium by gene amplification and metagenomics of universal 16S rRNA failed. Finally, the isolation in culture of a fastidious bacterium not detected by using universal PCR was successfully achieved by using a BCYE agar plate with CO2 atmosphere at 30 °C. We performed genome sequencing of this bacterium using a strong extraction procedure. The genomic comparison allowed us to classify this bacterium as Klenkia terrae. And finally, it was also detected in the stool and kibble that caused the contamination by using specific qPCR against this bacterium. The elucidation of this contamination provides additional evidence that DNA extraction could be a bias for the study of the microbiota. Currently, most studies that strive to analyze and compare the gut microbiota are based on metagenomics. In a gnotobiotic mice unit contaminated with the fastidious Actinobacteria Klenkia terrae, standard culture, 16S rRNA gene amplification and metagenomics failed to identify the micro-organism observed in stools by gram-staining. Only a procedure based on culturomics allowed us to identify this bacterium and to elucidate the mode of contamination of the gnotobiotic mice unit through diet.

Discovery and Further Studies on Giant Viruses at the IHU Mediterranee Infection That Modified the Perception of the Virosphere.

The history of giant viruses began in 2003 with the identification of Acanthamoeba polyphaga mimivirus. Since then, giant viruses of amoeba enlightened an unknown part of the viral world, and every discovery and characterization of a new giant virus modifies our perception of the virosphere. This notably includes their exceptional virion sizes from 200 nm to 2 µm and their genomic complexity with length, number of genes, and functions such as translational components never seen before. Even more surprising, Mimivirus possesses a unique mobilome composed of virophages, transpovirons, and a defense system against virophages named Mimivirus virophage resistance element (MIMIVIRE). From the discovery and isolation of new giant viruses to their possible roles in humans, this review shows the active contribution of the University Hospital Institute (IHU) Mediterranee Infection to the growing knowledge of the giant viruses' field.

Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere.

Here we report the discovery of two Tupanvirus strains, the longest tailed Mimiviridae members isolated in amoebae. Their genomes are 1.44-1.51 Mb linear double-strand DNA coding for 1276-1425 predicted proteins. Tupanviruses share the same ancestors with mimivirus lineages and these giant viruses present the largest translational apparatus within the known virosphere, with up to 70 tRNA, 20 aaRS, 11 factors for all translation steps, and factors related to tRNA/mRNA maturation and ribosome protein modification. Moreover, two sequences with significant similarity to intronic regions of 18 S rRNA genes are encoded by the tupanviruses and highly expressed. In this translation-associated gene set, only the ribosome is lacking. At high multiplicity of infections, tupanvirus is also cytotoxic and causes a severe shutdown of ribosomal RNA and a progressive degradation of the nucleus in host and non-host cells. The analysis of tupanviruses constitutes a new step toward understanding the evolution of giant viruses.

Pacmanvirus, a New Giant Icosahedral Virus at the Crossroads between Asfarviridae and Faustoviruses.

African swine fever virus, a double-stranded DNA virus that infects pigs, is the only known member of the Asfarviridae family. Nevertheless, during our isolation and sequencing of the complete genome of faustovirus, followed by the description of kaumoebavirus, carried out over the past 2 years, we observed the emergence of previously unknown related viruses within this group of viruses. Here we describe the isolation of pacmanvirus, a fourth member in this group, which is capable of infecting Acanthamoeba castellanii Pacmanvirus A23 has a linear compact genome of 395,405 bp, with a 33.62% G+C content. The pacmanvirus genome harbors 465 genes, with a high coding density. An analysis of reciprocal best hits shows that 31 genes are conserved between African swine fever virus, pacmanvirus, faustovirus, and kaumoebavirus. Moreover, the major capsid protein locus of pacmanvirus appears to be different from those of kaumoebavirus and faustovirus. Overall, comparative and genomic analyses reveal the emergence of a new group or cluster of viruses encompassing African swine fever virus, faustovirus, pacmanvirus, and kaumoebavirus.IMPORTANCE Pacmanvirus is a newly discovered icosahedral double-stranded DNA virus that was isolated from an environmental sample by amoeba coculture. We describe herein its structure and replicative cycle, along with genomic analysis and genomic comparisons with previously known viruses. This virus represents the third virus, after faustovirus and kaumoebavirus, that is most closely related to classical representatives of the Asfarviridae family. These results highlight the emergence of previously unknown double-stranded DNA viruses which delineate and extend the diversity of a group around the asfarvirus members.

Updating strategies for isolating and discovering giant viruses.

Almost fifteen years ago, the discovery of Acanthamoeba polyphaga mimivirus, the first giant virus, changed how we define a virus. It was discovered incidentally in a process of isolating Legionella sp. from environmental samples in the context of pneumonia epidemics using a co-culture system with Acanthamoeba. Since then, much effort and improvement has been put into the original technique. In addition to the known families of Mimiviridae and Marseilleviridae, four new proposed families of giant viruses have been isolated: Pandoravirus, Pithovirus, Faustovirus and Mollivirus. Major improvements were based on enrichment systems, targeted use of antibiotics and high-throughput methods. The most recent development, using flow cytometry for isolation and presumptive identification systems, opens a path to large environmental surveys that may discover new giant virus families in new protozoa supports used for culture support.