Marine DNA virus and Metagenomics

1. Under
Dr. I.S Bright Singh
Dr. Swapna P. Antony

2. Introduction
Why????

3. Why marine
Virus???
Why Metagenomics??
 Currently
best
estimation
is
concentrations
of
approximately
~106
viruses ml-1 of surface
seawater, ~3×106 viruses
ml–1 in the deep sea to
~108 viruses ml–1 in
productive
coastal
waters.
 DNA containing viruses
are abundant in Marine
system.
 Metagenomics study came
in light to address the
challenge
of
studying
unculturable viral particles
(<99%).
 Metagenomics
is
an
alternative
cultureindependent and sequenceindependent approach that
does not rely on the
presence of any particular
gene in all the subject
entities.

4. Virus looks like!!!!!!
A virus particle, called a virion, can be thought of as a delivery
system that surrounds a payload.
The delivery system consists of structural components used by
the virus to survive in the environment and bind to host cells
The payload contains the viral genome and often includes
enzymes required for the initial steps in virus replication
Virus as “a piece of bad news wrapped in a protein
coat.”

5. Examples of the main types of viruses:
Tailed phage that infect bacteria .
Filamentous viruses that infect all domains of life, and
Enveloped viruses that infect animal and plant cells.

6. Origin of Viruses:
Alternative
hypothesis
• DNA part coding for important cellular machinery
escape from cellular control gained essential viral
gene. These genes replicated by cellular host and
form virus like particles.
Reductive
evolution
• Primitive cells having essential cellular machinery
decreased in size and genome get encapsulated by
endosymbiont that develop in primitive viral like
particles
Origin
based on
micelles
• Primitive atmosphere contain micelles (can trap
nucleic acid like particles), with the passing of time
as trapped material in micelles increased ribozyme
activity evolved and micelles become protovirus,
form protein and fused to share information, this
way slowly-slowly protovirus evolved in to modern
viru

7. Escape hypothesis: unequal cell division produces minicells with single chromosome
(a or b) but no translation apparatus. The chromosome a will be eliminated but
chromosome b will survive because it is associated with a proteins coat that allows its
transfer into a new RNA cell, it becomes a virus.
Reduction hypothesis: a small RNA cell became an endosymbiont of a larger RNA
cell. It looses its translation apparatus but continue to replicate autonomously and
become infectious (similar to some pathogenic bacteria in eukaryotic cells).

8. Last Universal Cellular Ancestor (LUCA)
Virus Research 2006

11. Life Cycle:

12. Marine Viruses:
Spencer 1955- The first phage isolated from the marine
environment was reported more than 50 years ago but the
abundance of viruses was recognized in the late 1980s.
Bergh et al. 1989 - Published a paper in journal Nature “High
abundance of viruses found in aquatic environments”.
This paper gives insight that viruses are abundant and
ecologically important components of the marine environment.
Marine viruses includes eukaryotic viruses, phage and
generalized transducing agents (GTAs) and infect all organisms
from bacteria to whales.
Pascal Hingamp et. al. 2013: Exploring nucleo-cytoplasmic
large DNA viruses in Tara Oceans microbial metagenomes.
Nature (2013).

13. Most viruses in the ocean are
thought to infect prokaryotes.
They are typically DNAcontaining viruses.
Found deep ocean to
hydrothermal vents.
In surface waters, very large
viruses can be found, They
probably infect single-celled
photosynthetic
organisms
(phytoplankton) and Eukarya
(cells with a nucleus).
Viruses also infect metazoans
and benthic plants from
prokaryotes to whales.

14. Major breakthrough in study of Marine Viruses:

15. Abundance and distribution of marine viruses
Surface seawater ~106 viruses ml-1
~3×106 viruses ml–1 in the deep sea
~108 viruses ml-1 in productive
coastal waters [Danovaro et.al.
2003].
If it is assumed that volume
of the oceans is 1.3×1021~1022 l
average abundance of viruses
is 3×108~109 l–1, then ocean
waters contain~4×1030 ~ 1031
viruses.
Black- Virus, white - Bacteria
The highest viral abundances are usually observed, euphotic
zone and in rich coastal surface waters decrease exponentially
with increasing depth and increases exponentially as number
of bacteria and chlorophyll increases.

