Infectious Pancreatic Necrosis Virus VP5 Is Dispen
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病菌学杂志 2005年第14期
Section for Pathology, National Veterinary Institute, 0033 Oslo, Norway
Center for Biosystems Research, University of Maryland Biotechnology Institute
Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland 20742
Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Sciences, 0033 Oslo, Norway
ABSTRACT
Infectious pancreatic necrosis virus (IPNV) is the causative agent of infectious pancreatic necrosis (IPN) disease in salmonid fish. Recent studies have revealed variation in virulence between isolates of the Sp serotype, associated with certain residues of the structural protein VP2. The isolates are also highly heterogenic in the coding region of the nonstructural VP5 protein. To study the involvement of this protein in the pathogenesis of disease, we generated three recombinant VP5 mutant viruses using reverse genetics. The "wild-type" recombinant NVI15 (rNVI15) virus is virulent, having a premature stop codon at nucleotide position 427, putatively encoding a truncated 12-kDa VP5 protein, whereas rNVI15-15K virus encodes a 15-kDa protein. Recombinant rNVI15-VP5 virus contains a mutation in the initiation codon of the VP5 gene that ablates the expression of VP5. Atlantic salmon postsmolts were challenged to study the virulence characteristics of the recovered viruses in vivo. The role of VP5 in persistent infection was investigated by challenging Atlantic salmon fry with the recovered viruses, as well as with the low-virulence field strain Sp103 and a naturally occurring VP5-deficient mutant of Sp103. The results show that VP5 is not required for viral replication in vivo, and its absence does not alter the virulence characteristics of the virus or the establishment of persistent IPNV infection.
INTRODUCTION
Infectious pancreatic necrosis virus (IPNV) is a well-known cause of acute contagious disease in salmonid fish. The mortality of an outbreak varies considerably, depending on the species, age, and physical condition of the fish, as well as the virulence of the viral strain (4, 17, 40, 46). Only Sp strains are reported to cause disease in seawater-adapted salmonid fish (postsmolts) (28, 47). A high percentage of infected fish become long-term carriers of the virus (2, 35). In acutely diseased salmonids, IPNV induces lesions principally in exocrine pancreatic tissue, while in persistently infected fish, the virus is found in macrophages (29, 38). So far, little is known about the molecular and immunological mechanisms involved in the establishment of an IPNV carrier state.
IPNV is the prototype virus of the family Birnaviridae and belongs to the genus Aquabirnavirus (10). The genome consists of two segments of double-stranded RNA packaged in a nonenveloped icosahedral shell 60 nm in diameter (7). The smaller genomic segment, B, is 2,784 nucleotides (nt) long and encodes VP1, the virion-associated RNA-dependent RNA polymerase (9, 14). The larger segment, A, is 3,097 nucleotides long and encodes a 107-kDa precursor protein in a single large open reading frame (ORF), which is cotranslationally cleaved by the viral nonstructural (NS) protease, VP4, generating VP2 and VP3 structural proteins (8, 12, 13, 34, 41). Segment A also encodes a 15-kDa arginine-rich protein from a small ORF partly preceding and overlapping the polyprotein ORF (24, 25). This protein, designated VP5, has been detected in virus-infected cells (34), but so far there is no evidence of the protein being found in purified virions. It has recently been shown that the initiation of translation for VP5 starts at the second in-frame start codon in the Sp and VR-299 strains (45, 51), and the absence of expression does not influence virus growth in vitro (51). The existence of field strains lacking the initiation codon for VP5 protein is also well documented (25, 43).
We have recently characterized the virulence of nine IPNV serotype Sp strains by challenging Atlantic salmon (Salmo salar L.) fry (43). Some strains were highly virulent, causing up to 90% mortality and severe necrosis of the liver and pancreas of infected fry, while other strains produced low mortality (up to 6%) and no detectable lesions. Nucleotide sequencing of the viral genomes revealed heterogeneity in the open reading frame encoding VP5 and in certain VP2 amino acids potentially associated with the virulence of the virus. Four of the strains encoded the full-length 15-kDa protein, while one strain had a mutation in the start codon of the VP5 protein. Three strains had premature stop codons at nt 427, encoding a putative 12-kDa VP5, and yet another strain had a premature stop codon at nt 496; all strains encoding a truncated form of VP5 were virulent.
IPNV is known to induce apoptosis in infected CHSE-214 cells (27). The suicide of a cell in response to viral infection is postulated to be an important mechanism of host defense (15). Viruses have evolved various mechanisms to evade the apoptotic response of the host cell (1), and some viruses encode homologues of cellular antiapoptotic Bcl-2 proteins (vBcl-2s) (6). The Bcl-2 family of intracellular proteins includes both pro- and antiapoptotic molecules and plays a pivotal role in regulation of apoptotic processes (5, 22). IPNV VP5 contains Bcl-2 homology domains, and overexpression in CHSE-214 cells enhances host cell viability following IPNV infection, likely because of antiapoptotic activity and by limiting viral protein expression (26). However, the physiologic role of this protein during infection is unknown, and there are no reports of in vivo studies of IPNV VP5-deficient mutants.
For Sindbis virus, Semliki Forest virus, and Japanese encephalitis virus, it has been shown that Bcl-2 overexpression facilitates persistent in vitro infection (32, 33, 44). The in vivo role of a viral Bcl-2 was recently identified, as -herpesvirus 68 vBcl-2 was found to be dispensable for acute replication and disease but important for persistent replication (20). Since IPNV VP5 possesses Bcl-2 homology domains, it is conceivable that this protein may play a role in persistent infection.
A reverse-genetics technique for the Birnaviridae was first described by Mundt and Vakharia (37), using synthetic RNAs derived from cDNA clones to generate infectious bursal disease virus. The same approach was employed to recover IPNV from transfected CHSE cells (52). Genetically engineered IPNV VP5 mutants of the VR-299 serotype have been studied in vitro (51), demonstrating that the absence of this protein does not influence replication in cell culture. In this paper, we describe the generation of three VP5 mutants of a virulent IPNV of the Sp serotype using reverse genetics and the first application of this technique to study the function of VP5 protein in IPNV virulence and persistence in vivo.
MATERIALS AND METHODS
Cells, viruses, and serum. Chinook salmon embryo cells (CHSE-214; ATCC CRL-1681) were used for transfection experiments and for detection, propagation, and quantification of virus. The cells were maintained at 20°C in minimal essential medium containing Hanks' salts and supplemented with 10% fetal bovine serum (FBS) (transfection experiments) or in Leibovitz's L-15 medium supplemented with 5% FBS and 50 μg ml–1 gentamicin (challenge studies). Rainbow trout gonad (RTG-2) cells (ATCC CCL-55) were grown in L-15 medium supplemented with 10% FBS at 15°C. IPNV field isolates Sp103, Sp103-VP5, NVI-015, and NVI-020, which belong to the Sp serotype, were used in this study. NVI-015 and NVI-020 cause high mortality in Atlantic salmon fry challenge, while Sp103 (also called NVI-025) is less virulent (43). Analysis of the small ORFs showed that Sp103 and NVI-020 encode the full-length 15-kDa VP5 while NVI-015 encodes a putative truncated 12-kDa VP5 (Table 1). A naturally occurring VP5-deficient virus, Sp103-VP5, was isolated by plaque purification of Sp103 (Table 1). The complete nucleotide sequence of segment A has been determined for all isolates, and the VP1-encoding sequence of segment B has been determined for NVI-015, Sp103, and Sp103-VP5 (GenBank accession no. AY379740 and AY379741 for NVI-015, AY379736 for NVI-020, AY354519 and AY354520 for Sp103, and AY823632 and AY823633 for Sp103-VP5).
To prepare monospecific antiserum against VP5, a peptide corresponding to the N-terminal part of the protein (NH2-20RDWTSKHPGRHNGETHLKT38-COOH) was custom synthesized (Bio-Synthesis) and used for repeated immunization of a rabbit to obtain anti-VP5 antibody.
Construction of full-length cDNA clones. Construction of full-length cDNA clones containing the entire coding and noncoding regions of IPNV NVI-015 RNA segments A and B was performed by standard cloning procedures as described previously (52). On the basis of the published IPNV sequence of the Sp strains, several primer pairs were synthesized and employed in reverse transcription (RT)-PCR amplifications (Table 2). To generate cDNA clones of segment A of NVI-015, two primer pairs (A-A5'NC plus A-KpnR and A-KpnF plus A-SpPstR) were used for RT-PCR amplification (Table 2). Using genomic RNA as a template, the desired overlapping cDNA fragments of segment A were synthesized and amplified in accordance with the supplier's protocol (Perkin-Elmer). The amplified fragments were cloned into the EcoRI site of the pCR2.1 vector (Invitrogen Corp.) to obtain plasmids pCR15A5' and pCR15A3', which were used for sequence analysis. Then, plasmids pCR15A5' and pCR15A3' were double digested with the restriction enzyme pairs XbaI plus KpnI and KpnI plus PstI to release 5'-end and 3'-end fragments. These fragments were then cloned between the XbaI and PstI sites of the pUC19 vector to obtain plasmid pUC19NVI15A. This plasmid contains a full-length copy of segment A, which encodes all the structural proteins (VP2-VP3), the NS protease (VP4), and the 12-kDa polypeptide (VP5) (Fig. 1.). To construct a cDNA clone of NVI-015 segment B, two pair of primers, B-B5'NC plus B-BIR and B-BIF plus B-Bgl3'NC, were synthesized and used for RT-PCR amplification (Table 2). The amplified fragments were cloned into the pCR2.1 vector as described above to obtain plasmids pCR15B5' and pCR15B3'. To construct a full-length cDNA clone of this segment, the 5'-end fragment of IPNV (from the plasmid pCR15B5' fragment) was first cloned into the EcoRI site of the pUC19 vector to obtain pUC15B5'. Then, the 3'-end fragment of IPNV (from plasmid pCR15B3') was inserted between the unique MfeI and SphI sites of plasmid pUC15B5' to obtain plasmid pUC19NVI15B, which encodes VP1 (Fig. 1).
