- Email: [email protected]
Identification and isolation of infective filamentous particles in Infectious Salmon Anemia Virus (ISAV)
Accepted Manuscript Identification and isolation of infective filamentous particles in infectious salmon anemia virus (ISAV) Ramón Ramírez, Sergio H. Marshall PII:
S0882-4010(17)31771-0
DOI:
10.1016/j.micpath.2018.02.029
Reference:
YMPAT 2795
To appear in:
Microbial Pathogenesis
Received Date: 27 December 2017 Revised Date:
5 February 2018
Accepted Date: 13 February 2018
Please cite this article as: Ramírez Ramó, Marshall SH, Identification and isolation of infective filamentous particles in infectious salmon anemia virus (ISAV), Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.02.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Identification and isolation of infective filamentous particles in Infectious
2
salmon anemia virus (ISAV).
3
Ramón Ramírez1, 3; Sergio H. Marshall1,2,3, *.
4
1. Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Facultad
5
de Ciencias, Pontificia Universidad Católica de Valparaíso, Campus Curauma
6
Valparaíso, Chile.
7
2. Laboratorio de Patógenos Acuícolas, Núcleo Biotecnología Curauma, Pontificia
8
Universidad Católica de Valparaíso, Valparaíso, Chile.
9
3. Unidad de Microscopía Confocal.
SC
Núcleo Biotecnología Curauma. Pontificia
Universidad Católica de Valparaíso, Valparaíso, Chile.
M AN U
10
RI PT
1
11
*Corresponding author, Sergio H. Marshall. Mailing address: Laboratorio de
13
Genética e Inmunología Molecular. Instituto de Biología. Facultad de Ciencias.
14
Campus Curauma Pontificia Universidad Católica de Valparaíso. Avenida
15
Universidad 330. Curauma. Valparaíso, Chile. Phone: (56-32) 2274866. Fax: (56-
16
32) 227-4835. E-mail: [email protected].
17
First Author email: Ramón Ramírez, [email protected]
20 21 22 23 24 25 26
EP
19
AC C
18
TE D
12
ACCEPTED MANUSCRIPT
Abstract
28
The infectious salmon anemia virus (ISAV) is an aquatic pathogen that is a
29
member of the Orthomyxoviridae family with lethal hemorrhagic potential. Although
30
it affects other species of salmonid fish, ISAV only causes disease in Atlantic
31
salmon (Salmo salar) specimens in sea water. In spite of the fact that the virus has
32
been described as enveloped with icosahedral symmetry, viral like particles with
33
anomalous morphology have been observed in field samples, this we have not
34
been able to recover then in adequate quantities for full demonstration. We report a
35
procedure to concentrate and recover these novel forms of the virus, comparing
36
two cell lines from different origins, demonstrating that these forms were
37
preferentially expressed in cells of epithelial origin.
M AN U
SC
RI PT
27
38 39
Keywords:
40
Salmo salar; ISAV; morphology; epithelial cells; filamentous particles.
44 45 46 47 48 49 50 51 52 53
EP
43
AC C
42
TE D
41
ACCEPTED MANUSCRIPT
54
1. Introduction
56
Infectious Salmon Anemia (ISA) is a highly contagious disease that preferentially
57
affects Atlantic salmon (Salmo salar), the main fish species cultivated in Chile. Its
58
etiologic agent is a virus belonging to the Orthomyxoviridae family, known as
59
Salmon Infectious Anemia Virus (ISAV), which presents a wide number of variants
60
with different degree of virulence [1] [2] [3] [4].
61
The virus is morphologically described as enveloped spherical particles ranging in
62
size from 45 to 140 nm in diameter[2] [3] [4]. In addition filamentous particles have
63
also been reported although their role in infection has not been elucidated [5] 7.
64
These large-sized filamentous particles are highly pleomorphic, encountered in
65
small amounts measuring up to 700 nm in length and covered with spikes of 10
66
nm in length [5].
67
While the structures of ISAV have been described in many reports [2]-[5], the role
68
that these structures might have in the biology and/or pathogenesis of ISAV has
69
not been experimentally elucidated, likely because the viral load is low in infected
70
cells, and the virus is extremely fragile[6]. Indeed, the most recent studies tend to
71
ignore these anomalous structures, defining ISAV exclusively as a pleomorphic
72
virus that is usually spherical or ovoid in shape and covered in 10nm long spikes
73
that are evenly distributed in the envelope, with a 90 ± 130 nm diameter [7–16].
74
Based on the lack of understanding in the field, we aimed to develop a procedure
75
to enrich these large anomalous structures in an in vitro system, to evaluate their
76
infective potential compared with standard, normal-sized particles. The procedure
77
involves growing the virus in two cell lines of different origins, concentrating the
78
large forms via differential filtration, followed by differential gradient purification,
79
and evaluation of their comparative infective potentials in the same cell line of
80
origin.
81
To obtain a high enough concentration of the filamentous virus particles for the
82
procedure, it was necessary to increase the viral load during the infective process.
AC C
EP
TE D
M AN U
SC
RI PT
55
ACCEPTED MANUSCRIPT
In order to achieve this, we designed a strategy derived from information available
84
from the influenza virus (IAV), a well-studied pathogen that is closely related to
85
ISAV. In IAV, similar filamentous morphologies have been observed in vitro only at
86
low numbers of passages, while standard spherical morphology remain constant in
87
time [17]. On the other hand, the filamentous forms are preferentially expressed
88
when grown in tissue culture cells of epithelial origin, while virus produced by
89
infected non-epithelial cells yield almost exclusively particles of spherical
90
morphology [18].
91
2. Material and Methods
92
2.1 Cell line
93
An epithelial fish-cell line (ASK; Atlantic Salmon Kidney; ATCC® CRL-2747™) was
94
infected with ISAV in parallel with a cell line derived from Atlantic salmon peripheral
95
blood leucocytes [19] (SHK-1; Salmon Head Kidney-1; SIGMA). Specific antibodies
96
were used to ensure the epithelial origin of the corresponding cell line (figure 1B; 1-
97
4); (anti-cadherin; 1:500; Thermo, Waltham, MA USA: clone # PA5-19479 and anti-
98
cytokeratin [AE1+AE3]; 1:200, ab961, Abcam, Cambridge, UK).
99
The cell lines were maintained at 17°C in Leibovitz medium (L-15; Gibco,
100
Invitrogen, United Kingdom) supplemented with penicillin (100 IU ml−1),
101
streptomycin (100 g ml−1), and 15% fetal calf serum (FCS; Gibco).
102
2.2 Viral infection
103
Each cell line was infected at a multiplicity of infection of 1 (MOI 1.0), with the
104
virulent HPR3 ISAV variant passaged only twice in vitro before infection. Cells
105
were incubated at 17°C for two weeks.
106
2.3 Confocal scanning microscopy (CSM).
107
In order to determine the type of morphology existing and remaining in the different
108
cell lines, the attached cells were exposed to specific antibodies to evaluate the
AC C
EP
TE D
M AN U
SC
RI PT
83
ACCEPTED MANUSCRIPT
predominant viral morphology. The cultures were incubated at 17°C, and
110
completed the post-infection time, cells were fixed with 4% formaldehyde in 0.13 M
111
phosphate buffer at pH 7.4 and at room temperature for 15 min and washed with
112
PBS. Cells were then permeabilized for 1 h at room temperature with 0.1% Triton
113
X-100 in 1× PBS. The cells were fixed, and cell sections were blocked with 3%
114
bovine serum albumin (BSA) containing 0.5% Tween 20 for 30 min at 37°C.
