1 Title: Bioinformatic Analysis of the Genome of Infectious Salmon Anemia Viruses 1 associated ...

43 Pages·2010·3.2 MB·English

JVI Accepts, published online ahead of print on 1 September 2010
J. Virol. doi:10.1128/JVI.01202-10
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Title: Bioinformatic Analysis of the Genome of Infectious Salmon Anemia Viruses
2 associated with outbreaks of high mortality in Chile
3
4 Authors: Cottet L.*1, Cortez-San Martin M.*1, Tello M.1, Olivares E.1, Rivas-
5 Aravena A.2, Vallejos E.2, Sandino AM.1,2, and Spencer E.1
6
D
o
7 1Centro de Biotecnología Acuícola, Laboratorio de Virología, Facultad de Química w
n
lo
8 y Biología, Universidad de Santiago de Chile. Avenida Libertador Bernardo a
d
e
9 O’Higgins 3363, Santiago, Chile d

f
r
o
10 m

h
11 2Research and Development Laboratories, Laboratorios GAM S.A., Santiago, Chile ttp
:
/
/
12 jv
i.
a
s
13 Corresponding Author: m
.
o
r
14 Cortez-San Martin M. g
/

o
15 email: [email protected]. Telephone: [phone] n

A
p
16 r
il
3
,
17 * Both authors contributed equally to this work. 2
0
1
9

b
y

g
u
e
s
t
1
18 ABSTRACT
19 The Infectious Salmon Anemia Virus (ISAV), an Orthomyxovirus, is at present the
20 major cause of the outbreaks of high mortality rates of salmon in Chile. It has been
21 proposed that the virulence of ISAV isolates lies mainly in hemagglutinin-esterase
22 and fusion glycoproteins. However, based on the current information, the
23 contribution of other viral genes cannot be ruled out. To study this aspect, we
D
o
24 isolated and determined the complete coding sequence of two high-prevalence w
n
lo
25 Chilean isolates associated with outbreaks of high mortality rates: ISAV752_09 and a
d
e
26 ISAV901_09. These isolates were compared to fifteen Norwegian isolates that d

f
r
o
27 exhibit differences in their virulence. For this purpose, we performed: bioinformatic m

h
t
28 analysis of i) functional domains, ii) specific mutations, iii) Bayesian phylogenetic tp
:
/
/
29 and, iv) structural comparison between ISAV and Influenza glycoproteins using jv
i.
a
s
30 molecular modelling. Phylogenetic analysis shows two genogroups for each m
.
o
r
31 protein, one of them containing the Chilean isolates. The gene sequence of the g
/

o
32 polymerase complex and nucleoprotein indicated that they are closely related to n

A
p
33 homologues from high-pathogenic Norwegian viruses. r
il
3
,
34 Notably, seven of the eight mutations that are present only in the Chilean isolates 2
0
1
35 are on the polymerase complex and nucleoprotein. 9

b
y
36 Structural modelling of hemagglutinin-esterase shows patches of variable residues g
u
e
37 on its surface. Fusion protein modelling shows that insertions are flexible regions s
t
38 that could affect proteolytic processing, increasing either accessibility or the
39 number of recognition sites for specific proteases. We found antigenic drift
40 processes related to insertion into the isolated segment 5 of the ISAV752_09. Our
2
41 results confirm the European origin of Chilean isolates as the result of
42 reassortments from Norwegian ancestors.
43 INTRODUCTION
44 The Infectious Salmon Anemia Virus (ISAV) is a pathogen that principally affects
45 Atlantic salmon, causing multi-systemic disorders. It has been associated with high
46 mortality in the aquaculture industry since 1984 (38). The cumulative mortality of
D
o
47 each outbreak of ISAV in Norway and other countries is very high, reaching 100% w
n
lo
48 in some cases (8, 25, 38, 45, 67). ISAV is a member of the Orthomyxoviridae a
d
e
49 family, and its only member belongs to the Isavirus genus (41, 52). ISAV shows a d

f
r
o
50 pleomorphic structure, with spiky projections composed of hemagglutinin-esterase m

h
t
51 protein. It interacts with the sialic acid receptor (28) and a fusion protein that tp
:
/
/
52 induces the fusion between the virus and endosomal membranes (4). Similar to jv
i.
a
s
53 influenza A and B viruses, ISAV displays eight segments of negative single- m
.
o
r
54 stranded RNA (9), and it has been suggested that ISAV uses its own polymerase g
/

o
55 to copy and transcribe its genome. The function of most proteins encoded by the n