The 5' end of NVI-020 segment A was also amplified by RT-PCR as described above, and the amplified fragment was cloned into pCR2.1 to obtain plasmid pCR20A5'. Plasmid pUC19NVI15-15K was prepared by replacing a BstEII fragment in plasmid pUC19NVI15A with the respective BstEII fragment derived from plasmid pCR20A5' (Fig. 1).
Two primer pairs (pUCNdeF plus A-VP5R and A-VP5F plus A-BstER) were designed to construct a mutant cDNA clone of segment A lacking the initiation codon of VP5. These primers were used for PCR amplification of the parent plasmid, pUC19NVI15A, which yielded DNA fragments of 353 bp and 463 bp, respectively. These fragments were combined and subsequently amplified by PCR, using the flanking primers (pUCNdeF and A-BstER) to produce an 816-bp fragment. This fragment was cloned into the pCR2.1 vector to obtain plasmid pCRVP5. This plasmid was digested with NdeI and BstEII, and the resulting fragment was cloned into appropriately cleaved pUC19NVI15A. The final mutant clone of segment A was designated pUC19NVI15-VP5 (Fig. 1).
DNA from the above-mentioned plasmids was sequenced by the dideoxy chain termination method, using an automated DNA sequencer (Applied Biosystems), and the sequence data were analyzed using PC/Gene (Intelligenetics) software. The integrity of the full-length constructs was tested by an in vitro transcription-translation-coupled reticulocyte lysate system using T7 RNA polymerase (Promega).
Transcription and transfection of synthetic RNAs. Plasmids pUC19NVI15A, pUC19NVI15-15K, and pUC19NVI15-VP5 were linearized by PstI, whereas, pUC19NVI15B was digested by BglII. Further treatments were carried out as described previously (52). The linearized DNA was used to produce in vitro transcripts with a T7 mMessage mMachine kit (Ambion) according to the manufacturer's instructions. Briefly, approximately 3 μg linearized DNA template was added to the transcription reaction mixture (20 μl) containing 40 mM Tris-HCl (pH 7.9); 10 mM NaCl; 6 mM MgCl2; 2 mM spermidine; 0.5 mM (each) ATP, CTP, and UTP; 0.1 mM GTP; 0.25 mM cap analogue [m7G (5') ppp (5') G], 120 units of RNasin, and 150 units T7 RNA polymerase and incubated at 37°C for 90 min.
CHSE-214 cells, grown to 90% confluence in a T-25 flask, were transfected with cRNAs of both segments as described previously (52). Briefly, the cells were washed once with phosphate-buffered saline (PBS). Three milliliters of OPTI-MEM I (GIBCO/BRL) was added to the monolayer, and the cells were incubated at room temperature for 1 h. Simultaneously, 0.15 ml of OPTI-MEM I was incubated with 12.5 μg of Lipofectin reagent for 45 min in a polystyrene tube at room temperature. Equimolar amounts of RNA transcripts of segments A and B (8 μg each), resuspended in 0.15 ml of diethyl pyrocarbonate-treated water, were added to the OPTI-MEM-Lipofectin mixture, mixed gently, and incubated on ice for 5 min. After the OPTI-MEM I was removed from the monolayers in the T-25 flask and replaced with 1.5 ml of fresh OPTI-MEM, the nucleic acid-containing mixture was added dropwise to the CHSE cells and swirled gently. After 3 h of incubation at room temperature, the mixture was replaced with minimal essential medium containing Hanks' salts and 10% FBS (without rinsing the cells). The cultures were incubated at 15°C for 5 days, and the cell supernatant was harvested by freeze-thawing twice and passed onto fresh CHSE monolayers. Cytopathic effect (CPE) was usually visualized 4 days after the second pass. All the recombinant viruses were then passed into CHSE cells once more, the cells were freeze-thawed twice, and the cell-free supernatants were stored at –70°C for use as virus stocks in this work. The identities of recovered viruses were verified by sequencing the DNA products obtained after RT-PCR amplification, using specific primers for IPNV segment A.
Immunofluorescence. Infection of CHSE cells by the recombinant viruses was analyzed by immunofluorescence assay using rabbit anti-VP5 specific serum. Briefly, CHSE cells were infected with IPNV at a multiplicity of infection (MOI) of 1.0 and incubated at 15°C. At 16 h postinfection (p.i.), the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were permeabilized with 0.1% Triton 100 for 10 min at room temperature and then incubated with anti-VP5 diluted 1:100 in PBS containing 2% bovine serum albumin for 1 h. After being washed with PBS, the cells were treated with fluorescein-labeled goat anti-rabbit antibody at 1:1,000 dilution (Kirkegaard & Perry Laboratories) and examined by fluorescence microscopy.
Growth curve and plaque assay. To analyze the growth characteristics of IPNV, confluent CHSE or RTG-2 cells (in 35-mm dishes) were infected with the recombinant viruses at an MOI of 1.0. Aliquots collected at various time points were stored at –70°C. The supernatants were centrifuged and titrated on CHSE cells by plaque assay. Briefly, confluent monolayers of CHSE cells grown in six-well plates were infected with serially diluted supernatants from virus stock. After 1 h of incubation at 15°C, the cells were washed once with PBS and overlaid with 0.6% SeaPlaque Agarose (Difco) in Eagle's minimal essential medium containing 5% FBS. After 3 days of incubation at 15°C, the overlays were removed and the cells were fixed and stained with a solution containing 25% formalin, 10% ethanol, 5% acetic acid, and 1% crystal violet for 5 min at room temperature. After the cells were rinsed with distilled water, the plaques were counted.
Analysis of viral protein expression. To analyze viral protein synthesis, RTG-2 cells grown in 60-mm dishes were mock infected or infected with rNVI15, rNVI15-15K, or rNVI15-VP5 at an MOI of 1.0. The infected cells were incubated at 15°C until 8, 16, 24, and 32 h p.i. At the completion of each incubation period, the culture medium was aspirated, and the cells were washed with PBS and lysed. Proteins in the obtained cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 5% stacking and 12.5% resolution gels, and proteins were electrophoretically transferred to a nitrocellulose membrane. The membrane was probed with a polyclonal rabbit anti-IPNV serum, and virus-specific proteins were detected with streptavidin-alkaline phosphatase and naphtholphosphate fast red color development reagents.
Virulence study of Atlantic salmon postsmolts. The three recombinant isolates rNVI15, rNVI15-15K, and rNVI15-VP5 were propagated by inoculation of the second-passage supernatant onto CHSE-214 cells. The cell culture supernatant was obtained after a brief centrifugation, and the infectious titer was determined by end point dilution on CHSE cells grown in 96-well plates. The 50% tissue culture infective dose (TCID50) was estimated by the method of K?rber (30). The challenge was conducted at VESO Vikan's research facility in Namsos, Norway. Approximately 2,200 Atlantic salmon (Salmo salar L.) parr of an average weight of 70 g going through smoltification at the time of challenge were included in this study. The fish were obtained from a commercial supplier (AquaGen) at the eyed-egg stage and kept at VESO Vikan's commercial hatchery until smoltification. There were no records of disease during the freshwater period. The fish were exposed to 24 h of daylight the last 7 weeks before challenge and were tested for seawater tolerance before transportation to the research station 5 days before challenge. The smolts were randomly divided into 11 tanks 1 m in diameter, with 200 smolts in each tank. The day before challenge, the water supply was changed from freshwater to seawater. The fish were challenged by 1-hour immersion with IPNV at a concentration of 104 TCID50/ml in a total of 200 liters of water. Fish in three parallel tanks were infected with each of the three IPNV strains, while fish in two parallel tanks were given cell culture medium only and kept as controls. One tank in each group was reserved for sampling, from which three fish were collected each day, and organ specimens of the pancreas, liver, stomach, and kidney were fixed in 10% phosphate-buffered formalin. Mortality was recorded on a daily basis in the remaining tanks. The experiment was terminated at 30 days postchallenge. Five fish from every group were humanely killed, and kidney samples were collected on the last day of challenge. The samples were stored at –70°C before preparation of a tissue homogenate (10% [wt/vol] in Leibovitz's L-15 medium with 50 μg ml–1 gentamicin) and inoculation onto CHSE cells to check if IPNV could be recovered. The samples were considered negative when no CPE was observed after two passages. The same procedure was employed to check if virus could be recovered from control fish and from fish sampled prior to challenge. Formalin-fixed tissue samples collected at peak mortality were stained with hematoxylin and eosin and examined by light microscopy. Immunohistochemical detection of IPNV antigen in parallel sections was performed as previously described (43).
