RISK ANALYSIS 

FOWL LARYNGOTRACHEITIS-MAREK'S DISEASE VACCINE, 

SEROTYPE 3, LIVE MAREK'S DISEASE VECTOR, 

PRODUCT CODE 16J1.R1 (UNLICENSED) 

March 6, 2009 

Supersedes 

March 3, 2008 

Biomune Company 

8906 Rosehill Road 

Lenexa, Kansas 66215 

U.S. Vet Lic. No. 368 
Biomune Co. 

U.S. Vet. Lie. No. 368 

TABLE OF CONTENTS' 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

RISK ANALYSIS 

SUMMARY OF INFORMATION FORMAT 

SUMMARY OF INFORMATION FORMAT APPENDIX 

RISK ASSESSMENT 

PAGE 

1 - 20 

21 -43 

44-56 

1 The blackened areas in this document contain Confidential Business Information (CBI) of a proprietary 

nature, the disclosure of which under the Freedom of Information Act (FOIA) is likely to cause our firm 

substantial competitive harm. Therefore, we request not be disclosed under FOIA. 
BiomuneCo. March 6, 2009 

U.S. Vet. Lic. No. 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

I. Introduction 

A. Objective 

1. Fowl Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live 

Marek's Disease Vector, Product Code 16J1.R1 (Unlicensed) was 

constructed from the agents listed below in the Research and 

Development Department, Biomune Company, Lenexa, KS. If 

approved, the final vaccine will be made, tested and manufactured 

in the Manufacturing Department, Biomune Company, Lenexa, KS, 

U.S. Veterinary License No. 368. Biomune Company has Biosafety 

Level 2 animal containment facilities available for testing of live 

vectors. 

2. The Regulated Biological Agent (RBA), 

contains a turkey h<>.,..t,.f",",Q 

and expresses a 

B. Proposal 

1. Species: Chickens, embryos at 18 days of incubation and day of 

age or older. 

2. Proposed claim: Fowl Laryngotracheitis-Marek's Disease Vaccine, 

Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 

(Unlicensed) is recommended for use in embryos at 18 days of 

incubation and in healthy day of age chicks as an aid in the 

prevention of laryngotracheitis (L T) and Marek's disease (MD), 

which is caused by virulent Marek's disease virus (MDV). 

3. Geographic area: United States (all states) 

4. Route of administration: In ovo to embryos at 18 days of incubation 

and subcutaneous to day of age chicks. 

5. 

Attenuation of the HVT strain 

has not been observed. Administration of the MSV _ is 

expected to cause a transient viremia in the chick that does not 

shed to contact chickens. Hatchability problems are not expected 

when administered in ovo and no adverse tissue reaction is 

expected at the site of inoculation when administered 

subcutaneously to day of age chicks. 

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Biomune Co. Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

March 6, 2009 

u.s. Vet. Lic. No. 368 

II. Description of the Regulated Biological Agent Construction 

A. The Backbone Biological Agent 

1. Turkey herpesvirus is a double-stranded linear DNA virus in the 

Herpesviridae family and Alphaherpesvirinae subfamily. HVT is 

ubiquitous and non-oncogenic in domestic turkeys and it is 

classified as serotype 3 of MDV. The HVT parent strain, _, 

has been used commercially to vaccinate chickens against Marek's 

disease since 1972 (Witter and Schat, 2003). HVT is classified in 

the Biosafety Level 1 category. 

2. Physical Characteristics of the Backbone Agent 

B. Donor Biological Agents and Donor DNA or Genes 

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BiomuneCo. 

U.S. Vet. Lie. No. 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

March 6, 2009 

a. The. gene was isolated from the virulent LTV field isolate 

632 (Poulsen et al., 1991). The. gene from this isolate is 

used in a USDA-licensed vaccine, Fowl Pox-Laryngotracheitis 

Vaccine, Live Fowl Pox Vector, Product Code 1 F31.RO 

(Licensed) . 

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Biomune Co. March 6, 2009 

U.S. Vet. Lie. No. 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

C. Construction and Characterization of the Regulated Biological Agent 

1. Diagrams for the construction of the Regulated Biological Agent are 

provided as described below. 

2. 

e. After transfection, virus successfully grown _ was 

separated by limiting dilutions. Wells containing plaques were 

expanded in duplicate and screened for expression of the _ 

gene. The recombinant virus was passed seven times to isolate 

a clone of MSV _ and _ to expand the clone that 

was designated as master seed virus (MSV). 

Procedures and results of 

n the Appendix. 

3. Physical Characterization of the Regulated Biological Agent 

a. Southern blot analysis and PCR/sequencing of important 

regions were conducted to characterize the physical map of the 

RBA. _ Southern blotting schemes are provided in Figure 8, 

as well as • PCR schemes to amplify. regions. The 

sequence of each of these PCR products was analyzed. 

Procedures and results of these experiments are described in 

the Appendix. Sequences of primers used in the. 

PCR/sequencing schemes and sequencing are provided in 

Table 1, Appendix. 

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Biomune Co. March 6, 2009 

u.s. Vet. Lic. No. 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

c. The. Southern blots and the. PCR schernes described in 

Figure 8 were used to test MSV nand n+5. Procedures and 

results of these experiments are described in the Appendix. 

d. An electronic file providing the genetic sequence for the 

homology plasmid was provided in a diskette. 

4. Because the. gene and the _ gene do not provide any new 

virulence factors to the HVT backbone and MSV _ is 

expected to be biologically the same as the parent virus, the 

recommended NIH/CDC biosafety level will be the same as the 

parent virus (Biosafety Level 1). . 

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Biomune Co. March 6, 2009 

u.s. Vet. Lic. No. 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

III. Biological Properties or Virulence for the Regulated Biological Agent used 

for Master Seed 

A. Phenotypic Characteristics 

1. The cloning site is thought to be nonessential for HVT repl replicates similar to the 

HVT parent strain in vitro and in vivo. This insertion site has been 

used previously in two of our USDA-licensed HVT recombinant 

products, which are (1) Bursal Disease-Marek's Disease Vaccine, 

Serotype 3, Live Marek's Disease Vector, Product Code 1ABB.RO 

(Licensed) and (2) Marek's Disease-Newcastle Disease Vaccine, 

Serotype 3, Live Marek's Disease Vector, Product Code 16N1.RO 

(Licensed). 

