Phenotypic Characterization of Recombinant Marek’s Disease Virus in Live Birds Validates Polymorphisms Associated with Virulence
Abstract
:1. Introduction
2. Materials and Methods
2.1. Viruses
2.2. Mutagenesis of 686-BAC
2.3. Reconstitution of the 686 Mut Viruses
2.4. In Vivo Pathogenicity Study
2.5. Pathotyping Assay
2.6. Determination of Pathotype
2.7. Statistical Analysis
3. Results
3.1. Modified Recombinant MDVs
3.2. Pathogenicity Analysis of the Recombinant MDVs
3.3. Pathotype Analysis of the Recombinant MDVs
3.4. Graphical Display of the Recombinant MDV Pathotypes
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | vv+MDV | vMDV | Amino Acid Change | Protein Position |
---|---|---|---|---|
UL22 | C | A | R to M | 603 |
UL36 | C | T | R to K | 35 |
UL37 | A | G | V to A | 3 |
UL41 | G | T | P to H | 136 |
UL43 | A | G | I to V | 399 |
R-LORF8 | T | C | M to V | 73 |
R-LORF7 | C | T | R to C | 119 |
R-LORF7 | A | C | Q to P | 153 |
ICP4 | A | G | S to P | 181 |
ORF | Primer | Sequence (5′ to 3′) * |
---|---|---|
UL22 | UL22mut.FP | ACAAGTCACAACTTCTGATGCATATATAGTCAACCTTCTCATCAATGGTGTTTTAAGCTTtagggataacagggtaatcgattt |
UL22mut.RP | AGCAGAATTGGTAATCACCAAGCTTAAAACACCATTGATGAGAAGGTTGACTATATATGCgccagtgttacaaccaattaacc | |
UL36 | UL36mut.FP | TTTCGATCAAGCGAACAGAGTCCAGACGTCGATTTATCTCTTAACACGTTCGCAGCTAGCtagggataacagggtaatcgattt |
UL36mut.RP | CTACATCGAACGCACCCATGCTAGCTGCGAACGTGTTAAGAGATAAATCGACGTCTGGACgccagtgttacaaccaattaacc | |
UL37 | UL37mut.FP | TCCAATAAAACTTTCAGTGGCCATATTTCATCGGTCGTTACGGCAGACATTATTCAGGCtagggataacagggtaatcgattt |
UL37mut.RP | ACAGAGGGTTGGCGATAGTTGCCTGAATAATGTCTGCCGTAACGACCGATGAAATATGGCgccagtgttacaaccaattaacc | |
UL41 | UL41mut.FP | TCAAACCTTTCGCTCCATCTACAAACCTTCTTCCCGGGGTGTTTCCTCTTACACCGCGCCtagggataacagggtaatcgattt |
UL41mut.RP | GAAGTACGGATGTTGGAGAGGCGCGGTGTAAGAGGAAACACCCCGGGAAGAAGGTTTGTAgccagtgttacaaccaattaacc | |
UL43 | UL43mut.FP | AATGCAAATAAGGGAATTAAACAATTAGCAGCTGCCTATGTAGTGAAATCTATACTGGGAtagggataacagggtaatcgattt |
UL43mut.RP | GTAAACTAGTTATGATAAATCCCAGTATAGATTTCACTACATAGGCAGCTGCTAATTGTTgccagtgttacaaccaattaacc | |
R-LORF8 | R-LORF8mut.FP | CTTCACAGGGGACATTCAAAACAAGCCCAGAGCCGTCACGTGGAACACGTCTCGAGTCGAtagggataacagggtaatcgattt |
R-LORF8mut.RP | GCTTTCTTGAGGGGAGCGATCGACTCGAGACGTGTTCCACGTGACGGCTCTGGGCTTGTTgccagtgttacaaccaattaacc | |
R-LORF7 | Meqmut.FP | CTAAGGACTGAGTGCACGTCCCTGCGTGTACAGTTGGCTTGTCATGAGCCAGTTTGCCCTtagggataacagggtaatcgattt |
