Unexpected thermal stability of two enveloped megaviruses, Emiliania huxleyi virus and African swine fever virus, as measured by viability PCR.

BACKGROUND: The particle structure of Emiliania huxleyi virus (EhV), an algal infecting member of nucleocytoplasmic large DNA viruses (NCLDVs), contains an outer lipid membrane envelope similar to that found in animal viruses such as African swine fever virus (ASFV). Despite both being enveloped NCLDVs, EhV and ASFV are known for their stability outside their host environment. METHOD: Here we report for the first time, the application of a viability qPCR (V-qPCR) method to describe the unprecedented and similar virion thermal stability of both EhV and ASFV. This result contradicts the cell culture-based assay method that suggests that virus "infectivity" is lost in a matter of seconds (for EhV) and minutes (for ASFV) at temperature greater than 50 °C. Confocal microscopy and analytical flow cytometry methods was used to validate the V-qPCR data for EhV. RESULTS: We observed that both EhV and ASFV particles has unprecedented thermal tolerances. These two NCLDVs are exceptions to the rule that having an enveloped virion anatomy is a predicted weakness, as is often observed in enveloped RNA viruses (i.e., the viruses causing Porcine Reproductive and Respiratory Syndrome (PRRS), COVID-19, Ebola, or seasonal influenza). Using the V-qPCR method, we confirm that no PRRSV particles were detectable after 20 min of exposure to temperatures up to 100 °C. We also show that the EhV particles that remain after 50 °C 20 min exposure was in fact still infectious only after the three blind passages in bioassay experiments. CONCLUSIONS: This study raises the possibility that ASFV is not always eliminated or contained after applying time and temperature inactivation treatments in current decontamination or biosecurity protocols. This observation has practical implications for industries involved in animal health and food security. Finally, we propose that EhV could be used as a surrogate for ASFV under certain circumstances.

Analysis of microRNA expression profile in Emiliania huxleyi in response to virus infection.

The interactions between Emiliania huxleyi and E. huxleyi virus (EhV) regulate marine carbon and sulfur biogeochemical cycle and play a prominent role in global climate change. As a large DNA virus, EhVs have developed a novel "virocell metabolism" model to meet their higher metabolic needs. However, the regulatory mechanism of this metabolic model is still largely unclear. MicroRNAs (miRNAs) can regulate biological pathways through targeting hub genes in the metabolic processes. Here, we performed high-throughput small RNA sequencing to analyse miRNA expression in EhV99B1 infected E. huxleyi BOF92. A total of 26 miRNAs (including 2 virus-derived miRNAs) were identified, including four up-regulated and one down-regulated miRNAs. These results were further validated through quantitative real-time PCR. Functional enrichment analysis showed that five differentially-expressed miRNAs might be involved in the regulation of carbohydrate metabolism, lipid metabolism and amino acid metabolism. Moreover, the expression levels of differentially-expressed miRNAs were negatively correlated with that of several lipid metabolism-related genes, such as ACC-1, SPT, ACOX, ACAT, CERS and ACADS, indicating that these miRNAs might play an important regulatory role in virus-mediated lipid metabolism.

Infection of phytoplankton by aerosolized marine viruses.

Marine viruses constitute a major ecological and evolutionary driving force in the marine ecosystems. However, their dispersal mechanisms remain underexplored. Here we follow the dynamics of Emiliania huxleyi viruses (EhV) that infect the ubiquitous, bloom-forming phytoplankton E. huxleyi and show that EhV are emitted to the atmosphere as primary marine aerosols. Using a laboratory-based setup, we showed that the dynamic of EhV aerial emission is strongly coupled to the host-virus dynamic in the culture media. In addition, we recovered EhV DNA from atmospheric samples collected over an E. huxleyi bloom in the North Atlantic, providing evidence for aerosolization of marine viruses in their natural environment. Decay rate analysis in the laboratory revealed that aerosolized viruses can remain infective under meteorological conditions prevailing during E. huxleyi blooms in the ocean, allowing potential dispersal and infectivity over hundreds of kilometers. Based on the combined laboratory and in situ findings, we propose that atmospheric transport of EhV is an effective transmission mechanism for spreading viral infection over large areas in the ocean. This transmission mechanism may also have an important ecological impact on the large-scale host-virus "arms race" during bloom succession and consequently the turnover of carbon in the ocean.

Hijacking of an autophagy-like process is critical for the life cycle of a DNA virus infecting oceanic algal blooms.

Marine photosynthetic microorganisms are the basis of marine food webs and are responsible for nearly 50% of the global primary production. Emiliania huxleyi forms massive oceanic blooms that are routinely terminated by large double-stranded DNA coccolithoviruses. The cellular mechanisms that govern the replication cycle of these giant viruses are largely unknown. We used diverse techniques, including fluorescence microscopy, transmission electron microscopy, cryoelectron tomography, immunolabeling and biochemical methodologies to investigate the role of autophagy in host-virus interactions. Hallmarks of autophagy are induced during the lytic phase of E. huxleyi viral infection, concomitant with up-regulation of autophagy-related genes (ATG genes). Pretreatment of the infected cells with an autophagy inhibitor causes a major reduction in the production of extracellular viral particles, without reducing viral DNA replication within the cell. The host-encoded Atg8 protein was detected within purified virions, demonstrating the pivotal role of the autophagy-like process in viral assembly and egress. We show that autophagy, which is classically considered as a defense mechanism, is essential for viral propagation and for facilitating a high burst size. This cellular mechanism may have a major impact on the fate of the viral-infected blooms, and therefore on the cycling of nutrients within the marine ecosystem.

