### RiskAnalysis

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

USDA ProductCode: 16N1.RO (Unlicensed)

Updated

Merial, Inc. U.S. Veterinary License Number298

Confidential Business Information Redacted

### Merial, Inc. ·RiskAnalysis May 18, 2014 Page 2 of67 U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype

### 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)



### Table ofContents I. INTRODUCTION ................................................................................................................................... 6 

## A. Objective........................................................................................................................................... 6 

## B. Proposal............................................................................................................................................. 7 II. DESCRIPTION OFTHE REGULATED BIOLOGICALAGENT CONSTRUCTION ..............................

### ?

A. The Backbone Biological Agent........................................................................................................ 

### ? 

## B. Donor Biological Agents and Donor DNA orGenes .......................................................................11 

### Ill. BIOLOGICAL PROPERTIES OR VIRULENCE FOR THE REGULATED BIOLOGICALAGENT USED

A. FOR MASTER SEED........................................................................................................................... 34 What are the known phenotypic characteristics or effects ofthe Regulated Biological Agent identified as Master Seed? (e.g., expresses envelope gene/sequence, lacks thymidine kinase activity, lower titer in MA105 cells, etc.) ................................................................................................................ 34 B. What arethe virulence characteristics ofthe Regulated Biological Agent used forthe Master Seed?



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..35 C. Is the Regulated Biological Agent used forthe Master Seed virulentfortarget animals, non-target animals? ......................................................................................................................................... 35 D. Do the Donor DNA sequences enhance the virulence orthe ability ofthe MasterSeed to survive in thetargetanimals, non-target animals? .........................................................................................36 E. Discuss the potential for horizontal gene transfer or recombination ofthe Regulated Biological Agent.

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.......:...37 F. Describe the shed/spread capabilities ofthe Regulated Biological Agent. .....................................38 G. Discuss the expected environmental impact or survivability ofthe Regulated Biological Agent, and provide available scientific evidence...............................................................................................39 IV. RISKASSESSMENT...........................................................................................................................40

A. Procedure .......................................................................................................................................40 B. 

### Hazard identification .......................................................................................................................41 1. Animal safety ...............................................................................................................................41 2. Public health safety...................................................................................................................... 46 3. Environmental Safety...................................................................................................................47 C. ReleaseAssessment Prior to a Proposed Field Safety Trial..........................................................49 D. Risk characterization ........................................................,.............................................................53 Confidential Business Information Redacted

### Merial, Inc. Risk Analysis U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed) May 18,2014 Page 3 of67

1. Likelihood ratings ......................................................................................................................... 53 2. Consequence ratings ··································'······························'·················································53 3. Degree ofcertainty rating.............................................................................................................53 4. Calculating the expected risk.......................................................................................................54 5. Risk ratings ...................................................................................,..............:...............................54 V. RISK MANAGEMENT RECOMMENDATIONS .:.·.............,..................................................................59 Tables 

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### Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed) Appendices 

# I May 18, 2014 Page 

### 5of67

2. Purchase, H.G. and R.L. Witter, "Public health concerns from human exposure to oncogenicavian herpes 

### viruses: 

### Areview'' 

### References 1. Afonso,C.L., Tulman,E.R., Lu,Z., Zsak,L., Rock,D.L. and Kutish,G.F. (2001), 

### J. Viral. 75(2):971-978 2. Calnek, B.W. and Witter, R.L., Diseases ofPoultry, 9:342-385, 1991 3. Cho, B.R. 1976. Poultry Science 55:1830-1833 4. Cho, B.R. and S.G. Kenzy. 1975. Poultry Science 54:109-115 5. Cho, B.R., S.G. Kenzy, and S. A. Haider. 1971. Poultry Science 50:881-887 6. Ebeling etal., 

### J. Viral. 47:421-433, 1983 7. H.G. Purchase etal., Am. 

### J. Vet. Res., 32:1811-1823, 1971 8. K. Dorsch-Hasleretal., Proc. Natl. Acad. Sci. USA, 82:8325-8329, 1985 9. Merceretal.., Virology 129:94-1061983 10. N. Zygraich and C. Huygelen, Avian Diseases, 16:793-798, 1972 11. Osterrieder and Vautherot, ElsevierAcademic Press,p.17-31, 2004 12. P.A. Phillips

**~nd** P.M. Biggs, 

### J. Natl. Cancer lnst. 49:1367-1373, 1972 13. Taylor, 

### J. etal., 

### J. Viral., 64: 1441-1450, 1990 14. Tripathy, Deoki: Pox. Diseases ofPoultry (1991) Ed. Calnek, B.W., Iowa State University Press, Ames, Iowa; pp583-596 15. Witteret al,, Am. 

### J. Vet. Res., 31:525-538, 1970

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### Merial, Inc. RiskAnalysis U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek'sbiseaseVector, Code: 16N1.RO (Unlicensed)



### I. INTfKlDUCTION
A. Objective May 18,2014 Page 6 of67

The objective ofthis analysis is to characterize risks associated with the proposal to conduct experimental 

### vaccination underfield study conditions in chickens using a live Marek's Disease, Serotype 3, HVTvirus which contains a Newcastle disease virus (NOV- gene cassette derived from-**ND~** 

### and subsequent commercialization and distribution ofthe product, pending CVB acceptance ofdata in support oflicensure for use in chickens. 1. Identifywhere the Regulated Biological Agentwas constructed and where the productwill be made, tested and manufactured.Address the available level ofcontainment. Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, unlicensed, USDA product code 16N1.RO (herein identified as-,was constructed'

. _, ''"· -.-·- 

### ... ,.. ' .. - 

### ··-

-

- ------- ----- ------ ------ -



### 2. Provide a brief(one sentence) description ofthe Regulated BiologicalAgent. The Regulated Biological Agent (RBA), designated-,will contain a Marek's Disease, Serotype 3, HVT

### virus which contains a gene cassette derived

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### Merial, Inc. RiskAnalysis U.S. 

### Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

B. Proposal 

### May 18,2014 Page 7 of67

The proposal is to conduct experimental vaccination studies in chickens in the field to determinethe safety of a live Marek'sdisease, Serotype 3, HVT virus which contains a NOV- gene cassette derived from

- NOV,- and subsequent commercialization and distribution ofthe product.
1. What isthe intended use ofthe product? Species: Avian; 18-day-old or older embryonated eggs orday-old chickens Proposed claim: Thevaccine is indicated for use in healthy 18-day-old or older embryonated eggs orday-old chickens as an aid in the prevention ofMarek's disease and Newcastle disease. Geographic area: United States (all states), 

# - Route ofadministration: in ova, subcutaneous Briefdescription ofthe expected safety profile: The RBAvaccine is expected to elicit an immune response in the chiCken to Marek's disease virus and Newcastle disease virus without causing gross observable lesions as directed by 9CFR113.330 (c) for Marek's or signs of Newcastle disease such as severe respiratory signs or death as directed by 9CFR 113.329 (c). 

### II. DESCRIPTION OF THE REGULATED BIOLOGICALAGENT CONSTRUCTION
A. The BackboneBiologicalAgent
1. Whatorganism was used forthe Backbone Biological Agent?Are there any known virulence features associatedwith the Backbone BiologicalAgent? What happens In the targetspecies? The organism used forthe backbone is a Marek's Disease, Serotype 3, HVTvirus belonging to: Family: Herpesviridae Subfamily: Alphaherpesvirinae Genus: Species: Strain: Meleagrid herpesvirus Meleagrid herpesvirus 1orturkey herpesvirus (HVT) FC126
The parent strain is avirulent in chickens and does not cause gross observable lesions associatedwith Marek's diseasevirus. Only limited contact spread occurs among chickens buttransmission could not be demonstrated by the airborne route

### (Dise~ses of Poultry,

### 12~ Edition, Chapter 15, page 491). The results of Study 12-027entitled "Ability ofMarek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector-,X+5, Product Code: 16N1.RO (Unlicensed) Administered Subcutaneous to Day-Old SPF Chicks to Spread to NaiVe Day-Old Turkey Poults, Naive Day-Old Chicks, HVT-Infected Turkey Poults, and HVT-Infected Chicks When Housed Together" showed thatthere was no spread ofthe RBAto HVT infected turkeys, naiveturkeys, HVT infected chickens or narve chickens. This studywas accepted by the CVB on December 5, 2013

Confidential Business Information RedactedMerial, Inc. RiskAnalysis U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle DiseaseVaccine, Serotype 3, Live Marek's DiseaseVector, Code: 15N1.RO (Unlicensed)
What Is the previous safe use ofthe Backbone Biological Agent? May 18, 2014 Page 8 of67

(I) If

**avail~ble** provide historyofprevious safe use using published literature or Internal documents. Includethe recommended CDC/NIH biosafety level for use ofthe Backbone Biological Agent. The vectorvirus is the FC-126 strain ofturkey herpesvirus (HVT). This strain has been widely used as a vaccine against Marek's disease (MD) inchickens since 1971. It has contributed to a significant 

### reduction in carcass condemnation rates in chickens.
The FC-1.26 strain was isolated from turkey_s 

### bY R.L. Witter(Regional Poultry Research Laboratoi"\', USDA,



### Ea~t Lansing, Michigan USA) in 1970 (Witteret at., "Isolation from turkeys ofa celt-associated herpesvirus antigenicatly related to Marek's disease virus," Am. 

### J. Vet. Res.,"197D, 31, 525-538). Vaccine strain FC-126 of HVT, grown in chicken embryo fibroblasts, is use.d to produce L YOMAREX and CRYOMAREX, the livevaccines against

**M~rek's** disease marketed l)y Merial SAS, Lyon, France; and Marek's Disease Vaccine, Serotype 3, Live Virus, product code 1641.00, marketed by Merial, Inc. It has also been used as the backbone for USDA licensed product code 1A88.RO, Bursal DiseaseMarek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector. Many billions ofchickens have been vaccinated with these vaccines. The biosafety level for manipulating HVTvirus is BSL1. 2. Physical characteristics ofthe Backbone Agent

a. Provide aflow diagram orexplanation ofthe process ofhowthe Backbone BiologicalAgent was constructed. (i) The Hvivectorwas derived from the vaccine strain of FC-126 strain ofMarek's {see Section II.A.1.(i), page 8}Describethe proposed sitefor Donor DNA insertion. HVT exhibits characteristics of herpesviruses: icosahedral (162 capsomeres), enveloped virus with a

**diam~ter**of 150 to 170 nm. The diameter

**o~**the capsid measures 90 to 100 nm and it contains dsDNA (approximately 160 kbp). The sequence ofthe whole FC126 strain of HVT (159160bp) has been published (Afonso,C.L., Tulman,E.R., Lu,Z., Zsak,L., Rock,D.L. and Kutish,G.F. (2001)The genome ofturkey herpesvirus 

### J. ViraL 75,(2), 971-978) and is

**availabl~**_in Genbank (NC_002641).

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### Lie. No. 298 Risk Analysis Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

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### U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease'vaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed) May 18,2014 

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(ii) Do the flanking regions ofthe proposed insertion site Backbone Biological Agent have any known regulatory elements thatcould moderate the expression ofthe Inserted Donor DNA?

flanking regions are not thought to have regulatory elements that could moderate the expression of the inserted donor DNA. The only modification introduced regarding the parental virus HVT is the deletion No 

### open reading frames of HVT have been modified. The exact same locus of insertion was used to insert an infectious bursal disease virus. gene in the USDA licensed product code 1A88.RO, Bursal Disease-Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector. (iii) Identify unique restriction endonucleases (not morethan five) thatwill give identifiable digestion patterns useful forcharacterizing thefinal Backbone DNA

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. Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

B. Donor BiologicalAgents and Donor DNA orGenes 

### May 18,2014 Page 11 of

### 67

1. Whatare the Donor Biological Agents used tosource each Donor DNA sequence inserted intothe Backbone Biological Agent?
Gene insert: native

# I gene from NOV 

### Family: Subfamily: Genus: 

### Species: Strain: Paramyxoviridae 

### Paramyxovirinae Avulavirus 

### Avian Paramyxovirus 1 (APMV1) or Newcastle Disease Virus (NDV) 

# - NOV is an enveloped virus containing a non-segmented negative strand RNA genome. It contains several genes including the NP (nucleoprotein), P(phosphoprotein), M(matrix), F(fusion), HN (hemagglutininNeuraminidase) and 

### l (large polymerase subunit) genes. The gene- was used as the protective gene ofNOV. eDNA clones for the NOV- gene were derived from a- NDV,- The gene cloning and sequence that had homologyto the NOV- gene has been described byThe exactsame donor NDV- sequence has been used in USDA licensed product 17C1.RO, Newcastle Disease- Fowl Pox Vaccine, live Fowl PoxVector, licensed October27, 

### 1995.

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### Vet. Lie. No. 298 

### Marek's Disease-Newcastle DiseaseVaccine, Serotype 3, 

### Live 

### Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

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### Page 12 of67

- - - -- --- - -- - - - -- -- -- - - 

### = --- ::;,__--__-_--_**~---_-----_** 

### -_**--_----~~-::--** -=----_----:::--= -- exact same promoterwas used gene in the USDA licensed product code 1A88.RO, Bursal Disease-Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector.

seijUeJnce was used downstream from the infectious bursaldiseasevirus. gene in the USDA licensed product code 1A88.RO, Bursal Disease,Marek's

### Dise~se Vaccine, Serotype 3, Live Marek's Disease Vector.
a. Has there been safe use ofthe DonorSequences orDonor Genes as well as Safe use ofthe DonorBiologicalAgent? 

### (i)**provideJ·~Ievant ref~rences**  forsafe use.

### T~e donor

### NOV~ sequence has been used in USDA licensed product 17C1.RO, Newcastle Disease-Fowl Pmivaccine, Live Fowl PoxVector, licensed October27, 1995.
The locus ofinsertion, promoter and poly-A signal iiave been used in USDA licensed product code 1A88.RO, Bursal bisease-Marek's DiseaseVaccine, Serotype 3, Live Marek's"DiseaseVector.
In orderto identify any impact this insert may have on current National Veterinary Services Laboratories (NVSL) Newcastle disease virus (NOV).testing, the vaccine was evaluated using a realtime reverse transcriptase PCR for the detection and differentiation ofvelogeriic NOV as utilized in the NVSL NOV surveillance program. The primers and probes used in this assay are identical to those used in the NVSLprotocol SOP-AV-1505.05 (Attachment 2). The NOV

....gene did not reactwith the oligonuCleotides used in the NVSL real-time RT-PCR protocol for aetection of virulent NOV. ·Additionally, the RBA does not contain the NDVmatrixgene, and will not reactwith the primers setdesigned for

# Hs detection. The RBA

# does nottest as exotic

# Nov.

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### of67

b. Are there specific parts ofthe DonorGene(s) orSequences thatwere used for insertion? Show pertinentsequences orrestriction endonuclease sites.

C. Construction and Characterization ofthe Regulated**Biologi~al** Agent The RBAwas obtained by homologous recombination between the parental HVT and adonorplasmid

. 

### . . 

# Eii5S

imrnurlOfiuoi·es,::en,ce,, using an antibody that recognizes the product ofthe inserted gene.. No resistance factors are used as a means of selection. 

### As explained above, the donorplasmid- was constructed from the donor plasmid- used to generate the USDA licensed product code 1A88.RO, Bursal Disease-Marek's Disease Vaccine, Serotype 3, live Marek's Disease Vector. The IBDV. gene was removed from the-and replaced bythe NDVI -gene

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### Merial, Inc. Risk Analysis U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, 

# ·uve Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

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- -------------- --- -- --

- -=:::=- 

### --=--- __ 

## --= --=--**~-** 

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## -=-=--=-=--=--

__ _ -=-::::-.=-=-=----

# ---=---=-=-=-=---==-=--------=---

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### Lie. No. 298 Risk Analysis Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

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### Merial, Inc. Risk Analysis 

### U.S. Vet. Lie. 

### No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, 

## live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

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### Merial, Inc. U.S. Vet. 

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### Merial, Inc. RiskAnalysis U.S. 

### Vet. Lie. No. 298 Marek's Disease-Newcastle DiseaseVaccine, Serotype 3, Live Marek's DiseaseVector, c'ode: 16N1.RO (Unlicensed)

**·-~** 

### .

**--~** 

### . May 18, 2014 Page 

### 18 of67

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----- - - --

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-------- ----=----- --- ------- -- - --------

**-~--** ----- --- --- - ----

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1. Provide aflow**diagr~.m** on the construction ofthe Regulated Biological Agent Includethe following: Final Backbone Biological Agent Donor DNA or gene All shuttlevectors Host cell lines used Selection techniques and methods used to constructthe final Regulated Biological Agent
The general flowchart used to generate the RBA is shown in Figure 10.

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### Merial, Inc. U.S. Vet. Lie. No. 298 Risk Analysis Marek's Disease-Newcastle Disease Vaccine, Serotype 

### 3, live Marek's Disease Vector, Code: 16N1.RO (Unlicensed) May 18,2014 Page 21 of

### 67

2. Describe the laboratorymethods or criteria usedto evaluatethe Regulated Biological Agent. The genomicstructure at"the recombinant virus, as well as the absence of parental HVT, has been verified.by (1) Southern blot analysis, (2) PCR analysis and (3) lmmunoprecipitation and Western blotanalysis. A double immunofluorescence assay using a chicken anti-HVT polyclonal antibody and the rabbit anti-NDVI polyclonal antibody labeled with an anti-chicken serum conjugated with FITC (green) and anti-rabbit serum conjugated with Cy3 (red) fluorochrome was performed on plaques obtained from the

- 924 plaqueswere screened and all 924 HVT plaques (green)were found to express NDVI (red).

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### Lie. No. 

### 298 RiskAnalysis Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector; Code: 16N1.RO (Unlicensed)

3. Physical characterization ofthe Regulated Biological Agent 

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### 18, 2014 Page 

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a. Characterize the pliyslcal rtap uslng unique bonorDNA and Backbone.Biologic;!