Persistence study in Atlantic salmon fry. Fifteen hundred Atlantic salmon fry were obtained as eyed eggs from a commercial supplier (AkvaGen) and held at VESO Vikan's hatchery until the start of feeding. There were no records of disease during or after hatching. Thirty fry (average weight, 0.1 g) were sampled and frozen at –70°C. Before challenge, these fry were tested by RT-PCR to ensure that they were IPNV negative. The fry were then moved to VESO Vikan's research facility and randomly distributed into six tanks of 250 fry each. After 1 week of acclimatization, the fry were challenged with IPNV strains Sp103, Sp103-VP5, rNVI15, rNVI15-15K, and rNVI15-VP5 or mock infected with cell culture medium (control group). The viruses had been propagated by inoculation of the third-passage supernatant onto CHSE cells and quantified by titration as described above. Challenge was performed at a dose of 105 TCID50/ml of IPNV at a total volume of 4 liters per tank. The water was aerated during the challenge, and after 3 h, normal flow was resumed. The water temperature was approximately 13°C throughout the experiment. Mortality was recorded on a daily basis, and dead fish were collected and frozen at –70°C. Ten dead fry from each group were sampled at the height of infection and fixed by immersion in 10% phosphate-buffered formalin, and sagittal sections of whole fry were subsequently prepared for histopathological examination and IPNV antigen detection by immunohistochemistry. From 5 weeks p.i., 10 fry in each group were humanely killed every other week and frozen individually at –70°C. The experiment was terminated 5 months after challenge. For detection of IPNV in persistently infected fish, whole fry were homogenized 1:5 (wt/vol) in PBS using a stomacher. One hundred microliters of this homogenate was transferred to RLT buffer containing 2-mercaptoethanol (RNeasy Mini kit; QIAGEN) and stored at –70°C. The rest of the homogenate was diluted by adding 5 volumes of Leibovitz's L-15 medium supplemented with 50 μg ml–1 gentamicin, and the supernatant was obtained after a brief centrifugation. The tissue homogenates were inoculated onto CHSE-214 cells grown in 24-well tissue culture plates in final dilutions of 1 and 0.1%, and incubated for 1 week at 15°C. The cell culture medium after first passage was used to infect new monolayers. The samples were considered negative when no CPE was observed after 1-week incubation of the second passage. RNA was isolated from all negative samples using the RNeasy Mini Kit in accordance with the supplier's protocol (QIAGEN), and RT-PCR was performed to amplify a 224-bp IPNV-specific DNA fragment, as described by Taksdal et al. (49) with minor modifications. QIAGEN's OneStep RT-PCR kit was used according to the manufacturer's instructions, with 0.5 μg RNA and 15 pmol each of primers IPNV1 and IPNV2 (Table 2) in a total reaction volume of 25 μl. The cycling conditions were 60°C for 30 min and 95°C for 15 min, followed by 45 cycles at 94°C for 45 s, 57°C for 45 s, and 72°C for 1 min and finally 72°C for 10 min. The PCR products were visualized by agarose gel electrophoresis. Three controls were included for each RT-PCR run: one positive and one negative tissue sample and one negative control in which water was substituted for RNA.
Sequencing of the viral genome from persistently infected fish. Cell culture supernatant after one passage in CHSE cells was harvested from one positive fish in each group at the 13-week time point. In addition, cell culture supernatants were harvested after inoculation of RTG-2 cells with tissue homogenates of three fish each from tanks infected with rNVI15, rNVI15-15K, and rNVI15-VP5 sampled at 2 and 19 weeks. Viral genomic RNA was isolated using a QIAamp Viral RNA Mini Kit in accordance with the protocol supplied by the manufacturer. A segment of the viral genome coding for VP5 and VP2 was subsequently amplified by RT-PCR, using primers A-A5'NC and Sp1689R (Table 2). The RT-PCR products were purified and sent to a commercial company (MWG Biotech AG) for sequencing. The sequence data were analyzed using BioEdit software (23).
Nucleotide sequence accession numbers. Sequences for Sp103-VP5 have been dpeosited in the GenBank database as AY823632 and AY823633.
RESULTS
Construction of full-length cDNA clones. To determine the role of VP5 in virulence, we constructed full-length cDNA clones of segments A and B of IPNV Sp strain NVI15 as shown in Fig. 1. Using the parental clone pUC19NVI15A as a backbone, two mutant cDNA clones of NVI15 segment A were prepared. In plasmid pUC19NVI15-15K, a BstEII fragment (nucleotides 24 to 586) of the parental plasmid was replaced with a similar BstEII fragment of IPNV strain NVI-020, which does not contain a premature stop codon at nt 427 (TGACGA) (43). Thus, this chimeric clone encodes a 15-kDa VP5 but does not contain any additional mutation in either VP2 or VP5. Previously, we showed that one of the field isolates, NVI-010, does not encode the VP5 protein, as it contains ATA instead of an ATG initiation codon (43). Using site-directed mutagenesis, we prepared a plasmid, pUC19NVI15-VP5, which lacks the initiation codon for VP5. The functionalities of all these clones were tested by in vitro transcription-coupled translation reactions, which yielded protein products that comigrated with the marker IPNV proteins after separation on SDS-PAGE and autoradiography (data not shown).
Transfection and recovery of the mutant IPNVs. In order to recover the recombinant viruses, we transfected CHSE cells with combined plus-sense transcripts of segments A and B derived from various plasmids, as described previously (52). As expected, rNVI15, rNVI15-15K, and rNVI15-VP5 were rescued from the transcripts derived from wild-type or modified segment A and NVI15 segment B. To verify that the mutant viruses were indeed recovered from cRNAs, the genomic RNA was isolated and analyzed by sequencing the DNA products obtained after RT-PCR amplification, using a primer pair specific for segment A. Sequence analysis of the cloned PCR products confirmed the expected nucleotide mutations in the mutant viruses. To detect expression of VP5, CHSE cells were infected with the recovered viruses and analyzed by immunofluorescence assay using VP5-specific antiserum. Cells infected with rNVI15 and rNVI15-15K gave positive immunofluorescence signals that confirmed the expression of VP5. The fluorescence was seen in the cytoplasm, mainly within the perinuclear region (Fig. 2b and c). Cells infected with rNVI15-VP5 did not give any fluorescence signal, nor did mock-infected cells (Fig. 2a and d).
Characterization of the recombinant viruses in vitro. To compare the replication kinetics of the recovered viruses, both CHSE and RTG-2 cells were infected with these viruses at an MOI of 1.0. At the indicated time points, the supernatants from infected cells were collected and titrated in CHSE cells by plaque assay. Figure 3 depicts the growth curves of rNVI15, rNVI15-15K, and rNVI15-VP5 in CHSE (Fig. 3A) and RTG-2 (Fig. 3B) cells, respectively. These viruses grew to similar titers in CHSE cells, which is in accord with the results of Weber and coworkers, who could not detect any difference in the growth of VP5-deficient mutants and the wild-type IPNV strain VR-299 (51). The recovered viruses showed a slight delay in growth in CHSE cells compared to RTG-2 cells. Cytopathic effects appeared earlier in RTG-2 cells than in CHSE cells, but the final titers of all viruses were 1 log unit higher in CHSE cells than in RTG-2 cells.
The protein expression profiles of RTG-2 cells infected with the different recombinant IPNVs were compared, but no difference in virus-specific protein expression was detected (Fig. 4).
Virulence study in Atlantic salmon postsmolts. To assess the virulence and the pathogenic phenotype of the recovered viruses in fish, Atlantic salmon postsmolts were infected or mock infected with either rNVI15, rNVI15-15K, or rNVI15-VP5 viruses. Ten days after challenge, fish started dying in all infected groups. A parallel course of mortality was recorded for these viruses, and at the conclusion of the study 30 days postchallenge, the cumulative mortality had reached 81, 81, and 86% for rNVI15, rNVI15-15K, and rNVI15-VP5 viruses, respectively (Fig. 5). In the control group, mortality was 9%. Virus was recovered from all five fish challenged with rNVI15-15K and rNVI15-VP5 and from four out of five fish challenged with rNVI15 but not from the five control fish examined. Histopathological examination demonstrated the characteristic lesions of IPN, with necrosis of hepatic and exocrine pancreatic tissues (data not shown). The presence of virus within lesions was verified by immunohistochemical detection of IPNV antigen in sections done in duplicate. There were no pathological changes and no positive detection of viral antigen in control fish. Viral genomic RNA was extracted directly from fish challenged with the three recombinant viruses and amplified with a primer pair encompassing the VP5 gene. The nucleotide sequences of the resulting PCR products were determined, which confirmed the mutations in the VP5 gene for the two mutant viruses and a lack of mutation in the unmodified recombinant virus.
Persistence study in Atlantic salmon fry. To evaluate the role of VP5 in persistent IPNV infection and to examine if there are differences among high- and low-virulence viruses in the ability to induce persistent infections, Atlantic salmon fry were challenged with five IPNV strains and observed for the following 5 months. Viruses rNVI15, rNVI15-15K, and rNVI15-VP5 are derived from the virulent field strain NVI-015 and encode 12-kDa, 15-kDa, and no VP5 proteins, respectively. Sp103 is a low-virulence field strain encoding a 15-kDa VP5, whereas Sp103-VP5 is a VP5-deficient variant isolated by plaque purification of Sp103. Figure 6 shows cumulative mortality in fry challenged with different viruses. During the second week after challenge, mortality started to increase in all infected groups. At this time point, IPN characteristic lesions were detected in dead fry sampled from all challenged groups, but not in the control group. The presence of virus within the lesions was verified by immunohistochemistry (data not shown). From the third week p.i., the mortality in fry decreased and then remained low throughout the rest of the study (Fig. 6). From 5 weeks p.i. on, 10 fish from each group were sampled every other week for the detection of IPNV carriers by inoculation in cell culture and RT-PCR techniques. No IPNV-positive fish were detected in the control group at any time point (Fig. 7). At the first sampling (5 weeks), all challenged fish were IPNV positive. Subsequent samplings showed that most of the Sp103-VP5- and rNVI15-VP5-infected fish became carriers, and IPNV was detected in all 10 fish examined 19 weeks after challenge. Most of the fish infected with rNVI15 and rNVI15-15K also remained IPNV carriers, as 8 out of 10 fish examined were virus positive after 19 weeks. Only in the group infected with Sp103 was a decline in IPNV-positive fish observed, and at 19 weeks p.i., none of the 10 fish examined were carriers. These results indicate variation among the strains in the ability to establish persistent infection.
Sequencing of the viral genome from persistently infected fish. Viral RNA was isolated from CHSE-214 cells inoculated with tissue homogenates from fish sampled 13 weeks after challenge and was used for RT-PCR amplification of a segment covering the VP5- and VP2-encoding region. For the field viruses Sp103 and Sp103-VP5, no differences in the sequence were observed from fish sampled at 13 weeks postchallenge. In the case of recombinant viruses, an AlaThr substitution at amino acid 221 of VP2 was detected. Therefore, viral RNA was isolated from RTG-2 cells inoculated with tissue homogenates from fish sampled both 2 and 19 weeks after challenge and used for RT-PCR amplification of a segment covering amino acid 221 of VP2. The obtained sequence was identical to the input virus sequence during the acute phase of infection (2 weeks postchallenge), as expected (Table 3). However, a single amino acid substitution at position 221 of VP2 (AlaThr) was detected in all three viruses recovered from fish sampled at 19 weeks postchallenge, suggesting that in the carrier state, the dominant virus has a Thr residue at this position (Table 3).