115
Evaluation was done via confocal microscopy using a 1:500 dilution of a
116
monoclonal antibody (clone 8D2/E9, Austral Biologicals) against the ISAV
117
Hemagglutinin-Esterase protein, to visualize and distinguish types of viral
118
morphologies produced by each cell line [18] [20] [21].
119
Slides were prepared by using Vectashield mounting medium. Image acquisition
120
was performed with a Leica TCSSP5 II confocal microscope, and samples were
121
observed under a 100× lens.
SC
2.4 Viral isolation
M AN U
122
RI PT
109
After two weeks, the supernatant of infection was recovered, centrifuged at 3,000
124
rpm for 15 minutes to remove cell debris, and then further centrifuged at 10,000
125
rpm for another 15 minutes. The resulting supernatants were pooled for each cell
126
line and filtered through Centricon Amicon Ultra-15® centrifugal filter units
127
(Millipore, USA), and centrifuged at 4,000xg four times for 5 minutes with short
128
intermediate pauses to enhance concentration efficiency. The use of Centricon®
129
centrifugal filter units is the key to obtaining enough biomass, since they
130
concentrate viral particles to a minimal final volume (figure 1 A).
131
To further characterize the Centricon®-enriched material, procedures validated for
132
IAV were used to resolve the anomalous particles from the standard types, as the
133
filamentous particles should migrate faster than the spherical ones [18]. 1.5 mL of
134
the remaining filtered material from each cell line were resolved through a 20-60%
135
continuous sucrose gradient and centrifuged for 2 hours, at 100,000xg, 4°C.
136
Nevertheless, to verify true mobility of the putative ISAV anomalous particles, a
AC C
EP
TE D
123
ACCEPTED MANUSCRIPT
parallel procedure was evaluated. This procedure involved labeling the Centricon®-
138
filtrate with an FITC-HE polyclonal mono-specific antibody and running it in a
139
sucrose gradient to visualize the different viral forms resolved. Visualization of viral
140
forms in a sucrose gradient was done via fluorescent light source (488 nm).
141
To evaluate the infective potential of each viral form resolved though the sucrose
142
gradient, each recovered fraction (the gradients were fractionated into 28 aliquots
143
of 0.5 mL) from each gradient was submitted to three different analyses: A: qRT-
144
PCR; B: plaque assay, and C: TEM.
145
2.5 qRT-PCR.
146
Specific primers for ISAV segment 8 and PCR conditions were as described by
147
Snow et al., the official procedure validated by Sernapesca-Chile [22]. Samples
148
were considered ISAV-positive based on cycle threshold (Ct) values <30.
149
confirm the virulence potential of ISAV, specific primers for segment 6 were used
150
as previously described [23]. The RNA was extracted with the RNA easy MiniKit
151
(Qiagen, MD, USA).
152
2.6 Virus Titration.
153
Infectivity titers were determined as described previously by plaque assay [24]. The
154
cells were seeded into 6-well plates at a density of 1 x 105 cells cm-2, and cells
155
were incubated for three days at 17 °C. The viral inoculum was tenfold serially
156
diluted, ranging from 1/10 (10-1) to 1/100.000 (10-5) in L15 medium (2X) without
157
FBS. The culture medium was removed from the seeded cells, and the virus was
158
added in a volume of 500 uL, which was absorbed on the cellular monolayer for
159
four hours at 17 °C. The inoculum was removed and 3 mL of semi-solid medium
160
was added to each well. The semi-solid medium was composed of L15 medium
161
supplemented with FBS (10%), and LMP agarose (0.5%) (UltraPureTM Low
162
Melting Point Agarose (Invitrogen cat. 16520-050). The plates were incubated for
163
15 days post-infection at 17 °C. For visualizing, 2 mL of crystal violet (1%) was
164
added for 1 h at 25 °C, and finally the excess crystal violet was removed.
To
AC C
EP
TE D
M AN U
SC
RI PT
137
ACCEPTED MANUSCRIPT
2.7 Transmission electron microscopy (TEM)
166
To evaluate the content in the filtered supernatant, 100 µL aliquot of each sample
167
from the Centricon® filtrate were independently loaded onto Formvar-coated carbon
168
grids for 1 min, excess liquid was carefully withdrawn with Whatman no. 1 filter
169
paper, and the cells were stained with 10 µl of 1% aqueous uranyl acetate followed
170
by a 10-min incubation at 37°C before visualization under the EM (141 Phillips
171
Tecnai electron microscope; at a range of 12 to 80kV).
172
2.8 Freezing and thawing assay.
173
Two cycles of freezing and thawing were carried out from the same aliquot of
174
purified virus. From each sample 100 µl were removed to perform negative staining
175
and visualized by TEM. The remaining volume was used to infect the ASK cell line
176
with an MOI of 1 for 15 days and the viral titer was measured by plaque assay.
177
3. Results and Discussion
178
Figure 1A summarizes the procedure to concentrate the different types of viral
179
morphologies. When evaluating viral morphology by means of CSM, filamentous
180
particles associated with the surface of the epithelial cell line could be visualized
181
(Figure 1B; 6), condition that was not observed in non-epithelial cells (figure 1B; 5).
182
From the epithelial cells, two types of morphologies were exclusively visualized.
183
One of the EM-visualized cell types were filamentous particles between 700 to
184
2500 nm in length and 70 to 120 nm in width, as well as the typical smaller
185
spherical in morphology, as demonstrated in figure 1C. In the non-epithelial cell
186
line, only spherical smaller-sized particles were visualized (data not shown). Figure
187
1 summarizes the enrichment procedure. Figure 2 shows two bands only in the
188
epithelial extract, in agreement with the resolution observed for IAV, in which the
189
lighter band corresponds to the standard spherical particles, and the faster moving
190
band to the filamentous anomalous. With this information in hand, the standard
191
gradients were fractionated into 28 aliquots of 0.5 ml.
AC C
EP
TE D
M AN U
SC
RI PT
165
ACCEPTED MANUSCRIPT
Figure 3-A shows the results of RNA extracted from each fractionated gradient to
193
confirm the presence of ISAV by standard qRT-PCR procedures [22]. The highest
194
Ct value observed with the filamentous particles implies a lower viral RNA load, in
195
accordance with the low titer obtained. In contrast, the lower Ct values obtained
196
with the spherical particles perfectly matches with the higher viral titer obtained.
197
Notwithstanding, the decrease in RNA content might be also due to the fragility of
198
these filamentous particles that are highly sensitive to ultracentrifugation.
199
The plaque assay results shown in Figure 3-A, indicates positive responses in
200
fractions 17 and 21 for the epithelial extract, while positiveness was only detected
201
in fraction 17 from the non-epithelial cells.
202
morphologies were detected in the epithelial cells, the viral titer detected in fraction
203
17 (spherical particles) was higher than that of fraction 21 (filamentous particles)
204
probably due to the fragility of the latter as previously described.
205
Figure 3 shows the results of the TEM analyses. Consistent with our previous
206
observations and expectations, fractions 17 from both gradients contained the
207
spherical classical particles (figure 3-B) while only fraction 21, exclusively from the
208
epithelial cell line, was enriched with the large-sized filamentous virus particles
209
(figure 3-B). Finally, to further confirm that the filamentous particles corresponded
210
to ISAV, an immuno-gold assay was performed using an anti HE protein as the
211
primary antibody, and an anti-mouse IgG conjugated with gold particles (Sigma),
212
as the second antibody, confirming their ISAV nature (figure 3 B and C; lower
213
sections).
