CHAPTER 3
SEQUENCE OF THE EPIZOOTIC HEMATOPOIETIC NECROSIS VIRUS
GENOME: INSIGHT INTO RANAVIRUS EVOLUTION
ABSTRACT
Members of the genus Ranavirus (family Iridoviridae) have been
recognized as major viral pathogens of cold-blooded vertebrates. Ranaviruses
(RVs) have been associated with amphibians, fish and reptiles. At this time, the
relationship between RV species is still unclear. Previous studies suggest that
RVs from salamanders are more closely related to RVs from fish than they are to
RVs from other amphibians, such as frogs. Therefore, to gain a better
understanding of the relationship among RV isolates the genome of epizootic
hematopoietic necrosis virus (EHNV), an Australian fish pathogen, was
sequenced. EHNV is more similar in size, G+C content and number of open
reading frames (ORFs) to the other amphibian-like ranaviruses (ALRV) such as
Frog virus 3 (FV3), the type species of the genus Ranavirus, tiger frog virus
(TFV) and Ambystoma tigrinum virus (ATV) than it is to the grouper RVs.
Phylogenetic analysis of 16 conserved iridovirus ORFs show that EHNV is more
closely related to ATV than to FV3/TFV and that the grouper iridoviruses are a
more distantly related group of RVs. Further support of this relationship comes
from whole genome dotplots. EHNV and is co-linear with ATV and FV3 is colinear with TFV. However, there are two major genomic inversions between
FV3/TFV and EHNV/ATV. There is limited co-linearity between EHNV/ATV and
the grouper iridoviruses; however, co-linear segments are observed when
84
comparing EHNV/ATV and the grouper iridoviruses while a rearrangement of
these segments are observed between FV3/TFV and the grouper iridoviruses.
Therefore, the ALRVs, and EHNV, must have evolved from a common ancestor.
During the evolution of the RVs, a genomic rearrangement occurred in the
FV3/TFV-like RVs that separated them from the EHNV/ATV-like RVs. This
genomic rearrangement was followed by genomic deletions in all of the ALRVs
that removed a number of ORFs initially present in the ancestral RV. These
deletions likely occurred with the speciation of the ALRVs. These findings
suggest that the ancestral RV was a fish virus and that a recent host shift and
subsequent speciation of the ALRVs has taken place and suggest that host shifts
among RV species may be possible.
85
INTRODUCTION
Iridoviruses are large dsDNA viruses that infect both vertebrate and
invertebrate hosts (43). The family Iridoviridae contains 5 genera, the genera
Iridovirus and Chloriridovirus associated with insects, the genera
Lymphocystivirus and Megalocytivirus that infect fish species, and the genus
Ranavirus whose members have been associated with mortality events in
amphibians, fish and reptiles (43). At this time, the type isolates for each genus in
the family Iridoviridae have been sequenced (Table 3.1) and recently all of the
iridovirus genomes were re-annotated (12). This re-annotation set the
parameters for predicting iridovirus open reading frames (ORFs), ORFs that are
necessary for an iridovirus, ORFs that are shared among individual iridovirus’
genera and ORFs that are specific to a particular group or isolate were also
defined.
Members of the genus Ranavirus have been recognized as major
pathogens of cold-blooded vertebrates (4, 43). For example, ranaviruses (RVs)
have been isolated from amphibians in North America (3, 10, 15, 22, 23), Asia
(18, 44), Australia (38) and the United Kingdom (5, 11), fish (1, 28, 31), and
reptiles (2, 6, 19, 25, 29, 30). As interest in RVs has grown, the number of
ranaviruses that have been completely sequenced has also increased. These
include frog virus 3 [FV3; (39)], the type virus of the genus Ranavirus, tiger frog
virus [TFV; (18)], a closely related RV to FV3 that was isolated from frogs in Asia,
and Ambystoma tigrinum virus [ATV; (24)], a RV associated with salamander
86
mortalities in North America. In addition, two grouper iridoviruses, also members
of the genus Ranavirus, the grouper iridovirus [GIV; (41)] and the Singapore
grouper Iridovirus [SGIV; (35)] have recently been sequenced. Information
obtained by comparing ranavirus genomic sequences offers insight into RV
evolutionary history, identifies core groups of genes, and gives insight into the
genes responsible for viral immune evasion and pathogenesis.
Previous studies have shown that RV isolates can be translocated across
large distances in infected salamanders that are used as bait for sport fishing
(23). Phylogenetic analysis of the major capsid protein (MCP) sequence from
salamander RV isolates from the southern Arizona border to Canada were
compared to other RV MCP sequences (23). The data suggest that salamander
RV isolates are more closely related to fish RV isolates, such as epizootic
hematopoietic necrosis virus (EHNV), than to other amphibian (frog) RV isolates
like FV3 (23). Dotplot analysis comparing the genomic sequence of ATV to FV3
and TFV show two major genomic inversions (24) while the FV3 and TFV
genomes show complete co-linearity. These data suggest that at some point in
virus evolutionary history an ancestral virus diverged into the salamander virus
and frog virus lineages. A genomic rearrangement occurred in one of the
lineages at the time of divergence or after. Subsequent host specific evolution
occurred, limiting cross transmission among isolates, in such a way that frog RVs
do not cause disease during laboratory infection of salamanders and vice versa
(22). There is some evidence that salamander RV isolates can be isolated or
87
detected from laboratory infected frogs (34) and that a pathogen host shift is the
result of the movement of these pathogens (23). Therefore, the ecological and
economic consequences of RVs moving in the environment include the potential
of these pathogens infecting and decimating new amphibian populations.
