# The reverse genetics applied to fish RNA viruses

Stephane Biacchesi

► To cite this version:

Stephane Biacchesi. The reverse genetics applied to fish RNA viruses. Veterinary Research, 2011, 42, pp.1-14. 10.1186/1297-9716-42-12. hal-02651429

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# The reverse genetics applied to fish RNA viruses

Stéphane Biacchesi

## Abstract

Aquaculture has expanded rapidly to become a major economic and food-producing sector worldwide these last 30 years. In parallel, viral diseases have emerged and rapidly spread from farm to farm causing enormous economic losses. The most problematic viruses encountered in the field are mainly, but not exclusively, RNA viruses belonging to the *Novirhabdovirus*, *Aquabirnavirus*, *Alphavirus* and *Betanodavirus* genera. The recent establishment of reverse genetics systems to recover infectious fish RNA viruses entirely from cDNA has made possible to genetically manipulate the viral genome. These systems have provided powerful tools to study all aspects of the virus biology and virus-host interactions but also gave the opportunity to use these viruses as live vaccines or as gene vectors. This review provides an overview on the recent breakthroughs achieved by using these reverse genetics systems in terms of viral protein function, virulence and host-specificity factor, vaccine development and vector design.

## Introduction

Fish farmers are faced worldwide to viral infections which destroy each year a significant part of the production and thus induce important economic losses [1,2]. In addition to direct losses and delay in the aquaculture expansion, viral diseases also cause additional impacts including shortage of food and jobs as well as social and environmental costs. Major viral diseases are mainly due to two antigenetically distinct *Novirhabdoviruses* which can coexist in the same fish farms: the viral hemorrhagic septicemia virus (VHSV) and the infectious hematopoietic necrosis virus (IHNV). Other viruses have also a serious impact in the field such as the infectious pancreatic necrosis virus (IPNV), an *Aquabirnavirus* infecting for instance salmonid and viruses belonging to the *Betanodavirus* genus which cause serious diseases in several marine fish species. The genome of these viruses consists of either a negative-sense single-stranded RNA molecule for the *Novirhabdoviruses* of about 11 kilobases (kb) [3,4], two segments of double-stranded RNA (A and B) of about 2.8 and 3.1 kilobase pairs (kbp) each for the *Aquabirnavirus* [5,6] or two positive-sense single-stranded RNA (RNA1 and RNA2) of about 1.4 and 3.1 kb each for the *Betanodaviruses* [7,8].

These viral diseases are the most frequent but due to the expansion of the salmonid aquaculture and the

development of new fish species production, new diseases have emerged in the recent years [2]. One of the most striking examples is undoubtedly the emergence of the sleeping disease in farmed rainbow trout (*Oncorhynchus mykiss*) and the pancreas disease in marine-reared Atlantic salmon (*Salmo salar*). These diseases are known since the 80s and have now spread in most of the European countries and in North America. For instance, the sleeping disease may affect 30 to 40% of the French fish farms. Viruses responsible for these diseases, the sleeping disease virus (SDV) and the salmon pancreas disease virus (SPDV) belong to the *Alphavirus* genus, with a genome which consists of a positive-sense single-stranded RNA molecule of about 12 kb [9,10].

Since many years, goals of different laboratories around the world have been mainly but not exclusively devoted to the development of new vaccine approaches based in part on live attenuated viruses to prevent viral diseases in aquaculture [11,12] and the determination of potential genetic factors that could explain the differences of virulence existing between virus strains and their host range restriction. For that, a way allowing the manipulation of the viral genome is required. A major step in engineering a viral RNA genome is the ability to recover live virus from a DNA copy of the RNA genome: this is called “reverse genetics”. First success in reverse genetics for a positive-stranded RNA virus, the poliovirus (*Picornaviridae* family), was in 1981 [13]. Thirteen years later, a reverse genetics system was
established for the recovery of a nonsegmented negative-
stranded RNA virus, the rabies virus (RV) [14]. The abil-
ity to manipulate a cDNA intermediate, exact copy of
the viral RNA genome, opens the possibility of deleting
genes for studying their function, introducing targeted
mutations to determine potential genetic factors of viru-
lence or inserting heterologous genes of interest and
using these recombinant viruses as gene vectors.

Reverse genetics systems have been now established
for several fish RNA viruses such as the *Novirhabdo-
viruses* IHNV and VHSV [15-18], the *Alphavirus* SDV
[19], the *Aquabirnavirus* IPNV [20] and also for several
*Betanodavirus* species such as for the striped jack ner-
vous necrosis virus (SJNNV) [21]. A large number of
various recombinant viruses have been generated by dif-
ferent groups. Some of them are currently being evalu-
ated as live vaccines in field trials and some others are
being used as gene vectors in fish. Some examples of
such recombinant viruses will be described and the
advances in terms of vaccine development, virulence
and host-specificity factor mapping and vector design
will be discussed.

Reverse genetics systems: description and
technical tricks

Alphavirus

Salmonid *Alphaviruses* are recognized as serious patho-
gens of farmed Atlantic salmon and rainbow trout in
Europe [9]. Sleeping disease (SD) is an infectious disease
of rainbow trout reared in fresh water while salmon pan-
creas disease (SPD) has been recorded in farmed Atlantic
salmon. The aetiological agents responsible for these dis-
eases, namely SDV and SPDV, respectively, are closely
related viruses that belong to the *Alphavirus* genus of the
*Togaviridae* family [10,22]. Sindbis virus (SINV), one the
most studied virus among the *Alphavirus* genus, is trans-
mitted by mosquitoes, and its alternate vertebrate host is
usually a bird or a mammal. Like all Alphaviruses, SDV
and SPDV virions are spherical (70 nm in diameter), with
a lipid envelop containing heterodimeric glycoprotein
spikes composed of two virus glycoproteins (E1 and E2).
Some alphaviruses may contain a third envelop protein
(E3). The envelope is tightly organized around an icosaha-
dral nucleocapsid which contains the genomic RNA. The
viral genome consists of a positive-sense single-stranded
RNA molecule of about 12 kb in length, which is capped
at the 5'-terminus and polyadenylated at the 3'-terminus,
and contains two open reading frames (ORF). The 5' two-
thirds of the genome encode four non-structural proteins
(nsP1 to nsP4) that are translated directly from the geno-
mic RNA as a polyprotein precursor, which undergoes
subsequent proteolytic cleavage. The four non-structural
proteins are involved in genome replication and the synth-
esis of a subgenomic RNA from the negative-strand copy

of the genome that corresponds to the 3' third of the viral
genome and encodes the second ORF. This second ORF is
translated as a polyprotein, which is processed to produce
the viral structural proteins: the capsid, the glycoproteins
E3, E2, E1 and the 6K protein. In contrast to the other
Alphaviruses, an arthropod-independent transmission has
been demonstrated for salmonid Alphaviruses by cohabi-
tation experiments [9].

Based on the observation that genomic RNA from most
of the positive-stranded RNA viruses could serve as mes-
senger RNA (mRNA) and initiate infection upon transec-
tion in a permissive cell line, the recovery of infectious
virus from complementary DNA (cDNA) was first
described for poliovirus by Racaniello and Baltimore,
almost 30 years ago [13]. The first infectious cDNA for a
member of the *Alphavirus* genus was established for SINV
few years later by Rice et al. [23]. The recovery of recombi-
nant alphavirus from cDNA was typically based on the
transfection into cells of synthetic capped RNA transcripts
generated by in vitro transcription from SP6- or T7-driven
full-length viral cDNA constructs. Although less efficient
than transfection of in vitro-synthesized RNA, transfection
of viral genome cDNA copy under the control of a RNA
polymerase II promoter (the cytomegalovirus (CMV)
immediate early promoter) was also described (for review
[24]). Similar approaches for SDV failed to produce infec-
tious recombinant virus [19,25]. The key to recover SDV
was found by fusing a self-cleaving hammerhead ribozyme
sequence at the 5'-end of the cDNA copy of SDV full-
length genome [19]. Indeed, a precise 5'-end of SDV RNA
genome appears to be crucial to initiate an infectious
cycle. This cDNA construct was then cloned under the
control of a T7 RNA polymerase (T7RNAP) promoter
(Figure 1) and recombinant SDV was recovered upon
transfection of this cDNA plasmid into fish cells (bluegill
fry BF-2 cells) previously infected by a recombinant vacci-
nia virus (vTF7-3; [26]) expressing the T7RNAP. Because
of the temperature requirement of each virus (37°C and
10°C for vTF7-3 and SDV, respectively), cells were first
incubated at least 7 h at 37°C to allow their infection by
the vTF7-3 and the expression of enough functional
T7RNAP before being shifted at 10°C following the proto-
col previously described for fish rhabdovirus minigenome
rescue ([27] and section 2.4). This system was further sim-
plified by the exchange of the T7RNAP promoter by that
of the CMV avoiding the restrictive use of the vTF7-3
[19]. A similar cDNA construct was recently used with
success for the expression of a salmon alphavirus subtype
3 replicon in fish cells [28].

