2013 International Symposium on Ranaviruses by Yumi Une

1. Distribution of ranavirus
in Japan
The 2013 International Symposium on Ranaviruses
Yumi Une Laboratory of Veterinary Pathology,
School of Veterinary Medicine, Azabu
University

2. The current state of ranaviral disease in Japan
Outbreaks of ranavirus infection in Japan
13 episodes (12 locations )
In nature : 8 episodes, only bullfrogs
In captivity : 5 episodes, various species
： outbreak points in captivity
： outbreak points in nature
year in nature in captivity
2008 Bullfrog Hynobius nebulosus
2009 Bullfrog
Bullfrog
Bullfrog
Bullfrog
2010 Bullfrog
2011 Bullfrog
Bullfrog
2012 Tylototriton (2 spices)
Poison Dart Frog (num erous)
Poison Dart Frog (num erous)
Hynobius hidam ontanus

3. Bullfrog
Our aim is to predict the impact of ranavirus on native amphibians
and formulate measuresfor its prevention.
Habitation area
● Outbreaks often occur in
nature.
● Distribution is very wide.
● Exotic species in Japan.
(This species was introduced from North
America in 1918)
To clarify the prevalence and origin of ranavirus in Japan
Kidneys of Bullfrog tadpoles.

4. Mao et al.(1997)
M 10 11 12 13 14 15 M 10 11 12 13 14 15 M 10 11 12 13 14 15
Result of PCR methods.
Left: FV3 primer,Center: JP primer, Right; M68F primer
M; molecular-weight marker, Number; case number
Profile of primer sets
Primer for the major capsid protein gene.
FV3 FV3M C P4F 5'-G AC TTG G C C AC TTATG AC -3'
530bp FV3M C P5R 5'-G TC TC TG G AG AAG AAG AA-3'
JP RanaJP556F 5'-G G TTC TTC C C C TC C C ATTC TTC TT-3'
217bp RanaJP772R 5'-G G TC ATG TAG AC G TTG G C C TC G AC -3'
M 68Ｆ M 68F 5'-G C AC C AC C TC TAC TC TTATG -3'
230bp B IVM C P154 5'-C C ATC G AG C C G TTC ATG ATG -3'

5. Jan Feb M ar A pr M ay Jun Jul A ug Sep O ct N ov D ec
Atsugi
Chiba
Tokyo
Fukui
Shiga
Fukui
Ehime
Hiroshima
Saitama
Kouchi Gunma
Ebina
17.5% 7.5% 0%
28%
Infectious prevalence of ranavirus in Bullfrogs
(No disease outbreak )
11 locations, 15 times, n=407, positive 24 (5.9%)

6. 0
10
20
30
40
50
60
70
80
90
100
4/9/2010 5/9/2010 6/9/2010 7/9/2010 8/9/2010 9/9/2010 10/9/2010 11/9/2010 12/9/2010 1/9/2011 2/9/2011 3/9/2011 4/9/2011
n= 429, 4 positive, 0.93% (range 0-5.1%)
30 Sep
24 Dec
Monthly collection
Infectious prevalence of ranavirus in Bullfrogs
(no disease outbreak )
monthly collection

7. Infectious prevalence of ranavirus in Bullfrogs
( With disease outbreak )
0
10
20
30
40
50
60
70
80
90
100
n= 410, 102 positives
mean25%(range 0-96%)
2nd outbreak (2010)
no outbreak (2011)
monthly collection

8. Conclusion regarding Bullfrogs
• Ranaviral disease occurs often in Bullfrog, but
this species is not suitable to understand the
situation of ranavirus in Japan.
• Reason:
Infection prevalence is very low.
The appropriate time for sampling could not be
determined.
Only RCV-JP was detected in Bullfrogs.

