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Virology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y v i r o
Virulence correlates with fitness in vivo for two M group genotypes of Infectious
hematopoietic necrosis virus (IHNV)
Andrew R. Wargo a,c,⁎, Kyle A. Garver a,b, Gael Kurath a,c
a
b
c
Western Fisheries Research Center, 6505 NE 65th Street, Seattle, WA 98115-5016, USA
Pacific Biological Station, 3190 Hammond Bay Road, Nanaimo, B.C., Canada V9T 6N7
University of Washington, Seattle, WA, USA
a r t i c l e
i n f o
Article history:
Received 23 February 2010
Returned to author for revision
17 March 2010
Accepted 22 April 2010
Available online xxxx
Keywords:
Virulence
Fitness
Evolution
Ecology
In vivo
Vertebrate
Virus
a b s t r a c t
The nature of the association between viral fitness and virulence remains elusive in vertebrate virus systems,
partly due to a lack of in vivo experiments using statistically sufficient numbers of replicate hosts. We
examined the relationship between virulence and fitness in Infectious hematopoietic necrosis virus (IHNV), in
vivo, in intact living rainbow trout. Trout were infected with a high or low virulence genotype of M
genogroup IHNV, or a mixture of the two genotypes, so as to calculate relative fitness and the effect of a
competition environment on fitness. Fitness was measured as total viral load in the host at time of peak viral
density, quantified by genotype-specific quantitative RT-PCR (qRT-PCR). The more virulent IHNV genotype
reached higher densities in both single and mixed infections. There was no effect of competition on the
performance of either genotype. Our results suggest a positive link between IHNV genotype fitness and
virulence.
© 2010 Elsevier Inc. All rights reserved.
Introduction
Viral fitness, here defined as the relative ability of viruses to
produce infectious progeny in a given environment, plays a critical
role in viral evolution (Domingo and Holland, 1997; Domingo et al.,
1997; Holland et al., 1991). The overall selection of viral strains within
and between hosts is likely to depend heavily on their relative fitness
(Novella et al., 1995; Quinones-Mateu et al., 2000; Troyer et al., 2005).
It is also theorized that an association between fitness and viral traits,
such as virulence, might exist (Alizon et al., 2009; Ebert, 1998; Troyer
et al., 2008). Understanding the nature of these associations could
provide valuable insights into how the traits themselves evolve
(Brown et al., 2006; Bull, 1994; Frank, 1996; Levin, 1996; Read, 1994).
Few in vivo studies have been conducted in vertebrate systems
examining the relationship between viral fitness and in particular, the
virulence trait, and therefore the nature of this association remains
elusive. Here we developed and carried out a methodology to quantify
viral fitness in vivo, in a vertebrate virus system, and shed light on the
association between fitness and virulence. We defined virulence as
mortality caused to the host.
The bulk of research examining the relationship between virulence
⁎ Corresponding author. Western Fisheries Research Center, 6505 NE 65th Street,
Seattle, WA 98115-5016, USA.
E-mail address: [email protected] (A.R. Wargo).
and viral fitness has relied on bacteriophage, plant host systems, or
vertebrate viruses in cell culture. Although crucial for establishing the
foundation of virulence evolution theory, the findings from these
studies often differed from each other. In bacteriophage a positive
association between virulence and viral density was found under
certain modes of transmission (Messenger et al., 1999). Whereas, in
plant viruses, research suggested that virulence was positively
correlated with the infectivity but not the density of barley stripe
mosaic virus (Stewart et al., 2005), and work on cucumber mosaic
virus revealed genotypes with intermediate levels of virulence had
highest transmissibility (Escriu et al., 2003). Furthermore, in vertebrate viruses, in vitro studies on FMDV, poliovirus, and SIV indicated
that in some cases more virulent genotypes had the fitness advantage
and in others they did not, depending on the viral genotype or growth
conditions examined (Chumakov et al., 1991; Herrera et al., 2007;
Voronin et al., 2005).
Ultimately, since the host immune response is likely to play an
important role in virulence evolution (Alizon et al., 2009; Frank and
Schmid-Hempel, 2007), the relevance of in vitro studies to intact
vertebrate systems is unknown. To more closely reflect the vertebrate
host, an ex vivo system was developed for HIV using human peripheral
blood mononuclear cells. This system, in conjunction with epidemiological studies, revealed that viral fitness and transmission potential
are important factors for the success and spread of different HIV
genogroups (Arien et al., 2005) and may be positively correlated with
0042-6822/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
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Please cite this article as: Wargo, A.R., et al., Virulence correlates with fitness in vivo for two M group genotypes of Infectious hematopoietic
necrosis virus (IHNV), Virology (2010), doi:10.1016/j.virol.2010.04.023
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disease progression in the field (Frasher et al., 2007; Quinones-Mateu
et al., 2000; Sagar et al., 2003; Troyer et al., 2005).
Few truly in vivo empirical studies examining the association
between vertebrate viral virulence and fitness, in intact hosts, are
available; likely due to the difficulty of conducting research in such
systems. Of the studies conducted, many were not specifically aimed
at testing the virulence–fitness association and, similar to in vitro
work, produced incongruous results. An examination of foot-andmouth disease in swine and influenza virus in chickens both
suggested more virulent genotypes had a replication advantage
(Carrillo et al., 1998; Suzuki et al., 2009). Alternatively, investigations
of SIV in primates indicated that virulence was not associated with
viral replication (Pandrea et al., 2008) and work on hepatitis B in
ducks alluded that less virulent strains had the fitness advantage
(Lenhoff et al., 1998). A limitation to these in vivo studies was the
small number of infected hosts examined; typically less than 10
animals. Natural viral infections are likely to show high levels of
variation in viral dynamics, and indeed this phenomenon was
observed in the above mentioned, as well as other in vivo studies
(Burke et al., 2006; Munster et al., 2009; Troyer et al., 2008). As such,
conclusions about population level processes are likely to be more
accurate when greater host numbers are analyzed.
Recently, an in vivo fitness assay was developed for Infectious
hematopoietic necrosis virus (IHNV) in intact living rainbow trout
(Oncorhynchus mykiss) hosts. Using this assay it was found that two
genetically distinct IHNV isolates (referred to as genotypes from here
forward) with equal virulence had equal fitness (Troyer et al., 2008).
To more fully elucidate the nature of the virulence relationship in
IHNV, we expanded this previous study by comparing the relative
fitness of two genotypes which differ in virulence, while also
examining the fitness parameter in more detail.
