______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 Role as “bridge vectors” - Culiseta morsitans unresolved Key EEE Historical Events – Northeastern US

• 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 • 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 Resurgence and Expansion of EEE Activity in NE US

• 1964 to 2002 – ME sporadic outbreaks with no apparent VT NH pattern NY

• 2003 – resurgence Northward and expansion MA CT RI Expansion Human Cases NJ - 1964 – 2002 = 47 - 2003 - 2019 = 79 Resurgence

25

20

15 < 1 case / yr 4-5 cases / yr

10

5

0

1966 1968 1970 1972 1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018

Armstrong & Andreadis NE J Med 2013 EEE Veterinary Cases Northeastern US ME 2003 - 2019 NH VT

NY

MA

CT N = 309 RI NJ Ave = 18 / yr

50 45 40 35 30 25 20 15 10 5

0

2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Human and Veterinary Cases of EEE in the Northeastern US - 2019

14 Human Cases = 23 12 Fatalities = 8 (35%) EEE Activity = 30 Counties, 7 States 10 Human cases = 23 (8 fatalities, 35%) 8 Veterinary cases = 42

6 ME

4

2 VT 0 NH CT MA ME NH NJ NY RI NY

MA Veterinary Cases = 42 CT

RI

NJ

CT MA ME NH NJ NY RI Factors Contributing to the Resurgence of EEE in the Northeastern US

• Reforestation and wetland restoration – - Increased habitat for Culiseta by mid 1800’s much of the forests in the melanura northeastern US were stripped and - Proliferation of wetland roosting sites cedar swamps were destroyed for birds (e.g. robins, wood thrush)

• Suburban development near critical - Increasingly expose people to the wetland mosquito habitat threat of EEE infection

- Enhance overwintering survival • Changes in average temperatures and precipitation events related to climate - Extend transmission season change - Accelerate generation time - Milder winters - Increase frequency of blood feeding - Warmer summers - Accelerate virus replication within - Extremes in both precipitation and mosquito drought - Allow mosquitoes to extend northward range

Komar & Speilman NY Acad Sci 1994 Historical Risk Factors for EEE in the Northeastern US

Pre-Season In-Season • Significant EEE activity in the previous a• Above average Culiseta melanura year populations a • Mild winters with insulating snow cover a • EEE virus isolations from mosquitoes in a • High water table in enzootic swamps June or early July a • Above average rainfall in the prior a • Isolations of EEE virus from a mammal- fall/winter and spring biting mosquitoes – Cq. perturbans a• Numerous EEE isolations > 30 – 50 a • High MIR in Culiseta melanura >1:1000 a • EEE activity beyond traditional areas a• Early and above average equine cases a• Infection of a human prior to August EEE isolations and abundance of Cs. melanura – Connecticut, 1996-2019

90 20 No. EEE virus isolations 80 Mean no. Cs. melanura / trap EEE activity associated with 70 Cs. melanura abundance 15

60 / trap /

50 Cs.melanura 10

40 Cs. melanura Cs.

30 Mean 5

EEE Isolates from from Isolates EEE 20

10

0 0

2006 2011 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2007 2008 2009 2010 2012 2013 2014 2015 2016 2017 2018 2019 Year 45

Culiseta melanura Abundance 2019 40 Connecticut 22 Year Mean

35

30

25

20

15

Ave. No Mosquitoes per Light Trap Light per Mosquitoes No Ave. 10

5

0 June July August September October 120 Weekly Isolations of EEE virus 1996-2018 from field collected mosquitoes in Connecticut

80

2013

No. EEE virus isolations virus EEE No. 40

0 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 Week 120 2019 Weekly Isolations of EEE virus 1996-2018 from field collected mosquitoes in Connecticut 2019

2019 80 2019

Earlier EEE virus 2013

No. EEE virus isolations virus EEE No. 40 amplification in 2019

0 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 Week 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)

Phenology of Overwintering Development of Cs. melanura adults P L4 L3 L2 * L1 1.0

0.8

0.6

0.4

0.2 Relative proportion of eachstage

0.0 * * * * * * DEC JAN FEB MAR APR MAY

16.0 Crypt water temperature

12.0 C

0 0 8.0

4.0

0.0

Andreadis et al JAMCA 2012 EEE Virus Isolations from Mosquitoes in Connecticut 1996 - 2018

Species (n = 19) No.