Aquabirnavirus

Infectious pancreatic necrosis (IPN) is a serious disease
of salmonid and a number of other fish species that can
lead to high mortality (70%) in first feeding fry and
<figure><img src="image_2.png" /></figure>

fingerlings of most salmonids. Furthermore, the aetiol-
gical agent responsible for the disease (IPNV) can be
vertically transmitted via the egg and broodstock carrier
fish facilitating its rapid spread to new geographic area
and its expansion to other farmed fish species [29].
IPNV is the type species of the *Aquabirnavirus* genus of
the *Birnaviridae* family. Infectious bursal disease virus
(IBDV), another member of *Birnaviridae* family that has
been studied in the greatest detail is the causative agent

of highly immunosuppressive disease in young chickens
and belongs to another genus, *Avibirnavirus* [5]. IPNV
is a non-enveloped icosahedral capsid virus (60 nm in
diameter) that contains two segments (A and B) of dou-
ble-stranded RNA [30]. Segment A (about 3.1 kbp) con-
tains two ORF. ORF2 encodes a 106-kDa precursor
protein, which is autoproteolytically cleaved by the viral
encoded protease (VP4 or NS for non structural) to gen-
erate the viral capsid proteins VP2 and VP3. Segment A
also encodes an additional non structural protein VP5
from the small ORF1, which precedes and partly over-
laps the large ORF2. Segment B (about 2.8 kbp) encodes
a 94-kDa protein, called VP1, which is the viral asso-
ciated RNA-dependant RNA polymerase (RdRp).

The first description of segmented double-stranded
RNA virus recovery entirely from cDNA was given by
Mundt and Vakharia for IBDV [31]. Few years later, Yao
and Vakharia used a similar approach to recover IPNV
and thus reporting the first reverse genetics system for a
RNA virus infecting an aquatic species [20]. In both
cases, synthetic positive-sense RNA transcripts of seg-
ments A and B were transcribed in vitro from linearized
full-length cDNA plasmids with the T7RNAP in the
presence of a synthetic cap analogue and used to trans-
fect permissive cells (chinook salmon CHSE-214 cells)
(Figure 1). IPNV recovery was also achieved upon trans-
fection of vTF7-3-infected fish cells (fathead minnow
EPC (epithelioma papulosum cyprinid) cells) with seg-
ment A and B cDNA copies (Biacchesi S., unpublished
data). Segments A and B were cloned under the control
of a T7RNAP promoter and upstream the antigenomic
sequence of a self-cleaving ribozyme derived from the
hepatitis delta virus (δ) allowing the expression by the
T7RNAP of RNA molecules displaying a precise 3'-end
without any additional nucleotide [32].

Betanodavirus

Viral nervous necrosis (VNN) and viral encephalopathy and retinopathy (VER) have emerged as major constraints on culture of larval and juvenile and sometimes older marine fish worldwide leading to behavioural abnormalities and high mortalities [2,7]. The aetiological agents responsible for these diseases are small icosahedral viruses belonging to the *Betanodavirus* genus, within the *Nodaviridae* family. *Betanodavirus* genus is further divided in four species based on similarities in the variable region of the RNA2 and designated as Barfin flounder nervous necrosis virus (BFNNV), Red-spotted grouper nervous necrosis virus (RGNNV), Striped jack nervous necrosis virus (SJNNV, the type species of the *Betanodavirus* genus) and Tiger puffer nervous necrosis virus (TPNNV) [8]. Black Beetle virus (BBV) and Flock House virus (FHV), the most studied viruses among the *Nodaviridae* family, infects insects and belongs to the second genus, *Alphanodavirus*. Fish nodaviruses are icosahedral, non-enveloped viruses with a commonly reported diameter of about 25 nm. The virus contains two single-stranded positive-sense RNA molecules, namely RNA1 (3.1 kb) and RNA2 (1.4 kb). Both RNA molecules are capped at their 5'-ends and lack poly(A) tails at their 3'-ends. The larger genomic segment, RNA1, encodes the RdRp (or protein A) and the smaller genomic segment, RNA2, encodes the coat

protein. In addition, subgenomic RNA3, which is
synthesized from RNA1 during RNA replication and not
packaged into virions, encodes the two non-structural
proteins B1 and B2.

The first recovery from cDNA of a virus belonging to the *Nodaviridae* family was reported by Kaesberg's group for BBV [33]. Because of the lack of an appropriate cell culture system, the establishment of such reverse genetics system for betanodaviruses was hampered. The first system was reported by Iwamoto and colleagues for SJNNV in 2001 [21]. This system was similar to that previously described for BBV and IPNV. It was based on the transfection into fish cells (E-11 cells cloned from striped snakehead SSN-1 cells) of synthetic RNA molecules of viral RNA 1 and 2 transcribed in vitro from linearized full-length cDNA plasmids with the T7RNAP and in the presence of a synthetic cap analogue (Figure 1). This system was further simplified by the use of a stable mammalian cell line expressing constitutively the T7RNAP, the BSR T7/5 cells [34]. In that goal, the cDNA plasmids encoding the RNA 1 and 2 were slightly modified by the fusion of a self-cleaving ribozyme δ at the 3'-end of each genome cDNA copy. Because of the temperature requirement of the mammalian cells and the betanodaviruses, 37°C versus 20-30°C, respectively, transfected-cells were incubated at 28°C for 48 h and then the cell lysates were inoculated on permissive fish cells at the optimal temperature for the viral replication [35,36]. Reverse genetics systems are now available for several betanodavirus species [21,35-37].

Novirhabdovirus

IHNV and VHSV are causative agents of acute systemic
diseases leading to high mortality, mainly, but not exclu-
sively, in young cultured and wild salmonid fish all over
the world and are listed as notifiable by the World
Organisation for Animal Health (OIE) [2,38]. IHNV and
VHSV are both enveloped bullet-shaped viruses (about
180 nm long and 70 nm in diameter) with a genome
consisting of a non-segmented negative-sense single-
stranded RNA molecule of about 11 kb [3,4]. These
viruses belong to the *Rhabdoviridae* family within the
*Mononegavirales* order. The most extensively studied
mammalian rhabdoviruses are the rabies virus (RV) that
belongs to the *Lyssavirus* genus and the vesicular stoma-
titis virus (VSV) that belongs to the *Vesiculovirus* genus.
Similar to the mammalian rhabdoviruses, the IHNV and
VHSV genomic RNA which encodes five structural pro-
teins, is tightly encapsidated with a nucleoprotein (N), a
polymerase-associated phosphoprotein (P) and the large
RdRp (L) to form the helical ribonucleoprotein complex
(RNP). The two other structural proteins are the matrix
protein (M), which interacts with the RNP and the viral
envelope and is involved in the budding step, and the
unique viral surface glycoprotein (G) which is implicated
in the entry step. In contrast to other rhabdoviruses,
IHNV and VHSV genomes encode a small non struc-
tural NV (Non Virion) protein. Due to the presence of
the NV gene, localized between the G and L genes,
IHNV and VHSV are separated from the other rhabdo-
viruses and belong to the *Novirhabdovirus* genus [4].

The recovery of rhabdoviruses from cDNA presents a unique challenge because neither their genomic RNA nor their antigenomic complements serve as mRNA, and thus cannot be used directly to recover infectious viruses. In other words, the RNA of negative-stranded viruses (NSV) are not infectious upon transfection into a permissive cell line in contrast to those of positive-stranded viruses. The minimal infectious unit of NSV is the RNP complex which is able to drive both the sequential transcription of each viral gene and the replication of the full-length RNA genome. Based on this observation, the feasibility of producing infectious NSV entirely from cDNA was demonstrated by Conzelmann's group for RV, 15 years ago [14]. The goal of this system is to reconstruct into permissive cells a functional RNP complex. In that goal, cells are co-transfected with expression plasmids encoding N, P and L proteins together with a plasmid encoding the full-length viral antigenome, all under the control of the T7RNAP, which is provided to the cells by an infection with a recombinant vaccinia virus (vTF7-3; [26]) (Figure 1). In the co-transfected cells, the antigenome RNA is encapsidated by the N protein to form a suitable template for the RdRp (L and P) to drive the replication and the synthesis of genomic RNP. Once genomic RNP are available, an infectious cycle can be initiated with the expression of all viral proteins followed by the production of new infectious virions. Two tricks have contributed to the success of this system. The first one was the expression of an antigenome instead of a genome to prevent any hybridization with the mRNA expressed from the helper plasmids (N, P and L), which could prevent the initial encapsidation of the genome into a functional RNP complex and/or induce the host innate response mediated by the type I interferon. The second trick was the use of a self-cleaving ribozyme δ sequence fused to 3'-end of the antigenome cDNA allowing the expression of an antigenome RNA molecule having a precise 3'-end without any additional nucleotides, which were found deleterious for the rescue.