IHNV is a single-stranded, negative-sense RNA rhabdovirus,
endemic in salmonid fishes throughout western North America
(Bootland and Leong, 1999; Kurath et al., 2003). The virus typically
causes acute infection, with an often rapid onset of disease and
mortality (Bootland and Leong, 1999). Hundreds of isolates of the
virus have been obtained from the field and characterized, showing
wide ranging levels of virulence, sometimes reaching close to 100%
mortality of the host fish population (Garver et al., 2006; LaPatra et al.,
1994). Field studies indicate that multiple genotypes of IHNV can cocirculate and that newly emerged genotypes can displace resident
ones, suggesting possible fitness differences between genotypes
(Anderson, 2000; Troyer and Kurath, 2003; Troyer et al., 2000).
Phylogenic analyses of field isolates have revealed three major
genogroups of IHNV in North America, designated U, M, and L (Kurath
et al., 2003). Among them the M genogroup is hypothesized to have
arisen in rainbow trout (Troyer and Kurath, 2003; Troyer et al., 2000).
We examined the fitness of a high and low virulence IHNV genotype, both from the M genogroup, when in the fish host alone
in a single infection environment, and in the presence of the other
genotype in a mixed infection competition environment. This experimental design enabled us to determine relative fitness and the impact
competition had on a genotype's fitness (de Roode et al., 2005). The
comparison of single versus mixed infections was not done in our
previous study (Troyer et al., 2008) and to our knowledge, has not
been explored when examining the virulence–fitness association
in vertebrate virus systems. To improve the quantification of viral
fitness, we developed genotype-specific real-time quantitative PCR
(qPCR) assays, making it possible to measure the absolute viral load of
each genotype in both single and mixed infections, in individual fish.
To fully capture the high levels of anticipated fish-to-fish variation,
23–28 fish per treatment group were utilized, providing robust fitness
estimates.
Our experiments confirmed the virulence difference between the
two M group IHNV genotypes (Garver et al., 2006; Troyer et al., 2000)
and revealed that the more virulent genotype reached higher den-
sities in the within-host virus populations in both single and mixed
infections, suggesting it had a fitness advantage. Both genotypes
performed equally well in single infections as they did in mixed
infections, implying that there was no direct competition for a limited
resource in the fish host. Our results clearly demonstrated a positive
association between virulence and viral fitness as defined here.
Results
Virulence
To demonstrate the relative virulence of viral genotypes HV and
LV, we compared the mortality induced in triplicate batch challenges
of 20 fish infected with genotype HV, to those infected with genotype
LV. The replicate tanks all showed the same characteristic IHNV acute
infection dynamic (Fig. 1). Mortality began between days 5 and 7 for
HV-infected fish and 7 and 9 for LV-infected, continued through day
17, and quickly tapered off, with the last LV-infected fish dying on day
24 and the last HV-infected on day 26. Over the 28 day monitoring
period, fish infected with genotype HV had an average median
survival of 11 ± 1 days, which was significantly shorter than the 23 ±
1 day median survival of fish infected with genotype LV (Fig. 1; Logrank = 40.35, d.f. = 1, P b 0.001). Fish infected with genotype HV also
experienced a significantly higher cumulative percentage of mortality
(CPM, 85 ± 5%) than the fish infected with genotype LV (30.5 ± 3%),
over the monitoring period (Fig. 1; X21 N 25, P b 0.0001). Thus, these
results are consistent with previous observations that HV is the more
virulent of the two IHNV genotypes, as defined by infection-induced
mortality (Garver et al., 2006). When examining only the fish that
died, there was no difference in the mean day to death between HVinfected fish (12 ± 1 days) compared to LV-infected fish (13 ± 1 days),
suggesting that the kinetics of disease progression was approximately
the same for both virus genotypes.
Viral fitness
To determine the relative fitness of genotypes HV and LV, the total
viral load of each genotype was compared in fish infected with either
HV alone, LV alone, or a mixture of the two, in three independent
experiments. This made it possible to determine the relative fitness of
each genotype alone versus in a competition environment and
examine the direct effect of competition on the two genotypes.
Across all three fitness experiments there was a substantial degree of
Fig. 1. Experimental batch immersion challenge defining virulence difference between
IHNV genotypes HV (high virulence, black line) and LV (low virulence, gray line) in
triplicate groups of 20 juvenile rainbow trout. Lines are daily mean cumulative percent
mortality (± 1 S.E.M) over 30 days of infection. Mock exposed fish showed no mortality
(not shown).
Please cite this article as: Wargo, A.R., et al., Virulence correlates with fitness in vivo for two M group genotypes of Infectious hematopoietic
necrosis virus (IHNV), Virology (2010), doi:10.1016/j.virol.2010.04.023
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3
fish-to-fish variation in viral load within treatment groups, such that
viral load ranged well over 4 orders of magnitude between the fish
(Fig. 2, S2, S3). Despite this level of variation, significant differences
were observed between the treatment groups. When comparing
mean viral loads for all fish in an experiment, genotype HV produced
more viral copies than LV in both single and mixed infections, across
all experiments (Fig. 3, genotype effect: F1,233 = 60, P b 0.001). On
average the viral production of genotype HV was 1–2 orders of
magnitude higher than that for genotype LV (Fig. 3). Ultimately, viral
genotype HV had greater fitness than LV to such a degree that overall,
approximately 76% of the total viral population produced in the mixed
infection groups was HV (Fig. 4A), thus, HV maintained its fitness
advantage in both single and mixed infections. Despite the general
dominance of HV, the less virulent genotype was not completely
Fig. 3. Mean viral loads (± 1 S.E.M) of HV (black bars) or LV (gray bars) determined by
qPCR in virus-infected treatment groups from three independent viral fitness
experiments (A–C).
Fig. 2. Viral loads of genotypes HV (black bars) and LV (gray bars) determined by qRTPCR in individual fish from experiment 1, three days after exposure to A) HV alone,
B) LV alone, C) a 1:1 mixture of HV:LV. Analogous data from experiments 2 and 3 can be
found in the Supplemental materials.
excluded and there were 14 out of 81 individual hosts where LV was
the majority genotype in fish receiving a mixed infection (Fig. 4B).
When examining the impact of competition on genotype performance, there was no significant difference in the viral copy output of
either genotype in single versus mixed infections, despite a suggestive
trend of a small decrease during competition for HV in experiments 2
and 3 (Fig. 3, competition effect: F1,232 = 2.3, P = 0.13). Furthermore,
the total virus produced in mixed infections (HV + LV) was not
significantly different than that produced in single infections of HV.
The three replicate experiments produced very similar results,
although total viral production decreased with subsequent experiments such that the most virus was produced in experiment 1 and the
least in experiment 3 (Fig. 3, experiment effect: F2,233 = 18, P b 0.001).