Therefore, a more complete understanding of the genetic determinants that make
up RVs would help predict future transmission events.
Epizootic hematopoietic necrosis virus (EHNV) was isolated in Australia
from redfin perch, (Perca fluviatilis), and rainbow trout, (Oncorhynchus mykiss).
EHNV can be classified as an indiscriminate pathogen of fresh water fin fish as it
readily kills juvenile redfin perch and rainbow trout in inland water bodies of
throughout Australia (42). In addition, challenge experiments showed that
following bath inoculation other fish species are also susceptible to infection with
EHNV including the Macquarie perch (Macquaria australasica), silver perch
(Bidyanus bidyanus), mosquito fish (Gambusia affinis) and mountain galaxias
(Galaxias olidus). In contrast, Murray cod (Maccullochella peeli), golden perch
(Macquaria ambigua), Australian bass (Macquaria novemaculeata), Macquarie
perch, silver perch and Atlantic salmon (Salmo salar) were susceptible only by
intraperitoneal (i.p.) injection of virus. Serological surveys (A. Hyatt, unpublished
data) show that redfin perch and rainbow trout can also be carriers of EHNV.
Virus was re-isolated from animals not showing clinical signs of disease, making
them likely vehicles for the translocation and introduction of EHNV into naive host
populations. Preliminary and unpublished data (A. Hyatt) have shown that i.p.
88
inoculation of Australian frogs with EHNV and the cane toad Bufo marinus results
in seroconversion but no signs of clinical disease (46). While EHNV has been not
been identified in fish populations in North America it is possible that this
pathogen could be translocated, via movement of animals for food, bait or
scientific purposes, thereby infecting and potentially decimating naïve fish
populations. Since the recognition of disease due to EHNV in Australia in 1986,
similar systemic necrotizing iridovirus syndromes have been reported in farmed
fish. These include catfish (Ictalurus melas) in France (European catfish virus)
(26), sheatfish (Silurus glanis) in Germany (European sheetfish virus) (2, 3),
turbot (Scophthalmus maximus) in Denmark (5) and others in Finland (4, 31). In
addition, while EHNV has been classified as a RV, the relationship between this
fish pathogen and amphibian RVs is poorly understood. Therefore, in order to
better understand the relationship among RV isolates the complete sequence of
EHNV genomic DNA was determined. The characteristics of the EHNV genome,
its relatedness to other iridoviruses and insights into RV evolution are the focus
of this study.
89
MATERIALS AND METHODS
Generation of the EHNV genomic DNA library. EHNV viral DNA was
isolated and kindly provided by Dr. Michael Bremont, Institut National de la
Recherche Agronomique, France. The EHNV viral shotgun library was
constructed by shearing 10 g of viral DNA in 200 l of TE (10 mM Tris-HCl; 1
mM EDTA) buffer. The sheared DNA was ethanol precipitated and the pellet
containing DNA was end repaired using the T4 DNA polymerase, concentrated
by ethanol precipitation, and quantified. Bst X1 adaptors were added to the end
repaired viral DNA and the sheared, repaired viral DNA was size selected by gel
electrophoresis. DNA from 2 to 4 kbp was extracted from the gel and ligated into
the pOT13 DNA plasmid. Plasmid DNA was transformed into DH10B competent
cells by electroporation and plated onto pre-warmed agar plates containing 50
g/ml chloramphenicol and incubated overnight at 37oC. Colonies containing
plasmid were selected using automated equipment, and plasmid DNA was
isolated using solid phase reversible immobilization technology. Isolated
plasmids were sequenced using automated equipment (ABI 3730XL; Applied
Biosystems). Sequences were aligned and assembled using Phred/Phrap
(http://www.genome.washington.edu) and finished with Consed (Gordon et al.
1998) and primer walking to fill in the gaps.
Genome annotation. The newly sequenced genome was annotated
using similar procedures as described previously (24). Using the BLASTP,
BLASTX, TBLASTX procedures (32, 33), all open reading frames (ORFs) with
90
sequence similarity to any other closely related viral ORF and/or containing
domain(s) or homology with any known protein were identified. Identified ORFs
were confirmed using the Genome Annotation Transfer Utility program (GATU;
http://www.biovirus.org/), a program that uses previously annotated genomic
DNA as a reference for annotating a newly sequenced genomic DNA, by using
all of the completely sequenced iridoviruses as a reference sequence. The
iridoviruses used in this analysis were (also see Table 1): Ambystoma tigrinum
virus [ATV; (24)]; frog virus 3 [FV3; (39)]; tiger frog virus [TFV; (18)]; grouper
iridovirus [GIV; (41)]; Singapore grouper iridovirus [SGIV; (35)]; lymphocystis
disease virus-1 [LCDV-1; (40)]; lymphocystis disease virus-China [LCDV-C; (45)];
infectious spleen and kidney necrosis virus [ISKNV; (17)]; orange spotted
grouper iridovirus [OSGIV; (27)]; rock bream Iridovirus [RBIV; (8)]; insect
iridovirus 6 (IIV-6) or Chilo iridovirus [CIV; (21)] and invertebrate iridovirus 3 (IIV3) or mosquito iridovirus [MIV; (7)]. ORFs in the family Iridoviridae are presumed
to be non-overlapping; however, ORFs were considered overlapping if both
ORFs have high sequence identity (i.e. BLASTP expected score) to other
sequenced iridoviruses.
