# RISKANALYSIS

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

TABLE OF CONTENTS' Fowl Laryngotracheitis-Marek's Disease Vaccine Serotype 3, Live Marek's DiseaseVector


### RISK ANALYSIS

SUMMARY OF INFORMATION FORMAT SUMMARY OF INFORMATION FORMAT APPENDIX RISKASSESSMENT PAGE 1

- 20 21 -43 44-56

1 The blackened areas inthis document contain Confidential Business Information (CBI) ofa proprietary nature, the disclosure ofwhich underthe Freedom of Information Act (FOIA) is likelyto cause ourfirm 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 containmentfacilities availablefortesting of live vectors. 2. The Regulated Biological Agent (RBA), contains aturkey h<>.,..t,.f",",Q 

### and expresses a

B. Proposal 1. Species: Chickens, embryos at 18 days ofincubation 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 ofage chicks as an aid in the prevention of laryngotracheitis (LT) and Marek's disease (MD), which is caused by virulent Marek's disease virus (MDV). 3. Geographic area: United States (all states) 4. Route ofadministration: In ovo

### to embryos at 18 days of incubation and subcutaneous to day ofage chicks. 

### 5. Attenuation ofthe HVT strain has not been observed. Administration ofthe MSV

_ is expected to cause atransientviremia 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 atthe site ofinoculation when administered subcutaneouslyto day ofage chicks.



### 1Biomune Co. Fowl Laryngotracheitis-Marek's DiseaseVaccine Serotype 3, Live Marek's DiseaseVector

SUMMARY INFORMATION FORMAT March 6, 2009 

### u.s. Vet. Lic. No. 368 

### II. Description ofthe Regulated BiologicalAgentConstruction
A. The Backbone Biological Agent 1. Turkey herpesvirus is a double-stranded linear DNAvirus in the Herpesviridae

### family and Alphaherpesvirinae

### subfamily. HVT is ubiquitous and non-oncogenic in domesticturkeys and it is classified as serotype 3 of MDV. The HVT parent strain, _, has been used commerciallyto vaccinate chickens against Marek's disease since 1972 (Witter and Schat, 2003). HVT is classified in the Biosafety Level 1category. 2. Physical Characteristics ofthe BackboneAgent

B. Donor Biological Agents and Donor DNA or Genes



### 2BiomuneCo. U.S. Vet. Lie. No. 368 Fowl Laryngotracheitis-Marek's DiseaseVaccine 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 etal., 

### 1991). The.gene from this isolate is used in a USDA-licensed vaccine, Fowl Pox-Laryngotracheitis Vaccine, Live Fowl PoxVector, Product Code 1F31.RO (Licensed)

.



### 3Biomune Co. March 6, 2009 U.S. Vet. Lie. No. 368 Fowl Laryngotracheitis-Marek's Disease Vaccine Serotype 3, Live Marek's DiseaseVector

SUMMARY INFORMATION FORMAT C. Construction and Characterization ofthe Regulated Biological Agent
1. Diagrams forthe construction ofthe Regulated Biological Agent are provided as described below.



### 2. e. Aftertransfection, virus successfully grown

_ was separated by limiting dilutions. Wells containing plaques were expanded in duplicate and screened for expression ofthe

_ gene. The recombinantvirus 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 ntheAppendix.
3. Physical Characterization ofthe Regulated Biological Agent

a. Southern blot analysis and PCR/sequencing of important regions were conducted to characterize the physical map ofthe RBA.

_ Southern blotting schemes are provided in Figure 8, as well as • PCR schemes to amplify. regions. The sequence ofeach ofthese PCR products was analyzed. Procedures and results ofthese experiments are described in the Appendix. Sequences of primers used in the. PCR/sequencing schemes and sequencing are provided in Table 1, Appendix.

4Biomune Co. March 6, 2009 

### u.s. Vet. Lic. No. 368 Fowl Laryngotracheitis-Marek's DiseaseVaccine Serotype 3, Live Marek's DiseaseVector

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 ofthese experiments are described in theAppendix. d. An electronic file providing the genetic sequenceforthe 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 biologicallythe same as the parent virus, the recommended NIH/CDC biosafety level will be the same as the parentvirus (Biosafety Level 1).

.



