CHARACTERIZATION OF EELPOUT RHABDOVIRUS (EpRV) ON

CHARACTERIZATION OF EELPOUT RHABDOVIRUS
(EpRV) ON CELL CULTURE
E Blomkvist*1, A Alfjorden1, M Hakhverdyan1, T S Boutrup2, H Ahola1, F Ljunghager3, Å Hagström1, N J Olesen2, M Juremalm1, M Leijon1, J-F Valarcher1, C Axén1
1 National Veterinary Institute, Uppsala, Sweden; 2 Technical University of Denmark, Copenhagen, Denmark; 3 Swedish Agency for Marine and Water Management, Gothenburg, Sweden
Conclusion
1 day p.i.
7 days p.i.
13 days p.i.
Control
BF-2 is the best of four tested cell cultures for EpRV
isolation. We recommend a combination with FHM
culture and if CPE does not occur on FHM,
inoculation should be done again using BF-2
supernatant.
Figure 2. Cell culture results for EpRV inoculation in Outbreak#1
The CPE was not similar to other viruses. Cell
cultures were full of debris, holes only appeared
on BF-2 cells. For FHM and EPC, cells suddenly
detached when the amount of debris was massive.
BF-2
CPE appeared day 5 post inoculation (p.i.),
with full CPE day 7 p.i. Titration identified a
2.7 x 105 TCID50/ml
Inoculate
Introduction
In 2014 mass mortalities occurred in eelpout (Zoarces
viviparous) along the Swedish south-east coast (Figure
1). A new rhabdovirus, subsequently named eelpout
rhabdovirus (EpRV) was identified in two investigated
outbreaks.
Results
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Figure 1.
Right: Reported eelpout
mass mortalities (yellow
dots) along the Swedish
coast in 2014. Two sites
(orange/yellow dots) were
sampled
Below: Diseased eelpout
at Outbreak#2
Bothnian
Bay
Inoculate
Control
FHM
For Outbreak#1, CPE only arose after
inoculation with BF-2 culture supernatant.
In Outbreak#2, CPE appeared on FHM
cells day 7 p.i, with full CPE day 14 p.i.
Titration identified a 1.9 x 104 TCID50/ml
Bothnian
Sea
EPC
CPE did not occur for Outbreak#1 (Figure
2), whereas full CPE was produced for
Outbreak#2 day 7 p.i. Titration not done
North
Sea
Outbreak#1
Baltic
Sea
Control
Outbreak#2
To characterize EpRV growth on four cell lines used
for piscine virus isolation.
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Control
• Kidney, spleen and heart from three eelpouts
(Outbreak#1) were pooled to one sample.
• Kidney, spleen, heart and brain from four eelpouts
(Outbreak#2) were pooled per fish.
Virus isolation was done according to Commission
Decision 2001/183/EC (1):
• Inoculation onto BF-2 and FHM cells, with
dilution in two steps.
• Incubation 14-21 days at 15°C, with passage each 7
days.
• Additional culture on RTG-2 cells (Outbreak#1)
and EPC (Outbreak#1-2) - supernatant from 7day-old BF-2 cell culture used as inoculum.
• Plates monitored daily from day 3 for cytopathogenic effects (CPE).
RTG-2
Some debris but no obvious CPE produced
Summary
Eelpout Rhabdovirus (EpRV), associated
with mortalities in eelpout, can be propagated and isolated on BF-2 and FHM cell cultures
before further analysis.
Inoculate
Method
Inoculate
Aim
What´s new
Virus identification through deep sequencing is described on poster P-117
A manuscript describing the outbreak and investigations has been submitted for publication. A
qPCR assay for detection of EpRV has been developed and a manuscript will be prepared
Eva Blomkvist
Animal Health and Antimicrobial Resistance
NATIONAL VETERINARY INSTITUTE
post. SE-751 89 Uppsala, Sweden
phone. +46 18 67 40 00 fax. +46 18 30 91 62
e-mail. [email protected]
web. www.sva.se
2015-08-31
Reference
Directive 2001/83/EC of the European Parliament and of the Council; 6 Nov 2001. http://
eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2001:311:0067:0128:en:PDF
Acknowledgements
The authors would like to thank Daniel Sebring, Anders Sjölin, Lars Förlin
and Jonas Nilsson for help with collection of fish.
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