DISCUSSION
The reverse-genetics system for IPNV was first developed in 1998 and has been applied to in vitro studies of viral replication (51, 52). This report describes for the first time challenge experiments in both fry and postsmolts using recovered IPNVs of Sp serotype to evaluate the role of the VP5 nonstructural protein.
Previous studies have shown that the expression of VP5 in VR299 and Sp strains is initiated from the second in-frame start codon, yielding a 15-kDa protein (45, 51). However, results from our laboratories indicate that Sp strains of low virulence encode a 15-kDa protein, whereas more virulent strains often contain a truncated VP5 protein gene (43, 45). In order to study the expression of this 12- to 15-kDa VP5 protein, we constructed infectious cDNA clones of the virulent field stain NVI-015 and generated VP5 mutant viruses, one encoding a truncated 12-kDa VP5 and a second encoding a full-length VP5, whereas the third was deficient in VP5 expression. The rescue of the recombinant IPNVs was verified by sequencing of the DNA products obtained after RT-PCR amplification and by immunofluorescence assay using anti-15-kDa specific antibody. The growth kinetics of the VP5 mutant viruses in CHSE and RTG-2 cells were similar, which confirmed that VP5 does not affect the rate of replication and that it is dispensable for viral replication. These results are in agreement with those of Weber and coworkers, who also reported similar findings in CHSE cells (51).
The virulence characteristics of the recovered viruses were subsequently tested by challenging Atlantic salmon postsmolts, which is technically more demanding than challenging fry. In Norway, IPN outbreaks in seawater are economically more important than freshwater outbreaks in fry. Melby and colleagues (36) concluded that all commercial seawater salmon farms in Norway harbor IPNV carriers. According to national surveys, outbreaks are seen in 40 to 70% of all seawater sites every year. In addition, extensive sequencing of Norwegian IPNV field isolates carried out in our laboratories has shown that strains closely related to NVI-015 dominate in postsmolt outbreaks of IPN. The experimental challenge gave cumulative mortalities above 80% for the three genetically engineered viruses, which is relatively high compared to what was found in previous postsmolt IPN cohabitation challenges (3, 48). The high mortality figures were not solely due to the use of recovered IPNV strains, since two additional field strains were included in the study, ending with 76 and 86% cumulative mortalities (data not shown). This study demonstrates that Atlantic salmon postsmolt IPNV challenges can be successfully performed with bath exposure. Among the fish infected with rNVI15, rNVI15-15K, and rNVI15-VP5 viruses, 81, 81, and 86% succumbed to IPNV infection, respectively. The results clearly indicate that VP5 is not required for viral replication in vivo and that all VP5 variant strains are able to induce IPN disease. It can also be concluded that VP5 has no function as a virulence factor, since rNVI15-VP5 was no different from the other strains. The VP5-deficient mutant gave a slightly higher mortality than the VP5-producing strains, although the difference was not statistically significant.
The slightly higher mortality figures observed in the rNVI15-VP5-challenged fish could indicate a certain replicative advantage of VP5-deficient strains over VP5-encoding strains. The absence of the small upstream VP5 ORF might affect the translation efficacy of the major ORF encoding the polyprotein. Translation initiation site selection is determined in part by the context of the nucleotide sequence surrounding the first AUG codon encountered by the scanning ribosomal unit (31). The initiation codon (uppercase) of the small segment A ORF starting at nt 112 is in a weak context (34, 51) compared to the large ORF starting at nt 119 (NVI-015 nt 106 to 122: auaucaAUGcaagAUGa). As a consequence, the scanning ribosome will probably initiate translation from this weak AUG at a low frequency compared to the stronger downstream AUG of the large ORF encoding the polyprotein. This mechanism, referred to as leaky scanning, enables the virus to maximize its genome coding capacity by encoding functionally distinct proteins from a common mRNA (19). Consequently, loss of the small ORF could lead to increased translational efficiency of the polyprotein, again resulting in slightly increased replication of VP5-deficient IPNV strains. However, the virtually overlapping growth curves of the VP5-encoding and VP5-deficient viruses obtained from two different cell lines does not lend support to an idea of replicative advantage of VP5-deficient strains. In addition, the viral capsid proteins VP2 and VP3 (encoded by the large ORF) appeared simultaneously and in comparable amounts in cells infected with VP5-encoding or VP5-deficient strains.
In the second challenge study, acute mortality measured at 5 weeks p.i. ranged from 5 to 27%. The low mortality in this experiment could result from higher genetic resistance to IPN in the challenged fish (39, 40). The fry were provided by AquaGen, a breeding company that has implemented selection for increased genetic resistance of Atlantic salmon to IPN since 2001 (A. Storseth, personal communication). Salmon full-sibling families have been tested for IPN resistance since 1997, demonstrating that mortalities range from 0 to 96% between families (A. Storseth, T. Aasmundstad, K. O. Ali, S. Kj?glum, S. A. Korsvoll, B. H?yheim, and A. Guttvik, poster presentation at Aquaculture Europe 2003, Trondheim, Norway, 8 to 12 August 2003). Again, the mortality in the group of fish infected with rNVI015-VP5 (27%) was slightly higher than in fish infected with rNVI15-15K (18%) and rNVI15 (20%). However, this experiment was designed to study the establishment of persistent infections and not to compare virulence characteristics for different strains. As a consequence, fish were infected in a single tank for each virus strain, and frequent samplings were performed. Statistical comparison of mortality figures is therefore not relevant.
VP5 was found to be dispensable for virus virulence, but it is still reasonable to believe that the protein has a specific function, particularly since the majority of IPNV strains encode the protein (25). As described by Hong and colleagues (26), IPNV VP5 has Bcl-2 homology domains and is antiapoptotic, delaying apoptotic cell death in the early replication cycle of IPNV infection. The early cell response to viral infection involves a multitude of reactions, including induction of apoptosis in infected cells, and viral inhibition of apoptosis could allow sustained viral replication in cells that are destined to commit suicide. Several viruses encode Bcl-2-homologous proteins, and although their physiological functions are not completely elucidated, they might be involved in the establishment of persistent infections (6, 20). To investigate whether IPNV VP5 is involved in the development of a carrier state, Atlantic salmon fry were infected with the virulent recombinant viruses rNVI15, rNVI15-15K, and rNVI15-VP5, along with two field strains of low virulence, Sp103 and Sp103-VP5. Five months after challenge, all examined fry infected with the two VP5-deficient mutant strains were IPNV carriers, and 80% of the fish infected with rNVI15 and rNVI15-15K were carriers. The results suggest that the establishment of persistent IPNV infection is independent of VP5 expression. It is not possible to develop conclusions about the effect on the length of the carrier state, since the experiment was terminated 5 months after challenge, and it is anticipated that IPNV-infected fish become life-long carriers.
In the group of fish infected with IPNV strain Sp103, there was a steady decline in the number of IPNV-positive fish, and at 19 weeks after challenge, no carriers were detected. Interestingly, although this strain encodes Ala at residue 221 of VP2, it seems to be less prone to mutate to Thr at this position following cell culture passage. Even after 10 passages in CHSE cells, approximately half the virus population still encoded Ala (47a). All three NVI-015-derived strains gave an AlaThr mutation after a few passages in CHSE cells, originally thought to be a cell culture adaptation phenomenon. The accumulation of this mutation in persistently infected fish was thus an unexpected finding. It may imply that Thr at VP2 residue 221 is a prerequisite for persistent infection and that Sp103, encoding a genetically relatively stable Ala at this position, is less able to establish long-term persistent infection. Sp103-VP5, on the other hand (encoding Thr at position 221), induced a carrier state in 79 of 80 fish examined. To the best of our knowledge, this is the first finding of a molecular determinant of persistent IPNV infection.
RNA viruses, due to the absence or the low efficiency of proofreading activities associated with RNA polymerases, have a high mutation rate during genome replication and are thought to replicate as complex and dynamic mutant swarms called viral quasispecies (11). For hepatitis C virus (HCV), quasispecies appearance is associated with evasion of the immune response and progression to chronicity, whereas genetic stability is linked to self-limiting disease (16). The emergence of virus mutants with Thr at position 221 of VP2 could be a result of selective pressure on the viral population by the immune response on epitopes recognized either by neutralizing antibodies or by cytotoxic T lymphocytes. Indeed, residue 221 lies within the central variable domain of VP2 containing the major conformational epitopes recognized by neutralizing monoclonal antibodies (18, 50). Another possibility to consider is selection due to different cell tropism in acute infection (exocrine pancreatic and liver tissue) (43) compared to persistent infection (macrophages) (38). A preferential tropism of certain viral variants for specific cell types could explain the selection of this mutant in CHSE cells, a phenomenon not observed in RTG-2 cells (47a). Again, for HCV, it was recently demonstrated that dendritic cells can be an extrahepatic reservoir of the virus and that the HCV genomic sequence derived from dendritic cells differed from that found in the plasma of the same patient (21). We have done some further experiments demonstrating that the AlaThr substitution at position 221 of VP2 leads to attenuation of the virus (Song et al., submitted). The persistence of this attenuated virus in covertly infected fish is interesting, since reemergence of the virulent variants in the carrier fish could be involved in stress-mediated recurrence of IPN (42, 48).
Having established that VP5 is not essential for virulence or for the establishment of persistent IPNV infection, the natural follow-up would be to perform in vitro and in vivo studies of the apoptosis-inducing effects of the VP5 variant viruses to confirm the apoptosis-inhibiting effect of VP5 proposed by Hong and colleagues (26). Furthermore, the most probable candidate for causing differences in virulence among closely related isolates of the Sp serotype has been mapped to the VP2 protein (43, 45), which has been confirmed using reverse genetics (47a).
ACKNOWLEDGMENTS
This study was supported by Norwegian Research Council grant 134136/120 to ?.E. and by National Research Initiative of the USDA Cooperative State Research, Education, and Extension Service grant 2001-35204-10065 to V.N.V.
We thank A. Ramstad, study director for the challenge experiments at VESO Vikan, Namsos, Norway. Thanks also go to Gerard H. Edwards at the Center for Biosystems Research, University of Maryland, for technical assistance.