Phylogenetic and dotplot analysis. Phylogenetic analysis was
conducted by obtaining the homologues of the EHNV ORFs 8L
(NTPase/helicase), 10L (RAD2), 11R, 13L (ICP-46), 14L (MCP), 16L (thiol
oxidoreductase), 24R (RNase III), 43R (RNA polymerase  subunit), 44L (DNA
polymerase), 48L (phosphotransferase), 53L (myristylated membrane protein),
91
72R, 77R, 92L (NTPase), 95R and 100R (putative replication factor) from
representative members of the sequenced iridoviruses in GeneBank by BLASTP
analysis (http://www.ncbi.nlm.nih.gov/). All sequences were concatenated using
BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The sequences were
aligned and the neighbor-joining analysis was conducted using the MEGA 3.1
software (26) with the default options. Dot plots comparing all of the sequenced
iridoviruses (Table 1) to EHNV were generated using JDotter
[http://www.biovirus.org/; (36, 37)] using the default settings.
92
RESULTS
EHNV genome characteristics. An EHNV genomic DNA library was
successfully generated and produced over 1,800 EHNV specific sequences. The
sequences were aligned and assembled into the complete genomic sequence
with an average coverage of ~4 fold redundancy per base pair (bp).
The finished EHNV genomic sequence (127,011 bp) is larger than the
amphibian RVs ATV, TFV and FV3 (average 105,754 bp) but smaller than the
grouper RVs GIV and SGIV (average 139,962 bps; Table 3.1). In addition, the
EHNV genome is larger than the other fish pathogens ISKNV, OSGIV and RBIV
(average 112,026 bp) but smaller than the insect viral genomes CIV and MIV
(average 201,307 bp). The lymphocytiviruses, LCDV-1 and LCDV-C, have very
different sized genomes (Table 3.1). EHNV genomic DNA is smaller than the
average of these two viral genomic sequences (average 144,450 bp). EHNV has
a similar G+C content (54%) as TFV, FV3, ATV, ISKNV, OSGIV and RBIV (5355%), a slightly higher G+C content than the grouper iridoviruses GIV and SGIV,
and the insect Iridovirus MIV (48-49%) and a much higher G+C content as
compared to LCDV-1, LCDV-C and CIV (27-29%).
Open reading frame analysis. One hundred open reading frames
(ORFs) were predicted in the EHNV genome based on the annotation criteria
described in the materials and methods (Fig. 3.1 and Table 3.2). The number of
ORFs predicted in EHNV (100) is larger than the number of ORFs predicted in
ATV (92) but close to the number of ORFs predicted in FV3 (97) and TFV (103);
93
however, all of these RVs have considerably fewer ORFs than the 139 ORFs
seen in the fish RVs GIV and SGIV (Table 3.1). In addition, the number of EHNV
ORFs is relatively close to the number of ORFs predicted in the other fish
iridoviruses ISKNV, OSGIV, RBIV and LCDV-1 while the number of ORFs in
LCDV-C, CIV and MIV are much larger, corresponding to their larger genome
sizes (Table 3.1). The variation in genome size and corresponding numbers of
ORFs suggests that not all RVs have the same coding capacity. At this time it is
unclear why this variation is observed. Having more sequence information may
help explain this phenomenon.
Of the 100 EHNV ORFs, 26 ORFs are conserved throughout the family
Iridoviridae (Table 3.3 and 3.4). These ORFs can be defined as the core
iridovirus genes since they are present in every iridovirus sequenced to date and
confirm published reports that all iridoviruses contain these 26 ORFs (12). The
majority of these conserved ORFs (21/26) have a predicted function based on
sequence homology to other characterized proteins or have been identified
based on experimental data (Table 3.4). In contrast, only 4 of the 27 additional
ORFs that are conserved throughout the genus Ranavirus have a predicted
function (Tables 3.2 and 3.5) and only one of the 13 amphibian RV specific
genes has a predicted function, the vIF2 (ORF 61R) (Table 3.6). Further
experimental analysis of these RV ORFs is necessary to identify the function(s)
of these RV specific ORFs.