### 5Biomune 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 orVirulence forthe Regulated BiologicalAgent used for MasterSeed 

### A. Phenotypic Characteristics
1. The cloning site is thought to be nonessential for HVT repl replicates similarto the HVT parentstrain 

### in vitro and 

### in vivo. This insertion site has been used previously in two ofour USDA-licensed HVT recombinant products, which are (1) Bursal Disease-Marek's DiseaseVaccine, Serotype 3, Live Marek's Disease Vector, Product Code 1ABB.RO (Licensed) and (2) Marek's Disease-Newcastle DiseaseVaccine, Serotype 3, Live Marek's DiseaseVector, Product Code 16N1.RO (Licensed). 2. The RBA expresses the.gene of LTV and the

__gene. Expression ofthe.protein was demonstrated by black plaque assay and western blot using

_ antibodies. Expression ofthe from the detected Procedures and results ofthese**rl"",r.ri~)"rl** 

### in the Appendix. B. Virulence Characteristics
1. There are no known virulence characteristics ofthe.gene orthe

_ gene, so addition ofthe genes to HVT is not expected to be virulentfor chickens. 2. The RBA does not contain any genetic elements ortoxin genes that are known to be inherentlyvirulent. C. Virulence in Target and Non-TargetAnimals 1. In orderto address virulence in target animals,

_ MSVwas 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 byAPHIS on August7, 200B. Safety of

_ MSV in chickenswas also tested in a backpassage study.

_ MSVwas passed five times in chickens and tested forvirulence and genetic stability. No adverse vaccine reactions, orclinical signs of MD or L T, were observed during each passage orfor45 days in the fifth backpassage group.



### 6Biomune Co. Fowl Laryngotracheitis-Marek's Disease Vaccine March 6, 2009 U.S. Vet. Lie. No. 368 Serotype 3, Live Marek's DiseaseVector SUMMARY INFORMATION FORMAT 

### The in vivo 

### genetic stability of MSV

_ was confirmed using moleculartests 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 DiseaseVector, Uncoded (Unlicensed)", which was approved by APHIS on August 11, 2008. 2. Safetywas demonstrated in otheravian species (turkeys, quail, pheasants, and pigeons) by: (1) inoculation with 

### MSV_or the HVT parent strain, and (2) comparison ofclinical signs, gross lesions, adverse vaccine reactions, and virus isolation between thesetwo groups. Results demonstrated that other avian species inoculated with MSV

_ showed no clinical signs, no gross lesions or adverse vaccine reactions. The host range ofMSV was similarto that of HVT parent. Therefore, MSV is safe in these avian species and use in chickens poses no safety riskto otheravian 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. Safetywas 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 thatthe host range of MSV similarto that ofthe HVT parentstrain. 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 DiseaseVector, Product Code 16J1.R1 (Unlicensed)", which was approved by APHIS on December 16, 2008. D. TissueTropism 1. The tissue tropism ofMSV

_ was determined and compared to the HVT parent strain.

a.**~hickens** 

### were vaccinated with a 10Xdose of MSV

_ or an equivalentamount of HVT parent strain, and



### 7Biomune Co. March 6, 2009 U.S. Vet. Lic. No. 368 Fowl Laryngotracheitis-Marek's Disease Vaccine Serotype 3, Live Marek's DiseaseVector

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 ofage,**~**lesions ofMD or LT. At 10 and 21 days ofage, 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 atthesetime points. Based on these results, itwas concluded thatthe tissue tropism of MSV

_ was similarto that ofthe 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. Safetywas demonstrated in other avian species (turkeys, quail, pheasants and pigeons) by: (1) inoculation with 

### MSV_ orthe HVT parent strain, and (2) comparison ofclinical 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 similarto that ofthe 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 DiseaseVector, Product Code 16J1.R1 (Unlicensed)", which was approved by APHIS on December 15, 2008. 2. Turkey herpesvirus is ubiquitous in domesticturkeys and is also ubiquitous in commercial chickens dueto vaccine use since the 1970s. The literature indicates that HVT replicates in turkeys, but is non-oncogenic (Witter eta/., 1972; Witter and Schat, 2003). The host range of HVT has been defined byexperimental infections of various birds, but has been betterdefined in tissue culture. HVT is known to replicate in primary chicken, duckand 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 eta/., 1972). Also, investigatorstrying to replicate HVT in mammalian primary cultures and cell lines were unable to detect evidence ofvirus replication even after performing



### 8Biomune Co. March 6, 2009 U.S. Vet. Lic. No. 368 Fowl Laryngotracheitis-Marek's DiseaseVaccine Serotype 3, Live Marek's DiseaseVector

