Guidance :

Dr. Swapna P. Antony

DST-INSPIRE Faculty

NCAAH, CUSAT, Kochi

Presented By:

Dhirendra Kumar SinghReg. No: 25212002

M. Tech., NCAAH

Molecular identification and Phylogenetic

analysis of marine DNA viruses

Introduction

Why????

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??

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!!!!!!

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.

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 viru

Origin based on micelles

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

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).

R Danovaro et al. Nature .2008

Relationships between viruses and prokaryotes in deep-sea sediments worldwide.

Importance of Viruses

Phage TherapyAtmospheric

C02Viral 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

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

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 isometricMarseillevirus Acanthamoeba 370 Nucleus and

cytoplasm

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%).

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.

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.

NCAAH

Why metagenomics for my study?????

Current openion in Virology 2012

Reason 2………

Overview of the present work

New sequence and Viral relation

Virus

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

Nature protocol 2009

Hose clamp

>0.2 μm bacteria, protists

Flow

Retentate

Peristaltic pump

Pressure gaugeFiltrate

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

8 L

SG John et al - 2011

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

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                        = 0.18CONCENTARTION 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 MILLIQO.D AT 260 nm = 0.14

                                         = 350 nanog/microlconcentration required for PCR = 100ng DNA

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.

PCR conditions:

Target Virus Annealing Temperature

Expected Size

1. CYANO-PD-S 2. PHYCO-DSD-CAP

3. IRIDO-MCP3

55220900

350-400

1. ADENO DSD 2. CYNO PD

3. IRIDO-DSD-CAP-L 4. IRIDO-TNFR2

60300250300300

1. IRIDO-MCP32. CYANO-POL

65 350250-300

94 – 5 min94 - 30 sec55 – 30 sec  ( 35 cycles)72 – 30 sec72 – 10 min

Cloning and Sequencing

TA CLONING (PGEMT VECTOR) , COLONY PCR, PLASMID EXTRACTION

SEQUENCING AT SCIGENOM, KOCHI

SEQUENCE ANALYSIS PERFORMEDUSING

GENETOOL, BIOEDIT, BLAST, EXPASY, CLUSTALW, GENEDOC AND MEGA 5.0

SUBMITTED TO GENBANK

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.

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

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

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/).

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.

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)

Adenovirus: Nucleotide & Amino acid sequence of hexon protein

Results:

Results will not be shared here Sorry , Hope soon you will se them in Publication………..

BLASTp ANALYSIS OF HEXON PROTEIN OF ADENOVIRUS

BLASTn RESULT OF HEXON SEQUENCE OF ADENOVIRUS

MULTIPLE SEQUENCE ALIGNMENT OF NUCLEOTIDE SEQUENCE OF ADENOVIRUS HEXON GENE

GROUP-I

GROUP-II

PHYLOGENETIC TREE OF ADENOVIRUS HEXON PROTEIN GENE BASED ON THE NUCLEOTIDE SEQUENCE

Multiple alignment analysis of hexon protein of adenovirus

GROUP- II

GROUP- I

PHYLOGENETIC TREE BASED ON THE AMINO ACID SEQUENCES OF ADENOVIRUS HEXON PROTEIN

GROUP- II

Iridovirus: Nucleotide & Amino acid sequence of Major Capsid Protein

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

MULTIPLE ALIGNMENT ANALYSIS OF MAJOR CAPSID PROTEIN OF IRIDOVIRUS

GROUP- I

GROUP- II

Phycodnavirus : Nucleotide & Amino acid sequence of Major Capsid Protein

Continued……

BLAST n

BLASTp

NCAAH

Multiple Sequence Alignment

Phylogenetic Analysis

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.

Paper Communicated in Peer-viewed Journals

Marine Virus: Larger genome with bigger impact - A reviewDhirendra 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, IndiaSubmitted in: Archives in Virology - Elsevier. Impact Factor- 2.6

Review’s

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 geneDhirendra Kumar Singh1 and Swapna P. Antony1*1National Center for Aquatic Animal Health, Cochin University of Science and Technology, Cochin-682016, Kerala, IndiaCommunicated in: Journal of Experimental Marine Biology and Ecology, Impact- 2.4

MORE PUBLICATION……..

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.

My special Thanks goes to….

thanks to Ramya Chechi andall NCAAH scholars for their kind help and

support.