The total drop in viral load between experiment 1 and 3 was less than
Please cite this article as: Wargo, A.R., et al., Virulence correlates with fitness in vivo for two M group genotypes of Infectious hematopoietic
necrosis virus (IHNV), Virology (2010), doi:10.1016/j.virol.2010.04.023
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A.R. Wargo et al. / Virology xxx (2010) xxx–xxx
Fig. 4. IHNV genotype dominance in fish exposed to a mixed infection. (A)The mean percent (± 1 S.E.M) of total virus produced that was HV across all fish receiving a mixed infection
for the three experiments. (B)The number of individual fish where HV (black bars) or LV (gray bars) was dominant in a mixed infection for the three experiments.
an order of magnitude (Fig. 3). In summary, our results consistently
suggest that HV had generally higher fitness than LV.
Discussion
The association between virulence and viral fitness is a heavily
debated topic (Alizon et al., 2009; Brown et al., 2006; Bull, 1994; Ebert,
1998; Levin, 1996; Read, 1994). In vertebrate virus systems, the
ambiguity in this subject is partly due to a lack of convincing data,
particularly from in vivo experiments, with statistically sufficient
numbers of replicate hosts. Here we have shown a positive
relationship between virulence and the fitness of the salmonid virus
IHNV in its natural host, rainbow trout, using in vivo experiments. The
more virulent IHNV genotype (HV) consistently produced greater
numbers of infectious progeny than the less virulent genotype (LV) in
both single and mixed infections in trout.
Typically, experiments examining viral fitness have focused on coinfection to estimate relative fitness differences between genotypes
(reviewed in Troyer et al., 2008). We added a further aspect by
comparing the performance of viral genotypes alone as well as in coinfection. This made it possible to directly assess how the performance of each genotype was influenced by the competition
environment and what impact this had on overall fitness (de Roode
et al., 2005). Interestingly, both genotypes performed equally well in
co-infection as they did alone. These findings suggest that the higher
viral production of HV compared to LV is largely due to differences in
viral replication rather than exploitation competition governed by
limited resources or other similar mechanisms (Brudeseth et al., 2002;
Read and Taylor, 2001). This is despite intentionally observing viral
dynamics on day 3 at the peak of within-host replication, when
resources available to the virus are expected to be most strained
(Troyer et al., 2008). Given these results, one would expect that the
total virus produced in a mixed infection would be slightly greater
than that of HV single infections, due to the added presence of LV.
However, the total virus in mixed infections (HV + LV) was not found
to significantly differ from that produced in single infections of HV.
This was likely due to the very small contribution of genotype LV to
the total virus in mixed infections, combined with the slight, although
non-significant, drop in viral production for genotype HV in mixed
infections in experiments 2 and 3. Thus, there also appeared to be no
facilitation response by either genotype to the presence of the other,
such as increased replication, as postulated in other systems
(Hodgson et al., 2004) and predicted by some theoretical models
(van Baalen and Sabelis, 1995).
We cannot rule out the possibility that a more pronounced
competition dynamic may appear for IHNV later in the infection, after
the host immune response develops, if immune mediated apparent
competition occurs in the IHNV-trout system, such that the immune
response to one genotype has a negative effect on the other (LaPatra
et al., 1994; Read and Taylor, 2001). Fish are known to develop both
innate and adaptive immunity to IHNV, however, previous studies
indicate that viral load dynamics are determined very early in the
infection process, suggesting adaptive immunity may play a minor
role in shaping the observed virulence differences between IHNV
genotypes (Lorenzen and LaPatra, 1999; Peñaranda et al., 2009;
Purcell et al., 2009c).
It is plausible that the competition dynamic in mixed IHNV
genotype infections of trout ultimately depends on whether or not the
genotypes co-infect the same host cell. A recent study of interference
competition in vitro, in foot-and-mouth disease virus (FMDV),
revealed that a less virulent mutant had the advantage in high-MOI
conditions, where the probability of cellular co-infection was high;
whereas the high virulence mutant had the advantage under low MOI
conditions, where the probability of cellular co-infection was low
(Ojosnegros et al., 2010). If cellular co-infection does not occur for
IHNV, this could explain why genotype LV is out competed by HV,
given the findings of Ojosnegros et al. It may be that the IHNV viral
load of in vivo infections never reached a level where cells became
limited, and thus low MOI conditions were maintained such that
competition for cells and the likelihood of cellular co-infection were
rare. In general, IHNV is a relatively low titer virus, reaching titers of
only 102–107 infectious virus particles per gram of tissue during in
vivo infections, but the MOI during in vivo infections is not known.
Some evidence of a block to cellular co-infection in IHNV was
observed in a study that found cells bound with one species of
rhabdovirus had reduced affinity for another species (de las Heras et
Please cite this article as: Wargo, A.R., et al., Virulence correlates with fitness in vivo for two M group genotypes of Infectious hematopoietic
necrosis virus (IHNV), Virology (2010), doi:10.1016/j.virol.2010.04.023
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al., 2008), however, co-infection with different genotypes of the same
virus species was not examined. A further reduction in the probability
of IHNV cellular co-infection could occur if genotypes compartmentalize in different tissues as they spread throughout the host. The
course of IHNV infection begins with viral entry via gills, esophagus, or
skin, followed by systemic infection, with most virus replication
focused in the hematopoietic tissue of the kidney and spleen (Drolet
et al., 1994; Harmache et al., 2006; Helmick et al., 1995). Evidence for
compartmentalization during this process is lacking. Ultimately, the
absence or presence of cellular co-infection is beyond the scope of the
work presented here and does not detract from our observation that
HV consistently produced more viral copies than LV in both single and
mixed infections.
It should be noted that in this study our goal was to use genotypespecific qPCR to quantify the dominant consensus component of the
HV and LV genotypes in single and mixed infection populations. As a
negative-sense RNA virus, IHNV populations exist as quasi-species
mixtures of variants, and it is possible that some mutations in the
qPCR primer or probe sequence sites could have occurred during
these experiments, resulting in a small number of genomes that were
not detected. However, the region chosen for quantification showed
no sequence variation in 3 clones for both genotypes HV and LV (data
not shown), and previous studies indicate that the mutant spectra
within natural quasi-species of IHNV is low for an RNA virus
(Emmenegger et al., 2003). In addition, given that they are singlestranded, negative-sense RNA viruses, recombination is extremely
rare or non-existent for rhabdoviruses. Thus we are confident that our
methods are valid for quantifying the two genotypes.