2. The RBA expresses the. gene of LTV and the __ gene. 

Expression of the. protein was demonstrated by black plaque 

assay and western blot using _ antibodies. 

Expression of the from the 

detected 

Procedures and results of these 

rl"",r.ri~)"rl in the Appendix. 

B. Virulence Characteristics 

1. There are no known virulence characteristics of the. gene or the 

_ gene, so addition of the genes to HVT is not expected to be 

virulent for chickens. 

2. The RBA does not contain any genetic elements or toxin genes that 

are known to be inherently virulent. 

C. Virulence in Target and Non-Target Animals 

1. In order to address virulence in target animals, _ MSV was 

tested for safety in chickens according to 9 CFR 113.330(b). 

Procedures and results are described in a report entitled "120-Day 

Safety of Fowl Laryngotracheitis-Marek's Disease Vaccine, 

Serotype 3, Live Marek's Disease Vector, Uncoded (Unlicensed)", 

which was approved by APHIS on August 7, 200B. 

Safety of _ MSV in chickens was also tested in a 

backpassage study. _ MSV was passed five times in 

chickens and tested for virulence and genetic stability. No adverse 

vaccine reactions, or clinical signs of MD or L T, were observed 

during each passage or for 45 days in the fifth backpassage group. 

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Biomune Co. Fowl Laryngotracheitis-Marek's Disease Vaccine March 6, 2009 

U.S. Vet. Lie. No. 368 Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

The in vivo genetic stability of MSV _ was confirmed using 

molecular tests on virus isolated from the fifth backpassage group. 

Procedures and results are described in a report entitled 

"Backpassage: Genetic Stability and Safety of Fowl 

Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live 

Marek's Disease Vector, Uncoded (Unlicensed)", which was 

approved by APHIS on August 11, 2008. 

2. Safety was demonstrated in other avian species (turkeys, quail, 

pheasants, and pigeons) by: (1) inoculation with MSV_ or 

the HVT parent strain, and (2) comparison of clinical signs, gross 

lesions, adverse vaccine reactions, and virus isolation between 

these two groups. Results demonstrated that other avian species 

inoculated with MSV _ showed no clinical signs, no gross 

lesions or adverse vaccine reactions. The host range of MSV 

was similar to that of HVT parent. Therefore, MSV 

is safe in these avian species and use in chickens poses 

no safety risk to other avian species. This study is described in a 

report entitled "Safety in Avian Species: Fowl LaryngotracheitisMarek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, 

Product Code 16J1.R1 (Unlicensed)", which was approved by 

APHIS on December 15,2008. 

Safety was also tested in mammalian cell lines. 

cell lines were in this study, Mouse Porcine 

_, and Canine These mammalian cell 

lines were inoculated with MSV and passed five times. No 

cytopathic effects were observed any cell line or any passage. 

Also, similar results were obtained when these species were 

inoculated with the parent HVT strain. Based on these results, it 

was concluded that the host range of MSV similar to 

that of the HVT parent strain. Therefore, MSV is safe in 

these mammalian species and use in chickens poses no safety risk 

to mammalian species. This study is described in a report entitled 

"Safety in Non-Avian Species: Fowl Laryngotracheitis-Marek's 

Disease Vaccine, Serotype 3, Live Marek's Disease Vector, 

Product Code 16J1.R1 (Unlicensed)", which was approved by 

APHIS on December 16, 2008. 

D. Tissue Tropism 

1. The tissue tropism of MSV _ was determined and compared 

to the HVT parent strain. 

a. ~hickens were vaccinated with a 10X dose of MSV 

_ or an equivalent amount of HVT parent strain, and 

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Biomune Co. March 6, 2009 

U.S. Vet. Lic. No. 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

virus isolations were conducted on various tissues. Chickens 

vaccinated with MSV _ showed no adverse vaccine 

reactions, no clinical signs of MD or L T through 21 days of age, 

~ lesions of MD or LT. At 10 and 21 days of age, MSV 

_ was isolated from purified white blood cells from the 

blood, spleen, thymus and bursa. Similarly, the HVT parent 

strain was isolated from these same tissues at these time 

points. Based on these results, it was concluded that the tissue 

tropism of MSV _ was similar to that of the HVT parent 

strain. Therefor~ is safe for use in chickens and 

poses no safety risk. This study is described in a report entitled 

"Tissue Tropism of Fowl Laryngotracheitis-Marek's Disease 

Vaccine, Serotype 3, Live Marek's Disease Vector, Product 

Code 16J1.R1 (Unlicensed)", which was approved by APHIS on 

December 10, 2008. 

b. Safety was demonstrated in other avian species (turkeys, quail, 

pheasants and pigeons) by: (1) inoculation with MSV_ 

or the HVT parent strain, and (2) comparison of clinical signs, 

gross lesions, adverse vaccine reactions, and virus isolation 

between these two groups. Results demonstrated that other 

avian species inoculated with MSV _ showed no clinical 

signs, no gross lesions or adverse vaccine reactions. The host 

range of MSV was similar to that of the HVT parent. 

Therefore, is safe in these avian species and use 

in chickens poses no safety risk to other avian species. This 

study is described in a report entitled "Safety in Avian Species: 

Fowl Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

(Unlicensed)", which was approved by APHIS on December 15, 

2008. 

2. Turkey herpesvirus is ubiquitous in domestic turkeys and is also 

ubiquitous in commercial chickens due to vaccine use since the 

1970s. The literature indicates that HVT replicates in turkeys, but is 

non-oncogenic (Witter et a/., 1972; Witter and Schat, 2003). The 

host range of HVT has been defined by experimental infections of 

various birds, but has been better defined in tissue culture. HVT is 

known to replicate in primary chicken, duck and quail cells, as well 

as quail cell lines (Cowen and Braune, 1988; Lee, 1971; SamorekDziekanowska,1977). Unsuccessful attempts to replicate HVT in 

several mammalian species such as newborn hamsters, rhesus 

monkeys and marmoset monkeys have been conducted (Witter and 

Schat, 2003; Sharma et a/., 1972). Also, investigators trying to 

replicate HVT in mammalian primary cultures and cell lines were 

unable to detect evidence of virus replication even after performing 

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Biomune Co. March 6, 2009 

U.S. Vet. Lic. No. 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

six to 10 blind passages (Meulemans et a/., 1973; Witter and 

Sharma, 1974). 

