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1 Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE C1A 4P3, Canada
2 Department of Computer Science and Information Technology, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE C1A 4P3, Canada
3 New Brunswick Department of Agriculture and Aquaculture, PO Box 6000, Fredericton, NB E3B 5H1, Canada
Correspondence
Frederick S. B. Kibenge
kibenge{at}upei.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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13 aa with the presence of a specific motif 352FNT354 in the highly polymorphic region spanning residues 337V to M372 in the HE protein stalk, in combination with a specific motif 265YP266 very close to the trypsin-cleavage site 267RA/G268 of the precursor F0 protein were correlated with reduced cytopathogenicity and reduced virulence for Atlantic salmon. Phylogenetic analysis suggests that the original ancestral ISAV was virulent. The virulence of the North American genotype has not changed much, whereas the European genotype evolved into two genogroups, the real-European genogroup that is still virulent and the European-in-North America genogroup, which is of lower virulence. A novel phylogenetic software program, BACKTRACK, estimated that the North American and European genotypes diverged between 1879 and 1891, whereas the European-in-North America genogroup diverged from the real-European genogroup between 1976 and 1988. This direction of evolution supports insertion of specific motifs in the HE protein, resulting in ISAV attenuation.
Present address: 17 Rosedale Drive, Charlottetown, PE, Canada. 
Present address: Rolling Hills Pet Hospital, Chula Vista, CA, USA.
A supplementary table listing the oligonucleotide primers used is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Viruses in the genus Isavirus are enveloped particles of 90–140 nm diameter with surface projections consisting of a combined haemagglutinin-esterase (HE) protein encoded on segment 6 (Falk et al., 2004) and a separate fusion (F) protein encoded on segment 5 (Aspehaug et al., 2005). ISAV therefore represents a unique orthomyxovirus having its fusion activity localized on a separate protein. The genome consists of eight segments of linear, single-stranded negative-sense RNA ranging in length from 1.0 to 2.4 kb with a total molecular size of approximately 14.3 kb (Clouthier et al., 2002), and different researchers have collectively sequenced all eight RNA genomic segments.
It has been hypothesized that the ISAV variant without any deletion in the HE protein, designated HPR0 to indicate a full-length highly polymorphic region (HPR) in HE, represents an avirulent phenotype of the virus and the source of the virulent ISAV isolates. The avirulent nature of HPR0 viruses was indicated by the lack of disease in the host fish, and by their failure to replicate in cell culture (Cunningham et al., 2002; Cook-Versloot et al., 2004), but the molecular bases for these limitations are not well defined.
Studies on factors that affect influenza A virus host range and virulence have indicated that alteration in any of the 10 genes can affect virulence or host range, depending on the system under investigation (Webster et al., 1992; Subbarao et al., 1993; Brown, 2000; Zhirnov et al., 2002). In the case of avian influenza A viruses, one virulence factor is correlated with the haemagglutinin cleavage site, allowing the grouping of the viruses in chickens and turkeys into highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) viruses (Suarez et al., 2004). The LPAI viruses, which have only two basic amino acids at the cleavage site, are only cleaved by trypsin-like enzymes and are thus restricted to replication at sites in the host where such enzymes are found, i.e. respiratory and intestinal tracts. On the other hand, the HPAI viruses with multiple basic amino acids at the cleavage site (Senne et al., 1996) allow the precursor haemagglutinin (HA0) molecule to be cleavable by ubiquitous host proteases (Stieneke-Grober et al., 1992) and the viruses to replicate systemically, resulting in multiple organ and tissue damage with severe disease and death (Rott, 1992). The counterpart of the influenza A virus HA protein in ISAV are two proteins, the HE protein which is not cleaved (Krossøy et al., 2001a) and the F protein which is cleaved (Aspehaug et al., 2005). Determinants of virulence on the HE gene have yet to be investigated in any detail. In the F protein, which is synthesized as a precursor protein, F0, proteolytic cleavage to F1 and F2 is essential for virus fusion to cell membranes, but the apparent lack of multiple basic amino acids at the cleavage site of virulent isolates suggested that the cleavage site structure was not as important in the pathogenicity of ISAV as in other orthomyxoviruses (Aspehaug et al., 2005).
