Electron microscopy is a powerful tool in the field of microbiology. It has played a key role in the rapid diagnosis of viruses in patient samples and has contributed significantly to the clarification of virus structure and function, helping to guide the public health response to emerging viral infections. In the present study, we used scanning electron microscopy (SEM) to study the infectious cycle of SARS-CoV-2 in Vero E6 cells and we controlled some key findings by classical transmission electronic microscopy (TEM). The replication cycle of the virus was followed from 1 to 36 h post-infection. Our results revealed that SARS-CoV-2 infected the cells through membrane fusion. Particles are formed in the peri-nuclear region from a budding of the endoplasmic reticulum-Golgi apparatus complex into morphogenesis matrix vesicae. New SARS-CoV-2 particles were expelled from the cells, through cell lysis or by fusion of virus containing vacuoles with the cell plasma membrane. Overall, this cycle is highly comparable to that of SARS-CoV. By providing a detailed and complete SARS-CoV-2 infectious cycle, SEM proves to be a very rapid and efficient tool compared to classical TEM.

Viruses generally are defined as lacking the fundamental properties of living organisms in that they do not harbor an energy metabolism system or protein synthesis machinery. However, the discovery of giant viruses of amoeba has fundamentally challenged this view because of their exceptional genome properties, particle sizes and encoding of the enzyme machinery for some steps of protein synthesis. Although giant viruses are not able to replicate autonomously and still require a host for their multiplication, numerous metabolic genes involved in energy production have been recently detected in giant virus genomes from many environments. These findings have further blurred the boundaries that separate viruses and living organisms. Herein, we summarize information concerning genes and proteins involved in cellular metabolic pathways and their orthologues that have, surprisingly, been discovered in giant viruses. The remarkable diversity of metabolic genes described in giant viruses include genes encoding enzymes involved in glycolysis, gluconeogenesis, tricarboxylic acid cycle, photosynthesis, and β-oxidation. These viral genes are thought to have been acquired from diverse biological sources through lateral gene transfer early in the evolution of Nucleo-Cytoplasmic Large DNA Viruses, or in some cases more recently. It was assumed that viruses are capable of hijacking host metabolic networks. But the giant virus auxiliary metabolic genes also may represent another form of host metabolism manipulation, by expanding the catalytic capabilities of the host cells especially in harsh environments, providing the infected host cells with a selective evolutionary advantage compared to non-infected cells and hence favoring the viral replication. However, the mechanism of these genes' functionality remains unclear to date.

The discovery of Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3 µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5 Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that eight putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all eight viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question “what is a virus?”.

COVID-19 is the biggest pandemic the world has seen this century. Alongside the respiratory damage observed in patients with severe forms of the disease, gastrointestinal symptoms have been frequently reported. These symptoms (e.g., diarrhoea), sometimes precede the development of respiratory tract illnesses, as if the digestive tract was a major target during early SARS-CoV-2 dissemination. We hypothesize that in patients carrying intestinal SARS-CoV-2, the virus may trigger epithelial barrier damage through the disruption of E-cadherin (E-cad) adherens junctions, thereby contributing to the overall gastrointestinal symptoms of COVID-19. Here, we use an intestinal Caco-2 cell line of human origin which expresses the viral receptor/co-receptor as well as the membrane anchored cell surface adhesion protein E-cad to investigate the expression of E-cad after exposure to SARS-CoV-2. We found that the expression of CDH1/E-cad mRNA was significantly lower in cells infected with SARS-CoV-2 at 24 hours post-infection, compared to virus-free Caco-2 cells. The viral receptor ACE2 mRNA expression was specifically down-regulated in SARS-CoV-2-infected Caco-2 cells, while it remained stable in HCoV-OC43-infected Caco-2 cells, a virus which uses HLA class I instead of ACE2 to enter cells. It is worth noting that SARS-CoV-2 induces lower transcription of TMPRSS2 (involved in viral entry) and higher expression of B⁰AT1 mRNA (that encodes a protein known to co-express with ACE2 on intestinal cells). At 48 hours post-exposure to the virus, we also detected a small but significant increase of soluble E-cad protein (sE-cad) in the culture supernatant of SARS-CoV-2-infected Caco-2 cells. The increase of sE-cad release was also found in the intestinal HT29 cell line when infected by SARS-CoV-2. Beside the dysregulation of E-cad, SARS-CoV-2 infection of Caco-2 cells also leads to the dysregulation of other cell adhesion proteins (occludin, JAMA-A, zonulin, connexin-43 and PECAM-1). Taken together, these results shed light on the fact that infection of Caco-2 cells with SARS-CoV-2 affects tight-, adherens-, and gap-junctions. Moreover, intestinal tissues damage was associated to the intranasal SARS-CoV-2 infection in human ACE2 transgenic mice.

