Risk Analysis 

Marek's Disease-Newcastle Disease Vaccine, 

Serotype 3, Live Marek's Disease Vector 

USDA Product Code: 16N1.RO (Unlicensed) 

Updated 

Merial, Inc. 

U.S. Veterinary License Number 298 

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Me rial, Inc. ·Risk Analysis May 18, 2014 

U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, Page 2 of67 

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

Table of Contents 

I. INTRODUCTION ................................................................................................................................... 6 

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

B. Proposal ............................................................................................................................................. 7 

II. DESCRIPTION OF THE REGULATED BIOLOGICAL AGENT CONSTRUCTION .............................. ? 

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

B. Donor Biological Agents and Donor DNA or Genes ....................................................................... 11 

Ill. BIOLOGICAL PROPERTIES OR VIRULENCE FOR THE REGULATED BIOLOGICAL AGENT USED 

A. 

FOR MASTER SEED ........................................................................................................................... 34 

What are the known phenotypic characteristics or effects of the 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 are the virulence characteristics of the Regulated Biological Agent used for the Master Seed? 

. " ... " .................................... " ......... " ....................... " .. " .............. " ... "."." ... " ... ". " ... " ................. " .. 35 

C. Is the Regulated Biological Agent used for the Master Seed virulent for target animals, non-target 

animals? ......................................................................................................................................... 35 

D. Do the Donor DNA sequences enhance the virulence or the ability of the Master Seed to survive in 

the target animals, non-target animals? ......................................................................................... 36 

E. Discuss the potential for horizontal gene transfer or recombination of the Regulated Biological Agent. 

. " ........ "" ................ " .... " ..... " .... " ... "." ..... " ... " ....... " ........................... "." ........... " ... "." ..... " ....... : ... 37 

F. Describe the shed/spread capabilities of the Regulated Biological Agent. ..................................... 38 

G. Discuss the expected environmental impact or survivability of the Regulated Biological Agent, and 

provide available scientific evidence ............................................................................................... 39 

IV. RISK ASSESSMENT .......................................................................................................................... .40 

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

B. Hazard identification ...................................................................................................................... .41 

1. Animal safety .............................................................................................................................. .41 

2. Public health safety ...................................................................................................................... 46 

3. Environmental Safety .................................................................................................................. .47 

C. Release Assessment Prior to a Proposed Field Safety Trial .......................................................... 49 

D. Risk characterization ........................................................ , ............................................................. 53 

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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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1. Likelihood ratings ......................................................................................................................... 53 

2. Consequence ratings ··································'······························'·················································53 

3. Degree of certainty rating ............................................................................................................. 53 

4. Calculating the expected risk ....................................................................................................... 54 

5. Risk ratings ................................................................................... , .............. : ............................... 54 

V. RISK MANAGEMENT RECOMMENDATIONS .:.· ............. , .................................................................. 59 

Tables 

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Table 7: Risk Ratings 

Figures: 

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U.S. Vet. Lie. No. 298 Marek's Disease-l'·iewcastle Disease Vaccine, Serotype 3, 

Live Marek's blsease 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) 

Appendices 

I 

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Page 5 of67 

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

viruses: A review'' 

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 of Poultry, 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 et al., J. Viral. 47:421-433, 1983 

7. H.G. Purchase et al., Am. J. Vet. Res., 32:1811-1823, 1971 

8. K. Dorsch-Hasleretal., Proc. Natl. Acad. Sci. USA, 82:8325-8329, 1985 

9. Merceret al.., Virology 129:94-1061983 

10. N. Zygraich and C. Huygelen, Avian Diseases, 16:793-798, 1972 

11. Osterrieder and Vautherot, Elsevier Academic Press,p.17 -31, 2004 

12. P.A. Phillips ~nd P.M. Biggs, J. Natl. Cancer lnst. 49:1367-1373, 1972 

13. Taylor, J. et al., J. Viral., 64: 1441-1450, 1990 

14. Tripathy, Deoki: Pox. Diseases of Poultry (1991) Ed. Calnek, B.W., Iowa State University Press, Ames, Iowa; 

pp 583-596 

15. Witteret al,, Am. J. Vet. Res., 31:525-538, 1970 

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U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, 

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

I. INTfKlDUCTION 

A. Objective 

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

vaccination under field study conditions in chickens using a live Marek's Disease, Serotype 3, HVT virus which 

contains a Newcastle disease virus (NOV- gene cassette derived from-ND~ 

and subsequent commercialization and distribution of the product, pending CVB acceptance of data in support 

of licensure for use in chickens. 

1. Identify where the Regulated Biological Agent was constructed and where the product will be made, 

tested and manufactured. Address the available level of containment. 

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 of the Regulated Biological Agent. 

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

B. Proposal 

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Page 7 of67 

The proposal is to conduct experimental vaccination studies in chickens in the field to determine the safety of 

a live Marek's disease, Serotype 3, HVT virus which contains a NOV- gene cassette derived from 

- NOV,- and subsequent commercialization and distribution of the product. 

1. What is the intended use of the product? 

Species: Avian; 18-day-old or older embryonated eggs or day-old chickens 

Proposed claim: The vaccine is indicated for use in healthy 18-day-old or older embryonated eggs or day-old 

chickens as an aid in the prevention of Marek's disease and Newcastle disease. 

Geographic area: United States (all states), 

-

Route of administration: in ova, subcutaneous 

Brief description of the expected safety profile: The RBA vaccine 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 BIOLOGICAL AGENT CONSTRUCTION 

A. The Backbone Biological Agent 

1. What organism was used for the Backbone Biological Agent? Are there any known virulence features 

associated with the Backbone Biological Agent? What happens In the target species? 

The organism used for the backbone is a Marek's Disease, Serotype 3, HVT virus belonging to: 

Family: Herpesviridae 

Subfamily: Alphaherpesvirinae 

Genus: 

Species: 

Strain: 

Meleagrid herpesvirus 

Meleagrid herpesvirus 1 or turkey herpesvirus (HVT) 

FC126 

The parent strain is avirulent in chickens and does not cause gross observable lesions associated with 

Marek's disease virus. Only limited contact spread occurs among chickens but transmission could not be 

demonstrated by the airborne route (Dise~ses of Poultry, 12~ Edition, Chapter 15, page 491). The results of 

Study 12-027 entitled "Ability of Marek'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 that there was no spread of the RBA to 

HVT infected turkeys, naive turkeys, HVT infected chickens or narve chickens. This study was accepted by the 

CVB on December 5, 2013 

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Live Marek's Disease Vector, Code: 15N1.RO (Unlicensed) 

What Is the previous safe use of the Backbone Biological Agent? 