SUMMARY INFORMATION FORMAT six to 10 blind passages (Meulemans 

### eta/., 1973; Witter and Sharma, 1974). E. Potential for Horizontal GeneTransfer
1. The chance of

### in vivo recombination between MSV

_ and field strains of LTV and otherviruses was considered. To date, no recombination events between MDV, including HVT, and LTV have been reported underfield 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 ofan 

### in vivo recombination event between MSV

_ and field MDVvirus is unknown because the occurrence of horizontal genetransfer among the various MDVserotypes underfield conditions is not known and is considered very rare (Witterand Schat, 2003). Common MDVvaccination 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 ofthese vaccines or with field MDVviruses (serotype 1) have been reported. One case ofexperimentally produced MDVserotype 2 recombination with MDVserotype 1was reported, butwas not repeatable (Hirai 

### eta/., 1990). The chance ofan 

### in vivo recombination event is unknown, but a remote possibility. F. Shed/Spread
1. Shed/spread capability ofthe MSV

_ was evaluated. Briefly, chicken embryoswere vaccinated 

### in ova with a 10Xdoseof MSV

_ or an equivalent amount of HVT parent strain. After hatch, for each vaccine, a group ofnon-vaccinated chickens was placed in the same isolation unitto observe the potential shed/spread ofthe vaccine. At 10, 14 and 21 days ofage, virus reisolation was attempted from the blood ofall treatment groups. Virus was reisolated from the vaccinated groups, while no virus was reisolated from any contact control group. Based on these itwas concluded thatthe shed/spread capability of MSV similarto 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 DiseaseVaccine, Serotype 3, Live

9

### ! Biomune Co. Fowl Laryngotracheitis-Marek's DiseaseVaccine 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 byAPHIS on December 15, 2008. 3. Thetransmissibility ofthe recombinant is thoughtto be similartothe

_**HVT~t**strain. Previously reported transmissibility results ofthe HVT

_ strain (Marek's Disease 

### Vaccine, Serotype 3, Live Virus, Product Code 16L1.06 (Unlicensed» demonstrated thatthe

.. • strain did not spread to non-inoculated contact control chickens. These results aredescribed in the report entitled "Shed/Spread of Bursal Disease-Marek's DiseaseVaccine, Serotype 3, Live Marek's Disease 

### Vector", which was**appro~USDAAPHIS** on July 11, 2002. Reports in the literaturefor

_ demonstratethat transmissibility of HVT is limited from chicken to chicken duetothe limited virus present in thefeatherfollicle epithelium (Cho, 1975; Zygraich and Huygelen, 1972). G. Environmental Impactor Survivability To evaluatethe environmental stability, thesurvival of MSV

# 1IIIIIIII in sterile wood shavings and cotton swabs at 37 

### ±3°C or25 

### ±3°Cwas compared tothat ofthe HVT parentstrain. No significant differencewas seen between the recombinant and the parent strain, therefore 

### MSV

_ was considered to be safe forthe environment.

10Biomune Co. Fowl Laryngotracheitis-Marek's DiseaseVaccine Serotype 3, Live Marek's DiseaseVector

SUMMARY INFORMATION FORMAT March 6, 2009 U.S. Vet. Lic. No. 368 REFERENCES
1. Cho, B. 

### R. Horizontal transmission ofturkey herpesvirus to chickens. IV. Viral maturation in thefeatherfollicle epithelium. Avian Dis. 19:136-141. 1975. 2. Cowen, B. S., and M. O. Braune. The propagation ofavian viruses in a continuous cell line (QT35) ofJapanese quail origin. Avian Dis. 32:282-297. 1988. 3. Griffin, B. E. Sequence and analysis ofpolyoma virus DNA. In: DNAtumor 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 DNAwith inserted MDVserotype 1 DNAsequences 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 ofmammalian 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 ofthe infectious laryngotracheitis virus glycoprotein gB gene by the polymerase chain reaction. Virus Gene. 514:335-347. 1991. 8. Samorek-Dziekanowska, E. Production ofthe high virus concentration ofturkey herpesvirus (HVT strain FC-126) propagated in Japanesequail embryofibroblast culture. Part 

### I. Bulletin ofVeterinary 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 diseasevirus 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 diseasevirus challenge by a single vaccination with an avian herpesvirus vectorexpressing 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 ofturkeys. 

