Viruses under the sea

CORKsViruses infect every living organism on the planet, but not every habitat has been explored for their presence. The igneous ocean crust had not yet been examined for viruses, but seek and ye shall find: there are plenty of viruses under the seas.

The oceanic basement is an enormous ecosystem that lies at the bottom of the seas, beneath a thick layer of sediment. It is composed of igneous rock through which percolates 20 million cubic kilometers of water. Previous study of cores from this region has revelealed the presence of prokaryotes, but no one had looked for viruses.

Facilitating the study of the oceanic basement are seafloor observatories that have been placed into existing boreholes. Two have been placed in 3.5 million year old rock in the northeastern Pacific Ocean. They penetrate hundreds of meters through sediment and into the basement (illustrated; image credit) and are fitted with plumbing that allows sampling of uncontaminated fluids from different depths in the basement rock.

Analysis of fluids recovered from these sites revealed both prokaryotes (8,000 per ml) and virus particles (90,000 per ml). Ribosomal RNA sequence analysis showed that bacteria dominated these communities, with some Archaea but virtually no eukaryotes.

Examination of the fluids by electron microscopy showed virus particles of different kinds: tailed and untailed icosahedral particles, untailed globular particles, and rod and spindle shaped. My favorite is the lemon shaped particle, for its form and implied taste.

To provide information on the viral genomes in the oceanic basement, sequences were determined from total cellular DNA (material retained on a 0.2 micron filter) extracted from the samples. Most viral sequences likely had archaeal hosts. Some prophage sequences were identified – viral genomes integrated into host DNA – which allowed more certain identification of the infected cell.

Most of the identified archaeal and bacterial virus sequences came from the families Myoviridae and Siphoviridae (think tailed, icosahedral viruses). One complete circular DNA genome identified is 55,906 nucleotides in length with 81 open reading frames. Twenty of these encode proteins with recognizable functions, such as capsid proteins, a primase and a DNA polymerase. No genes encoding tRNAs, such as those found in giant viruses, were identified.

Some sequences were similar to those of giant viruses like mimiviruses and phycodnaviruses. These viruses are known to only infect eukaryotes. Eukaryotic genomes were rare in the basement metagenome collections (1% of the community in one location).

I am not surprised that viruses have been found in ocean’s basement. Still, I’m amazed when I think about how far down they are, in warm water (65 degrees C) in 3.5 million year old rock.

To paraphrase Samuel E. Wright (Under The Sea):

The viruses are always greener
In somebody else’s lake
You dream about going up there
But that is a big mistake
Just look at the viruses around you
Right here under the ocean floor
Such wonderful viruses surround you
What more is you lookin’ for?


TWiV 436: Virology above Cayuga’s waters

At Cornell University in Ithaca, New York, Vincent speaks with Susan, Colin, and Gary about the work of their laboratories on parvoviruses, influenza viruses, and coronaviruses that infect dogs, cats, horses and other mammals.

You can find TWiV #436 at, or listen below.

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Forget the fourth domain of life

three domains of lifeWhen giant viruses were discovered – with genomes much larger than any previously seen – some suggested that they had descended from a fourth domain of life (the current three are bacteria, archaea, and eukaryotes). Part of the reason for such a claim was the finding of homologs of bacterial and eukaryotic genes, including molecules involved in translation. Analysis of new giant viruses encoding even more components of the translation machinery has thrown cold water on the fourth domain hypothesis.

Klosneuvirus, with a 1.57 million base pair DNA genome, was discovered in a wastewater treatment plant in Austria, and three related viruses – Indivirus, Hokovirus, and Catovirus – were found in environmental samples.  Sequence analyses suggests that these viruses should be classified in a subfamily of the Mimiviridae.

The Klosneuviruses encode far more components of the translational machinery than do mimiviruses – 25 tRNAs, 19 aminoacyl tRNA synthetases, 11 initiation and elongation proteins, a chain release factor, and tRNA modifying enzymes.

Phylogenomic analyses demonstrate that the aminoacyl tRNA synthetase and translation factor genes are likely derived from protists. This finding is not compatible with the hypothesis that these viruses are derived from a fourth domain of life. It is more likely that smaller ancestors of giant viruses acquired these genes from known eukaryotes.

Why these components of the translational system have been maintained in these giant virus genomes is an excellent question. They might confer some advantage to the viruses, for example when host translation is shut off as a viral defense. Having components of the translational apparatus might allow viral protein synthesis to proceed.