May 18, 2014 

Page 8 of67 

(I) If avail~ble provide history of previous safe use using published literature or Internal 

documents. Include the recommended CDC/NIH biosafety level for use of the Backbone 

Biological Agent. 

The vector virus is the FC-126 strain of turkey herpesvirus (HVT). This strain has been widely used 

as a vaccine against Marek's disease (MD) in chickens 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 (Witter et at., "Isolation from turkeys of a 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 live vaccines 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 of chickens have 

been vaccinated with these vaccines. The biosafety level for manipulating HVT virus is BSL 1. 

2. Physical characteristics of the Backbone Agent 

a. Provide a flow diagram or explanation of the process of how the Backbone Biological Agent 

was constructed. 

(i) The Hvi vector was derived from the vaccine strain of FC-126 strain of Marek's {see Section 

II.A.1.(i), page 8}Describe the proposed site for 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 of the 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 of turkey herpesvirus J. ViraL 75,(2), 971-978) and is availabl~ _in Genbank (NC_002641). 

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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) 

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

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(ii) Do the flanking regions of the proposed insertion site Backbone Biological Agent have any 

known regulatory elements that could moderate the expression of the 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 more than five) that will give identifiable 

digestion patterns useful for characterizing the final Backbone DNA 

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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) 

B. Donor Biological Agents and Donor DNA or Genes 

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Page 11 of 67 

1. What are the Donor Biological Agents used to source each Donor DNA sequence inserted into the 

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 

of NOV. eDNA clones for the NOV- gene were derived from a-NDV,-The gene 

cloning and sequence that had homology to the NOV- gene has been described byThe exact same donor NDV- sequence has been used in USDA 

licensed product 17C1.RO, Newcastle Disease- Fowl Pox Vaccine, live Fowl Pox Vector, licensed October 27, 

1995. 

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

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

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

exact same promoter was 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 bursal disease virus. 

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 of the Donor Sequences or Donor Genes as well as Safe use of the 

Donor Biological Agent? 

(i) provideJ·~Ievant ref~rences for safe use. 

T~e donor NOV~ sequence has been used in USDA licensed product 17C1.RO, Newcastle 

Disease-Fowl Pmivaccine, Live Fowl Pox Vector, licensed October 27, 1995. 

The locus of insertion, promoter and poly-A signal iiave been used in USDA licensed product code 

1A88.RO, Bursal bisease-Marek's Disease Vaccine, Serotype 3, Live Marek's "Disease Vector. 

In order to 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 of velogeriic NOV as utilized in 

the NVSL NOV surveillance program. The primers and probes used in this assay are identical to 

those used in the NVSL protocol SOP-AV-1505.05 (Attachment 2). The NOV .... gene did 

not react with the oligonuCleotides used in the NVSL real-time RT-PCR protocol for aetection of 

virulent NOV. ·Additionally, the RBA does not contain the NDV matrix gene, and will not react with the 

primers set designed for Hs detection. The RBA does not test as exotic Nov. 

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

May 18, 2014 

Page 13 of 67 

b. Are there specific parts of the Donor Gene(s) or Sequences that were used for insertion? 

Show pertinent sequences or restriction endonuclease sites. 

C. Construction and Characterization of the Regulated Biologi~al Agent 

The RBA was obtained by homologous recombination between the parental HVT and a donor plasmid . . . 

Eii5S 

imrnurlOfiuoi·es,::en,ce,, using an antibody that recognizes the product of the 

inserted gene.. No resistance factors are used as a means of selection. 

As explained above, the donor plasmid- 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 by the NDVI 

-gene 

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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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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) 

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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. Lie. No. 298 

Risk Analysis 

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

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

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l-' 

Merial, Inc. Risk Analysis 

U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, c'ode: 16N1.RO (Unlicensed) ·-~ . --~ . 

May 18, 2014 

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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) 

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1. Provide a flow diagr~.m on the construction of the Regulated Biological Agent Include the following: 

Final Backbone Biological Agent 

Donor DNA or gene 

All shuttle vectors 

Host cell lines used 

Selection techniques and methods used to construct the final Regulated Biological Agent 

The general flowchart used to generate the RBA is shown in Figure 10. 

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

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U.S. Vet. Lie. No. 298 

Risk Analysis 

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

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2. Describe the laboratory methods or criteria used to evaluate the Regulated Biological Agent. 

The genomic structure 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 blot analysis. 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 plaques were screened and all 924 HVT plaques (green) were found to express NDVI (red). 

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Risk Analysis 

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

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3. Physical characterization of the Regulated Biological Agent 

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

~qent reStrl~tion endO_nuc_lea!?e sit2s and describe re~Uit~ng reslrlcfion fragments and 

··'; 

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

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b. Devise a PCR .or restriction endonUclease· test baSed on the Backbone -Biological Agent 

sequence and the Donor Sequence that will identify and characterize the Donor 

DNA/Backbone Biological Agent construct. 

Several PCR's have been developed to characterize and identify the RBA. The localization of the 

primers on the HVT and the RBA genome is shown in Figure 13. The sequence of the primers is 

shown in Table 2 and the size of the amplification product is shown in Table 3. 

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

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c. What will be the criteria for stability and purity of the Regulated Biological Agent Master Seed 

nand n+5? 

PCR pattern and plaque immunoscreen results were used to determine both stability and purity of the 

recombinant virus. 

d. Provide the genetic sequence in eiectronlc format for any new or altered genetic sequences, 

inciuding iri-~~i1ion ~-nd flanking sequerl~e . . . _, .. - -

The DNA sequenc~ of the parental HVT-fragment and the corr~sponding fragment of the 

RBA were su6mitted as l('lord files on a daia disk on June 24, 2011. 

4. What is the recoorim~nded NIHJCDC biosafety level for the. 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 a reseafCh ~~tti~g: At thiS' 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 new virulence factor t~ the backbone virus w~ w.!i~idcor1S,ider BSL 1 s~fficient'l~r th!'> 

RBA, Similar to the parental organism. 

5. PrOvide:_.a shOrt ~Ummary or d~SCriPtion of geiletiC 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~tifs that may promote homologoi.~ ·- . - - - . . . . ·-·· - • • - _f.V • - - - .' 

recombination, DNA insertion, or-gene -~~_pression bf existing 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) confirmed that \he homologous 'r~c;:cimbination 

Occurred as eXpected._There a~e rio knowh mOtifS lhat may promote homologous reCOrilbination, 

DNA inSertion or gene expression of existin;~ ciP~n ~eading fr~~es. Only 01)8 foi'eign open reading 

frame was inserted and coding for the 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 of the 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 is identical to the I gene inserted in the USDA 

licensed ·product 17C1.RO, Newcastle Disease- Fowl Pox Vaccine, Live Fowi Pox Vector, licensed 

October 27, 1995. 