# ~qent**reStrl~tion** endO_nuc_lea!?e 

### sit2s and describe

# re~Uit~ng reslrlcfion fragments and

··'; 

### '·

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### 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed) May 18,2014 Page 23 of67

b. Devise a PCR.or restriction endonUclease·test baSed on the Backbone-Biological Agent sequence and the Donor Sequence thatwill identify and characterize the Donor DNA/Backbone Biological Agentconstruct. Several PCR's have been developed to characterize and identifythe RBA. The localization ofthe primers on the HVT and the RBA genome is shown in Figure 13. The sequence ofthe primers is shown in Table 2 and the size ofthe amplification product is shown in Table 3.

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### No. 298 Risk Analysis Marek's Disease-Newcastle Disease Vaccine, Serotype 

### 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

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### Merial, Inc. Risk Analysis May 18,2014 Page 26 of67 U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 1SN1.RO (Unlicensed)

c. Whatwill be the criteria forstabilityand purityofthe Regulated Biological AgentMasterSeed nand n+5? PCR pattern and plaque immunoscreen resultswere used to determine both stability and purity ofthe 

### recombinantvirus. d. Provide the genetic sequence in eiectronlc formatforany new oraltered genetic sequences, inciuding

## iri-~~i1ion**~-nd** flanking

## sequerl~e. 

### . . _,..- - The DNA

### sequenc~ ofthe parental HVT-fragmentand the

### corr~sponding fragment ofthe RBAwere su6mitted as l('lord files on a daia disk on June 24, 2011. 4. Whatisthe

## recoorim~nded NIHJCDC biosafety levelforthe. kegulated Biological Agent?

.**~** 

### . . . - •AccoiTiing

## t~ NIH/CDC definition, Marek's disease ili(ll; is a Risk Group·1 agent since it is not

## ad;~ciated with

## di~ea;~

# fn healthy adult

## hu~~ns..·BSL2

## pra;ti~es arid procedures

## w~re utii;ied to



### .

## en~~re·**~tJ;~r-~ontainllJent of~_ihe** **~;perime.nt~-i C~o.st~~Ct** iil areseafCh

## ~~tti~g: 

# AtthiS'

## ti~e.**consld~;jfig**

# ihe**gener~ted** safety data and the (aci,that

## th~**~o\ti!ilill** gene

## in~ert**do~spot** •.

P[OVide

## ~ny newvirulence factor

## t~ the backbone virus

## w~**w.!i~idcor1S,ider** BSL1

## s~fficient'l~rth!'> RBA, Similarto the parental organism.
5. PrOvide:_.a shOrt

### ~Ummary or

### d~SCriPtion ofgeiletiC motifs

### tb_~·t

## may have

### rl~sulted as a·consequence-Of the geiietic reco.ii'binatio·n.(ll.c.3.d). Are tliere any known

# m~tifsthat may promote

# homologoi.~ ·- . - 

### - - 

### . 

# . . .

·-·· - • • - _f.V • - - - .' recombination, DNA insertion, or-gene**-~~_pression**

## bfexisting Or new**op~n** reading**fr~mes?**


### a. ••-< Pre-Master Seed'vini·s

# Produce~--~~liililliill

# T~e insert19n

# bit~e**.ND'!illlflil.-e;~.r~s_~,iRHca~~ette·.was** pl'et:isS_Iy

# t~rgeted**tot~~~** 

# I and

## the.~nalyticaljesti~g (PCRand

## Soufh~ril blot) confirmedthat

## \he homologous

## 'r~c;:cimbination Occurred as eXpected._There

### a~e rio knowh mOtifSlhat may promote homologous reCOrilbination, DNA inSertion orgene expression of

## existin;~**ciP~n ~eading** **fr~~es.** Only 01)8foi'eign open reading frame

## was inserted and coding forthe

## NDV~~~~~~ gene. No significant (>60 amino acid) open reading frames

### wer~ created

## by the

### inserts~**S~tju~nce.** The locus of insertion, the promoter and the polyadeliylation signal ofthe RBA are identical to the -USDAiicensed product code 1A88.RO, Bursal

### Disease-M~rek's Disease Vaccine, Serotype 

### 3, Live Marek's Disease Vector. The NDV- gene isidentical to the

# I gene inserted in the USDA licensed ·product 17C1.RO, Newcastle Disease- Fowl Pox Vaccine, Live Fowi Pox Vector, licensed October27, 1995.

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### Merial, Inc. U.S. Vet. Lie. No. 298 RiskAnalysis Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's DiseaseVector, Code: 16N1.RO (Unlicensed) May 18, 2014 Page 28 of67

performed on HVT 

### FC126, RBA MSV+5 and BP5 viral genomic DNA showed the expected hybridization pattern [see Figures 15 and 

### 16].

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### May 18, 2014 Page 29 of67

### Merial, Inc. U.S. Vet. Lie. No, 298 'RiskAnalysis Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector/Code: 16N1.RO (Unlicensed)

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### May 18,2014 Page 30 of67

### Merial, Inc. Risk Analysis U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle DiseaseVaccine, Serotype 3, Live Marek's DiseaseVector, Code: 16N1.RO (Unlicensed)

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### May 18, 2014 Page 31 of67---·,_



## .,-.

-;o:

## ·•· Merial, Inc. Risk Analysis U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)
(iii) lmmunopreclpitation and Western Blot

.; '

j,_

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### May 18,2014 Page 32 of67

### Merial, Inc. U.S. Vet. Lie. 

### No. 298 Risk Analysis Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's DiseaseVector, Code: 16N1.RO (Unlicensed)

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### May 18,2014 Page 

### 33 of67

### Merial, Inc. "Risk Analysis U.S. Vet. Lie. No. 

### 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed) May 

### 18, 2o14 Page 34 of67

- -. -. Ill. BIOLOGICAL PROPERTIES OR VIRULENCE FCJR THE REGULATED BIOLOGICALAGENT USED FOR MASTER SEED
A. Whatarethe known phenotypic characteristics oreffects ofthe Regulated Biological Agentidentlfied as

### M~ster Seed? (e.g., expresses envelope gene/sequence, lac_ks thymidine kinase activity, lowertiter ln.MA105 cells, etc.) ,_1;**-~** 

### . 

# . . The double label immunofluorescence test described in Section II.C.2 (page 21) abovewas performed in order to analyze the

# ex~re~sion of

# I protein through

# i~itial virus passage. The lysis plaque.s are reveale.d with 

### a .p61yclonal antibody produced i.n chickens immwnized with 

### HVT and an anti-chicken-lg conjugated with fluoff::Scefh

# (Qr~eM·fl~oreScence). The

# S~Pr:esSiOn·.Qfl**RrO~in** is 

### revea1Sa through

# mo~oCI~rial**anti6~Ciies direci~d** **~gainst**the

# 1gene of

### NOV, and a conjugal!!

## m~\fe With anti-mouse-lgG,

## coupl~d with Cy3

# .fiuor6ci~fq"m~(red

# fiu6res,~nee).·The red

# fiuqre;b~i\cie is therefore searched (or

# in'e~~iy green plaque. This

## 'tesi~llp~**"aemo~str~tio~** ofthe phen6typic.stabiiityorllie

## r~~ombih~~~**vii~..** 

# .. . .'.'

- - . - •'' ';·- . . ':· iiT1rnurlopreciP,ii"tatic>n and

### \IVestern blottei;hnique deiectedan aplproxinl.'it<ely!

# th~i**c?r[e~ponds totlle"exp~Ste:~** **;~e** cii.

# com~onentof!he >[cE>nt••ae· ofthe RBA

### pl~ques expressing

### thelp~?teili atvarious

### ~Liture pa";ssarge,s,

## .a"§d~l~nmirled by analyzingthe titer ofthe

## vaccine~dqseif used 

## iii the 

## 120"•:•·"'''"', 

# .3ctepte.dby th;:-cVB

## {)'~ Apti,l 7,

## d~~11**}8ever~ioi"\:** "VirllleOII:< ;I'-_,._",,-, ,____ ;-. - CVB On 9, 2011) and.an effi¢acy '-

-- ,_,_**~** _, .-,);,--,·!_ '
. CVB on August 23, 2012) are

. 

### . -**-~--** - .

.The

## ~arehMECc\2,6**st~ain.Ws*i•P!•l!>~JrprTI.!\i.rk~y~by**

## fl.L. WJtter.JRegipn_al Poultry B,esearch Laboratory, USDA, East Lansing, Mlchigan, USA) in 1970. The

## v~ccine strain FC-126 ofHVT,

## grow~ in chicken embryo fibroblasts, is used to produce CYROMAREX® and L YOMAREX®, the live vaccines against Marek's disease marketed by Merial SAS, Lyon, France; and Marek's Disease Vaccine, Serotype 3, LiveVirus, product code 1641.00, marketed by Merial, Inc. Many billions ofchickens have been vaccinated with these vaccines. The HVTvirus is a non-pathogenic virus ofturkeys. No HVTgene was modified 

### by the insertion ofthe NDVI expression cassette and therefore, no biological effect is exp8cted dUe to the genetic marlipulation at the cloning site. Furthermore, the insertion of a NDVI expression cassette into the HVT vector had no or minimal effect on the replication ofthe vector as determined during viral passage from MSV to MSV+5.

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### 35 of 67

The NDVI protein is likely expressed at the surface (anchored in the cell membrane) of infected cells; it may also be present in the envelope ofviral particles. However, shed and spread (see Section III.C, page 35-36), aswell as tissue tropism studies (see Section Ill.D. page 36-37) have demonstrated that the phenotypic characteristics ofthe RBAwere similarthan those ofthe parental HVT indicating that the NDVI expression did not change the behaviorofthe parental virus. B. Whatare thevirulence characteristics ofthe Regulated Biological Agent used forthe MasterSeed? The recipient virus was derived from the FC-126 strain of HVT, which is recommended for use in day ofage chickens or in ovo vaccination. This strain is avirulent forchickens and its natural host is turkeys. No HVT genewas modified and therefore, no biological effect are expected due to the genetic manipulation at the cloning site. The same vector and cloning site is used in USDA licensed product code 1A88.RO, Bursal Disease-Marek's Disease Vaccine, Serotype-3, Live Marek's Disease Vector. C. Isthe Regulated Biological Agent used forthe MasterSeed virulentfortargetanimals, non-target 

### animals?

-wasaccepted by the CVB on March 9, 2011. The study satisfied the recommendations of Veterinary Services Memorandum 800.201 forevaluating the lack of reversion to virulence in chickens and the limitations ofviral shed and spread to unvaccinated (na·lve) contacts. One-day-old chickens were inoculated with the RBA MSV; blood was taken at seven days post-inoculation and used to inoculate anothergroup of one-day-old chickens. Four additional passageswere made. Na"ive birds were placed in contactwith each group receiving the backpassed virus. At each passage, bully coat extractions were inoculated on chicken embryo fibroblast monolayers to monitorvirus recovery for both the vaccinates and the contacts. At seven weeks post-inoculation, 10 birds from each passage were necropsied and examined for lesions typical of Marek's Disease. Afinal passage was conducted using 25 one-day-old chickens, with one control group receiving the MSV and a second control group receiving a placebo. These birds were also necropsied at seven weeks post-inoculation; lesions were not observed in any ofthe birds. Virus was recovered from all passages, as well as from the positive control birds. No virus was recovered from the contact controls. The recovered virus from the backpass birds was also tested forexpression ofthe

# I protein and found to be positive.
The safety study in mice- showed no recovery ofvirus after subcutaneous injection and epidermal scarification of 15 mice. Marek's Disease Virus, SR-3 (HVT) vaccine, USDA product code 1641.00, was administered to 15 additional mice and 6 mice were left untreated to se!Ve as negative controls. On days 3, 7, and 14, the trachea, brain, spleen, heart, liver, lungs, kidneys and skin at the site of Inoculation were removed from 5vaccinates and 2 controls. The buffy coatwas extracted from the tissue homogenates and evaluated for presence of Marek's disease virus on chicken embryo fibroblast monolayers. This study supports the lackofa negative effect ofthe product in non-target animals due to no virus replication in murine tissue. The study was accepted by the CVB on October25, 2011.

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### U.S. Vet. Lie. No. 

### 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 

### 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)
The**·~!ely** study in non-target avian spe1c1es, May 

### 18, 2014 Page 

### 36 of67

--------- --- -

- - -- - - --= =----_- - -- - _- - ----- -

- --- -- - ----------- was accepted-by the CVB on March 8, 2012. The study contained groups of40 quail, turkeys, ducks, pheasants orpigeons that received either Marek's diluent or a 10Xdose ofthe RBAvaccine. The birds were observed for signs of Marek's disease (MD) or lesions associated with MD at necropsy. No birds exhibited signs or lesions associated with MD. Safety studies were performed using both the in avo and subcutaneous routes of inoculation with observance for 120 days Each chicken and/or 18-day-old embryo received

### betW~e~-- pfu 

### Ofthe RBA MSV. Controls groups including non-vaccinated shams and non-vaccinated/challenged controls were included in the study as directed by 9 CFR 113.330. No untoward signs were seen in**t~e** chickens inoculated with the RBA MSV. At50 days 95.8% to 100% ofthe nonvaccinated, challenged controis inoculated with MDV RB1 Bstrain developed Marek's lesions. At 120 days, none ofthe vaccinated birds and none ofthe birds that received a placebo showed signs of Marek's disease. "The average body weightsof

### th~ birds in these two groups did not differ. According to the CVB, these studies satisfied

### iheofequii~;.,'~nts of9.CFR 113.330(b) io·rthe subcutaneous

## aiiii in avo. routes ofadministration (CVB

## a6il~~feo

# an Aprii 7, 2011). D. Dothe Donor DNA sequences enhancethevirulence orthe ability ofthe MasterSeed to survive In the targetanimals1**non~target** animals? 

### 1. What isthetissuetropism ofthe Regulated Biological Agent in targetand non-targetanimals? HVT replicates in chickens and induces a detectable level ofviremia. Leukocytes become infected and provide the main mechanism of**cell~associated** virus transport tb othertissues. Cell-free infectious virus occurs only in the featherfollicle epithelium between the second and third week of infection, butthere is little or no natural spread ofHVT between chickens (P.A. Phillips and P.M. Biggs, "Course ofinfection in tissues of 

### susceptible chickens afterexpoSure to Strains of Marek's disease virus and turkey Herpesvirus," 1972, J. 

### Natl. Cancer Inst. 49, 1367-1373; N. Zygraich and C. Huygelen, "Inoculation ofone-day-old chicks with different strains ofturkey herpesvirus. 

### 11. Virus replication in tissues of inoculated animals," 1972, Avian Diseases 16, 793-798).
HVT replicates exclusively in cells ofavian origin (chicken, turkey, duck, quail, pheasant, pigeon, or goose) 

### (H.G. Purchase et al., "Responses ofcell cultures from various avian species to Marek's disease virus and turkey Herpesvirus," 1971 Am. 

### J. Vet. Res. 32, 1811-1823). The vaccine strain is produced in primary cells of 

### chicken embryos. 

## Hvr is cell associated, and is present in very small concentration in the supernatant of infected cell cultures.

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the birds were inoculated with the RBAvaccine or the FC-126 parental virus. An additional group ofbirds received diluent only to setVe as negative controls. On days 7 and 28 post-inoculation, thespleen, liver, lung, kidney, and gonads were isolated from five birds ofeach group ofvaccinated birds and the spleen was isolated from three ofthe negative control birds. Buffy coat extractions from each organ were evaluated for virus isolation on chicken embryo fibroblasts monolayers. The presence ofthe virus was identified by the obsetVation ofcytopathic effect. NDVI protein expression was evaluated by immunofluorescence using at least one plate perorgan per bird. Both the RBA and FC-126 were isolated from the spleen, liver, lung, and kidney samples; the gonad sample from one bird was positive forthe RBA. Plaques obtained from birdsthat had been inoculated with lhe RBAwere positive forthe expression ofthe NDVI protein, providing evidence of 

### no loss ofexpression aftersingle in vivo passage (note: the reversion to virulence study proved no loss of expression afterfive passes in vivcf- CVB accepted on March 8, 2011). The data from this study supportthe conclusion that the tissue tropism ofthe vaccine construct and the parental virus do not differ. The study was accepted by CVB on March 30, 2011. E. Discuss the potential for horizontal gene transferor recombination ofthe Regulated Biological Agent.
1. Is there any reason to believe thatthe potential for horizontal gene transfer or recombination is different in targetand non-targetanimals? Horizontal gene transfer between HVT and other Marek's disease viruses (MDV) has never been reported. This is likely due to the low percentage ofhomology between the 3 MDV serotypes (Osterrieder and Vautherot, 2004The genome content ofMarek's disease-like viruses. In F. Davison 

### &V. Nair(Eds.) Marek's Disease. An Evolving Problem, ElsevierAcademic Press, pp 17-31). Furthermore, the NDVI gene has no known toxic effect when inserted in a Marek's disease virus, and therefore, even ifa horizontal gene transfer were to occur, 

### it should not increase the virulence ofthe recipient virus.

was conducted to investigate the possibility ofincreased pathogenicity following co-administration of the RBA with other Marek's disease viruses from other serotypes. To determinethe stability ofthe gene insert in the face ofpathogenic MD, the RBAwas combined with a virulent MDV strain (GA 22) and wilh serotypes 1and 2 ofMarek's disease virus. This co-administration should favor recombination between viruses if itoccurs. There was no increase in virulence observed in the GA22, Rispens orSB-1 groups co-inoculated with the RBA vaccine based on gross MDVlesions. There were no statistical differences in average body weight between the GA 22 and the -/GA22- 

# ! 5555555555555& Rispens and the-/Rispens- or SB-1 and the-/SB-1- combinalion groups at the end ofthe 120 day obse!Vation period. The results showed no increase in pathogenicity of any ofthe MD strains when combined with the RBA, indicating that recombination either did not occuror if it occurred (unlikely), it did not generate a Confidential Business Information RedactedMerial, Inc. Risk Analysis U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code:"16N1.RO (Unlicensed)

more virulent virus. This report was accepted by the CVB on

### Ja~u·ary 3, 2013 F. Describethe

### shed/spr~ad**capab\li\1~~** ofthe. Regulate<l Biological Agent. May 18,2014 Page 38 of67

Study

- --- --- ----- ------------ 

### -- --**~--------=----_--------------** --- -_-- ---_ -::::--------::;-- was accepted by the CVB on March 13, 2012. ino•cul<lted with eitherthe RBAX+5, licensed HVT, or Marek's diluent. One ofthe groups that received Marek's diluent only were inoculated with the very virulent MDV Serotype1, RB1 Bstrain by IP injection at hatch.

