Viruses are the most common biological entities in the oceans by an order of magnitude. However, very little is known about their diversity. Here we report a genomic analysis of two uncultured marine viral communities. Over 65% of the sequences were not significantly similar to previously reported sequences, suggesting that much of the diversity is previously uncharacterized. The most common significant hits among the known sequences were to viruses. The viral hits included sequences from all of the major families of dsDNA tailed phages, as well as some algal viruses. Several independent mathematical models based on the observed number of contigs predicted that the most abundant viral genome comprised 2-3% of the total population in both communities, which was estimated to contain between 374 and 7,114 viral types. Overall, diversity of the viral communities was extremely high. The results also showed that it would be possible to sequence the entire genome of an uncultured marine viral community.

Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including Acinetobacter baumannii. We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year-old diabetic patient with necrotizing pancreatitis complicated by an MDR A. baumannii infection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a 4-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for an A. baumannii isolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patient's downward clinical trajectory, clearance of the A. baumannii infection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted.

Metagenomic sequence analysis is rapidly becoming the primary source of virus discovery 1–3. A substantial majority of the currently available virus genomes comes from metagenomics, and some of these represent extremely abundant viruses even if never grown in the laboratory. A particularly striking case of a virus discovered via metagenomics is crAssphage, which is by far the most abundant human-associated virus known, comprising up to 90% of the sequences in the gut virome 4. Over 80% of the predicted proteins encoded in the approximately 100 kilobase crAssphage genome showed no significant similarity to available protein sequences, precluding classification of this virus and hampering further study. Here we combine comprehensive search of genomic and metagenomic databases with sensitive methods for protein sequence analysis to identify an expansive, diverse group of bacteriophages related to crAssphage and predict the functions of the majority of phage proteins, in particular, those that comprise the structural, replication and expression modules. Most if not all of the crAss-like phages appear to be associated with diverse bacteria from the phylum Bacteroidetes, which includes some of the most abundant bacteria in the human gut microbiome and are also common in various other habitats. These findings provide for experimental characterization of the most abundant but poorly understood members of the human-associated virome.

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. “Fluorescent timer” protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of “fluorescent timer” protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. “Fluorescent timer” protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.