Given our results, an obvious question remains, why does HV have
higher fitness than LV? The specific factors that provide the more
virulent strain with higher viral fitness in the IHNV system remain
under investigation. The molecular basis of virulence is unknown for
IHNV, but preliminary results suggest that it involves more than one
viral gene (unpublished data). Previous studies comparing the course
of infection for two IHNV genotypes from different phylogenetic
genogroups, indicate that factors such as host entry ability, replication
rate, transcription, and innate immune evasion, could all potentially
regulate fitness differences (Park et al., in press; Peñaranda et al.,
2009; Purcell et al., 2009a). In these previous studies the two IHNV
genotypes were from genogroups M and U, that differ in host
specificity, presumably due to evolutionary adaptation to rainbow
trout and sockeye salmon respectively (Kurath et al., 2003). A key
difference between this previous work, and the present study, is that
the two IHNV genotypes used here are both in the M genogroup, and
thus have the same rainbow trout host specificity (Kurath et al.,
2003). Furthermore, the other studies did not examine co-infection
and were primarily interested in the kinetics of viral infection over
time. Interestingly, in regards to the present study, in 17% of cases the
less virulent strain was dominant in the mixed infections. This
suggests that part of the general mechanism involved in regulating
fitness may involve a significant advantage for the first virus genotype
to establish an infection. In the future, mapping the location of fitness
and virulence determinants on the viral genome could provide further
insight into why genotypes differ in fitness.
The broader question is how the fitness differences observed here
will influence the evolution of viral virulence at the host population
level. Our findings are consistent, although not conclusive, with the
much debated trade-off hypothesis: the cost of shortened infection
duration due to higher virulence comes with the benefit of increased
viral fitness (Alizon et al., 2009). To fully understand the evolution of
virulence on a host population scale, and accurately assess the tradeoff hypothesis, requires data on transmission of individual genotypes,
and ultimately a measurement of lifetime fitness. The present study
does not attempt to provide such information but is rather aimed at
setting the foundation for understanding the nature of the virulence–
viral fitness association. The logical progression from this work would
5
be an examination of IHNV genotype transmission and fitness over the
entire course of infection.
How our conclusions transcend across different host-pathogen
taxa is also unknown. Our results agreed with some studies where a
correlation between fitness and virulence was found (Arien et al.,
2005; Carrillo et al., 1998; Frasher et al., 2007; Messenger et al., 1999;
Sagar et al., 2003; Stewart et al., 2005; Suzuki et al., 2009; Troyer et al.,
2005; Voronin et al., 2005) and differed from some where such a
correlation was not found (Escriu et al., 2003; Herrera et al., 2007;
Lenhoff et al., 1998; Pandrea et al., 2008). We suspect that to some
degree these phenomena may be taxon dependent. Here we
attempted to mimic field conditions by conducting all infections in
vivo, in a naturally co-evolved host-pathogen association. Our work
represents one of the few studies to examine the association of
virulence with replication in a vertebrate virus system, using high
numbers of intact living hosts. Rainbow trout mount well characterized innate, humoral, and cellular immune responses to IHNV
infection (Lorenzen and LaPatra, 1999; Purcell et al., 2009b), an
important factor for shaping the evolution of virulence across all
vertebrates (Alizon et al., 2009; Frank and Schmid-Hempel, 2007). It is
interesting to note that this study revealed a high level of fish-to-fish
variation (Fig. 2, S2, S3), and indeed even a small degree of variability
in the mean virus produced was seen between experiments, possibly
due to differences between the fish lots used. The observation of up to
four orders of magnitude difference in viral load between individual
fish in these experiments is similar to the variation in virus titres
found in the field (Mulcahy et al., 1982), thus indicating that our
experiments mimic natural field infections. Furthermore, this variability exemplifies how powerful host genetic diversity as well as
stochastic processes may be for shaping pathogen fitness, and
reaffirms the importance of using a large, naturally diverse host
population when empirically exploring virulence evolution theory.
Given the large sample sizes in this study, this work offers a solid
foundation to begin developing population level estimates of
virulence evolution for further validation against field data and
experimentation.
Materials and methods
Virus and host
We used two genetically distinct IHNV isolates (genotypes)
labeled HV (high virulence) and LV(low virulence), previously
referred to as 220-90 and WRAC/039-82 respectively, which were
originally obtained from farm cultured rainbow trout in the field and
subsequently characterized for virulence (Garver et al., 2006). Over
the entire viral G gene there is 3.6% (58/1621 nucleotides) sequence
divergence between LV and HV. Both genotypes belong to the M
genogroup of IHNV and thus have evolved with host specificity for
rainbow trout (Kurath et al., 2003). The virus was propagated on EPC
cells (Fijan et al., 1983) to create viral stocks stored at − 80 °C, then
titered by replicate plaque assay (Batts and Winton, 1989; Troyer
et al., 2008) as HV: 2.71 ± 0.067 × 107 pfu/ml and LV: 2.6 ± 0.69 ×
107 pfu/ml (mean ± 1 S.E.M). The viral load of the virus stocks was
determined by qRT-PCR on 8 independent replicates as HV: 0.906 ±
0.0832 × 109 and LV: 1.00 ± 0.221 × 109 viral copies/ml (mean ± 1 S.E.
M). The ratio of viral load (qPCR) to pfu (plaque assay) was therefore
HV: 33.6 ± 3.86 and LV: 43.8 ± 20.1 (mean ± 1 S.E.M). The hosts for all
experiments were 1–3 g research grade juvenile rainbow trout,
Oncorhynchus mykiss (Clear Springs Food, Inc.), maintained in flowthrough, pathogen-free, sand-filtered and UV-irradiated fresh water
at 15 °C as described elsewhere (Troyer et al., 2008). All trout came
from the same source, however the fish for experiment 1 were
obtained at a different time of the year and therefore were a different
lot. All animal procedures were approved under the University of
Washington IACUC protocol 3042-11.
Please cite this article as: Wargo, A.R., et al., Virulence correlates with fitness in vivo for two M group genotypes of Infectious hematopoietic
necrosis virus (IHNV), Virology (2010), doi:10.1016/j.virol.2010.04.023
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Virulence challenges
The relative virulence of IHNV genotypes HV and LV was
determined using standard viral challenges of fish in batch. Triplicate
groups of 20 juvenile (2.5 g) rainbow trout were infected by immersion in water containing virus at a concentration of 2 × 105 pfu virus/
ml H2O of each genotype, or mock infected as a control. The challenge
dose was chosen to be consistent with numerous previous batch
virulence challenge studies of IHNV in juvenile salmonids (Garver
et al., 2006). After a 1 hour exposure to virus in static water the fish
were maintained as separate groups, in water flow-through conditions, and daily mortality was monitored for a period of 28 days as
outlined elsewhere (Garver et al., 2006).
In vivo viral competition
To determine if virulence correlates with viral fitness in IHNV
we quantified the within-host viral load of genotypes HV and LV in
single and mixed infections, as a proxy for genotype fitness. Juvenile
rainbow trout were infected by batch immersion in water with
1 × 104 pfu virus/ml H2O of each genotype alone, a mixed infection of
1 × 104 pfu virus/ml H2O of each genotype at a 1:1 ratio (2 × 104 pfu/
ml total virus), or mock infected (no virus), in group sizes of 23–28
fish per viral treatment as outlined in Table 1. We used twice as much
virus in the mixed infection group so we could compare the
performance of a viral genotype alone verses in competition at the
same input dose. Previous studies indicated that this doubling of virus
has no consequence for the viral dynamics (Peñaranda and Kurath,
unpublished). The fish were exposed to virus for 12 h in static water
conditions and then washed in flowing water for one hour and
separated into individual beakers to prevent cross-infection (Troyer
et al., 2008). The viral dosage for fitness challenges was chosen as the
minimum dose at which all fish become infected with either viral
genotype when held in isolation as previously described (Troyer et al.,
2008).