This system was then adapted to numerous viruses
belonging to the *Mononegavirales* order [39] and was
the starting point of various genetic manipulations (for
review: [40,41]). Concerning the fish NSV, the descrip-
tion of a similar system, including plasmid design and
T7RNAP supply by a recombinant vaccinia virus, was
first given by Leong's group for the snakehead

rhabdovirus (SHRV), a Novirhabdovirus which replicates
at an elevated temperature of 31°C (Figure 1) [42]. This
system was further adapted to low-temperature growing
Novirhabdoviruses, such as IHNV and VHSV for
which the optimal temperature of replication is 14°C
[17,18,27]. This low-temperature requirement made the
development of IHNV and VHSV reverse genetics more
complex since vaccinia virus needs a temperature closed
to 37°C to be able to express the T7RNAP. This obsta-
cle was overcome by the selection of an IHNV and
VHSV permissive cell line (fathead minnow EPC cells)
able to undergo an incubation at 37°C for at least seven
hours, a minimal incubation time required to express
enough T7RNAP into the cells to drive the expression
of each plasmid DNA before shifting the cells at 14°C
(for review: [43]). Two kinds of vaccinia-virus-free
reverse genetics systems for Novirhabdoviruses were
then described removing the biosafety requirement
imposed by the vaccinia virus manipulation and its
highly recombinogenic property leading to nucleotide
exchange between the transfected plasmids which can
make difficult the recovery of certain mutants [18,44]
(Figure 1). Leong's group described the SHRV recovery
from a fish cell line constitutively expressing the
T7RNAP (EPC-T7, derived from EPC cells) [45] and
Vakharia's group demonstrated the recovery of both
IHNV and VHSV using the cellular RNA polymerase II
[15,16]. In this latter system, the cDNA encoding the
IHNV N, P and L proteins and the viral antigenome
were cloned under the control of a CMV promoter
instead of that for the T7RNAP. In addition, a hammer-
head ribozyme was fused to the 5'-end of the antige-
nome cDNA allowing the expression by the RNA
polymerase II of an antigenome RNA molecule display-
ing a precise 5'-end.

A wide panel of applications and breakthroughs

Reverse genetics makes it possible to manipulate viral
RNA genomes through its cDNA copy and thus to eval-
uate genomic changes, such as gene deletion, heterolo-
gous gene exchange or addition and nucleotide
substitution, on the biology and the pathogenesis of
these recombinant viruses. All these reverse genetics-
based studies over the last twelve years have provided
new insights on fish RNA viruses that are presented
above.

Analysis of viral proteins of unknown function

Novirhabdovirus NV protein

The ability to generate recombinant viruses by reverse
genetics allows the opportunity to examine the role of
the non-structural proteins in virus biology and patho-
genic mechanisms. For instance, the NV proteins (111
to 122 amino acids), expressed by IHNV and VHSV in
GGNNV-ΔB2 virus mutant using their reverse genetics system [35]. Indeed, although virus replication could be detected by indirect immunofluorescence assay after five consecutive passages on permissive cells, the genome analysis of these recovered viruses demonstrated that all of them had a C-to-T reversion at the B2 start codon restoring its expression. These results underline that betanodavirus B2 protein has an essential function at least during the virus replication. Additional data showed that GGNNV B2 binds long double-stranded RNA (dsRNA) preventing their recognition by the siRNA machinery and thus blocking the subsequent production of virus-derived small interfering RNA [58,59]. In contrast, B2 protein is unable to antagonize the dsRNA-mediated interferon response, as monitored by the *Mx* gene induction which is proportional to RNA1 accumulation in fish cells. Thus it seems that for betanodaviruses, which cause high mortalities in larval and juvenile marine fish, the most important host response at these fish developmental stages is RNA silencing [60]. To further complicate the understanding of B2 involvement in betanodavirus replication, recent studies indicate that B2 may play a dual role during the viral infection by first modulating the host siRNA response and then inducing mitochondria-mediated necrotic cell death [61]. The existence of a B1 ORF encoded by the betanodavirus subgenomic RNA3 is controversial. Although several groups reported that the B1 ORF may be absent in RNA3 [56,57], Chen and colleagues recently detected the early expression of B1 protein in RGNNV-infected fish cells using a B1 polyclonal antibody and showed that B1 may have an antagonist effect on B2 function by enhancing host cell viability [62]. Recovery of a recombinant virus in which the B1 ORF will be ablated (RGNNV-ΔB1) and observation of an enhancement of cell death in RGNNV-ΔB1-infected cells could give an additional argument to this new B1 function.

## Aquabirnavirus VP5 protein

The reverse genetics has also been used to investigate the potential role of VP5 on IPNV replication and virulence. Indeed, studies on recombinant IBDV lacking expression of VP5 showed that VP5 was nonessential for productive virus replication in cell culture, but played a key role in pathogenicity of IBDV [63]. Several recombinant IPNV, encoding for different size variants of VP5 or ablated for VP5 expression (IPNV-ΔVP5), were then successfully recovered. These virus mutants demonstrated that VP5 protein is not required for efficient virus growth in vitro, as previously observed for IBDV, but also in vivo in Atlantic salmon fry and post-smolt which differs from IBDV [64,65]. IPNV-ΔVP5 mutant was able to cause IPN disease in challenged

Atlantic salmon post-smolts, leading to final cumulative mortality of 86% compared to 81% recorded with the wild-type virus [64]. Moreover, VP5 does not seem to be involved in the establishment of a carrier state since surviving salmon fry infected by IPNV-ΔVP5 were all IPNV carrier at least five months after being challenged as observed with those infected with the wild-type virus [64]. Altogether, these results strongly suggest that VP5 has no function as a virulence factor, but it can reasonably be assumed that VP5 provides a selective advantage since the majority of IPNV strains isolated to date encode the protein. It was previously reported that IPNV VP5, from the E1S strain, contains Bcl-2 homologous domains and that its stable expression in salmon cells enhances cell viability following exposure to apoptotic inducers [66]. However, this function is not conserved among IPNV strains. Indeed, IPNV serotype Sp strain and its VP5-deletion mutant derivative did not show any anti-apoptotic effect of VP5 expression in infected cell culture and in fish [67]. Finally, VP5 could be involved in the inhibition of interferon-stimulated gene expression [68]. But, based on the fact that inhibition of the host innate immune defence by a virus is required to establish a productive infection, the role of VP5 in the blockage of the interferon signalling does not appear to be crucial since IPNV-ΔVP5 was as virulent as its wild-type parent [64].

## Virus vectors

With efficient reverse genetics systems available for several major fish pathogens, the degree of flexibility of the genome of these viruses to genetic engineering and their ability to express foreign genes of interest were verified by several groups. For this purpose, negative-stranded viruses (NSV), such as rhabdoviruses, appear to be good candidates as vectors for transient gene expression compared to positive-stranded viruses (PSV) for many reasons (for review on mammalian virus-derived vectors [24,40,41]). Indeed, NSV genomes present a modular organization with individual cistrons defined by conserved gene start and gene end signals recognized by the viral RdRp and leading to the transcription of separate mRNA. In contrast, PSV genomes are expressed from unique polycistron mRNA which are translated into polyprotein precursors that follow a subsequent maturation by cellular or viral proteases. Moreover, the helical structure of the RNP complex of NSV brings two additional interesting features. First, due to the helical structure of the RNP, there is no defined size limit for the genomes in contrast to viruses with icosahedral capsids such as aquabirnaviruses, fish alphaviruses and betanodaviruses, in which the genome size is constrained. Nevertheless, although no size limit for NSV genome has been clearly defined, this rise of the
genome size is always correlated with an increasing
attenuation of the NSV vector. Second, the tight encap-
sidation of the NSV genome in the RNP reduces the
chance of recombination as observed at high frequency
with PSV, and prevents access of reverse transcriptases
to the RNA avoiding any risk of cellular transformation.
Finally, due to a decreasing gradient of gene expression
along the NSV genome from the 3'-end to the 5'-end
(i.e. the expression of the first gene is the most abun-
dant and the last gene the least); the level of expression
of the gene of interest can be modulated by moving its
position in the gene order. Thus, NSV modular organi-
zation can be readily manipulated for the insertion and
stable maintenance of nonessential sequences such as
foreign genes. In both cases, the host restriction has to
be considered for a wider use of the NSV and PSV vec-
tors to other fish species.

