Mosquito-borne infectious diseases measures in Tokyo · Global brief on vector-borne diseases（WHO,2014） Countries of areas at risk of Dengue Fever In 1942-1944 Nagasaki, Hiroshima,

Mosquito-borne infectious diseases

measures in Tokyo (From the field of microbiological testing)

Tokyo Metropolitan Institute of Public Health

Department of Microbiology

Kenji Sadamasu

Global brief on vector-borne diseases（WHO,2014）

Countries of areas at risk of Dengue Fever In 1942-1944

Nagasaki,

Hiroshima,

Kobe, Osaka

More than

24,000 people

Past outbreak cases

Mosquito species that cause infectious diseases

Pathogenic agents Mosquito species in Japan

Dengue fever Family Flaviviridae

Dengue virus Aedes albopictus

Chikungunya fever Family Togaviridae

Chikungunya virus Aedes albopictus

Zika virus infection Family Flaviviridae

Zika virus Aedes albopictus

Japanese

encephalitis Family Flaviviridae

Japanese encephalitis virus Culex tritaeniorhynchus

Yellow fever Family Flaviviridae

Yellow fever virus Aedes aegypti*

West Nile fever Family Flaviviridae

West Nile virus Aedes albopictus 、Culex

tritaeniorhynchus、Anopheles

Malaria Plasmodium Marchiafava et

Celli Anopheles.sp

* not inhabited in Japan

Year 2013 2014 2015 2016 2017 2018

Dengue fever 249

(67) 341

(162) 293

(93) 335

(92) 245

(65) 201

(61)

Chikungunya fever 14

(3) 16

(5) 17

(7) 13

(5) 5

(3) 4

(0)

Zika virus infection 12 (3)

5 (0)

0 (0)

Japanese

encephalitis 9

(0) 2

(0) 2

(0) 11 (0)

3 (0)

0 (0)

Yellow fever 0

(0) 0

(0) 0

(0) 0

(0) 0

(0) 0

(0)

West Nile fever 0

(0) 0

(0) 0

(0) 0

(0) 0

(0) 0

(0)

Malaria 40

(12) 57

(27) 37

(16) 53

(18) 61

(27) 50

(14)

_：Including domestic infections in Japan

Mosquito-borne infectious diseases incidents

in Japan / Tokyo

Flow of reporting Notifiable Diseases in Tokyo

（including mosquito-borne infectious diseases)

Health center

Doctor

Report incidents.

Viral confirmatory tests of blood samples Input the reported incidents in NESID.

① Confirm on NESID and report to the government ② Feedback by weekly

report etc.

Tokyo Metropolitan Institute of Public Health (Infectious Disease Information Center)

(Division of virology) Virus tests / epidemiological information

Flow of capturing the total incidents

The doctor who diagnosed the infected patient reports to the regional health center.

National Institute of Infectious Diseases

Share info.

Infection Control Div., Bureau of

Social Welfare and Public Health

Incident notification fax

Nine days before admission, she had returned from a two-week trip to

Japan, during which she visited Ueda, Fuefuki, Hiroshima, Kyoto and

Tokyo in August.

Eurosurveillance, Volume 19, Issue 3, 23 January 2014

In September 2013, the case infected Dengue virus type 2

In Ｊａｐａｎ? I could not believe it.

Dengue Fever in 2014 Domestic infections in Japan

（Ministry of Health, Labor and Welfare report）

• Teenage women (Saitama prefecture) was the

first patient, a total of 163 cases

Tokyo Metropolitan patients（108 cases）

• About 230 blood samples were tested

（PCR-sequencing analysis、NS1、IgM）

• Mosquitoes captured in Yoyogi Park

（ＰＣＲ）

2014,Tokyo

Patient Ｎｏ． age Gender residence Onset date

Did you go to Yoyogi Park?

Were you bitten by a mosquito?

1 10's Female Saitama 20 August Yes Yes

2 20's Male Tokyo 24 August Yes Yes

3 20's Female Saitama 18 August Yes Yes

- - - - - - -

138 50's Male Tokyo 10 September Near the park Yes

139 20's Male Tokyo 11 September Shinjuku chuo

Park Yes

140 20's Male Tokyo 13 September Near the park Yes

Profile of Dengue Fever Patients

Yoyogi Park⇒

Yoyogi Park,Tokyo

新宿御苑

http://3.bp.blogspot.com/-mIWbs62En2g/UdP-Jr8_42I/AAAAAAAACh4/SqvXe0p84Ww/s1600/P6234379.JPG

Method of catching mosquitoes (Light trap)

