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'! viii
TABLE OF CONTENTS
LIST OF TABLES
........................................................................................................... xi LIST OF FI
GURES
....................................................................................................... xiii
CHAPTER 1
...................................................................................................................... 1 INTRODUCTION
............................................................................................................ 1 1.1. Bioinvasion through ballast water
........................................................................... 2 1.2. Status of ballast water management guidelines and standards
................................ 3 1.3. Knowledge gaps in the curr
ent understanding of viruses in ballast water
............... 6 1.4. Dissertation outline
.................................................................................................. 6 REFERENCES
............................................................................................................... 8 CHAPTER 2
.................................................................................................................... 12 LITERATURE REVIEW
.............................................................................................. 12 2.1. Overview
................................................................................................................ 13 2.2. Occurrence of viruses in ballast water
................................................................... 13 2.3. Methods for the detection of viruses in aquatic environments
.............................. 17 2.3.1. Electron microscopy
....................................................................................... 17 2.3.2. Cell culture
...................................................................................................... 17 2.3.3. Polymerase chain reaction (PCR)
................................................................... 18 2.3.4. Metagenomics
................................................................................................. 19 2.4. Application of metagenomics to study virus
es in aquatic environments
............... 21 2.5. Methods used to prepare viromes in aquatic environments
................................... 27 2.6. Summary of literature review
................................................................................ 33 REFERENCES
............................................................................................................. 34 CHAPTER 3
.................................................................................................................... 41 RESEARCH QUESTIONS AND OBJECTIVES
........................................................ 41 CHAPTER 4
.................................................................................................................... 44 MATERIALS AND METHODS
................................................................................... 44 4.1. Overview
................................................................................................................ 45 4.2. Evaluation and optimization of sample preparation methods
................................ 45 4.2.1. Evaluation of the tangential flow filtration method
using groundwater and
surface water
............................................................................................................. 45 4.2.1.1. Tangential flow filtration setup and procedure
........................................ 45 4.2.1.2. Data analysis and statistics
....................................................................... 49 4.2.1.3. Virus recovery by tangential flow filtration
............................................. 50 4.2.2. Evaluation of nucleic acid extraction methods for simultaneous recovery of
viral DNA and RNA
................................................................................................. 53 4.2.2.1. Standard curve generation for quantitative PCR assay
............................ 53 ix 4.2.2.2. Viral nucleic acid extraction and quantitative PCR detection
................. 58 4.2.2.3. Data analysis an
d statistics
....................................................................... 58 4.2.2.4. Comparison of viral nucleic acid extraction methods
.............................. 59 4.3. Description of materials and methods used for preparing and analyzing viromes
62 4.3.1. Viro
me preparation
......................................................................................... 64 4.3.1.1. Sample collection
..................................................................................... 64 4.3.1.2. Variable measurement and estimation
..................................................... 64 4.3.1.3. Primary concentration of viral particles
................................................... 65 4.3.1.4. Secondary concentration of viral particles
............................................... 65 4.3.1.5. Pu
rification of viral particles
................................................................... 65 4.3.1.6. Viral nucleic acid extraction
.................................................................... 66 4.3.1.7. Random transcription/amplification of viral nucleic acid extracts
.......... 66 4.3.2. High
!through
put sequencing
.......................................................................... 68 4.3.3. Bioinformatics analyses
.................................................................................. 68 4.3.3.1. Preproce
ssing and quality control of raw sequence reads
....................... 68 4.3.3.2. De novo
assembly of sequence reads
....................................................... 69 4.3.3.3. Taxonomic classification
......................................................................... 69 REFERENCES
............................................................................................................. 72 CHAPTER 5
.................................................................................................................... 75 METAGENOMIC INVESTIGATION OF BALLAST WATER VIRAL
COMMUNITIES IN THE GREAT LAKES
................................................................ 75 Abstract
......................................................................................................................... 76 5.1. Introduction
............................................................................................................ 77 5.2. Materials and methods
........................................................................................... 80 5.2.1. Sample collection
............................................................................................ 80 5.2.2. Preparation and sequencing of viromes
.......................................................... 85 5.2.3. Analysis of viromes
........................................................................................ 85 5.2.4. Data access
