Orbivirus activities at NVSL

Eileen N. Ostlund Associate Director

National Veterinary Services Laboratories

U.S. Department of Agriculture Animal and Plant Health Inspection Service

Veterinary Services Science, Technology and Analysis Service

October, 2015

Veterinary Services

Topics

• 2014 Calendar Year and 2015 Year-to-

date Bluetongue and Epizootic

Hemorrhagic Disease Virus observations

at NVSL

• African Horse Sickness Virus PCR

International Ring Trial

2

3

BTV Identifications 2014 (NVSL, except where noted)

BTV not typed

State No. Species

ID 2 alpaca, deer*

MO 1 cattle

WA* 7 cattle, sheep, deer

BTV-11

State No. Species

CO 1 goat

BTV-17

State No. Species

NE 2 WTD

NJ 3* WTD

*2 isolates from SCWDS BTV-10

State No. Species

NE 1 bighorn sheep

ID (WI)† 1 cattle

†WI cattle in quarantine recently

shipped from ID

BTV-18

State No. Species

FL 1* WTD

*Isolate from SCWDS, first report of

BTV-18 in the U.S.

*testing done at WADDL

4

BTV Identifications 2015 (NVSL, except where noted)

BTV not typed

State No. Species

ID* 15 deer, sheep,

cattle, bighorn

NV* 1 cattle

OR* 1 sheep

TX* 3 WTD

WA* 20 WTD, mule deer,

elk, bighorn

BTV-13

State No. Species

OK 1 sheep

NV* 1 cattle

BTV-10

State No. Species

AZ 1 bighorn sheep

CA 5 sheep

FL 1 WTD *BTV identified by WADDL, typing NVSL

BTV activity has also been reported

in TX, but information regarding

numbers and types is not available.

*BTV identified by WADDL, typing NVSL

BTV-17

State No. Species

CA 3 sheep/ *mule

deer

ID* 8 cattle, WTD,

sheep, yak

NV* 3 bighorn sheep

WA* 9 WTD, mule deer

*BTV identified by WADDL

5

BTV Identifications 2015 (NVSL, except where noted)

BTV-3

State No. Species

TX 1* Cattle

TX 1* White-tailed deer

BTV-19

State No. Species

FL 1 White-tailed Deer

*BTV identified at TVMDL, typing NVSL

First report of BTV-3 in TX

BTV-24 previously identified in FL in

2007

BTV-22

State No. Species

FL 1* White-tailed Deer

*BTV identified at TVMDL, typing

NVSL; BTV-22 previously identified

in FL in 2002 & 2005 BTV-6

State No. Species

FL 1* White-tailed Deer

BTV-19 previously identified in FL in

2003

*Also positive EHDV-6; BTV-6

previously identified in FL in 2006

BTV-24

State No. Species

FL 1 White-tailed Deer

NVSL Bluetongue Virus Identification

(endemic serotypes) 1992-2015

FLORIDA

MISSISSIPPI

WEST

VIRGINIA

RHODE ISLAND

WASHINGTON

OREGON

IDAHO

MONTANA

WYOMING

NORTH

DAKOTA

SOUTH

DAKOTA

NEBRASKA IOWA

MINNESOTA

WISCONSIN

ILLINOIS

INDIANA OHIO

MISSOURI KANSAS

COLORADO

UTAH

NEVADA

CALIFORNIA

ARIZONA NEW MEXICO

OKLAHOMA ARKANSAS

KENTUCKY

VIRGINIA

TEXAS

MASSACHUSETTS

GEORGIA ALABAMA

SOUTH

CAROLINA

NORTH CAROLINA TENNESSEE

PENNSYLVANIA

MARYLAND

NEW JERSEY

NEW YORK

CONNECTICUT

VERMONT

NEW HAMPSHIRE

MAINE

DELAWARE

LOUISIANA

MICHIGAN

2*

10

11

13

17

2, 10

11

10,11, 17

13

17

13

17

11, 17

11,17

10

13, 17

11, 17

10, 11,

13, 17

11, 13, 17 13, 17

11, 17

11, 13, 17

10, 11,

17

BTV isolate / PCR positive

17 BTV serotype identified

11, 17

11, 13, 17

17

11, 13,

17

11

13, 17 11

13,

17

*BTV-2 isolated and identified by UC Davis, CA

17 11

17

10†

10†

†BTV-10 WI, cattle originated in ID

13, 17

‘Exotic’ BT serotypes in U.S. (1999-2015)