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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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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) 

Confidential Business Information Redacted 

May 18,2014 

Page 30 of 67 
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) 

Confidential Business Information Redacted 

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Page 31 of 67 
---·,_ 

.,-. 

-;o: 

·•· 

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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) 

(iii) lmmunopreclpitation and Western Blot 

.; ' 

j,_ 

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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) 

Confidential Business Information Redacted 

May 18,2014 

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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) 

May 18, 2o14 

Page 34 of67 

- -. -. 

Ill. BIOLOGICAL PROPERTIES OR VIRULENCE FCJR THE REGULA TED BIOLOGICAL AGENT USED FOR 

MASTER SEED 

A. What are the known phenotypic characteristics or effects of the Regulated Biological Agentidentlfied 

as M~ster Seed? (e.g., expresses envelope gene/sequence, lac_ks thymidine kinase activity, lower titer 

ln.MA105 cells, etc.) ,_1; 

-~ . . . The double label immunofluorescence test described in Section II.C.2 (page 21) above was 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 1 gene 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~ of the phen6typic. stabiiityorllie r~~ombih~~~ vii~.. .. . . '. ' - - . - •'' ';·- . . ':· 

iiT1rnurlopreciP,ii"tatic>n and \IV estern blot tei;hnique deiected an 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 at various ~Liture pa"; ssarge,s, 

.a"§d~l~nmirled by analyzing the titer ofthe vaccine~dqseif used iii the 120 "•:• ·"'''"', 

.3ctepte.d by 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 of HVT, 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, Live Virus, product code 

1641.00, marketed by Merial, Inc. Many billions of chickens have been vaccinated with these vaccines. The 

HVT virus is a non-pathogenic virus of turkeys. No HVT gene was modified by the insertion of the 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 of the vector as determined during viral passage from MSV to MSV+5. 

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

May 18,2014 

Page 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 of viral particles. However, shed and spread (see Section III.C, page 35-36), 

as well as tissue tropism studies (see Section Ill. D. page 36-37) have demonstrated that the phenotypic 

characteristics of the RBA were similar than those of the parental HVT indicating that the NDVI expression 

did not change the behavior of the parental virus. 

B. What are the virulence characteristics of the Regulated Biological Agent used for the Master Seed? 

The recipient virus was derived from the FC-126 strain of HVT, which is recommended for use in day of age 

chickens or in ovo vaccination. This strain is avirulent for chickens and its natural host is turkeys. No HVT 

gene was 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. Is the Regulated Biological Agent used for the Master Seed virulent for target animals, non-target 

animals? 

-was accepted by the CVB on March 9, 2011. The study satisfied the recommendations of 

Veterinary Services Memorandum 800.201 for evaluating the lack of reversion to virulence in chickens and the 

limitations of viral 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 another group of 

one-day-old chickens. Four additional passages were made. Na"ive birds were placed in contact with each 

group receiving the backpassed virus. At each passage, bully coat extractions were inoculated on chicken 

embryo fibroblast monolayers to monitor virus 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. A final 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 of the 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 for expression of the I protein and found to be 

positive. 

The safety study in mice- showed no recovery of virus 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 5 vaccinates and 2 controls. The buffy coat was extracted from the tissue homogenates and 

evaluated for presence of Marek's disease virus on chicken embryo fibroblast monolayers. This study supports 

the lack of a negative effect of the product in non-target animals due to no virus replication in murine tissue. 

The study was accepted by the CVB on October 25, 2011. 

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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 of 40 quail, turkeys, ducks, pheasants or pigeons that received either Marek's 

diluent or a 10X dose of the RBA vaccine. 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 Of the 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. At 50 days 95.8% to 100% of the nonvaccinated, challenged controis inoculated with MDV RB1 B strain developed Marek's lesions. At 120 days, 

none of the vaccinated birds and none of the 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 of 9 .CFR 113.330(b) io·r the subcutaneous aiiii in avo. routes of administration 

(CVB a6il~~feo an Aprii 7, 2011). 

D. Do the Donor DNA sequences enhance the virulence or the ability of the Master Seed to survive In the 

target animals1 non~target animals? 

1. What is the tissue tropism of the Regulated Biological Agent in target and non-target animals? 

HVT replicates in chickens and induces a detectable level of viremia. Leukocytes become infected and 

provide the main mechanism of cell~associated virus transport tb other tissues. Cell-free infectious virus 

occurs only in the feather follicle epithelium between the second and third week of infection, but there is little 

or no natural spread of HVT between chickens (P.A. Phillips and P.M. Biggs, "Course of infection in tissues of 

susceptible chickens after expoSure to Strains of Marek's disease virus and turkey Herpesvirus," 1972, J. Natl. 

Cancer In st. 49, 1367 -1373; N. Zygraich and C. Huygelen, "Inoculation of one-day-old chicks with different 

strains of turkey herpesvirus. 11. Virus replication in tissues of inoculated animals," 1972, Avian Diseases 16, 

793-798). 

HVT replicates exclusively in cells of avian origin (chicken, turkey, duck, quail, pheasant, pigeon, or goose) 

(H. G. Purchase et al., "Responses of cell 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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Live Marek's Disease Vector, Code: 16N1.RO (Unlicensed) 

May 18, 2014 

Page 37 of67 

the birds were inoculated with the RBA vaccine or the FC-126 parental virus. An additional group of birds 

received diluent only to setVe as negative controls. On days 7 and 28 post-inoculation, the spleen, liver, lung, 

kidney, and gonads were isolated from five birds of each group of vaccinated birds and the spleen was 

isolated from three of the negative control birds. Buffy coat extractions from each organ were evaluated for 

virus isolation on chicken embryo fibroblasts monolayers. The presence of the virus was identified by the 

obsetVation of cytopathic effect. NDVI protein expression was evaluated by immunofluorescence using at 

least one plate per organ 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 for the RBA. Plaques obtained from birds that 

had been inoculated with lhe RBA were positive for the expression of the NDVI protein, providing evidence of 

no loss of expression after single in vivo passage (note: the reversion to virulence study proved no loss of 

expression after five passes in vivcf- CVB accepted on March 8, 2011 ). The data from this 

study support the conclusion that the tissue tropism of the 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 transfer or recombination of the Regulated Biological Agent. 

1. Is there any reason to believe that the potential for horizontal gene transfer or recombination is 

different in target and non-target animals? 