## Pr~sence of HVTwas determined by cell culture, followed by fluorescent antibody staining. Presence ofthe RB1 Bstrain was confinned by polymerase chain reaction (PCR) spe"cific for Serotype 1virus. Tracheal and cloacal swabs were collected from the birds to examine for viral presence. Featherfollicles Were also examined for virat presence. Virus could not be recovered from eithercloacal ortracheal swabs. Virus was i'e?Dvered from feather follicles at some time points during the 28- day study. The RBA viral plaques recovered retained the 

### inserted gene. The 

### patterh ofdisSemination ofthe RBA MSV+S was similar to the HVTvacCinated birds.
A shed/spread study was conducted as part ?Ithe reversion to virulence study- CVB accepted on March 9, 2011 -See Section Ill.C., page 35-36). No virus was recovered from "na"ive birds placed with the backpass bi_rds at any time points evaluated, norwere any**lesi~ns** seen in the contacts at necropsy. 1. Include shed/spread potential in non-target animal species.

any ofthe non-target species.
As discussed in Section Ill.C. (page 35-36), Study- shoWed no recovery ofvirus after subcutaneous injection and epiderrTial scai"ificiition of 15 mice.

HVT infected turkeys, na"lve turkeys, HVT infected chickens nor na"ive chickens.

Confidential Business Information RedactedMerial, Inc. RiskAnalysis U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed) May 18, 2014 Page 39 of67

2. Include inthe discussion anycontributionsto shed/spread capabilities that mightbe associated with particularattributes ofthe Backbone Biological Agentand Donor DNA sequence. It is not expected that insertion ofthe NDVI genewill contribute tothe shed/spread properties ofthe backbone virus. The HVTvirus is not efficient in viral spread and has only shown very limited spread to contacts in the environment (see Section II.A.1, page 7). The limited shed is mainly through feather follicles and the insertion oftheI gene into a non-essential site of HVT does not impactthe shed pattern ofthe HVT virus (no viral shed seen as discussed in Section III.F, page 38-39) 3. Reference relevantscientific publications. a. Cho,B.R 1976. Horizontal transmission ofturkey herpesvirus to chickens. 5. Airborne transmission between chickens. Poultry Science 55:1830-1833. b. Cho, B.R. and S.G. Kenzy. 1975. Horizontal transmission ofturkey herpesvirusto chickens. 3. Transmission in three different lines ofchickens. Poultry Science 54:109-115. c. Cho, B.R., S.G. Kenzy, and S. 

### A. Haider. 1971. Horizontal transmission ofturkey herpesvirus to chickens. 1. Preliminary observation. Poultry Science 50:881-887. G. Discuss the expected environmental impactorsurvivability ofthe Regulated Biological Agent, and provide available scientific evidence. Turkeys are the natural host ofHVT. HVT is shed into the environment by feather follicles ofinfected turkeys. Prevalence of infection in turkeys approaches 100 percent. To our knowledge, natural HVTinfection in chickens from infected turkeys has not been reported. Chickens do not appearto be infected except through vaccination, afterwhich the virus replicates. The survivability ofthe RBA in the environment is expected to be similarto that ofthe parental HVT. The RBA vaccine will be frozen in a cell-associated form. This form is poorly resistant in the environment since it is dependent on the viability ofthe cells. The RBA may be shed as cell-free virus by the feather follicle ofthe vaccinated chickens. The resistance ofthis cell-free virus present in danderis much higherthan the cell-associated form. In our studies, the RBA is nottransmitted horizontally among chickens indicating thatthe amount ofvirus shed is not sufficient to infect naTve chickens and/orthat chickens are very resistant to HVT infection by the mucosal route (Study-discussed in Section Ill.C., page 35-36 ofthis report).
The host/range specificity ofthe experimental vaccine was evaluated with the non-target avian species safety test, Study-,discussed in Section III.C. (page 35-36) ofthis report.
A study design that includes the length oftime the virus can be recovered from sterile shavings (same as those found in commercial chicken houses) saturated with the RBA was conducted-.The test included the survivability ofthe virus in shavings at room temperature(- 25'C). Two hundred grams (g) of pine shavings were obtained from the Merial Inc.- The shavings were divided into two equal 100g samples. Each sample was saturated at a rate of 1ml ofsterile diluentto 1 g ofshavings. The shavingswere autoclaved for sterility.

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## u.s.Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype

## 3, Live Marek's Disease

## Vector, Code: 16N1.RO (Unlicensed) May 18,2014 Page 40 of67

Afterautoclaving and cooling to room temperature, 100 ml ofdiluted RBA containing- pfulml was added to onesample, and 100 ml ofdiluted licensed 

### HVT containing- pfulmlwas added to the other sample. The inoculated shavings were held at room temperature forthe i:luiation ofthe study.
On days 0, 1, 2, 3, and 7 post-inoculation (orwhen three consecutive days read no virus recovery), 5 g from the RBA shaving sampleswere collected aseptically and placed in a sterile sample bag containing 20 ml of sterile diluent. The contents were mixed and placed in a blender, then poured through a double layerofsterile gauze. The collected fluid was used forvirus recovery 

### by placing one millimeterofa 

### second filtrate onto chicken embryo fibroblasts monolayers. The procedure was repeated with the 

### HVT shavingssample.
On Day 0, the RBA samples resulted in an average of4 positive 

### HVTI protein expressed plaques and the 

### HVT samples showed an average of 1.7 plaques. No virus was recovered on days 1-7. This report was accepted bythe CVB on November 6, 2013. 1. Do the Donor DNA sequences orgene sequences associated withthe Backbone BiologicalAgent enhancethe ability to survive in the environment or increase resistance to therapeutic agents? The insertion ofthe NDVI geneshould notcontribute to the ability ofthe backbonevirus to survive in the environment since the insertion ofthe NDVI gene is in a

### non-e~sentia1, non-regula_torY'i"egion ofthe HVr vector. The survivability of

### HVT virus with orwithout the NDVI gene described in Section III.G. (page 39-40) 

### Showed no virUs recovery afterthe day of inoculation.
IV. RISKASSESSMENT Risk assessment 

### m8y be defined as the likelihood ofan 

### adVerse event occurring and the consequences ifthat adverse eVent occur's. An adverse event is

### deflf~ed as safety hazards to animals, public health, orthe environment. A safety hazard is defined as a danger, risk, or peril; absence ofpredictability associated with an event; oran expected or unpredictei:l event. A. Procedure The risk assessment is conducted based on safety characteristics ofthe vaccine. The safety characteristics are based on empirical data and established scientific facts. The completion ofarisk assessment requires that the vaccine microorganism is properly characterized. This information is included in the Veterinary Biologics Summary Information Format Section Ill that is provided by Merial, Inc.

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B. Hazard identification May 18, 2014 Page 41 of67

Hazard identification consists ofidentifying all possible adverse events to animal safety, public health safety, and environmental safety relative to the recommended use ofthis experimental vaccine, Marek's DiseaseNewcastle Disease, Serotype 3, Live Marek's Disease Vector, Product Code 16N1.RO, unlicensed.
Thevaccine thatwill be used in this proposal is very closely related to the USDA licensed vaccine product code 1A88.RO. The only difference between the RBA and the licensed construct- is the gene insert. This modification did not introduce any increased riskto animal safety, publichealth safety or environmental safety. Merial has submitted considerable in vivo and in vitro data for the RBA. These data demonstrate this construct is safe in mice, chickens, ducks, quail, pigeons, turkeys and pheasants. The safety profile ofthe RBA in mice and the other non-target avian species is the same as that seen previouslywith 

### the- construct (Licensed, Code 1A88.RO). 1. Animal safety

a. Summary No animal safety problems have been identified with this experimental vaccine. The vaccine was safe when tested in one-day-old chicks. The vaccine was also safe when tested in several non-target avian species and one mammalian species suggesting that the vaccine would be safe ifother nontargetspecies were exposed. The vaccine did not revert to virulence and was genetically stable through five backpassages in chickens (See Section Ill.C., page 35-36). Studies indicated that the vaccine is genetically stable at MSV, MSV+5 and backpass 5 (bpS) {See Section II.C.5.b.(i) and (ii), page 27-28}. b. Targetanimal safety: (i) Vaccination ofchickens The Master Seed Virus did not demonstrate any virulent properties when inoculated into one-day-old chicks at 10times the recommended field dose as written in 9CFR 113.330. A testwas carried out to study the possibility of reversion to virulence ofthe vaccine strain. Five passages were carried out. Infected leukocyteswere found in each passage. No abnormal signs were noted in the birds during the passages. NaiVe hatchmates were placed in contact with each group ofbirds receiving the backpassed virus. No virus was recovered from those contacts, nor any lesions seen at necropsy (See Section Ill.C., page 35-36).

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### Risk Analysis U.S. Vet. Lie. No. 298 

### Marek's Disease-Newcastie blsease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 1SN1.RO (Unlicensed)
(ii) Vaccinatlon/chal!enge May 18, 2014 Page 42 of67

No adv8rse immune interactions are expected with the use ofthis vaccine.
In Study-,susceptible chicks vaccinated by the SQ route with**~ --=---~**  ----

- - -

- X+5 vaccine were protected against GB Texas Newcastle disease virus when·challenged intramuscularly (1M) at 28 dayswith 104'5EIDsa/0.1 ml·dose. Thisstudy was accepted bythe CVB on August 21, 2012.
In Study- chicks hatched from embryos ------ ---- --

- were protected against Newcastle disease challenge when challenged IM on
·. 39
. - : - Day 28 With 10 · EIDso/0.1 ml-dose ofGB Texas NDV. Th1s studywas accepted bythe CVB on August 23, 2012.
In Study-. chicks hatched from susceptible 18- to 19-day-old embryos thatwere inoculated by the in ovo vaccine were protected against MDV challenge. 2013.

, suSceptible chickS vaccinated 

### by the**sa~**roUte 1 vaccine were protected against MDV challenge. This study was aceepted by the CVB oh December23, 2013.
(iii) Reversion to virulence Backpassage studies demonstrated that the RBA does not revertto virulence when administered to chicks. Supportive data includes: 

### • Study-, Reversion to virulence-

### five passages on chickens (See Section Ill.C., page 35-36) • Recombinant virus in vivo genetic stability analysis by Southern Blot and PCR, n, n+5 and bpS {See Section II. C.S.b.(i) and (ii), page 27-28}.
These studies also demonstrate the genetic and phenotypic stability ofthe RBA MSV in vivo [See Sections II.C.S.b., page 27 and Sections liLA, page 34-35 and III.C, page 35-36] (iv) Puritytesting The MSV and the prelicensing serials were tested by the applicant and found to meet all standard requirements of9CFR113.300 and the additional testing ofthe MSV as found in Sections II.C.3.c (page 26) and II.C.S.b.(i) and (ii) (page 27).

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(v) Identitytesting May 18, 2014 Page 43 of67

The expression oftheI genewas demonstrated by using fluorescent antibodytesting utilizing a.monoclonal antibody. The employed immunoprecipitation and Western blot technique detected an approximately- protein in the- sample that corresponds to the expected size protein. {See Section II.C.S.b.(iii), page 32}. (vi) Effectofgene manipulation on pathogenicity The RBAwas derived from the FC-126 strain of HVT, which is recommended for use in day ofage chickens or in avo 

### vaccination. The strain is avirulent for chickens.
The donor gene

# II has no known pathogenic ortoxic properties. No pathogenic ortoxic properties have been identified forthe productwhen expressed in the experimental 

### recombinant- vaccine (see Section Ill). (al Phenotvpiceffectofthe gene modification!s) (i} Attenuation ofwild-type HVTvirus The parental FC-126 strain was isolated from turkeys by 

### R.L Witter (Regional Poultry Research Laboratory, USDA, East Lansing, Michigan, USA) in 1970. Thevaccine strain FC126ofHVT, grown in chicken embryo fibroblasts, is used to produce CYROMAREX® and L YOMAREX®, the live vaccines against Marek's disease marketed by Merial SAS, Lyon, France; and Marek's Disease Vaccine, Serotype 3, Live Virus, product code 1641.00, marketed by Merial, Inc. Many billions ofchickens have been vaccinated with these vaccines (see Section II.A.1, page 7). (ii) Insertion ofthe I gene into a putative ORF The locus ofinsertion into the HVT genome is located in an intergenic region. No HVT gene was modified by the insertion and therefore, no biological effect are expected due to the genetic manipulation atthe cloning site. The same vector and cloning site is used in USDA licensed product code 1A88.RO, Bursal Disease-Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector. (see Sections II.A.1., page 7 and 11.8.1., page 11). (iii) 

# I gene modifications The NDVI gene (cloned as eDNA from NOVviral RNA) has not been modified. The exact same donor NDVI sequence has been used in USDA licensed product 17C1.RO, Newcastle Disease- Fowl Pox Vaccine, Live Fowl PoxVector, licensed October27, 1995. (Section 11.8.1., page 11).

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### 298 Marek's Disease-Newcastle Diseal;e Vaccine, Serotype 3, Live Marek;s DiseaseVector, Code: 15N(RO (Unlicensed)



# . '

. lbl Previous use ofdnnor genes and recipient microorganism

(i) Donor Sequences cir Donor Genes 

### May 18, 2014 Page 44 of67

The exactsame.donor NDVI gene sequence has been used in USDA licensed product 17C1.RO, Newcastle Disease-Fowl PoxVaccine, Live Fowl PoxVector. Licensed October 27, 

### 1995. (ii)
- promoter corresponds to the promoter/enhancerof

has been safely used in: 1) USDA**li~nsed** vaccine, Bursal Disease-Marek's Disease, Serotype 3, Live Marek's Disease Vector, product code 1A88.RO and 2) Combination licensed vaccine, BursBI**Diseas~~M~f6Ws** Disease, Serotypes 2&3, Live Virus, Live Marek's Disease Vector, product code 1A88.R2).
tel Phenotvpic effectofany genetic marker(sl The RBA MSVdoes not contain any genetic markers. (vii) Genetic s.tabillty The RBA isgenetically stable in vitro {see Section II.C.5.b.(i) and (ii), page 27-28}.
The genetic stability ofthe RBA in vivo was demonstrated through five backpassages in reversion to virulence studies in chickens (see Sections Ill.C., page 35-36) (viii) Phenotypic stability Experiments conducted by Merial, Inc. indicate thatthe RBA is phenotypicallystable in cell culture (see Section III.A., page 34). The phenotypic stability ofthe RBA MSVin vivo was studied in a reversion to virulence studythrough five backpassages in chickens (see Section Ill.C., page 35-36). The expression oftliel genewas expressed through five backpassages. The expression was maintained at 100 percent{see Sections III.A. (page 34) and II.C.5.b. (i) and (ii), page 27-28}. (ix) Alteration in tissuetropism The results oftransmissibility studies indicatethatthe proliferation/tissue tropism ofthe experimental vaccine virus is similarto its parental HVTvaccine virus {Section !II.D., page 36-37). (x) Effectofoverdosing Target host animals were inoculated with 10 times more virus than the recommended field dosewith no adverse effects observed (see Sections Ill.C., page 35-36).

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### Merial, Inc. RiskAnalysis U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

c. Non-targetanimal safety (i) Susceptible non-targetanimals 

### May 18,2014 Page 

### 45 of67

The safety ofthe RBA has been confirmed in non-target species in which the parental HVT may potentially replicate (turkey, quail, pheasant). The data indicate that recombinant RBA vaccine strain was perfectly safe in these species, as is the parental HVTvirus (see Section Ill.C., page 35-36). (ii) Virulence in non-targetanimals The results ofthe safety studies indicate that the host/range specificity ofthe RBA MSV has not been altered from that ofthe parental FC-126 vaccine strain, and thatthe recombinantvaccine is safe in non-target avian species (see Study- in Section Ill.C., page 35-36). 

### Itwas also shown to be 

### safe in a non-target mammal species (mouse). In virus proliferation studies, no virus recovery was detected from tissue samples ofmice inoculated with the RBA (see Section Ill.C., page 35-36).

In this study48 one-day-old chickens were inoculated with 0.2 ml ofl··· • diluted to contain -/dose. Eight one-day-old turkey_poults and 8 one-day-old chickens were inoculated with- of HVTvaccine diluted to contain-/dose.Twenty-fourone-dayold chickenswere inoculated with- ofHVTvaccine diluted to contain-/dose. Sham vaccinates included 21 one-day-old turkey poults and 26 one-day-old chicks inoculated with- of Marek's disease vaccine diluent.. The vaccinated or sham vaccinated birds were placed into units, with each unit housing six vaccinated day-old chicks and fourvaccinate or sham vaccinated day-old chicks orturkey poults. At 28 days the spleens were removed from 24 RBA vaccinated chickens and 12 HVT vaccinated chickens and the 10 sham vaccinated chicks and aqualitative analysis conducted on each spleen for Marek's virus recovery and NDVIstaining. On Day 42 the remaining turkey poults and chickenswere splenectomized and evaluated forvirus recovery. The results showed no spread ofthe RBA to HVT infected turkeys, na"Ne turkeys, HVT infected chickens or na"ive chickens. The study results also showed that there was no spread of HVTvirus from infected chickens to sham chickens but did spread to na"ive turkeys at a limited rate 

### (-38%). (iii) Possible outcomes of non·targetanimal exposure There are no expected safety issues with this construct in non-target species. The parental virus is derived from a commercially available vaccine strain. The studies discussed above suggest that these safety characteristics have not been altered in the RBA (see Seclion Ill.C. (page 35-36) and IV.B.1.c.(i), page 45).