3

3

3

3

3

3 3

3 3

3

22

22

14

14

19

5 1

1 6

24

12

12

9

9

BTV-1: 2004, 2010, 2013

BTV-3: 1999, 2001, 2002, 2003, 2006,

2008, 2009, 2012, 2013, 2015

BTV-5: 2003

BTV-6: 2006, 2015

BTV-9: 2008, 2012

BTV-12: 2008, 2010, 2012

BTV-14: 2003, 2009

BTV-18: 2014

BTV-19: 2003, 2015

BTV-22: 2002, 2005, 2015

BTV-24: 2007, 2015

12

12

12

12

1

3

3 3

18

19

3 22

6

24

BTV-1: 10, 13

BTV-2: 99, 03, 05, 06, 13

BTV-3: 99, 01, 02, 03, 13

BTV-5: 03

BTV-6: 06, 15

BTV-9: 08, 12

BTV-10: 02, 03, 15

BTV-11: 13

BTV-12: 10, 12

BTV-13: 99

BTV-14: 03

BTV-17: 99

BTV-18: 14

BTV-19: 03, 15

BTV-22: 02, 05, 15

BTV-24: 07, 15

1 2

3

3

5 6

9

9

11 12

12

14

19

22

22

24

Florida 1999-2015:

BT serotype identifications

(NVSL)

2

2

3

17

13 3

3 10 3

3 10

2

2

1 18

19

22

10

6

24

9

EHD Virus Identifications, 2014

EHD 2

State No. Species

FL 1 deer

TX 1 Eld’s deer

EHD 6

State No. Species

FL 1 WTD

NC 2 WTD

EHD not typed

State No. Species

ID 3* WTD

NE 1† bison

WA 3* WTD

*EHD identified by WADDL

†Suspect

• Sept 23, 2015 ProMed

reports EHD outbreak in

Illinois, with 47 suspected

cases in white-tailed deer.

Type not reported.

• Increased EHD 2 activity in

IA and OK in 2015; has not

been observed to date in NE

or SD.

10

EHD 2

State No. Species

IA 7 WTD, cattle

IL 1 WTD

KS 1 WTD

OK 3 elk, WTD

EHD not typed

State No. Species

KY 1* cattle

*EHD identified by WADDL

EHD Virus Identifications, 2015

EHD 6

State No. Species

FL 1 WTD

NVSL Epizootic hemorrhagic disease virus (EHDV)

identification 1992-2015

FLORIDA

MISSISSIPPI

WEST

VIRGINIA

RHODE ISLAND

WASHINGTON

OREGON

IDAHO

MONTANA

WYOMING

NORTH

DAKOTA

SOUTH

DAKOTA

NEBRASKA IOWA

MINNESOTA

WISCONSIN

ILLINOIS

INDIANA OHIO

MISSOURI KANSAS

COLORADO

UTAH

NEVADA

CALIFORNIA

ARIZONA NEW MEXICO

OKLAHOMA ARKANSAS

KENTUCKY

VIRGINIA

TEXAS

MASSACHUSETTS

GEORGIA ALABAMA

SOUTH

CAROLINA

NORTH CAROLINA TENNESSEE

PENNSYLVANIA

MARYLAND

NEW JERSEY

NEW YORK

CONNECTICUT

VERMONT

NEW HAMPSHIRE

MAINE

DELAWARE

LOUISIANA

MICHIGAN

WASHINGTON, DC

EHDV 2

EHDV 1 and 2

EHDV 6

EHDV type unknown

NVSL EHD/BT Identifications (by county), 2013

(U.S. cases)

2 6

FLORIDA

MISSISSIPPI

WEST

VIRGINIA

RHODE ISLAND

WASHINGTON

OREGON

IDAHO

MONTANA

WYOMING

NORTH

DAKOTA

SOUTH

DAKOTA

NEBRASKA IOWA

MINNESOTA

WISCONSIN

ILLINOIS

INDIANA OHIO

MISSOURI KANSAS

COLORADO

UTAH

NEVADA

CALIFORNIA

ARIZONA NEW MEXICO

OKLAHOMA ARKANSAS

KENTUCKY

VIRGINIA

TEXAS

MASSACHUSETTS

GEORGIA ALABAMA

SOUTH

CAROLINA

NORTH CAROLINA TENNESSEE

PENNSYLVANIA

MARYLAND

NEW JERSEY

NEW YORK

CONNECTICUT

VERMONT

NEW HAMPSHIRE

MAINE

DELAWARE

LOUISIANA

MICHIGAN

BT

EHD

EHD

2

10

BT

2

2

2

2

17

BT

2

17

2

2

17 2 2

2

2 2

17

17 17

2 2 2

2

2

2

1

11

11

13

13

2 17

11

2

2 2

2

2 17

13

2

11

1

6

2

2

2

2

2 2

2

2

17

13

BT

2

11

2

11

2

1

13

1 2

2 2

2 2

17 13

2

11

13

11

EHD

2

3

11

3

BT

2

EHD/BT Identifications (by county), 2014 (U.S. cases, identified by, or reported to the NVSL)