It is accepted that there is variation in pathogenicity among ISAV isolates. The present study used 13 different isolates of ISAV of known in vivo pathogenicity (Kibenge et al., 2006) and compared their replication in three permissive fish cell lines in an attempt to identify and characterize the correlates of ISAV virulence. Thus, this study utilized our understanding of the pathogenicity mechanisms of avian influenza A viruses (a well studied orthomyxovirus) to begin to decipher the correlates of pathogenicity of ISAV. Previous studies had focused exclusively on the HPR of the HE gene as the determining factor because of the receptor-binding function of the HE protein, and the detection of avirulent HPR0 viruses without deletions in the HE gene (Cunningham et al., 2002; Nylund et al., 2003; Plarre et al., 2005). However, the F gene of HPR0 viruses has never been reported. Thus, we targeted ISAV RNA segments 5 and 6 as key potential virulence genes, particularly since we had clearly identified highly pathogenic ISAV and low pathogenic ISAV isolates (Kibenge et al., 2006). The results showed candidate motifs in the surface glycoproteins of ISAV isolates that probably play a similar role to the virulence factor at the HA cleavage site of avian influenza viruses. The sequence variation between ISAV isolates in the two viral genes correlated not only with geographical origin but also with cytopathogenicity in cell culture and pathogenicity for fish.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) and Atlantic salmon kidney (ASK-2) cells (Rolland & Winton, 2003) were grown as described previously (Kibenge et al., 2006). Chinook salmon embryo cells (CHSE-214) were grown at 16 °C in Hanks' minimal essential medium (Invitrogen) supplemented with 10 % fetal bovine serum, 100 IU penicillin G ml–1, 100 µg streptomycin ml–1 and 0.25 µg amphotericin B ml–1, the monolayer cultures were used 24 h after seeding. The source, genotype and pathotype of the 13 ISAV isolates used in this study are summarized in Table 1. The viruses were propagated and titrated in TO cells as described previously (Kibenge et al., 2001) prior to use in the subsequent studies. The viruses were passaged once in ASK-2 or CHSE-214 cells before use in the respective cell lines.
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). The developments of cytopathic effect (CPE) and growth curves of the ISAV isolates were compared in TO, ASK-2 and CHSE-214 cell lines. Each 25 cm2 flask of confluent cell monolayers grown was drained of medium and then inoculated with 1.0 ml 105 TCID50 ml–1 of the different ISAV isolates. Virus was adsorbed for 1 h at room temperature and then 5 ml of maintenance medium was added and the flasks were incubated at 16 °C. Flasks were inspected daily for CPE and for each virus; one flask was harvested at 9–10 days post-infection (p.i.) (and at 14 and 21 days p.i. in case of CHSE-214 cells) by freezing at –20 °C. The culture lysates (i.e. total virus) were then titrated to determine growth cycles of virus on the respective cell lines. Virus titration utilized serial 10-fold dilutions of virus suspensions inoculated on 48-well plates containing TO, ASK-2 or CHSE-214 cell monolayers from which the TCID50 was determined as described previously (Kibenge et al., 2001).
RT-PCR and molecular cloning.
Total RNA from the 13 ISAV isolates grown in TO cells was separately extracted from 300 µl volumes of cell culture lysates using TRIzol LS Reagent (Invitrogen) prior to RT-PCR amplification. RT-PCR amplification was performed with the Titan One Tube RT-PCR System kit (Roche Diagnostics) in a PTC-200 DNA Engine Peltier thermal cycler (MJ Research) using cycling conditions as described previously (Kibenge et al., 2007). The oligonucleotide primers used are listed in Supplementary Table S1 (available in JGV Online). The PCR products were then cloned into the pCRII vector using a TOPO TA cloning kit (Invitrogen) in preparation for nucleotide sequencing.