Note that genes encoding ribosomal RNAs or proteins have not been found in any virus. In fact no virus encodes a complete protein synthesis machinery. Maybe they have yet to be discovered? Or perhaps these energetically costly activities are best left to the cell?


TWiV 435: Two virus particles walk into a cell

The TWiVome discuss the blood virome of 8,420 humans, and thoroughly geek out on a paper about the number of parental viruses in a plaque.

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The purity of plaques

dose-response-plaque-assayThe plaque assay – my favorite assay in the world – is a time-honored procedure to determine the number of viruses in a sample, and to establish clonal virus stocks. The  linear relationship between the number of infectious particles and the plaque count (illustrated; image credit) shows that one infectious particle is sufficient to initiate infection. Despite the one-hit kinetics of plaque formation, could more than one virus contribute to a plaque?

To answer this question, ten genetically marked polioviruses were mixed and subjected to plaque assay. Of 123 plaques, 6 (4.9%) contained more than one virus. Similar results were found when polioviruses with phenotypic markers were studied.

Examination of poliovirus stocks by electron microscopy revealed both single particles and aggregates of 2 to 10 particles. Increasing particle aggregation by treatment of viruses with low pH increased co-infection frequency, indicating that aggregation of particles leads to multiply infected cells.

When these experiments were repeated with mutagenized polioviruses, the co-infection frequency increased – probably because recombination and complementation between two defective genomes leads to rescue of the defects.

Do these findings indicate that poliovirus plaque formation does not follow one-hit kinetics? The results do not prove that, in unmutagenized virus stocks, more than one poliovirus is needed to form a plaque. They only show that a small percentage of plaques contain more than one poliovirus. The presence of more than one poliovirus in 5-7% of plaques is likely a consequence of virion aggregation. It would be informative to prepare poliovirus stocks with no aggregates, and determine if co-infected plaques are still observed.

Some viruses of plants and fungi follow two-hit kinetics: two virus particles, with two different genomes, are needed for infection (illustrated). Assuming that 4-7% of poliovirus plaques are initiated by multiple viruses, the resulting plots deviate only slightly from a straight line, and do not resemble the curves of two-hit kinetics.

What are the implications of these findings for the use of plaque assays to produce clonal virus stocks? Even though the frequence of multiply infected plaques is low, the possibility of producing a mixed population is still possible, if only one plaque purification is done. In our laboratory we have always repeated the plaque purification three times, which should ensure that no multiply infected plaques are isolated.

Update 3/31/17: I would like to see similar experiments done with other viruses, to see how often multiple viruses can be found in a plaque. Examples included hepatitis A virus, which is released from cells in membranous vesicles containing multiple virus particles; and enveloped viruses, which might aggregate more frequently than naked viruses.

I looked back at the 1953 publication in which Dulbecco and Vogt first described the plaque assay for poliovirus, and demonstrated one hit kinetics. The dose-response curve clearly shows one-hit kinetics with little deviation of the individual data points from a straight line.

plaque dose response

Linear relationship between the number of plaques and the virus concentration. Image credit.


TWiV 434: Live long and pupate

The esteemed TWiVumvirate reveal the discovery of a new negative stranded RNA virus of wasps that regulates longevity and sex ratio of its parasitoid host.

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The viruses in your blood

blood bagIf you have ever received a blood transfusion, along with the red blood cells, leukocytes, plasma and other components, you also were infused with a collection of viruses. A recent study of the blood virome of over 8,000 healthy individuals revealed 19 different DNA viruses in 42% of the subjects.

Viral DNA sequences were identified among the genome sequences of 8,240 individuals that were determined from blood. Of the 1 petabyte (1 million gigabytes) of sequence data that were generated, about 5% did not correspond to human DNA. Within this fraction, sequences of 94 different viruses were identified. Nineteen of these were human viruses. The method is not expected to reveal RNA viruses except retroviruses which are integrated as DNA copies in the host chromosomes.

The most common human viruses identified were herpesviruses, including cytomegalovirus, Epstein-Barr virus, herpes simplex virus, and human herpesvirus 7 and 8, found in 14-20% of individuals. Anelloviruses, small viruses with a circular genome, were found in 9% of the samples. Other viruses found in less than 1% of the samples included papillomaviruses, parvoviruses, polyomavirus, adenovirus, human immunodeficiency virus and human T-lymphotropic virus (the latter two integrated into the host DNA).

