### Risk Analysis

Marek's Disease Vaccine,

Serotype 1, LiveVirus

USDA ProductCode: 16L1.RO (Unlicensed)

Updated

Merial, Inc. U.S. Veterinary License Number298

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### 298 Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed)



### Table ofContents
I. Introduction
.......................................................................................................................................................... 

### 6 

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

### 6 B. Proposal
........................................................................................................................................................... 

### 6 II. Description ofthe Regulated Biological Agent Construction
................................................................................

### 7

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

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

### 10 C. Construction and Characterization ofthe Regulated Biological Agent
...........................................................

### 13 Ill. Biological Properties orVirulence forthe Regulated Biological Agent used for Master Seed
.............................21

A. What are the known phenotypiccharacteristics or effects ofthe Regulated Biological Agent identified as Master Seed? (e.g., expresses envelope gene/sequence, lacks thymidine kinase activity, lowertiter in MA105 cells, etc.)
............................................................................................................................................ 21 

### B. Whatare the virulence characteristics ofthe Regulated Biological Agent used forthe Master Seed?
..........21 C. Is the Regulated Biological Agent used forthe Master Seed virulent fortarget animals, non-target animals?
..

........................................................................................................................................................................ 21 D. Do the Donor DNA sequences enhance the virulence orthe ability ofthe MasterSeed to survive in the target animals, non-target animals? ..........................................................................................................................23 

### E. Discussthe potential for horizontal gene transfer or recombination ofthe Regulated Biological Agent..
.......24 

### F. Describethe shed/spread capabilities ofthe Regulated Biological Agen\...
...................................................

### 26 G. Discussthe expected environmental impact orsurvivability ofthe Regulated Biological Agent. and provide available scientific evidence........................................... ............................................................................ ...28 IV. RiskAssessment............................................ ...................................................................................... 

### ...29



### A. Procedure .............................................. 

### ....................................................................29 B. Hazard identification
......................................................................................................................................30

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### Lie. No. 298 Serotype 

### 1, Live Virus, Code: 

### 16L1.RO (Unlicensed)



### 1. Animal safety..............................................................................................................................................30 

### 2. Public health safety .................................................................................................................................... 34 

### 3. Environmental Safety .................................................................................................................................34 

### C. Release Assessment Prior to a Proposed Field SafetyTrial ..........................................................................36 D. Risk characterization ......................................................................................................................................40 

### 1. Likelihood ratings ....................................................................................................................................... 40 

### 2. Consequence ratings ................................................................................................................................. 40 

### 3. Degree ofcertainty rating ...........................................................................................................................40 

### 4. Calculatingthe expected risk......................................................................................................................40 5. Risk ratings......................................................................................., ........................................................41 V. Risk Management Recommendations ...............................................................................................................44 

### A. Procedure .....................................................................................................................................................44 B. Recommendations.......................................................................................................................................... 44

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Tables 

### Page Table 1: Sequence ofthe primers used in PCR reactions and expected size (in bp) obtained after PCR 15 performed with different primer pairs on the- candidate, 

### the-parental strain and amixture of- and Rispens. Table 2: Calculating the Expected Risk 45 Table 3: Risk Ratings 46

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### Appendices 

# I Risk Analysis Marek's DiseaseVaccine, Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed) May 19, 2014 Page 5 of74

2. Purchase, H.G. and R.L. Witter(\986),. "Public health concerns from human exposure to oncogenicavian herpesviruses: A revieW' J. Am. Vet Med Ass.\89:1430-6 

### References 1. Afonso et al., J. Viral., 74:3815-31, 2000 2. Borenstein and Davidson, JViral Methods, 82:119-127, 1999 3. Bublot Mand Sharma J, Marek's Disease. An Evolving Problem, p.\68-185, 2004 4. F. Davison 

### &V. Nair (Eds.), Marek's Disease. An Evolving Problem, p. 17-31, 2004 5. H.G. Purchase et al., Am. J. Vet. Res., 32:1811-1823, 1971 6. Hertig et al., Virology 235:367-76, 1997 7. lsfort R.J. et al., Virology, 302:125-33, 1994 8. Jarosinski KW et al, 84:7911-7916, 2010 9. Jarosinski KW, etal, J. Viral. 81:10575-10587, 2007 10. Jones ei al., J. Viral., 70:2460-2467, 1996 11. Jones etal., Proc Natl Acad Sci, 90:3855-9, 1993 12. Kaleta, E. F. and Neumann, Diseases of Poultry, 12th Edition, p. 461, 2008 13. Kung etal., Microbiollmmunol., 171:1-25, 1991 14. Laidlaw and Skinner, J. Gen. Viral., 85:305-22, 2004 15. Moore et al, Avian Dis., 44:827-41, 2000 16. Morgan etal, Avian Dis. 36:858-870, 1992 17. Okamuraa H. et al, Vaccine, 20: 483-489, 2002 18. Osterriederand Vautherot, Marek's Disease. An Evolving Problem, p.\7-31, 2004 19. Purchase, H.G. and R.L. Witter, J. Am. Vet Med Ass. 189:1430-6, 1986 20. Reddy etal., PNAS, 99:7054-9, 2002 21. Rispens et al, Avian Dis., 16:108-25, 1972 22. Schat & Nair, Diseases ofPoultry, 12th Edition, p. 452-514, 2008 23. Singh etal., J. Viral, 77:5855-62, 2003 24. Spatz SJ and Schat K.A., Virus Gene, 42:331-8, 2011 25. Spatz SJ, Petherbridge L, Zhao Y and NairV., J Gen Viral, 88:1080-1096, 2007 26. Sun et al., Virology, 397:270-6, 2010 27. Thiryetal., Rev. Med. Virol.\5:89-103, 2005 28. Thiry etal., Vet. Microbial., 113:171-177,2006 29. Witteretal., Avian Dis., 41:407-21, 1997 30. Zhang and Cui, Sci China C Life Sci, 48:81-8, 2005

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I. Introduction A. Objective 

### RiskAnalysis Marek's Disease Vaccine, Serotype 1, LiveVirus, Code: 16L1.RO (Unlicensed) May 19, 2014 Page 6 of74

1. Identifywherethe Regulated Biological Agentwas constructed and where the productwill be made, tested and manufactured. Address the available level ofcontainment. The parental virus is an RN strain ofa Marek's Disease Serotype 1vaccine with the insertion ofthe long terminal repeat (LTR) ofthe reticuloendotheliosis virus (REV). This construct was developed atThe virus was transferred to Merial Select, Gainesville, GA

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

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

# -

2. Provide a brief(one sentence) description ofthe Regulated Biological Agent. The Regulated Biological Agent contains 

### Marek's diseasevaccine (RN strain), serotype 1which comprises a LTR sequence of REVB. Proposal 1. Whatisthe intended use ofthe product?
a. Species: Avian; day-old chickens b. Proposed claim: Marek's DiseaseVaccine, Serotype 1, Live Virus, Code 16L1.RO (Unlicensed) is indicated for use in healthy day-old chickens as an aid in the prevention of Marek's disease caused 

### by very virulent Marek's disease virus c. Geographic area: United States (all states),
d. Route ofadministration: Subcutaneous e. Briefdescription ofthe expected safety profile: The Marek's Disease Vaccine is expected to elicit an immune response in the chicken to Marek's Disease Virus without causing gross observable lesions as directed by 9CFR113.330 (c) (3).

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11. Description ofthe Regulated Biological Agent Construction A. The Backbone BiologicalAgent May 19, 2014 Page 7 of74

1. Whatorganism was used forthe Backbone BiologicalAgent? Are there any known virulence features associated with the Backbone BiologicalAgent?What happens inthetargetspecies?

Whatis the previous safe use ofthe Backbone Biological Agent? (i) Ifavailable provide historyof previous sa_fe use using published literature orinternal documents. Includethe recommended CDC/NIH biosafety level for use ofthe Backbone Biological Agent.

2. Physical characteristics ofthe Backbone Agent

a. Provide a flow diagram or explanation Ofthe process of howthe Backbone Biological Agentwas constructed.

- ----- --------------------------- -Theviruswas produced by infecting specific pathogen free (SPF) primary cell culture (chicken embryo fibroblasts, CEFs) and observing the cytopathic effect (CPE). The virus/cells were then harvested

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using atrypsin solution and then frozen back in acell-associated form using a specificfreezing solution containing DMSOthat protects the viability ofthe cell/virus. Viral DNAwas extracted from infected CEFs and was used to generate the biological agent. (i) Describe the proposed site for Donor DNA insertion
.



### .

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

# --

-- - - 

### --

The sequence ofCVI988 vaccine strain has been deposited into Genbank (Accession 00530348). This is oursequence ofreference forthe CVI988 genome forthe present SIF.

- - - - -

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

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(ii) Do theflanking regions ofthe proposed insertion site Backbone Biological Agent have any known regulatoryelements that could moderate the expression ofthe inserted Donor PNA?

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### B. Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed)

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

- - - - -- -

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

### (IIi) Identifyunique restriction endonucleases (not more than five) that

### will give identifiable digestion "patterns useful forcharacterizing the final Backbone DNA



### Don~r BiologicalAgents and Dono";DNA or



### Gene~ 1. Whaiarethe DonorBiologicalAgents ilsedto source each Donor DNA sequence inserted intothe



## B~ckbone Biological



## Ag~~t? '·
. . . . . . j_'''

. ,,

**-,._~- -~""**  

### tJ:om

**paren~a-1-** 

### MDV

**~8¢cine** 

### strain Snd



## ...we.therefore·Eansicie"red

**lh~--** as 

## The~virus



## wa~oiJI:ai~<>d DNAfrom 

### ii ·

In summary, the Donor Biological Agents used to source Donor DNAsequences are the following:

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### a. Has there been safe use ofthe DonorSequences orDonorGenes as wellas Safe use ofthe Donor BiologicalAgent? {i) Provide relevant references forsafe uSe. 

### No liCensed vaCcine h'8s incorporated the donorsequence

Forboth examples, the parental MDV1 virus was avirulent strain. In- virus,

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i' the backbone is the non-p'athogenic .......strain 

### and



## therefor~, th.\-'virus is expected to

, .":·:·: _-. · --" --· ._ - - :r-:- ·_- .-,_T-_-.;:_,-,_,...,,_.,_.,:r,-·
- - - -· · be further.attenuaJed compared to

**th~se** examples.
. · '··· · ·•... · .

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

Donor sequence. No identified virulence genes were therefore brought from the .......Donor 

### sequenceinto the- candidate. The Donorsequence had a long homologous sequence with the parentaliiiiiill genome. A sequence analysis ofthe genomic regions flanking the indicated that this sequence is identicalto the Donor sequence. In particular, the sequenced region contains

the Donorsequence does not contain genes involved in MDV_virulence.

.InSl!mmary, the genomic partthat is known to 

### Contain MDVvirulence genes is

**o~!ginat~ng frQ_~**  the:-

- in the

# 11!11111 candidate. The Donorsequence includes · ' · -
--------- - -----
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- asequence where no gene involved in virulence has been identified. The resulting chimera is 

### therefore expected to conseiVe the safety ofthe- backbone.

Figure4: Localization of majorgenomic differences between attenuated CVI988 

### strain and oncogen.ic· on the Genbank 00530348 CVI988 

### sequence.



# - b. Are there specific parts ofthe Donor Gene(s) orSequences thatwere used for insertion? Show pertinentsequences or restriction endonuclease sites. 

### The DonorSequence-containsMDVsequences flanking- 

### insertthatwere used for targeting insertion ofthe- into the exactsame locus ofthe- 

### genome by 

### homologous recombination. As explained above and shown in figure 3, the MDV flanking sequence encompasses

# I These flanking sequences do not contain genes known to play a role in MDVvirulence. 

### C. Construction and Characterization ofthe Regulated Biological Agent
1. Provide aflow diagram on the construction ofthe Regulated Biological Agent. Include the following:

a. FinalBackbone BiologicalAgent: b. DonorDNA orgene c. Allshuttle vectors d. Hostcelllines used e. Selection techniques andmethods usedto construct the final RegulatedBiologicalAgent See Section liB for flow diagram.
The flow diagram on the construction of- agent is shown in Figure 3. The Backbone Biological Agent is

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the- Gallid herpesvirus 2 (Marek's disease virus serotype 1)- strain .•••••••

- isthe Donorsequence. The Host cell line used is primary chicken embryo fibroblasts (CEFs). Genomic DNA isolated from purified > ---------- --



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

### ------

Pathogenesis experiments in- chickens showed that, like parental- virus,- was fully attenuated and unable to transform T-lymphocytes. 

### Since- virus was avirulent in chickens, its protection efficacy against challenge with- a highly pathogenic field strain, was evaluated

# at-
Results showed protection compared to. protection 

### by commercial CVI988 vaccine indicating

A

### virus suspension of- virus was sent from- to Merial Select forevaluation. The material was 

### subjected to plaque purification and 3clones that contain

- were selected.



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

### - - 

## - - 

### - - - 

# - - 

# -- ---- -- - 

### - - 

### --- -- 

### ------ -- 

# -

2. Describethe laboratory methods orcriteria used to evaluate the flanking regions of

### the. include 

### the sequences

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### Live Virus, Code: 16L1.RO (Unlicensed) May 19, 2014 Page 16 of74

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### -

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a. Characterizethe physical map using unique Donor DNA and Backbone Biological Agent restriction endonuclease sites and describe resulting restriction fragments and digestion patterns. 

### The restriction map of- is shown in the- genetic stability report (Appendix 1, figure 2). A Southern blot analysiswas performed on the- DNAdi11ge,:ted



# ======Obse~rved~ hybridization patternswere identical to the expected ones based on the sequence ofthe Donor DNA.
b. Devisea PCRor restriction endonuclease test based on the Backbone Biological Agent sequence 

### and the DonorSequence thatwill identifyand characterize the Donor DNA/Backbone Biological Agentconstruct.



### As described above, the PCR using prim"ers.can differentiate the- agent from the parental

- virus and any other MDV1 isolates. Results ofthe PCRwith primers-areshown on Figure 6. This PCR can clearly distinguish- from otherserotype 1Marek's disease viruses (Rispens, GA-22 and

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c. What

### .will be the criteria forstabilityand purity ofthe Regulated Biological Agent MasterSeed n and n+5? The genomicstability was evaluated by

# (1)-.:~~~================ - (2) by Southern blotting and (3) by sequencing (1) the- masterseed virus (MSV), (2) the 

### in vitro passages from the MSV (X+5), and (3) the**5~** 

### in vivo back-passage in chickens (BPS). Results were exactly as expected and identical forthe 3 tested samples confirming identity and showing the genetic stability ofthecandidate. Results are described in the- genetic stability report (Appendix 1)

d. Provide the genetic sequence in electronic formatforany new oraltered genetic sequences, including insertion and flanking sequence. Enclosed with this submission is an electronicfile containing the- sequences described in the -geneticstability report: the sequence from the

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

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4. Whatis the recommended NIH/CDC biosafety level forthe Regulated Biological Agent? According to NIH/CDC definition, Marek's disease virus is a Risk Group 1agent since it is not associated with disease in healthy adult humans. BSL2 practices and procedures were utilized to ensure proper containment ofthe experimental construct in a research setting. At this time, considering the generated safety data, we would consider BSL1sufficient for- similarto the parental organism. 5. Provide a shortsummaryordescription ofgenetic motifs thatmay have resulted as a consequence ofthe genetic recombination (II.C.3.d). Are there any known motifsthatmay promote homologous 

### recombination, DNA insertion, or gene expression ofexisting or new.open reading frames?



### inserted atthe exactlysame location in thegenome. TtfeiVirus has beEm plaque purified and a homogenous viral

### pOpt.ilati~ri havingConfidential Business Information Redacted

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### 74**~** -------**-----~--------** 

### -----

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

## -------

- - ---- -- 

### - - 

### --- - 

### --- - --- 

# -- ------ --

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

-- --------- potentially betransferred to anotherfield MDV isolate by homologous recombination. Such genetictransfer requires having both viruses infecting the same cells. MDVvaccines are administered 

### by a parenteral route to the birds before (in ovo) or at hatch, whereas the natural infection byfield MDV1 (wtMDV) isolates occurred bythe respiratory tract MD vaccines induce a rapid onset of immunitythat restrictswild type MDVinfection and replication. The likelihood of having a cell co-infected with 

### vaccine and wtMDV at the same time is therefore very low. If nevertheless co-infection occurs, different chimeras could be generated 

### by single or double crossing-over

### between- and wtMDV1 genomes that could lead to the generation ofa Such new virus would emerge in avaccinated host and therefore its replication would be inhibited 

### by the vaccine-induced immune response. 

### It mayfail to reach and replicate in the featherfollicie sufficiently to have enough virus shed that could be successfully persist into the environment and transmitted to other chickens. Based on the current should not increase MD condemnations since itshould showattenuated phenotype forbodyweight loss, mortality and MD lesions compared to the parental wtMDV. The risk ifit occurs, the consequences would be negligible. In summary, we consider that the insertion recombination or DNA insertion.