94
There appears to be 9 ORF clusters, containing a minimum of 4
consecutively oriented ORFs, throughout the genome. These clusters of
consecutively orientated ORFs (CCOO) all have a similar orientation, either right
or left, with the majority of the CCOOs going in the right orientation. While the
genomes of iridoviruses are circularly permutated and terminally redundant (43)
and therefore the orientation of these ORFs was an arbitrary decision as to the
orientation of the start of the genome, it is surprising the amount of conservation
among RV isolates within these regions. For example, the region between ORFs
54 and 77 contains 21 of the 24 predicted ORFs in the right orientation, while in
ATV 19 of the 20 ORFs in this region (ATV ORFs 52 – 69) are oriented in the
same direction (24). FV3 and TFV also have similarly oriented ORFs in this
region (18, 39). However, the orientation of the ORFs in these viruses is to the
left due to a major genomic rearrangement that inverted this cluster of genes.
Within the region described above, the largest CCOO is comprised of 11
ORFs (ORFs 62 – 70R; Fig. 1). Only two of these 11 ORFs have a predicted
function, ORF 61R which codes for the viral homologue of the eukaryotic
initiation factor  (vIF2) and ORF 62R which codes for the predicted tyrosine
kinase/lipopolysaccharide modifying enzyme that is found in all other iridoviruses
(Table 3.2). Open reading frame 64R, a unique EHNV ORF, has a predicted
capsid maturation protease function and the remaining 7 ORFs in the CCOO
have no predictable function based on homology/identity to any protein in the
database (Table 2). It is unclear at this time the role of the majority of ORFs in
95
this region. This clustering of genes has also been observed previously (12) and
the sequence of EHNV follows a similar pattern. The role of these clusters in viral
fitness and survival is still unclear.
In overall appearance the EHNV CCOOs are reminiscent of pathogenesis
islands (PAIs) found in pathogenic bacteria. The bacterial PAIs, mobile genetic
elements that contribute to rapid changes in virulence potential (9, 13, 14, 16),
contain ORFs that are in the same orientation and code for genes that have been
correlated to increased pathogenesis. These “islands” are suggested to be
transmitted in plasmids that incorporate into the host’s genome by homologous
recombination. While this scenario seems unlikely for EHNV, it is reasonable to
hypothesize that these regions have been maintained within the RVs and must
be present for RV virion formation and/or pathogenesis. Poxviruses, a group of
closely related DNA viruses (20), contain groups of ORFs at the hairpin ends of
their genomes that are associated with virulence. While this region of the poxviral
genome does not look like the CCOOs found in EHNV, it is interesting to note the
similarity between these related vertebrate pathogens in that ORFs correlating
with pathogenesis are in close proximity to each other. Further analysis of this
region of the EHNV genome will shed light into the specific function of these
ORFs.
Phylogenetic analysis. Sixteen EHNV ORFs and the EHNV homologues
in 9 other iridoviruses were used to generate a concatenated phylogeny (Fig 3.2).
The phylogenetic tree shows high bootstrap support (100%) for EHNV being a
96
member of the genus Ranavirus, family Iridoviridae. In addition, EHNV is more
closely related to ATV, a salamander RV, than it is to FV3 and TFV, frog RVs,
supporting previously published analysis (23). EHNV is more distantly related to
LCDV-1, LVDC-C, ISKNV and CIV (members, respectively, of Lymphocytivirus,
Megalocytivirus and Iridovirus genera). The grouper iridoviruses, GIV and SGIV,
also members of the genus Ranavirus, are more closely related to EHNV than
the previously mentioned iridoviruses and these viruses appear to have diverged
at some point in evolutionary history. Based on these data and that of others (35,
41), the grouper iridoviruses, or GIV-like isolates, should be considered a
distantly related species within the genus Ranavirus. This phylogenetic analysis
also supports the classification of the other genera in the family Iridoviridae, as all
genera are separated into individual clades with high bootstrap support (100%;
Fig. 3.2) consistent with previous reports (43).
While the iridovirus lineage has been documented (20) the information
used to distinguish members of the genus Ranavirus are limited to one or few
genes. The power of using whole genomic sequences for viral comparisons is
overwhelming. Based on these observations and those of others (12) the RVs
are divided into two sub-species, the GIV-like and Amphibian-like ranavirus
(ALRV) sub-species. The ALRVs can be further classified as being ATV-like or
FV3-like.
Dotplot analysis. The EHNV genome was compared to itself by dotplot
analysis (Fig. 3.3A). Dotplot analysis is a way to analyze and compare genomic
97
sequences. This type of analysis utilizes the entire genomic sequence and the
plots generated can reveal information on the entire genome and how genomic
sequences are organized. The dotplot shows the -45o co-linear line plus
horizontal and vertical “dots” (Fig 3.3A). These “dots” indicate repeat sequences
found throughout the EHNV genome. Closer examination of one of these regions
shows the repeated sequences, not as dots, but more like dark lines (Fig 3.3B).
These lines are runs of sequence that are similar throughout the EHNV genome.
Interestingly, one can almost map out the EHNV ORFs between these “dots”
suggesting that these repeated regions may be involved in the regulation of gene
expression. The “dots” also appear to be running in the lighter streaks running
vertically and horizontally in the dotplot (Fig 3A – C). These lighter streaks
represent regions of different G/C content. The EHNV genome has an overall
G+C content of 54% (Table 3.1), but this does not mean the entire genome has a
uniform G/C content, as some regions are more G/C rich than others. The A/T
rich regions appear as lighter streaks on the dotplot. Similar patterns have been
observed previously in dotplots of the ATV, TFV and FV3 (12, 24, 39).