Rain cover

(Airflow)

Dry battery

Motor

Net

(traps mosquitoes in)

Cool bag (with dry ice)

Midget lamp

Yoyogi Park Map

Dengue Virus detected

traps

2-3,Sep,2014

①

②

③

④ ⑤

⑥

⑦

⑧

⑨

⑩

set places of light traps

11/16-17

Daily reports of Dengue Fever in 2014, Tokyo

Yoyogi Park

Shinjuku chuo

Park

Sep Aug Examination period of mosquito test

Yoyogi Park closed

Yoyogi Park(area A) closed

Ministry of Health, Labor and Welfare announced at the first time

(151 cases)

Number of reports

The practice of viruses

testing from mosquitoes

Aedes albopictus

Mosquitoes

①Mosquitoes in1.5mL tubes Max 30 mosquitoes/tube 35 mosquitoes ⇒ 2 tubes 30/tubes and ５/tubes

Aedes albopictus

② 300μL of Sterile phosphate buffer(PBS) in tube

③ Use Micro multi mixer

④ Crushing for 1 minute with Micro multi mixer

⑤ Centrifugation of 1.5ｍLtube (8,000rpm,10min）、 Take 140μL of the supernatant

QIA cube

（Nucleic acid extraction machine）

12 samples/hour

Nucleic acid test results of Dengue virus

of mosquitoes captured in Yoyogi Park

Collection date

tub

es

Dengue virus（＋）

※1sample：30 mosquitoes /1.5mL tube

※

4 / 10 points

4 / 20 points

3 / 20 points

（21,3,15）

（30,5,30,12,19,5）

（28,30,29,30,16）

Nucleic acid test results of Dengue virus

of mosquitoes collected in Yoyogi Park

Points

Mosquitoes

number

NAT

positives

Mosquitoes

number→

Under bar : samples of the same point

Aedes aegypti （1-5days） 108～109copy number/Mos

Mosquitoes are said to be sucking their blood( may be 2-2.5μL)

Dengue virus viral load(copy number) in mosquitoes

Quantitative results of Dengue virus type 1

in Yoyogi mosquitoes ( in 2014)

Real-time PCR amplification curve

Standard solution

1：ATCC Synthetic Dengue virus type 1 RNA （1x105～1x106 copies/μL)

2：AMPLIRUN DENGUE 1 VIRUS RNA CONTROL ,Vircell （ 12500～

20000 copies/μL)

・Dengue virus type1

・Match the First patient’s sequence（LC002828）

Dengue virus type 1 envelope gene analysis

LC006123_NIID106/Tokyo2014

mosquitoes１

mosquitoes２

mosquitoes３

LC002828_NIID100/Saitama2014（First patient）

Patients A

Patient B

Patient C C

KJ545459_GD-D13202/China(Guangzhou)2013

JN415489_Bali_2010a/Indonesia2010

EU081238_05K3301DK1/Singapore2005

EU069623_T3352/04_Singapore2004

EU448399_0508aTw/Singapore2005

EU069621_T3206/04_Singapore2004

FR666924_36000/05/Malaysia2005

JF967888_0910aTw/Thailand2009

AB111076_02-20-1HuNIID/Thailand(J)2002

AB111071_01-61-1HuNIID/Cambodia(J)2001

AB873104_NRTqs/Cambodia(J)2013

HM181953_BID-V4254/Cambodia2007

JF967858_0904aTw/Vietnam(Taiwan)2009

JF937618_BID-V4081/Vietnam-South2008

AB111066_01-15-1HuNIID/India-Thai(J)2001

AB111065_99-36-1HuNIID/Paraguay(J)1999

AY153755_cesara1/Costa_Rica1993

AF425631_CAREC780572/Trinidad-Tobago1978

AF226685_Den1BR/90/Brazil1990

AF513110_BR/01-MR/Brazil2001

AF311956_BR/97-111/Brazil1997

AF311957_BR/97-409/Brazil1997 89

100

70

80

92

100

69

80

98

96 100

57

47

97 74

68

64

46

65

92

79

96 Phylogenetic analysis of Dengue virus type 1

envelope genes detected from patients/mosquitoes

The same viruses belong to the same cluster in the phylogenetic tree.