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111. Biological Properties orVirulence forthe Regulated Biological Agent used for MasterSeed A. Whatare the known phenotypiccharacteristics oreffects ofthe Regulated BiologicalAgent identified as MasterSeed? (e.g., expresses envelope gene/sequence, lacks thymidine kinase activity, lowertiter In MA105 cells, etc.) The insertion ofthe. expression cassette into the serotype 1- strain vector,

- had no negative effect on the in vitro replication ofthe vector as determined during

The cytopathiceffect ofthe MSV to MSVX+S of- showed typical herpesvirus infection oichicken embryo fibroblast distinguished by foci of infection with rounded refractive cells resulting in plaque formation.
B. Whatare thevirulence characteristics ofthe Regulated Biological Agent used forthe MasterSeed? 

### The- virus used asthe Backbone BiologicalAgentwas isolated from

- No known virulence feature is associated with the Backbone Biological Agent.

- As described in Section 11.8.1, Studies**~ere** conducted to demonstrate that the MDV- 

### SR-1 virus was not pathogenic 

### induding a reversion to virulence study, safety in mice, atrophy of lymphoid organs such as thymus, spleen and bursa, and safety in non-target avian species (See Section Ill Cfor individual study design and results). No difference in virulence was observed between the parental- strain and the MDVSR-1 virus.
C. Is the Regulated Biological Agent used forthe Master Seed virulentfortargetanimals, non-target animals? The regulated biological agent, MDV SR-1- is avirulentforchickens. The ability ofthe virus to revertto avirulentfonn was evaluated in

inoculated intraperitoneallyWith the MDV SR-1- MSV; blood was taken at seven days postinoculation and used to inoculate another group of one-day-old chickens. Four additional passageswere made. At each passage, narve hatchmates were placed with the inoculated birds to serve as contact controls to monitorviral spread. Also at each pa:ss<1ge

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from each passage were necropsied and examined for lesions typical ofMarek's Disease. A final passage was conducted using 25 one-day-old chickens. A control group receiving the MSVdiluted the same way as that given to the initial vaccinates and a second control group receiving a placebo were evaluated with the final product passage. Na"ive hatchmates were also placed in contact with birds ofeach ofthe three groups: final backpass, MSV inoculates and the birds receiving the placebo. These birds were also necropsied at seven weeks post-inoculation; lesions were not observed in any ofthe birds. MDV SR-1- MSV was recovered from BP1, BP2, BP4 and BPS. Virus was not recovered at BP3. No virus was recovered from the contact controls at any point evaluated in the study. Underthe conditions ofthis study the MDV SR-1,

- MSV did not revertto virulence nordid it spread to non-vaccinated contacts. The study satisfied the recommendations ofVeterinary Services Memorandum 800.201 for evaluating the lack of reversion to virulence in chickens. This study reportwas accepted by CVB on 20 December2011. The safety of MDV SR-1- experimental X+5 (final product passage) as evaluated in mice- -showed no recovery ofvirus after subcutaneous injection and epidermal scarification of 15 mice. On days 3, 7, and 14, the trachea, brain, spleen, heart, liver, lungs, kidneys and skin at the site ofinoculation were removed from 5vaccinates and 2 naiVe controls that received a placebo only.•••••••

- from the tissue homagenates and evaluated for presence of Marek's disease virus on chicken embryo fibroblast monolayers. All tissueswere negative forvirus recovery. This study supportsthe lack ofa negative effect ofthe MDV SR-1 •••virus in non-target animals. This study reportwas accepted by CVB on 16April 2012. Safety in non-target avian 

### species,-,was evaluated using the following avian species: quall, turkey, duck, pheasant and pigeon. Each group ofbirds was inoculated with experimental X+5 (final product passage). A group ofna"ive birds ofeach species were inoculated with a- placebo. On studydays46 and 47 birds from each species were bled and tested for Marek's disease antibodies. The birds from all the species were necropsied at eitherday 46 or47 and observed forgross observable lesions ofMarek's disease. The MDV SR-1- experimental X+5 vaccine was shown to be safe for non-target avian species. No lesions were seen and no humoral antibody to Marek's disease was induced by the vaccination. This study report was accepted by CVB on 19 April 2012. A 120day safety study- was conducted according to 9CFR 113.330 Section (b). Two hundred (200) one-day-old, SPF chickens were randomly assigned to one offour groups offifty and to housing in negative pressure isolation units. 

### Fifty chickens were vaccinated SQ with the MDV, SR-1, -MasterSeed Virus (MSV),- 10Xconcentrated on Study Day 0. The remaining 150 non-vaccinated chickens were divided into three groups of 50 chickens each in order to serve as challenge controls, sham-vaccinated and/or sham-vaccinated contact controls forthe SQ vaccinated group. None of the chickens vaccinated with the 1OX dose ofMDV SR-1,- MSV, negative controls orcontact controls had gross lesions ofMarek's disease during the study oratthe final necropsy on Study Day 120. The incidence of Marek's disease in the challenge control group was 100 percent. There were no significant

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differences in body weights between the MDV SR-1,- MSV 1 OX vaccinates and the negative control birds- between the contactcontrols and the negative controls- or between the MDV SR-1,- MSV 10X vaccinates+ the contactcontrols and the negative controls- One hundred percent ofthe birds in all the groups survived to five-days-of-age. The MDV, SR-1,- Master Seed Virus (MSV 10X)- proved to be safe throughout the 120 day observation period when administered SQ to one-day-old chicks. This study report was accepted by CVB on 08 May 2012.

D. Dothe DonorDNA sequences enhancethevirulence orthe abilityofthe MasterSeed to survive in thetargetanimals, non-targetanimals?


### 1. What isthetissuetropism ofthe Regulated Biological Agent intargetand non-targetanimals? MDV replicates in chickens and induces adetectable level ofviremia. Leukocytes become infected and provide the main mechanism ofcell-associated virus transport to othertissues through formation of intracellularbridges (Schat & Nair (2008) Marek's Disease 

### in Diseases of Poultry, Ed. Y.M. Sail, 121 " Edition, page 461-3). MDV replicates exclusively in cells ofavian origin (chicken, turkey, duck, quail, pheasant, pigeon, or goose) (H.G. Purchase eta/., "Responses ofcell cultures from various avian speciesto Marek'sdisease virus and turkey Herpesvirus," 1971 Am. 

### J. Vet. Res. 32, 1811-1823). MDV is typically characterized by proliferation in lymphoid cells in various tissues and organs, primarily blood and spleen. The vaccine strain is produced in primary cells ofchicken embryos. MDV is cell associated, and is present in very small concentration in the supernatant of infected cell cultures.
SPF chickens were inoculated with MDV SR-1- experimental X+5 vaccine ora licensed Rispensvaccine. An additional group of birds received diluent only to serve as negative controls. On days 7 and 28 postinoculation, the spleen, liver, lung, kidney, and gonads were isolated from five birds from each group of vaccinated and the spleen was isolated from three ofthe negative control birds. from each organ were evaluated forvirus isolation on chicken embryo fibroblasts monolayers. The presence of the virus was identified by the observation ofcytopathiceffectfrom one spleen taken from the MDV SR-1

- vaccinates on study day 28. Rispens virus was isolated from multiple birds and multipletissues tested. The results ofthis study support the conclusion that the tissue tropism ofthe vaccine construct and the parentalvirus are similar (spleen isollati•on) CVB forreviewon 20 December 2013.

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### was conducted to re-evaluate potential to recover virus from multiple tissues. On days 7, 14 and 49 post-inoculation, birds vaccinated with either- ora 

### licensed Rispens vaccine were tested forvirus recoveryfrom the following tissues: spleen, liver, lungs, kidneys, and feathertips and from blood. The results ofthe study showed the same type ofvirus recovery from- with the exception that more ofthe tissues showed virus recovery of- at day 14. No virus was recovered at day 49 from the- vaccinates. This study reportwas accepted by CVB on 23 December2013.

E. Discuss the potential for horizontal gene transfer or recombination ofthe Regulated Biological Agent. 1. Isthere any reason to believe thatthe potential for horizontal gene transfer or recombination is different in targetand non-targetanimals? Gene transfer among herpesviruses may occur by homologous recombination. Since the genomes ofthe three MDVserotypes [serotype 1(MDV1) or Gallid herpesvirus 2 including the CVI988 vaccine strain, serotype 2 (MDV2)) orGallid herpesvirus 3 including SB-1 vaccine strain, and serotype 3or Meleagrid herpesvirus 1or herpesvirus ofturkey (HVT)) share little nucleotide sequence homology (Osterrieder and Vautherot, 2004The genome content of Marek's disease-like viruses. In F. Davison &V. Nair (Eds.) Marek's Disease. An Evolving Problem, ElsevierAcademic Press, pp 17-31), recombination events between MDVserotypes is unlikely to occur. It has indeed never been reported despite the wide use of combination vaccines such as HVT+881, HVT+CVI988 or HVT+SB1+CVI988. The potential for horizontal genetransferfrom- is therefore restricted among MDV1 viruses. To occur, such potential homologous recombination requires: (1) co-infection ofthe same cell with 2 different viruses atthe sametime and (2) replication in this cell. Since Marek's disease virus infection is restricted to chickens and occasionally some related avian species including quails, turkeys, pheasants (Schat & Nair (2008) Marek's Disease, in A.M. Fadly, Diseases ofPoultry,

**12~** Edition, pp452-514), MDV recombination can potentially occur in these species only. The results ofthe non-target safety in avian species and mice did not showvirus replication in the main tissues targeted by Marek's disease virus confirming the very low risk ofsuch event to occur. The- virus contains a- DNA sequence inserted into the- strain genome. While unlikely, there is potential forthe transferof to the genome ofa MDV1 isolate by homologous recombination. lithe- vaccine is administered together with the -vaccine,there is a potential of- transfer from- into the- genome. Homologous recombination will insert the. at the same locus ofthe- genome as in that of- Since the backbone of- is the- genome, this horizontal transferwill not create a newvirus with a

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### RiskAnalysis Marek's Disease Vaccine, Serotype 1, LiveVirus, Code: 16L1.RO (Unlicensed)

different phenotype thanMay 19, 2014 Page 25 of74

Horizontal transfer may also occur between- and field MDV1. As stated above, both viruses need to be present in the same cell at the same time for arecombination to occur. MDV vaccines are administered 

### to the birds before natural infection and the route ofvaccination (subcutaneous or in ova

### administration) differsfrom that of natural infection (respiratory infection by infected danders). The- virus will therefore replicate earlierthan the field MDV1 and in differenttissues. In addition, MD vaccines induce a rapid onset ofimmunitythatwill restrictwild type MDV infection and replication. It is known also for herpesviruses that once acell is infected with avirus, it subsequently blocks co-infection with another virus (Thiry

**~tal.,** 2005
.. Recombination in alphaherpesviruses. Rev. Med. Viral. 15, 89-103; Thiry et al., 2006. Recombination in the alphaherpesvirus bovine herpesvirus 1. Vet. Microbial. 113, 171-177). The likelihood ofhaving acell co-infected with vaccine and wtMDV atthe same time, and, subsequently, ofgenetransfer is therefore very low. In the unlikely event

three hundred (300) one-day-old SPF chickswere randomized into six differentgroups and inoculated subcutaneously (SO), in the nape ofthe neck, 0.2 ml per bird, with either: Group 1-MDV SR-1 Group2- MDVSR-1- (X+5)plusvMDVSR-1 GA22,- Group3-vMDVSR-1 GA22,- Group 4- MDV SR-1- (X+5) plus MDV SR-3 HVT,- Group 5- MDV SR-3 HVT,

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### Merial, Inc. RiskAnalysis Marek's.·Disease Vaccine, May 19, 2014 Page 26 of74 

### U.S. Vet. Lie. 

### No. 298 Serotype 1, LiveVirus, Code: 16L1.RO (Unlicensed)

(See Section 

### 5 Table: 1ofthe study report forthe inoculum titers) Afterthe inoculations, the chicks were placed into their respective units. On study Day 21, ten randomly selectedbirds fr?m the-GA22 and-HVTvaccinates were bled for·····

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

### from these groups were inoculate•d tissue culture and the

### vir~s recovered was confirmed as being GA22 from ihe-/GAZ2vaccinates and HVTvirus from the-/HVTvaccinates. No- was isolated at Day 21. On Study Day 

### 35, all the

### r~maining bi_l-ds in 

### Grou-Ps 1-6were terminated, 

### sexed, Weighed and 

### obser:,-ed for gross

### Ma~ek;s

### Dis8~~9**lesioi1:S~** There was no increase in virulence observed in the GA 

# grou_P_ 

# illiillllllll as compared to the GA22 group

## Th~ birds containing 

### ·-HVTor HVT

### only 

### had no gross lesioils associati9d
___ '
.There Was 

### no significant group effect on the body weight between the GA22 and the-GA22 inoculated groups-

- and HVT and the-/HVTinoculated

### g~oup.~-- at end ofthe 

### 35 day



## --------------**--~0-:t)Se~atiail_periOd.**

### this**stuaY.~eP6rt was·?ccep~ed** **by-~he**

## cVB

## 6n fS

## JU1Y**201~.-**

.. -- . ,,**~- -~--**  - '";;!· 

### F.

## oeS'~~ib~ ttie shed/spread capabiiitti!s oftheRegulated Bioiogicl!l

## A~ent.

# Viru~'6ou~E.not**b:re~d~;r~i:t** irom

# eit~~rcloacal or .. , '' the results obtained with the disseihlni:itioh ;;:21.
__**-_~:;·_-** _**'><<-.-~.c-,;.** _·1**-~--~,--,.:·.c.** 

### ··· ·**~·:.;t~'i-·-~·,'~'(-'S;:· ~** **~** 

### - -- - ------------------- ------

random!y selep.ted vaccinated**an~** five 

### non-vaccina1ed**conta9~s** per**~r.o~p** were**,termin~.ted** and necropsied,

## Wit~;_siSiee'iis iMdividually naJVesteC!. on

### the

## la~t**day~~**the

## stu~\, (D..i9 49), all the remaining birds were eUtffaOI2:ed

### ~hd necropsied 

### fofsigns of MDV and

### th·.~**~tUdy** v#s temiiiiated. Spleens-frpiil 

### fiVe randomly

## ~eiecte~ vaccinated

## b1rd~

# and the 

### remafnirlg five

## .non-VB.ccinat~d contacts per

### grOuP'-WSre individually 

### harvested. Virus isoiation was only attempted on the spleens harvested from the non-vaccinated contacts. All tracheal and cloacal swabs were negative forvirus recovery. 