214
While upon initial investigation, the data appear to suggest that the filamentous
215
particles could be exclusively generated in epithelial cells, figure 4 shows that this
216
is not the case, as filamentous particles are visualized in the non-epithelial cell line
217
as well, but in very low quantity. Notwithstanding, the fragility of these large
218
filamentous particles described, constitutes a real problem in regard to the
219
reproducibility of the proposed enrichment procedure. In fact, more than one round
220
of ultracentrifugation significantly decreases the amount of filamentous viral
221
particles recovered, and losing the entire content is possible. Additionally, extreme
SC
RI PT
192
AC C
EP
TE D
M AN U
In spite of the fact that both
ACCEPTED MANUSCRIPT
care must be taken to perform freezing and thawing cycles of the aliquots
223
containing these particles, because they lose infectivity easily. This was confirmed
224
by an additional experiment described in figure 5, which showed that when purified
225
particles were exposed to two cycles of freezing and thawing, they changed in
226
morphology and decreased in infectivity. This occurred only in the filamentous
227
structures and less prominently in the regular spherical ones (A and B,
228
respectively).
229
4. Conclusions
230
We conclude that the large-size infective filamentous structures described in IAV
231
literature also exist in ISAV, and although they might exist in cells of different
232
origins, they are particularly enriched in epithelial cells in vitro. The meaning of this
233
is currently not understood. The described structures are extremely fragile, and
234
appear to co-exist with the standard-size virus particles, with equivalent infectivity,
235
demonstrated by the fact that when parallel infections are carried at equivalent
236
MOIs, similar results are observed. In order to elucidate the role, these particles
237
might have in the infectivity potential of ISAV, biomass analysis would be required,
238
which was outside of the scope of this novel study on enriching for these
239
anomalous particles in epithelial cell lines.
SC
M AN U
TE D
EP AC C
240
RI PT
222
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
241 242
Figure 1.
AC C
EP
TE D
243
244 245
Figure 2.
ACCEPTED MANUSCRIPT
246 247 248
RI PT
249 250 251 252
SC
253 254
M AN U
255 256 257 258
TE D EP
260
Figure 3.
AC C
259
ACCEPTED MANUSCRIPT
261 262 263 264
RI PT
265 266 267 268 269
SC
270 271 272
M AN U
273 274 275 276
EP
TE D
Figure 4.
AC C
277
ACCEPTED MANUSCRIPT
278 279 280 281 282
RI PT
283 284 285 286
SC
287 288 289
M AN U
290 291 292 293 294
Figure 5.
295
299 300 301 302
EP
298
AC C
297
TE D
296
ACCEPTED MANUSCRIPT
Figure 1: Procedure to enrich anomalous filamentous viral particles from
304
ISAV-infected epithelial cell line culture media. A: Culture media was clarified
305
by light centrifugation (3000 rpm, 15 min), further cleaned (10,000 rpm 15 min),
306
and concentrated by Centricon to enrich for putative anomalous viral particles. B:
307
Demonstration of the epithelial origin of the target cell line (3,4) versus the control
308
(1,2); B1: Immuno-fluorescence for Cadherin (anti-cadherin (1:500; Thermo,
309
Waltham, MA USA: clone # PA5-19479) in non-epithelial cell line; B2: immuno-
310
fluorescence for Cytokeratin (anti-cytokeratin [AE1+AE3] (1:200, ab961, Abcam,
311
Cambridge, UK) in non-epithelial cell line; B3: immuno-fluorescence for Cadherin in
312
epithelial cells; B4: immuno-fluorescence for Cytokeratin in epithelial cells. Used
313
antibodies were labeled in green, while in blue we can see nuclear labeling TO-
314
PRP-3 iodide (643/661) (Life Technologies, USA); B5 and B6: The attached cells
315
of each bottle were exposed to specific antibodies to evaluate the predominant
316
viral morphology. The epithelial cells presented a distribution of HE Protein (in
317
green) in a filamentous form (B6) unlike non-epithelial cell line (B5). C: Enrichment
318
of the anomalous viral particles post-Centricon® in epithelial cells. C2; Zoom,
319
Comparison between filamentous and spherical virus membranes.
320
Figure 2: Visualization of morphologies observed in the continuous 20-60%
321
sucrose gradient. A. Schematic: transfer of Centricon-enriched viral structures
322
resolved by sucrose gradient. B: Visualization of viral forms via fluorescent light
323
source (488 nm); B1: Supernatant of a non-infected, non-epithelial cell line in the
324
presence of HE/FITC; B2: Supernatant of a non-infected, epithelial cell line in the
325
presence of HE/FITC; B3: Supernatant of an infected, non-epithelial cell line in the
326
presence of HE/FITC; B4: Supernatant of an infected epithelial cell line in the
327
presence of HE/FITC. C: Fluorescence analysis of each fractionated gradient. The
328
graph shows two peaks at fractions 17 and 21 in the epithelial cell line, and only
329
one peak at fraction 17 in the non-epithelial cell line. Control supernatants (non–
330
infected cells) did not show fluorescence. Upper 9 fractions from the gradient were
331
not graphed; Abbr. On graph: N-EC: non-epithelial cell; EC: epithelial cell.
AC C
EP
TE D
M AN U
SC
RI PT
303
ACCEPTED MANUSCRIPT
Figure 3: Multiple analyses from selected fractions of the processed
333
epithelial cell line. A: Summary table of the viral titer and indication of the Ct
334
values for viral segments 6 and 8 from fractions 17 and 21. B and C: TEM results
335
for fractions of 17 and 21; scale bar: 100 nm; B: enrichment of spherical particles
336
as opposed to C, where filamentous particles are enriched. Bottom figures B and
337
C: immuno-gold assay demonstrating the ISAV nature of the particles.
RI PT
332
Figure 4: Visualization through TEM and confocal microscopy of filamentous
339
particles in a non-epithelial cell line. A: TEM analysis, scale bar: 500 nm. B:
340
visualization of HE protein labeled with HE/Alexa 543 (red color) antibody
341
distributed as filaments on the cell surface, in green color immune-fluorescence
342
Nucleoprotein (clone 2C2/H4; Austral Biologicals) Labeled with antibodies
343
NP/Alexa 488 and blue color: nuclear labeling con TO-PRO-3 iodide (643/661)
344
(Life Technologies, USA). Scale bar 10µm.
345
Figure 5: High fragility of the ISAV filamentous particles: Effect of freezing
346
and thawing in morphology and infectivity. A1-3 and B1-3: diagrams of two
347
Freezing-Thawing cycles to purified viral morphologies; A4 - A6 and B4 - B6: TEM
348
of each condition. As a result, fraction 21 (filaments), suffered morphological
349
changes and accumulations, which could explain the decrease in infectivity by
350
each condition (A7 - A9). Fraction 17 spherical particles are more resistant to
351
treatment and retain infectivity.
M AN U
TE D
EP
352
SC
338
Acknowledgments:
354
This research was supported by PUCV Scholarship (Ph.D. Thesis to R.R), since
355
2014 until 2017 and also supported by Molecular immunology and genetics
356
laboratory; PUCV.
357
AC C
353
ACCEPTED MANUSCRIPT
358
References
359
[1]
K. Falk, E. Namork, E. Rimstad, S. Mjaaland, B.H. Dannevig, Characterization of infectious salmon anemia virus, an orthomyxo-like virus isolated from Atlantic salmon (Salmo salar L.)., J. Virol. 71 (1997) 9016– 9023.
364 365 366 367
[2]
B.H. Dannevig, K. Falk, E. Namork, Isolation of the causal virus of infectious salmon anaemia (ISA) in a long-term cell line from Atlantic salmon head kidney, J. Gen. Virol. 76 (1995) 1353–1359. doi:10.1099/0022-1317-76-61353.