Nina Santi and Haichen Song contributed equally to this work.
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Center for Biosystems Research, University of Maryland Biotechnology Institute
Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland 20742
Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Sciences, 0033 Oslo, Norway
ABSTRACT
Infectious pancreatic necrosis virus (IPNV) is the causative agent of infectious pancreatic necrosis (IPN) disease in salmonid fish. Recent studies have revealed variation in virulence between isolates of the Sp serotype, associated with certain residues of the structural protein VP2. The isolates are also highly heterogenic in the coding region of the nonstructural VP5 protein. To study the involvement of this protein in the pathogenesis of disease, we generated three recombinant VP5 mutant viruses using reverse genetics. The "wild-type" recombinant NVI15 (rNVI15) virus is virulent, having a premature stop codon at nucleotide position 427, putatively encoding a truncated 12-kDa VP5 protein, whereas rNVI15-15K virus encodes a 15-kDa protein. Recombinant rNVI15-VP5 virus contains a mutation in the initiation codon of the VP5 gene that ablates the expression of VP5. Atlantic salmon postsmolts were challenged to study the virulence characteristics of the recovered viruses in vivo. The role of VP5 in persistent infection was investigated by challenging Atlantic salmon fry with the recovered viruses, as well as with the low-virulence field strain Sp103 and a naturally occurring VP5-deficient mutant of Sp103. The results show that VP5 is not required for viral replication in vivo, and its absence does not alter the virulence characteristics of the virus or the establishment of persistent IPNV infection.
INTRODUCTION
Infectious pancreatic necrosis virus (IPNV) is a well-known cause of acute contagious disease in salmonid fish. The mortality of an outbreak varies considerably, depending on the species, age, and physical condition of the fish, as well as the virulence of the viral strain (4, 17, 40, 46). Only Sp strains are reported to cause disease in seawater-adapted salmonid fish (postsmolts) (28, 47). A high percentage of infected fish become long-term carriers of the virus (2, 35). In acutely diseased salmonids, IPNV induces lesions principally in exocrine pancreatic tissue, while in persistently infected fish, the virus is found in macrophages (29, 38). So far, little is known about the molecular and immunological mechanisms involved in the establishment of an IPNV carrier state.
IPNV is the prototype virus of the family Birnaviridae and belongs to the genus Aquabirnavirus (10). The genome consists of two segments of double-stranded RNA packaged in a nonenveloped icosahedral shell 60 nm in diameter (7). The smaller genomic segment, B, is 2,784 nucleotides (nt) long and encodes VP1, the virion-associated RNA-dependent RNA polymerase (9, 14). The larger segment, A, is 3,097 nucleotides long and encodes a 107-kDa precursor protein in a single large open reading frame (ORF), which is cotranslationally cleaved by the viral nonstructural (NS) protease, VP4, generating VP2 and VP3 structural proteins (8, 12, 13, 34, 41). Segment A also encodes a 15-kDa arginine-rich protein from a small ORF partly preceding and overlapping the polyprotein ORF (24, 25). This protein, designated VP5, has been detected in virus-infected cells (34), but so far there is no evidence of the protein being found in purified virions. It has recently been shown that the initiation of translation for VP5 starts at the second in-frame start codon in the Sp and VR-299 strains (45, 51), and the absence of expression does not influence virus growth in vitro (51). The existence of field strains lacking the initiation codon for VP5 protein is also well documented (25, 43).
We have recently characterized the virulence of nine IPNV serotype Sp strains by challenging Atlantic salmon (Salmo salar L.) fry (43). Some strains were highly virulent, causing up to 90% mortality and severe necrosis of the liver and pancreas of infected fry, while other strains produced low mortality (up to 6%) and no detectable lesions. Nucleotide sequencing of the viral genomes revealed heterogeneity in the open reading frame encoding VP5 and in certain VP2 amino acids potentially associated with the virulence of the virus. Four of the strains encoded the full-length 15-kDa protein, while one strain had a mutation in the start codon of the VP5 protein. Three strains had premature stop codons at nt 427, encoding a putative 12-kDa VP5, and yet another strain had a premature stop codon at nt 496; all strains encoding a truncated form of VP5 were virulent.
IPNV is known to induce apoptosis in infected CHSE-214 cells (27). The suicide of a cell in response to viral infection is postulated to be an important mechanism of host defense (15). Viruses have evolved various mechanisms to evade the apoptotic response of the host cell (1), and some viruses encode homologues of cellular antiapoptotic Bcl-2 proteins (vBcl-2s) (6). The Bcl-2 family of intracellular proteins includes both pro- and antiapoptotic molecules and plays a pivotal role in regulation of apoptotic processes (5, 22). IPNV VP5 contains Bcl-2 homology domains, and overexpression in CHSE-214 cells enhances host cell viability following IPNV infection, likely because of antiapoptotic activity and by limiting viral protein expression (26). However, the physiologic role of this protein during infection is unknown, and there are no reports of in vivo studies of IPNV VP5-deficient mutants.
For Sindbis virus, Semliki Forest virus, and Japanese encephalitis virus, it has been shown that Bcl-2 overexpression facilitates persistent in vitro infection (32, 33, 44). The in vivo role of a viral Bcl-2 was recently identified, as -herpesvirus 68 vBcl-2 was found to be dispensable for acute replication and disease but important for persistent replication (20). Since IPNV VP5 possesses Bcl-2 homology domains, it is conceivable that this protein may play a role in persistent infection.
A reverse-genetics technique for the Birnaviridae was first described by Mundt and Vakharia (37), using synthetic RNAs derived from cDNA clones to generate infectious bursal disease virus. The same approach was employed to recover IPNV from transfected CHSE cells (52). Genetically engineered IPNV VP5 mutants of the VR-299 serotype have been studied in vitro (51), demonstrating that the absence of this protein does not influence replication in cell culture. In this paper, we describe the generation of three VP5 mutants of a virulent IPNV of the Sp serotype using reverse genetics and the first application of this technique to study the function of VP5 protein in IPNV virulence and persistence in vivo.
MATERIALS AND METHODS
Cells, viruses, and serum. Chinook salmon embryo cells (CHSE-214; ATCC CRL-1681) were used for transfection experiments and for detection, propagation, and quantification of virus. The cells were maintained at 20°C in minimal essential medium containing Hanks' salts and supplemented with 10% fetal bovine serum (FBS) (transfection experiments) or in Leibovitz's L-15 medium supplemented with 5% FBS and 50 μg ml–1 gentamicin (challenge studies). Rainbow trout gonad (RTG-2) cells (ATCC CCL-55) were grown in L-15 medium supplemented with 10% FBS at 15°C. IPNV field isolates Sp103, Sp103-VP5, NVI-015, and NVI-020, which belong to the Sp serotype, were used in this study. NVI-015 and NVI-020 cause high mortality in Atlantic salmon fry challenge, while Sp103 (also called NVI-025) is less virulent (43). Analysis of the small ORFs showed that Sp103 and NVI-020 encode the full-length 15-kDa VP5 while NVI-015 encodes a putative truncated 12-kDa VP5 (Table 1). A naturally occurring VP5-deficient virus, Sp103-VP5, was isolated by plaque purification of Sp103 (Table 1). The complete nucleotide sequence of segment A has been determined for all isolates, and the VP1-encoding sequence of segment B has been determined for NVI-015, Sp103, and Sp103-VP5 (GenBank accession no. AY379740 and AY379741 for NVI-015, AY379736 for NVI-020, AY354519 and AY354520 for Sp103, and AY823632 and AY823633 for Sp103-VP5).
To prepare monospecific antiserum against VP5, a peptide corresponding to the N-terminal part of the protein (NH2-20RDWTSKHPGRHNGETHLKT38-COOH) was custom synthesized (Bio-Synthesis) and used for repeated immunization of a rabbit to obtain anti-VP5 antibody.
Construction of full-length cDNA clones. Construction of full-length cDNA clones containing the entire coding and noncoding regions of IPNV NVI-015 RNA segments A and B was performed by standard cloning procedures as described previously (52). On the basis of the published IPNV sequence of the Sp strains, several primer pairs were synthesized and employed in reverse transcription (RT)-PCR amplifications (Table 2). To generate cDNA clones of segment A of NVI-015, two primer pairs (A-A5'NC plus A-KpnR and A-KpnF plus A-SpPstR) were used for RT-PCR amplification (Table 2). Using genomic RNA as a template, the desired overlapping cDNA fragments of segment A were synthesized and amplified in accordance with the supplier's protocol (Perkin-Elmer). The amplified fragments were cloned into the EcoRI site of the pCR2.1 vector (Invitrogen Corp.) to obtain plasmids pCR15A5' and pCR15A3', which were used for sequence analysis. Then, plasmids pCR15A5' and pCR15A3' were double digested with the restriction enzyme pairs XbaI plus KpnI and KpnI plus PstI to release 5'-end and 3'-end fragments. These fragments were then cloned between the XbaI and PstI sites of the pUC19 vector to obtain plasmid pUC19NVI15A. This plasmid contains a full-length copy of segment A, which encodes all the structural proteins (VP2-VP3), the NS protease (VP4), and the 12-kDa polypeptide (VP5) (Fig. 1.). To construct a cDNA clone of NVI-015 segment B, two pair of primers, B-B5'NC plus B-BIR and B-BIF plus B-Bgl3'NC, were synthesized and used for RT-PCR amplification (Table 2). The amplified fragments were cloned into the pCR2.1 vector as described above to obtain plasmids pCR15B5' and pCR15B3'. To construct a full-length cDNA clone of this segment, the 5'-end fragment of IPNV (from the plasmid pCR15B5' fragment) was first cloned into the EcoRI site of the pUC19 vector to obtain pUC15B5'. Then, the 3'-end fragment of IPNV (from plasmid pCR15B3') was inserted between the unique MfeI and SphI sites of plasmid pUC15B5' to obtain plasmid pUC19NVI15B, which encodes VP1 (Fig. 1).