Confidential Business Information Redactedrvierial, Inc. Risk Analysis U.S. Vet. Lie. 

# No. 298 Marek's Disease-Newcastle DiseaseVaccine, Serotype 3, Live Marek's DiseaseVector, c·ode: 16N1:Ro (Unlicensed)

2. Public health safety

a. Summary 

### May 18,2014 

### Page 46 of67

Hazards to public health are not expected. There are no reports ofHVTcausing productive infection in any species otherthan avian species, and no infection of humans has been reported despite accidental inoculation occurring during vaccine administration. The parental HVT vaccine strain from which this experimental vaccine was derived has been used as a commercial HVTvaccine for chickims in Europe without any adverse effects orhazards to public health. b. Probabilityofhuman exposure Hurilan exposure Will be limited to personnel administering the vaccine and conducting the field**Studies.~** 'C. · PafhOQenicity of parent miCroorganiSm

## ·rn humans



### ·- ,_ ,. -· - ' -t···

### ThSr~'are

## iiO

## repOrts·of HVT

### c8U~in9· ProdUdive infeCtion 

### in_ariY

## spedes otherthan avian species and n6 ibfection of

### hum~ns has beenreported [Tnpathy, Deoki: Pox.

### Dis~ases of Poultrv (1991). Ed. C'ainek, B.W., Iowa State UniverSity Press, Ames, iowa; pp 583-596]. The parent HVTvaccine has been us'ed as a cbmmefcial poultry vaccine in Europe with hb reported adverse effects or hazards to public health. d. Virulenc_e ofthe vaccine microor_ganisrn in humans The RBA has never been ihtentionally inoculated into humans. Thus the actual outcome of an accidental human inoculation is unknown. Based on the current knowledge and wide use of 

### HVT parental virus and HVT-vectored vaccines, the constructis likely non-virulent in humans.
e. Possible outcome of human exposure There are ho expected safety concernS associated with hurilan expos·ure based on the biological properties ofwildtype HVT and the parental vaccine strain. It is the opinion ofexperts in the human and avian cancerfield that Marek's disease and vaccines against MD pose no human health hazards [Purchase, H.G. 8nd R.L Witter,**~Public** health co'ricerns from human exposure to oncogenic aviat1 herpes viruses: A review'' (Appendix 2)].

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### Merial, Inc. Risk Analysis U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 

### 16N1.RO (Unlicensed)

3. Environmental Safety

a. Summary 

### May 18, 2014 Page 47 of67

The risk to the environment is low. The shed/spread capability ofthe RBA vaccine is limited. Some virus may be spread to the environmentthrough the dander ofvaccinated chickens butthe shed virus cannot infect other chickens indicating thatthe NDVI gene insertion did not provide an increased spreading ability to the virus. The hosUrange specificity ofthe experimental vaccine does notappear to have been altered from the wild-type HVT, which is restricted to avian species. The safety characteristics ofthe experimental vaccine in non-target avian species do not appearto have been altered from that ofthe recipient virus which was derived from acommercial HVTvaccine strain. Although the distribution ofHVT strains in the environment has not been reported, no adverse effects have been attributed to the use ofthese vaccines in the field. There are no expected adverse ecological effects associated with the proposed field trials,with this experimental vaccine. b. Environmental distribution There are 2 HVTvectored NDV vaccines currently licensed and being distributed globally. To our knowledge the RBA does not behave differently in terms ofenvironmental distribution than these 2 HVT-vectored NDVvaccines.

c. Shed/spread capabilities The results ofstudies indicate thatthe shed/spread capabilities ofthe RBA do not differfrom that of the parental virus. This study was part ofthe reversion to virulence Study-. CVB approved on March 9, 2011.The contact controls placed with each group ofchickens receiving the backpassed virus 

### did not show any virus recovered from spleens norwere any lesions noted at necropsy (see Section lll.C. page 35-36). In the non-target avian species studies described in Section Ill.C. no adverse events attributable to HVTwere reported forany ofthe vaccinates or contact control birds. All HVT recovery attempts from tracheal and cloacal swabs from the vaccinates and contacts were negative. d. Horizontal gene transmission/recombination potential The potential for in vivo recombination with field and othervaccine viruses was evaluated forthis vector in licensing- USDA product code 1A88.RO,

- was combined with virulent MDV, GA 22 strain and with serotypes 1and 3 of MDV's. This allowed for an evaluation of recombination between the viruses. The results showed there was no difference in pathogenicity ofany ofthe MD virus strains when combined with- and that no recombinant virus was detected. Study- was conducted with was combined with avirulent MDV strain (GA 22) and with serotypes 1and 2 ofMarek's disease virus. This allowed for potential recombination between viruses. There was no increase in virulence observed in the GA 22, Rispens or SB-1 groups co-inoculated with the- vaccine based on gross MDV lesions.

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### Lie. No. 

### 298 Marek's Disease-Newcastle DiseaseVaccine, Serotype 3, Live Marek's Disease Vector, Code: 

### 16N1.RO (Unlicensed) 

### May 18, 2014 Page 

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There were no statistical differences in average body weight between the GA 22 and the- ./GA22 vaccinates, the Rispens and the-/Rispensvaccinates orthe SB-1 and the -/SB-1 vaccinates atthe end ofthe 120 day observation period. The results showed no increase in palhogenicity ofany ofthe MD strains when co-inoculated with- (see Section III.E., page 37). 

# ' e. Potential fortransmission to Invertebrates: The potential for transmission ofthe RBA is expected to be minimal. The limited shed/spread properties ofthe virus should reduce the probability ofmechanical transmission post vaccination (see Section IV.B.3.c., page 47). Even iftransmission did occur, the attenuated properties ofthe RBA are not expected to be altered (see Section III.C., page 35-36). f. HostI range specificity: 

### Thereare no reports ofHVTcausing·productive infection inanyspeciesotherthanthe avianspeciesand no infection ofhumanshas been reported [Calnek, Witter: Marek's Disease. Diseases ofPoultrv(1991), Iowa State University Press, Ames, Iowa; pp 342-385; and H. G. Purchase et al., "Responses ofcell culturesfrom various avian speciesto Marek'sdiseasevirus arid turkey herpesvirus" (1971), Am. 

### J. Vet. Res., 32, 1811-1823]. Ournon-targetstudiesshowedthatthe insertionofthe NDVIgenedid notmodify the host range specificity ofthe vector. 

### g. Survivability ofthe 

### microorganism in the environrrient: The very limited amount of virus shed by the feather follicles via dander may potentially survive for months in theenvironment, similartothe parental HVTvirus and other licensed HVT-vectored vaccines. Thisshed virus is, however, notinfectiousforchickensbythe natural route. It may potentiallyinfect HVTila"iveturkeys. However, the prevalenceof HVTinfectiOn inturkeys approaches 100percentandwe have demonstrated that HVT-infected turkeys cannot be horizontally infected with the RBA. Experimental vaccine from vaccinated chickens did notspread to HVfinfected turkeys, na'ive turkeys, HVT infected chickens or na"ive chickens (Study-.The study results also showed thatthere was no spread of HVT virus from infected chickens to sham chickens but did spread to na"ive turkeys at a limited rate (-38%). h. Physical and/orchemical factors affecting dispersal in the environment: No physical and/or chemical factors have been identified that would enhance the dispersal ofthe RBA in the field i. Adverse ecological effects The demonstrated safety and lack ofproliferation ofthe RBA in other non-target avian and mammalian species (see Section III.C, page 35-36), and the established limited host range ofHVT reduces the risk ofspread to non-target animal species.

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### Inc. U.S. Vet. Lie. 

### No. 298 

### RiskAnalysis Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's DiseaseVector, Code: 16N1.RO (Unlicensed)

C. Release Assessment Priorto aProposed Field SafetyTrial Environmental release proposal Summarv 

### May 18,2014 Page 49 

### of67

The safety characteristic ofthe vaccine microorganism must be evaluated within the context ofthe target environment. Thus, the release assessment consists ofacomprehensive evaluation ofthe proposed release so asto detenmine 1}the location and characteristics ofthe release site; 2} the test dose and total amount of the experimental biologicto be used in the proposed study; 3) the frequency and duration ofresponse to the test material; 4} potential escapes into occupational, residential, or outdoorenvironments; and 5} the individuals, populations, or ecosystems thatwill be, or may be, exposed to the experimental biologic. Forthe purpose ofrisk assessments, proposed studies

### with live experimental vaccine microorganisms are classified as either contained studies orenvkonmental releases. AContained Study is defined as an 

### experiment in laboratory contained or physically contained animal facilities. An Environmental Release refers tothe release ofthe vaccine microorganism into the accessible environment. Planned introduction into the 

### environment refers to the deliberate release oflive microorganisms from contained facilities to the environment. Differences between small-scale field tests and commercial releases are considered in the 

### release assessment. VB-APHIS recommends and encourages the use ofsmall-scale field tests as appropriate-forthe experimental veterinary biologic. 

### Contained release Not applicable. 1. Environmental release Summart 

### Merial, Inc. proposes to conduct safety field tests to determine the safety ofthe Marek's Disease-Newcastle Disease, Serotype 3, liveMarek's Disease Vectorvaccine, unlicensed, USDA product code 16N1.RO in the target animal species, chickens, with the objective of licensing and commercial distribution. The Field Safety Studies will be conducted in at leastthree regions ofthe USA. A listoftentative recipient states is included below. These studieswill be conducted in chicken houses and hatcharies that are typical ofthe commercial poultry industry. The potential forescape and dispersal from the studysites is low. There are no known biological organisms, physio-chemical factors, ortoxic chemicals thatwould contribute to the establishment of the RBA in the environment. Personnel experienced in poultry health managementwill be conducting the proposed study.

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### Merial, Inc. 

### RiskAnalysis U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype3, Live Marek's DiseaseVector, Code: 16N1.RO (Unlicensed)

2. Tentativelocation oftestsites Arkansas, Georgia, Indiana, Iowa, Missouri, Ohio, Oklahoma, Pennsylvania, and Virginia 3. Characteristics oftestsites. 

### May 18, 2014 Page 50 of 67

Thetest sites include commercial poultry houses, multiple houses persite. Each housewill hold approximately 17,000-22,000 birds. The eggs/chicks will be inoculated atthe cooperator's hatcheries and then trucked to the house(s). The details ofeach test sitewill be included in the 9

### CFR 103.3 authorization requestforthe Field Safety Study.
4. Personnel Personnel administeringthe vaccine will consist of hatchery personnel underthe direction ofthe participating cooperator. Merial veterinary staff, and/orqualified Merial R&D staffwill bepresentforthevaccination and placementofthe chickens. Merial personnel will be involved in data management and in studyfollow-up. The Veterinary Scientist in charge ofthis Field Safety Studywill 5. Experimental design Numberofanimals Atleast20,000 birds will be vaccinated with thetestvaccine. Description ofanimals Eighteen-day-old embryonated commercial eggs orone-day-old commercial layer/breeder/broilerchicks of mixed breedswill be used forthis study. Route ofadministration In avo inoculation ofthe eggs or

**subcutan~ous** inoculation into the nape ofthe

**n~ck** of the chicks. Dosage Thevaccine for subcutaneous or in ova injection

### will be prepared for use in the mixing room ofthe hatchery. Thevaccine containerwill be asealed plastic IV bag, which will be fitted to a Marek's diseasevaccinating machine using sterile plastic IV tubing. Theserials ofproductthatwill be used in this studywill have been satisfactorilytested for purity before use in thefield studies.

COnfidential Business Information RedactedMerial, Inc. 

### U.S. Vet. Lie. No. 298 RiskAnalysis Marek's Disease-Newcastle DiseaseVaccine, Serotype 3, Live Marek's DiseaseVector, Code: 16N1.RO (Unlicensed)
Total amount oftest material May 18,2014 Page 51 of67

The vaccine is frozen in 2.0 ml glass vials each containing 2,000 doses. The vaccine will be reconstituted and added to sterile diluent at the ratio ofone (1) ampule per 400 ml ofsterile diluent forsubcutaneous injection and one (1) vial per 100 ml sterile diluentfor in ovo inoculation. Subcutaneous injection of0.2 ml or in ovo inoculation of0.05 ml constitutes a single field dose. With the exception of NOV and Marek's products, the chickswill receive all othervaccines according to each company's vaccination program. Approximately 100,000 doseswill be required forthe trial. Protocol The field safety protocol will be submitted to CVB for approval. Frequency and duration ofexposure Directexposure of people to vaccine could potentially occurat the hatchery. The animal caretaker/grower responsible for the**managerr1~nt** ofthe test flocks will be exposed to the inoculated chickens, but not the vaccine, forthe length ofthe field test period, approximately 60 days or less. The employees at the slaughter house are not expected to be exposed to the vaccine virus. No evidence exists to showthat HVT can be transmitted to humans. The recombinant virus cannot cause NDVeither in chickens or in humans because it does not contain complete NOV, only a gene from that organism. The duration ofexposure is not expected beyond the day ofvaccination. The method ofdisposing ofwaste All vaccine containers, diluentcontainers, tubing, needles, and gloves will be placed in waste containers (i.e., biohazard bags) and autoclaved or incinerated, or destroyed on site by industry accepted methods. The vaccinating machineswill be disinfected according to standard hatchery procedures. Dead birds and litterwill be treated according to each cooperating company's program. Decontamination oftest site Following vaccination, the vaccination area atthe hatchery

### will be disinfected and washed according to standard procedures. Crates used to transport the chickens will be returned to the hatcherywhere they will be washed and disinfected. The broiler/layer houses will be cleaned and made ready forthe placement ofthe nextflockof chicks according to each company's program. 6. Potential for escape and dispersal Lowshedding was seen but spread transmission studies were negative. Therefore, no escape or dispersal into the environment is anticipated.

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### Merial, Inc. RiskAnalysis U.S. Vet. 

### Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

7. Potential forestablishment In the environment 

### May 18, 2014 Page 52 of67

Marek's disease virus already exists in the environment as apoultry pathogen. Several live Marek's disease vaccines including HVTvectored NDV are used commercially in the United States under USDA license. The vector organism is derived from a licensed vaccine strain of HVT. The use ofsubcutaneous administration 

### and in ovo inoculation will minimize the chance ofescape into the environment. It is not anticipated that conditions will existoutside the laboratorythat will support perpetuation ofthe recombinant virus. No toxic chemicals or metabolites are known to be produced bythe virus, which could endangerthe environment. 8. Monitoring Monitoring methods 

### The monitoring ofthe field testwill consist ofmaintaining the records associated with the production of commercial chickens. Mortality and morbiditywilt be recorded bythe growerateach test location. The flocks will be observed daily for seven weeks 

### fOr adverse events related to the vaccine bY

### the grower or field 

### personnel ofthe cooperating company. Merial'sveterinary·staffand/or research department staffwill visit at leastonce during this period. These potential adverse events may include, but are not limited to, unusual postinoculation 

### reactionS and/or Marek's disease. 

### Any birds dying with clinical signs or lesions of Marek's diseasewill be necropsied and appropriate tissues examined for histopathology and/or retained forviral isolation. Any adverse clinical signs associated with the vaccination will be immediately reported to the veterinarian in charge. Ifthere is any evidence ofdecreased hatchability associated With the vaccine, diagnosticworkwill be performed to d6termine the cause ofthe 

### decreased hatch. Frequency of monitoring 

### Each flockwill be observed daily bythe growerorcaretakerduring the growout, which is expected to last no more than 60 days for broilertype birds (layers/breeders will growout for a longer period oftime). Birds will be observed at least once bythe Veterinary or Research staffand more frequently ifnotified that adverse events have been observed in the flock. Birds will be observed for clinical signs of Marek's disease. The safety ofthe vaccines will be reported in the field safety study report afterseven weeks ofobservation. 9. Contingency plans in case ofadverse events. No adverse events are expected but 

### ifadverse events attributable to the Marek's disease vaccine are 

### observed, the primary investigatorwill be notified immediately and the event documented in the study log. The grower, the cooperator's veterinarian and Merial veterinary staffwill workjointlyto determine a plan of action, ifnecessary.

