Avian Disease & Oncology Lab (ADOL) Research Update

John Dunn, Hans Cheng, Mohammad Heidari, Huanmin Zhang

USDA-ARS-USPNRC

Research Programs at ADOL1) Genomics (Cheng, Zhang, Vacant SY)Title: Employing genomics, epigenetics,

and immunogenetics to control diseases induced by avian tumor viruses

2) Tumor Virus (Dunn, Heidari, Vacant SY)Title: Genetic and biological determinants

of avian tumor virus pathogenicity, transmission, and evolution

Improved Genetic Map for Chicken Genome Assembly

• Despite chicken being the first agricultural animal with a genome assembly (2004) and several updates, it is still incomplete, e.g., 8 of the 38 autosomes lack any sequence information.

• To aid in the most recent assembly (galgal5), genetic markers from unplaced sequence contigs were added to the East Lansing genetic map.

Chicken assembly galgal5.0 status

• Assigned sequence to 33 of 38 autosomes

• 29 new linkage groups defined• Contiguity is 10-fold better• Increased representation by

~180Mb• Yet, 138 Mb of sequence

unassigned

Blautia spp. Succinate Lactate

Lactobacillus spp.

Days 14, 21

AcetateDay 28

Faecalibacterium spp. Butyrate

Streptococcus spp.

Days 35, 42

Under MDV infection

Marek’s disease virus infections influence chicken gut colonization

Promotes colon health in humans against cancers

Healthy Chicken

Identify Driver Genes for Marek’s Disease Tumors

Tumor Samples

Transformation and

Tumorigenesis

1,000 pfuMDV

(JM/102W)

4-8 Weeks

200 Birds

Line 6 (resistant) x 7 (susceptible)

IKZF1IKZF1: IKAROS Family Zinc Finger 1 • Associated with T-cell development

– Important role in CD4 vs. CD8 lineage commitment

• Tumor Suppressor Gene– Acute Lymphoblastic Leukemia (ALL)

• Significantly mutated across samples (19%)• Deleterious mutations (dominant negative)

12-M400

Farms with Any Poultry Inventory on December 31, 2012

1 Dot = 20 Farms

U.S. Department of Agriculture, National Agricultural Statistics Service

United States Total233,7700 100

Miles0 100

Miles

0 200

Miles

2012 Census of A

griculture

Recent MDV isolates

0

10

20

30

40

50

60

70

80

90

100 747B

738

747A

JM

/102

W

571

617A

74

7C

596A

583A

56

8A

656C

587

586

653A

608

Md5

598

610B

54

9A

Md5

61

2 56

4A

595

615K

568B

61

5J

584C

611

643G

64

3P

656A

686

645

722D

73

9A

610A

730B

739B

584B

58

4A

730A

73

0C

652

648B

66

0A

722B

64

8A

722C

Virulence Ra

nk

New strains not more pathogenicVirulence rank (red = new cases)

v

vv+

vv

UL US

32 genomic regions used for targeted sequencing

R-LORF1 UL15 UL41 R-LORF7 R-LORF5 UL15 UL43 R-LORF7 R-LORF7 UL22 UL47 R-LORF5 R-LORF7 UL36 UL52 R-LORF1 R-LORF8 UL36 LORF11 ICP4

UL6 UL36 R-LORF14a SORF1 UL6 LORF6 R-LORF14a ? UL9 LORF7 R-LORF8 ICP4

Projected gene of targeted regions

Amplicons 200-400bp, Illumina MiSeq (2x250bp PE reads)

Md5/Md11

232

287L

295549A12

568AB

571

583A

584AB

595

596A

608610A

610B

611

615K

617A

643GP

648AB/660A

652

653A/674

656C

612/670

645/676

685/691690

701 709A709B/738

718AB/723

722ABCD/730ABC

739A/B

747ABC

754AB754C

GA/22/RPL39

JM102W

MIS-X

MSU-2

Md3/Md8

RB1B686

vvvvv+

1960-19691970-19791980-1989

1990-19992000-20092010-2015

Diversity of sequenced isolatesLocation, virulence & time

• 15 vMDV (1962-2014)• 24 vvMDV (1977-2011)• 25 vv+MDV (1990-2013)

phyMLTreeView

vMDVvvMDVvv+MDV

CC

C

C

C

C

C

C

A

AAA

B

B

B

CC

C

C

C

C

C

C

Preliminary association results

Low Vr Base

% of strains with Low Vr Base High Vr

Base Gene AAProtein Positionv vv vv+

A 94 63 40 C UL22 M->R 602

G 94 42 16 A UL36 H

T 94 29 0 C UL36/LORF6 K->R/L 34

G 67 17 0 A UL37 A->V 2T 83 29 4 G UL41 H->P 135G 83 21 16 A UL43 V->I 398C 100 38 8 T R-LORF8 V->M 72T 61 17 0 C R-LORF7 C->R 118C 94 46 24 A R-LORF7 P->Q 152G 78 21 12 A ICP4 P->S 180C 72 21 8 T ICP4 P->S 180

Effect of probiotics on MD vaccination

Company A Live lactic acid bacteria (LAB) probiotic

Company B Yeast fermentation product (YFP)(Saccharomyces cerevisiae)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

HVT (in ovo) HVT + LAB (in ovo) HVT (in ovo) + YFP (Feed)

% M

arek's

disease

%MD (challenged with 583A)

p=0.1850 p=0.0785

Trials 1 & 2: %MD reduced

Trial 3: Less effect with more virulent strains

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

HVT (in ovo) HVT + LAB (in ovo) HVT (in ovo) + YFP (Feed)

% M

arek's

disease

%MD (challenged with Md5 or 612)

Md5

612

N/A

• All genes chosen for deoptimization were– Unique– Annotated at both the gene and protein level– Of manageable length for de novo synthesis and cloning

MDV Codon de-optimization

UL19(MCP/VP5)

UL27(glycoprotein B)

UL53(glycoprotein K)

UL54(ICP27/ DNA pol)

UL US

• The initial ~40 bases and the final ~40 bases were left in the wild type sequence

UL54-wt

UL54-M1

invariant region codon pair deoptimized

MDV attenuation strategy

0 20 40 60 80 100 120

B40 BAC

UL54 M1

UL54 M2

gK M1

UL19 M1

gB M1

#trials #birds

5 67

4 60

3 48

1 33

1 50

1 50

• Codon pair deoptimization in UL54 significantly reduces the incidence of Marek’s disease

**

P = 0.004

P = 0.003

UL54 recoding reduces Marek’s incidence in inoculated birds

AcknowledgementsADOL & Mich. State Univ.• Robert Silva• Steve Conrad• Sudeep Perumbakkam• Alec Steep• Lonnie Milam• Laurie Molitor• Pam Campbell• Morgan Lewis• William Ratledge• Chris Kellogg

Funding

Washington Univ.• Wes Warren

Penn State Univ.• Andrew Read• David Kennedy• Moriah Szpara

Field samples• Eric Gingerich• Patty Dunn• Kenton Kreager• Jack Rosenberger• Enrique Montiel• Many others

Thank you for your attention