A
p
56 segments of ISAV has been assigned according to their similarities to the proteins r
il
3
,
57 encoded by the influenza A virus. Thus, polymerase, which in influenza A 2
0
1
58 synthesizes both mRNA and vRNA, is constituted by three putative proteins coded 9

b
y
59 by segment 1 (polymerase basic 2, PB2 (73), segment 2 (polymerase basic 1, PB1 g
u
e
60 (41)), and segment 4 (polymerase acid, PA (3, 65, 73)). Segment 3 codes for s
t
61 nucleoprotein, NP, which participates in vRNA transport to the nucleus (3).
62 In the influenza A virus, hemagglutinin (HA) protein is coded by segment 4 and is
63 responsible for sialic acid recognition (28), erythrocyte agglutination (20), and
64 fusion between the viral membrane and the endosome (4). Unlike influenza A
3
65 viruses, where hemagglutinin and fusion activity are present in the same
66 polypeptide chain, fusion and hemagglutinin activity in ISAV correspond to two
67 independent proteins coded by segments 5 and 6, respectively, making ISAV the
68 first member of the Orthomyxoviridae family that has shown these activities in
69 different proteins (4). Furthermore, for ISAV, hemagglutinin protein displays a
70 receptor-destroying activity in the same protein, like Influenza C, hence it is called
D
o
71 hemagglutinin-esterase (HE) (40). The fusion protein in ISAV is synthesized as a w
n
lo
72 precursor protein designated as F0. For fusion between viral and cellular a
d
e
73 membranes, F0 must be cleaved by cellular proteases to generate F1 and F2, d

f
r
o
74 which are held together by di-sulfur bridges. Fusion activity is significantly m

h
t
75 improved by HA (4). Segment 7 codes for two nonstructural proteins. The NS1 tp
:
/
/
76 protein is coded by ORF1 and exhibits interferon antagonist activity (24). As with jv
i.
a
s
77 influenza A, NS2 is a splicing product of the same transcript, which may m
.
o
r
78 correspond to a nuclear export protein since it contains nuclear export signals. A g
/

o
79 third protein was detected in TO cells infected with the Canadian RPC/NB 980- n

A
p
80 049-1 isolate that can be recognized by antibodies against NS1. Furthermore, the r
il
3
,
81 analysis of North American isolates seems to suggest the theoretical existence of 2
0
1
82 this third protein, while the European isolates are predicted to generate a truncated 9

b
y
83 protein (39, 48). g
u
e
84 It has been suggested that segment 8 codes for 2 proteins from a bicistronic s
t
85 mRNA. The first ORF codes for the matrix protein, which is the major structural
86 protein of the virion (5, 24, 78), and the second ORF codes for an M2 protein that
87 displays two nuclear localization signals. M2 is able to bind RNA and shows anti-
88 interferon activity (24).
4
89 Through the sequence comparison of segments 2 and 8, ISAV isolates were
90 classified into two genotypes, European and North American. Both genotypes
91 probably diverged in the year 1900, coinciding with the beginning of European
92 salmon exports to America. (6, 31, 42). With the appearance of more isolates, it
93 was established that strains from Norway, Scotland, the Faroe Islands, and Nova
94 Scotia belong to the European genotype. On the other hand, Canadian and North
D
o
95 American isolates are considered North American genotypes (13, 36, 64). Based w
n
lo
96 on the similarity to segments 2 and 8 of Canadian virus isolates in 2001, Chilean a
d
e
97 ISAV isolated from infected Coho salmon was initially classified as the North d

f
r
o
98 American genotype (34, 57). However, comparisons made in 2009 of 51 m

h
t
99 sequences of segment 5 and 78 sequences of segment 6 from Chilean isolates, tp
:
/
/
100 obtained from Atlantic salmon since 2007, showed that Chilean isolates have a jv
i.
a
s
101 Norwegian origin. Evidence strongly suggests that ISAV was introduced in Chile as m
.
o
r
102 an avirulent strain that mutated into virulent (35). g
/

o
103 The most variable genomic elements of ISAV are located in segments that code for n