In addition to the -45o line observed in the dotplot, in one region of the
EHNV genome it is possible to observe repeated ORFs (Fig 3.3C). This region,
containing a CCOO described above, consists of 5 ORFs that are hypothesized
to be the result of gene duplication events followed by divergent gene evolution.
At some point in the gene duplication process, 2 ORFs were inverted. Support for
this hypothesis comes from the fact that EHNV ORFs 55R, 56R and 57R have
98
homology to two ALRV ORFs (e.g. ATV 53R and 54R; TFV ORF 23R and 24R;
Table 3.2). EHNV ORF 58L and 59L, inverted in respect to ORFs 55 – 57R in
this region, have homology to the same two RV ORFs. When these EHNV ORFs
are aligned there is little conservation at the DNA level (<20% identity), but
conservation is higher at the amino acid level (>20% identity). The repeated and
inverted ORFs can be observed in ATV, FV3 and SGIV. However, ATV and FV3
have 2 copies of this repeated ORF while the fish RVs (GIV/SGIV) have 5 copies
of this repeated ORF (data not shown). In the GIV-like RVs, one of the repeated
ORFs is located at the beginning of the genome (ORF 4L, SGIV) and does not
cluster with the other 4 copies. Interestingly, this gene, SGIV ORF 4L, is adjacent
to the vHSD gene (ORF 3R). This is identical to the order of the homologues in
EHNV and ATV (ORFs 54R and 55R and ORFs 52R and 53R, respectively). The
repeated ORFs offer insight into the evolution of the genus Ranavirus. Based on
preliminary phylogenetic analysis of these ORFs it appears that the ancestral RV
was a fish virus containing multiple copies of these ORFs (data not shown).
Subsequent convergent deletion evolution through speciation deleted 3 of these
ORFs in the ATV-like and FV3-like RVs. This event must have occurred
separately for the ATV- and FV3-like viruses as these viruses adapted to their
amphibian hosts. An alternative hypothesis would be that gene transfer between
the ATV-like and FV3-like viruses occurred giving them identical repeated ORFs.
As observed in other dotplot comparisons [(12, 24);Fig. 3.4] the typical
RV repeat patterns can be seen when comparing EHNV to ATV, FV3 and TFV
99
(Fig. 3.4A – B). Comparing EHNV to ATV by dotplot showed co-linearity between
these two RVs (Fig. 3.4A). This is a surprising result as these two viruses infect
very different hosts and have been isolated on different continents. This
observation suggests that that these two different RV pathogens are very closely
related, confirming the phylogenetic analysis above (Fig 3.2). There are regions
of the dotplot that show unique sequences in EHNV as compared to ATV and
these regions correlate to the unique EHNV ORFs (Table 3.2) or extra noncoding DNA sequence. These extra sequences are visualized as breaks in the 45o co-linear line and a shift in this line to the right. This shift represents
sequence that is in EHNV but not present in ATV.
In contrast to previous reports (39), no inversions were observed between
FV3 and TFV using the same program (MacVector) or the dotplot program used
in this study (JDotter; data not shown). In addition, dotplots of SGIV and GIV
reveal complete co-linearity, although the start of these RV genomes differ (data
not shown). Therefore, FV3 and TFV are grouped together and SGIV and GIV
are grouped together for the following dotplot analysis (Fig 3.4B). Two major
genomic inversions in FV3 and TFV, as compared to EHNV/ATV, can be
visualized on the dotplot as a +45o line (Fig. 3.5B). This is similar to previously
published dotplots (12, 24). When comparing EHNV to GIV or SGIV, no long
stretches of co-linearity exist between these sequences, although small sections
of co-linearity remain as seen through a dotplot analysis (Fig. 3.4C). The short
diagonal lines on the dotplot are indicative of groups of ORFs (2 – 4 ORFs long)
100
that are scattered throughout the genome. The dotplot correlates with the
phylogeny in that EHNV is more closely related to the amphibian RVs and that
the GIV-like viruses are more distantly related. These results have been
observed by others (12) and suggest a more distant relationship of EHNV and
the GIV-like viruses than EHNV and the other RVs.
No co-linearity was observed when comparing EHNV to all of the other
completely sequenced iridovirus isolates (Fig 3.4F – J). Short stretches of colinearity can be observed in these dotplot comparisons, but the number, intensity
and length of these lines suggest that EHNV is more distantly related to these
iridoviruses. Differences in G+C content can be visualized by the intensity of the
dotplot. For example, the EHNV genome has a 54% G+C content while the G+C
content of LCDV-1, LCDV-C and CIV is approximately 30% (Table 3.1). Islands
of A/T rich sequences of the EHNV genome are visualized as grey streaks on the
dotplot running in the vertical direction (Fig. 3.4D – E). The lighter regions,
looking vertically on the dotplot, represent the G/C regions of the EHNV genome,
as the LCDV genomes are more A/T rich. There is one major G/C rich region of
the LCDV-1 and –C genomes that can be observed as a dark streak across the
dotplot on the horizontal plane (Fig. 3.4D – E). In contrast, when comparing
dotplots of similar G/C content, the A/T rich regions are observed as lighter
streaks throughout the dotplot (Fig 3.4F – H).
