1. health and fitness
2. disease

Viral Diseases in Zebrafish: What Is Known and Unknown
Marcus J. Crim and Lela K. Riley
Abstract
Key Words: Danio rerio; infectious disease; ornamental
fish; pathogen-free; pet trade; virus; zebrafish
Introduction
A
nimal models for biomedical research “evolve” over
time as species initially obtained from a nonlaboratory source, such as agriculture, wildlife, or the pet
trade, are adapted to the laboratory, experimental tools and
methodologies are developed, improvements are made in
Marcus J. Crim, MBA, DVM, is a research fellow in the Comparative
Medicine Program at the University of Missouri, Columbia. Lela K.
Riley, PhD, is the Director and General Manager of IDEXX-RADIL in
Columbia, MO.
Address correspondence and reprint requests to Dr. Marcus J. Crim,
Comparative Medicine Program, University of Missouri, 4011 Discovery
Drive, Columbia, MO 65201 or email [email protected].
Volume 53, Number 2
2012
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Naturally occurring viral infections have the potential to
introduce confounding variability that leads to invalid and
misinterpreted data. Whereas the viral diseases of research
rodents are well characterized and closely monitored, no
naturally occurring viral infections have been characterized
for the laboratory zebrafish (Danio rerio), an increasingly
important biomedical research model. Despite the ignorance
about naturally occurring zebrafish viruses, zebrafish models are rapidly expanding in areas of biomedical research
where the confounding effects of unknown infectious agents
present a serious concern. In addition, many zebrafish research
colonies remain linked to the ornamental (pet) zebrafish
trade, which can contribute to the introduction of new pathogens into research colonies, whereas mice used for research
are purpose bred, with no introduction of new mice from the
pet industry. Identification, characterization, and monitoring
of naturally occurring viruses in zebrafish are crucial to the
improvement of zebrafish health, the reduction of unwanted
variability, and the continued development of the zebrafish
as a model organism. This article addresses the importance of
identifying and characterizing the viral diseases of zebrafish
as the scope of zebrafish models expands into new research
areas and also briefly addresses zebrafish susceptibility to
experimental viral infection and the utility of the zebrafish as
an infection and immunology model.
husbandry and biosecurity, and, finally, laboratory breeding colonies are developed to maintain pathogen-free,
purpose-bred research animals. For example, the bestdeveloped and most widely used species in animal-based
laboratory research is the mouse, Mus musculus (Wade and
Daly 2005). The development of the mouse as a laboratory
model began in the 1910s and 1920s as American mouse
fanciers provided a ready source of multiple lineages to
researchers searching for a genetic basis for cancer (Wade
and Daly 2005; Wade et al. 2002). As mouse-based research expanded into other health-related fields, outbreaks
of infectious disease confounded some research studies.
To counter the deleterious effects of disease outbreaks,
research investigations were initiated to identify mouse
pathogens. Since that time, a large number of naturally
occurring viruses and other pathogens of mice have been
described, and discoveries about their transmission and
pathogenesis have contributed to the understanding of infectious disease. Understanding the transmission and pathogenesis of murine diseases facilitated the elimination of many
pathogens from research mice, directly improving animal
health as well as the utility of the mouse model. Husbandry,
biosecurity, and health monitoring have since continued to
improve in the rodent world, reducing variability and the
number of animals necessary for individual experiments.