To assess genotype fitness in the fitness challenges, fish were
euthanized and the viral load of each viral genotype in individual fish
was quantified using genotype-specific quantitative reverse transcription PCR (qRT-PCR), after a 3 day period of in vivo growth and
competition in static water. Day three was chosen as the point of fish
harvest because it is known from viral growth kinetics studies to be
when viral load reaches peak levels (Peñaranda et al., 2009; Troyer
et al., 2008).
RNA extraction and cDNA synthesis
Viral RNA was extracted from whole fish as previously outlined
(Troyer et al., 2008) with the following modifications. Denaturing
solution (D.S. — 4 M guanidinium thiocynate, 25 mM sodium citrate
pH7, 0.1 M 2-mercaptoethanol, 0.5% N-lauroylsarcosine) was added
to each fish in individual whirl-packs™ at a ratio of 4 ml D.S. per gram
Table 1
Treatment groups for the 3 experiments of the study.
Treatment group
(genotype, dose)
LV, 1 × 104 pfu/ml
HV, 1 × 104 pfu/ml
LV:HV mixed, 2 × 104 pfu/ml
Mock control (no virus)b
Number of fish analyzeda
Exp. 1
Exp. 2
Exp. 3
27
28
26
6
27
23
27
4
27
26
28
4
Genotype-specific
qRT-PCR analysis
LV assay
HV assay
LV and HV assays
LV and HV assays
a
Excludes fish that died during the experiment. Mortalities were: experiment 1:1
(mix); experiment 2:0; experiment 3:1(HV).
b
Three of the fourteen mock samples were positive for HV by qPCR and two for LV, at
virus concentrations an order of magnitude or greater below fish exposed to virus. This
level of possible contamination, which likely occurred during sample processing, did
not significantly affect the results.
of fish (1/5 dilution). The fish were homogenized in a Seward
Stomacher® 80 (Biomaster) at high speed for 2 × 60 s, or until completely liquefied, at room temperature. A 1 ml aliquot of the fish homogenate was then transferred to a 15 ml tube on ice for subsequent
phenol-chloroform extraction as outlined elsewhere (Troyer et al.,
2008) (Supplemental materials). To assess RNA extraction and concentration, spectrophotometry readings were taken of each sample.
The RNA extraction method has proven to be highly repeatable with
no significant difference in viral load quantified by qPCR of multiple
aliquots of a sample extracted independently (data not shown).
Extracted RNA was subsequently converted to cDNA by combining
0.125 µg oligo-dT, 0.125 µg random hexamers (Promega), 2 µl–3 µl
RNA sample (∼1–2 µg total RNA), and enough H2O to bring the final
volume to 13 µl. The mix was incubated at 70 °C for 5 min, held on ice
and the volume brought to 20 µl with: 1 mM dNTPs, 4 µl 5× M-MLV
reaction buffer, 100 U MMLV RT, 10 U RNAsin, and H2O (Promega).
The sample was then incubated at 42 °C for 1 h, 70 °C for 15 min, and
held at 4 °C until storage at −80 °C.
Quantitative PCR
The genotype-specific qPCR assays utilized genotype differentiating forward primers (HV: 5′-CCC GAT GCC AAT GGT ACA CT-3′; LV:
5′-CCG ATG CCA ATG GTA CAC C-3′), MGB TaqMan probes (HV: 5′MGB-VIC-CCC CAA AGA GTG TTC TGA-NFQ-3′; LV: 5′-MBG-6′FAMCAA CAC AAA AGA GTG TTC TTA-NFQ-3′; Applied Biosystems), and
reverse primers (HV: 5′-GTG CAT TTT CCT CCA ATA AAG TCC-3′, LV:
5′-TTG GTA CAT TTT CCT CCA ATA AAA TCT-3′). Reliable quantification
was obtained for HV and LV in artificial mixtures spanning ratios of
10,000:1–1:1–1:10,000 HV:LV (HV: Log (observed quantity) = Log
(expected quantity) ⁎ (1.005 ± 0.01) − 0.03 ± 0.06, F1,29 = 8400, P b
0.001, R2 = 0.99; LV: Log(observed quantity) = Log(expected quantity) ⁎ (1.002 ± 0.01) + 0.01 ± 0.05, F1,32 = 9700, P b 0.001, R2 = 0.99)
with no detection of non-target at 1 × 107 copies and target minimum
detection limits of 10 copies/reaction (104 viral copies/g fish). All
qPCR reactions were at a total volume of 12 µl, containing 900 nM
each primer, 200 nM probe, 6 µl 2× TaqMan Universal PCR Master Mix
(Applied Biosystems), 0.328 µl H2O, and 5 µl of a 1/10 dilution of
sample cDNA, and run on a 7900HT ABI Prism machine. Cycling conditions were 50 °C for 2 min, 95 °C for 10 min, then 40 cycles of 95 °C
for 15 s and 60 °C for 1 min.
Given the robust nature of the qPCR methodology to detect all viral
RNA, it quantified total viral genetic material, including both genome
copies and mRNA, and thus indicates “viral load” rather than numbers
of virus particles. A consistent relationship between viable virus
quantities found in plaque assays and qPCR viral load has previously
been observed for IHNV in rainbow trout (Purcell et al., 2006) and
qPCR viral load has regularly been used to quantify viral replication
(Peñaranda et al., 2009; Purcell et al., 2009c).