The other 75 viruses are likely contaminants from laboratory reagents or from the environment. These include sequences from non-human retroviruses, four different giant DNA viruses, and a virus of bees, all found in less than 10 samples. These findings illustrate the challenge in distinguishing bona fide human viruses from contaminants.

Identifying viruses in blood is an important objective for ensuring the safety of the blood supply. Donor blood is currently screened for HIV-1 and 2, human T-lymphotropic virus-1 and 2, hepatitis C virus, hepatitis B virus, West Nile virus, and Zika virus. These viruses are pathogenic for humans and can be transmitted via the blood. Some viruses, such as anelloviruses and pegiviruses, are in most donated blood, yet their pathogenic potential is unknown. It is not feasible to reject donor blood that contains any type of viral nucleic acid – if we did, we would not have a blood supply.

Continuing studies of the blood virome are needed to define which viruses should be tested for in donated blood. The human papillomavirus (17 people), Merkel cell polyomavirus (49 people), HHV8 (3 people) and adenovirus (9 people) detected in this study could be transmitted in the blood and their presence should be monitored in future studies.

It’s important to emphasize that this work describes only viral DNA sequences, not infectious viruses. The blood supply is screened by nucleic acid tests, but it is important to determine if infectious virus is also present. If viral DNA is present in blood but infectious virus is never found, then it might not be necessary to reject blood based on the presence of certain sequences.

Image credit.

TWiV 433: Poops viruses and worms

The lovely TWiV team explore evolution of our fecal virome, and the antiviral RNA interference response in the nematode C. elegans.

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TWiV 432: Conjunction junction, what’s your function?

The TWiVites discuss Zika virus seroprevalence in wild monkeys, Zika virus mRNA vaccines, and a gamete fusion protein inherited from viruses.

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Did viruses enable sex?

Dengue virus E glycoproteins (monomer at top) lie flat on the virus particle as dimers (middle). At endosomal low pH, three monomers reorient to place the fusion peptide (orange) into the cell membrane. Image credit.

A key step in sexual reproduction is the fusion of haploid cells to form a diploid zygote, yet the molecular mechanism underlying this joining of cells is poorly understood. Two studies reveal amazing similarities between proteins required for fusion of sperm and egg, and virus with host cells.

A screen for genes that cause male sterility in the flowering plant Arabidopsis led to the identification of the HAP2 protein. This protein was later found to be important for sperm-egg fusion in Arabidopsis and in the unicellular algae Chlamyodomonas. 

Homology modeling shows that the HAP2 protein looks very much like a class II viral fusion protein (illustrated). Found in dengue virus and many related viruses, dimers of these viral glycoproteins lie flat on the viral membrane, and are comprised largely of beta-strands. At one end of the protein is a fusion loop which allows the virus and cell membranes to join at the start of infection.

The HAP2 protein also has what looks to be a viral fusion loop. Removal or alteration of this sequence in Tetrahymena prevents fusion of mating cells. The fusion loop of the dengue virus E glycoprotein cannot substitute for the HAP2 sequence. Furthermore, vesicular stomatitis viruses with HAP2 in place of the viral glycoprotein cannot enter cells. However the results of biophysical experiments indicate that the HAP2 fusion loop can interact with membrane lipids in ways reminiscent of viral fusion peptides.

Solution of the atomic structure of HAP2 reveals a trimer with protein folds and an upright ‘hairpin’ configuration (illustrated for dengue virus) typical of class II fusion proteins. While acidification of viral type II fusion proteins is required for rearrangement to the post-fusion form, the trigger for HAP2 is not known.

These results clearly show that HAP2 is a type II fusion protein that mediates the joining of haploid gametes in the first step of sexual reproduction. These viral and cell proteins are so similar that it is highly improbable that they arose by convergent evolution. HAP2 is ancient: besides green algae and plants, it is also found in unicellular protozoa, cnidarians, hemichordates, and arthropods, indicating that it was likely present in the last common ancestor of eukaryotes. But viruses existed before the evolution of eukaryotic sex, raising the scenario that type II fusion proteins first arose in viruses, which provided them to eukaryotic cells for use in gamete cell fusion.

Without viruses, there would be no sex, and therefore no humans, or many other animals on Earth.

We continue to recognize new ways that the evolution of eukaryotic life has depended on viruses. These include a viral gene used to produce the placenta; enhancer elements for innate immunity; prions; and the nucleus. What exactly did eukaryotes invent?