A closer examination of the genomic dotplots of EHNV verses the SGIV
and FV3 verses the SGIV gave insight into the ALRV’s evolution. By
101
consecutively numbering the small stretches of co-linear ORFs on the dotplots, it
was possible to observe an inversion of segments in the FV3 genome (Fig 3.5A –
B). EHNV has segments 1 through 6 oriented together in consecutive order (Fig.
3.5A), while FV3 has a rearrangement of these segments (Fig. 3.5B). This
rearrangement of segments corresponds to the inversion observed when
comparing the EHNV/ATV and FV3/TFV genomic sequences (Fig 3.4B).
Therefore, these data suggest that the inversion observed when comparing
EHNV/ATV and FV3/TFV occurred in the FV3-like lineage. Sequence analysis of
more RV isolates will be necessary to fully investigate this hypothesis.
102
DISCUSSION
The genomic sequence of EHNV has been confirmed as a member of the
genus Ranavirus (family Iridoviridae). Genetic analysis using 16 conserved ORFs
show EHNV more closely related to ATV than any other iridovirus sequenced to
date. This relatedness is supported by dotplot analysis, where EHNV and ATV
show completely co-linear genomes. Only FV3 and TFV genomic sequences are
similar, having 2 inversions as compared to EHNV. This relatedness correlates
with the similarity observed in the phylogeny. In addition, the fish RVs appear to
contain 5 copies of a repeated ORF while the ALRVs contain 2 copies.
Based on the data obtained in this study, it is most likely that the ALRVs
have evolved from an ancestral fish virus. In addition, a recent host shift from fish
to amphibians likely occurred. After the species jump, the ALRVs lost several
non-essential genes/sequences. A second event occurred that rearranged the
FV3-like virus genomic DNA relative to ATV/EHNV causing a separation of the
ALRVs into ATV-like and FV3-like viruses. The amphibian viruses subsequently
lost genes and this led to adaption to their amphibian hosts. This hypothesis
relies on both the ATV-like and FV3-like RVs losing the same ORFs as they
adapted to their amphibian host independently. While this seems nonparsimonious, the alternative view is even less parsimonious. The alternative
hypothesis suggests that the ancestral RV, also a fish virus, had only 2 copies of
the repeated ORFs and that the divergent fish RVs (i.e. EHNV and the GIV-like
RVs) both duplicated these ORFs after divergence had occurred. To have two
103
independent groups of viruses undergo identical genomic changes is less likely
to happen. This insight into the evolution of RVs suggests that other host shifts
are possible and that it is important to monitor RVs in the bait, pet and wild fish,
amphibian and reptile populations.
104
REFERENCES
1.
Ahne, W., M. Bremont, R. P. Hedrick, A. D. Hyatt, and R. J.
Whittington. 1997. Iridoviruses associated with epizootic haematopoietic
necrosis (EHN) in aquaculture. World Journal of Microbiology &
Biotechnology 13:367-373.
2.
Allender, M. C., M. M. Fry, A. R. Irizarry, L. Craig, A. J. Johnson, and
M. Jones. 2006. Intracytoplasmic inclusions in circulating leukocytes from
an eastern box turtle (Terrapene carolina carolina) with iridoviral infection.
Journal of Wildlife Diseases 42:677-684.
3.
Bollinger, T. K., J. Mao, D. Schock, R. M. Brigham, and V. G.
Chinchar. 1999. Pathology, isolation, and preliminary molecular
characterization of a novel iridovirus from tiger salamanders in
Saskatchewan. J Wildl Dis 35:413-29.
4.
Chinchar, V. G. 2002. Ranaviruses (family Iridoviridae): emerging coldblooded killers. Arch Virol 147:447-70.
5.
Cunningham, A. A., T. E. S. Langton, P. M. Bennett, J. F. Lewin, S. E.
N. Drury, R. E. Gough, and S. K. MacGregor. 1996. Pathological and
microbiological findings from incidents of unusual mortality of the common
frog (Rana temporaria). Philosophical Transactions of the Royal Society of
London Series B-Biological Sciences 351:1539-1557.
6.
De Voe, R., K. Geissler, S. Elmore, D. Rotstein, G. Lewbart, and J.
Guy. 2004. Ranavirus-associated morbidity and mortality in a group of
captive eastern box turtles (Terrapene carolina carolina). J Zoo Wildl Med
35:534-43.
7.
Delhon, G., E. R. Tulman, C. L. Afonso, Z. Lu, J. J. Becnel, B. A.
Moser, G. F. Kutish, and D. L. Rock. 2006. Genome of invertebrate
iridescent virus type 3 (mosquito iridescent virus). J Virol 80:8439-49.
8.
Do, J. W., C. H. Moon, H. J. Kim, M. S. Ko, S. B. Kim, J. H. Son, J. S.