### J. Natl. Canc. Inst. 53:1731-1742. 1974. 13.Witter, 

### R. L., and K. A. Schat. Marek's disease. In: Diseases ofpoultry, 11th 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 ofafunctional promoter in the long terminal repeat of Rous sarcomavirus. Cell 22:787-797. 1980. 15.Yanagida, N., 

### R. Ogawa, Y. Li, L. F. Lee, and 

### K. Nazerian. Recombinantfowl pox virus expressing the glycoprotein B homolog and the pp38 gene of Marek's diseasevirus. 

### J. Virol. 66:1402-1408. 1992.

11BiomuneCo. 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 ofMarek's diseasevirus homologues ofherpes simplex virus majortegument proteins. 

### J. Gen. Viral. 74:1837-1845.1993. 17.Zygraich, N., and C. Huygelen. Inoculation ofone-day old chicks with different strains ofturkey herpesvirus. II. Virus replication in tissues of inoculated animals. Avian Dis. 16:793-798. 1972.



### 12BiomuneCo. U.S. Vet. Lic. No. 368 Fowl Laryngotracheitis-Marek's DiseaseVaccine Serotype 3, Live Marek's Disease Vector SUMMARY INFORMATION FORMAT March 6, 2009BiomuneCo. 

### u.s. Vet. Lic. No. 368 Fowl Laryngotracheitis-Marek's DiseaseVaccine Serotype 3, Live Marek's Disease Vector SUMMARY INFORMATION FORMAT March 6, 2009BiomuneCo. U.S. Vet. Lie. No. 368 Fowl Laryngolracheilis-Marek's DiseaseVaccine Serotype 3, Live Marek's DiseaseVector SUMMARY INFORMATION FORMAT March 6, 2009

15BiomuneCo, U,S, Vet Lic, No, 368 Fowl Laryngotracheitis-Marek's Disease Vaccine Serotype 3, Live Marek's Disease Vector SUMMARY INFORMATION FORMAT March 6, 2009BiomuneCo. U.S. Vet. Lic. No. 368 Fowl Laryngotracheitis-Marek's Disease Vaccine Serotype 3, Live Marek's Disease Vector SUMMARY INFORMATION FORMAT March 6, 2009Biomune Co, U,S, Vet. Lic, No, 368 Fowl Laryngotracheitis-Marek's Disease Vaccine Serotype 3, Live Marek's DiseaseVector SUMMARY INFORMATION FORMAT March 6, 2009

18Biomune Co. U.S. Vet. Lic. No. 368 Fowl Laryngotracheitis-Marek's Disease Vaccine Serotype 3, Live Marek's DiseaseVector SUMMARY INFORMATION FORMAT March 6, 2009Biomune Co. U.S. Vet. Lie. No. 368 Fowl Laryngotracheitis-Marek's Disease Vaccine Serotype 3, Live Marek's DiseaseVector SUMMARY INFORMATION FORMAT March 6, 2009



### 20SUMMARY INFORMATION FORMAT APPENDIX



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43Biomune Company Fowl Laryngotracheitis- March 6, 2009 Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 RISKASSESSMENT I. Background Laryngotracheitis (LT) is an acute respiratory disease ofchickens characterized by signs of respiratory depression, gasping, swollen heads, and expectoration of bloody exudate (Guy and Bagust, 2003). L T is responsiblefor egg production losses and mortality. The etiologic agent of L T in chickens is laryngotracheitisvirus (LTV) or Gallid herpesvirus 1. LTV is a memberofthe family Herpesviridae in the subfamilyAlphaherpesvirinae. Like most herpesviruses, LTV may become latent in the trigeminal ganglia and be reactivated from latency. The latency of LTV may be afactor in the reappearance ofthis disease in intensive poultry production areas ofthe 

## u.s. The glycoproteins of LTV are important immunogens that can elicit both humoral and cell-mediated immunity. Ofthe LTVglycoproteins, gene. has been used in recombinantfowlpox viruses and shown to provide protective immunity in chickens vaccinated with these viruses (Tong etaI., 2001 and Sun et

### a/., 200S). The LTV.gene is also used in a USDA-licensed vaccine, Fowl Pox-LaryngotracheitisVaccine, Live Fowl PoxVector, Product Code 1F31.RO (Licensed). Turkey herpesvirus, or Meleagrid herpesvirus 1, is classified in the family Herpesviridae in the subfamilyAlphaherpesvirinae. Non-oncogenicturkey herpesvirus (HVT) is considered part ofthe Marek's disease virus (MDV) group and designated as serotype 3 MDV. HVT has been used extensively as a safe and efficaciousvaccine 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 vectorforvarious poultry viral diseases (Darteil etal., 1995; Morgan eta/., 1993; Ross etal., 1993). Vaccines forwhich 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 DiseaseVaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16N1.RO (Licensed).
In an effortto produce an L T vaccine that protects chicks early in life, we have developed a recombinant vaccine in which the.genefrom LTVwas inserted intothe genome ofthe HVT