Confidential Business-Information-RedactedMerial, Inc. U.S. Vet. Lie. No. 298 Risk Analysis Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 

### 16N1.RO (Unlicensed)

D. Risk characterization May 18,2014 Page 53 of67

Risk characterization integrates the results of hazard identification and the release assessment into a risk statementthat includes: 1) a likelihood rating; 2) a consequence rating; 3) a risk rating; and4) a discussion of risk.
Each likelihood and consequence rating is qualified by a degree ofcertainty rating and includes ajustification for the rating. The justification forthe ratings consists ofidentifying the applicable sections in the hazard identification and the release assessmentthat support the assigned rating. The risk rating is based upon the likelihood, consequence, and degree ofcertainty ratings. 1. Likelihood ratings Likelihood ratings are assigned foranimal safety, public health safety, and environmental safety based on the following criteria: Low= Medium= High= 2. Consequence ratings An adverse event is unlikely to occur An adverse event could possibly occur An adverse eventwill most probably occur
Consequence ratings are also assigned foranimal safety, public health safety, and environmental safety based on the following criteria: Low=
Medium= High= The consequences 

### ifthe adverse event occurswill not be severe (the adverse event is self-limiting and would have negligible impact). The consequences 

### ifthe adverse event occurs is moderately severe (the adverse eventwill have an impact, but it is not permanent, and can be treated). The consequences ifthe adverse event occurs is severe (the adverse event will have an impact, is permanent, and cannot be treated). 3. Degree ofcertainty rating Each likelihood and consequence rating is qualified by a degree ofcertainty rating that is based on the following criteria: Certain= Moderately certain = Uncertain=

Confidential Business Information Redacted The rating is supported by directscientific evidence. The rating is supported by indirect scientificevidence. the rating is not supported 

### by scientific evidence.Merial, Inc. U.S. Vet. 

### Lie. No. 

### 298 Risk Analysis Marek's Disease-Newcastle DiseaseVaccine, Serotype 3, Live

## Ma~ek;s Disea;.ieVecfor, Code: 16N1.RO (Unlicensed) 

### May 18, 2014 Page 

### 54 of67

4. Calculating the expected risk
Numerical values have been assigned to the likelihood, consequence, and degree ofcertainty ratings described above (referto Table 6). Each numerical value rating was derived from the importance placed on the rating ofeach category. The assigned numerical values are weighed to place emphasis on the seventy of expected risk. These values reflect the professionaljudgment ofthe applicant (Merial, Inc.).
5. Risk ratings The risk ratings are based upon the likelihood, consequences and degrees ofcertainty ratings, and the expected risk for each category. A total of 81 rating combinations are possible; e.g., likelihood lowmoderately certain, consequences low- moderately certain (see Table 7). Each combination has been assigned a riskrating of low, medium, or high. The assigned risk ratings wereweighed to place emphasis on the severity ofthe expected risk. Again the severity ofthe risk reflects thejudgment ofthe applicant. The low, medium, and high risk ratings are defined forthe purpose ofdecision-making as follows:
Low= Medium; High; AcCepiabl9 riSk; ver'y few concerns are associated with the proposal (does not justifydenying the proposal). Unacceptable risk; moderate concerns are associated with the proposal (either identify valid mitigative procedures or denythe prop-osal). Unacceptable risk; majorconcerns a:re assOciated with the ·prbposal (denythe proposal).

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### Merial, 

### Inc. Risk Analysis U.S. Vet. Lie. No. 298 

### Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

a. Animal safety Likelihood rating: Low (LL) Degree ofcertainty rating: Certain (C) Justification for ratings: IV. B. 

### t. b. i. IV. 

### B.1. b. ii. IV. 

### B.1. b. iii. IV. 

### B. 1. b. iv. IV. 

### B.1. b. 

### v. IV. B. 1. b. vi. IV. B. 1. b. vii.

. IV. 

### B. 1. b. ix.

IV. 

### B. 1. c. i. IV. 

### B. 3. b. IV. B. 3. d. IV. 

### C. 2. d. IV. C. 2. 

### f. Consequence rating: Low (CL) Degree ofcertainty rating: Moderately certain (MC) Justification for ratings: IV. 

### B.1. b. ii. IV. 

### B.1. b. iii. IV. 

### B. 3. b. IV. 

### C. 2. h. IV.C.2. i. Expected Risk Rating: 0.7500 Risk Characterization: LL.C.CL.MC

Confidential Business Information Redacted 

### May 18,2014 Page 55 of67

### Merial, Inc. RiskAnalysis 

## u.s. Vet. 

### Lie. No. 298 Marek's**Disease~Newcastle** DiseaseVaccine, Serotype 

### 3, 

### Live Marek's Disease\/ector, 

### Code: 16N1.RO (Unlicensed)
Risk rating: Low May 18, 2014 Page 56 of67

The safety risks to the environment are low. The host/range specificity ofthe RBA does not appearto have been altered from the wild-type HVT, which is restricted to avian species. Although the protocol ofthe field safety study does not include procedures for monitoring the shed or spread ofthe RBA into the environment during the course ofstudy, studies previously conducted bythe applicant indicate that the shedding capabilities ofthe experimental vaccine are limited, and the shed virus is unable to spread to otherchickens or HVT-infected turkeys. Furthermore, the proposed release will be confined to poultry houses. Thus, exposure ofnontarget animals and the dissemination ofthe experimental vaccine virus in the environment during the course ofthe field testswill be restricted by its use in poultry houses. This assessment indicates that one-day-old chicks or 18-day-oid chicken embryos will be vaccinated in commercial hatcheries, and subsequentiytranspOrted to commercial chicken 

### houses forthe duration ofthe field trials. All the waste material from the vaccination procedure will be properly disposed. The vaccination area at the hatChery and the crates used to transport 

### the chickens from the hatcheryto the poultry houseswill also be decontaminated. The RBAwill be used in combination with other Marek's disease vaccines (serotype 1and 2). Our recombination safety study has shown that co-administration ofthese vaccines did not generate a more virulent virus. 

### Anotherconcern could bethe recombination event

### beiw~en the RBA and a field HVT strain resulting in the horizontal transferofthe

# I gene. Since natural HVT infection ofchickens has 

### never been described, it is unlikely that the chickenswill be co-infected with the RBA and field HVT. Even if parental HVT is accidentally administered togetherwith the RBA, the homologous recombination that may occur could potentiallytransferthe NDVI expression cassette from the RBAto the parental HVf. The newly created vfrus woUld be verysimilarto and have the same 

### properties as the RBA. Furthermore, this newly created HVT may be shed at low levels but it is unlikely to be spread to contact chickens. The consequences ofsuch recombination events is 

### negligible. Conclusions and discussion of risk 

### The risk to animal safety associated with this proposal to conduct afield trial with experimental Marek's Disease-Newcastle Disease Vaccine, Live Marek's Disease Vector, is low. The recipient 

### virus used to construct the RBA MSVwas derived from an HVTvaccine strain that has been used as acommercial poultry vaccine in the United States and Europe. Safety studies conducted bythe applicant demonstrate the safety ofthe experimental vaccine in chickens and in several non-target avian and mammalian species. The proposed study will be conducted by personnel that have extensive experience in poultry health management. b. Public health safety 

### Likelihood rating: Low (LL) Degree ofcertainty rating: Moderately certain (MC) Confidential Business Information Redacted

### Merial, Inc. Risk Analysis U.S. Vet. 

### Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 

### 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed) May 18, 2014 Page 57 of67

Justification for ratings: IV. B.1. b. iv. IV. 

### B. 2. b. IV. 

### B. 2. 

### c. IV. 

### B. 2. d. IV. 

### B. 3. b. IV. B. 3.d. IV. 

### C. 2. d. IV. C. 2. e. vii Consequences rating: Low (CL) Degree ofcertainty rating: Moderately certain (MC) Justification for ratings: IV. B. 2. 

### c. IV. B. 2. d. IV. B. 2. e. Expected Risk Rating: 0.5625 Risk Characterization: LL.MC.CL.MC Risk rating: low Conclusion and discussion of risk There are no reports of productive infections caused 

### by Marek's disease in any species other than avian. The RBAwas derived from an HVTvaccine strain that has been used as a commercial poultryvaccine the United States and Europe, with no adverse effects on public health. Studies conducted by the applicant indicate that the safety characteristics ofthe parent vaccine strain have been preseJVed in the RBA. Only the personnel administering the vaccine will be exposed to the experimental vaccine virus. The other personnelwill be exposed onlyto the inoculated chickens. Studies conducted bythe applicantdemonstratethatthe shed/spread property ofthe experimental vaccine from vaccinated chickens is limited.

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### Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

c. Environmental safety Likelihood rating: Low (LL) Degree ofcertainty rating: Moderately certain (MC) Justification for rating: 

### IV. B. 1. b. 

### v. (b) 

### IV. B. 1. b. 

### vii. IV. B.1. 

### c. i. 

### IV. B. 1. 

### c. ii. IV. B. 3. b. IV. B. 3. d. 

### IV. B. 3. 

### e. IV. B. 3. g. 

### IV. C. 2. d. 

### IV. C. 2. 

### e. 

### iii. IV. C. 2. 

### e. 

### vii. IV. C. 2. f. Consequences ifadverse event occurs: Low (CL) Degree ofcertainty rating: Moderately certain (MC) Justification for ratings: 

### IV. B.1.

### c. iii. IV. B. 3. 

### e. IV. B. 3. h. IV.C.2.c. 

### IV. C. 2. 

### e. vi. IV. C. 2. 

### e. viii. IV. C. 2. 

### e. ix. 

### IV. C. 2. g. IV. C. 2. h. IV.C.2. i. Expected Risk Rating: 0.5625 RiskCharacterization: LL.MC.CL.MC Risk rating: Low

Confidential Business Information Redacted 

### May 18,2014 Page 

### 58 of67Merial, Inc. Risk Analysis 

### U.S. Vet. 

### Lie. No. 298 Marek's Disease-Newcastle DiseaseVaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed) May 18, 2014 Page 59 of67



### Conclusion and discussion of risk The safety risks to the environment are low. The host range specificity ofthe RBA does not appearto have been altered from the wild-type HVT, which is restricted to certain avian species. Although the Field Safety Study protocol will not include procedures formonitoring shed or spread ofthe experimental vaccine into the environment during the course ofthe study, studies conducted bythe applicant indicate that the shed/spread capabilities ofthe experimental vaccine are limited. Furthenmore, the proposed release will be confined to poultry houses. Thus, the 

### exposure of non-target animals and the dissemination ofexperimental vaccine virus in the environmentduring the course ofthe field tests will be restricted by its use in poultry houses.
V. RISK MANAGEMENT RECOMMENDATIONS 

### A. Procedure 

### Risk management uses information from the previous section to determine means of reducing or eliminating safety risks to animals, public health, orthe environment. 

### B. Recommendations Thesafety risks to animals, public health, and the environment are low. A risk rating of lowis acceptable risk, with very little concern associatedwith the proposal. The applicant recommends the approval ofthe proposed field test byVB, APHIS provided the study is conducted in accordance with the requirements of9CFR 103.3, and the filed protocol ofthe study.

Confidential Business Information Redacted

### Merial, Inc.

. RiskAnalysis U.S. Vet. 

### Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's DiseaseVector, Code: 16N1.RO (Unlicensed)
Table 6. Calculatingthe Expected Risk
VALUE RATINGS
Likelihood (L)
Low (L) LL = 1.00 Medium (M) LM =0.50 High (H) LH =0.10 Consequence (C)
Low (L) CL = 1.00 Medium (M) CM=0.10 High (H) CH =0.01 •tfthe Likelihood rating is Medium or High and the Consequence rating is also Medium or High

use Degree ofCertainty Ratings I; forall othercombinations use Degree ofCertainty Ratings II. Degree ofCertainty Ratings I Certain (C) 

## c =0.50 Moderately Certain (MC) MC 

### =0.75
Uncertain (U) 

## u=

### 1.00 Degree ofCertainty Ratings II Certain (C) 

## c = 1.00 Moderately Certain (MC) MC 

### =0.75 Uncertain (U) 

## u=

### 0.50 EXPECTED RISK [(likelihood) x(degree ofcertainty)] x](consequence)x(degree ofcertainty)]= Risk Rating

Confidential Business Information Redacted 

### May 18,2014 Page 60 of67

### Merial, Inc. 

### Risk Analysis 

### U.S. Vet. Lie. No. 298 

### Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed)

Table 7. RiskRatlngs
Risk Expected Risk Risk Characterization Expected Characterization Risk Rating Risk LL.C.CL.C. 

### 1.0000 L LM.U.CM.C. 

### .0250 LL.C.CL.MC. 

### .7500 L LM.C.CM.MC. 

### .0188 LL.MC.CL.C. 

### .7500 L LM.MC.CM.C. 

### .0188 LL.MC.CL.MC. 

### .5625 L LM.C.CM.C. 

### .0125 LL.C.CL.U. 

### .5000 L LH.U.CM.U. 

### .0100 LL.U.CL.C. 

### .5000 L LL.C.CH.C. 

### .0100 LM.C.CL.C. 

### .5000 L LH.MC.CM.U. 

### .0075 LL.MC.CL.U. 

### .3750 L LH.U.CM.MC. 

### .0075 LL.U.CL.MC. 

### .3750 L LL.C.CH.MC. 

### .0075 LM.C.CL.MC. 

### .3750 L LL.MC.CH.C. 

### .0075 LM.MC.CL.C. 

### .3750 L LH.MC.CM.MC. 

### .0056 LM.MC.CLMC. 

### .2813 L LL.MC.CH.MC. 

### .0056 LL.U.CL.U. 

### .2500 L LH.C.CM.U. 

### .0050 LM.C.CL.U. 

### .2500 L LH.U.CM.C. 

### .0050 LM.U.CL.C. 

### .2500 L LL.C.CH.U. 

### .0050 LM.MC.CL.U. 

### .1875 L LL.U.CH.C. 

### .0050 LM.U.CL.MC. 

### .1875 L LM.U.CH.U. 

### .0050 LM.U.CL.U. 

### .1250 L LH.C.CM.MC. 

### .0038 LH.C.CL.C. 

### .1000 L LH.MC.CM.C. 

### .0038 LL.C.CM.C. 

### .1000 L LL.MC.CH.U. 

### .0038 LH.C.CL.MC. 

### .0750 L LL.U.CH.MC. 

### .0038 LH.MC.CL.C. 

### .0750 L LM.MC.CH.U. 

### .0038 LL.C.CM.MC. 

### .0750 L LM.U.CH.MC. 

### .0038 LL.MC.CM.C. 

### .0750 L LM.MC.CH.MC. 

### .0028 LH.MC.CL.MC. 

### .0563 M LH.C.CM.C. 

### .0025 LL.MC.CM.MC. 

### .0563 M LL.U.CH.U. 

### .0025 LH.C.CL.U. 

### .0500 M LM.C.CH.U. 

### .0025 LH.U.CL.C. 

### .0500 M LM.U.CH.C. 

### .0025 LL.C.CM.U. 

### .0500 M LM.C.CH.MC. 

### .0019 LL.U.CM.C. 

### .0500 M LM.MC.CH.C. 

### .0019 LM.U.CM.U. 

### .0500 M LM.C.CH.C. 

### .0013 LH.MC.CL.U. 

### .0375 M LH.U.CH.U. 

### .0010 LH.U.CL.MC. 

### .0375 M LH.MC.CH.U. 

### .0008 LL.MC.CM.U. 

### .0375 M LH.U.CH.MC. 

### .0008 LL.U.CM.MC. 

### .0375 M LH.MC.CH.MC. 

### .0006 LM.MC.CM.U. 

### .0375 M LH.C.CH.U. 

### .0005 Confidential Business Information Redacted 

### May 18,2014 Page 61 of67

Risk Rating 

### M M M 

### M M 

### M M M M M 

### M M M M M M 

### M M M M M M M M M ·M



### M M H H H H H H H H

### Merial, Inc. Risk Analysis U.S. Vet. Lie. No. 298 Marek's o·isease-Newcastle Disease Vaccine, Serotype 3, live Marek's DiseaseVector, Code: 15N1.RO (Unlicensed)
LM.U.CM.MC. .0375 

### M LH.U.CH.C. .0005 

### LM.MC.CM.MC. .0281 

### M LH.C.CH.MC. .0004 

### LH.U.CL.U. .0250 

### M LH.MC.CH.C. .0004 

### LLU.CM.U. .0250 

### M LH.C.CH.C. .0003 

### LM.C:CM.U. .0250 

### M

Confidential Business Information Redacted 

### H H H H 

### May 18,2014 Page 62 of67

### Merial, Inc. U.S. Vet. 

### Lie. No. 298 RiskAnalysis Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek's DiseaseVector, Code: 16N1.RO (Unlicensed) 

# Appendix 1 May 18,2014 Page 

### 63 of67



## Pages 63-66 redacted duetoconfidential business information

Confidential Business Information Redacted

### Merial, Inc. RiskAnalysis May 18, 2014 U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, · Page 67 of67 Live Marek's Diseasevedtor, Code: 16N1.RO (Unlicensed) 

# Appendix2 Purchase, H.G. and R.L. Witter,"Public health concerns from human exposure to oncoge11ic avian herpes viruses: A review"

Confidential Business Information Redactedi'



### PUBLIC REALTH

**GO~C~S** 

### FROM l!UM!\N EXPOSURE TO ONCOGENIC AVIAN lfllRl'ES V!RUSES : 

## AREVIEW by 

### H. G. l'UIWIIASE



## R. L. WlTTER 

# DEC 12 

# 1985

1

### From the 

# u.s. Department of Agriculture, Agricultural Research Service.,

Jleltsville Area, llAR,C-west, Beltsville, Haryland ;W705 

### and 

# u.s. Department of Agticnlture; Agricultural Research Service, Regional l'ou1try Research Laboratory, 3606 East 

## Mt. Hope Road, East Lansing, Michigan 48823 Presented at the 

### Annual 11eeting

**o~** 

### the A,mer:l.can Veterinary 11edieal Association, Las Vegas, NE. 

### July 23, 19.85.

'•

### 2



## Ever eince tile**iilent1f1~atic;m** 

## of a**herpesvi;r~.~s** 

## as ·the**~!.llle** 

## of 

## Ma:r;eit•s disease (MD) ani;! 

## the isolat:i.oq 

## and lliie 

## of lhre vaccines against MD, there waa**con~ern** 

### among 

## some of 

## the lay 

## public and some saientiatil 

## t;het there lll!3,y 

## have hl!!e!'l a**~a1111al** 

## association bebi'een these viruses and hJ1lD8.11**~aneer,** 

## This.
concern was 

## projected as 

## an ino;rilinate 

## nwnber of 

## reports, pro and con, in the 

## scienti£1.c 

## Util:tature ove:r the past 

## two**dt~<::ades.** 

## l!.esol11:t:toXl. 

## of 'thia q!J'eat:ioll ¥18.11 

## needed tQ 

## make illforllled :decisions 

## about li¢ensing biQJ.()gics. 

## to ill!tdtet 

## safe a.nd 

## wholel!ollle :fo!ld 

## prodqcts ill, 

## !:he United St:ate11 l!lld :for**~qrtc** 

## a!:\1!. tll provide ln.t'omation fot conslltllers. Resu1ts .from statistical epidell!iology, serological epidemiology, ¢omp<Jtiaon o"f eti<1logic 