### All ofthe featherfollicle samples ofthe MDV SR·1- vaccinates tested negative forvirus recovery. Only at day21 cduld vtrus be recovered from featherfollicles oftheMDV SR-1- vaccinated birds. All ofthe feather**f~_llicle** samples tested**from_~he** sham**v~ccinated** birds were negative forvirus recovery. Therewas no virus recovered from any ofthe spleens harvested from the sham-vaccinated contacts at 21 and 49 days using either virus recovery or PCR. There was no evidence ofclinical disease in any ofthe

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### Lie. No. 298 Risk Analysis Marek's DiseaseVaccine, Serotype 1, LiveVirus, Code: 16L1.RO (Unlicensed) May 19, 2014 Page 27 of74

non-vaccinated contact birds. Underthe conditions ofthis study, birds vaccinated SQ with the MDV, SR-1,

- experimental vaccine showed similardissemination patterns to birds vaccinated SQ wfth the commercially available MDV, SR-1 Rispens vaccine. There was no evidence of shed/spread to nonvaccinated contact birds, demonstrated by the lack ofclinical disease and negative virus recovery in the contact birds housed with the MDV- vaccinated birds. The study reportwas accepted by CVB on 27 December2011. 1. Includeshed/spread potential in non-targetanimal species. As discussed in Section IIIC, Safety in Non-targetAvian Species,-· no lesions and no humoral antibody attributable to Marek's diseasewas induced by thevaccination. As discussed in Section IIIC, the safety ofMDV SR-1- experimental X+5 (final product passage) as evaluated in mice- showed no recovery ofvirus after subcutaneous injection and epidermal 

### scarification of 

### 15 mice. 2. Include inthe discussion any contributionsto shed/spread capabilities that mightbe associated with particularattributes ofthe Backbone Biological Agentand Donor DNA sequence. A study perfonned on th**~** - -- - --- - -- - ----

1\lt<Jge•th<lf,, these studies do not indicate an increased risk ofhorizontal transmission due to the insertion of. In agreementwith these published data, ourstudies indicate that the- virus did not shedto contacts as discussed in Section Ill.F.

Confidential Business Information RedactedMerial, 

### Inc. · ,. ·RiskAnalysis ·Marek's

### Diseas~Vaccine, May 19, 2014 Page 28 of74 U.S. Vet. 

### Lie. No. 298



### G. Serotype 1, Llve Virus, Code: 1si..1.RO (uniicensed)

3. Ref'7rence**re~evant ~ciEmtlfic**  publications. Jarosinski twJ, et al. 2007. Horizontal transmission of Marek's disease virus requires US2, the UL13 protein kinase, and gC. 

### J. Viral. 81:10575-10587. Jarosinski twJ, Osterrieder N. 201 0. Further analysis ofMarek's disease virus horizontal transmission confirms that UL44 (gC) and UL13 protein kinase activity are essential, while US2 is nonessential. J. Viral. 84:7911-7916.
.. Jones D., Brunovskis P., Witter

### R. and Kung H.J. (1996) Retroviral insertional activation in a herpesvirus: transcriptional activation of US genes 

### by an_integratecl_ !eng terminal repeat in a Marek's disease virus clone. J. Viral. 70, 2460-2467. · · · · Okamuraa H., M. Sakaguchi, K yokoga...,a, E. Jokunaga,S. Abeb, S. Tokiyoshi, K,**Mizuno~. L~Ekof**  

### contact transmission of recombin-ant Marek's-disease Virus type 1expres-sing the fusion protein--of Newcastle diseasevirus Vaccine 20 (2002) 483-489

## S~n

# AJ,

# Xuxv; Pelherbridge L, Zhao YG.'NairV, Cui

## zz. (2010) Functional evaluation ofthe role of reticuloendotheliosis virus long terminal repeat (L TR) integrated intothe genome,ofa·field strain ofMarek's disease virus. Virology 397(2):270-6. ' · ·· ' Witter RL, Li D, Jones D, Lee LF, Kung HJ. Retroviral insertional mutagenesis ofa herpesvirus: a Marek's 

### disease.virus mutant attenuated foroncogenicity but notfor jmmunosuppre_ssion or in vivo replication. Avian Dis. 1997Apr-Jun;41(2)'407-21. · ' ' · ·' · · Zhang Z., Cui Z. (2005) Isolation of recombinant field strains of Marek's disease virus integrated with reticuloendotheliosis virus genome fragments:Sci China C Life Sci 48, 81-8. :(-J. , ' ;-.. ·r p- <

. ' ;u_,_

• - - • - ' _., -· :"' "-_· • •**-,,.·,,-{~·-** _- ·:';, -_ '_"'-'•_: • .>- ,_ - - -- -'_ ---- -'_ -- -__, ---- - _<

.biscusi!'ihe

### ~Jipected environrrleri!ai linpa'ct

## Ci'i**survivabili\Y.~f**

# theReg1hated**Biologi~'al**

## Agent, and

·,**._.'\;~-;:~-·-::··. :~**  __**~;':~_,,..-**...'.i'\;_:

- _,,--:__**·_-'_·;·.~~-** ;_ :i!;"'
__ '.;
..**~_,;** ''?-_-**~-,~~~** ··,·-·- ' 

## ·Provide**·a\i~illibiEt S~il~tific** 

## e'vid·ence. · ··'.t·· ' · · ·' \f ·';Y · 

# . -_-_**,~.-·)- :;.~~~_.**  

# ,-_._·.**)~** 

# _:-,

_ _ -. .-**~---~>~·** 

# .._;,__> .,_· .,.. < • -- .-' ,:.-;-:· ,;,;·_ ' 

# -'<· ' .· -, :; ': Chickens are the natural host ofMDV SR-1**v~ruses.** MDV is shed mto the erivironineht bYfeather follicles



### . ·-.'.

- 

# . ,, . . : ·.:• 

### .**·~** 

### .. of infected chickens. Prevalence of infection in chickens is dependent upon the control measures used to

### ,~ont\?,1 the dis7ase in

### th,~.field<Th7,.s,urviv.~~]\r**~f** MDV SR-1- in the environmentisflXpected to be ,·· similarto,

# or less,th.an.. that ofthe_ parental- strain Besed on th,e resulls 

### o,t.the**dis~eminati9n** study

.>•.·..· _ ,_ -"
_. .-**~-"~-- .·--?--·~** 

### .**..,-,·-·~··"'·!_,~--'"-** -'•-t**·t·,~=(~** .••. -- _._.':T·-r-·-.-••,.-: · . .: • •:-• :;,; ·"' ., , ,. 

### ·.r '! :·--lt·- , .;···- :-:
,·

### (Se~**-~ectio~** Ill

### ~·-**th~t sh~~ed**  nO

### ,~!~!!~·re,~ovefl!**~rpm ~at~-~r** f9.Jiicles:

### ~9r any

### _yir'~s 

## . -..**ieco~rred**

## frdrn**ri_a_rve·~~ptact_~~- -~The** **Ml?~**

## SR-:1-**V8?d~~--~lil**

## ..be**.~r,q~6h ir\~-~fi-~~~of~i9t.~,Q}orm.** 

-**-~** > :• 0-': , ._**-~-•o-.--'-''** ,_ >_"--
__**~'-~'f''-:** r**_-.-,.-~_--** :--:· _" , -**-~•** , --,-- "7**~--- _-:,----;---=~**  --- .,, __ --**:,-,-~-** --d,-.o'T- •--:",•{-,. ,,_ •, ,..,.,1 - .c._'----_-. , 00**-'-"'-~'c** ;,·This,f9itn is poorly resistant iii thoi_enviroriment siriQe.it is.depehdeHI pn the Viabiliiy pfth.e cells.**Th~** virus**!(~·o,;,** :•_-c- _ ,- __ ,. • >**~--,.** -- {.;
_ ., _ , ,_,, ' , ,., - ----":
_**-~t:"~J;7** ,.**'L-:~,:~** ·,;•_,:_ ..'· '-

. may be**~hed ~s** **cell-frei~** virus pythe**feath.~r**follicle ofthe yaccin11ted chic.kens:, blit:?CcOrding to !he results**~-:-** i ._,,._ ..,,_,,__ - -___ .·:.;-, ·. . !( .-**~··** ...,_. ,f
.**.T~.;.;--** ,, :·=, .·-"· ;,'-!:.
. ofthe dissemination study- and shed/spread study- MDV SR-1.- is not \: .transmitted h_orizontally among

### chick~n·s'indicating that the amount

# or infectiviiy ofvirus sheet

### rs--~bt**-~·· ~**  

### ---· ., 

### \._. '· . _,;-_ . .. 

### . '•" . sufficientto infect na'fVe chickens. ' 

### "'

### Th~**qosUra~ge** specificity bfthe

### experime~tal vaccine was evaiJated with the non-target avia_n species safety**t~st,-** discussed in Section IILC of lhis report. A study design that includes the length oftime the virus can be recovered from sterile shavings (same as those found in commercial chicken houses) saturated with ....was conducted .... The test

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### Merial, Inc. 

### U.S. Vet. 

### Lie. No. 298 

### RiskAnalysis Marek's Disease Vaccine, Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed)

inoculated shavings were held at room temperature forthe duration ofthe study.

--..,f May 19, 2014 Page 29 of74

On days 0, 1, 2, 3, and 7 post-inoculation (orwhen three consecutive days read no virus recovery),- -shavingsample were collected aseptically and placed in a sterile sample bag containingThe procedurewas repeated with the Rispens shavings sample. On Day 0,- showed an average of3.3 plaques per ml ofshaving solution and the licensed Rispens vaccine showed an average of2 plaques per ml of shaving solution. Therewas no virus recovery afterday 0. This study was accepted by the CVB on 01 January 2013. 1. Dothe Donor DNA sequences orgene sequences associated withthe Backbone BiologicalAgent enhancethe abilityto survive in the environment or increase resistance to therapeutic agents? The insertion ofthe. should not contribute to the ability ofthe backbone virus to survive in the environment. is not located in agenomic position close to MDV1 genes contributing to the horizontal transmission The survivability of

- virus with the. insert described in Section Ill. Gshowed no virus recovery afterthe day of inoculation. There is no therapeutic agents for MDV and therefore, the potential effect on resistance to therapeutic agents is not applicable. IV. Risk Assessment Risk assessment may be defined as the likelihood ofan adverse event occurring and the consequences 

### if that adverse event occurs. An adverse event is defined as safety hazards to animals, public health, orthe environment. Asafety hazard is defined as adanger, risk, or peril; absence of predictability associated with an event; oran expected or unpredicted event. 

### A. Procedure The risk assessment is conducted based on safety characteristics ofthe vaccine. The safety characteristics are based on empirical data and established scientific facts. The completion ofa risk assessment requires that the vaccine microorganism is properly characterized. This infonmation is included in theVeterinary Biologics Summary Information Format Section Ill (page 21) that is provided by Merial, Inc.

Confidential Business Information RedactedMerial, Inc. 

### U.S. Vet. 

### Lie. No. 

### 298

B. Hazard

### iden,tiflc~tlon Risk Analysis 

### Marek's Disease Vaccine, Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed) May 19, 2014 Page 30 of74

Haza;8 identification consists ofidentifying all possible

## adver~e events to anima_! safety, public health safety, and environmental safety relative to the recommended use ofthis experimental vaccine, Marek's Disease Vaccine,**~erptype** 1, Live Virus, Product.Code 16l1.RO, unlicensed. The vaccine thatwill be us,ed in thisproposal is ve;':Oib"sely

# i~i,3led to a USDA licensed

# VOCoC;n~.·- ···Such 

### r· modification is not known to increase risk to animal safety, public health safety or environmental safety, '· ,, '

- ' ..:·.-,·.. ,_ ·,**sine~ si~_il~f** 

### _inserts are present in other USDA licensed vaccin_es 

# lillilliililii!..'"
. tJerial hassubmitted

### c~nsiderablio in vivo\md 

### in vit?o data on·- These data shqwed·thatthe**,,-,~~---** '-- _.,:;;...._. >/ ' ._:. -- -,._\;;.:

- -**~.** '.**--~·'•'** - _ _.. ·'·:: • -- - ' :- c9hstructwas safe in mice, chickens, ducks, quail,

### pi§~~ri!i, turkeys arid pheasabts_.,Jhe safety 

### prOfile of- in mice and the other non-target

### avi_an

# sp'edes is tHe 

### same asthe

## com;;,erC-i81 Rispens**va~cine.** 1. Animal safety 

### a. sUmmary No ariimal 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 species and one mammalian species suggesting thatthe va·C9in8 Vfbuld be safe if**o~her_n9n-target** species were exposed. The vaccine d_id not revert to virulence and was genetically stable through five backpassages in chickens (See Section Ill C). Studies in cell culture indicated thatthe vaccine is genetically stable at n, n+5, and backpass 5 (bp5) passages (See Sections II.C.3.c)}.

b. Targetanimal safety: (i) Vaccination ofchickens The Master Seed Virus did not demonstrate any virulent propertieswhen inoculated into one-dayold chicks at 10times the recommended field dose aswritten in 9CFR 113.330. A testwas carried out to study the pOssibility of reversion to virulence ofthe vaccine strain. Five passages were carried out. Infected leukocyteswere fourid in each passage. No abnormal signs were noted in the birds during the passages. Naive hatchmateswere placed in contact with each group ofbirds receiving the backpassed virus. No·virus was recovered from those contacts, nor any lesions seen atnecropsy (See Section Ill C). 

### {ii) Vaccination/challenge No <=!dverse immune interactions are expected with the use ofthis vaccine. In vaccination/challenge studies, susceptible chicks vaccinated by

-- 

### -- ------

- vaccine protected against

# 1!!11 when challenged- at 5 days of

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### Merial, Inc. U.S. Vet. Lie. No. 298 RiskAnalysis Marek's Disease Vaccine, May 19, 2014 Page31 of74 Serotype 1, LiveVirus, Code: 16L1.RO (Unlicensed)

age- accepted by CVB on 20 December 2011 ). These results supportthe product claim to "aid in the prevention of Marek's disease caused byveryvirulent Marek's disease virus". 

### (iii) Reversion to virulence Backpassage studies demonstrated that the- virus does not revertto virulence when administered to chicks. Supportive data include: • -Reversionto virulence -five passages on chickens (See Section Ill C) • Recombinant virus 

### in vivo genetic stability analysis by Southern Blot and PCR, n, n+5 and bp5 (See Section II.C.3.c). These studies also demonstrate the genetic and phenotypic stability ofthe- MSV 

### in vivo [See Sections II.C.3.cand Sections III.Aand Ill.B) (iv) Puritytesting The MSVwas tested bythe applicant and found to meet all standard requirements of 9CFR113.300. (v) 

### Identitytesting

(vi) The presence ofthe

# Ill, has been confirmed 

### by the use ofconventional PCR as described in 

### Section II.C.3.b. It is proposed to test each serial for identity using- to confinm the presence 

### Effectofgene manipulation on pathogenicity
. 

### The recipient- virus Was derived recommended for use in day ofage chickens. The strain is 8.Virulentfor chickens. which is



### The donor insert,. has no known pathogenic ortoxic properties and it does not code for any polypeptide. No pathogenic ortoxic properties have been identified for- when presentin the experimental- vaccine (see Section Ill).

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### Merial, Inc. U.S. Vet. Lie. No. 298 RiskAnalysis Marek's Disease Vaccine, May 19, 2014 

### Page 32 of74 Serotype 1, LiveVirus, Code: 16L1.RO (Unlicensed)

(a) 

### Phenotypic effectofthe gene modification(s) The- virus had no clear phenotypic difference compared to parental virus. In vitro 

### replication and safetywere similar. The level of- in the blood and in different tissues was lower and no infectious virus could be isolated from the featherfollicles. In contrast, the MD protection level induced by- was at least equivalent to that induced by- (b) 

### Pr<>vi<lUS use o.fdqnor genes and _re.pipient**mi~roorga[lism** 

# •.**~·~ii-~·~~~iili'~=·~·~i'~liil~ll**

# ...................................................

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

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

# RecipientMicroorqa~is~:the

# par~ntal strain of
....:irus is thE;IIIIil vaccine strain

-· • -.-: - ,.- »-- .•1·.

- _.c. ·--."---·"·-- ,_._ ...,., .-:o-«-- --.-. 