Horizontal gene transfer between HVT and other Marek's disease viruses (MDV) has never been reported. 

This is likely due to the low percentage of homology between the 3 MDV serotypes (Osterrieder and 

Vautherot, 2004 The genome content of Marek's disease-like viruses. In F. Davison & V. Nair (Eds.) Marek's 

Disease. An Evolving Problem, Elsevier Academic Press, pp 17-31). Furthermore, the NDVI gene has no 

known toxic effect when inserted in a Marek's disease virus, and therefore, even if a horizontal gene transfer 

were to occur, it should not increase the virulence of the recipient virus. 

was conducted to investigate the possibility of increased 

pathogenicity following co-administration of the RBA with other Marek's disease viruses from other serotypes. 

To determine the stability of the gene insert in the face of pathogenic MD, the RBA was combined with a 

virulent MDV strain (GA 22) and wilh serotypes 1 and 2 of Marek's disease virus. This co-administration 

should favor recombination between viruses if it occurs. There was no increase in virulence observed in the 

GA 22, Rispens or SB-1 groups co-inoculated with the RBA vaccine based on gross MDV lesions. There were 

no statistical differences in average body weight between the GA 22 and the -/GA 22-

!

5555555555555& Rispens and the-/Rispens- or SB-1 and the-/SB-1-

combinalion groups at the end of the 120 day 

obse!Vation period. The results showed no increase in pathogenicity of any of the MD strains when combined 

with the RBA, indicating that recombination either did not occur or if it occurred (unlikely), it did not generate a 

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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. Describe the shed/spr~ad capab\li\1~~ of the. Regulate<l Biological Agent. 

May 18,2014 

Page 38 of67 

Study - --- --- ----- ------------ -- --

~--------=----_-------------- --- -_-- ---_ -::::--------::;--

was accepted by the CVB on March 13, 2012. 

ino•cul<lted with either the RBA X+5, licensed HVT, or Marek's 

diluent. One of the groups that received Marek's diluent only were inoculated with the very virulent MDV 

Serotype1, RB1 B strain by IP injection at hatch. Pr~sence of HVT was determined by cell culture, followed by 

fluorescent antibody staining. Presence of the RB1 B strain was confinned by polymerase chain reaction 

(PCR) spe"cific for Serotype 1 virus. Tracheal and cloacal swabs were collected from the birds to examine for 

viral presence. Feather follicles Were also examined for virat presence. Virus could not be recovered from 

either cloacal or tracheal 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 of disSemination of the 

RBA MSV+S was similar to the HVT vacCinated birds. 

A shed/spread study was conducted as part ?I the 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, nor were any lesi~ns seen in the contacts at necropsy. 

1. Include shed/spread potential in non-target animal species. 

any of the non-target species. 

As discussed in Section Ill. C. (page 35-36), Study- shoWed no recovery of virus after subcutaneous 

injection and epiderrTial scai"ificiition of 15 mice. 

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

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May 18, 2014 

Page 39 of67 

2. Include in the discussion any contributions to shed/spread capabilities that might be associated with 

particular attributes of the Backbone Biological Agent and Donor DNA sequence. 

It is not expected that insertion of the NDVI gene will contribute to the shed/spread properties of the 

backbone virus. The HVT virus 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 of the I gene into a non-essential site of HVT does not impact the shed pattern of the HVT 

virus (no viral shed seen as discussed in Section III.F, page 38-39) 

3. Reference relevant scientific publications. 

a. Cho,B.R 1976. Horizontal transmission of turkey herpesvirus to chickens. 5. Airborne transmission 

between chickens. Poultry Science 55:1830-1833. 

b. Cho, B.R. and S.G. Kenzy. 1975. Horizontal transmission of turkey herpesvirus to chickens. 3. 

Transmission in three different lines of chickens. Poultry Science 54:109-115. 

c. Cho, B.R., S.G. Kenzy, and S. A. Haider. 1971. Horizontal transmission of turkey herpesvirus to 

chickens. 1. Preliminary observation. Poultry Science 50:881-887. 

G. Discuss the expected environmental impact or survivability of the Regulated Biological Agent, and 

provide available scientific evidence. 

Turkeys are the natural host of HVT. HVT is shed into the environment by feather follicles of infected turkeys. 

Prevalence of infection in turkeys approaches 100 percent. To our knowledge, natural HVT infection in 

chickens from infected turkeys has not been reported. Chickens do not appear to be infected except through 

vaccination, after which the virus replicates. The survivability of the RBA in the environment is expected to be 

similar to that of the 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 of the cells. The RBA may be shed as 

cell-free virus by the feather follicle of the vaccinated chickens. The resistance of this cell-free virus present in 

dander is much higher than the cell-associated form. In our studies, the RBA is not transmitted horizontally 

among chickens indicating that the amount of virus shed is not sufficient to infect naTve chickens and/or that 

chickens are very resistant to HVT infection by the mucosal route (Study-discussed in Section Ill. C., 

page 35-36 of this report). 

The host/range specificity of the experimental vaccine was evaluated with the non-target avian species safety 

test, Study-, discussed in Section III.C. (page 35-36) of this report. 

A study design that includes the length of time 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 of the virus in shavings at room temperature(- 25'C). Two hundred grams (g) of 

pine shavings were obtained from the Me rial Inc.- The shavings were divided into two equal 

100 g samples. Each sample was saturated at a rate of 1 ml of sterile diluent to 1 g of shavings. The 

shavings were autoclaved for sterility. 

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Page 40 of67 

After autoclaving and cooling to room temperature, 100 ml of diluted RBA containing- pfulml was added 

to one sample, and 100 ml of diluted licensed HVT containing- pfulml was added to the other sample. 

The inoculated shavings were held at room temperature for the i:luiation of the study. 

On days 0, 1, 2, 3, and 7 post-inoculation (or when three consecutive days read no virus recovery), 5 g from 

the RBA shaving samples were 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 layer of sterile 

gauze. The collected fluid was used for virus recovery by placing one millimeter of a second filtrate onto 

chicken embryo fibroblasts monolayers. The procedure was repeated with the HVT shavings sample. 

On Day 0, the RBA samples resulted in an average of 4 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 by the CVB on November 6, 2013. 

1. Do the Donor DNA sequences or gene sequences associated with the Backbone Biological Agent 

enhance the ability to survive in the environment or increase resistance to therapeutic agents? 

The insertion of the NDVI gene should not contribute to the ability ofthe backbone virus to survive in the 

environment since the insertion of the NDVI gene is in a non-e~sentia1, non-regula_torY' i"egion of the HVr 

vector. The survivability of HVT virus with or without the NDVI gene described in Section III. G. (page 39-40) 

Showed no virUs recovery after the day of inoculation. 