368 369 370
[3]
A.I. Sommer, S. Mennen, Multiplication and haemadsorbing activity of infectious salmon anaemia virus in the established Atlantic salmon cell line, J. Gen. Virol. 78 (1997) 1891–1895.
371 372 373 374
[4]
D. Bouchard, W. Keleher, H.M. Opitz, S. Blake, K.C. Edwards, B.L. Nicholson, Isolation of infectious salmon anemia virus (ISAV) from Atlantic salmon in New Brunswick, Canada, Dis. Aquat. Organ. 35 (1999) 131–137. doi:10.3354/dao035131.
375 376 377
[5]
C.W.R. Korenl, A. Nylund, Morphology and morphogenesis of infectious salmon anaemia virus replicating in the endothelium of Atlantic salmon Salmo salar, Dis. Aquat. Organ. 29 (1997) 99–109.
378 379 380
[6]
P.C. Roberts, R. a Lamb, R.W. Compans, The M1 and M2 proteins of influenza A virus are important determinants in filamentous particle formation., Virology. 240 (1998) 127–137. doi:10.1006/viro.1997.8916.
381 382 383 384
[7]
T.M. Eliassen, M.K. Frøystad, B.H. Dannevig, M. Jankowska, a Brech, K. Falk, K. Romøren, T. Gjøen, Initial events in infectious salmon anemia virus infection: evidence for the requirement of a low-pH step., J. Virol. 74 (2000) 218–227. doi:10.1128/JVI.74.1.218-227.2000.
385 386 387 388
[8]
F.S. Kibenge, O.N. Gárate, G. Johnson, R. Arriagada, M.J. Kibenge, D. Wadowska, Isolation and identification of infectious salmon anaemia virus (ISAV) from Coho salmon in Chile., Dis. Aquat. Organ. 45 (2001) 9–18. doi:10.3354/dao045009.
389 390 391 392
[9]
393 394 395
[10]
AC C
EP
TE D
M AN U
SC
RI PT
360 361 362 363
a. Nylund, H. Plarre, K. Hodneland, M. Devold, V. Aspehaug, M. Aarseth, C. Koren, K. Watanabe, Haemorrhagic smolt syndrome (HSS) in Norway: Pathology and associated virus-like particles, Dis. Aquat. Organ. 54 (2003) 15–27. doi:10.3354/dao054015.
R.J. De Groot, Structure, function and evolution of the hemagglutininesterase proteins of corona- and toroviruses, Glycoconj. J. 23 (2006) 59–72. doi:10.1007/s10719-006-5438-8.
ACCEPTED MANUSCRIPT
[11]
K. Watanabe, M. Karlsen, M. Devold, E. Isdal, a. Litlabø, a. Nylund, Viruslike particles associated with heart and skeletal muscle inflammation (HSMI), Dis. Aquat. Organ. 70 (2006) 183–192. doi:10.3354/dao070183.
399 400 401 402
[12]
S.T. Workenhe, D.W. Wadowska, G.M. Wright, M.J.T. Kibenge, F.S.B. Kibenge, Demonstration of infectious salmon anaemia virus (ISAV) endocytosis in erythrocytes of Atlantic salmon., Virol. J. 4 (2007) 13. doi:10.1186/1743-422X-4-13.
403 404 405 406
[13]
B.L. Schiøtz, N. Roos, A.L. Rishovd, T. Gjøen, Formation of autophagosomes and redistribution of LC3 upon in vitro infection with infectious salmon anemia virus, Virus Res. 151 (2010) 104–107. doi:10.1016/j.virusres.2010.03.013.
407 408 409 410 411
[14]
L.S. Ulanova, G. Isapour, A. Maleki, S. Fanaian, K. Zhu, A. Hoenen, C. Xu, Ø. Evensen, G. Griffiths, B. Nyström, Development of methods for encapsulation of viruses into polymeric nano- and microparticles for aquaculture vaccines, J. Appl. Polym. Sci. 131 (2014) 8785–8796. doi:10.1002/app.40714.
412 413 414 415
[15]
S.H. Marshall, R. Ramirez, a. Labra, M. Carmona, C. Munoz, Bona Fide Evidence for Natural Vertical Transmission of Infectious Salmon Anemia Virus in Freshwater Brood Stocks of Farmed Atlantic Salmon (Salmo salar) in Southern Chile, J. Virol. 88 (2014) 6012–6018. doi:10.1128/JVI.03670-13.
416 417 418 419
[16]
A. Kavaliauskis, M. Arnemo, A.-L. Rishovd, T. Gjøen, Activation of unfolded protein response pathway during infectious salmon anemia virus (ISAV) infection in vitro an in vivo, Dev. Comp. Immunol. 54 (2015) 46–54. doi:10.1016/j.dci.2015.08.009.
420 421 422
[17]
J. Seladi-Schulman, J. Steel, A.C. Lowen, Spherical influenza viruses have a fitness advantage in embryonated eggs, while filament-producing strains are selected in vivo., J. Virol. 87 (2013) 13343–53. doi:10.1128/JVI.02004-13.
423 424
[18]
R.I.W.C. Ompans, Host cell dependence of viral morphology, 95 (1998) 5746–5751.
425 426 427 428 429
[19]
B.H. DANNEVIG, B.E. BRUDESETH, T. GJØEN, M. RODE, H.I. WERGELAND, Ø. EVENSEN, C.M. PRESS, Characterisation of a long-term cell line (SHK-1) developed from the head kidney of Atlantic salmon (Salmo salarL.), Fish Shellfish Immunol. 7 (1997) 213–226. doi:https://doi.org/10.1006/fsim.1996.0076.
430 431 432
[20]
J.S. Rossman, G.P. Leser, R. a. Lamb, Filamentous Influenza Virus Enters Cells via Macropinocytosis, J. Virol. 86 (2012) 10950–10960. doi:10.1128/JVI.05992-11.
433 434
[21]
J.C. Cox, A.W. Hampson, R.C. Hamilton, An immunofluorescence study of influenza virus filament formation, Arch. Virol. 63 (1980) 275–284.
AC C
EP
TE D
M AN U
SC
RI PT
396 397 398
ACCEPTED MANUSCRIPT
doi:10.1007/BF01315033.
435
[22]
M. Snow, P. McKay, A.J.A. McBeath, J. Black, F. Doig, R. Kerr, C.O. Cunningham, A. Nylund, M. Devold, Development, application and validation of a Taqman real-time RT-PCR assay for the detection of infectious salmon anaemia virus (ISAV) in Atlantic salmon (Salmo salar)., Dev. Biol. (Basel). 126 (2006) 133-45–6.
441 442 443 444
[23]
D. Sepúlveda, H. Bohle, A. Labra, H. Grothusen, S.H. Marshall, Design and evaluation of a unique RT-qPCR assay for diagnostic quality control assessment that is applicable to pathogen detection in three species of salmonid fish., BMC Vet. Res. 9 (2013) 183. doi:10.1186/1746-6148-9-183.
445 446 447 448
[24]
M.T. Castillo-Cerda, L. Cottet, D. Toro-Ascuy, E. Spencer, M. Cortez-San Martín, Development of plaque assay for Chilean Infectious Salmon Anaemia Virus, application for virus purification and titration in salmon ASK cells, J. Fish Dis. 37 (2014) 989–995. doi:10.1111/jfd.12198.
M AN U
SC
RI PT
436 437 438 439 440
AC C
EP
TE D
449
ACCEPTED MANUSCRIPT
Highlights:
AC C
EP
TE D
M AN U
SC
RI PT
1. An anomalous viral morphology is described for Infectious Salmon Anemia Virus. 2. Anomalous particles are preferentially expressed in cells of epithelial origin. 3. Description of a method to concentrate and isolate anomalous viral particle. 4. These described particles co-exist with the standard-size virus particles.
