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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
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