E. Potential for Horizontal Gene Transfer 

1. The chance of in vivo recombination between MSV _ and 

field strains of LTV and other viruses was considered. To date, no 

recombination events between MDV, including HVT, and LTV have 

been reported under field conditions. HVT infects and replicates in 

lymphocyte cells, while LTV infects and replicates in respiratory 

organs, such as the larynx and trachea, and goes latent in 

trigeminal ganglia. Therefore, an encounter between HVT and LTV 

in host animals~are and the chance of in vivo recombination 

between MSV _ and field LTV strains is extremely limited. 

2. The chance of an in vivo recombination event between MSV 

_ and field MDV virus is unknown because the occurrence 

of horizontal gene transfer among the various MDV serotypes 

under field conditions is not known and is considered very rare 

(Witter and Schat, 2003). Common MDV vaccination practices in 

the United States involve the mixture of HVT (serotype 3), SB-1 

(serotype 2), and Rispens (serotype 1) vaccines. To date, no 

natural recombination events between any of these vaccines or 

with field MDV viruses (serotype 1) have been reported. One case 

of experimentally produced MDV serotype 2 recombination with 

MDV serotype 1 was reported, but was not repeatable (Hirai et a/., 

1990). The chance of an in vivo recombination event is unknown, 

but a remote possibility. 

F. Shed/Spread 

1. Shed/spread capability of the MSV _ was evaluated. Briefly, 

chicken embryos were vaccinated in ova with a 10X dose of MSV 

_ or an equivalent amount of HVT parent strain. After hatch, 

for each vaccine, a group of non-vaccinated chickens was placed in 

the same isolation unit to observe the potential shed/spread of the 

vaccine. At 10, 14 and 21 days of age, virus reisolation was 

attempted from the blood of all treatment groups. Virus was 

reisolated from the vaccinated groups, while no virus was reisolated 

from any contact control group. Based on these it was 

concluded that the shed/spread capability of MSV 

similar to the HVT parent strain and neither MSV 

HVT parent strain was transmissible. Therefore, is 

safe for use in chickens and poses no safety risk. This study is 

described in a report entitled "Shed/Spread of Fowl 

Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live 

9 
! 

Biomune Co. Fowl Laryngotracheitis-Marek's Disease Vaccine March 6, 200S 

U.S. Vet. Llc. No. 368 Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

Marek's Disease Vector, Product Code 16J1.R1 (Unlicensed)", 

which was approved by APHIS on December 15, 2008. 

3. The transmissibility of the recombinant is thought to be similar to the 

_ HVT~t strain. Previously reported transmissibility results 

of the HVT _ strain (Marek's Disease Vaccine, Serotype 3, Live 

Virus, Product Code 16L 1.06 (Unlicensed» demonstrated that the .. 

• strain did not spread to non-inoculated contact control chickens. 

These results are described in the report entitled "Shed/Spread of 

Bursal Disease-Marek's Disease Vaccine, Serotype 3, Live Marek's 

Disease Vector", which was appro~USDAAPHIS on July 11, 

2002. Reports in the literature for _ demonstrate that 

transmissibility of HVT is limited from chicken to chicken due to the 

limited virus present in the feather follicle epithelium (Cho, 1975; 

Zygraich and Huygelen, 1972). 

G. Environmental Impact or Survivability 

To evaluate the environmental stability, the survival of MSV 1IIIIIIII in 

sterile wood shavings and cotton swabs at 37 ± 3°C or 25 ± 3°C was 

compared to that of the HVT parent strain. No significant difference was 

seen between the recombinant and the parent strain, therefore MSV 

_ was considered to be safe for the environment. 

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Biomune Co. Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

March 6, 2009 

U.S. Vet. Lic. No. 368 

REFERENCES 

1. Cho, B. R. Horizontal transmission of turkey herpesvirus to chickens. IV. Viral 

maturation in the feather follicle epithelium. Avian Dis. 19:136-141. 1975. 

2. Cowen, B. S., and M. O. Braune. The propagation of avian viruses in a 

continuous cell line (QT35) of Japanese quail origin. Avian Dis. 32:282-297. 1988. 

3. Griffin, B. E. Sequence and analysis of polyoma virus DNA. In: DNA tumor 

viruses, J. Tooze, ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 

NY. pp. 843-913.1981. 

4. Hirai, K., M. Yamada, Y. Aral, S. Kato, and S. Nii. Replicating Marek's disease 

virus (MDV) serotype 2 DNA with inserted MDV serotype 1 DNA sequences in a 

Marek's disease Iymphoblastoid cell line MSB1-41C. Arch. Virol. 114:153-165. 

1990. 

5. Lee, L. F. Large-scale production of Marek's disease virus. Avian Dis. 15:565-

571.1971. 

6. Meulemans, G., P. Halen, and P. Schyns. Susceptibility of mammalian and avian 

cell cultures to infection with cell-free turkey herpes virus. J. Compo Pathol. 

83:605-608. 1973. 

7. Poulsen, D. J., C. R. A. Burton, J. J. O'Brian, S. J. Rabin, and C. L. Keeler, Jr. 

Identification of the infectious laryngotracheitis virus glycoprotein gB gene by the 

polymerase chain reaction. Virus Gene. 514:335-347. 1991. 

8. Samorek-Dziekanowska, E. Production of the high virus concentration of turkey 

herpesvirus (HVT strain FC-126) propagated in Japanese quail embryo fibroblast 

culture. Part I. Bulletin of Veterinary Inst. Pulawy. 21:10-16.1977. 

9. Sharma, J. M., R. L. Witter, G. Shramek, L. G. Wolfe, B. R. Burmester, and F. 

Deinhardt. Lack of pathogenicity of Marek's disease virus and herpesvirus of 

turkeys in marmoset monkeys. J. Natl. Canc. Inst. 49:1191-1197.1972. 

10. Tsukamoto, K., S. Saito, S. Saeki, T. Sato, N. Tanimura, T. Isobe, M. Mase, T. 

Imada, N. Yuasa, and S. Yamaguchi. Complete, long-lasting protection against 

lethal infectious bursal disease virus challenge by a single vaccination with an 

avian herpesvirus vector expressing VP2 antigens. J. Virol. 76:5637-5645. 2002. 