S0882-4010(17)31771-0
DOI:
10.1016/j.micpath.2018.02.029
Reference:
YMPAT 2795
To appear in:
Microbial Pathogenesis
Received Date: 27 December 2017 Revised Date:
5 February 2018
Accepted Date: 13 February 2018
Please cite this article as: Ramírez Ramó, Marshall SH, Identification and isolation of infective filamentous particles in infectious salmon anemia virus (ISAV), Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.02.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Identification and isolation of infective filamentous particles in Infectious
2
salmon anemia virus (ISAV).
3
Ramón Ramírez1, 3; Sergio H. Marshall1,2,3, *.
4
1. Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Facultad
5
de Ciencias, Pontificia Universidad Católica de Valparaíso, Campus Curauma
6
Valparaíso, Chile.
7
2. Laboratorio de Patógenos Acuícolas, Núcleo Biotecnología Curauma, Pontificia
8
Universidad Católica de Valparaíso, Valparaíso, Chile.
9
3. Unidad de Microscopía Confocal.
SC
Núcleo Biotecnología Curauma. Pontificia
Universidad Católica de Valparaíso, Valparaíso, Chile.
M AN U
10
RI PT
1
11
*Corresponding author, Sergio H. Marshall. Mailing address: Laboratorio de
13
Genética e Inmunología Molecular. Instituto de Biología. Facultad de Ciencias.
14
Campus Curauma Pontificia Universidad Católica de Valparaíso. Avenida
15
Universidad 330. Curauma. Valparaíso, Chile. Phone: (56-32) 2274866. Fax: (56-
16
32) 227-4835. E-mail: [email protected].
17
First Author email: Ramón Ramírez, [email protected]
20 21 22 23 24 25 26
EP
19
AC C
18
TE D
12
ACCEPTED MANUSCRIPT
Abstract
28
The infectious salmon anemia virus (ISAV) is an aquatic pathogen that is a
29
member of the Orthomyxoviridae family with lethal hemorrhagic potential. Although
30
it affects other species of salmonid fish, ISAV only causes disease in Atlantic
31
salmon (Salmo salar) specimens in sea water. In spite of the fact that the virus has
32
been described as enveloped with icosahedral symmetry, viral like particles with
33
anomalous morphology have been observed in field samples, this we have not
34
been able to recover then in adequate quantities for full demonstration. We report a
35
procedure to concentrate and recover these novel forms of the virus, comparing
36
two cell lines from different origins, demonstrating that these forms were
37
preferentially expressed in cells of epithelial origin.
M AN U
SC
RI PT
27
38 39
Keywords:
40
Salmo salar; ISAV; morphology; epithelial cells; filamentous particles.
44 45 46 47 48 49 50 51 52 53
EP
43
AC C
42
TE D
41
ACCEPTED MANUSCRIPT
54
1. Introduction
56
Infectious Salmon Anemia (ISA) is a highly contagious disease that preferentially
57
affects Atlantic salmon (Salmo salar), the main fish species cultivated in Chile. Its
58
etiologic agent is a virus belonging to the Orthomyxoviridae family, known as
59
Salmon Infectious Anemia Virus (ISAV), which presents a wide number of variants
60
with different degree of virulence [1] [2] [3] [4].
61
The virus is morphologically described as enveloped spherical particles ranging in
62
size from 45 to 140 nm in diameter[2] [3] [4]. In addition filamentous particles have
63
also been reported although their role in infection has not been elucidated [5] 7.
64
These large-sized filamentous particles are highly pleomorphic, encountered in
65
small amounts measuring up to 700 nm in length and covered with spikes of 10
66
nm in length [5].
67
While the structures of ISAV have been described in many reports [2]-[5], the role
68
that these structures might have in the biology and/or pathogenesis of ISAV has
69
not been experimentally elucidated, likely because the viral load is low in infected
70
cells, and the virus is extremely fragile[6]. Indeed, the most recent studies tend to
71
ignore these anomalous structures, defining ISAV exclusively as a pleomorphic
72
virus that is usually spherical or ovoid in shape and covered in 10nm long spikes
73
that are evenly distributed in the envelope, with a 90 ± 130 nm diameter [7–16].
74
Based on the lack of understanding in the field, we aimed to develop a procedure
75
to enrich these large anomalous structures in an in vitro system, to evaluate their
76
infective potential compared with standard, normal-sized particles. The procedure
77
involves growing the virus in two cell lines of different origins, concentrating the
78
large forms via differential filtration, followed by differential gradient purification,
79
and evaluation of their comparative infective potentials in the same cell line of
80
origin.
81
To obtain a high enough concentration of the filamentous virus particles for the
82
procedure, it was necessary to increase the viral load during the infective process.
AC C
EP
TE D
M AN U
SC
RI PT
55
ACCEPTED MANUSCRIPT
In order to achieve this, we designed a strategy derived from information available
84
from the influenza virus (IAV), a well-studied pathogen that is closely related to
85
ISAV. In IAV, similar filamentous morphologies have been observed in vitro only at
86
low numbers of passages, while standard spherical morphology remain constant in
87
time [17]. On the other hand, the filamentous forms are preferentially expressed
88
when grown in tissue culture cells of epithelial origin, while virus produced by
89
infected non-epithelial cells yield almost exclusively particles of spherical
90
morphology [18].
91
2. Material and Methods
92
2.1 Cell line
93
An epithelial fish-cell line (ASK; Atlantic Salmon Kidney; ATCC® CRL-2747™) was
94
infected with ISAV in parallel with a cell line derived from Atlantic salmon peripheral
95
blood leucocytes [19] (SHK-1; Salmon Head Kidney-1; SIGMA). Specific antibodies
96
were used to ensure the epithelial origin of the corresponding cell line (figure 1B; 1-
97
4); (anti-cadherin; 1:500; Thermo, Waltham, MA USA: clone # PA5-19479 and anti-
98
cytokeratin [AE1+AE3]; 1:200, ab961, Abcam, Cambridge, UK).
99
The cell lines were maintained at 17°C in Leibovitz medium (L-15; Gibco,
100
Invitrogen, United Kingdom) supplemented with penicillin (100 IU ml−1),
101
streptomycin (100 g ml−1), and 15% fetal calf serum (FCS; Gibco).
102
2.2 Viral infection
103
Each cell line was infected at a multiplicity of infection of 1 (MOI 1.0), with the
104
virulent HPR3 ISAV variant passaged only twice in vitro before infection. Cells
105
were incubated at 17°C for two weeks.
106
2.3 Confocal scanning microscopy (CSM).
107
In order to determine the type of morphology existing and remaining in the different
108
cell lines, the attached cells were exposed to specific antibodies to evaluate the
AC C
EP
TE D
M AN U
SC
RI PT
83
ACCEPTED MANUSCRIPT
predominant viral morphology. The cultures were incubated at 17°C, and
110
completed the post-infection time, cells were fixed with 4% formaldehyde in 0.13 M
111
phosphate buffer at pH 7.4 and at room temperature for 15 min and washed with
112
PBS. Cells were then permeabilized for 1 h at room temperature with 0.1% Triton
113
X-100 in 1× PBS. The cells were fixed, and cell sections were blocked with 3%
114
bovine serum albumin (BSA) containing 0.5% Tween 20 for 30 min at 37°C.
115
Evaluation was done via confocal microscopy using a 1:500 dilution of a
116
monoclonal antibody (clone 8D2/E9, Austral Biologicals) against the ISAV
117
Hemagglutinin-Esterase protein, to visualize and distinguish types of viral
118
morphologies produced by each cell line [18] [20] [21].