### which is - -**-=---=---~--** --- - - -- =---- - ---=-=---

- - - - - 

### (c) Phenotypic effect ofany genetic marker(s) 

### The- MSV does notcontain any geneticmarkers.

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

### Lie. No. 298 RiskAnalysis Marek's DiseaseVaccine, May 19, 2014 Page 33 of74 Serotype 1, LiveVirus, Code: 16L 1.RO (Unlicensed)
(vii) Genetic stability -isgeneticallystable 

### in vitro {see Section II.C.3.c}. The genetic stability of- 

### in vivowas demonstrated through five backpassages in reversion to virulence studies in chickens (see Sections Ill.C). The presence of conserved throughoutthe five backpassages. (viii) Phenotypicstability Experiments conducted by Merial, Inc. indicate that- is phenotypically stable in cell culture (see Section Ill. A.). 

### The phenotypicstability ofthe- MSV 

### in vivowas studied in a reversion to virulence studythrough five backpassages in chickens (see Section III.C). No reversion to virulence was observed after backpassages. 

### (ix) Alteration intissue tropism The results oftransmissibility studies indicate that the proliferation/tissue tropism ofthe experimental v'accine virus is similarto its parental- virus (Section Ill.D). (x) Effectofoverdosing Target host animals were inoculated with 10times more virus than the recommended field dose with no adverse effects observed (see Sections Ill.C).


### c. Non-targetanimal safety (i) Susceptible non-targetanimals The safety of- has been confirmed in non-target species. The data indicatethatthevaccine strain was safe in these species (see Section Ill. C.). (ii) Virulence in non-targetanimals The results ofthe safety studies indicate thatthe hosUrange specificity ofthe- MSV has not been altered fromthatofthe parental- strain, and thatthevaccine issafein non-target 

### avian species (see- in Section Ill.C). 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 ofmice inoculated with- (see Section Ill. C.). 

### (iii) Possible outcomes ofnon-targetanimal exposure

- is not expected to pose a safety risk in non-target species. The parental virus is derived 

### strain. The studies discussed above suggestthat these safety characteristics have not been altered in- (see Section III.C.).

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

2. Public health safety
a. Summary Risk Analysis Marek's Disease Vaccine, ' ·J • Serotype 1, Live Virus, Coile: 16L1.RO (Unlicensed) May 19, ·2014 Page 34 of74

Hazardsto public health are not expected. There are no reports of Marek's disease causing productive infection in any species otherthan avian species, and no infection ofhumans has been reported despite accidental inoculation occurring during vaccine administration. The parental serotype 1vaccine strain from which this experimental vaccine was derived has been used as a commercial vaccine for chickens in Europe/Asia/US/Latin American without any adverse effects or hazardsto public health. b. Probabilityofhuman exposure Human exposure

### will be limited to personnel administering the vaccine and conducting the field studies.

c. Pathogenicityofparent microorganism in humans There are no reports ofSerotype 1Marek's disease causing productive infection in any species otherthan avian species and no infection ofhumans has been reported (Schat & Nair (2008) Marek's Disease in .-. 

### -·

**.~** .. ,-, Diseases of F'9)Jitry, Ed,



### 2~ Edition, page, 452).

d. Virulence ofthe vaccine microorganism in humans -hasneverbeen inoculated into humans. Thusttie actual outcome ofan accidental human inoculation is unknown. Based on the current knowledge and wide use ofSerotype 1 Rispens vaccines and thesafety data, the- is likely non-virulent in humans.
-e. Possible outcome of human exposure There are no expected safety concerns associated with human expoSure based on the biological properties ofwildtype Serotype 1 Marek's disease viruses. It is the opinion ofexperts in the human and avian cancer field that Marek's disease and vaccines against MD pose no human health hazards [Purchase, H.G. and R.L.



### Witt~~·(1986), "Public health concerns from human exposure to oncogenic avian herpesviruses: A review" 

### J. Am. Vet MedAss.18g:1430:6 (Appendix 2)]. 3. Environmental Safety

a. Summary The safety risks to the environment are low. The shed/spread capabilities ofthe experimentalvaccine are limited. Some- virus may potentially be spread to the environmentthrough the danders ofvaccinated chickens but the shed virus unlikely to infect other chickens indicating thattheinsertion did not provide an increased spreading ability to the virus. The hosUrahge specificity ofthe experimental vaccine does not appear to have been altered from the Serotype 1Marek's disease virus, which is restricted to avian species. The safety characteristics ofthe experimental vaccine in non-target avian species do not appearto have been altered from that ofthe recipient virus which was derived from a

Confidential Business Information RedactedMerial, Inc. U.S. Vet. 

### Lie. No. 298 Risk Analysis Marek's DiseaseVaccine, Serotype 1, LiveVirus, Code: 16L1.RO (Unlicensed) May 19,2014 Page 35 of74

No adverse effects have been attributed to the use of licensed Serotype 1, Rispens 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 multiple Serotype 1vaccines currently licensed and being distributed globally. To our knowledge the- experimental vaccine should not behave differentlyin terms ofenvironmental distribution than currently licensed vaccines.
c. Shed/spread capabilities The results ofstudies indicate that the- vaccine microorganism was limited in its shed/spread capabilities since viable virus could not be isolated from featherfollicles and the vaccine did notspread to non-vaccinated contact birds. This studywas part ofthe reversion to virulence- 

### CVB accepted on 20 December 2011 and the dissemination/shed/spread, 

### -CVBaccepted on 27 December 2011. The contact controls placed with each group ofchickens receiving the backpassed virus and the birds placed with the vaccinates in the dissemination study did not show any virus recovered from spleens nor were any lesions noted at necropsy (see Section II_I.C). In the non-target avian species studies described in 

### Section III.C no adverse reactions attributable to- were

**report~d** 

### for.any ofthe vaccinates orcontact control birds. All- recovery attempts from tracheal and cloacal swabs from the vaccinates and contacts were negative, as were the featherfollicle epithelial cells. d. Horizontal gene transmission/recombination potential The potential for 

### in vivo recombination with field and othervaccine viruses was evaluated in- (see Section III.E.). Therewas no increase in pathogenicity seen when combined with avirulent GA22 Marek's disease virus. The PCR results ofthe recovered virus showed only GA22 virus present with no. insert.
e. Potential for transmission to invertebrates: The potential fortransmission of- is expected to be minimal. The limited shed/spread properties of

- should reduce the probability of mechanical transmission for the- vaccine microorganisms (see Section Ill.F.}. Even iftransmission did occur, the attenuated properties of- are notexpected to be altered (see Section Ill.C.}. f. HostI range specificity: There are no reports of- or any Serotype 1 Marek's disease virus causing productive infection in any species other than the avian species and no infection of humans has been reported (Schat 

### & Nair (2008} Marek's Disease in Diseases of Poultry, Ed. Y.M. Sail, 121 h Edition, page 452}. Our non-target studies showed that the insertion ofthe- did not modify the host range specificity ofthe vector.

Confidential Business Information Redacted

### Merial, Inc. 

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

### 298 · RiskAnalysis Marek's Disease Vaccine, Serotype 1, Live Virus, Code:.16L1.RO (Unlicensed)

g. Survivability ofthe microorganism in the environment: -. -·J..c: 

### May 19, 2014 Page 36 of74

The very limited amount ofvirus shed bythe featherfollicles via dander may potentially survive for months in the environment, similar to the parental type virus and other licensed Serotype 1 vaccines. However, the amount and/orinfectivityof
.. shed from vaccinated chickens appearvery lowsincethe vaccine did not spread to nalve chickensh. Physical and/orchemical factors affecting dispersal in the environment: No physical and/orchemical factors have been identified thatwould enhance the dispersal of !hein the field. 

### i. Adverse ecolOgical effects The demonstrated safety

## a~d lack ofproliferation of- in other non-target avian and mammalian species (see

## S~ction Ill.C),
.. and the

## esta~li;shed limited

## h~st range of- reduces the risk ofspread to non-target animal species:
....- 

## c. Release Assessment

### Pri~rto a Proposed Field SafetyTrial Environmental releaSe propOsal Summarv The safety characteristic ofthe vaccine microorganism must be evaluated within the context ofthe target environment. Thus, the release assessment consists ofa comprehensive evaluation ofthe proposed release so as to detenmine 1) the location and characteristics ofthe release site; 2)the test dose and total amountofthe _experimental biologicto 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. Forthe purpose ofrisk asses_sments, proposed studies with**li~~** experimental vaccine microorganisms are classified as e"fthercontairied stu-dies orenvironnlental**rele~ISeS.** A**Contain~d** StUdyis**define~** as!an experiment in

### lab~i-a:tory contained or physically

### corlt~ined animal facilities. An EnviryJnmentafRelease refers tothe release ofthe vaccine microorganism into ttie accessible

### enviro~me_nt. 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**assessme~t.** VB-APHIS recommends and encourages the use of small-scale field tests as appropriate forthe experimental veterinary_biologic. Contained release Not applicable.

Confidential Businsss Information RedactedMerial, Inc. 

### U.S. Vet. 

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

### 1, Live Virus, Code: 

### 16L1.RO (Unlicensed)

1. Environmental release Summarv May 19, 2014 · Page 37 of74

Merial, Inc. proposes to conduct safety field tests to determine the safety ofthe Marek's DiseaseVaccine, Serotype 1, Live Virus, unlicensed, USDA product code 16L1.RO in the target animal species, chickens, with the objective of licensing and commercial distribution. The Field Safety Studies will be conducted in at leastthree regions ofthe USA. A list oftentative recipient states is included below. These studieswill be conducted in chicken houses that are typical ofthe commercial poultry industry. The potential forescape and dispersal from the release sites is low. There are no known biological organisms, physiochemical factors, ortoxic chemicals thatwould contribute to the establishment of- in the environment. Personnel experienced in poultry health managementwill be conducting the proposed study. 2. Tentative location oftestsites Arkansas Georgia Kentucky North Carolina Tennessee Texas 3. Characteristics oftestsites. Thetest sites include commercial poultry houses, multiple houses per site. Each housewill hold approximately 17,000-22,000 birds. The chicks will be inoculated at the cooperators hatcheries and then trucked tothe house(s). The details of each test site will be included in the 9 CFR 103.3 authorization request forthe Field Safety Study-. 4. Personnel Personnel administering the vaccine will consist of hatcher personnel underthe direction ofthe participating cooperator. Merial veterinary staff, and/orqualified Merial R&D staffwill be present forthe vaccination and placement ofthe chickens. Merial personnel will be involved in data management and in study follow-up. TheVeterinary Scientist in charge ofthis Field Safety Study will be Dr 5. Experimental design Number ofanimals At least 20,000 birds will be vaccinated with the test vaccine. Descriotion ofanimals One-day-old commercial layer/breeder/broiler chicks ofmixed breeds will be used forthis study.

Confidential Business Information RedactedMerial, Inc. 

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

### RiskAnalysis Marek's Disease·vaccine, Serotype 1, 

### Live Virus, Code: 16L1.RO (Unlicensed)

Route ofadministration Subcutaneous inoculation into the nape ofthe neck ofthe chicks. Dosage May 19, 2014 Page 

### 38 of74

The vaccine forsubcutaneous injection will be prepared for use in the mixing room ofthe hatchery. The 

## ., vaccine containerwill be

# asealed plastic IV bag, that will be fitted to a Marek's disease vaccinating machine using sterile plastic IVtubing. The serials ofproduct that will be used in this**stu~y** will have been satisfactorily tested for"purity before releasing into the environment. Total amount oftest material Thevaccine is frozen in 2.0 ml glassvials each containing 1,000 doses. The vaccine will be reconstituted and added to sterile diluent at the ratio ofone (1) ampule per200 ml ofsterile diluent for subcutaneous injection of0.2 ml per chick. With the exception of Marek's products, the chicks can receive all other vaccines according to each company's vaccination program. Approximately 100,000 doseswill be required forthe trial. Protocol The Field Safety Protocol- was submitted to CVB for approval. Frequencv and duration ofexposure Directexposure ofpeople to vaccine could potentially occur atthe hatchery. The animal caretaker/grower responsible for the management ofthe test flocks will be exposed to the inoculated chickens, but not the vaccine, forthe length ofthe field test period, approximately 60 days or less. The employees atthe slaughterhouse are notexpected to be exposed to the vaccine virus. 

## No evidence exists to showthat

- can be transmitted to humans and therefore will not cause disease in**h~mans.** The duration of exposure is notexpected beyond the day ofvacCihation. The method ofdisposing ofwaste 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 ori site by industry accepted methods. The vaccinating IT)achines will be disinfected according to standard hatchery procedures. Dead birds and litterwill be treated acCording to each cooperating company's program. Decontamination oftest site Following vaccination, the vaccination area at the hatchery will be disinfected and washed according to standard procedures. Crates used to transport the chickens will be returned to the hatcherywhere they will be washed and disinfected. The broiler/layer houses will be cleaned and made ready forthe placementofthe nextflock ofchicks

Confidential Business Information Redacted

### Merial, Inc. U.S. Vet. Lie. No. 298 RiskAnalysis Marek's DiseaseVaccine, Serotype 1, LiveVirus, Code: 16L1.RO (Unlicensed)
according to each company's program. 6. Potential forescape and dispersal May 19, 2014 Page 39 of74

Spread transmission studieswere negative. Therefore, no escape or dispersal into the environment is anticipated. 7. Potential forestablishment in the environment Marek's**diseas~** virus already exists in the environment as a poultry pathogen. Several live Marek's disease vaccines including Serotype 1vaccines are used commercially in the United States under USDA license. The vector organism is derived from a licensed vaccine strain of- The use of subcutaneous administration will minimize the chance ofescape into the environment. It is not anticipated that conditions will exist outside the laboratorythatwill support perpetuation ofthe recombinant virus. No toxic chemicals or metabolites are known to be produced bythe virus, which could endangerthe environment. 8. Monitoring Monitoring methods The monitoring ofthe field testwill consist ofmaintaining the records associated with the production of commercial chickens. Mortality and morbidity will be recorded bythe grower at each test .location. The flocks will be observed daily for seven weeks foradverse reactions to the vaccine 

### bythe groweror field personnel ofthe cooperating company. Meriel's veterinary staff and/or research department staffwill visit at least once during this period. These potential adverse reactions may include, but are not limited to, unusual post-inoculation 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/orretained for viral isolation. Any adverse clinical signs associated with the vaccination will be immediately reported to the veterinarian in charge. Frequency of monitoring Each flock will be observed daily bythe grower or caretakerduring the growout, which is expected to last no morethan 60 days for broiler type birds (layers/breeders will growoutfor a longer period oftime). Birdswill beobserved at least once bythe Veterinary or Research staffand more frequently ifnotified that adverse events have been observed in the flock. Birds will be observed forclinical signs ofMarek's disease. The safety ofthe vaccineswill be reported in the field safetystudy report afterseven weeks ofobservation. 9. Contingency plans in case ofadverse events. No adverse events are expected but ifadverse events attributable to the Marek's diseasevaccine are observed, the primary investigatorwill be notified immediately and the event documented in the study log. The grower, the cooperator's veterinarian and Merial veterinary staffwill workjointlyto determine a plan of action, ifnecessary.

Confidential Business Information RedactedMerial, Inc. 

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

## RiskAnalysis Marek's Disease Vaccine, Serotype 1, live'Virus, Code: 16L1.RO (Unlicensed)

D. Risk characterization May 

### 19, 2014 Page 40 of74

Risk characterization integrates the results of hazard identification and the release assessment into a risk statemen!that includes: 1) a likelihood rating; 2) a consequence rating; 3) arisk rating; and 4) adiscussion ofrisk. Each likelihood and consequence rating is qualified by

# a degree ofcertainty rating and includes a justificationforthe rating. Thejustification forthe ratings consists ofidentifying the·applicable sections in the hazard identification and.