Meqmut.RP | CCGTTAGGGGTACCGCCATAGGGCAAACTGGCTCATGACAAGCCAACTGTACACGCAGGGgccagtgttacaaccaattaacc | |
R-LORF7 | Meqmut2.FP | CGCACGATCCCGTTCCTGAACCTCCCATTTGCACTCCTCCACCTCCCTCACCGGATGAACtagggataacagggtaatcgattt |
Meqmut2.RP | GAGCAATGTGGAGCGTTAGGTTCATCCGGTGAGGGAGGTGGAGGAGTGCAAATGGGAGGTgccagtgttacaaccaattaacc | |
ICP4 | ICP4mut.FP | CCTCACCAAAATCGGCTGCCAGGATCTCCAGTAGAGGAGGACTGGATGTCCCGCCGCTTCtagggataacagggtaatcgattt |
ICP4mut.RP | GACATCGAGTCCGCTTCCGGAAGCGGCGGGACATCCAGTCCTCCTCTACTGGAGATCCTGgccagtgttacaaccaattaacc |
Gene | vv+MDV Allele | vMDV Allele | 686 | 686∆TRS-TRL | 686 mut1 | 686 mut2 | Average Depth |
---|---|---|---|---|---|---|---|
UL22 | C | A | C | C | A | A | 126 |
UL36 | C | T | C | C | T | T | 133 |
UL37 | A | G | A | A | G | G | 131 |
UL41 | G | T | G | G | T | T | 124 |
UL43 * | G | T | G | G | T | T | 131 |
UL43 | A | G | A | A | G | G | 133 |
R-LORF8 | T | C | T | T | C | C | 174 |
R-LORF7 | C | T | C | C | T | T | 186 |
R-LORF7 | A | C | A | A | C | C | 157 |
ICP4 | A | G | A | A | G | G | 97 |
LORF2 * | A | G | A | G | A | A | 129 |
Clinical Trial | Challenge Virus | Birds at Risk 1 | MD-Positive 2 | % MD | Differences 3 |
---|---|---|---|---|---|
1 | None | 15 | 0 | 0 | a |
v686-BAC | 15 | 10 | 67 | b | |
v686∆TRS-TRL | 12 | 11 | 92 | b | |
v686 mut1 | 8 | 7 | 88 | b | |
v686 mut2 | 14 | 12 | 86 | b | |
2 | None | 12 | 0 | 0 | a |
v686-BAC | 9 | 9 | 100 | b | |
v686∆TRS-TRL | 8 | 8 | 100 | b | |
v686 mut1 | 14 | 11 | 79 | b | |
v686 mut2 | 15 | 10 | 67 | b |
Replicate | Group Vaccine/Challenge | Birds at Risk 1 | MD Positive 2 | % MD | Differences 3 |
---|---|---|---|---|---|
1 | None/JM/102W | 16 | 15 | 94 | a |
None/Md5 | 16 | 14 | 88 | a | |
None/648A | 16 | 16 | 100 | a | |
None/v686∆TRS-TRL | 15 | 15 | 100 | a | |
None/v686 mut1 | 16 | 15 | 94 | a | |
None/v686 mut2 | 16 | 16 | 100 | a | |
HVT/JM/102W | 9 | 2 | 22 | a | |
HVT/Md5 | 9 | 3 | 33 | b | |
HVT/648A | 9 | 8 | 89 | b | |
HVT/v686∆TRS-TRL | 10 | 6 | 60 | a,b | |
HVT/v686 mut1 | 10 | 6 | 60 | a,b | |
HVT/v686 mut2 | 9 | 4 | 44 | a,b | |
HVT+SB1/JM/102W | 9 | 1 | 11 | a | |
HVT+SB1/Md5 | 9 | 3 | 33 | a,b | |
HVT+SB1/648A | 9 | 6 | 67 | b | |
HVT+SB1/v686∆TRS-TRL | 10 | 7 | 70 | b | |
HVT+SB1/v686 mut1 | 8 | 2 | 25 | a,c | |
HVT+SB1/v686 mut2 | 6 | 3 | 50 | a,b | |
2 | None/JM/102W | 13 | 6 | 46 | a |
None/Md5 | 13 | 13 | 100 | b | |
None/648A | 11 | 11 | 100 | b | |
