______________________________________

Reflections on the Ecology and Epidemiology of

Eastern Equine Encephalitis in the Northeastern

United States

________________________________________________

Theodore G. Andreadis

Center for Vector Biology & Zoonotic Diseases and

Northeast Regional Center for Excellence in

Vector-Borne Diseases

The Connecticut Agricultural Experiment Station

New Haven, CT

Eastern Equine Encephalitis

• Most pathogenic arthropod-borne virus in North

America (Alphavirus: Togaviridae, SSRNA)- ~ 6-7 human cases / year

- 40% case fatality rate

- Neurological impairment in survivors (35%)

- No commercial vaccine or effective treatment

• Activity is most common in and around freshwater

hardwood swamps – highly focal

• Perpetuates in an enzootic cycle involving wild

Passeriformes birds and ornithophilic mosquitoes

• Principal enzootic vectors in the northeastern US

- Culiseta melanura

- Culiseta morsitansRole as “bridge vectors”

unresolved

• 1831 - First equine outbreak of EEE virus in Massachusetts - “horses dying

of a brain disease”

• 1933 - First isolation of EEE virus from horse brain during an outbreak in

coastal areas of Delaware, Maryland, New Jersey and Virginia

• 1934 - Mosquitoes first incriminated as potential vectors in a series of vector

competence studies with species of Aedes, Culex and Coquillettidia

• 1935 - Birds considered as reservoir hosts; 1950 - first isolation of EEE made

from a wild bird

• 1938 - The first human cases confirmed – 35 (25 fatal) human cases, > 300

horse cases in Massachusetts; 38 horse cases in Connecticut

Key EEE Historical Events – Northeastern US

• 1938 - Shown that virus could cause of encephalitis in wild and

domestic pheasants in Connecticut

• 1949 - First isolation of EEE virus from mosquitoes – Cq. perturbans

• 1951 - First isolation of EEE virus from Culiseta melanura

• 1959 - Major outbreak in New Jersey – 33 human cases

• 1971 - EEE discovered in Central New York – 1st human case

Human Cases of EEE in the United States 1964 - 2019

N = 347

NY

NJ

MA

NH

VT

CT RI

ME

0

5

10

15

20

25

19

66

19

68

19

70

19

72

19

74

19

76

19

78

19

80

19

82

19

84

19

86

19

88

19

90

19

92

19

94

19

96

19

98

20

00

20

02

20

04

20

06

20

08

20

10

20

12

20

14

20

16

20

18

< 1 case / yr

Resurgence

4-5 cases / yr

Northward

Expansion

Human Cases

- 1964 – 2002 = 47

- 2003 - 2019 = 79

• 1964 to 2002 –

sporadic outbreaks

with no apparent

pattern

• 2003 – resurgence

and expansion

Resurgence and Expansion of EEE Activity in NE US

Armstrong & Andreadis NE J Med 2013

RI

NY

ME

NJ

NHVT

MA

CT

0

5

10

15

20

25

30

35

40

45

50

20

03

20

04

20

05

20

06

20

07

20

08

20

09

20

10

20

11

20

12

20

13

20

14

20

15

20

16

20

17

20

18

20

19

EEE Veterinary Cases

Northeastern US

2003 - 2019

N = 309

Ave = 18 / yr

Human and Veterinary Cases of EEE in the Northeastern US - 2019

CT MA ME NH NJ NY RI

Veterinary Cases = 42

0

2

4

6

8

10

12

14

CT MA ME NH NJ NY RI

Human Cases = 23

Fatalities = 8 (35%)

NY

NJ

MA

NH

VT

CT

RI

ME

EEE Activity = 30 Counties, 7 States

Human cases = 23 (8 fatalities, 35%)

Veterinary cases = 42

• Reforestation and wetland restoration –

by mid 1800’s much of the forests in the

northeastern US were stripped and

cedar swamps were destroyed

Factors Contributing to the Resurgence of EEE in the Northeastern US

- Increased habitat for Culiseta

melanura

- Proliferation of wetland roosting sites

for birds (e.g. robins, wood thrush)