It is well documented and widely accepted by the biomedical research community that naturally occurring viral
infections can introduce significant confounding variability,
which ultimately results in invalid and misinterpreted data as
well as increased animal numbers. Laboratory mice are
housed in vivariums with conditions that are carefully controlled and limit pathogen transmission. Mice used for research are also purpose bred, with no introduction of new
mice from the pet industry. In contrast, the development of the
zebrafish (Danio rerio) model for many types of biomedical
research is in its infancy. Standard controls used to prevent
pathogen introduction and transmission among rodents—such
as approved vendor lists, pathogen exclusion lists, and health
monitoring programs—are not widely practiced in the management of zebrafish colonies (Lawrence et al. 2012). The viral
infections of laboratory rodents have been extensively studied,
yet virtually nothing is known about naturally occurring viruses of laboratory zebrafish. The growing importance and
expansion of zebrafish models in biomedical research necessitate improvements in the standards of husbandry and biosecurity in laboratory zebrafish colonies. Improved biosecurity
of zebrafish colonies requires a better understanding of natural
Table 1 Viruses described in teleost fishes
Viruses
Orthomyxoviridae
Paramyxoviridae
Picornaviridae
Reoviridae
Retroviridae
Rhabdoviridae
Benko et al. 2002; Harrach and Benko 2007
Lorincz et al. 2011
Choi et al. 2004; Graham et al. 2004; Jeffery et al. 2007; Shlapobersky et al. 2010
Go et al. 2006; Kitamura et al. 2006; Tsai et al. 2005; Weber et al. 2009; Whittington et al. 2010
Essbauer and Ahne 2001
Nylund et al. 2008b
Zhao et al. 2008
Smith et al. 1980a,b
Miyazaki et al. 2000; Schutze et al. 2006
Batts et al. 2011
Bigarre et al. 2009; Gomez et al. 2008; Hegde et al. 2003; Montes et al. 2010; Moody et al.
2009; Nylund et al. 2008a
Falk et al. 1997; Mjaaland et al. 1997
Falk et al. 2008
Berthiaume et al. 1982; Robin and Dery 1982; Robin and Lariviere-Durand 1983
Goodwin et al. 2010; LaPatra et al. 1995; Mohd Jaafar et al. 2008
Francis-Floyd et al. 1993
Gagne et al. 2007; Tao et al. 2008; Teng et al. 2007
occurring zebrafish pathogens, including viruses, their diagnosis, and how they are transmitted.
Information about the confounding effects of naturally
occurring pathogens in zebrafish used for biomedical research lags far behind the information available for both
aquaculture fishes and mammalian laboratory species. In
fact, few naturally occurring zebrafish pathogens of any
kind have been well characterized. Most of the pathogens
that are known represent bacterial, fungal, or parasitic agents
that were previously identified in commercially important
fish species and later recognized in zebrafish. The lack of
information about naturally occurring viral infections in
zebrafish reflects a lack of investigation in this area rather
than an inability of viral pathogens to infect zebrafish (Kent
et al. 2009), as evidenced by studies documenting the experimental infection of zebrafish with viruses isolated from
other fish species (LaPatra et al. 2000; Lopez-Munoz et al.
2010; Lu et al. 2008; Ludwig et al. 2011; Novoa et al. 2006;
Phelan et al. 2005b; Sanders et al. 2003; Seeley et al. 1977;
Xu et al. 2008) and the presence of multiple endogenous retroviruses, retrotransposons, and other retroid agents in the
zebrafish genome (Basta et al. 2007; Shen and Steiner 2004).
Many aquatic viruses are not host-specific, possibly reflecting evolutionary access to a wider range of potential
hosts when virions are distributed in the water column. For
example, viral hemorrhagic septicemia virus (VHSV1) infects
1Abbreviations that appear ≥3x throughout this article: IPNV, infectious
pancreatic necrosis virus; IHNV, infectious hematopoietic necrosis virus;
SHRV, snakehead rhabdovirus; VHSV, viral hemorrhagic septicemia virus
136
dozens of species of both marine and freshwater fish, and
some aquatic viruses can naturally infect both fishes and amphibians or both fishes and aquatic invertebrates. Because
many aquatic viruses can infect multiple species, zebrafish
are likely to be susceptible to viral pathogens that have already been identified in other fish species. The number and
the diversity of viruses that have been described in other
teleost fishes are quite large (Table 1). Research efforts to
identify naturally occurring viruses in zebrafish should
therefore include a search for novel viruses and a search for
viruses previously isolated from other teleost fishes, especially tropical cyprinid species.