11. Witter, R. L., K. Nazerian, and J. J. Soloman. Studies on the in vivo replication of 

turkey herpesvirus. J. Natl. Canc.lnst. 49:1121-1129.1972. 

12. Witter, R. L., and J. M. Sharma. Transient infectivity and heterokaryon formation 

in hamster cell cultures inoculated with cell-associated stocks of Marek's disease 

virus and herpesvirus of turkeys. J. Natl. Canc. Inst. 53:1731-1742. 1974. 

13. Witter, R. L., and K. A. Schat. Marek's disease. In: Diseases of poultry, 11 th ed. 

Y. M. Saif, H. J. Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald, and D. E. 

Swayne, eds. Iowa State University Press, Ames, IA. pp. 407-465. 2003. 

14. Yamamoto, T., B. de Crombrugghe, and I. Pastan. Identification of a functional 

promoter in the long terminal repeat of Rous sarcoma virus. Cell 22:787-797. 

1980. 

15.Yanagida, N., R. Ogawa, Y. Li, L. F. Lee, and K. Nazerian. Recombinant fowl pox 

virus expressing the glycoprotein B homolog and the pp38 gene of Marek's 

disease virus. J. Virol. 66:1402-1408. 1992. 

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BiomuneCo. Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

March 6, 2009 

u.s. Vet. Lic. No. 368 

16.Yanagida, N., S. Yoshida, K. Nazerian, and L. Lee. Nucleotide and predicted 

amino acid sequences of Marek's disease virus homologues of herpes simplex 

virus major tegument proteins. J. Gen. Viral. 74:1837-1845.1993. 

17.Zygraich, N., and C. Huygelen. Inoculation of one-day old chicks with different 

strains of turkey herpesvirus. II. Virus replication in tissues of inoculated animals. 

Avian Dis. 16:793-798. 1972. 

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BiomuneCo. 

U.S. Vet. Lic. No. 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

March 6, 2009 
BiomuneCo. 

u.s. Vet. Lic. No. 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

March 6, 2009 
BiomuneCo. 

U.S. Vet. Lie. No. 368 

Fowl Laryngolracheilis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

March 6, 2009 

15 
BiomuneCo, 

U,S, Vet Lic, No, 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

March 6, 2009 
BiomuneCo. 

U.S. Vet. Lic. No. 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

March 6, 2009 
Biomune Co, 

U,S, Vet. Lic, No, 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

March 6, 2009 

18 
Biomune Co. 

U.S. Vet. Lic. No. 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

March 6, 2009 
Biomune Co. 

U.S. Vet. Lie. No. 368 

Fowl Laryngotracheitis-Marek's Disease Vaccine 

Serotype 3, Live Marek's Disease Vector 

SUMMARY INFORMATION FORMAT 

March 6, 2009 

20 
SUMMARY INFORMATION FORMAT APPENDIX 

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Biomune Company Fowl Laryngotracheitis- March 6, 2009 

Marek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

I. Background 

Laryngotracheitis (L T) is an acute respiratory disease of chickens characterized 

by signs of respiratory depression, gasping, swollen heads, and expectoration 

of bloody exudate (Guy and Bagust, 2003). L T is responsible for egg 

production losses and mortality. The etiologic agent of L T in chickens is 

laryngotracheitis virus (LTV) or Gallid herpesvirus 1. LTV is a member of the 

family Herpesviridae in the subfamily Alphaherpesvirinae. Like most 

herpesviruses, LTV may become latent in the trigeminal ganglia and be 

reactivated from latency. The latency of LTV may be a factor in the 

reappearance of this disease in intensive poultry production areas of the u.s. 

The glycoproteins of LTV are important immunogens that can elicit both 

humoral and cell-mediated immunity. Of the LTV glycoproteins, gene. has 

been used in recombinant fowlpox viruses and shown to provide protective 

immunity in chickens vaccinated with these viruses (Tong et aI., 2001 and Sun 

et a/., 200S). The LTV. gene is also used in a USDA-licensed vaccine, Fowl 

Pox-Laryngotracheitis Vaccine, Live Fowl Pox Vector, Product Code 1 F31.RO 

(Licensed). 

Turkey herpesvirus, or Meleagrid herpesvirus 1, is classified in the family 

Herpesviridae in the subfamily Alphaherpesvirinae. Non-oncogenic turkey 

herpesvirus (HVT) is considered part of the Marek's disease virus (MDV) group 

and designated as serotype 3 MDV. HVT has been used extensively as a safe 

and efficacious vaccine against Marek's disease (Witter and Schat, 2003). Due 

to the large DNA genome, herpesviruses have been evaluated for use as a viral 

vector carrying foreign gene(s). For poultry, HVT has been evaluated as a 

vector for various poultry viral diseases (Darteil et al., 1995; Morgan et a/., 

1993; Ross et al., 1993). Vaccines for which HVT is the vector have been 

licensed by the USDA and include (1) Bursal Disease"Marek's Disease Vaccine, 

Serotype 3, Live Marek's Disease Vector, Product Code 1ASS.RO (Licensed) 

and (2) Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's 

Disease Vector, Product Code 16N1.RO (Licensed). 

In an effort to produce an L T vaccine that protects chicks early in life, we have 

developed a recombinant vaccine in which the. gene from LTV was inserted 

into the genome of the HVT _ strain. The name of the vaccine is Fowl 

Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live Marek's Disease 

Vector, Product Code 16J1.R1 (Unlicensed), which is referred to here as MSV -. 

44 
Biomune Company Fowl Laryngotracheitis- March 6, 2009 

Marek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

II. Hazard Identification 

As stated in the "Risk Assessment Outline For Use in Preparing Risk Analyses 

For Biotechnology-Derived Products" that the Center of Veterinary Biologics 

published on October 1, 2005, hazard identification consists of identifying all 

possible adverse events to (1) animal safety, (2) public health safety, and (3) 

environmental safety. Biomune Co. has conducted a risk analysis to assess 

whether there are risks associated with the use of this vaccine in the United 

States. Following are the conclusions from the risk analysis for each of the areas 

of concern: 

A. Animal Safety 

The safety characteristics of Fowl Laryngotracheitis-Marek's Disease Vaccine, 

Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 

(Unlicensed) have been rigorously evaluated. Safety studies conducted to 

evaluate animal safety are described below. The risk to animal safety 

associated with _ Master Seed Virus (MSV) is low. 

