MCB3026F
Viromics
Lecture 1: Diversity of viruses and influence of carbon cycling
- the study of an organism’s entire genome
- virome: the total viral genome content of the DNA and/or RNA found in certain biological sampling areas such as entire
oceans or just in humans
- there is a largely undescribed universe of viruses out there, in everything from seawater to duck ponds, biofilms to
beehvies, colons to parrots
- cataloguing some of that diversity can only lead to new insights, which will lead to the kinds of questions that can be
answered by ‘metagenomics approaches’
- viromics: oramics systems virology (the interaction of viral genomes with their environments)
Viruses in the water
- shows virus particles suspended in water (natural water sample), take a drop of the water, fuse it with a
dye that binds directly to the double-stranded DNA to the double-stranded DNA, put it under a
fluorescent microscope which illuminates the particles
- the rectangle contains two prokaryotes, the ellipse indicates less than 25 virus like particles and the
circle indicates one protist
Important roles in the oceans
- viruses are by far the most abundant biological entities in the oceans,
comprising approximately 94% of the nucleic-acid-containing particles,
however because of their small size they represent only 5% of the biomass
- in spite of their great abundance, only since 1990s that the importance of
marine viruses has been accepted by the scientific community
- are now conscious that marine viruses play important roles in the oceans
- a compelling argument can be made that the biggest reservoir of unidentified genetic information is all around us, in the
global population of bacteriophages
- it has been calculated that the total number of phage particles in the biosphere is 10^31
- suggests that there are more phage particles that all other biological forms added together
- if abundance can be equated with success, then phages represent the result of natures’ most successful experiment
Viruses in seawater 1
- viruses exist wherever life is found
- they are a major cause of morality, a driver of global geochemical cycles and a reservoir of the greatest genetic diversity
on earth, and in the oceans, viruses infect all the living things from bacteria to whales, effect the form of available nutrients
and the termination of algal blooms
- viruses can more between marine and terrestrial reservoirs, raising the specter of emerging pathogens
- our understanding of the effect of viruses on global systems and processes continues to unfold
Viruses in seawater 2
- viruses are extremely abundant in aquatic systems
- TEM indicated that 3.10^6 viruses ml^-1 in the deep sea to 10^8 viruses ml^-1 in productive coastal waters
- assuming the volume of the ocean is 1.3x10^21 liters and the average abundance of viruses is 3x10^9 liters ^-1, the ocean
waters contain 4x10^30 viruses
- a marine virus containing about 0.2 of of carbon and is 100 nm long, this is 200 Mt of carbon, equivalent to 75 million
blue whales (10% carbon by weight)
- viruses lysing bacteria is a major way of moving carbon between live bacteria and the water
Viruses ins seawater 3
- viruses are catalysis for biochemical cycle
- viruses short-circuit the flow of carbon and nutrients from phytoplankton and
bacteria to higher trophic levels by causing the lysis of cells and shunting the flux
to the pool of dissolved and particulate organic matter
- this result is that more of the carbon is respired, thereby decreasing the trophic
transfer efficiency of nutrients and energy through the marine food web
- viruses that infect microbes in deep-sea sediments may be a key driver in the
world’s largest ecosystem and integral to the global carbon cycle
- lytic viruses kill about 80% of the single-celled organisms in the sediment and
sub-surface ocean layers, thereby releasing large amounts of dissolved carbon into the deep seas
, - every year this ‘viral shunt’ releases up to 630 million tons of the carbon sequestered by particles sinking into these deep-
sea benthic zones
- viruses should be included in ocean carbon management models
Methods used to look for marine viruses
Count virus particles by:
- transmission electron microscopy
- epifluorescence microscopy
- flow cytometry
Metagenomic analysis:
- total DNA/cDNA sequencing from seawater
- metagenomics of fractionated water
Example of dsDNA phages that infect bacteria
a) Myoviruses are often the most commonly isolated phage from natural marine communities,
have contractile tails, typically lytic and often have relatively broad host ranges
b) podoviruses have a short non-contractile tail, typically lytic and have very narrow host ranges,
are less commonly isolated from seawater
c) siphoviruses have long non-contractile tails, are frequently isolated from seawater, often have a
relatively broad host range and many are capable of integrating into the host genome
Lecture 2: Viromics example: viruses found in stressed corals
Eukaryotic viruses
- metagenomic analysis indicates that stressors induce production of herpes-like viruses in the coral Porites compressa
- at least one-third of all coral species are now threatened with extinction as oceans are getting hotter and more anoxic,
causing coral bleaching and causing corals to die (global warming is a major factor)
- coral disease has been a major contributor to this threat, but little is known about the responsible pathogens
- to date, most research has focused on bacterial and fungal diseases, however, viruses may also be important for coral
health
- using a combination of empirical viral metagenomics and real-time PCR, we show that Porites compressa corals contain a
suite of eukaryotic viruses, many related to Herpesviridae
- purified virus-sized particles from sea water
- extracted DNA, verified there was no cellular DNA present by PCR for 16S and 18S rRNA and amplified the viral DNA
using a GenomiPhi phi29 polymerase-based genome amplification kit from GE Healthcare
Sciences
Metagenomic Sequencing
- the 454 system relies on an emulsion (an aqueous solution in oil suspension by extreme agitation or sonication which
ensures that only one or two DNA molecules plus the necessary enzymes in each droplet that is coupled to the
pyrosequencing) polymerase chain reaction (PCR) step that is coupled to pyrosequencing
- individual fragments of DNA, 300-500 base pairs long, are attached to beads in vitro and amplified with PCR to generate
millions of identical copies on each bead
- fragments are sequenced by use of a massively parallel reaction format in 1.6 million wells on a picotitre plate
- this system can generate around 250 base pairs of sequence per reaction while performing 400,000 reads in a single
instrument tun
= 100 million base pairs per instrument run
GenomPhi genome amplification
- random-primed DNA synthesis
- isothermal phage polymerase
- multiple priming and long extensions lead to effective whole-genome
amplification
- going from 1 microliter which contains between 1 and 10 nanograms of
input DNA down to 4-7 micrograms of product that is just a linear
amplification scheme using a phage derived polymerase