Transcript standard development
RNA transcripts of the full viral glycoprotein (G) gene (target of
qPCR) were developed for HV and LV and used as qRT-PCR standards
at known concentrations. The development of the LV-G plasmid
(pWracG) has previously been described (Corbeil et al., 2000), pHV-G
was developed as outlined here. As a template for the pHV-G plasmid,
HV viral RNA was extracted from a 400 µl volume of virus in cell free
media with 500 µl of Tri® Reagent (Sigma-Aldrich) plus 100 µl EPC
cells as a carrier according to the manufacturer's protocol (supplemental materials). The viral RNA was converted to cDNA using
SuperScript II™ reverse transcriptase (Invitrogen) as suggested by the
manufacturer, with 1.6 µg RNA and 2 pmol of a G gene specific primer
(5′-GGA CTA GTA TGG ACA CCA CGA TCA CCA CTC CG-3′). The cDNA
was then PCR amplified to produce the full 1.6 kb G gene product
using Taq DNA polymerase (Promega) in a 100 µl reaction with: 10 µl
Please cite this article as: Wargo, A.R., et al., Virulence correlates with fitness in vivo for two M group genotypes of Infectious hematopoietic
necrosis virus (IHNV), Virology (2010), doi:10.1016/j.virol.2010.04.023
ARTICLE IN PRESS
A.R. Wargo et al. / Virology xxx (2010) xxx–xxx
10× PCR buffer, 0.2 mM DNTPs, 2.5 mM MgCl2, 25 U Taq, 7.5 mg RNase
A (Promega), 5 µl cDNA, 1 pmol primers (forward: 5′-GGA CTA GTA
TGG ACA CCA CGA TCA CCA CTC CG-3′; reverse: 5′-GGC CCG GGT TAG
GAC CGG TTT GCC ACG TGA T-3′). Cycling conditions were 94 °C
2 min, 30 cycles: 94 °C 30 s, 50 °C 30 s, 72 °C 2 min; followed by 72 °C
10 min, 4 °C hold.
The HV PCR product was gel purified using the QIAquick® gel
extraction kit according to the manufacturer's protocol (Qiagen) and
then inserted into pSTBlue-1 vector using the AccepTor® vector kit as
outlined by the manufacturer (Novagen). Appropriate colonies were
sequenced to ensure full G gene insertion and a stock of plasmid pHVG was prepared using the EndoFree® Plasmid Maxi kit (Qiagen).
To obtain negative-sense transcript RNA, 20 µg of plasmids pHV-G
and pLV-G were digested with HindIII and BamHI respectively and RNA
transcripts were synthesized with T7 RNA polymerase (Promega).
Residual DNA was digested with RQ1 DNase and DNA contamination
was subsequently found to be less than 0.1%. The concentrations and
gene copy numbers of transcript standards rHV-G and rLV-G (length
1626 bp and 1745 bp respectively) were determined by spectrophotometry as outlined elsewhere (Purcell et al., 2006). We also used a
general non-genotype-specific IHNV qPCR reaction to further calibrate
the standards against each other (Purcell et al., 2006). Every set of cDNA
reactions included an RNA transcript standard (rHV or rLV) which was
utilized for qPCR in a 10 fold cDNA dilution series with G gene copies
ranging from 5 × 108 to 1 × 101 (standard curves: HV: R2 = 0.997 ±
0.001, slope = −3.48 ± 0.04, intercept = 45.36 ± 0.31; LV: R2 = 0.998 ±
0.001, slope= -3.49±0.03, intercept =44.49 ± 0.13).
Statistics
Virulence data was tested with a Cox-test (SPSS), revealing that
replicate tank was not a significant predictor, which was thus dropped
from all further analyses (P N 0.10). Kaplan–Meier and log-rank tests
(SPSS) were conducted to determine differences in survivorship. Data
divided or pooled by tank showed the same result. Mean day to death
and percent mortality were analyzed using Chi-square tests in excel.
Analysis of genotype viral production was carried out in SPSS using
general linear models (GLM) starting with the maximum model (viral
load = viral genotype (HV or LV) + competition (alone versus mixed) +
experiment (I, II, III) + viral genotype ⁎ competition + viral genotype⁎ experiment+ competition ⁎ experiment+ viral genotype⁎ competition ⁎ experiment) and non-significant terms were dropped until the
minimal model was reached (supplemental materials). Tukey's posthoc test was utilized for multiple comparisons within groups. To avoid
violating independence assumptions, for statistical analysis we randomly assigned half the mixed infection fish to the HV group and the
other half to the LV group. The same results were obtained when
analyzing all fish (Tables S1–S6, Fig. S1) and therefore these data are
shown in the figures. All data were log transformed to meet the test
assumptions and dead fish were excluded from the GLM viral production analysis because exact time of death could not be determined
and may influence viral replication (Table 1).
Acknowledgments
We thank B. Batts and J. Ranson for technical assistance and J.
Winton and B. Kerr for valuable discussions, as well as two
anonymous reviewers for thoughtful comments. We are grateful to
S.E. LaPatra of Clear Springs Foods, Inc., for providing research grade
rainbow trout. This work was supported by the USGS Western
Fisheries Research Center, and funding for A. Wargo was provided by
the National Institute of Health R.L. Kirschestein NRSA University of
Washington institutional service award 5 T32 AI007509 and National
Science Foundation Ecology of Infectious Diseases grant 0812603. The
funders had no role in study design, data collection and analysis,
7
decision to publish, or preparation of the manuscript. Mention of
trade names does not imply U.S. Government endorsement.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.virol.2010.04.023.
References
Alizon, S., Hurford, A., Mideo, N., Van Baalen, M., 2009. Virulence evolution and the
trade-off hypothesis: history, current state of affairs and the future. J. Evol. Biol. 22
(2), 245–259.
Anderson, E.D., 2000. Molecular epidemiology reveals emergence of a virulent
infectious hematopoietic necrosis (IHN) virus strain in wild salmon and its
transmission to hatchery fish. J. Aquat. Anim. Health 12 (2), 85–99.
Arien, K.K., Abraha, A., Quinones-Mateu, M.E., Kestens, L., Vanham, G., Arts, E.J., 2005.
The replicative fitness of primary human immunodeficiency virus type 1 (HIV-1)
group M, HIV-1 group O, and HIV-2 isolates. J. Virol. 79 (14), 8979–8990.
Batts, W.N., Winton, J.R., 1989. Enhanced detection of infectious hematopoietic necrosis
virus and other fish viruses by pretreatment of cell monolayers with polyethylene
glycol. J. Aquat. Anim. Health 1 (4), 284–290.
Bootland, L.M., Leong, J.C., 1999. Infectious hematopoietic necrosis virus. In: Woo, P.T.K.,
Bruno, D.W. (Eds.), Fish diseases and disorders: CAB International, Vol. 3, pp. 57–112.
Brown, N.F., Wickham, M.E., Coombes, B.K., Finlay, B.B., 2006. Crossing the line:
Selection and evolution of virulence traits. PLoS Pathog. 2 (5), e42.
Brudeseth, B.E., Castric, J., Evensen, O., 2002. Studies on pathogenesis following single
and double infection with viral hemorrhagic septicemia virus and infectious
hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss). Vet. Pathol.
39 (2), 180–189.
Bull, J.J., 1994. Perspective: Virulence. Evolution 48 (5), 1423–1437.
Burke, B., Derby, N.R., Kraft, Z., Saunders, C.J., Dai, C., Llewellyn, N., Zharkikh, I., Vojtech,
L., Zhu, T., Srivastava, I.K., Barnett, S.W., Stamatatos, L., 2006. Viral evolution in
macaques coinfected with CCR5- and CXCR4-tropic SHIVs in the presence or
absence of vaccine-elicited anti-CCR5 SHIV neutralizing antibodies. Virology 355
(2), 138–151.