# the release assessment that support··ihe assigned rating. The risk rating is ' -i•
.• . i**·~** . ·, ·• based upon the likelihcioa, consequence, and degree ofcertainty ratings. 1. Likelihood ratings •Likelihood ratings are assigned for animal safety, public health safety, and environmental safety based on thefollowing criteria: Low= Medium= High= 

### 2. Consequence ratings An adverse event is unlikelyto occur An adverse event could possibly occur An adverse event will most probably occur Consequence ratings are also assigned foranimal safety, public health safety, and environmental.safety based onthe following criteria: Low=. Medium= High= The consequences ifthe adverse eventoccurswiJI not be severe (the adverse event is self-limiting and would have negligible impact). The consequences ifthe adverse event occurs is moderately severe (the adverse event will have an impact, but it is not permanent, and can be treated). The consequences 

### ifthe adverse event occurs is severe {the adverse event will have an impact, is permanent, and cannot be treated). 3. Degree ofcertainty rating Each likelihood and consequence rating is qualified by adegree ofcertainlY rating that is base9 on the following criteria: Certain= Moderately certain = Uncertain= 4. Calculating the expected risk The rating is supported bydirect sqientific evidence. The rating is supported by indirect scientific evidence. the rating is notsupported by scientificevidence.

Numericalvalues have been

### assign~d to the likelihood, conSequence, and

### degre~ ofcertainty ratings described above (referto Table 2). Each numerical value rating was derived from the importance placed on the rating ofeach category. The assigned numerical values are weighed to place emphasis on the severity ofexpected risk. These values reflect the professional judgment ofthe applica-nt (Merial, Inc.).

Confidential Business Information Redacted

### Merial, 

### Inc. U.S. Vet. 

### Lie. No. 298 

### RiskAnalysis Marek's Disease Vaccine, May 19, 2014 Page 41 of74 

### Serotype 

### 1, Live Virus, Code: 

### 16L1.RO (Unlicensed)

5. Risk ratings The risk ratings are based upon the likelihood, consequences and degrees ofcertainty ratings, and the expected risk for each category. Atotal of81 rating combinations are possible; e.g., likelihood lowmoderately certain, consequences low- moderately certain (see Table 3). 

### 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 ofthe expected risk. Again the severity ofthe risk reflects thejudgment ofthe applicant. The low, medium, and high risk ratings are defined forthe purpose ofdecision-making as follows: Low= Acceptable risk; very few concerns are associated with the proposal (does not justify denying the proposal). Medium= 

### High=

a. Animal safety Unacceptable risk; moderate concerns are associated with the proposal (either identifyvalid mitigative procedures ordenythe proposal). Unacceptable risk; majorconcerns are associated with the proposal (denythe proposal).
Likelihood rating: Low (LL) Degree ofcertainty rating: Certain (C) Justification for ratings: 

### IV. 8.1.b. 

### i. IV. 8.1. b. ii. IV. 

### B. 1. b. iii. IV. 8.1. 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. 

### Consequence rating: Low (Cl) Degree ofcertainty rating: Moderately certain (MC) Justification for ratings: 

### IV. B.1. b. ii. IV. B.1. b. iii. IV. B. 3. b. IV. B. 3. h. Expected Risk Rating: 0.7500 Risk Characterization: LL.C.CL.MC

Confidential Business Information RedactedMerial, Inc. 

### U.S. Vet. Lie. 

### No. 298 RiskAnalysis Marek's DiseaseVaccine, May 19, 2014 Page 42 of74 Serotype 1, LiveVirus, Code: 16L1.RO (Unlicensed)

Risk rating: Low The safety risks to the animals and environment are low. The host/range specificity of

- does not appearto have been altered from the parental type virus, which is restricted to avian species. Although the protocol ofthe field safety study does not include procedures for monitoring the shed orspread o- into the environment during the course ofstudy, studies previously conducted by the applicant indicatethatthe shedding capabilities ofthe experimental vaccine are limited, and the virus is unable to spread to other chickens. Furthermore, the proposed release will be confined to poultry houses. Thus, exposure ofnontarget animals and the dissemination ofthe.experimental vaccine virus in the environment during the course ofthe field testswill be restricted by its use in poultry houses. This assessment indicates that one-day-old chickenswill be vaccinated in commercial hatcheries, and subsequently transported to commercial chicken houses forthe· duration of the field trials. All the waste material from the vaccination procedure will be decontaminated priorto disposal. The vaccination area at the hatchery and the crates used to transportthe chickens from the hatcheryto the houses will also**~e decon~aminated.**  The- vaccine will be used in combination with other Marek's disease vaccines (Serotypes 3and 2). Our recombination safety study has shown that co-administration of these vaccines did not generate a more virulent virus. Conclusions and discussion of risk The risk to animal safety associated with this proposal to conduct a field trial with experimental Marek's Disease Vaccine, Serotype 1, Live Virus, is low. The recipient virus used to construct the- MSVwas derived from a licensed vaccine strain that has been used as a commercial poultry vaccine in the United States and Europe, as well as Asia and Latin America. Safety studies conducted by the applicant demonstratethe safety ofthe experimental vaccine in chickens and in several non-target avian and mammalian species. The proposed study will be conducted by personnel that have extensive experience in poultry health management. b. Public health safety Likelihood rating: Low (LL) Degree ofcertaintv ratina: Moderately certain (MC) Justification for ratings: IV. B. 1. b. iv. IV. B. 2. b. IV. 

### B. 2. 

### c. IV. 

### B. 2.d. IV. B. 3. b.

Confidential Business Information RedactedMerial, 

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

### RiskAnalysis Marek's Disease Vaccine, May 

### 19, 2014 Page 43 of74 

### Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed)

IV. B. 3. d. Consequences rating: Low(CL) Degree ofcertainty rating: Moderately certain (MC) Justification for ratings: IV. 

### B. 2. 

### c. IV. 

### B. 2. d. 

### IV. B. 2. 

### e. Expected Risk Rating: 

### 0.5625 Risk Characterization: LL.MC.CL.MC Risk rating: Low Conclusion and discussion of risk There are no reports of productive infections cau.sed by Marek's disease in any species other than avian. The recipient- virus was derived from avaccine strain that has been used as acommercial poultry vaccine the United States and Europe, aswell as, Asia and Latin America, with no adverse effects on public heallh. Studies conducted bythe applicant indicate that the safety characteristics ofthe parental type vaccine strain have been preserved in the recombinant- MSV. Only lhe personnel administering thevaccine will be expose.d to the experimental vaccine virus. The other personnel will be exposed onlyto the inoculated chickens. Studies conducted by the applicant demonstratethatthe shed/spread property ofthe experimental vaccine from vaccinated chickens is limited.


### c. Environmental safety Likelihood rating: Low (LL) Degree ofcertainty raling: 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. Consequences ifadverse event occurs: Low (CL) Degree ofcertainty rating: Moderately certain (MC) Justification for ratings: 

### IV. B. 1. c. iii.

Confidential Business Information Redacted

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

### Lie. No. 298 RiskAnalysis Marek's Disease Vaccine, May 19, 2014 Page 44 of74 

### Serotype 1, 

### Live Virus,Code: 16L1.RO (Unlicensed)

IV. 

### B. 3. 

### e. IV. 

### B. 3. h. IV. C. Expected Risk Rating: 0.5625 Risk Characterization: LL.MC.CL.MC Risk rating: Low Conclusion and discussion ofrisk The safety risks to the environment are low. The host range specificity of- does not appearto have been altered from the parental type vaccine strain, which is restricted to certain avian species. Although the Field Safety Study protocol will not include procedures for monitoring shed or spread of- into the**en~ironment** duringthe course ofthe study, studies conducted by the applicant indicate that the shedlsprea'd capabilities ofthe experimental vaccine are limited. Furthermore, the proposed release will be confined to pOultry houses. Thus, the exposure Of non-target animals and the dissemination of experimental vaCcine virus ii"l the 

### environment during the course ofthe field tests will be restricted by its use in poultry houses. V. Risk Management Recommendations A. Procedure Risk management uses information from the previous section to determine means of reducing or eliminating safety risks to animals, public health, orthe environment. B. Recommendations The safety risks to animals, public health, and the environment are low. A risk rating oflow is acceptable risk, with very little concern associated with the proposal. The applicant recommends the approval ofthe proposed field test by VB, APHIS provided the study is conducted in accordance with the requirements of 9CFR 103.3, and the filed protocol ofthe study.

Confidential Business Information Redacted

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

### No. 298 Risk Analysis Marek's DiseaseVaccine, Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed)


## Table 2. Calculating the Expected Risk VALUE RATINGS Likelihood 

### (L) Low (L) Medium (M) High (H) Consequence (C) 

### Low (L) Medium (M) High (H) LL = 

### 1.00 LM = 

### 0.50 LH = 

### 0.10
CL 

### = 1.00 CM = 

### 0.10 CH 

### = 0.01 May 19, 2014 Page45 of74



### *If

### the Likelihood rating is Medium or High and the Consequence rating is also Medium or High

use Degree ofCertaintyRatings I; 

### for all other combinations use Degree ofCertainty Ratings II. 

### Degree ofCertainty Ratings I 

### Certain (C) Moderately Certain (MC) Uncertain (U) Degree ofCertainty Ratings 

### II Certain (C) Moderately Certain (MC) Uncertain (U) EXPECTED RISK 

# c = 

### 0.50 MC = 

### 0.75 

## u = 

### 1.00



# c = 

### 1.00 MC = 

### 0.75 

## u = 

### 0.50 [(likelihood) x (degree ofcertainty)] x [(consequence) x (degree of certainty)] = 

### Risk Rating

Confidential Business Information Redacted

### Merial, Inc. U.S. Vet. Lie. No. 298 RiskAnalysis Marek's Disease Vaccine, Serotype 1, LiveVirus, Code: 16L1.RO (Unlicensed)



## Table 3. Risk ratings Risk Expected Risk Risk Expected Characterization Risk Rating Characterization 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.CL.MC. 

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

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

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

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

### .25QO 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 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 LL.U.CM.U. 

### .0250 M LH.C.CH.C
. 

### .0003

Confidential Business Information Redacted 

### May 19, 2014 Page 46 of74



### 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 H H H H

### Merial, Inc. U.S. Vet. Lie. No. 298 RiskAnalysis Marek's DiseaseVaccine, Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed)

Appendix 1 May 19, 2014 

### Page47 of74



### Pages 47-73 redacted due to confidential business information

Confidential Business Information Redacted

### Merial, Inc. U.S. Vet. Lie. No. 298 RiskAnalysis Marek's ·Disease Vaccine, Seri:ityp'e 

### 1, LiveVirus, Code: 

### 16L1.RO (Unlicensed)



### Appendix 2 May 19, 2014 Page 74 of74



### Purchase, H.G. and R.L. Witter 

### (1986), "Public health concerns from human exposure to oncogenic avian herpesviruses: A review"

### J. Am. Vet Med Ass. 

### 189:1430-6

Confidential Business Information Redacted

### l'!JBLIC !lMLTH CON'CF;RNS 

## FROM HUM<\N EXPOSURE TO ONCOGENIC AVIAN 

## l!ERJ?l::S VlRUSES: AltEVlEW by 

## 1!. G. PURCHASE

R. L. WITTER 

# DEC 12 

# 1985

1

### FrQlll the U.S. Department of 

### Agriculture, Agricultural Research Service,

Beltsville Area, BA.RC-t;rast, lleltsv:l.llt;, Maryland ;W705 

### and 

# u.s. Depart:Jnent of Agriculture; Agricultural Research Service, Regional l'ou1try ll.eaearch Laboratory, 3606 East 

## Mt. Hope Road, East Lansing, Michigan 48823 Presented at the Annual 11eet:l.ng of the A,nterican Veterinary Medical Association, taa Vegas, NE. July 23, 1985.

'•

### 2 

## Summs.ry
Ever sio.ce tbe 

## ident.l.f:lc:ation of a herpesVirus as !:he 10<1.11se**o~** 

## Marek•a disease (liD) 

## lind the 

## isol.atioll, and lille**o~** 

## live vaccines against MD,**th~re** 

## was concern among some of the lay public and 

## some scienti.sts ;:hat 

## there 

## may have 

## b!!en a qauaal sssociatiPn**betve;~n** 

## thes!ll vi):llses**~,tnd** 

## b,WDan 

## cancer, 'l.'llis. 

## concern was 

## projected as an 

## inorditlllte n1l!llber of 

## terwrts, Pro 

## and 

## con, in the scientific 

## l:l.teratute over 

## the past two 

### deca!le"· 

## ltesolut:ion o£ this qtie!ltiPt\ 

## was needed 

## to Jnak·e irifotmed 

## decision,s 

## about lJ:censing 

## bi()J.pgf,cs, to lll!itkE\t 

## safe and whol.e!!Qlllii! 

## foQd 

## prpdqcts ill, the 

## Udted Statell 

## and tor**~Qrt'** 

## I!,Q,d to proVide information :for consllllle·rs. Results from 

## sta.Ustical epidemiology, serological epid!!ndology, eo1!1pariso:n 

## of etloloJ!ic a$ent!l, laboratocy**lnMUJ~Sl** 

## and**pri~~U~te** 

## inoculation, culture i:n mall!lllalian c!!lls, 

## and**eJ:l!llli:ttst~c;on** 

## of !1llPQt:t1miatic**11U'ect~o:n** 

## a:nd drll.1!ma.tantia1 evidence W:!ll:!l 

## reView!!d .l.n this pa}1er. 

## The data**iljail:a~!!d** 

## that no clear 

## !!vidence of 

## ail li'tio1.:>g1c rel<\tiO)lshtp existed and, although addi1;1ona1 st11dies were poas:tbJ.!!, there was 

## en()UJlh evide.nc!! to support a 

## i!irm .conclnslon. On this basis 

## expert co!IUJiittees sponsored by 

## the government con:cluded that MD was n.ot 

## a pul;lltc health proPlem.

··.,**Clln<~el;'** 

### is 

## one of tM most dreaded diseases in modern times because of its high frequency, unpredictabU:i.ty, inevitable occurrence, prolonged duration, and chronic and painful co11rse Qften leading to death. Tl\ere has been extensive research· in this century to 

## try to identify the causes of cancer, dhemica.l ca;;Cinogens, such liS coll.l tar which iu<hlcell cance:J: in chimney sweeps, have been elearly showll. to·be one of the causes of cancer. Similarl]l', ionizing radiation csn cause cancer in persona t:nl'l:posely or :Lnedve:rte1ltl]1 <!;r;posed to it. Agriculturll.l chemicals lilte chlorinated bydroc11rbon insecticides, ;Lndustrial chendcais like polychlorinated biphenyls (l.'C8s) 

## and chemical contaminsnta lllte dioxins have become widely dist1;ibuted in the environment and sometimes in the food chain before they were shown to.**c~use** 

## cancer in 

## e.>;perimental anima.ls. Wi<l,e:t.y used fdods like grains and**oilse<~d.s** 

## have become ·contaminated with mycoto:dn.s, some of the most powerful carc.inogenl! 

## k.now. l!ven nat)lral constituents of the diets 

# ox most people such as 111eata are irrcrtminated with caus;Lng humau ceucer, Aberp<!svirus 

## b presumed to cause Burkitt's lymphoma, first descrlbed in Africa, and**ll~sopharyngeal** 

## cat.cinoil!B., first described in 

## Chins. M:>re recently RNA viruses such as hum.au T-c<!ll**leuket~~ia** 

## virus have been it;criminated as the cause of acquired immunodeficiency syndrome in

## ~n.**1** 

## Enormous efforts have been

## dtr!lcted t(.> 

## understanding at the nu>lec\llar level the sequence oJ: events that results in cancer. UO;til 

## these events are fully underqtooil. by 

## sdedt::l.sts and the infomation is disseminated 

## to the general public there will be 

## a c.ollc,;,:rn 

## by the**gen~:i:a;l.** 

## .pqblic 

## tJml: items :l:n their food 

## and enviroli.mellt to11ld ind11ce

A 

## review of 

## the public health aspects 

## of aniDlal t\Uilo:r 

## viruses was 

## publi&hed in. 19(io,2 

## !n ;l.:t,

## ~:telen. examined thi! 

## rela.tionsh:tp bel:W!!en a 

## var:l,ety uf viruses knOW!\ 

## at**~hat** 

## time and 

## humim disease including canc.er .lind iJmnquqsuppressiQn. 

## He concluded tha.t "over 100 anii!i<ll tilmor viruses 

## ha:ve 'Peen iaqla.te<l, 

## and ()illY l;lt;e {Yaba .Tillllqr 

## Virus) has**~inor** 

## public significance.• However, the heJ:pesvirus 

## of l'fll.rek'

## s**duea~e** 

### (t!D) W<I,Jo 

## n_ot**isoA;~.'t;eq** 

## u.n.t:J:lr J.:967l'4 and the herpe:sv:t:r\lll of 

## tl;rkeys (HVT) was p_at isolate:i:l \Ill

## til

## :!.~$9.5


## W;ith lihe 

## ident1!ieadon of 

## a v,t:rqs 

## as the**callll~>** 

### <:>f 

## MD 

## l!.nd publicity

## ~urroundi~g the development; of vac.niues against 110,6 there has beau 

## .concern among soine 

# otiiha la;y publ.ic and cSOille. 

## silietitists "that exppaqte to the 

## 110 virti>! 

## an.d J:Oil.ated 

## Jl;patho$eli..lc 

# Yl!ccine viruses in 

## th<> l!nv.trot11ll,e:nt, or 

## eat::l.ng po!l.l.f!'tr infected 

## w:l,th 

## ·these viruses, could .contribute 

## to the cause 

## .o£**.ca.nc~r** 

## in man. The question ()f 

## wh.etller 

## thet!! 'l.s 

# l! relationship betw!!en taalle 

## infectototis of 

## poultry and diaease in man needs to be 

## resolved so tha.t 

### :l.nfonei:l 

## decisions can

"• 

# ! 

# .i l ' 

# I ' ' 

# *

### 5 

## be made about licensing of biologics for domestic animals and man and about the

**~af<i!tY** 

## and wholesom<i!Uess of poultry and poultry product$, Also, the pre.,<i!nce of; .,uch lt1ruse" in meat e>;n be used as a non-tariff trade barrier to prevent the export of poultry and poultry producta.