IV. RISK ASSESSMENT 

Risk assessment m8y be defined as the likelihood of an adVerse event occurring and the consequences if that 

adverse eVent occur's. An adverse event is deflf~ed as safety hazards to animals, public health, or the 

environment. A safety hazard is defined as a danger, risk, or peril; absence of predictability associated with an 

event; or an expected or unpredictei:l event. 

A. Procedure 

The risk assessment is conducted based on safety characteristics of the vaccine. The safety characteristics 

are based on empirical data and established scientific facts. The completion of a risk 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 of 67 

Hazard identification consists of identifying all possible adverse events to animal safety, public health safety, 

and environmental safety relative to the recommended use of this experimental vaccine, Marek's DiseaseNewcastle Disease, Serotype 3, Live Marek's Disease Vector, Product Code 16N1.RO, unlicensed. 

The vaccine that will 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 risk to animal safety, public health 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 of the RBA in mice 

and the other non-target avian species is the same as that seen previously with 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 if other nontarget species 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. Target animal safety: 

(i) Vaccination of chickens 

The Master Seed Virus did not demonstrate any virulent properties when inoculated into 

one-day-old chicks at 1 0 times the recommended field dose as written in 9CFR 113.330. A 

test was carried out to study the possibility of reversion to virulence of the vaccine strain. 

Five passages were carried out. Infected leukocytes were found in each passage. No 

abnormal signs were noted in the birds during the passages. NaiVe hatchmates were placed 

in contact with each group of birds 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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(ii) Vaccinatlon/chal!enge 

May 18, 2014 

Page 42 of67 

No adv8rse immune interactions are expected with the use of this 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 days with 104

'

5 EIDsa/0.1 ml·dose. This study 

was accepted by the 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 of GB Texas NDV. Th1s study was accepted by the CVB 

on August 23, 2012. 

In Study-. chicks hatched from susceptible 18- to 1 9-day-old embryos that were 

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 December 23, 2013. 

(iii) Reversion to virulence 

Backpassage studies demonstrated that the RBA does not revert to 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 of the 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) Purity testing 

The MSV and the pre licensing serials were tested by the applicant and found to meet all 

standard requirements of 9CFR113.300 and the additional testing of the 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) Identity testing 

May 18, 2014 

Page 43 of67 

The expression of the I gene was demonstrated by using fluorescent antibody testing 

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) Effect of gene manipulation on pathogenicity 

The RBA was derived from the FC-126 strain of HVT, which is recommended for use in day 

of age chickens or in avo vaccination. The strain is avirulent for chickens. 

The donor gene II has no known pathogenic or toxic properties. No pathogenic or toxic 

properties have been identified for the product when expressed in the experimental 

recombinant- vaccine (see Section Ill). 

(al Phenotvpic effect ofthe gene modification!s) 

(i} Attenuation of wild-type HVT virus 

The parental FC-126 strain was isolated from turkeys by R.L Witter (Regional Poultry 

Research Laboratory, USDA, East Lansing, Michigan, USA) in 1970. The vaccine strain FC126 of HVT, 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 of chickens have been vaccinated with these 

vaccines (see Section II.A.1, page 7). 

(ii) Insertion of the I gene into a putative ORF 

The locus of insertion 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 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. (see Sections II.A.1., page 7 and 11.8.1., page 11 ). 

(iii) I gene modifications 

The NDVI gene (cloned as eDNA from NOV viral 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 Pox Vector, licensed October 27, 1995. 

(Section 11.8.1., page 11 ). 

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. ' . 

lbl Previous use of dnnor genes and recipient microorganism 

(i) Donor Sequences cir Donor Genes 

May 18, 2014 

Page 44 of67 

The exact same. donor NDVI gene sequence has been used in USDA licensed product 

17C1.RO, Newcastle Disease-Fowl Pox Vaccine, Live Fowl Pox Vector. Licensed October 

27, 1995. 

(ii) - promoter 

corresponds to the promoter/enhancer of 

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 effect of any genetic marker(sl 

The RBA MSV does not contain any genetic markers. 

(vii) Genetic s.tabillty 

The RBA is genetically stable in vitro {see Section II.C.5.b.(i) and (ii), page 27-28}. 

The genetic stability of the 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 Me rial, Inc. indicate that the RBA is phenotypically stable in cell 

culture (see Section III.A., page 34). The phenotypic stability of the RBA MSV in vivo was 

studied in a reversion to virulence study through five backpassages in chickens (see Section 

Ill. C., page 35-36). The expression oftliel gene was expressed through five 

backpassages. The expression was maintained at 100 percent{see Sections II I.A. (page 

34) and II.C.5.b. (i) and (ii), page 27-28}. 

(ix) Alteration in tissue tropism 

The results of transmissibility studies indicate that the proliferation/tissue tropism of the 

experimental vaccine virus is similar to its parental HVT vaccine virus {Section !II.D., page 

36-37). 

(x) Effect of overdosing 

Target host animals were inoculated with 10 times more virus than the recommended field 

dose with no adverse effects observed (see Sections Ill. C., page 35-36). 

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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) 

c. Non-target animal safety 

(i) Susceptible non-target animals 

May 18,2014 

Page 45 of67 

The safety of the 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 HVT virus (see Section Ill. C., page 35-36). 

(ii) Virulence in non-target animals 

The results of the safety studies indicate that the host/range specificity of the RBA MSV has not been 

altered from that of the parental FC-126 vaccine strain, and that the recombinant vaccine is safe in 

non-target avian species (see Study- in Section Ill. C., page 35-36). It was also shown to be 

safe in a non-target mammal species (mouse). In virus proliferation studies, no virus recovery was 

detected from tissue samples of mice inoculated with the RBA (see Section Ill. C., page 35-36). 

In this study 48 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 HVT vaccine diluted to contain-/dose. Twenty-four one-dayold chickens were inoculated with- of HVT vaccine 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 four vaccinate or sham vaccinated day-old 

chicks or turkey 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 a qualitative analysis conducted 

on each spleen for Marek's virus recovery and NDVI staining. On Day 42 the remaining turkey 

poults and chickens were splenectomized and evaluated for virus recovery. The results showed no 

spread of the 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 HVT virus from infected chickens to sham 

chickens but did spread to na"ive turkeys at a limited rate (-38%). 

(iii) Possible outcomes of non·target animal 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). 

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U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, 

Live Marek's Disease Vector, 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 of HVT causing productive infection 

in any species other than 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 HVT vaccine for 

chickims in Europe without any adverse effects or hazards to public health. 

b. Probability of human 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 other than avian species and 

n6 ibfection of hum~ns has been reported [Tnpathy, Deoki: Pox. Dis~ases of Poultrv (1991 ). Ed. 