Antigenic chimeric viruses: example of Novirhabdovirus
glycoprotein gene exchange

An attractive method to express foreign antigens by a recombinant virus is to replace its major protective surface antigen with that of another virus of interest. Such constructs have been generated by exchanging the unique surface glycoprotein of different distantly-related and antigenically-distinct fish rhabdoviruses [45,69]. The IHNV genome was engineered such that the IHNV G gene was replaced by those of VHSV (another fish *Novirhabdovirus*, 51% homology between both proteins) and the spring viremia of carp virus (SVCV; a fish vesicularovirus, 29% homology between both G) [69]. Both chimeric recombinant viruses, IHNV-Gvhsv and IHNV-Gsvcv, were successfully recovered and were found as efficient as IHNV for multicycle replication in cell culture. The only remarkable feature was the plaque morphology induced by IHNV-Gvhsv which was of intermediate size compared to those induced by both parental viruses (IHNV, small plaques and VHSV, large plaques). However, although IHNV-Gvhsv was as virulent as its biological parents (IHNV and VHSV) in rainbow trout, the histological lesions induced by this recombinant virus in trout were more similar to those obtained with IHNV [70,71]. Interestingly, although SVCV is not pathogenic in rainbow trout, a high cumulative mortality could be observed with IHNV-Gsvcv by bath immersion (93% of cumulative mortality with IHNV-Gsvcv versus 95% with IHNV), demonstrating that SVCV G is fully functional in the IHNV background and that another SVCV protein should be involved in the host restriction [70]. The efficient recovery of these chimeric viruses could be explained by the effective incorporation of the foreign glycoprotein into the viral particles as demonstrated on sucrose-gradient purified IHNV-Gvhsv and IHNV-Gsvcv virions. This

observation shows that IHNV does not require any spe-
cific IHNV G-derived sequence to efficiently incorporate
a heterologous glycoprotein. This is similar to previous
observations with the mammalian rhabdovirus VSV for
which a correct length of the cytoplasmic tail is more
important than specific amino acid sequences (for
review [41]). In contrast, efficient incorporation of for-
eign glycoprotein into RV virions requires that the cyto-
plasmic tail of the RV glycoprotein is conserved. The
IHNV flexibility for heterologous glycoprotein incor-
poration was further demonstrated by the IHNV G
exchange with the mammalian VSV counterpart [70].
The recombinant IHNV-Gvsv was fully efficient for
replication in fish cells at low temperature (14°C).
A similar observation was performed with a recombinant
SHRV, a warm-water fish Novirhabdovirus, in which the
SHRV glycoprotein was exchange by that of IHNV
(SHRV-Gihnv; 52% of homology between both G) [45].
Finally, the high flexibility of IHNV was further demon-
strated by simultaneous exchange of IHNV G and M
genes with those of VHSV [69]. In addition, these recom-
binant viruses were useful to investigate whether the
glycoprotein was the only protein responsible for the
temperature growth restriction of IHNV. Indeed, IHNV
and VHSV cannot grow at a temperature above 20°C,
whereas SVCV, SHRV and VSV replicate well up to 28°C,
30°C and 37°C, respectively. However, SHRV-Gihnv only
gives limited replication at 30°C pointing out the confor-
mational instability of the IHNV glycoprotein at this
temperature [45]. Moreover, IHNV-Gsvcv and IHNV-
Gvsv do not replicate at temperatures higher than 20°C,
demonstrating that the G is not the only protein involved
in the temperature growth restriction and that probably
the polymerase complex requires an optimal temperature
for its efficient activity [69,70].

Insertion of a foreign gene

As explained above (see section 3.2), the strategy used
to insert an additional ORF in RNA viruses depends on
the genome organization. Concerning NSV, such as
rhabdoviruses, the sequential transcription is initiated by
the RdRp upstream of each ORF by the recognition of a
gene start signal and ended at a gene end signal that
serve as transcription stop and polyadenylation signals
(for review on mammalian NSV [40,41]). A functional
cassette expressing a foreign gene has to be engineered
as a cDNA including the desired ORF flanked by the
viral gene start and gene end signals. These signals can
be added artificially or recovered from an existing gene
by exchanging the viral ORF by that of the gene of
interest or by engineering a fusion protein. In the case
of an artificial cassette, the cDNA is then inserted into a
non-coding intergenic region found between each viral
gene. Due to the gradient of expression in NSV genome,
the insertion site into the viral gene order will influence
the level of expression of the foreign gene as well as the
level of attenuation of the virus vector. Indeed, the
insertion of a foreign sequence decreases the expression
of the following genes and has more or less effect on
the replication of the recombinant virus.

Both strategies were tested for IHNV and VHSV
[15-17,47,72]. First, the IHNV NV ORF was replaced by
different reporter genes as CAT, the green fluorescent
protein (GFP), the *Renilla* luciferase (LUC), the VHSV
G and different other virus-antigen encoding genes
([18,47,73,74], Harmache A. and Brémont M., unpublished
data). Although these viruses were highly attenu-
ated by the deletion of the NV gene (see section 3.1),
they were all able to efficiently express detectable
amount of the foreign protein in cell culture. In vivo,
these viruses were non-pathogenic in rainbow trout and
too much attenuated to be used as vaccine vectors (see
below) [47,48,74]. Second, artificial cassettes of expres-
sion were then constructed for both IHNV and VHSV
[15-17,47,72]. IHNV and VHSV share similar consensus
sequences for the gene end and gene start signals except
for the hairpin loop structure shown to be part of
the end of the IHNV gene start signal: (UCURUC
(U)₇RCCGUG(N)₄CACR versus UCUAUC(U)₇RCCGUG,
respectively). These signals were successfully used to
express a large panel of reporter genes: LUC, GFP and
red fluorescent proteins such as Cherry and Tomato
[15-17,47] and viral antigens from ISAV, IPNV and SDV
([72], Harmache A. and Brémont M., unpublished data).
All these recombinant viruses were readily recovered at
high titer demonstrating that the insertion of an addi-
tional gene in the intergenic region between P-M genes
(IHNV), M-G genes (IHNV and VHSV) and N-P genes
(VHSV) have a limited effect on the efficacy of replication
at least in cell culture. Moreover, recombinant IHNV
viruses with insertion between M-G genes were shown
fully pathogenic in rainbow trout by bath immersion
inducing final cumulative mortalities similar to that
induced by IHNV [47]. As compared to other RNA
viruses, foreign sequence introduced into IHNV and
VHSV genomes were surprisingly stable after multiple
passages in cell culture (up to 20 passages) and in rain-
bow trout (Biacchesi S. and Brémont M., unpublished
data). For a mammalian rhabdovirus (RV), it was shown
that the expression of a reporter gene that does not affect
the virus replication was stable over 25 passages in cell
culture [75].

These recombinant viruses, expressing a foreign gene,
were used for several applications that led to amazing
observations. The most interesting example was
achieved with a recombinant IHNV expressing the LUC
gene and a non-invasive bioluminescence imaging device
[47]. Indeed, this technology provided evidence that the

fins were the portal entry of IHNV into the rainbow
trout. The authors also showed that a non-pathogenic
IHNV-ΔNV, in which the NV gene was replaced by the
LUC gene, had a limited replication exclusively in the
fins and could persist in fins up to 3 weeks postinfection
without any further propagation into the infected fish.
These observations explained why IHNV-ΔNV could
not be detected by RT-PCR in internal organs and in
part the absence of induced-antibody expression [48,74].
Although IHNV-ΔNV-immunized fish were fully pro-
tected against a subsequent challenge with a highly-
pathogenic IHNV strain at 30 days post-immunization
[48], mortalities could be recorded for challenge per-
formed at a later period post-immunization (Harmache
A. and Brémont M., unpublished data). Altogether, the
results suggest that the early protection was the conse-
quence of an interference phenomenon in the fins
between the IHNV-ΔNV and the wild-type virus.