DNA sequencing and analysis of sequence data.
Plasmid DNA for sequencing was prepared as described before (Kibenge et al., 1991). DNA sequencing was performed as described previously (Kibenge et al., 2007) by the ACGT Corporation. Sequence analysis used the BLAST program (Altschul et al., 1990), the Sequence Manipulation suite version 2 (http://www.ualberta.ca/
stothard/javascript/index.html) and the FASTA program package for personal computers (Pearson & Lipman, 1988).
Phylogenetic analysis.
Sequences were aligned by using CLUSTAL_X with the default settings (Thompson et al., 1997). Phylogenetic trees were generated using PHYLIP 3.66 (Felsenstein, 2006). Alignment regions containing gaps were excluded from the analysis. The results were analysed by using the bootstrap method (1000 replicates) to provide confidence levels for the tree topology. We then used different outgroup sequences to determine and verify the root of each tree.
Rate of nucleotide substitution.
The method of nucleotide substitution rate estimation as described by Saitou & Nei (1986) was applied to ISAV as described previously (Krossøy et al., 2001b). This method recommends examining the evolutionary relationship of genes obtained from different isolates first and then using only isolates that are closely related by descent. In this study, we used it to estimate the rates of nucleotide substitutions in RNA segments 5 and 6 of the 13 ISAV isolates. Because the evolution of the HPR in segment 6 does not occur at the same general rate of evolution as the rest of the gene, this region was excluded from the phylogenetic analysis of the HE gene.
Divergence time estimation in a rooted phylogenetic tree.
To apply the method introduced by Saitou & Nei (1986) in a rooted phylogenetic tree, we needed to modify this method in the following aspects. First, we make use of the existing information in a rooted phylogenetic tree so that we can estimate the divergence time using only one strain. Suppose ancestor A diverges into B and something else at time t1 and B exists in time t2. Then, the expected evolutionary distance (no. substitutions per site) from A to B may be expressed as d=(t2–t1)x
, where is the rate of nucleotide substitution per site per year. Therefore, t1 can be estimated by the equation
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), we had determined the pathogenicity of ISAV for Atlantic salmon, rainbow trout and coho salmon, allowing us to group the 13 ISAV isolates into three pathogenicity phenotypes (high, intermediate and low pathogenicity) (summarized in Table 1
). Several fish cell lines were investigated in this study since variation in replication rate of the virus could be a result of selective adaptation to a particular cell line (Mjaaland et al., 2002). The development of CPE and growth curves of the 13 ISAV isolates were compared in TO, ASK-2 and CHSE-214 cell lines, and the results are summarized in Table 2. We show in this study that in vivo virulence characteristics can be correlated with the development of CPE and the total virus production in two Atlantic salmon macrophage-like cell lines TO and ASK-2, and the CHSE-214 cell line. The less virulent isolates, which were mostly of the European genotype, took longer to cause CPE; CPE did not involve all cells in the monolayer, and produced lower virus titres than more virulent isolates. On this basis, the highly pathogenic ISAV isolates replicated more aggressively in cell culture (grew well in more cell lines, produced CPE sooner more completely and grew to higher titres) than less pathogenic isolates. In Atlantic salmon, ISAV seems to target endothelial cells (Falk & Dannevig, 1995) and macrophage-like cells (Moneke et al., 2003), suggesting that the key pathogenic event in ISA is the infection of these cells. However, no significant differences were seen in the growth curves of the high-pathogenicity and low-pathogenicity isolates in TO and ASK-2 cells (data not shown). Among Canadian ISAV isolates, the severity of CPE in CHSE-214 cell line correlated with the ability to cause mortality in rainbow trout. ISAV isolate NBISA01, which was the most virulent, also replicated the best in the CHSE-214 cell line.
