1

The Molecular Ecology of Viruses in the SeaThe Molecular Ecology of Viruses in the Sea

Steven M ShortOcean Sciences Department

University of California Santa Cruz

Steven M ShortOcean Sciences Department

University of California Santa Cruz

What are viruses?• Smallest, simplest microorganisms

• just a genome in a protein coat• genome may be single- or double-stranded

RNA or DNA

• Obligate intracellular “parasites”• no inherent metabolism• rely on host cell energy and materials• generally host specific

lysogenic

lyticinduction

?

productive

Virus replicationVirus replication

Centrifugation for TEM counts of viruses

EM grid

Seawater

Acrylic holderor

Epoxy Plug

Ultracentrifuge

100,000 x Gravity!

photo by A. Chan & C. A. Suttle

Viruses in a natural seawater sampleViruses in a natural seawater sample

Eye

Mercury Arc Lamp

Exciter filter(removes long wavelenghts)

Barrier filter

Dichroic mirror

size of a virus is below the limit of optical resolution(i.e. < 400 nm)

Therefore, must use fluorescent DNA stains to see them

Eye

barrier filter

mercury arc lamp

excitation filter(removes long wavelengths)

dichroicmirror

Viral counts by epifluorescence microscopy

2

MO

0.02 µm FilterSYBR Green I stain

BacteriumVirus

photo by Grieg Steward

Bacteria and viruses viewed by epifluorescence microscopy

Courtesy of Grieg Steward

Viruses vs. BacteriaLiterature Summary

Viral infection can facilitate species successions

Bratbak et al. 1993 MEPS 93: 39-48

(putative E. Huxleyi virus)

Viral infection can affect carbon flow

Bratbak et al. 1998 AME 16:1-9

• Viruses cause transfer of organic carbon from algal cells to DOC

• Bacterial growth is stimulated by the DOC

-virus

+virus

Wommack et al. 1999 Microbiol Mol Biol R 64:69-114

Virus/host dynamics: ‘kill the winner’Virus/host dynamics: ‘kill the winner’

Toxins?

Immunity?

Enzymes?

Lysogenic conversion

Estimate: 40% of marine isolates harbor prophagesJiang and Paul 1998 AEM 64:2780-2787

Other consequences of infection

3

Consequences of viral infections

• influence species successions• Increase respiration & remineralization

in the food web• Enhance microbial diversity• Mediate transduction and change host

phenotype through lysogenic conversion

Marine food webs

Illustration by S. Cook, Scripps Institution of Oceanography

Microbial LoopMicrobial Loop

Classic food chainClassic food chain

Viral LoopViral Loop

from IGBP Science no 2. (JGOFS)

Global carbon cycleGlobal carbon cycle

from IGBP Science no 2. (JGOFS)

Ocean surface CO2 exchangeOcean surface CO2 exchange

Provided by the SeaWiFS Project, NASA/Goddard Space Flight Center and ORBIMAGE

11.1 10>.01 50Chlorophyll a concentration (mg/m3)

Ocean Chlorophyll a concentrationOcean Chlorophyll a concentration

Photos by Sharyn Hedrick at Smithsonian Environmental Research Center (www.serc.si.edu/algae)

Phytoplankton: aquatic 1° producers

4

Viral infections of phytoplanktonMicromonas pusilla virus

Mayer & Taylor 1979 Nature 281: 299-301

VIRUSES

Cottrell & Suttle 1991 Mar Ecol Prog Ser 78: 1-9

Viral infections of phytoplanktonMicromonas pusilla virus

50 nm

Van Etten et al. 1991 Microbiol Rev 55: 586-620

Viral infections of phytoplanktonChlorella sp. virus

Suttle & Chan 1995 Mar Ecol Prog Ser 118: 275-282

Viral infections of phytoplanktonChrysochromulina sp. virus

VIRUSES

Nagasaki et al. 1994 Mar Biol 119: 307-312

Viral infections of phytoplanktonHeterosigma akashiwo virus

VIRUSES

uninfected

infected

Viral infections of phytoplanktonEmiliania huxleyi virus

Brussaard et al. 1996 Aquat Microb Ecol 10:105-113

1 µm

uninfected infected

VIRUSES

SEM

Coccolithophorids in the Bering Sea from http://seawifs.gsfc.nasa.gov

5

Tarutani et al. 2001 Aquat Microb Ecol 23: 103-111

Viral infections of phytoplanktonHeterocapsa circularisquama virus

VIRUSES

uninfected infected

Research questionsResearch questions

1. How diverse are marine phytoplankton viruses?

2. Are algal-virus communities dynamic?

3. Are changes in the composition of algal-virus communities related to changes in the environment?

Molecular biology & marine viruses• viruses are morphologically simple

• different viruses cannot be distinguished by morphological characteristics alone