This article addresses the importance of identifying
and characterizing the naturally occurring viral infections
of zebrafish as the scope of zebrafish models expands into
new research areas, and it briefly discusses zebrafish susceptibility to experimental viral infection and the characteristics of zebrafish models that enable the study of host
factors and viral immunity.
Importance of Identifying Naturally
Occurring Viruses in Zebrafish
Historically, the zebrafish has served as a model for vertebrate
development and genetics, with virtually all experimentation
occurring during the first few days postfertilization; thus, adult
zebrafish have been traditionally maintained exclusively as
breeding stock to produce embryos for experimentation. The
lack of information about viral infections in zebrafish can be
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DNA viruses
Adenoviridae
Circoviridae
Herpesviridae
Iridoviridae
Polyomaviridae
Poxviridae
RNA viruses
Birnaviridae
Calciviridae
Coronaviridae
Hepeviridae
Nodaviridae
References
Volume 53, Number 2
2012
subsequently infected with Edwardsiella tarda, Streptococcus iniae, or VHSV, the flounder birnavirus–infected
flounder were more resistant to VHSV infection than controls but less resistant to bacterial infections (Pakingking
et al. 2003). Later experiments showed that flounder birnavirus–infected flounder exhibited significant protection from
VHSV when they were exposed 3, 7, or 14 days following
flounder birnavirus exposure, but there was no significant
mortality difference from controls when the VHSV exposure occurred 21 days after flounder birnavirus infection
(Pakingking 2004). Similarly, flounder birnavirus infection conferred complete protection in sevenband grouper
(Epinephelus septemfasciatus) to subsequent infection
with red-spotted grouper nervous necrosis virus, whereas
80% mortality was observed in sevenband grouper not
exposed to flounder birnavirus (Pakingking et al. 2005).
Regardless of whether clinical signs are evident, underlying infection with an unknown viral agent may confound
experimental infection studies with a different pathogen.
Flounder birnavirus does not normally cause disease in infected flounder, but it profoundly influences the outcome of
infection studies on other pathogens. This set of studies illustrates the type of confounding effects that might be expected when infection studies are carried out in zebrafish
that have been unknowingly exposed to subclinical viral infections. Similarly, it is well documented in salmonid fishes
that recent or simultaneous viral infection can dramatically
alter the outcomes of other infections. If these viral infections had been unrecognized, the vastly different experimental outcomes would have been difficult to interpret and may
have erroneously been attributed to other factors. For example, Atlantic salmon (Salmo salar) acutely infected with infectious pancreatic necrosis virus (IPNV1) and infectious
salmon anemia virus display significantly lower mortality
than salmon infected with infectious salmon anemia virus
only (Johansen and Sommer 2001), but Atlantic salmon
coinfected with IPNV and either of the bacterial pathogens
Vibrio salmonicida or Aeromonas salmonicida exhibited significantly higher mortality than salmon infected with V. salmonicida or A. salmonicida alone (Johansen and Sommer
2001; Johansen et al. 2009). Rainbow trout coinfected with
IPNV and infectious hematopoietic necrosis virus (IHNV1)
displayed 50% less mortality than trout infected with either
virus individually (Alonso et al. 2003). In a similar study,
coinfected trout displayed a reduced tissue distribution of
IHNV among the internal organs compared with trout infected with IHNV alone (Brudeseth et al. 2002). Notably,
rainbow trout infected with IHNV 14 days after infection
with IPNV exhibited only 2% mortality, whereas trout infected with IHNV alone exhibited 72% mortality (Byrne
et al. 2008). Finally, Hedrick and colleagues (1994) showed
that preexposure to Cutthroat trout virus protected rainbow
trout from subsequent IHNV challenge for up to 4 weeks. If
extrapolating from salmonids to zebrafish, unrecognized viral
infections in zebrafish could be anticipated to interfere with
subsequent experimental infections. It therefore seems prudent to search for naturally occurring viruses in zebrafish
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partially explained by the fact that, in the absence of an epizootic event, chronic morbidity and mortality in a zebrafish colony generally have not prevented researchers from
collecting a sufficient number of zebrafish embryos to conduct their studies. Consequently, many researchers are accustomed to accepting a level of morbidity and mortality.