Pandoraviruses are giant viruses of ameba with 1 μm-long virions. They have an ovoid morphology and are surrounded by a tegument-like structure lacking any capsid protein nor any gene encoding a capsid protein. In this work, we studied the ultrastructure of the tegument surrounding Pandoravirus massiliensis virions and noticed that this tegument is composed of a peripheral sugar layer, an electron-dense membrane, and a thick electron-dense layer consisting in several tubules arranged in a helicoidal structure resembling that of cellulose. Pandoravirus massiliensis particles were stained by Calcofluor white, a fluorescent dye of cellulose, and the enzymatic treatment of particles by cellulase showed the degradation of the viral tegument. We first hypothesized that the cellulose tegument could be synthesized by enzymes encoded by the virus. Bioinformatic analyses revealed in P. massiliensis, a candidate gene encoding a putative cellulose synthase, with a homology with the BcsA domain, one of the catalytic subunits of the bacterial cellulose synthase, but with a low level of homology. This gene was transcribed during the replicative cycle of P. massiliensis, but several arguments run counter to this hypothesis. Indeed, even if this gene is present in other pandoraviruses, the one of the strain studied is the only one to have this BcsA domain and no other enzymes involved in the synthesis of cellulose could be detected, although we cannot rule out that such genes could have been undetected among the large proportion of Orfans of pandoraviruses. As an alternative, we investigated whether P. massiliensis could divert the cellulose synthesis machinery of the ameba to its own account. Indeed, contrary to what is observed in the case of infections with other giant viruses such as mimiviruses, it appears that the transcription of the ameba, at least for the cellulose synthase gene, continues throughout the growth phase of particles of P. massiliensis. Finally, we believe that this scenario is more plausible. If confirmed, it could be a unique mechanism in the virosphere.

The ongoing outbreak of novel coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 infection has spread rapidly worldwide. The major transmission routes of SARS-CoV-2 are recognised as inhalation of aerosol/droplets and person-to-person contact. However, some studies have demonstrated that live SARS-CoV-2 can be isolated from the faeces and urine of infected patients, which can then enter the wastewater system. The currently available evidence indicates that the viral RNA present in wastewater may become a potential source of epidemiological data. However, to investigate whether wastewater may present a risk to humans such as sewage workers, we investigated whether intact particles of SARS-CoV-2 were observable and whether it was possible to isolate the virus in wastewater. Using a correlative strategy of light microscopy and electron microscopy (CLEM), we demonstrated the presence of intact and degraded SARS-like particles in RT-qPCR SARS-CoV-2-positive sewage sample collected in the city of Marseille. However, the viral infectivity assessment of SARS-CoV-2 in the wastewater was inconclusive, due to the presence of other viruses known to be highly resistant in the environment such as enteroviruses, rhinoviruses, and adenoviruses. Although the survival and the infectious risk of SARS-CoV-2 in wastewater cannot be excluded from our study, additional work may be required to investigate the stability, viability, fate, and decay mechanisms of SARS-CoV-2 thoroughly in wastewater.