Dengue virus is divided

into four serotypes, and

serotype 1 is further

divided into four

genotypes.

http://www.metro.tokyo.jp/INET/KONDAN/2014/12/40oco100.htm

Tokyo metropolitan Mosquito-mediated infectious

disease measures meeting report

December 24, 2014

・Surveillance of infectious disease

vector mosquitoes in the parks

（Mosquito type classification and virus testing）

・Virus NAT of Patient's blood and

sequence analysis of viruses

・Infectious diseases information on the website

（Countermeasures against infectious

disease vector mosquitoes in Tokyo）

Role of Tokyo Metropolitan Institute of Public Health

A

B

C

D

E

F

G

H

G

I

G

Facility name Facility name

1 Oi Central Seaside Park 9 Inokashira Park

2 Odaiba Marine Park 10 Tama Zoological Park

3 Aoyama Cemetery 11 Sayama Park

4 Yanaka Cemetery 12 Oyamada Green Area

5 Somei Cemetery 13 Hachioji Cemetery

6 Shakujii Park 14 Medical Botanical Garden

7 Toneri Park 15 Jindai Botanical Park

8 Kinuta Park 16 Mizuho Nogei High School

Facility name

A Yoyogi Park (Districts A and B)

B Hibiya Park

C Hamarikyu Gardens

D Toyama Park

E Ueno Park

F Komazawa Olympic Park

G Hikarigaoka Park

H Sarue Onshi Park

I Kasai Rinkai Park

Facilities for wide-area surveillance

Facilities for focused surveillance

16 9

Early detection of mosquito-borne infections Surveillance of infectious disease vector mosquitoes in the parks

Surveillance of infectious disease vector mosquitoes in the parks

Wide-area surveillance (16points/16 places)

June to October

Focused surveillance (50points/9 places)

April to November

Tests items - Infectious agents-carrying

mosquito monitoring

- Monitoring of virus-carrying mosquitoes

- Adult mosquito classification and Virus survey

- Virus survey of mosquito larvae

Test pathogens

Dengue virus Chikungunya virus Zika virus West Nile virus Plasmodium falciparum

Dengue virus Chikungunya virus Zika virus

Type and number of mosquitoes

captured [Data in 2018]

Aedes albopictus 2,563 Culex tritaeniorhynchus 311 Anopheles.sp 1 Others 206

Adults

Aedes albopictus 3,851 Aedes aegypti 0 Others 2,739 Larvae 4,292 There were no mosquitoes with the viruses(2015-2018).

Virus NAT of Patient's blood and sequence analysis of viruses

Variability of Dengue virus type 1 envelope sequences from the Patients

○ All Dengue fever patients (2015-2018) reported in Japan were infected abroad and there were

no domestic infections.

〇 None of the DENV1 strains(from 2015 to 2018) had a same nucleotide sequence of Yoyogi strain in 2014.

○ Dengue viruses were divided into two groups (Lineage-1 and 2).

○ Strains from PHL and IND belong to Lineage-2 and strains from other regions belong to Lineage-1.

LC011945/ 2014 Saitama

(Yoyogi)

15-DP28

15-DP88

15-DP5

15-DP17

15-DP20

15-DP11

15-DP68

LC011949/2014 Shizuoka

15-DP32

15-DP50

15-DP60

15-DP44

15-DP51

15-DP42

15-DP57

15-DP45

15-DP58

15-DP67

15-DP87

100

100

100

100

99

58

100

97

90

84

65

100

0.01

PHL, IND

IDN, TAH, SGP, MMR, etc. http://www.freemap.jp/

IND

SLI

TAH

Lineage-2

Lineage-1

Lineage-1

Lineage-2

Patient-derived DENV type 1sequence analysis

Takaya.S.,et al,Emerging Infectious Diseases . 23, 156-158，2017

Chikungunya ｖｉｒｕｓ

March, 2016

45 years old、Male

Travelling overseas ：India

Rash, joint pain

Serum and urine samples

( health center brought in )

Chikungunya virus

sequences Positive

Virus isolated (Vero cell)

Asian Lineage

September, 2016

39 years old、Male

Travelling overseas ：Dominican Republic

Red-toned eczema with itching, conjunctivitis

Serum and urine samples( health center brought in )

ZIKA-V sequences Positive

Virus isolated (Vero cell)

ZIKA virus KY014304_Zikavirus_DOM_2016

KY014304.1_Zika_virus_isolate_Zika_virus/H.sapiens-wt/DOM/2016/BB-0180-SER_polyprotein_gene_complete_cds

16-DP137

16-DP137-2

KX673530.1_Zika_virus_isolate_PHE_semen_Guadeloupe_complete_genome

KY014295.1_Zika_virus_isolate_Zika_virus/H.sapiens-wt/USA/2016/FL-010-URI_polyprotein_gene_complete_cds