16. Relationships between viruses and prokaryotes in deep-sea sediments
worldwide.
R Danovaro et al. Nature .2008

17. Importance of Viruses:
Phage Therapy
Aquaculture
as
disease
controlling
weapon.
Detection
and
diagnostics of disease
causing agents, as
antimicrobial agent
Carbon , Fe and
Nitrogen cycle
Viral lysis diverts carbon
from the POC to the DOC
pool, effectively “shortcircuiting” the microbial
loop away from the
grazers.
Atmospheric C02
Viral lysis of phytoplankton
DMS- gas that influences cloud
formation. Viral lysis releases
organic Fe complexes which is
1000 times more bioavailable
and efficiently assimilated by
bacterial cells than Fe(III)

18. Importance of phages in Gene transfer and diversity:

19. Marine Viruses: Truth or Dare. Annual Review of Marine Science 2012
The transferred genes
can allow a cell to
expand into different
niches (for example,
through the activation of
photosynthetic genes,
changing the life cycle
of
biogeochemically
important
phytoplankton such as
cyanobacteria
and
coccolithophorids).
Studies shows Escherichia coli O157 strain represented up to 16% of the
chromosomal DNA as Prophage DNA.
Microarray demonstrated that 50% of the strain specific DNA from
Lactobacillus gut commensal is represented by prophage DNA and Salmonella
strains belonging to serovars Typhimurium and Typhi.

21. Nuclear cytoplasmic large DNA virus (NCLDV):
Eukaryotic viruses having large double
stranded DNA (dsDNA) genome ranging
from 100kb up to 1.26Mb.
Virus of theses group replicate either
exclusively in the cytoplasm, or in both
cytoplasm and in nucleus of host cell
Host range of these viruses is from
microscopic unicellular eukaryotes to
larger animals, including humans.

22. Virus family
Host range
Phycodnaviridae
Green algae; algal
symbionts of
paramecia and
hydras
Animals: insects,
reptiles, birds,
mammals
Mammals
Genome
size range,
kb
150-400
Replication site Virion
Nucleus and
cytoplasm
isometric
130-380
Cytoplasm
isometric
170
Cytoplasm
150-190
Nucleus and
cytoplasm
Nucleus and
cytoplasm
Mimiviridae
Insects, mainly,
Noctuids
Insects, coldblooded
vertebrates
Acanthamoeba
1,180
Cytoplasm
Marseillevirus
Acanthamoeba
370
Nucleus and
cytoplasm
Poxviridae
Asfarviridae
Ascoviridae
Iridoviridae
100-220
isometric

24. LAKSHMINARAYAN M. IYER Journal of Invertebrate Pathology (2009)

25. NCAAH

26. CPV- Iridovirus, Ehv – phycod, Asfv- asfavirus, CIVAscoviridae, VIV- Poxviridae
Lakshminarayan M. Iyer ,Virus Research .2006

27. Phylogenetic reconstruction of NCLDV members.
EhV- E. huxleyi virus, ASFV- African swine fever virus, APMVA. polyphaga mimivirus, FPV, Fowlpox virus etc.
Fischer M G et al. PNAS 2010

28. Metagenomics:
Metagenomics is an alternative culture-independent and sequenceindependent approach that does not rely on the presence of any
particular gene in all the subject entities.
Why Metagenomics???
Metagenomics study came in light to
address the challenge of studying
unculturable prokaryotes (<99%).

29. The Global Ocean Sampling
Expedition (GOS)
Begun as a Sargasso Sea
pilot sampling project in
August 2003.
1,800 microbial species
were discovered including
148 novel phylotypes,
encoding more than 1.2
million genes.
This study expanded
our knowledge of ocean
photobiology, microbial
diversity, and evolution.

30. NCAAH

32. Why metagenomics for my study?????
Current openion in Virology 2012

33. Reason 2………

35. Overview of my work:
New
sequence and
Viral relation
Phylogeny
DNA
Virus

36. Cloning
NCAAH

37. Sampling and Ultrafiltration
Water sample collection( 50 -100 liter) from open ocean.
Concentration of Viral Particles:
10-20 liters of sea water filtered through .2µm filters and
spiral cartridge with a molecular weight cut off by 100 kDa.
For further concentration of viruses (ml) concentrated
sample can be centrifuged and pellet can be stored at 4º c
after adding distilled water.
Table: Range of viral nucleic acid recovery from different sources.

38. Filtration using TFF filter:
Principle:
Tangential-flow filtration has been
used to isolate variety of particles from
a variety of environments.
For the purposes of this method, we
will focus on the 0.2-µm and 100-kDa
filters.
Tubing is attached to either end of
the TFF and the sample is run from the
collection
reservoir
through
a
peristaltic pump and a pressure gauge
and then across the TFF, concentrating
the microbes (0.2 mm; Fig. 2a) or
microbes and viruses (100-kDa TFFs) in
the ‘retentate.’
It is important to always keep the
pressure within the tubes less than 10
p.s.i. (B 62 kPa), as higher pressures
can compromise the viral particles.
Nature protocol 2009