Cs. melanura 264 Ae. canadensis Ae. canadensis 32 7.8% Ae. cinereus 18 Cs. melanura Ae. vexans 15 Ae. cinereus 64.1% 4.0% Cx. salinarius 12

Ur. sapphirina 12 Ae. vexans 3.6% Cx. pipiens 10

Ae. trivittatus 9 Cx. salinarius 2.9% An. punctipennis 8 Ae. cantator 5 Ur. sapphirina 2.9% Cx. pipiens Cs. morsitans 5 2.4% Cq. perturbans 4 Ae. trivittatus 2.2% Others (7 species) 18 Minor species (10) An. punctipennis 7.8% 1.9% EEE Virus Detections from Mosquitoes in CT, MA, NJ and NY - 2019

Connecticut New York Ur. sapphirina 5.7%

Cq. perturbans Cs. melanura Cs. melanura 4.0% 88.9% 65.6%

Ae. canadensis 4.1%

Cx. salinarius 3.3% Ae. vexans 3.3% Cq. perturbans Ps. ferox 4.8% 3.3% Ae. vexans Cx. salinarius Minor species (5) An. punctipennis 1.6% 4.8% 4.1% Cx. pipiens 2.5% 1.6% Ae. cinereus 1.6% Massachusetts New Jersey

Cq. perturbans 33.4% Cs. melanura Ae. canadensis 74.0% Cs. melanura 2.7% 45.9% Ae. canadensis Ae. triseriatus 5.6% 2.7%

Ae.albopictus 2.7% Cx. salinarius 10.6% Cx. pipiens/restuans Cx. pipiens/restuans Ae. vexans 17.8% 2.8% 1.6% Mean EEE Virus Titers in Field-Collected Mosquitoes by • There are major Plaque Assay 1 differences in the quantity of virus found in EEE Ae. cinereus 2.9 virus-positive, field- Ae. vexans 1.2 collected mosquitoes An. punctipennis 1.7

An. quadrimaculatus 1.7 • Cs. melanura appears to be the only species in An. walkeri 1.4 which virus titers are Cs. melanura 6.6 sufficiently high enough to Cx. restuans 0.8 support efficient Cx. salinarius 1.3 transmission Cq. perturbans 2 >3.0 Ae. canadensis 2.8 • Other species include Ae. Ae. cantator 1.5 cinereus, Cq. perturbans, Ae. triseriatus 1.6 Ae. canadensis

Ae. trivittatus <0.8 Required for transmission Ps. ferox <0.8 1,000 – 10 million PFU’s • Important to consider virus titers when implicating Ur. sapphirina 1.0 other mosquito vectors 0 1 2 3 4 5 6 7

Mean Log10 PFU’s/mosquito pool 1Armstrong & Andreadis EID 2010 2Nasci & Mitchell JAMCA 1996 Host Feeding Patterns of Culiseta melanura and Potential Bridge Vectors of EEE in the Northeastern US (CT, MA, NY, VT)

100 Bird Mammal 80 Bird & Mammal

60 0.2% 5.1% 3.7% 2.0% 8.4% Human Human Human Human Human

Percent 40

20

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

Molaei & Andreadis EID 2006, Molaei et al AJTMH 2006, JME 2008, VBZD 2013 Proportion of Avian and Mammalian Derived Blood Meals in Culiseta melanura populations in the Northeastern US

Mammal Bird 6.0% 1.3% Vermont Human 94.0%

5.8% 3.7% New York Equine 94.2%

1.1% 0.3% Massachusetts Human 98.9%

0.3% Connecticut 99.7%

0 20 40 60 80 100

Molaei & Andreadis EID 2006, Molaei et al AJTMH 2006, JME 2008, VBZD 2013 Connecticut Massachusetts N = 42 species Scarlet Baltimore N = 55 species Red-eyed Tanager Oriole Red-eyed Vireo Vireo Wood Northern Thrush American Cardinal Wood Thrush Robin Northern Black-capped 18% Cardinal 22% Chickadee