KU853012.1_Zika_virus_isolate_Dominican_Republic/2016/PD1_complete_genome

LC190723.1_Zika_virus_genomic_RNA_complete_genome_strain:_ZIKV/Hu/Yokohama/1/2016

LC190723_ZIKV/Hu/Yokohama/1/2016

KU647676.1_Zika_virus_strain_MRS_OPY_Martinique_PaRi_2015_polyprotein_gene_complete_cds

KX280026.1_Zika_virus_isolate_Paraiba_01_complete_genome

KU926310.1_Zika_virus_isolate_Rio-S1_complete_genome

KU926309.1_Zika_virus_isolate_Rio-U1_complete_genome

KU527068.1_Zika_virus_strain_Natal_RGN_complete_genome

KU527068.1_Zika_virus_strain_Natal_RGN_complete_genome(2)

KY003153.1_Zika_virus_strain_ZIKV/34997/Pavia/2016_polyprotein_gene_complete_cds

KU729217.2_Zika_virus_isolate_BeH823339_polyprotein_gene_complete_cds

KR872956.1_Zika_virus_strain_17829_polyprotein_gene_complete_cds

KU321639.1_Zika_virus_strain_ZikaSPH2015_complete_genome

KU497555.1_Zika_virus_isolate_Brazil-ZKV2015_complete_genome

KX766029.1_Zika_virus_isolate_R116265_complete_genome

KY328289.1_Zika_virus_isolate_HN16_complete_genome

KU501216.1_Zika_virus_strain_103344_polyprotein_gene_complete_cds

KX156774.2_Zika_virus_strain_ZIKV/Homo_sapiens/PAN/CDC-259359_V1-V3/2015_complete_genome

KU501215.1_Zika_virus_strain_PRVABC59_complete_genome

KU312312.1_Zika_virus_isolate_Z1106033_polyprotein_gene_complete_cds


KU365778.1_Zika_virus_strain_BeH819015_polyprotein_gene_complete_cds

LC219720_ZIKV/Hu/NIID123/2016(NCGHM)

KU707826.1_Zika_virus_isolate_SSABR1_complete_genome


KU509998.3_Zika_virus_strain_Haiti/1225/2014_complete_genome

KJ776791.2_Zika_virus_strain_H/PF/2013_complete_genome

KX447516.1_Zika_virus_isolate_1_0111_PF_polyprotein_gene_complete_cds



KU509998.3_Zika_virus_strain_Haiti/1225/2014_complete_genome(2)

LC191864_ZIKV/Hu/Chiba/S36/2016

KM078929.1_Zika_virus_strain_CHI1805214_NS5_protein_gene_partial_cds

KF993678.1_Zika_virus_strain_PLCal_ZV_from_Canada_polyprotein_gene_partial_cds

EU545988.1_Zika_virus_polyprotein_gene_complete_cds

KU681082.3_Zika_virus_isolate_Zika_virus/H.sapiens-tc/PHL/2012/CPC-0740_complete_genome

HQ234499.1_Zika_virus_isolate_P6-740_polyprotein_gene_partial_cds

NC_012532.1_Zika_virus_complete_genome

LC002520.1_Zika_virus_genomic_RNA_complete_genome_strain:_MR766-NIID

HQ234498.1_Zika_virus_isolate_MR_766_polyprotein_gene_partial_cds

LC002520_MR766-NIID

KF383086.1_Zika_virus_strain_ArA975_nonstructural_protein_5_gene_partial_cds

KF383115.1_Zika_virus_strain_ArB1362_polyprotein_gene_complete_cds

KF268949.1_Zika_virus_isolate_ARB15076_polyprotein_gene_complete_cds


HQ234500.1_Zika_virus_isolate_IbH_30656_polyprotein_gene_partial_cds


KF383088.1_Zika_virus_strain_ArD30101_nonstructural_protein_5_gene_partial_cds

KF383116.1_Zika_virus_strain_ArD7117_polyprotein_gene_complete_cds


HQ234501.1_Zika_virus_isolate_ArD_41519_polyprotein_gene_partial_cds



KF383084.1_Zika_virus_strain_HD78788_nonstructural_protein_5_gene_partial_cds




82

34

78

51

54

78

54

40

56

88

99

97

99

71

66

53

100

69

100

96

65

64

87

68

63

87

65

62

50

48

40

92

29

6

12

16

17

11

0.02

Asian

Lineage

African

Lineage

Asian

Lineage

KY014304_Zikavirus_DOM_2016

KY014304.1_Zika_virus_isolate_Zika_virus/H.sapiens-wt/DOM/2016/BB-0180-SER_polyprotein_gene_complete_cds