## agents,**1abo~al:ocy** 

### l!l!l,l!lllllll 

## a!ld 

## primate inoculation, 

## culture in mammalian cells., and**eUlllinst~c;on** 

## c;of**~;~ppPI:twisti~** 

## iJ:Uecdlln llll.<l. eitl:\llllstantial.**eV~<l.enee** 

## were 

## rev:l.ewei!. in 

## thi.a paper, the data**indit:a~ed** 

## that .no t:laar 

## evtd!!nce "of an !l!tiCll!:>&ic 

## re4tiopsh'lp existed 

## and, a,lthoug'h 

## addi1;ional studies 

## were possib:I.e, 

## there was enQUfih evidence 

## to support a .firlll .conclusion. on this btcsiS expert co!lllllittees sponsor<=d by 

## the goverlltllent cop:clud:ed tha.t MD was llot; 

## a puhli<:: )lealtb. probl<;llll.

## 3



## Cancer is one of tM m<>st dreaded diseases in modern times because of its high frequency, unpred.ictabUity, inevitable occ:urrenc:e, prolonged duration, and chronic and painful course often leading to deatb.. Tb.ere has been extensive research·in this centucy to try to identify the causes of cancer. Chel:dlcal c.atcinogens, such as eoal Q.r which induces eanc:e.t in chimney aweeps, have been clearly shown to·be one of the causes of cancer. Similarly, ionizing radiation can cause cancer in persona -pur)losely or inadverte11tJ.y**e~!lsed** 

## to 

## ;tt:. Agricu1tural chemicals like chlorinated bydr!lcarbon insecticides, industrial che!Dic:als like polychlorinated. biphenyls (PCBe) and. c.bemics;l c:onta!llinants like dioxins have become videly d:l.stribul:ed in the envirotl.mellt and sometimes in the food chain before they were shown to cause cancer in el!;pstimental anii!Uils. Wi<l.el:r used foods like: grains and o!l!feeds have become contaminated with mycoto:dns, some of the most powerful cardnogens krto'Wlr. Evev. natUtill cons·tituents o.f the dieU 

## QX most peopie such
as 01eats are incrim;i,nated wit4 causi11g lluman cancer. A h.arpeavirus 

## b presumed to cause Burkitt's lyl:dphoma, first described in Africa, and. nilsopharynge!il. cal:ciiloma, first described in Chi!lll. M:>te 

## recently RNA virusen such as human 'l!-cell leukemia virus have been ii\criminllted 1111 

## the cause of

. . 1
. . 

## acquired immunodeficiency syndrome in man. Enormous efforts have been

## 4 d:!,.r<;octed 

## to. understanding 

## at the molecular level the 

## sequence 

# ·Or events 

## that results in ca.Mer. Until t!Jeae events 

## are fully 

## understood l>y**~>detit:l.sts** 

## ap.d the 

## i;o.fqrmation is disseminated to 

## the general public**th~re** 

## wi11 bE! a c()l,l.¢!lfil. by the**g!ln~ta;l.** 

## Public 

## that items :l.n theit food 

## lind envir()nment could induce

A 

## review of :the public. health 

## aspects of animal tullior viru$e,s waf! publ:.b'hell itt

## 1!!~~.;! lp. 

## it, Wl\:l.elen exam.i;1ed tht\ 

## re,lationsh:!.p bel:w!!en a 

## vad,ety 

## of viruses knoWll. at that 

## time and human disease 

## i:aeluding cancer .and 

## :1u1Jllqncysuppression. l!e 

## eoltelU:ded that "ovet 

## 100 .!lllil!llll 

## tllmot 

## viruses have been ia()lateii !llld. onlY one (Yaba Tllll!or 

## Virus) has 'l!lino-r 

## pubtic sd,gni£ical1ce•" However.• ·the herpesvirus of M;;r.<!.k'l! !Uaease (M!)) 

## ·wa.a t!9t**iso:l.~•:te!:\** 

## i.ln.til 

## 1967' 1•4 and the.

## herpesviruli! of 

## turkeys (HVT)

## wa~ not

## is~>lat!>d Qntil .1969,5

With the idendficad<lil of a 

## virus as the .;ause !:If 

## Mil 

## and publie:lty surrounding 

## the devll!lopment< .!lf vaccines 

## against 

# w,6 theta 

## has been concern ilnlong s()l!le of the 

## lay public and some. aeietrt:bts that exp<:>sute to the Mil 

## virus and**t~lsted** 

## apatho$enic vl1ccil1e vltu.sea 

## in**·~he** 

## envlrot\ilient, or eating pollltey 

## :tllfected with 

## these vinses, could ,contribute to the 

## l!aUse .o.f 

## C!lnCeli ill. 

## :ma11. poUlt-ry and disease in man needs to be resolved so that 

## infomed d,eci!lions can

### 5



### b~ 

### made about licensing of biologics for domestic animals and man and about the $af!i!tY and wholesomeness of poultcy and poultcy products, Also, the presence of such

**virt~ses** 

### in meat can be used as a non-tariff trade barrier to prevent the export of poultry and poultty products, X.aatly; such infotlnetion is needed to assu:te poultry :faoaers and process,Lng plant workers of occupational

**saf~ty** 

### and tQ 

### assure

**consum~rs** 

### that poultry and pouitcy products !i:te safe and wholesome to eat. Mimy 

### &dentists have attempted to examine the association between cancer virt!llea in animals and cancer in man using diverse approaches. The 

## MD virus is ubiquitous and vaccines against 

## MD are used woddWide (approximately 

## 3 b;lllion

**doae~>** 

### of

**l:tv~** 

### vaccin!i! !lr!l U1fed in

**th~**

# v.s. alone) on meat'"'grodUc:lng (broilers) and egg-producing animals. llUllli.ns have been e:a:p<Jsed to 

## _MD virus since dilmestication of the chickan and to vacJ'!ines against 

## MD f<Jr almost 

## 2 decades. There has been ample oppot"tunity for humans to contl;l<:t the virt!Bea. Thus an association between these

**Virt~ses** 

### and human 

## cancer could be J:evealed in epidemiologic studies. There bas 11.lso been e_xtenaive exper;l;mentation with these viruses in 

### chickens, laboratory anituls, primates 

## add humans.

### hUJ!18.n 

## disease is 

## often contradictory and has 

## never b!lett

**~:eviewed** 

## in depth, 'the 

## last review focused 

## almost excluSively on 

## RNA l:llmot 

## Yiruaell, ,partil!uiady 2
.... ·. .· . . . . . . .. . .... 

## feline leukemia virus. Thill comprehenSive rsv:l:ew 

## of the literatute 

## will provide r!i!levant 

## data to 

## guide sde.ntists and 

## resl'l!:r;c.h 

## !ldmiuiat,rat:ors ;in 

## deCJ.ding where add1tiona1 'rea·earcih 

## is neeaed .and what 

## rtllJearch approaehes tl> 

## take, It Jrl.ll provide adlll:t;nistratora of ;t'edew and st!lte 

## llcUon a.nd regul;ato:ey 

## agencies Jrl..th t:'he, 

## most c:ompl;ete comp:J:la.tiou 

## or infotmat:l:oa 

## published 

## so far to det:etlidlle whether 

## a hUillan health 

## hazal:d ensts, the nature 

## and extent 

## of the hstal7d an.d :!fh!tther :ae.tions are n.ecesaaty. 'llhb :review will be !lf 

## inte:t:est to 

## the poult:t:y 

## ;tndustcy, Whl;ch 

## would !luf:fer 

## severe 

## <fconomic

lil. th;l.s 

## TeViewwe b11'le '111.1lllllllltizlld the 

## d11ta, developed conclusio11s that follow 1ogically from 

## the data, and discus.aed ph:l.losophically 

## sol!le of 

## the dUell!lllas in 

### the scientific process. Mention :l;a 

## made of 

### efforts 

## !J.y the G.overrunen.t 

### to 

## deal w:l.th the 

## issue by 

## e$tablishing colllniittees and eonill:dasions 

## td study t.he 

## avdlsble 

## data. We wlll 

## eon.f.';tne our

**O\>li!~n:·vat:l.<lns** 

## and temarks to the public bealth 

## B!!peets of: 

## the avian

**he~:peavixuaes** 

### of MD 

## and the 

## li 

## VEl vit'lls vaccines, including HVT, 

### that 

## preve!lt MD.



# -. i 

# 1

**~** 

# 1 % 

# i

### 7 

## Avian Oncogenic "!lidRe:tated Viroses

Marek's disease is a lympho-proliferative disease of chickens caused by 

## a he:flleilvitt!113•4 and- characterized by a

# mononuclea~; cell infiltration and 6 

## proliferation in peripheral nerves, gonad, iri!J., Viscera, musc:te and !Jkin. lt is probably the most commonly occUrring neoplasm <if 

## any aoi1l!41. In :1.!!67 it caused annual losses in the United State11 in exc,ess of $150 mi:Llion su<i similar :losses were incurred in other countries of the world. Losses at'e <iue to <ieereased egg production, mortality from. MD, incl:'e:ased mortalitY from ot:her causes, and condemnations of carcasses in 

## the poultry processing plant. The diSee..se and losaes from it occur wo:rldwide

# J

The herpes '{ictus thal: 

## eause·s MD is not transm:l,tt_ed vert-ically from parent to offsp'fing.6 Therefore eggs are free of the virus, It 

## is transmi.tted horizontally in the dust atld dander that sloughs off from the feather follicle epithelium in which the virus matures to its infectious form. The virus

.

# -'

- - -- - 

## survives ·1n the environment for several mol.lths at room temperat.lire, !-bat flocks become infected With the virus within 2 to 3 

## weeks after hatching and

### 6 

## the virus spreads to most bitds with:!.!l the flock by -a:pprpximate).y 7 

## weeks of age--tile age When 

## most broile.-s are**!!lau~:~htered** 

## for human consumption. ln live b:!.rda, the 

## virua persists in i!lfected cells in the blood ror several years and probably indel;initely. Virus can be isolated from virtually all body tissues, including muscle (meat), for ptolonged 'periods. llowever., S:ggs
are consi¢ered free of v!ru!l• Tits 

## vitlls :!.8 

## h:!.ghl:t ceU-aasociated**i~** 

## a;ll. organs 

## except 

# the f9th<at folliCle !I 

## pitheU!llli. Tlte eell•e!lsoe1ated 

## viru-s .i!J**l1!)l1'"'~1iiC tio~s**  

## o)lce the eell11 die, but the •eelt...,free Virus 

## iu the skin remain.a iniect:i()us. Because 

## the vi:rus l_a fou11d wolildwiile 111 

## chickens and is found. 

## in. jungle fi»rl iii 

## lihei-r natural 

## 'hl!.bitat:, it :!.a**l.:Ue~l.y** 

## that**~nk:l.n<i** 

## 'h,as**eot~s~ed**

### v1rus~in£eete<! 

## ehick:en meat Since long before domest:ication of 'the ehiekan.**Vaeai~es** 

## are uaeli to control. MD 

## in all eoun:trJ.I!is 1>f the world thll.t 

# h?.ve intensive pou:ltl:JI' production systems. Vaccilles 

## are <:omposed 

## by the eel1-ifree 

## l.yopM,l1z;ll'd**v:l.l;'\1~** 

## or eeU-•ssaoeiated vtrus in 

## ¢ell \"!ultures 

## :Whi.eh 'h,ave 

## "been froz:en in Uquid nitrogen. The virus most co!IWiotily used is 

## HVT, wh:!.dh is a ndn<-pstliogen:!.e 'litua anl::!.geuieally rel«t:ed to MD 

## vit\ls wi!:tch WJl.!l 

## isolated. from

## turkey~.5 Other vaccines :luclu.de atte11usted ID viroses llu4 na.t\lraUy apathogenic MP vi.ruses; The uaturaily apsthogenic virus is gaining more

## 9 acceptance as a vacci.ne worldwide. A bivall!lnt vaccine incorpora.ting .!IVT and a naturally !lpiithogeni<! Virus wall :l-icensed in the United Stl!teli! in October 1983.

All vaccines are injeeted into**ch1c\t~** 

## on the day tb,ey hl>.tcb, The vscc!ne V!rusea repli<:a.te in the chickens and persist in them for life. 0£ the three vaccine viruses only the naturally apllthogenic one spreads readily horizontally from chiclten to <;hicken though some o£ the attenuated viruses spread poorly. The vaccines are highl.y effeetive in prev¢ntina the l.esiona ·and economic losses from MD; hQWever, they do not prevent infection w.!.th Ml V!rua nor the .spread of MD virus. horizontally. M:)st of .the·egg-type chickens throughput the world .and. !!lost of the m.eat-type chickens :!.u some eount:riea suc.h
as**~:he** 

## lillited States, Canada, and Italy are vaccinated. Thus, lllost c.hi.ekens. at slaughter epnta:!.n bpth the virulent MD vi.us and the vaccine vi.ua.

Nomenclatur.e

The pathogenic MD Viruses of chickens, the apathogenic Sill):!.lar viruses of chickens, and the li\1T are serPlPgically distinct from oM another. Because 

## Wlt and the apathogen:!.e viroses do not induce MD, they cannot be tefetred to
as MD Viruses or as a'lhn oncogenic herpesviruses, Also, because HVT occurs naturally :!.n turkeys, it.cannot 

## be referred to as a chicken virus. However,

> '

## 10 t:he**virus~&** 

## are antigetdell:lly e1.osely**~~!lated** 

## :though distinct from 

## one another, 'i.rhey**hllv~** 

## been ea.1led serotype 1, serotype 2, and serotype 3 MD 

## viruses,**reJ~Jpeet;L**

## ve1.y. For 

## :the purpose of this papet', we will teret 

## to tlielil


## as avian (onoogen;tc or**oneogenically~re.J.ated)** 

## hetpesViruses (AOHV),


## Review b:r Expert**C®~~J~ittees**


### bi. 

## tne Udt:ell 

## Stat!!$ o:f AD!eri<1a**(USA):~** 

## veterinary bil>los1c.a 

## are 

## re$Ula,ted ltnder the authority l;lf 

## tile 'Vi;rus

.. $erwn 

## Toxin 

## 1\;c.t.
. '!)he l1**~s,** 

## De-partll!ent :Q:f 

## Ag:i:lc,u;lt:uJ:e (USDA) 

## is 

## responsibl-e 

## for**~rsllluring** 

### thlll: !lllch 

## biolog:i.:¢s ate pure;, 

## $life, patent and**~ff:l.oac..ioill!l·** 

## With 

## l:ne disl!nve:t:r of the eaullativi\ he:t-pe's v,irtis' o£ W 

## iD. 1llli7 and t:ne tso1a:d:<:in 

## of t:he. 

## l!etpeav'irus of 

## tuik,eye (lm) 

## in 

## 19&9,15

# qQ~stit;ill$ were :t.a.iaed 

## about 

## tlle safety of 'Mil 

## virua-:tnfeoted e'htekene,
Dr,**J~i~sd.e** 

# t,. St<!:Lnfe1d, S.n'!."ge_on General or the 

## Pu'liUc. Health 

## Ser'ltLce, c.onve11ed a 

## cqmluit:tee. 'ln 1970 

## t.o re'Vil2W tlle atatus an<l. current 

## re1ati<lnllhip of



# I

## 11 

## a . .· viruses of the avian .leukosis

**comple;~t** 

## to h\llll8n llisM.ae. Dr. Steinfeld made the folloWing specific pointll•
"Even thqugh there is no known S!>SOciation between viruses which are colllllton in fowl and in chicken eggs to human disease or any known haeard to human hell.lth, l, 

## lieli!!ve t!le only rational. goal l(e l!ho.ul4 at:rive £ot is elimination of those viruses from. J;oth eggs anl,i poultry

**~o t~f:**  

## the:rte can be n-o posa.fbl.e human health .hazard associated ltith their .i.ngestion and so the fowl pop\U.ation will be a more heal.thy one."
Attached to the lett;e:r was a repore which. stated in part 

## "« large ntllli'Per of key investigators who have apenf: many Years atydying the av:l,an

**at~,d** 

## other animal cancer viruses looking for li.nks with human cancer having encounteted
no instances which could reasonably lead. to the postulate that; these

**~::ancer**

**virus~s** 

## have 'been naturally transmitted to lllSn OJ:" 

## even, for that: mat:ter, to other animal species. Thus the

**evidet:~ce** 

## and scientific literature, as well as the opinions of the beat scientistll<.in thb 

## field, all add up to one aj. L. Steinfield, Surgeon General of the Public Health Service, Public Health Service, Department of Health and Human Services, Washington DC. Personal communication.