9. Distribution of ranavirus in Japan
Indian rice frog
(Fejervarya kawamurai)
• Classification: Ranidae
• Small and brown
• A common species in rice fields in
western Japan
• Southern strain
• Hibernates
• Breeding season: long, from April to
August
★
Currently, its habitats are expanding
in Japan. It is considered a
domestic exotic’ species.
Distribution of in Indian rice frog
■ Naturally habitation area
■ Area of invasion
Infection prevalence is high in the Indian rice frogs

10. Material & Methods
1. Survey in 13 locations in 8 Prefectures between May 2011 and October 2012
（Tochigi, Tokyo, Aichi, Kyoto, Hyogo, Okayama, Ehime, Nagasaki)
2. 3 seasons（ spring, summer, autumn)
3. Maximum 30 individuals per collection time
4. PCR method (3 primer sets)
5. Kidneys (plus liver, and spleen)
Shikoku island
Kyushu island
Main island

11. Results
1. All 8 Japanese Prefectures: Ranavirus (+)
2. Infection prevalence of ranavirus ：12.9% (152/1,177)
3. Annual variability: 2011 8.7 % (47/541)
2012 16.5 % (105/636) ↑
↑
4. Seasonal variation:
prevalence greatest
during autumn

12. Seasonal variation in each location
(8 locations that could be surveyed in two successive years)
Tochigi
Tokyo
Aichi
Hyogo
Kyoto
Okayama
Ehime 1
Ehime 2
Rapid rise
in prevalence in autumn

13. 0
10
20
30
40
50
60
70
80
90
100
Spring Summer Autumn
0
10
20
30
40
50
60
Spring Summer Autumn
0
5
10
15
20
25
30
Spring Summer Autumn
0
5
10
15
20
25
Spring Summer Autumn
2011
2012
2013
Ehime 2
Tochigi Ehime 1
Okayama
Annual variability of prevalence in each location

14. 0
10
20
30
40
50
60
Spring Summer Autumn
0
10
20
30
40
50
60
70
80
90
100
Spring Summer Autumn
0
5
10
15
20
25
30
Spring Summer Autumn
0
5
10
15
20
25
Spring Summer Autumn
2011
2012
2013
Ehime 2
Tochigi Tokyo
Okayama
Annual variability of prevalence in each location
0％
10.7％
31.3％

15. 1. Rana catesbeiana ranavirus (RCV-JP)
Une(2009)
2. Hynobius nebulosus ranavirus (HNV)
3. Fejervarya kawamurai ranavirus (FKV)
4. Tiger frog ranavirus (TFV) He (2002)
RCV-JP
HNV
FKV
TFV
The phylogenetic tree of ranavirus

16. Tochigi
Tokyo
Aichi
KyotoHyogo
Okayama
EhimeNagasaki
RCV-JP
HNV
FKV
TFV
Virus
type by
location

17. 0% 20% 40% 60% 80% 100%
2012
2011
Hyogo
0% 20% 40% 60% 80% 100%
2012
2011
0% 20% 40% 60% 80% 100%
2012
2011
Tokyo
0% 20% 40% 60% 80% 100%
2012
2011
Kyoto
Okayama
0% 20% 40% 60% 80% 100%
2012
2011
Aichi
0% 20% 40% 60% 80% 100%
2012
2011
Ehime
■ RCV-JP ■ HNV ■ FKV

18. Nagasaki
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Autumn
Summer
Spring
RCV-JP
FKV
TFV
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Autumn
Summer
Spring
Autumn
RCV-JP
FKV
HNV
Aichi
2012
2012
2011

19. Conclusion
Our results show that
1. Ranavirus is distributed widely in Japan.
2. In nature, the prevalence of ranavirus is rising steadily.
3. Multiple variations of the virus are circulating in amphibian
communities.
4. Within a short period, the virus type has changed in wild
Indian rice frog communities.
Regarding the spread of ranavirus in nature, the role of this frog
has not been determined.
But given that the Indian rice frog is an invasive species with
rapidly expanding distribution and also a suitable host for
ranavirus, we postulate that it may contribute, or have
contributed, to the emergence of ranavirus in native amphibian
communities in Japan.

20. Thank you for your attention.
Contact：une@azabu-u.ac.jp