Recombinant VHSV and IHNV, expressing Tomato
and GFP reporter gene, respectively, were simulta-
neously used to coinfect fish cell monolayers [17]. It was
observed that up to 74% of the cell monolayer could be
coinfected by both viruses demonstrating limited super-
infection exclusion between these two Novirhabdo-
viruses. These results suggest that fish living in endemic
region for IHNV and VHSV are likely to be coinfected
by both viruses, as previously observed during experi-
mental coinfection [76]. As mentioned above (in section
3.2.1), IHNV has an extreme flexibility to accommodate
heterologous structural proteins, such as foreign glyco-
proteins. So, it could be assumed that during coinfection
such events occur and lead to chimeric and/or pseudo-
typed viruses with unique phenotype in fish. In contrast,
it is less probable that recombination occurs between
both viruses since IHNV and VHSV display a high
genetic divergence (57% of position identity between
both genomes) and recombination is extremely rare in
NSV [77,78].

Finally, Harmache et al. investigated the potential of
IHNV as vaccine vehicle against other fish viral diseases
([72] and Harmache A. and Brémont M., unpublished
data). They reported the production of recombinant
IHNV based vaccine vectors that carry three expression
cassettes of foreign antigens derived from four of the
most devastating fish viral pathogens: VHSV G glyco-
protein, IPNV VP2, SDV C-E3-E2 and infectious salmon
anaemia virus (ISAV) hemagglutinin-esterase glycopro-
tein and fusion protein. They found that the three for-
eign antigens were efficiently and stably expressed by
the recombinant IHNV vector although its genome was
increased in size more than 50% compared to the wild-
type genome (about 5.5 kb of foreign sequences).
Because of this important increase of the genome size
together with the effect on the expression of the
downstream viral genes, these IHNV vectors were unsurprisingly highly attenuated in trout leading to only 10% of cumulative mortality. Nevertheless, surviving trout were protected against subsequent challenges with not only the pathogenic IHNV but also with the targeted viruses. Thus, genome size increase has an effective attenuation effect which can be assumed relatively stable due to the low rate of recombination observed in NSV genomes [77,78]. However, it cannot be totally excluded that the foreign gene sequences by themselves or their products are somewhat responsible for the observed attenuation.

The use of fish PSV as gene vector has been less explored compared to fish NSV. Indeed, the flexibility of the PSV genomes for genetic engineering and their ability to stably express foreign genes of interest are impaired for several reasons. PSV genomes are expressed as polyprotein precursors that require a subsequent maturation by cellular or viral proteases. Moreover, because of packaging constraints (icosahedral capsids), large inserts (> 2 kb) are not stable upon passaging and rapidly removed by recombination which is observed at high frequency with PSV (for review on mammalian alphavirus [24]). Therefore, a foreign gene has to be engineered as a cDNA including the desired ORF flanked by a protease cleavage site and inserted in the genome in frame with the viral ORF without ablating a potential overlapping ORF. The gene or the antigen of interest can also be expressed as a fusion protein. These kinds of genome manipulation are generally lethal for the vector and thus various insertion sites have to be tested in the goal to generate an infectious virus. Concerning the alphaviruses, the identification of the subgenomic RNA promoter element allowed the construction of viral genomes with additional subgenomic RNA promoters leading to the synthesis of additional subgenomic mRNA. This strategy was applied to SDV by Moriette and colleagues [19]. The SDV replication was not drastically impaired when an extra gene (GFP or LUC) was present in the viral genome. The second subgenomic mRNA was inserted either upstream or downstream to the structural protein genes and up to three additional subgenomic mRNAs were tested. These data demonstrated that SDV is able to encapsidate a genome that could be more than 20% longer than that of the wild-type virus. However, expression of GFP was highly unstable and no longer detected after one additional passage of the recombinant virus in cell culture. However, the long-term success of developing mammalian alphavirus as vaccine vector should not be underestimated. Further development should be necessary to increase the stability of the foreign gene such as expression of the foreign protein as a cleavable component of the viral structural polyprotein or insertion of the

foreign protein in a viral nonstructural protein as described for SINV [79,80]. No example of betanodavirus and aquabirnavirus used as gene vector are available in the literature underlying the difficulty to generate gene vectors with PSV.

## Virulence and host specificity factors

The reverse genetics is a powerful tool to map in the virus genome virulence and host specificity factors based on previous putative nucleotide changes determined after genome sequence comparisons of virus strains displaying different phenotypes. For instance, amino acid sequence comparisons of IPNV VP2 protein from various field isolates belonging to the Sp serotype, which exhibit different mortality rates in Atlantic salmon fry, identified putative residues responsible for modulating virulence between strains [81,82]. The significance of these amino acid changes in VP2 were later confirmed with recombinant reassortant and chimeric viruses generated by reverse genetics. Indeed, Song et al. demonstrated that virulence and also adaptation to cell culture are controlled by two VP2 amino acids at positions 217 and 221 [83]. Highly virulent strains encode Thr217 and Ala221, while moderate to low virulent strains have Pro217 and Ala221 and variants having Thr221 are almost avirulent, irrespective of the amino acid in position 217. In parallel, these observations allowed better understanding of the molecular basis of the cell culture attenuation by detecting the Thr221 substitution in VP2 after 10 passages of a virulent strain in a certain fish cell line. This virus became highly attenuated and induced only 15% of cumulative mortality in Atlantic salmon fry, compared to 68% mortality recorded with its virulent parent strain. The recent publication of the crystal structure of IPNV virion demonstrated that these two amino acid residues at position 217 and 221 are localized in the most peripheral loop at the top of the VP2 spike [30]. Since these two residues are exposed and do not participate in VP2 folding and interactions between subunits, their major role in IPNV strain virulence suggest that they may be involved in attachment to the target cell.

Similar approaches were undertaken to study betanodavirus host specificity and temperature restriction. Although a high overall percentage of homology is found between the four types of betanodavirus isolates, some isolates have marked host specificity. For example, SJNNV causes disease only in the striped jack (*Pseudocarax dentex*), whereas members of RGNNV type, such as sevenband grouper nervous necrosis virus (SGNNV), has a broad host range and causes diseases for a variety of warm-water fish species, particularly for sevenband grouper (*Epinephelus septemfasciatus*). Recombinant reassortant and chimeric viruses were generated by
using reverse genetics systems established for SJNNV, RGNNV and SGNNV [37,84]. The authors of these two reports showed that the variable region of the RNA2, which encodes the C-terminal region of the coat protein, controls host specificity in betanodaviruses. Indeed, RNA2 chimeric viruses from SJNNV and RGNNV demonstrated that SJNNV mutants containing the variable region of RGNNV RNA2 infected sevenband grouper larvae as efficiently as RGNNV, while RGNNV mutants containing the variable region of SJNNV RNA2 infected striped jack larvae similarly to SJNNV. Since the C-terminal of the coat protein was shown by electron cryomicroscopy to form the surface-protruding domain of virus-like particles [85], its major role in betanodavirus host specificity suggests that this region may be involved in attachment to the target cell. These two reverse genetics systems were further used to investigate the temperature restriction observed between SJNNV and RGNNV [86]. Whereas the optimal temperature for the growth of SJNNV is 20-25°C, that of RGNNV is 25-30°C. Thus, a set of recombinant reassortant and chimeric viruses from SJNNV and RGNNV were recovered and their optimal temperature for replication in cell culture was determined. Although both RNA1 and RNA2 seem to control this temperature sensitivity as shown by reassortant viruses, chimeric viruses indicate that the RGNNV RNA1 region, encoding amino acid residues 1 to 445 of the RdRp was implicated in this restriction. Interestingly, this region encodes a mitochondrial-targeting signal and is located outside of the catalytic domain of the RdRp.