None/v686∆TRS-TRL | 13 | 13 | 100 | b | |
None/v686 mut1 | 13 | 9 | 69 | a,b | |
None/v686 mut2 | 12 | 12 | 100 | b | |
HVT/JM/102W | 16 | 0 | 0 | a | |
HVT/Md5 | 15 | 1 | 7 | a | |
HVT/648A | 15 | 10 | 67 | b | |
HVT/v686∆TRS-TRL | 16 | 2 | 13 | a,b | |
HVT/v686 mut1 | 16 | 4 | 25 | a,b | |
HVT/v686 mut2 | 16 | 3 | 19 | a,b | |
HVT+SB1/JM/102W | 16 | 0 | 0 | a | |
HVT+SB1/Md5 | 15 | 3 | 20 | a | |
HVT+SB1/648A | 16 | 4 | 25 | a | |
HVT+SB1/v686∆TRS-TRL | 16 | 2 | 13 | a | |
HVT+SB1/v686 mut1 | 16 | 3 | 19 | a | |
HVT+SB1/v686 mut2 | 16 | 2 | 13 | a |
Non-Vaccinated | Vaccinated | Virulence by Weighted Mean | Pathotype | ||||||
---|---|---|---|---|---|---|---|---|---|
Virus | # of Birds | # of MD | % of MD | # of Birds Vaccinated | % of Total MD | ||||
HVT | HVT+SB1 | HVT | HVT+SB1 | ||||||
JM/102W | 29 | 21 | 72 | 25 | 25 | 8 | 4 | 5 | |
Md5 | 29 | 27 | 93 | 24 | 24 | 17 | 25 | 22 | |
648A | 27 | 27 | 100 | 24 | 25 | 75 | 40 | 52 | |
v686∆TRS-TRL | 28 | 28 | 100 | 26 | 26 | 31 | 35 | 33 | vv or vv plus |
v686 mut1 | 29 | 24 | 83 | 26 | 24 | 38 | 21 | 27 | vv |
v686 mut2 | 28 | 28 | 100 | 25 | 22 | 28 | 23 | 24 | vv |
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Kim, T.; Hearn, C.J.; Mays, J.; Velez-Irizarry, D.; Reddy, S.M.; Spatz, S.J.; Cheng, H.H.; Dunn, J.R. Phenotypic Characterization of Recombinant Marek’s Disease Virus in Live Birds Validates Polymorphisms Associated with Virulence. Viruses 2023, 15, 2263. https://doi.org/10.3390/v15112263
Kim T, Hearn CJ, Mays J, Velez-Irizarry D, Reddy SM, Spatz SJ, Cheng HH, Dunn JR. Phenotypic Characterization of Recombinant Marek’s Disease Virus in Live Birds Validates Polymorphisms Associated with Virulence. Viruses. 2023; 15(11):2263. https://doi.org/10.3390/v15112263
Chicago/Turabian StyleKim, Taejoong, Cari J. Hearn, Jody Mays, Deborah Velez-Irizarry, Sanjay M. Reddy, Stephen J. Spatz, Hans H. Cheng, and John R. Dunn. 2023. "Phenotypic Characterization of Recombinant Marek’s Disease Virus in Live Birds Validates Polymorphisms Associated with Virulence" Viruses 15, no. 11: 2263. https://doi.org/10.3390/v15112263
APA StyleKim, T., Hearn, C. J., Mays, J., Velez-Irizarry, D., Reddy, S. M., Spatz, S. J., Cheng, H. H., & Dunn, J. R. (2023). Phenotypic Characterization of Recombinant Marek’s Disease Virus in Live Birds Validates Polymorphisms Associated with Virulence. Viruses, 15(11), 2263. https://doi.org/10.3390/v15112263