C'ainek, B.W., Iowa State UniverSity Press, Ames, iowa; pp 583-596]. The parent HVT vaccine has 

been us'ed as a cbmmefcial poultry vaccine in Europe with hb reported adverse effects or hazards to 

public health. 

d. Virulenc_e of the 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 of experts in the human 

and avian cancer field 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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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 of 67 

The risk to the environment is low. The shed/spread capability of the RBA vaccine is limited. Some 

virus may be spread to the environment through the dander of vaccinated chickens but the shed virus 

cannot infect other chickens indicating that the NDVI gene insertion did not provide an increased 

spreading ability to the virus. The hosUrange specificity of the experimental vaccine does not appear 

to have been altered from the wild-type HVT, which is restricted to avian species. The safety 

characteristics of the experimental vaccine in non-target avian species do not appear to have been 

altered from that of the recipient virus which was derived from a commercial HVT vaccine strain. 

Although the distribution of HVT strains in the environment has not been reported, no adverse effects 

have been attributed to the use of these 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 HVT vectored NDV vaccines currently licensed and being distributed globally. To our 

knowledge the RBA does not behave differently in terms of environmental distribution than these 2 

HVT-vectored NDV vaccines. 

c. Shed/spread capabilities 

The results of studies indicate that the shed/spread capabilities of the RBA do not differ from that of 

the parental virus. This study was part of the reversion to virulence Study-. CVB approved on 

March 9, 2011.The contact controls placed with each group of chickens receiving the backpassed 

virus did not show any virus recovered from spleens nor were 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 HVT were reported for any of the 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 other vaccine viruses was evaluated for this 

vector in licensing- USDA product code 1A88.RO, 

- was combined with virulent MDV, GA 22 strain and with serotypes 1 and 3 of MDV's. This 

allowed for an evaluation of recombination between the viruses. The results showed there was no 

difference in pathogenicity of any of the MD virus strains when combined with- and that no 

recombinant virus was detected. Study- was conducted with was 

combined with a virulent MDV strain (GA 22) and with serotypes 1 and 2 of Marek'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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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 48 of67 

There were no statistical differences in average body weight between the GA 22 and the-

./GA 22 vaccinates, the Rispens and the-/Rispens vaccinates or the SB-1 and the 

-/SB-1 vaccinates at the end of the 120 day observation period. The results showed no 

increase in palhogenicity of any of the MD strains when co-inoculated with-(see Section 

III.E., page 37). 

e. Potential ' for transmission to Invertebrates: 

The potential for transmission of the RBA is expected to be minimal. The limited shed/spread 

properties of the virus should reduce the probability of mechanical transmission post vaccination (see 

Section IV.B.3.c., page 47). Even if transmission did occur, the attenuated properties of the RBA are 

not expected to be altered (see Section III.C., page 35-36). 

f. Host I range specificity: 

There are no reports of HVT causing ·productive infection in any species other than the avian species and 

no infection of humans has been reported [Calnek, Witter: Marek's Disease. Diseases of Poultrv (1991 ), 

Iowa State University Press, Ames, Iowa; pp 342-385; and H. G. Purchase et al., "Responses of cell 

cultures from various avian species to Marek's disease virus arid turkey herpesvirus" (1971 ), Am. J. Vet. 

Res., 32, 1811-1823]. Our non-target studies showed that the insertion ofthe NDVI gene did not modify 

the host range specificity of the vector. 

g. Survivability of the microorganism in the environrrient: 

The very limited amount of virus shed by the feather follicles via dander may potentially survive for 

months in the environment, similar to the parental HVTvirus and other licensed HVT-vectored vaccines. 

This shed virus is, however, not infectious for chickens by the natural route. It may potentially infect HVTila"ive turkeys. However, the prevalence of HVT infectiOn in turkeys approaches 100 percent and we have 

demonstrated that HVT-infected turkeys cannot be horizontally infected with the RBA. Experimental 

vaccine from vaccinated chickens did not spread to HVf infected turkeys, na'ive turkeys, HVT infected 

chickens or na"ive chickens (Study-. The study results also showed that there 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/or chemical factors affecting dispersal in the environment: 

No physical and/or chemical factors have been identified that would enhance the dispersal of the 

RBA in the field 

i. Adverse ecological effects 

The demonstrated safety and lack of proliferation of the RBA in other non-target avian and 

mammalian species (see Section III.C, page 35-36), and the established limited host range of HVT 

reduces the risk of spread to non-target animal species. 

Confidential Business Information Redacted 
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) 

C. Release Assessment Prior to a Proposed Field Safety Trial 

Environmental release proposal 

Summarv 

May 18,2014 

Page 49 of67 

The safety characteristic of the vaccine microorganism must be evaluated within the context of the target 

environment. Thus, the release assessment consists of a comprehensive evaluation of the proposed release 

so as to detenmine 1} the location and characteristics of the release site; 2} the test dose and total amount of 

the experimental biologic to be used in the proposed study; 3) the frequency and duration of response to the 

test material; 4} potential escapes into occupational, residential, or outdoor environments; and 5} the 

individuals, populations, or ecosystems that will be, or may be, exposed to the experimental biologic. 

For the purpose of risk assessments, proposed studies with live experimental vaccine microorganisms are 

classified as either contained studies or envkonmental releases. A Contained Study is defined as an 

experiment in laboratory contained or physically contained animal facilities. An Environmental Release refers 

to the release of the vaccine microorganism into the accessible environment. Planned introduction into the 

environment refers to the deliberate release of live 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 of small-scale field tests as 

appropriate- for the experimental veterinary biologic. 

Contained release 

Not applicable. 

1. Environmental release 

Summart 

Merial, Inc. proposes to conduct safety field tests to determine the safety of the Marek's Disease-Newcastle 

Disease, Serotype 3, live Marek's Disease Vector vaccine, 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 least three regions of the USA. A list of tentative recipient states is included 

below. These studies will be conducted in chicken houses and hatcharies that are typical of the commercial 

poultry industry. The potential for escape and dispersal from the study sites is low. There are no known 

biological organisms, physio-chemical factors, or toxic chemicals that would contribute to the establishment of 

the RBA in the environment. Personnel experienced in poultry health management will be conducting the 

proposed study. 