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). The coding regions of the two genes (in RNA segments 5 and 6) of the 13 ISAV isolates were sequenced from plasmid DNA of cloned RT-PCR products. All sequences were examined for authenticity by looking for the respective ORFs using the ORF Finder program in the Sequence Manipulation suite (Stothard, 2000). The nucleotide and predicted protein sequences were used to search the GenBank database using BLASTX and BLASTP (Altschul et al., 1990) to confirm the identity of the cloned ISAV genes. The new sequences have been deposited in GenBank under the accession numbers listed in Table 3.
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) was used to perform pairwise comparisons of the nucleotide and deduced amino acid sequences, and the identities for each of the two genes among the 13 isolates are listed in Tables 4 and 5. In both cases, the sequence identity data revealed two genotypes, one European (containing isolates 810/9/99, 390/98, 485/9/97, U5575-1 and 04-085-1) and one North American (containing isolates 01-0593-1, 98-049-1, 02-0775-14, 01-0973-3, 02-1179-4, 7833-1, NBISA01 and 98-0280-2). The two genotypes had a nucleotide sequence identity of 76.5 % and amino acid sequence identity of 84.5 % on the F gene (Table 4); and nucleotide sequence identity of 79.3 % and amino acid sequence identity of 84.5 % on the HE gene (Table 5), very similar to those previously found among 24 ISAV isolates (Kibenge et al., 2001). Thus, the isolates were more variable in the F gene at the nucleotide level but of similar variation in both F and HE genes at the amino acid level. Others have made similar observations (Devold et al., 2006).
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). The amino acid changes in the putative proteins of these genes in the viruses are listed in Table 6, identifying the global amino acid differences that are genotype-specific and the local amino acid differences unique to individual virus isolates. In the F protein, the global amino acid differences characteristic of the European (five isolates) and North American (eight isolates) genotypes were mainly concentrated in the areas spanning aa positions 21–37, 54–79, 104–130, 162–196, 220–226, 250–268, 367–397 and 429–434. Four of these areas (21–37, 104–130, 250–268 and 429–434) correspond to known structural/functional roles in the protein and may therefore be the basis for some of the phenotypic differences between the two ISAV genotypes: aa region 21–37 is immediately after the signal peptide at aa region 1–17 (Clouthier et al., 2002) and includes the motif 32IPRTGYVRSA41 that may occur as an insert, IN3, close to the proteolytic cleavage site of the precursor F0 protein in some ISAV isolates (Devold et al., 2006); aa region 104–130 contains a potential glycosylation site at 110NLT112 (Devold et al., 2006); aa region 250–268 is the C terminus of the F1 subunit with the proposed trypsin-cleavage site at 267RA/G268 (Aspehaug et al., 2005); and aa region 429–434 is at the C terminus of the F2 subunit in the primary transmembrane region spanning aa 417–439 (Aspehaug et al., 2005; Devold et al., 2006). Interestingly, the proposed fusion peptide at the N terminus of the F2 subunit spanning aa 277–292, which is highly conserved within virus families (Aspehaug et al., 2005), had only 1 aa difference between the two ISAV genotypes at V284I. The local amino acid differences were unique to individual viruses, except for isolates 01-0973-3, 98-049-1 and 7833-1 of the North American genotype, which had no unique amino acid changes at all. Two local amino acid changes in isolate 04-085-1, N265Y and L266P, are at the C terminus of the F1 subunit very close to the proposed trypsin-cleavage site at 267RA/G268 (Aspehaug et al., 2005). Fig. 1(a) shows the alignment of amino acids of various ISAV isolates around this proteolytic cleavage site of the precursor F0 protein. Isolate 04-085-1 is a low pathogenic strain of the European genotype found in eastern Canada, and experimentally shown to induce very low mortality in Atlantic salmon (18.2 %) (Kibenge et al., 2006). Therefore, the local amino acid changes in this isolate at aa positions 265 and 266 may be characteristic of the low-pathogenicity phenotype of ISAV by influencing the accessibility of this site to proteolytic enzymes. This mutation is just upstream of the trypsin-cleavage site 267RA/G268 in the area where proteolytic cleavage of the precursor F0 protein is expected to occur (Aspehaug et al., 2005). It is interesting to note that this mutation also occurs at the same location as in inserts IN1, IN2 and IN3 found in the F protein of some ISAV isolates (Fig. 1a; Devold et al., 2006).