The 5' end of NVI-020 segment A was also amplified by RT-PCR as described above, and the amplified fragment was cloned into pCR2.1 to obtain plasmid pCR20A5'. Plasmid pUC19NVI15-15K was prepared by replacing a BstEII fragment in plasmid pUC19NVI15A with the respective BstEII fragment derived from plasmid pCR20A5' (Fig. 1).
Two primer pairs (pUCNdeF plus A-VP5R and A-VP5F plus A-BstER) were designed to construct a mutant cDNA clone of segment A lacking the initiation codon of VP5. These primers were used for PCR amplification of the parent plasmid, pUC19NVI15A, which yielded DNA fragments of 353 bp and 463 bp, respectively. These fragments were combined and subsequently amplified by PCR, using the flanking primers (pUCNdeF and A-BstER) to produce an 816-bp fragment. This fragment was cloned into the pCR2.1 vector to obtain plasmid pCRVP5. This plasmid was digested with NdeI and BstEII, and the resulting fragment was cloned into appropriately cleaved pUC19NVI15A. The final mutant clone of segment A was designated pUC19NVI15-VP5 (Fig. 1).
DNA from the above-mentioned plasmids was sequenced by the dideoxy chain termination method, using an automated DNA sequencer (Applied Biosystems), and the sequence data were analyzed using PC/Gene (Intelligenetics) software. The integrity of the full-length constructs was tested by an in vitro transcription-translation-coupled reticulocyte lysate system using T7 RNA polymerase (Promega).
Transcription and transfection of synthetic RNAs. Plasmids pUC19NVI15A, pUC19NVI15-15K, and pUC19NVI15-VP5 were linearized by PstI, whereas, pUC19NVI15B was digested by BglII. Further treatments were carried out as described previously (52). The linearized DNA was used to produce in vitro transcripts with a T7 mMessage mMachine kit (Ambion) according to the manufacturer's instructions. Briefly, approximately 3 μg linearized DNA template was added to the transcription reaction mixture (20 μl) containing 40 mM Tris-HCl (pH 7.9); 10 mM NaCl; 6 mM MgCl2; 2 mM spermidine; 0.5 mM (each) ATP, CTP, and UTP; 0.1 mM GTP; 0.25 mM cap analogue [m7G (5') ppp (5') G], 120 units of RNasin, and 150 units T7 RNA polymerase and incubated at 37°C for 90 min.
CHSE-214 cells, grown to 90% confluence in a T-25 flask, were transfected with cRNAs of both segments as described previously (52). Briefly, the cells were washed once with phosphate-buffered saline (PBS). Three milliliters of OPTI-MEM I (GIBCO/BRL) was added to the monolayer, and the cells were incubated at room temperature for 1 h. Simultaneously, 0.15 ml of OPTI-MEM I was incubated with 12.5 μg of Lipofectin reagent for 45 min in a polystyrene tube at room temperature. Equimolar amounts of RNA transcripts of segments A and B (8 μg each), resuspended in 0.15 ml of diethyl pyrocarbonate-treated water, were added to the OPTI-MEM-Lipofectin mixture, mixed gently, and incubated on ice for 5 min. After the OPTI-MEM I was removed from the monolayers in the T-25 flask and replaced with 1.5 ml of fresh OPTI-MEM, the nucleic acid-containing mixture was added dropwise to the CHSE cells and swirled gently. After 3 h of incubation at room temperature, the mixture was replaced with minimal essential medium containing Hanks' salts and 10% FBS (without rinsing the cells). The cultures were incubated at 15°C for 5 days, and the cell supernatant was harvested by freeze-thawing twice and passed onto fresh CHSE monolayers. Cytopathic effect (CPE) was usually visualized 4 days after the second pass. All the recombinant viruses were then passed into CHSE cells once more, the cells were freeze-thawed twice, and the cell-free supernatants were stored at –70°C for use as virus stocks in this work. The identities of recovered viruses were verified by sequencing the DNA products obtained after RT-PCR amplification, using specific primers for IPNV segment A.
Immunofluorescence. Infection of CHSE cells by the recombinant viruses was analyzed by immunofluorescence assay using rabbit anti-VP5 specific serum. Briefly, CHSE cells were infected with IPNV at a multiplicity of infection (MOI) of 1.0 and incubated at 15°C. At 16 h postinfection (p.i.), the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were permeabilized with 0.1% Triton 100 for 10 min at room temperature and then incubated with anti-VP5 diluted 1:100 in PBS containing 2% bovine serum albumin for 1 h. After being washed with PBS, the cells were treated with fluorescein-labeled goat anti-rabbit antibody at 1:1,000 dilution (Kirkegaard & Perry Laboratories) and examined by fluorescence microscopy.
Growth curve and plaque assay. To analyze the growth characteristics of IPNV, confluent CHSE or RTG-2 cells (in 35-mm dishes) were infected with the recombinant viruses at an MOI of 1.0. Aliquots collected at various time points were stored at –70°C. The supernatants were centrifuged and titrated on CHSE cells by plaque assay. Briefly, confluent monolayers of CHSE cells grown in six-well plates were infected with serially diluted supernatants from virus stock. After 1 h of incubation at 15°C, the cells were washed once with PBS and overlaid with 0.6% SeaPlaque Agarose (Difco) in Eagle's minimal essential medium containing 5% FBS. After 3 days of incubation at 15°C, the overlays were removed and the cells were fixed and stained with a solution containing 25% formalin, 10% ethanol, 5% acetic acid, and 1% crystal violet for 5 min at room temperature. After the cells were rinsed with distilled water, the plaques were counted.
Analysis of viral protein expression. To analyze viral protein synthesis, RTG-2 cells grown in 60-mm dishes were mock infected or infected with rNVI15, rNVI15-15K, or rNVI15-VP5 at an MOI of 1.0. The infected cells were incubated at 15°C until 8, 16, 24, and 32 h p.i. At the completion of each incubation period, the culture medium was aspirated, and the cells were washed with PBS and lysed. Proteins in the obtained cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 5% stacking and 12.5% resolution gels, and proteins were electrophoretically transferred to a nitrocellulose membrane. The membrane was probed with a polyclonal rabbit anti-IPNV serum, and virus-specific proteins were detected with streptavidin-alkaline phosphatase and naphtholphosphate fast red color development reagents.
Virulence study of Atlantic salmon postsmolts. The three recombinant isolates rNVI15, rNVI15-15K, and rNVI15-VP5 were propagated by inoculation of the second-passage supernatant onto CHSE-214 cells. The cell culture supernatant was obtained after a brief centrifugation, and the infectious titer was determined by end point dilution on CHSE cells grown in 96-well plates. The 50% tissue culture infective dose (TCID50) was estimated by the method of K?rber (30). The challenge was conducted at VESO Vikan's research facility in Namsos, Norway. Approximately 2,200 Atlantic salmon (Salmo salar L.) parr of an average weight of 70 g going through smoltification at the time of challenge were included in this study. The fish were obtained from a commercial supplier (AquaGen) at the eyed-egg stage and kept at VESO Vikan's commercial hatchery until smoltification. There were no records of disease during the freshwater period. The fish were exposed to 24 h of daylight the last 7 weeks before challenge and were tested for seawater tolerance before transportation to the research station 5 days before challenge. The smolts were randomly divided into 11 tanks 1 m in diameter, with 200 smolts in each tank. The day before challenge, the water supply was changed from freshwater to seawater. The fish were challenged by 1-hour immersion with IPNV at a concentration of 104 TCID50/ml in a total of 200 liters of water. Fish in three parallel tanks were infected with each of the three IPNV strains, while fish in two parallel tanks were given cell culture medium only and kept as controls. One tank in each group was reserved for sampling, from which three fish were collected each day, and organ specimens of the pancreas, liver, stomach, and kidney were fixed in 10% phosphate-buffered formalin. Mortality was recorded on a daily basis in the remaining tanks. The experiment was terminated at 30 days postchallenge. Five fish from every group were humanely killed, and kidney samples were collected on the last day of challenge. The samples were stored at –70°C before preparation of a tissue homogenate (10% [wt/vol] in Leibovitz's L-15 medium with 50 μg ml–1 gentamicin) and inoculation onto CHSE cells to check if IPNV could be recovered. The samples were considered negative when no CPE was observed after two passages. The same procedure was employed to check if virus could be recovered from control fish and from fish sampled prior to challenge. Formalin-fixed tissue samples collected at peak mortality were stained with hematoxylin and eosin and examined by light microscopy. Immunohistochemical detection of IPNV antigen in parallel sections was performed as previously described (43).
Persistence study in Atlantic salmon fry. Fifteen hundred Atlantic salmon fry were obtained as eyed eggs from a commercial supplier (AkvaGen) and held at VESO Vikan's hatchery until the start of feeding. There were no records of disease during or after hatching. Thirty fry (average weight, 0.1 g) were sampled and frozen at –70°C. Before challenge, these fry were tested by RT-PCR to ensure that they were IPNV negative. The fry were then moved to VESO Vikan's research facility and randomly distributed into six tanks of 250 fry each. After 1 week of acclimatization, the fry were challenged with IPNV strains Sp103, Sp103-VP5, rNVI15, rNVI15-15K, and rNVI15-VP5 or mock infected with cell culture medium (control group). The viruses had been propagated by inoculation of the third-passage supernatant onto CHSE cells and quantified by titration as described above. Challenge was performed at a dose of 105 TCID50/ml of IPNV at a total volume of 4 liters per tank. The water was aerated during the challenge, and after 3 h, normal flow was resumed. The water temperature was approximately 13°C throughout the experiment. Mortality was recorded on a daily basis, and dead fish were collected and frozen at –70°C. Ten dead fry from each group were sampled at the height of infection and fixed by immersion in 10% phosphate-buffered formalin, and sagittal sections of whole fry were subsequently prepared for histopathological examination and IPNV antigen detection by immunohistochemistry. From 5 weeks p.i., 10 fry in each group were humanely killed every other week and frozen individually at –70°C. The experiment was terminated 5 months after challenge. For detection of IPNV in persistently infected fish, whole fry were homogenized 1:5 (wt/vol) in PBS using a stomacher. One hundred microliters of this homogenate was transferred to RLT buffer containing 2-mercaptoethanol (RNeasy Mini kit; QIAGEN) and stored at –70°C. The rest of the homogenate was diluted by adding 5 volumes of Leibovitz's L-15 medium supplemented with 50 μg ml–1 gentamicin, and the supernatant was obtained after a brief centrifugation. The tissue homogenates were inoculated onto CHSE-214 cells grown in 24-well tissue culture plates in final dilutions of 1 and 0.1%, and incubated for 1 week at 15°C. The cell culture medium after first passage was used to infect new monolayers. The samples were considered negative when no CPE was observed after 1-week incubation of the second passage. RNA was isolated from all negative samples using the RNeasy Mini Kit in accordance with the supplier's protocol (QIAGEN), and RT-PCR was performed to amplify a 224-bp IPNV-specific DNA fragment, as described by Taksdal et al. (49) with minor modifications. QIAGEN's OneStep RT-PCR kit was used according to the manufacturer's instructions, with 0.5 μg RNA and 15 pmol each of primers IPNV1 and IPNV2 (Table 2) in a total reaction volume of 25 μl. The cycling conditions were 60°C for 30 min and 95°C for 15 min, followed by 45 cycles at 94°C for 45 s, 57°C for 45 s, and 72°C for 1 min and finally 72°C for 10 min. The PCR products were visualized by agarose gel electrophoresis. Three controls were included for each RT-PCR run: one positive and one negative tissue sample and one negative control in which water was substituted for RNA.