119
Slides were prepared by using Vectashield mounting medium. Image acquisition
120
was performed with a Leica TCSSP5 II confocal microscope, and samples were
121
observed under a 100× lens.
SC
2.4 Viral isolation
M AN U
122
RI PT
109
After two weeks, the supernatant of infection was recovered, centrifuged at 3,000
124
rpm for 15 minutes to remove cell debris, and then further centrifuged at 10,000
125
rpm for another 15 minutes. The resulting supernatants were pooled for each cell
126
line and filtered through Centricon Amicon Ultra-15® centrifugal filter units
127
(Millipore, USA), and centrifuged at 4,000xg four times for 5 minutes with short
128
intermediate pauses to enhance concentration efficiency. The use of Centricon®
129
centrifugal filter units is the key to obtaining enough biomass, since they
130
concentrate viral particles to a minimal final volume (figure 1 A).
131
To further characterize the Centricon®-enriched material, procedures validated for
132
IAV were used to resolve the anomalous particles from the standard types, as the
133
filamentous particles should migrate faster than the spherical ones [18]. 1.5 mL of
134
the remaining filtered material from each cell line were resolved through a 20-60%
135
continuous sucrose gradient and centrifuged for 2 hours, at 100,000xg, 4°C.
136
Nevertheless, to verify true mobility of the putative ISAV anomalous particles, a
AC C
EP
TE D
123
ACCEPTED MANUSCRIPT
parallel procedure was evaluated. This procedure involved labeling the Centricon®-
138
filtrate with an FITC-HE polyclonal mono-specific antibody and running it in a
139
sucrose gradient to visualize the different viral forms resolved. Visualization of viral
140
forms in a sucrose gradient was done via fluorescent light source (488 nm).
141
To evaluate the infective potential of each viral form resolved though the sucrose
142
gradient, each recovered fraction (the gradients were fractionated into 28 aliquots
143
of 0.5 mL) from each gradient was submitted to three different analyses: A: qRT-
144
PCR; B: plaque assay, and C: TEM.
145
2.5 qRT-PCR.
146
Specific primers for ISAV segment 8 and PCR conditions were as described by
147
Snow et al., the official procedure validated by Sernapesca-Chile [22]. Samples
148
were considered ISAV-positive based on cycle threshold (Ct) values <30.
149
confirm the virulence potential of ISAV, specific primers for segment 6 were used
150
as previously described [23]. The RNA was extracted with the RNA easy MiniKit
151
(Qiagen, MD, USA).
152
2.6 Virus Titration.
153
Infectivity titers were determined as described previously by plaque assay [24]. The
154
cells were seeded into 6-well plates at a density of 1 x 105 cells cm-2, and cells
155
were incubated for three days at 17 °C. The viral inoculum was tenfold serially
156
diluted, ranging from 1/10 (10-1) to 1/100.000 (10-5) in L15 medium (2X) without
157
FBS. The culture medium was removed from the seeded cells, and the virus was
158
added in a volume of 500 uL, which was absorbed on the cellular monolayer for
159
four hours at 17 °C. The inoculum was removed and 3 mL of semi-solid medium
160
was added to each well. The semi-solid medium was composed of L15 medium
161
supplemented with FBS (10%), and LMP agarose (0.5%) (UltraPureTM Low
162
Melting Point Agarose (Invitrogen cat. 16520-050). The plates were incubated for
163
15 days post-infection at 17 °C. For visualizing, 2 mL of crystal violet (1%) was
164
added for 1 h at 25 °C, and finally the excess crystal violet was removed.
To
AC C
EP
TE D
M AN U
SC
RI PT
137
ACCEPTED MANUSCRIPT
2.7 Transmission electron microscopy (TEM)
166
To evaluate the content in the filtered supernatant, 100 µL aliquot of each sample
167
from the Centricon® filtrate were independently loaded onto Formvar-coated carbon
168
grids for 1 min, excess liquid was carefully withdrawn with Whatman no. 1 filter
169
paper, and the cells were stained with 10 µl of 1% aqueous uranyl acetate followed
170
by a 10-min incubation at 37°C before visualization under the EM (141 Phillips
171
Tecnai electron microscope; at a range of 12 to 80kV).
172
2.8 Freezing and thawing assay.
173
Two cycles of freezing and thawing were carried out from the same aliquot of
174
purified virus. From each sample 100 µl were removed to perform negative staining
175
and visualized by TEM. The remaining volume was used to infect the ASK cell line
176
with an MOI of 1 for 15 days and the viral titer was measured by plaque assay.
177
3. Results and Discussion
178
Figure 1A summarizes the procedure to concentrate the different types of viral
179
morphologies. When evaluating viral morphology by means of CSM, filamentous
180
particles associated with the surface of the epithelial cell line could be visualized
181
(Figure 1B; 6), condition that was not observed in non-epithelial cells (figure 1B; 5).
182
From the epithelial cells, two types of morphologies were exclusively visualized.
183
One of the EM-visualized cell types were filamentous particles between 700 to
184
2500 nm in length and 70 to 120 nm in width, as well as the typical smaller
185
spherical in morphology, as demonstrated in figure 1C. In the non-epithelial cell
186
line, only spherical smaller-sized particles were visualized (data not shown). Figure
187
1 summarizes the enrichment procedure. Figure 2 shows two bands only in the
188
epithelial extract, in agreement with the resolution observed for IAV, in which the
189
lighter band corresponds to the standard spherical particles, and the faster moving
190
band to the filamentous anomalous. With this information in hand, the standard
191
gradients were fractionated into 28 aliquots of 0.5 ml.
AC C
EP
TE D
M AN U
SC
RI PT
165
ACCEPTED MANUSCRIPT
Figure 3-A shows the results of RNA extracted from each fractionated gradient to
193
confirm the presence of ISAV by standard qRT-PCR procedures [22]. The highest
194
Ct value observed with the filamentous particles implies a lower viral RNA load, in
195
accordance with the low titer obtained. In contrast, the lower Ct values obtained
196
with the spherical particles perfectly matches with the higher viral titer obtained.
197
Notwithstanding, the decrease in RNA content might be also due to the fragility of
198
these filamentous particles that are highly sensitive to ultracentrifugation.
199
The plaque assay results shown in Figure 3-A, indicates positive responses in
200
fractions 17 and 21 for the epithelial extract, while positiveness was only detected
201
in fraction 17 from the non-epithelial cells.
202
morphologies were detected in the epithelial cells, the viral titer detected in fraction
203
17 (spherical particles) was higher than that of fraction 21 (filamentous particles)
204
probably due to the fragility of the latter as previously described.
205
Figure 3 shows the results of the TEM analyses. Consistent with our previous
206
observations and expectations, fractions 17 from both gradients contained the
207
spherical classical particles (figure 3-B) while only fraction 21, exclusively from the
208
epithelial cell line, was enriched with the large-sized filamentous virus particles
209
(figure 3-B). Finally, to further confirm that the filamentous particles corresponded
210
to ISAV, an immuno-gold assay was performed using an anti HE protein as the
211
primary antibody, and an anti-mouse IgG conjugated with gold particles (Sigma),
212
as the second antibody, confirming their ISAV nature (figure 3 B and C; lower
213
sections).