• Suburban development near critical

wetland mosquito habitat

- Increasingly expose people to the

threat of EEE infection

• Changes in average temperatures and

precipitation events related to climate

change

- Milder winters

- Warmer summers

- Extremes in both precipitation and

drought

- Enhance overwintering survival

- Extend transmission season

- Accelerate generation time

- Increase frequency of blood feeding

- Accelerate virus replication within

mosquito

- Allow mosquitoes to extend northward

range

Komar & Speilman NY Acad Sci 1994

Pre-Season

• Significant EEE activity in the previous

year

• Mild winters with insulating snow cover

• High water table in enzootic swamps

• Above average rainfall in the prior

fall/winter and spring

Historical Risk Factors for EEE in the Northeastern US

In-Season

• Above average Culiseta melanura

populations

• EEE virus isolations from mosquitoes in

June or early July

• Isolations of EEE virus from a mammal-

biting mosquitoes – Cq. perturbans

• Numerous EEE isolations > 30 – 50

• High MIR in Culiseta melanura >1:1000

• EEE activity beyond traditional areas

• Early and above average equine cases

• Infection of a human prior to August

aaa

a

aaaaa

a

a

0

5

10

15

20

0

10

20

30

40

50

60

70

80

901

99

6

199

7

199

8

199

9

200

0

200

1

200

2

200

3

200

4

200

5

200

6

200

7

200

8

200

9

201

0

201

1

201

2

201

3

201

4

201

5

201

6

201

7

201

8

201

9

Me

an

Cs

. m

ela

nu

ra/ tr

ap

EE

E Is

ola

tes

fro

m C

s.

me

lan

ura

Year

No. EEE virus isolations

Mean no. Cs. melanura / trap

EEE isolations and abundance of Cs. melanura – Connecticut, 1996-2019

EEE activity

associated with

Cs. melanura

abundance

0

5

10

15

20

25

30

35

40

45A

ve

. N

o M

osq

uito

es p

er

Lig

ht T

rap

Culiseta melanura AbundanceConnecticut

2019

22 Year Mean

June July August September October

0

40

80

120

7/1 7/8 7/15 7/22 7/29 8/5 8/12 8/19 8/24 9/2 9/9 9/16 9/23 9/30 10/7 10/14 10/21 10/28

No

. E

EE

vir

us

is

ola

tio

ns

Week

1996-2018

2013

Weekly Isolations of EEE virus

from field collected mosquitoes

in Connecticut

0

40

80

120

7/1 7/8 7/15 7/22 7/29 8/5 8/12 8/19 8/24 9/2 9/9 9/16 9/23 9/30 10/7 10/14 10/21 10/28

No

. E

EE

vir

us

is

ola

tio

ns

Week

1996-2018

2019

2013

2019

2019

2019

Weekly Isolations of EEE virus

from field collected mosquitoes

in Connecticut

Earlier EEE virus

amplification in

2019

Culiseta melanura

• Habitat: Densely wooded freshwater swamps

(red maple and white cedar) and sphagnum

bogs

• Development: Develop in subterranean

“crypts” in deep shaded cavities under tree

roots

• Seasonal Distribution: mid-May – October

• Feeding Preference: Primarily birds with

occasional feeding on mammals including

humans

• Number of Generations: 2-3 per year

• Adult Flight Range: > 2 miles

• Overwintering Stage: Larvae (all instars)

0.0

4.0

8.0

12.0

16.0Crypt water temperature

0 C

0.0

0.2

0.4

0.6

0.8

1.0

P L4 L3 L2

* * * ***

adults

Rela

tive p

roport

ion o

f each s

tage

DEC JAN FEB MAR APR MAY

* L1

Phenology of Overwintering Development of Cs. melanura

Andreadis et al JAMCA 2012

Cs. melanura64.1%

Ae. canadensis7.8%

Ae. cinereus4.0%

Ae. vexans3.6%

Cx. salinarius2.9%

Ur. sapphirina2.9%

Cx. pipiens2.4%

Ae. trivittatus2.2%

An. punctipennis1.9%

Minor species (10)7.8%

Species (n = 19) No.