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; Klenk & Garten, 1994), which is essential for fusion of the virus to cell membranes (Perdue & Suarez, 2000). Virions with uncleaved HA0 are non-infectious (Steinhauer, 1999). We speculate that the local amino acid changes in ISAV isolate 04-085-1 at aa positions 265 and 266 in the F protein (Fig. 1a) play a similar role to the virulence factor at the HA0 cleavage site of avian influenza viruses, making them (i.e. motif 265YP266) a putative molecular marker of virulence in ISAV. In this case, the 265YP266 mutation probably makes the cleavage site less accessible to proteases and so prevents cleavage or makes cleavage less efficient.
The amino acid differences in the HE protein among the isolates were similar to previous descriptions (Kibenge et al., 2001; Devold et al., 2001; Mjaaland et al., 2002; Nylund et al., 2003), differentiating ISAV into European (five isolates) and North American (eight isolates) genotypes with an HPR spanning residues 337–372 (Table 6). The global differences were not absolute as some changes characteristic of the European genotype involved a change to either of 2 aa or a change that included an isolate belonging to the North American genotype or both. Among the local amino acid differences, the most noteworthy was N164D, which was unique to isolate 04-085-1 of the European genotype found in North America; this isolate is of lower virulence than those found in Europe.
HPR of the HE gene as virulence determinant
The HPR is located in the stem region of the ISAV HE protein (Kibenge et al., 2001). Therefore, deletions or insertions in the HPR may affect several properties of HE, including flexibility and receptor-binding specificity and affinity, antigenicity, esterase activity, interaction with other proteins (e.g. matrix protein) and possibly the polymerization of the protein (Aspehaug, 2005), potentially affecting all functions of the protein. The widely held model suggests that virulent variants of the HPR0 archetype arise by deletion of several nucleotides in the HPR, probably due to a strong functional selection pressure (Cunningham et al., 2002; Mjaaland et al., 2002; Nylund et al., 2003). To determine the molecular basis for the limitations of the HPR in the HE protein, the predicted amino acid sequences of the HPR of various strains of ISAV were compared (Fig. 1b). Twelve of the isolates used in this study belonged to six HPR groups reported previously (Nylund et al., 2003; Plarre et al., 2005). Isolate 04-085-1 did not belong to any of the existing European HPR groups. While this isolate resembled isolate U5575-1 in having the second putative N-glycosylation site 349NQT351 in the European genotype, it was different in having an adjacent motif 352FNT354 commonly found in HPR0 viruses or those virus strains that are difficult to isolate in cell culture (Mjaaland et al., 2002). Fig. 1(b) shows the alignment of amino acids characteristic of the different HPR groups compared with the ability of the different viruses to be isolated in cell culture (an indication of cytopathogenicity of the viruses). Of all HPR groups reported to date, the new HPR group of ISAV isolate 04-085-1 is the closest to HPR0 in terms of the pattern of deletion/insertion associated with this unique phenotype, characterized by slow or poor replication in cell culture, subclinical infection and carrier status. These data suggest that the stalk length of the HE protein in the HPR is correlated with the ability of the virus to be isolated (i.e. consistently induce CPE) in cell culture. On the basis of these data, we hypothesize that the number of amino acids deleted/inserted and/or mutation of the 352FNT354 motif in HPR of the HE stalk are determinants of pathogenicity because they are necessary for multiple cycles of virus infection essential for virus isolation in cell culture. As evidenced in Fig. 1(b), the non-cultivable, non-pathogenic ISAV strains (HPR0 viruses) possess the HE protein without any amino acid deletion. Isolates (like 04-085-1) with 13 