214
While upon initial investigation, the data appear to suggest that the filamentous
215
particles could be exclusively generated in epithelial cells, figure 4 shows that this
216
is not the case, as filamentous particles are visualized in the non-epithelial cell line
217
as well, but in very low quantity. Notwithstanding, the fragility of these large
218
filamentous particles described, constitutes a real problem in regard to the
219
reproducibility of the proposed enrichment procedure. In fact, more than one round
220
of ultracentrifugation significantly decreases the amount of filamentous viral
221
particles recovered, and losing the entire content is possible. Additionally, extreme
SC
RI PT
192
AC C
EP
TE D
M AN U
In spite of the fact that both
ACCEPTED MANUSCRIPT
care must be taken to perform freezing and thawing cycles of the aliquots
223
containing these particles, because they lose infectivity easily. This was confirmed
224
by an additional experiment described in figure 5, which showed that when purified
225
particles were exposed to two cycles of freezing and thawing, they changed in
226
morphology and decreased in infectivity. This occurred only in the filamentous
227
structures and less prominently in the regular spherical ones (A and B,
228
respectively).
229
4. Conclusions
230
We conclude that the large-size infective filamentous structures described in IAV
231
literature also exist in ISAV, and although they might exist in cells of different
232
origins, they are particularly enriched in epithelial cells in vitro. The meaning of this
233
is currently not understood. The described structures are extremely fragile, and
234
appear to co-exist with the standard-size virus particles, with equivalent infectivity,
235
demonstrated by the fact that when parallel infections are carried at equivalent
236
MOIs, similar results are observed. In order to elucidate the role, these particles
237
might have in the infectivity potential of ISAV, biomass analysis would be required,
238
which was outside of the scope of this novel study on enriching for these
239
anomalous particles in epithelial cell lines.
SC
M AN U
TE D
EP AC C
240
RI PT
222
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
241 242
Figure 1.
AC C
EP
TE D
243
244 245
Figure 2.
ACCEPTED MANUSCRIPT
246 247 248
RI PT
249 250 251 252
SC
253 254
M AN U
255 256 257 258
TE D EP
260
Figure 3.
AC C
259
ACCEPTED MANUSCRIPT
261 262 263 264
RI PT
265 266 267 268 269
SC
270 271 272
M AN U
273 274 275 276
EP
TE D
Figure 4.
AC C
277
ACCEPTED MANUSCRIPT
278 279 280 281 282
RI PT
283 284 285 286
SC
287 288 289
M AN U
290 291 292 293 294
Figure 5.
295
299 300 301 302
EP
298
AC C
297
TE D
296
ACCEPTED MANUSCRIPT
Figure 1: Procedure to enrich anomalous filamentous viral particles from
304
ISAV-infected epithelial cell line culture media. A: Culture media was clarified
305
by light centrifugation (3000 rpm, 15 min), further cleaned (10,000 rpm 15 min),
306
and concentrated by Centricon to enrich for putative anomalous viral particles. B:
307
Demonstration of the epithelial origin of the target cell line (3,4) versus the control
308
(1,2); B1: Immuno-fluorescence for Cadherin (anti-cadherin (1:500; Thermo,
309
Waltham, MA USA: clone # PA5-19479) in non-epithelial cell line; B2: immuno-
310
fluorescence for Cytokeratin (anti-cytokeratin [AE1+AE3] (1:200, ab961, Abcam,
311
Cambridge, UK) in non-epithelial cell line; B3: immuno-fluorescence for Cadherin in
312
epithelial cells; B4: immuno-fluorescence for Cytokeratin in epithelial cells. Used
313
antibodies were labeled in green, while in blue we can see nuclear labeling TO-
314
PRP-3 iodide (643/661) (Life Technologies, USA); B5 and B6: The attached cells
315
of each bottle were exposed to specific antibodies to evaluate the predominant
316
viral morphology. The epithelial cells presented a distribution of HE Protein (in
317
green) in a filamentous form (B6) unlike non-epithelial cell line (B5). C: Enrichment
318
of the anomalous viral particles post-Centricon® in epithelial cells. C2; Zoom,
319
Comparison between filamentous and spherical virus membranes.
320
Figure 2: Visualization of morphologies observed in the continuous 20-60%
321
sucrose gradient. A. Schematic: transfer of Centricon-enriched viral structures
322
resolved by sucrose gradient. B: Visualization of viral forms via fluorescent light
323
source (488 nm); B1: Supernatant of a non-infected, non-epithelial cell line in the
324
presence of HE/FITC; B2: Supernatant of a non-infected, epithelial cell line in the
325
presence of HE/FITC; B3: Supernatant of an infected, non-epithelial cell line in the
326
presence of HE/FITC; B4: Supernatant of an infected epithelial cell line in the
327
presence of HE/FITC. C: Fluorescence analysis of each fractionated gradient. The
328
graph shows two peaks at fractions 17 and 21 in the epithelial cell line, and only
329
one peak at fraction 17 in the non-epithelial cell line. Control supernatants (non–
330
infected cells) did not show fluorescence. Upper 9 fractions from the gradient were
331
not graphed; Abbr. On graph: N-EC: non-epithelial cell; EC: epithelial cell.
AC C
EP
TE D
M AN U
SC
RI PT
303
ACCEPTED MANUSCRIPT
Figure 3: Multiple analyses from selected fractions of the processed
333
epithelial cell line. A: Summary table of the viral titer and indication of the Ct
334
values for viral segments 6 and 8 from fractions 17 and 21. B and C: TEM results
335
for fractions of 17 and 21; scale bar: 100 nm; B: enrichment of spherical particles
336
as opposed to C, where filamentous particles are enriched. Bottom figures B and
337
C: immuno-gold assay demonstrating the ISAV nature of the particles.
RI PT
332
Figure 4: Visualization through TEM and confocal microscopy of filamentous
339
particles in a non-epithelial cell line. A: TEM analysis, scale bar: 500 nm. B:
340
visualization of HE protein labeled with HE/Alexa 543 (red color) antibody
341
distributed as filaments on the cell surface, in green color immune-fluorescence
342
Nucleoprotein (clone 2C2/H4; Austral Biologicals) Labeled with antibodies
343
NP/Alexa 488 and blue color: nuclear labeling con TO-PRO-3 iodide (643/661)
344
(Life Technologies, USA). Scale bar 10µm.
345
Figure 5: High fragility of the ISAV filamentous particles: Effect of freezing
346
and thawing in morphology and infectivity. A1-3 and B1-3: diagrams of two
347
Freezing-Thawing cycles to purified viral morphologies; A4 - A6 and B4 - B6: TEM
348
of each condition. As a result, fraction 21 (filaments), suffered morphological
349
changes and accumulations, which could explain the decrease in infectivity by
350
each condition (A7 - A9). Fraction 17 spherical particles are more resistant to
351
treatment and retain infectivity.
M AN U
TE D
EP
352
SC
338
Acknowledgments:
354
This research was supported by PUCV Scholarship (Ph.D. Thesis to R.R), since
355
2014 until 2017 and also supported by Molecular immunology and genetics
356
laboratory; PUCV.
357
AC C
353
ACCEPTED MANUSCRIPT
358
References
359
[1]
K. Falk, E. Namork, E. Rimstad, S. Mjaaland, B.H. Dannevig, Characterization of infectious salmon anemia virus, an orthomyxo-like virus isolated from Atlantic salmon (Salmo salar L.)., J. Virol. 71 (1997) 9016– 9023.
364 365 366 367
[2]
B.H. Dannevig, K. Falk, E. Namork, Isolation of the causal virus of infectious salmon anaemia (ISA) in a long-term cell line from Atlantic salmon head kidney, J. Gen. Virol. 76 (1995) 1353–1359. doi:10.1099/0022-1317-76-61353.
368 369 370
[3]
A.I. Sommer, S. Mennen, Multiplication and haemadsorbing activity of infectious salmon anaemia virus in the established Atlantic salmon cell line, J. Gen. Virol. 78 (1997) 1891–1895.