Although the zebrafish research community has not historically exhibited the level of concern for eliminating infectious diseases that is now common in the rodent research
community, the advantages of zebrafish as a model organism
have resulted in its recent expansion in areas of biomedical
research where the confounding effects due to unknown infectious agents are a serious concern. Research areas such as
aging, cancer, immunity, infection, and toxicology often require that the zebrafish be maintained for a much greater
portion of their life span and that the histopathologic changes
in adult animals be assessed. Increased mortality, underlying
chronic inflammation, altered cytokine levels, tissue damage, and tissue repair resulting from natural infections are
likely to be important confounding variables in these types
of studies.
Although no cases of naturally occurring viral infections
have been reported in zebrafish research facilities, such infections probably occur, considering what is known about
other fishes held in captivity. If viral infections occur, they
may seriously affect many types of research. The task of identifying and characterizing naturally occurring viral infections in zebrafish is thus critically important. The potential
cost of undiagnosed viral infections may be increasing as the
nature of zebrafish research changes and as zebrafish facilities become more centralized. Although some viral infections in any host species may produce epizootic events, high
mortality, or noticeable clinical signs, many viral infections
cause only subclinical or low-grade infections. Moreover,
the negative effects of subclinical infections on research
are rarely reported (see Kent et al. 2012, in this issue). Even
unnoticed viral infections may alter the immune system and
confound research (Baker 1998). The risk of confounding
effects is also related to the type of experimentation. Because
zebrafish models have expanded from developmental biology
and genetics to include models for toxicology, aging, cancer,
infection, and immunology, many kinds of research may be
seriously and adversely affected by unidentified underlying
viral infections.
Because no naturally occurring viral infections have
been reported in zebrafish, it is not possible to provide specific examples of how naturally occurring viral infections
have confounded zebrafish research. However, examples
demonstrating the significant effects of viral infections on
subsequent infections in other fish species illustrate the
potential confounding effects of unrecognized viral infections on the use of zebrafish as an infection model. For example, multiple instances of viral infections nonspecifically
conferring either increased susceptibility or increased resistance to a subsequent infection with other pathogens have
been reported. When Olive flounder (Paralichthys olivaceous) were infected with flounder birnavirus and then
138
fish species (Bowser et al. 2005; Francis-Floyd et al. 1993).
Spontaneously occurring neoplastic lesions are relatively
common in laboratory zebrafish. Most commonly reported
are spermacytic seminomas affecting the testes, spindle cell
sarcomas (malignant nerve sheath tumors), ultimobranchial
gland tumors, and gastrointestinal tumors, which may have a
neoplastic or preneoplastic prevalence of greater than 30%
in some zebrafish facilities (M.L. Kent, personal communication). These gastrointestinal tumors display no sex predilection, occur in multiple background strains, and appear to
be confined to recirculating systems. To date, no dietary or
waterborne carcinogens have been identified, suggesting the
possibility of an infectious component. Florida strain wildtype zebrafish treated with N-nitroso-N-ethylurea in one
study exhibited 100% incidence of cutaneous papillomas
(Beckwith et al. 2000); however, no cutaneous papillomas
were observed in similar experiments conducted at several
other research institutions, suggesting the possibility of an
unrecognized oncogenic virus (Kent et al. 2009). Thus,
zebrafish are subject to neoplasia, which varies by population, suggesting that zebrafish may be hosts to unknown oncogenic viruses. Oncogenesis due to unrecognized viruses
may therefore be a confounding variable in cancer research
using zebrafish. A detailed review of neoplasia in zebrafish is
included in this issue (Spitsbergen et al. 2012).
The importance of pathogen-free animals for experimentation is widely accepted for mammalian laboratory models.