Sequencing of the viral genome from persistently infected fish. Cell culture supernatant after one passage in CHSE cells was harvested from one positive fish in each group at the 13-week time point. In addition, cell culture supernatants were harvested after inoculation of RTG-2 cells with tissue homogenates of three fish each from tanks infected with rNVI15, rNVI15-15K, and rNVI15-VP5 sampled at 2 and 19 weeks. Viral genomic RNA was isolated using a QIAamp Viral RNA Mini Kit in accordance with the protocol supplied by the manufacturer. A segment of the viral genome coding for VP5 and VP2 was subsequently amplified by RT-PCR, using primers A-A5'NC and Sp1689R (Table 2). The RT-PCR products were purified and sent to a commercial company (MWG Biotech AG) for sequencing. The sequence data were analyzed using BioEdit software (23).
Nucleotide sequence accession numbers. Sequences for Sp103-VP5 have been dpeosited in the GenBank database as AY823632 and AY823633.
RESULTS
Construction of full-length cDNA clones. To determine the role of VP5 in virulence, we constructed full-length cDNA clones of segments A and B of IPNV Sp strain NVI15 as shown in Fig. 1. Using the parental clone pUC19NVI15A as a backbone, two mutant cDNA clones of NVI15 segment A were prepared. In plasmid pUC19NVI15-15K, a BstEII fragment (nucleotides 24 to 586) of the parental plasmid was replaced with a similar BstEII fragment of IPNV strain NVI-020, which does not contain a premature stop codon at nt 427 (TGACGA) (43). Thus, this chimeric clone encodes a 15-kDa VP5 but does not contain any additional mutation in either VP2 or VP5. Previously, we showed that one of the field isolates, NVI-010, does not encode the VP5 protein, as it contains ATA instead of an ATG initiation codon (43). Using site-directed mutagenesis, we prepared a plasmid, pUC19NVI15-VP5, which lacks the initiation codon for VP5. The functionalities of all these clones were tested by in vitro transcription-coupled translation reactions, which yielded protein products that comigrated with the marker IPNV proteins after separation on SDS-PAGE and autoradiography (data not shown).
Transfection and recovery of the mutant IPNVs. In order to recover the recombinant viruses, we transfected CHSE cells with combined plus-sense transcripts of segments A and B derived from various plasmids, as described previously (52). As expected, rNVI15, rNVI15-15K, and rNVI15-VP5 were rescued from the transcripts derived from wild-type or modified segment A and NVI15 segment B. To verify that the mutant viruses were indeed recovered from cRNAs, the genomic RNA was isolated and analyzed by sequencing the DNA products obtained after RT-PCR amplification, using a primer pair specific for segment A. Sequence analysis of the cloned PCR products confirmed the expected nucleotide mutations in the mutant viruses. To detect expression of VP5, CHSE cells were infected with the recovered viruses and analyzed by immunofluorescence assay using VP5-specific antiserum. Cells infected with rNVI15 and rNVI15-15K gave positive immunofluorescence signals that confirmed the expression of VP5. The fluorescence was seen in the cytoplasm, mainly within the perinuclear region (Fig. 2b and c). Cells infected with rNVI15-VP5 did not give any fluorescence signal, nor did mock-infected cells (Fig. 2a and d).
Characterization of the recombinant viruses in vitro. To compare the replication kinetics of the recovered viruses, both CHSE and RTG-2 cells were infected with these viruses at an MOI of 1.0. At the indicated time points, the supernatants from infected cells were collected and titrated in CHSE cells by plaque assay. Figure 3 depicts the growth curves of rNVI15, rNVI15-15K, and rNVI15-VP5 in CHSE (Fig. 3A) and RTG-2 (Fig. 3B) cells, respectively. These viruses grew to similar titers in CHSE cells, which is in accord with the results of Weber and coworkers, who could not detect any difference in the growth of VP5-deficient mutants and the wild-type IPNV strain VR-299 (51). The recovered viruses showed a slight delay in growth in CHSE cells compared to RTG-2 cells. Cytopathic effects appeared earlier in RTG-2 cells than in CHSE cells, but the final titers of all viruses were 1 log unit higher in CHSE cells than in RTG-2 cells.
The protein expression profiles of RTG-2 cells infected with the different recombinant IPNVs were compared, but no difference in virus-specific protein expression was detected (Fig. 4).
Virulence study in Atlantic salmon postsmolts. To assess the virulence and the pathogenic phenotype of the recovered viruses in fish, Atlantic salmon postsmolts were infected or mock infected with either rNVI15, rNVI15-15K, or rNVI15-VP5 viruses. Ten days after challenge, fish started dying in all infected groups. A parallel course of mortality was recorded for these viruses, and at the conclusion of the study 30 days postchallenge, the cumulative mortality had reached 81, 81, and 86% for rNVI15, rNVI15-15K, and rNVI15-VP5 viruses, respectively (Fig. 5). In the control group, mortality was 9%. Virus was recovered from all five fish challenged with rNVI15-15K and rNVI15-VP5 and from four out of five fish challenged with rNVI15 but not from the five control fish examined. Histopathological examination demonstrated the characteristic lesions of IPN, with necrosis of hepatic and exocrine pancreatic tissues (data not shown). The presence of virus within lesions was verified by immunohistochemical detection of IPNV antigen in sections done in duplicate. There were no pathological changes and no positive detection of viral antigen in control fish. Viral genomic RNA was extracted directly from fish challenged with the three recombinant viruses and amplified with a primer pair encompassing the VP5 gene. The nucleotide sequences of the resulting PCR products were determined, which confirmed the mutations in the VP5 gene for the two mutant viruses and a lack of mutation in the unmodified recombinant virus.
Persistence study in Atlantic salmon fry. To evaluate the role of VP5 in persistent IPNV infection and to examine if there are differences among high- and low-virulence viruses in the ability to induce persistent infections, Atlantic salmon fry were challenged with five IPNV strains and observed for the following 5 months. Viruses rNVI15, rNVI15-15K, and rNVI15-VP5 are derived from the virulent field strain NVI-015 and encode 12-kDa, 15-kDa, and no VP5 proteins, respectively. Sp103 is a low-virulence field strain encoding a 15-kDa VP5, whereas Sp103-VP5 is a VP5-deficient variant isolated by plaque purification of Sp103. Figure 6 shows cumulative mortality in fry challenged with different viruses. During the second week after challenge, mortality started to increase in all infected groups. At this time point, IPN characteristic lesions were detected in dead fry sampled from all challenged groups, but not in the control group. The presence of virus within the lesions was verified by immunohistochemistry (data not shown). From the third week p.i., the mortality in fry decreased and then remained low throughout the rest of the study (Fig. 6). From 5 weeks p.i. on, 10 fish from each group were sampled every other week for the detection of IPNV carriers by inoculation in cell culture and RT-PCR techniques. No IPNV-positive fish were detected in the control group at any time point (Fig. 7). At the first sampling (5 weeks), all challenged fish were IPNV positive. Subsequent samplings showed that most of the Sp103-VP5- and rNVI15-VP5-infected fish became carriers, and IPNV was detected in all 10 fish examined 19 weeks after challenge. Most of the fish infected with rNVI15 and rNVI15-15K also remained IPNV carriers, as 8 out of 10 fish examined were virus positive after 19 weeks. Only in the group infected with Sp103 was a decline in IPNV-positive fish observed, and at 19 weeks p.i., none of the 10 fish examined were carriers. These results indicate variation among the strains in the ability to establish persistent infection.
Sequencing of the viral genome from persistently infected fish. Viral RNA was isolated from CHSE-214 cells inoculated with tissue homogenates from fish sampled 13 weeks after challenge and was used for RT-PCR amplification of a segment covering the VP5- and VP2-encoding region. For the field viruses Sp103 and Sp103-VP5, no differences in the sequence were observed from fish sampled at 13 weeks postchallenge. In the case of recombinant viruses, an AlaThr substitution at amino acid 221 of VP2 was detected. Therefore, viral RNA was isolated from RTG-2 cells inoculated with tissue homogenates from fish sampled both 2 and 19 weeks after challenge and used for RT-PCR amplification of a segment covering amino acid 221 of VP2. The obtained sequence was identical to the input virus sequence during the acute phase of infection (2 weeks postchallenge), as expected (Table 3). However, a single amino acid substitution at position 221 of VP2 (AlaThr) was detected in all three viruses recovered from fish sampled at 19 weeks postchallenge, suggesting that in the carrier state, the dominant virus has a Thr residue at this position (Table 3).