aa deletions but with the 352FNT354 motif have reduced cycles of virus infection. In contrast, most ISAV isolates have >13 aa deletions (or if less, with deletion or mutation of the 352FNT354 motif) in the HPR and consequently replicate efficiently both in vitro and in vivo. Our hypothesis assumes that the amino acid deletions/insertions and/or mutations in the HPR are the major structural properties that determine flexibility of the HE stalk. Other structural changes such as the second putative N-glycosylation site at 349NQT351 may also affect the flexibility of the HE stalk and contribute to reduced virus virulence (Kibenge, 2004). HE sequences without any putative glycosylation sites have been found, which were from ISAV RT-PCR-positive fish from which no virus could be isolated (Fig. 1b). This concept is also supported by the observation that in avian influenza viruses, glycosylation of the haemagglutinin and stalk-length of the neuraminidase combine as a determinant of virus virulence (Baigent & McCauley, 2001). Since the receptor destroying enzyme (esterase) in ISAV is on the same protein with the receptor-binding site (haemagglutinin) in the HE protein, the stalk-length of the HE protein in the HPR may influence pathogenicity and the ability of the virus to undergo multiple cycles of infection in cell culture with production of CPE by affecting the efficiency of esterase activity.
In Newcastle disease virus (family Paramyxoviridae), virulence is determined in part by both the cleavage site of the fusion protein, and the stem region and globular head of the haemagglutinin-neuraminidase protein (de Leeuw et al., 2005). Thus, a combination of motifs in the HPR affecting flexibility of the HE protein, and the specific motif 265YP266 very close to the proposed trypsin-cleavage site in the precursor F0 protein (Fig. 1) may similarly be virulence determinants in ISAV. However, it is also evident that other viral genes contribute to ISAV pathogenicity, since seven of the ISAV isolates of North American genotype used in the present study belonged to the same HPR group (e.g. HPR21), and yet differed in their pathogenicity in Atlantic salmon and rainbow trout (Kibenge et al., 2006).
Evolutionary trees of ISAV
In the present study, we used three steps to analyse the evolutionary relationships of the 13 ISAV strains. First, we generated unrooted phylogenetic trees of the ISAV isolates for the two genes (HE and F). We used the corresponding genes of other members of the family Orthomyxoviridae as outgroup sequences to determine the root for each tree. These phylogenetic trees are shown in Fig. 2(a) and (b). These trees are balanced and more meaningful from the evolutionary point of view. Both the F and HE phylogenetic trees consistently classified the 13 ISAV isolates into two major genotypes identified previously by pairwise comparisons of the sequences (Tables 4 and 5): North American genotype containing isolates 01-0593-1, 98-049-1, 02-0775-14, 01-0973-3, 02-1179-4, 7833-1, NBISA01 and 98-0280-2; and European genotype containing isolates 810/9/99, 390/98, 485/9/97, U5575-1 and 04-085-1. Both trees further classified the European genotype into two further genogroups. The first genogroup contained 810/9/99, 390/98 and 485/9/97, and these are all isolates from Europe (real-European genogroup). The second genogroup contained U5575-1 and 04-085-1, and these isolates are from North America, but they are very similar to those isolates from Europe (European-in-North America genogroup). The F tree also classified the North American genotype into two genogroups consisting of the two North American HPR groups, HPR20 (isolate 98-0280-2) and HPR21 (isolates 01-0593-1, 98-049-1, 02-0775-14, 01-0973-3, 02-1179-4, 7833-1 and NBISA01).
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). The influenza A virus HA gene, in which both the receptor and fusion activities are in one protein, has an even higher rate of mutation estimated at about 2x10–3 base substitutions per position per virus generation (Webster et al., 1992).