1. Target animal safety 

a. Vaccination 

Fowl Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live 

Marek's Disease Vector, Product Code 16J1.R1 (Unlicensed) was 

demonstrated to be safe in the target animal, chickens, in a trial 

conducted in accordance with 9 CFR 113.330(b). Briefly, MSV 

_ MSV was inoculated at a 10X dose to embryos at 18 days 

of incubation by the in ovo route or to day of age chicks by the 

subcutaneous route. The chickens were observed for 123 days for 

clinical signs of Marek's disease (MD) or LT. As controls, a group of 

non-vaccinated chickens were observed as negative controls and 

another group of chickens was not vaccinated and was inoculated 

with the MDV very virulent challenge RB1 B. Of these nonvaccinated, RB1 B-challenged controls, chickens 

exhibited gross lesions of MD. After 123 days, neither the negative 

control group nor the MSV _ vaccinate groups had grossly 

observable lesions of MD. Also, the average body weights of the 

_ MSV groups and negative control groups were not 

stati~ifferent from one another. Therefore, it was concluded 

that _ MSV is safe for use in chickens and poses no safety 

risk. This study is also described in a report entitled "120-Day Safety 

of Fowl Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live 

Marek's Disease Vector, Uncoded (Unlicensed)", which was 

approved by APHIS on August 7,2008. 

45 
Biomune Company Fowl Laryngotracheitis- March 6, 2009 

Marek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

b. Vaccination/Challenge 

The safety of Fowl Laryngotracheitis-Marek's Disease Vaccine, 

Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 

(Unlicensed) was also evaluated during efficacy trials. In the first 

efficacy study against LTV challenge, either embryos at 18 ~ 

incubation or day of age chicks were vaccinated with MSV_. 

These chickens were held for. weeks to develop immunity before 

challenge. During this period, chickens were observed daily and no 

adverse vaccine reactions or clinical signs of MD or L T were 

observed. After this observation period, these chickens were 

challenged with LTV and this vaccine was efficacious. No adverse 

effects due to the vaccine were observed during the observation 

period after challenge. In the second efficacy study against MDV 

challenge, either embryos at~ of incubation or day of age 

chicks were vaccinated with _ MSV. At five days old, 

chickens were challenged with the MDV GA strain and observed for 

seven weeks. No adverse effects due to the vaccine were observed 

during the observation period after challenge. Therefore, this 

vaccine is efficacious and safe for use in chickens and poses no 

safety risk. These studies are described in a report entitled "Efficacy 

in SPF Chickens: Fowl Laryngotracheitis-Marek's Disease Vaccine, 

Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 

(Unlicensed) (Laryngotracheitis Virus Challenge)", which was 

submitted to APHIS on Decymber 12, 2008, and in a report entitled 

"Efficacy in SPF Chickens: Fowl Laryngotracheitis-Marek's Disease 

Vaccine, Serotype 3, Live Marek's Disease Vector, Product Code 

16J1.R1 (Unlicensed) (Marek's Disease Challenge - GA Strain)", 

which was submitted to APHIS on December 12, 2008. 

c. Reversion to virulence 

The inability of _ MSV to revert to virulence was established 

in a backpassage study. The _ MSV was passed five times in 

chickens. No adverse vaccine reactions or clinical signs of MD or L T 

were observed during each passage or for 45 days in the fifth 

backpassage group. This study is described in a report entitled 

"Backpassage: Genetic Stability and Safety of Fowl 

Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live 

Marek's Disease Vector, Uncoded (Unlicensed)", which was 

approved by APHIS on August 1-1, 2008. 

d. Purity tes~ 

Purity of _ MSV was satisfactory in accordance with 9 CFR 

113.27,113.28,113.30,113.31,113.34,113.37, and 113.47. The 

polymerase chain reaction (PCR) assays for chicken anemia virus 

and reticuloendotheliosis virus indicated that neither chicken anemia 

46 
Biomune Company Fowl Laryngotracheitis- March 6, 2009 

. Marek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

virus nor reticuloendotheliosis virus were present in _ MSV. 

Procedures and results of purity testing are described in Appendix 1 

of the Summary I nformation Format. 

e. Effect of gene manipulation on pathogenicity 

The flanking regions of the insertion site of MSV 

non-coding regions between 

the genome of the HVT 

to be non-essential for HVT replication since 

Similarly to the HVT parent strain in vitro and The parent 

strain and the insertion site have previously been used in two of our 

USDA-licensed HVT recombinant products, which are (1) Bursal 

Disease-Marek's Disease Vaccine, Serotype 3, Live Marek's Disease 

Vector, Product Code 1ABB.RO (Licensed) and (2) Marek's DiseaseNewcastle Disease Vaccine, Serotype 3, Live Marek's Disease 

Vector, Product Code 16N1.RO (Licensed). 

(P(llll!~An et 

The amino 

sequence does not have any significant sequence Similarity to 

any known protein. Expression of the LTV III protein was 

demonstrated by black plaque assay and western blot analYSis using 

_ antibodies. Procedures and results of these assays are 

described in Appendix 1 of the Summary Information Format. The III gene from the same LTV isolate is used in a USDA-licensed 

vaccine, Fowl Pox-Laryngotracheitis Vaccine, Live Fowl Pox Vector, 

Product Code 1F31.RO (Licensed). 

~ 

The __ gene is used in MSV'" as a _ Q!lne. 

the _ gene was confirmed by detecting .. 

__ activity in the plaque assay. Procedures and results of 

this assay are described in Appendix 1 of the Summary Information 

Format. The __ gene is used in a USDA-licensed vaccine, 

Fowl Pox-Laryngotracheitis Vaccine, Live Fowl Pox Vector, Product 

Code 1 F31.RO (Licensed). 

47 
Biomune Company 

f. 

Fowl LaryngotracheitisMarek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

March 6, 2009 

The polyadenylation 

signal the gene a USDA-licensed vaccine, 

Bursal Disease-Marek's Disease Vaccine, Serotype 3, Live Marek's 

Disease Vector, Product Code 1ABB.RO (Licensed). 