A
p
104 surface glycoproteins HE and F. The segment that encodes for HE has a mutation r
il
3
105 rate of 1.13x10-3 nucleotides per site per year, and is the major determinant of , 2
0
1
106 variability. The variability of HE is located mainly in two regions, one localized in 9

b
y
107 the N-terminal end, in the extracellular region of the protein. This region allows for g
u
e
108 classifying European isolates into three groups: Group 1 contains only a few s
t
109 Norwegian isolates; group 2 contains isolates from Norway, one from the Faroe
110 Islands and one from Scotland; group 3 contains Scottish and Norwegian isolates
111 (57).
5
112 The second region located in the C-terminal end of HE, in the extracellular portion
113 of the protein, close to a transmembrane domain (63), is considered the major
114 determinant of variability. This region is highly variable among genotypes, so it was
115 therefore defined as a highly polymorphic region, HPR (13). Although it has been
116 proposed that HPR is the result of recombination (13), the most plausible
117 mechanism is that each HPR comes from partial deletions of precursor HPR0 (11).
D
o
118 This is supported by the fact that HPR0 contains every HPR sequences described w
n
lo
119 until now, with HPR0 being the longest sequence. In addition, HPR0 has been a
d
e
120 reported in healthy fish, suggesting that it is a non-virulent precursor that could d

f
r
o
121 generate the virulent strains described for ISAV (10, 11, 50, 57) through multiple m

h
t
122 deletions of its sequence. Nearly 30 different HPRs have been identified. It has tp
:
/
/
123 been suggested that the diversity of HPR sequences provides antigenic variability jv
i.
a
s
124 (57). HPR was proposed as a virulence marker, with the most virulent strains m
.
o
r
125 containing HPR4 (36, 37, 50, 51), but some authors do not agree with that g
/

o
126 conclusion (66). Although every HPR in Chile has been identified, the most n

A
p
127 abundant is the genotype 7b, which originates from Norway (35). r
il
3
,
128 The other surface protein, the fusion protein, is also used to determine genetic 2
0
1
129 variation in ISAV. The segment 5 sequence has a mutation rate of 0.67x10-3 9

b
y
130 nucleotides per site per year (37), lower than that of HE. Close to the cleavage site, g
u
e
131 several isolates display an insertion of 8 to 11 amino acids (IN). To date, four s
t
132 insertions called IN1, IN2, IN3 and IN4 have been described. At the genomic level,
133 IN1 is identical to a sequence of segment 3 from positions 1100 to 1123; IN2 is
134 identical to a sequence from segment 5 located between nt 123 to 155, IN3 is
135 identical to nt 93-122 of segment 5, and IN4 is identical to nt 399 to 429 of segment
6
136 2. This supports the notion of recombination between ISAV segments (14) as a
137 source of variability. Notably, in an analysis of 51 Chilean isolates, 43 showed 11
138 amino acid insertions identical to IN4. This insertion has only been found in Chilean
139 isolates (35). Recently, the IN region has been associated with virulence (25).
140 Little is known about the function of the proteins of ISAV; most of the conclusions
141 are based on their homology with their putative counterparts in influenza viruses.
D
o
142 This paper describes the sequencing of the coding region and genetic w
n
lo
143 characterization of ISAV752_09 and ISAV901_09, which are the most common a
d
e
144 Chilean ISAV isolates. ISAV752_09 displays HPR 7b genotype and ISAV901_09 d

f
r
o
145 displays HPR 1c genotype. These viruses, which display different HPR and IN m

h
t
146 sequences, were analyzed for the presence of conserved domains, predicted tp
:
/
/
147 structural domains, folding homologues, and phylogenetic relationships, compared jv
i.
a
s
148 to sequences of coding regions of the European isolates. m
.
o
r
149 g
/

o
n

A
p
r
il
3
,

2
0
1
9

b
y

g
u
e
s
t
7
150 MATERIAL AND METHODS
151 Bioinformatic analysis.
152 Homologues of proteins coded by ISAV genomes were analyzed at three levels:
153 sequence, structure, and folding. Homologous sequences were identified using
154 BLAST (2) against a non-redundant database of sequence protein deposited in a
155 gene bank. Structural homologues were identified through BLAST against the PDB
D
o
156 database. Folding homologues were identified using PHYRE w
n
lo
157 (http://www.sbg.bio.ic.ac.uk/phyre/) (32) and ROBETTA (http://robetta.bakerlab.org/) a
d
e
158 servers (12). A cutoff of 60% certainty was used with PHYRE as a limit to folding d

f
r
o
159 homology. This means that at least 60% of amino acids are able to fold into this m

h
t
160 structure. ROBETTA server was used to perform a folding homology search with tp
:
/
/
161 BLAST, PSIBLAST, HHSEARCH and pFAM. An E-value cutoff of 0.001 was used jv
i.
a
s
162 for BLAST, PSIBLAST and pFAM, and a cutoff prob of 85 was used for m
.
o
r
163 HHSEARCH. Homology modeling was performed using Modeller 9v6 Linux 386 g
/

o
164 (18). Structural homologues were identified using BLAST against the PDB databank n