## l;!oncl.uaion.--not .only is there no eit.!dence !:hat poultry**produc~ll** 

## .ar10 'a**hs~ard** 

## to man 

## as a possible cause 

## of cancer, but 

## that the 

## data strongly 

## suppo;i:t a

Prior 

## to**~h'i'** 

## ol11gbal Ucel\llllre 

## o:l; the herpesvirus 

## o.f tutkeys 

## (I;lvT) .in:

## 1r;~to.,**6** 

# tne USDA 'r'equested 

## the Dep;u:tllienll 

# t:l£ l!eaith 

# and**Hu~n** SerVices t;:i 

## reV'lel1 the pubU:e**ll~~>l;~l\** 

## imp\lii.cl(t::l.ons 

## 9£ l!ti':¢11 

## an a:cdon.**l~J~:perta** 

## were**~al;ted** 

## to recview 

## the Uteraja,n:e 

## and 

## all infotmation 

## .avaiiable. cThey 

## ·conoiuded tnat 

## Ucendng 

## the vaccine woUld pol!a no 

## public 

## health. problem.b



## In 1983, 

## the USDA 

## raised Sim:l:lar concerns regardcing 

## tb.e 

# licensure '0£ 

## the SB-i M> 

## va<:eine. 

# An A<fHoc Commlttee ·of experts again reviewed the present s•ate of 

## l<liowledge, ''It was 

## t;.he**co)lill!iJ:tee•~** 

## opfni<l'Q.**~het** 

## wilth:l.p: !:he 

## 'l'hd>t;$ o•f

bPersonal 

### cO!lllllunication. D. E. Espeseth, 

### Veterinary !Uo:logics 

### 'Staff, 

## Animal and 

## Plant lledth In:feC.:tlon Sex;vice, l;!yat.tsv11ie, 

## MD 2071$2.

### the 

## a,va:l.l.able data there was 

## no r.euon to anticipate any public health

•l>. :C 

## problems from tilling thea<!! vaaeines."

ll.eV.i<!!W of the Lite!:at11r!! !jtatistical Epidemiology

If there were a statist:l,cally signHicant correlation between the denf!ity of potilttY (ntllllher produced in a country) and a pJlttictilar type of cancer in humans. one·might .infer that 

## there was an etiologic relationship between t:he two. Several adentists have attempted to show such a correiat:l.on (Table 1),

The death certificate files for the state of Washington 

# tor 19.50 to l.967 and the state of Oregon for 1950 ·to 1964 were uamiMd using a case-controlled methQd,8 Among 5368 case-control pairs, there 

## ·wa.a •a etathticslly significant association between fa:rm'ing occupations and death from leuke!l!ia
<!Personal cO!IIlllu!l.iMtion. G. F. Vande Woude, Laboratory of Molecular Ontology, NatiQttal Institutes of Health, Bethesda, MD. Currently at NC! 

## .Ft'eduiek Caneer Research Facility, P.O, l!ox B, Frederic:k, MD 21.701.

## and multiple lll.yeloma. No 

## such aallociation was seen ior: 

## Hodgkin's di.;leaae, 

## reticulum. cell sarcoma, or other lylll.phomas. Poultry**fa~era** 

## s\l,awe>d 

## thE>**hij!ihe~Jt** 

## propartionste cl!,lle excess, i.e., ten c/ilses dyina fral!l l.eukem:l.a li!lld 

## onlY' 

## 'Qile tlu!;tched control, 

## a difference that 

## was statistically sJ,¢ficant at P.i.05, There wete h:l.gbet frequenciea 

## pf leukemia deEJ.thl! amon11 orc:hard;l.ste

## ,wb.aat faJ:!llll:i:'ll, dairy**fa:~ers,** 

## ci!.tt:le**r@ch•~:rs, n!lr!le~n,**  

## faJ:ll!'!!rll (1!-o 

## explanat:l.on given), and farm 

## lahor.ats though the 

## difference$ 

## wete 11Q,t, 

## statist:l,cally**signif:lc~t.** 

## Ail @AlYI!il.s 

## Qf 'de!lth 

## in, 

## Pa.litorn.ia ,fal:llt sQ.d 

## npA-fllrm**reliiJA~ts b~1:wee~**  

## 1959 lind 1961**shaw<~d** 

## a l!:l.gnificant**(P~0.05)** 

## reduction 'in mort!l11!;y**f~olll** 

## all
cancer 1n 

## fatm residents though 'the 

## t<>tl!i fno,rta:Lity was si!D:ll;i.'l:' ill 

## fartl! !lnd ' ' 9 ' ' ' ' ,' ', '**non-fa~ r~l!li!i.ents.** **~rtality** 

## ·frqm leukelllica was fn,:r!!aliled tl!ou;gh 'ti.Q,t 

## signii;ic"'!ntly so. 

## ;the**pr<~~eedures** 

## uaed involved calculating,

# average 'l\l,ortality 

## ratea ftom the 

## 19'60 

## li.S. cCensus for Cl!J.:I.f(lrnia and lilli!'lg 

## these rates llfl 

## s!:<!n\ia.rd!l 

# to**~,:omput~ a~jll!ct!l!i**  

## numbers of 

## dea:tha for**Jla~** 

## \liA<I 

## non.,.f,arm populations ,and to calculate atanda:tdbed motta:lity rati(>s**betw~en ~eated**  

## lllld accrual d!laths.

### Th~ 

## possibl,e**r~l;ttionstup** 

### be

## tween h!l!ll4n eance'!1 d,eatha and**~xpoaure** 

## to poultcy was studied in 1,019 coll11ties in ten southeastern states for 1950 to -1969.10 Deaths from uterine cervical and ovarian cancers and from multiple myeloma were si$U:I.ficantly**(P~.**

## 01) higher in counties with high poultry popu1atione than in counties with low poultry populations. However, the sigtt:i£icauee for the 1a,tter two cancers disappeared ..men the data w!lrl!! compared 

## w1th data from the total USA. No excesa ri!lk of lymphomsa, Hodgkin'a dis$i!ae, .attd leJllcemia in people oc.eupationa11y exposed to poultry Wall 

## dete_cted.

In another stUdy, records of death certificates ft:olii Nebraal<a for 1957 to 1974 were exam:I.Jled.11

. LeukeiJU.a death3 Cl084) were each matched with two deaths (2168) from other causes for age at death, year of death, county of reSidence, ra.ee, and sex. Thera was an elevated dsk 

## C>f ll!!ukemia**amo~g** 

## farmt<rs which was statistically significant (P<0.05) for farmers boru
3

- - - 

## between 1901 and 1943 

## or who died between 1957 and :!-965. When the mortality
was st:rati.,fied by county of resi.dence and :comvared with various agricultural**p~toduc.t191l** 

## fsct()ra, there was a significatttly**(P~O,()S)** 

## increa.sed incidence of .leukemia among farll!ers from heavy corn-producing counties. Also**the~te** 

## was a higher risk from acute**unspecifi~d** 

## leukemia lll!loug famera from heavy corn, hog, and**chicke~** 

## producing and herbicide-using counties. The study using s:l:milar methods was conducted in Iowa12 and involved 1675 leukemic deaths

## 16



## leUkemia. was 

## also higher 

## llli!Ong certain 

## age groups oJ; faP!lers in <:OU!ll;ies 

## wLth high soybean, :i!otn end egg prod11ction and largest nill!lllai1 <if 

## acre$ t;t:eata\1 with h<>rbillides. U:>rtality from 

## unspecified lymphatic le\ikel!!!a Walil 

### assocll;.j;ed 

## wii;h


## corn per acre; nlllllber of 

## milk l!ows and numb.er of 

## eag-lay!ng chickens.**~not:hllr** 

## aase-conl:ro1l,e4 

## study 

## was conducted 

## in 

## Wiat.ons;l.n tieing I!IOI:ta1ity 

## data from 

## 1968' t{) 

## 197613 il\"vol

## vtng 411 ileath_s from ®:l.til)1e 

## m:Yellllllil 

## and' 926 

## carEifUll,y aelect_ed ,cQtltr!lll!.**~rtal:!.**

## ty from multiple myeloma was signHicantly (1'<.1.15) 

## higher <Uilbng**far~~~ers** 

## than**non~fatmers** 

## pll.rticu.liltly 

## those residing in**c-o~,~nties** 

## high in chicken inve)l.t:ory, 

## fertili_zi!t< 

# use• ·and 

## acres treii.ted -with 

## Jusectic:ides.


## An iutetesting E!p:l.d<lllli<:>log:l.cal**~>urvey** 

## o;f**~aophag!Hil .c:an;:eJ:_~** 

## waa condue:ted

# it~ North Ctdna lletwe<l!l 

## 1969 111td 1971.:1'1,d !he 

## mortality

# xat~a in 

## .the two :highest**eout~ti.,s** 

## wa11 Ii,ear1y 97 

## d111ea t)!E! 

## !llorta::Ut:y t<rte 

## hPIII t'he 

## esophllgeal

-, d

## Miller RW1 

## Vital diseases. 

## Childhood 

## Cancer Etiology Newsletter 

## 1977; November 15, 844.



# I ! 

# l

## 11 alllong humans pa]:alleled the incidence of esophageal cancer among chickens. there was a higher incidelfce of nitrosamilfas in food samples £roll! the high :!,nddence county than the low incidence county. No statistical analyses 'Were performed.

From the shave,. there are three possible conclusions. The first is that the relationship betwee.n h1l!Jian cancer and cancer in chickeos (including W} is coincidental; the second is that an unknown factor such as a chemical or envirotllllentsl factor on chicken farms is responsible for both the cancer in ch;l.<;kena .and the cancer in humansl and the third is that there is a ea.usal rel.atlonsl:dp between csn¢er in chickens and <::ancer in humans. From theae data, one cannot select l!lllong these alternatives and so no conelusio'!lS with

**reg~rd** 

## to the public health

**implic~tiona** 

## of 

## AOHV can be drawn.

Serolozical Epidemiology

The exlllllination of human sera for antibody to AOHV and the examination 

# or avian sera for antibody to human putative caneer viruses have also been used t -_ - - 

## to show the relationship between cancer in chickens and cancer in humans. Of 64 healthy human ;Jubjects examined by the indirect fluorescent antibody test for antibody to MD virus, all 

## but four, who were young subjects twenty years

### 1.8



## . . 15 old or younger, were**pos:l.t~v~~.** 

## · Conve.rsely, of ·eleven ·ch:lcken sera**e~nl!!!l** 

## for ant;!:b0dy .to Ell virus·, eight were positive.**Anti~M!l** 

## titers ranged from 

## lUll to 

## >1Hi40 .and ant:l.-EB titer$ 

## va,ry f;tom 

## 1:.10 to 

## 1:40, A subseq.u-ent**-~-tl.lOy** 

### ;f.rom :the .SB.Jliti 

### l&bora_tv_r-f examined seta 

### from 

## .7.2 _humans -whose ·work wau 

## intiliUttely**~oune~ted** 

## vlth chi.l!ltens 

## i!lld £rom 

## 72 of'f:i.ee WQ;tketa 

## ·WhO ·d:i.d not bave much contact 

## with c:hiekens. Seta from 

## .70•.9% and 

## /'6.4% of the wqrk!!r& 'in 

## pouJ.try :t'ltms hild anti...;)>l) 

## and al:lti-EB 

## v!tus ant:ibody tite'rs 

## of 

## 1:160, while only 

## 20.8% cand 

## 32.()% of the cont:r:o1 sera had ·antibody·respect;ivi!ly,

## 16 Also, of the 48 

## seta from cynomologus monkeys, twelve had antibody to MD 

## virus and



## In a**sub•u~queJit** 

## l!tudy \l,n 

## 'the 

## l1SA, '205 hW!Ian 

## sera were examined fO.r '1:1 an'tibody 

## to HVT and 

## MD 

## virus, The sera came frol!! :J,>eri!QilS 

## w;lth a, 

## v!rr;l.ed amouqt 

## of exposure to av:t!ln**h~qr~~** 

## viruses, from BuJ;k:tt:t'a**l,ymph'Q~** 

## pa.t:lents and from students. Sporadic aam,p!es had 'titers ·in 

## the fluorescent antibody 

## tes,'t 

## :of .1:5 

## or hlQ. 

## Only 

## two l!<llllpl:i!a had 

## a titett of 1:;!!), 

## Neue of the sera posit! 

## ve 

## hy the' fluorescent an;tibody 

## tel!t had li<!Ut.-aJ,izif!,g a!Xtibody,**Th<~;r¢** 

## was 

## no 

## obvious re!ationship 'llel:Ween the 

## extent 

## of .contact With 

## .AO!IV .and 

## d\e 

# '

. ' 

## presence of ·detectable antibody,

## Th,e reasons for the cross reactions between human 11era and AOHV and vice ve:rsa are



## un~wn. They coul.d have been



## ~aused by exposure of the chickens and h9'Jilll18 

## to an !lgent or !lgents 1fi

## th a Cqmmon antigen. The low in.::idenee and J,.ow titer of antibody :i.n

**b<~th** 

## chickens and humans against the virus 

## of the qther species makes 

## it unlikely that diseases in 

## both chickens and humans are caused by 

## the same·viru.s. However, a

**co~~t~~~on** 

## sntigen between the avian and human 

## viruseJI 

## is a 

## pqasibiUtY•
Comlfatison o:f

**;Etiol<~gic** 

## Agents
There are colll!llon antigens detected by different immunologic.al testa 11.111ong 111any of 

## the het:pes vitu.se11< ShC!rtly a£ter the ;taolatio.n o£ li.VT, it 

## was shown ·to have colll!llon. utigen$ with W virus in both the

**flt.~onacent ~~t~.t:l.body**  

## and :Lmmunpdiffmlion tests.5 

## Infectious bovine rhinotracheitis, Ell virus, and MD v;l.:t"Us



# al~;.o have common antigens )ly these



# t!lst~;.

**11• 19**  

## !Jo one s.et o£ 

## l!>qterJ!llenl;.s MD

**Vi:r~~s,** 

## but not 

## I!VT 1 

## cross-tee,¢te!i 

## 'l!i1;4

**!)I!I!UQ.orl!lb~es** 

## virus by the fluorescent antibody tes·t20 whereas in 

## other experiments both viruses 

## 21
.. reacted. Some workers were UMble to show a cross reaction b_etWeen MD 

## '. d
. 22 virns an Ell 

## virus. Comm\)n grou.p-specific and \lllique typ<rspecHic .antigens have been demonatra'ted between

**Lt.~ck~** 

## renal adenocarcinoma o:f froga, MD 9,nd EB 

## viruses, herpes· S'implex and cytomegalovirus.23 It is not

### 20 

## SU't'prts:!..!lS that viruses tha.t 

## are**evol~it:I.Oi14dly** 

## elo'lely related share common antigens. Some 11£ the!!e viruses, like MD virus and EB virus 

## share the ability to**tr~sform** 

## cells. However, infli!ctiV;l.ty iilnd repllc:;•ti<!v. aPPII•r to be 

## .!lighl.;r

ti.uoora iii 

## blilnans.
Tll!llo:l.' polysaccl!iu:ide l!UPl;l:allC!!B 

### Qn 

## Mil t:l1DlQJ:ts And 

## Mn infellted 

## cells h41l

# e~:~mmun. ·glycoprQt:eii! anti,geul! 

## with a variety of hl!lfllJn ·tWIIOl:l!.

## '24,

## Z,'S Virgin 

## !eii1Sle gutiil,la pigs were illul!un.ized with tumor po1ysa:ccll.ar:lde 

## substances £roll! thl! av'illil or human tl1Dlors. 

## Uterine segments were 

## then exposed ·ta the .exi:rsct: ;md 

## contraction of the segments ·lllessuted. the 

## uteri were carefully 

## i:l.esens1died t<ll 

### '!I<>tmll 

## Ktitis..,lii! ·prior 

## to i;estii1S• 

## ·stat;;J:qtii:!aily •lan:l.fliiltll,t 

## (l'<;;.O;L) 

## rsl!\c.tlon!l we.te \;lltained 

## w1tb**~** 

## la,;ge variety o£ 

## hl1Dlan 

## c.aneen**~** 

## ineludin$**ltll\lk~,.ia,** 

## l}'ll,tphoma, Hodgkin'!I 

## d:l,aer.stll, 

## eatcinon18 of 

## t;he pllnereaa, stomadt, 

## co1on, k'id.ney ani! 

## l\ln$ an,d satcoma o£ tihe 

## bladder 

## but ·fiOt 

## w.tt!h n:orma1 eh:lciken, 

## mc>uaEt, or hll»lan 

## J:isques. Clllly 

# )!; o£ th.e tel!

## 'I: t.eatll.tl'J wers posit'!. ve when nonns.l. ant:tgeM 

## were used wherea!l 20% .of 

## ths earc:tnomaa, 60%' o£ the sarcomas 

## and 85% of**.th~>** 

## :l.)t!llp1wmas wej;e poeltive. Thus**t1u~re** 

## ate comnton

\

-- - - 

### - -**:~** -**croat~-x:e-a_etbre** 

## antigens 

## betw!!en Mil and human**t!lll!ots~** 

## the MtUte o£ 

## t:helle aut:tgelill 

## :ls uilk:uown. How.aver, this observation does not necessarily 

# link 

# W virua 

## a:ud human 

## cancer.**·~.** 

# I ' I I

## 21 

## Passtve ifiU!lunity could be transferred to guinea pig uterine segments with
serum from patients with !lancer. However, using this test 63% of subject:'! with diseases other than caucer, 74% of subjects wil;h_ 

## carcinomas a:nd 57% 

### of subject!! with i!lelanomas and sarcomas reacted l;o Mil 

## virus antigens. Onl.y 

### 6% of

# tlu~ae sera reacted with herpes simplex virus or J;:B virus.26 These findings eend to b:riug into_ questiou the specif:lcity of the l::e!lt.