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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) 

2. Tentative location of test sites 

Arkansas, Georgia, Indiana, Iowa, Missouri, Ohio, Oklahoma, Pennsylvania, and Virginia 

3. Characteristics of test sites. 

May 18, 2014 

Page 50 of 67 

The test sites include commercial poultry houses, multiple houses per site. Each house will hold approximately 

17,000-22,000 birds. The eggs/chicks will be inoculated at the cooperator's hatcheries and then trucked to the 

house(s). The details of each test site will be included in the 9 CFR 103.3 authorization request for the Field 

Safety Study. 

4. Personnel 

Personnel administering the vaccine will consist of hatchery personnel under the direction of the participating 

cooperator. Merial veterinary staff, and/or qualified Merial R&D staff will be present for the vaccination and 

placement of the chickens. Merial personnel will be involved in data management and in study follow-up. The 

Veterinary Scientist in charge of this Field Safety Study will 

5. Experimental design 

Number of animals 

At least 20,000 birds will be vaccinated with the test vaccine. 

Description of animals 

Eighteen-day-old embryonated commercial eggs or one-day-old commercial layer/breeder/broiler chicks of 

mixed breeds will be used for this study. 

Route of administration 

In avo inoculation of the eggs or subcutan~ous inoculation into the nape of the n~ck of the chicks. 

Dosage 

The vaccine for subcutaneous or in ova injection will be prepared for use in the mixing room of the hatchery. 

The vaccine container will be a sealed plastic IV bag, which will be fitted to a Marek's disease vaccinating 

machine using sterile plastic IV tubing. The serials of product that will be used in this study will have been 

satisfactorily tested for purity before use in the field studies. 

COnfidential Business Information Redacted 
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) 

Total amount of test material 

May 18,2014 

Page 51 of 67 

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 of one (1) ampule per 400 ml of sterile diluent for subcutaneous injection 

and one (1) vial per 100 ml sterile diluent for in ovo inoculation. Subcutaneous injection of 0.2 ml or in ovo 

inoculation of 0.05 ml constitutes a single field dose. With the exception of NOV and Marek's products, the 

chicks will receive all other vaccines according to each company's vaccination program. Approximately 

100,000 doses will be required for the trial. 

Protocol 

The field safety protocol will be submitted to CVB for approval. 

Frequency and duration of exposure 

Direct exposure of people to vaccine could potentially occur at the hatchery. The animal caretaker/grower 

responsible for the managerr1~nt of the test flocks will be exposed to the inoculated chickens, but not the 

vaccine, for the length of the 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 show that HVT can be 

transmitted to humans. The recombinant virus cannot cause NDV either in chickens or in humans because it 

does not contain complete NOV, only a gene from that organism. The duration of exposure is not expected 

beyond the day of vaccination. 

The method of disposing of waste 

All vaccine containers, diluent containers, 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 machines will be disinfected according to standard hatchery procedures. Dead birds and litter will 

be treated according to each cooperating company's program. 

Decontamination of test site 

Following vaccination, the vaccination area at the hatchery will be disinfected and washed according to 

standard procedures. Crates used to transport the chickens will be returned to the hatchery where they will be 

washed and disinfected. 

The broiler/layer houses will be cleaned and made ready for the placement of the next flock of chicks 

according to each company's program. 

6. Potential for escape and dispersal 

Low shedding was seen but spread transmission studies were negative. Therefore, no escape or dispersal 

into the environment is anticipated. 

Confidential Business Information Redacted 
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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) 

7. Potential for establishment In the environment 

May 18, 2014 

Page 52 of67 

Marek's disease virus already exists in the environment as a poultry pathogen. Several live Marek's disease 

vaccines including HVT vectored 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 of subcutaneous administration 

and in ovo inoculation will minimize the chance of escape into the environment. It is not anticipated that 

conditions will exist outside the laboratory that will support perpetuation of the recombinant virus. No toxic 

chemicals or metabolites are known to be produced by the virus, which could endanger the environment. 

8. Monitoring 

Monitoring methods 

The monitoring of the field test will consist of maintaining the records associated with the production of 

commercial chickens. Mortality and morbidity wilt be recorded by the grower at each test location. The flocks 

will be observed daily for seven weeks fOr adverse events related to the vaccine bY the grower or field 

personnel of the cooperating company. Merial's veterinary· staff and/or research department staff will visit at 

least once 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 disease will be necropsied and appropriate tissues 

examined for histopathology and/or retained for viral isolation. Any adverse clinical signs associated with the 

vaccination will be immediately reported to the veterinarian in charge. If there is any evidence of decreased 

hatchability associated With the vaccine, diagnostic work will be performed to d6termine the cause of the 

decreased hatch. 

Frequency of monitoring 

Each flock will be observed daily by the grower or caretaker during the growout, which is expected to last no 

more than 60 days for broiler type birds (layers/breeders will growout for a longer period of time). Birds will be 

observed at least once by the Veterinary or Research staff and more frequently if notified that adverse events 

have been observed in the flock. Birds will be observed for clinical signs of Marek's disease. The safety of the 

vaccines will be reported in the field safety study report after seven weeks of observation. 

9. Contingency plans in case of adverse events. 

No adverse events are expected but if adverse events attributable to the Marek's disease vaccine are 

observed, the primary investigator will be notified immediately and the event documented in the study log. 

The grower, the cooperator's veterinarian and Merial veterinary staff will work jointly to determine a plan of 

action, if necessary. 

Confidential Business-Information -Redacted 
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) 

D. Risk characterization 

May 18,2014 

Page 53 of67 

Risk characterization integrates the results of hazard identification and the release assessment into a risk 

statement that includes: 1) a likelihood rating; 2) a consequence rating; 3) a risk rating; and 4) a discussion of 

risk. 

Each likelihood and consequence rating is qualified by a degree of certainty rating and includes a justification 

for the rating. The justification for the ratings consists of identifying the applicable sections in the hazard 

identification and the release assessment that support the assigned rating. The risk rating is based upon the 

likelihood, consequence, and degree of certainty ratings. 

1. Likelihood ratings 

Likelihood ratings are assigned for animal 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 event will most probably occur 

Consequence ratings are also assigned for animal safety, public health safety, and environmental safety 

based on the following criteria: 

Low= 

Medium= 

High= 

The consequences if the adverse event occurs will not be severe (the adverse 

event is self-limiting and would have negligible impact). 

The consequences if the adverse event occurs is moderately severe (the adverse 

event will have an impact, but it is not permanent, and can be treated). 

The consequences if the adverse event occurs is severe (the adverse event will 

have an impact, is permanent, and cannot be treated). 

3. Degree of certainty rating 

Each likelihood and consequence rating is qualified by a degree of certainty rating that is based on the 

following criteria: 

Certain= 

Moderately certain = 

Uncertain= 

Confidential Business Information Redacted 

The rating is supported by direct scientific evidence. 