_ strain. The name ofthe vaccine is Fowl Laryngotracheitis-Marek's DiseaseVaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 (Unlicensed), which is referred to here as MSV 

# -.

44Biomune Company Fowl Laryngotracheitis- March 6, 2009 Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 RISKASSESSMENT 

### II. Hazard Identification As stated in the "RiskAssessment Outline For Use in Preparing Risk Analyses For Biotechnology-Derived Products" thatthe Center ofVeterinary 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 whetherthere are risks associated with the use ofthis vaccine in the United States. Following are the conclusions from the risk analysis foreach ofthe areas ofconcern: 

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

_ MSVwas inoculated at a 10Xdose to embryos at 18 days of incubation bythe in ovo route orto day of age chicks bythe subcutaneous route. The chickens were observed for 123 daysfor clinical signs of Marek's disease (MD) or LT. As controls, a group of non-vaccinated chickens were observed as negative controls and anothergroup of chickens was notvaccinated and was inoculated with the MDVvery virulent challenge RB1 B. 

### Ofthese nonvaccinated, RB1 B-challenged controls, chickens exhibited gross lesions ofMD. After 123 days, neitherthe negative control group northe MSV

_ vaccinate groups had grossly observable lesions of MD. Also, the average bodyweights ofthe

_ MSV groups and negative control groupswere not**stati~ifferent** from one another. Therefore, it was concluded that

_ MSVis 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 DiseaseVector, Uncoded (Unlicensed)", which was approved byAPHIS on August 7,2008.

45Biomune Company Fowl Laryngotracheitis- March 6, 2009 Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 RISKASSESSMENT b. Vaccination/Challenge The safety of Fowl Laryngotracheitis-Marek's DiseaseVaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 (Unlicensed) was also evaluated during efficacy trials. In the first efficacy study against LTVchallenge, eitherembryos at 18**~** incubation or day ofage chicks were vaccinated with 

### MSV_. These chickens were held for.weeks to develop immunity before challenge. During this period, chickenswere observed daily and no adverse vaccine reactions orclinical signs of MD or L T were observed. Afterthis observation period, these chickens were challenged with LTV and this vaccine was efficacious. No adverse effects dueto the vaccine were observed during the observation period after challenge. In the second efficacy study against MDV challenge, either embryos**at~** of incubation orday of age chicks were vaccinated with

_ MSV. Atfive days old, chickens were challenged with the MDVGA strain and observed for seven weeks. No adverse effects dueto 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 DiseaseVaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 (Unlicensed) (Laryngotracheitis Virus Challenge)", which was submitted toAPHIS 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

_ MSVto revertto virulence was established in a backpassage study. The

_ MSVwas passed five times in chickens. No adversevaccine reactions or clinical signs of MD or L T were observed during each passage orfor

### 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 byAPHIS on August 

### 1-1, 2008. d. Purity**tes~** Purity of

_ MSVwas 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) assaysfor chicken anemiavirus and reticuloendotheliosis virus indicated that neither chicken anemia
46Biomune Company Fowl Laryngotracheitis- March 6, 2009

. Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 RISKASSESSMENT virus nor reticuloendotheliosis viruswere present in

_ MSV. Procedures and results ofpuritytesting are described in Appendix 1 ofthe Summary Information Format.

e. Effect of gene manipulation on pathogenicity The flanking regions ofthe insertion site of MSV non-coding regions between the genome ofthe HVT to be non-essential for HVT replication since Similarlytothe HVT parent strain 

### in vitro and The parent strain and the insertion site have previously been used intwo ofour USDA-licensed HVT recombinant products, which are (1) Bursal Disease-Marek's DiseaseVaccine, Serotype 3, Live Marek's Disease Vector, Product Code 1ABB.RO (Licensed) and (2) Marek's DiseaseNewcastle DiseaseVaccine, Serotype 3, Live Marek's Disease Vector, ProductCode 16N1.RO (Licensed).**(P(llll!~An** et

The amino sequence does not have anysignificant sequenceSimilarityto any known protein. Expression ofthe LTV

# IIIprotein was demonstrated by black plaque assayand western blot analYSis using

_ antibodies. Procedures and results ofthese assays are described in Appendix 1ofthe Summary Information Format. The 

# III gene from the same LTVisolate is used in a USDA-licensed vaccine, Fowl Pox-LaryngotracheitisVaccine, Live Fowl PoxVector, Product Code 1F31.RO (Licensed). The

__gene is used in MSV'"as a _ Q!lne.

### ~the

_ genewas confirmed bydetecting

..