• therefore, molecular techniques (PCR and gene sequencing) were needed to distinguish different viruses, but…

• viruses don’t encode ribosomes• can’t use rDNA sequences as molecular markers

(e.g. 16S rDNA for prokaryote phylogeny)• other molecular markers (pol) had to be used

Target DNA

Denature DNA, 95 oC

Primer annealing, 50 oC

Extend DNA, 70 oC

Repeat Cycles (30)

exponential amplification of target sequence

N

nN o

N = No x 2n

Polymerase chain reaction (PCR)

II VI III I V

Catalytic site

dNTP binding sites

N CI II III

ExonucleaseDomain (3’-5’)

Polymerasedomain

The catalytic site is universally conserved among B-family DNA polymerases and contains the amino acids sequence YGDTDS

Generic map of DNA polymerasesGeneric map of DNA polymerases

AVS1 AVS2

3 kb

3’ 5’

~500 bp

catalytic site

~700 bp

Virus DNA polymerase genes and locations of conserved regions

Virus DNA polymerase genes and locations of conserved regions

Chen & Suttle 1995 AEM 61:1274-1278

EGATVLDA YSKKRYAAYGDTDS

6

Upstream: AVS1Upstream: AVS1E G A T V L D AE G A T V L D A

5’ GAAGGTGCTACTGTTTTTGATGCT 3’5’ GAAGGTGCTACTGTTTTTGATGCT 3’Amino acid sequenceAmino acid sequence

G C C C CC C C CG C C C CC C C CAGAG

AGAG

AGAG

AGAG

AGAG

AGAG

Decoded sequenceDecoded sequence

5’ GA(A/G)GGIGCIACIGTI(T/C)TIGA(T/C)GC 3’5’ GA(A/G)GGIGCIACIGTI(T/C)TIGA(T/C)GC 3’Sense primerSense primer

Downstream: AVS2Downstream: AVS2Y S K K R Y A AY S K K R Y A A

5’ TATAGTAAAAAACGTTATGCTGCT 3’5’ TATAGTAAAAAACGTTATGCTGCT 3’Amino acid sequenceAmino acid sequence

3’ AT(A/G)(T/A)(C/G)ITT(T/C)TT(T/C)(G/T)CIAT(A/G)CGICG 5’3’ AT(A/G)(T/A)(C/G)ITT(T/C)TT(T/C)(G/T)CIAT(A/G)CGICG 5’