For alphaviruses, incidental mutations introduced during the RT-PCR steps needed to construct infectious cDNA for SDV have also contributed to identify potential attenuating position in their genomes. Concerning SDV, several nucleotide substitutions have been mapped in the structural and non-structural genes of the infectious cDNA compared to the published sequence (see details in [19,25]). Although the recombinant SDV (rSDV) was fully efficient for replication in cell culture reaching a final titer of 3 × 10⁸ PFU/mL similar to the field isolate, the virus was totally attenuated in rainbow trout when administrated by either bath immersion or injection. Nevertheless, high titers (about 10⁸ PFU/mL of serum) of rSDV could be harvested from sacrificed fish up to three weeks post-infection. Immunized rainbow trout acquired long-lasting (at least seven months) and complete protection against challenges with a highly virulent SDV strain by bath immersion or injection. In addition, rSDV-vaccinated fish were also protected from a challenge with the wild-type SPDV, a closely related but distinct salmonid alphavirus. It has to be mentioned that in addition to the genome sequence mutations, the temperature of virus adaptation to the cell culture

seems to play a role in the SDV attenuation. Indeed, naïve trout infected with the wild-type SDV adapted to 14°C (SDV14) or with that adapted to 10°C (SDV10) exhibited different cumulative mortality rates two months postinfection, roughly 80% and 8%, respectively. The rSDV was also recovered at 10°C which could explain in part its in vivo attenuated phenotype. The effect of the temperature at which the viruses were produced on the virulence was established by adapting rSDV and SDV10 to grow at 14°C. Indeed, the resulting viruses induced higher cumulative mortality rates in trout: from 0 to 24% for rSDV and 7% to 30% for SDV10, respectively. The comparison of the nucleotide sequences encoding the structural proteins pinpointed six major amino acid changes (three in E2, two in 6K and one in E1) between rSDV and its 14°C-adapted derivative. Moreover, as SDV10 was derived from SDV14 after plaque purification, it could be assumed that SDV14 inoculum represents a quasispecies population pathogenic for trout and that the viral heterogeneity was greatly reduced for SDV10 and even more for the rSDV derived from a single cDNA clone. This hypothesis was in part demonstrated by the partial sequencing of the three virus genomes which pinpointed a large number of amino acid changes, roughly 60 and 20 in the non-structural genes of SDV14 and SDV10 compared to the counterpart region in rSDV [25].

Concerning IHNV, the introduction of four incidental mutations during the construction of the full-length cDNA construct was enough to highly attenuate the recovered virus. This recombinant IHNV, containing 2 amino acid substitutions in the N, one in the P and one in the M, induced less than 10% of cumulative mortality in trout by bath immersion and was found to be a strong inducer of IHNV-specific antibodies [70,71]. Surprisingly, when two additional mutations, previously described in the G of a neutralization-escape IHNV mutant (RB1 strain) as highly attenuating (amino acids 78 and 218) [87], were introduced in this virus, virus-induced mortality was not decreased [71]. This observation together with the fact that a recombinant IHNV containing G78 and G218 as unique mutations was only slightly attenuated suggest that the RB1 strain, for which the G was the only gene fully sequenced, have other changes in its genome [71]. No example of putative virulence factor screening by reverse genetics has been reported for Novirhabdoviruses in the literature yet. However, recombinant IHNV viruses containing the G and/or NV genes from North American U or M IHNV strains have been constructed and tested for host-specific virulence in both rainbow trout and sockeye salmon (*Oncorhynchus nerka*) (Kurath G., Harmache A., and Brémont M., unpublished data). The U and M IHNV strains differ in host specific virulence: U strain is
virulent in sockeye salmon but not rainbow trout, and the M strain is virulent in rainbow trout but not sockeye salmon. These studies revealed that the G and NV genes do not contain the determinants of host-specific virulence for these IHNV strains, suggesting that the molecular mechanism(s) of host-specificity in this case must be different from the viral coat protein host cell attachment mechanism described for IPNV and betanodaviruses. Finally, in recent decades, several VHSV and IHNV strains have been isolated and fully sequenced. For instance, several VHSV strains have been isolated from several marine fish species (for review see [38]). The interesting observation was that VHSV isolates originating from marine fish show low pathogenicity to rainbow trout and Atlantic salmon, although some of these isolates are pathogenic for turbot (*Scophthalmus maximus*). Several nearly complete genome sequences from marine and freshwater isolates displaying variable level of virulence in rainbow trout are now available in GenBank. From the analysis of these sequences, several groups have already pinpointed putative amino acid residues that could be involved in VHSV strain virulence [88,89]. Interestingly, these authors point out that as few as 4 to 10 amino acid changes were identically substituted between the marine and freshwater strains suggesting that only a limited number of amino acid residues might be involved in the level of virulence between isolates. Using the newly described reverse genetics system based on the hypervirulent freshwater VHSV 23-75 strain [17], these amino acid residues could be easily tested in the goal to determine their biological relevance in VHSV virulence.

## Conclusion remarks

Since the description of the first reverse genetics system for a fish RNA virus in 1998 [20], several other systems have been established for the major fish RNA virus pathogens. These technologies have largely contributed to a better understanding of the biology of these viruses in term of virulence and host specificity factors as well as virus-host interactions and virus entry in the host. They also allowed the genomic manipulation of these viruses and thus the generation of gene-deletion mutants, attenuated virus vaccines and gene vectors, such as multivalent live attenuated vaccines and tracer viruses expressing reporter genes. Live attenuated virus vaccines would be the optimal fish vaccines in term of cost, protective efficacy and ease of administration (bath immersion instead of injection for traditional vaccines). But, additional works have to be undertaken to remove any existing residual virulence and insure their stability avoiding any reversion to a wild-type phenotype in the goal to address the safety concerns of the consumers and to the environment (for review on development of

safer live attenuated virus vaccines see [90]). Regulatory constraints are the main obstacle for this kind of vaccines especially in view of aquaculture practices where cultured fish live in ponds or nets with no physical barriers to wild stocks. Indeed, even if non-pathogenic for the targeted animal, the live virus vaccine has to be safe to all other species in the aquatic environment. One alternative would be the use of these live virus vaccines in a confined environment during the immunization protocol and then assure the absence of any persistent virus infection before housing the immunized fish in an open aquatic environment. Furthermore, reverse genetics systems could be used to generate single-replication cycle virus-based vaccine vectors such as alphavirus replicons. Although this kind of vaccine was found safe and protective in mammals after injection, the immunogenicity in fish of such vaccines should be demonstrated when administrated by bath immersion. The next several years will clearly be very exciting time in fish RNA virus research since advances in virus genome manipulation, fish genome sequencing and fish immune system characterization have greatly increased these last years.

## Acknowledgements

The author received financial support from the French National Institute for Agricultural Research (INRA), the European Community and Interact International (Boxmeer, the Netherlands). The fish facility staff (INRA) is gratefully acknowledged for the fish experiments. The author thanks all the members of the Fish Molecular Virology group (INRA) who contributed to this work and more especially Michel Brémont, Monique LeBerre, Annie Lamoureux, Emilie Mérouer, and Julie Bernard. The author thanks Michel Brémont and Emilie Mérouer for critically reading the manuscript.

## Competing interests

The authors declare that they have no competing interests.

Received: 23 August 2010 Accepted: 18 November 2010

Published: 24 January 2011

## References

1. Ariel E, Olesen NJ: Finfish in aqualculture and their diseases - A retrospective view on the European Community. *Bull Eur Ass Fish Pathol* 2002, 22(2):72-84.

2. Walker PJ, Winton JR: Emerging viral diseases of fish and shrimp. *Vet Res* 2010, 41(6):51.

3. Hoffmann B, Beer M, Schutze H, Mettenleiter TC: Fish rhabdoviruses: molecular epidemiology and evolution. *Curr Top Microbiol Immunol* 2005, 292:81-117.

4. Tordo N, Benmansour A, Calisher C, Dietzgen RG, Fang R-X, Jackson AO, Kurath G, Nadin-Davis S, Tesh RB, Walker PJ: Rhabdovidae. In *Virus Taxonomy VIII*. Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. London: Elsevier Academic Press; 2005:623-644.

5. Delmas B, Kibenge FSB, Leong JA, Mundt E, Vakharia VN, Wu JL: Birnaviridae. In *Virus taxonomy VIII*. Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. London: Elsevier Academic Press; 2005:561-569.

6. Rodriguez Saint-Jean S, Borrego JJ, Perez-Prieto SI: Infectious pancreatic necrosis virus: biology, pathogenesis, and diagnostic methods. *Adv Virus Res* 2003, 62:113-165.

7. Munday BL, Kwang J, Moody N: Betanodavirus infections of teleost fish: a review. *J Fish Dis* 2002, 25:127-142.

8. Schneemann A, Ball LA, Delsert C, Johnson JE, Nishizawa T: Nodaviridae. In *Virus taxonomy VIII*. Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. London: Elsevier Academic Press; 2005:865-872.
9. McLoughlin MF, Graham DA: Alphavirus infections in salmonids-a review. *J Fish Dis* 2007, **30**(9):511-531.

10. Weaver SC, Frey TK, Huang HV, Kinney RM, Rice CM, Roehrig JT, Shope RE, Strauss EG: Togaviridae. In *Virus taxonomy VIII*. Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. London: Elsevier Academic Press; 2005:999-1008.

11. Biering E, Villoing S, Sommerset I, Christie KE: Update on viral vaccines for fish. *Dev Biol (Basel)* 2005, **121**:97-113.

12. Sommerset I, Krossoy B, Biering E, Frost P: Vaccines for fish in aquaculture. *Expert Rev Vaccines* 2005, **4**(1):89-101.