The only current effort to generate pathogen-free zebrafish is
being conducted at the Sinnhuber Aquatic Resources Laboratory at Oregon State University. To date, this laboratory has
eliminated one pathogen, the microsporidian Pseudoloma
neurophilia (Kent et al. 2011). For development of pathogen-free zebrafish to be successful, however, naturally occurring viral pathogens affecting zebrafish colonies must be
identified and diagnostic assays to detect viral infection must
be developed to facilitate the elimination of viral pathogens.
Importance of Biosecurity for Laboratory
Zebrafish
Because naturally occurring viral infections can confound
research and therefore pose risks to zebrafish facilities, it is
important to prevent the introduction of new viruses into naive fish colonies and to identify and manage existing pathogens within a colony. Biosecurity of laboratory zebrafish is
affected not only by the current lack of knowledge regarding
naturally occurring zebrafish viruses but also by the introduction of zebrafish raised by the pet and aquarium trade
into laboratory zebrafish facilities. In contrast to purposebred research mice, ornamental zebrafish are often raised
together with other species that harbor numerous pathogens.
A recent example of pathogen transmission from the pet
trade to laboratory zebrafish is neon tetra disease, caused by
the microsporidian Pleistophora hyphessobryconis, which
was detected in three unrelated laboratory zebrafish facilities
(Sanders et al. 2010). These parasitic outbreaks were directly
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because the zebrafish is an important infection model for
aquaculture and biomedical research and uncovering any underlying viral infections is critical to a correct interpretation
of experimental infections.
The potential effects of unrecognized viral infections
may, in some cases, be similar to the confounding effects
documented for parasitic and bacterial infections in zebrafish
and other laboratory fishes. Mycobacterium spp. are the most
important bacterial pathogens of laboratory zebrafish and cause
a wide array of chronic inflammatory lesions that complicate
histopathologic interpretation (see Kent et al. 2012, in this
issue; Sanders et al. 2012, in this issue). Similarly, viral
infections may confound toxicologic studies or other studies that rely on histopathology by introducing unexplained
histopathologic changes. Underlying parasitic infections
have also been shown to complicate the interpretation of
histopathologic changes in fish toxicologic studies. Parasitic infections make fish more susceptible to the toxic effects of zinc (Boyce and Yamada 1977), cadmium chloride
(Pascoe and Cram 1977), and petroleum hydrocarbons
(Moles 1980). Similarly, detrimental effects of polychlorinated biphenyls on anterior kidney leukocytes were more
severe in parasitized juvenile salmon (Jacobson et al.
2003). Underlying infections may also alter immune function; for example, chronic subclinical infections contribute
to stress, which can subsequently contribute to immunosuppression. When experimentally parasitized zebrafish were
subjected to other stressors, they suffered increased mortality, earlier onset of infection, reduced weight, and reduced
fecundity compared with unparasitized zebrafish (Ramsay et
al. 2009). Subclinical infections probably also alter the
type or amount of inflammation. For example, increased
inflammation and altered cytokine production caused by
underlying infections can affect cell proliferation rates, creating a confounding variable in tumorigenesis models. In
fact, the incidence of intestinal neoplasia following treatment with dimethylbenzathracine was significantly higher in
zebrafish infected by the intestinal nematode Pseudocapillaria tomentosa than in similarly treated uninfected zebrafish (Kent et al. 2002).
In recent years, researchers have developed immunocompromised zebrafish to aid in the study of hematopoiesis,
tumorigenesis, infection, and immunity. As with other species, immunocompromised zebrafish are likely to be more
susceptible to viral infections than wild-type fish, exhibiting
higher mortality, higher morbidity, more clinical signs, and
more severe histopathologic lesions. Thus, in experimental
models where immunocompromised zebrafish are required,
the use of virus-free fish will play a major role in reducing
the variability in data because of confounding factors, such
as inflammation and other host responses to infection, and
will simultaneously reduce the number of fish required to
achieve adequate statistical power.
