Metagenomics and Viral study

1. Guidance :
Dr. Swapna P. Antony
DST-INSPIRE Faculty
NCAAH, CUSAT, Kochi
Presented By:
Dhirendra Kumar Singh
Reg. No: 25212002
M. Tech., NCAAH
Molecular identification and
Phylogenetic analysis of
marine DNA viruses

2. Introduction
Why??

3. Why marine
Virus???
 Higher Concentration
(~108 viruses ml–1 ) but less
is known.
 DNA containing viruses
are abundant in Marine
system.
 Causative agent of disease
in aquaculture.
 Less in known about
diversity and evolution.
 Important for
biogeochemical cycles.
 Metagenomics (Jo
Handelsman, Jon Clardy et
al. 1998) study came in light
to address the challenge of
studying unculturable viral
particles (<99%).
 Metagenomics is an
alternative culture-
independent and sequence-
independent approach that
does not rely on the
presence of any particular
gene in all the subject
entities.
Why Metagenomics??

4. A virus particle, called a virion, can be thought of as a delivery
system that surrounds a payload.
Virus as “a piece of bad news wrapped in a protein
coat.”
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 looks like!!!!!!

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
• 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.
Alternative
hypothesis
• Primitive cells having essential cellular machinery decreased in
size and genome get encapsulated by endosymbiont that
develop in primitive viral like particles.
Reductive
evolution
• 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 virus.
Origin
based on
micelles

7. Virus Research 2006Last Universal Cellular Ancestor (LUCA) : 3.8 billion yrs ago

11. 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).

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

13. Importance of Viruses
Phage Therapy Atmospheric C02
Viral lysis diverts carbon
from the POC to the DOC
pool, effectively “short-
circuiting” the microbial
loop away from the
grazers.
Aquaculture as
disease controlling
weapon.
Detection and
diagnostics of disease
causing agents, as
antimicrobial agent
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)
Carbon , Fe and
Nitrogen cycle

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

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

16. Metagenomics
Metagenomics is an alternative culture-independent and sequence-
independent 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%).

17. The Global Ocean Sampling Expedition (GOS)
With the goal of assessing the genetic diversity in marine
microbial communities and to understand their role in
nature's fundamental processes in Sargasso Sea.
Started in August 2003.
The GOS datasets were submitted to
both NCBI and Community Cyber
infrastructure for Advanced Marine
Microbial Ecology Research and
Analysis (CAMERA)
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.

18. Sampling Route of The Sorcerer II
Onboard scientists take a 200 to 400 liters water sample
approximately every 200 miles, filter it through progressively
smaller filters to capture the various sized organisms, and then freeze
the filters with the captured microorganisms.

19. NCAAH

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

21. Reason 2………

22. Overview of the present work
New sequence
and Viral
relation
Phylogeny
DNA
Virus

23. Study Site & Samples used:
50 liters of surface sea water sample was
collected in polyethylene containers from
the Cochin Barmouth region, India (at
latitude 9° 58' 0" North and longitude 76°
15' 0" East) during September 2013.
Purification and Concentration of Samples:
Using tangential flow filtration system of 0.2 µM filters and
flocculation process.
Using this system ~50 liters of pre-filtered sea water was
concentrated down to ~20 ml from which viral particles were
pelleted by ultracentrifugation at 14, 000 x g. The viral pellet was re-
suspended with ~2 ml of sterile PBS buffer.
Sampling and purification

24. Nature protocol 2009
Hose clamp
>0.2 μm bacteria,
protists
Flow
Retentate
Peristaltic
pump
Pressure gauge
Filtrate
0.2 μm(viral
particles
Whole water sample
>0.2 μm (bacteria, protists
<0.2 μm (viral particles
Tangential Flow Filtration Unit
50 liter of
sea water
Next Step

25. 8 L
SG John et al - 2011

26. Due to small genomic content in virus, we followed QIAGEN MiniElute virus spin
procedure rather than rigorous manual laboratory protocol for viral DNA extraction.
Viral sample (already kept in -20oC) was lysed by pulse-vortexing with 25 µl of
QUIAGEN protease and 200 µl of buffer AL for 15 sec in 2 ml centrifuge tubes.
This sample was incubated at 56 oC for 15 minute and mixed with 250 µl of 100%
ethanol and kept 5 minute at room temperature now this lysate was carefully
transferred in QUIAGEN mini elute column.
Buffer AW1 and AW2 (provided with kit) was added on column sequentially and
centrifuged 800 rpm for 1 minute with each. Filtrate was discarded and column was
placed in fresh collecting tube.
The column was washed by adding 500 µl 100% ethanol and centrifuging at 8000 rpm,
collection tube was discarded.
QUIAGEN mini elution kit was dried by centrifuging at 14,000rpm for 3 minutes and
incubating at 56oC for 3 minutes.
For elution of DNA, 150 µl of elution buffer was applied on dried column and column
was centrifuged at 14,000 rpm.
Filtrate containing viral DNA sample was collected in autoclaved microcentrifuge tube
and stored at -20oC.
Viral DNA Isolation