13. Racaniello VR, Baltimore D: Cloned poliovirus complementary DNA is infectious in mammalian cells. *Science* 1981, **214**(4523):916-919.

14. Schnell MJ, Mebatsion T, Conzelmann KK: Infectious rabies viruses from cloned cDNA. *EMBO J* 1994, **13**(18):4195-4203.

15. Ammayappan A, Kurath G, Thompson TM, Vakharia VN: A reverse genetics system for the Great Lakes strain of viral hemorrhagic septicemia virus: the NV gene is required for pathogenicity. *Mar Biotechnol (NY)* 2010, Epub ahead of print.

16. Ammayappan A, Lapatra SE, Vakharia VN: A vaccinia-virus-free reverse genetics system for infectious hematopoietic necrosis virus. *J Virol Methods* 2010, **167**(2):132-139.

17. Biacchesi S, Lamoureux A, Merour E, Bernard J, Brémont M: Limited interference at the early stage of infection between two recombinant novirhabdoviruses: viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus. *J Virol* 2010, **84**(19):10038-10050.

18. Biacchesi S, Thoulouze MI, Bearzotti M, Yu YX, Brémont M: Recovery of NV knockout infectious hematopoietic necrosis virus expressing foreign genes. *J Virol* 2000, **74**(23):11247-11253.

19. Moriette C, Leberre M, Lamoureux A, Lai TL, Brémont M: Recovery of a recombinant salmonid alphavirus fully attenuated and protective for rainbow trout. *J Virol* 2006, **80**(8):4088-4098.

20. Yao K, Vakharia VN: Generation of infectious pancreatic necrosis virus from cloned cDNA. *J Virol* 1998, **72**(11):8913-8920.

21. Iwamoto T, Mise K, Mori K, Arimoto M, Nakai T, Okuno T: Establishment of an infectious RNA transcription system for Striped jack nervous necrosis virus, the type species of the betanodaviruses. *J Gen Virol* 2001, **82**(Pt 11):2653-2662.

22. Weston J, Villoing S, Bremont M, Castric J, Pfeffer M, Jewhurst V, McLoughlin M, Rodseth O, Christie KE, Koumans J, et al: Comparison of two aquatic alphaviruses, salmon pancreas disease virus and sleeping disease virus, by using genome sequence analysis, monoclonal reactivity, and cross-infection. *J Virol* 2002, **76**(12):6155-6163.

23. Rice CM, Levis B. Strauss JH, Huang HV: Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. *J Virol* 1987, **61**(12):3809-3819.

24. Frolov I, Hoffman TA, Pragai BM, Dryga SA, Huang HV, Schlesinger S, Rice CM: Alphavirus-based expression vectors: strategies and applications. *Proc Natl Acad Sci USA* 1996, **93**(21):11371-11377.

25. Moriette C: Le virus de la maladie du sommeil des salmonidés: mise au point d'un ADNc infectieux et obtention d'anticorps monoclonaux. *Orsay: Paris XI Orsay*; 2005.

26. Fuerst TR, Niles EG, Studier FW, Moss B: Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. *Proc Natl Acad Sci USA* 1986, **83**(21):8122-8126.

27. Biacchesi S, Yu YX, Bearzotti M, Tafalla C, Fernandez-Alonso M, Brémont M: Rescue of synthetic salmonid rhabdovirus minigenomes. *J Gen Virol* 2000, **81**(Pt 8):1941-1945.

28. Karlsen M, Villoing S, Rimstad E, Nylund A: Characterization of untranslated regions of the salmonid alphavirus 3 (SAV3) genome and construction of a SAV3 based replicon. *ViroJ* 2009, **6**:173.

29. Roberts RJ, Pearson MD: Infectious pancreatic necrosis in Atlantic salmon, *Salmo salar* L. *J Fish Dis* 2005, **28**(7):383-390.

30. Coulibaly F, Chevalier C, Delmas B, Rey FA: Crystal structure of an Aquabirnavirus particle: insights into antigenic diversity and virulence determinism. *J Virol* 2010, **84**(4):1792-1799.

31. Mundt E, Vakharia VN: Synthetic transcripts of double-stranded Birnavirus genome are infectious. *Proc Natl Acad Sci USA* 1996, **93**(20):11131-11136.

32. Perrotta AT, Been MD: A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. *Nature* 1991, **350**(6317):434-436.

33. Dasmahapatra B, Dasgupta R, Saunders K, Selling B, Gallagher T, Kaesberg P: Infectious RNA derived by transcription from cloned cDNA copies of the genomic RNA of an insect virus. *Proc Natl Acad Sci USA* 1986, **83**(1):63-66.

34. Buchholz UJ, Finke S, Conzelmann KK: Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. *J Virol* 1999, **73**(1):251-259.

35. Fenner BJ, Thiagarajan R, Chua HK, Kwang J: Betanodavirus B2 is an RNA interference antagonist that facilitates intracellular viral RNA accumulation. *J Virol* 2006, **80**(1):85-94.

36. Takizawa N, Adachi K, Kobayashi N: Establishment of reverse genetics system of betanodavirus for the efficient recovery of infectious particles. *J Virol Methods* 2008, **151**(2):271-276.

37. Iwamoto T, Okinaka Y, Mise K, Mori K, Arimoto M, Okuno T, Nakai T: Identification of host-specificity determinants in betanodaviruses by using reassortants between striped jack nervous necrosis virus and sevenband grouper nervous necrosis virus. *J Virol* 2004, **78**(3):1256-1262.

38. Skall HF, Olesen NJ, Mellergaard S: Viral haemorrhagic septicaemia virus in marine fish and its implications for fish farming-a review. *J Fish Dis* 2005, **28**(9):509-529.

39. Conzelmann KK: Reverse genetics of mononegavirales. *Curr Top Microbiol Immunol* 2004, **283**:1-41.

40. Bukreyev A, Skiadopoulos MH, Murphy BR, Collins PL: Nonsegmented negative-strand viruses as vaccine vectors. *J Virol* 2006, **80**(21):10293-10306.

41. Finke S, Conzelmann KK: Recombinant rhabdoviruses: vectors for vaccine development and gene therapy. *Curr Top Microbiol Immunol* 2005, **292**:165-200.

42. Johnson MC, Simon BE, Kim CH, Leong JA: Production of recombinant snakehead rhabdovirus: the NV protein is not required for viral replication. *J Virol* 2000, **74**(5):2343-2350.

43. Brémont M: Reverse genetics on fish rhabdoviruses: tools to study the pathogenesis of fish rhabdoviruses. *Curr Top Microbiol Immunol* 2005, **292**:119-141.

44. Garcin D, Pelet T, Calain P, Roux L, Curran J, Kolakofsky D: A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back nondefective interfering virus. *EMBO J* 1995, **14**(24):6087-6094.

45. Alonso M, Kim CH, Johnson MC, Pressley M, Leong JA: The NV gene of snakehead rhabdovirus (SHRV) is not required for pathogenesis, and a heterologous glycoprotein can be incorporated into the SHRV envelope. *J Virol* 2004, **78**(11):5875-5882.

46. Schutze H, Enzmann PJ, Mundt E, Mettenleiter TC: Identification of the non-virion (NV) protein of fish rhabdoviruses viral haemorrhagic septicaemia virus and infectious haematopoietic necrosis virus. *J Gen Virol* 1996, **77**(Pt 6):1259-1263.

47. Harmache A, LeBerre M, Droineau S, Giovannini M, Brémont M: Bioluminescence imaging of live infected salmonids reveals that the fin bases are the major portal of entry for Novirhabdovirus. *J Virol* 2006, **80**(7):3655-3659.

48. Thoulouze MI, Bouguyon E, Carpentier C, Brémont M: Essential role of the NV protein of Novirhabdovirus for pathogenicity in rainbow trout. *J Virol* 2004, **78**(8):4098-4107.

49. Lauring AS, Andino R: Quasispecies theory and the behavior of RNA viruses. *PLoS Pathog* 2010, **6**(7):e1001005.

50. Venter PA, Schneemann A: Recent insights into the biology and biomedical applications of Flock House virus. *Cell Mol Life Sci* 2008, **65**(17):2675-2687.

51. Galiana-Arnoux D, Dostert C, Schneemann A, Hoffmann JA, Imler JL: Essential function in vivo for Dicer-2 in host defense against RNA viruses in drosophila. *Nat Immunol* 2006, **7**(6):590-597.

52. Li H, Li WX, Ding SW: Induction and suppression of RNA silencing by an animal virus. *Science* 2002, **296**(5571):1319-1321.