...................................................................................................... 86 5.3. Results
.................................................................................................................... 87 5.3.1. General water quality
...................................................................................... 87 5.3.2. Overview of the virome data sets
.................................................................... 89 5.3.3. Taxonomic profile of the Great Lakes ballast and harbor water viromes
....... 92 5.3.4. Viral pathogens of fish and shrimp in the Great Lakes ballast and harbor
waters
...................................................................................................................... 107 5.3.5. Comparison among the Great Lakes ballast and harbor water viromes
....... 112 5.3.6. Comparison of the Great Lakes ballast and harbor water viromes with other
aquatic viromes
....................................................................................................... 117 5.4. Discussion
............................................................................................................ 120 5.4.1. Diversity of viruses in ballast water
.............................................................. 120 5.4.2. Implications and control of viruses in ballast water
..................................... 123 APPENDIX
................................................................................................................. 127 REFERENCES
........................................................................................................... 129 CHAPTER 6
.................................................................................................................. 136 TRANSPORTING OCEAN VIROMES: INVASION OF THE AQUATIC
x BIOSPHERE
................................................................................................................. 136 Abstract
....................................................................................................................... 137 6.1. Introduction
.......................................................................................................... 138 6.2. Materials and methods
......................................................................................... 139 6.2.1. Sample collection
.......................................................................................... 139 6.2.2. Preparation and sequencing of viromes
........................................................ 145 6.2.3. Analysis of viromes
...................................................................................... 145 6.2.4. Data access
.................................................................................................... 146 6.3. Results and discussion
......................................................................................... 147 6.3.1. Influence of global shipping on transport of the ocean virome
.................... 147 6.3.2. Effect of engineered, management
, and environmental variables on the ocean
virome
..................................................................................................................... 165 6.3.3. Potential invasion by rare viral pathogens
.................................................... 173 6.4. Conclusions
.......................................................................................................... 182 APPENDIX
................................................................................................................. 183 REFERENCES
........................................................................................................... 187 CHAPTER 7
.................................................................................................................. 191 CONCLUSIONS
........................................................................................................... 191 7.1. Summary
.............................................................................................................. 192 7.2. Implications for policy and technological development
...................................... 194 7.3. Limitations and recommendations for future research
........................................ 196 REFERENCES
........................................................................................................... 200 xi LIST OF TABLES
Table 1.1.
Numeric ballast water discharge standards at the federal and state level
......... 5! Table 2.1.
Viral levels found in ballast water
.................................................................. 15! Table 2.2.
Metagenomic studies in aquatic viral ecology
................................................ 24! Table
2.3. Methods used in published studies to prepare viromes from aquatic
environments
..................................................................................................................... 28! Table 4.1.
Percent recovery efficiencies (average ± standard deviation) of phages in 20
ÐL groundwater and 20
ÐL surface water samples
.................................................................. 52! Table 4.2.
Sequence of the primers and probes and PCR protocol used for quantitative
PCR assay
......................................................................................................................... 55! Table 5.1.
Identification, description, and collection information for the ballast and
harbor water samples
........................................................................................................ 83! Table 5.2.
Water quality of the ballast and harbor water samples
................................... 88! Table 5.3.
Summary of virome datasets
........................................................................... 90! Table 5.4.
Sample
Ðspecific PE reads mapped to individual assemblies
.......................... 91! Table 5.5.
Distribution of dsDNA phage hosts identified in the ballast and harbor water
viromes
.............................................................................................................................. 99! Table 5.6.
Relative abundance of the viral families in the ballast and harbor water
viromes
............................................................................................................................ 104! Table 5.7.
Contigs identified as viral pathogens of fish and s
hrimp by BLASTX search
against the NCBI RefSeq database
................................................................................. 109! Table 5.8.
Summary statistics of read mapping to five reference genomes of koi
herpesvirus
...................................................................................................................... 110! Table 5.9.
Similarity matrix of contigs between ballast and harbor water viromes using
TBLASTX
....................................................................................................................... 115! Table 5.10.