DISCUSSION
The reverse-genetics system for IPNV was first developed in 1998 and has been applied to in vitro studies of viral replication (51, 52). This report describes for the first time challenge experiments in both fry and postsmolts using recovered IPNVs of Sp serotype to evaluate the role of the VP5 nonstructural protein.
Previous studies have shown that the expression of VP5 in VR299 and Sp strains is initiated from the second in-frame start codon, yielding a 15-kDa protein (45, 51). However, results from our laboratories indicate that Sp strains of low virulence encode a 15-kDa protein, whereas more virulent strains often contain a truncated VP5 protein gene (43, 45). In order to study the expression of this 12- to 15-kDa VP5 protein, we constructed infectious cDNA clones of the virulent field stain NVI-015 and generated VP5 mutant viruses, one encoding a truncated 12-kDa VP5 and a second encoding a full-length VP5, whereas the third was deficient in VP5 expression. The rescue of the recombinant IPNVs was verified by sequencing of the DNA products obtained after RT-PCR amplification and by immunofluorescence assay using anti-15-kDa specific antibody. The growth kinetics of the VP5 mutant viruses in CHSE and RTG-2 cells were similar, which confirmed that VP5 does not affect the rate of replication and that it is dispensable for viral replication. These results are in agreement with those of Weber and coworkers, who also reported similar findings in CHSE cells (51).
The virulence characteristics of the recovered viruses were subsequently tested by challenging Atlantic salmon postsmolts, which is technically more demanding than challenging fry. In Norway, IPN outbreaks in seawater are economically more important than freshwater outbreaks in fry. Melby and colleagues (36) concluded that all commercial seawater salmon farms in Norway harbor IPNV carriers. According to national surveys, outbreaks are seen in 40 to 70% of all seawater sites every year. In addition, extensive sequencing of Norwegian IPNV field isolates carried out in our laboratories has shown that strains closely related to NVI-015 dominate in postsmolt outbreaks of IPN. The experimental challenge gave cumulative mortalities above 80% for the three genetically engineered viruses, which is relatively high compared to what was found in previous postsmolt IPN cohabitation challenges (3, 48). The high mortality figures were not solely due to the use of recovered IPNV strains, since two additional field strains were included in the study, ending with 76 and 86% cumulative mortalities (data not shown). This study demonstrates that Atlantic salmon postsmolt IPNV challenges can be successfully performed with bath exposure. Among the fish infected with rNVI15, rNVI15-15K, and rNVI15-VP5 viruses, 81, 81, and 86% succumbed to IPNV infection, respectively. The results clearly indicate that VP5 is not required for viral replication in vivo and that all VP5 variant strains are able to induce IPN disease. It can also be concluded that VP5 has no function as a virulence factor, since rNVI15-VP5 was no different from the other strains. The VP5-deficient mutant gave a slightly higher mortality than the VP5-producing strains, although the difference was not statistically significant.
The slightly higher mortality figures observed in the rNVI15-VP5-challenged fish could indicate a certain replicative advantage of VP5-deficient strains over VP5-encoding strains. The absence of the small upstream VP5 ORF might affect the translation efficacy of the major ORF encoding the polyprotein. Translation initiation site selection is determined in part by the context of the nucleotide sequence surrounding the first AUG codon encountered by the scanning ribosomal unit (31). The initiation codon (uppercase) of the small segment A ORF starting at nt 112 is in a weak context (34, 51) compared to the large ORF starting at nt 119 (NVI-015 nt 106 to 122: auaucaAUGcaagAUGa). As a consequence, the scanning ribosome will probably initiate translation from this weak AUG at a low frequency compared to the stronger downstream AUG of the large ORF encoding the polyprotein. This mechanism, referred to as leaky scanning, enables the virus to maximize its genome coding capacity by encoding functionally distinct proteins from a common mRNA (19). Consequently, loss of the small ORF could lead to increased translational efficiency of the polyprotein, again resulting in slightly increased replication of VP5-deficient IPNV strains. However, the virtually overlapping growth curves of the VP5-encoding and VP5-deficient viruses obtained from two different cell lines does not lend support to an idea of replicative advantage of VP5-deficient strains. In addition, the viral capsid proteins VP2 and VP3 (encoded by the large ORF) appeared simultaneously and in comparable amounts in cells infected with VP5-encoding or VP5-deficient strains.
In the second challenge study, acute mortality measured at 5 weeks p.i. ranged from 5 to 27%. The low mortality in this experiment could result from higher genetic resistance to IPN in the challenged fish (39, 40). The fry were provided by AquaGen, a breeding company that has implemented selection for increased genetic resistance of Atlantic salmon to IPN since 2001 (A. Storseth, personal communication). Salmon full-sibling families have been tested for IPN resistance since 1997, demonstrating that mortalities range from 0 to 96% between families (A. Storseth, T. Aasmundstad, K. O. Ali, S. Kj?glum, S. A. Korsvoll, B. H?yheim, and A. Guttvik, poster presentation at Aquaculture Europe 2003, Trondheim, Norway, 8 to 12 August 2003). Again, the mortality in the group of fish infected with rNVI015-VP5 (27%) was slightly higher than in fish infected with rNVI15-15K (18%) and rNVI15 (20%). However, this experiment was designed to study the establishment of persistent infections and not to compare virulence characteristics for different strains. As a consequence, fish were infected in a single tank for each virus strain, and frequent samplings were performed. Statistical comparison of mortality figures is therefore not relevant.
VP5 was found to be dispensable for virus virulence, but it is still reasonable to believe that the protein has a specific function, particularly since the majority of IPNV strains encode the protein (25). As described by Hong and colleagues (26), IPNV VP5 has Bcl-2 homology domains and is antiapoptotic, delaying apoptotic cell death in the early replication cycle of IPNV infection. The early cell response to viral infection involves a multitude of reactions, including induction of apoptosis in infected cells, and viral inhibition of apoptosis could allow sustained viral replication in cells that are destined to commit suicide. Several viruses encode Bcl-2-homologous proteins, and although their physiological functions are not completely elucidated, they might be involved in the establishment of persistent infections (6, 20). To investigate whether IPNV VP5 is involved in the development of a carrier state, Atlantic salmon fry were infected with the virulent recombinant viruses rNVI15, rNVI15-15K, and rNVI15-VP5, along with two field strains of low virulence, Sp103 and Sp103-VP5. Five months after challenge, all examined fry infected with the two VP5-deficient mutant strains were IPNV carriers, and 80% of the fish infected with rNVI15 and rNVI15-15K were carriers. The results suggest that the establishment of persistent IPNV infection is independent of VP5 expression. It is not possible to develop conclusions about the effect on the length of the carrier state, since the experiment was terminated 5 months after challenge, and it is anticipated that IPNV-infected fish become life-long carriers.
In the group of fish infected with IPNV strain Sp103, there was a steady decline in the number of IPNV-positive fish, and at 19 weeks after challenge, no carriers were detected. Interestingly, although this strain encodes Ala at residue 221 of VP2, it seems to be less prone to mutate to Thr at this position following cell culture passage. Even after 10 passages in CHSE cells, approximately half the virus population still encoded Ala (47a). All three NVI-015-derived strains gave an AlaThr mutation after a few passages in CHSE cells, originally thought to be a cell culture adaptation phenomenon. The accumulation of this mutation in persistently infected fish was thus an unexpected finding. It may imply that Thr at VP2 residue 221 is a prerequisite for persistent infection and that Sp103, encoding a genetically relatively stable Ala at this position, is less able to establish long-term persistent infection. Sp103-VP5, on the other hand (encoding Thr at position 221), induced a carrier state in 79 of 80 fish examined. To the best of our knowledge, this is the first finding of a molecular determinant of persistent IPNV infection.
RNA viruses, due to the absence or the low efficiency of proofreading activities associated with RNA polymerases, have a high mutation rate during genome replication and are thought to replicate as complex and dynamic mutant swarms called viral quasispecies (11). For hepatitis C virus (HCV), quasispecies appearance is associated with evasion of the immune response and progression to chronicity, whereas genetic stability is linked to self-limiting disease (16). The emergence of virus mutants with Thr at position 221 of VP2 could be a result of selective pressure on the viral population by the immune response on epitopes recognized either by neutralizing antibodies or by cytotoxic T lymphocytes. Indeed, residue 221 lies within the central variable domain of VP2 containing the major conformational epitopes recognized by neutralizing monoclonal antibodies (18, 50). Another possibility to consider is selection due to different cell tropism in acute infection (exocrine pancreatic and liver tissue) (43) compared to persistent infection (macrophages) (38). A preferential tropism of certain viral variants for specific cell types could explain the selection of this mutant in CHSE cells, a phenomenon not observed in RTG-2 cells (47a). Again, for HCV, it was recently demonstrated that dendritic cells can be an extrahepatic reservoir of the virus and that the HCV genomic sequence derived from dendritic cells differed from that found in the plasma of the same patient (21). We have done some further experiments demonstrating that the AlaThr substitution at position 221 of VP2 leads to attenuation of the virus (Song et al., submitted). The persistence of this attenuated virus in covertly infected fish is interesting, since reemergence of the virulent variants in the carrier fish could be involved in stress-mediated recurrence of IPN (42, 48).
Having established that VP5 is not essential for virulence or for the establishment of persistent IPNV infection, the natural follow-up would be to perform in vitro and in vivo studies of the apoptosis-inducing effects of the VP5 variant viruses to confirm the apoptosis-inhibiting effect of VP5 proposed by Hong and colleagues (26). Furthermore, the most probable candidate for causing differences in virulence among closely related isolates of the Sp serotype has been mapped to the VP2 protein (43, 45), which has been confirmed using reverse genetics (47a).
ACKNOWLEDGMENTS
This study was supported by Norwegian Research Council grant 134136/120 to ?.E. and by National Research Initiative of the USDA Cooperative State Research, Education, and Extension Service grant 2001-35204-10065 to V.N.V.
We thank A. Ramstad, study director for the challenge experiments at VESO Vikan, Namsos, Norway. Thanks also go to Gerard H. Edwards at the Center for Biosystems Research, University of Maryland, for technical assistance.
Nina Santi and Haichen Song contributed equally to this work.
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