39. DNA extraction :
Rebecca V Thurber et al. nature 2009

40. Primer Design and PCR amplification:
Host and discription
ORF
Primer
Sequence
Reference
CPS1
CPS8
F
R
5′-GTAG[T/A]ATTTTCTACATTGA[C/T]GTTGG-3′
5’-AAATA(C/T)TT(G/A/T)CCAACA(A/T)ATGGA-3’
Zhong et al. 2002
AVS1
AVS2
F
R
5’-GA[A/G]GGIGCIACIGTI[T/C]TIGA[T/C]GC-3’
5’GCIGC[A/G]TAIC[G/T][T/C]TT[T/C]TTI[G/C][A/T][A/G]TA-3’
Chen, F., and C. A. Suttle 1996
Chen, F., and C. A. Suttle 1995
DNA pol gene of Virus
AVS1
family infecting microalgae
POL
F
R
5’-GA[A/G]GGIGCIACIGTI[T/C]TIGA[T/C]GC-3’
5’-[G/C][A/T][A/G]TCIGT[A/G]TCICC[A/G]TA-3’
F
R
F
R
F
R
F
R
F
R
F
R
5’-GGT CCC TGA C-3’
5’-GGT CCC TGA C-3’
5’-GGG TAA CGC C-3’
5’-GGG TAA CGC C-3’
5’-CAG CAC CCA C-3’)
5’-CAG CAC CCA C-3’)
5’-CCG CAG CCA A-3’)
5’-CCG CAG CCA A-3’)
5’-GCG ATC CCC A-3’)
5’-GCG ATC CCC A-3’)
5’-CCAAAYCTYGCMCARGT-3’
5’-CTCGTCRTGSACRAASGC-3’
Marine Cyanophage
capsid protein (CPS ) of
viral
infecting
Synechococcus spp. (gp20)
OPA-6
RAPD Study and random
primer for Virus
OPA-9
(Chesapeake Bay water )
OPA-13
CRA-22
CRA-23
Cyanobacterial podoviruses pol gene
DNA pol gene
CP-DNAP
Poxviridae
A32L
MotifI
AYDG
F
R
F
R
5’-GGAGATATGGATCCGGATGTCGTGCAG-3’
5’-GGCGCTCAGCGGCCGCATTGCCGCCG-3’
5’-GAGGCGCCCTTCAGGGCCTACCTGCTCTCG-3’
5’-CGAGAGCAGGTAGGCCCTGAAGGGCGCCTC-3’
5’-CCCGTCTACAACGCCTTCGTGTGGCCGGAC-3’
5’-GTCCGGCCACACGAAGGCGTTGTAGACGGG-3’
Danielle M. Winget et al.
2008
Sijun Huang 2010
Fong-Yuan Lin et al
2011

41. PCR frequently employed in this project on a range of templates and using
a range of conditions. PCR primers used in this study will be acquired from
the literature (Table ) or alternatively designed.
DNA pol gene in viruses:
F Chen and C A Suttle 1995 Appl. Environ. Microbiol

42. Clone library construction:
Ligation:
PCR product will be ligated in to pGEM-T Easy
cloning vector.
Transformation in E. Coli competent cells.
Positive clones picked up ( colony PCR) .
Ligations -using the pGEM-T cloning kit according
to the manufacturer’s instructions.
Escherichia coli DH5 α chemically competent cells.
The resultant E. coli colonies were screened by PCR
(colony PCR).

43. pGEM-T Easy Vector:
pGEM-T Easy Vector systems are
convenient systems for cloning PCR
products.
Vectors
have
3 terminal
thymidine to both ends. These single 3 T
overhangs at the insertion site greatly
improve ligation efficiency of a PCR
product into the plasmid by preventing
recircularization of the vector and
providing a compatible overhang for PCR
products with 5′ A overhangs.
Properties:
The high-copy-number.
Inserts of several kilobases can be successfully cloned.
Strong Promoter: T7 and SP6 RNA polymerase promoters flanking a
multiple cloning region within the coding region for the α-peptide of β
galactosidase. Insertional inactivation of the α-peptide allows
recombinant clones to be directly identified by color screening on
indicator plates containing

44. Nucleic acid amplification Sequencing and
Phylogenetic analysis::
To amplify the DNA conditions will be applied based
on previous references cited in the table.
Aligning of sequences using software ( Ex.
Sequencer 3 software).
Sequence alignment and similarity analysis.
Phyogenetic reconstruction using (NJ method)
Calculation of evolutionary distance ( Jukes contor
method).
Nucleotide sequence submission in Gen Bank.

45. Phylogenetic:
NCAAH

46. Conclusion
 This work will give an in sight into diversity of
DNA viruses of cochin barmouth region.
Reveal the Phylogenetic relation of DNA virus.
Determination of genetic diversity of marine
viruses.
 Search for new gene sequence.
 Gen bank submission.