16-DP137

16-DP137-2

KX673530.1_Zika_virus_isolate_PHE_semen_Guadeloupe_complete_genome

KY014295.1_Zika_virus_isolate_Zika_virus/H.sapiens-wt/USA/2016/FL-010-URI_polyprotein_gene_complete_cds

KU853012.1_Zika_virus_isolate_Dominican_Republic/2016/PD1_complete_genome

LC190723.1_Zika_virus_genomic_RNA_complete_genome_strain:_ZIKV/Hu/Yokohama/1/2016

KU647676.1_Zika_virus_strain_MRS_OPY_Martinique_PaRi_2015_polyprotein_gene_complete_cds

KX280026.1_Zika_virus_isolate_Paraiba_01_complete_genome

KU926310.1_Zika_virus_isolate_Rio-S1_complete_genome

KU926309.1_Zika_virus_isolate_Rio-U1_complete_genome

KU527068.1_Zika_virus_strain_Natal_RGN_complete_genome

KU527068.1_Zika_virus_strain_Natal_RGN_complete_genome(2)

KY003153.1_Zika_virus_strain_ZIKV/34997/Pavia/2016_polyprotein_gene_complete_cds

KU729217.2_Zika_virus_isolate_BeH823339_polyprotein_gene_complete_cds

KR872956.1_Zika_virus_strain_17829_polyprotein_gene_complete_cds

KU321639.1_Zika_virus_strain_ZikaSPH2015_complete_genome

KU497555.1_Zika_virus_isolate_Brazil-ZKV2015_complete_genome


KY328289.1_Zika_virus_isolate_HN16_complete_genome

KU501216.1_Zika_virus_strain_103344_polyprotein_gene_complete_cds

KX156774.2_Zika_virus_strain_ZIKV/Homo_sapiens/PAN/CDC-259359_V1-V3/2015_complete_genome

KU501215.1_Zika_virus_strain_PRVABC59_complete_genome

KU312312.1_Zika_virus_isolate_Z1106033_polyprotein_gene_complete_cds



LC219720_ZIKV/Hu/NIID123/2016(NCGHM)

KU707826.1_Zika_virus_isolate_SSABR1_complete_genome


KU509998.3_Zika_virus_strain_Haiti/1225/2014_complete_genome

KJ776791.2_Zika_virus_strain_H/PF/2013_complete_genome




KU509998.3_Zika_virus_strain_Haiti/1225/2014_complete_genome(2)

LC191864_ZIKV/Hu/Chiba/S36/2016

KM078929.1_Zika_virus_strain_CHI1805214_NS5_protein_gene_partial_cds

KF993678.1_Zika_virus_strain_PLCal_ZV_from_Canada_polyprotein_gene_partial_cds

EU545988.1_Zika_virus_polyprotein_gene_complete_cds

KU681082.3_Zika_virus_isolate_Zika_virus/H.sapiens-tc/PHL/2012/CPC-0740_complete_genome

HQ234499.1_Zika_virus_isolate_P6-740_polyprotein_gene_partial_cds

NC_012532.1_Zika_virus_complete_genome

LC002520.1_Zika_virus_genomic_RNA_complete_genome_strain:_MR766-NIID

HQ234498.1_Zika_virus_isolate_MR_766_polyprotein_gene_partial_cds

LC002520_MR766-NIID


KF383115.1_Zika_virus_strain_ArB1362_polyprotein_gene_complete_cds



HQ234500.1_Zika_virus_isolate_IbH_30656_polyprotein_gene_partial_cds





HQ234501.1_Zika_virus_isolate_ArD_41519_polyprotein_gene_partial_cds



KF383084.1_Zika_virus_strain_HD78788_nonstructural_protein_5_gene_partial_cds




84

33

77

51

55

78

54

40

58

85

99

98

99

71

66

61

99

71

99

95

65

65

88

72

66

87

62

64

48

50

42

92

30

7

13

15

16

10

0.02

Infectious diseases Information on the website Countermeasures against infectious disease vector mosquitoes in Tokyo

Weekly reports of dengue fever patients

Information on mosquito-borne infections

① Posters

② Ad-rapped buses

③ Leaflets

④ Panel display ⑤ Motion picture distribution

If you lose water, mosquitoes will not come.

Is stagnant water remaining?

Documents

Mosquito-borne infectious diseases measures in Tokyo · Global brief on vector-borne diseases（WHO,2014） Countries of areas at risk of Dengue Fever In 1942-1944 Nagasaki, Hiroshima,