__activity in the plaque assay. Procedures and results of this assay are described in Appendix 1 ofthe Summary Information Format. The

__gene is used in a USDA-licensed vaccine, Fowl Pox-LaryngotracheitisVaccine, Live Fowl PoxVector, Product Code 1F31.RO (Licensed).



### 47Biomune Company

f. Fowl LaryngotracheitisMarek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 RISKASSESSMENT March 6, 2009

The polyadenylation signal the gene a USDA-licensed vaccine, Bursal Disease-Marek's Disease Vaccine, Serotype 3, Live Marek's DiseaseVector, Product Code 1ABB.RO (Licensed). Genetic stability and**phenotypic~** The

_ MSVwas passed

_ 

### in vitro resulting in MSVII, which represents the highest level to be used in production ofthis vaccine. To verifythe

### in vitro stability of MSV_,several moleculartests were conducted. Southern blot analysis and PCR/sequencing ofimportant regions ofthe genome were performed to determinethe stability ofthe inserts. Western blot analysis and black plaque assay, conducted to confirm the expression ofthe gene**in",~rt"**
_. Results ofthese moleculartestsfrom were indistinguishable to those of MSV. Therefore, it was concluded that

_ MSVwas confirmed to be genetically and phenotypically stable 

### in vitro. Procedures and results ofthese tests are described in Appendix 1ofthe Summary Information Format. To confirm the

### in vivo genetic stability of

_ MSV, virus isolated from the fifth backpassage was examined bythe same molecular tests described above to verify stability ofthe gene insert (Southern blot analysis and PCR/sequencing ofimportant**regions~ene** (West,ern blot analysis, black plaque assay

_ Results ofthese moleculartests from virus isolated from thefifth 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 byAPHIS on August 11, 2008.
g. Alteration oftissue tropism To address the possible concern thatthe L T gene insert into the HVT genome could cause variations to the HVTtropism, the tissue tropism ofthe

_ MSVwas evaluated. Briefly, chickens were


### 48Biomune Company Fowl Laryngotracheitis- March 6, 2009 Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 RISKASSESSMENT vaccinated with a 10Xdose of

_ MSVor an equivalent amount of HVT parent strain, and virus isolationswere conducted on various tissues. Chickens vaccinated with

_ MSV showed no adverse vaccine reactions, no clinical signs of MD or L T through.**~ge,** orgross lesions of MD or LT. At

_ days ofage,

_ MSVwas 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 thatthe tissue tropism MSVwas similarto the HVT parent strain. Therefore, is safefor 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 byAPHIS on December 10, 2008. h. Effect ofoverdosing In several studies described in sections II. 

### A. 1. a., II. 

### A. 1. g. and II. C. 1., 10Xdose of

_ MSVdid 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 10Xdose of

_ MSV in turkeys, quail, pheasants and pigeons. The avian species used in this studywere selected dueto reports in the literature and possible contact ofthese species with commercial chicken farms. HVTwas originally isolated from turkeys (Witter etaI., 1970), and is ubiquitous in domesticturkeys. HVT is also known to replicate in turkeys, but is non-oncogenic (Witter etaI., 1972; Witter and Schat, 2003). Quail are susceptible to MDVand use of HVT vaccination in commercial quail flocks is common (Kobayashi etal., 1986). Experimental infections of MDVserotype 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). Safetywas demonstrated in other avian species**~** quail, pheasants, and pigeons) by: (1) inoculation with

_ MSVorthe HVT parent strain, and (2) comparison ofclinical signs, gross lesions, adverse vaccine reactions, and virus isolation between these two**grou~ults** demonstrated that other avian species inoculated with

_ MSVshowed no clinical signs,**n~esions** or adverse vaccine reactions. The host range of