Genetic stability and phenotypic~ 

The _ MSV was passed _ in vitro resulting in MSVII, 

which represents the highest level to be used in production of this 

vaccine. To verify the in vitro stability of MSV _, several 

molecular tests were conducted. Southern blot analysis and 

PCR/sequencing of important regions of the genome were performed 

to determine the stability of the inserts. Western blot analysis 

and black plaque assay, 

conducted to confirm the expression of the gene in",~rt" 

_. Results of these molecular tests from were 

indistinguishable to those of MSV. Therefore, it was concluded that 

_ MSV was confirmed to be genetically and phenotypically 

stable in vitro. Procedures and results of these tests are described in 

Appendix 1 of the Summary Information Format. 

To confirm the in vivo genetic stability of _ MSV, virus isolated 

from the fifth backpassage was examined by the same molecular 

tests described above to verify stability of the gene insert (Southern 

blot analysis and PCR/sequencing of important regions~ene 

(West,ern blot analysis, black plaque assay _ 

Results of these molecular tests from 

virus isolated from the fifth backpassage were indistin~e from 

those of MSV. Therefore, it was concluded that MSV _ was 

confirmed to be genetically and phenotypically stable in vivo. This 

study is described in a report entitled "Backpassage: Genetic Stability 

and Safety of Fowl Laryngotracheitis-Marek's Disease Vaccine, 

Serotype 3, Live Marek's Disease Vector, Uncoded (Unlicensed)", 

which was approved by APHIS on August 11, 2008. 

g. Alteration of tissue tropism 

To address the possible concern that the L T gene insert into the HVT 

genome could cause variations to the HVT tropism, the tissue 

tropism of the _ MSV was evaluated. Briefly, chickens were 

48 
Biomune Company Fowl Laryngotracheitis- March 6, 2009 

Marek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

vaccinated with a 10X dose of _ MSV or an equivalent 

amount of HVT parent strain, and virus isolations were conducted on 

various tissues. Chickens vaccinated with _ MSV showed no 

adverse vaccine reactions, no clinical signs of MD or L T through. 

~ge, or gross lesions of MD or LT. At _ days of age, 

_ MSV was isolated from purified white blood cells from the 

blood, spleen, thymus and bursa. Similarly, the HVT parent strain 

was isolated from these same tissues at these time points. Based on 

these results, it was concluded that the tissue tropism 

MSV was similar to the HVT parent strain. Therefore, 

is safe for use in chickens and poses no safety risk. This study is 

described in a report entitled "Tissue Tropism of Fowl 

Laryngotracheitis-Marek'.s Disease Vaccine, Serotype 3, Live 

Marek's Disease Vector, Product Code 16J1.R1 (Unlicensed)", which 

was approved by APHIS on December 10, 2008. 

h. Effect of overdosing 

In several studies described in sections II. A. 1. a., II. A. 1. g. and II. 

C. 1., 10X dose of _ MSV did not cause any adverse vaccine 

reactions or clinical signs of MD and LT. 

2. Non-Target animal safety 

a. Safety in non-target avian species 

~et animal safety studies were conductlld with a 10X dose of 

_ MSV in turkeys, quail, pheasants and pigeons. The avian 

species used in this study were selected due to reports in the 

literature and possible contact of these species with commercial 

chicken farms. HVT was originally isolated from turkeys (Witter et aI., 

1970), and is ubiquitous in domestic turkeys. HVT is also known to 

replicate in turkeys, but is non-oncogenic (Witter et aI., 1972; Witter 

and Schat, 2003). Quail are susceptible to MDV and use of HVT 

vaccination in commercial quail flocks is common (Kobayashi et al., 

1986). Experimental infections of MDV serotype 1 have shown 

pheasants to be susceptible to infection (Witter and Schat, 2003). 

The host range of HVT has been defined by replication in primary 

chicken, duck, and quail cells, as well as quail cell lines (Cowen and 

Braune, 1988; Lee, 1971; Samorek-Dziekanowska, 1977). 

Safety was demonstrated in other avian species ~ quail, 

pheasants, and pigeons) by: (1) inoculation with _ MSV or the 

HVT parent strain, and (2) comparison of clinical signs, gross lesions, 

adverse vaccine reactions, and virus isolation between these two 

grou~ults demonstrated that other avian species inoculated 

with _ MSV showed no clinical signs, n~esions or 

adverse vaccine reactions. The host range of _ MSV was 

49 
Biomune Company Fowl Laryngotracheitis- March 6, 2009 

Marek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

similar to that of HVT parent. Therefore, _ MSV is safe in 

these avian species and use in chickens poses no safety risk to other 

avian species. This study is described in a report entitled "Safety in 

Avian Species: Fowl Laryngotracheitis-Marek's Disease Vaccine, 

Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 

(Unlicensed)", which was approved by APHIS on December 15, 2008. 

b. Safety in non-avi~s 

A safety study of _ MSV in non-avian species was conducted 

by evaluating its host range in non-avian species using mammalian 

cell lines and comparing its admissibility to that of the turkey 

herpesvirus parent strain. Unsuccessful attempts to replicate HVT in 

several mammalian species such as newborn hamsters, rhesus 

monkeys and marmoset monkeys have been conducted (Witter and 

Schat, 2003; Sharma et al., 1972). Also, investigators trying to 

replicate HVT in mammalian primary cultures and cell lines were 

unable to detect evidence of virus replication even after six to 10 

blind passages (Meulemans et aI., 1973; Witter and Sharma, 1974). 

Three mammalian cell lines were used in the present study and they 

are Mouse Porcine _ and _ Canine 

_. These mammalian cell lines were inoc~ 

MSV and passed. times. No cytopathic effects were observed in 

any cell line or any passage. Also, similar results were obtained 

when these species were inoculated with the parent HVT strain. 

Based on these results, it was concluded that the host range of 

_ MSV was similar to that of the HVT parent strain. 

Therefore, _ MSV is safe in these mammalian species and 

use in chickens poses no safety risk to mammalian species. This 

study is described in a report entitled "Safety in Non-Avian Species: 

Fowl Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live 

Marek's Disease Vector, Product Code 16J1.R1 (Unlicensed)", which 

was approved by APHIS on December 16, 2008. 