CTCC G GA C C C CCTCC G GA C C C CAGAG

AGAG

AGAG

AGAG

Decoded sequenceDecoded sequence

Antisense primerAntisense primer

Degenerate algal-virus-specific primersDegenerate algal-virus-specific primers

AcNPVBmNPV

HzNPVLdNPV

MpV-SP1MpV-PB8MpV-PL1

CbV-PW1CbV-PW3

PBCV-1NY-2A

CVA-1

MCMV

GPCMVHCMV

HSV-1HSV-2

PrVVZV

EBV

ASFVVacV

FPVCbV

AcNPVBmNPVHzNPVLdNPVMpV-SP1MpV-PB8MpV-PL1CbV-PW1CbV-PW3

PBCV-1NY-2A

CVA-1

MCMV

GPCMVHCMV

HSV-1HSV-2PrVVZV

EBV

ASFVVacVFPVCbV

88

98

99

100

100

100

100100

99

100

100

100100

79

100

97

92

94

99

89

60

77

98

100

100100

100

100

97

100

100

100

98

5750

52

5855

50

A. neighbor-joining tree B. parsimony tree

Chen & Suttle 1996 Virology 219:170-178

dsDNA virus polymerase genesdsDNA virus polymerase genes

MethodsMethods

0.02 - 0.45 µm

200 l

~ 500 µl

DNA extractionconcentration PCR amplification of pol

700bp clone PCR products withblue/whitevector

RFLP analysis

reamplification to confirm pol insert

sequence analysis and phylogeny construction

OTU1

OTU2

OTU3

OTU4

OTU5

MpV-SP1MpV-SP2

MpV-GM1

MpV-PB6MpV-PB7

MpV-PB8

MpV-PL1

MpV-SG1

CbV-PW1CbV-PW3

PBCV-1NY-2A

CVA-1

HSV-1

0 0.1

69

100

97

94

100

100

100

100

90

9176

63

96

100

Neighbor-joining treen = 100

Chen et al. 1996 AEM 62:2869-2874

Phylogeny of algal viruses from amplified pol sequences

Phylogeny of algal viruses from amplified pol sequences

New and improved methodsNew and improved methods

0.02 - 0.45 µm

~ 500 µl

DNA extraction

concentrationPCR amplification of pol

Sequence analysis and community comparison

200 l

700bp

Agarose gel electrophoresis

DGGE analysis

Agarose gel electrophoresisAgarose gel electrophoresis

• DNA molecules are negatively charged

• DNA is attracted to the anode of an electrical field

• different sized DNA migrate at different rates in a gel

++ ++ +

300 bp 600 bp 600 bp

7

++ ++ +

600 bp 600 bp 600 bp

Denaturing gradient gel electrophoresisDenaturing gradient gel electrophoresis

• gels are polyacrylamide with linear gradients of denaturants (urea and formamide)

• equal length DNA fragments with different sequences can be resolved

Elec

trop

hore

tic M

obili

ty

100 %

40 %

5 %

• dsDNA has discrete domains that denature (melt) at different conditions

• domain melting temperature is sequence dependant

• gel mobility is reduced as dsDNA partially denatures

DNA sequence resolution by DGGE

low

high

denaturant

MpV

-SP1

2000

600

50

Ladd

er

Ladd

er

CVA

-1

nega

tive

VC 2

71

com

bine

d

MpV

-PB

8

Agarose gel electrophoresisAgarose gel electrophoresis

Short & Suttle 2000BioTechniques 28: 20-26

8 h 6 h 4 h

0

60

7 h

com

bine

dM

pV-S

P1M

pV-P

B8

CVA

-1

com

bine

dM

pV-S

P1M

pV-P

B8

CVA

-1

com

bine

dM

pV-S

P1M

pV-P

B8

CVA

-1

VC 2

71

Perc

ent d

enat

uran

t

Denaturing gradient gel electrophoresisDenaturing gradient gel electrophoresis

Short & Suttle 2000BioTechniques 28: 20-26

Spatial variability of algal virusesSpatial variability of algal viruses

P25

PCR products amplified from virus samples collected from several locations

PCR products amplified from virus samples collected from several locations

ladd

erA B

a

P19

Mb5

Mb2

2S4S1

5H

6M

a4B

b

P S4*

ladd

erne

g

A – Antarctic B - Barkley Sound M - Malaspina Inlet H - Howe Sound S - Salmon Inlet P - Pendrell Sound* - primer AVS1 only

legend1500

600

8

Mb2

2

A Ba

P19

P25

Mb5

S4S15

H6

Ma4

Bb

Psfc

S4*

20

55

Perc

ent d

enat

uran

t

A – Antarctic B - Barkley Sound M - Malaspina Inlet H - Howe Sound S - Salmon Inlet P - Pendrell Sound* - primer AVS1 only

legend

Denaturing gradient gel of PCR products

Denaturing gradient gel of PCR products

ASFV

Phycodnaviridae

Baculoviridae

Herpesviridae

Branch Support – ML/NJ

93/100

99/100

96/93

96/88

Maximum likelihood tree of dsDNA viruses

Maximum likelihood tree of dsDNA viruses

Phylogeny of unknown algal virusesPhylogeny of unknown algal virusesShort & Suttle 2002 AEM 68: 1290-1296