When deolcyribose-puriue fragmeots from various _cence;ous tisaues were used. to :tll1llllll:liZe guinea pigs and the uteri were tested with tUlllor polysaccharide substance as rE!leasing antigen, react:I.(Jus WE!re obtained between cl1nee:rous t;tssue$ and v1td antigens at dilutioM of 0.8 to 4,9 

## :x: 1012 whereas rea 

## c.tiona with control audgen were 0.4 )( 1012 and uorms.l lung tissue did not reaet with either viral or c.qutrol anUgen.26

## ~n viral polysac.eharide substance ane:Lgens from Ml) 

### virus, Ell virus, herpes s:l.mpl':!X Viruses eypes l and II, and
mouse hepatitis Vitus were used as releasing antigens sud MD virus and EB virus used as illllilujlizing aneigens, cross reactivity wM obtained.· The highest titers were obt:ain<ld with EB virus and MD virus and t:be lowest t1ters with these two Viruses and herpes simplex aud mouse hepatitis virus,

## The above resilltll eonfirm 

## the presence of common antigEUllil amo11g 

## many herpesviruse$ from 

## different 11pecies of amphibians, mammals and birds. l!owever, they do not 

## relate to the public helllth**:J;mplioaUo~:~>s** 

## o'f AOl!V•

Attempts have been made to shgw that**aVi~tn** 

## herj)esv:truses have broader l:!.ost range 'J:han 

## just avia11 speeies 

## by 

## inoculating them 

## ;tnto labor!Atory 

## atfimab, particUlarly mamli!il1s. Successful 

## inauc.t±on or malignaucy 

## :l.n mice 

## ,£oiJ,otdng inocula:don 11£ tUlllQ!: 

# mater:l.lll ]!ro111 

## ch;i!!keas 

## rd.th 

## J1li w!ls ,

# il.e$cri~~ in_ 

# a 11hort: 

## al>straet.e

## ~e material :i.uoreaseil the iucideuee of lymphomas f:tom 

# !3% to 

## against eh;llleuge with mouse tU!llor material ·induced by 

## Mil t\UIIor lnoeulation.e No subsequent 

## studies hlive been 

## reported, Others have 

## taile<i

eAnloa :Mt\, 

## N;telsen 

## Alt, Morae PA, 'Werder AAJ Increase<i number qf lymphomas in nr:J.ce inocplated 

## wlth Hatek's disease**t~U~~Q:t.** 

## In A'bstracts of the 

## 71th annual l!leetin,g 

## vf the :Allied

## can Society fol'.' Microbiqlogy, New Odeans, 

### LA. 

## 8-13, pp 92, 

## 197/.

.,..

## 23 to induce tuiQors in Chinese a,p,d Syrian hamsters, and Le_wis rats inoculated when new borp, and kept for six months.27 No viremia or antibody cleti\Ctable by fluorescent antibody immunodiffusion testa was present.
Inoc.U.atiol;! of ten cynomolgus, 8 rhesus, and 2 bonnet monkeys '01ith cell-assoclated or; cell-free

# wrr or cell-assoc:!.at.ed WV £aUecl to induce tumors ox- other diaea.se afte:r 

# ii to 31 months ob,.erva.tion.

## 17 .UJ>o n<> virelllia wall cleteeted. Sera of 3 HVl' inoculated monkeys reacted With HVT anti$en i!n a flqorescent antibody test, '!<hereall 8 )'Ill virus ill.Oculated monkl!iys remained negative. No neutTalizing antibody was detected. In a follow-up s'tll.dy 14 new bom, 25 juvenile, and 24 adul.t matwoset 111onkeys were injected with llimUar



# ~~~aterial and. observed for 12 to 18 mot\th,s•28 No cliUica,l sign& of' disease attributable 

## to the inoculum were seen. None of the treated lllonkeys was Viremic when testecl approximately 4 week1> post inoculation. Sera from ()ne of 34 marmosets iMculated with IN! and of 2 of 29 .mamoset.s given .MD virp,s reacted,against HVT o.r liD vit:lls in a fluoTescent antibody test. However, pre:tnuculation sera from 2 of 3 

## reactor marmosets were also p'ositive by this

**tea~;.** 

## These results could not be confirmed by the vtrus neut:r-alitation tellt wd thus the specificity of the serologic reactions remains equivocal. A similar absence of tumors and virell!ia was reported in 5 adult rhesus monkeys

### 24 

## illo.culat.l!d '!f:!:th 

## a non-l!M9g'eflic i!t:tain i:lf 

## MD v:l.:rus.29 P:r1lll!lte 

## inoeulationa also yieided ilegativ!B 

## results

# ~lth an attenuat::e'd lltr!lin of 

## MD virull,;,f

.Thl! abs<\n<:!B of tum_qr 

## 1,nductioll 

## in pril)l<!;t!Bs**~ett:dilly** 

## lends**~uppod:** 

## to**~he** 

## .conclusio.n 

## !;!tat 

## .MD v:l.tis 

## ·.and l!VT 

## are**iio'n"'~;>athogeu:l.e** 

## in 

## primates lind; •by**ext:S~Ul:l,t:ib.,** 

## .in 

## hUllla!ls. 

## However• ·because of tne nature of the 

## expe#m!!nl;$, iio 

## definitive 

## eOX>Clu$lotis can 

# be.ii-tawn. The 

## AOHV ba;vi! ·a

# ~t'row hoet

# rli-i~;se •nd «<i!.ly 

## infect and 

## replicate in 

## a few species 

## of birds. 'they 

## do not 

## appeat to infect and 

## replicate in mallllllllls_, in(!liiding**p~im~t'tes.** 

### It is 

## thati!fore WU.ikllly 

## tba;t they 

## w!U 'infect llnd 

## repl!¢ate 

## :ln h!.1ll!llns.

U 

## AoHV e.ould**propasat~ r~ad:l;ly**  

## tn**waminaU~J-n cells~**  

## thell 

## .they mi)iht 

## ia.t'eet ll)llllllDalli, 'llhe 

## llVi' produte<t .a eytopat!).ogeni¢ 

## eJ;fect ±11 

## hamster Mdney 

## l).gq 'blaMer 

## epi 

## dte!ial

# c~11s.)JQ 

## ·Tl:te virus was 

## panaged succ.ess±vely 

## in hamster cells alid to ¢h'1cken 

## embryo ceUs, io.t>:et#er\®-**pr!i!pa-r~d** 

## tn**~~:nickena** 

## against 

## this 'Virus ·n.eutra:H.zed it 

## 'but 

## antiserum to 

## herpes almple;c Y:I.:Ns t}"pe 

## l 

## or type


## J:delloer <,;; <lentral !tleregeneeskundug 'Instituut;, Lelystad, 

## The Nethetlanda, Petso11al 

## colJUI!Un:l.cation,

## 2.5 2 did not neutr.l!l.be it. The W 

## virus and !NT 

## produced a cytopathic effect in hamster kidney cella even a:f;ter 6 to 7 passages onto newly prepared hamster 31
. . 

## kidney monolayars. Virus particles were f.9und in infected cell cuJ.turea and the cultures contained e:xtendve polyka.ryocytes,32 The v:i.rus propl(lgating in hamster kidney cella was identical to the 

## .IIVT used to inoculate those .cells by complement; fi%ll.tion, fluorescent antibody and serum**n<~utra.liza.Uon** 

## tests. Subsequently, Ml) 

## v:i.rus was also snown to replicate :i.n hamster kidney.cells by


## serum neut;:tall,z,.tion, c<llllplement .fixation, and fluorescent antibody tests.

## 33 Similarly, 

# mrr and MD virue also propagated in primary guinea pig kidn.ay :tn an ute1lsiYe ;;md 

## detailed examination of this phenomenon, MD virus or IIVT ili,fe¢dVit:y wail! f<>l.lJJci 1:<> 

## persist fax up !:P ten serial passages in pr4,mat:y hlllllater kidn!!Y cultures iMcuJ,ated. With cell-a!lsoc;iated MD Virus or 

## il'IT. HoweVer, ;Ln!ecd.Vity was only present in these p\ISSaged cultures when avill.ll cells, which were present in the inocqlum, .persisted 

## 'l.tl the c1,11tures. l:nfeetiv:l.ty could be eliminated by treatment with cytotoxic**anti~avian** 

## cell ant:L:body a!ld tell-free vit'lls was unable to infect thg 

## mammalian cultures alone.36 Others h<1ve been unable tQ infect prima.ry hsmstli!r kian,ey cultures.27

l 

## W virus was u1lable to produce a cytopathic effect in cultures of .15 

## ma.nmuilian species 11lcluding dog, cat, mink, swine, bovine, goat, equine,

## hamater, mouse, bat, 

## 'ribb:J.t 1 

## potoroo,

**spot~ted** 

## dclph:J;n,

**:gre~en** 

## molil<:eY i\lnd hllllllln.

## 37 S:J.mi.lsr re11\!lts were obtained with 

## HVT on 

## human, dolJ:, 

## cuf, baboon, rabbit and pig 

## <:el11;.30



# :ll.v~n after



# t~ HV'l' 

## was pl;'opl!.gsl:ed in 

## hwnater ki<h!ey ceUs 

## for several passages, 

## it wes unable to 

## :ini!eet these t;!.ssue 

## cultures. 

## 26

Neither 

# m

**v.r:~a** 

# not ll:Vl' would 

## i'epl;:J.cate in ceU 

## t:Ultni'es of

**~~ol!.et** kidney, IIUl:r;mi:lae,t 

## ilkin and 

## mliscle,, 'li.Ume.n embryonic; kidney; a,M the

**huJ~~a,n** 

## ee11 

## l:!.ne '11!
......38.28 'rreatllieut ·with il'l!!:thY:tlllil:ttioethyidextran 

## or ihtrliv:tal.et 

## 1nadiate4 Sen<!ai 'Y'lt1111 or

**cq~el.l,ltivstion'w:tth X'"i.rJ;adi~!l:ei:l**  **Vlm"':l.~ect:ed**


# w Virus and 

## cell-£ree aVT dl.d 

## ntn: infect 

## Clibese 

## nall1ste'i',

**syr:i.~U~ b:mt~al!,**  

## boVine, doJ!, human, 

# rabb:tt or pot(lrQlle 

## ¢eUs;27 Similat" 

## '!ieaults ,have



# h~~n obtdned by othet:s.

## 3'll,,.9'J,AO

Once again mammalian 

## calls were refraQtory to infection even under 

## conditions of ard.£id.a.l

**~ell** 

## 'hybr'l;d:tzatfiin 

## between

**virus~infected** 

## celia and the 

## llla!lllllalian eells wfth ultraviolet 

## inactivated Sendai virus. Tll.u!i AP!W failed 

## to ipfe<:t ·and 

## replicate :tn 

## lll8mmi!.lian cells, but: could cont:Ln111! to 

## replicate in hybrid cells composed of mammalian and avian hetet(lk,atyons.

## 2.7 Viruses that fail to infect**ma~lisn** 

### cell**cultur~s ar~** **l~ss** 

### likely to infect 

## mannnals than are**'lirus~s** 

## that infect and grpw 

## well iti mammalian cell 

## cultures.
OpPQrtunist.ic lnfections
Birds, !lffectl!d 

## with MD and healtP,y 

## bit:ds were e:r;unined for the presence of 

## bacteria tha.l: might pose a 

## public health hazard. Ert.tetobacter:l.a were three to 

## 111.ne till)es more 

## ftequently eucoUlltered in the ViS\'l!Ora oJ; 

### dipeased birds than 

## in the 

## !Jame 

## ()tgana of 

## controls.. In two 

## eases Salmonella 

## were**:l.sols.t~d** 

## from affected cltickens and 

## ill. tnree cases 

## eliterobac1;e,r;l.a were isolated rl:om the 

## museul.ature 

# or affected birds.41 The 

## bird·a with overt disease, 

## in this cal!fe 

## MD, would be expected to be less resistant to bacteriel infection. H.::>wever, these inf'ec,tiona**ar~** 

## l.il<;ely no more eolll!llon**~tll!ong** 

## bir<;ls affected witll W than 

## among birds with 

## other a:l,lm.-;nta 

## such as injuries. 

## Cltc\!Distantial. Evidence

There have been many reports· 

## of an asspcia

## tioo between cancer in 

## animals altd cancer j,n man. There have also been reports of <ll'gaqbms ii!Qlated from human and 

## anima,l maligrum.cies which are highl.y pleomorphic and variable 

## io. many characteristics and which are capable of 

## develPping viru!!-l:lkO!

# 2S bodies,42 However, controlled e:a:perimental eVidence o:f 

# ail etiologic rdatlon!lhip 

## J>etween these Drg!!nis111s 

## and human 

## eaacer is**1ac~ins•** 

## Similsr;Ly there have been l!ll!.UY 

## Jteports o$1 cancer 

## va.eeines and 

## cures. The only tnforl!lf.!tioll**t~va;1.1able** 

### 1t!ls 

## bee11 through the pnblie 

## media, 't:h:rtobsh**:l.ntem~ws** 

### bY 

## the proponents, or 

## fn 

## privately pr!1;1ted 

## pubUca.tiQne that an l\l:!t 

## avail.l!bl.e to Ul!rariee 

# o;r: the 

## general. pnh.lt.c. 

## The 

## :i.nfotma.tiou is 11ot: puhU11hed 

## in the open 

## medical. or scient:ffic 

## literature 11'h.ere it 

## can be**rerl~ed** 

## an'd va11daJ;ed 

## hY other 

## scientists,

. Current tec.htilques 

## or 

## 11pproaches J:or 

## <!!ttell)pt:l.ng 

## to 

## resl)l.ve tlle puhl.lc 

## health eonce:rnll ltnd 

## the ·results <lf 

## 'ulling 

## thea!! tech!l.iques !Ire i11JJi!mat'fi:l!d ill. 

## Table 2, Unequivocal.l.y 

## positiva fiudiugs !Ising e!Leh Of 

## these too1!1 coW.tl 

## ·tmve inerill){ll$ted ·.liD 

## or it11 vaec:l.ne**vi~;Uselil** 

### QJ:' 

## 'both 

## as public h'lalth eoP.cern,s. t)lu11 a hishlY**sJ.jPI:!:~ieAnt eorr<~J.af:iou**  

## betwe.en 11 

## p®l.ie 

## he11lth**~:~:~ndil:ion~** 

## e.g•., 

## leukemia, ·and .exposure to 

## an ·AOllV, e.g., MD virus, in the absence of a 

## correlation be

## !:Ween the cond:Ltioli and 

## other factors, 

## e.g., cQtn 

## prOduedon 

## and fertilizer ·ulle, 

## could have 

## strongly 'supported 

## the hypothesis that W 

## virus 'bM 

## .something to d.o 

## wlth the human leukelliia. 

## Simila'r exampleil' of 

## findings usi!lg 

## other techn:iqUes which would st):'ongly .support 

## this hypothesta ate 

## in table 2,

'·

## 29 Negative findings**us~:l.ng** 

## each of the techniques currently available, namely aJ;atlst:l.c.al epidemiology, serological epidi!lli1ology1 

## lillllmn&l inoculatiou, cell culture inoculation, and comparison of etiologic agents cannot refute the h}'Potlieais that the public health condition is caused by e:s;posure to the A.OHV. Thus, for example, the absenca. of a correlat.ton between a public health condition and expo.aure to AOHV d()es 119t 

## confirm that there is no relationship between the two. One can conclude only that this technique failed to find a relationrllup under the :conditions and in tb.e circUlltstauces iu vhich it vas used. This_ia the basis for the conclusions of the expert committees and commissions re.fetred to prl!vi.ously ;in this paper.

rrom the above discun:l.on it appears unlfkely that additional efforts to
use the techniques currently a:vailebls w:l.ll :reeo1vl! the public**he~altl:lc** 

## concerns of MD end its vaccines, Certainly research resu.lta of the last 8 years have fa:U.ed to hti_n& Ul! l!.nY closer to a resolution of the issue,2 A fUtther way of s)ledding light on the possible relationship il! 

## i.n the use of the nev molecular b:ioiogy tools to study the pathogenesis .o.t'**~cancer** 

## in man and animala. 

## lf a unique r:ole for the AOHV can be. found in the molecular pathQgenesis of h!JIUBn cancer, then the public heal.t4 hazard of the virus will be confi:tllled. It is important to note thst just finding portions of a viral genome :l.n human tumors, e.g., thl! IDY" and 

## !!!:. oncogenes of avian leUkosis



# ....

### 1 

## viruses, is iusuf'fieient if 

## J:bl!se geqes are also present in normal e.ells. 

# 30
Conversely, 

# if the molecular pathogenesis 

## of all hlltti!lll**ean~er** 

## is**e:~~plaiaed** 

## and no role 1li 

## found 'fcrr any unique AOHV 

## ganes or gene products, the11. the**absen~e** 

## of a**publi~** 

## health hazard**~n** 

### h!il 

## confirmed.

This**nomp:reh~n.sl.ite** 

## rev:i.ew 

## of the literature 'b:d.ngs 

## together, 

# for the £1rll't time, a laz:ge 

## body of**infotn~ation** 

## relatiD,g t.o 4QHV el!.d 

## human health**ha~~:aras.,** 

## J?srtl:!!tilarl,y**l\u~ll** 

## canc!!r.. :n.is ·review s)lol!lt:! he 

## U!lefl!l to acieAe:f;ats all.d 

## adm:f.q;istrators in public reseateh and**<~.e.tion** 

## a.ge1leies and 

## J.n p:dvate industry. 'l:he reView**~overs** 

## ell:!! nUiael:'ous**s~eempt!!** 

## .to**te!!ol~** 

## t® '():Uesl.i.t'on o£ whetlup; AOHV 

## pose any ptibl:l.c health hazard to huma11s. !L'he large body qf experimental <ntidence in both avian 

## imd hlllil<m. virol()gy, eel'o1ogy, pathology, and epidemiology stron$1,Y auppot't.!r 

## the eonClua:l.on t!mt p;o 

## et:l.ologi.c rda!;:l.qn!ihip e:<;:Ls'ta bei:)feen AOHV and hW!lAD. 

## cancer• Thqa, in our opinion !l!l.d in the 

## opinion o£ experts 

## in the human and avian**canc~r** 

## £ield, MD 

## and vaccines pos10ible, there is**~ufflc:ient** 

## evidence reviewed in this 

## paper to 

## wa:r.ts'llt: th.is 

## finn couc:luslon.**·~-_. ~-**  '

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19, Qnd

# x. 'l:atllibe S, 

### Nai:to 

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# 's dis<!ase 

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'1,7, 

## Hlozanek SV. :Lack of 

## pathOgenicity 

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## virus arid 

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### :f()it' 

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# ·cell culturea. FOlia JUol 

## (Praha) 1974;**2tf:S1~5il,**


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### Natl 

## Gancer Inst 

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.,,