Distribution of ranavirus in Japan

The 2013 International Symposium on Ranaviruses

Yumi Une 　 Laboratory of Veterinary Pathology, 　　　　　　　　 School of Veterinary Medicine, Azabu University

The current state of ranaviral disease in JapanOutbreaks 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 captivity2008 Bullfrog Hynobius nebulosus2009 Bullfrog

BullfrogBullfrogBullfrog

2010 Bullfrog2011 Bullfrog

Bullfrog2012 Tylototriton (2 spices)

Poison Dart Frog (numerous)Poison Dart Frog (numerous)Hynobius hidamontanus

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.

Mao et al.(1997)

M 　 10 　 11 　 12 13 　 14 　 15M 　 10 　 11 12 13 14 15M 　 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 FV3MCP4F 5'- GACTTGGCCACTTATGAC- 3'530bp FV3MCP5R 5'- GTCTCTGGAGAAGAAGAA- 3'J P RanaJ P556F 5'- GGTTCTTCCCCTCCCATTCTTCTT- 3'217bp RanaJ P772R 5'- GGTCATGTAGACGTTGGCCTCGAC- 3'M68Ｆ M68F 5'- GCACCACCTCTACTCTTATG- 3'230bp BIVMCP154 5'- CCATCGAGCCGTTCATGATG- 3'

J an Feb Mar Apr May J un J ul Aug Sep Oct Nov Dec

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%)

4/9/2

010

4/19/2

010

4/29/2

010

5/9/2

010

5/19/2

010

5/29/2

010

6/8/2

010

6/18/2

010

6/28/2

010

7/8/2

010

7/18/2

010

7/28/2

010

8/7/2

010

8/17/2

010

8/27/2

010

9/6/2

010

9/16/2

010

9/26/2

010

10/6/2

010

10/16/2

010

10/26/2

010

11/5/2

010

11/15/2

010

11/25/2

010

12/5/2

010

12/15/2

010

12/25/2

010

1/4/2

011

1/14/2

011

1/24/2

011

2/3/2

011

2/13/2

011

2/23/2

011

3/5/2

011

3/15/2

011

3/25/2

011

4/4/2

011

4/14/2

011

4/24/2

0110

10

20

30

40

50

60

70

80

90

100

n= 429, 4 positive, 0.93% (range 0-5.1%)

30 Sep24 Dec

Monthly collectionInfectious prevalence of ranavirus in Bullfrogs

(no disease outbreak )

monthly collection

Infectious prevalence of ranavirus in Bullfrogs ( With disease outbreak )

6/20/2

010

7/3/2

010

7/16/2

010

7/29/2

010

8/11/2

010

8/24/2

010

9/6/2

010

9/19/2

010

10/2/2

010

10/15/2

010

10/28/2

010

11/10/2

010

11/23/2

010

12/6/2

010

12/19/2

010

1/1/2

011

1/14/2

011

1/27/2

011

2/9/2

011

2/22/2

011

3/7/2

011

3/20/2

011

4/2/2

011

4/15/2

011

4/28/2

011

5/11/2

011

5/24/2

011

6/6/2

011

6/19/2

011

7/2/2

011

7/15/2

011

7/28/2

011

8/10/2

011

8/23/2

011

9/5/2

011

9/18/2

0110

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

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.

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

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

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 　　

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

Spring Summer Autumn0

10

20

30

40

50

60

70

80

90

100

Spring Summer Autumn0

10

20

30

40

50

60

Spring Summer Autumn0

5

10

15

20

25

30

Spring Summer Autumn0

5

10

15

20

25

2011

2012

2013

Ehime 2

Tochigi Ehime 1

Okayama

Annual variability of prevalence in each location

Spring Summer Autumn0

10

20

30

40

50

60

Spring Summer Autumn0

10

20

30

40

50

60

70

80

90

100

Spring Summer Autumn0

5

10

15

20

25

30

Spring Summer Autumn0

5

10

15

20

25

2011

2012

2013

Ehime 2

Tochigi Tokyo

Okayama

Annual variability of prevalence in each location

0％

10.7％

31.3％

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

Tochigi

Tokyo

Aichi

KyotoHyogo

Okayama

EhimeNagasaki

RCV-JPHNVFKVTFV

Virus type by location

2012

2011

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Hyogo

2012

2011

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

2012

2011

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Tokyo

2012

2011

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Kyoto

Okayama

2012

2011

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Aichi

2012

2011

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Ehime

■ RCV-JP 　■ HNV 　■ FKV

Nagasaki

Autumn

Summer

Spring

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

RCV-JP

FKV

TFV

Autumn

Summer

Spring

Autumn

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

RCV-JP FKV

HNV

Aichi

2012

2012

2011

ConclusionOur 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.

Thank you for your attention.

Contact ： une@azabu-u.ac.jp