Naturally occurring infections along with oncogenic viruses
may also play a role in confounding experiments, including
cancer studies using zebrafish (Kent et al. 2009), because
viruses are associated with tumorigenesis in several other
Zebrafish as a Viral Infection and Host
Defense Model
The zebrafish is a useful and attractive model for infectious
disease and immunity research and is considered a refinement
over the use of mammalian infection models (Burgos et al.
2008). The zebrafish model boasts the capacity to allow investigation of specific immune system components at various
stages of immunologic development, and extensive molecular,
genetic, and imaging tools are available for this species.
The utility of the zebrafish model for infection and immunity
experiments has been extensively reviewed (Meeker and Trede
2008; Phelps and Neely 2005; Sullivan and Kim 2008; Trede
et al. 2004; van der Sar et al. 2004; Yoder et al. 2002) and
hinges on the functional similarity of the zebrafish immune
system and the mammalian immune system.
Briefly, zebrafish exhibit both the innate and adaptive
arms of the immune system, including leukocyte populations, inflammatory mediators, and signaling molecules
that are similar to those of the mammalian immune system.
Zebrafish have B and T lymphocytes, monocytes, and phagocytic tissue macrophages that are similar to their mammalian counterparts, as well as at least two granulocyte lineages
Volume 53, Number 2
2012
(Phelps and Neely 2005). Of these granulocyte lineages,
zebrafish neutrophils exhibit segmented nuclei and cytoplasmic granules that are myeloperoxidase positive, whereas the
second granulocyte lineage displays characteristics of both
eosinophils and basophils (Bennett et al. 2001). Zebrafish B
and T lymphocytes have many similarities to their mammalian counterparts; both rag1 and rag2 have been identified in zebrafish (Willett et al. 1997a,b), and both T-cell
receptor genes and B-cell immunoglobulin genes exhibit
V(D)J recombination (Haire et al. 2000). Whereas mammalian adaptive immune systems include five immunoglobulin (Ig) classes—IgA, IgD, IgE, IgG, and IgM—the zebrafish
exhibits three known immunoglobulin classes—IgD, IgM,
and IgZ—as well as a second IgZ-like immunoglobulin,
IgZ-2 (Danilova et al. 2005; Hu et al. 2010b).
The innate arm of the zebrafish immune system likewise
bears considerable resemblance to the mammalian system.
For example, 24 putative toll-like receptor (TLR) variants
have been identified in zebrafish, including at least one homologue for each of the following mammalian TLRs: TLR1,
TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9, in addition to fish-specific TLRs (Jault et al. 2004; Meijer et al.
2004). Gene homologues encoding adaptor proteins important for signal transduction also have been identified
in zebrafish, including toll-interleukin 1 receptor domain–
containing adaptor protein (TIRAP), myeloid differentiation
primary response gene 88 (MyD88), sterile alpha and
Armadillo motif–containing protein (SARM), and tollinterleukin 1 receptor–containing adapter molecule (TICAM)
(Jault et al. 2004; Meijer et al. 2004). Homologues have also
been identified for IRAK-4 and TRAF6, key proteins in the
TLR signaling pathway (Phelan et al. 2005a; Sullivan and
Kim 2008). When ZFL cells were infected with snakehead
rhabdovirus (SHRV1), the zebrafish homologues for TLR3
and TRAF6 were upregulated, demonstrating conserved
TLR signaling in zebrafish (Phelan et al. 2005a).
Many components of the complement system have been
identified in zebrafish, including B (Gongora et al. 1998),
C1q (Hu et al. 2010a), C1s (Nakao et al. 2011), C3 (Samonte
et al. 2002; Vo et al. 2009a,b), C4 (Samonte et al. 2002), C6,
C7, C8a, C8b, C8y, and C9 (Nakao et al. 2011), H (Sun et al.
2010), I, mannose-binding lectin–associated serine protease
(MASP)-1, MASP-2, MASP-3, and mannose-binding lectin
(Nakao et al. 2011). The classical pathway, alternative
pathway, and lectin pathways all activate complements in
teleost fishes (Holland and Lambris 2002; Nakao et al.