B. Public Health Safety 

The risk to public health is low for this vaccine. Human exposure will be 

limited to persons administering the vaccine or handling vaccinated chickens. 

Turkey herpesvirus is known to be insignificant with regard to public health 

(Witter and Schat, 2003) and no known infection in humans has been 

reported. We concluded that _ MSV is of no public health significance 

due to the narrow host range of HVT to avian species (Witter and Schat, 

2003) and to the non-target animal ~dies described here. Based on 

the confirmed narrow host range of _ MSV, there are no expected 

safety concerns associated with human exposure. 

50 
Biomune Company Fowl Laryngotracheitis- March 6, 2009 

Marek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

C. Environmental Safety 

The risk to the environment is low and there are no expected adverse 

ecological effects of _ MSV on the environment. Exposure to nontarget animals and dissemination of the vaccine into the environment will be 

restricted by its use in poultry houses. 

1. Shed/Spread capabilities 

Reports in the literature for _ demonstrate that transmisSibility of 

HVT is limited from chicken to chicken due to the limited virus present in 

the feather follicle epithelium ~75; Zygraich and Huygelen, 1972). 

Shed/spread capability of the _ MSV was evaluated. Briefly, 

chickens were vaccinated in ovo with a 10X dose of _ MSV or an 

equivalent amount of HVT parent strain. After hatch, for each vaccine, a 

group of non-vaccinated chickens was placed in the same isolation unit 

to observe the potential shed/spread of the vaccine. At 10, 14 and 21 

days of age, virus reisolation was attempted from the blood of all 

treatment groups. Virus was reisolated from the vaccinated groups, 

while no virus was reisolated from any contact control group. Based on 

these results, it was concluded that the shed/spread of MSV 

_ was similar to the HVT parent strain and MSV 

nor the HVT parent strain was transmissible. Therefore, is 

safe for use in chickens and poses no safety risk. This study is 

described in a report entitled '"Shed/Spread of Fowl LaryngotracheitisMarek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, 

Product Code 16J1.R1 (Unlicensed)", which was approved by APHIS on 

December 15, 2008. 

2. Horizontal gene transmission/recombination potential 

The chance of in vivo recombination between _ MSV and field 

strains of LTV and other viruses was considered. To date, under field 

conditions, no recombination events between MDV, including HVT, and 

LTV have been reported. HVT infects and replicates in lymphocyte cells, 

while LTV infects and replicates in respiratory organs such as the larynx 

and trachea, and goes latent in trigeminal ganglia. Therefore, an 

encounter between HVT and LTV in host animals is very rare, and the 

chance of in vivo recombination between _ MSV and field LTV 

strains is extremely limited. 

The chance of an in vivo recombination event between _ MSV 

and field MDV virus is unknown because the occurrence of horizontal 

gene transfer among the various MDV serotypes under field conditions is 

not known and is considered very rare (Witter and Schat, 2003). 

Common vaccination practices in the United States involve the mixture of 

HVT (serotype 3), SB-1 (serotype 2), and Rispens (serotype 1) vaccines. 

51 
Biomune Company Fowl LaryngotracheitisMarek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

March 6, 2009 

To date, no natural recombination events between any of these vaccines 

or with field MDV viruses (serotype 1) have been reported. One case of 

experimentally produced MDV serotype 2 recombination with MDV 

serotype 1 was reported but was not repeatable (Hirai etal., 1990). The 

chance of an in vivo recombination event is unknown, but a remote 

possibility. 

3. Host/Range specificity 

The host range of HVT is limited to certain avian species (Witter and 

Schat, 2003). HVT is not known to replicate in mammalian species. No 

changes in the host range of _ MSV compared to HVT were 

observed in the non-target animal safety studies described here. 

4. Survivability of the microorganism in the environment 

To evaluate the environmental stability, the survival of _ MSV in 

sterile wood shavings and cotton swabs at 37 ± 3°C or 25 ± 3°C was 

compared to that of the HVT parent strain. No significant difference was 

seen between the recombinant and the parent strain, therefore _ 

MSV was considered to be safe for the environment. 

5. Potential for transmission to invertebrates 

HVT is not known to be transmitted to invertebrates. Oncogenic MDV-1 

has been shown to be passively carried by darkling beetles (Alphitobius 

diaperinus), but free-living litter mites, mosquitoes, and coccidial oocysts 

could not be associated with transmission (Witter and Schat, 2003). 

6. Physical and/or chemical factors affecting dispersal in the environment 

No known physical and/or chemical factors are known to enhance the 

dispersal of_ MSV in the environment. 

7. Adverse ecological effects 

Because of limited shed/spread limited environmental 

survivability and narrow host ranges of MSV, there are no 

expected adverse ecological effects of MSV on the 

environment. Exposure to non-target animals and dissemination of the 

vaccine into the environment will be restricted by its use in poultry 

houses. 

52 
Biomune Company Fowl Laryngotracheitis- March 6, 2009 

Marek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

III. Risk Characterization 

A. 

B. 

Animal Safety 

1. Likelihood rating 

2. 

3. 

4. 

i. Low (L)= An adverse event is unlikely to occur. 

ii. Certain (C)= The rating is supported by direct 

scientific evidence. 

Consequence rating 

i. Low (CL)= The consequence, if the adverse event 

Occurs, is not severe. 

ii. Moderately certain (MC) = The rating is supported by 

direct and indirect scientific evidence. 

Expected risk 

i. [(Iikelihood)x(degree of certainty)]X[(consequence)x 

(degree of certainty)]=Risk rating 

ii. [(1.00)x(1.00)]X[(1.00)x(O.75)]=O.75 

Risk rating 

Public Health Safety 

1. Likelih90d rating 

i. Low (L)= An adverse event is unlikely to occur. 

ii. Certain (C)= The rating is supported by direct 

scientific evidence. 

2. Consequence rating 

i. Low (CL)= The consequence, if the adverse event 

Risk Rating 

LL=1.00 

C=1.00 

CL=1.00 

MC=O.75 

Low 

Risk Rating 

LL=1.00 

C=1.00 

Occurs, is not severe. CL=1.00 

ii. Moderately certain (MC) = The rating is supported by 

direct and indirect scientific evidence. MC=O.75 

3. Expected risk 

i. [(Iikelihood)x(degree of certainty)]X[(consequence)x 

(degree of certainty)]=Risk rating 

ii. [(1.00)x(1.00)]X[(1.00)x(O.75)]=O.75 

4. Risk rating Low 

53 
Biomune Company Fowl Laryngotracheitis- March 6, 2009 

C. 