53. Wang XH, Aliyari R, Li WX, Li HW, Kim K, Carthew R, Atkinson P, Ding SW: RNA interference directs innate immunity against viruses in adult Drosophila. *Science* 2006, **312**(5772):452-454.

54. Delsert C, Morin N, Comps M: Fish nodavirus lytic cycle and semipermissive expression in mammalian and fish cell cultures. *J Virol* 1997, **71**(7):5673-5677.

55. Nagai T, Nishizawa T: Sequence of the non-structural protein gene encoded by RNA1 of striped jack nervous necrosis virus. *J Gen Virol* 1999, **80**(Pt 11):3019-3022.
56. Sommerset I, Nerland AH: Complete sequence of RNA1 and subgenomic RNA3 of Atlantic halibut nodavirus (AHNV). *Dis Aquat Organ* 2004, **58**(2-3):117-125.

57. Iwamoto T, Mise K, Takeda A, Okinaka Y, Mori K, Arimoto M, Okuno T, Nakai T: Characterization of Striped jack nervous necrosis virus subgenomic RNA3 and biological activities of its encoded protein B2. *J Gen Virol* 2005, **86**(Pt 10):2807-2816.

58. Fenner BJ, Goh W, Kwang J: Sequestration and protection of double-stranded RNA by the betanodavirus b2 protein. *J Virol* 2006, **80**(14):6822-6833.

59. Fenner BJ, Goh W, Kwang J: Dissection of double-stranded RNA binding protein B2 from betanodavirus. *J Virol* 2007, **81**(11):5449-5459.

60. Lu MW, Chao YM, Guo TC, Santi N, Evensen O, Kasani SK, Hong JR, Wu JL: The interferon response is involved in nervous necrosis virus acute and persistent infection in zebrafish infection model. *Mol Immunol* 2008, **45**(4):1146-1152.

61. Su YC, Wu JL, Hong JR: Betanodavirus non-structural protein B2: A novel necrotic death factor that induces mitochondria-mediated cell death in fish cells. *Virology* 2009, **385**(1):143-154.

62. Chen LJ, Su YC, Hong JR: Betanodavirus non-structural protein B1: A novel anti-necrotic death factor that modulates cell death in early replication cycle in fish cells. *Virology* 2009, **385**(2):444-454.

63. Mundt E, Kollner B, Kretzschmar D: VP5 of infectious bursal disease virus is not essential for viral replication in cell culture. *J Virol* 1997, **71**(7):5647-5651.

64. Santi N, Song H, Vakharia VN, Evensen O: Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence. *J Virol* 2005, **79**(14):9206-9216.

65. Weber S, Fichtner D, Mettenleiter TC, Mundt E: Expression of VP5 of infectious pancreatic necrosis virus strain VR299 is initiated at the second in-frame start codon. *J Gen Virol* 2001, **82**(Pt 4):805-812.

66. Hong JR, Gong HY, Wu JL: IPNV VP5, a novel anti-apoptosis gene of the Bcl-2 family, regulates Mcl-1 and viral protein expression. *Virology* 2002, **295**(2):217-229.

67. Santi N, Sandro A, Sindre H, Song H, Hong JR, Thu B, Wu JL, Vakharia VN, Evensen O: Infectious pancreatic necrosis virus induces apoptosis in vitro and in vivo independent of VP5 expression. *Virology* 2005, **342**(1):13-25.

68. Skjesol A, Aamo T, Hegseth MN, Robertsen B, Jorgensen JB: The interplay between infectious pancreatic necrosis virus (IPNV) and the IFN system: IFN signaling is inhibited by IPNV infection. *Virus Res* 2009, **143**(1):53-60.

69. Biacchesi S, Bearzotti M, Bouguyon E, Brémont M: Heterologous exchanges of the glycoprotein and the matrix protein in a Novirhabdovirus. *J Virol* 2002, **76**(6):2881-2889.

70. Biacchesi S: Recovery of recombinant piscine rhabdovirus by reverse genetics. *Orsay: Paris XI Orsay*; 2002.

71. Romero A, Figueras A, Tafalla C, Thoulouze MI, Brémont M, Novoa B: Histological, serological and virulence studies on rainbow trout experimentally infected with recombinant infectious hematopoietic necrosis viruses. *Dis Aquat Organ* 2005, **68**(1):17-28.

72. Harmache A, LeBerre M, Lamoureux A, Brémont M: Recombinant IHNV expressing three foreign expression units an an efficient bath immersion vaccine in salmonids. *XIII International conference on negative strand viruses* Salamanca, Spain; 2006.

73. Boudinot P, Bernard D, Boubekeur S, Thoulouze MI, Brémont M, Benmansour A: The glycoprotein of a fish rhabdovirus profiles the virus-specific T-cell repertoire in rainbow trout. *J Gen Virol* 2004, **85**(Pt 10):3099-3108.

74. Romero A, Figueras A, Thoulouze MI, Brémont M, Novoa B: Recombinant infectious hematopoietic necrosis viruses induce protection for rainbow trout Oncorhynchus mykiss. *Dis Aquat Organ* 2008, **80**(2):123-135.

75. Mebatsion T, Schnell MJ, Cox JH, Finke S, Conzelmann KK: Highly stable expression of a foreign gene from rabies virus vectors. *Proc Natl Acad Sci USA* 1996, **93**(14):7310-7314.

76. Brudeseth BE, Castric J, Evensen O: 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* 2002, **39**(2):180-189.

77. Chare ER, Gould EA, Holmes EC: Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses. *J Gen Virol* 2003, **84**(Pt 10):2691-2703.

78. Spann KM, Collins PL, Teng MN: Genetic recombination during coinfection of two mutants of human respiratory syncytial virus. *J Virol* 2003, **77**(20):11201-11211.

79. Atasheva S, Gorchakov R, English R, Frolov I, Frolova E: Development of Sindbis viruses encoding nsP2/GFP chimeric proteins and their application for studying nsP2 functioning. *J Virol* 2007, **81**(10):5046-5057.

80. Thomas JM, Klimstra WB, Ryman KD, Heidner HW: Sindbis virus vectors designed to express a foreign protein as a cleavable component of the viral structural polyprotein. *J Virol* 2003, **77**(10):5598-5606.

81. Santi N, Vakharia VN, Evensen O: Identification of putative motifs involved in the virulence of infectious pancreatic necrosis virus. *Virology* 2004, **322**(1):31-40.

82. Shivappa RB, Song H, Yao K, Aas-Eng A, Evensen O, Vakharia VN: Molecular characterization of Sp serotype strains of infectious pancreatic necrosis virus exhibiting differences in virulence. *Dis Aquat Organ* 2004, **61**(1-2):23-32.

83. Song H, Santi N, Evensen O, Vakharia VN: Molecular determinants of infectious pancreatic necrosis virus virulence and cell culture adaptation. *J Virol* 2005, **79**(16):10289-10299.

84. Ito Y, Okinaka Y, Mori K, Sugaya T, Nishioka T, Oka M, Nakai T: Variable region of betanodavirus RNA2 is sufficient to determine host specificity. *Dis Aquat Organ* 2008, **79**(3):199-205.

85. Tang L, Lin CS, Krishna NK, Yeager M, Schneemann A, Johnson JE: Virus-like particles of a fish nodavirus display a capsid subunit domain organization different from that of insect nodaviruses. *J Virol* 2002, **76**(12):6370-6375.

86. Hata N, Okinaka Y, Iwamoto T, Kawato Y, Mori K, Nakai T: Identification of RNA regions that determine temperature sensitivities in betanodaviruses. *Arch Virol* 2010, **155**(10):1597-1606.

87. Kim CH, Winton JR, Leong JC: Neutralization-resistant variants of infectious hematopoietic necrosis virus have altered virulence and tissue tropism. *J Virol* 1994, **68**(12):8447-8453.

88. Betts AM, Stone DM: Nucleotide sequence analysis of the entire coding regions of virulent and avirulent strains of viral haemorrhagic septicaemia virus. *Virus Genes* 2000, **20**(3):259-262.

89. Campbell S, Collet B, Einer-Jensen K, Secombes CJ, Snow M: Identifying potential virulence determinants in viral haemorrhagic septicaemia virus (VHSV) for rainbow trout. *Dis Aquat Organ* 2009, **86**(3):205-212.

90. Lauring AS, Jones JO, Andino R: Rationalizing the development of live attenuated virus vaccines. *Nat Biotechnol* 2010, **28**(6):573-579.

doi:10.1186/1297-9716-42-12

Cite this article as: Biacchesi: The reverse genetics applied to fish RNA viruses. *Veterinary Research* 2011 **42**:12.

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