Similarity matrix of contigs between ballast and harbor water viromes using
QUAST ........................................................................................................................... 116! xii
Table 5.11.
Accession number
of the Illumina HiSeq sequencing data
......................... 128! Table 6.1.
Summary of sampling information
............................................................... 141! Table 6.2.
Summary of engineered, management, and environmental variables
........... 143! Table 6.3.
Overview of the sequence reads and the assembled contigs of the virome
libraries
........................................................................................................................... 150! Table 6.4.
Summary of Similarity Percentage (SIMPER) analysis
............................... 158! Table 6.5.
Identified viral pathogens by performing BLASTX searches against
non!redundant (nr) d
atabase
.......................................................................................... 176! Table 6.6.
Accession number of the Illumina HiSeq sequencing data
........................... 184! xiii
LIST OF FI
GURES
Figure 4.1.
Experimental setup for the tangential flow filtration using ultrafilters with
30,000 Dalton molecular weight cutoff. The retentate is recirculated until the volume
remained is less than 500 mL.
........................................................................................... 47! Figure 4.2.
A procedure for virus concentration from the groundwater and surface water
samples.
............................................................................................................................. 48! Figure 4.3.
Standard curve
s for the quantification of MS2 cDNA (A) and PhiX174 DNA
(B) using the LightCycler Instrument and the LightCycler 480 Probe Master mix.
........ 56! Figure 4.4.
Comparison
of qPCR C
T value for viral nucleic acid extracts from seeded
water with 10
5 and 10
6 PFUs/mL of MS2 (A) and PhiX174 (B). The error bars represent
standard deviation of the C
T value calculated from six replicates. Different letters in the
figure indicate si
gnificant differences according to Bonferroni LSD multiple comparison
tests (
p < 0.05). ................................................................................................................. 60! Figure 4.5.
A pipeline for the viral metagenomic study with high
Ðthroughput sequencing
used in this research.
......................................................................................................... 63! Figure 5.1.
Google Earth maps showing the Great Lakes (left) and ballast water source
and discharge ports of the five vessels (right). Ballast waters (AB, BB, IB, MB, and PB)
were originated from different parts (Lakes Erie, Huron, Michigan, and Ontario) of the
Great
Lakes but all discharged in three terminals (BH, IH, and MH) of the Port of
Duluth
ÐSuperior.
............................................................................................................... 82! Figure 6.1.
Relative distribution of viromes from ballast and ha
rbor waters. Pie charts
represent a mean relative abundance of assigned viral families (three replicates from 24
samples). ÔOthersÕ are viral families whose maximum relative abundances across viromes
are less than 3% (including RNA viruses). Vessels with b
allast waters arriving in the Port
of Los Angeles/Long Beach are shown as a green star and the Port of Singapore as a red
star. Circles and squares in the map indicate ballast waters exchanged beyond and within
200 nautical miles from nearest shoreline, re
spectively.
................................................ 148! Figure 6.2.
Influence of geography on virome composition. 72 virome data sets were
compared with each other using the Bray
!Curtis similarity matrix based on relative
abundance of viral families.
A, Principal Coordinates Analysis (PCoA) plot presenting
the difference in the virome composition. Convex hulls were used to group observations
by ocean. Closed and open symbols repres
ent ballast and harbor waters, respectively.
B, Analysis of similarity (ANOSIM) result to identify the difference in the virome
composition. Bold text indicates a significant difference between ocean viromes.
....... 156! xiv
Figure 6.3.
Response of the top three viral families contributing most to the differences
between oceans to geographical variation. Relationship between relative abundances of
Microviridae
, Podoviridae
, and Myoviridae
and samplesÕ geographic origin was
examined. Latitude and longitude are expressed in decimal scale. R was the Pearson
correlation coefficient for the relative abundance of viral families against the either
latitude or longitude in 72 data set
s. Bold text indicates a statistical significance.
Green
and red dots represent vessels with ballast waters arriving in the Port of Los
Angeles/Long Beach and the Port of Singapore, respectively.
....................................... 163! Figure 6.4.
Comparison of virus richness between ballast and harbor waters. Virus
richness was defined as a total number of identified viral families.
A, Boxplot presenting
virus richness of individual sample.