2011; Sunyer and Lambris 1998). Similarly, homologues
to many mammalian cytokines have been identified in
zebrafish, including interleukin 1␤ (Pressley et al. 2005),
type I (Altmann et al. 2003) and type II (Igawa et al. 2006)
interferons, tumor necrosis factor ␣ (Praveen et al. 2006;
Pressley et al. 2005), and several interleukins (Sullivan and
Kim 2008).
Moreover, several innate immune system components
with antiviral properties have been identified in zebrafish.
For example, as in the mammalian antiviral response, zebrafish
type I interferon directly induces myxovirus resistance (Mx)
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linked to zebrafish purchased from the pet trade (Sanders
et al. 2010). In addition, the diagnostic pathology service at
the Zebrafish International Resource Center occasionally detects common aquarium fish helminth parasites, indicating
that the submitted zebrafish were raised in outdoor ponds
(M.L. Kent, personal communication). The often unrestricted
relationship between the pet trade and research colonies undoubtedly facilitates the introduction of viral pathogens, as
well as parasites and bacteria, into laboratory zebrafish
colonies. Although knowledge of the viruses that infect the
aquarium fish species that are raised with zebrafish in the pet
trade is limited, the viruses that have been described include
herpesviruses (Gilad et al. 2002; Jeffery et al. 2007), iridoviruses (Go et al. 2006; Hossain et al. 2008; Jeong et al. 2008a,b),
a nodavirus (Hegde et al. 2003), and a reovirus (Seng et al.
2004). This research not only demonstrates that laboratory
zebrafish may still harbor a variety of pathogens from their
origin in the pet trade but also provides evidence that zebrafish raised in outdoor ponds continue to directly enter laboratory colonies, providing a constant source of new pathogens
from other pet and aquarium species. It is, therefore, also
likely that at least some laboratory zebrafish are currently
infected with viruses prevalent in the ornamental fish trade.
Moreover, many newer zebrafish facilities are centralized; therefore, an epizootic event caused by breaches in
biosecurity has the potential to be devastating by exposing
the zebrafish colonies of multiple investigators to pathogens during an outbreak of disease (Kent et al. 2009). The
risk of viral spread between zebrafish populations within a
centralized facility is increased by the close proximity of
aquatic systems, increased traffic, and overlap in personnel
and other resources.
Experimental Viral Infections in Zebrafish
Infection studies designed to develop the zebrafish as a
model for viral infection in commercially important fish species have demonstrated the experimental susceptibility of
zebrafish to infection by several families of viruses, including Birnaviridae (IPNV) (LaPatra et al. 2000; Seeley et al.
1977); Rhabdoviridae (IHNV) (LaPatra et al. 2000; Ludwig
et al. 2011), Spring viremia of carp virus (Lopez-Munoz
et al. 2010; Sanders et al. 2003), VHSV (Novoa et al. 2006)
and SHRV (Phelan et al. 2005b); Nodaviridae (Malabar
grouper nervous necrosis virus) (Lu et al. 2008); and Iridoviridae (infectious spleen and kidney necrosis virus) (Xu et al.
2008). The susceptibility of zebrafish to experimental infections with this broad range of viruses suggests not only that
naturally occurring viruses occur in zebrafish but also that
many viral families may be represented among the as-yet
unidentified zebrafish viruses.
Zebrafish have also been used as models of mammalian
viral infection, although there are some limitations to their use.
For example, zebrafish are often maintained at approximately
28°C, whereas some mammalian viruses adapted to replicate
at 37°C may not be pathogenic at 28°C. However, advantages to zebrafish models for mammalian viral infections
include the capacity for live imaging, whole-organism histo140
pathology and immunohistochemistry, and temperature-shift
experiments. Because zebrafish larvae are small and transparent, every tissue in the zebrafish can be observed while
still functioning in the live animal (Ludwig et al. 2011). To
accomplish a similar objective in mice would require mice to
be sacrificed at multiple time points with multiple harvests
of selected tissues in an attempt to piece together the overall
picture. Furthermore, as poikilotherms, zebrafish can survive over a range of temperatures, allowing some viral infections to be “halted” by shifting the temperature so that viral
replication does not continue, as was recently accomplished
with IHNV (Ludwig et al. 2011). By shifting the temperature
of live infected embryos from 24°C to 28°C, the infectious
process was halted at various points, facilitating the characterization of the course of infection (Ludwig et al. 2011).