27. Viral DNA quantified by spectrophotometer
Viral DNA was quantified and qualified by spectrophotometric
analysis at 260 & 280 nm in a 1-cm light-path length quartz
cuvette.
SAMPLE IN ELUTION BUFFER
O.D AT 260 nm = 0.18
CONCENTARTION OF DNA = 0.D AT 260nm X 50 X DILUTION FACTOR
(IN OUR CASE 50)
= 0.18 X 50 X 50
= 450 MICROG / 1 ML
= 450 nanog/microl
SAMPLE IN MILLIQ
O.D AT 260 nm = 0.14
= 350 nanog/microl
concentration required for PCR = 100ng DNA

28. 40 primers were designed based on conserved regions of marine
DNA viruses viz. DNA polymerase gene, major capsid proteins,
Hexon Protein gene etc. were synthesized at Sigma Inc.
Primer Length: optimal length of PCR primers is 18-22 bp.
Primer Melting Temperature: Primers with melting temperatures
in the range of 52- 65 oC.
GC Content: should be 40-60%.
Primer Designing and Synthesis
Gene Tool Software
Standardization of PCR conditions:
PCR conditions were standardized by varying annealing
temperatures for various primer sets.

29. PCR conditions:
Target Virus Annealing
Temperature
Expected
Size
1. CYANO-PD-S
2. PHYCO-DSD-CAP
3. IRIDO-MCP3
55
220
900
350-400
1. ADENO DSD
2. CYNO PD
3. IRIDO-DSD-CAP-L
4. IRIDO-TNFR2
60
300
250
300
300
1. IRIDO-MCP3
2. CYANO-POL
65 350
250-300
94 – 5 min
94 - 30 sec
55 – 30 sec ( 35
cycles)
72 – 30 sec
72 – 10 min

30. Cloning and Sequencing

31. Cloning, Ligation and Sequencing
The amplified DNA fragments
were cloned in pGEM-T Easy
vector by TA cloning.
The ligation reaction mixture
contained 3.5µl of PCR product
and .5µl of pGEM-T Easy vector,
5µl 2x rapid ligation buffer and
1µl of T4 DNA ligase in a final
volume of 10 ml.
Positive amplicons were
cloned on to pGEMT Easy
vector.

32. 1.5 ml bacterial culture was transferred to a 2 ml autoclaved eppendorf tube, and
centrifuged at 6,000 rpm for 5 min, supernatant was removed.200 μl of resuspension
solution (1M Tris-cl,pH-8.0,0.5M EDTA,1M Glucose, RNase, MilliQ) was added into
each tube, and vortex to completely resuspend cell pellet.
Plasmid was extracted from positive clones using standard laboratory procedure.
Immediately after adding 200 μl of lysis buffer (10N NaOH,25% SDS, MilliQ) tubes
were gently inverted 4-5 times and 350 μl of neutralizing solution (5M Pottassium
acetate, Glacial acetic acid, MilliQ) was added (mixed gently by inverting the tubes 4-5
times).The tubes were centrifuged at 12,000 rpm for 10 min. The supernatant was transferred to
a new labeled 1.5 ml Eppendorf tube containing 600 μl of ice cold isopropanol (mixed by
gentle taping) and tubes were placed in ice for 10 min.
Plasmid DNA precipitate (transparency pellet) was done by centrifuging tubes at 12,000
rpm for 10 min. Supernatant was discarded and the pellet was washed with 70% ethanol
by centrifuging at 12000 rpm for 10 mint.
Tubes were air dried by keeping invert position on a piece of paper towel for 10-20 min.
20-30 μl of 10Mm Tris-cl was added in air dried DNA pellet and the tubes were kept in
4°C for overnight to completely dissolve the pellet and stored at -200C.
Presence of plasmid was also confirmed by running plasmid sample on 1% agarose gel
and visualised under UV light.
Plasmid isolation and Amplification

33. Sample Product Size (bp)
ADENO DSD 300
CRYSO-DSD 525
CYANO PD -L 220
IRIDO-DSD-CAP-L 350
PHYCO-DSD-CAP 400
CYANO-POL 300
IRIDO-TNFR2 350-400
PHYCO-MCP2 900
Plasmid sample given for sequencing

34. Sequencing and Analysis of Sequence
 10µl of plasmid was handed over for sequencing at
SciGenom, Cochin, India.
 The sequences were analysed, trimmed and assembled
using GeneTool software.
 The nucleotide sequence homology and the translated
amino acid sequence comparisons were performed
using BLAST algorithm (BLASTn and BLASTp) of the
National Center for Biotechnology Information (NCBI)
(http://www.ncbi.nlm.nih.gov/blast).
 Gene translation and prediction of deduced proteins
were performed with ExPASy
(http://www.au.expasy.org/).

35. Continued……
 The multiple sequence alignments of nucleotide and amino
acid sequence were performed with sequences retrieved from
NCBI and multi-aligned using ClustalW and GeneDoc
computer programmes.
 Phylogenetic and molecular evolutionary analyses was
conducted by the Neighbor-Joining (NJ) and Maximum-
Likelihood (ML) methods using MEGA version 5.
 The nucleotide sequences described in this study were
deposited in to GenBank and were assigned accession numbers
KJ958986 and KJ958987.