B, Boxplot presenting virus richness of ballast and
harbor water groups.
Bonferroni
!corrected
pairwise
t!tests were conducted to test for
significant differences in virus richness between groups. Black lines within boxplots
represent median values and whiskers ind
icate minimum and maximum values. CABW,
ballast water from the Port of Los Angeles/Long Beach (LA/LB); CAHW, harbor water
from the Port of LA/LB; SGBW, ballast water from the Port of Singapore; SGHW,
harbor water from the Port of Singapore.
....................................................................... 167! Figure 6.6.
Global distribution of eukaryotic viral pathogens. Samples containing viral
pathogen
!associated contigs were represented in the map. B, ballast water; H, harbor
water; D, where viral pathogen
!induced disease was found.
......................................... 175! !!J!!!!!!!!!!!!!!!!!!!!!5).1("*'8'
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57 Figure 4.3
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60 Figure 4
.4. Comparison of
qPCR
CT value for viral nucleic acid ex
tracts from seeded
water with 10
5 and 10
6 PFUs/mL of MS2 (A) and PhiX174 (B).
The error bars represent
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61 Figure 4.4
(cont
Õd) standard deviation of the C
T value calculated from six
replicates. Different letters in the
figure indicate significant differences according to
Bonferroni LSD multiple comparison
tests (p < 0.05). Abbreviation
s: qPCR, quantitativ
e PCR;
CT, cycle threshold;
PFUs, plaque
Ðforming
units
; LSD; least significant difference
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82 Figure 5
.1. Google Earth maps showing the Gre
at Lakes (left) and ballast water source and discharge ports of the five vessels (right).
Ballast waters (AB, BB, IB, MB, and PB) were originated from different parts (Lakes Erie, Huron, Michigan, and Ontario) of th
e Great Lakes but all discharged in three
terminals (BH, IH
, and MH) of the Port of Duluth
ÐSuperior.
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!!!! 94 Figure 5
.2 (cont
Õd) parameters were placed under ÒNot assigned,Ó and contigs that did not have any hits to known sequences in t
he databases were placed
under ÒNo hits.Ó Types of viral genomes (B), types of virus hosts (C), and contigs assigned to viral families (D) in the ball
ast and
harbor water viromes. Viral families whose maximum relative abundances across eight viromes less t
han 3% were represented as
ÒOthers
aÓ. Unassigned contigs at the family level were represented as ÒOthers
b.Ó 95 Figure 5
.2 (cont
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96 Figure 5
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'>:'M!
111 Table
5.8 (cont
Õd) b Number of bases of a reference genome sequence
aligned by reads.
Bold letters indicated
complete genome sequences of Koi herpesvirus retrieved from the NCBI RefSeq database.
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!!!!!!!! 114 Figure 5
.3 (cont
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:!R=S'!G=@%>!=>7!6;'!+'&86A8B;%:!
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>7!=>!=D(=:()6(A'!B8>7!<'>'A=6'7!=!
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118 Figure 5.4.
Comparison of the ballast and harbor water viromes with published freshwater viromes based on sequence similarity with
a cross
-TBLASTX search.
The six viromes (AB, BH, IB, IH, MH, and PB) from this
research
are highlighted in blue. TBLASTX
Ð2008OMZst3viral200m
2008OMZst5viral200m
2008OMZst3viral10m
2008OMZst5viral55m
2008OMZst5viral90m
2008OMZst3viral90m
British_Columbia___96_2004
Gulf_of_Mexico___96_01
Arctic_Vir___2002Coral_Atoll_Kiritimati
Coral_Atoll_Palmyra
Coral_Atoll_Tabueran
Sargasso_Sea___2005Coral_Atoll_Kingman
Tampa_Bay_induced
GS122GS117GS112GS108RW_Park_DNA
RW_Effluent_DNA
RW_Nursery_DNA
Potable_DNA
RW_Effluent_RNA
RW_Nursery_RNA
Antarctic_Lake_Spring
Antarctic_Lake_Summer
Lake_Pavin
Lake_Bourget
IB_fa
PB_fa
IH_fa
MH_fa
AB_fa
BH_fa
ElBerbera
IlijMolomharTilapia_Channel___0806Tilapia_Channel___1105Tilapia_Channel___0406Prebead_pond___0406!0.50.00.51.0distance ! hclust("average")
Type of virome
Freshwater
Private
Seawater
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119 Figure 5
.4 (cont
Õd) based comparison was not available for the other two viromes (BB and MB), due to insufficient numbers of input contigs. Virom
e names were retrieved from the MetaVir. Sam
pling site, virome fraction, and sequencing platform of each virome were shown in the
parentheses.