Cs. melanura 264

Ae. canadensis 32

Ae. cinereus 18

Ae. vexans 15

Cx. salinarius 12

Ur. sapphirina 12

Cx. pipiens 10

Ae. trivittatus 9

An. punctipennis 8

Ae. cantator 5

Cs. morsitans 5

Cq. perturbans 4

Others (7 species) 18

EEE Virus Isolations from Mosquitoes in Connecticut 1996 - 2018

Cs. melanura65.6%

Ur. sapphirina5.7%

Cq. perturbans4.0%

Ae. canadensis 4.1%

Cx. salinarius3.3%

Ae. vexans3.3%

Ps. ferox3.3%

An. punctipennis2.5%

Ae. cinereus 1.6%Cx. pipiens

1.6%

Minor species (5)4.1%

EEE Virus Detections from Mosquitoes in CT, MA, NJ and NY - 2019

Connecticut

Cs. melanura45.9%

Cq. perturbans33.4%

Ae. canadensis5.6%

Cx. salinarius10.6%

Ae. vexans1.6%

Cx. pipiens/restuans2.8%

Massachusetts

New Jersey

Cs. melanura74.0% Ae. canadensis

2.7%

Ae. triseriatus2.7%

Ae.albopictus2.7%

Cx. pipiens/restuans17.8%

Cs. melanura88.9%

Cq. perturbans4.8%

Cx. salinarius4.8%

Ae. vexans1.6%

New York

1.0

<0.8

<0.8

1.6

1.5

2.8

>3.0

1.3

0.8

6.6

1.4

1.7

1.7

1.2

2.9

0 1 2 3 4 5 6 7

Ur. sapphirina

Ps. ferox

Ae. trivittatus

Ae. triseriatus

Ae. cantator

Ae. canadensis

Cq. perturbans

Cx. salinarius

Cx. restuans

Cs. melanura

An. walkeri

An. quadrimaculatus

An. punctipennis

Ae. vexans

Ae. cinereus

Mean Log10 PFU’s/mosquito pool

Required for transmission

1,000 – 10 million PFU’s

1Armstrong & Andreadis EID 2010

2Nasci & Mitchell JAMCA 1996

2

• There are major

differences in the quantity

of virus found in EEE

virus-positive, field-

collected mosquitoes

• Cs. melanura appears to

be the only species in

which virus titers are

sufficiently high enough to

support efficient

transmission

• Other species include Ae.

cinereus, Cq. perturbans,

Ae. canadensis

• Important to consider virus

titers when implicating

other mosquito vectors

Mean EEE Virus Titers in Field-Collected Mosquitoes by

Plaque Assay 1

0

20

40

60

80

100

Cs. melanura Cq. perturbans Ae. canadensis Cx. salinarius Ae. cinereus

Bird

Mammal

Bird & Mammal

Host Feeding Patterns of Culiseta melanura and Potential Bridge Vectors of EEE in the Northeastern US (CT, MA, NY, VT)

Perc

ent

0.2% Human

5.1% Human

2.0% Human

3.7% Human

Molaei & Andreadis EID 2006, Molaei et al AJTMH 2006, JME 2008, VBZD 2013

8.4% Human

0 20 40 60 80 100

Connecticut

Massachusetts

New York

Vermont

Mammal

Bird

94.2%

94.0%

98.9%

99.7%

6.0%

5.8%

1.1%

0.3%

Proportion of Avian and Mammalian Derived Blood Meals in Culiseta melanura populations in the Northeastern US

1.3% Human

0.3% Human

Molaei & Andreadis EID 2006, Molaei et al AJTMH 2006, JME 2008, VBZD 2013

3.7% Equine

Wood Thrush

American Robin

Tufted Titmouse

Common Grackle

Chipping Sparrow

Black-capped Chickadee

Northern Cardinal

Red-eyed Vireo

Scarlet Tanager

ConnecticutN = 42 species

Molaei & Andreadis EID 2006, Molaei et al AJTMH 2006, JME 2008, VBZD 2013

18%

14%

13%

Wood Thrush

American Robin

Song Sparrow

Ovenbird

Red-eyed Vireo

Scarlet Tanager

Common Yellowthroat

Baltimore Oriole

Black-Capped

Chickadee

New YorkN = 52 species

24%

9%8%

American Robin

Tufted Titmouse

Black-capped

Chickadee

Scarlet Tanager

Field Sparrow

Northern Cardinal

Wood Thrush

Red-eyed Vireo

Baltimore Oriole

Massachusetts N = 55 species

22%

9%

9%

Green Heron

American RobinCommon

Yellowthroat

Black-capped Chickadee

Savannah Sparrow

Common Grackle

Northern Cardinal

Rose-breasted Grosbeak

Veery

VermontN= 49 species

18%

12%

10%

Culiseta melanuraAvian-Derived Blood Meals

EEE Antibody Prevalence in Wild Birds: Regional Comparisons

0

20

40

60

80

100

Wood Trush American Robin Gray Catbird Ovenbird Song Sparrow

NJ (Crans et al. JME 1994)

MA (Main et al. AJTMH 1988)

NY (Emord and Morris, JME 1984)

NY (Howard et al. JME 2004)

ME (Elias et al. VBZD 2017)