The rating is supported by indirect scientific evidence. 

the rating is not supported by scientific evidence. 
Merial, Inc. 

U.S. Vet. Lie. No. 298 

Risk Analysis 

Marek's Disease-Newcastle Disease Vaccine, 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 of certainty ratings 

described above (refer to Table 6). Each numerical value rating was derived from the importance placed on 

the rating of each category. The assigned numerical values are weighed to place emphasis on the seventy of 

expected risk. These values reflect the professional judgment of the applicant (Merial, Inc.). 

5. Risk ratings 

The risk ratings are based upon the likelihood, consequences and degrees of certainty 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 risk rating of low, medium, or high. The assigned risk ratings were weighed to place emphasis on 

the severity of the expected risk. Again the severity of the risk reflects the judgment of the applicant. The low, 

medium, and high risk ratings are defined for the purpose of decision-making as follows: 

Low= 

Medium; 

High; 

AcCepiabl9 riSk; ver'y few concerns are associated with the proposal (does not 

justify denying the proposal). 

Unacceptable risk; moderate concerns are associated with the proposal (either 

identify valid mitigative procedures or deny the prop-osal). 

Unacceptable risk; major concerns a:re assOciated with the ·prbposal (deny the 

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 of certainty 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 of certainty 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. Risk Analysis 

u.s. Vet. Lie. No. 298 Marek's Disease~Newcastle Disease Vaccine, 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 of the RBA does not 

appear to have been altered from the wild-type HVT, which is restricted to avian species. 

Although the protocol of the field safety study does not include procedures for monitoring the 

shed or spread of the RBA into the environment during the course of study, studies previously 

conducted by the applicant indicate that the shedding capabilities of the experimental vaccine 

are limited, and the shed virus is unable to spread to other chickens or HVT-infected turkeys. 

Furthermore, the proposed release will be confined to poultry houses. Thus, exposure of nontarget animals and the dissemination of the experimental vaccine virus in the environment during 

the course of the field tests will 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 subsequentiy transpOrted to commercial chicken 

houses for the duration of the 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 hatchery to the poultry houses will also be decontaminated. 

The RBA will be used in combination with other Marek's disease vaccines (serotype 1 and 2). 

Our recombination safety study has shown that co-administration of these vaccines did not 

generate a more virulent virus. 

Another concern could be the recombination event beiw~en the RBA and a field HVT strain 

resulting in the horizontal transfer of the I gene. Since natural HVT infection of chickens has 

never been described, it is unlikely that the chickens will be co-infected with the RBA and field 

HVT. Even if parental HVT is accidentally administered together with the RBA, the homologous 

recombination that may occur could potentially transfer the NDVI expression cassette from the 

RBA to the parental HVf. The newly created vfrus woUld be very similar to 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 of such recombination events is 

negligible. 

Conclusions and discussion of risk 

The risk to animal safety associated with this proposal to conduct a field trial with experimental 

Marek's Disease-Newcastle Disease Vaccine, Live Marek's Disease Vector, is low. The recipient 

virus used to construct the RBA MSV was derived from an HVT vaccine strain that has been 

used as a commercial poultry vaccine in the United States and Europe. Safety studies 

conducted by the applicant demonstrate the safety of the 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 of certainty rating: Moderately certain (MC) 

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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) 

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 of certainty 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 RBA was derived from an HVT vaccine strain that has been used as a 

commercial poultry vaccine the United States and Europe, with no adverse effects on public 

health. Studies conducted by the applicant indicate that the safety characteristics of the 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 personnel will be exposed only to 

the inoculated chickens. Studies conducted by the applicant demonstrate that the shed/spread 

property of the experimental vaccine from vaccinated chickens is limited. 

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) 

c. Environmental safety 

Likelihood rating: Low (LL) 

Degree of certainty 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 if adverse event occurs: Low (CL) 

Degree of certainty 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 

Risk Characterization: LL.MC.CL.MC 

Risk rating: Low 

Confidential Business Information Redacted 

May 18,2014 

Page 58 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, 2014 

Page 59 of67 

Conclusion and discussion of risk 

The safety risks to the environment are low. The host range specificity of the RBA does not 

appear to 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 for monitoring shed or 

spread of the experimental vaccine into the environment during the course of the study, studies 

conducted by the applicant indicate that the shed/spread capabilities of the experimental vaccine 

are limited. Furthenmore, the proposed release will be confined to poultry houses. Thus, the 

exposure of non-target animals and the dissemination of experimental vaccine virus in the 

environment during the course of the 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, or the environment. 

B. Recommendations 

The safety risks to animals, public health, and the environment are low. A risk rating of low is acceptable risk, 

with very little concern associated with the proposal. The applicant recommends the approval of the proposed 

field test by VB, APHIS provided the study is conducted in accordance with the requirements of 9CFR 1 03.3, 

and the filed protocol of the study. 

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) 

Table 6. Calculating the 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 

•tf the Likelihood rating is Medium or High and the Consequence rating is also Medium or High 

use Degree of Certainty Ratings I; for all other combinations use Degree of Certainty Ratings II. 

Degree of Certainty Ratings I 

Certain (C) c =0.50 

Moderately Certain (MC) MC = 0.75 

Uncertain (U) u = 1.00 

Degree of Certainty Ratings II 

Certain (C) c = 1.00 

Moderately Certain (MC) MC = 0.75 

Uncertain (U) u = 0.50 

EXPECTED RISK 

[(likelihood) x (degree of certainty)] x ](consequence) x (degree of certainty)]= Risk Rating 

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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) 

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 

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May 18,2014 

Page 61 of 67 

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 Disease Vector, 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 

Risk Analysis 

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

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

Appendix 1 

May 18,2014 

Page 63 of67 

Pages 63-66 redacted due to confidential business information 

Confidential Business Information Redacted 
Merial, Inc. Risk Analysis May 18, 2014 

U.S. Vet. Lie. No. 298 Marek's Disease-Newcastle Disease Vaccine, Serotype 3, · Page 67 of 67 

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

Appendix 2 

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

oncoge11ic avian herpes viruses: A review" 

Confidential Business Information Redacted 
i' 

PUBLIC REALTH GO~C~S 

FROM l!UM!\N EXPOSURE TO ONCOGENIC AVIAN 

lfllRl'ES V!RUSES : A REVIEW 

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. Resu1 ts .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 (PCB e) 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 "!lid Re: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 pi theU!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~edv1rus~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.nges tion 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<f Hoc 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 w1 th 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 humans l 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 rhino tracheitis, 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 frog a, 

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!lli1ology 1 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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