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148 Figure 6.1.
Relative distribution of viromes from ballast and harbor waters.
Pie chart
s represent
a mean
relative abundance of
assigned
viral families
(three replicates
from 24 samples)
. ÔOthersÕ
are v
iral families whose maximum relative abundances across
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149 Figure 6
.1 (cont
Õd) viromes
are
less than 3% (including RNA viruses)
. Vessels with ballast waters arriving in the Port of L
os Angeles/Long Beach
are
shown as a green star and
the Port of Singapore
as a red star.
Circles and squares in the map indicate ballast waters exchanged beyond
and wit
hin 200 nautical miles from
nearest shoreline,
respectively.
Abbreviations:
ds,
double
!stranded; ss, single
!stranded.
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156 Figure 6.2.
Influence of geography on virome composition.
72 virome
data sets
were
compared with each other
using the Bray
!Curtis similarity matrix based on relative
abundance of viral families.
A, Principal Coordinates Analysis (PCoA) plot presenting
the d
ifference in the virome composition
. Convex hulls were used to group observations
by ocean. Closed and open symbols represent ballast and harbor waters, respectively.
B, Analysi
s of similarity (ANOSIM) result
to identify the difference in the virome
composition. Bold text indicates a significant difference between
ocean viromes
. -0.2
-0.1
0.00.10.20.3Coordinate 1 (52.93%)
-0.3
-0.2
-0.1
0.00.10.2Coordinate 2 (12.84%)
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164 Figure 6
.3 (cont
Õd) examined.
Latitude and longitude are expressed in decimal scale.
R was the Pearson
correlation coefficient for the
relative abundance of viral families
against the
either
latitude or longitude in 72 data sets
. Bold text indicates a
statistical sign
ificance
. Green
and red dots represent vessels with ballast waters arriving in the Port of Los
Angeles/Long Beach and the P
ort of Singapore, respectively.
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171 Figure 6.5.
Effect of engineered, management, and environmental variables on the differences in the ocean virome.
Response of virus
richness to
engineered,
management,
and environmental variables was examined.
A, Relationship between virus richness and
engineered
and management
variables.
B, Relationship between virus richness and
environmental
variables.
R was the Pearson
correlation coefficient for the
virus richness
against the
variables
. Bold text indicates a
statistical significance
. Green and red dots
represent ballast and harbor waters collected from the Port of Los Angeles/Long Beach and the Port of Singapore, respectively
. !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!02040608010001020304050Time in ballast tanks (day)
Virus richness
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!02040608001020304050Distance from the nearest shoreline (nautical mile)Virus richness
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!0200400600800100001020304050Distance from the nearest shoreline (nautical mile)Virus richness
!!!!!!!!
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!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!0200400600800100001020304050Distance from the nearest shoreline (nautical mile)Virus richness
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172 Figure 6
.5 (cont
Õd) !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!0102030405001020304050LatitudeVirus richness
!"#"$"%&'()"
!"*"%&%%%+""
!!!!!!!!
,-./01-23-456".50/57638"
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!152025303501020304050SalinityVirus richness
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!010203040500246810LatitudeVirus richness (phage)
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!0102030405005101520LatitudeVirus richness (vertebrate virus)
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!0102030405005101520LatitudeVirus richness (invertebrate virus)
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!0102030405005101520LatitudeVirus richness (plant virus)
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! 175 Figure 6.6.
Global distribution of eukaryotic viral pathogens.
Samples containing
viral pathogen
!associated contigs were represented
in the map.
B, ballast water; H, harbor water; D, where
viral pathogen
!induced
disease was found
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