_ MSVwas


### 49Biomune Company Fowl Laryngotracheitis- March 6, 2009 Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 RISKASSESSMENT similarto 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 byAPHIS 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 ofthe turkey herpesvirus parentstrain. 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 etal., 1972). Also, investigatorstrying to replicate HVT in mammalian primary cultures and cell lines were unable to detect evidence ofvirus replication even after sixto 10 blind passages (Meulemans etaI., 1973; Witter and Sharma, 1974). Three mammalian cell lines were used in the presentstudy and they are Mouse Porcine

_ and

_ Canine

_. These mammalian cell lines were**inoc~** MSVand 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, itwas concluded thatthe host range of

_ MSVwas similarto that ofthe 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 byAPHIS on December 16, 2008. B. Public Health Safety The risk to public health is lowforthis vaccine. Human exposure will be limited to persons administering the vaccine or handling vaccinated chickens. Turkey herpesvirus is known to be insignificantwith 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 duetothe narrow host range of HVTto 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.



### 50Biomune Company Fowl Laryngotracheitis- March 6, 2009 Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 RISKASSESSMENT C. Environmental Safety The risk to the environment is low and there are no expected adverse ecological effects of

_ MSVon the environment. Exposure to nontarget animals and dissemination ofthe vaccine into the environmentwill be restricted by its use in poultry houses.
1. Shed/Spread capabilities Reports in the literature for

_ demonstratethattransmisSibility of HVT is limited from chicken to chicken due tothe limited virus present in the featherfollicle epithelium**~75;** Zygraich and Huygelen, 1972). Shed/spread capability ofthe

_ MSVwas evaluated. Briefly, chickens were vaccinated 

### in ovo with a 10Xdose of

_ MSVor an equivalent amount ofHVT 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 ofthe vaccine. At 10, 14 and 21 days ofage, virus reisolation was attempted from the blood ofall treatment groups. Virus was reisolated from the vaccinated groups, while no virus was reisolated from any contact control group. Based on these results, itwas concluded thatthe shed/spread of MSV

_ was similarto the HVT parent strain and MSV northe HVT parent strain was transmissible. Therefore, is safefor use in chickens and poses no safety risk. This study is described in a report entitled '"Shed/Spread of Fowl LaryngotracheitisMarek's DiseaseVaccine, Serotype 3, Live Marek's DiseaseVector, Product Code 16J1.R1 (Unlicensed)", which was approved byAPHIS on December 15, 2008.
2. Horizontal gene transmission/recombination potential The chance of

### in vivo recombination between

_ MSVand field strains of LTV and otherviruses was considered. To date, underfield 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

_ MSVand field LTV strains is extremely limited. The chance ofan 

### in vivo recombination event between

_ MSV and field MDVvirus is unknown because the occurrence of horizontal genetransfer among the various MDVserotypes underfield 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.
51Biomune Company Fowl LaryngotracheitisMarek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 RISKASSESSMENT March 6, 2009

To date, no natural recombination events between any ofthese vaccines orwith field MDVviruses (serotype 1) have been reported. One case of experimentally produced MDVserotype 2 recombination with MDV serotype 1was reported butwas not repeatable (Hirai etal., 1990). The chance ofan 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

_ MSVcompared to HVTwere observed in the non-target animal safety studies described here.
4. Survivability ofthe 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 ofthe HVT parent strain. No significantdifferencewas seen between the recombinant and the parent strain, therefore

_ MSVwas considered to besafe forthe environment.
5. Potential fortransmission 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), butfree-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 enhancethe 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 ofthe vaccine into the environment will be restricted by its use in poultry houses.

52Biomune 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, ifthe 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 ofcertainty)]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 unlikelyto occur. ii. Certain (C)= The rating is supported by direct scientific evidence.
2. Consequence rating i. Low (CL)= The consequence, ifthe 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 ofcertainty)]X[(consequence)x (degree ofcertainty)]=Risk rating ii. [(1.00)x(1.00)]X[(1.00)x(O.75)]=O.75
4. Risk rating Low

53Biomune Company Fowl Laryngotracheitis- March 6, 2009



### C. Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 RISKASSESSMENT Environmental Safety 1. Likelihood rating i. Low (L)= An adverse event is unlikelyto occur. ii. Certain (C)= The rating is supported by direct scientific evidence.
2. Consequence rating i. Low (CL)= The consequence, ifthe 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 ofcertainty)]=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 ofthe vaccine in field safetytrials will not result in a significant impact on animal safety, public health or environmental safety. The risk rating for Fowl Laryngotracheitis-Marek's DiseaseVaccine,**Serotyp~** 3, Live Marek's DiseaseVector, Product Code 16J1.R1 (Unlicensed) Master Seed is low. We request authorization to conductfield safety trials with Fowl LaryngotracheitisMarek's Disease Vaccine, Serotype 3, Live Marek's DiseaseVector, Product Code 16J1.R1 (Unlicensed) upon acceptance from APHIS.