The first zebrafish infection studies using a mammalian
virus demonstrated dose-dependent infection of the adult
zebrafish nervous system with herpes simplex virus type 1
(Herpesviridae) (Burgos et al. 2008; Hubbard et al. 2010).
Zebrafish treated with the antiviral acyclovir exhibited significantly reduced viral load in all examined regions (head,
dorsal midbody, and ventral midbody), whereas zebrafish
treated with cyclophosphamide exhibited significantly higher
mortality and increased viral loads (Burgos et al. 2008).
In summary, zebrafish are experimentally susceptible to
a variety of viruses described in other fish species. The viruses include the following: IPNV, IHNV, Malabar grouper
nervous necrosis virus, Spring viremia of carp virus, SHRV,
VHSV, infectious spleen and kidney necrosis virus, and
herpes simplex virus type 1. These experimental infections
document zebrafish susceptibility to five viral families:
Birnaviridae, Rhabdoviridae, Nodaviridae, Iridoviridae,
and Herpesviridae. These experiments provide evidence that
laboratory zebrafish are infected with naturally occurring
viruses.
Conclusion
The similarity between the zebrafish and mammalian immune systems, the capacity of the zebrafish model to allow
investigation of specific immune system components at different stages of immunologic development, and the molecular, genetic, and imaging tools available for this species
make the zebrafish particularly useful and attractive as a
model for infectious disease and immunity research. However, zebrafish studies of infection and immunity, as well as
other types of zebrafish research, are at risk of being confounded by unrecognized naturally occurring viral infections. To date, what is known about viral disease in zebrafish
is entirely the result of experimental infections using viruses
isolated from other species; no naturally occurring viral infections have been reported in the zebrafish. However, the lack
of information regarding naturally occurring viral infections
in zebrafish does not reflect an inability of viral pathogens to
infect zebrafish because zebrafish are easily experimentally
infected with a variety of viruses isolated from other fishes.
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expression (Haller et al. 2007). Mx proteins are a group of
high molecular weight dynamin-like proteins whose antiviral properties were first recognized when inbred mouse
strains with mutations at the Mx locus were found to be susceptible to influenza A virus, an orthomyxovirus (Haller
et al. 1979; Lindenmann 1962, 1964; Workenhe et al. 2010).
Since then, Mx proteins have been shown to have antiviral effects against several viral families, including Bunyaviridae,
Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, and
Togaviridae. Because the Mx gene is not normally expressed
in the absence of viral infection, Mx expression is commonly
used as a marker for type I interferon activity (Haller et al.
2007) and can be used as evidence of viral infection in the
zebrafish, as demonstrated by the upregulation of interferon
and Mx RNA in zebrafish experimentally infected with
SHRV (Phelan et al. 2005b).
Thus, the similarity between the zebrafish immune system and the mammalian immune system, the genetic and
molecular tools available for zebrafish, and the ability to
image an entire infected embryo or fish over the course of
infection make the zebrafish an excellent model to investigate viral pathogenesis and host defenses. Investigating
natural infection with zebrafish viruses in addition to experimental infections will improve the usefulness of the
zebrafish as a model organism, not only elucidating routes
of infection, virulence factors, host defenses, and viral countermeasures to host defenses but also providing information
about potential confounding effects, modes of transmission,
and necessary biosecurity measures for improved operation
of bioaquatics facilities.
References
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It is imperative that naturally occurring viruses of zebrafish
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implemented. Furthermore, continued research to elucidate
the specific pathogenesis and transmission of each virus will
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for various areas of research, and such research will aid in
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of the zebrafish as a model organism. Moreover, the connection between laboratory zebrafish and the ornamental fish
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