Molecular identification and phylogenetic

relationship of Marine DNA Virus

Under Dr. I.S Bright Singh

Dr. Swapna P. Antony

Introduction

Why????

Why marine Virus???

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 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

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).

Virus Research 2006Last Universal Cellular Ancestor (LUCA)

Life Cycle:

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).

Most viruses in the ocean are thought to infect prokaryotes.They are typically DNA-containing 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.

Major breakthrough in study of Marine Viruses:

Abundance and distribution of marine viruses

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.

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

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

Importance of phages in Gene transfer and diversity:

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.

Marine Viruses: Truth or Dare. Annual Review of Marine Science 2012

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

Ascoviridae Insects, mainly, Noctuids

150-190 Nucleus and cytoplasm

Iridoviridae Insects, cold-blooded

vertebrates

100-220 Nucleus and cytoplasm

Mimiviridae Acanthamoeba 1,180 Cytoplasm isometricMarseillevirus Acanthamoeba 370 Nucleus and

cytoplasm

LAKSHMINARAYAN M. IYER Journal of Invertebrate Pathology (2009)

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

Fischer M G et al. PNAS 2010

Phylogenetic reconstruction of NCLDV members.

EhV- E. huxleyi virus, ASFV- African swine fever virus, APMV- A. polyphaga mimivirus, FPV, Fowlpox virus etc.

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)

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.

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Why metagenomics for my study?????

Current openion in Virology 2012

Reason 2………

Overview of my work:

New sequence and Viral relation

Virus

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Cloning

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.

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

DNA extraction :

Rebecca V Thurber et al. nature 2009

Primer Design and PCR amplification:Host and discription ORF Primer Sequence Reference

Marine Cyanophage capsid protein (CPS ) of viral infecting Synechococcus spp. (gp20)

CPS1 F 5′-GTAG[T/A]ATTTTCTACATTGA[C/T]GTTGG-3′ Zhong et al. 2002CPS8 R 5’-AAATA(C/T)TT(G/A/T)CCAACA(A/T)ATGGA-3’

DNA pol gene of Virus family infecting microalgae

AVS1 F 5’-GA[A/G]GGIGCIACIGTI[T/C]TIGA[T/C]GC-3’ Chen, F., and C. A. Suttle 1996Chen, F., and C. A. Suttle 1995AVS2 R 5’GCIGC[A/G]TAIC[G/T][T/C]TT[T/C]TTI[G/C][A/T][A/G]TA-3’

AVS1 F 5’-GA[A/G]GGIGCIACIGTI[T/C]TIGA[T/C]GC-3’POL R 5’-[G/C][A/T][A/G]TCIGT[A/G]TCICC[A/G]TA-3’

RAPD Study and random primer for Virus(Chesapeake Bay water )

OPA-6 F 5’-GGT CCC TGA C-3’ Danielle M. Winget et al.2008R 5’-GGT CCC TGA C-3’

OPA-9 F 5’-GGG TAA CGC C-3’R 5’-GGG TAA CGC C-3’

OPA-13 F 5’-CAG CAC CCA C-3’)R 5’-CAG CAC CCA C-3’)

CRA-22 F 5’-CCG CAG CCA A-3’)R 5’-CCG CAG CCA A-3’)

CRA-23 F 5’-GCG ATC CCC A-3’)R 5’-GCG ATC CCC A-3’)

Cyanobacterial podovirusesDNA pol geneCP-DNAP

pol gene F 5’-CCAAAYCTYGCMCARGT-3’ Sijun Huang 2010R 5’-CTCGTCRTGSACRAASGC-3’

Poxviridae Fong-Yuan Lin et al2011A32L F 5’-GGAGATATGGATCCGGATGTCGTGCAG-3’

R 5’-GGCGCTCAGCGGCCGCATTGCCGCCG-3’MotifI F 5’-GAGGCGCCCTTCAGGGCCTACCTGCTCTCG-3’

R 5’-CGAGAGCAGGTAGGCCCTGAAGGGCGCCTC-3’

AYDG 5’-CCCGTCTACAACGCCTTCGTGTGGCCGGAC-3’5’-GTCCGGCCACACGAAGGCGTTGTAGACGGG-3’

DNA pol gene in viruses:

F Chen and C A Suttle 1995 Appl. Environ. Microbiol

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.

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).

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.

pGEM-T Easy Vector:

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

Properties:

Nucleic acid amplification Sequencing and Phylogenetic

analysis::

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.

To amplify the DNA conditions will be applied based on previous references cited in the table.

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Phylogenetic:

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.