Cloning, expression and characterization of serine palmitoyltransferase (SPT)-like gene subunit (LCB2) from marine Emiliania huxleyi virus (Coccolithovirus).

The authors have isolated and characterized a novel serine palmitoyltransferase (SPT)-like gene in marine Emiliania huxleyi virus (EhV-99B1). The open-reading frame (ORF) of EhV99B1-SPT encoded a protein of 496 amino acids with a calculated molecular mass of 96 kDa and Ip 6.01. The results of sequence analysis showed that there was about 31%-45% identity in amino acid sequence with other organisms. The maximum likelihood phylogenetic tree suggested that the EhV99B1-SPT gene possibly horizontally transferred from the eukaryote. Hydrophobic profiles of deduced amino acid sequences suggested a hydrophobic, globular and membrane-associated protein with five transmembrane domains (TMDs) motifs. Several potential N-linked glycosylation sites were presented in SPT. These results suggested that EhV99B1-SPT was an integral endoplasmic reticulum membrane protein. Despite lower sequence identity, the secondary and three-dimensional structures predicted showed that the "pocket" structure element composed of 2α-helices and 4ß-sheets was the catalytic center of this enzyme, with a typical conserved "TFTKSFG" active site in the N-terminal region and was very close to those of prokaryotic organisms. However, the N-terminal domain of EhV99B1-SPT most closely resembled the LCB2 catalysis subunit and the C-terminal domain most closely resembled the LCB1 regulatory subunit of other organisms which together formed a spherical molecule. This "chimera" was highly similar to the prokaryotic homologous SPT. For a functional identification, the EhV99B1-LCB2 subunit gene was expressed in Escherichia coli, which resulted in significant accumulation of new sphingolipid in E. coli cells.

Survival of a surrogate African swine fever virus-like algal virus in feed matrices using a 23-day commercial United States truck transport model.

African swine fever virus (ASFV) is a member of the nucleocytoplasmic large DNA viruses (NCLDVs) and is stable in a variety of environments, including animal feed ingredients as shown in previous laboratory experiments and simulations. Emiliania huxleyi virus (EhV) is another member of the NCLDVs, which has a restricted host range limited to a species of marine algae called Emiliania huxleyi. This algal NCLDV has many similar morphological and physical characteristics to ASFV thereby making it a safe surrogate, with results that are applicable to ASFV and suitable for use in real-world experiments. Here we inoculated conventional soybean meal (SBMC), organic soybean meal (SBMO), and swine complete feed (CF) matrices with EhV strain 86 (EhV-86) at a concentration of 6.6 × 107 virus g-1, and then transported these samples in the trailer of a commercial transport vehicle for 23 days across 10,183 km covering 29 states in various regions of the United States. Upon return, samples were evaluated for virus presence and viability using a previously validated viability qPCR (V-qPCR) method. Results showed that EhV-86 was detected in all matrices and no degradation in EhV-86 viability was observed after the 23-day transportation event. Additionally, sampling sensitivity (we recorded unexpected increases, as high as 49% in one matrix, when virus was recovered at the end of the sampling period) rather than virus degradation best explains the variation of virus quantity observed after the 23-day transport simulation. These results demonstrate for the first time that ASFV-like NCLDVs can retain viability in swine feed matrices during long-term transport across the continental United States.

Elucidating the composition and conservation of the autophagy pathway in photosynthetic eukaryotes.

Aquatic photosynthetic eukaryotes represent highly diverse groups (green, red, and chromalveolate algae) derived from multiple endosymbiosis events, covering a wide spectrum of the tree of life. They are responsible for about 50% of the global photosynthesis and serve as the foundation for oceanic and fresh water food webs. Although the ecophysiology and molecular ecology of some algal species are extensively studied, some basic aspects of algal cell biology are still underexplored. The recent wealth of genomic resources from algae has opened new frontiers to decipher the role of cell signaling pathways and their function in an ecological and biotechnological context. Here, we took a bioinformatic approach to explore the distribution and conservation of TOR and autophagy-related (ATG) proteins (Atg in yeast) in diverse algal groups. Our genomic analysis demonstrates conservation of TOR and ATG proteins in green algae. In contrast, in all 5 available red algal genomes, we could not detect the sequences that encode for any of the 17 core ATG proteins examined, albeit TOR and its interacting proteins are conserved. This intriguing data suggests that the autophagy pathway is not conserved in red algae as it is in the entire eukaryote domain. In contrast, chromalveolates, despite being derived from the red-plastid lineage, retain and express ATG genes, which raises a fundamental question regarding the acquisition of ATG genes during algal evolution. Among chromalveolates, Emiliania huxleyi (Haptophyta), a bloom-forming coccolithophore, possesses the most complete set of ATG genes, and may serve as a model organism to study autophagy in marine protists with great ecological significance.

Dip in the gene pool: metagenomic survey of natural coccolithovirus communities.

Despite the global oceanic distribution and recognised biogeochemical impact of coccolithoviruses (EhV), their diversity remains poorly understood. Here we employed a metagenomic approach to study the occurrence and progression of natural EhV community genomic variability. Analysis of EhV metagenomes from the early and late stages of an induced bloom led to three main discoveries. First, we observed resilient and specific genomic signatures in the EhV community associated with the Norwegian coast, which reinforce the existence of limitations to the capacity of dispersal and genomic exchange among EhV populations. Second, we identified a hyper-variable region (approximately 21kbp long) in the coccolithovirus genome. Third, we observed a clear trend for EhV relative amino-acid diversity to reduce from early to late stages of the bloom. This study validated two new methodological combinations, and proved very useful in the discovery of new genomic features associated with coccolithovirus natural communities.