371 372 373 374
[4]
D. Bouchard, W. Keleher, H.M. Opitz, S. Blake, K.C. Edwards, B.L. Nicholson, Isolation of infectious salmon anemia virus (ISAV) from Atlantic salmon in New Brunswick, Canada, Dis. Aquat. Organ. 35 (1999) 131–137. doi:10.3354/dao035131.
375 376 377
[5]
C.W.R. Korenl, A. Nylund, Morphology and morphogenesis of infectious salmon anaemia virus replicating in the endothelium of Atlantic salmon Salmo salar, Dis. Aquat. Organ. 29 (1997) 99–109.
378 379 380
[6]
P.C. Roberts, R. a Lamb, R.W. Compans, The M1 and M2 proteins of influenza A virus are important determinants in filamentous particle formation., Virology. 240 (1998) 127–137. doi:10.1006/viro.1997.8916.
381 382 383 384
[7]
T.M. Eliassen, M.K. Frøystad, B.H. Dannevig, M. Jankowska, a Brech, K. Falk, K. Romøren, T. Gjøen, Initial events in infectious salmon anemia virus infection: evidence for the requirement of a low-pH step., J. Virol. 74 (2000) 218–227. doi:10.1128/JVI.74.1.218-227.2000.
385 386 387 388
[8]
F.S. Kibenge, O.N. Gárate, G. Johnson, R. Arriagada, M.J. Kibenge, D. Wadowska, Isolation and identification of infectious salmon anaemia virus (ISAV) from Coho salmon in Chile., Dis. Aquat. Organ. 45 (2001) 9–18. doi:10.3354/dao045009.
389 390 391 392
[9]
393 394 395
[10]
AC C
EP
TE D
M AN U
SC
RI PT
360 361 362 363
a. Nylund, H. Plarre, K. Hodneland, M. Devold, V. Aspehaug, M. Aarseth, C. Koren, K. Watanabe, Haemorrhagic smolt syndrome (HSS) in Norway: Pathology and associated virus-like particles, Dis. Aquat. Organ. 54 (2003) 15–27. doi:10.3354/dao054015.
R.J. De Groot, Structure, function and evolution of the hemagglutininesterase proteins of corona- and toroviruses, Glycoconj. J. 23 (2006) 59–72. doi:10.1007/s10719-006-5438-8.
ACCEPTED MANUSCRIPT
[11]
K. Watanabe, M. Karlsen, M. Devold, E. Isdal, a. Litlabø, a. Nylund, Viruslike particles associated with heart and skeletal muscle inflammation (HSMI), Dis. Aquat. Organ. 70 (2006) 183–192. doi:10.3354/dao070183.
399 400 401 402
[12]
S.T. Workenhe, D.W. Wadowska, G.M. Wright, M.J.T. Kibenge, F.S.B. Kibenge, Demonstration of infectious salmon anaemia virus (ISAV) endocytosis in erythrocytes of Atlantic salmon., Virol. J. 4 (2007) 13. doi:10.1186/1743-422X-4-13.
403 404 405 406
[13]
B.L. Schiøtz, N. Roos, A.L. Rishovd, T. Gjøen, Formation of autophagosomes and redistribution of LC3 upon in vitro infection with infectious salmon anemia virus, Virus Res. 151 (2010) 104–107. doi:10.1016/j.virusres.2010.03.013.
407 408 409 410 411
[14]
L.S. Ulanova, G. Isapour, A. Maleki, S. Fanaian, K. Zhu, A. Hoenen, C. Xu, Ø. Evensen, G. Griffiths, B. Nyström, Development of methods for encapsulation of viruses into polymeric nano- and microparticles for aquaculture vaccines, J. Appl. Polym. Sci. 131 (2014) 8785–8796. doi:10.1002/app.40714.
412 413 414 415
[15]
S.H. Marshall, R. Ramirez, a. Labra, M. Carmona, C. Munoz, Bona Fide Evidence for Natural Vertical Transmission of Infectious Salmon Anemia Virus in Freshwater Brood Stocks of Farmed Atlantic Salmon (Salmo salar) in Southern Chile, J. Virol. 88 (2014) 6012–6018. doi:10.1128/JVI.03670-13.
416 417 418 419
[16]
A. Kavaliauskis, M. Arnemo, A.-L. Rishovd, T. Gjøen, Activation of unfolded protein response pathway during infectious salmon anemia virus (ISAV) infection in vitro an in vivo, Dev. Comp. Immunol. 54 (2015) 46–54. doi:10.1016/j.dci.2015.08.009.
420 421 422
[17]
J. Seladi-Schulman, J. Steel, A.C. Lowen, Spherical influenza viruses have a fitness advantage in embryonated eggs, while filament-producing strains are selected in vivo., J. Virol. 87 (2013) 13343–53. doi:10.1128/JVI.02004-13.
423 424
[18]
R.I.W.C. Ompans, Host cell dependence of viral morphology, 95 (1998) 5746–5751.
425 426 427 428 429
[19]
B.H. DANNEVIG, B.E. BRUDESETH, T. GJØEN, M. RODE, H.I. WERGELAND, Ø. EVENSEN, C.M. PRESS, Characterisation of a long-term cell line (SHK-1) developed from the head kidney of Atlantic salmon (Salmo salarL.), Fish Shellfish Immunol. 7 (1997) 213–226. doi:https://doi.org/10.1006/fsim.1996.0076.
430 431 432
[20]
J.S. Rossman, G.P. Leser, R. a. Lamb, Filamentous Influenza Virus Enters Cells via Macropinocytosis, J. Virol. 86 (2012) 10950–10960. doi:10.1128/JVI.05992-11.
433 434
[21]
J.C. Cox, A.W. Hampson, R.C. Hamilton, An immunofluorescence study of influenza virus filament formation, Arch. Virol. 63 (1980) 275–284.
AC C
EP
TE D
M AN U
SC
RI PT
396 397 398
ACCEPTED MANUSCRIPT
doi:10.1007/BF01315033.
435
[22]
M. Snow, P. McKay, A.J.A. McBeath, J. Black, F. Doig, R. Kerr, C.O. Cunningham, A. Nylund, M. Devold, Development, application and validation of a Taqman real-time RT-PCR assay for the detection of infectious salmon anaemia virus (ISAV) in Atlantic salmon (Salmo salar)., Dev. Biol. (Basel). 126 (2006) 133-45–6.
441 442 443 444
[23]
D. Sepúlveda, H. Bohle, A. Labra, H. Grothusen, S.H. Marshall, Design and evaluation of a unique RT-qPCR assay for diagnostic quality control assessment that is applicable to pathogen detection in three species of salmonid fish., BMC Vet. Res. 9 (2013) 183. doi:10.1186/1746-6148-9-183.
445 446 447 448
[24]
M.T. Castillo-Cerda, L. Cottet, D. Toro-Ascuy, E. Spencer, M. Cortez-San Martín, Development of plaque assay for Chilean Infectious Salmon Anaemia Virus, application for virus purification and titration in salmon ASK cells, J. Fish Dis. 37 (2014) 989–995. doi:10.1111/jfd.12198.
M AN U
SC
RI PT
436 437 438 439 440
AC C
EP
TE D
449
ACCEPTED MANUSCRIPT
Highlights:
AC C
EP
TE D
M AN U
SC
RI PT
1. An anomalous viral morphology is described for Infectious Salmon Anemia Virus. 2. Anomalous particles are preferentially expressed in cells of epithelial origin. 3. Description of a method to concentrate and isolate anomalous viral particle. 4. These described particles co-exist with the standard-size virus particles.