Opening Pandora’s Box to find the largest virus on Earth

法国发现新巨型病毒 可能来自外太空(图)
Colorized Pandoravirus electron microscopy(图) (Photo credit: 禁书网中国禁闻)

This last year has had many important developments in the field of microbiology, but by far one of the most interesting is the publication discovery of the Pandoravirus genus of viruses. Published in the July 19th 2013 issue of Science Magazine, it is undeniably the biggest virus ever discovered; both in terms of its genome (2.5 MILLION base pairs in length) and it’s physical size ( at 1 micrometer in length it can be seen with a LIGHT microscope!).

So why do these new viruses matter? Why is it so groundbreaking to find a virus of this size? Read on to learn more about the importance of the discovery of these giant viruses.

For roughly the last decade the Mimivirus was considered to be the largest known virus, with over 1000 genes and a particle size of about 0.7 micrometers (that’s 0.7 millionths of a meter).  This discovery was enough of a shock to make many scientists wonder what the upper limit in size for a true virus could be. The new Pandoravirus genus (they found 2 distinct different species to put in the Pandoravirus genus, one from Chile: P. salinus (Fig 1 A1, B1), and one from Australia: P. dulcis (Fig1 A2, B2)) radically expands upon the discovery of the Mimivirus. These new Pandoraviruses also infect Acanthamoeba (a type of amoeba, a single-celled organism) but are much larger than the previously discovered Mimivirus (Fig 1 F). In fact, at 2.5 Megabases in length, the P. dulcis genome is on par with the size of the smallest parasitic eukaryotes, which is a stunning discovery. Many scientists did not expect that an obligate intracellular parasite such as a virus could attain a size this large.

Fig. 1 Images of Pandoravirus particles and their proteomic profiles. Light microscopy (A) and electron microscopy images (B) of P. salinus (1) and P. dulcis (2) purified particles. (C) Electrophoresis profiles of P. salinus (lane 1) and P. dulcis (lane 2) extracted proteins. (D) Internalized P. salinus particle in the host vacuole. Once fused with the vacuole membrane (arrow), the virion internal membrane creates a continuum with the host cytoplasm. The particles are wrapped into a ~70-nm-thick tegument-like envelope consisting of three layers. (E) Magnified image of the opened ostiole-like apex: from the inside out, a layer of light density of unknown composition (~20 nm, marked “b”), an intermediate dark layer comprising a dense mesh of fibrils (~25 nm, marked “a”), and an external layer of medium density (~25 nm, marked “c”). This tegument-like envelope is interrupted by the ostiole-like pore measuring ~70 nm in diameter. As shown in (B1) and (B2), the lipid membrane internal to the particle encloses a diffuse interior devoid of visible substructure, except for a spherical area of electron-dense material (50 nm in diameter, arrowhead) seen episodically but in a reproducible fashion. (F) Ultrathin section of an Acanthamoeba cell filled with P. salinus at various stages of maturation.
Fig. 1 Images of Pandoravirus particles and their proteomic profiles.
Light microscopy (A) and electron microscopy images (B) of P. salinus (1) and P. dulcis (2) purified particles. (C) Electrophoresis profiles of P. salinus (lane 1) and P. dulcis (lane 2) extracted proteins. (D) Internalized P. salinus particle in the host vacuole. Once fused with the vacuole membrane (arrow), the virion internal membrane creates a continuum with the host cytoplasm. The particles are wrapped into a ~70-nm-thick tegument-like envelope consisting of three layers. (E) Magnified image of the opened ostiole-like apex: from the inside out, a layer of light density of unknown composition (~20 nm, marked “b”), an intermediate dark layer comprising a dense mesh of fibrils (~25 nm, marked “a”), and an external layer of medium density (~25 nm, marked “c”). This tegument-like envelope is interrupted by the ostiole-like pore measuring ~70 nm in diameter. As shown in (B1) and (B2), the lipid membrane internal to the particle encloses a diffuse interior devoid of visible substructure, except for a spherical area of electron-dense material (50 nm in diameter, arrowhead) seen episodically but in a reproducible fashion. (F) Ultrathin section of an Acanthamoeba cell filled with P. salinus at various stages of maturation. Ref #1

So how exactly did they find these new viruses? When these particles kept appearing in the Acanthamoeba cultures and killing off the host cell in the presence of antibiotics, it indicated that these particles were viral in nature. This was a bit of a puzzle because no one had ever seen viral particles even close to this size. However, when the whole infectious cycle was observed (about 15 hours), the researchers saw that once these particles were internalized they released their contents into the cell and a true “eclipse” phase occurred where there the contents of the particle were lost in the cell. Roughly 4 hours later though, the nucleus of infected cells began to deteriorate until 8-10 hours after infection, when new Pandoravirus particles begin to form in the cytoplasm of the infected cell. After a few more hours the infected cells lyse and release about 100 new Pandoravirus particles into the environment. This observation coupled with the antibiotic resistance showed the group that they were indeed working with a new, very large, virus.

To distinguish between the two forms, the researchers did a very basic electrophoresis test where the viral particles were digested by a protease and then run on a gel and stained. As you can see above in Figure 1C these two related Pandoaviruses have different proteomic profiles, indicating they are indeed unique species.

To truly distinguish between the two new viruses, the research group also sequenced the genome of both viruses. Both are composed of double stranded DNA but P. salinus has genome of 2.47-2.77 Mb while P. dulcis is 1.97 Mb in length. When they delved deeper into the genomic analysis it was found that P. salinus has a minimum of 2556 protein coding sequences, making it the most complex virus observed to date.

Stranger still, when they searched for these genes in all of the known sequence data produced by the many different sequencing endeavors they found that only a few of these genes had any known homologue. The other 93% percent (!) of the P. salinus genome has no known similarity to ANY organism that has been sequenced before. This is an incredible discovery as it shows just how far removed this organism is from anything else science has ever encountered before.

This distance from all known organisms is what has given the Pandoravirus its name. Just like the Greek myth of Pandora’s Box, we’re not quite sure what kind of knowledge will come from this unprecedented discovery now that it’s been made, making the Pandoravirus one of the newest and most exciting areas of virology research.

References:

1.           Philippe, N. et al. Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes. Science (New York, N.Y.) 341, 281–6 (2013).

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