**~stly;** 

## such infoP»ation 

## i" needed to assur<i! poultry termers and processing plant workers of occupational aa:f.'ety and to assure conslllllers that p!ltdtry and p!luitry products a:re safe and wholelfome to eat. 

# Many sc:l.ent:i.ats hsve attempted to examine th<i! association between canci!r viruses in animals and cancer in man using diverse approaches. the MD virus is ubiquitous and vaccines against MD are used woddwide (appro:dma.tely 

## 3 billion do!!es of live vaccine

**ar~** 

## Use<;! in the 11.S. alotte) on !l!eat
...producing (bro!lers) and egg-producing

**anitlla~s.** 

## Humi.ns haV<i! been exposed to _MD virus ilirice dilmestication of the chicken and to vaccines agdnilt MD for al!i!ost 2. 

## decades. There has been atnpl<i! opportunity for hnma.ns to

**cont~ct:** 

## the virusea. thus an asl!lociation between these vi_ruses and human cancer could be revealed in epidemiologic studies, There hss also been extensive experimentation with these viruses in chickens,

**~aboratory** 

## animals, primates 

## an'd humans•

## The voluminous

**lite~atut:e** 

## rdating 

## MD virus and 

## vacciMs againat MP to 

## human disease is 

### often 

## contradictory and has 

## neve;r been reviewed in depth, 'l'he 1aet:

**~:eview** 

## focused almost 

## exclusively ,on RNA t:lllllOr 

## Viruaes', .part:tcu1a,-1y 

## feUne leukemia virus.2 

## l'lrl& comprehensive raview of 

## the. literature will proVide :re'levani: data 

## to guide 

## scientists and resea:rc.h l!dm;l.nist;r:ato:rs 

## ;l.n deciding where 

## additional 

## t-eaeat'eh is needed and nfult 

## res.earcih app:roaehea to take. It Wi.l.l pltoVids 

## !l<:bl:l.nlstrato;t's o:f federal. and 

## state !lction 

## and

**regul.al:~lli'Y** 

## agencies With 

## the most complete compilation 

## tlf informstion publiahed so fat to 

## detetm:!.ne whether a 

## hilllllln health haza'l:li 

## exiBts, 

## the natu;re and 

## extent of 

## the ba:r<al:d 

## and 

## whether actiCI!ls are 

## necessary. 

## 'mlb 'revlew w:tU 

## be 

## Qf !ntexest 

## to the 

## p:oult;r:y

**:tndt~stty,** 

## wh:teh 

## would suffe;r severe

**~co;~;omie**



## 111 

## this review we hllV<a :SillljJnat'iZlld 

## the da1:a, developed 

## Mnclus:tons that 

## follow logically from 

## the data, 11111d discussed ph:l.losophically!!(l!tte of 

## the dilell!!!Uls 

## :in 

## the scientific process. Mention is 

## made of efforts by 

## tbe Government 

## to deal with 

## the 'issue by

**e~tablishlng** 

## committees 

## and co!!illiissions 

## td study 

## t.he llVsilable data. We 

## Will conf:tne our ol>servat:f.(lne 

## and r-enurrl.ts 

## to the public heal;:.!\ B;!!peeta 

## ol: !;he avian 

## hei:peaviruslls nf MO and the 1:1.Ve

**Vitt~s** 

## vaccines, including HVT, 

## that prewmt MO.

'• ' 

# ' '

# ' 

# i ? 

### r 

# I

### 7 

## Avian ()neogenic and Related Viruses.

Marek's disease is a lympho-proliferative disease of chickens caused by a berpesvirus3•4 and· clla:racter:l.zed by a mononuclear -cell infiltration and proliferation in peripheral nerves, gonad, iri!l, via.cera, muscle and skin.6 It 

## :J.s ptobabl.t the most colilmonly occurring neoplasm l)f anY anilll4l. tn**1!1~7** 

## it cauaed annual lo!lses in the United States in**ex~;ess** 

## 9£ $1.50 mill;!.on ami silllilar losses 

# w•u:e incurred in other countdes of the wotld. Losses ate d.ue to**dectt~ased** 

## agg '{lroduction, mortality from MD., incl;eJi&ed mottlility fro111**otbe~r** 

## causes, and condemnstions of carcasses in 

## the poultry processing plant. '.the diSease and losses from it oc.cur worldwide.7

The herpes virus t:hal: 

## causes MD is ndt transmitt.ed vertically fro•l parent to oft'sp1iing.6 There£ote eggs are free of the virus. It is transmitted hoi'ii'iontally in the dust and dander that 

## sloughs of£ from the feather follicle epitheliWII :l.n which the virus matures to its infectious form. The virus 'shrvives ·in the environment for several months at room temperature, Moat f1deks become infected With the virus within 2 to 3 

## weeks after ha1:ching and

## the 

## virus spreads to most birda within the flock by**a:ppr!):o;~m!lte;Ly** 

## 1 weeks of age--the 

## aae wnen mQet 

## 1n:oilers are 9laughtered for 

## human consumption. 

## ln 11ve 

## birds, the 

## virull persists in infected cells 

## in the blood for 

## .Bever!il years 

## and pr<ibably :tn,deUnitely, 

## Virus can be isolated J;rom virtually all 

## body tissues, inCluding muscle (meat), for ptolonged 

## ll.BdQds, However, 

## egg!! are considered f1:ee 

## of 

## v:l.ruG• Tlua**v~rus** 

## is h:l,gbly**ceu~sssuciated** 

## in a.ll

...



## infectious. 

## Beeause ths virus 'la found 

## worldwicle in chickens anll is 

## found ill 

## jungle £owl in 

## theil' nat11rs'l**~!Yl,tat:,** 

## :l.t is 

## 11\t\'!ly tha:t manldn4 l:las eonsumed virus-infl'lcted 

## chicken meat- 

## since long before domeatication 

## of 'the C.hiclten,



## Vaccines are .uaed to control •MD" 

### in 

## all {'!onn:tr:l,"e of 

## the worii!. tha:t P;l,ve. 

## intensive poultry produet:l.on 

## systems. Vaccines 

## a::re composed by 

## ehe**eeil~free lyophUi;~:ed**  

## v:l.!;us Ill: 

## ceU-iaSsociated virus 

## in !!ell cultures 

## #hich haVe bl!eu frozen 

## in liquid. nitrogen. 

## The virus most co!m!lonly used. ia HVT, 

## which is a**ncln~pathogenic vi.~s**  

## salliSen:l.eally r!Ola.t:ed 

## t.o MD vims 

## w'hleb.**w;;~~** 

## :Lsolated from 

## 5 

## turkeYil• Other vaccinea include attellUll.ted MD virnl!es 

## llntl lllltura.lly apathogenic 

## MD viruses; The natunily 

## apathogenic virus is gaining 

## more

''

## 9 acceptance as a vaccine worldwide. A**l;lival!!n~** 

## vaccine incorporating HV'l' and a naturally apathogeuic virus was licensed in the United States in October 1983.
All vaccinee are**i~:~.jeei:ed in~o**  

## chiclt!! on the day they hatch, The vacct'lle VirulleS replicate in the chickelll! and persist in thl:!lll for life. Of the three vaccfue viruses only the na..turally a.pathogeuie one spruds rudily horhontally from ch:i.clten to chicken thOugh aome of the attenuated viruses spread poorly. 'l'he vaceil!.es are highly effective in prevl!nting i:be lesions and economic**las~es** 

## from MD; however, they do nat prevent infection with MD Virus nor the :spread of MP vUus. hodzontaUy. !>bat of the·egg-type chickens throughout the world and moat of the 

## mest-.type ehickens 1i1 

## some count:tiea such as the United States, Canada, and Ita1y are vaccinated. Thus, most chi.ckens at sla:ughter contain both the virulent MD virus and the vaccine virus.

The pathogenic MP viruses of chickens, the apathogenic similar viruses of chickens, and the l!VT are serologically distinct from one a!lother. Because 

## wtr and the apathogenic viruses do not induce MD, they eannot be referred to as MD Viruses or as avian oncogenic herpesviruses, Also, because HVT occurs naturally in turkeys, it 

## ·cannot be referred to as a chicken virus. However,

' '-.;. 

## the 

## Yirus:es ate an.ld.gellic.B:11y closely**~;ela~ed** 

## though 

## diatinct from one al10l:her. 

## ·'fhey 

# have been 

## called serotype 1; serotype 

## 2, 

# and serotype 

## 3 MD 10



## vi!;'Uses, reepeeti veiy. Fo-,: .the pu-,:pose of 

## tllis papel:', we wUi tefet to t:he!il 

## as 

## avian. (oneogen;to:: 

## or oncogeniaally..,re1a.ted) herpesVirua.es (;AOHV).
Review by Expert Commit·tees
In 

## the Unit:e.d Statl!s of ·AII!etit\s (USA), 

## veterinaty bioloS:ie!l &t'e 

## te$UU·te4 under 

## the authOrity l;>.f 

## t!1@**'Vi:tus~serum ~o:z;in**  

## Act•. The ·u.s, DepaJ;tll!en!: ilf 

## Agricul.tu:re (USDA) 

## is resPonsible £or assuring thst snc.h biologies a'j:e puJ:e;, 

## safe, poteut ani;i 

## e'fficacio\Ul. With the dtac.ovety 

## <>f the c.auliative he1:pea v;l.rtis of M:l :l.n ·1967 anti tlte 

## ia.o1atf9n o.f the herpeaVitus o£**tur~eYII** 

### {lM) ;l.n

# 191:i~,0 

## <l,l!est:tpM were

# t~lised aboU:t l:l!e aa.fety 

## Pf MD

# virua~i!lfected chickeilS, 

## l)r. ,Jesde

# t, Steinfeld, SuJ:geon General of the Public: Health Service, 

## conv•,med 

## a committee 'l,u 

## 1970 to :review 

## the !lt;at\ls 11n<'! 

## cutrent :relationdhip 

## of

-

# ...

# u viruses of the avian leukosis complex t_o h!llMn dis!lase.a Dr. Steinfeld made the folloWing 

## apeeif:l,c point!l•
"Even though there 1s no known association between viruses which are common in 

## fowl and.in chicken eggs 

## to human disease or any known hazard to hllllllUi 

## haalth, 

## l pel_ieve 

## tile o)l].y ratieinal_ goal we l!hould .strive for is eliminat:l,ou of thoae

**virus&~.~ £rot~~c**  

## both 

## eggs and. poultry 1;10 

### th;li: 

## there 

## can be uo 

## poas:i.ble human health hazard associated with t:he:tl' 

## ingestion 411d 

## so the 

## fowl pcrpulation will be 

## a more hesl.thy oll!'l." A,ttach<!d tO 

## the letter 

## was a report wh:!.ch seated in part "a large number of k,ey invel!tigatora who have spent many yl;!ars st_lldying_ 

## the avlan and 

## other animal cancer viruses looking 

## for liuks. with 

## human cancer having encouuteted 

## M inst11nces wh:l.ch 

## C:Puld re!laOU!Ibly lead to the poat1;1lste that: these. cancer virua11s have been 

## naturally transmitted 

## to msn or even, 

## !or that matter, 

## to 

## other animal species. Thus the evide!lce sud s<;ient!flc

**literaturE~,** 

## as well as the Pp:l.nions of the best sd.entists in this 

## field, all 

## add up to one

11

# J. L. Steinfield, Surgeon General of the Public Health Service, Public Health Service, Department of Health and Human Services, Washington DC. Personal communication.

## 12 

## cqncl!la:l.on--not only 1s 

## there no eid.de!lce !:hat "poultry ·product$ 

## .are 11.**ll!i~ard** 

## to mali 

## all a 

## possible 

## cause of 

## cancer, but 

## that the 

## data strongly 

## suppoJ:t: a 

## positivi!! l!tatelll=t 

# that t'l."ans,lllil!a!on of 

## cancar f):Qlll 

## chickens to man 

## does 

# not**occur.~~** 

## Priol: to**~he** 

## odg;l.na.l 

## licensure of 

## the 

## herpesvirus of tutkeys 

## (Ji,VT) .in 

## 1!110,6 the iiSliA requested the.

# De!>art~nt 

# pf Health and !ill!il!ln Serv!c.es 

## to 

## :rev:l;ew the**pul>li~** 

## b.l!a.:li.l:lt imp':U,alitt<lns <>f 

## su:C:h ·a.n a:tid<lli· 

## 'Experts**w~re** 

## J:al1ed to recview the 

## "U:tersJ;1,1re and 

## all infotmation 

## avaiia:ble. They conc:iuded that 

## ltcensing the vac;c:tne woUld pon no 

## public health 

## prohJ.em.b


## In 1983, 

## the USDA raised aim:l.lar concerns regarding the 

## licensure 

## of the SB-i MD 

## vacofne. 

# An Ad' Hoc Coriun:l.tt'ee or 

## experts 

## again reviewed the 

## pteaen:t. state of l<l>.owleiige, 

## ":Lt**w~;~a** 

## tile tl11!lmittee'Jl 

## optni!!n th?;t 

## wtthfn the**'Umt~s** 

## of

bPeraonal collllllUII.1cation. D. E• 

## .Eapeae·th, Veterinary Bio:logica 'Sta££. Animal and 'l'lant ·Healtll 

## Inf.ecdon Service, 

## I;yat.t!lville, 

## f.ID 20782
..

### 13 

## the available data there was up realloll. to anticipatl! any public health 

## '

-- ""'

- ...c problema from using theal!**vacc:ln~>a.** 

## Review of**th~>** 

## Literature !)tatiatical Epidemiology
If there were a atatbtic:ally significant correlation between the denScity of poultry (numbet produced in a country) and a patticulat type of cancer in }\umanSc, oJle·might infer that there was an etiologic relationSchip between the .two. Several sCientists have attempted to show such a correlation (Table 1),
The death certificate files for the state of Washington for 1950 to 196.7 and t:he 81:ate of Oregon for 1950 to 1964 were examined using .a.case-controlled method,8 Among 5368 

## c:ase•control pair!!, there ·was a statistical:!.y significant :association between :fa:rmlng occupations and death :fro!ll leulc.emia
ll.Personal communication, G. 