Kim, E. J. An, M. K. Kim, S. K. Lee, M. S. Han, S. J. Cha, M. S. Park, M.
A. Park, Y. C. Kim, J. W. Kim, and J. W. Park. 2004. Complete genomic
DNA sequence of rock bream iridovirus. Virology 325:351-63.
9.
Dobrindt, U., B. Hochhut, U. Hentschel, and J. Hacker. 2004. Genomic
islands in pathogenic and environmental microorganisms. Nature Reviews
Microbiology 2:414-424.
105
10.
Donnelly, T. M., E. W. Davidson, J. K. Jancovich, S. Borland, M.
Newberry, and J. Gresens. 2003. What's your diagnosis? Ranavirus
infection. Lab Anim (NY) 32:23-5.
11.
Drury, S. E. N., R. E. Gough, and A. A. Cunningham. 1995. Isolation of
an Iridovirus-Like Agent from Common Frogs (Rana temporaria).
Veterinary Record 137:72-73.
12.
Eaton, H. E., J. Metcalf, E. Penny, V. Tcherepanov, C. Upton, and C.
R. Brunetti. 2007. Comparative genomic analysis of the family
Iridoviridae: re-annotating and defining the core set of iridovirus genes.
Virol J 4:11.
13.
Gerlach, R. G., and M. Hensel. 2007. Salmonella Pathogenicity Islands in
host specificity host pathogen-interactions and antibiotics resistance of
Salmonella enterica. Berliner Und Munchener Tierarztliche Wochenschrift
120:317-327.
14.
Gerlach, R. G., D. Jackel, B. Stecher, C. Wagner, A. Lupas, W. D.
Hardt, and M. Hensel. 2007. Salmonella Pathogenicity Island 4 encodes
a giant non-fimbrial adhesin and the cognate type 1 secretion system.
Cellular Microbiology 9:1834-1850.
15.
Greer, A. L., M. Berrill, and P. J. Wilson. 2005. Five amphibian mortality
events associated with ranavirus infection in south central Ontario,
Canada. Dis Aquat Organ 67:9-14.
16.
Hacker, J., B. Hochhut, B. Middendorf, G. Schneider, C. Buchrieser,
G. Gottschalk, and U. Dobrindt. 2004. Pathogenomics of mobile genetic
elements of toxigenic bacteria. International Journal of Medical
Microbiology 293:453-461.
17.
He, J. G., M. Deng, S. P. Weng, Z. Li, S. Y. Zhou, Q. X. Long, X. Z.
Wang, and S. M. Chan. 2001. Complete genome analysis of the
mandarin fish infectious spleen and kidney necrosis iridovirus. Virology
291:126-39.
18.
He, J. G., L. Lu, M. Deng, H. H. He, S. P. Weng, X. H. Wang, S. Y.
Zhou, Q. X. Long, X. Z. Wang, and S. M. Chan. 2002. Sequence
analysis of the complete genome of an iridovirus isolated from the tiger
frog. Virology 292:185-97.
19.
Hyatt, A. D., M. Williamson, B. E. H. Coupar, D. Middleton, S. G.
Hengstberger, A. R. Gould, P. Selleck, T. G. Wise, J. Kattenbelt, A. A.
106
Cunningham, and J. Lee. 2002. First identification of a ranavirus from
green pythons (Chondropython viridis). Journal of Wildlife Diseases
38:239-252.
20.
Iyer, L. M., L. Aravind, and E. V. Koonin. 2001. Common origin of four
diverse families of large eukaryotic DNA viruses. J Virol 75:11720-34.
21.
Jakob, N. J., K. Muller, U. Bahr, and G. Darai. 2001. Analysis of the first
complete DNA sequence of an invertebrate iridovirus: coding strategy of
the genome of Chilo iridescent virus. Virology 286:182-96.
22.
Jancovich, J. K., E. W. Davidson, J. F. Morado, B. L. Jacobs, and J. P.
Collins. 1997. Isolation of a lethal virus from the endangered tiger
salamander Ambystoma tigrinum stebbinsi. Diseases of Aquatic
Organisms 31:161-167.
23.
Jancovich, J. K., E. W. Davidson, N. Parameswaran, J. Mao, V. G.
Chinchar, J. P. Collins, B. L. Jacobs, and A. Storfer. 2005. Evidence
for emergence of an amphibian iridoviral disease because of humanenhanced spread. Mol Ecol 14:213-24.
24.
Jancovich, J. K., J. Mao, V. G. Chinchar, C. Wyatt, S. T. Case, S.
Kumar, G. Valente, S. Subramanian, E. W. Davidson, J. P. Collins,
and B. L. Jacobs. 2003. Genomic sequence of a ranavirus (family
Iridoviridae) associated with salamander mortalities in North America.
Virology 316:90-103.
25.
Johnson, A. J., A. P. Pessier, and E. R. Jacobson. 2007. Experimental
transmission and induction of ranaviral disease in western ornate box
turtles (Terrapene ornata ornata) and red-eared sliders (Trachemys
scripta elegans). Veterinary Pathology 44:285-297.
26.
Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: Integrated software
for molecular evolutionary genetics analysis and sequence alignment.
Briefings in Bioinformatics 5:150-163.