Perc

ent

Hillsborough NH 2005

Merrimack NH 2005

New Haven CT 2006

Rockingham NH 2006

Rockingham NH 2005

Hillsborough NH 2005

Rockingham NH 2006

Rockingham NH 2006

Hillsborough NH 2006

Fairfield CT 2003

New London CT 2004

New London CT 2003

New London CT 2003

New London CT 2004

New London CT 2003

New London CT 2003

Fairfield CT 2003

Windham CT 2003

Fairfield CT 2003

New London CT 2003

Fairfield CT 2003

Fairfield CT 2003

New London CT 2001

New Haven CT 2001

Fairfield CT 2001

New London CT 2001

Middlesex CT 2001

Fairfield CT 2000

New Haven CT 2003

NH

2005-06

CT

2003-04

CT

2001 & 03

CT

2000-01

Oswego NY 2007

Oswego NY 2005

Oswego NY 2004

Oswego NY 2004

Oswego NY 2004

New London CT 2006

New London CT 2007

New London CT 2007

New London CT 2007

New London CT 2006

New London CT 2004

Fairfield CT 2003

New London CT 2003

New London CT 2003

Fairfield CT 2003

New London CT 1998

Fairfield CT 1998

Middlesex CT 1998

New London CT 2003

Georgia 1997

New London CT 2003

New London CT 2000

Hartford CT 2000

Florida 1991

Florida 1982

New London CT 1996

Florida 1993

NY

2004-07

CT

2006-07

CT

2003

CT

2000

• EEE viruses group into

temporally discrete genetically

diverse clades by year -

suggests separate annual

introduction events into the

region

− Migrating viremic birds

• Some strains persist into 2nd

year - provides evidence for

local overwintering

− Vertical transmission in

mosquitoes (Philbrook et al. CDC TR

1961)

− Recrudescence in chronically

infected birds (Crans et al. JME 1994)

− Reptiles or amphibians (?)

• Support for both hypotheses

Tracking Eastern Equine Encephalitis Virus

Perpetuation in the Northeastern United States by

Phylogenetic Analysis

PM Armstrong, TG Andreadis, JF Anderson, JW Stull, CN Mores.

2008. Am. J. Trop. Med. Hyg.

• Sequenced complete genomes of 433

EEEV strains collected within the U.S.

from 1934 to 2014

• EEEV evolves relatively slowly and that

transmission is enzootic in Florida,

characterized by higher genetic diversity

and long-term local persistence

• EEEV in CT, MA and NY were

characterized by lower genetic diversity,

multiple introductions, and shorter local

persistence

• Supports a source-sink model in which FL

is the major source of EEEV

No.

sequences

No.

humancases

160

120

40

80

24

15

7

9

1-4

Journal of Virololgy 92 (12), 2018, 1-18.

Large-Scale Complete-Genome Sequencing and

Phylodynamic Analysis of Eastern Equine Encephalitis

Virus Reveals Source-Sink Transmission Dynamics in

the United States

Tan et al. (19 authors)

Northeastern US EEE Virus Transmission Cycle

Virus

EnzooticCycle

Wild Passerine Bird Reservoir

and Amplifying Hosts

Juneto

October

Culiseta melanura

August to October

Epidemic /

Epizootic

Transmission

“Bridge Vectors”

Coquillettidia perturbans

Aedes canadensis

Culex salinarius

Culiseta melanura

Local

Overwintering

& Annual

Introduction

• High likelihood that the EEE virus will

reemerge

- EEE usually persists after a major outbreak

- Have consistently experienced equine

and/or human cases every year since 2004

• Unlikely that we will experience as

high a level of EEE virus activity

- Herd immunity in reservoir birds – dampen

enzootic transmission

• Remains to be seen how widespread

activity will be

- Will we see further expansion into NH, ME

and VT?

Human case

Veterinary case

Expectations for 2020

0

10

20

30

40

50

60

70

20

04

20

05

20

06

20

07

20

08

20

09

20

10

20

11

20

12

20

13

20

14

20

15

20

16

20

17

20

18

20

19

2019

Human cases

Veterinary cases

2004 - 2019

• Human serosurvey - human exposure ?

- EEE antibodies detected in 0.7% of persons with no history of

encephalitis after 1955 outbreak in Massachusetts (Feemster et

al NEJM 1958)

- Inapparent infections ranged from 3.1% to 7.6% after the

1959 outbreak in New Jersey (Goldfield et al. Am J Epidem 1968)

• Identification of Cs. melanura breeding sites in newly

recognized foci of human and animal infection

• Screening larvae for virus – overwintering

- One reported isolation of EEE virus from Cs. melanura larvae (Philbrook et al CDC Tech Rep 1961)

- Never been duplicated or confirmed

• Pre-season treatment of Cs. melanura breeding sites

• Methoprene has been shown to be an effective larvicide

when applied by fixed wing aircraft (Woodrow et al JAMCA

1995)

• Enhanced mosquito surveillance – in season

Research – Surveillance Priorities

EEE Challenges and Issues

1. Risk assessment and communication

• How do best assess human risk and communicate it to the public

• Analysis and interpretation of surveillance findings

• Triggers for response

2. Sharing samples for genetic analysis and validation

• Virus availability – virus isolation vs PCR detection

3. Delays in laboratory diagnosis of human infection

• Concerns with commercial labs – serology and false negatives

4. Prevention and control

• Personal protective measures – effectiveness?

• Preseason preemptive treatments of Cs. melanura breeding sites with larvicides

• Truck-mounted and aerial adulticides – how do we evaluate effectiveness

• Difficulties with public acceptance – environmental issues

• Delays in implementation and high costs

• What level of control is needed to reduce human risk of infection

Questions?

Alexander Skochkov “Old Mosquito”