36. Results
Sequence analysis completed for 3 sequences:
1. Adenovirus (Target gene–Hexon protein gene)
2. Iridovirus (Target gene–Major capsid protein)
3. Phycodnavirus (Target gene –Major capsid protein)

37. Adenovirus: Nucleotide & Amino acid sequence of hexon protein
Results:
Results will not be shared here
Sorry , Hope soon you will se
them in Publication………..

38. BLASTp ANALYSIS OF HEXON PROTEIN OF ADENOVIRUS

39. BLASTn RESULT OF HEXON SEQUENCE OF ADENOVIRUS

40. MULTIPLE SEQUENCE ALIGNMENT OF NUCLEOTIDE
SEQUENCE OF ADENOVIRUS HEXON GENE

41. GROUP-I
GROUP-II
PHYLOGENETIC TREE OF ADENOVIRUS HEXON PROTEIN GENE BASED ON THE
NUCLEOTIDE SEQUENCE

42. Multiple alignment analysis of hexon protein of adenovirus

43. GROUP- II
GROUP- I
PHYLOGENETIC TREE BASED ON THE AMINO ACID SEQUENCES OF ADENOVIRUS HEXON PROTEIN

44. GROUP- II

45. Iridovirus: Nucleotide & Amino acid sequence of
Major Capsid Protein

46. Accession Description Query cover E value Ident
GQ273492.1 Turbot reddish body iridovirus, complete genome 11% 0.003 100%
KC244182.1 Rock bream iridovirus isolate RBIV-C1, complete genome 10% 0.032 100%
AB104413.1
Red sea bream iridovirus genomic DNA, circular physical
map, complete sequence
11% 0.032 100%
AY779031.1 Large yellow croakeriridovirus, complete genome 11% 0.032 100%
AF371960.1
Infectious spleen and kidney necrosis virus, complete
genome
10% 0.032 100%
AY532606.1
Rock bream iridovirus strain RBIV-KOR-TY1 from South
Korea, complete genome
10% 0.032 100%
AY894343.1 Orange-spotted grouper iridovirus, complete genome 10% 0.032 100%
AY150217.1 Ambystomatigrinumstebbensi virus, complete genome 5% 1.4 90%
BLASTn RESULT OF MAJOR CAPSID PROTEIN OF IRIDOVIRUS

47. MULTIPLE ALIGNMENT ANALYSIS OF MAJOR CAPSID PROTEIN OF
IRIDOVIRUS

48. GROUP- I
GROUP- II

49. Phycodnavirus : Nucleotide & Amino acid
sequence of Major Capsid Protein

50. Continued……

51. BLAST n

52. BLASTp
NCAAH

53. Multiple Sequence Alignment

54. Phylogenetic Analysis

55. Future Dimensions and Use of this
work
Open the door of metagenomic viral study in Cochin bar
mouth region since my work clearly indicate presence of
large viruses in this area.
Discovery of new viral gene.
The application of metagenomic sequence information will
facilitate the design of better culturing strategies to link
genomic analysis with pure culture studies.
Reassembly of multiple genomes will provide insight into
energy and nutrient cycling within the community, genome
structure, gene function, population genetics and
microheterogeneity, and lateral gene transfer among
members of an uncultured community.

56. Paper Communicated in Peer-viewed Journals
Marine Virus: Larger genome with bigger impact - A review
Dhirendra Kumar Singh1, Swapna P. Antony1*and I.S. Bright Singh1
1National Center for Aquatic Animal Health, Cochin University of Science
and Technology, Cochin-682016, Kerala, India
Yes, I am a virus and I can encode eukaryotic proteins.
Dhirendra Kumar Singh1 and Swapna P. Antony1*
1National Center for Aquatic Animal Health, Cochin University of Science
and Technology, Cochin-682016, Kerala, India
Submitted in: Archives in Virology - Elsevier. Impact Factor- 2.6
Review’s

57. Continued…..
Research Article for Publication:
International Conference:
Major Capsid Protein Gene: A reliable phylogenetic biomarker for marine
viruses“
Abstract Submitted for Power Point Presesntation in “International Conference
on emerging trend in Biotechnology” (ICETB-2014) going to held in JNU-
Delhi on 6-9 November 2014
Molecular characterization and phylogenetic analysis of Family
Iridoviridae from marine environment as inferred from the major capsid
protein gene
Dhirendra Kumar Singh1 and Swapna P. Antony1*
1National Center for Aquatic Animal Health, Cochin University of Science and
Technology, Cochin-682016, Kerala, India
Communicated in: Journal of Experimental Marine Biology and Ecology, Impact- 2.4

58. Molecular characterization and phylogenetic
analysis of Family Adenoviridae major capsid
protein has been done, writing of paper is
remaing.
Analysis of Some other sequences might be
analysed and cab be written as paper.

59. My special Thanks goes to….
thanks to Ramya Chechi and
all NCAAH scholars for their kind help and support.