17

6

FLORIDA

MISSISSIPPI

WEST

VIRGINIA

RHODE ISLAND

WASHINGTON

OREGON

IDAHO

MONTANA

WYOMING

NORTH

DAKOTA

SOUTH

DAKOTA

NEBRASKA IOWA

MINNESOTA

WISCONSIN

ILLINOIS

INDIANA OHIO

MISSOURI KANSAS

COLORADO

UTAH

NEVADA

CALIFORNIA

ARIZONA NEW MEXICO

OKLAHOMA ARKANSAS

KENTUCKY

VIRGINIA

TEXAS

MASSACHUSETTS

GEORGIA ALABAMA

SOUTH

CAROLINA

NORTH CAROLINA TENNESSEE

PENNSYLVANIA

MARYLAND

NEW JERSEY

NEW YORK

CONNECTICUT

VERMONT

NEW HAMPSHIRE

MAINE

DELAWARE

LOUISIANA

MICHIGAN

BT

EHD

BT

BT

11

17

6

2 6

2

17

10

EHD

10

18

BT

BT

BT

EHD

EHD

EHD/BT Identifications (by county), 2015 (U.S. cases, identified by, or reported to the NVSL)

6

FLORIDA

MISSISSIPPI

WEST

VIRGINIA

RHODE ISLAND

WASHINGTON

OREGON

IDAHO

MONTANA

WYOMING

NORTH

DAKOTA

SOUTH

DAKOTA

NEBRASKA IOWA

MINNESOTA

WISCONSIN

ILLINOIS

INDIANA OHIO

MISSOURI KANSAS

COLORADO

UTAH

NEVADA

CALIFORNIA

ARIZONA NEW MEXICO

OKLAHOMA ARKANSAS

KENTUCKY

VIRGINIA

TEXAS

MASSACHUSETTS

GEORGIA ALABAMA

SOUTH

CAROLINA

NORTH CAROLINA TENNESSEE

PENNSYLVANIA

MARYLAND

NEW JERSEY

NEW YORK

CONNECTICUT

VERMONT

NEW HAMPSHIRE

MAINE

DELAWARE

LOUISIANA

MICHIGAN

BT

EHD

2 2

2

2

BT

10

2

2

19

3

2 17

17

17

17

17

17

17

17

17

17

BT

BT 17

13

10 22

2

2

2

BT

BT

EHD

10

10

6

24

6

13

10

17

African Horse Sickness

International Ring Trial

AHSV detection by PCR

15

African Horse Sickness

• African Horse Sickness (AHS) is an acute or subacute Culicoides-borne, viral disease of horses and other equids that is often fatal. Clinical signs and lesions are compatible with impaired respiratory and circulatory function.

• There are 9 serotypes of AHSV

• AHSV is not present in the Americas

• If introduced, could be spread by native Culicoides spp.

• Current import requirements for equids from affected areas = 60 day quarantine

– Impetus for decreasing quarantine with AHSV PCR tests

Host susceptibility • All solipeds are susceptible to infection.

Susceptibility to disease varies by species. Horses > Mules > Donkeys > Zebras

75-90% 50% 5-10% no illness

• Dogs can become infected and ill by eating infected horse meat.

Clinical Disease

• The incubation period is usually 5-14 days

• AHSV replicates in endothelium, especially heart and lung

• Fever up to 41C (106F), congestion of conjunctivae

• Pulmonary, cardiac, mixed forms of disease

• Differentia Diagnosis includes EIA, EVA, Piroplasmosis, etc.

2015 AHS PCR Ring Trial

• OIE AHS ad hoc group requested opinion on adoption of an RT-PCR as a prescribed test for international trade

• Ring Trial administered by AHS OIE Reference Laboratories in the UK and Spain

• Variety of methods being used by AHS testing labs – compare results on provided sample set.

• Participants from 4 OIE AHS reference laboratories: South Africa, UK, Spain (2), plus EU reference center for equine infectious diseases (other than AHS), Deltammune in South Africa and NVSL = 7 laboratories

19

2015 AHS PCR Ring Trial • Panel 1 (not tested by NVSL)

• Panel 2 = 45 samples (40 positive)

– Inactivated AHSV-spiked horse blood samples

– Panel encompassed all AHSV serotypes

• Reference strains and recent outbreak isolates

– Serial dilutions of some AHSV serotypes

– One equine encephalosis virus spiked sample

– AHSV-negative horse blood samples

• Each laboratory used their own internally validated

and accredited test

– Laboratories could also provide results for additional

methods

– 10 different PCR methods reported 20

2015 AHS PCR Ring Trial • NVSL tested by 3 methods

– Fernández-Pinero (FP) [2009] conventional RT-PCR

• FP conventional missed several positive samples

– Fernández-Pinero (FP) [2009] real time RT-PCR

• Correct results on nearly all samples (a few highly diluted samples were missed by most labs).

– Guthrie [2013] real time RT-PCR

• Correct results on nearly all samples (a few highly diluted samples were missed by most labs).

• The Guthrie method was slightly more sensitive.

• Current NVSL method

• NVSL performed serotyping PCRs on positive samples

– All AHSV typing correct

21

2015 AHS PCR Ring Trial

• Aguero 2008 method is used by many

EU laboratories

• Dossier presented to OIE Biological Standards

Commission in September 2015

– Accepted Aguero 2008 method as validated test with

understanding that other PCR tests may yield equivalent results

– Will be incorporated in OIE manual pending May 2016 OIE

meeting

• Guthrie 2013 method undergoing separate validation

testing

22

Questions?