Field Sparrow Chipping 14% 9% Sparrow 13% American 9% Common Scarlet Tufted Robin Titmouse Grackle Tanager Black- Tufted capped Chickadee Titmouse Culiseta melanura New York Avian-Derived Blood Meals Vermont N = 52 species Black- Rose-breasted Baltimore Veery N= 49 species Capped Grosbeak Common Oriole Chickadee Northern Yellowthroat Green Wood Cardinal Heron Scarlet Thrush Common Tanager Grackle 18% 24% Savannah Red-eyed Sparrow Vireo 12% Black-capped 10% 8% 9% Ovenbird Chickadee American Common Robin American Song Yellowthroat Sparrow Robin Molaei & Andreadis EID 2006, Molaei et al AJTMH 2006, JME 2008, VBZD 2013 EEE Antibody Prevalence in Wild Birds: Regional Comparisons

100 NJ (Crans et al. JME 1994) MA (Main et al. AJTMH 1988) NY (Emord and Morris, JME 1984) NY (Howard et al. JME 2004) 80 ME (Elias et al. VBZD 2017)

60 Percent

40

20

0 Wood Trush American Robin Gray Catbird Ovenbird Song Sparrow Hillsborough NH 2005 Tracking Eastern Equine Encephalitis Virus Oswego NY 2007 Perpetuation in the Northeastern United States by Merrimack NH 2005 Oswego NY 2005 Phylogenetic Analysis New Haven CT 2006 NY Rockingham NH 2006 Oswego NY 2004 2004-07 PM Armstrong, TG Andreadis, JF Anderson, JW Stull, CN Mores. NH Rockingham NH 2005 Oswego NY 2004 2008. Am. J. Trop. Med. Hyg. 2005-06 Hillsborough NH 2005 Oswego NY 2004 Rockingham NH 2006 New London CT 2006 Rockingham NH 2006 • EEE viruses group into New London CT 2007 Hillsborough NH 2006 temporally discrete genetically CT Fairfield CT 2003 New London CT 2007 2006-07 diverse clades by year - New London CT 2007 suggests separate annual New London CT 2004 New London CT 2006 New London CT 2003 New London CT 2004 CT introduction events into the Fairfield CT 2003 New London CT 2003 2003-04 region New London CT 2004 New London CT 2003 CT − Migrating viremic birds 2003 New London CT 2003 New London CT 2003 New London CT 2003 nd Fairfield CT 2003 • Some strains persist into 2 Fairfield CT 2003 Windham CT 2003 New London CT 1998 year - provides evidence for CT Fairfield CT 2003 Fairfield CT 1998 2001 & 03 local overwintering New London CT 2003 Middlesex CT 1998 Fairfield CT 2003 − Vertical transmission in Fairfield CT 2003 New London CT 2003 Georgia 1997 mosquitoes (Philbrook et al. CDC TR New London CT 2001 1961) New London CT 2003

New Haven CT 2001 New London CT 2000 − Recrudescence in chronically CT Fairfield CT 2001 2000 infected birds (Crans et al. JME 1994) Hartford CT 2000 New London CT 2001 CT Florida 1991 2000-01 − Reptiles or amphibians (?) Middlesex CT 2001 Florida 1982

New London CT 1996 • Support for both hypotheses Fairfield CT 2000 New Haven CT 2003 Florida 1993 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)

• 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, No. No. multiple introductions, and shorter local sequences human cases

persistence 40 1-4

80 7 • Supports a source-sink model in which FL 120 9 is the major source of EEEV 15 160 24 Northeastern US EEE Virus Transmission Cycle

Epidemic / Epizootic Transmission

Culiseta melanura

Enzootic June “Bridge Vectors” Cycle to Coquillettidia perturbans October Aedes canadensis Culex salinarius Virus Culiseta melanura

Local Overwintering & Annual Wild Passerine Bird Reservoir August to October Introduction and Amplifying Hosts Expectations for 2020

2019 • High likelihood that the EEE virus will Human case reemerge Veterinary case - 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 70 60 2004 - 2019 Human cases 50 Veterinary cases • Remains to be seen how widespread 40 activity will be 30 20 - Will we see further expansion into NH, ME 10

and VT? 0

2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Research – Surveillance Priorities

• 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 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”