## F. V'ande Woude, Laboratory of Moleclilar (jncology, National Institutes of liealth, Bethesda, MD. Currently at NCI Frederick Cancer Research Facility, P.O. :Box 11, Frederick, MD 21701.

## and nrul.tiple myeloma,. No such association Was 

## seen for Hodgkin's d:l.l;leaae, 

## reticulWll cell 

## sarcoma, or 'other lymphomas. 

## PoulJ;ry famers showe4 

## the h:l.gh<1.11t proportionate <;:ase 

## excess, :l..e., ten cl!aes dyina from leUkemia and 0nty 

## one m4tched 

## control, a difference that 

## was 

## stat:l.sdcally**a~gnifl.cant:** 

## at P<

## .05, 

## 'Xhere were 

## h:l.!!bet; hequf!ncies 

## !lf leukemia deaths among 

## orcbardiats,wheat 

## fatme:t's, dairy tamers, 

## ¢a,t1:1e r@cher!l,**>turae~n,** 

## ;l;aru!era (no explanatl411 given), and 

## farn**lahol:'er;~** 

## thOugh 

## the d:l.fJ;ereneee we:re 

## not stat:l.sti.ceUy s:l.gniticsnt,,


## Ail ap.a,l.ysis qf**~eath** 

## 'in**C1.!1Uo~~** 

### tarlll. and 

## non-farDl.**re,s:l.d~ts b-et:wl!e~**  

## 1959 lind X9ll1 snowed a significant <P.£0.05) reduction ·in 

## m:or'taiity :trol!i all cancer :l.n farm re:s:l.deuts thl>ugh the toliJ!.l mortal:!.

## ty was s1mi1ar in**f~rm** 

## and 

## non-fllr!ll

# residt~nta.9 <J'fprJ:ality <from leukemia vaa

# i;l!)r~as~d though no<t 

## s1,gt!:l.fieii!ntly sp, 

## '):he proe$dures 

## used invoived**caicul,atin~** 

## average 

## 1\!0rtal:l.ty ratea Uoilt tl\e l!!li(} 

## ti

# .s. Census for 

## Calirotuia 

## and using 

## these 

# rateB as populations and 

## to calculate standardized mortality ratios between**~ected** 

## lind actual d!laths.

## 1.5**Th~** 

## posaib),e relationship beti<een h1l!ll411 eane.et d,eatha and exposure to pQultry was studied in 1,019 collll.ties in l:en so.utheastern states for 19.50 to 1969.10 Deaths from uterine cervical and ovarian cancers and from multiple myeloma were sign:l..ficantly (1'.5_;.01) 

## higher in counties with high 

## poultry poJ?ulations than in counties with low poultry populations. However, the sigidfieance for the latter ti<o cancers disappeared when the data were compared with data from the total USA. No excess risk l)f 

## lymphomas• Hodgkin's disesll&, and lell\(emi!l in Jleople oeeupatiow(ily exposed to poultry was detected.
In another study, records of death certificates 

## fr0111 Nebraalca for 19.57 to 1974 wer.e elCallU,n.ed.n

## Leuk~a deaths (1.084) 

## were each matched with t:wo dea.ths (2168) 

## from other causes for age at death, year of death, county of residence, race, and sex. There was .an elevated rtsk of leukemia among fanners which was statistically signific!lnt (P_50.05) for fanners born

- 

### . 

## between 1901 

## and 1943 

## or who died beti<een 1957 and :!.965. When t;he mortality was atrat:l,fied by county of reai.dence and compared with various agricultural pJtoductipn factQrs, there was a significantly**(P~O,()s)** 

## incre;:>.sed i!lcidence of .leukemia among farmers 

## from heavy com-producing counties. Also there was a higher risk from acute unspecified leUkemia. !ll)lOng farmers from heavy corn, hog, and chicken producing and herbicide-using counties. The study using similar methods wall conducted in Iowa.12 and invol.ved 1675 leukemic deaths



# -.

## 16 lil:>d .l3,50 c<>ntrola.**I.~itikemia.** 

## mortality was 

## higher among 

## :famers (P.:5_,-'\)S)• 

### Leukemia was 

## also higher 

## among certain 

## age groups o:f**.:farmer~>** 

### in <;OU!ltiea 

## with high 

## soybean, co:i:n and 

## egg p:i;Od!lction and largest**li\llllba~** 

## Of**a~:res** 

### treateit 

## with l;erbicides. )t)rtal:t

## ty from 

## unspecified lymphatic le\ikel!!ia was 

## assoe!e:ted with cotn 

## per 

## acre. nlli!lber 

## of !Dilk: 

## cows and 

## nlli!lb.er 

## of egg-laying chickens.**~not:her** 

## ·case-controlled study was conducted 1n 

## Wiaconain uaing mortality data from 1968.to :1117!113 invoivin$ All deaths from 

## iiidd.ple myeloma and' 

## 926 careft.Jll,y lleleeted 

## cQ'Iltrolll.**}~lrtality** 

## f:rom multiple 

## mYiil.O!!U!,**W~J.s** 

### sigo.lficati.tly (P.:5..0!'i) 

## higher slilbng 

### .fa:t'ml!':ts 

## than non-farmers 

## particularly those :tesiding 

## in col,lllties )l.igh in .chicken. 1nven.tcn:y,**~er~iUJ~;I!r** 

## us"• ·and 

## acrres 

## t.-eii.ted with Jnaeeti<:.fde.l!.

An 

### intenl!ting J>pidf!!lli!'ilogical !inrvey of 

## e!lophllgeal <;anceJ:;.waa 

### eqnduct;ed in No:tth (Jtdna 

## betwe!!!l :1.969 and 

## 1971l4•ti 

### !he 

## mort!liity rat:e 

## 11'1. !:he tw.o 

## highest 

## eotiuties waa**t~e;rdy** 

## 97 t;imes the moJ:tallty 

## rate hom the esophageal cancer in· 

## the two lowest rate counties. 

## The io.c:l,dence 

## Of esopb;\j!e&l caJICel!'



## dMiUer RW: .Yira.i <liseases. 

## Childhood Cancer 

## Etiology Newalette:t 

## l977; November 15, 

## 044.

-

## ... 

# ' 

# j I ;

## Qong humans paralleled the incidence of esophageal cancer among chickens, there was a higher incide)lce of n:ttroaam:l.nea in food samples from the high ;l,ncidence county than the low incidence county. l{o statistical analyses were perforD!ed.
Fro111 the ab_ove; there are three possible conclusions. The first is that the relationship between h1lll)sn cancer and cancer in chickens (including W) is -coincidental; the second is that an unknown factor such as a chemical or envirolll!lental factor on chicken farms is responsible for both the cancer in ch;!.ckens and the cancer in humans; and the third is that there is a ea_usal relationship between cancer in chickens and cancer in humans. From these data, one C<!litlol; select

**~ong** 

## these alte:rn,aUves and so n:o conelusio1lS with regard to the public health implic<!tions of AOHV can be drawn, Serological Epidemiology
'l'he examination of human sera for antibody to AO!f!i and the examination of avian sara for antibody to human putative cancer viruses have also been used \

- - - 

### - 

## to show the relationship between cancer in chickens and cancer in hUlllans. Of 64 healthy human subjects examined by the indirect fluorescent antibody teat for antibody to MD virus, all but four, who were young subjects twenty ye,ars

## 18 old or )'Ounger, -were posl.t:lvt1!.15 Converae1y, of <'ileven ·ch:l,cken sera**~txa!ltined** 

# tor l!nt:lbody 

## _to 

## Ell virus-. eight were positive. Ant1-'MD 

## titers ra,nged fr= 

## lUO to 

## >1#640 

## _and**and~Ell** 

## titer$ 

## va,ry from 

## 1:10 to 

## 1:40, A aubaeq\!a!lt**:~-J:;ud.y** 

### ,from :the**.s~** 

### l&bora.tv_i""f 

### exarnine_d seta 

### from 7_2 

## humans whose 

## work was 

## inti!IIStely conne.c:ted V;!.th 

## chlekens aJXd from

# n. office workers who -did not have much contact w:lth chickens. Seta from 

## .70.9% and 

## 16.4% of the workers in 

## polil.try 

## fatms 114!1 IU!.ti..,Hl ani! 

## al:lti-tili v:tt'tia 

## .ant:ibPdy t:f.tets ·Of 

## 111601 

## while only 

# 20.8% sud 

# 32.ii:t of the controi sera had -ant:tbody·rel!peetivaly,

# 16 Also, of the 48 

## sera from ·cynomologus monkeys, twelve had antibody to MD 

## virus and

In a 

## subsequent l;tudy ;tn the USA, ·205 

## h1llllan se>;a were examine.d foA '11 antibody to HVl' 

## and MD 

## virus, 

## l'he sara 

## came fro111**"er~ons** 

### lf:tth 

## a Vl!ried 

## SlDOUI\t Of eJCVOSUre 

## tO aVi_!ltl 

### harpe!! 

## '(iruses,_ );rom Bul;k;l,tt'!1 

## lymphQ)lla**p;~,ti!i!JXt$** 

## and from students. Sporadic samples had tit-ers ·in the fluorescent autihody positive by the fluo-rescent an:tibody 

## test had ·MUt:rali:zing sutibody,**Th~l:ti!** 

## was no pbvious te1ationabi_p beween 

## the extent of contact with .AOHV .and ·the 

# '

- - . 

## presence of detectable antibody.

## The reasons for the cross reactions between hwnan sera and AOHV and vice 

## !!!!!. are un:lqwwn. 

## They could have been caused by exposure o£ the chickens and lluma:ns tQ an !'gent or agents with a common antigen. The low incidence and 

# low titer of antibody in both chickens and humans against the virus of the other species makes 

## it unlikely that diseases in both chickens and humans are caused by 

## the aame·viru_s. However, a common antigen between the avian and h\llll.!ln v!rus$ is !I 

## pQSsibil:!.ty, Compatist,ln o£ ;Etiologic. Agents
There al:'e common !lntigens detected by different immunologic_al tests among many o£ the

**he:~:pes** 

## viruses, Shortly lifter the ;taolation of 

## liVT, it was shown to have commoa. antigens with MD virus i1;1 

## botb the fluo.res·eent antibody and 5 

## illlliiWlodiffusion tests. Infectious bovine rhin(ltl:'acheitis, EB virus, and MD v:Lrus



# ~1'1.0 .have common ut:igens ]ly these t!lsta.18•19 In one set of exper.i!nel;lt;8 MD virus, b!it not HVT, cross-reaete!l With_

**paeu!;ior<~bies** 

## virus by

. . . . 20 

## the fluorescent antibody test whel:'eas in other experiments both viruses reacted.21 Some workers were uaable to show a croas reaetion between MD vibJs and EB virus.22 Collllllpn group-apedfie an.d \lllique type-'!pec.ific antigens have been demonstrated between Luck' renal adenocarcinoma of frogs,



## . . . . . . . 23 MD and EB v:Lruses, hl!rpes dmplex and cytomegalovirus. It is not

### 20 

## I!Urpt'ia:!,p.g that viruses tha.t are evollltioilarily :c.lo11e1y :r:elated share common antigens. Sollie o£ tllel'!e vil;uses, like MD virus and EB virus share the 

## ability to 

## traha:fom cells. However, :i.rrfeo:.tiv:f.ty and 

## replication a,ppaa.r**~o** 

## lle 

## !lighl;r hol!lt-speu:ific. Thus, these data provide no 

## evidence that the AOHV 'l!!i!Y cause



## TU!liOl" polyll4cclla:ride lltll>l!tancee on 

## MD tumoJ;a and 

## MD l¥tfllicted 

## cells had common ·g,I.yccproteiA aut:l.genl! with a variety of htiWln twnor11.24•25 Virgin fewde guinea pigs· wl!re iliiDlunized with tUJDor polysacCharide subs

## tancea from 

## the .avian or 

## hU!lllln tt!lllt!rs. 

## Uterine sepents were then exposed 

## ·to the ·eXtract 

## and con'tract;ion of th'e sepen,ts iueaaured. The utet'i we:r:e carefully 

## desensitized to 

## !torma1 

## autlgenl! •prip;; t:o 

## testing, 

## Sti!,t':bticall;y si81i'tfii:ant 

## (l',$,01)**rea~;.t:tt!M W~~<:te.**  

## ill>t;t.ined with a 

## la:!:j!e variety of human 

## cancers including leukemia, lYJ!!plioma, 

## I!odskin's dise<>ae, ca:r.ciJ!Plllll 

## of' the 

## P'll).C.!:'eaa, stonm()h, colon, kidney ¢.lid 

## lung 

## and 

## sal.'<:olllli. of 

## the bladder lntt 

## not 

## w:i.J;:'b. no.rl!lal ch:t.ciketJ,, 

## lli:Pna_e, or 

## I:IU!li8U !;issues. Only 

## 11% 

## o:f; the 

## t;e!Jt i"eslllct:s 

## Jl'are poaid.ve when 

## uo:cm111 antigens were 

## u.sed whereaa 

## 20.% of the 

## carcinPmas,**~0%** 

## of the 

## sarcomas and 85% o:f the 

# lympnontas wei'e positive. Thus there are 

## common d:oss-teS.et:tve 

## l!lltigenl.l between 

## MD sud humap, 

## t!lll!ots; the nlltute t!f 

## th!lse 

## antigens 

## b uuknown. However, this observation does 

## not necessarily 11.nk MD vtrua and**h~an** 

## eancet.


# I ' ' 

# i f

## 21 

## Pass:i.ve illllllw.tity could be transferred to guinea p:i.g uterine segments with serum from patients with

**~81\Cer.** 

## However, using this test 63% of subjects with diseases other than cancer, 74% of aubjects with. carcinomas a:nd 5.7% 

## of subjects 'il'itll !llelanoua and sarcomas reacted t:o MD virus antigens. Only 6% of 

## 26 these sera reacted with herpes simplex virus or 

## l!:ll: virus. Theile findings t:end to bring into question the specificity of the test.
'When deoxyribosl!-purine fragments from variou.a cancerous tis.sues were use<!. to :i.mmani:te guinea !'ig11 and the uteri war!> tested with tumor pol.ysaccharide substance as rfl!ieasing anti3en, reactions were obta:l.ned between cancerous ti!lsUes and vird antigens at dilutions of 

## 0.8 to 4,9 x 1012 whereas reactions with control al\tigen were 0.4 x 1012 and normal iung tissue did not react with either viral. Pr epntrol aut;tgen.26



## ~en **vir~:~.l** ppl.ysaccharide substance ant;l.gens fro111 MD virus, Ell virus, herpes simpl:u: vituaes types l and II, and mouse hepatitis virus were used as releasing antigens and MD virus and EB virus used as immunizing antigens, cr<Jss

**~:esctivity** 

## was obtained. The highest 

## t:!.terll were obtained with EB virus and MD virus and the lowest titers with these two viruses all.d herpes simplex and mouse hepatitis virus,

## The above results confir,:n the presence of common**antigen~** 

## amou11 ma,ny herpesvituse$ from diffetent species of amphibians, mammals and birds. f!oweve:r, they do not relat!\1 

## tp tl!e 

## ptib:Uc l!ealt:h ;tmp!i<::ati<liJJ:J of AOHV,



## AtteQlpts have be-en 

## made t9 sh<;>W 

## that avian herpeeviruses .have 1ir9ader hpf!t range th,an 

## just aVian species by 

## inQculating**the~~~** 

## :tnto laborl!tory .anin!alli 1 

## part:tcU1ai.l;y 

## m!UIIlMls. Succ.essrul 

## induction of malignancy 

## in 111ice 

## :foi1otif.tng inocul,ati<ln qf

## t~o.t ma,1:J>rl<il 

## 'b:o1l! cMekenl! wihlt ·110·

## w~s iiMedbe!l in A 

## $hart abst:raet.e l'AI! 

## mat11tial increasl!i! 'the iuc:idence of lymphomas £tom 

# 3i to apptoximately 30%. 

## Additionally, bununi.tation or mice with HV'r 

## protec:ted them against challenge with mouse ·tumor JUate:rial induced by 

## MD tumor inoculation.e No subsequent 

## studies have been reported, Others have '!:ailed



## eA,tuoa JM,**~;telsen** 

## Ail, Morse PA, "Wetder AA: 

## Increased n.umher o'!: 

## ,lympboma·s ii1 

## udce inoculated with 

## Ma.tek'a disease tuinot. 

## In AbstJ:acts of 

## the 

## 71th 

# -.-

.. 