Estimation of divergence time of ISAV
The third step in our evolutionary relationship analysis was to calculate the divergence time for each inner node of a rooted phylogenetic tree. We fed the BACKTRACK program with the phylogenetic trees shown in Fig. 2(a) and (b), and the year of isolation data shown in Table 1. This program generated divergence time estimation for all the inner nodes in these phylogenetic trees. The results are shown as year intervals in parentheses in Fig. 2. It must be noted that while the estimations of BACKTRACK may not be perfect, they can be used to gain some insights of the complex evolutionary mechanism of ISAV. Fig. 2(a) shows the estimated divergence time for the F gene for the real-European genogroup and the European-in-North America genogroup to be between 1977 and 1991; this estimate conforms to the estimate based on the HE gene in Fig. 2(b) (1976 and 1988). Fig. 2(a) also shows the divergence of two genogroups within the North American genotype: one genogroup is represented by ISAV isolate 98-0280-2 of HPR20; the second genogroup contains all the other isolates of the North American genotype belonging to HPR21. We estimated that this separation occurred from 1993 to 1996. The estimated divergence time for the European genotype and the North American genotype is 1758 to 1781.
The divergence time estimation of the HE gene is shown in Fig. 2(b). According to our estimation, the European genotype and the North American genotype diverged between 1879 and 1891. This is much later than the estimated result based on the F gene in Fig. 2(a) (1758–1781). However, it is earlier and more specific than the previous estimated period that was based on evolution of the ISAV PB1 gene (Krossøy et al., 2001b). We believe that these discrepancies are related to the uniformed mutation rate model. In our estimation, we assume the mutation rate for one gene is the same during the evolution process. This model works fine for short-time divergence time estimation, but for long-term divergence time estimation, the accumulation of errors could make the estimation inaccurate, particularly if there are also virus protein-specific functions constraining the evolution of certain genes. A variable-rate mutation model needs to be developed for more precise divergence time estimation.
Fig. 2(b) also shows the estimated divergence time for the real-European genogroup and the European-in-North America genogroup to be between 1976 and 1988. Interestingly, this newly introduced virus from the Bay of Fundy, New Brunswick, Canada, is of lower virulence compared with the real-European genogroup and the North American genotype viruses (Tables 1 and 2, Fig. 1). Given the fact that the European-in-North America genogroup is associated with the presence of the 349NQT351 and 352FNT354 motifs in the HE protein stalk, the analyses suggest that the original ancestral ISAV did not have these motifs and was therefore virulent. This direction of evolution supports insertion of specific motifs resulting in ISAV attenuation, which is in contrast to the widely held deletion theory. At the present time we cannot categorically state the source of the inserted genetic material in the HPR of HE. However, such an event is consistent with the recombination events on segment 5 in the F protein (Fig. 1a; Devold et al., 2006).
In conclusion, we identified putative molecular markers of ISAV virulence by comparing the sequences of potential virulence genes (F and HE) in 13 different isolates of ISAV. The results showed candidate motifs in the surface glycoproteins of ISAV isolates that probably play a similar role to the virulence factor at the HA cleavage site of avian influenza A viruses. We found that sequence variation between ISAV isolates in the two surface glycoprotein genes correlated not only with geographical origin but also with cytopathogenicity in cell culture and pathogenicity for fish. To our knowledge, this is the first systematic demonstration for ISAV of a direct molecular relationship between the HE gene (HE protein stalk length and 352FNT354 motif in HPR), the F gene (motif 265YP266 very close to F protein cleavage site), ISAV cytopathogenicity in cell culture and the ability to cause clinical disease in Atlantic salmon. Furthermore, development and use of a novel computer program, BACKTRACK, allowed us to put a timeline on the divergence between the different genotypes and genogroups of ISAV in the northern Hemisphere.
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ACKNOWLEDGEMENTS |
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Received 17 April 2007;
accepted 20 July 2007.
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