the story of mimivirus: a giant virus 1992, bradford, england

The story of Mimivirus: a giant virus

1992, Bradford, England

amoeba

Pneumonia outbreak

Cooling tower

Hospital cooling tower

Gram-positive particles were found in the amoeaba

It was also found that it does not pass through a filter of 0.2 micron (200nM): probably a bacteria…

BUT: PCR failed to amplify the 16S rRNA sequence, suggesting that this is not a bacteria… but a virus

Nu=nucleolus

Cm=Cell membrance

Vp=virus particle

Nm=nucleic membrane

The next step was to try and look at things under the electron microscope (inside Acanthamoeba polyphaga)

Looking closer the particle has a characteristic viral morphology. Icosahedral capsid of 400nm diameter.

Confocal microscopy with specific mimivirus monoclonal antibodies.

Phagocytized mimivirus particles are visible within the cytoplasm of amoebae (arrows)

Eclipse phase:the time between infection of a virus, and the appearance of mature virus within the cell; an interval of time during which viral infectivity cannot be recovered.

At the forth hour (H4), we can see an eclipse phase: the virus is not detected.

H8: perinuclear (near the nucleus) particles become visible.

Conclusion from microscopy: life cycle resembles a virus

Because it mimics a bacterium in gram staining it was called Mimivirus: mimicing microbe virus.

The genome was found to be a circular double stranded DNA of 1,181,404 bp (1,181 kbp).

The genome is bigger than that of a few bacteria:

Mycoplasma genitalium (580 kbp)A parasitic bacterium which lives in the primate genital and respiratory tracts. It is of special interest for biologists because it is considered to be the organism with the smallest genome, aside from viruses and Nanoarchaeum discovered in 2002.

Ureaplasma urealyticum (752 kbp)This microorganism is part of the normal genital flora of both men and women. It is found in about 70% of sexually active humans. It can also cause disease, including urethritis (inflammation of the urethra, a tube which connects the urinary bladder to the outside of the body), infertility…

Ureaplasma urealyticum vs. MimivirusThe size of the particle is approximately the same

The most boring man on earth is not boring.It is the second most boring man that is boring…

The second biggest known virus is phage D of Bacillus megateriumSize = 670 kbp (Mimivirus = 1181 ).

http://www.giantvirus.org/news.html

The third biggest virus is Phycodnavirus.Infecting algaeDouble stranded DNASize = 250-560 kbp (Mimivirus = 1181 kbp)

A phycodnavirus at cell surface

Shotgun sequencing of Mimivirus

DNA -> mechanical shearing. Output is random fragments (in this case two libraries, one of 5kb and one of 9kb).

Fixing sticky ends

Ligating the fragments with an adaptor in which the end is a Bst XI restriction fragment

Cloning.

Shotgun sequencing of Mimivirus

Assembly programs: Phred/Phrap

Sequencing was done from both strands

Assembly to 6X coverage (each nucleotide on average appears in 6 reads).

The genome

1,262 ORFs longer than 100bp.

The virus shares some sequences with other Nucleocytoplasmic Large DNA Viruses (NCLDV viruses).

Phylogeny within viruses

Based on MSA of ribonucleotide reductase (NJ tree using Clustal).

Ribonucleotide reductase: catalyzes the formation (reduction) of deoxyribonucleotides from ribonucleotides. One of the enzyme found in all organisms.

A new family: Mimiviridae

Phylogeny with respect to the tree of life

Based on concatenating 7 universally conserved genes from the three domain of life the tree and bootstrap values were estimated using ML.

Mimivirus = forth domain of life???

Highly debated.