### atinua! 

## meeting of the Auledcan 

## Socl.ety £01; 

## Ml.crobioJ.ogy, 

## New Orlesna, 

## LA. 

## 8-13, pp 

## 92, 1971.

...... 

## '



# .I



# l l 

# ' t

## 23 to induce tumors in Chinese a!ld Syrian hamsters, and. Lewis rats inoculated 27 

## when new born and kept far six months. .No viremia or antibody deteetable by £1uarescent antibody immunodiffusion tests was present. Inoel(l.at;l.a!;! or t!ln cynomolgns, 8 rhesus, and 2 bonnet monkeys with cell-associated or. C:ell-'free l!VT or cell-!ls!lociated MDV failed to induce

. . .. . . . 17 

## tumors or other diaea.se after ll to 31 months

**ob~;ervation.** 

## Also no Viremia was detected. Sera of 3 HVT inoculated monkeys t:eacted With HVT antifilll:t 

## iln a fluorescent anUbody test, whereas 8 Ill). 

## virus inoculated monkeys reuined negative. No neutralizing sntib!'dy was detacted. In .a f!'ll.ow-up st!ldy 14 new born, 25 juvenile, and 24 adult mat!lloaet l!lankeys were :tnjected With similar matarial and observed for 12 to 18 months.2

## li No Q.Unica.l signs o! di!!ealle att:ribut:able to tha inoculum were seen. None of the treated monlteya wa.s vmmic when tested approximately 4 weeks post inoculation. Sera from

**o~** 

## .Qf l4 marmoseta inQculated With liV:r and of 2 of 2.9 .mamoset.s given MD 

## vi:rusc reacted,against HVT or .MD 

### vii'liS 

## in a fluorescent antibody test. Howavet:, pre:l:noculat:ion s.ers from 2 of 3 reactor marmosets were also positive by 

## this test. these results could

**.t~ot** 

## be confitmed by the v:l.rus neutral.itat!.on

**t~st** 

## an.d thus the specificity ·of the

**s~rologic** 

## reaction.s remains equivocal. A similar absence of tumors and viremia was reported in 5 adult rhesus monkeys

## 24 inoculatll.d 

## w:!:l;h a

# non-Qnco~tet~ic $train of MD virus.

# i!? l'r1nllite inoeulation111 also yielded

# Mgativ~ resul.tll

# ~ith an attenuated stratn of MD viruaJ


## .The absence oJ 

## tumtn:- lnduction in 

## pril)l<ltes certainly 

## lends sup!lort .to 

## J;:he conclusion that 'ldJ)·· 

## ·vil."lls and IIV'l' are**iion~pathogenie** 

## in primates l!.nd, by 

## extensioU., in bUllll!.'!rs. 

## Hol@Ver, :because 

## of the iiature 

## of the e:r;J)etfme1lts, 

## iio definitiVe 

## conolus:l.ons can be 

## iiraw. 'l'ha AOHV'

## hav~ ·a rkrr61<!' hp11t r.l:n;ge and· qnly 

## infect and 'i"l'!pllcate in a few sped.es 

## of 

## birds. 

## 'they do not 

## a.ppear 

## to infect and 

## replieate in malll.lllltls., including 

## primates. It is 

## there.fora UiU.ikely 

## that l:hey will 

## infect. llnd repli¢ate in hUllll!.ns.



## lf 

## AOHV' coilld prop!lgate read!1.y !n mammaUan cella" then they might 

## in£ect

- ' ' 

### l!lllllillUtll!, 

## The IIVT produte<l .a eytopatbqgenic. eJ;fect !11**hillill~t;er** 

### kid.11ey 

## agd,

# l!iada~r eplthe1ta1 cells:.410 ·Tl.te vlrus :was paaaage4 successively 

## in hamster .ceUs and tQ 

## ¢hieken embcy!l cells, io.t>t:laet\J'!ll p:repa:t!')d :l!l 

## J::hililtena agaJ.nst 

## this Virus ·nelit:rldized it 

## but antiserum t:o herpes simplex 

## v:ttus type l. or type


## .fdeBoer G: Central llieregeneeskundug Instituut, Lelystad., The Netherlands. 

## Personal communication.



# . 

# ' ! I 

## j ' 

## • 

# l

# ~ '

## 25 2 did nut neutraUze it. The MD virus and HVt 

## produced a cytopathic effect in hal!lstet kidney cell.s even after 6 to 7 passages onto newly prepared hall!ster 

## 31
. . . kidney monolayers. Virus -particles were found in infected cell cultures and the cultures contai.ned ext:emaive polykaryocyte.a.32 the virus prop';igating in hamster kidney cells was iaentical to the HVT used to inoculate those cells by complement; fiXIltion, fluorescent antibody and serum neutt'alization tests. Subsequently, Ml) 

## virus was also shown to replicate in hamst.er kidney cells by l!erum nsuttalization, complement fixation, and fluorescent antibOdy testa.

## 33 S:!mila:rly, HV'l' and MD viz:us also propasated in p:ri!llary gu:f.nea pig kidney 

## 11 . 34,35 ce s. ln an**e;~ttensive** 

## and detailed examination of. this phenomenon., MD Vi:ttiS 

## or 

## ll'Vt' J.nf.ect;l.Vil:y 

## was fo!.lnd to persist for up 1:0 ten serial passages in 

## pt;!.lllary hamster kidnli!Y cultures inocul,ated with cell-assoc;iated MD Virus or 

## ltVT. However, infect:iV'ity was only present in these passaged cultures when avian cell·s, which were**pr~sent** 

## ill the inocjllum, persisted :lu the c.,Ut:ure.a. tnfectiv:lty could be eliminated by treatment with cytotoxic**anti~avian** 

## cell antibody and .:ell-free virus was unable to infect th<l mallll'l1alian cultures aloue.36 Others have been unable to infect primary hamster kidney cultures.27

MD virus was unable to produce a cytopathic effect in cultures of 15 m4ID!llalian species including dog, cat, m.ink, swine, bovine, goat, equine,

## namster, mou.se, 

## ·bat, -ra'bbit 1 

## potprqo, sp()t,teq dolphin, 

## sreen moti.key ·and hU!l!8.n.;l

## 7 S;l.l!lilar reaul:ts were obtained 

## with 

## HVT on 

## human, dog, 

## cdi!, baboon, rabbit 

## and pig



# cell~,

## 30 Even after 

## the llvt 

## was



# p~opa~t..d :i.n hamat:er 

## ki<luey cells for 

## aevetal passages, 

## it was unable to inreet these 

## tissue cuitures. 

# .• 26

Neither 

## MD vltil's nor 

## liVl' would

**repl;i~ate** 

## :i.n. cell cilltl.!ril'i! 

# tit u,ttiiol!!!!t kidney, Ullll;lnoset

**~l<;in** 

## and muscle,, hlilnan 

## -embryonic kidneY> 

## and 

## the hUiila..n -cell line



# ·wl~3a.Z **8**



# 'l!reat~teut with ililethy:tlll'n:f.tioeth:iiidextran or 6ltrliv;ta1et 

## lrradiJ,ated 

## SeMai 

# 'Yit1111 ·or

**co~.c\llti**

## v!!Hon 'wit:h X"--irl;'adiated 

## iv-i.m"':i.l\feeted 

## avian cel'la.

## <Hii not tesillt 

## :tn teplitation. ln 

## another 

## .stull.y,ceil"'sa.shc:litted W ViJ;us and 

## ci!il.l-ftee llVT 

## dtd n()t 

## infect Chinese ha!l!ste't, Syrian

**:b.SUIIlt~r.** 

## bovine, do)!, human, 

## rabbit t!r ,!)OtOl"!fU$ 

## ¢<al,ls.27 Simil.a"!.' "!.'eelllts

## ,have heel! obtained 

## by othet's.Jll,S9•40


## Once 

## again.mamma,lian 

## caUs were 

## refractory to 

## infection even 

## under conditiollil of 

## artlfiC:i.d ce:11 'hyl:f'tidtzati()11 

## 'between v'lrus-infectei! 

## ce1is and the lliSIIWUiliali 

### c;ells-

## With t!lt"!.'aVio,l.et iMC'itiVated 

## send!;i 

## virus. Thuli AO!W 

## replicate 

## in hybrid 

## cells composed of'·mammalian 

## ad avian

**hetel':()~aty.ona**

## •

. -,,,

## 1..7 

## Viruses that fail**~o** 

## infect**~ammal~sn** 

## cell cultures are less likely to infect nu1llltnB.ls than are v:irus<aa that infect and grow well iri ma.nunal.ian ceU cultures.**OpPQ~tunistic.** 

## !nfections
Birds effectii!d with ID and heaJ.thy birds were eL!llllined for the presence of bacteria that might pos11 a public health 11<\zard.**~-te_tobacteria** 

## were three to**~ne** 

## times more freqne'(ltly encountered in the visl.'lers. of diseased birds than in the sue 

## organs of controls. In two cases Saimonella were isolated from affected chickens and in three cases enterobacteria were isolated from the musc.u1ature 

# o£ affectea birds.

# 41 The

# b~rds with overt disease, in thl.s case MD, would be eXpected to be less**re!!~Stant** 

## to bacteria,!. infect;ion. J!qwe:ve:r, these infection& are likely no more**~ommon a~ong**  

## birds affected with MD than among b:l,rds with ot]J.er ailments such as injuries.
Circumstantial Evf,dence
There have been many reports- of an association between cancer in animals and cancer :l,n man. There have also been reports of organisms bolated from human and animal nmlipancies which are highly pleo!l!Orphic and variable in many characteristic!! and which are capable of developing virul!-lik!'!

# 28 bodies.42 However, controlled ell:perimental evidence 

## of an etio:lo,gic J:elatio1.l!!hip l>etween these prg11nisms and human can.cer is 

## lacUng. S:illlill!ltly there 

## b.l!lve**'b~>en** 

## many 

## teports o;f cancer vaccines 11nd cures. The ol11y info:t111atio11 avai_iable l1as 

## :heen thtQugh the pnblic media, through interv:l.i!ws 

## l>y the proj)ommts, 

## or fn :privately printed 1>1ibliaatiQ11s that an**~:~at** 

## avail11.hle 

## to li'brad.es ¢%' 

## the gen.erd**pu:b1~c.** 

## T!)e in.fotmatiQn is n.ot publi11hed 

## in the open medical Qt 

## !l"cieudlJ.c literature whel:e it 

## can he reviewed and v!llidated )ly 

## pther Mied_iJJts.

Current techniques Qr appJ:oaches for attempHug 

## to relil.olve the public health**concern~;~** 

## and the -resW.ts 

## of using these technique" 

## s're**s~iiiz·ed** 

### in. 

## Table 2, Unequ!vocallY positive**f:l.~dings** 

## using e11eh of 

## theae toola coulil ·l;ulve 

## incriminated MD o.- itt> 

## vae_c!ne v:I.:Nae!! or both a11 public health concerns. 'thus a highly s;!,jJliifJ.cant cotre:t.af:ion betwe.en il. 

## pUblic hel\lth (!.Qn(iij:ion**~** 

## lhJh• 

## leukemia, and exposure to an AOHV, e.g., 

## MD vi:rua, in the a'baence of a correlation between 

## tih"' cond:l.!;lon and other factors, e.g., c<>tnpJ;iJdncdoii: a:nd, fertilizer use, coUld have strongly ·suppo-rted the hypothesis that 

## MD v1ru11 

## ·had something to do with the human leukemia. S:f.m:f.lat 

## exa!Dples· 

## o.f findings using other techll,iques which would st:tongly support this hypothesis ate in**~able** 

## 2•

... 

### -, 

# !

## 29 Negative findil!gS using each of the techniques currently available, namely statistical epidemiology, serological epidemiology,**~1** 

## inoculation, cell culture inoculation, and compal:ison of etiologic agents cannqt refute the nypothe:sis that the public health condition is caused by exposure to the AO.HV. Thus, for example, the absence of a correlation betll'een a public hl!lalth condition and expo.sure to AOHV does not confirm that there is no relationship between the two. One can conclude only that thb technique failed to f:i.ud 

## a relationllhip uuder tile conditions and :l.n the circWIIstances in**whi~** 

## it was Ui!led, This is the basis for the conclusions of the expert committees and eomi1111ioUiil referretl to previ.ousl.y in this paper.

rrom the above discusslon it appears unlfkely that additional efforta to use the t!!.chniquea c\lrrently a'lailable w:l.ll. 

## reeol.v!l the public he.al.th concel;'lls of 11D and 

## :Us**va~:<:~ines,** 

## Certainly research resu.lts of the last 8 years have :failed t<> 

## brinl:l: u!! a,ny closer to a resolution of the J;l!aue.2 A ftitther way of ahe!idilll:l: l.ight on the possible relationsh:l.p il! 

## ,in the use of the uew molecular :biology ·tools to study the .Pathogenesis of cancer in man and an:imals. 

## If a unique ::o1e for the AOHV can be found in the molecular pathc;>geues1s of htm!an cancer, then the public heal.tl'; hazard of the virus will be co.Uined. It is important to note that just finding portions of a viral genome in htilllan tumors, e.g., the mye and arc. oncogenes of avian leukosis

# 30 ' ' 1 viruses, is inauf:ficien-t 

# it '!:hue 

## gen-es are also presen-t in Ml:!!lal 

## cells. Coaversely, if the 

## molecular pathogenesis of all lil.lill<!ll. 

## ¢iltl.cer is ellplain-ed 

## and no 

## role is 

## found 'fl:fr any 

## unique AOHV 

## genes or gene 

## products, theu. 

## the abseuce of a public health hazard 

## call, be confirmed.



## This comprehetuiiire 'tev:l.ew 

## of the. 

## t:l.terllture 'bdn.gs 

## together, for the ii:t:lit time, 

## a laz::ge body o'f 

## :l.nfo:rlllation •relating t.o AQ!{V 

## Sll.d hlllll!ln health hazards., patt:l.tl!l4tlY 

## hull!l!'u**canc~.r..** 

### 'l'l!'is 

## -review shol!ld be u11e'fl!l J;o 

## scielltiJ!ts and adm!q;isnators in public res!!at:ch 

## and action a,geneiell and 

## 1.n private industry. 

## 'l'lle rav:::tew Cl.lvera 

## the nUII1eroUII 

## al:hli!pt!! .to 

## resolve the. q,ueation 

## ·of tthel:her AORV pose any ptiblie health hazard 

## to htlll!aus. 'fhe large 

## body q£ 

## experimental evidence 

## in both

## .avian 

## and hwar. 

## virolpgy, sero1og;<, 

## pathology• 

## and epidellllology nrongly aupp<:>rts .the 

## eouclua:ton 'tMt 

## .P:o 

## et!ol(lgic :relatioaship 

## exiata bel:)faen AOHV 

## and human <;llllcer• Thqa, 

## in our 

## optnion 11nd 

## in the 

## opinion of experts in 

## the human and avian cancer 

## field, MD all.d vaccines posaible, 

## there is suffic:ienl: 

## ev:l;dence reviewed iu this 

## paller to warr;tnt 

## ·thia firm 

## oouc1us1on.



## ···-·

### REFERENCES


### 1. 

## Marx JL, Kolata G., Lewin 

# a. l'umoi: vi.ruses and oncogenes, Am. 

## As

## soc Advancement of 

## Sci, 1984;31.
2. 

## l'heileu Ga:. /.'Ublic: 

## t!ealth aspects of animal tumor viruse!!. l'heriogenology 

## 1976;6:331-342.
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