27.
Lu, L., S. Y. Zhou, C. Chen, S. P. Weng, S. M. Chan, and J. G. He.
2005. Complete genome sequence analysis of an iridovirus isolated from
the orange-spotted grouper, Epinephelus coioides. Virology 339:81-100.
28.
Mao, J. H., R. P. Hedrick, and V. G. Chinchar. 1997. Molecular
characterization, sequence analysis, and taxonomic position of newly
isolated fish iridoviruses. Virology 229:212-220.
107
29.
Marschang, R. E., P. Becher, H. Posthaus, P. Wild, H. J. Thiel, U.
Muller-Doblies, E. F. Kalet, and L. N. Bacciarini. 1999. Isolation and
characterization of an iridovirus from Hermann's tortoises (Testudo
hermanni). Arch Virol 144:1909-22.
30.
Marschang, R. E., S. Braun, and P. Becher. 2005. Isolation of a
Ranavirus from a gecko (Uroplatus fimbriatus). Journal of Zoo and Wildlife
Medicine 36:295-300.
31.
Qin, Q. W., S. F. Chang, G. H. Ngoh-Lim, S. Gibson-Kueh, C. Shi, and
T. J. Lam. 2003. Characterization of a novel ranavirus isolated from
grouper Epinephelus tauvina. Diseases of Aquatic Organisms 53:1-9.
32.
Schaffer, A. A., L. Aravind, T. L. Madden, S. Shavirin, J. L. Spouge, Y.
I. Wolf, E. V. Koonin, and S. F. Altschul. 2001. Improving the accuracy
of PSI-BLAST protein database searches with composition-based
statistics and other refinements. Nucleic Acids Research 29:2994-3005.
33.
Schaffer, A. A., Y. I. Wolf, C. P. Ponting, E. V. Koonin, L. Aravind, and
S. F. Altschul. 1999. IMPALA: matching a protein sequence against a
collection of PSI-BLAST-constructed position-specific score matrices.
Bioinformatics 15:1000-1011.
34.
Schock DM, B. T., Chinchar VG, Jancovich JK, Collins JP. 2007.
Experimental evidence that amphibian ranaviruses are mutli-host
pathogens. Copeia in press.
35.
Song, W. J., Q. W. Qin, J. Qiu, C. H. Huang, F. Wang, and C. L. Hew.
2004. Functional genomics analysis of Singapore grouper iridovirus:
complete sequence determination and proteomic analysis. J Virol
78:12576-90.
36.
Sonnhammer, E. L. L., and R. Durbin. 1995. A Dot-Matrix Program with
Dynamic Threshold Control Suited for Genomic DNA and ProteinSequence Analysis. Gene-Combis 167:1-10.
37.
Sonnhammer, E. L. L., and J. C. Wootton. 2001. Integrated graphical
analysis of protein sequence features predicted from sequence
composition. Proteins-Structure Function and Genetics 45:262-273.
38.
Speare, R., and J. R. Smith. 1992. An Iridovirus-Like Agent Isolated from
the Ornate Burrowing Frog Limnodynastes ornatus in Northern Australia.
Diseases of Aquatic Organisms 14:51-57.
108
39.
Tan, W. G. H., T. J. Barkman, V. G. Chinchar, and K. Essani. 2004.
Comparative genomic analyses of frog virus 3, type species of the genus
Ranavirus (family Iridoviridae). Virology 323:70-84.
40.
Tidona, C. A., and G. Darai. 1997. The complete DNA sequence of
lymphocystis disease virus. Virology 230:207-16.
41.
Tsai, C. T., J. W. Ting, M. H. Wu, M. F. Wu, I. C. Guo, and C. Y. Chang.
2005. Complete genome sequence of the grouper iridovirus and
comparison of genomic organization with those of other iridoviruses. J
Virol 79:2010-23.
42.
Whittington, R. J., C. Kearns, A. D. Hyatt, S. Hengstberger, and T.
Rutzou. 1996. Spread of epizootic haematopoietic necrosis virus (EHNV)
in redfin perch (Perca fluviatilis) in southern Australia. Aust Vet J 73:1124.
43.
Williams, T., V. Barbosa-Solomieu, and V. G. Chinchar. 2005. A
decade of advances in iridovirus research. Adv Virus Res 65:173-248.
44.
Zhang, Q. Y., F. Xiao, Z. Q. Li, J. F. Gui, J. H. Mao, and V. G. Chinchar.
2001. Characterization of an iridovirus from the cultured pig frog Rana
grylio with lethal syndrome. Diseases of Aquatic Organisms 48:27-36.
45.
Zhang, Q. Y., F. Xiao, J. Xie, Z. Q. Li, and J. F. Gui. 2004. Complete
genome sequence of lymphocystis disease virus isolated from China. J
Virol 78:6982-94.
46.
Zupanovic, Z., C. Musso, G. Lopez, C. L. Louriero, A. D. Hyatt, S.
Hengstberger, and A. J. Robinson. 1998. Isolation and characterization
of iridoviruses from the giant toad Bufo marinus in Venezuela. Diseases of
Aquatic Organisms 33:1-9.