The Molecular Ecology Seawater of Viruses in the Seakudela/migrated/OS130/Lectures/OS130...The...
Transcript of The Molecular Ecology Seawater of Viruses in the Seakudela/migrated/OS130/Lectures/OS130...The...
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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
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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
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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
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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
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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
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++ ++ +
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