Marek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

Environmental Safety 

1. Likelihood rating 

i. Low (L)= An adverse event is unlikely to occur. 

ii. Certain (C)= The rating is supported by direct 

scientific evidence. 

2. Consequence rating 

i. Low (CL)= The consequence, if the adverse event 

Risk Rating 

LL=1.00 

C=1.00 

Occurs, is not severe. CL=1.00 

ii. Moderately certain (MC) = The rating is supported by 

direct and indirect scientific evidence. MC=O.75 

3. Expected risk 

i. [(likelihood)x(degree of certainty)]X[(consequence)x 

(degree of certainty)]=Risk rating 

ii. [(1.00)x(1.00)]X[(1.00x(O.75)]=O.75 

4. Risk rating Low 

IV. Conclusions 

Based on this risk analysis, Biomune Co. concludes that release of the 

vaccine in field safety trials will not result in a significant impact on animal 

safety, public health or environmental safety. The risk rating for Fowl 

Laryngotracheitis-Marek's Disease Vaccine, Serotyp~ 3, Live Marek's 

Disease Vector, Product Code 16J1.R1 (Unlicensed) Master Seed is low. We 

request authorization to conduct field safety trials with Fowl LaryngotracheitisMarek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Product 

Code 16J1.R1 (Unlicensed) upon acceptance from APHIS. 

54 
Biomune Company Fowl Laryngotracheitis- March 6, 2009 

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Marek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

1. Cho, B. R. Horizontal transmission of turkey herpesvirus to chickens. IV. Viral 

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2. Cowen, B. S., and M. O. Braune. The propagation of avian viruses in a 

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Swayne, eds. Iowa State University Press, Ames, IA. pp. 121-134.2003. 

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6. Lee, L. F. Large-scale production of Marek's disease virus. Avian Dis. 15:565-

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7. Meulemans, G., P. Halen, and P. Schyns. Susceptibility of mammalian and avian 

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8. Morgan, R W., J. Gelb, Jr., C. R. Pope, and P. J. A. Sonderneijer. Efficacy in 

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9. Poulsen, D. J., C. R A. Burton, J. J. O'Brian, S. J. Rabin, and C. L. Keeler, Jr. 

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10. Ross, L. J. N., M. M. Binns, P. Tyers, J. Pastorek, V. Zelnik, and S. Scott. 

Construction and properties of a turkey herpesvirus recombinant expressing the 

Marek's disease virus homologue of glycoprotein B of herpes simplex virus. J. 

Gen. Virol. 74:371-377. 1993. 

11. Samorek-Dziekanowska, E. Production of the high virus concentration of turkey 

herpesvirus (HVT strain FC-126) propagated in Japanese quail embryo fibroblast 

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12.Sharma, J. M., R L. Witter, G. Shramek, L. G. Wolfe, B. R. Burmester, and F. 

Deinhardt. Lack of pathogenicity of Marek's disease virus and herpesvirus of 

turkeys in marmoset monkeys. J. Natl. Cane. Inst. 49:1191-1197. 1972. 

13.Sun, H. L., Y. F. Wang, G. Z. Tong, P. J. Zhang, D. Y. Miao, H. D. Zhi, M. Wang, 

and M. Wang. Protection of chickens from Newcastle disease and infectious 

laryngotracheitis with a recombinant fowlpox virus co-expressing the F, HN 

genes of Newcastle disease virus and gB gene of infectious laryngotracheitis 

virus. Avian Dis. 52:111-117. 2008. 

55 
Biomune Company Fowl Laryngotracheitis- March 6, 2009 

Marek's Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, Product Code 16J1.R1 

RISK ASSESSMENT 

14.Tong, G. Z., S. J. Zhang, S. S. Meng, L. Wang, H. J. Qiu, Y. F. Wang, L. Yu, and 

M. Wang. Protection of chickens from infectious laryngotracheitis with a 

recombinant fowlpox virus expressing glycoprotein B of infectious 

laryngotracheitis virus. Avian Pathol. 30:143-148. 2001. 

15.Tsukamoto, K., S. Saito, S. Saeki, T. Sato, N. Tanimura, T. Isobe, M. Mase, T. 

Imada, N. Yuasa, and S. Yamaguchi. Complete, long-lasting protection against 

lethal infectious bursal disease virus challenge by a single vaccination with an 

avian herpesvirus vector expressing VP2 antigens. J. Virol. 76:5637-5645. 2002. 

16. Witter, R. L., K. Nazerian, H. G. Purchase, and G. H. Burgoyne. Isolation from 

turkeys of cell associated herpesvirus antigenically related to Marek's disease 

virus. Am. J. Vet. Res. 31:525-538.1970. 

17. Witter, R. L., K. Nazerian, and J. J. Soloman. Studies on the in vivo replication of 

turkey herpesvirus. J. Natl. Canc. Inst. 49:1121-1129.1972. 

18.Witter, R. L., and J. M. Sharma. Transient infectivity and heterokaryon formation 

in hamster cell cultures inoculated with cell-associated stocks of Marek's disease 

virus and herpesvirus of turkeys. J. Natl. Canc. Inst. 53:1731-1742. 1974. 

19. Witter, R. L., and K. A. Schat. Marek's disease. In: Diseases of poultry, 11th ed. 

Y. M. Saif, H. J. Bames, J. R. Glisson, A. M. Fadly, L. R. McDougald, and D. E. 

Swayne, eds. Iowa State University Press, Ames, IA. pp. 407-465. 2003. 

20. Yamamoto, T., B. de Crombrugghe, and I. Pastan. Identification of a functional 

promoter in the long terminal repeat of Rous sarcoma virus. Cell 22:787-797. 

1980. 

21.Zygraich, N., and C. Huygelen. Inoculation of one-day old chicks with different 

strains of turkey herpesvirus. II. Virus replication in tissues of inoculated animals. 

Avian Dis. 16:793-798. 1972. 

56