Carrillo, C., Borca, M., Moore, D.M., Morgan, D.O., Sobrino, F., 1998. In vivo analysis of the
stability and fitness of variants recovered from foot-and-mouth disease virus
quasispecies. J. Gen. Virol. 79 (7), 1699–1706.
Chumakov, K.M., Powers, L.B., Noonan, K.E., Roninson, I.B., Levenbook, I.S., 1991.
Correlation between amount of virus with altered nucleotide sequence and the
monkey test for acceptability of oral poliovirus vaccine. Proc. Natl. Acad. Sci. U. S. A.
88 (1), 199–203.
Corbeil, S., LaPatra, S.E., Anderson, E.D., Kurath, G., 2000. Nanogram quantities of a DNA
vaccine protect rainbow trout fry against heterologous strains of infectious
hematopoietic necrosis virus. Vaccine 18 (25), 2817–2824.
de las Heras, A.I., Saint-Jean, S.R., Perez-Prieto, S.I., 2008. Salmonid fish viruses and cell
interactions at early steps of the infective cycle. J. Fish Dis. 31 (7), 535–546.
de Roode, J.C., Pansini, R., Cheesman, S.J., Helinski, M.E.H., Huijben, S., Wargo, A.R., Bell,
A.S., Chan, B.H.K., Walliker, D., Read, A.F., 2005. Virulence and competitive ability
in genetically diverse malaria infections. Proc. Natl. Acad. Sci. U. S. A. 102 (21),
7624–7628.
Domingo, E., Holland, J.J., 1997. RNA virus mutations and fitness for survival. Ann. Rev.
Microbiol. 51, 151–178.
Domingo, E., Menéndez-Arias, L., Holland, J.J., 1997. RNA virus fitness. Rev. Med. Virol. 7
(2), 87–96.
Drolet, B.S., Rohovec, J.S., Leong, J.C., 1994. The route of entry and progression of
Infectious Hematopoietic Necrosis Virus in Oncoryhynchus-mykiss (Walbaum)- A
sequential immunohistochemical study. J. Fish Dis. 17 (4), 337–347.
Ebert, D., 1998. Evolution - Experimental evolution of parasites. Science 282 (5393),
1432–1435.
Emmenegger, E.J., Troyer, R.M., Kurath, G., 2003. Characterization of the mutant spectra
of a fish RNA virus within individual hosts during natural infections. Virus Res. 96,
15–25.
Escriu, F., Fraile, A., Garcia-Arenal, F., 2003. The evolution of virulence in a plant virus.
Evolution 57 (4), 755–765.
Fijan, N., Sulimanovic, E., Bearzotti, M., Muzinic, D., Zwillenberg, L.O., Chilmonczyk, S.,
Vautherot, J.F., de Kinkelin, P., 1983. Some properties of the epithelioma papulosum
cyprini (EPC) cell line from carp Cyprinus carpi. Annales de l'Institut Pasteur
Virologie 134 (2), 207–220.
Frank, S.A., 1996. Models of parasite virulence. Q. Rev. Biol. 71 (1), 37–78.
Frank, S.A., Schmid-Hempel, P., 2007. Mechanisms of pathogensis and the evolution of
parasite virulence. J. Evol. Biol. 21 (2), 396–404.
Frasher, C., Hollingsworth, T.D., Chapman, R., de Wolf, F., Hanage, W.P., 2007. Variation
in HIV-1 set-point viral load: Epidemiological analysis and an evolutionary
hypothesis. Proc. Natl. Acad. Sci. U. S. A. 104 (44), 17441–17446.
Garver, K., Batts, W., Kurath, G., 2006. Virulence comparisons of infectious hematopoietic necrosis virus U and M genogroups in sockeye salmon and rainbow trout. J.
Aquat. Anim. Health 18 (4), 232–243.
Harmache, A., LeBerre, M., Droineau, S., Giovannini, M., Bremont, M., 2006.
Bioluminescence imaging of live infected salmonids reveals that the fin bases are
the major portal of entry for Novirhabdovirus. J. Virol. 80 (7), 3655–3659.
Please cite this article as: Wargo, A.R., et al., Virulence correlates with fitness in vivo for two M group genotypes of Infectious hematopoietic
necrosis virus (IHNV), Virology (2010), doi:10.1016/j.virol.2010.04.023
ARTICLE IN PRESS
8
A.R. Wargo et al. / Virology xxx (2010) xxx–xxx
Helmick, C.M., Bailey, J.F., LaPatra, S., Ristow, S., 1995. The esophagus/cardiac stomach
region: Site of attachment and internalization of infectious hematopoietic necrosis
virus in challenged juvenile rainbow trout Oncorhynchus mykiss and coho salmon
O-kisutch. Dis. Aquat. Org. 23 (3), 189–199.
Herrera, M., Garcia-Arriaza, J., Pariente, N., Escarmis, C., Domingo, E., 2007. Molecular
basis for a lack of correlation between viral fitness and cell killing capacity. PLoS
Pathog. 3 (4), 498–507.
Hodgson, D.J., Hitchman, R.B., Vanbergen, A.J., Hails, R.S., Possee, R.D., Cory, J.S., 2004.
Host ecology determines the relative fitness of virus genotypes in mixed-genotype
nucleopolyhedrovirus infections. J. Evol. Biol. 17 (5), 1018–1025.
Holland, J.J., Delatorre, J.C., Clarke, D.K., Duarte, E., 1991. Quantitation of relative fitness and
great adaptability of clonal populations of RNA viruses. J. Virol. 65 (6), 2960–2967.
Kurath, G., Garver, K.A., Troyer, R.M., Emmenegger, E.J., Einer-Jensen, K., Anderson, E.D.,
2003. Phylogeography of infectious haematopoietic necrosis virus in North
America. J. Gen. Virol. 84 (4), 803–814.
LaPatra, S.E., Lauda, K.A., Jones, G.R., 1994. Antigenic variants of infectious hematopoietic necrosis virus and implications for vaccine development. Dis. Aquat. Org. 20
(2), 119–126.
Lenhoff, R.J., Luscombe, C.A., Summers, J., 1998. Competition in vivo between a
cytopathic variant and a wild-type duck hepatitis B virus. Virology 251 (1), 85–95.
Levin, B.R., 1996. The evolution and maintenance of virulence in microparasites.
Emerging Infect. Dis. 2 (2), 93–102.
Lorenzen, N., LaPatra, S.E., 1999. Immunity to rhabdoviruses in rainbow trout: the
antibody response. Fish Shellfish Immunol. 9 (4), 345–360.