A
p
165 or folding recognition servers. A total of 50 models were generated and one of them r
il
3
,
166 was chosen according to their lowest value of the objective function. The quality of 2
0
1
167 the models was evaluated using PROSA 3.0 (77) 9

b
y
168 (https://prosa.services.came.sbg.ac.at/prosa.php). Structures were visualized with g
u
e
169 the VMD software (30). Global multi-alignment was performed with ClustalW (76), s
t
170 using the BLUSOM 62 or BLUSOM 45 matrix. Local multi-alignments were
171 performed using DIALIGN2 (http://bibiserv.techfak.uni-bielefeld.de/dialign/) (53).
172 RPS-BLAST (46) was used to analyze the conserved domains, with the lowest E-
8
173 value as a cutoff (0.001). PHYRE server was used to predict secondary protein
174 structures.
175 Cloning and sequencing genomes from ISAV752_09 and ISAV901_09
176 isolates.
177 Viral RNAs were isolated from kidneys of Salmo salar infected with ISAV, and the
178 isolates were named ISAV752_09 and ISAV901_09 (Gift GAM Laboratory S.A).
D
o
179 Total RNA was extracted using the total RNA I kit (E.Z.N.A, Omega Bio-tek). RT- w
n
lo
180 PCR for each ISAV segment was carried out separately using the One-Step RT- a
d
e
181 PCR System with Platinum® Taq DNA Polymerase (Superscript III, Invitrogen), d

f
r
o
182 according to the manufacturer’s conditions. The following thermal program was m

h
t
183 used: 30 min at 50 °C, 2 min at 94 °C, followed by 39 cycles of 15 s at 94 °C, 30 s tp
:
/
/
184 at 55 °C and 30 s at 68 °C, and a final extension o f 5 min at 68 °C. The primers jv
i.
a
s
185 used in each RT-PCR are listed in Table 1. m
.
o
r
186 PCR products for each segment were cut from 1% agarose gel, and cDNA was g
/

o
187 purified using the E.Z.N.A (Omega Biotek) gel extraction kit. Purified cDNA was n

A
p
188 cloned in pGEMT Easy (Promega) following the manufacturer’s instructions and a r
il
3
,
189 previously described method (70). The identity of each cloned segment was further 2
0
1
190 confirmed by DNA sequencing of both strands (Macrogen, USA). 9

b
y
191 Sequences and GenBank access number. g
u
e
192 The genomic sequences of ISA viruses chosen for this study came from isolates s
t
193 with a completely sequenced coding region which includes the sequences of 15
194 previously reported Norwegian viruses, plus the complete sequences of two
195 Chilean ISA viruses presented in this work. The GenBank access numbers of the
196 sequences used in this work are GU830895 to GU830902 for the ISAV752_09
9
197 isolate, and GU830903 to GU830910 for the ISAV901_09 isolate. The other
198 sequences used in this work correspond to those previously reported by
199 Markussen et al. 2008, (GenBank access numbers from DQ785175 to DQ785285),
200 and the sequences of the SK77-06 isolate (GenBank access numbers EU118815
201 to EU118822). The sequences used in this work are summarized in the
202 supplementary material (A-1).
D
o
203 Bayesian analysis. w
n
lo
204 In order to conserve the proper reading frame, each gene sequence was converted a
d
e
205 into amino acids using the AlignmentHelper 1.0 program (49). A copy of the d

f
r
o
206 original nucleotide sequence was kept. Then each gene was aligned separately m

h
t
207 using MUSCLE (17), with default parameter settings. The aligned amino acid tp
:
/
/
208 sequences were back translated into their original nucleotide sequence using jv
i.
a
s
209 AlignmentHelper. Model selection for each gene was determined using the Akaike m
.
o
r
210 Information Criterion (1) as implemented in jModelTest (26, 60). The best-fit g
/

o
211 models for most of the partitions corresponded to General Time Reversible (GTR) n

A
p
212 + G. Phylogenies and were estimated using Bayesian Inference as implemented in r
il
3
,
213 BEAST 1.5.3 (16), with a relaxed uncorrelated lognormal molecular clock (15). 2
0
1
214 Bayesian Posterior probabilities were determined by running 200 million 9

b
y
215 generations with a discarded burn-in of 10% and the trees were sampled every 20 g
u
e
216 thousand generations. Tracer 1.5 (62) was used to check for convergence and s
t
217 mixing. Branch support values are reported as posterior probabilities.
218
10