54Biomune Company Fowl Laryngotracheitis- March 6, 2009

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

1. Cho, B. R. Horizontal transmission ofturkey herpesvirusto chickens. IV. Viral maturation in thefeatherfollicle epithelium. Avian Dis. 19:136-141. 1975. 2. Cowen, B. S., and M. O. Braune. The propagation ofavian viruses in a continuous cell line (QT35) ofJapanese quail origin. Avian Dis. 32:282-297. 1988. 3. Darteil, 

### R, M. Bublot, 

### E. Laplace, J. 

### F. Bouquet, J. C. Audonnet, and M. Riviere. Herpesvirus ofturkey recombinantviruses expressing infectious bursal disease virus (IBDV) VP2 immunogen induce protection against an IBDVvirulent challenge in chickens. Virology 211 :481-490. 1995. 4. Guy, J. S., and T. J. Bagust. Laryngotracheitis

..In: Diseases ofpoultry, 11th 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. 121-134.2003. 5. Hirai, K., M. Yamada, Y. Aral, S. Kato, and S. NiL Replicating Marek's disease virus (MDV) serotype 2 DNAwith inserted MDVserotype 1 DNAsequences in a Marek's disease Iymphoblastoid cell line MSB1-41C. Arch. Virol. 114:153-165. 1990. 6. Lee, L. F. Large-scale production of Marek's disease virus. Avian Dis. 15:565- 571.1971. 7. Meulemans, G., P. Halen, and P. Schyns. Susceptibility of mammalian and avian cell cultures to infection with cell-free turkey herpesvirus. J. Compo Pathol. 83:605-608. 1973. 8. Morgan, R W., J. Gelb, Jr., C. R. Pope, and P. J. A. Sonderneijer. Efficacy in chickens ofherpesvirus ofturkeys recombinant vaccine containing thefusion gene of Newcastle diseasevirus: Onset of protection and effect of maternal antibodies. Avian Dis. 37:1032-1040. 1993. 9. Poulsen, D. J., C. 

### R A. Burton, J. J. O'Brian, S. J. Rabin, and C. L. Keeler, Jr. Identification ofthe infectious laryngotracheitisvirus glycoprotein gB gene bythe polymerase chain reaction. Virus Gene. 514:335-347.1991. 10.Ross, L. J. N., M. M. Binns, P. Tyers, J. Pastorek, V. Zelnik, and S. Scott. Construction and properties ofa turkey herpesvirus recombinant expressing the Marek's disease virus homologue ofglycoprotein B of herpes simplexvirus. J. Gen. Virol. 74:371-377. 1993. 11.Samorek-Dziekanowska, 

### E. Production ofthe highvirus concentration ofturkey herpesvirus (HVTstrain FC-126) propagated in Japanese quail embryofibroblast culture. Part 

### I. Bulletin ofVeterinary Inst. Pulawy. 21:10-16. 1977. 12.Sharma, J. M., 

### R 

### L. Witter, G. Shramek, 

### L. G. Wolfe, B. R. Burmester, and F. Deinhardt. Lack ofpathogenicity of Marek's diseasevirus 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 ofchickens from Newcastle disease and infectious laryngotracheitiswith a recombinant fowlpox virus co-expressing the F, HN genes of Newcastle diseasevirus and gB gene of infectious laryngotracheitis virus. Avian Dis. 52:111-117. 2008.



### 55Biomune Company Fowl Laryngotracheitis- March 6, 2009 Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Product Code 16J1.R1 RISKASSESSMENT 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 chickensfrom infectious laryngotracheitiswith a recombinantfowlpox 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 diseasevirus challenge bya 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 ofcell 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 ofturkeys. 

### J. Natl. Canc. Inst. 53:1731-1742. 1974. 19.Witter, 

### R. L., and K. A. Schat. Marek's disease. In: Diseases ofpoultry, 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 ofa functional promoter in the long terminal repeat of Rous sarcoma virus. Cell 22:787-797. 1980. 21.Zygraich, N., and C. Huygelen. Inoculation ofone-day old chicks with different strains ofturkey herpesvirus. II. Virus replication in tissues of inoculated animals. Avian Dis. 16:793-798. 1972.

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