ASFVMCMVHCMV

EBVHSV - 1

HSV - 2

MpV – SP1OTU1

OTU3MpV – GM1MpV – PB8MpV – PL1MpV – SG1PSB99-1 PSC99-2

SO98-5BSA99-1

OTU5CVA-1

PBCV-1NY2A

LdNPVAcNPVBmNPV99 / 100

98 / 100

87 / 76

84 / 71

50 / NA

89 / 6954 / 100

92 / 99

86 / 74

91 / 47

89 / 36

98 / 10093 / 100

96 / 93

99 / 10094 / 64

96 / 97

70 / NA

86 / 98

62 / 45

83 / NA

OTU4OTU2

BSA99-2

PSC99-1

SIA99-1BSA99-5

SO98-3SO98-2SO98-1BSB99-2

CbV – PW3100 / 100

0.1

CbV – PW1

96 / 88

MIB99-2

Barkley Sound

Southern Ocean

Jericho Pier,Vancouver

British Columbia

Temporal variability of algal viruses

• Nonspecific 500 bp product due to upstream primer only

• Classic PCR optimization: ↑ stringency = ↑ specificity, but…

• gradient PCR revealed that lower annealing temperatures increased reaction specificity

• Thus, in this case, stringency ≠specificity

• because primer melting temps are mismatched?

L N42 5643 44 46 48 50 52 5554

Annealing temperature (ºC)

AVS PCR optimization

Optimized PCR: 45 °C annealingOptimized PCR: 45 °C annealing

474

475

480

481

482

483

484

485

486

487

400

477

478

479

Ladd

er

Ladd

er

9

01-F

eb-0

0

01-M

ar-0

0

01-A

pr-0

0

01-M

ay-0

0

01-J

un-0

0

01-J

ul-0

0

01-A

ug-0

0

01-S

ep-0

0

01-O

ct-0

0

01-N

ov-0

0

01-D

ec-0

0

01-J

an-0

1

01-F

eb-0

1

01-M

ar-0

1

01-A

pr-0

1

01-M

ay-0

1

01-J

un-0

1

Tem

pera

ture

( °C

)

0

5

10

15

20

25

Chl

orop

hyll a

( mg

l-1 )

0

10

20

30

40

50

60

70

Salin

ity (

‰ )

0

5

10

15

20

25

30

35TemperatureChlorophyll aSalinity

Jericho Pier, Vancouver, BC00

/03/

10

00/0

3/24

00/0

4/07

00/0

4/21

00/0

5/05

00/0

5/19

00/0

6/02

00/0

6/15

00/0

6/29

00/1

0/19

00/1

1/02

00/0

7/13

00/0

7/27

00/0

8/10

00/0

8/24

00/0

9/07

00/0

9/21

00/1

0/05

00/1

1/16

01/0

1/11

00/1

1/30

01/0

1/25

01/0

2/21

01/0

3/07

01/0

3/22

01/0

4/04

01/0

4/18

01/0

5/03

00/1

2/14

00/1

2/28

01/0

2/08

Tide

Hei

ght (

m)

1

2

3

4

5

Algal viruses and tidal cycles

Algal viruses and phytoplankton blooms

0.45

–1.2

µm

>1.2

µm

Viru

ses

18S

rDNA (eu

kary

otes

)

Viruses and 18S rDNA fingerprints

90

70

50

30

0

80

60

40

20

80

60

40

20

0

Algal viruses (DNA pol)

0.45 – 1.2 µm 18s rDNA

> 1.2 µm 18s rDNA

Cluster analysis of communities

Summary

• Closely related algal viruses were identified in samples from geographically distant locations

• At times, changes in algal virus community composition were coincident with changes in the environment

• Through one year, algal virus communities at Jericho Pier were more stable than eukaryote communities

• Some algal viruses persisted through fluctuating environmental conditions suggesting that the production of, and mortality from some algal viruses is constant

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Phytoplankton Chaos

J Huisman & F J Weissig (1999) Nature 402: 407-410

Transient Chaos & fractal boundaries

J Huisman & F J Weissing 2001The American Naturalist 157: 488-494

Final comments on aquatic viruses

• Viruses are a ubiquitous, abundant and dynamic component of aquatic food webs

• Viruses affect elemental cycles in the ocean and host community composition

• Viruses may complicate what is already a nearly intractable ecology