Messenger, S.L., Molineux, I.J., Bull, J.J., 1999. Virulence evolution in a virus obeys a
trade-off. Proc. R. Soc. London Ser. B. 266 (1417), 397–404.
Mulcahy, D., Burke, J., Pascho, R., Jenes, C.K., 1982. Pathogenesis of infectious
hematopoietic necrosis virus in adult sokeye salmon (Oncorhynchus nerka). Can.
J. Fish. Aquat. Sci. 39 (8), 1144–1149.
Munster, V.J., de Wit, E., van den Brand, J.M.A., Herfst, S., Schrauwen, E.J.A., Bestebroer,
T.M., van de Vijver, D., Boucher, C.A., Koopmans, M., Rimmelzwaan, G.F., Kuiken, T.,
Osterhaus, A., Fouchier, R.A.M., 2009. Pathogenesis and transmission of swineorigin 2009 A(H1N1) influenza virus in ferrets. Science 325 (5939), 481–483.
Novella, I.S., Duarte, E.A., Elena, S.F., Moya, A., Domingo, E., Holland, J.J., 1995.
Exponential increases of RNA virus fitness during large population transmissions.
Proc. Natl. Acad. Sci. U. S. A. 92 (13), 5841–5844.
Ojosnegros, S., Beerenwinkel, N., Antal, T., Nowak, M.A., Escarmis, C., Domingo, E., 2010.
Competition-colonization dynamics in an RNA virus. Proc. Natl. Acad. Sci. U. S. A.
107 (5), 2108–2112.
Pandrea, L., Sodora, D.L., Silvestri, G., Apetrei, C., 2008. Into the wild: simian immunodeficiency virus (SIV) infection in natural hosts. Trends Immunol. 29 (9), 419–428.
Park, J.W., Moon, C.H., Wargo, A.R., Purcell, M.K., and Kurath, G., in press. Differential
growth of U and M type Infectious hematopoietic necrosis virus in a rainbow trout
derived cell line, RTG-2. J. Fish Dis. doi:10.1111/j.1365-2761.2010.01153.x.
Peñaranda, M.M.D., Purcell, M.K., Kurath, G., 2009. Differential virulence mechanisms of
infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus
mykiss) include host entry and virus replication kinetics. J. Gen. Virol. 90 (9),
2172–2182.
Purcell, M.K., Hart, S.A., Kurath, G., Winton, J.R., 2006. Strand-specific, real-time RT-PCR
assays for quantification of genomic and positive-sense RNAs of the fish
rhabdovirus, infectious hematopoietic necrosis virus. J. Virol. Methods 132 (1–2),
18–24.
Purcell, M.K., Garver, K.A., Conway, C., Elliott, D.G., Kurath, G., 2009a. Infectious
haematopoietic necrosis virus genogroup-specific virulence mechanisms in
sockeye salmon, Oncorhynchus nerka (Walbaum), from Redfish Lake, Idaho.
J. Fish Dis. 32 (7), 619–631.
Purcell, M.K., Laing, K., Woodson, J., Thorgaard, G., Hansen, J., 2009b. Characterization of
the interferon genes in homozygous rainbow trout reveals two novel genes,
alternate splicing and differential regulation of duplicated genes. Fish Shellfish
Immunol. 26 (2), 293–304.
Purcell, M.K., Lapatra, S.E., Woodson, J.C., Kurath, G., Winton, J.R., 2009c. Early viral
replication and induced or constitutive immunity in rainbow trout families with
differential resistance to Infectious hematopoietic necrosis virus (IHNV). Fish
Shellfish Immunol. 28 (1), 98–105.
Quinones-Mateu, M.E., Ball, S.C., Marozsan, A.J., Torre, V.S., Albright, J.L., Vanham, G., van
der Groen, G., Colebunders, R.L., Arts, E.J., 2000. A dual infection/competition assay
shows a correlation between ex vivo human immunodeficiency virus type 1 fitness
and disease progression. J. Virol. 74 (19), 9222–9233.
Read, A.F., 1994. The evolution of virulence. Trends Microbiol. 2 (3), 73–76.
Read, A.F., Taylor, L.H., 2001. The ecology of genetically diverse infections. Science 292
(5519), 1099–1102.
Sagar, M., Lavreys, L., Baetan, J.M., Richardson, B.A., Mandaliya, K., Chohan, B.H., Kreiss, J.
K., Overbaugh, J., 2003. Infection with multiple human immunodeficiency virus
type 1 variant is associated with faster disease progression. J. Virol. 77 (23),
12921–12926.
Stewart, A., Logsdon, J.J., Kelly, S., 2005. An empirical study of the evolution of virulence
under both horizontal and vertical transmission. Evolution 59 (4), 730–739.
Suzuki, K., Okada, H., Itoh, T., Tada, T., Mase, M., Nakamura, K., Kubo, M., Tsukamoto, K.,
2009. Association of increased pathogenicity of Asian H5N1 highly pathogenic
avian influenza viruses in chickens with highly efficient viral replication
accompanied by early destruction of innate immune responses. J. Virol. 83 (15),
7475–7486.
Troyer, R.M., Kurath, G., 2003. Molecular epidemiology of infectious hematopoietic
necrosis virus reveals complex virus traffic and evolution within southern Idaho
aquaculture. Dis. Aquat. Org. 55 (3), 175–185.
Troyer, R.M., LaPatra, S.E., Kurath, G., 2000. Genetic analyses reveal unusually high
diversity of infectious haematopoietic necrosis virus in rainbow trout aquaculture.
J. Gen. Virol. 81 (12), 2823–2832.
Troyer, R.M., Collins, K.R., Abraha, A., Fraundorf, E., Moore, D.M., Krizan, R.W., Toossi, Z.,
Colebunders, R.L., Jensen, M.A., Mullins, J.I., Vanham, G., Arts, E.J., 2005. Changes in
human immunodeficiency virus type 1 fitness and genetic diversity during disease
progression. J. Virol. 79 (14), 9006–9018.
Troyer, R.M., Garver, K.A., Ranson, J.C., Wargo, A.R., Kurath, G., 2008. In vivo virus
growth competition assays demonstrate equal fitness of fish rhabdovirus strains
that co-circulate in aquaculture. Virus Res. 137 (2), 179–188.
van Baalen, M., Sabelis, M.W., 1995. The dynamics of multiple infection and the
evolution of virulence. Am. Nat. 146 (6), 881–910.
Voronin, Y., Overbaugh, J., Emerman, M., 2005. Simian immunodeficiency virus variants
that differ in pathogenicity differ in fitness under rapid cell turnover conditions.
J. Virol. 79 (24), 15091–15098.
Please cite this article as: Wargo, A.R., et al., Virulence correlates with fitness in vivo for two M group genotypes of Infectious hematopoietic
necrosis virus (IHNV), Virology (2010), doi:10.1016/j.virol.2010.04.023